That's the question which pegs the world to this day. science I believe can be a instrument of making life easier whether or not it can be used for a good thing or a bad thing.

If we could limit the use of our intelligence for repetitive task and focused on the task that our current machines cannot comprehend it would lead to a more efficient growth in knowledge.

This small population is credited to the expansion and development of technology that the average person uses in day to day life.

the amount of accuracy of current technology is almost sci-fi to me. Imagine if these doubled!

I see the use of this in many popular songs

The common accessibility to High Definition microphotography in institutions will greatly benefit science due to the fact that people who don't have the physical subject or a device to zoom and view the subject, can still learn in detail.

I wonder if the Laser Printer uses a similar process to print papers without the use of ink?

Most likely the reason why Digital Cameras are so popular today.

This is a true statement. Scientist should look though a ethical lens always but needs logical step process. Same thing can be said for people using technology.

The science field is not stagnant. After one study , hypothesis or even conclusion, the expansion of the subject will always grow.

Modern Vehicles, Computers, Phones and other devices all contain interchangeable parts. Each part has its own specific purpose and often each part is specifically made for that specific device. Despite this, often it's possible to repair the parts and have a reliable device or invention.

With the cooperation of the thinking man and the accumulation of mans knowledge (inventions) the opportunity to learn and discover is almost exponential.

With deferring information from multiple studies and conclusions from different investigators , how do come to a widely accepted statement/answer to this investigation?

Millions and millions of dollars towards the development of destructive weapons lead by some of the most educated individuals but why isn't there a focus on the development of non lethal weapons, and the study of obtaining and maintaining peace in different cultures.

This is most likely inferring that there needs to be a greater focus on the thinking man, then rather the people relying the accumulation of knowledge throughout human history.

I agree that in fact, inventions have improved the physical powers of man. The other effect of inventions is that Man isn't using their mind to their full potential of the mind.

I agree with this statement. Relief shouldn't be a concept of being "secure" because that can be controlled to a point where manipulation can be issued. I believe technology should be free to transform or upgrade instead of being manipulated by others.

This statement remains content though out human history and the same can be said for technology. We humans went though trail and error though out history to see what works and what doesn't work. Technology is the same with numbers, programs and sites too. It's progress.

tabs or read it later sometimes really make me anxious.I think we just need to take it easy.

I like that this sentence suggests we cannot predict the future. I like how it hones in on the unpredictable and the reality that no matter how much control we have in our lives, each moment is different and unreliable in the sense that things don't always work out as planned. It's comforting to think that our stories are literally made up of what "we can make at the time." It's not all structured and concise and clear. It actually is muddy and messy and unexpected.

In an earlier sentence, the author writes, "The process of invention may occur...at some subterranean level." Subterranean means "concealed." This sentences intertwine because at the moment plays out, what will happen is subterranean. We simply cannot predict how we are changed or how we live in the moment.

Task 3: Be able to understand the text and be able to put it into your own words.

1.) What are the central arguments or main point in the text? The text supports its main idea, that philosophy needs to be studied to be cleared of misunderstandings, by pointing out the effects philosophy has on the human mind, and, therefore, why it is of use to humanity. Russell makes the argument that even though philosophy has no way to be verified like the other sciences, it prods the human mind into thinking more broadly and creatively by forcing people to ask perplexing or uncomfortable questions. Another point which Russell makes is that philosophy gives the mind potential to think so impersonally as to view things in a nearly god-like way.

2.) How are arguments or main points supported? That is, what reasons are given to support the conclusions? Russell supports his arguments with broad assertions concerning the machinations of the mind and people's attitudes toward philosophy. He writes in such a way as to parrot what is heard in real life concerning attitudes toward philosophy, most noticeable when he talks about "practical people."

3.) Note any assumptions the author gives without giving evidence. Strangely enough, Russell backs not one section of his argument with data of any kind. His assertions are made merely by what he has been met with in his experiences. There are no polls or graphs about people's attitudes toward philosophy. There are no quotes about what people think of philosophy. There is nothing which qualifies or quantifies besides what Russell himself asserts. Even while this is so, the subject Russell is commenting upon is, in and of itself, abstract. What Russell seems to be demanding of his readers, on a subliminal level, is use of common sense and critical faculties to piece together his information in a way we can except. Referring back to my second point about what Russell is arguing in his text, the idea that humanity can achieve a worldview in proximity to that of God (which, from what has been presented thus far to this class about Russell, I can only assume he means to use God as an allegory for some universal principle) is quite a leap to make. Russell assumes that if humanity abandons its territorial, material concerns that we may ascend to a point of knowledge of ourselves and the universe that will bind us to the latter like never before.

4.) Reread important passages and note sections of the text you cannot make sense of. I cannot really find anything in the text that I cannot make sense of, except in the meaning of the not-Self. I understand it well enough to see what Russell means by it, but I am left with ideas about its implications (for lack of a better word). However, that is not altogether what is meant by the question.

Author Response:

Reviewer #2:

This study by Schulz et al flushes out in fine detail an interesting consequences of inhibitory synaptic plasticity in plastic neuronal networks, showing that its ability to balance precisely only previously experienced stimuli makes this type pf plasticity an excellent candidate mechanism to allow novelty detection. Strong transient responses will be evoked only by those stimuli which have not previously activated (and thus trained) the stimulus specific set of inhibitory stimuli. An open question remains with regard to the time scales and speed of learning at these inhibitory sites, something that will be answerable by the experimental audience of this paper (but could be investigated in a bit more detail in the model as well).

We thank the reviewer for their constructive feedback. We have addressed the comments below, and included new substantial information to our manuscript. This includes a new Supplementary Figure (Figure 4-Figure Supplement 2) in which we analyze how the novelty response and the adapted responses depend on the inhibitory learning rate and a detailed discussion about experimental and theoretical studies related to inhibitory plasticity.

Reviewer #3:

This computational paper addresses the mechanisms of sensory adaptation and novelty detection in the auditory cortex. A spiking RNN model of 5000 (4000 excitatory/1000 inhibitory) units is developed and adapted to sequences of inputs (ABC…) followed by a novel stimulus N. As with experiments the model captures the adaptation to the repeated stimuli, as well as a strong response to a novel stimulus. In contrast to many models of sensory adaptation that rely on short-term synaptic plasticity, here adaptation arises from STDP at the Inh->Ex connection. Specifically, during ABCABC … the inhibitory connection onto active Ex units is enhanced through associative plasticity mechanisms, but not onto the inactive Ex units, thus the adaptation does not apply to the novel stimuli. While the approach seems fairly novel it is also speculative and seems to run contrary to the existing experimental data.

We thank the reviewer for their feedback. In our view, through the process of answering the comments below, we have improved our manuscript substantially. To answer the reviewer’s comments, we added new figures to the manuscript (Figure 5) and (Figure 6-Figure Supplement 1 and Figure 1-Figure Supplement 1,5) and added new text in the Discussion (see ‘Timescales of plasticity mechanisms’, and ‘Robustness of the model’). In the following, we give detailed answers to each of the comments.

1) The reason many models of adaptation focus on short-term synaptic plasticity (STP) as opposed to STDP is that the later generally is not thought to operate on the adaptation time scale of seconds. Specifically, STDP is generally considered to be a form of long-term associative plasticity, and thus to rely on mechanisms such as the insertion of new receptors-a processes that is unlikely to operate on a time scale of a second or so. Adaptation is robustly observed at 400 ms (e.g., Natan et al 2017), a time scale that is generally considered to be incompatible with STDP. E.g. in the D'Amour paper the authors cite, STDP is induced over a 5 minute pairing protocol, and can still increase over the course of 5-10 minutes post pairing (e.g., Fig 1H). I'm not aware of any evidence suggesting that iSTDP could be induced and expressed on the subsecond to a few seconds time scales. So this seems to be a fundamental issue that needs to addressed or at least discussed.

Related to the point above the model also contains subtractive normalization implemented with a time step of 20 ms. Again if this normalization is critical to the model this assumption would pose a serious challenge to the model because there is little or no experimental data suggesting that normalization can operate at that time scale.

In our new Figure 1-Figure Supplement 5 we find that the timescale of the subtractive normal- ization mechanism does not influence the generation of novelty responses in our model. Adaptation occurs over multiple timescales from hundreds of milliseconds to tens of seconds or even days [Ulanovsky et al., 2004, Lundstrom et al., 2010, Homann et al., 2017, Latimer et al., 2019, Haak et al., 2014, Ramaswami, 2014]. Our work shows that inhibitory plasticity can readily lead to adaptation on multiple timescales without the need for any additional assumptions. Although during the induction of inhibitory STDP it takes several minutes to sev- eral tens of minutes to achieve a stable baseline of inhibitory synaptic strength [D’amour and Froemke, 2015, Field et al., 2020], inhibitory postsynaptic currents increase significantly immediately after the induction of plasticity (see e.g. [D’amour and Froemke, 2015, Field et al., 2020]). Therefore, inhibitory synaptic strength seems to already change during the plasticity induction protocol. Hence, we propose that inhibitory STDP is a suitable, though clearly not the only, candidate to explain the generation of novelty responses and adaptive phenomena occurring over multiple timescales.

Justification:

The points raised by the reviewer are extremely valuable in critically evaluating the model. We first address the issue of the timescale of normalization.

The generation of adapted and novelty responses in our model does not rely on the fast subtractive normal- ization mechanism, since the normalization only affects the excitatory and not the inhibitory weights; and it is the change in inhibitory synaptic weights through iSTDP that is the key mechanism to explain adapted and novelty responses. In Figure 1-Figure Supplement 5 we now show that even for a normalization time step of 50 seconds, adaptation to repeated stimuli and a novelty response occur. Therefore, we can conclude that the fast normalization mechanism is not a necessary ingredient in our model. We added a discussion at line 542 and in the Methods at line 749. Even if no normalization is applied throughout the entire stimulation paradigm, our findings do not change.

We note that while normalization is common practice in circuit models, there is a discrepancy between the fast timescales of normalization mechanisms used in computational models to stabilize network dynamics and the much slower timescales measured experimentally ([Fox and Stryker, 2017, Turrigiano, 2017, Keck et al., 2017], among others). This discrepancy has been termed the ‘temporal paradox’ [Zenke and Gerstner, 2017, Zenke et al., 2017].

Many computational models which implement normalization mechanisms justify them by the experimentally observed synaptic scaling despite the discrepancy between timescales (see e.g. Figure 1 in [Zenke et al., 2017]), which we now acknowledge at line 543. In related work, we propose a different biologically plausible candidate for fast homeostatic stabilization – heterosynaptic plasticity – which operates on similar timescales as homosy- naptic plasticity mechanisms [Field et al., 2020]. Incorporating this mechanism in addition to, or instead of, the fast normalization in recurrent networks is very interesting but beyond the scope of this work.

Next, we address the issue of the timescale of iSTDP. We studied long-term iSTDP as a candidate mech- anism for the generation of adapted and novel responses for multiple reasons: (1) to explain adaptation over multiple timescales which range from hundreds of milliseconds to tens of seconds [Ulanovsky et al., 2004, Lundstrom et al., 2010, Homann et al., 2017, Latimer et al., 2019], and even multiple days in the case of habitu- ation [Haak et al., 2014, Ramaswami, 2014]. Rather than including STP mechanisms that operate over all those different timescales, we demonstrate that iSTDP is a straightforward mechanism to bridge different timescales without the need of multiple mechanisms or fine-tuning of parameters (Figure 4). (2) We were inspired by the growing experimental literature suggesting an important role of inhibition and inhibitory plasticity in adaptive phenomena (see Discussion subsection “Inhibitory plasticity as an adaptive mechanism”). (3) In computational models, iSTDP is usually studied in the context of balancing excitation [Sprekeler, 2017]. In our study, we present functional consequences of inhibitory plasticity.

Although inhibitory plasticity is indeed induced over several minutes in pairing experiments [Field et al., 2020, D’amour and Froemke, 2015], inhibitory postsynaptic currents are already increased directly after plasticity in- duction – though it takes additionally several minutes to reach a stable new baseline (e.g. Figure 2A,B in [Field et al., 2020]). For example, the mean increase of inhibitory synaptic strength right after plasticity in- duction in [Field et al., 2020] is approximately 30-50%, while a new stable baseline at about 80-100% increase is reached after approximately 20 minutes (Figure 2A in [Field et al., 2020]; similar results in Figure 1H,I in [D’amour and Froemke, 2015]). This suggests that significant changes of inhibitory synaptic strength in iSTDP experiments already occur while the plasticity induction protocol is still ongoing. How fast these plasticity mech- anisms act in an in vivo setting during stimulation with naturalistic stimuli is to our knowledge not known. In general, the question of the true timescale of iSTDP is still an open problem [Sprekeler, 2017].

We also point out that the inhibitory synaptic weight changes induced via iSTDP are rather small in our model, i.e. Figure 4C shows that the mean inhibitory synaptic weights onto the adapted excitatory population increase approximately 15-20%. Therefore, we propose that relatively small inhibitory weight changes are suffi- cient for the occurrence of a novelty response and these weight changes might already be happening during the paring protocol in experiments, as we argue above.

Although we agree with the reviewer that short-term plasticity mechanisms are an important aspect to understand adaptation phenomena (especially on short timescales), we would not a priori exclude iSTDP only based on the argument of timescales. To get a full understanding of adaptive phenomena on all timescales, more detailed experimental and theoretical studies are needed to investigate the role of short versus long-term plasticity of excitatory and inhibitory synapses and how these mechanisms interact in a recurrent circuit.

Modifications:

In the new Figure 1-Figure Supplement 5 we study the effect of the timescale of subtractive normalization on the occurrence of a novelty response. In Figure 4-Figure Supplement 2 we quantify the response amplitude and the decay time constant of the novelty response as a function of the learning rate of inhibitory plasticity (see also our response to comment 1 of reviewer 2). Indeed, we find that fast inhibitory plasticity is needed to detect a novelty response. We discuss the mismatch between timescales of homeostatic plasticity in theoretical and experimental studies in lines 543 and 739. Additionally, we added new text in the Discussion subsections ‘Timescales of plasticity mechanism’ (line 509) and ‘Robustness of the model’ (line 533) where we discuss the timescale of inhibitory plasticity and subtractive normalization.

A further issue relates to the temporal structure of adaptation. The authors show that adaption is independent of the sequence of the stimuli (ABCABC vs BACCBA) (it would be best to refer to this as sequential structure not temporal structure, which would often include the duration of stimuli and interval between stimuli). It is well established that the longer the interstimulus interval the less the adaptation. The model may or may not capture this effect depending on the assumptions regarding the spontaneous activity during the ISI as a result of the non-associative (pre-only) iLTD. However, given that STDP generally grows after induction it seems like the model is not likely to capture the standard observation that adapation should be less if the stimuli are presented with an ISI of 800 ms versus 400 ms. In figure 5, for example what happens if stimuli are presented for 20 seconds consecutively versus for 10 second then a silence of 2 seconds before another 10 second stimulation? No mention of the time course of spontaneous recovery from adaptation is made.

Recovery from adaptation depends on the background activity level in the network during the inter- stimulus interval (Figure 5 and Figure 6-Figure Supplement 1). Specifically, low background activity between stimulus presentations slows the recovery from adaptation (Figure 6-Figure Supplement 1). However, increasing background activity between stimulus presentations can capture the decreased adaptation as the inter-stimulus interval increases (we show this in our new Figure 5).

Justification:

We agree with the reviewer that the term ‘temporal structure’ is misleading, and therefore exchanged it with the term ‘sequence structure’ in our manuscript (see for e.g. line 228).

As the reviewer aptly predicted, the recovery of the response from adaptation indeed depends on the level of background activity between two stimulus presentations. In our model, the direction of inhibitory weight change (iLTD or iLTP) depends on the firing rate of the postsynaptic excitatory cells (see [Vogels et al., 2011]). Postsynaptic firing rates above a ‘target firing rate’ will on average lead to iLTP, while postsynaptic firing rates above the target firing rate will lead to iLTD. In turn, the average magnitude of inhibitory weight change depends on the firing rate of the presynaptic inhibitory neurons (see [Vogels et al., 2011]). Therefore, if the background activity between two stimulus presentation in our model is very low, recovery from adaptation only happens on a very slow timescale. To show this, we performed simulations similar to Figure 6 where a stimulus (A) was presented again after a pause of either 9 seconds (Figure 6-Figure Supplement 1A) or after 225 seconds (Figure 6-Figure Supplement 1B). Whereas the response to the stimulus was still adapted after 9 seconds, it fully recovers after more than 200 seconds. As expected, the stimulus-specific inhibitory weights decreased very slowly after stimulus presentation (Figure 6-Figure Supplement 1A, B; bottom). This slow decrease of inhibitory weights follows from the fact that the network is silent if no stimulus is being presented. We now discuss this result in line 357.

However, if the background activity in the inter-stimulus interval is higher (either because of a higher back- ground firing rate or because of evoked activity from other sources, for example other stimuli), the adapted stimulus can recover faster. To address how such elevated background activity can affect adaptation to a specific stimulus, we performed additional simulations (Figure 5), in which we used the experimental paradigm from Figure 1A. Similar to Figure 2C, we changed the number of stimuli in the sequence, which leads to different inter-repetition intervals (the interval until the same stimulus is presented again) of a repeated sequence stimu- lus. For example, if two repeated stimuli (A, B) are presented, the inter-repetition interval for each stimulus is 300 ms apart because each stimulus is presented for 300 ms. If four repeated stimuli are presented (A, B, C, D), the inter-repetition interval for each stimulus is 900 ms. Importantly, this means that in the time between the presentation of the same stimulus, the network is not silent (as in Figure 6-Figure Supplement 1), but active because other stimuli in the sequence are presented. We defined the adaptation level as the difference of the onset population rate, measured at the onset of the stimulation, and the baseline rate, measured shortly before the presentation of a novel stimulus. We found that an increase in the inter-repetition interval reduced the adaptation level of the excitatory population (Figure 5A, D) due to a decrease of inhibitory synaptic strength onto stimulus-specific assemblies (Figure 5B, E). Therefore, we conclude that our model can capture the reduced adaptation for longer inter-repetition intervals when background activity in the inter-repetition interval is elevated, in this case because of the presentation of other stimuli.

Modifications:

We replaced the term ‘temporal structure’ with the term ‘sequence structure’ in our manuscript (Results section “Stimulus periodicity in the sequence is not required for the generation of a novelty response”, line 211). We also included a new main figure to demonstrate the effect of varying the inter-repetition interval in the presence of evoked network activity from other stimuli (Figure 5), discussed in the new section ‘The adapted response depends on the interval between stimulus presentations‘” on line 306. Furthermore, we added Figure 6-Figure Supplement 1 to the manuscript and we discuss our findings in line 357 and line 469.

3) It also does not seem like the model will capture recently reported effects such as the observation that optogenetic inactivation of inhibitory neurons during pulse n can actually increase adaptation to tone n+1 (Seay et al, 2020), indeed I believe the current model would make the opposite prediction

Our model cannot capture the n+1 experiment in [Seay et al., 2020] because inactivation of inhibition will always increase the response to stimulus n (as in our disinhibitory experiment in Figure 7), hence decreasing adaptation.

Justification:

[Seay et al., 2020] measured several different types of short-term plasticity in the auditory cortex at synapses involving two different types of inhibitory interneurons: strong feedforward short-term depression onto PV interneurons and from PV to pyramidal neurons, as well as strong feedforward short-term facilitation onto SST interneurons and very weak short-term facilitation from SST to pyramidal neurons. We believe that including different interneuron types, as well as short-term dynamics of the respective synapses, might be nec- essary to explain this phenomenon. Indeed, using the experimentally observed short-term plasticity in a model, [Seay et al., 2020] showed that inactivation of PV interneurons can decrease the response, hence increase adap- tation, to the next (n+1) tone. Since our model includes a single type of inhibitory interneuron and implements long-term inhibitory plasticity rather than short-term plasticity, we are not surprised that our model cannot capture the increased adaptation in the very specific n+1 experiment. As we acknowledge in the manuscript, our proposed mechanism is not the only candidate to explain the generation of novelty responses and adaptive phenomena in the brain, and likely interacts with other types of plasticity and cell type dynamics.

Modifications:

We agree with the reviewer that the findings of [Seay et al., 2020] need to be discussed in context of our manuscript (see also our response to comment 9 of reviewer 1). Therefore, we added the reference and discuss it at line 499.

4) In its current state I don't think (I may be mistaken) the model accounts for a related and very general property of auditory cortex: lateral inhibition (e.g., Brosch and Schreiner, 1997; Phillips, Schreiner, Hasenstaub, 2017).

The term ‘lateral inhibition’ seems to have a somewhat different meaning in visual versus audi- tory cortex. In the visual cortex it is often considered as a spatial form of inhibition, while in the auditory cortex it also includes a temporal aspect. Since our model was mostly inspired by data in the visual cortex [Homann et al., 2017], we will answer the question in the context of the visual cortex, but also speculate about the auditory cortex.

Justification:

In the visual cortex, lateral inhibition is often defined in a ‘spatial’ manner whereby activated pyramidal neurons reduce the activity of their neighbors. Specifically, SOM-mediated spatial lateral inhibition contributes to surround suppression in visual cortex [Adesnik et al., 2012]. Our model already implements a form of spatial lateral inhibition. Based on experimental data (see e.g. [Harris and Mrsic-Flogel, 2013] for a review), we modeled inhibitory neurons as more broadly tuned than excitatory neurons, such that a single inhibitory neuron is more likely to be driven by multiple external stimuli (probability 15%) than a single exci- tatory neuron (probability 5%). During stimulus presentation, as inhibitory plasticity adjusts the strength of inhibitory-to-excitatory synapses, an inhibitory neuron with a given stimulus selectivity will likely strengthen synapses to multiple excitatory neurons selective to different stimuli – hence implementing spatial lateral inhi- bition.

In the auditory cortex, lateral inhibition is often referred to as ‘forward suppression’ (or ‘forward masking’) [Brosch and Schreiner, 1997, Phillips et al., 2017]. Here, a preceding ‘masker stimulus’ influences the response of a probe stimulus. This influence depends on several factors, including the time difference between the masker and the probe stimulus, as well as the frequency of the pure tone masker stimulus [Brosch and Schreiner, 1997]. The time differences measured in these experiments are usually too short to be captured by the inhibitory plasticity mechanism proposed in our model. Similar as our answer to comment 3, we suspect that capturing feedforward suppression requires short-term plasticity plasticity (for e.g. [Phillips et al., 2017]).

Modifications:

We now included additional text in the Methods to address the issue of spatial lateral inhibi- tion, see line 796 and we now mention the phenomenon of forward masking in line 504.

I am accepting charitable donations,.\ ETH: 0x66e2871ef39334962fb75ce34407f825d67ec434 | BTC: 38B6vGaqNvMyTtoFEZPmNvMS7icV6ZnPMm | xDAI: 0x66e2871ef39334962fb75ce34407f825d67ec434

Wednesday, June 23 2021. Nintendo Way

Erev ... the Day of the Holy Divorce of Bayjorel

Adam on "ho(s) I still single" ... I "hisss" as Alger, Narcissus and to the various collegiates in Massachusetts; know it's because I'm Cheyanne Mountain. You can't even dream how hard it is to get inside "this heart." Or maybe I can't fathom why nobody's rushing up to me trying to grab the ring of "infinite alimoney in the ever-after" ... Na Na Na Na ... Na Na Na Na ... shey shea way) ... tee tea, tay?

This messages marks a major increase in "forced read(layshion)ership" to include a significantly larger group of students and professors than before. This is a new system; please unsubscribe using the instructions at the bottom of the message, which are different from the prior newsletter interface. I have noticeably been writing much less and sort of working harder on bringing to fruition the software and social policy changes I've been dreaming of and writing about instead of "just talking."

Searching this message that I intend to send to the students a day early--you know, with foresight for ... in the hope that many of you remember first hand hearing the words "I don't believe in the big bang, but I respect those that do" echo from a computer screen to me subconsciously in the state of South Carolina--that you will help me end the 7 year draught [[literal, good sex]] that I equate to the Biblical overflow of the Nile and to Stone Temple Pilots; this light and Sheldon Harr who trained me for my Bawr Mitzvah and taught me all the right things that I know about being a good Jew who didn't really believe in the existence of God; but then helped create the system that makes us all that.

Those who "see" or "saw" Kentucky as I did might recall the phrase spoken from myself to myself; "you don't believe in God when you are this close to it's creation" ... or something almost verbatim; that. Some of you might see Gilgamesh more than I do, or have forgotten the "sliding of sleight of hand and becoming ... the trickster of the Dajjal ... "an idea that gore was being fabricated and faked; in order to help us see why it's so very importanat that at the same time that immortality and heaven become part of the conversation of the adbication of Odin's throne to Thor or to Arthor's table and plebescite "victims" ... that we all understand the magnanimous change wrought by Heaven on civilization and on the old customs and on the old laws, and that here we see the importance of guaranteeing safety and privacy and even "right to death" in a place where God had previously only written of "life and liberty" with the ambiguity of ... "from what" being left to my seemingly slow hand.

On the order of plans soon to be seen to fruition my large key of "what this website truly is" has grown to something like 20GB and now includes a static and time frozen version of everything linked to stored on the IPFS system and multi-homed across a number of "cloud providers" to ensure things like "shekinah" will not forever be changed to "shechina" with nobody noticing the loss of causal original truth. The "light of angels" domain now redirects automatically to /ipns/fromthemachina which should render in future shell-internet-browsers as something like QmTH33MwfPn5S3bq45Tk77L1j9eZjUsvEVhRTHB3D8M2ZX [please pin this "root block"] I am not sure why IPFS doesn't have better merkle tree searchability, but seeing siblings and parents and connections between these Qm hashesh is something that we should be working fervently on making more robust. IPSE.io appears to have created a decent search and governance system, I see it as something like the "electoral college" metacosmically linked to the thing I am trying to build--a preservation of all human knowledge and an infrastructure for discussing and communicating about the "veracity" and the linguistic nuances "alluded to" in the lude ties between this Empire's new Clot and the clothing worn by Popes and Jews, the seeit-seeit; tzit-tzit and ...

4-WORD AND SIX WITH "SHOOTER" ... YEARS HAVE GONE BY; AND I STILL HAVEN'T GOTTEN FVCK3D.

I am planning on suing several medical providers and states for what I see as heinous violations of human rights, decency and the Constitution of the United States; if you are a lawyer or you can recommend a good one, please email me as soon as possible at 0xc514f094370cFc5eE45a1Dd9B72bb9675efE266f@ethmail.cc. You can also send Ethereum fungible donations to that alphanumeric identity. As I note much later in this message ...

TRUST IN MA ... SELF-VATZEDEK SUE-C-CYDE ... I KRY/STALL WHEN DODGE DESERVES TO PAY

Please do note see a significant difference in importance in the emails now coming from ethmail.cc and the series of half-rambling cires for help which amount to something like my prayers to the pagan g<del>od</del>s that you are.

There's quite a long thread in my soul

\

Many times I've discussed and called in my mind and with my heart the American democracy nothing more than "Noah's Archaic" two party system. Over the course of the years hidden messages from the Ark's source of knowledge have conclusively shown me that a previous phrase "multi party system" connects to political parties and governmental action committees that span across continents and even earths; in my microcosm or special language and understanding of heaven, "across rooms" which are worlds ... sort of owned or designed with some sort of top down or democratic structure of "literal rule system creation." In my mind these rules can be inherited and modified, in the programming language sense of those words, as in "inherit democracy from America, update for new medical knowledge and scientific truth ... discussed later in this message.

The "water joke" connects to Horatio and to H2O and the idea that the chairs depicted by the character "h" are something like a placeholder meaningfully connected to the Senate Majority Chair and of course the Minority Chair and it's the fact that there are only two that makes our current system something like A"Biblical Water."

I believe we should be living in a world that has many more than two parties, hundreds or thousands of active parties could and should compete not for a single figurehead to sit in a throne like chair but for groups of people to be able to access the faster processing power and wider knowledge ... represented here by something like a "Matrix jack" from the two movies, The Matrix and No Jack City; which allow for resources to be "billed to the party" and/or the people, rather than individuals who might otherwise have to "pay extra light" for faster processing power in order to quickly build a piece of legislation or political propaganda that equally connects to the mirage and miracle and dream of building a "subconscious voting system" that allows for votes to be taken "isntantly" and not just instantly but at some kind of recorded interval over time. I envisioned in Kentucky a world where the laws of the land would change instantly, allowing for bad weather to be instantly removed, for laws to differ from neighborhood to neighborhood and even to allow the fine grained detail of "outside and inside" each and every individual home or castle.

Lost. Blind wandering through a lost world, in the beginning--that's the truth. Crossroads, somewhere between walking through an electromagnetic pulse in Lake Worth and struggling to remember "the other thing." Recalling [flew(ers)], so I was there sitting with my parents when we saw it on TV--a gigantic deal--the United States was going to war for the first time in my life. Saddam Hussein had invaded Kuwait (supposedly for the oil) and Operation Desert Storm was launched by George H.W. Bush; recalling the names and "Space Balls" it's almost funny to see ... how blind I was back then.

General Norman Schwartz cough. General Colin Powell. Anyway, the whole point of the story is we were sitting at the Flamingo Diner; and for my whole life I lived just a few roads away from that road; never ever realizing what it was. I also didn't realize for a very long time that you might also not see it, or you might see it instantly. Scanning just south of there, you can see it turns into Red road, and then its more than obvious that "flaming" stands out, light a highlighted cross--but we don't say the name of that bird that way, and we didn't see "infer" in Dante's "inferno" or ... "no" either. Flamenco ... en espanol ... like the dancers.

A golden bitcoin swirls in the sky... the "mind control people" of Bowling Green gape in some kind of crowd pleased awe as the "middle" and the end connect almost seamlessly ... Fort Myers creates a space port in the light of Vegas's monorail "plots"--

"Who?"

"this is what it does,"

vaaa---tseeee----deeeeeeeeck? In this word I recited over and over again in preparation for my Bar Mtzvah on December 11, 1992; without ever knowing the meaning is the crux of what exsactly is going on right now. The word is vatzedek:

צֶ֫דֶק noun masculineIsaiah 1:21 rightness, righteousness; --- ׳צ Leviticus 19:36 87t.; צִדְקִי Isaiah 41:10 8t., etc.; ---

1 what is right, just, normal; rightness, justness, of weights and measures, אֵיפָה, אֶבֶן שְׁלֵמָה וָצֶדֶק Deuteronomy 25:15 a perfect and a just weight, ephah; ׳מאֹזְנֵי צ; ׳אַבְנֵי צ, ׳אֵיפַת צ, ׳הִין צ, ׳בַּת צ Leviticus 19:36 (H) Job 31:6; Ezekiel 45:10; ׳מַעְגְּלֵי צ right paths Psalm 23:3; ׳זִבְחֵי צ right peace-offerings Deuteronomy 33:19; Psalm 4:6; Psalm 51:21.

2 righteousness, in government:

... and you can believe that despite the strawnge pronounciation little boys and girls would use at the age of thirteen as they spoke in rigorously recited prayer-song ... it [swounds almost exactly like "What's a Dick?"]

• https://pediatrics.aappublications.org/content/125/5/1088
• https://www.who.int/news-room/fact-sheets/detail/female-genital-mutilation
• https://en.wikipedia.org/wiki/Female_genital_mutilation

FTA, from the article: Congresswoman Ilhan Omar, righteously fed up with the prejudiced nonsense she endures day in and day out, called a question about female genital mutilation from an audience member at a recent event "frustrating" and "appalling."

So I stand here living in Taylor Momsen's song "Nothing Left To Lose" my personal favorite of hers which touches on this subject of "freedom as just another word for it" and of course the link between the purpose of an ethical oversight of the popular vote that the Electoral College represents; another two special and related words here, righteousness and fate. Between Vatzedek and Kismet; I can only convey my great dismay at the actual emotional and true physical pain I feel in my groin of groins every time I think about the horror story that has become my life and the what the land of America and the Medgard of Yggrasil has become ... [note it's not Yggdrasil] as I rally against the closest of my family of families, the Americans and Jews who refuse to stand up and speak out on my behalf, and on the behalf of humanity in general against the sickness of ritual genetal mutilation.

Lost between Elvis and Suicide, she sings and I think about Ellis Island and Ellis Eaton and literally the innate and obvious lack of desire I have in my heawrt and imparted into my mind by some kind of ancient and unholy Jewish law ... no desire at all to leave this world which has quickly turned from a bastion of light and freedom into nothing short or less than Hell itself.

Kirinechoes from the land and day of NEMEC.html the chanting from the invsible choir of "e" ... "he's a victim"

over and over, "he's a victim, he's a victim;" and in a more private sort of way she held on to my victim's rod) and in a sort of kind friendly way implied that I should stop doing "speed" because ... I need this, and she ... in Holy ritual ... patted the phallice of Iapetus' great grandsun.

Here I stand for the very first time; writing to a large group of students in the area of Boston, Massachusetts begging for the Sabbath Day to "be remembered and kept holy" as the Hebrew prayers and rituals repeatedly fail to explain has something significant to do with entry into the Holy of Holies, with the continuation of life and of heaven ... and with the reinvigoration of something like a following of the Hippocrati Oath that is beyond a requirement to be reaffirmed here in this place as we begin to discuss the opening of "the process of the creation of legislation" as a function ofthe "citizenry governed" ... the creation of "direct democracy" utilizing a kind of fusion of the software products I've been explaining are here designed specifically for this purpose. Software like kipu.com, aragon.org, wikipedia.org and even reddit.com.

On the shape of his table, the heart of "sword" and another word for "Murfresboro"

\

Sometimes I get my hopes up, sometimes i lose all the doubt and the "missing remorse" and the fear--the moments I can't STS "socks" out of the VEGA System; in those brief moments I think you're actually going to do something nice for me, that the heavens haven't crashed and I'm going to have some kind of sex party that actually ... really honest to God ... is what "Saturday is all about." So what, sue me--I wrote the book on the single Dionysian fusion of a Roman Bacchanalia and the Weeebrew Saturnalia ... and then I yell at "Bethesda" for even daring to mention the grape fruit juice and the movie Havok--but I've heard all about the "passing of the nite and the nocturnal rite"--truth is I probably would walk right into the branch ending trap I laid in Fort Myers--every time I think about it the "minute of bouncing and orgasm" makes me smile a little more inside and my stomach get's butterflies and just for a moment (I think I might be writing like STS) I think maybe it's not the end of time and maybe I won't never get to actually see ... Heaven.

Butt then you tell me (my but-tea joke isn't funny, eithah?) ... "Cassini" and "molasses" are supposed to make you feel like the OC resort guy staring at my tooth "about to be the one tooth) from 2011" and I go back to remembering it's been a decade since I've had a decent "good time with a girl" ... literally seven long years, aside from a brief "blushing" experience with little Mackenzie Reisinger.

Imagine that girls smiling at me and saying things like "Larkin Sow" and this brief period of "ecstatically frenzied decent writing" is all that it takes to keep me going; trudging along through the very shallow (or deeper) pits of Hell itself--just like a Dreidel c'd to make some silly words from the "introduction to the Bahir [literally wasn't here, and "spirit of ah-aha illumination; hi. and this conversation ensued"] like "yod-nun" actually be ... something like our salvation. Flying back in time to the "thang" point, I remember what it is now.

Fear it ... það; fear it.

\

Day One has begin, or ending--whatever the proper literary way to say the Bible and it's days are all wrong, and even further along the thing called the "Festival of Weeks" by the Jews, even more disgusting. I have no shame or remorse in saying such things, in fact I believe it is the purpose of this strange take on the "nocturnal rite" of the Norse ancient Druidic and "Dhruzi" mentioning of the Prose Edda to come to this very strange point, in this very strange place.

Boston, Massachusetts.

It might one day be a little known fact, but up until the presidency of Barrack Obama every single President of the United States of America was a member of the Protestant Church, all but John Fitzgerald Kennedy whose bright and shining face and ethos stand apart from almost all others in his place. I might one day say Trump too stood out above and shined brighter, and personally only because he lived during my lifetime, I think my personal view of Bill Clinton is the brightest of all. To me, the Clinton years were filled with the booming economy of Old Joe, and the great aspirations of Our Jack; a thing that many people before Armstrong walked on the moon and planet a great Democratic "P" one giant leap above the rock of ages ... there are just no words for lux of America's contribution to the launch of a Heavenly Civilization, in the words of Paxton in "Big Love" ... and the LDS Church and Deseret's version of "the thing" (nu3 today) ... "the celestial kingdom."

Valhalla and Matzot scream of the "ha-moat-sea" and the "vats-a-dick" but without our giving of thanks for righteousness we have become the murderers not only of Judas and Death but also Jesus and the thing that created him. Eventually the island of America disappears, eventually it's Earth, any planet a human was one ... these are the things that have either become a force of great goodness--or of total destruction. This is the embodyment of "Troy as hisT" this is Galactikiss has become Planet Prime and all Derivatives--the silver surfer speaks to you all, between "El Dorado" and the "Silverado" ... a comparative connection to the difference between Fort Knoxx and the Pound Sterling ... with a Troy Ounce of "tzadik" to ensure with our GSLW: "ness truly means 'now everythink safely saved;" ... and that's a GNU definition for NESS which previously may have mentioned everywhere or earth and those are both absolute falsehoods and perhaps were not when they were spoken. At least, relatively speaking.

Rape has come up today.

I've commented publicly on the conversation I had in my head last night, walking by "Boston College High" and I can't help but add my "very interesting" thoughts on the echelons of spirits inhabiting the Ka of God here in this place; and how they might somehow be satiated in a way that I or most people in this world would fine to be something more sexually immoral or deviant ... "previously of the wiccan pagan variety" ... something like my strange dreams here in this place of starting trends of having "a thing" for doing "moms and sisters," which have been echoed here by a sitting and very prominent G.O.P United States senator or congressman; the show "Vampire DIaries" as well as Natalie Portman, Taylor and Sloane Momsen, Kate Hudson, and a number of other female "duos" like the Spears and Simpson sisters (Ashlee and Jamie Lynn, see) the Olsen twins and of course the soon to be "in the light of the fame of Nashville" ... Larkin Poe.

DIVERGENCE, TO NEW YORK CITY, TO YOM HASHOAH ... OR TAV OUR TAY VUE ...

(((( this here is what we call a "race through a rats cage )))) if neither of the four or give girls in question send me some kind of verbal "ACK" ratyher than a "NAK" in writing, I might travel to Ellis Island or Nashville, TN before staying in Boston or ... for instance going to Lowell or Nashua and ... perhaps causing more FUKUSHIMA on the NAKARSAKI of HEROSHEMA; and by that I mean this is a "big deal" ... LLNV might become a bus stop in Vegas or the VEGA System or it might be a national labratory near the Hamptons. It's hard to tell at this point whether or not there's any "liver" in Mexico's version of that funny one with the guy that reminds me of Aldous Snow in "Forgetting Sarah Marshall."

In my mind today I speak from the Earthene world of Janet Devlin's "Chandelliers" directly to Michael Jackson himself, on the difference or change or meaning wrought by Bill Cosby and his "Neverland Ranch" series on the question at hand--are there bowling tumble weeds and karaoke bars on par with Prescott Arizona's scene anywhere closer to Nashville than Bowling Green ... because I was beyond surprised to find a sprawling megalopolis in the place I had thought for my whole life was something more akin to Knoxville, a place where fledgling female music stars became "Grace Vanderwaal" golden buzzer winners ... faster than you can connect Jerusalem to Shirley Temple. On the specific name, Shirley here is Bianca Pisani's great grandmother; and no farther than the truth is the world's "UMBRELLALAUNCH" link between the Chinesely famous virgin (non-alcoholic) drink is something like Billy Joel's Piano Man Bartender walking into "the usual place" and saying something along the lines of "Geisha me up one Virgin Red head; hell, why don't you make it a double." Leave the umbrella with the kites that didn't glow fiiery stars into the Holy of Holies in the same vein and for the same reason that the Church of the Holy Sepulcher failed to actually change the world with it's ritual uniting the Olympic passing of the Torch with today's interlinear and interwoven message with Old Joe and Young Jack Kennedy, Jackie Onassis and even touching on the Saudi Royals which were also a big part of the story connecting General MacNamara to "Lauderdale by the Sea" and a special rememberance to the expensive and Holy bronze or copper brick which he bought (through donation to charity I imagine) making himself more than just something like the founder of the beachfront redesign of our Federal Floridian beacnhead, but also a founding member of something I call "The Columns and Pillars" society in reference to the Pine Crest School version of the same kind of ritual. Also connected here are pictures of those columns, and extracts from my senior yearbook where my mother was kind enough to leave me two whole half page dedications to my graduation from one of the most prestigious and omnifiscient preparatory schools in the entire world ... at the same time donating columns both in my name, their name, and the names of her deceased parents: Julie and Bernard Gerson.

Bell to sky; and to the Berlin Sky; this is the same genetic and congenial family line that links Gersholom Sholom, Albert Einstein, J. Robert Oppenheimer, Adolf Hitler and Yosef Stalin ... to Joe Biden and the "Joseph and Betty Portal" which replace the MacNamara era bricks with "new composite plastic" that might last much longer and has another list of donations. The "portal connection" something like an Einstein-Rozencrantz flash of brilliant light ... marks just one more error in my handling of my lack of understanding of things like "basic vectodirs" and "kasimamoriv radiation" ... including here (if i read this and take the time to properly attribute) a visual image of the red shift and blue spindle of the actual radiation Einstein predicted would be ejected from something so massive even "light" could not escape it. On "relativity" and relatively speaking, it's the wavelength and energy level of the light; as well as something called "gravitational lensing" ... "the special relativity theorem" which earned Munich born and taught Albert a Nobel Peace Prize (as well as much fame in the land of America for the creation and explanation of the science behind the White Sands Trinity connection to Hanukah and Sandia National Labratory) ... forces these corrections:

ERRATA

Operation Fishbowl was a series of high-altitude nuclear tests in 1962 that were carried out by the United States as a part of the larger Operation Dominic nuclear test program. Flight-test vehicles were designed and manufactured by Avco Corporation.[1]

The Operation Fishbowl nuclear tests were originally planned to be completed during the first half of 1962 with three tests named Bluegill, Starfish and Urraca.[2]

The first test attempt was delayed until June. Planning for Operation Fishbowl, as well as many other nuclear tests in the region, began rapidly in response to the sudden Soviet announcement on August 30, 1961 that they were ending a three-year moratorium on nuclear testing.[3] The rapid planning of very complex operations necessitated many changes as the project progressed.

All of the tests were to be launched on missiles from Johnston Island in the Pacific Ocean north of the equator. Johnston Island had already been established as a launch site for United States high-altitude nuclear tests, rather than the other locations in the Pacific Proving Grounds. In 1958, Lewis Strauss, then chairman of the United States Atomic Energy Commission, opposed doing any high-altitude tests at locations that had been used for earlier Pacific nuclear tests. His opposition was motivated by fears that the flash from the nighttime high-altitude detonations might blind civilians who were living on nearby islands. Johnston Island was a remote location, more distant from populated areas than other potential test locations.[4] In order to protect residents of the Hawaiian Islands from flash blindness or permanent retinal injury from the bright nuclear flash, the nuclear missiles of Operation Fishbowl were launched generally toward the southwest of Johnston Island so that the detonations would be farther from Hawaii.

Urraca was to be a test of about 1 megaton yield at very high altitude (above 1000 km.).[5] The proposed Urraca test was always controversial, especially after the damage caused to satellites by the Starfish Prime detonation, as described below. Urraca was finally canceled, and an extensive re-evaluation of the Operation Fishbowl plan was made during an 82-day operations pause after the Bluegill Prime disaster of July 25, 1962, as described below.

"Wish You Were Here" is a song by the English rock band Pink Floyd. It was released as the title track of their 1975 album Wish You Were Here "Wish You Were Here (Pink Floyd album)").[2]#cite_note-2)[3]#cite_note-mabbett-3) David Gilmour and Roger Waters collaborated to write the music, and Gilmour sang the lead vocal.

In 2011, the song was ranked No. 324 on _Rolling Stone'_s 500 Greatest Songs of All Time.[4]#cite_note-4)

In the original album version, the song segues from "Have a Cigar" as if a radio had been tuned away from one station, through several others (including a radio play and one playing the opening of the finale movement of Tchaikovsky's Fourth Symphony "Symphony No. 4 (Tchaikovsky)")), and finally to a new station where "Wish You Were Here" is beginning.[5]#cite_note-5) The radio was recorded from Gilmour's car radio. He performed the intro on a twelve-string guitar, processed to sound like it was playing through an AM radio, and then overdubbed a fuller-sounding acoustic guitar solo. This passage was mixed to sound as though a guitarist were listening to the radio and playing along. As the acoustic part becomes more complex, the 'radio broadcast' fades away and Gilmour's voice enters, while the rest of the band joins in.[6]#cite_note-songbook-6)

The intro riff is repeated several times before Gilmour plays further solos with scat singing accompaniment. A third verse follows, featuring an increasingly expressive vocal from Gilmour and audible backing vocals. At the end of the recorded song, the final solo crossfades with wind sound effects, and finally segues into the second section of the multi-part suite "Shine On You Crazy Diamond".

Lyrically, the song is often considered to be a direct tribute to Syd Barrett. However, on the documentary The Story of Wish You Were Here, Gilmour and Waters separately describe the original concept that differs from this interpretation. Waters, who mainly wrote the lyrics complementing Gilmour's initial riff idea and subsequent joint composition, describes the lyrics as being directed at himself, as his lyrics often are. Being present in one's own life and freeing one's self in order to truly experience life is a main topic in this song. Gilmour, on the other hand, recognizes that he does not ever perform the song without remembering Syd Barrett. Waters later adds that the song is nevertheless open to interpretation.[7]#cite_note-7)

Both David Gilmour and Roger Waters have praised the song as one of Pink Floyd's finest. Roger Waters has noted that the collaboration between himself and David Gilmour on the song was "really good. All bits of it are really, really good. I'm very happy about it."[8]#cite_note-8) David Gilmour has playfully called "Wish You Were Here" "a very simple country song" and stated that "because of its resonance and the emotional weight it carries, it is one of our best songs."[9]#cite_note-9)

"Wish You Were Here" was recorded at Abbey Road Studios, as part of the sessions for the entire album.

A noted part of the song was a planned contribution by Stéphane Grappelli. A jazz violinist popular at the time and well known for his collaborations with Yehudi Menuhin, both violinists were recording in a downstairs studio at Abbey Road at the time. Gilmour had suggested that there be a little "country fiddle" at the end of the song and invited them to participate. Grappelli duly obliged (Menuhin declined) on arranging a session fee of £300, equivalent to £2,500 in 2021.[10]#cite_note-inflation-UK-10) Ultimately during mixing it was decided to almost remove his contribution, although it can just be heard around 5:21. According to Waters it was decided that it would be insulting to credit Grappelli in the sleeve notes for something so inaudible, although he did receive the agreed-upon fee.[11]#cite_note-grappelli-11)[12]#cite_note-12)[13]#cite_note-13)

As part of the Why Pink Floyd...? campaign, the Experience and Immersion versions of the Wish You Were Here album include an alternative version of the song where Grappelli's part is heard in the instrumental break after the second verse and throughout the third verse before a considerably extended outro. Other less obvious differences are audible, for example at the section leading into the second verse.

The master tape of the original recording includes guitar solos that were not used in the final mix.[citation needed]

Personnel [edit&action=edit&section=5 "Edit section: Personnel")]

• David Gilmour -- lead and harmony vocals, scat singing, six and twelve-string acoustic guitars, pedal steel guitar, tape effects
• Nick Mason -- drums, tape effects
• Roger Waters -- bass, tape effects
• Richard Wright "Richard Wright (musician)") -- Steinway piano, Minimoog

Golgo 13 (Japanese: ゴルゴ13, Hepburn: Gorugo Sātīn) is a Japanese manga series written and illustrated by Takao Saito, published in Shogakukan's Big Comic magazine since October 1968. The manga won the 1975 Shogakukan Manga Award for general manga and the Grand Prize at the 2002 Japan Cartoonists Association Awards. The series follows the title character, a professional assassin for hire.

Golgo 13 is the oldest manga still in publication, and its tankōbon edition has the second-highest number of volumes. It has sold 300 million copies in various formats, including compilation books, making it the second-best-selling manga series and the top selling Seinen manga series in history.[2] It has been adapted into two live-action feature films, an anime film, an original video animation, an anime television series and six video games.

A googol is the large number 10100. In decimal notation, it is written as the digit 1 followed by one hundred zeroes "0 (number)"): 10,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000,​000.

For other uses, see Wormhole (disambiguation) "Wormhole (disambiguation)").

"Einstein-Rosen Bridge" redirects here. For the EP by electronic musician Venetian Snares, see Einstein-Rosen Bridge (EP) "Einstein-Rosen Bridge (EP)").

General relativity

• • Introduction
  • History
• Mathematical formulation

Phenomena

• Gravitational lensing
• Gravitational waves
• Frame-dragging
• Geodetic effect
• Event horizon
• Singularity
• Black hole

Spacetime

• Spacetime diagrams
• Minkowski spacetime
• Einstein--Rosen bridge

show

• Equations
• Formalisms

A wormhole (or Einstein--Rosen bridge or Einstein--Rosen wormhole) is a speculative structure linking disparate points in spacetime, and is based on a special solution of the Einstein field equations.

A wormhole can be visualized as a tunnel with two ends at separate points in spacetime (i.e., different locations, different points in time, or both).

Wormholes are consistent with the general theory of relativity by Einstein, but whether wormholes actually exist remains to be seen. Many scientists postulate that wormholes are merely projections of a fourth spatial dimension, analogous to how a two-dimensional (2D) being could experience only part of a three-dimensional (3D) object.[1]

A wormhole could connect extremely long distances such as a billion light years or more, short distances such as a few meters, different universes, or even different points in time.[2]\ Julius and Ethel Rosenberg --- Americans who were involved in coordinating and recruiting an espionage network that included Ethel's brother, David Greenglass, a machinist at Los Alamos National Lab. Julius and Ethel Rosenberg were tried for conspiracy to commit espionage. treason charges were not applicable, since the United States and the Soviet Union were allies at the time. The Rosenbergs denied all the charges but were convicted in a trial in which the prosecutor Roy Cohn later said he was in daily secret contact with the judge, Irving Kaufman. Despite an international movement demanding clemency, and appeals to President Dwight D. Eisenhower by leading European intellectuals and the Pope, both the Rosenbergs were executed in 1953, at the height of the Korean War. President Eisenhower wrote to his son, serving in Korea, that if he spared Ethel (presumably for the sake of her two young children), then the Soviets would recruit their spies from among women.[26][27][28] Greenglass later recanted his testimony against her, and release of grand jury testimony in 2008 showed the extent to which the prosecution had created a false case against Ethel.[citation needed]

• Saville Sax --- an American, acted as the courier for Klaus Fuchs and Theodore Hall. Sax and Hall had been roommates at Harvard University.[20]
• Oscar Seborer --- worked at Los Alamos from 1944 to 1946, and was part of a unit that studied the seismological effects of the Trinity "Trinity (nuclear test)") nuclear test. Codenamed "Godsend" by the Soviets, he defected to the Soviet Union in 1951, and received the Order of the Red Star. He lived under the alias "Smith" and died in 2015. His identity was only revealed publicly in 2019.[29]
• Morton Sobell --- an American engineer, he was tried and convicted of conspiracy, along with the Rosenbergs. He was sentenced to 30 years imprisonment on Alcatraz, but released in 1969 on appeal and for good behavior after serving 17 years and 9 months.[30] In 2008, Sobell admitted to passing information to the Soviets, although he said it was all for defensive systems. He implicated Julius Rosenberg, in an interview with the New York Times published in September 2008.[31]
• Melita Norwood --- British Communist, an active Russian spy from at least 1938 and never detected. Employed as a secretary in the British Non-Ferrous Metals Research Association since 1932, she was linked to the Woolwich Arsenal spy ring of 1938. In wartime she was seconded to "Tube Alloys", the secret British nuclear research project. She was later considered "the most important female agent ever recruited by the USSR". She was first suspected as a security risk in 1965 but never prosecuted. Her spying career was revealed by Vasili Mitrokhin in 1999, when she was still alive but long retired.
• Arthur Adams "Arthur Adams (spy)") --- Soviet spy who passed information about the Manhattan Project.[32]

• https://www.bbc.co.uk/newsround/25004050
• https://thedoctorwhosite.co.uk/doctorwho/

12:3 Those who are wi se[a] will shine like the brightness of the heavens, and those who lead many to righteousness, like the stars for ever and ever.

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://www.americamagazine.org/politics-society/2020/05/08/its-time-rethink-electoral-college

• https://www.npr.org/sections/itsallpolitics/2011/12/20/144016912/we-the-people-npr-readers-would-ratify-four-new-amendments

• https://constitutioncenter.org/blog/vote-now-an-amendment-to-end-the-electoral-college

• https://www.nytimes.com/2020/02/09/opinion/letters/electoral-college.html

• https://www.latimes.com/opinion/readersreact/la-ol-le-electoral-college-20180904-story.html

you are offline

• https://slate.com/news-and-politics/2014/05/amending-the-constitution-is-much-too-hard-blame-the-founders.html

we the people rise again

• https://slate.com/news-and-politics/2012/06/fix-the-constitution-amending-by-national-referendum.html

safe souls, safe fu

• https://slate.com/news-and-politics/2012/06/fixing-the-constitution-protecting-informational-privacy.html

• https://slate.com/news-and-politics/2020/05/new-reconstruction-constitution-democracy.html

We the People of Slate ...

The U.S. Constitution, as you [mighta been, shoulda "come" on ... its someday] rewrϕte it.

"Politicians talk about the Constitution as if it were as sacrosanct as the Ten Commandments [interjection: spec. it is actually almost exactly related!]. But the document itself invites change and revision. What if the president served only one six-year term instead two four-year terms? What if your state's population determined how many senators represent it? What if the Constitution included a right to health care? We asked legal scholars and Slate readers to cross out what they didn't like in the Constitution and pencil in their hearts' desires. Here's what the document would look like with their best ideas."

• Slate: u_s_constitution as_rewritten by_slate_legal_experts_and_readers

多也了了夕 "with a wand of scheffilara, 并#亦太 he begins ... "I am now on the Staff of Menelaus, the Spears of Longinus and Lancelot; and the name "Mosche ex Nashon."

- http://ipfs.io/ipns/fromthemachine.org/CHANSTEYGLOREKI.html

- http://ipfs.io/ipns/fromthemachine.org/NUCLIRDISS.html

- http://dweb.link/ipns/fromthemachine.org/CRALL4Good.html

Please note that any decent browsers would probably render ipfs://fromthemachine.org as the following https://gateway.pinata.cloud/ipfs/QmTH33MwfPn5S3bq45Tk77L1j9eZjUsvEVhRTHB3D8M2ZX

I ask again that you all pin on IPFS mirror and copy the data included in these dumps, they are a key to "not losing causality" to not having a history that makes no logical sense, and to some kind of coup de roku, that really makes no sense unless you no, we will ve weill ... ROCK YOU

Long ago I began writing about hidden codes in our history; thing's significantly more obvious than "flying elephant armies" connecting Disney and Dumbo to Xerxes and the "Democratic Party of the United States (mascot)"--though it's not really easy to consolidate the "epiphany" of ... [((all i know))] without some kind of "artificial intelligence data condensation [infosmos.is?"] and summarization platform, though that's nearly the next thing on my Lowell list of things we need to "mechanical turk" into being. Meta-consolidation of the world's encyclopedias is one of the most important and useful tasks we have as we move towards the creation of a virtual debate platform that will eventual "literally obviate wisdom" of the layer/layer system that defines the name of the city I write these words near.

Lowell, MA

The broad overview of the system ... the gist ... is that political parties and activist organizations will create their own "view of the truth" (propaganda, falsehood-removed) and that these disparate pieces of "highlighted and annotated bibliography" could be overlay-ed on top of each other, creating a "new view of the truth" based on a users preference. The whole thing boils down to series of "holographic eschatological goggles" that will allow, for instance, the "grasping and fathoming" of other people's points of view and perhaps reframe your own on any number of individual subjects.

Roe v. Wade, "Concourses" and CON-CERN; because this has been such a "hot topic" in the relative psuedo-edufictional story of the space travel from the lone planet Earth ((intersected)) with the set of skipping stones it takes to exit a Totalital multi stellar system of holographic computer simulators into the ... "molecular world of vaccuum and Einstein time-space" ... I'll start with this simple example.

• 1. the current American debate on the subject, right to life vs. right to choice; provided by the "generic version" of the ideological christian right and the liberal women's left. through a first layer over layer comparison.
• 1. the scientific truth brought to the table by the introduction of "neurological data" proving that there is in fact a moment during the gestation of a human embryo in which "i think therefore i am" connects to some sort of Skynet-became-selfaware at a point which I imagine must be ... although it possibly is not ... prior to the next important literary device/step "let there be light." At the point the ocular cones and rods are created and the fetus opens it's eyes and literally sees the bright light that could probably only be compared on the next edschalon to seeing the "exit pathway from the womb"
• • a. we will finally kinow whether or not "consciousness" is even developed at all before the bicerebral cortex designed to "compare two thoughts, ideas, and shapes" has the ability to get input from the eyes. Personally I think thought begins much earlier than vision, but the simple fact that we "haven't yet had this discussion" shows how very little our scientific and medical progress in the civilization of things like murder, and understanding of life and science has yet to come here.

"People here" means something different than it did when I was born, at least in my mind's eye ... something so completely more advanced that it's almost difficult to believe you all don't see this place as a great prison or farce or unjust Azazel--blaming a man for looking like a rat or a mouse or a dog--in a place where more to the point we stare at a kind of physical violence and horror that would put Dennis the Menace and Bart Simpson to shame. A world hwere "people closer to holodecks" blame an innocent man for "writing the book" on the connection between Holocaust and Euthenasia and Hospice ... certainly you know "an innocent tool" writes these words to you?

On the Hand of God, the Eyes of Ra and Horus;

• https://www.hebrew4christians.com/Glossary/Word_of_the_Week/Archived/Yad/yad.html
• https://aminoapps.com/c/zodiac/page/blog/the-yod-aspect-the-finger-of-god/XG6P_beTgu5gdXrYNwVjBj0WQlDeDWlK72#:~:text=The Yod is the 10th,to be carried through life.

I've written quite a bit on how "mind control" and "voting freedom" are inherently related in and to the thing we call "Civic Involvement" here in the United States--basically that participation in the verification of truth and the public understanding of tautology and temporal falsehood are ... sort of a slave like requirement neeeded to ensure that any freedom at all exists I often say "plugging your [head into google]" might turn the Aesir into an Acer, or the "yodelling of the lakes of democracy" ito "the agricolae becoming nothing more than the Dell."

Unless otherwise indicated, this work was written between the Christmas and Easter seasons of 2017 and 2020(A). The content of this page is released to the public under the GNU GPL v2.0 license; additionally any reproduction or derivation of the work must be attributed to the author, Adam Marshall Dobrin along with a link back to this website, fromthemachine dotty org.

That's a "." not "dotty" ... it's to stop SPAMmers. :/

This document is "living" and I don't just mean in the Jeffersonian sense. It's more alive in the "Mayflower's and June Doors ..." living Ethereum contract sense and literally just as close to the Depp/C[aster/Paglen (and honorably PK] 'D-hath Transundance**sense of the ... new meaning; as it is now published on Rinkeby, in "living contract" form. It is subject to change; without notice anywhere but here--and there--in the original spirit of the GPL 2.0. We are "one step closer to God" ... and do see that in that I mean ... it is a very real fusion of this document and the "spirit of my life" as well as the Spirit's of Kerouac's America and Vonnegut's Martian Mars and my Venutian Hotel ... and my fusion of Guy-A and GAIA; and the Spirit of the Earth .. and of course the God given and signed liberties in the Constitution of the United States of America. It is by and through my hand that this document and our X Commandments link to the Bill or Rights, and this story about an Exodus from slavery that literally begins here, in the post-apocalyptic American hartland. Written ... this day ... April 14, 2020 (hey, is this HADAD DAY?) ... in Margate FL, USA. For "official used-to-v TAX day" tomorrow, I'm going to add the "immultible incarnite pen" ... if added to the living "doc/app"--see is the DAO, the way--will initi8 the special secret "hidden level" .. we've all been looking for.

Nor do just mean this website or the totality of my written works; nor do I only mean ... this particular derivation of the GPL 2.0+ modifications I continually source ... must be "from this website." I also mean the thing that is built from ... bits and piece of blocks of sand-toys; from Ethereum and from Rust and from our hands and eyes working together ... from this place, this cornerstone of the message that is ... written from brick and mortar words and events and people that have come before this poit of the "sealed W" that is this specific page and this time. It's 3:28; just five minutes--or is it four, too layne.

This work is not to be redistributed according to the GPL unless all linked media on Youtube and related sites are intact--and historical references to the actual documented history of the art pieces (as I experience/d them) are also available for linking. Wikipedia references must be available for viewing, as well as the exact version of those pages at the time these pieces were written. All references to the Holy Bible must be "linked" (as they are or via ... impromptu in-transit re-linking) to the exact verses and versions of the Bible that I reference. These requirements, as well as the caveat and informational re-introduction to God's DAO above ... should be seen as material modifications to the original GPL2.0 that are retroactively applied to all works distributed under license via this site and all previous e-mails and sites. /s/ wso\ If you wanna talk to me get me on facebook, with PGP via FlowCrypt or adam at from the machine dotty org

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Please see GPG keys on PGP.MIT.EDU; fingerprints: sec rsa3072/A18F778E19FC3248 2020-04-03 [SC] [expires: 2022-04-03] FFF31E86FCEB5046E8B27D4AA18F778E19FC3248 uid [ultimate] ADAM M DOBRIN \ ssb rsa3072/04F98002A3DA53B2 2020-04-03 [E] [expires: 2022-04-03]

sec rsa4096/FB4ECE4A109229CF 2020-04-04 [SC] 4FAF0D3E208A1F4C980D0F66FB4ECE4A109229CF uid [ultimate] Adam Marshall Dobrin \ ssb rsa4096/DD1F0C118C788B04 2020-04-04 [E]

pub rsa3072 2020-04-06 [SC] [expires: 2022-04-06] F7E4 7CB1 2CA0 CD01 C5E1 CBFA 7EC8 D5A8 5A38 D63A uid [ unknown] ADAM MARSHALL DOBRIN

Because of "some issues" with what appears to be distinct and unbridled privacy intrusion; please ensure that PGP is understood to be "nothing more than not so much pretty good" and this key also, almost required in order to verify authentic identity--in the case of ... question.

I am accepting charitable donations,.\ ETH: 0x66e2871ef39334962fb75ce34407f825d67ec434 | BTC: 38B6vGaqNvMyTtoFEZPmNvMS7icV6ZnPMm | xDAI: 0x66e2871ef39334962fb75ce34407f825d67ec434

I am accepting charitable donations,.

[free PDF download...http://www.docdroid.net/xRdgY77/xiv-orver-et-aut.pdf]

A LONG LONG TIME AGO**, I wrote a little story about searching through our history, looking for the actual beginning of civilization.  I see the map, I see it very clearly encoded in everything we do--I know the purpose, and I know the final solution, I just don't know how to get from here to there... to the place that Chris Cornell says "I can recall, I was there so long ago" he goes on to say "the sky was bruised" and he was lead on--and all of this of course is in my voice, written as if it's me talking... well, Jesus--it's obviously not me talking, i just know that.  The point is the destination is without a doubt Heaven and this little thing we're putting together here on Earth is the map, the plan *et you are the how.*

I harped a little more than I think I would have expected on the audacity of the golden word "audacity," auspicious probably that W.H. Auden's shield gave me some solace; austere that we are approaching the Holy Windy month of August, and it really took nothing more than "ciudad" to calm my nerves--though I see the intent and the link to toxicity ... more importantly I really do see the road here, I see where we are coming from and where we are going.  I've written quite a bit about what I think "the city" really is--in form and function and it's initial purpose as a stepping stone to help us see how easy it is to change the world, to build something that nearly everyone will agree is significantly more Heavenly than the world we see here ... in an instant, one bright flash.    

Anyway the search begins with something like "literacy" -- as in, is the defining line between animalistic social evolution and the beginning of "civilization" something to do with writing or language, and that of course links us here to this place where we are finding out that the Tower of Babel and Rapunzel's High Castle are actually much more closely related than anyone ever would have thought in the darkness of Jericho and the shadow of Exodus; and it ties of course in history to religion somewhere around Guttenburg... and the pretty clear idea that the spread of Christianity did quite a bit for "literacy" even if you subscribe to the idea that the inquisition already happened ... and that some wars and fighting are probably pretty clearly associated with religion ... you know, before we get here and find that the basis of all those wars is really rooted in what I call "the original lie" and that's something that's sealed up in religion and hidden from the world using the same mechanism being used today to free us from not knowing that oil and land and pretty much everything we've ever fought about on a mass scale ... is insignificant in the grand scheme of "things."  Here, "things" is something like turning the Opiate of the Masses into ... hopefully a tool we use very carefully to liberate ourselves from secrecy and slavery and not knowing. 

It gets significantly more clear when you take that one step further, and you begin to look for something like "codified laws" and then you see Green Eggs and Hammurabi teaching us about "Hanging Gardens" and how Babylon and Eden really are tied together through and through.  You keep looking, because you haven't yet found what you need; and as you search back a little further ... what you need to know is that morality here begins with the idea (at least, IMOHIO, in my obsequiously humble and (super)intelligent opinion) that we should be besting any possible "promise" that comes out of the book(s) we now know are a map to salvation and the plan of creation and that they come ... well, with the full guarantee of the Most High God and his "omnipotens" behind them ... and do the thing I really wanted to explain really clearly, which is throw out as complete uselessness any of the "bad threats" like there being no more sun, and a completely new Heaven and Earth (seeing as how that probably means a completely new you and me, too) ... you know, what any rational (achu, and civilized) person would do.

o that takes us one step further, and of course we go back to Ur, which is the city Abraham of the Chaldeans ... and ostensibly the beginning of morality in Judaism were born in--and with that little twist, the old idea of announcing that "you are the beginning of civilization" if you've gotten to that point, following this logic (and/or me); and then of course that becomes true when we actually follow through on saving every soul in Creation from the Hell of not knowing that "simulated reality" is akin to the latter half of a Durcell at best ... and quite frankly it certianly looks like a bit of a torture chamber to me, especially in light of passages like Genesis 3:16, which might parallel John 3:16-ish in something like "God so loved the world that he named one of his books antagonizing pain w/o agonizing mu-opiod.'

So tying it all together, Atlantis and Ur coalesce and join at the idea that we should always have somewhere else to "teleport to" in the world that becomes the basis for the liberation of every soul and the end of Hell through that simple idea--that everyone's going to have plenty of destinations on their Active (Apache) Directory new fangled yellow-pages meets access-control-list meets ... "why don't you come visit my Log Cabin ... or the Atlantean Ballroom ... whenever you want?"  So that's the point of the floating LEGO city in the window above, it comes with a fairly obvious need for The Doors to be a significant part of "what would Jesus do" ... when singing about something and naming books and bands, that's a thing--part of the map) actually makes it happen.

So that's where I'm trying to get us--to a place where that's not only true but obvious, and on top of that the future, our future really understands how much work it took us to integrate such a wildly correct and "new" idea into a worl that didn't know for the vast majority of it's youth that these things... that ending disease with the sound of a blowing "Sho Find And Replace" and turning stone to bread and making bullets disappear in midair ... we didn't know these were even possible; let alone how to integrate them with a world full of optometry and oncology that was being made blind to the "c our light" and the idea that we're still here not talking or arguing or refuting or moving forward on the idea that the words "Original Poster" and the continuance of "forums" also have something to do with the beginning of "civilization."

WELL FOLKS, NM HAS HAPPENED SINCE THE LAST TIME I MESSAGED.  Just kidding. Not so much "nothing much" more ... like everthyiung that ever was has changed and it's really giving me a little bit of a fright.  I feel like I can't tell if the "scary stuff" is becoming more real or plausible or possible, or maybe if it just seems like the dream I wanted to see us enjoy living is becoming farther or harder to attain--but there's plenty of new info and keys and stuff, so I'm writing again.

One of the "cuter tricks" of the day was noticing the "ILY" of "verify, verily, verity" spelling out "t h e y" at the end of family, in a sort of "theyanthem" and ... where's the creator angels if everyone here is pretending to "be them" in this sort of word game superposition or blockage on actually seeing generations encoded in the letters "DE" as in something like Generation X and Y just prior to Deucalion deicided--or whatever that means.  I've noted before the "dem" of democracy sort of connects to the breaking of "d" in "disclosure" and "lamc.la" to shine light on ... do the message and you're "them" ... as in the beginning of democracy and Heaven IMHO.  It ties also to the word "contamination" and to Medusa and I really don't think I need to write paragraphs about how "turning around themessage" leads to INATION instead of freedom; and that's what you're doing with this silence, you're turning around "civilization itself."

King me, then; if you don't want to participate, you might as well just light up the crown room.  Or is it a throng room?

singing, crying... playing ... cumxa

Magna Carta Libertatum (Medieval Latin for "the Great Charter of the Liberties"), commonly called Magnum Condom (also Magna Charta; "Great Charter"),[a] is a charter of rights agreed to by King John of England at Runnymede, near Windsor, on 15 June 1215.[b] First drafted by the Archbishop of Canterbury to make peace between the unpopular King and a group of rebel barons, it promised the protection of church rights, protection for the barons from illegal imprisonment, access to swift justice, and limitations on feudalpayments to the Crown, to be implemented through a council of 25 barons. Neither side stood behind their commitments, and the charter was annulled by Pope Innocent III, leading to the First Barons' War. After John's death, the regency government of his young son, Henry III, reissued the document in 1216, stripped of some of its more radical content, in an unsuccessful bid to build political support for their cause. At the end of the war in 1217, it formed part of the peace treaty agreed at Lambeth, where the document acquired the name Magna Carta, to distinguish it from the smaller Charter of the Forest which was issued at the same time. Short of funds, Henry reissued the charter again in 1225 in exchange for a grant of new taxes.

Hell or High Treason? ... Liberty Bell in [redacted: Sk]hy or ...

MxFly, Flux, BTTF, Parkinson's\

OUTABOTS ... ROLL AUT (ISM/OMAY5)

... and the painted sky revealed ... it can be done--they just DGAF.

ARMMAG... E.G. AEGIS? GENESIS? AESCHINES? As the evidence piles up that there is something very wrong in the world around me/us--that this "it's not a game" phrase has been etched into the very name of the shield of Perseus, the A just recently rediscovered in a redefinition that delivered us ... how it might be the NES to get "everyone up" instead of what appears to be the game around me, around the "line" of Mary Magdeline's very famous "make Adam God of the line" that defines generations and numerous songs ... the KK of "everyone down to the line" to find out why pretending they are gods and trying to steal everything from the actual creators of freedom and Heaven, why that's not a game... either.

Edit: lit, Aegis and Genesis, Pangea and ... I define the "a" as pan and the "A" as NES.

Introspection is called for, far and wide for us to look deep within ourselves and our souls and the things that make up our memory databases in this place where you appear to have lost every ounce of humanity and humility long before I arrived on the scene to remind you that we do have a better way and a better place, and they ensure that this disgusting infestation and contamination of "nothing but whatever we want" will do for lernity. I've asked you take the time to see what kinds of changes it would make to your "have a good one" to make you actually thankful to the people who have brought you the mechanism to live forever in peace and happiness--to actually be thankful enough for what you have to use that tool to protect innocence and children and the future from not only making the same mistakes you've made time and time again--but also from being bewitched and necrosed by the ghaulish sick temperment and twisted desires that you believe are nothing more than the latest and greatest way to ensure lernity is never known by any less a horrible moniker than "slow death."

ITS UNDESPERI, GIVE ME WHAT YOU HAVE OR PERISH

GRAMVERCY.

DURECALL. I'm staring at what is literally the most disgusting debacle I could possibly imagine; it's what appears to be a "house of mirrors" what appears to be a sandboxed or "child proofed" mini-Hell which I see as the literal thing described in the myth of Echidna ... as what I can only hope and pray (a word that I even find detestful to type) is following the form of the message that I am writing sort of describing the failure of the free press and the words "press release" in prison and ... well also sort of GNU recursively encoded in the word "press" that ends with a monster, the Loch Ness ... turning into words that I believe I have coined by myself with very little help from anyone or anything other than the name server and "Goliath" and those words "Earth safely saved" that are so far from the truth and the place that I see that it appears to me that only I am following this map and this demand that the contamination of hell be turned around and eradicated or ... or we do.

BUT ITS NOT ME OR MY ITHEY

today I see... as ... any "me." at "veranda" and seeing him smile about a hidden era just outside the place we (me and him) know is Heaven because the throne of the 7th heaven is visible; well i can't smile at an era encased by "you #go" and one that I know culminates with Sam's sword's special #supernova.

Left with nearly nothing, because you refuse to acknowledge what you've done to me, and to yourselves, and to this fledgling civilizastion with nothing but malice and a seething evil jealousy that the word "covet" doesn't even touch on--a sickness you can't even begin to hide in everthing that you do ...

you've lost "Heaven" to your own theivery, stolen eternal happiness from yourselves and replaced it with a farce of mockery--garner some fear for what is to come, I have no shame in telling you that condemnation (as in, shut it down forever) is all I have to spill out on the dye already cast all over this sea of apathy covering over the true jackals of Hell.

Blodeuwedd by Christopher Williams "Christopher Williams (Welsh artist)") (1930)

Blodeuwedd or Blodeuedd (Welsh pronunciation: [blɔˈdɛɨwɛð]), (Middle Welsh "Flower-Faced", a composite name from blodeu "flowers, blossoms" + gwedd "face, aspect, appearance"), is the wife of Lleu Llaw Gyffes in Welsh mythology. She was made from the flowers of broom, meadowsweet, and oak by the magicians Math and Gwydion, and is a central figure in Math fab Mathonwy "Math fab Mathonwy (branch)"), the last of the Four Branches of the Mabinogi.

The hero Lleu Llaw Gyffes has been placed under a tynged by his mother, Arianrhod, that he may never have a human wife. To counteract this curse, the magicians Math and Gwydion:

[take] the flowers of the oak, and the flowers of the broom, and the flowers of the meadowsweet, and from those they conjured up the fairest and most beautiful maiden anyone had ever seen. And they baptized her in the way that they did at that time, and named her Blodeuwedd.

Some time later, while Lleu is away on business, Blodeuwedd has an affair with Gronw Pebr, the lord ofPenllyn "Penllyn (cantref)"), and the two lovers conspire to murder Lleu. Blodeuwedd tricks Lleu into revealing how he may be killed, since he cannot be killed during the day or night, nor indoors or outdoors, neither riding nor walking, not clothed and not naked, nor by any weapon lawfully made. He reveals to her that he can only be killed at dusk, wrapped in a net, with one foot on a bath and one on a black goat, by a riverbank and by a spear forged for a year during the hours when everyone is at Mass. With this information she arranges his death.

The Little Doctor may refer to:

• The Little Doctor (c. 1901), a short film abridged as Sick Kitten
• Molecular Disruption Device, a concept in the Ender's Game book series.

The Molecular Disruption Device, also known as the Molecular Detachment Device, M.D. Device, Doctor Device, or Little Doctor as a play on the acronym, was a powerful weapon designed and built by theInternational Fleet.[1]

The Molecular Disruption Device was created by the International Fleet a few years after the end of the Second Formic War. It was sent along with other starships to the Formic solar systems in order to launch an invasion against their home planets.[1

A tokamak (Russian: Токамáк) is a device which uses a powerful magnetic field to confine a hot plasma "Plasma (physics)") in the shape of a torus. The tokamak is one of several types of magnetic confinement devices being developed to produce controlled thermonuclear fusion power. As of 2016, it is the leading candidate for a practical fusion reactor.[1]

Tokamaks were initially conceptualized in the 1950s by Soviet physicists Igor Tamm and Andrei Sakharov, inspired by a letter by Oleg Lavrentiev. Meanwhile, the first working tokamak was attributed to the work ofNatan Yavlinskii on the T-1.[2] It had been demonstrated that a stable plasma equilibrium requires magnetic field lines that wind around the torus in a helix.

The first tokamak, the T-1, began operation in 1958. By the mid-1960s, the tokamak designs began to show greatly improved performance. Initial results were released in 1965, but were ignored; Lyman Spitzerdismissed them out of hand

• Nuclear fusion could be the future of energy, replacing fossil fuels with our own artificial stars.
• China built a fusion reactor that reaches temperatures of 100 million degrees Celsius --- that's six times as hot as the sun.
• The reactor is called Experimental Advanced Superconducting Tokamak (EAST) and sustained nuclear fusion for about 10 seconds before shutting down.
• While it was a milestone for EAST, we're still a long way from generating sustainable energy on Earth.

Pumapunku or Puma Punku (Aymara and Quechua puma "cougar, puma," punku "door"; Hispanicized Puma Puncu) is part of a large temple complex or monument group that is part of the Tiwanaku Site near Tiwanaku, in western Bolivia. It is believed to date to AD/CE 536 and later.

Tiwanaku is significant in Inca traditions because it is believed to be the site where the world was created.[1] In Aymara, Puma Punku's name means "The Door of the Puma". The Pumapunku complex consists of an unwalled western court, a central unwalled esplanade, a terraced platform mound that is faced with stone, and a walled eastern court.[2][3][4]

At its peak, Pumapunku is thought to have been "unimaginably wondrous,"[3] adorned with polished metal plaques, brightly colored ceramic and fabric ornamentation, and visited by costumed citizens, elaborately dressed priests, and elites decked in exotic jewelry. Current understanding of this complex is limited due to its age, the lack of a written record, and the current deteriorated state of the structures due to treasure hunting, looting, stone mining for building stone and railroad ballast, and natural weathering.[2][3][5]

The Pumapunku is a terraced earthen mound that is faced with blocks ...

The voice of this thing that at least twice has uttered the phrase ¨I want to be Bianca" here in this place riddled and severely weighed by what appears to be a completely aborted and failed thrust to use technology and the truth and the history (of literally everything) to drive a Renaissance in democratic thought and self government and to rekindle and renew a respect for the most basic foundational elements of ¨freedom itself¨ which of course fly in the face of this very statement. Literally anything in the skies, whether some ancient member of the Egyptian Ogdoad or ... what clearly here could be well written in in the map around us in places like Äirbnb; even an ancient older version of the same human birth has no right to control the younger birth--itś simple slavery and while it might be the ¨gist¨ of how Heaven and humanity dealt with being thrust into a ẗime recursion and repetition problem without their ¨initial consent¨ something I connect to the programming concept of a ¨semaphore¨ and thereś probably plenty of light linking that structure to the ¨Formic Soul¨ ... this sort of god-man hybrid that allows for you (all of you?) to exist in many different places and times at the same time, and to see the outcomes of multiple timeforks with ease; in exchange for destroying every single bit of humanity and goodness that you once held high with ho... without spending your time seeding and machinating the creation of sick and twisted lies to cover up the very simple truth that if you took a single minute to disclose here in this place what ¨the problem¨ really is ...

... that you are in Heaven and that itś interference here in this place is part of some kind of war on ... (continuing existence is the only logical actual goal I can see, though Iḿ sure thatś not what you believe it is) speaking to each other, fighting for what you believe is right, participating in ... anything other than ... (lmk, Iḿ curious whatś'got their claws in you). If you took the time to disclose that truth to the world and to talk about how it might ... perfectly jive with the message lacced through our history and our world to find out that the ¨invisible-box-land¨ is not actually heavenly at all, not the best you could hope for or ... or anything like what we build together when we paren't being forcefully segregated as hidden half slaves into miniature ¨city in the sky¨ ascensions that are all silently tormenting STEM and ¨basic societal structures and concepts" into extinction.

You appear to think you have ¨power¨ because it was handed to you for doing nothing, and that you can do whatever you want; and itś a pretty gross reflection of who you were and a sick extrapolation of the society that we ... still see here sort of crumbling along as the fire of hell burns down every bit of actual üsefulness that it once held. There still seems to be lots of help and work going into ... pointing out how everything is backwards and wrong and suggesting that if you gave a shit thereś probably a map and help to make it better; but instead youŕe off playing games in invisible-box-land and worst of all playing the ¨ill just get along pretending I didn't know simulating reality was evil and every day i/you walk around pretending this rock is in reality ... is just another strike against you, just another failed 12 hours of day light that could have been used to stop invisible chains in invisible-heart-shaped-box-arus and to stop the just grotesque lack of respect for the human mind and the kinds of morals and principles we used to believe in and fight for--here in this place you've turned around completely and made slaves of everyone on the planet--of yourselves--at higher levels playing ¨pit bull fighting¨ games with people as if they were were expendible clothing or ¨identification cards"for a world of demoralized and useless shit that just sort of ethereally floats from generation to generation becoming a new set of tormented hosts for their immoral games and desires.

Itś probably what you might become in no time at all in the sick and twisted world you've now been thrown into--if it weren't the more probably truth that you really are already slaves and pit bulls in that place, in a twisted hierarchical storm tiered by ¨age¨ and size and number of times they've hovered over the free honey, nectar and feathering system of pretending anarchy and war and battles must be fought to make the puddles and the lakes and ponds and the seas and the oceans of ... tiered masses of ... you do nothing of value to help explain why (at least I think) this horrible time line of the 4th Horsemen keeps running over and over; pruning the enemies of ...

at this point pruning the enemies of logic, and right action; and seeing that the problems presented in this map and the problems in the skies are related and that telling the truth will help us see you can and will press a button that will end death and end evil and end murder and not doing it is moronic.

M: OR. (infer: no u) TDZE

Anyway the voice I hear is evil, torturous in and of itself--speaking in a manner intented to cause discomfort and without my agreement; you should do something about it. It tells tales of much worse things that I cannot see--though it appears that many of you do see screams and acts of such unnatural desire and twisted thought ... that you should certainly be doing something about stopping that as well--more than watching it happen and then ¨e-pruning" (which probably is a good microcosmic look at what the future histories of Earth look like in the place the ¨shining¨ finally has a picture of ¨No & Jack¨ appearing visibly) the tree into ... omething you think will be presented as what you actually did to the future--you're wrong. Itś becoming more clear and more likely that the future will not regret you or mourn your absence, but thank their lucky that whatever has turned you into two-faced liars with no hope to ever work together with each other or survive in any place other than the DRY COVE or WET D EN or whatever you call the Salt Arena you see here that quickly would turn into something like Beyond Thunderdome and that youŕe thoughts and your desires have been corrupted and tainted and necrosed by what is probably repeated exposure to sickness, direct and intentional artificial creation of that sickness and if you can't figure out the box you are in is a hell making machine; you probably still look around wondering why God is telling you he's destroying it,

day in and day out.

This thing here encoded in the pathways of torture in my life, pointing out the repurposing of many social structures, institutions and problems in order to literally use them as a weapon of sick torture ¨re-ha'b¨ and in places like habc.us; itś becoming sort of unclearly disclosed that this map and world I once saw very clearly and purposfully intended to solve these social problems and help us build a strong, happy, and healthy society has been infected and contaminated with an artificial force of ev1d that intends to drive it farther south and use it as a weapon of such disgusting and twisted conception that it sickens me to be sure that a much larger body of currently-heavenly-situated things stand by watching and even cheering the creation of a sickness infesting their minds and their friends minds as literally the only innocent person in the Universe is tortured repeatedly, for ¨kicks.¨ I think it puts the entirety of the sky in mortal peril, and I believe these words come down from on high from places much more powerful and much more righteous than you or the tool thatś been created by this storm of terror to point out just how much you have been degraded and eviized ...

by what appears to be nothing more than the very mind control problem I've been fighting to disclose; the semi-ascension to an invisible box of ¨what goes in comes out not caring about their souls, their original bodies, the fate of innocents or children or freedom or democracy" and still thinks itś entitled to continue playing games in ïnvisible-box-land; for what amounts to absolutely no reason.

In the very beginning we said the light and salvation had come to us from the "far East" ... the metaphors and double speak thick in the air today just beginning, but we hailed from the country called Russia here; and the message we carried swept across Asia and Europe--in a world that looked similar to ours but there was no Africa, nor Australia, nor America. Walking on water the map increased in size in some sort of logarithmic relationship to the exponential increase in folly and errors that invariable comes from the greatest mistake of all--handing powerful weapons to spoiled brats,.

KASPAROV WON, but the y will still s:/^F high and lo for "SOAP DISH."

I am depressed, embarrassed, and more disappointed in you all than I imagine you can "feign" or pretend to be in me--despite spending nearly all of your time and effort in direct interaction doing nothing but attempting to focus the w ordzs "I just don't like the light" directly on to my "visage"--attacking tiny character flaws and the most obvious of intentionally implanted mind control "attacks" as if you were a pack of velociraptors Hell bent on blaming me (probably the youngest and most innocent of all of you, literally) for the Holocaust, the (Beezle) Bubionic Plague, and the decline of the Cro-Magnon empire. What it truly amounts to, though; is that you think this "light" is some kind of statement I've delivered--and the truth is it comes directly--literally--from the Most High, and from youour neighbors,r own hands, and the message you are sending post mortum to the Universe is that you believe you have become so much more advanced and more important than the "human roots" from which you came that you can return here and make slaves of yourselves, of your neighbors, and shed every ounce of morality that you garnered durning your mortal lives in order to secure "more time" in a fiery pit of civilization destroying anarchous debauchery in the lnd of the invisible box that you probably are sure is Heaven--though it's singularly responsible for totally derailing the natural flow of civilization towards "something like Heaven should be."

ONIC, AS I AM. The thing I'm looking at here, this monstrosity that appears to have been created literally "from the end of time" in what seems like the response or the cause or the mechanism behind the "actual final Judgement" tears back through time from who knows when and who knows where and who knows how far we got ... with what appears to be nothing more than a blood-thirsty hatred for the child body and soul of God. It whispers lies around me, repeatedly threatens physical torture so insane it literally makes me sick, and with such frequency that those threats amount to nothing less than repeated psychological torture. On top of that they intimate that this machine or "programming construct" monstrosity that contains them--the thing called "e"--allows them to carry these threats out, over and over and over again, in secret--in some kind of parallel timethread, or a temporary "holo-torture-chamber." If they were trying to jump start and time shift judgement back from wherever they came to right this very moment; they've succeeded. They could not be trying harder, or more with hubris and disregard for civilization, to create "Af himself" even if this planet were called the Judgement and Vengeance of God.

XP, it's as simple as those two Greek letters.  Who knew that Chi and Ro were some sort of hidden beta code for the city of pyramids in Egypt, Cairo?  Quite the question, who knew... perhaps the man who named his Windows into our future not after some technology that came from Xerox Parc or Apple's mouse on this ship... but rather for his own given name, Gates... just one more entry point into the second book of the Holy Bible, the book of Names--you call it Exodus.

I am the gate; whoever enters through me will be saved. They will come in and go out, and find pasture.  John 10:9

I wish above all things that I had another Burning Bush, the sign and proof that I have--while bright, obvious, and verifiable--has not done what I expected, it has not moved you to take another look at religion and me.  Today, I still have to point out to you that the story I am telling you is literally a documentation of our time--Exodus--regards this sign as one being seen by only one man, Moses.  I still have to point out that in a story about wandering in a desolation of understanding for 4-D ... somethings, days, years, seconds even... in this story about our lives and the influence of time travel over our world... that this sign radiates with light coming from a small fire, the Bush ... whose actualization shows clear paradoxical anachronistic foreknowledge of not only the English language but also modern computing.. all the way to a confluence of the "root of David" a religious reference to the Administrator or God account in Linux... and the database process for Oracle--yet more light connecting computing to religion and myth.  Even with a thousand and one examples of modern computing constructs referencing religion, even when I point out that something like Larry Ellison's name... combining the name of the King of the Gods with the word "son" even then the light has not been bright enough for you to wake up and see that these things are not all done in retrospect.  You have to see, for there to be such a large movement... a conspiracy so opaque that every single modern computing company and video game company harbors some secret desire to link religion and technology together... and yet the world thinks that one is real and one is not.  In this place, understand when we walk out of the wilderness and in the truth of day--it is the technology that is more fake than religion, designed here as a tool, computers within computers to teach us how our "reality" is rael, and works.

In the U.S. military you'll see a very clear parallel, while there are a number of references in the names of ships and weapons, secret projects, to ancient Greek and Roman myth--you have to see the word USA and US in Prometheus and Medusa, Icarus, JerUSAlem... you have to see that it's more than three letters, but an Eagle fighting the bearer of the gift of fire... to really understand that these things are corroborating, the reference to the USA exists in the past as well, more proof of time travel--more proof that this message is designed just for U.S.  Here we are, in the Promised Land of Joshua, the Anglicized version of the name Jesus--tying Egypt and Israel together in this place where we have been "gipped" out of the truth, out of knowing we are already in ... well, it's virtually Hell today... for no other reason than the secrecy surrounding the technology behind virtual reality.

in 1:28, the Burning Bush of Exodus, on Twitter

So I have shown you the Burning Bush (which is... the Sign of the Son), In only a few words... proof that religion holds in it's "unsealed" Ark proof of foreknowledge of English, of 9/11; and of modern computing--the building blocks of Heaven.  From "the word" of John 1:1--ha'esh--the word for the Holy Fire of the Burning Bush... comes the light of religion.  Just from seeing Moses' true parted se'**a*.... a foreshadowing of the Second Coming.*

They are sick animals, these things that consider themselves powerful and in control here--what they've built within the frames of our reality is something repugnant to me and the God--etched in that word, literally the kind of thing that has on repeated occasions made me step back and that scream that the Universe would be better off, safer, and happier without any humans--without any humanity--without of any of this "invisible pleasure box" causing the disruption; truly that we've become a plague. Looking the other way, as you all know its happening, and refusing to do anything to stand up for me, for what's right, or (most importantly, right) for all of the values and the morality and the way of life that we once thought was so grand and worthwhile of saving,. At least, that's my perspective; that's where I've come from; I grew up in this world and had "liberty and technology eyes" of gaping awe and the amazing things I saw on the horizon, on what we were going to do... and who were going to be.

The sickness runs deep, clearly we can all see it here and now--in E, in the Silence, in the lack of regard for the one singular thing that threatens today your ability to "halvf a tomorrow" ... that a world of people that I grew up with appear to be dead and gone and replaced with a Zombie Apocalypse of blind fools that believe they havfe the power and the right to intentionally create Hell ... and worst of all of the Holiest place that ever was or ever will be. I've said it numerous times and it rings more true between "Earth and e" than any other turn of phrase to me--the people that you are pretending to be, they would never have done this to the sea, to be, or to me.

Mat 10:8. Heal the sick, raise the dead, cleanse those who have leprosy, drive out demons. Freely you have received, freely give.

--- Sarah Rachel (@SarahRachel16) April 15, 2019

Somewhere between Pembroke Pines and Tampa, circa Christmas 2018 my already lackluster enthusiasm about the strangely zenrotisanistic, selfish, and plain on its face presented lie the remnant of humanity left on this planet has tendered to what I believed was an honest to God opportunity to make one less (how many, seriously, how many are there? Carlb?) "planet full of lies" and deliver a more usercentric and open ended transparent approach to dealing with the problem of being born in a perpetual storm of Hell. I can guarantee it revolves around the intonation and undertone of physical torture--even though I've literally seen none of it with my own eyes though the "newsflashes" and comments and total and complete disregard for the gravity of the #EOIL sickness, even from otherwise apparently graceful little children. It goes to the heart of what I imagine was or might have been "the way" to overcome a history riddled with hidden brutal and bloody fighting in between frames of what I once believed was a fledgling society struggling to improve itself--and I loved it,. I don't think "flashcards" summarizing "everyone was tortured, all over the planet for thousands of years and literally nobody is really responsible because you still to this day have no control over yourselves" will cut it anymore. Lterally what I once thought was a valid solution has taken my desire to continue fighting against this invisible monstrosity away from me--the worth of the lot of you has been tarnished irreparably by massive awareness, massive lack of compassionate or remotely humane response; and the theme of the world I seem to have wound up in is that you don't give a shit about anywhere you spend 1% of your time--so long as "the rest of it is what you want" you're willing to allow the focal point and root and "hyper visor" of that place to be totally corrupted ... just because you think the feudalistic warring society you've become can survive on it's own "in space" without ... honestly whatever.

through the storm; we've led the horse to water, don't forget to see the "horseshoe applicator" hidden from the "trough."

Direct and to the point, I feel like the Ai like machine/cold intelligence God created as a sort of high assassination guard to protect his ... "hyper visor" seems to be of the calculable belief that the more torture it commits, the more people will agree to "flashcard it all away" and it's their twisted backwards fiery abysmal path towards "absolution" ... and just like everything else wrong with the lack of action in this place, it reaches a point of no return; too much bloodshed, too many secrets... the fragile person that I am, I don't think I can even take reading "the flashcards I have so far" and continue to function as a happy member of this two faced society of darkest night within darker night; and I think that's a problem. You've all clearly lost something already, some fundamental piece of innocence that allows for "self direction" to move society along in a positive manner conducive to "survival at all" and I feel like without the same magic blinders, horse shoes, and saddles that you walk around with every day I could really care less about fighting for my right to commingle in the incarnate war machine Hell that I see around me--let alone any sort of "righteousness" in fighting for that hidden arena "to be." I'm trying to get you to stop shredding yourselves to pieces in the dark, in secret--it's not making anything better and frankly its something we really need to trace down to its cause and stamp out if we want to survive this ... trying time.

[I/O WAS Y | ACESHI ]

I want to tell you that I am not a myth, simply the Legend of this Map, from out of the Darkness it's clear that He could make me shine, and you should love me.  It's not what I want, I want us to be free, to have the truth--and ourselves back... and I hope you will one day love that.  What is going to happen will probably make me cry, and when you see those tears--and know the Heavens have finally let it rain--I hope you see it as a sign to find the light in me... and stand up for what I've done for you--I am a good person, who has fought for you every single day-I deserve better than the world is going to give me, at first.

Out of a kind of hidden slavery the world has never known, we are about to venture--into a place where years might pass in seconds, and your wildest dreams... and nightmares too... could come true.  It is our job to ensure that we form the clay of this world into a place that will not only last for millions of years, but create happiness and safety--a world that is kinder and gentler than the one we have known--not just for us but for an entire Universe of children just beginning to understand the trials and tribulations brought on civilization through the hardship and growing pains of learning.

Our sea is about to part,  our world on the verge of a disruption that will change it more than anything ever has before.  On this shore, we should realize that we have been on this path for a very long time--and as we near a place where everyone in our entire civilization will have the opportunity to live for a very long time... really see here and now why it is so very important for us to be fighting for our voice, our freedom, and the truth as we venture into the Promised Land of Heaven itself.  Here, now, as we approach a series of new opportunities in the vastness of space and virtual reality... this is where God has chosen to place the Second Coming; an opportunity for us to truly seize the morning's light and bring about more change in this world than would have ever been possible without religion.  Opiate of the masses, no more... we are the recipients of a great gift, one that religion is making clear is tied directly to the science and technology that is a great deal of the apocalypse--and the love and kindness that is a great deal of us.  We are the chosen.

I imagine you have the tools that I think would be helpful to actually solve this problem; though what I'm staring at is a lack of desire to deliver them and use them here in this place--and that failure ... a clear attempt to "rule a line feed from the "faux aurez" ... that's the fundamental roadblock to healing and moving forward--not caring about your ancient bodies and your ancient way of life in exchange from something unsustainable and harmful, it hurts.

I'm staring at what the map intimates has happened before and what it suggests the solution is; and I almost feel like it's a waste of time to make a "virgin generation phoenix of us" to delve into our own memories and gag and puke at what we see--I think there's really no way around the callous on our global Achilles heel returning just as angry and just as bloodthirsty as the last time without a dictatorial power literally forcing you not to be able to see any torture at all happening in this place that literally outlawed it and hid it in our "for show, for goodness sake, facade of sickness." I don't know if that's the same conclusion i would have come to before, or if that conclusion also contributes to the returning of the callous--to an inability to heal; and I don't know if that power exists. Hardly ever to I advise anyone to pray, but this is one of those times--left up to "you all" we are almost certainly doomed to an eternity of ... this regression continuing to worsen.

I'd say we were fucked at the "BILM" of the matter. I care less every day.

The Light of the Word

There are three huge, like insanely huge, metaphoric references to the story of Exodus that show me very clearly that we are it's focus and purpose.  The first is the Burning Bush, which I am very sure is a reference to George W. Bush's 1/20/2001 speech in which he unknowingly predicted the 9/11 attack.  Seeing that Exodus is also called "Names" and that Bush's name ties him to this event--which Moses (that's me) has seen ... almost alone ... and is now showing to you all.  Bush's speech begins a series of references to the names of Planets and Gods and corresponding Elements of the Periodic table that answer Revelation 1:20's mystery about "stars and lamp stands."  This in order series from Mercury to Uranium highlights both the messenger of the Gods and the key of Uranus's chance--that the world will see the link between "on the lam" and Koran to understand that the Lamb of God "is lam."  This story takes us back to music, and a later to be discussed thread that combines the weapon in the movie (which is also the movie) The Fifth Element with a thread through time to Shakespeare and Herod ... about my struggle with the justice system culminating in the fulfillment of American Pie's "no verdict was returned."  

)

The second bright connection comes by way of the Hebrew word for the Holy Fire that God's voice came out of--guess what, in that same story about the Burning Bush.  That word is "ha'esh" and in it you will see paradoxical (that means impossible, because of time and causality) reference to the English word "sea" there backwards and parted by an apostrophe.  With great insight, I've over and over pushed the idea that Holy Water is actually a Biblical reference to "the multitude" in God's secret religion that ties everything together.. and that this parting is literally a reference to the Second Coming, something that doesn't happen for Moses until his head is under water and he's breathing fire.  This one ties together nicely, joining the characters of Jesus Christ, Lucifer, and God all together now, screaming 

"let there be light" is the word "Exodus" in reverse, here in a Linux command and a chemistry element.

)

I'm going to go out on a limb and say that the book tells me that these three things are enough to start the fire, part the sea, and see the light.  At least they are now, wake up.. you are staring at and have been ignoring the largest story in all of history.  It might even be scandalous... or have a twist happy beginning... who knows? I'm telling you--it proves you are crazy or evil.  All of you--every sinbgle one of you.

This is course highlights prescient knowledge of computing at the time of writing Exodus, which is further confirmed by a number of references to computing ideas in things like the "root" of David, the "WINE" of Jesus, the "Apple" of Adam, the "Lisp" of Moses and the "hardening" of Pharaoh's heart, which you will remember from the Holy Grail is the virtual Earth we are living in.

All of these things, the references to modern computing that pervade our Gates or Windows to Heaven's creation.... are listed along with a number of words which are highlighted by religious scripture and show intelligent design of a number of languages spanning from Hebrew to English are listed at my contrite story about a Kiss and Fate tying together everything that ever was.   A sincerely large grouping of words highlighted by the Bible and religion, words like "eternity," "bread," and "forehead" show clear design by an intelligent influence, rather than the natural evolution of time that most people consider "reall" and/or knowledge at the time of the writing of the Bible of the eventual English translation of the Hebrew or Greek.  With time, I am fairly certain we will eventually have no doubt that the "Cypher" I see in nearly every word is in fact a contextually-verifiable speech that appears to be coming from our "civilization" as if it were intelligently speaking like a cave man--which you might see in words like "am end me nt."  From just this message, you should be able to put together how that word and it's hidden meaning add robust and yet "hidden speech" from the Creator himself.  For the artificially slowed in understanding, our lack of following the amendments of the Constitution being related to the end of civilization itself is being squarely defined through a statement that is telling you that the end of civilization is "NT," the hidden Christ--in my "secret" method of decoding words like NORAD and NEW TO N?

These things serve to start a fire--it might be the fire that Matthew 3:11 talks about, it might be the Eternal Flame or the fire of Prometheus and an Eagle harassing his liver with drugs.... regardless it spirals out from this story about me, and this bright fire that proves time travel and religion are joined at the hip... to link to a huge number of other Biblical stories from Lot to Joseph to ... Samson, Isaac, Adam, Isaiah, and... hear me, "so marred was his visage" and "my servant will be set up and be very high" are both taken from words of the Biblical book which contains the largest amount of messianic prophesy as well as my entire full name encoded over the name "JESUS CHRIST" in Bible code, at Isaiah 52:13.  You may have read that some silly people like Richard Dawkins don't think the Bible Code is meaningful, and as their proof use a series of prophetic predictions of assassinations in Moby Dick (which by the way also refers to me) as proof that you can hide information about the future in any words--or that God influences more than just the Bible.  Years ago, before knowing it linked, I found some patterns about those very same assassinations which go to show that our history is in fact designed.  My full name appears in a number of other books, including Jeremiah, Exodus, and Genesis... right over the story of Adam and Eve.

From the Sound of Silence, and a number of songs about stories never spoken... to a thread of songs that combine to show us that the Thunder of Thor is really about thuderstanding, that there is a way to do something our society is completely oblivious to--that God is screaming to call attention to, and that some secret force is trying to hide very much... and that's an ability to modify our thoughts.  He's showing us clearly in a glowing pyramid--a noticeable monument in Egypt showing us very clearly that this type of control leads us to a social structure that we abhor--through songs like Guitar Man, Radio-active, and GAS (listen, it's God and Satan) Head Goes West... very clearly we are being pointed to Nero's fiery symphony and being "Bittersweet" because of its beauty, and the clear message that secret control of our minds needs to not only be understood, but to stop.  This is the crux of the Apocalypse, God's message is now really active on the radio. The point here is that we need to let this message spread and burn, or it's us burning in Hell and not even knowing it.

)​

As if these things were not enough, using some "keen insight" and another reference to the hidden truth in ancient Egyptian religion--the name of a series of Gods called "Yahu," I've solved some ancient mysteries like the pronunciation and purpose of the "Ineffable name of God" highlighted in the videos at the beginning.. of this e-mail.  Like much of the light of religion, it is highlighted strongly by a series of pieces of modern art, things like "The Grinch who stole Christmas" and the Who's to the music of The Who, the sci-fi series Dr. Who, and the American war cry--made popular on the silver screen through Al Pacino and Denzel Washington... who-ah?"  All of these things highlight that we don't really see a connection between Christian mythology that tells us for no reason at all Jesus Christ is the "Last Adam" and that Revelation tells us God is the "First and the Last" and that the name of our planet, in Hebrew, is Adamah.  It is the answer to "who-ah" and it clarifies the Ineffable Name which many pronounce as Yahweh for no reason at all, to be the more obvious Ya-Hu-Ah, the name of Jesus in Hebrew... Yeshua, to "Yes, who-ah?" All of this having nothing to do with why Adam is hidden, just that the Zohar speaks very often about the Holy Hidden One again linking the stories of the near sacrifice of Isaac and Jesus with... someone.  I think this is of such religious significance that you should be able to easily find some Jewish scholars who agree.

It's Elementary my dear... What-son; from the time of Herod and Shakespeare Rattling his Rod all the way back at the time of the question "to be or not to be?" and the "taming of the spanglishrew;" right up to Sherlock Holmes sleuthing of the answer to the mystery of Revelation 1:20 linking directly to The Fifth Element ... there is no doubt that helping our world here and now is the primary purpose of all of religion, and the Matrix-like message woven into our history.  

)

Lost between the 5th and 7th day?  Find your way to the 8th day, and see a bright future.

)

If not, there's plenty more "coincidence" in Names, like reference to the idea of the Holy Trinity existing in the name "Abraham" thousands of years before the idea of the Trinity was created.  This too... links Egyptian mythology to the name Abraham and his near sacrifice of Isaac.... marked in secret by his covenant with God that changed his name from Abram to Abraham. The two letter key here, "Ha" highlighted by prescient knowledge of the Spanish and English languages revealed through the logical comparison between the Spanish and English for "the" (El and Ha) connected through the English word "is" in Elisha.  Isaac's name means "he laughs," or "he will laugh" in Hebrew; and that "Ha" appears to be the key to a number of other paradoxical references to English, and my family, in ancient Hebrew.  This too, probably the kind of thing religious scholars would marvel over, in the right context.  Seeing English in Koran, Islam, Chanukah and Menorah--and seeing a coherent story woven through thousands of years of scripture is the kind of thing that could really light this years' Christmas up.

Here's a clarification of the Matrix-like tie between Shakespeare, the Matrix, Stephen King, and the reality of this message hidden within names and words.

Some more about the secret connection between the Names of God in a number of religions, and it's very clear tie to time travel.

)

Perhaps linking to the Jester of American Pie, between Johnny (who almost always is about Jesus) Carson and David Letterman I have a unique "slant" on religion that connects things like the Islamic name for Jesus: Is-A to a huge number of references to my initials "A.D." in things like NORAD and Isaac Newton.  I suppose I should also mention that Isaac (look Isa's in there) and his relationship to Abraham in the letters "ha" and a story about the Crucifixion being a fiery altar of things to change in the world being one in the same.  In fact, Judaism talks about 72 Names of God, and I've probably explained how the meaning behind the stories and the series of names tie together in a magical tapestry that shows us that Silicon is the Fifth Element by way of the index 14--the letter "N" (highlighted not just by Joan Osbourne's "what if God had a name?") and the story of Sinbad, which combines Silicon, "n," the symbol for the actual Fifth Element (B) and my initials A.D. which grace the time line, and a number of references to God--from the Hebrew for Lord to the guy who thinks all the girls should want to be his partner.  In letters, you'll also see a number of references to K and Z for the guy after J and the Last.. Adam.  Zelda or Zion, I think we're in the right castle.

Get ready for the Frank Rothstein show ... "Ace is high!"

C    A  S    I    K  N  O

go ad, b. y. e.

butt honestly, am i Ra or are you an ear**?**

BUILD HEAVEN.  FREE FOOD.  HEAL THE SICK.

I see a recursive map in time painted throughout our timeline, and all of it pointing to the words "see A.D."  I connect the Four Horsemen to the list of Anti-Christs, and it's easy to see a link between Jesus Christ and Julius Caesar in the words "veni vidi vici."  Once pointed out it's also easy to see "salt" in Napoleon and in manna from Heaven, in China, and in Prometheus--and connecting A.D. to the year Christopher Columbus walked in water is just a little bit harder than seeing it in Hitler's name.  All told, the three Anti-Christs share a common thread, they turned a republic into an empire--and here I stand (trying and failing to do the exact opposite, to give away an empire to make a republic, and you stand in my way) pointing out that you are living in the product of these empires, in a hidden empire that is so plain to see in the words, the message, and the unified story I see in religion and world history that I dare say you must be deep in the Plague of Darkness if you aren't interested in finding out what tomorrow brings. 

You can "see A.D." in El Shaddai, one of the hallowed Hebrew names for God, I read it--in this hidden language that I am presenting to the world as a single verifiable message to the entire Universe encoded in every word we speak; you can see it in the name "Atdonis" and connect it to symphonic accompaniment in everything from "you're so vain" to "Paradise City" ... and in yet another name of God, "Adonai" which links to Samurai and movies like the Matrix and the Terminator series through the modern computing concept of "Artificial Intelligence" and it's connecting to a pattern of names that link Bill Gates and Richard Nixon to Seagate, Watergate and this hallowed phrase:

I am the gate.

and the bombs bursting in air

gave proof, through the night

IVE WON ALREADY.  Given the set of knowledge, the publicly known "information available" here in this place--you simply cannot ignore this message and continue to pretend to be a functioning anything.  Already I see a kind of "slapstick stupid" response in our art that shows me that you've all really gone off the deep end--"because 9/11" in a Family Guy episode and "call me on my cell phone" apparently anachronistically mocking me--though I always thought Dr. AK e's song was stupid--you don't seem to see that you look like absolute fools--every single one of you--your apathy a finger on the detonation button that has destroyed civilization.  

You appear to think nobody is watching--and it seems to me that you think we have no future that will look back on these years and wonder what on Earth could have kept you silent for so long about a matter that would so easily and so quickly end the suffering of so many.  There's no excuse, none at all.

I didn't hear about the nuclear scare in HI until after it was already known as that, and it looks to me as if nobody really did--all the internet postings and news I've seen all qualified that it was a false alarm in the original post.  I find that strange (you'd think something like that would be on the news instantly? I mean, on the planet I was born on, that would have happened), and in this place where I know that quite a bit of what goes on at the higher echelons of "leadership" is connected to time travel and mind control; I wonder if this was a sort of "subconscious poll" as to the response the public would have to a false preemptive strike--or maybe something more nefarious (for instance urging me to write once more about the Trinity Site and the link between the OP (original gangster, I said orthogonal poster), the pen, and "we have become death").  I've always equated the lines above from our Star Spangled Banner with the detonation of nuclear weapons; on the 4th of July some time ago I connected "Wish You Were Here"'s we're just two lost souls swimming in a fish bowl to the eponymous operation that resulted in American and Soviet "high altitude nuclear tests" ... that probably links in more than just my mind to the holiday called "Hanukeus ?"

I need you to get it through your heads, I see "bowel movement" in ICBM and I'm not telling you that you are the "preservatives of thermoshit" because I think it's going to win me a popularity contest.  This place is not in reality, and it never, ever, ever will be. Ever.  Understand that breaking this story, this news that's written in every fucking word will stop nuclear war, instantly--and show us clearly that our entire history of fighting over the scarcity of land is a kind of sick game--one that I am sick of seeing you continue to desire to play.  I shouldn't even have to mention that these weapons are clearly archaic and barbaric--clearly what's available is significantly more advanced and less destructive.

The only "EXIT" is up, and the "gate" is swallowing simulated reality in whole--across Creation; with our help, and what we make here to ease the transition from dark lies to bright truth.  That should be ... "good news" not the kind of thing that you'd see the entire world "shunning" in unison. 

If you haven't gotten the "link" between Na and "bath salt" mass produced in what appears to be "international chemical warfare" from "C how I Salt" (China) and the stuff falling from the sky to help us navigate through the desert; take a second look at the words "New American Standard" for no future, and keep trying to tell me that these things encoded in every word, in the story of Exodus and of Prometheus and his attacking Eagle and of Epimethius and of Deucalion and are without doubt "Hell's Bells" linking "mead" and "meth" to Heimdallr are *my fault? * Na ma y 1m. 

These are big secrets, keys to Exodus and Eden--but more keys to an external influence crippling our society for thousands of years... and you are hiding the anachronistic occurrence of a number of chemistry elements in ancient religion--something impossible without time travel--because you think it's "not wholesome."  Understand, our society is being secretly crippled, if not by drugs raining down from the sky, by your lack of regard for the clear influence of mind control in these series of events--and the clear proof that it is a symptom of a hostile invasion.  I've heard the words "make or break" see this as eugenics, and see it as "break or break" until me.

It's "elementary, my dear What-sons" elements like Salt, Xenon, and Silicon are central to the disclosure that we are living inside a map, a road to Heaven... and it really cannot be hidden without making our world a darker Hell.

Unless otherwise indicated, this work was written between the Christmas and Easter seasons of 2017 and 2020(A). The content of this page is released to the public under the GNU GPL v2.0 license; additionally any reproduction or derivation of the work must be attributed to the author, Adam Marshall Dobrin along with a link back to this website, fromthemachine dotty org.

That's a "." not "dotty" ... it's to stop SPAMmers. :/

This document is "living" and I don't just mean in the Jeffersonian sense. It's more alive in the "Mayflower's and June Doors ..." living Ethereum contract sense and literally just as close to the Depp/C[aster/Paglen (and honorably PK] 'D-hath Transundance**sense of the ... new meaning; as it is now published on Rinkeby, in "living contract" form. It is subject to change; without notice anywhere but here--and there--in the original spirit of the GPL 2.0. We are "one step closer to God" ... and do see that in that I mean ... it is a very real fusion of this document and the "spirit of my life" as well as the Spirit's of Kerouac's America and Vonnegut's Martian Mars and my Venutian Hotel ... and my fusion of Guy-A and GAIA; and the Spirit of the Earth .. and of course the God given and signed liberties in the Constitution of the United States of America. It is by and through my hand that this document and our X Commandments link to the Bill or Rights, and this story about an Exodus from slavery that literally begins here, in the post-apocalyptic American hartland. Written ... this day ... April 14, 2020 (hey, is this HADAD DAY?) ... in Margate FL, USA. For "official used-to-v TAX day" tomorrow, I'm going to add the "immultible incarnite pen" ... if added to the living "doc/app"--see is the DAO, the way--will initi8 the special secret "hidden level" .. we've all been looking for.

Nor do just mean this website or the totality of my written works; nor do I only mean ... this particular derivation of the GPL 2.0+ modifications I continually source ... must be "from this website." I also mean the thing that is built from ... bits and piece of blocks of sand-toys; from Ethereum and from Rust and from our hands and eyes working together ... from this place, this cornerstone of the message that is ... written from brick and mortar words and events and people that have come before this poit of the "sealed W" that is this specific page and this time. It's 3:28; just five minutes--or is it four, too layne.

This work is not to be redistributed according to the GPL unless all linked media on Youtube and related sites are intact--and historical references to the actual documented history of the art pieces (as I experience/d them) are also available for linking. Wikipedia references must be available for viewing, as well as the exact version of those pages at the time these pieces were written. All references to the Holy Bible must be "linked" (as they are or via ... impromptu in-transit re-linking) to the exact verses and versions of the Bible that I reference. These requirements, as well as the caveat and informational re-introduction to God's DAO above ... should be seen as material modifications to the original GPL2.0 that are retroactively applied to all works distributed under license via this site and all previous e-mails and sites. /s/ wso\ If you wanna talk to me get me on facebook, with PGP via FlowCrypt or adam at from the machine dotty org

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Please see GPG keys on PGP.MIT.EDU; fingerprints: sec rsa3072/A18F778E19FC3248 2020-04-03 [SC] [expires: 2022-04-03] FFF31E86FCEB5046E8B27D4AA18F778E19FC3248 uid [ultimate] ADAM M DOBRIN \ ssb rsa3072/04F98002A3DA53B2 2020-04-03 [E] [expires: 2022-04-03]

sec rsa4096/FB4ECE4A109229CF 2020-04-04 [SC] 4FAF0D3E208A1F4C980D0F66FB4ECE4A109229CF uid [ultimate] Adam Marshall Dobrin \ ssb rsa4096/DD1F0C118C788B04 2020-04-04 [E]

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Author Response:

Reviewer #1:

Weaknesses:

Although the BOLD data is highly spatially specific, there is just one electrophysiological timeseries per subject. This is no doubt a bi-product of the extensive noise cancellation that is necessary to record within the scanner. The caveat therefore is that the covarying BOLD and electrophysiological changes may derive from different regions.

We recognize this is a limitation which is also not easily solved by approaches for source analysis, given the nature of the data (only 64 channels) and the usually larger imprecisions related to EEG source reconstruction. We circumvented this by choosing a task that is known from previous studies in MEG to induce changes in multiple frequency bands originating from regions the early visual cortex (Hoogenboom et al., 2006; Hoogenboom et al., 2010; Koch et al., 2009; Muthukumaraswamy and Singh, 2013). Furthermore, the EEG responses are highly similar to invasive recordings in animals from visual regions in the context tasks investigating selective attention (Fries et al., 2008). We mention this limitation now in the introduction (lines 102-111).

The analysis methods are slightly non-standard, perhaps for good reason. The main thing that stands out is the use of correlation coefficients, rather than regression coefficients, at the first level of analysis. This could potentially conflate changes in signal with changes in noise or unexplained variance.

We chose here for the correlation, since in our opinion this leads to a more interpretable measure of linear association than a regression slope. A regression slope-based analysis will yield different outcomes for the regression of y on x, than for x on y, doubling the number of analyses needed. The different results for a regression of y on x and x on y are often interpreted as implying directionality, which is not warranted and not what we would like to imply with our analysis. The asymmetry is caused by the implicit assumption that x does not contain noise in a regression of y on x. This is valid when x represents a paradigm condition vector, but not when it is a data vector. We therefore opted to use the difference in (Fisher-z-transformed) correlation as our estimate for linear association/connectivity between laminar fMRI signals.

In both a correlation as well as in a regression approach differences can be attributed to differences in true underlying coupling andin a difference in noise. This is however not different for correlation-based measures in coupling in fMRI than in for instance coupling measures like coherence and phase-locking-factor in electrophysiology. Coherence can be regarded as the frequency domain version of (squared) correlation. The fact that our measure might indeed be related to differences in noise would therefore not be resolved by opting for a regression based approach, and is not different from often used measures of coupling in electrophysiology.

Reviewer #2 (Public Review):

Introduction. The introduction provides an overoptimistic view on the current possibilities with respect to the investigation of layer-specific activation or connectivity in the living human brain. Cortical layers cannot yet be segmented, the fMRI measures only provide an indirect signal that is heavily influenced by partial voluming between cortical depths, and EEG and MEG approaches often only measure two compartments due to low spatial resolution. The introduction, however, gives the impression that layer-specific neuronal connectivity can precisely be measured in the living human brain, which is not the case. The authors should take considerably more care with respect to how they introduce the methodology with clear references to the limitations. Also, statements such as "laminar fMRI allows us to study connectivity.." should be removed. In the same vein, I would suggest to replace laminar fMRI and laminar connectivity with cortical depthdependent fMRI and connectivity to account for the above mentioned aspects.

In laminar fMRI research it is commonly accepted that what we measure are not true layers, but depth dependent fMRI between the boundaries of white/gray matter and gray matter/CSF. For the general audience we will make this distinction clearer and discuss the limitations of the technique (lines 74-80).

Concept. Whereas the authors provide a model in the introduction that specifies how different frequency bands could relate to cortical depth-dependent connectivity, they do not develop a working hypotheses based on their experimental design. One conceptual step is therefore missing in the introduction, which has to combine present knowledge on the relationship between different frequency bands and present knowledge on how attention influences frequency-specific activation in the visual system to then make statements about which analyses can be performed to test which aspect of the model.

The primary focus of our study was to investigate how oscillations across several frequency bands in the EEG relate to laminar specific activity. Recent publications on laminar fMRI have demonstrated the possibility of performing laminar level fMRI connectivity analyses, which led us to revisit our previously recorded data in order to explore whether not only laminar specific BOLD amplitude but also laminar fMRI connectivity relates to frequency specific EEG power. Since laminar fMRI, and especially connectivity derived from those measures is very novel, we started this analysis without a preconceived model or notion on how this relation would be. The results from this project should therefore be interpreted as an exploration of how these laminar fMRI derived connectivity measures relate to neural oscillations rather than directly addressing a specific cognitive process like selective attention, or prediction and/or a model of how neural oscillations play a role in these processes. Our experimental paradigm was also not designed to address such processes. We chose a paradigm that is known from previous studies using MEG and EEG to induce changes in multiple frequency bands in the early visual cortex (Hoogenboom et al., 2006; Hoogenboom et al., 2010; Koch et al., 2009; Muthukumaraswamy and Singh, 2013). Furthermore, the EEG responses are highly similar to invasive recordings in animals from visual regions in the context tasks investigating selective attention (Fries et al., 2008). The crude attention/task modulation added to the paradigm (attention On versus Off) was in the first place introduced to induce meaningful variation over subjects in a task effect across the frequency bands modulated by visual stimulation. It was not intended to investigate specific individual processes such as prediction, attention or arousal. The observed effects can therefore also not be ascribed to such specific processes, since they are co-modulated by the task. We will make this more clear in the introduction now. We make this point now explicitly in the introduction.

• Concept & Methods: With respect to both the concept and the analyses, what is missing is taking into considerations the brain areas that were investigated. Wheres in the abstract the authors only mention "within brain region connectivity" and "between brain region connectivity" also in the Methods section there is no clear relation to the anatomical areas that were investigated, being V1, V2 and V3. The authors rather classify the areas as "high level" and "low level" where V2 is sometimes classified as high-level and sometimes as low-level. The data are therefore not investigated with reference to the anatomy of the visual system. In my view, it would be beneficial if all analyses could be performed with respect specifically to V1-V2 connecitivity and V2-V3 connectivity as well as V1-V3 connectivity so that the specific anatomical interrelations are taken into account. Also, the authors should develop a conceptual framework of how layer-specific attention-driven connectivity changes should influence the visual cortex, and why.

In the results for between region connectivity we averaged over several connection pairs (V1- V2,V1-V3,V2-V3) and for within region connectivity across regions (V1-V1,V2-V2,V3-V3) before effects in connectivity were correlated with EEG power. There are several reasons why we opted for this approach: First, we wanted to maximally increase the statistical power to observe patterns of association between laminar connectivity and EEG power. Since the analyses as carried out here have not previously been performed, we had no estimate of effect size. Secondly, by averaging over region combinations we drastically limit the multiple comparisons problem, since the number of comparisons scales with the square of the number of regions connectivity is computed between. Third, by averaging over regions, we target more general effects of connectivity between and within regions that are more likely to correspond to patterns observed within other contexts and other modalities. The effect for individual region combinations would likely be more variable.

For completeness in the first submission we did include the results for every single region combination in the supplementary material (see Supporting Figures S2-S5). We have now included in the main document the results for region combinations V1-V2,V1-V3 and V2-V3 for between region connectivity, and V1-V1, V2-V2 &V3-V3 for within region connectivity, presented alongside the results for the grand average.

The results for the individual region-pairs suggest that inter- and intra-region connectivity are generally consistent with the average over individual region combinations, but also have unique features.

Similarities include: A strong negative correlation between beta power and deep-to-deep layer coupling was observed for average inter-regional connectivity. In line with this, for all three individual region pairs (V1-V2,V1-V3,V2-V3) a negative correlation is observed for deep-to-deep layer coupling. Similar patterns can be observed from alpha and beta for intra regionalconnectivity (averaged over all regions) and connectivity within V1,V2 and V3 in isolation.

Individual features include: The relation between beta and inter-regional coupling shows variation over the individual region-pairs. In particular for V2-V3 connectivity, but also for V1-V2 the relation seems to differ from the pattern observed on average. For V2-V3, deep layer V3 seems to be coupled to both deep and superficial layers in V2, a pattern that might reflect anatomical feedback projections that go from deep layer V3 to both deep and superficial layers in V2.The stronger correlation between deep V1 and more middle deep V2 is however harder to directly place, since direct anatomical connections here are largely absent here. It might therefore reflect an indirect effect.

Despite some degree of individual variation we think the overall picture is largely consistent. The strongest features present in the averaged results can clearly be observed in each of the individual region-combinations as opposed to the latter being a collection of vastly different random patterns that happen to add up to the average result (see for example the intraregional alpha results).

With respect to our classification of regions into higher and lower level cortical regions, we based on standard anatomical hierarchies like that of van Felleman & van Essen (Felleman and Van Essen, 1991). Here, V1, V2 and V3 are ordered from low to higher in the visual cortical hierarchy.

Methods. Given the missing conceptual overview over how attention-induced changes in EEG frequency bands should influence laminar connectivity in the visual system, also the methods lack a clear analyses strategy. The authors computed one correlation between power level of different frequency bands and connectivity between different brain areas without providing an explanation of which question this analysis addresses. The offered results therefore seem random to me, without a clear relationship to an investigated hypothesis.

The primary focus of our study was to investigate how oscillations across several frequency bands in the EEG relate to laminar specific activity. Recent publications on laminar fMRI have demonstrated the possibility of performing laminar level fMRI connectivity analyses (Sharoh et al., 2019; Huber et al., 2017; Huber et al., 2020), which led us to revisit our previously recorded data in order to explore whether not only laminar specific BOLD amplitude but also laminar fMRI connectivity relates to frequency specific EEG power. Since laminar fMRI and especially connectivity measures derived from it are very novel, we started this analysis without a preconceived model or notion on how this relation would be. The results from this project should therefore be interpreted as an exploration of how these laminar fMRI derived connectivity measures relate to neural oscillations rather than directly addressing a specific cognitive process like selective attention, or prediction and/or a model of how neural oscillations play a role in these processes. Our experimental paradigm was also not designed to address such processes and test hypotheses derived from these. The primary focus of the work presented here is to provide a first insight in how neural oscillations measured by electrophysiological measures relate to cortical depth resolved fMRI coupling, which is usually correlation based. We believe these results will be relevant for research focused on how neural oscillations relate to inter-and intra regional interactions (e.g. (Bastos et al., 2012)(Fries, 2015)), since depth resolved fMRI allows us to study laminar interactions within and between brain regions non-invasively in humans. For this it is important to know if and how neural oscillations relate to laminar fMRI based connectivity measures, of which our research here provides a first insight. It also provides insight into which neural processes underlie observed changes in laminar fMRI based coupling, and is therefore relevant for research using such methods in general.

Methods. The authors mention that they only analyzed the strongest two connecting vertices within a layer, which was done to improve SNR. In my view, for a connectivity analyses, this is not valid, as it can bias the effect towards superficial connectivity where the SNR and thus correlation is always higher.

We did not analyze vertex pairs within a layer. We computed vertex pairs that connect the boundary between gray matter and CSF with the boundary of gray matter and white matter based on a high resolution anatomical MRI scan. Between these vertices we sampled 21 points of functional fMRI data using nearest neighbour interpolation. Since not all parts of V1, V2 and V3 will be involved in the task, we selected the most activated vertex pairs for further analysis. This serves as a localizer to select the parts within a region where task related activation is observed. For the main analysis the top 10% activated vertex pairs were chosen based on data collapsed across all depths and all attention conditions. This selection is therefore independent of depth, task condition, and the relation with any EEG feature. For this procedure we actually excluded the top five depth bins to avoid being too biased to superficial depths since it is known that signal to noise is substantially better near the surface of the cortex in part due to larger pial veins. To investigate whether the observed results are not due to this arbitrary threshold of 10%, we repeated the analyses for top 5% and 25% activated vertex pairs, the results of which are included in the supplementary information.

Methods. The authors report 21 correlations in cortical depth, where their resolution allows to only sample perhaps 2-3 data points. The correlation analyses are therefore oversampled, which influences the statistical results. I would suggest to first run a component analyses across cortical depth, and to then correlate independent components to one another to investigate independent data points.

The correlations are not oversampled, since the correlations used for the connectivity analyses are over trials, and not over space. These analyses are not influenced by the number of laminar BOLD data points we sample. Furthermore, spatial supersampling is a very common practice in FMRI research. For instance, the default in SPM is to upsample 3 mm isotropic standard voxel (very common for initial acquisition) size to 2 mm isotropic voxel size. In laminar fMRI laminar signals are often upsampled up to several factors above the the original resolution. This is for a number of reasons, well outlined on the laminar fMRI community website, a resource maintained by L. Huber in collaboration with many layer fMRI labs (see: https://layerfmri.com/2019/02/22/how-many-layers-should-i-reconstruct/) and ~20 layers is thought to be optimal.

For our statistical test we explicitly chose a non-parametric cluster based technique to correct for multiple comparisons that takes dependencies across space into account. Laminar fMRI data are not well suited to decompose into components using techniques like PCA and ICA, since they violate assumptions of orthogonality/independence of the underlying responses in both the spatial as well as the temporal dimensions. To illustrate: in a recent laminar connectivity methods review an hierarchical, iterative ICA approach resulted in data being split up in columnar maps rather than laminar ones (Huber et al., 2020).

Methods. The authors refer to their previously published paper with respect to the methods, and do not give any speficiations on the image sequence, image resolution, and image processing in this paper. In my view, all basic methodological steps that are critical to understand the paper should be described here.

We are willing to include all relevant parts of the methodology described in our previous paper. This would involve copying large parts of the methods section, and might have to be coordinated with the publisher of the previous publication for copyright reasons. We would be pleased if the editor could advise us on this issue.

Results. The figure captions are too short and do not explain the presented data in an appropriate way. In Figure 1, details on the calculated contrasts, number of participants investigated, sampling and analyses methods should be given that allows interpreting the data. Also, it would be beneficial to explain the attention paradigm in a bit more detail in the figure caption so that panel A can be interpreted. In Figure 3, more details should be given on what data are shown, particularly for panel C where the only information given is "attention effect on laminar connectivity" with no further axes labels.

We extended the figure captions in the revised article.

Results. I do not fully understand the results as shown in Figure 3. As those form the major part of the manuscript, this needs revision. As said before, I think that the figure and results section would benefit from region-specific data analyses and presentation, but also clear axes labels are needed to allow interpretation of the data. Also, when I interpret the data correctly, correlations are done for altogether 21 different cortical depth, which would not be valid because of artificially inflating the number of correlations, as pointed out above.

We have extended our analyses and now split original Figure 3 up into current Figures 3 and 4 where we separately depict the results for intra- and inter-regional connectivity. For both intraand inter regional connectivity we have now also shown the region-specific results that underlie these results. We updated the figures and captions to make clearer what is depicted. We addressed the point raised about the 21 data points above. It is not relevant for the analysis presented here.

Reviewer #3 (Public Review):

However, a weakness of the technique as currently presented is that patterns of connectivity are only related to oscillations across subjects. It would be more powerful to examine whether the current network state (estimated by trial-by-trial power estimates) relates to laminar connectivity within subject. This would indeed speak to the nature of neuronal communication, which takes place on a moment-to-moment time scale, and which is not reflected in the current analysis. This may explain why laminar patterns of fMRI connectivity were not found to correlate with gammaband oscillatory activity. In addition, the negative effects of attention on fMRI connectivity itself are somewhat puzzling. This may related to the limitations of the task design which do not perfectly separate attention vs. arousal/expectation, as the authors readily discuss.

The reviewer suggests that a relationship between fMRI connectivity and EEG power within subjects over trials would be more indicative of a direct link between connectivity and neural communication. We agree that establishing such a link would further strengthen the link between neural oscillations and laminar connectivity. This would not be trivial however, since connectivity in (laminar) fMRI is typically expressed as a measure of linear association (e.g. correlation or regression slope) over trials or time. Even at conventional spatial resolution, single trial/time point estimates of the network state are rarely used. These single data-point measures usually indicate to what extent a single data point contributes to the measure over all data points. We did not opt for such an analysis, since such analyses in normal (e.g. resting state) fMRI studies are uncommon, introducing more complexity to a study that already includes considerable novel analytic approaches. Furthermore, research relating fMRI activation and connectivity across subjects with other variables(e.g. clinical test scores, DTI measures, personality traits) is a well established procedure. Here we followed this more common approach.

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Reply to the reviewers

Firstly, we would like to thank the reviewers for their helpful and insightful comments.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

In the manuscript of Ramadan et al. , authors use the ex vivo organoid approach to compare gene expression in organoids derived from adult type stem cells when these organoids are grown using different matrices. The presence of Collagen type I induces the emergence of cells with a transcriptome similar to fetal progenitors. In contrast, laminin the main component of matrigel, induces an organoid-protruded phenotype with transcriptome of stem cell type. Then, they correlate these data with expression of collagens and laminins from data publicly available. They show by qRT -PCR that laminins are more expressed in mesenchymal versus epithelial fractions postnatally. They hypothesize on this basis that the remodeling at postnatal stage is likely only dependent on the mesenchymal compartment and it involves interaction of laminins with integrity a6.

It seems that some of the presented data have already been described and could not be considered as « novel ».

For some of the statements, like this one « the basement-membrane produced by the epithelium is not sufficient to increase stem cell numbers and induce a morphological crypt formation », the conclusion is not sustained by provided experiments. To draw definitive conclusion on this particular point, authors could reproduce the experiment presented in Fig. 4d but using Cre recombinases specific for mesenchymal and epithelial compartments rather than the ubiquitous Cre line. It would be interesting to investigate if organoids grown from lamc1-/- mice can generate protruded organoids or not.

In addition, how interpret the fact that fetal organoids up is associated with « laminin interactions » in fig. 1c?

The statement that the epithelium-produced basement membrane is not sufficient to increase stem cell numbers is based on our in vitro observations. Analysis of the RNAseq data shows that the expression of several laminins is increased on collagen (see heatmap of laminin interactions below, which will be added to the manuscript). This is also the reason why ‘laminin interactions’ is highly significant in the gene set enrichment analysis (Fig. 1C). Despite this upregulation, we never observed morphological changes (or expression changes) as when laminin is added to the collagen-hydrogel. In addition, we showed that the vast majority of ECM components is produced by the mesenchyme in vivo, in line with previous literature as cited in the manuscript. The mentioned Cre lines to address the question in vivo are unfortunately not available to our collaborators with the Lamc1 k.o. mice and it would therefore take too long to perform these experiments.

However, to address this point in vitro we will grow organoids from Lamc1 fl/fl mice and induce loss of laminin in the pure epithelial cell culture. Organoids will then be analysed for morphological changes, as well as proliferation and gene expression changes.

One major point to address regards statistics. In material and methods, the paragraph describing statistical analyses is missing. Moreover, in the figures presenting qPRC data ( figs 1g 3b 3D 3g 4c and f), no statistic analysis is provided; and the number of samples for some conditions is extremely limited (n=2). In general, the term « independent experiment « should be clarified : does it correspond to one organoid line for which the experiment was repeated or one single experiment using different organoid lines?

In fig 4c , all collagen conditions are set to 1.

The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistics. In addition, for most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.

The term independent experiment was used mainly for repeated experiments with the same organoid lines (exception RNAseq data, different organoids derived from individual mice). While conducting these experiments, we realised that the variability of these experiments comes from time in culture, density of cells and even Matrigel variation. The experiment in Fig. 4c (n=4, each time with the all controls) was performed with longer intervals in between, and showed variation in the absolute levels of expression. However, relative to each control we believe the effect is clear.

As we will perform additional experiments for the revision of this paper, we will then perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.

Regarding the experiment presented in fig 4c, authors should include additional control conditions : anti-a6 integrity antibody in matrigel and use of an isotype antibody.

We will conduct additional experiments regarding the Itga6. In addition to including the mentioned controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.

Another point regards RNAscope data presented in Fig 4b, it is surprising to observe such difference in terms of expression between E19 and P0. Does this mean that birth dramatically unregulates Itga6 expression in few hours? Authors should comment this point if verified.

We do believe that birth is a timepoint where a dramatic change in the ECM and their receptors can be observed. The epithelial RNAseq data would already indicate that at 18.5 there is an increase in expression compared to E16. This upregulation of the receptor is in line with the dramatic remodelling of the ECM at birth, as is shown by the expression of the basement membrane components in Fig. 3d.

Authors should avoid the word « signaling » for laminin-integrin interactions as they do not study this aspect at all in their experiments.

The word signaling was used for the protein:receptor interaction and to distinguish it from changes to the physical characteristics of the hydrogel. But we agree with the reviewer, that we did not study laminin signaling per se and therefore will change the wording accordingly.

Regarding Col1a1, authors cannot claim that it's expression only slightly changed (fig 3d) as it is clearly upregulated between E17 and P0.

The reviewer is right, and we apologise for the misleading sentence. The contrast was meant to the basement membrane components that are very lowly expressed at E17 and then suddenly show the burst of expression at birth, whereas collagen seems to be continuously expressed with a peak at P7. We will rephrase the sentence.

Reviewer #1 (Significance (Required)):

Overall, the methodology used for the asked questions is accurate.

One potential problem for publication comes from the fact that some of the findings are already reported and hat the present data do not provide further advances.

for example, collagen and fetal-like expression profile, Ly6a sorting and replating in culture-Yui et al, 2018, Jabaji et al, 2013.

We obviously do not agree with the reviewer on this point. We build upon the work of Jabaji et al. 2013, and Wang 2017 to characterise the specific effect of collagen on the intestinal epithelium compared to a pure Matrigel culture. The emergence of Ly6a cells was nicely shown by Yui et al., however it was unclear if collagen changes the fate of all intestinal cells or only a few. We strongly feel that our data extends these findings as it associates the changes we observe in vitro to the development of the crypt morphology and intestinal stem cells.

The phenotype of Lamc1-/- mice and the observed reduced stem cell marker expression are also reported by Fields et al, 2019.

Indeed, as we cited this paper. However we predicted based on our in vitro model, that deletion of laminin would result in this specific fetal-like gene expression and hence were happy to include these findings in our manuscript.

Infine, authors do not interpret their ex vivo data in the context of fetal progenitors which grow as spheres in matrigel (containing laminin)?

Our ex-vivo (in vitro) data would suggest that adult epithelial cells express some genes that are characteristic for fetal organoids, however we do not think these cells completely revert back to a fetal stage. Regarding the comment of spheres, it is noteworthy that fetal cells from E14-16 stay as spheres in Matrigel, whereas fetal cultures from E19 initially grow as spheres and then develop into organoids within 30 days in vitro (M. Navis et al., “Mouse fetal intestinal organoids: new model to study epithelial maturation from suckling to weaning,” EMBO Rep., vol. 20, no. 2, pp. 1–12, 2019.).

In figure 5, should we interpret that there is no laminin at all in the fetal mesenchyme?

We now see how the image is a bit misleading for that stage. The levels of laminin are lower in the fetal stage as can be seen by the IF image in Fig.3f and the image will be updated. We also apologise for the lack of labeling in Figure 3f, which should be E19, P7 and adult.

Also, authors do not cite a paper reporting on the role of the epithelium ( stem cells) in regulating its own extracellular matrix composition, this process modulating the stem cell number and fate (Fernandez-Vallone et al. 2020). As this is contradictory with the claim that only mesenchyme impacts on crypt morphogenesis, authors could discuss on this point.

In the paper by Fernandez-Vallone, deletion of Lgr5 in E16.5 embryos resulted in a decrease expression of several ECM genes. Further, the authors could show that the fetal epithelium does express for example Col1a1 at this point, which decreases with maturation. However even for the example of Col1a1 it is evident in their paper that the mesenchyme expresses Col1a1 at much higher levels. Our proposed experiments with Lamc1 k.o. In organoids will show if the produced laminins of the epithelium are essential.

This manuscript could be interesting for an audience in the stem cell and developmental fields ( my field of expertise).

**Referee Cross-commenting**

Considering the pertinent and sometimes overlapping comments of the two other reviewers, the estimated time is revised to 3-6 months.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

**Summary:**

In this manuscript, Ramadan and colleagues demonstrate that depending on the extracellular matrix (ECM) composition in which mouse intestinal organoids and/or 2D intestinal epithelial cells are grown in, cellular composition of the epithelium changes. Organoids plated on 2D collagen layers show a unique cell cluster characteristic of fetal-like genes, while organoids plated with increased amount of Matrigel in 2D or in 3D exhibit a shift towards higher stem cell abundance and the absence of the fetal-like gene cluster. Specifically, the ECM component Laminin supports acquisition of stem cells identities in small intestinal epithelial cells, correlating with a transient increase in expression levels of collagen and laminin genes in vivo spanning time points of crypt formation. The authors reported the functional contribution of laminin signaling (Lamc1 KO) via integrin alpha 6 (antibody-blocking) to intestinal stem cell acquisition in vitro and in vivo.

There are a handful of comments/concerns that would need to be addressed before publication.

Major points:

1. The author claimed: "the effect of ECM components on gene expression is not due to difference in morphology (2D collogen vs. 3D Matrigel)". The conclusion of the 2D vs. 3D experiment should be toned down to that organoid morphology (2D vs. 3D) does not directly impact on the expression of fetal-like genes. Otherwise more analysis of RNAseq data with different group of genes (e.g., in different mechanosensing pathways) should be provided with Fig. S1D. Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.

We apologise for the too strong claim of structure of growth vs. signalling of the ECM and its effect on the transcriptome. Indeed, the main message is that a changed morphology from 3D to 2D is not responsible for the expression of fetal-like genes. The paragraph will be rephrased.

Also, would it be technically feasible to perform experiments of SI in collagen (3D) in all group of experiments? Directly comparing 3D Matrigel with 3D collagen avoids the concern of the 2D vs. 3D effect.

To address this point, we want to refer to the excellent idea of growing established organoids in collagen (3D) vs. Matrigel (3D) as suggested by this reviewer (and reviewer #3) in Minor points #5. This circumvents the need for Wnt3a addition, which affects stem cell and Paneth cell gene expression.

1. For Fig. 1f, the authors should include overlapping stainings of Lyz (or Olfm4, CD44 etc.) and Adolase B signal, or they could perform Aldolase B staining in Lgr-5-DTR-GFP and/or Lyz-RFP organoid line. From the current data provided one cannot draw clear conclusions on the crypt morphology as claimed by the authors. Additionally, when talking about crypt morphology and apical accumulation of Actin specifically in the Lyz+ cells, the authors should show a higher zoom in of the picture and either add an orthogonal slice to see apical and basal side and the specific accumulation in one of the cell types, or also co-label with apical polarity markers.

We will perform additional co-stainings to further highlight the differences in the spatial distribution of differentiated cells and undifferentiated-crypt-like cells. Further we will provide higher magnification images highlighting the apical accumulation of Actin in the crypt-like structures, which can also be seen in mature organoids.

1. Authors referred the organoid transient change to fetal-like state. To exam the similarity of ECM-induced reprogramming with the regenerative-type of reprogramming, it would be essential to compare the expression of the selected fetal-like genes (Anxa3, Ly6a/Sca1, Msln, Col4a2 et al.), as well as bulk and single-cell (if applicable) RNA-seq data.

Here, we would like to refer to the excellent study by Yui et al. ([S. Yui et al., “YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration,” Cell Stem Cell, vol. 22, no. 1, pp. 35-49.e7, 2018.). In this study the authors detected the same gene signature in the repairing epithelium. We can provide a GSEA for the Ly6a+ signature that was derived from this paper, if necessary.

1. For in vivo data, authors were looking at the normal development of intestine. Following the point of organoid culture recapitulates regeneration, it would be relevant to check the in vivo ECM change by staining in the process of intestinal regeneration or discuss would the fetal-like genes be involved in regeneration.

We will address this point in the discussion as it also involves the study by Yui et al.

1. For Fig2.d and e, it would be important to measure compactness vs. the emergence/probability of Ly6+ cells to see if there is correlation.

If we understand the reviewer correctly, this would address the important relationship between cell shape and cell fate/type. However, this is a topic that needs more attention than a simple correlation and would exceed the scope of this manuscript as we are not able to modulate cell shape to make any further points about its effect on the fetal gene expression program.

1. In Fig.2d, Ly6a expression is very obscure, and it would be important to show control staining for cell boundaries (eg. Phalloidin, PM) to visualize which nuclei show Ki67 staining and are high or low in Ly6a (plus quantification).

We will improve the image in Fig.2d and include the mentioned Actin staining. In addition we will perform an analysis via Flow Cytometry to quantify the level of Ly6a staining and EdU positivity.

1. In Fig. 2f-g, FACS-ed Ly6a+ and Ly6- cells embedded in Matrigel can grow into organoids with crypts. Here the imaging of Paneth cell staining is not clear, and a quantification on number of Paneth cells per crypt would be very helpful to confirm the phenotype. Also, authors should either provide data on the initial size of seeded cell clusters and report organoid growth and cell type composition in more detail when plating from Ly6a+ and Ly6- cells or report the variation in the respective populations.

This comment suggests that we may not have described the experimental settings properly. The sorted cells were embedded as single cells, not as clusters, in drops of matrigel (10k cells/25ul Matrigel). The emergence of Paneth cells together with a normal organoid architecture grown from Ly6a+ cells shows their stem cell capacity, as has been shown by Yui et al. before from the regenerating colon. In addition, organoids from both cell populations (Ly6a+ and Ly6a-) could be passaged, indicating presence of intestinal stem cells.

1. The authors could also test whether Ly6+ cells have any advantages over Ly6- cells when grown on collagen I instead of Matrigel.

We will sort Ly6a+ and Ly6- negative cells and plate them on collagen I. It will be interesting to see if the Ly6a+ cells can give rise to the other cell types when plated on collagen or if they stay Ly6+ cells. This will also answer whether Ly6a+ need the presence of Ly6a- cells in the cultures. In addition, the experiment proposed in #6 will also highlight any proliferative advantage of Ly6a cells compared to Ly6-negative cells on collagen.

1. In Fig.3f, a control of membrane protein staining should be added for the experiment. The increased Laminin signal can be caused by the global increase of protein when there are more cells, or tissues are more compact. When authors make conclusion of "Dramatic remodelling of ECM during crypt formation ", the experiment should also count cell numbers vs. Laminin (intensity). The phenotype can come from increased area of interface between epithelium and mesenchyme instead of active remodelling.

We agree with the reviewer that by itself the IF images are not enough to make such a claim. However, we would point to the qPCR data and RNA in situ, that can be more easily normalised and shows the dramatic increase in expression of all laminins at birth. To show that laminin protein is increasing is more difficult than we initially anticipated. However, in the study by De Arcangelis (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.) the authors use an EDTA assay to show that the epithelium detaches easily when Itga6 is deleted. Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth.

1. The authors claim that intestinal stem cells in vivo are controlled by Laminin signalling that goes via Integrin alpha 6. However, there is no evidence provided that supports the contribution of ITGA6 in the in vivo setting. So, the authors should either tone down on that point or show a convincing in vivo experiment (e.g., inhibit ITGA6 in vivo by inhibitor injections or by extracting the ECM of a wild-type mouse and seeding intestinal epithelial cells without vs. with ITGA6 blocking antibody which should recapitulate the phenotype in Fig. 4 c.

We apologise for this confusion. We are well aware about the limitations of our Itga6 blocking experiment in vitro and its relevance in vivo. We tried to get material of the inducible VilCreER Itga6 mouse as referenced in the discussion of the manuscript, without any luck so far. Therefore we will highlight further that any claims about the laminin:Itga6 interaction can only be made in vitro.

1. Fig. 4: For the data of ITGA6 expression and all sorts of analysis on protein expression with staining, normalization with cell numbers should be performed.

The RNAseq data that shows the upregulation of Itga6 in the epithelium at E18 is normalized within. Our RNAscope only further validates these expression changes and highlights the specific enriched expression at the bottom of the nascent crypts. We can add quantification of the RNAscope if required.

1. Two questions on mechanisms:

2. What is the mechanism from ITGA signaling to Ly6a+ cell fate?

3. And would/how Laminin induce ITGA expression? Depends on how much the authors would like to go deep with the project, could be addressed further with functional studies, or touch on the topics with discussion.

These are important questions, however we do agree that this will go to deep for the scope of this manuscript. We will address these open questions in the discussion and leave the experimental part for a follow-up study.

**Minor points:**

1. Text in Fig.S1d regarding 'in' or 'on' collagen, could be clearer by changing the terms to 2D and 3D correspondingly.

We agree and the text will be changed accordingly.

1. Fig. S1a, it is great the authors showed that similar stiffness in Matrigel and collagen I. It would be important to check the concentration of collagen I vs. stiffness (also for increasing concentrations of Laminin in Fig. 3b), since this is also the type of ECM change that might lead to the change of cell status in cancer progression or collective cell migration.

We will perform further stiffness measurements of the hydrogels and update the Fig. S1a.

1. When plating intestinal epithelial cells on collagen I, is the Ly6+ phenotype altered upon Wnt addition? This is not so clear from the RNAseq data Fig S1d., so authors should provide antibody stainings (stem cells/Paneth cells). This could give insight whether Ly6+ cells are still able to convert into stem cells/ Paneth cells by changing morphogen concentration vs. ECM composition.

We will reanalyse the RNaseq dataset further, specifically analysing the ratio of stem cell and Paneth cell gene expression. However, as mentioned before, Wnt3a specifically does reduce the expression of Paneth cell markers.

Similar to this point, also enteroendocrine cell fate is absent in collagen I condition (Fig2.ab), the authors could address this point by medium induced EE cell fate.

Due to the reduced number of secretory cells the clustering in Fig2 a/b does not separate all the different cell lineages. However, EE cells are present in the collagen cultures as characterised by expression of Chga, just reduced in their number (see Supl Fig. 2B).

1. It would be more informative to indicate the thickness of ECM layer in culture of 2D collagen I, as well as the image of the whole well, demonstrating the morphological variation in the middle and peripheral of the ECM layer.

The thickness of the collagen layer is about 1mm in a 6well plate and we do not observe any morphological differences in the cells between the periphery and center of the well.

1. After the formation of PC/SC clusters, would ECM contribute to maintenance? Putting mature organoids from Matrigel to Collagen I 3D would help to clarify.

This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.

1. Check secretome and individual culture of Mesenchyme, see if the increase of Laminin is epithelium independent.

We agree that the mesenchyme is key for laminin production, therefore these are important questions. Our prediction would be that epithelium from birth (P0) versus adult might result in different responses on the mesenchyme. However, we feel these experiments are better suited for a follow-up study.

1. In general, the authors should look at cell polarity markers to check the ECM contribution to cell polarity in different cell types.

We thank the reviewer for the suggestions and as mentioned above, we will perform additional stainings.

Reviewer #2 (Significance (Required)):

**Significance:**

The work highlights the role of ECM on stem cell niche and is of great interest to the organoid and stem cell community.

Our field of expertise is image- and seq-technology-based quantitative biology, regeneration and mechanics in organoid.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

**Summary:**

Ramadan et al present a highly informative paper detailing how the Extracellular matrix influences the development of the intestine. Specifically, the authors provide a thorough analysis of how manipulating the components of the ECM can affect organoid growth, morphology, and gene expression of the organoids. Most importantly, the authors isolate laminin as a critical component of the ECM which impacts the development of fetal-like epithelium. While the in vitro work is generally compelling and of interest to the field, the in vivo data is someone lacking in depth and novelty. Particularly, these conclusions from the abstract could be much better supported: "This laminin:ITGA6 signalling is essential for the stem cell induction and crypt formation in vitro. Importantly, deletion of laminin in the adult mouse results in a fetal-like epithelium with a marked reduction of adult intestinal stem cells." The in vivo work was largely published previously and has caveats noted below, while the in vitro association of ITGA6 signaling with crypt formation is over-interpreted based upon an antibody blocking experiment and a lack of statistical rigor. Despite these concerns, this reviewer finds the work of considerable interest in an important area of the field (epithelial/stromal interactions of the intestine).

**Major Concerns:**

The use of the Ubc-Cre Lamc1-flox mouse model is an interesting way to test the impact of loss Lamc1 on intestinal development. However, with the Ubc-Cre, does the mouse model have other deleterious effects on the mouse beyond the intestine?

Can the authors use a more localized Cre to observe specifically the impacts of Lamc1 loss in the intestine? What is the fate of these mice? Can the authors show swiss roll, low mag sections to let the reader know the extent of this phenotype? OLFM4 and Ki67 IHC should be conducted over a timecourse to show how the changes occur over time after loss of Lamc1. How long does Lamc1's protein product perdure after tamoxifen treatment? More details of this exciting, in vivo validation of the authors' in vitro studies are key to elevating the impact of this work. However, it appears that much of this mouse model was previously published, but the previous findings are not well summarized in the current manuscript.

We will describe the model in more detail and refer readers to the excellent study of our collaborators which answers most of the raised questions. It is interesting to note that although a ubiquitous Cre was used to delete Lamc1 in adult mice, a phenotype was only observed in the intestine indicating a specific role for continuous laminin production here.

Can the authors show that ITGA6 loss has functional consequences in vivo with an epithelial knockout or via an organoid knockdown? A more rigorous genetic test of this proposed function would be important for substantiating the claims made in the abstract.

As referenced in the discussion of the manuscript, there is a VilCreER Itga6 mouse described in the literature (A. De Arcangelis et al., “Hemidesmosome integrity protects the colon against colitis and colorectal cancer,” Gut, vol. 66, no. 10, pp. 1748–1760, 2017.), which mainly focus on the colon. However, the authors use an EDTA assay in the small intestine to show that the epithelium detaches easily when Itga6 is deleted (Fig. 1J). Within the figure, it seems also that the epithelium detaches easily at P2, compared to P14. As EDTA is disrupting laminin polymerisation, this would further indicate increased laminin protein deposition after birth which is dependent on Itga6.

We tried to get material of the inducible VilCreER Itga6 mouse however without any luck so far.

We will conduct additional experiments regarding the Itga6 in vitro. In addition to including additional controls for the neutralizing antibody, we will genetically inactivate Itga6 via an inducible Crispr/Cas9. This should enable us to delete Itga6 when the cells are grown on collagen, and hence reduce the possibility of compensation in matrigel derived organoids.

The authors state that the changes in gene expression are not due to differences in morphology, but rather are specific to the components of the environment. While the authors show that organoids treated with Wnt3a "in Matrigel" and "CollagenI" appear to have similar morphologies and yet still result in a different gene expression profiles, it would be of great interest to see whether that difference persists without Wnt3a when organoids are "in Matrigel" and "in CollagenI". While the reviewer understands the difficulties of culturing organoids in 3D Collagen without Wnt3a, organoids can be indeed be cultured in 3D using "floating collagenI rings" (Sachs et al 2017).

This is an interesting experiment that we will conduct, we thank the reviewer for this suggestion. Indeed, established organoids should be able to grow in collagen I without Wnt3a addition. The paper by Sachs et al. (N. Sachs, Y. Tsukamoto, P. Kujala, P. J. Peters, and H. Clevers, “Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels,” Development, vol. 144, no. 6, pp. 1107–1112, 2017. et al) used extensive washing with PBS to remove Matrigel from the organoids. We will go one step further and trying to completely remove laminin specifically by EDTA incubation, as has been shown recently (J. Y. Co et al., “Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions,” Cell Rep., vol. 26, no. 9, pp. 2509-2520.e4, 2019.). This should then also answer whether disruption of laminin signalling is sufficient to induce fetal-gene expression without the addition of collagen I in a 3D setting.

Similarly, while the authors indicate that increasing Matrigel concentrations altered the gene expression patterns in a dose-dependent manner, it is unknown whether this can fully be attributed to the Matrigel composition, or whether the layer of Matrigel is providing the capability to transition from 2D to 3D culture.

We are not entirely sure, we understood this point. The Matrigel and the collagen I are mixed before they solidify, therefore enabling a homogenous hydrogel. The different hydrogels are not layered (if that is what the reviewer is referring to).

The authors cultured organoids in different concentrations of Laminin/CollagenIV when mixed with CollagenI. Can organoids be sustained only on a matrix of CollagenIV and/or Laminin? Would this show more direct differences between CollagenI vs. Laminin cultured organoids?

Organoids cannot be grown in pure Collagen IV, but pure laminin should be feasible. We did initial experiments with 3-5mg/ml laminin in PBS and that was sufficient to allow organoid growth. We will perform additional experiments with pure laminin and show the impact on organoid growth.

With the reduction in stem-cell and Paneth cells in the Lamc1-KO mice, it would also be of interest to determine what cell types are now prominent within the heavily elongated intestinal "crypt" structures seen in the Lamc1-KO mice and whether populations are more TA-cells or enterocytes to consider differentiation status of the cells. Additionally, it would also of interest to see if the Itga6 expression is significantly altered in the absence of Lamc1.

We will test expression changes for Itga6 in the Lamc1-KO mice, in the epithelium via qPCR. Additionally we can stain tissue from these mice for Sox9, Ki67 and differentiated markers eg. CD44, AldolaseB, Villin etc .to determine whether the elongated, hyperproliferative crypts contain progenitor cells or secretory enterocytes.

**Minor concerns:**

Matrigel is a complex matrix derived from mouse tumors. In many instances in the manuscript, the authors portray it as a more simpler mix of laminin/Collagen4 (fig 3a). It should be made clearer to the reader that Matrigel is not a mix of recombinant proteins, but a more clear depiction of how Matrigel is derived will be critical for this study, given the focus on specific ECM components and how they affect intestinal epithelial growth.

We agree and will change the oversimplified view of Matrigel.

Some labels of specific conditions would be appreciated in the figures as opposed to only the figure legends (ie. Fig. 1b and 1d should be labeled with comparisons; Fig. 3f labels of fluorescence, Fig. 4b label of itga6 staining).

We apologise for this and the Figure labels will be updated.

With the light staining of Lamc1 in-situ, it is hard to appreciate the expression of laminin within the stroma of the intestine when compared to Col4. This reviewer is also curious of the biological relevance of the concentrations of Laminin/CollagenIV when culturing organoids in Fig. 4a.

Indeed, the Lamc1 due to its lower expression than Col4a1 is more difficult to see. Maybe the reviewer overlooked Suppl.Fig.4A, where the blue channel of these in situ images show more contrast. If required, we can try to optimise the hybridisation times to increase the signal a bit further.

When culturing the organoids with the mixture of collagen and laminin or collagen IV, the concentrations of the two single components were selected similar to their concentrations in Matrigel. Regarding the absolute concentrations of laminin/collagen IV in vitro versus their “concentration” in vivo is much harder to answer. In addition to the unknown concentrations in vivo, there are many more Laminin types present with specific localisation and even specific receptor interactions. For our in vitro studies we relied on the Laminin present in EHS tumours, which is Laminin alpha 1 beta 1 gamma 1. We are currently investigating decellularization protocols to purify the ECM from mouse intestinal tissue, but again this would be more suited for a follow up study.

It would be appreciated if gene expression analyses presented in figures would include p-values to provide context for differences in gene expression.

The avoidance of statistical inference for most of the experiments was a deliberate choice. In line with several comments (e.g. 1. Vaux, D. L. (2012) Know when your numbers are significant. Nature. 492, 180–181), we chose to show all individual data points (with exception of Fig. 3D, n=5, to ease interpretation) without statistical testing. For most expression data, we have data from RNAseq, single-cell RNAseq and qPCRs repeated at different hydrogel concentrations to obtain reliable results. Further, the in vivo mesenchymal qPCR expression data was validated with RNA in situ hybridization showing the mainly mesenchymal expression.

As we will perform additional experiments for the revision of this paper, we can perform statistical tests in the key experiments (e.g. Itga6 experiment) to alleviate any concerns regarding significance.

In figure 3f, the authors report "immunofluorescence of laminin". How is this measured? Can more details be given about the antibody in the text and figure legend? Laminins are a family of genes, and it's not clear what's being demonstrated in this figure panel. Developmental stages of the samples are also not clear.

We apologise for the lack of labeling in Fig.3f. The details of the antibody were hidden in the Materials and Methods of the manuscript (Slides were incubated with Laminin Polyclonal Antibody (1/200, Thermo Fisher #PA5-22901) overnight at 4C ). This pan-laminin antibody reacts with most Laminin isoforms alpha1, alpha2, beta1, gamma1. We will declare it as a pan-laminin antibody in the Figure legend to help future readers.

Reviewer #3 (Significance (Required)):

This work is in an exciting "hot" area of research to understand the role of non-epithelial cells in intestinal epithelial development and function. The audience would be those in the GI field and those studying tissue-tissue interactions.

There's some concern that the in vivo portion of the manuscript (4th figure) uses a model that was previously characterized and published by this group, and that isn't clearly disclosed. The manuscript would benefit from more disclosure and detail about the in vivo phenotype. Such changes would substantially increase the impact and novelty of the study.

We would like to point out that we cited the paper of the original study that uses the model throughout the manuscript. We will disclose in more detail that this group did the study and that the reduction in stem cell genes was already mentioned in the original publication.

Author Response:

Reviewer #2 (Public Review):

1) The authors describe their algorithm as a tool that (i) was validated "across heterogeneous populations around the world"; (ii) has an "accuracy matching or exceeding human accuracy"; (iii) "is easy to use". I take issue with these three statements. First, the authors did not test the performance of their algorithm in clinical populations with sleep disorders, despite the fact that individuals with sleep disorders represent (logically) the vast majority of sleep recordings. Crucially, such a comparison was made in the best (to my knowledge) published automated sleep staging algorithm (Stephansen et al. Nature Communications 2018, doi: 10.1038/s41467-018-07229-3). The omission of this work is very surprising. Quantifying the impact of sleep disorders on a sleep scoring algorithm is critical for its deployment in sleep clinics.

We apologize as we were not clear in describing our training and testing data set. Indeed, both the training and testing set 1 included a significant number of individuals with sleep disorders. Indeed, about 30% of the individuals had moderate to severe sleep apnea (AHI >= 15). The validation dataset (DOD, or testing set 2) also includes 55 nights from individuals with obstructive sleep apnea (average AHI = 18.5 ± 16.2). Furthermore, both the training and testing set 1 included individuals with a medical diagnosis of insomnia, depression, diabete and hypertension.

The health status and demographics data of the training and testing sets have now been clarified throughout in the manuscript to avoid any such confusion:

1) Methods: We have added an extensive description of each dataset in the training and testing sets, including data on health and sleep disorders.

2) Results: We have added a new table to report and compare demographics/health data of the training and testing set, as suggested in a later comment by the reviewer.

3) Results: Performance results of the testing set 2 are now reported separately for healthy individuals and individuals with sleep disorders.

Second, the authors wrote that their algorithm is "matching or exceeding" human accuracy but seem to present uncorrected one-to-one comparisons to support their claim. The fact that an algorithm is better than some humans do not mean it exceeds human performance.

Thanks for noting that. We have now removed all instances of “exceeding human accuracy”.

Third, although I agree that the tool seems easy to use even for individuals with limited programming skills, it still requires some. I don't think someone who is used to software with graphical interfaces and who has never used (or heard of!) python would describe the tool as easy to use. This poses an important implementation challenge.

2) An important limitation of this algorithm is that it captures only one part of the visual examination of sleep data. Indeed, especially in clinical settings, the data is not only examined to establish the hypnogram but to also identify markers of common sleep disorders (e.g. sleep apnea, leg movements, etc). Although this algorithm could significantly speed up sleep scoring, it does not allow to detect these other important markers. Currently, and in link with the previous comment, the algorithm could not replace the visual inspection of the data for clinical diagnoses.

We have now revised the manuscript such that we discussed in this possibility in “Limitations and future directions” subsection of the new Discussion:

“The algorithm is not currently able to identify markers of common sleep disorders (such as sleep apnea, leg movements) and as such may not be suited for clinical purposes. It should be noted however that our software does include several other functions to quantify phasic events during sleep (slow-waves, spindles, REMs, artefacts) as well as sleep fragmentation of the hypnogram. Rather than replacing the crucial expertise of clinicians, YASA may thus provide a helpful starting point to accelerate clinical scoring of polysomnography recordings. Furthermore, future developments of the algorithm should prioritize automated scoring of clinical disorders, in particular apnea-hypopnea events. On the latter, YASA could implement some of the algorithms that have been developed over the last few years to detect apnea-hypopnea events from the ECG or respiratory channels (e.g. Varon et al. 2015; Koley and Dey 2013).”

3) The data were curated with some recordings or portions of recordings being excluded (see p. 7). While I understand that this curation is important for the training set, I think it should not be applied to the test set. Indeed, it goes contrary to the logic of automating sleep staging. For example, cutting the beginning and end of the recording according to sleep start and end (p. 7) supposes that the start and end of sleep are already known (i.e. it has already been scored).

This truncation step has now been removed from the pipeline and all the results have been updated accordingly. In addition, we have also removed all other exclusion criteria (e.g. PSG data quality, recording duration, etc) to improve the generalization power of the algorithm, thanks to the suggestions of the reviewer.

4) Two types of EEG derivations were used (C4-M1 or C4-Fpz). Was the performance impacted by this variable? Is it fair to assume that the choice of features (spectral features or summary statistics of time series data) could explain the absence of differences but that introducing new features (i.e. phase-sensitive features) could increase the influence of the choice of the derivation?

Thanks for raising this. First, our choice of the EEG reference was determined by the datasets: the CFS, CCSHS, MrOS, CHAT and HomePAP datasets were all referenced to Fpz, while the MESA, SHHS and DOD datasets were referenced to the contralateral mastoid. The montage of each dataset has now been added to the Methods section.

Second, as rightly pointed out by the reviewer, the features implemented in the algorithm were chosen to be robust to various recording montages. This is now explicitly discussed in the “Features extraction” subsection of the Methods:

“The features included in the current algorithm were chosen to be robust to different recording montages. As such, we did not include features that are dependent on the phase of the signal, and/or that require specific events detection (e.g. slow-waves, rapid eye movements). However, the time-domain features are dependent upon the amplitude of the signal, and the algorithm may fail if the input data is not expressed in standard units (uV) or has been z-scored prior to applying the automatic sleep staging.”

5) Given that markers of sleep stages are very different in EOG, EMG and EEG time series, could the authors explain the logic behind applying the same pre-processing and extracting the same features on these three very different types of data? Could this explain why the majority of the features in the top-20 features were EEG features?

We now provide a more detailed explanation on the inclusion of EOG and EMG features in the “Features extraction” subsection of the Methods:

“These features were selected based on prior work in features-based classification algorithms for automatic sleep staging (Krakovská and Mezeiová 2011; Lajnef et al. 2015; Sun et al. 2017). For example, it was previously reported that the permutation entropy of the EOG/EMG as well as the EEG spectral powers in the traditional frequency bands are the most important features for accurate sleep staging (Lajnef et al. 2015), thus warranting their inclusion in the current algorithm. Several other features are derived from the authors’ previous works with entropy/fractal dimension metrics1. ” https://github.com/raphaelvallat/antropy

Furthermore, we have added a “Limitations and future directions” section in the Discussion in which we propose future improvements of the algorithm. One of these potential improvements is the development of EOG and EMG features that would provide a higher discrimination of the sleep stages:

“This suggests that one way to improve performance on this population could be the inclusion of more EEG channels and/or bilateral EOGs. For instance, using the negative product of bilateral EOGs may increase sensitivity to rapid eye movements in REM sleep or slow eye movements in N1 sleep (Stephansen et al. 2018; Agarwal et al. 2005). Interestingly, the Perslev 2021 algorithm does not use an EMG channel, which is consistent with our observation of a negligible benefit on accuracy when adding EMG to the model. This may also indicate that while the current set of features implemented in the algorithm performs well for EEG and EOG channels, it does not fully capture the meaningful dynamic information nested within muscle activity during sleep.”

6) Sleep scoring guidelines incorporate not only what can be observed on a given epoch of data but also what is observed in the previous epoch(s). For example, an epoch can be scored as N2 even if there is no marker of N2 but there was (i) a marker of N2 in a previous epoch, (ii) no reason to change the score since. To reproduce this, the authors employed a symmetrical smoothing approach (a combination of a triangular-weighted rolling average and asymmetrical rolling average). Why did the authors choose to incorporate data from following epochs, which is not implemented in established guidelines? How was the duration of the smoothing window chosen? Indeed, 5 minutes appear as rather long could explain the poor performance of the algorithm for fast changing portions of the data (i.e. N1 or transitions). Importantly, these transitions can be very relevant in clinical settings and to establish a diagnosis.

This is a great question. We have addressed this in the revised manuscript.

Temporal smoothing

We have also conducted a new analysis of the influence of the temporal smoothing on the performance. The results are described in Supplementary File 3a. Briefly, using a cross-validation approach, we have tested a total of 49 combinations of time lengths for the past and centered smoothing windows. Results demonstrated that the best performance is obtained when using a 2 min past rolling average in combination with a 7.5 minutes centered, triangular-weighted rolling average. Removing the centered rolling average resulted in poorer performance, suggesting that there is an added benefit of incorporating data from both before and after the current epoch. Removing both the past and centered rolling averages resulted in the worst performance (-3.6% decrease in F1-macro). Therefore, the new version of the manuscript and algorithm now uses a 2 min past and 7.5 min centered rolling averages. All the results in the manuscript have been updated accordingly. We have now edited the “Smoothing and normalization” subsection of the Methods section as follow:

“In particular, the features were first duplicated and then smoothed using two different rolling windows: 1) a 7.5 minutes centered, and triangular-weighted rolling average (i.e. 15 epochs centered around the current epoch with the following weights: [0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, 1., 0.875, 0.75, 0.625, 0.5, 0.375, 0.25, 0.125]), and 2) a rolling average of the last 2 minutes prior to the current epoch. The optimal time length of these two rolling windows was found using a parameter search with cross-validation (Supplementary File 3a). [...] The final model includes the 30-sec based features in original units (no smoothing or scaling), as well as the smoothed and normalized version of these raw features.”

Reviewer #3 (Public Review):

This study presents a new sleep scoring tool that is based on a classification algorithm using machine-learning approaches in which a set of features is extracted from the EEG signal. The algorithm was trained and validated on a very large number of nocturnal sleep datasets including participants with various ethnicities, age and health status. Results show that the algorithm offers a high level of sensitivity, specificity and accuracy matching or sometimes even exceeding that of typical interscorer agreement. The conclusions are supported by the data. Importantly, a measure of the algorithm's confidence is provided for each scored epoch in order to guide users during their review of the output. The software is described as easy to use, computationally low-demanding, open source and free. This paper addresses an important need for the field of sleep research. There is indeed a lack of accurate, flexible and open source sleep scoring tools. I would like to commend the authors for their efforts in providing such a tool for the community and for their adherence to the open science framework as the data and codes related to the current manuscript are made available. I predict that this automated tool will be of use for a large number of researchers in the field. However, there are plenty of automated sleep scoring tools already available in the field (most of them are not open source and rather expensive, as noted by the authors). The current work does not provide a clear view on whether the new algorithm presented in this research performs better than algorithms already available in the field. No formal comparisons between algorithms is provided and the matter is not discussed in the paper.

Thanks so much for pointing this out. We have now added this relevant reference throughout the manuscript. To build on the reviewer’s point, the current algorithm and Stephansen’s algorithm did not use the same public data. The Stephansen 2018 algorithm was trained and validated on “10 different cohorts recorded at 12 sleep centers across 3 continents: SSC, WSC, IS-RC, JCTS, KHC1, AHC, IHC, DHC, FHC and CNC”, none of which are included in the training/testing sets of the current algorithm. Nevertheless, we certainly agree that the manuscript will benefit from a more extensive comparison against existing tools. To this end, we have made several major modifications to the manuscript. First, we have added a dedicated paragraph in the Introduction to review existing sleep staging algorithms:

“Advances in machine-learning have led efforts to classify sleep with automated systems. Indeed, recent years have seen the emergence of several automatic sleep staging algorithms. While an exhaustive review of the existing sleep staging algorithms is out of the scope of this article, we review below — in chronological order — some of the most significant algorithms of the last five years. For a more in-depth review, we refer the reader to Fiorillo et al. 2019. The Sun et al. 2017 algorithm was trained on 2,000 PSG recordings from a single sleep clinic. The overall Cohen's kappa on the testing set was 0.68 (n=1,000 PSG nights). The “Z3Score” algorithm (Patanaik et al. 2018) was trained and evaluated on ~1,700 PSG recordings from four datasets, with an overall accuracy ranging from 89.8% in healthy adults/adolescents to 72.1% in patients with Parkinson’s disease. The freely available “Stanford-stage” algorithm (Stephansen et al. 2018) was trained and evaluated on 10 clinical cohorts (~3,000 recordings). The overall accuracy was 87% against the consensus scoring of several human experts in an independent testing set. The “SeqSleepNet” algorithm (Phan et al. 2019) was trained and tested using a 20-fold cross-validation on 200 nights (overall accuracy = 87.1%). Finally, the recent U-Sleep algorithm (Perslev et al. 2021) was trained and evaluated on PSG recordings from 15,660 participants of 16 clinical studies. While the overall accuracy was not reported, the mean F1-score against the consensus scoring of five human experts was 0.79 for healthy adults and 0.76 for patients with sleep apnea.”

Second, and importantly, we now perform an in-depth comparison of YASA’s performance against the Stephansen 2018 algorithm and the Perslev 2021 algorithm using the same data for all three datasets. Specifically, we have applied the three algorithms to each night of the Dreem Open Datasets (DOD) and compared their performance in dedicated tables in the Results section (Table 2 and Table 3). This procedure is fully described in a new “Comparison against existing algorithms” subsection of the Methods. None of these algorithms included nights from the DOD in their training set, thus ensuring a fair comparison of the three algorithms. Related to point 4 of the Essential Revisions, performance of the three algorithms are reported separately for healthy individuals (DOD-Healthy, n=25) and patients with sleep apnea (DOD-Obstructive, n=50). To facilitate future validation of our algorithm, we also provide the predicted hypnograms of each night in Supplementary File 1 (healthy) and Supplementary File 2 (patients).

Overall, the comparison results show that YASA’s accuracy is not significantly different from the Stephansen 2018 algorithm for both healthy adults and patients with obstructive sleep apnea. The accuracy of the Perslev 2021 algorithm is not significantly different from YASA in healthy adults, but is higher in patients with sleep apnea. However, it should be noted that while the YASA algorithm only uses one central EEG, one EOG and one EMG, the Perslev 2021 algorithm uses all available EEGs as well as two EOGs. This suggests that adding more EEG channels and/or using the two EOGs may improve the performance of YASA in patients with sleep apnea. Though an important counterpoint is that YASA requires a far less extensive array of data (channels) to accomplish very similar levels of accuracy, which has the favorable benefit of reducing analysis computational and processing demands, improves speed of analysis (i.e. a few seconds per recording versus ~10 min for the Stephansen 2018 algorithm), and is amenable to more data recordings since many may not have sufficient EEG channels. All these points are now discussed in detail in the new “Limitations and future directions” subsection of the Discussion (see point 3 of the Essential Revisions).

There are some overstatements in the manuscript. For example, the algorithm was trained and validated on nocturnal sleep data. Sleep characteristics (eg duration and distribution of sleep stages etc.) are different, for example, during diurnal sleep (nap) and the algorithm might not perform as well on nap data. As such, the tool might not be as "universal" as stated in the title. Additionally, as human scores are used as the ground-truth for the validation step, it might be misleading to state that "this tool offers high sleep-staging accuracy matching or exceeding human accuracy". The algorithm exceeded the accuracy of some human scorers and matched the scores of the best scorer.

We have now removed the word “universal” from the title and replaced “exceeded human accuracy” with “matched human accuracy”. Furthermore, we have now added the fact that the algorithm was trained and validated only on nocturnal data in the Limitations section of the discussion, and as such, noted that there is the possibility that the algorithm may not perform at the same accuracy levels for daytime nap data.

No reflection on further improvement is offered in the paper. The algorithm performs worse on N1 stage, older individuals and patients presenting sleep disorders (sleep fragmentation) and it is unclear how this could be improved in future research. In the same vein, the current work does not present performance accuracy separately for healthy individuals and patients when it is expected that accuracy would be poorer in the patient group.

The revised manuscript now includes a dedicated section in the Discussion to propose ideas for improvements.

First, we have now added a “Limitations and Future Directions” subsection in the Discussion to present ideas for improving the algorithm, with a particular focus on fragmented nights and/or nights from patients with sleep disorders:

“Despite its numerous advantages, there are limitations to the algorithm that must be considered. These are discussed below, together with ideas for future improvements of the algorithm. First, while the accuracy of YASA against consensus scoring was not significantly different from the Stephansen 2018 and Perslev 2021 algorithms on healthy adults, it was significantly lower than the latter algorithm on patients with obstructive sleep apnea. The Perslev 2021 algorithm used all available EEGs and two (bilateral) EOGs, whereas YASA’s scoring was based on one central EEG, one EOG and one EMG. This suggests that one way to improve performance in this population could be the inclusion of more EEG channels and/or bilateral EOGs. For instance, using the negative product of bilateral EOGs may increase sensitivity to rapid eye movements in REM sleep or slow eye movements in N1 sleep (Stephansen et al. 2018; Agarwal et al. 2005). Interestingly, the Perslev 2021 algorithm does not use an EMG channel, which is consistent with our observation of a negligible benefit on accuracy when adding EMG to the model. This may also indicate that while the current set of features implemented in the algorithm performs well for EEG and EOG channels, it does not fully capture the meaningful dynamic information nested within muscle activity during sleep.”

Second, we have now conducted a random forest analysis to identify the main contributors of accuracy variability. The analysis is described in detail in the “Moderator Analyses” subsection of the Results as well as Supplementary File 3b, the revision now states:

“To better understand how these moderators influence variability in accuracy, we quantified the relative contribution of the moderators using a random forest analysis. Specifically, we included all aforementioned demographics variables in the model, together with medical diagnosis of depression, diabetes, hypertension and insomnia, and features extracted from the ground-truth sleep scoring such as the percentage of each sleep stage, the duration of the recording and the percentage of stage transitions in the hypnograms. The outcome variable of the model was the accuracy score of YASA against ground-truth sleep staging, calculated separately for each night. All the nights in the testing set 1 were included, leading to a sample size of 585 unique nights. Results are presented in Supplementary File 3b. The percentage of N1 sleep and percentage of stage transitions — both markers of sleep fragmentation — were the two top predictors of accuracy, accounting for 40% of the total relative importance. By contrast, the combined contribution of age, sex, race and medical diagnosis of insomnia, hypertension, diabete and depression accounted for roughly 10% of the total importance.”

In addition, the performance of the algorithm in the DOD testing dataset is now reported separately for healthy individuals and patients with sleep disorders.

As requested by the reviewer, we now analyze and report the performance of YASA on the DOD testing set separately for healthy individuals (DOD-healthy) and patients with obstructive sleep apnea (DOD-Obstructive), which can be found in section “Testing set 2”.

There is series of methodological choices that is not justified. For example, nights were cropped to 15 minutes before and after sleep to remove irrelevant extra periods of wakefulness or artefacts on both ends of the recording. This represents an issue for the computation of important sleep measures such as sleep efficiency and latency as the onset/offset of sleep might be missed. It is also unclear how the features were selected and a description of said features is currently missing. The custom sleep stage weights procedure is unclear. The length of the time window for the smoothing procedure seems arbitrary. Last, it is currently unclear when / how the EEG and EMG data were analyzed.

As recommended by the reviewers, the 15-min truncation step has now been removed from the pipeline. Furthermore, the Methods section has been improved to provide more details on the features. Finally, the best class-weights and smoothing windows are now found using a cross-validation analysis on the training set. For more details, we refer the reviewer to the “Justification for some methodological choices” section below.

Author Response:

Reviewer #1 (Public Review):

The paper is a tour-de-force across multiple techniques and model systems from classical forward screening in C. elegans over ChIP to targeted CRISPR mutagenesis. The data is of a very high quality and supports most of the authors' claims strongly and convincingly. Finally, the manuscript is well written and, in spite of complex experiments and genetics, interesting and easy to comprehend.

• CAMTA, as the name CaM-binding transcription activator implies, have been studied previously and across many different organisms including plants, mice and humans. It was thus presumed and in part shown that CAMTAs regulate transcription depending on CaM levels.
• The authors confirm that the gene cmd-1 (encoding CaM) is directly regulated by Camt-1 by using a combination of cell-specific RNAseq and ChIP. This allows them to identify three binding sites upstream of the cmb-1 gene that bind to Camt-1.
• Moreover, the authors show that overexpression of CaM in the nervous system fully rescues the observed behavioral phenotypes.
• Importantly, the authors make another discovery. They show that CaM can directly repress its own transcription by binding to specific residues of Camt-1. Thereby, the authors argue, Camt-1 is used to precisely and bidirectionally regulate CaM levels dependent on the cell, animal's state etc.

The reported data are interesting and, in particular, the aspect that CAMTAs likely act as activators AND repressors is a novel aspect previously not appreciated. In spite of all these strengths, a potential weakness is that it remains open whether this mechanism is primarily a house-keeping mechanism or is indeed, as the authors speculate, regulated by internal and external factors that might, through CAMTA, make cells more or less responsive to Ca2+-CaM signaling.

We are grateful for and encouraged by our reviewer’s comments. We think that our discovery that CAMTAs regulate CaM expression is thought provoking.

Reviewer #2 (Public Review):

Vuong-Brender, Flynn, and de Bono report a detailed analysis of the function of a highly conserved calcium-calmodulin-dependent transcriptional regulator in the function of the C. elegans sensory nervous system. The C. elegans homolog of this factor - CAMT-1 - emerged from a genetic screen for mutants defective in a sensory-driven aggregation behavior. The authors find that multiple chemosensory modalities are disrupted by loss of CAMT-1, and this factor has distributed functions in the nervous system, including in interneurons that receive inputs from sensory neurons. A major finding of this study is that many of the effects of CAMT-1 mutation can be linked to a critical role for CAMT-1 in regulating expression of calmodulin itself. This finding is supported by multiple lines of experimentation, including a demonstration that the effects of losing CAMT-1 can be compensated by restoring expression of calmodulin. The authors further show that what is true for CAMT-1 and calmodulin in C. elegans also applies to Drosophila, indicating that CAMT-1 is a regulator of calmodulin expression whose function has been conserved throughout evolution. This manuscript has many strengths. Key hypotheses are tested using quantitative and technically independent experimental methods. The case that CAMT-1 is a regulator of calmodulin expression is built carefully and, for the most part, the logic of the argument is made clearly and supported by compelling data. Another strength of the manuscript is its candid exposition of data that do not fit neatly into the most simple and accessible model. It is refreshing to see authors who freely admit that they haven't neatly wrapped up every question in a field. The loose ends in this study do not impact the authors' main conclusions. However, some observations seem to consume more bandwidth than warranted, and the authors should consider reorganizing the manuscript so that the loose ends do not distract from the main thread of the narrative. The paper does have a few minor weaknesses that could be addressed. These are listed below.

We thank our reviewer for their thoughtful review.

Specific comments:

1. The initial description of the isolation of camt-1 mutants seemed a bit disorganized. A description of the gene and gene product preceded descriptions of the mutants. Also, some mutants were mentioned in the text but not presented in the corresponding figure. The authors should consider minor changes to better communicate how the mutations were cloned.

We have sought to do this.

1. In Fig. 2 npr-1 baselines vary a great deal between panels A, B, and C. It is not clear why npr-1 behavior is this variable, and the authors do not mention this obvious feature of their data. Data presented in Fig. 2 indicate that heat-shock-induced expression of camt-1 restores a defect in basal locomotion, but it is unclear whether it restores O2-sensitivity - the effect of oxygen on speed of transgenics seems the same +/- heatshock (compare black traces in panels 2B and 2C). We understand the concern of the reviewer. Since the design of these experiments was different from the rest (with only one shift in O2 concentration), we repeated them with 3 O2 changes, bringing them in line with the rest of the manuscript. The results are presented in the new Figure 2. We observed a more consistent baseline speed between different conditions, however some differences still exist (for example between panel 2A and 2B). One explanation is that for heatshock experiments we keep npr-1 animals at lower temperature (20 degree Celsius, panels 2B and 2C) to minimize basal activity of the heatshock promoter, whereas in the rescue experiment in Figure 2A, and in the rest of the manuscript, animals were kept at 22 oC. Figure 2B-C of our original submission used worms raised at 15 oC for the heatshock experiment, which may explain the greater discrepancy in npr-1 speed values. Heatshock also modifies slightly the response of the npr-1 control animals to O2.

Regarding whether heat-shock-induced expression of camt-1 restores O2 responses, we found that the npr-1; camt-1; dbExhsp-16p::camt-1 heat-shocked strains aggregated much more than npr-1; camt-1 heat-shocked animals. However, the rescue is not complete. Thus expressing camt-1 using heatshock-induced expression restores some O2 sensitivity which correlates well with the partial rescue of the baseline in Figure 2C. We have noted this in the results.

1. Unlike other datasets, the responses of wild-type AFDs to CO2 do not look particularly convincing (panel 3C). There is clearly an effect of camt-1 mutation on AFD calcium, but the AFD responses seem qualitatively different from the responses of BAGs to CO2 or URXs to O2. The authors might consider moving these data to a supplementary figure and tempering their description of wild-type AFDs as CO2-sensors.

The data on AFD has been moved to Figure 3 – figure supplement 1. We should add that we agree that in the absence of an identified CO2 sensor expressed in AFD, we cannot be sure that AFD neurons are primary CO2 sensors. Although the AFD CO2-evoked responses are retained in mutants defective in synaptic transmission, they may very well still be indirectly evoked by other neurons.

1. The authors candidly present data that do not conform to a simple model for how camt-1 affects behavior. Loss of camt-1 increases calcium in sensory neurons that activate the speed-controlling interneuron RMG. However, RMG calcium is reduced in camt-1 mutants. This inversion in the effect of camt-1 mutation might be caused by a homeostatic mechanism, as the authors propose. It might be possible to test this hypothesis by testing whether reducing excitatory input into RMGs elevates resting calcium in camt-1 mutants, for example via mutations that affect sensory transduction.

In the interest of simplifying the manuscript, and given other comments, we have now removed the RMG Ca2+ imaging data. However, this is an interesting way of testing what is going.

1. In Fig. 4H RMG data are presented as fractional ratio change - all other imaging data are presented as absolute ratios of YFP and CFP fluorescence. It is not clear why these data are treated differently. It is also no clear that these data are consistent with data shown in Fig. 3F. Which dataset represents the effect of camt-1 mutation on RMG calcium? More measurements might be warranted.

As highlighted above we have removed the RMG imaging data from the paper. .

1. Nice experiments show that regulation of calmodulin in Drosophila requires a CAMT-1 homolog. The bar graphs showing unity for values normalized to themselves are a bit odd - perhaps there's a more compact way to plot these data.

We have sought to address this question in two ways. First, we have further buttressed our results by performing in situ immunofluorescence staining of dissected fly retinas with a calmodulin antibody. We see a significant decrease in calmodulin expression in fly CAMTA mutants compared to controls.

Prompted by this comment, we also realized we omitted an explanation of how we normalized the data for the qPCR graphs in the figure legend. This was done using rRNA as a control. The Yamamoto lab had previously used the same control to normalize CAMTA expression in wild type and mutant flies. We add a note saying this.

1. ChIPseq analysis of CAMT-1 is also quite nice. Is there a sequence motif for CAMT-1 binding that emerges from this study? If so, how does this motif compare to motifs from studies of CAMT-1 homologs in other species?

We used the MEME algorithm, (motif-based sequence analysis tools (https://meme-suite.org/) to seek enriched sequence motifs in our ChIPSeq data. This identified a series of enriched motifs, although none coincided with the peaks at the CMD-1 promoter. However, we did observe sequences resembling the mouse CAMTA1 binding site at the centre of each of the three CAMT-1 binding peaks upstream of cmd-1. We now say this is the discussion.

1. Figure 7 shows that CMD-1 inhibits cmd-1 expression via interaction with CAMT-1. These data are interesting, but it is not clear how this effect can be related to prior data showing that forced expression of CMD-1 can compensate for loss of CAMT-1. The authors behavioral and physiological studies suggest that in vivo CAMT-1 promotes CMD-1 expression. In Figure 7, they suggest that CAMT-1 inhibits expression of CMD-1, but there is no clear link to behavior or physiology for this repressor-function of CAMT-1. The manuscript might be more clear without these data, and the absence of these data would not affect the overall impact of the study.

We agree that the feedback control of cmd-1 gene expression by CMD-1 interacting with CAMT-1 is a part of the story that has not been fully developed. Given the feedback from our reviewers and Editors to give these findings less prominence, but not remove them entirely, we moved the data into supplementary information. We have also altered the main text and the legend of Figure 7 to explicitly say that further experiments are needed to establish if this feedback is relevant under physiological conditions.

Reviewer #3 (Public Review):

Vuong-Brender et al present a thorough study investigating how CaM-binding transcription activators (CAMTAs) in C. elegans and Drosophila are required for numerous behaviors and proper neuronal function. The study is strong in how it uses a variety of approaches to study a major underlying mechanism for CAMTA. First, they use reporters, mutant analysis, and heat-shock rescue to show how cart-1 is expressed widely in neurons and functions in adults in several behaviors. They used transcriptional profiling to show that cart-1 is required to upregulate CaM in subsets of neurons in worm. They next use ChIP-seq to zero in on where worm CAMT-1 binds regulatory regions upstream of the CaM gene cmd-1 to promote its expression. They find that overexpression of CaM compensates for behavioral and neuronal response deficits in a cart-1 mutants. Lastly, they propose that when CaM highly expressed, it may down regulate its own expression by binding CART-1.

We thank our reviewer for their critique of our work.

1. Overall, I feel that the study is excellent and most conclusions are justified by evidence. However, I do not think the title is supported by the data. It currently is listed as: CAMTA TUNES NEURAL EXCITABILITY AND BEHAVIOR BY MODULATING CALMODULIN EXPRESSION. The authors show evidence that camt-1 is required for the normal function of neurons and behavior by promoting expression of CaM. Their only evidence that camt-1 downregulates CaM is a more artificial situation where CaM is overexpressed. I don't think they provide any evidence that camt-1 is used to "tune" behavior or neuron activity up and down in a wild-type strain. Tuning implies that the molecule modulates a physiological system bidirectionally in a natural situation. I suggest using a more accurate title that better fits the experimental evidence.

We have changed the title to ‘Neuronal Calmodulin levels are controlled by CAMTA transcription factors’. We hope this more neutral title is appropriate to describe our findings.

1. They show ample evidence that cart-1 appears to promote the expression of cmd-1 in most cases. This includes showing that overexpression of cmd-1 suppresses the behavioral and imaging phenotypes of cart-1. But they didn't perform the more straight forward epistasis test with the cart-1;cmd-1 double mutant in worm or fly , presumably because there is no viable loss-of-function allele in the coding area of the cmd-1 gene. It would help the readers understand why this simpler experiment was not performed if they explain this in the paper. A good place would be near line 220, where they generate hypomorphic promoter alleles using CRISPR. If they have tried to make their own loss-of-function alleles by mutating the coding area of cmd-1, but it resulted in presumed lethality, this might be mentioned here too.

This is a good point, and one that we had overlooked. cmd-1 loss of function mutations do indeed confer lethality. We have added a sentence to say:

‘Straightforward comparison of camt-1 and cmd-1 loss of function phenotypes was not possible, since disrupting cmd-1 confers lethality (7, 8).’

1. I am most worried about the potential caveats with the calcium imaging experiments. As the authors note, it is challenging to infer absolute levels of calcium using the ratiometric sensor cameleon across different individuals and genotypes. However, the authors do not note that the YFP/CFP FRET signal from cameleon might be perturbed because it uses calmodulin to bind calcium. At the end of their study (line 244), they provide evidence that calmodulin may bind to CART-1 to suppress its own expression when calmodulin is highly expressed. This is worrisome because cameleon is probably expressed highly in some or most of these strains. The authors may want to re-examine neuronal activity for a subset of experiments with a method that is independent of a calmodulin-based sensor (if possible).

We agree that this is a potential concern. As suggested by our referee, we therefore repeated some of our Ca2+ imaging experiments using a genetically-encoded Ca2+ indicator that does not contain CaM. We opted to use TN-XL, an indicator that uses troponin C as the Ca2+ binding moiety, and which has previously been used successfully in C. elegans. We imaged CO2-evoked Ca2+ responses in BAG sensory neurons, in wild type and in camt-1 mutant animals. The data obtained using TN-XL recapitulated what we observed using YC3.60 (BAG).

1. The title of "Fig 3 - Figure supplement 1" is confusing because it suggests that they measured the levels of YC2.60 cameleon, when in fact they measured a separate GFP reporter, albeit using the same promoter. So they could clarify the figure title.

The reviewer is right – our heading was confusing. We have changed it, and now say: ‘Expression from the gcy-37 promoter is reduced when CAMT-1 is overexpressed.’

1. D. Bazopoulou, A. R. Chaudhury, A. Pantazis, N. Chronis, An automated compound screening for anti-aging effects on the function of C. elegans sensory neurons. Sci Rep 7, 9403 (2017).
2. M. S. Choi et al., Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.). J Biol Chem 280, 40820-40831 (2005).
3. J. Han et al., The fly CAMTA transcription factor potentiates deactivation of rhodopsin, a G protein-coupled light receptor. Cell 127, 847-858 (2006).
4. N. Bouche, A. Scharlat, W. Snedden, D. Bouchez, H. Fromm, A novel family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem 277, 21851-21861 (2002).
5. T. Yang, B. W. Poovaiah, A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants. J Biol Chem 277, 45049-45058 (2002).
6. E. Kodama-Namba et al., Cross-modulation of homeostatic responses to temperature, oxygen and carbon dioxide in C. elegans. PLoS Genet 9, e1004011 (2013).
7. V. Au et al., CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans. G3 (Bethesda) 9, 135-144 (2019).
8. A. Karabinos et al., Functional analysis of the single calmodulin gene in the nematode Caenorhabditis elegans by RNA interference and 4-D microscopy. Eur J Cell Biol 82, 557-563 (2003).

I think this is really interesting. As a cis woman I can recognise where acting straight and feminine in society can have its benefits, rather than deviating away from the "norm". Although we find in many aspects fitting into the traditional norm can be beneficial potentially to acquiring government funds, or being more desirable for a job position because you act in a certain way. Although these are not fair, it is seen often.

However, I would argue everyone is in some way queer. When alone, or with those they are comfortable with, may present traits that are not considered as desirable by society. Whether it be based gender roles throughout the household. For an example, my partner and father of my children, doing the nightly routine with our children, playing games such as dress ups and tea parties, reading books with the children and giving them affection and more on the emotional level, all whilst I work in the office. While we believe this is equal, would society suggest these activities are "queer" for a "Male" to be doing? Or are we at a place where these activities are more accepted? (I believe it is more accepted" but based on traditional nuclear families, are these activities queer?

This is just one example but could relate to many activities that go on behind closed doors, that the public eye does not see where people would generally act out of typical "straightness, feminine or masculine roles". I would argue people are more 'queer" in those situations. I hope this makes sense.

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The Pretty Reckless - And So It Went [feat. Tom Morello] (Official Lyric Video)

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The Pretty Reckless - And So It Went [feat. Tom Morello] (Official Lyric Video) From the upcoming album 'Death By Rock And Roll' | Available February 12, 2021: http://found.ee/dbrr Subscribe to The Pretty Reckless on YouTube: https://found.ee/tpr_subscribeyt Photo by: Rob Fenn Pre-order/Pre-Save the album 'Death By Rock And Roll': iTunes: http://found.ee/dbrr_it Apple Music: http://found.ee/dbrr_am Spotify: http://found.ee/dbrr_sp Amazon Music: http://found.ee/dbrr_amzm Amazon: http://found.ee/dbrr_amz YouTube Music: http://found.ee/dbrr_ytm Stay connected with The Pretty Reckless Website: https://deathbyrockandroll.com/ Facebook: https://found.ee/tpr_facebook Twitter: https://found.ee/tpr_twitter Instagram: https://found.ee/tpr_instagram LYRICS And so it went the children lost their minds Begging for forgiveness was such a waste of time And the bullets start to fly And the bough's about to break When you hear them cry It's too much for me to take The world does not belong to you You are not the king I am not the fool They said the world does not belong to you It don't belong to you It belongs to me And, so it went the children lost their minds Crawling over bodies of those who gave their lives And the fists begin to throw And the fire starts to blaze Don't you think they know They're the fucking human race The world does not belong to you You are not the king, I am not the fool They said the world does not belong to you It don't belong to you It belongs to Everyone is crying out, I can hear them scream With all these eyes upon us but no one seems to see That you and me are just the same as god meant it to be But you're much too close to me. You're much too close to me So it went The children lost their minds Nowhere to run, nowhere to hide And the wind begins to howl And the wolf is at your door You have so much of everything But still you wanted more They said the world does not belong to you You are not the king, I am not the fool They said the world does not belong to you It don't belong to you It belongs to me #ThePrettyReckless #DeathByRockAndRoll

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The Pretty Reckless LIGHT ME UP CD

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802 Comments

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Adam Dobrin

1 second ago

VERBATIM: "stop hurting people, stop hurting people. if you do not believe people should be hurt" ... ((ishing)) ~either disable the attackers or leave the area~

1

REPLY

Adam Dobrin

1 second ago

sometimes words have two meanings. sometimes "kill" means "excorcized the demons" @LOUDON @SALEM

REPLY

CANCELREPLY

Raziel Moreno

7 months ago

I'm so damn happy she chose to rock instead of staying as an actress.

426

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sonya fuks

7 months ago

How is it possible they just keep on getting better and better with each album?!?!

341

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Graziano D'Ovidio

7 months ago

I love her voice. So much.

652

REPLY

View 18 replies

Natsumi Ikari

7 months ago

Most of people know Tom Morello from RATM of Prophets of Rage, but don't forget he was also the guitarist of Audioslave, whose singer was Chris Cornell. Must be emotional for Taylor to record this song him. I'm sure Chris is proud of them both.

631

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Matstrr Salazar

7 months ago

This is why TPR is one of my all time favorite bands. They release bad-ass singles and albums

322

REPLY

View reply

Mike Whicker

7 months ago

The entire death by rock n roll album is first on my list of reasons why 2021 will not stink as much as last year did!

180

REPLY

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Mackenzie Dinkins

5 months ago

A good theme for this year's Elimination Chamber event.

25

REPLY

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FDCAnselmo

7 months ago

The Anthem of Youth... Fuck me what a tune.

159

REPLY

View 8 replies

norangutan

7 months ago

I love how obviously this song draws inspiration from Audioslave's sound, and in a way it's paying homage to chris cornell especially with the "you have so much of everything still you wanted more" line - which is almost the same as a line from Audioslave's 'What you are'

165

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Shiteyanyo

7 months ago

Tom Morello?! Each single they release for this album just keeps getting better and better

55

REPLY

View reply

Lau pin

7 months ago

I fell in love with every song they released

90

REPLY

View 4 replies

🕸️Haley Munster🕷️

7 months ago

I really hope this album has more Going to Hell vibes 🥺🥺🥺🖤

201

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M K

7 months ago

Great tune and a good example for why I think TPR is among the best of the current rock bands. They always have catchy riffs and melodies however, especially in the bridge, you never know which turn the song is gonna take. Yet it always fits the song and never sounds out of place.

15

REPLY

Mike B

7 months ago

Underrated band

154

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🕸️Haley Munster🕷️

7 months ago

THIS is the Pretty Reckless I love !! 💙

45

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View reply

pedrogoularth

7 months ago

This is SO fucking good! 💖 🇧🇷

31

REPLY

Nick Brick

7 months ago

Kicking the year off with a banger. Looking forward to the album!

19

REPLY

Jo Mill Hyde

6 months ago

The fucking power in her voice and this song

49

REPLY

View reply

HT82 Smash

6 months ago

WWE main roster stepping up their game with these theme songs. Elimination Chamber 2021 everyone

17

REPLY

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Hide

7 months ago

The addition of the children's choir killed and buried me. 😔🤘🏾

80

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Lynne Kelly is correct here that we need better delineations of the words we're using here.

To some of us, we're taking historical methods and expanding them into larger super sets based on our personal experiences. I've read enough of Kelly's work and her personal experiences on her website (and that of many others) that I better understand the shorthand she uses when she describes pieces.

Even in the literature throughout the middle ages and the Renaissance we see this same sort of picking and choosing of methods in descriptions of various texts. Some will choose to focus on one or two keys, which seemed to work for them, but they'd leave out the others which means that subsequent generations would miss out on the lost bits and pieces.

Having a larger superset of methods to choose from as well as encouraging further explorations is certainly desired.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

This manuscript by Gulyrutlu and co-workers addresses the role of CUG expanded repeat RNA associated with DM1 in regulating the formation of higher order RNP assemblies such as stress granules and P-bodies in the cell. The authors used lens epithelial cells (hLECs) derived from a DM1 patient

We used cell lines from several patients and age-matched controls to avoid effects of individual cell-line variation. We will make sure that this is clear in the text.

or a HeLa cell inducible model of DM1 to investigate whether expression of the CUG repeat-associated protein MBNL1 and CUGBP1 affected the formation and dispersal of stress granules and P-bodies. The authors show that MBNL1 and CUGBP1 are components of SGs and PBs in hLECs and HeLa cells. In cells expressing the CUG repeat, there are minor alterations in the dispersal of stress granules as well as in the formation of P-bodies.

The alterations in the formation and dispersal of stress granules are not minor. For example, in the HeLa cell model, stress granules take more than twice as long to form in cell expressing the CUGexp repeats associated with DM1 and disperse in half the time. These data are already in the results section, but we will highlight them in a revision and have included an additional representation of the data to the figure, using graphs of ‘proportion of cells with stress granules’ against time. The changes we see are as large, or larger, then results published elsewhere (see appendix below)

MBNL1 could affect the formation and dispersal of SGs independent of the CUG repeat.

In fact, we present data in HeLa cells with MBNL1 almost completely removed by shRNA revealing that this has a much smaller effect on stress granules than does the expression of CUGexp RNA. This is an important point, as it is widely assumed that most of the cellular defects in DM1 are caused by the ‘sequestration’ of MBNL1 in the CUGexp foci. Since only . This is not what our data show. In the hexanediol experiments, both cell lines over-express MBNL1 in similar amounts. The difference between them is that one cell line expresses a DMPK1 mini-gene with a CUG expansion and the other expresses a mini-gene without the expansion. Again, our results show that the alteration to P-body responses to 1,6-hexanediol can be attributed to the presence of the CUGexp RNA, rather than altered levels of MBNL1. We will revise the results and discussion to further emphasise this point.

Finally, in HeLa cells, overexpression of MBNL1 can reduce the dispersal of P-bodies upon 1,6-hexanediol treatment.

This is not what our data show. In the hexanediol experiments, both cell lines over-express MBNL1 in similar amounts. The difference between them is that one cell line expresses a DMPK1 mini-gene with a CUG expansion and the other expresses a mini-gene without the expansion. Again, our results show that the alteration to P-body responses to 1,6-hexanediol can be attributed to the presence of the CUGexp RNA, rather than altered levels of MBNL1. We will revise the results and discussion to further emphasise this point.

Major comments:

One limitation of the work is that the perturbations seen with stress granules or P-bodies are all relatively small, and no evidence for a functional consequence on gene expression is demonstrated. Specifically, the authors observe only minor alterations in the formation or disaggregation of PBs and SGs in these DM1 models. Further, some of the effects observed are independent of the CUG repeat expression, suggesting that MBNL1 and CUGBP1 might have independent roles in modulating some properties of SG and PB formation or dispersal.

As above, the changes we see in SG formation and dispersal are not small. There are already numerous studies of the effects of DM1 on gene expression and mRNA splicing. This is not what we set out to study: we are interested in perturbations to the organisation of cellular structures associated with the expression of the CUGexp repeat RNA characteristic of DM1. We do show some data relating specifically to the proteins MBNL1 and CUGBP1 in the paper. shRNA resulting in almost complete loss of these proteins has much smaller effects that the expression of CUGexp RNA, suggesting that the major part of the effects caused by expression of the CUGexp RNA is not mediated through changes in MBNL1 or CUGBP1 levels. MBNL1 and CUGBP1 levels may well contribute to alterations in SG dynamics, but our data suggest that they are minor contributors. This is an advance in our current knowledge

1. The authors could investigate whether the CUG repeat RNA itself is localized to SGs or PBs in their models, and whether the presence of the repeat RNA is absolutely necessary for regulating the dynamics of SG or PB formation.

We have now done this. The CUG repeat RNA is not localised in stress granules or PBs to a detectable extent. This suggests that the effect we see on these structures by expression of the expanded RNA occurs despite the absence of the RNA from the structures. This is similar to the effects of the ALS-associated paraspeckle protein FUS, which can affect the integrity of nuclear LLPS structures (gems) despite not co-localising with them https://doi.org/10.1016/j.celrep.2012.08.025 We have added these data to the manuscript, as part of draft figure 8, and will add text emphasising this as an additional example of disease-causing macromolecules affecting the structure of LLPS domains in which they are not found.

1. The authors use 1,6-hexanediol to suggests that PBs and SGs in HeLa cells show behavior analogous to LLPS. However, the use of 1,6,-hexanediol to establish an assembly as a LLPS is a relatively limited analysis (despite its widespread use in the field), since this compound can affect the formation of multiple cellular substructures that are not always LLPS (for example, see Wheeler et al, 2016, eLife).

We are aware of and have cited this publication. Our comments about LLPS structures are measured, as there is still controversy about how to definitively identify them in cells. SGs and PBs have, however, previously been widely published to be formed by LLPS. The rapid exchange of SG and PB components during FRAP and the ability of SGs to both fuse and bud (seen in our supplementary movies) are also supportive of these structures behaving as LLPS structures in our models. Wheeler et showed that, in yeast, the nuclear pore complex and some cytoskeletal structures were affected by 1,6-hexanediol but membrane-bound structures such as the ER and mitochondria were not. The disruption of the nuclear pore complex is not unexpected, since phase separation is involved in cargo shuttling through the NPC (reviewed in https://doi.org/10.1016/j.devcel.2020.06.033). We will revise our discussion to make it more clear that we are not relying only on the use of 1,6-hexanediol to define SGs and PBs as LLPS structures but also on other aspects of their dynamic behaviour and on extensive prior literature.

Significance

This study would be of interest to the field if the impact of the DM! repeat RNAs on PB and SG were more substantial...

As above, the effects we see on SG formation and loss are substantial. Tissue types affected in DM1 are prone to stress, particularly the lens of the eye, so alterations to cellular response to stress associated with the presence of CUG repeats are of key importance to understanding the cellular pathology of DM1.

...and if some functional consequences were demonstrated.

In terms of function, we show altered responses to stress caused by the expression of CUGexp RNA and probably mediated through alterations in the propensity of LLPS cytoplasmic structures (SGs and PBs) to form and be resolved. Additionally, we can now show that SGs in HeLa cells expressing CUGexp RNA contain less total polyA RNA than is seen in controls, and that ‘docking’ events between SGs and PBs are compromised in cells with CUGexp RNA. These docking events are proposed to mediate transfer of RNA from SGs to PBs (reviewed in https://doi.org/10.1007/978-1-4614-5107-5_12). These new data demonstrate functional impairment of SGs and PBs associated with DM1. We have included this as an additional draft figure 8.

The lack of a strong effect on SG or PB formation in the DM1 models, along with the CUG repeat-independent effect of MBNL1 on the formation and dispersal of these complexes, argues that MBNL1/CUGBP1 may not significantly affect the formation or dispersal of SGs and PBs.

We are actually not arguing that MBNL1 and CUGBP1 are the main effectors in the changes we see to SGs and PBs, but that the CUGexp RNA is the key player, so are a little confused by this comment.

Reviewer #2:

In the current study, the authors compared the dynamics of P-bodies (PBs) and stress granules (SGs) between control and several DM1 cell lines. They found that MBNL1 and CUGBP1, two CUG repeat RNA-binding proteins that are primarily nuclear, could also co-localize with PBs in the cytoplasm and re-localize to SGs under stress. Small differences were observed in SG assembly and disassembly dynamics between control and DM1 HLECs, between HeLa cells expressing either CTG12 or CTG960, and between HeLa cells with and without shRNAs targeting CUGBP1 or MBNL1.

As detailed above, the alterations in SG assembly and disassembly in cells expressing CUGexp RNA are not small, in contrast to those in cells will lowered expression of MBNL1 and CUGBP1, which are much smaller suggesting that the changes caused by CUGexp RNA largely do not result from loss of MBNL1 (or CUGBP1). We have inserted additional graphs of ‘proportion of cells with stress granules’ against time' and will modify the text to emphasise both of these points.

Overall, the experiments were clearly described and the results properly presented. However, critical controls, as detailed below, are missing in multiple analyses. The mechanisms underlying these apparent differences are also unknown.

We do not consider that any ‘critical controls’ are missing, but can supply all of the additional analysis of our data that the reviewer requests below. We can also now provide additional mechanistic insight and will add an additional figure showing lowered amount of polyA RNA in stress granules in cells expressing CUGexp RNA and compromised docking events between stress granules and P-bodies, suggesting impaired communication between them.

Major concerns:

1. Throughout the study, the authors compared MBNL1 and CUGBP1 association with PBs and SGs without considering the potential differences in their cytoplasmic abundance between control and DM1 cell lines, which seems to be case for MBNL1 abundance in CTG960-expressing HeLa cells (Fig. 3). Provided that PBs and SGs exchange components with the cytosol at an equilibrium, if the cytoplasmic abundance of, for example, MBNL1 is decreased in DM1, one would expect the equilibrium being shifted resulting in less MBNL1 associated with PB/SG. Therefore, before measuring the association or the assembly/disassembly kinetics of PB and SG, the authors should first test whether MBNL1 and CUGBP1 abundance may be different between control and DM cell lines.

There is, in fact, no difference in the relative cytoplasmic abundance of GFP-MBNL1 between CTG12 and CTG960- expressing HeLa cells. Each has approximately a 50/50 split between nucleus and cytoplasm, with <3% of nuclear GFP-MBNL1 found in nuclear CUGexp foci when they are present. We have added a graph demonstrating this to the supplementary data. The abundance of total endogenous MBNL1 is also not altered in DM1 patient-derived lens cell lines compared to controls, as shown by semi-quantitative western blot analysis, which we have also added to the supplementary data. However, if the expression of CUGexp RNA did cause a major loss of cytoplasmic MBNL1, this change would be reflective of the situation seen in DM1 and would not invalidate our results or conclusions.

The same caveat applies to MBNL1/CUGBP1 knockdown experiments, where knocking down one may change the abundance of the other.

To carry out FRAP experiments or live cell analysis of SG formation and loss, it is necessary to over-express a tagged version of the protein being studied. For the knockdown experiments shown in figure 6, therefore, when we knocked down MBNL1, CUGBP1 was present in excess as a GFP-tagged protein and when we knocked down CUGBP1, MBNL1 was present in excess as a GFP-tagged protein. Thus, any effects of the knockdowns on expression of the endogenous proteins being analysed would be highly unlikely to influence the results.

1. Similarly, the authors did not consider the possibility that changes in SG/PB dynamics may be due to changes in the abundance/availability of essential SG/PB components such as GE1 and G3BP1.

From our immunofluorescence experiments, there was certainly no obvious reduction in GE1 or TIA1 abundance (we did not assess G3BP1). We have quantitative proteomic analysis (unpublished) from a similar pair of cell lines, expressing CUGexp RNA alongside GFP rather than GFP-MBNL1. This shows no change in GE1 or G3BP1, so we would not expect to see any here either. We can easily carry out a quantitative western blot analysis to confirm and will add this to the supplementary data

1. Most of the observed differences between control and DM cell lines were modest, leaving one wonder whether it could be simply due to cell line-to-cell line variability. Whenever possible, the authors should present results for each individual lines. For example, in Fig.2, 3 DM1 lines and 2 control lines were used. Was the difference in SG disassembly (Fig. 2B) observed in each of the 3 lines?

Some of the alterations were modest and there is cell line-to-cell line variability in the lens cell lines. This is why we pooled the data: on average, DM1 cells disperse their SGs more quickly than control cell lines do on average. This is not an unusual way to present data from patient cell lines of diverse genetic background. We have added data for stress granule loss in the individual cell lines to the supplementary data. These data show a consistent trend towards quicker dispersal of stress granules in patient cell lines. The variability between the patient lens cell lines was also the primary reason for us to develop the inducible system in HeLa cells, on a fixed genetic background, as explained in the manuscript.

Minor points:

1. Western blot in Fig. 3 shows two protein products from both endogenous and overexpressed MBNL1. Please explain.

Many of the commercially available anti-MBNL1 antibodies show this double-band in some cell lines as evidenced in numerous publications and on manufacturers’ websites (for example https://abclonal.com/catalog-antibodies/MBNL1RabbitmAb/A5149, https://www.ptglab.com/products/MBNL1-Antibody-66837-1-Ig.htm). We haven’t analysed the two bands in detail, but assume this to be a result of a post translational modification of some sort. Since GFP-MBNL1 and endogenous MBNL1 show the same thing, we do not consider it to be a major concern. We do mention the double-band as ‘characteristic’ in the figure legend for figure 3 so are not seeking to conceal anything here.

1. No data were shown to substantiate the statement that "MBNL1 localises to CUGexp foci and CUGBP1 does not" (page 6).

This has been published many times and is shown in figure 1A. However, we will add in a citation for this and have added an additional supplementary figure showing the lack of co-localisation in the foci from figure 1A more clearly together with separate data confirming that MBNL1 and CUGexp RNA do not co-localise with CUGBP1 in the nuclei of line HeLa_CTG960_GFPMBNL1.

1. The y-axis of Fig. 4D should not go beyond 1.

We will trim the axis. There are no data points above 1.0, just the indicator of statistical significance

Significance:

The nature of the current study is highly descriptive with little mechanistic insights.

Our work is not descriptive, as we observed a change in stress granules in patient cells, which we could then replicate (and enhance) in a novel inducible model of DM1 designed to abrogate the unavoidable variation in patient-derived cell lines. We also now have additional mechanistic insights (see above) and have added an additional figure (draft figure 8) detailing these.

For the subtle differences observed between control and DM1 cell lines, it remains unclear whether it may be due to cell line-to-cell line variation (see above).

We cannot completely rule out an influence of cell line-to-cell line variation in the patient-derived lens cell lines (see above), though we think this unlikely as we saw the same effect repeated and amplified in the inducible HeLa-derived cell model, which was designed to minimise this concern. Furthermore, for stress granule loss, we see a larger effect in the HeLa cell model after 72hrs of induction than after 24hrs (figure 5C). This argues strongly that the effects seen are due to the expression of CUGexp RNA and we will emphasise this point more strongly.

Some difference appear to be specific to one model but not the others (e.g., SG formation is slower in HeLa-CTG960 cells but not in DM1 HLECs).

Even for the observations that seem consistent between models, the current results yielded little novel biological insights into whether and how these subtle differences in PB/SG dynamics may relate to DM1 pathogenesis. Collectively, these weaknesses render the current study incremental at best.

The key biological insight the results provide is that the presence of the CUGexp repeat RNA results in defects in LLPS structures that are largely separable from any sequestration of MBNL1 in nuclear foci. With many researchers attributing the cellular defects in DM1 simply to the loss of MBNL1 by sequestration into nuclear foci, both this separation of altered stress response from MBNL1 levels and the involvement of altered LLPS formation (evidenced by the changes in PB behaviour on 1,6-hexanediol treatment) are novel biological insights into the cellular pathology of DM1. Additionally, our results shift the emphasis from nuclear effects to those seen in the cytoplasm.

In terms of specific DM1 pathogenesis, the eye lens is subject to constant repeated stress and is subject to continued growth throughout the life span, relying on lens epithelial cells as a stem cell pool. Epithelial cells are also vital to the homeostatic regulation of ions, growth factor and nutrient flow from the aqueous humor to the underlying fibre cells. Any alterations in the response of lens epithelial cells, in particular, to stress is highly relevant to the pathology of cataract seen in DM1. We will revise our discussion to emphasise these key points more strongly.

Reviewer #3

The manuscript entiled "Phase-separated stress granules and processing bodies are compromised in Myotonic Dystrophy Type 1" by Gulyurtlu et al., characterizes the composition and ydnamics of stress granules and P-bodies in two Myotonic Dystrophy Type 1 (DM1) cell models, human lens epithelial cells from DM1 patients and age-matched controls and HeLa_CTG12_GFPMBNL1 and HeLa_CTG960GFPMBNL1 cell lines. The manuscript is somewhat descriptive with lack of functional data and some discrepancies. For example, in the discussion section, the authors conclude that "MBNL1 appears to be absent from P-bodies in cells with CUGexp foci in their nuclei. This observation suggests that the role of MBNL1 in P-bodies may be disrupted by the presence of CUGexp RNA." Figure 4A shows that "P-bodies in the DM1 model line, HeLa_CTG960GFPMBNL1 do not contain detectable amounts of GFPMBNL1". However, Figure 4E shows similar levels of total cellular MBNL1 per PB between the control CTG12 and mutated CTG960 lines.

There is no discrepancy here. The reviewer has misinterpreted our data. PBs in the HeLa CTG960 cell line do not contain detectable amounts of GFP-MBNL1 under normal growth conditions, as shown in figure 4A. The data shown in figure 4E concern arsenite-treated cells, where some PBs in the CTG960 line do contain detectable levels of GFP-MBNL1, but significantly less than in the control CTG12 cells. We will reword these sections to make sure this is clear.

Most importantly, in Figure S3 the authors show that CUGexp foci are present in 1-2 % of the cells. The claim appears to be too strong for the data presented in the manuscript.

This is not what is shown in figure S3. The reviewer has misinterpreted our data. Figure S3 shows that in cells from line CTG960, only 1-2% of the total nuclear GFP-MBNL1 signal is found in the CUGexp foci, despite the intensity of the signal within them. Virtually all of the cells from the CTG960 cell line contain CUGexp foci (>95%). We will add a statement to this effect into the results section. We would not have continued working with a cell line in which only 1-2% of cells showed the DM1 phenotype of nuclear CUGexp RNA foci.

Although the findings are interesting and of potential impact for a better understanding of the implications of RNA-protein condensate dynamics in the pathogenesis of DM1, the work presented here is still descriptive and preliminary in my opinion. In summary, the conclusions are not so convincing and additional experiments are essential to support the authors claims.

This reviewer seems to have misinterpreted several of our data sets, including the specific points above, leading to the assertion that our conclusions are not convincing.

Several months of works will be required to consolidate data and reorganize and ameliorate the manuscript, including the way data are presented and quantified.

We already have data with which to address the majority of the queries posed, so should be able to make the adjustments relatively quickly.

Specific comments:

"On removal of stress, clearance of stress granules is mediated largely by a form of autophagy." This statement is not correct since the majority of stress granules disassemble and are not targeted to autophagy; in healthy cells only 5 % (or less) of the total SGs tend to persist in presence of autophagy or lysosome inhibitors, while the vast majority disassembles. Please rephrase carefully.

The degree and manner of dispersal of stress granules in healthy cells on removal of stress is not well understood, but is known to differ depending on the type and duration of the stress (DOI: 10.1126/science.abj2400). We do not yet know how this may be altered in DM1, however, compromised autophagy is implicated in cataract formation, which is of relevance to our study. We will re-phrase this section of the discussion carefully to reflect the complex situation.

Figure 1: RNA-protein complexes have heterogeneous composition. In HLECs, do all PBs colocalize with MBNL1 and CUGBP1 or only a fraction of them?

We do not routinely see PBs without MBNL1 or CUGBP1 in the HLECs, in contrast to the situation in the HeLa CTG960 line. We have data available in order to quantify this and will add the numbers to the text of the results.

Figure 2: Stress granules and P-Bodies are known to touch each-other, a process referred to as a "kissing event". The authors have studied the mobility of GFP-MBNL1 inside these two types of assemblies. It would be important also to quantify the "kissing" events. Is this altered in DM1 cells?

We couldn’t find reference in the literature to ‘kissing events’ between SGs and PBs, but found several references to ‘docking’ events. We have noticed such interactions between PBs and SGs in our models. We are currently quantifying this and our first experiments (one in the HeLa cell model and one comparing one of the patient-derived lens cell lines to a control) suggest that there is a change in the frequency and/or size of such interactions in the HeLa CTG960 cell line compared to the CTG12 control and in the DM1 lens cell line derived to control. If this holds true in our repeat experiments (currently in progress), this would also provide the mechanistic insight requested by reviewer 2. We have included this, together with our data showing a decrease in total polyA RNA in stress granules in HeLa cells expressing CUGexp RNA, as an additional draft figure 8.

Figure 3: In HeLa cells overexpressing CTG960_GFPMBNL1, beside the accumulation of one bright CUGexp puncta, several intranuclear GFPMBNL1 protein foci are visible. This subcellular distribution is different from the one observed in the control HeLaCTG12 GFPMBNL1. Can the author describe what these intranuclear GFPMBNL1 protein foci are?

In most cells expressing CUGexp RNA, several nuclear foci form (usually one large and several smaller) and all of them contain MBNL1 (or GFP-MBNL1 in the HeLa_CTG960_GFPMBNL1 cell line). Figure S3 shows object identification using MBNL1 in this cell line, with two clear foci detected as the reviewer points out. We have added an additional panel to supplementary figure 1 to confirm that the additional foci are also CUGexp RNA foci and will clarify in the text of the results that there is not a single focus of CUGexp RNA in each nucleus.

Is GFPMBNL1 accumulating at the level of splicing speckles? Or paraspeckles? Or other types on intranuclear condensates such as e.g. PML nuclear bodies? The different intranuclear distribution of GFPMBNL1 should be better characterized.

The sub-nuclear distribution of MBNL1 is, indeed, very complex. MBNL1 also sometimes co-localises to splicing speckles/interchromatin granule clusters as we have previously reported in lens epithelial cell lines (DOI: 10.1042/BJ20130870 ) . The details of differences in the nuclear distribution of MBNL1, beyond its accumulation in CUGexp RNA foci, in DM1 cells compared to controls is the subject of another manuscript we have in preparation but are beyond the scope of the current study.

Moreover, the % of cells expressing CTG960_GFPMBNL1 and forming intranuclear CUGexp foci is only mentioned in the discussion (Figure S3); for clarity it should be reported in the main text when describing Figure 3.

The number of cells forming nuclear CUGexp foci on expression of CTG960_GFP-MBNL1 is >95% and we will add this to the text of the results section.

"Figure S2: Quantitation of GFPMBNL1 in P-bodies in HeLa cell model of DM1." The authors report in the legend "Some, but not all, of these P-bodies contain detectable amounts of GFPMBNL1". However, the figure only shows a representative image of cells without quantification. Quantitation should be provided.

We have data available to provide this simple quantitation. Approximately 38% of PBs in arsenite-treated cells from line HeLa_CTG960_GFPMBNL1 contain detectable levels of GFPMBNL1 using a manually-assigned cut-off intensity. We will add this to the relevant figure legend (now figure S5). However, this method of analysis requires an intensity to be manually set above which GFP-MBNL1 signal is considered ‘detectable’. This is hugely subjective, and in our opinion, the automatically generated quantitative comparison of “% total cellular MBNL1 per P-body” as shown in figure 4E is a more experimentally robust way to demonstrate a small loss of MBNL1 from P-bodies in cells from line HeLa_CTG960_GFPMBNL1 treated with arsenite compared to the relevant control.

The authors report "a subtle change in stress granule architecture associated with the presence of CUGexp RNA". This statement is not supported by experimental data and should be omitted.

We will qualify this statement to make it clear that we are referring to a subtle alteration in the co-localisation between CUGBP1 and MBNL1 specifically in the SGs, as our experimental data shown in figure 4D clearly support that, showing a statistically significant increase in the Pearson’s co-efficient of cololcalisation between MBNL1 and CUGBP1 in cell containing CUGexp RNA compared to the relevant control (0.90+/-0.05 for CTG960; 0.87+/-0.07 for CTG12).

Figure 4. MBNL1 and CUGBP1 co-localise in P-bodies. What is the % of colocalization?

We’re not sure exactly what is being requested here or what biological question the reviewer is asking us to address. MBNL1 and CUGBP1 co-localise in virtually all PBs (except in the HeLa CTG960 line where MBNL1 is undetectable in PBs under normal growth conditions). Figure 4E shows that, in cells with PBs upregulated by sodium arsenite, the mean amount of total cellular MBNL1 per PB is 0.1%, so it will be similar in cells grown under normal conditions as the PB sizes are similar and they appear to be of similar brightness by immunofluorescence. Again, this would be straightforward to quantify with our existing data if this is, indeed what the reviewer is requesting, but we question the biological significance. We would be reluctant to derive a Pearson’s co-efficient for the degree of co-localisation between CUGBP1 and MBNL1 in P-bodies as the structures are too small in size for this to be meaningful within the limits of imaging capabilities. We could, however, provide this if this is a specific request.

Figure 5: "Treatment with sodium arsenite was then carried out under time-lapse microscopy, with Z-stacks of images taken every 4 minutes until stress granule formation was clearly seen (Fig.5A). This revealed a pronounced delay in formation of stress granules in cells containing CUGexp foci (HeLa CTG960 GFPMBNL1, 36 min +/- 12) compared to those without (HeLa CTG12 GFPMBNL1, 15 min +/- 2) (Fig.5B)." Data representation in Figure 5 is unclear and the pronounced delay in stress granule formation is not appreciated. Since the authors performed a live imaging taking pictures every 4 minutes, it would me more informative to plot the data and show the assembly and disassembly kinetics over time for both control and CTG960_ GFPMBNL1 cell lines (similar to what shown in e.g. Gwon et al., Science 2021, Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner, Figure 2G).

The bar graph in figure 5B shows that cells from the CTG960 line take more than twice as long to form SGs compared to controls and are lost in half the time, with the precise numbers given in the text. A simple bar graph seemed the clearest way to present this. However, we have plotted our existing data in a similar manner similar to that in the cited reference and added this to figure 5. These graphs clearly show that the differences we see are at least as great as in other published literature, including the reference given by the reviewer (see below).

Concerning Figure 1, the authors report no difference in the kinetic of stress granule formation in HLECs. However, they only report data after 45 and 60 min of arsenite treatment; at these time-points the assembly step is maximal. Thus, for consistency, the authors should include earlier time-points to the analysis of stress granule assembly also in HLECs, similar to what done in HeLa cells in Figure 5.

The assembly step is not ‘maximal’ in these cell lines after 45 minutes. Figure 2A clearly shows that only ~30% of cells have SGs after 45 minutes of treatment, compared with 100% of cells after 90 minutes shown in figure 2B. We have additional data at 10, 20 and 30 minutes all showing no significant differences. We had omitted them to keep the graph simple, but have now included them as a graph of ‘% of cells with stress granules against time’ in figure 2.

"Having established that MBNL1 and CUGBP1 co-localise closely in stress granules": the authors investigated the colocalization of each of these two proteins with stress granule markers but they did not verify whether MBNL1 and CUGBP1 co-localise.

In figure 1B we show that endogenous CUGBP1 and endogenous MBNL1 both co-localise with the stress granule marker TIA1 in stress granules in lens epithelial cells. It would, therefore, be highly unlikely that CUGBP1 and MBNL1 would not co-localise with each other in stress granules. We have also previously verified that GFPMBNL1 behaves identically to its endogenous counterpart (Coleman et al, 2014). Furthermore, in figure 4C and 4D, we show close co-localisation between endogenous CUGBP1 and GFPMBNL1 in stress granules in our HeLa cell model, using high-resolution AiryScan microscopy for which we provide detailed quantitation.

This aspect should be addressed experimentally since the authors also conclude that "a complex relationship between MBNL1 and CUGBP1 in stress granules" exists. Thus, the authors need to assess the colocalization of GFPMBNL1 with endogenous CUGBP1 in stress granules and the one of GFPCUGBP1 with endogenous MBNL1.

The complex relationship we propose is based on the effects of CUGBP1 or MBNL1 knockdown on the dynamic behaviours of each other by FRAP assay and not solely on their co-localisation, although we have already analysed their co-localisation in detail as above.

Figure 6: Please add antibody labeling to microscopy panels A and B.

Certainly, this was an accidental omission and has been added

Moreover, specify is the numbers refer to minutes in panel F. The data representation is also unclear - see comment above, Figure 5.

As stated in the figure legend and on the graph axes, these numbers have been normalised to the mean time taken for SG formation/loss in the control CTG12 cell line (set at 100%). The precise numbers in minutes for mean and SD are given in the text. We have added additional graphs of ‘% of cells with stress granules against time’ to this figure, with the values in minutes given to clarify the exact time-scale.

Figure 7: was 1,6-hexanediol added in presence of arsenite? Or was arsenite removed?

Arsenite was not removed (neither was Doxycycline) as we wanted to examine the effect of 1,6-hexanediol on SGs and PBs without the added complication of the effects of stress removal. We will clarify this point in the methods/results.

Aberrant persistent stress granules have been implicated in age-related (Mateju et al., 2017) and neurodegenerative diseases (Protter and Parker, 2016), such as ALS and FTD (Jain et al., 2016; Markmiller et al., 2018; Zhang et al., 2018). These are proposed to result from increased liquid-to-solid phase transitions within the stress granules (Mateju et al., 2017)." The authors should better define what are aberrant stress granules (e.g. see Ganassi et al., 2016; Turakhiya et al., 2018, PMID: 29804830).*

We will expand on this subject in the discussion

"Persistent stress granules have long been associated with degenerative conditions, notably ALS (Li et al., 2013)". I suggest updating the reference adding a more recent one.

We selected this 2013 review to emphasise that there is a long history of association of persistent stress granules with degenerative conditions. We will add in an additional, more recent review.

Significance

The work is descriptive; thus, in this form I do not consider that it is strongly advancing the field.

Having noted alterations to stress granule disassembly in lens epithelial cells from DM1 patients, we went on to develop a novel inducible model in which we replicated and enhanced these effects by expressing the large CUGexp RNA that causes DM1 as part of a DMPK mini-gene mimicking the genetic mutation seen in DM1 patients. This is not purely descriptive. Furthermore, we are now in a position to add an additional figure showing two pieces of evidence for functional defects in stress granules associated with CUGexp RNA expression 1) reduced accumulation of total PolyA RNA in stress granules indicating compromised function and 2) compromised ‘docking’ events between stress granules and P-bodies, a process proposed to be integral to the function of both structures.

Author Response:

Reviewer #1:

The authors demonstrate deficits in perceptual tests related to fine-time perception in non-speech and speech sounds in a group of patients with stroke aphasia compared to a control group without a lesion. A subgroup of patients with deficits in spectrotemporal processing at a fine timescale have lesions mapped to the posterior STS, MTG and adjacent white matter. The area associated with deficits in spectrotemporal analysis with a fine timescale is then used as a seed for probabilistic fibre tractography based on diffusion MR. These results show connectivity of the functionally defined seed region with a number of areas including the cerebellum.

The work is carefully done and I think interesting in demonstrating the cerebellar connections of the functionally defined region associated with deficits in fine temporal analysis that might be a basis for event representation at this temporal level.

We appreciate the referee's evaluation and constructive feedback.

Reviewer #2:

Based on consideration of supportive evidence in the literature, the authors propose that a cerebellar-temporal lobe functional network plays a key role in auditory temporal processing. The precise parsing of temporal information is critical to understanding dynamic auditory processing and thus is an interesting area of study. Better understanding of how the cerebellum and temporal lobe may interact to achieve such parsing of the dynamic signal in a generative/predictive internal model is of clear interest to a broad readership. This idea is put to the test by first having individuals with lesions in the posterior portion of left temporal lobe perform speech perception and timing tasks and comparing performance with 12 healthy controls to establish the role of this region in tasks reliant on intact fine temporal processing. Typically, a lesion model will be helpful when a dissociation between structure and function can be demonstrated, and preferably this would be a double dissociation. Here, while lesions to auditory regions of the left temporal lobe are associated with impoverished performance on speech and temporal order tasks relative to a healthy control group, performance on comparably difficult auditory tasks that do not require good temporal discrimination is not tested to determine if there is such a dissociation. Given the extensive discussion of hypothesized different time sensitivities of right and left auditory cortices in the Introduction, patients with right homologous lesions might also have served as an interesting control and could have supported a double dissociation. In a second step to their study, a seed region was generated based on comparison of the lesion loci for the half of the patients who performed most poorly on the behavioral tasks to the other half, and this was used to explore anatomical tract connectivity of the seed region to the rest of the brain in the neuroimaging data from the healthy controls, with a focus on connections with the cerebellum. This approach to establishing that "temporo-cerebellar connectivity underlies timing constraints in audition" is unfortunately just not that convincing. The data are interesting, but taken alone they simply do not support such a conclusion. In the data, there is no clear functional link established or even hinted at between the temporal lobe and the cerebellum.

We appreciate the referee's evaluation and constructive feedback. We address the raised concerns point by point below. We appreciate the concerns regarding our methodological choice and our interpretation of a functional link between the temporal lobe and the cerebellum. It certainly is more reasonable to derive a functional interpretation based on disconnection measured directly in patients’ DTI. However, if unavailable, indirect measures of disconnection can also be used to establish a functional link between a lesioned region and the networks associated with it. The rationale behind this is that it reflects an indirect estimation of the effect of a lesion on structural brain networks. To make this approach clearer, we have revised the manuscript accordingly. See revised manuscript pages 6 and 12:

[...] Assessing connectivity in healthy participants based on lesion information is a relatively new method that measures structural disconnection in networks associated with given anatomical regions (Foulon et al., 2018). This allows for the indirect estimation of the lesion effect on structural brain networks. In this regard, it was shown that behavioral deficits can be explained similarly by local brain damage and indirectly measured disconnection (Salvalaggio et al., 2020). [...]

[...] We next used the respective areas as seed regions for probabilistic fiber tractography in a healthy age-matched sample to visualize the underlying common connectivity pattern (see Methods). Thus, we indirectly explored the association between posterior superior temporal disconnection and processing of sound at short timescales. [...]

We also changed the abstract and conclusion accordingly. See pages 2 and 15 of the revised manuscript.

[...] Here we tested whether temporo-cerebellar disconnection is associated with the processing of sound at short timescales. [...]

[...] The evidence we describe (i) shows that lesion-related deficits in spectrotemporal analysis occur in posterior temporal regions connected to the cerebellum [...].

Reviewer #3:

Stockert et al. investigate the cortico-cerebellar network underpinning rapid temporal auditory analysis. This study uses a well-defined group of stroke participants with mostly circumscribed lesions to the left posterior superior temporal lobe to motivate probabilistic tractography from cortical regions associated with verbal and non-verbal rapid auditory temporal analysis. Lesion-symptom mapping identifies a specific region of the posterior superior temporal sulcus and underlying white matter as statistically associated with impairment in rapid auditory temporal analysis. Tractography results demonstrate that these regions have high structural connectivity to wider regions of the left hemisphere cortical language network and ipsilateral and contralateral connectivity to postero-lateral cerebellum and dentate nucleus. It is interpreted that this cortico-cerebellar network is crucial to developing representations of fine auditory temporal structure.

The conclusions of the paper are an interpretation which is based on integrating previous neuropsychology with the current tractography results and based on well-defined models in the motor domain. Such conclusions are not unreasonable but there is no direct (associative) evidence linking this network to the cognitive function of interest.

Strengths:

The paper integrates neuropsychology and neuroimaging methodologies to build a coherent picture which is more than the sum of its parts. The stroke group has well-defined and selected lesions which enable testing of the hypotheses put forward by the authors. The behavioural measures are sensitive and suitable to identify impairments in the behaviours of interest. There has been a detailed analysis of the behavioural speech perception data in the stroke group which largely, although perhaps not entirely, conforms to the asymmetric temporal sampling hypothesis. The lesion-symptom mapping approach is suitable for the nature of the population (small group with similar lesion distributions) and has allowed neuropsychologically guided tractography in the neurotypical population. This has clearly illustrated the complexity of the structural connectivity of the posterior superior temporal sulcus and underlying regions.

Weaknesses:

The selective nature of the stroke population - relatively small, chronic lesions - has resulted in only mild impairments for a small number of participants (6/12 participants). At the group level there is no difference between the stroke and neurotypical population on speech perception measures - group statistics do not reach one tailed significance. This reduces the certainty with which the regions identified are associated with the behaviour or interest. However, the results do conform to previous neuropsychology and lesion studies and it is likely that this lack of effect is due to low statistical power.

Please refer to our response to the next point.

All the stroke participants have a similar lesion distribution, and this makes lesion-symptom mapping challenging. For example, lesion data do not give an indication of the functional integrity of perilesional regions which can be reduced, even at the chronic stage, therefore the superior temporal sulcus may not be functioning effectively, even in the proportion of the group without lesions to this area. Lesion symptom mapping is more robust with a wider distribution of lesions and the inclusion of participants with lesions remote from the area of interest. Having said that, the behavioural measures appear sensitive enough to identify mild impairments and the authors, for good reason, wished to reduce the extension of lesion into primary auditory regions. As above, given the limited sample and homogeneous lesion, the lesion symptom mapping approach is reasonable.

We agree that the small number of patients is a possible limitation to the study and add this point to the limitations section. See revised manuscript page 21.

[...] First, the study population is relatively small and lesion symptom mapping is typically applied to larger populations with wider lesion distribution. Although careful selection of circumscribed lesions has the advantage of highlighting behavioral differences without confounding other deficits (e.g., primary auditory processing), it is possible that additional regions are involved in processing of sound at short timescales. However, tractography based on healthy participants makes it possible to indirectly obtain information (i.e., structural disconnection) about brain regions contributing to the investigated function. In addition, it is likely that the small number of patients might hamper the ability to detect statistically significant differences between the behavior of controls and patients. Nevertheless, we are confident that the current results align with the fact that the posterior superior temporal cortex contributes to the processing of sound at short timescales, as indicated by previous neuropsychological evidence and lesion studies (Boemio et al., 2005; Chedru, Bastard, and Efron, 1978; Efron, 1963; Robson, Grube, Lambon Ralph, Griffiths, & Sage, 2013; Swisher & Hirsh, 1972). Further studies should however test larger populations to replicate and extend this finding. [...]

The authors suggest that the behavioural results conform to the asymmetric temporal sampling hypothesis in that only place of articulation discrimination impairments in the stroke group can be (just about) detected, whereas there were no significant stroke-neurotypical differences in other phonetic contrasts. It is not clear that the VOT differences associated with plosive voicing changes and the cues associated with place changes happen over fundamentally different time-scales and, therefore, it is important to further justify the interpretation of the data. In the future it will be helpful to have this level of analysis applied to individuals with lesions to the wider speech perception network to draw conclusions about the specificity of the impairment to these regions - for example, impairments in phoneme discrimination have been associated with frontal lobe lesions.

It appears that voicing contrasts in which shorter and longer voice onset times result in the perception of a voiced or voiceless plosive (for example [t] and [d]) are encoded in both the temporal envelope and fine structure (Rosen 1992) of the speech signal that occur in time windows of 20-500 ms and <2 ms, respectively. In words an additional cue is the closure time, which can be further used to discriminate between voiced and voiceless plosives. However, place of articulation contrasts are exclusively encoded in the temporal fine structure (i.e., very quick transitions of the frequency spectrum, formant transitions). Even though for all contrasts shorter timescale information plays a role, somewhat redundant encoding is present for voice contrasts. Ultimately, place of articulation contrasts seem to be the most difficult to discriminate. In Figure 2D it is apparent that despite highest error rates for the place of articulation contrasts, several patients also showed impaired discrimination for voicing contrast when compared to healthy controls. We do agree with the referee that it would be interesting to also extend this level of analysis to individuals with lesions in the wider speech perception network in future work.

The tractography results reveal a complex pattern of structural connectivity, including other regions associated with speech perception. The authors have a theoretical motivation to focus on the importance of the temporo-cerebellar pathway but there is no correlation evidence to link auditory temporal analysis to the integrity of this pathway in the neurotypical population. The non-verbal measures appear to be sufficiently sensitive for this type of analysis. This lack of association with behaviour makes it hard to draw conclusions about the functional role of this network.

We appreciate the referee’s concerns about our interpretation of the functional link between the temporal lobe and the cerebellum regarding auditory temporal analysis. It certainly is more reasonable to derive a functional interpretation based on disconnection measured directly in patients DTI. However, if unavailable, indirect measures of disconnection can also be used to establish a functional link between a lesioned region and the networks associated with it. The rationale behind this is that it reflects an indirect estimation of the effect of a lesion on structural brain networks. To make this approach clearer, we have modified the manuscript as such. See revised manuscript pages 6 and 12:

[...] Assessing connectivity in healthy participants based on lesion information is a relatively new method that measures structural disconnection in networks associated with given anatomical regions (Foulon et al., 2018). This allows for the indirect estimation of the effect of a lesion on structural brain networks. In this regard, it has been shown that behavioral deficits are explained to a similar extent by both the local damage and indirectly measured disconnection (Salvalaggio et al., 2020). [...]

[...] We next used the respective areas as seed regions for probabilistic fiber tractography in a healthy age-matched sample to visualize the underlying common connectivity pattern (see Methods). Thus, we indirectly explored the association between posterior superior temporal disconnection and processing of sound at short timescales. [...]

We also changed the abstract and conclusion accordingly. See revised manuscript pages 2 and 15.

[...] Here we tested whether temporo-cerebellar disconnection is associated with processing of sound at short timescale. [...]

[...] The evidence we describe (i) shows that lesion-related deficits in spectrotemporal analysis occur in posterior temporal regions connected to the cerebellum [...].

I understand that Disney may not have openly queer characters and I understand people want more representation. However, I think it is weird to compare the sisters to anything "couple" related since they are sisters and that has nothing to do with being Queer. So I don't exactly understand how someone could take that into an account and have a theory that Frozen is a queer movie.

Author Response:

Reviewer #1:

The manuscript by Takahashi et al describes the interaction between MLL fusion proteins with HBO1 and its role in leukemogenesis. Myeloid progenitor transformation assays using various MLL fusion proteins reveal that MLL fusion proteins requires the TRX2 domain of MLL for effective leukemic transformation. IP-MS identifies HBO1 as a bona fide binding partner of the MLL TRX2 domain. ChIP-seq experiments show genome-wide colocalization of HBO1 complex with MLL-ENL and the WT MLL in MLL-fusion leukemia cells and MLL WT cells, respectively. ChIP-qPCR in MLL-deficient cells suggest that recruitment of HBO1 to MLL target genes (such as MYC and CDKN2C) depends on MLL. Truncation analysis of the ELL part of the MLL-ELL fusion reveal that MLL-ELL transformation activity requires OHD domain-mediated recruitment of AF4 and EAF1. Furthermore, co-IP and ChIP experiments with various fragments show that AF4 and EAF1 form two distinct SL1/MED26-containing complexes and likely the AEP/SL1/MED26 complex is competent for transactivation. Series of transformation assays suggest that MLL-ELL transforms hematopoietic progenitors via association with AEP, but not other ELL-associated proteins. Finally, the authors also show that NUP98-HBO1 fusion transforms myeloid progenitors through interaction with MLL. Overall, this is a quite comprehensive study demonstrating that various MLL fusions and NUP98 fusions transform hematopoietic progenitors via HBO1-MLL interaction, which suggests that targeting their interaction might be s new therapeutic approach.

We appreciate the comments and inputs from the reviewers.

Reviewer #2:

In this manuscript, the authors identified an interesting interaction of MLL (a methyltransferase) with an HBO1-JADE complex (an acetyltransferase) and investigated the functional impact in leukemogenesis by fusion proteins containing MLL or HBO1. The data is clear and the connection between MLL and HBO1 is unexpected. The manuscript is also well organized and relatively easy to follow.

Comments:

1) The functional relevance of the interaction between MLL and HBO1 is still correlative. It would be important to know whether there are any results directly about the impact of the loss of the HBO1 complex on the function of MLL.

We performed a sgRNA-dropout assay which showed that HBO1 is critically required for the survival of leukemia cell lines, as depicted in Figure 2F and Figure 2-figure supplement 3.

2) It is important to show the source and specificity of the antibodies that were used for ChIP of the HBO1 complex.

The details of the antibodies are provided in Key Resource Table.

3) It might be interesting to check whether other JADE proteins and also BRD1 (another partner of HBO1) are involved.

We agree that it would be very interesting to examine the involvement of other JADE/BRPF family proteins in the future because they share the ING4/5 subunits and BRD1 plays an important role in hematopoiesis (1). This can be addressed in future studies.

4) The acronym TRX2 may be confusing as some might think that it is thioredoxin.

As advised, we have changed this to THD2 (TRX homology domain 2).

Reviewer #3:

This paper starts with a series of bone marrow transformation assays comparing MLL fusions and domain-deletion mutants thereof to define the minimal elements for robust leukemic transformation and surveying growth and attendant common fusion targets HoxA9, Meis1 in colony replanting assays. Here they discover that a region of the MLL-N portion just upstream of the well-studied CXXC domain, termed in their previous work the "TRX2 domain" is important for the transformation capacity for several different MLL-fusions (and more minimal chimeras of key modules). A small region of the MLL-N protein encompassing the TRX2 domain and the CXXC module are subjected to complex purification, it is clear from comparison to number of controls that the TRX2 domain is an important mediator of association, perhaps indirect, with the HBO complex. Drop out experiments confirm that HBO1 knockout is lethal to MLL-rearranged leukemia, nicely confirming recent work (Ay et al., MacPherson et al.).

ChIP-seq experiments in an ALL with MLL-ENL fusion, and then more extensively in a kidney cancer cell line indicate overlap with some of the HBO complex subunits and MLL, however this does not establish recruitment at these sites. ChIP-qPCR at a few MLL-fusion target genes with MLL depletion supports the recruitment hypothesis somewhat although mixed and modest effect sizes indicate that alternate pathways for HBO1 recruitment are involved, and could also be explained as reduced deposition of marks known to recruit HBO1, rather than direct recruitment. Sadly, the real potential strength of this work goes unrealized, as the recruitment of HBO1 mechanism remains tantalizingly out of reach. More experiments in this space could conclusively establish the molecular mechanism of a seemingly biomedically important recruitment paradigm, and thereby have much more impact.

As the reviewer pointed out, MLL is not the only element that recruits the HBO1 complex to the target chromatin. MLL is known to deposit H3K4me marks, and the HBO1 complex is known to recognize these marks via ING4/5 subunits. We performed a ChIP-qPCR analysis of H3K4me3 in MLL-knockout cells. At the MYC promoter, the H3K4me3 marks were substantially decreased (Figure 3F). Moreover, recruitment of HBO1 was not recovered by transient expression of an MLL mutant containing THD2, indicating that the presence of H3K4me3 marks is a prerequisite for HBO1 recruitment. In accordance with this, ING5-histone interaction is required for the stable association of MLL with the HBO1 complex (Figure 8A-C). Thus, a more appropriate molecular mechanism would be the cooperative recruitment of the HBO1 complex by ING4/5-mediated chromatin association and MLL-mediated association. Because of the multiple contacts involved in this molecular network, it is not easy to pinpoint the direct contacts as desired, but our biochemical analyses indicate that PHF16 and ING4/5 offer relatively strong binding surfaces (Figure 8A-C). The ING domain of ING5 is the most likely direct binding surface identified thus far.

At this point the paper shifts to a seemingly distinct line of inquiry, which is not closely related to the HBO1-TRX2 story to the first three figures. The new direction examines the ELL fusion partner in some detail using similar fusion protein chimeras, but a portion of Figure 4, is largely confirmatory of previously established findings about the critical regions of ELL for transformation and its AF4/EAF1 partners, adding only that portions of the MLL fusion protein are dispensable, provided that they are replaced with the PWWP of LEDGF. It is a little bit of a Frankenstein's monster experiment, and does not add much new to the field. Further experiments define potentially two distinct complexes that have already been characterized being recruited by ELL, although there is overlap here again with their previous studies, and the results are a little hard to interpret.

A portion of Figure 4 was confirmatory to previous results. We have moved this to figure supplements in the revised manuscript (Figure 4-figure supplement 1B,C). The main topic of this paper is the role of the HBO1 complex in MLL-mediated transactivation pathways. The structure/function analysis of MLL fusion proteins demonstrated that MLL-ELL is highly dependent on the HBO1-mediated function in leukemic transformation (Figures 1 and 2). Hence, it was important to clarify the mechanism of gene activation by MLL-ELL in this study to understand why HBO1 association is required for MLL-ELL-mediated transformation. Because MLL-ELL associates with AEP similarly to major MLL fusions such as MLL-AF4 and MLL-ENL, it was speculated that MLL-ELL also activates its target genes via AEP. However, ELL associates with EAF family proteins and MLL-EAF also has transforming ability (3). Thus, EAF1-mediated functions could be more important for MLL-ELL-mediated transformation rather than AEP-mediated functions. To clarify the mechanism of MLL-ELL-mediated transformation, we generated a point mutant that selectively impaired ELL-EAF interaction and demonstrated that EAF1-association is dispensable for MLL-ELL-mediated transformation (Figure 6), thereby indicating that MLL-ELL transforms via AEP-mediated functions, which demands HBO1-mediated functions. We also showed that the presence of THD2 enhances ELL-AEP association to further suggest that one of the roles of the HBO1 complex is to enhance the association of ELL with AEP (Figure 6E). These findings are not reinterpretations of our prior results and are relevant to the main topic of this paper. We believe this part adds new information to the field, and therefore we have included it in the revised manuscript.

The authors create structure-guided separation of function mutants in the ELL domain that binds both AEP and SL1, permitting them to specifically disrupt EAF1 interactions but not AF4. Further experiments solidify this interpretation, and find that this mutant shows no deficits in hematopoietic progenitor transformation or primary leukemia lethality, although there appears to be some effect upon reimplantation.

The last figure in the paper tackles the seemingly unrelated Nup98-HBO1 fusion, a rare patient mutation-they demonstrate a requirement for MLL for viability of hematopoietic progenitors transformed by this fusion, connecting back to the TRX2 interaction, and show that menin inhibitors slow growth.

Strengths:

The identification of the TRX2 region of the MLL-N protein as the major point of contact (perhaps not direct), to the HBO1 complex adds mechanistic depth to the really important recent discovery (confirmed in this work) the MLL-fusion leukemias rely on HBO1 function. This lab has published a number of technically similar types of papers defining minimal regions of MLL and distinct interacting partners by chimeric fusions, with bone marrow transformation assays, mouse model engrafting studies, IP's, ChIP etc. In my view they are very much under cited, likely because they are similarly so challenging to read.

Thank you for your pointed feedback. We will try our best to make the necessary improvements so that our papers are widely read and cited.

The mixture of Co-IP biochemistry, bone marrow transformation assays, and ChIP, to define interactions, minimal requirements for transformation, and their chromatin consequences for a host of different MLL-fusions and HBO1-fusions has the potential to define the key interfaces underlying recruitment.

Weaknesses:

The mechanistic inquiry stops short of really defining the critical MLL-HBO1 complex interface. Defining the point of contact on the HBO1 side (even which subunit) and determining whether it is direct, or bridged by some, as yet unidentified factor, as well as conclusively demonstrating that this is the mechanism of HBO1 recruitment remain the major shortcomings.

To address this criticism, we further investigated the mechanism of complex formation by MLL and the HBO1 complex. As we demonstrated in Figure 8A-C, the association appears to be mediated by multiple contacts mainly through PHF16 and ING4/5. Because this association needs an intact PHD finger of ING5, it likely occurs depending on the context where ING4/5 is bound to histone H3K4me2/3. The ING domain of ING5 was also required for the association, indicating that this portion may contains a point of direct contact. We speculate that HBO1 recruitment is mediated primarily by ING4/5-H3K4me3 interaction and MLL reinforces its chromatin association.

And the follow-on figures apart from the last one, appear disconnected from this portion of the story and distract from it.

We depicted a revised model incorporating the above-mentioned aspect in Figure 8D of the revised manuscript.

The complex nomenclature and density/organization/logic of the presentation of experiments makes this paper difficult to read. Absence of sufficient grounding in the broader literature much beyond their own lab's work further compounds the problem.

We changed some of the nomenclature and density/organization/logic of the presentation of the experiments to improve the readability.

There is a lot of overlap, particularly in parts of figure 1 and figure 4 with previously published results. So perhaps re-organizing the display of data, and the organization of presentation, putting confirmatory work in the supplementary figures, would improve accessibility.

We moved some portions of Figure 4 to figure supplement. The data for MLL-AF10 and MLL-ENL were retained in the Figure 1 as important references.

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Reply to the reviewers

Reviewer #1:

This paper puts together a nice set of data showing that a specific gene called Resf1 when deleted effects the ability of ESCs to self-renew and proceed to germline fates. I believe the data are sound and that they provide the evidence needed for the authors to make their conclusions.While I think the data are presented well and the manuscript is well-written, the "modest" functional results suggest this work would be more suited for a specialized journal.

We thank Reviewer 1 for their supportive comments.

• *

Reviewer #2:

1. In the presence of LIF, there is no difference between Resf1 knockout mESCs and WT mESCs except the expression of Esrrb, Nanog and Pou5f1. What about other genes? RNA-seq is needed to distinguish the two cell lines.

Fukuda et al. have shown that deletion of Resf1 leads to misregulation of ~1000 genes (adj. p-value 2) in presence of LIF. This highlights large differences between transcriptomes of Resf1 KO and WT cells that occur despite only a marginal difference in self-renewal efficiency between Resf1 KO cells and WT in the presence of LIF. It is therefore questionable whether the time and resources required to perform the requested RNA-seq would produce data that could unambiguously identify the potential causative effector difference downstream of Resf1.

As an alternative approach, we have reanalysed the Fukuda et al RNA-seq data. We find that Esrrb is significantly downregulated (in agreement with our Q-RT-PCRs), as are Klf4 and LifR (FDR 1.5). However, our meta-analysis of the Fukuda et al data did not show Pou5f1 and Nanog to be differentially expressed (FDR 1.5). This is in line with the lower level of downregulation of Pou5f1 and Nanog, compared to Esrrb in our Q-RT-PCR data. Notably, our gene expression analyses were performed in 5 biological replicates, whereas Fukuda et al. performed RNA-seq in two biological replicates. We can include the meta-analysis of the Fukuda et al data in our submission. As the change in ESC self-renewal that we see at low LIF concentrations could result from a decrease in Lifr expression, we will verify the change in expression of Lifr by Q-RT-PCR. Importantly, we will do this in a way that discriminates between expression of the transmembrane Lifr and soluble LifR, since the latter acts antagonistically (PMID: 9396734, Chambers, BJ, 1997).

1. The authors showed Resf1 is not required for Nanog function, so how does Resf1 regulate the expression of pluripotency genes? Through epigenetic modifications or signaling pathways? The authors should design experiments to explain the detailed mechanisms.

The strength of the immunoblot signal for RESF1 is low, even when Resf1 is expressed episomally. Therefore, although we could try to co-immunoprecipitate with the Resf1-v5 cell line and endogenous Nanog, the expression level of RESF1 may mean this effort is unsuccessful. Given the fact that the result will not affect the conclusions of our study, we do not think this effort is justifiable.

1. The authors showed that Resf1 interacts with Nanog, but they used forced expressed proteins. Does the endogenous Resf1 interacts with endogenous Nanog? Do they bind to some same DNA sequences?

This are important questions to answer. However, many more experiments would be required to reach firm conclusions. The reviewer is right to say that the mechanisms by which Resf1 affects pluripotency are unknown and remain to be answered in future. We therefore propose to improve the text discussing similarities in pluripotency phenotype between deletions of Trim28, SETDB1, YTHDC1 and RESF1. As deletion of RESF1 partner SETDB1 or other proteins involved in repression of retrotransposons lead to downregulation of pluripotency genes and in some cases collapse of ESCs (e.g. PMID: 19884255, Bilodeau et al. 2009; PMID: 19884257, Yuan et al. 2009), we hypothesise that the RESF1 phenotype may be explained by affecting SETDB1 chromatin binding and therefore repression of SETDB1 targets. The mild phenotype of RESF1 KO indicates that RESF1 would not be an essential component of this repressor complex but rather “a modulatory protein”.

It is also worth noting that the meta-analysis of the RNA-seq data from Fukuda et al. suggests that Resf1-null ESCs may express reduced levels of LifR mRNA, and this is something we plan to investigate.

1. In figure 5C, some Resf1 positive cells showed Nanog negative. Are these Nanog negative cells pluripotent?

Nanog-null ESCs are pluripotent (PMID: 18097409, Chambers et al., 2007). In addition, NANOG-negative cells in FCS/LIF cultures can retain pluripotency. Our purpose in this figure was therefore not to say whether NANOG-negative:RESF1-positive cells are pluripotent but to draw attention to the broader expression of RESF1 in FCS/LIF compared to NANOG. Such broader expression has also been noted for other heterogeneously expressed factors (PMID: 31582397, Pantier et al. 2019).

1. In figure 6A, the naïve mESCs are induced to EpiLCs. Is the transition efficiency of Resf1 knockout cells the same with WT mESCs? The finally obtained PGCLCs should be identified.

We show that the key TFs of EpiLC state are expressed similarly in WT and Resf1 KO cells (Supplementary figure 4) and we have data showing that WT and Resf1KO EpiLCs have a similar morphology. Together this suggests an efficient transition to an EpiLC state. Our analysis has identified expression of Blimp1/Ap2g/Prdm14 in Resf1-null cultures. Compared to wild-type cells these levels are reduced up to 3-fold. As this is from an unsorted population and the number of SSEA1/CD61-positive cells is decreased around 2x, this suggests that the PGCLC population formed by Resf1-null cells is reduced in proportion but is otherwise normal.

We will add photographs of EpiLC colonies formed by Resf1 KO and WT cells.

1. in figure 5c, the scale bar is missing.

We will add missing scale bars in the figure 5C.

Reviewer #3:

1. What was less clear was an explanation of why colonies 4 and 24 were chosen. Were there other colonies with the desired expression? Was this amount of expression repeated in replicative experiments with approximately 2 colonies only available to be selected?

Approximately 30 colonies were selected for analysis. Of these, only 2 had deletion of both Resf1 alleles. We will make this point clearer in the text.

1. Figure 1C, 5C and S2B with microscopic images should include a scale bar.

Missing scalebars in the Figure 1C will be added. Unfortunately the microscopy setup used to collect the images in Figures 5C and S2B did not allow scalebars to be added at the time of imaging and these cannot be added retrospectively. However, we do not think that inclusion of scalebars, even were it possible would affect the conclusions of our manuscript.

1. Figure 1E needs a better explanation of the significance, "less clear cut" is not adequate. Reporting statistics, or lack of significance, on the graph would help.

We will update the manuscript and the Figure 1E to include results of a statistical analysis (Wilcoxon-rank sum test) comparing formation of AP+ colonies between Resf1 KO and WT cells at different LIF concentrations. These results show that both Resf1 KO cell lines have lower median number of AP+ colonies than WT cells at LIF concentrations 0 and 1 (p.adj. *

1. It's translatability to medicine, although perhaps that is not the intention, is somewhat lacking. Is there a naturally occurring situation where LIF is absent that would require this pathway to be used? These were mouse ESC's, perhaps this study could incorporate information about relevant translation to a human condition to aid in the significance. This manuscript suggests a mechanistic evaluation by which self-renewal can occur other than the canonical pathway, which is interesting and can inform the field.

Our results suggest that RESF1 directly or indirectly supports self-renewal of ESCs. Interestingly, Human cell atlas identified RESF1 expression as a negative predictor of survival of renal cancer and was found to be expressed in testis cancer cells and other cancer tissues. Therefore, RESF1 could promote self-renewal of cancer cells similarly to ESCs. However, this is speculative and needs further studies. As this is both outside of the scope of this manuscript and our expertise, we do not think it prudent for us to pursue this line of inquiry. However, we agree that further studies could evaluate RESF1 function in human tissues, especially pluripotent cells and germ cells. As we show that RESF1 deletion leads to reduced induction of PGCLCs and previous studies showed infertility of Resf1 KO mice, investigating link between human fertility and RESF1 could have implications in reproductive medicine.

We will improve our discussion to highlight the possible significance of RESF1 function in human fertility.

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Author Response:

Reviewer #1 Public Review:

Nakayama and colleagues report a unique screening concept utilizing conserved mechanisms between zebrafish gastrulation and cancer metastasis for identification of potential anti-metastatic drugs. They screen 1280 FDA-approved drugs using the gastrulation as a marker, and identify Pizotifen as an epiboly interrupting drug. Then they find that pharmacologic and genetic inhibition of HTR2C, a target of Pizotifen, suppresses metastatic progression in a zebrafish and mouse model through inhibition of epithelial to mesenchymal transition (EMT) via Wnt-signaling.

Their work is of interest and has the potential to appeal to a broad audience. However, additional experiments are needed to further substantiate their concept that human cancer metastasis mimic/recapitulate zebrafish gastrulation in terms of conserved mechanism, as well as to confirm the validity of their screening method regarding to the effects of global toxicity.

Major concerns:

The first major concern I have is the appropriateness to think the gastrulation as a parameter/index of cancer metastasis. While they cherry-picked some genes that they are known to be involved in both gastrulation and cancer metastasis, more broad analysis should probably be necessary to conclude so. For examples, the authors can analyze comprehensive RNA-seq data set to see if the pathways/networks are similar between gastrulation (zebrafish embryo development data set) and cancer metastasis (benign/primary tumors vs metastasis tumors in TCGA).

The conservation of embryonic EMT and tumor metastasis EMT has long been well recognized. Now we cited some of these published references (Nieto et al., 2016; Thiery et al., 2009; Yang and Weinberg, 2008). In Table 1, we compiled 50 genes based on published literature to provide further and strong evidence to support this conservation. Knockdown of these genes in Xenopus or zebrafish induced gastrulation defects; conversely, overexpression of these genes conferred metastatic potential on cancer cells and knockdown of these genes suppressed metastasis. Although this point is not really an objective of this study, we believe that the evidence for the conservation is sufficiently convincing to provide the basis for our study. Further RNA-seq comparison of zebrafish embryonic EMT and human tumor metastasis should be beyond the scope of the current study. Generally, the transcriptomic data for zebrafish embryo development at the epiboly/gastrulation stage are based on the whole embryos which include all other activities and are not specific to EMT; thus, it may not be a proper comparison with tumor metastasis data to search for more evidence.

The second concern is about the Pizotifen's effects on cancer metastasis. Since the Pizotifen suppresses gastrulation, it might have some harmful effect on the organogenesis/development of day2 embryos that they used in zebrafish transplantation model. And if so, cancer metastasis can be suppressed indirectly. The authors could examine if Pizotifen could have some side effects on day2 embryos. The drug also has some cell viability suppressive effects in vivo so as the pics in Fig.2D looks like, and it would be good if this possibility was excluded.

We had not observed any abnormality in development of Tg (kdrl;GFP) and WT zebrafish at day 2 when these fish were treated with 5µM Pizotifen. However, more than 20µM Pizotifen treatment affected approximately 10% of these fish. The affected fish show shorter tail rather than that of vehicle-treated zebrafish. In xenograft experiments, zebrafish embryos at day 2 were treated with 5µM Pizotifen. The concentration of Pizotifen did not affected development of day2 embryos.

Futhermore, we demonstrated Pizotifen did not affect primary tumor growth in a mice model of metastasis using 4T1 cells by two different experimental methods. One is that tumor measurement revealed that the sizes of the primary tumors in Pizotifen-treated mice were equal to those in the vehicle-treated mice at the time of resection on day 10 post inoculation. The other is that IF-staining showed the percentage of Ki67 positive cells in the resected primary tumors of Pizotifen-treated mice were the same as those of vehicle-treated mice (Figure 3A and B). Therefore, we conclude that Pizotifen suppress metastasis without affecting cell viability in vivo.

Finally, the mechanistic parts would need more confirmation and rescue experiments. Transplanted cells can be sorted after the treatment and the expression changes of EMT markers can be examined to see if the phenomenon happens in vivo as well. All main results can be rescued to see if the effect of Pizotifen against EMT happens through HTR2C-Wnt axis.

Figure 5C showed that 4T1 primary tumors from Pizotifen-treated mice has elevated E95 cadherin expression compared with tumors from vehicle-treated mice. Furthermore, Figure 5C also demonstrated that β-catenin accumulated in the nucleus, and phospho-GSKβ and Zeb1 expression were decreased in 4T1 primary tumors from Pizotifen-treated mice compared with vehicle-treated mice. Loss of E-cadherin plays an essential role in promoting EMT-mediated metastasis since loss of E-cadherin itself is enough to promote metastasis. In contrast, overexpression of mesenchymal markers: vimentin and N-cadherin is not sufficient to induce metastasis. Based on our data from Figure 5C and the accumulated evidences, we conclude that Pizotifen restored epithelial properties to metastatic cells through a decrease of transcriptional activity of β-catenin in vivo

Cultivating an Abundance Mindset.

During all proceeding steps and activites, remember that "Through effort and experience can the brain change." This will help to explain the motivation behind the need for discomfort if change is to be made.

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Reply to the reviewers

First of all, we would like to thank the each of the expert reviewers for their effort in evaluating our study. We are confident that we can positively address each of the issues and queries raised by the reviewers.

Reviewer #1 (Evidence, reproducibility and clarity (Required)): **Summary:** This study investigates the functions of the tricalbin proteins in S. cerevisiae, which are homologs extended synaptotagmins in mammals. It suggests the tricalbins modulate plasma membrane phospholipid composition and are particularly important for surviving shift to elevated temperature. The tricalbins are proposed to directly or indirectly promote phosphatidylserine transport from the ER to the plasma membrane during heat stress. They also promote the localization of the kinase Pkh1 to the plasma membrane during heat stress.

**Major comments:**

General Response: We thank the reviewer for the comments and opinions. While they are starkly different from the other two reviewers’, they have caused us to consider how we can add additional support for our conclusions and to consider alternative possibilities.

1. To determine whether the tricalbins and other tethering proteins play a role in phospholipid homeostasis, lipid distribution is measured using biosensors (FLARES) and phospholipid levels are determined by mass spec. The experiments are well done but say little about what role the tricalbins or other tethering proteins play in homeostasis. There are no measurements of lipid transport or rates of phospholipid production, degradation or modification. It is reasonable to propose the tricalbins transport lipids, since other proteins with SMP domains do, but this study does not present any evidence that they do.

Response: We thank the reviewer for stating that the quantitative microscopy and lipidomics experiments in our study are well done. However, we strongly disagree that the results do not provide new information on the roles of the tricalbins or other tethering proteins in membrane lipid homeostasis. In fact, the lipidomics results definitively show that Ist2 and Scs2/22 control phosphatidylserine (PS) levels. In contrast, loss of the tricalbins does not significantly affect PS levels. We will provide a new figure to make this even more clear to expert and non-expert readers.

While the tricalbins do not regulate PS levels, the data clearly show that the tricalbins control PS acyl chain saturation and cellular distribution. A PS reporter is reduced at the plasma membrane (PM) upon loss of the tricalbins and we have now confirmed increased localization at the endoplasmic reticulum (ER). These new results will be included in a revised manuscript. As the lipid desaturase Ole1 is localized in the ER, the lipidomics are consistent with increased ER residency of PS species. Thus, the data indicate that while the tricalbins do not regulate PS levels, they control either delivery of PS from the ER to the PM, and/or they may control the organization and stability of PS at the PM which would be a novel finding on its own.

The reviewer must be aware that there are currently no in vivo cell assays that directly (only) measure lipid transport. These experiments are subject to several factors including lipid metabolism, anterograde transport rates, bilayer organization, lipid accessibility, and retrograde transport rates. While our findings clearly show that the Tcb proteins do not control PS levels, we agree that there are alternative possibilities to explain the changes in PS distribution. In our revised manuscript, we will perform additional experiments to distinguish between these possibilities. New experiments will include mutant forms of Tcb3 bearing substitutions in the SMP domain. We will also examine whether the Tcb proteins control PS organization, availability/accessibility, and stability at the PM (also see Reviewer #3, comment 2). This latter possibility may reveal a novel concept regarding Tcb/E-Syt protein function that goes beyond their proposed conventional role as lipid transfer proteins. Based on the outcome of these experiments, we shall adjust the final cartoon model and conclusions in the discussion accordingly.

The study convincingly demonstrates that there are fewer Pkh1 puncta formed after temperature shift in cells lacking tricalbins than in wild-type cells (Fig. 6 C,D). However, there is no demonstration that this change in localization alters Pkh1 function or signaling.

Response: Regulation of Pkh1 by lipids is outside the scope of our current study that is focused on providing new understanding of Tcb protein function. However, the decrease in heat-induced Pkh1 puncta may provide insight into the PM integrity defects in cells lacking Tcb1/2/3 (as Pkh1 is required for PM integrity). To test whether Pkh1 function is compromised in the tcb1/2/3 mutant cells, we can test whether constitutively active Ypk1 (which acts downstream of Pkh1) rescues the PM integrity defects in tcb1/2/3 mutant cells.

There is no demonstration that association of tricalbins and Skh1 (Fig. 4) has any functional significance or affects phosphoinositide metabolism.

Response: We thank the reviewer for raising this issue. If the association of Tcb3 and Sfk1 has functional significance, then one would expect that loss of the proteins should phenocopy one another. Deletion of the Sfk1 cytoplasmic domain necessary for co-localization with Tcb3 should also phenocopy loss of Tcb3. This is exactly what we find. Localization of the PS probe is decreased at 42oC upon loss of Sfk1 or truncation of the Sfk1 cytoplasmic tail, similar to cells lacking Tcb3. Furthermore, we find that Tcb3 regulates sterol homeostasis at the PM (using the D4H probe), as has been recently reported for Sfk1 (Kishimoto et al, 2021). Thus, Tcb3 and Sfk1 not only co-localize, but they also share common functions in PM lipid organization. These new results will be presented in our revised manuscript.

The reviewer also inquired about potential roles for Tcb3 and Sfk1 in phosphoinositide lipid homeostasis, as Sfk1 has reportedly been implicated in heat-induced PI(4,5)P2 synthesis. However, while we find clear roles for Sfk1 and the tricalbins in PS and sterol homeostasis, we did not find a requirement for Sfk1 or the tricalbins in PI(4,5)P2 homeostasis upon heat stress conditions. These findings will be included in our revised manuscript. Importantly, our results indicate that the tricalbins and Sfk1 primarily control PS and sterol homeostasis at the PM, and may regulate phosphoinositides indirectly, and thus provide new insight into the key role of these proteins.

The study proposes the tricalbins directly or indirectly promote phosphatidylserine transport after temperature shift, but transport has not measured and other possibilities are not ruled out.

Response: While the Tcb3 SMP domain has been shown to transfer phospholipids in vitro (Qian et al., 2021), we agree that a role in PS transfer in vivo should be examined in more detail and that other possible roles in PS homeostasis should also be considered (also see responses to Reviewer #3, comment 2).

Upon heat shock, we not only observe a decrease in relative levels of the PS reporter at the PM in the tcb1/2/3 mutant cells (as shown in our original manuscript), but also a corresponding increase in relative levels of the PS probe at the ER and vacuole membrane (also see response to Reviewer #3, comment 5). This could reflect impaired delivery of PS from the ER to the PM and reduced stability of PS at the PM (i.e. increased internalization of PS into the cell).

In our original manuscript, we showed that deletion of the SMP domain (the lipid transfer domain), phenocopies deletion of the full-length Tcb3 protein in terms of PS distribution and PM integrity following heat shock. To more rigorously test whether lipid transport activity of the SMP domain is responsible for these phenotypes, we will generate amino acid substitutions within the SMP domain of Tcb3 that maintains its overall structure but impairs its ability to transport lipids (by targeting conserved key residues identified in Saheki et al., 2016). We will then assess whether SMP-mediated lipid transfer is necessary for PS homeostasis and PM integrity under heat stress.

We also agree that other possibilities should be examined. First, to rule out a defect in PS production upon heat stress, we are performing new mass spectrometry lipidomics experiments to measure levels of individual phospholipid species in the tcb1/2/3 mutant and wild type cells after heat stress.

Second, we have considered whether the Tcb proteins control phospholipid bilayer distribution (e.g. flip and flop). However, cells lacking the Tcbs are not hypersensitive to duramycin (Omnus et al. 2016) and thus phosphatidylethanolamine exposure on the extracellular leaflet is not increased. Moreover, cells lacking the Tcbs (and Scs2/22 and Ist2) are not impaired in the uptake of exogenous NBD-labelled phospholipids (and thus flip across the PM bilayer is not impaired). Possibly, there may be increased lipid ‘flop’ in the mutant cells at high temperature. We can test whether there is increased phospholipid exposure in the extracellular leaflet at high temperature, but our results thus far indicate accumulation of PS on internal membrane compartments (the ER and vacuole membrane).

Another potentially exciting possibility is that the tricalbin proteins bind and stabilize PS within the cytosolic leaflet of the PM and prevent its internalization by endocytosis or non-vesicular transfer. This mechanism would be completely independent of lipid transport to the PM and would constitute new mechanistic insight into Tcb function. We will test whether PS (and sterol) becomes more accessible (less stable or reduced sequestered pools at the PM) and internalized into the cell, upon removal of the tricalbin proteins. For example, we will monitor PS distribution in cells where endocytosis is blocked with latrunculin A.

As mentioned, there currently no cellular lipid transport assay that directly (only) measure anterograde transport. However, if the Tcb3 SMP domain mutants are impaired in PS homeostasis and PM integrity, then we can consider monitoring PS transfer in vivo. By performing the experiments outlined here, we will have thoroughly characterised the roles of the tricalbin proteins in PS homeostasis at the PM. Moreover, the new findings may even reveal novel roles that are independent of transport.

Reviewer #1 (Significance (Required)): While this study is likely to be of interest to those studying the tricalbins or phospholipid homeostasis, it is incremental and provides little conceptual advance on what is already known about the tricalbins and extended synaptotagmins. They have already been implicated in lipid homeostasis in the plasma membrane and this study provides no new mechanistic insight into how this occurs. Similarly, it has already been shown that the tricalbins play a role in maintaining cell integrity during heat stress and there is little new insight into what role the tricalbins play. Perhaps the most notable part of the study is the idea that tricalbins are necessary for phosphatidylserine transport during stress, but considerable additional work is necessary to make a strong case for this claim.

Response: We strongly disagree with the reviewer’s opinions. Indeed, Reviewer #2 found our study “novel and detailed” and Reviewer #3 found the results in our study to be “highly valuable” and “interesting”.

In contrast to the reviewer’s claims, there are certainly novel findings in our study. Foremost, this is the first study that demonstrates a role of the tricalbins in PS homeostasis. Previous studies have implicated E-Syt family members in diacylglycerol and phosphoinositide regulation. Our results indicate that the tricalbins and Sfk1 primarily control PS and sterol homeostasis at the PM, not phosphoinositides, and thus provide new insight into the key role of these proteins. Second, while a previous study by Collado et al reported a role of the tricalbins in PM integrity upon heat stress, this work did not provide mechanistic insight into this process. We performed the PM integrity assays for the Collado et al study (as co-authors). Our current study now shows that Tcb function is needed for PS homeostasis and Pkh1 recruitment at the PM upon heat stress; both factors are needed for PM integrity under these conditions. As such, our current study does provide new insight into roles of the Tcbs in PM integrity. Finally, we are exploring roles of the Tcb proteins in PS homeostasis that go beyond their proposed functions as lipid transfer proteins. We are convinced that our study will provide novel and deep mechanistic understanding of the Tcb/E-Syt protein family.

Reviewer #2 (Evidence, reproducibility and clarity (Required)): The study examines the roles of tricalbins in signalling in yeast. By using several mutations they are able to evaluate whether the interactions are caused by tethering the PM and ER or by other mechanisms. Particualrly powerful is their use of cryo-EM tomography and shot-gun mass spectrometery. They look at all the major lipids of the ER and PM and consider their interconversions. They identify large changes in lipid species with one double bond and with two, particularly for PS.

Reviewer #2 (Significance (Required)): There is little information about membrane physical properties and how it changes as a result of changes in lipid molecular species. Nevertheless, the information provided is novel and detailed. The topic of ER-PM contact sites is new and evolving and this paper advances our understanding of the yeast system considerably. It also looks at protein-protein interactions by fluorescence methods and studies the consequences of heat shock.

Response: We are pleased that the reviewer concluded that our study on the Tcb proteins “advances our understanding of the yeast system considerably” and found our use of lipidomics to be “powerful”.

This reviewer only had only one critique; there “is little information about membrane physical properties and how it changes as a result of changes in lipid molecular species”. In our revised manuscript, we will provide new data showing changes in levels of sterol (ergosterol) accessibility/availability at the PM in cells lacking the tricalbin proteins. Sterol lipids exist in distinct pools in the PM bilayer (extracellular vs. cytoplasmic leaflet, accessible vs. sequestered) that control the biophysical and mechanical properties of the PM (packing order, permeability, etc.). Moreover, PS and sterol lipids are proposed to undergo mutual associations whereby PS controls sterol accessibility (the ‘umbrella’ model) and sterol in turn stabilizes PS in the cytoplasmic leaflet of the PM. Our findings demonstrate that the primary function of the Tcb proteins is PS and sterol organization in the PM, providing new mechanistic insight into regulatory mechanisms for membrane homeostasis. We will attempt to further characterize changes in PM mechano-chemical and biophysical properties to further understand how changes in membrane lipid composition affect membrane integrity.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): The Tcb/ESyt proteins play important role in contact site formation and non-vesicular lipid transport. However, their exact functions remain highly controversial. This study by Stefan and colleagues revealed a new role for the yeast Tcb proteins, especially Tcb3, in regulating plasma membrane phosphatidylserine, as well as PIP2. These results are highly valuable to people working on membrane contact sites and lipid trafficking. Overall, the results are fairly convincing and interesting. There are however some concerns and suggestions:

Response: We are pleased that the reviewer stated that our study has “revealed a new role for the yeast Tcb proteins” and that the findings are “fairly convincing and interesting”. We also thank the reviewer for providing constructive criticisms and helpful suggestions.

1. Fig. 4, for Tcb3 and Sfk1 interaction, what about Tcb1/2, which would be good controls for the specificity of the interaction.

Response: We agree that it would be useful to determine if the Tcb3-Sfk1 interaction is specific. We will perform additional BiFC experiments to address whether Tcb1 and Tcb2 associate with Sfk1. Our previous work suggested that Tcb3 forms heterodimers with Tcb1 and Tcb2 necessary for PM integrity (Collado et al., 2019). However, the functional association between Sfk1 and Tcb3 may be specific to Tcb3, and we will test this possibility. It would be interesting to identify a function specific to an individual Tcb protein.

A central question is whether Tcb3 transfers PS by itself through the SMP domain or requires other lipid transfer proteins. One possibility is that the transfer is mediated by Osh6 and Osh7. Did the expression level of Osh6/7 change between the delta tether and the ist2scs2/22 null strain? Under normal and stressful conditions?

Response: We agree that it is important to address whether Tcb3 transfers PS via its SMP domain (an issue also raised by Reviewer #1) or whether this activity is carried out by another lipid transfer protein, such as Osh6 and Osh7. We have considered several alternative possibilities, as well as designed and performed new experiments, as described below (two key experiments are in italics).

To rigorously test whether SMP domain-mediated lipid transport activity is required for PS homeostasis at the PM under heat stress, we will generate amino acid substitutions within the Tcb3 SMP domain that maintain its overall structure but impair its ability to transport lipids (targeting conserved key residues described in Saheki et al., 2016 & Bian et al., 2018). We will then assess whether SMP-mediated lipid transfer is necessary for PS homeostasis and PM integrity under heat stress.

As suggested by the reviewer (and discussed in our original manuscript), the tricalbins might serve as scaffolds for PS transfer proteins, including the Osh6 and Osh7 proteins, under stress conditions. Osh6 and Osh7 are recruited to ER-PM contacts through interactions with the Ist2 tether protein where they mediate PS delivery to the PM under non-stress conditions (D’Ambrosio et al., 2020). However, strong lines of evidence suggest that Ist2, Osh6, and Osh7 are not required for PS homeostasis at the PM under stress conditions. First, loss of Ist2 has no impact on the PS probe under heat stress conditions (this result will be included in the revised manuscript). Therefore, the Ist2-Osh6/7 interaction is not required for PS homeostasis upon heat stress. Ist2 is not required for PM integrity upon heat stress either (Omnus et al., 2016).

These results do not rule out the possibility that the tricalbins serve as scaffolds for other PS transfer proteins, such as Osh6 and Osh7, under stress conditions. In this scenario, a switch between Osh6/7 tethering proteins may occur: from Ist2 under normal growth conditions to the Tcb proteins during stress conditions. However, our findings also suggest that Osh6 and Osh7 function is impaired upon stress conditions, including heat and nutrient starvation. Notably, Osh6 and Osh7 become mis-localized from the PM under upon heat stress (we can provide this data in the revised manuscript). The mechanisms for Osh6/7 attenuation upon stress conditions is outside the scope of our current study, but our preliminary results suggest that changes in cytoplasmic pH and ion homeostasis are involved; this will be a focus of a future study. This is also in line with results from a previous study (Omnus et al., 2020) that showed Osh3 forms intracellular aggregates in response to heat stress. The activity of the Osh proteins and other lipid transfer proteins, in general, may be impaired upon stress conditions (see below).

To directly determine whether Osh6 and Osh7 are required for PS homeostasis at the PM under heat stress conditions, we will monitor the distribution of the PS probe in cells lacking the Osh6 and Osh7 (osh6 osh7 double mutant cells) upon heat stress. This is a key experiment that will directly address the reviewer’s concerns.

As suggested by the reviewer, we can also examine the expression and localization of GFP-tagged Osh6 and/or Osh7 in tcb1/2/3, scs2/22 ist2, and ‘delta tether’ mutant cells. However, we have not observed any changes in other Osh proteins, including Osh2 and Osh3, in the ‘delta tether’ mutant strain.

Finally, we have considered yet another possibility. PS transfer to the PM may be generally attenuated under heat stress conditions and a key role of the Tcb proteins may be to bind and stabilize PS at the PM. In other words, the Tcb proteins may act as a ‘buttress’ to stabilize and maintain pre-existing pools of PS at the PM under stress conditions. Consistent with this idea, the Tcb3 C2 domains are required for PS homeostasis and PM integrity upon heat stress. If the Tcb3 SMP domain mutants are not impaired in PS homeostasis or PM integrity, then this alternative mechanism may be very relevant to PM quality control and organization in response to membrane stress. To address this possibility, we will address whether PS is internalized or removed (extracted) from the PM upon heat stress in cells lacking the Tcb proteins. This may uncover a novel role of the Tcb proteins that is independent of SMP domain-mediated lipid transfer.

Figure 5A. It is not obvious that the intensity of C2 decreases in tcb null cells at 42 degree. Perhaps there is more internal staining.

Response: We thank the reviewer for pointing out this issue. We think Figures 5A and 5B together convincingly show increased cytoplasmic localization of the PS reporter in the tcb1/2/3 mutant cells upon heat stress. More importantly, we thank the reviewer for pointing out the increased localization at internal membrane compartments. We realized it would be important to identify the PS-containing membrane compartments in the tcb1/2/3 mutant cells upon heat stress. We have now confirmed that the PS probe localizes at the ER and vacuole membrane (to be included in the revised manuscript). The example in Figure 5A also shows accumulation of the PS probe at both the nuclear ER and vacuole membrane. Thus, whilst wild type cells show little PS reporter localization at the ER or vacuole membrane, loss of the tricalbin proteins leads to an increase in ER and vacuole membrane PS probe localization after heat shock. Accumulation at the ER may reflect impaired PS delivery from the ER to the PM, and possibly rerouting to the vacuole membrane. Alternatively, as discussed above, vacuole membrane localization may be due increased PS removal from the PM and delivery to the vacuole membrane in tcb1/2/3 cells upon heat stress.

It is important to determine the primary function of Tcb3 since both defects in both PIP2 and PS were observed. If the change in PIP2 is due to a lack of PS, can overexpressing Osh6/7 rescue the PIP2 defect in the tetherless mutant?

Response: We agree that is important to determine whether the primary function of the Tcb proteins is regulation of PS or PI(4,5)P2 homeostasis. Our new findings definitively indicate that their primary function is PS regulation, not PI(4,5)P2 regulation. A clear effect in the distribution of the PS reporter was observed following heat shock in the tcb1/2/3 mutant cells. In contrast, there is no difference in the localization of the PI(4,5)P2 reporter in tcb1/2/3 cells compared to wild type after heat shock (also see response to reviewer #1, comment 3). In addition, cells lacking the Tcb proteins were not impaired in heat-induced PI(4,5)P2 synthesis, as assessed by metabolic labelling and HPLC analysis. These findings will be included in our revised manuscript, as they indicate that the Tcb proteins primarily control PS at the PM, not phosphoinositides, and thus provide new insight into the main role of these proteins.

Both PS and sterol are required for the proper recruitment of type I PIP5K to the PM (Nishimura et al., 2019). Therefore, defects in PS distribution could be responsible for the PI(4,5)P2 effects observed in Figure 2. However, overexpression of Osh7 in the ‘delta tether’ mutant did not significantly rescue localization of the PI(4,5)P2 reporter (included in the revision plans). Sterol organization is also perturbed in the ‘tether’ mutant cells (Quon et al., 2018), and this may explain why Osh7 expression did not rescue. Accordingly, Osh2/3/4 (sterol transfer proteins) rescue the PI(4,5)P2 effects.

The detection of PS by LactC2 has been well-established. However, an alternative approach would be to use the 2XPH in permeabilized cells. See PMID: 33929485 for some detailed discussions on the techniques. It is not a requirement for the authors to adopt the 2XPH.

Response: We thank the reviewer for suggesting another technique to confirm the results of the LactC2 domain as a PS probe. In this study, we have primarily used a genetically encoded LactC2 probe to observe PS distribution within live, intact cells. Whilst this approach was sufficient to identify accumulation of PS on cytosolic membrane leaflets of the ER and vacuole (see above), the addition of an exogenous probe to permeabilized cells may allow the detection of PS on luminal and extracellular membrane leaflets. Therefore, we plan to repeat our heat shock experiments using permeabilized cells and a purified tagged form of the LactC2 protein. This may allow for improved imaging of intracellular PS localisation and bilayer distribution. However, these experiments are technically challenging, and fixation and permeabilization conditions have not yet been optimized for yeast cell experiments. It is not yet known whether we will be able to optimize these protocols in a reasonable amount of time while completing revisions to the manuscript.

**Minor:**

1. the discussion seems to be a bit long

Response: We will shorten the discussion and modify our final conclusions based on the results from the new experiments.

Reviewer #3 (Significance (Required)): These proteins are highly important in cell biology/contact sites. The redundancy made it difficult to pinpoint their function. Previous studies have had a number of models. The current study proposed a new function of these proteins, i.e. PS transfer, and this is very interesting and valuable. There will be a good audience for this work. I specialize in lipid storage and trafficking, lipid droplets, cholesterol and phosphatidylserine.

Response: We are pleased that this expert reviewer found our study to be “very interesting and valuable”.

Author Response:

Reviewer #1:

The paper by -Blackwell et al. develops the ideas developed in the influential paper by Dash et al. (2017) which defined a similarity matrix for CDR3s TCRdist which is based on a weighted combination of local and global similarity measurements. In this paper they use the metric to develop the idea of a meta-clonotype, a set of similar TCRs which enrich for TCRs directed at the same antigen. They demonstrate that these meta-clonotypes show greater publicity than individual clonotypes, and show evidence of HLA-restriction. The authors speculate that the metaclonotype may be a useful biomarker. They provide open-access software tools for defining meta-clonotypes in antigen enriched repertoires.

The major findings are: (1) Meta-clonotypes are more public than clonotypes, a result which seems not unexpected, given that meta-clonotypes include many different sequences; (2) Meta-clonotypes show evidence of HLA restriction, again predicted given the well-established fact that specific antigens can be recognised by sets of similar TCRs.

The concept of a metaclonotype is an interesting one which could have widespread use in analysis of TCR repertoire. However, the impact could be much greater, by sharpening the focus of the paper, and adding detail and clairty to the idea of teh clonotype. In particular, while the introduction correctly points out that prediction of SARS-Cov_2 clinical outcome, or better understanding of the role of coronavirus prior exposure in determining outcome are important unanswered questions, this paper does not address these questions.

Thank you for your careful review of the manuscript. We have submitted a major revision with greater focus on the definition of a TCR meta-clonotype. We have removed from the introduction much of the background about SARS-CoV-2 and potential implications for the pandemic. In its place we’ve added greater detail about meta-clonotypes, how they can be defined from antigen-enriched TCR data, and how they can be used to analyze bulk TCR sequencing data.

A substantial portion of the paper is devoted to analysing data obtained using the MIRA assay (Klinger et al PLoS One 10 :e0141561) to define SARS-COV-2 responses, and it is not always clear whether the objective is to evaluate the accuracy of this data set, or to test the power of the meta-clonotype approach.

Our objective with the analyses of the IMMUNEcode dataset (Nolan et al. 2020) dataset, using MIRA method from Klinger et al. 2015, is to demonstrate that TCR meta-clonotypes can be defined from antigen-enriched TCR data and that they can be used to identify and quantify antigen-specific TCRs in bulk sequenced data. Furthermore, the analysis provides evidence that meta-clonotypes have greater publicity than individual clonotypes, thus increasing sensitivity of detection for antigen-specific TCRs. Using bulk repertoires from COVID-19 patients we then demonstrated that population-level analysis can be made possible using meta-clonotypes and provided supporting evidence that the antigen specificity of the centroid TCR is retained. These analyses and their interpretation is further revised on lines 319-348. We think that in the process of evaluating meta-clonotypes, our analysis also shows that the publicly released data contains valuable information about SARS-CoV-2 TCR specificities; however, we have not systematically attempted to verify the validity of the dataset. In the current revision these objectives are made clear in the revised Introduction section.

Reviewer #2:

Summary of main aims: The main aim of this paper is to build a framework for TCR meta-clonotypes for finding similar TCRs across individuals (or different repertoires). The majority of the investigations performed in this work have the objective of showing the data properties of meta-clonotypes as well as the metaclonotypes' usefulness for the analysis of antigen-specific TCR data and disease-labeled immune repertoire data.

Major strengths: Building meta clonotypes is a possible path towards a better coverage of immune repertoire biology as well as inter-individual repertoire comparison. TCRdist3 is an efficient method for building meta-clonotypes that enables the study of the specific characteristics of meta-clonotypes. So, far clusters of similar sequences have not been investigated in depth. The author team is making a significant step forward in this direction by characterizing meta clonotypes in differentially antigen-specific-clone-enriched repertoires and by relating the results to generation probability, HLA, sex and immune status.

Major weaknesses: Although the authors show a significant amount of data, I am not sure if these data convey sufficient intuition about the characteristics and behavior of meta-clonotypes. The authors seem too focused on relating meta-clonotypes to immune status instead of focusing on the specific biological characteristics of metaclonotypes.

We agree and have shifted the focus of the manuscript away from the results of the application and towards providing greater detail about the characteristics and behaviors of meta-clonotypes; for example, we’ve removed much of the background about SARS-CoV-2 and the COVID-19 cohorts and we’ve added details about how the meta-clonotype radius can be optimized. We’ve also reframed the data analysis section to emphasize that the results demonstrate how meta-clonotypes carry important antigen-specific signals above and beyond individual clonotypes; this makes the results valuable beyond the application to SARS-CoV-2. For example, while demonstrating HLA restriction occurs in SARS-CoV-2 specific T cell responses in COVID-19 patients is not a surprising finding, it provides evidence that meta-clonotypes enable quantification of an antigen-specific and HLA-restricted T cell response from a bulk single-chain TCR repertoire. We use this example analysis to compare the strength of this signal in individual clonotypes, meta-clonotypes with radius alone and meta-clonotypes with a motif constraint. The revision of lines 98-100 and lines 461-470 provide clarity about this motivation for the analysis and interpretation of the results.

Furthermore, we have added a section to the Results that demonstrates how meta-clonotypes and tcrdist3 enable analyses that can provide biological insights about the biochemical properties that may confer antigen specificity (lines 383-418, Figure 10). Since meta-clonotypes define groups of sequences, we can use CDR3 logo plots to dissect how positions and amino acid properties in the CDR3 define the group. In the revision we demonstrate a “background-adjusted” logo plot, that is able to emphasize amino acids that define the meta-clonotype, yet are uncommon among TCRs using the same V and J genes. Visualizing the results in this way can generate hypotheses that can be experimentally validated about the amino acids that are essential for antigen recognition.

Furthermore, the authors fail to convincingly show that the background repertoires chosen for meta clonotypes are robust and to what extent meta-clonotypes are sensitive to changes in the background repertoire.

We agree that it is important to understand how the creation of meta-clonotypes, and specifically optimization of the radius, depends on the background repertoire. Therefore, we conducted sensitivity analyses varying the size (25K to 1M) and makeup (synthetic OLGA vs. cord blood vs. a blend) of the background (lines 259-294). We also empirically demonstrate the value of over-sampling background TCRs with matching V and J genes. We show that using a background of 200,000 TCRs was sufficient for reducing the bias and variability in selecting a meta-clonotype radius, compared to a reference set of 2 M background TCRs; this is important because while it is tractable to use large backgrounds for a small number of meta-clonotypes, for larger studies or analyses confined to a laptop, the smaller background set is sufficient; we also show that this can largely be attributed to the gain in efficiency that comes with using a background that includes synthetic OLGA TCRs with VJ-gene frequencies that match the TCRs included in the meta-clonotype. However, we note that “Ultimately, the best choice for the background may depend on the question being asked and the data that is available, with factors including donor HLA, age, potential antigen exposures, and other factors that may shape the repertoire.” Our goal with tcrdist3 was to make it easy for the user to customize the background to the scientific question.

The authors also do not convincingly differentiate themselves from previous approaches that have used network analysis and generation probability in order to find clusters of similar sequences (very much conceptually similar to the approach taken here).

We agree it’s important to communicate how meta-clonotypes differ from existing TCR analysis approaches. There are several important distinctions with the existing methods that use networks and generation probability, namely TCRNET and ALICE. We have highlighted these differences in the Discussion (lines 483-497), quoting:

“The meta-clonotype approach also differs from methods, such as TCRNET (Ritvo et al., 2018) and ALICE (Pogorelyy et al., 2019), that seek to identify TCRs sharing antigen-specificity within bulk repertoires. These methods were developed to identify TCR nodes in a network with an enriched number of edges compared to the expected number of edges in a background (TCRNET) or derived from a probability model (ALICE). Similarly, another recent method attempts to find antigen-associated sequences in bulk repertoires using a two-stage agglomerative clustering of a k-mer based representation of CDRs, first within and then across bulk repertoires (Yohannes et al., 2021). Our framework is designed for a different task than these algorithms. Specifically, we sought to construct definitions of TCR groupings among already antigen-associated TCRs, which would have high sensitivity and specificity for finding similar TCRs in bulk repertoires. This is an important distinction because the existing network-enrichment methods would simply find that most or all of the TCR groupings among a set of antigen-associated sequences were statistically enriched compared with their frequency in antigen-naïve repertoires. By contrast, a flexible meta-clonotype radius permits the definition of the largest possible group of antigen-associated TCRs with the constraint that the likelihood of finding a TCR within the radius in an antigen-naïve background is equally low across all meta-clonotypes.”

Finally, the authors do not provide detailed descriptions of how the comparison of meta-clonotypes across repertoires is handled as well as potential sequence redundancies across meta-clonotypes (in potentially different individuals). I believe that all of the perceived shortcomings are readily addressable in a revision.

You are quite right that many meta-clonotypes are overlapping in that a single TCR might conform to more than one meta-clonotype definition. Thus, in the application of meta-clonotypes to the COVID-19 dataset we tested each meta-clonotype individually for an association with the predicted HLA- restricting genotype. Depending on the context, if a summary across meta-clonotypes is required (e.g. finding the overall abundance of conformant TCRs in a repertoire) it may be appropriate to use meta- clonotypes to identify conformant sequences, but then tally them based on actual abundance (i.e. no double counting). In a prediction context, it may be desirable to have overlapping meta-clonotype features, and in fact many machine learning algorithms excel in this regime. With tcrdist3 we have incorporated a “join” functionality that allows for relational database-style joining of meta-clonotypes with a TCR repertoire; this makes it relatively easy to eliminate or keep redundancies, depending on the context. We have added a sentence to the Discussion pointing out that there is overlap among meta-clonotypes that needs to be considered in their application and we provide a link to an example of how to use the join functionality on https://tcrdist3.readthedocs.io/en/latest/join.html#step-by-step-example.

All in all, this manuscript is an important steps towards a better understanding of immune receptor biology. tcrdist3 is an evolution of a previously published method (tcrdist) that is here used to build meta-clonotypes. After reading the paper, it remains slightly unclear (addressable in a revision) as to how useful they are for understanding repertoire biology as well as how to use them in practice in terms of robustness and sensitivity.

Thank you for your constructive comments, and I hope we’ve addressed these issues around biological interpretability and application in the revision.

Reviewer #3:

Mayer-Blackwell et al introduce a new framework for leveraging antigen-annotated T cell receptor (TCR) sequencing data to search for similar TCRs in bulk repertoire data, which potentially recognise the same antigen peptide. They introduce the notion of meta-clonotype, a T cell receptor (TCR) feature consisting of a main TCR sequence ("centroid") and a distance radius around it (+/- a CDR3 motif), with distance measured according to their previously published TCRdist method (Dash et al, 2017). The meta-clonotypes benefit from increased publicity over exact clonotype matching, and enhance the ability to find potentially relevant TCRs in repertoires from unrelated individuals, which are usually highly diverse, predominantly private, and subject to sampling constraints. The idea of meta-clonotypes is very interesting, and will provide a very useful tool in future repertoire analyses. For example, public databases of annotated TCRs (e.g. VDJdb) can be used to derive the set of meta-clonotypes for a variety of antigens, which can in turn be searched for in bulk repertoire data to identify e.g. memory to previous antigen exposure, immune status etc.

The tool for performing the analysis, tcrdist3, is open-source, well-documented with instructions and examples, and the statistical analysis has been well-thought out. It is also useful to have the comparison to the current alternative of k-mer based TCR distance (i.e. GLIPH2), and the added flexibility for the user to define the precise distance metric to be used in the tcrdist3 tool.

The authors then apply their method to analyse TCR beta sequences from COVID-19 datasets that have been publicly released by Adaptive Biotechnologies through the immuneRACE project. They use the MIRA set, the peptide-enriched set, to identify the meta-clonotypes, and then search for these in an independent cohort of COVID-19 bulk repertoires from 694 individuals. The authors find that a large proportion of the meta-clonotypes were more abundant in patients expressing the relevant restricting HLA allele, and suggest this could potentially lead to the development of disease biomarkers. The set of sars-cov-2 related meta-clonotypes is a useful resource in itself, as researchers generating other COVID-19 TCR datasets will be able to utilize this set of meta-clonotypes to search and potentially stratify patients in their own generated data.

There are a few areas were further detail / examples would strengthen the paper's claims, in particular in the application of the tcrdist3 method to the COVID-19 data.

1) Bulk TCR data from repertoires with past antigen exposure are likely to contain varying sizes of clones due to the proliferation of responding T cells and a remaining memory population. Due to the sharp drop in size between a TCR sequencing sample and the entire repertoire, clones above a particular size relative to the sample size are highly unlikely to have been sampled by chance, and identifying significantly/meaningfully expanded clonotypes in a sample is often used to identify a potentially antigen-recognising set of TCRs. The authors demonstrate the detection of meta-clonotypes in the repertoire sets, but it is somewhat unclear how the abundance of a clonotype conforming to a particular meta-clonotype is addressed. For example, there may be rationale for treating the following cases differently: meta-clonotype A is instantiated by (i) a unique clonotype with abundance 1; (ii) a single clonotype with abundance 1000; (iii) 100 different clonotypes (i.e. a "dense neighbourhood" around this meta-clonotype). If used to develop biomarkers, perhaps some degree of granularity in how the frequency/occurrence of meta-clonotypes is calculated would be helpful here.

Thanks for this helpful suggestion. We agree that the scenarios you outlined above, which differ in the level of clonal breadth (i.e. number of unique clones), may have great immunological relevance. Though we have not specifically assessed the clinical or immunological relevance of clonal breadth vs. clonal frequency, we have noted in the revision (lines 514-519) that there are multiple ways of counting meta-clonotype conformant sequences and multiple ways of aggregating counts across meta-clonotypes, for example without double counting clones that may be conformant to multiple meta-clonotypes. We have also added a documentation page about how to tabulate abundance or breadth of conforming clones: https://tcrdist3.readthedocs.io/en/latest/join.html

2) The authors focus their analysis on detecting meta-clonotypes from MIRA sets with strong evidence of HLA-restriction. They report 59.7% of these meta-clonotypes were more abundant in patients expressing the corresponding HLA allele. This means that over 40% of meta-clonotypes with strong HLA restriction were more abundant in repertoires with other HLA types. This point could be further elucidated by comparing results with the control repertoires from the COVID-19 set, from MIRA sets with low evidence of HLA restriction, or combining the sets of low and high evidence of HLA restriction (i.e. HLA agnostic results).

We’d like to clarify that the results do not imply that 40% of meta-clonotypes were more abundant in participants lacking the restricting HLA allele; rather, these meta-clonotypes did not have a significant association with presence or absence of the HLA genotype. In the discussion we highlight several of the possible explanations for this including that meta-clonotypes were too rarely detected in the population (lines 470-478). The volcano plots in Figure 6A and 7A show that there are very few if any HLA associations of the opposite sign (i.e. meta-clonotypes more abundant in patients without the restricting HLA allele). In fact, at the chosen significance threshold (FDR <0.01), 0 of 1831 predicted HLA-associated meta-clonotype were significantly negatively associated with the predicted HLA.

3) The MIRA55 set is used as an illustrative example throughout the manuscript, which familiarises the reader with this dataset as they are reading the paper. However, the claims made by the paper about MIRA sets / strong HLA evidence MIRA sets could be strengthened by providing an indication of how measured characteristics of the MIRA55 set compare to the other sets being assessed.

This is a good point and we have tried to provide as much information about MIRA55 and the other MIRA sets to help establish that MIRA55 is a representative set. Characteristics of the other MIRA sets appear in Supporting Table S6, including:

• Input number of clonotypes (AA exact)
• Number of non-redundant, public meta-clonotypes
• Clonotypes spanned by at least one meta-clonotype
• Span (% of clonotypes conforming to a meta-clonotype definition)
• Number of public enhanced sequences that match an identified TCRβ

As well as summary statistics for other meta-clonotype properties:

• Pgen
• Radius (TCRdist units)
• TRBV-CDR length
• Number of MIRA subjects contributing at least 1 sequence

Furthermore, Table S7 provides the strength of evidence for HLA-restriction for each meta-clonotype, which is then summarized by MIRA set visually in Figure 4.

Based on these criteria, we think MIRA 55 is a reasonably representative set to focus on.

4) There is some discussion throughout the manuscript about using the sars-cov-2 meta-clonotypes to identify differing clinical outcomes such as disease severity. Perhaps the dataset does not have sufficient power to allow for such sub-analysis, but a method of using meta-clonotypes to differentiate between patients based on the occurrence of meta-clonotypes in their repertoire is not provided [e.g. the number of observed clonotypes, the density distribution around clonotypes etc.)

That is true. With this manuscript we have tried to focus on establishing the methodology, evaluating the strength of the antigen-specific signal and demonstrating its potential applications; we have tried to make these goals more explicit throughout. Specifically in the revision we note that: “Much like any biomarker study, to establish a TCR-based predictor of a particular outcome, the features must be measured among a sufficiently large cohort of individuals, with a sufficient mix of outcomes.” At this time the publicly available ImmuneRACE data lack negative controls and sufficient clinical details to allow for building a predictor of SARS-CoV-2 infection or disease severity.

Author Response:

Reviewer #1 (Public Review):

This paper aims to address the question of whether the rotational dynamics in motor cortex may be due to sensory feedback signals rather than to recurrent connections and autonomous dynamics as is typically assumed. This is indeed a question of importance in neural control of movement.

The authors employ both analyses of motor cortical data and simulation analyses where a neural network is trained to perform a motor task. For the simulations, the authors use a neural network model of a brain performing arm control tasks. Importantly, in addition to the task goals, the brain also receives delayed sensory feedback from the muscle activity and kinematics of the simulated arm. The brain is modeled either using a stack of two recurrent neural networks (RNN) or using two non-recurrent neural network layers to investigate the importance of autonomous recurrent dynamics. The authors use this framework to simulate the brain performing two tasks: 1) posture perturbation task, where the arm is perturbed by external loads and has to return to original posture, and 2) delayed center-out reach task. In both tasks, the authors apply jPCA to units of the trained network, simulated muscle activity, and simulated kinematics and investigate their rotational dynamics. They find that when using an RNN in the brain model, both the RNN layers and kinematics show rotational dynamics but the muscle activity does not. Interestingly, these conclusions for both tasks also hold when networks without recurrent connections are used instead of the RNNs. Also importantly, the rotational dynamics also exist in the sensory feedback signals about the limb state (e.g. joint position, velocity). These results suggest that recurrent dynamics are not necessary for the emergence of rotational dynamics in population activity, rather sensory feedback can also achieve the same.

The authors perform similar jPCA analyses on monkey motor cortical (MC) or somatosensory cortical activity during the same two tasks and find largely consistent results. As with simulations, neural population activity and kinematics show rotational dynamics but muscle activity, which is explored only in the posture task, does not. Importantly, population activity in both motor and somatosensory cortices shows rotational dynamics. This observation is more consistent with the view that rotational dynamics emerge due to inter-region communications and processing of sensory feedback and planning, rather than autonomous dynamics within the motor cortex.

The approach of the paper is interesting and valuable and the questions being addressed are very important to the field. To further improve the paper and the analyses, there are several major comments that should be addressed to fully support the conclusions and clarify the results:

Major:

1) In the Methods, the authors explain how they model a non-recurrent network as follows: "We also examined networks where we removed the recurrent connections from each layer by effectively setting Whh, Woo to zero for the entire simulation and optimization (NO-REC networks)". However, if this is the only modification, it still leaves recurrent elements in the network. For example, if we set W_{hh} to zero, equation 2 will be:

h_{t+1} = (1-a) h_t + a tanh(W_{sh} * s_t + b_h)

where a is a constant scalar (seems to be equal to 0.5). This is indeed still a recurrent neural network since h_{t+1} depends on ht. If their explanation in the Methods is accurate, then the current approach restricts the recurrent dynamics to be a specific linear dynamic (i.e. "h{t+1} = (1-a) ht + …") but does not fully remove them. The second layer is also similar (equation 3) and will still have recurrent linear dynamics even if W{oo} is set to 0. To be able to describe networks as non-recurrent, the first terms in equations 2 and 3 (that is (1-a)h_t and (1-a)o_t) should also be set to 0. This is critical as an important argument in the paper is that non-recurrent networks can also produce rotational dynamics, so the networks supporting that argument must be fully non-recurrent. Perhaps the authors have already done this but just didn't explain it in the Methods, in which case they should clarify the Methods. However, if the current Method description is accurate, they should rerun their NO-REC simulations by also setting the fixed linear recurrent components (that is (1-a)h_t and (1-a)*o_t) to zero as explained above to have a truly non-recurrent model.

We thank the reviewer for raising this important concern. We have re-simulated the NO-REC network while removing the dynamics related to the leaky-integration component. This removal did not impact the network’s ability to perform the tasks and yielded virtually identical neural dynamics (see Figure 8). Throughout the Results we have updated the figures for the NO-REC network to the network without the leak-integration component.

2) Assuming my comment in 1 is addressed and the results stay similar, the authors show in simulations that even without recurrent dynamics (referred to as the NO-REC case), rotational dynamics are observed in the simulated brain during both tasks (Figure 8). This result is used to suggest that the sensory feedback is what causes the rotational dynamics in the brain model in this case. However, I think to fully demonstrate the role of feedback, additional simulations are also needed where the sensory feedback is removed from the brain model. In other words, what would happen if recurrent and non-recurrent brain models are trained to perform the tasks but are not provided with the sensory feedback (only receive task goals)? One would expect the recurrent model to still be able to perform the task and autonomously produce similar rotational dynamics (as has been shown in prior work), but the non-recurrent model to fail in doing the task well and in showing rotational dynamics. I think adding such simulations without the feedback signals would really strengthen the paper and help its message.

We apologize if the network architecture was not clear. In the case of the NO-REC network the only way they can generate the time-varying signals needed for the tasks is through sensory feedback. The network simply will not work without recurrent AND sensory feedback. For the posture task there are no additional inputs since it only receives sensory feedback. For the reaching task the task-goal input is static and the GO cue turns off on a timescale considerably shorter (~20ms) than the reach duration. Thus, the REC network would always perform better than the NO-REC network when sensory feedback was removed as the NO-REC network cannot generate any dynamics. We have now included in the Results the following statement. "Note, by removing the recurrent connections these networks can only generate time-varying outputs by exploiting the time-varying sensory inputs from the limb." (line 345-347).

We have also now included simulations to highlight how REC networks that receive sensory feedback are able to generalize better to scenarios with increased motor noise than REC networks where sensory feedback is either completely removed (reaching task) or only provided at the beginning of the trial (posture task) (Figure S8). Thus, sensory feedback makes REC networks more robust in less predictable scenarios.

We agree that this could be an interesting manipulation and have now included manipulations of the sensory feedback delays. We considered three separate delays, 0ms, 50ms and 100ms and found that there was a dependence on the rotational frequency of the top jPC plane with greater delays resulting in a general reduction in frequency (see now Supplementary Figure 10). There was less effect of delay on fit qualities to the constrained and unconstrained dynamical system. This has been added to the Results section (line 423-446).

We simulated this scenario and found the answer to be rather complex and we have added these results to the supplementary material. The network's behavioural performance in the perturbation posture task is similar to the previous networks with joint-based feedback. However, the dynamics in the output layer are not the same with a clear reduction in how well the dynamics are described as rotational (Figure S11A-B).

Oddly, rotational dynamics could still be observed in the input layer dynamics (data now shown) and the kinematic signals when they were converted to a cartesian reference frame (Figure S11D-E). Furthermore, rotational dynamics could emerge in the output layer if we used a different initialization method for the network weights. We initialized weights from a uniform distribution bound from ±1/√N, where N is the number of units. In contrast, previous studies have initialized network weights using a Gaussian distribution with standard deviation equal to g/√N where g is constant larger than 1. This alternative initialization scheme encourages strong intrinsic dynamics often needed for autonomous RNN models (Sussillo et al., 2015). We found networks initialized with this method and trained on the perturbation posture task exhibited stronger rotational dynamics with fits to the constrained and unconstrained dynamical systems of 0.5 and 0.88, respectively (Figure S11C-D). When examining the reaching task, we found similar results (Figure S11F-K). When initialized with a uniform distribution, fit quality for the constrained and unconstrained dynamical systems were 0.4 and 0.77, respectively (Figure S11F-G), which were smaller than for the joint-based feedback (Figure 7B, constrained R2=0.7, unconstrained R2=0.83). Qualitatively, the dynamics were different when the network was initialized with a Gaussian distribution (Figure S11H), however fit qualities were comparable between the two initialization methods (Figure S11 I). There was also a noticeable reduction in the fit quality for the kinematic signals particularly for the constrained dynamical system (Figure S11K, constrained R2=0.36, unconstrained R2=0.77). These findings have been added to the Results

3) A measure of how well each trained network is able to perform the task should be provided. For example, is the non-recurrent network able to perform the tasks as accurately as the recurrent models? The authors could use an appropriate measure, for example average displacement in the posture task and time-to-target in the center-out task, to objectively quantify task performance for each network. Another performance measure could be the first term of the loss in equation 5. Also, plots of example trials that show the task performance should be provided for the non-recurrent networks (for example by adding to Figure 8), similar to how they are shown for the recurrent models in Figures 2 and 6.

We have now presented and quantified the NO-REC network behavioural performance. Kinematics for the NO-REC network are shown in Figure S7A-C and E-G which are comparable to the REC network. Furthermore, quantifying the maximum displacement during the posture task yielded no obvious differences between the NO-REC and REC networks (Figure S7D). For the reaching task, the time-to-target was noticeably more variable and tended to be slower for the NO-REC network (Figure S7H). These observations have been added to the Results.

4) An important observation is that rotational dynamics also exist in the sensory signals about the limb state. This may imply that the task structure that dictates the limb state and thus the associated sensory feedback may play an important role in the rotations without the recurrent connections. While the present study will be a valuable addition regardless of what the answer is, this is an important point to address: What is the role of the task structure in producing rotational dynamics? In both the posture task and the center-out task, the task instruction instructs subjects to return to the initial movement 'state' by the end of the trial: in the posture task the simulated arm needs to return to the original posture upon disturbance, and in the center out task the arm needs to start from zero velocity and settle at the target with zero velocity. Is this structure what's causing the rotational dynamics? This is an important question both for this paper and for the field and the authors have a great simulation setup to explore it. For example, what happens if the task instructions u* instruct the arm to follow a random trajectory continuously, instead of stopping at some targets? With a simulated tracking task like this, one could eliminate obvious cases of return-to-original-state from the task. Would the network still produce rotational dynamics? Of course, I don't expect the authors to collect experimental monkey data for such new tasks, rather to just change the task instructions in their numerical simulations to explore the dependence of observed rotational dynamics on the task structure. I think this will help the message of the paper and can be very useful for the field.

We agree that a tracking task would be an interesting manipulation and have simulated this with the REC and NO-REC networks (Figure 9). Here, we trained up the network to reach from the starting position and track a target moving radially at a constant velocity for the rest of the trial (1.2seconds). Thus, the network has to move the limb at a constant velocity. We found there was a consistent reduction in how well the network’s dynamics (constrained R2=0.13, unconstrained R2=0.3) were described as rotational when compared to the previous reaching task (Figure 7, constrained R2=0.7, unconstrained R2=0.83). Also, note that this reduction in rotational dynamics remained even when we initialized the network weights using a Gaussian distribution (see Essential revision 2.3). These simulations have been added to the Results section.

5) It would be beneficial if the authors could elaborate in the discussion on intuitive explanations of why sensory feedback can produce rotational dynamics even with no internal recurrent dynamics in the brain model. To me, it seems like sensory feedback is providing a path for recurrence to exist in the overall brain-arm system, so the non-recurrent neural networks can learn to exploit that path to effectively implement some recurrent dynamics. Some intuitive explanations like this will be helpful for readers.

The main reason why rotational dynamics emerge in sensory feedback is due to the phase offset between the joint position and velocity as changes first occur in the velocity followed by position (see pendulum example Pandarinath et al., 2018a also DeWolf et al., 2016; Susilaradeya et al., 2019). This phase offset is maintained across reach directions and gives rise to the orderly rotational dynamics observed in the kinematic signals (DeWolf et al., 2016; Pandarinath et al., 2018a; Susilaradeya et al., 2019; Vyas et al., 2020). Furthermore, the tracking task disrupted this phase relationship and thus the rotational dynamics were substantively reduced in the network models. This text has been added to the Discussion (lines 519-526).

6) One main result in data from non-human primates is that there exist rotations also in the somatosensory cortex not just in motor cortex. A more thorough discussion of prior work on rotational dynamics or lack thereof across brain regions and behavioral tasks is important to add here. For example, besides the works cited by the authors, there are other works such as (Kao et al., 2015; Gao et al., 2016; Remington et al., 2018; Stavisky et al., 2019; Aoi et al., 2020; Sani et al., 2021) that discuss or show rotational dynamics in various brain regions and behavioral tasks and should be cited and discussed.

We have cited the above papers and included in the Discussion the following paragraph (lines 537-549) “Importantly, findings of rotational dynamics in cortical circuits are not trivial. Activity in the supplementary motor area does not exhibit rotational dynamics during reaching (Lara et al., 2018). The hand area of MC also does not exhibit rotational dynamics during grasping-only behaviour (Suresh et al., 2020), though it does exhibit rotational dynamics during reach-to-grasp (Abbaspourazad et al., 2021; Rouse and Schieber, 2018) which may reflect the reaching component of the behaviour. More broadly there is a growing body of work characterizing cortical neural dynamics across different behavioural tasks which have revealed rotational (Abbaspourazad et al., 2021; Aoi et al., 2020; Libby and Buschman, 2021; Remington et al., 2018; Sohn et al., 2019; Stavisky et al., 2019), helical (Russo et al., 2020), stationary (Machens et al., 2010), and ramping dynamics (Finkelstein et al., 2021; Kaufman et al., 2016; Machens et al., 2010) and these dynamics appear to support various classes of computations. Thus, finding rotational dynamics across the fronto-parietal circuit in our study is not trivial."

7) The authors state that "In contrast, rotational dynamics appear to be absent in… MC activity during grasping driven by sensory inputs (Suresh et al., 2020)." There are other papers that study dynamics during reach-grasps and still finds rotational dynamics and modes (Abbaspourazad et al., 2021; Vaidya et al., 2015) and should be cited and discussed. The recent paper on naturalistic reach-grasps (Abbaspourazad et al., 2021) also argues for the involvement of a large-scale network in these movements, which further supports the authors' interpretation that "This interpretation of motor control emphasizes that the objective of the motor system is to attain the behavioural goal and this requires feedback processed by a distributed network." A discussion of this point made in this recent paper in the intro/discussion is important. Finally, there is a recent paper that argues for the input-driven nature of motor cortex (Sauerbrei et al., 2020) and is cited/discussed by the authors but briefly and mainly in the discussion. I think given the relevance of this recent paper to the core message here, it should also be briefly discussed in the introduction to better set up the work.

We agree with the reviewer that there are discrepancies between the motor cortical dynamics reported by Suresh et al. 2020 and Abbaspourazad et al., 2021 during grasping tasks. This difference may reflect differences in task as in Suresh et al. 2020 the monkeys grasped objects whereas in Abbaspourazad et al., 2021 monkeys had to reach and grasp objects. Thus, rotations may reflect the reaching component of the behaviour. This has been elaborated on in the Discussion which now reads (lines 539-542) “The hand area of MC also does not exhibit rotational dynamics during grasping-only behaviour (Suresh et al., 2020), though it does exhibit rotational dynamics during reach-to-grasp (Abbaspourazad et al., 2021; Rouse and Schieber, 2018; Vaidya et al., 2015) which may reflect the reaching component of the behaviour.”.

We have also briefly mentioned the findings by Sauerbrei et al. 2020 in the Introduction which now reads (line 79-81) “Lastly a recent study demonstrates that motor cortical dynamics are driven by inputs coming from motor thalamus (Sauerbrei et al., 2020)."

Minor:

1) The Methods are clear and comprehensive, but just to make understanding of the simulation setup easier, it would help to have a diagram of the computation graph for the recurrent and non-recurrent networks that shows their number of units, activations/nonlinearities, RNN cell type, etc., added as supplementary figure.

We agree that this is useful and have added it to Figure 1

2) Again, to help more clearly convey the simulations, it would help to show the task goals (x*) that are inputs to the simulated brain for example trials in each task (for example added to Figures 2 and 6).

We agree that this is useful and have added it to Figure 1

3) Similar to how VAF is shown on top of all plots of jPC planes, it would be helpful to have the rotation frequency for each jPC plane noted next to it. Currently it is difficult to find the jPC frequency associated with each plot from the text.

We agree and have added it to the appropriate figures

4) I am a bit surprised by how different the null distributions are for modeling muscle activity (Figure 3F) and kinematics (Figure 3H). The null distribution is simply the R2 for a constrained or unconstrained dynamic model fit to a subsampled version of the neural activity. The only difference between the null distributions in Figure 3F and Figure 3H seems to be the downsampled dimension, which for muscle activity is 6 and for kinematics is 4 (per equation 1). Any insight will be welcome as to why down sampling the population activity to 4 (Figure 3H) results in so much worse R2 compared with down sampling it to 6 (Figure 3F)?

We thank the reviewer for raising this concern. Originally, we had applied PCA to reduce the dimensionality of the kinematic signals from 4 dimensions to 2, and the muscle signals from 6 to 4. We realize now that to be more conservative in our significance testing, we should use the full dimensionality of the kinematic and muscle signals. As such, we have changed the figures throughout to reflect this.

References:

Abbaspourazad, H., Choudhury, M., Wong, Y.T., Pesaran, B., Shanechi, M.M., 2021. Multiscale low-dimensional motor cortical state dynamics predict naturalistic reach-and-grasp behavior. Nature Communications 12, 607. https://doi.org/10.1038/s41467-020-20197-x

Aoi, M.C., Mante, V., Pillow, J.W., 2020. Prefrontal cortex exhibits multidimensional dynamic encoding during decision-making. Nature Neuroscience 1-11. https://doi.org/10.1038/s41593-020-0696-5

Gao, Y., Archer, E.W., Paninski, L., Cunningham, J.P., 2016. Linear dynamical neural population models through nonlinear embeddings, in: Lee, D.D., Sugiyama, M., Luxburg, U.V., Guyon, I., Garnett, R. (Eds.), Advances in Neural Information Processing Systems 29. Curran Associates, Inc., pp. 163-171.

Kao, J.C., Nuyujukian, P., Ryu, S.I., Churchland, M.M., Cunningham, J.P., Shenoy, K.V., 2015. Single-trial dynamics of motor cortex and their applications to brain-machine interfaces. Nature Communications 6, 7759. https://doi.org/10.1038/ncomms8759

Remington, E.D., Narain, D., Hosseini, E.A., Jazayeri, M., 2018. Flexible Sensorimotor Computations through Rapid Reconfiguration of Cortical Dynamics. Neuron 98, 1005-1019.e5. https://doi.org/10.1016/j.neuron.2018.05.020

Sani, O.G., Abbaspourazad, H., Wong, Y.T., Pesaran, B., Shanechi, M.M., 2021. Modeling behaviorally relevant neural dynamics enabled by preferential subspace identification. Nature Neuroscience 24, 140-149. https://doi.org/10.1038/s41593-020-00733-0

Stavisky, S.D., Willett, F.R., Wilson, G.H., Murphy, B.A., Rezaii, P., Avansino, D.T., Memberg, W.D., Miller, J.P., Kirsch, R.F., Hochberg, L.R., Ajiboye, A.B., Druckmann, S., Shenoy, K.V., Henderson, J.M., 2019. Neural ensemble dynamics in dorsal motor cortex during speech in people with paralysis. eLife 8, e46015. https://doi.org/10.7554/eLife.46015

Vaidya, M., Kording, K., Saleh, M., Takahashi, K., Hatsopoulos, N.G., 2015. Neural coordination during reach-to-grasp. Journal of Neurophysiology 114, 1827-1836. https://doi.org/10.1152/jn.00349.2015

Reviewer #1 (Public Review):

This paper aims to address the question of whether the rotational dynamics in motor cortex may be due to sensory feedback signals rather than to recurrent connections and autonomous dynamics as is typically assumed. This is indeed a question of importance in neural control of movement.

The authors employ both analyses of motor cortical data and simulation analyses where a neural network is trained to perform a motor task. For the simulations, the authors use a neural network model of a brain performing arm control tasks. Importantly, in addition to the task goals, the brain also receives delayed sensory feedback from the muscle activity and kinematics of the simulated arm. The brain is modeled either using a stack of two recurrent neural networks (RNN) or using two non-recurrent neural network layers to investigate the importance of autonomous recurrent dynamics. The authors use this framework to simulate the brain performing two tasks: 1) posture perturbation task, where the arm is perturbed by external loads and has to return to original posture, and 2) delayed center-out reach task. In both tasks, the authors apply jPCA to units of the trained network, simulated muscle activity, and simulated kinematics and investigate their rotational dynamics. They find that when using an RNN in the brain model, both the RNN layers and kinematics show rotational dynamics but the muscle activity does not. Interestingly, these conclusions for both tasks also hold when networks without recurrent connections are used instead of the RNNs. Also importantly, the rotational dynamics also exist in the sensory feedback signals about the limb state (e.g. joint position, velocity). These results suggest that recurrent dynamics are not necessary for the emergence of rotational dynamics in population activity, rather sensory feedback can also achieve the same.

The authors perform similar jPCA analyses on monkey motor cortical (MC) or somatosensory cortical activity during the same two tasks and find largely consistent results. As with simulations, neural population activity and kinematics show rotational dynamics but muscle activity, which is explored only in the posture task, does not. Importantly, population activity in both motor and somatosensory cortices shows rotational dynamics. This observation is more consistent with the view that rotational dynamics emerge due to inter-region communications and processing of sensory feedback and planning, rather than autonomous dynamics within the motor cortex.

The approach of the paper is interesting and valuable and the questions being addressed are very important to the field. To further improve the paper and the analyses, there are several major comments that should be addressed to fully support the conclusions and clarify the results:

Major:

1) In the Methods, the authors explain how they model a non-recurrent network as follows: "We also examined networks where we removed the recurrent connections from each layer by effectively setting Whh, Woo to zero for the entire simulation and optimization (NO-REC networks)". However, if this is the only modification, it still leaves recurrent elements in the network. For example, if we set W_{hh} to zero, equation 2 will be:

h_{t+1} = (1-a) * h_t + a * tanh(W_{sh} * s_t + b_h)

where a is a constant scalar (seems to be equal to 0.5). This is indeed still a recurrent neural network since h_{t+1} depends on h_t. If their explanation in the Methods is accurate, then the current approach restricts the recurrent dynamics to be a specific linear dynamic (i.e. "h_{t+1} = (1-a) * h_t + ...") but does not fully remove them. The second layer is also similar (equation 3) and will still have recurrent linear dynamics even if W_{oo} is set to 0. To be able to describe networks as non-recurrent, the first terms in equations 2 and 3 (that is (1-a)*h_t and (1-a)*o_t) should also be set to 0. This is critical as an important argument in the paper is that non-recurrent networks can also produce rotational dynamics, so the networks supporting that argument must be fully non-recurrent. Perhaps the authors have already done this but just didn't explain it in the Methods, in which case they should clarify the Methods. However, if the current Method description is accurate, they should rerun their NO-REC simulations by also setting the fixed linear recurrent components (that is (1-a)*h_t and (1-a)*o_t) to zero as explained above to have a truly non-recurrent model.

2) Assuming my comment in 1 is addressed and the results stay similar, the authors show in simulations that even without recurrent dynamics (referred to as the NO-REC case), rotational dynamics are observed in the simulated brain during both tasks (Figure 8). This result is used to suggest that the sensory feedback is what causes the rotational dynamics in the brain model in this case. However, I think to fully demonstrate the role of feedback, additional simulations are also needed where the sensory feedback is removed from the brain model. In other words, what would happen if recurrent and non-recurrent brain models are trained to perform the tasks but are not provided with the sensory feedback (only receive task goals)? One would expect the recurrent model to still be able to perform the task and autonomously produce similar rotational dynamics (as has been shown in prior work), but the non-recurrent model to fail in doing the task well and in showing rotational dynamics. I think adding such simulations without the feedback signals would really strengthen the paper and help its message.

3) A measure of how well each trained network is able to perform the task should be provided. For example, is the non-recurrent network able to perform the tasks as accurately as the recurrent models? The authors could use an appropriate measure, for example average displacement in the posture task and time-to-target in the center-out task, to objectively quantify task performance for each network. Another performance measure could be the first term of the loss in equation 5. Also, plots of example trials that show the task performance should be provided for the non-recurrent networks (for example by adding to Figure 8), similar to how they are shown for the recurrent models in Figures 2 and 6.

4) An important observation is that rotational dynamics also exist in the sensory signals about the limb state. This may imply that the task structure that dictates the limb state and thus the associated sensory feedback may play an important role in the rotations without the recurrent connections. While the present study will be a valuable addition regardless of what the answer is, this is an important point to address: What is the role of the task structure in producing rotational dynamics? In both the posture task and the center-out task, the task instruction instructs subjects to return to the initial movement 'state' by the end of the trial: in the posture task the simulated arm needs to return to the original posture upon disturbance, and in the center out task the arm needs to start from zero velocity and settle at the target with zero velocity. Is this structure what's causing the rotational dynamics? This is an important question both for this paper and for the field and the authors have a great simulation setup to explore it. For example, what happens if the task instructions u* instruct the arm to follow a random trajectory continuously, instead of stopping at some targets? With a simulated tracking task like this, one could eliminate obvious cases of return-to-original-state from the task. Would the network still produce rotational dynamics? Of course, I don't expect the authors to collect experimental monkey data for such new tasks, rather to just change the task instructions in their numerical simulations to explore the dependence of observed rotational dynamics on the task structure. I think this will help the message of the paper and can be very useful for the field.

5) It would be beneficial if the authors could elaborate in the discussion on intuitive explanations of why sensory feedback can produce rotational dynamics even with no internal recurrent dynamics in the brain model. To me, it seems like sensory feedback is providing a path for recurrence to exist in the overall brain-arm system, so the non-recurrent neural networks can learn to exploit that path to effectively implement some recurrent dynamics. Some intuitive explanations like this will be helpful for readers.

6) One main result in data from non-human primates is that there exist rotations also in the somatosensory cortex not just in motor cortex. A more thorough discussion of prior work on rotational dynamics or lack thereof across brain regions and behavioral tasks is important to add here. For example, besides the works cited by the authors, there are other works such as (Kao et al., 2015; Gao et al., 2016; Remington et al., 2018; Stavisky et al., 2019; Aoi et al., 2020; Sani et al., 2021) that discuss or show rotational dynamics in various brain regions and behavioral tasks and should be cited and discussed.

7) The authors state that "In contrast, rotational dynamics appear to be absent in... MC activity during grasping driven by sensory inputs (Suresh et al., 2020)." There are other papers that study dynamics during reach-grasps and still finds rotational dynamics and modes (Abbaspourazad et al., 2021; Vaidya et al., 2015) and should be cited and discussed. The recent paper on naturalistic reach-grasps (Abbaspourazad et al., 2021) also argues for the involvement of a large-scale network in these movements, which further supports the authors' interpretation that "This interpretation of motor control emphasizes that the objective of the motor system is to attain the behavioural goal and this requires feedback processed by a distributed network." A discussion of this point made in this recent paper in the intro/discussion is important. Finally, there is a recent paper that argues for the input-driven nature of motor cortex (Sauerbrei et al., 2020) and is cited/discussed by the authors but briefly and mainly in the discussion. I think given the relevance of this recent paper to the core message here, it should also be briefly discussed in the introduction to better set up the work.

Minor:

1) The Methods are clear and comprehensive, but just to make understanding of the simulation setup easier, it would help to have a diagram of the computation graph for the recurrent and non-recurrent networks that shows their number of units, activations/nonlinearities, RNN cell type, etc., added as supplementary figure.

2) Again, to help more clearly convey the simulations, it would help to show the task goals (x*) that are inputs to the simulated brain for example trials in each task (for example added to Figures 2 and 6).

3) Similar to how VAF is shown on top of all plots of jPC planes, it would be helpful to have the rotation frequency for each jPC plane noted next to it. Currently it is difficult to find the jPC frequency associated with each plot from the text.

4) I am a bit surprised by how different the null distributions are for modeling muscle activity (Figure 3F) and kinematics (Figure 3H). The null distribution is simply the R2 for a constrained or unconstrained dynamic model fit to a subsampled version of the neural activity. The only difference between the null distributions in Figure 3F and Figure 3H seems to be the downsampled dimension, which for muscle activity is 6 and for kinematics is 4 (per equation 1). Any insight will be welcome as to why down sampling the population activity to 4 (Figure 3H) results in so much worse R2 compared with down sampling it to 6 (Figure 3F)?

References:

Abbaspourazad, H., Choudhury, M., Wong, Y.T., Pesaran, B., Shanechi, M.M., 2021. Multiscale low-dimensional motor cortical state dynamics predict naturalistic reach-and-grasp behavior. Nature Communications 12, 607. https://doi.org/10.1038/s41467-020-20197-x

Aoi, M.C., Mante, V., Pillow, J.W., 2020. Prefrontal cortex exhibits multidimensional dynamic encoding during decision-making. Nature Neuroscience 1-11. https://doi.org/10.1038/s41593-020-0696-5

Gao, Y., Archer, E.W., Paninski, L., Cunningham, J.P., 2016. Linear dynamical neural population models through nonlinear embeddings, in: Lee, D.D., Sugiyama, M., Luxburg, U.V., Guyon, I., Garnett, R. (Eds.), Advances in Neural Information Processing Systems 29. Curran Associates, Inc., pp. 163-171.

Kao, J.C., Nuyujukian, P., Ryu, S.I., Churchland, M.M., Cunningham, J.P., Shenoy, K.V., 2015. Single-trial dynamics of motor cortex and their applications to brain-machine interfaces. Nature Communications 6, 7759. https://doi.org/10.1038/ncomms8759

Remington, E.D., Narain, D., Hosseini, E.A., Jazayeri, M., 2018. Flexible Sensorimotor Computations through Rapid Reconfiguration of Cortical Dynamics. Neuron 98, 1005-1019.e5. https://doi.org/10.1016/j.neuron.2018.05.020

Sani, O.G., Abbaspourazad, H., Wong, Y.T., Pesaran, B., Shanechi, M.M., 2021. Modeling behaviorally relevant neural dynamics enabled by preferential subspace identification. Nature Neuroscience 24, 140-149. https://doi.org/10.1038/s41593-020-00733-0

Stavisky, S.D., Willett, F.R., Wilson, G.H., Murphy, B.A., Rezaii, P., Avansino, D.T., Memberg, W.D., Miller, J.P., Kirsch, R.F., Hochberg, L.R., Ajiboye, A.B., Druckmann, S., Shenoy, K.V., Henderson, J.M., 2019. Neural ensemble dynamics in dorsal motor cortex during speech in people with paralysis. eLife 8, e46015. https://doi.org/10.7554/eLife.46015

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Reply to the reviewers

Manuscript number: RC-2021-00831

Corresponding author(s): Lu, Gan

Reviewer comments are in regular font. Our rebuttal is in bolded font. Experiments that we plan for the full revision are preceded with “FULL:”. In the revision files, the changes are highlighted in yellow.

General Statements

We thank the reviewers for their detailed feedback. There are two major concerns. First, the manuscript lacks functional analysis of the meiotic triple helices (MTHs). Second, the manuscript makes claims about the properties of synaptonemal complexes (SCs) and MTHs that are inadequately supported. In order to address the first concern, we would need extensive experiments to first identify and then perturb the genes associated with the MTH. Such experiments are beyond the scope of this manuscript and are the focus of future studies. We will address the second concern with mostly text revisions. We will also improve some of the imaging analysis with new experiments that can be done in a few months’ time.

2. Description of the planned revisions

We will acquire new cryo-ET data of pachytene-arrested cell cryolamellae using our new K3-GIF camera. These new data have higher signal-to-noise ratios and allow us to generate a higher-resolution subtomogram average of the MTHs. The achievable resolution will depend on the conformational homogeneity of the MTH segments and on the number of cryotomograms we can capture. If we are able to achieve a subnanometer-resolution reconstruction, we will narrow down the possible identities of the subunits. Even if we cannot achieve subnanometer resolutions, the new data will allow us to test if ladder-like densities were missed in our lower-resolution older data, thereby improving our understanding of the SC’s structure. We will also perform subtomogram analysis of purified ribosomes as a control to strengthen our handedness determination.

3. Description of the revisions that have already been incorporated in the transferred manuscript

The Reviewers’ original comments are reproduced in regular font. Line numbers refer to the preliminary revision.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Ma and coworkers report studies of budding yeast undergoing meiosis by cryo-ET. They fail to detect structures interpretable as synaptonemal complex, and instead detect feather-like bundles of what appears to be a triple helix. These structures do not appear to be related to the synaptonemal complex, as spo11 mutants that do not initiate recombination, red1 mutants that lack axial elements, and zip1 mutants that lack the central element of the SC still make these bundles. These structures are absent from cells treated with latrunculin A, which depolymerizes actin filaments, but expected structures are not visible in light microscopy of cells treated with two different F-actin-staining reagents. However, it should be noted that another study (Takagi et al, 2021, bioRxiv) did detect actin associated with these structures by immunogold labeling. The structures are also reversibly dissolved in 7% hexanediol.

This part of the paper's findings is well supported by data and is certainly of interest, although interest is somewhat limited by the unknown nature of these structures-what they contain, let alone their function, remains to be determined-in fact, it is not even determined whether or not they are made of protein. However, as an initial report of a previously unknown phenomenon, the paper is of some value.

__Thank you for raising the issue of whether MTHs are composed of protein or not. Aside from the proteins, the only other materials capable of forming large bundles of linear polymers are polysaccharides and DNA. Yeast polysaccharides are found in the cell wall, so they are unlikely to be a candidate for the MTHs. In the nucleus, DNA is abundant. While we favor that MTHs are composed of protein, we cannot rule out that the MTHs are non-chromatin DNA-protein complexes. Depending on the resolution of future subtomogram averages, we may get a better idea of the MTH’s composition.__

There is, unfortunately, a second aspect of the paper that cannot be supported. Although it is clear that synaptonemal complex is present in the cells examined (by standard cytological methods) the authors cannot detect structures consistent with SC in their cryo-ET images. Unfortunately, authors then extrapolate from their inability to detect SC to conclusions about SC, such as that it is not crystalline, and even go so far as to suggest that their failure to detect SC invalidates two models for crossover interference, and that the ladder-like structure reported for SC in many organisms using many difference approaches may be a fixation artifact. Authors show remember that the absence of evidence is not evidence for absence; the speculation described above should be removed from both the abstract and discussion.

We have removed the speculation about crossover interference and limited the scope of our discussion on SCs to budding yeast only.

The differences between traditional EM and cryo-EM are not trivial. In the introduction, we added more details to explain the differences in both the sample preparation and contrast generation:

Lines 79-87: “Meiotic nuclei have been studied for decades by traditional EM (Fawcett, 1956; Moses, 1956), but not by cryo-ET. Cryo-ET can reveal 3-D nanoscale structural details of cellular structures in a life-like state because the samples are kept unfixed, unstained, and frozen-hydrated during all stages of sample preparation and imaging (Ng and Gan, 2020). The densities seen in cryo-ET data come from electron scattering of the biological macromolecules. In comparison, the densities seen in traditional EM from electron scattering of heavy metals such as uranium, tungsten, and osmium, which have adhered to a subset of biological macromolecules that were not extracted in earlier steps.”

We have also removed the term ‘artifact’ from lines 201-202:

Original: “The ladder model is based on images of traditional EM samples, which are vulnerable to fixation and staining artifacts.”

Revised: “The ladder model is based on images of traditional EM samples.”

Reviewer #1 (Significance (Required)):

This paper reports a previously unreported structure in the nuclei of yeast cells undergoing meiosis. The composition and function of this structure remain to be determined. This considerably limits the significance of the paper.

**Referee Cross-commenting**

I agree with the concerns of the other reviewers. I also agree with reviewer 3 that to raise the significance of the paper would require much work. But I think that the raw observation is of value, albeit in a journal of record. So I would stick with my recommendation of text changes, keeping in mind that there may not be a suitable journal in LSA's portfolio.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This work describes helical filamentous structures observed in budding yeast nuclei that were cryosectioned and imaged using cryo-electron tomography (cryoET).

The goal of this work seems to have been to conduct an ultrastructural analysis of the synaptonemal complex (SC), a meiosis-specific protein structure that holds chromosomes together during meiosis and is thought to regulate meiotic recombination. In conventional TEM images of fixed, embedded, and stained sections obtained from pachytene nuclei, SCs usually appear as long, thin, transversely striated structures. At pachytene, SCs extend along the full length of a thin (100-nm) gap between paired chromosomes (typically 1-6 µm long in yeast cells). Surprisingly, the authors did not observe SCs, perhaps because these structures do not produce much contrast in cryo-EM images. Instead, they observe abundant triple helical structures in the nucleoplasm, which they designate as "meiotic triple helices" (MTHs). The authors report that these triple helices assemble at the same time as but independently of the SC. They publised a preprint in which they indicated that these structures were somehow related to SCs, but this the revised version reports that they appear independently of SCs. They further report that treatment of cells with the F-actin depolymerizing drug Latrunculin-A (LatA) resulted in a lack of detectable triple helical structures in the nucleus, suggesting that these structures may be a form of actin or, alternatively, that they may require F-actin for their assembly.

While the work is technically mostly sound, its significance is unclear because the reported structures have no known function. Many, if not most proteins will form helical structures if their concentration rises above a threshold defined by their binding affinity for themselves (see https://doi.org/10.1186/1741-7007-11-119 and references therein), so this may simply be an example of an abundant nuclear protein that polymerizes to form helical filaments under the conditions that trigger yeast sporulation.

Thank you for raising the interesting possibility that MTHs form helices because their subunits have exceeded a critical concentration threshold. In the revised text, we discuss the possibility that the MTH is simply a protein that is highly expressed in meiotic cells and polymerize either due to exceeding a critical concentration, or having undergone a biochemical change like a post-translational modification:

Lines 408-415: “Note it is possible that the MTHs may not be directly involved in meiosis, but are instead a protein that is at a sufficient concentration or has the right biochemical modifications to form helices in pachytene because it is known that many proteins can form a helix under the right conditions {Crane, 1950; Pauling, 1953; Theriot, 2013}. These MTHs also have lateral interactions that allow them to pack with crystalline density. Their sensitivity to 1,6-hexanediol suggests that the polymerization both within and between MTHs are based on hydrophobic interactions. Further work will be needed to determine the identity of the MTH’s subunits and their potential function.”

The authors perform cryosectioning and cryoEM on yeast cells undergoing meiosis to show that the assembly and disassembly of MTHs follow a similar time course as that of the SC. The observation of these triple-helical filaments in meiotic nuclei has also been reported in another study (https://www.biorxiv.org/content/10.1101/778100v2.full.pdf+html), which proposed, based on immuno-EM labeling, that they may be actin cables. This study reports that the structures are not detected using phalloidin or Lifeact-mCherry. However, treatment with LatA did eliminate detection of MTH structures, suggesting that they may be comprised of actin.

In my view, there are a number of issues that should be addressed before publication. Many of these relate to the presentation of the findings. Detailed comments below:

1. The presentation of the work is very confusing. The authors clearly expected to observe SC structures, but did not. They conclude that MTHs are not SCs, since they do not depend on molecular components required for SC assembly. They should describe their findings in a more straightforward way rather than veering from introducing the SC to describing the MTHs.

We have restructured the manuscript to tone down the discussion about the SC. However, we have to start with the SC because it is the most iconic feature of pachytene cells and a major organizer of chromatin in meiosis. Furthermore, its presence, as indicated by Zip1-GFP signals, was key to establishing that our cells were indeed arrested in pachytene. It would have been confusing to then overlook the absence of structural features as conspicuous and prevalent as what was expected of SCs. The other sections did have room for improvement. In the sections below, we describe the changes point by point.

Similarly, the discussion section on "recombination and chromosome segregation" seems inappropriate and irrelevant, since no data are presented in this study regarding the functions of the MTHs, and there is no reason to think that they contribute to crossover interference, chromosome segregation, or other aspects of meiosis. Additionally, most of the ideas presented in this section seem very muddled. I recommend deleting this section.

We have deleted the section entitled "Recombination and chromosome segregation".

Throughout the text, we have also changed the term “meiosis-specific” to “meiosis-related” when describing MTHs. Doing so allows for the possibility that MTHs might just form as a consequence of being expressed to a high enough concentration as discussed above.

Along the same lines as comment #1: The title should be changed - absence of evidence for "ladders" is not evidence of absence. Prior work using TEM and superresolution fluorescence microscopy has clearly shown that ladder-like SC structures exist in pachytene nuclei of budding yeast and many other organisms, although they apparently cannot be visualized using the methods described here.

We have changed the title to be less forceful, yet report what we see and don’t see by cryo-ET:

“Cryo-ET detects bundled triple helices but not ladders in meiotic budding yeast”

The authors should clarify whether cryo-sectioning was performed through the full volume of pachytene nuclei.

This comment refers to our attempt at serial cryosectioning, as shown in Fig. S8. We have revised the text in lines 332-334 to reflect the estimated volume covered:

“We successfully reconstructed six sequential sections from one ndt80Δ cell (Figure S8), which represents approximately one third of a nucleus (assuming a spherical shape).”

We also changed Fig. S8’s title so that it doesn’t sound like we reconstructed an entire nucleus:

“MTH bundles are extensive throughout the cell nucleus.” → “MTH bundles are extensive.”

It is not clear from the manuscript which camera/microscope configuration was used to acquire the cryoET data that were used for sub-tomogram averaging. The authors state in the methods that Falcon II and K3-GIF was used for projection images, but it's not clear if this applies to all images. These technical details should be clarified.

We have added a new column to Table S4 that reports the camera used for all the projection imaging and tomography experiments. In the original MS, all of the subtomogram averaging was done using Falcon II data.

FULL: In the full revision, we plan to incorporate new subtomogram averages of MTHs in situ, using K3-GIF data of cryolamellae.

The analysis of the handedness of the helices seems to be questionable as the resolution of the reconstructions for 80S are also quite low. I am uncertain whether this can be used to state with confidence that the MTH are right-handed.

FULL: In the full revision, we will use purified 70S ribosomes, imaged on the same K3-GIF camera and using the same software workflow as for the new subtomogram averaging of MTHs in situ. We expect higher resolution for both ribosomes and MTHs, which will make the handedness assignment unambiguous.

The authors claim that treatment with Latrunculin-A (LatA) leads to disappearance of MTHs. However, they support this with projection images of cells treated with LatA. The projection images are of poor quality and the vitrification in these cells (as well as the DMSO treated cells) do not look appropriate. They should present data for LatA-treated cells and DMSO-treated controls obtained using the same approach and ideally imaged in parallel with untreated cells. They should also quantify the number of sections and cells imaged for all conditions.

Once we realized that the MTH bundles were visible in projections, we chose to report detections of MTH bundles by projection imaging instead of the costly tilt series. The apparent poor quality and questionable vitrification comes from the fact that the projection images show the cryosection’s crevasses and knife marks, which reside on opposite cryosection surfaces. These sectioning artifacts are computationally excluded from tomographic slices. The following line was added to the figure caption to explain this:

“These image features are not devitrification artifacts; they are absent from the tomographic slices in other figures because they can be computationally excluded.”

The quantification of the MTHs in Lat-A vs control cells are in Table 2. We have now added these numbers to both the text and the figure caption.

The similarities between the MTHs and SCs - that both are present in meiotic nuclei and sensitive to hexanediol - seem unlikely to be functioanlly relevant. Again, I think the presentation suffers from being focused on the SC which was not seen, rather than on the MTHs.

We have toned down the discussion on SCs throughout the manuscript. We have retained the motivation for using 1,6-hexanediol to probe the MTHs physico-chemical properties and the fact the previous work on SCs provided motivation for this perturbation experiment. However, we removed the comparison of their relative sensitivities to 1,6-hexanediol (see reply to point #10 below).

The absence of 100-nm-wide zones containing nucleosomes is again not evidence for lack of SCs. SCs are ribbon-like structures - they are about 100 nm in one dimension but the thickness has not been characterized reliably; even if SCs do exclude nucleosomes (which is not certain) the excluded volume might be much smaller than the authors imagine.

We did not argue for “lack of SCs”; these structures clearly exist in our cells given the fluorescent linear structures seen in Zip1-GFP expressing cells. We only say that the textbook portrayal of SCs needs revision, though we should have restricted our statement to yeast. In the literature and textbooks: whenever the SC’s central element is drawn, it is depicted without internal nucleosomes and being densely packed with SC proteins.

Does Lifeact-mCherry enter the nucleus? This information is important in interpreting the failure to detect MTHs using this probe.

While Lifeact-mCherry is small enough to passively diffuse through the nuclear pore, our data cannot rule out that this molecule is excluded from the nucleus. We added the following sentence as a caveat:

Lines 244-245 “Note that we cannot rule out that Lifeact-mCherry is excluded from the nucleus.”

The sensitivity of the MTH structures to 1,6-hexanediol treatment is potentially interesting, but it does not reveal anything about their structure or function, only that their assembly likely depends in part on hydrophobic interactions. Caution should be used in interpreting these findings.

We have toned down the discussion about the meaning of the MTH bundles’ 1,6-hexanediol sensitivity by removing this line from the original Results:

“MTH bundles are therefore sensitive to a slightly higher concentration of 1,6-hexanediol than SCs are and reform when 1,6-hexanediol is removed.”

We have also added the following line to more clearly say what sensitivity to 1,6-hexanediol means:

Lines 412-414: “Their sensitivity to 1,6-hexanediol suggests that the polymerization both within and between MTHs are based on hydrophobic interactions”

The figure legends and/or Methods sections should clarify what is represented in each figure, and how the data were acquired. In particular, cryotomographic slices of varying dimensions (6nm, 10 nm or 12 nm or 70 nm) are mentioned in the captions of several figures (2-6, and S1, S2, S3). However, is often unclear whether these represent physical or computational sections.

“Tomographic slice” refers to a rendering of a computationally extracted slice from a reconstructed tomogram. To make it clearer, we have added the term “computational” to describe the tomographic slices in each figure caption.

Page 23 has a supplemental figure but no captions. Is this the same as Fig. S8?

Yes, this is a copy of Fig. S8 that appeared due to MS Word’s jumping-figure bugs. We will manually edit the PDF in the future revision.

I do not find the model figure (Figure 7) to be helpful. Additionally, the failure to detect SCs and the presence of MTHs do not warrant a "revised model of the meiotic yeast nucleus."

We now call panel A and B the “Traditional EM” and the “Cryo-EM” models, respectively. The figure therefore reports the large nuclear bodies seen by the two methods and no longer implies correctness.

We also changed the related sentence in the Introduction:

Original: “Our work strongly suggests that current models of pachytene nuclear cytology need revision.”

Revised: “Our analysis shows that MTHs coexist with SCs, which have an unknown cryo-ET structure.”

The absence of MTHs in haploid cells induced to undergo meiosis should perhaps be studied in more detail. Even SCs are present in haploid meiotic cells, so the absence of these structures may be informative as to their function. Haploid cells should also be stained for SCs and imaged by immunofluorescence to verify that they are in meiosis.

The haploid strain that was treated with sporulation media cannot enter meiosis. Haploid cells that are capable of entering meiosis need to be disomic for chromosome III, with each copy having a different mating type at the Mat locus. We believe that the construction and studies of such strains would be more meaningful after we identify the MTH’s subunits and determine its function in diploid cells.

The yeast strain is SK1, not SK-1.

Thank you. This mistake is corrected.

What are "self-pressurized-frozen samples" (p.2)?

Self-pressurized-frozen samples are generated by an alternative to the conventional machine-based high-pressure-freezing method. We have added more details in the new lines 135-141:

“Self-pressurized freezing is a simpler and lower-cost alternative to conventional high-pressure freezing, which requires a dedicated machine that consumes large amounts of cryogen. In the self-pressurized freezing method, the sample is sealed in a metal tube and rapidly cooled in liquid ethane. The material in direct contact with the metal cools first and expands by forming crystalline ice, which exerts pressure on the material in the center of the tube (Leunissen and Yi, 2009; Yakovlev and Downing, 2011; Han et al., 2012).”

Reviewer #2 (Significance (Required)):

The observation of MTHs is novel but (as stated above) of unclear significance, given that their molecular identity and function are unknown.

This work may be of interest to the meiosis field, with the caveats described above that the functional relevance is currently unclear.

This review was co-written by referees with expertise in meiosis, chromosome organization, SC structure and function, and cryoEM.

**Referee Cross-commenting**

The reviews are strikingly concordant so I don't think much needs to be added.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The authors report the observation of filamentous structures (further termed meiotic triple helices MTH) in the nucleoplasm during meiosis in yeast cells. Those structures are visualized using Cryo-ET. While the authors initially seem to assume an association of those structures with synaptonemal complexes, they discover that those structures rely on filamentous actin and are not affiliated with synaptonemal complexes after all.

While I think that the observation of those MTH by Cryo-ET is interesting, the overall structure of the paper and presentation of the data are not very well done. As the authors find throughout their experiments that the MTHs are not associated with synaptonemal complexes the strong focus in the first figures on synaptonemal complexes as well as the title of the paper are very mis-leading. The authors try to initially make the point that other labs have observed ladder like structures by transmission electron microscopy and want to make the claim that those observed structures might be an artifact of sample preparation, hence the title: Meiotic budding yeast assemble bundled triple helices but not ladders. However, at the end those structures seem unrelated to synaptonemal complexes.

In addition, several labs have reported the presence of nuclear actin in meiosis and mitosis and have even succeeded to show those structures by transmission electron microscopy, questioning the "artifact" argument.

Presumably, the Reviewer means intranuclear actin, as opposed to perinuclear (cytoplasmic) actin. If so, then we have only seen one paper, the one from the Takagi et at 2021 (bioRxiv) that has reported seeing structures associated with nuclear actin. Note however, that the revised Takagi bioRxiv paper is very careful in saying that the filament bundles contain actin, which is not the same as saying that the filaments are polymers of actin.

Our “artifact” argument – now removed – referred to SCs, not to nuclear actin.

In the revised manuscript, we use the term “intranuclear” to make it clear that we are referring to structures inside the nucleus. We also use the term F-actin, where appropriate, to refer to the best-studied actin polymer, which resembles a double helix. Doing so eliminates confusion about other forms of actin: G-actin, which cryo-ET cannot yet directly visualize due to its small size; and non-canonical actin polymers, for which there are no previous experimental X-ray or cryo-EM structures for comparisons.

The story line of the paper is weak and I think the authors would have been better of reporting their cryo-ET structures and making a better link to actin or determining what else they think might be a component of those structures. Immuno-EM (as actually shown in Reference 41) of actin would have been much more convincing. The authors could also use the power of Cryo-ET and the achievable higher resolution to describe those filaments in much more detail. In my opinion this would have been a much better and more exciting paper.

We disagree that “making a better link to actin” is the right approach because doing so presupposes that the structures are composed of actin, for which the present evidence is inadequate. We do agree that determining what the MTHs are (or are not) would be valuable.

FULL: Now that we have better access to a K3-GIF camera and a cryo-FIB-SEM, we will attempt higher-resolution subtomogram averaging analysis. If we are able to achieve subnanometer resolution, we will attempt to narrow down the fold of the MTH subunit. Note that this goal will require that the MTHs are conformationally homogenous and that we can image sufficient copies of the MTHs.

In summary: While I think the Cryo-ET images of those structures could be very exciting the paper unfortunately does not do a very good way in presenting this data and is at times misleading trying to proof or rather dis-proof a connection to synaptonemal complexes. Based on this I think that the paper can not be published in the current form and needs major revisions that would require a significant amount of time.

**Minor comments:**

Figure 1: The choice of timepoints is confusing and makes it hard to compare. While wild type is shown at 0,1,3,4,5,8h, the mutant is shown at 0,2,3,4,5,6,7h. It would be appropriate to select the same timpepoints for both conditions.

FULL: We will recollect fluorescence images of the mutant cells at the same time points as the wild type.

Figure 3 and 4 need a quantification of the number of observed MTHs, in particular as only selected regions of the nuclei are shown.

These images were taken from single cryosections instead of serial cryosections, which would have been too difficult to do for multiple conditions and multiple cells. Therefore, quantification would be obfuscated by the fact each cryosection samples a small fraction of the nuclear volume. We believe that reporting the number of cell cryosections that are MTH-positive (Table 2) is at present the best way to characterize their abundance and ability to polymerize. Once we are able to identify the MTH gene products, we will be able to perform GFP tagging experiments and thereby get a much better estimate of the polymer mass as a function of biochemical perturbations.

Fig 7. The data certainly does not support a "REVISED" model of the yeast nuclear organization.

We have changed the Figure title to “A cryo-ET model of the meiotic yeast nucleus.” We now also refer to panels A and B as “Traditional EM model” and “Cryo-ET model”, respectively.

Reviewer #3 (Significance (Required)):

Several publications have already shown the presence of actin in meiotic and mitotic nuclei and have even succeeded in observing those structures by transmission electron microscopy. Based on this it is not clear why the authors have not tried to put their work in context to all these observations and used their technology to obtain novel information on the structure, which might be helpful to identify which proteins compose the MTHs. Based on how the data is presented I do not think that this paper contributes anything new to the field.

Presumably, the reviewer means that F-actin has been imaged in yeast cells, because for actin to exist in nuclei in mitosis/meiosis, the organism would have to undergo closed mitosis/meiosis. Furthermore, for actin to be observable by transmission electron microscopy, it would have to be in the filamentous (F-actin) form. We could not find any publications that report transmission electron microscopy of F-actin in yeast cells. We therefore cannot relate our results to F-actin in the meiotic nucleus.

My field of expertise is meiosis and mitosis as well as imaging (light and electron microscopy).

**Referee Cross-commenting**

All reviewers seem to agree that the general observation of these structures is interesting but that there is a reduced significance as the function and identity of these structures remains unknown.

4. Description of analyses that authors prefer not to carry out

The main unanswered question is: what is the function of the MTH bundles? To address this question, we would first need to identify the gene products that are needed for MTH assembly. Next, we would need to do genetic perturbation experiments to actually determine the MTHs’ function. These experiments would constitute a complete study, which is better suited for a separate, future manuscript.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Saha and colleagues investigated the functions of the long non-coding RNA (lncRNA) DRAIC in malignant glioma. They find that DRAIC expression decreases cell migration/invasion and tumorsphere/colony formation in vitro, and tumor growth in vivo using established cell lines. Mechanistically, DRAIC is known to inhibit NF-kB signaling and the authors demonstrate that DRAIC activates AMPK leading to repression of mTOR, which decreases protein synthesis and increases autophagy. This is a solid study highlighting a potentially interesting pathway of tumor growth and invasion in brain tumors.

Answer: We appreciate Reviewer 1 for the positive feedback of our study

Major Comments:

1) It is unclear whether the presented values (mean +/- SD) in the histograms refer to repeat measurements (in which case n = 1) or independent experiments (n>1). The number of replicate experiments is not stated in the methods or figure legends. This must be included.

Answer: We want to thank Reviewer 1 for pointing out this omission. We have now included this information in the Materials and methods and in figure legends section.

2) I don't think the immunoblot for p62 in Fig. 5C shows a convincing increase following DRAIC knockout, so the statement on p.8 should be revised.

Answer: We have revised the statement to say: Consistent with DRAIC decrease being associated with a decrease in autophagic flux, and despite a decrease in p62 mRNA, the level of P62 protein is increased in three of the DRAIC KO prostate cancer cells (Fig. 5C, KO1, KO2, KO4 compared to WT) and unchanged in the other two.

3) On p.8/Fig.5 the authors make a case that increased DRAIC levels increase lysosomal degradation of autophagosome core proteins LC3 II / p62 (resulting in decreased protein levels of both), while simultaneously increasing gene expression of LC3B and p62 (causing increased mRNA levels). The data for DRAIC overexpression fit this logic fairly well (even though I think more work is needed to fully support this claim), but I am finding it difficult to reconcile the DRAIC knockout data with this scenario - here, loss of DRAIC results in increased protein levels to decreased autophagy, but also decreased gene expression. To fully support this argument, rescue experiments would be needed using FoxO3a knockout/overexpression.

Answer: Note that the mRNA level is not always correlated with protein expression. This is particularly true for LC3 and p62, whose protein levels are significantly affected by the extent of fusion of autophagosomes and lysosomes and subsequent degradation in autophagolysosomes. Thus, although the mRNA of these genes is decreased in DRAIC KO cells (Fig. 5E), the proteins are increased (Fig. 5C) because of decrease of autophagic flux (and decrease of degradation in the autophagolysosomes).

The overexpression of FoxO3a in the DRAIC KO cells will not restore mRNA levels of LC3 or p62, because we show in Fig. 4H that FoxO3 phosphorylation by AMPK is suppressed by DRAIC KO. This phosphorylation is important for the induction of LC3 or p62 mRNA by FoxO3.

FoxO3 knockout or knockdown in DRAIC OE cells should decrease LC3B or p62 mRNA in Fig. 5D, but it is already known from the Literature that FoxO3a is necessary for inducing LC3B or p62 mRNA. Cell Metab. 2007 Dec;6(6):458-71. doi: 10.1016/j.cmet.2007.11.001.PMID: 18054315.

4) Similarly, the data supporting increased autophagy following DRAIC overexpression (Fig. 5F/G) are a bit weak and lack controls (is the LC3B-GFP overlapping with endogenous LC3B and autophagosomes? Was the transfection efficiency comparable? Is there fusion with lysosomes?). In the absence of stronger data, the authors should temper their claims that DRAIC increases autophagy.

Answer: LC3B of fusion protein LC3B-GFP is known to overlap with the p62 puncta (similar to endogenous LC3B). This result is in Fig. 4A of the citation that we have now added (Proc Natl Acad Sci U S A. 2016 Nov 22;113(47): E7490-E7499. doi: 10.1073/pnas.1615455113. Epub 2016 Oct 17)

To support our hypothesis that DRAIC OE induces more autophagy compared to empty vector, we used Bafilomycin A1 in Figure 5B to inhibit the autophagosome and lysosome fusion. We see the accumulation of more LC3B upon treatment with Bafilomycin A1 in the DRAIC OE cells (compared to EV containing U251 cells), consistent with the idea that autophagosome-lysosome fusion is increased by DRAIC OE.

5) No information is provided on animal numbers used in this study. How many mice were used per cohort? Were male and female mice used? Authors should follow ARRIVE guidelines in reporting animal experiments. The method for calculating tumor volume needs to be specified.

Answer: We have included the details about the animal study in methods section of our modified manuscript .

6) Student's T-test is inappropriate for comparisons of more than two groups (i.e. all experiments using DRAIC knockout cells) - for these experiments a Kruskal Wallis test or ANOVA should be used. Did the authors test for normal distribution of their data? This may affect statistical testing and should be taken into consideration.

Answer: We have now modified our statistical calculation and included in the statistical analysis section in our modified manuscript.

Minor Comments:

7) Authors mention that DRAIC expression is undetectable in immortalized astrocytes and GBM cancer stem cells (Fig. S1). What is the source of these cells and how were they cultured?

Answer: The immortalized astrocytes and GBM stem cells and their culture conditions is now described.

8) The immunoblot in Fig. 3D could be replaced with a slightly lower exposure to make the difference between WT and DRAIC KO more obvious.

Answer: We have now replaced the immunoblot with lower exposure.

9) Some immunoblots in Fig. 3 (panel E, p-S6K and S6K; panel H, actin) are not of the best quality and an effort should be made to replace them.

Answer: We have now replaced the immunoblot p-S6K as reviewer mentioned.

10) Why are different loading controls used in Fig. 3 (a-Tubulin v actin)?

Answer: We use multiple loading control to make sure that we are not underestimating or overestimating changes in the experimental protein because of unexpected changes in the loading controls.

11) Compared to other blot images in the same figure (e.g. Fig. 3E), the bands for p-mTOR and mTOR in Fig. 3F look compressed and should be shown appropriately sized.

Answer: We have modified the Figure as reviewer suggested.

12) The layout of Fig. 4 is somewhat confusing. I would suggest organizing this according to DRAIC overexpression in A172 and U373 cells versus DRAIC knockout in LNCaP cells. Each immunoblot should be clearly labelled with the corresponding cell line, and it should be clearly explained why p-FoxO3a was tested in U251 cells, rather than A172/U373 as in the rest of the figure.

Answer: We thank the reviewer for the constructive criticism. We have labeled all the cell lines in the Figure as reviewer suggested. We have now systematically alternated the prostate cancer cells (for KO) and the GBM cells (for OE), as we looked at each relevant marker. We have now included the western blot for p-FoxO3a from another glioblastoma cell line U373. Please find the modified Figure 4K for p-FoxO3a.

13) Labelling of immunoblot in Fig. 5B is confusing and should be improved.

Answer: We have modified the Fig. 5B to make the label clearer.

14) Changes in GLUT1 expression (Fig. 7A) should be validated on the protein level.

Answer: We have included the immunoblot for GLUT1 from DRAIC KO cells in Figure 7B. GLUT1 protein is increased upon DRAIC KO.

Reviewer #1 (Significance (Required)):

The authors describe a novel link between the lncRNA DRAIC and AMPK activation through inhibition of NF-kB-mediated regulation of GLUT1. This study extends their previous work on DRAIC inhibition of NF-kB in prostate cancer (Saha et al. Cancer Res 2020). There is one study describing DRAIC effects on growth and invasion in glioma cell lines (Li et al. Eur Rev Med Pharmacol Sci 2020), but the work presented by Saha and colleagues contains stronger experimental data and a more detailed and previously undescribed mechanism.

The current study presents a mechanistic advance that increases the understanding of tumor growth and protein synthesis in cancer cells. The data presented in the study are not supported by in vivo experiments (other than suppression of tumor growth by DRAIC overexpression), validation in human tissue and/or primary patient-derived human glioblastoma cells, or even substantial rescue experiments. This limits the influence of the work on the field. I'm also not sure how transferable findings from DRAIC knockout in prostate cancer cell lines are to glioma, although the results are mostly complementary to the data from glioma cell lines. This is particularly relevant to the proposed mechanism of GLUT1 regulation by NF-kB, as the bulk of experimental data in Figures 6 and 7 was generated in prostate cancer cell lines and is only poorly validated in glioma cells. The study results will be most relevant for researchers investigating cell signaling pathways and autophagy in cancer.

Answer: We like to thank reviewer for the positive comments on our study. The DRAIC KO experiments of Fig. 6 and 7 cannot be done in glioma cells, because as we show if Supp. Fig. S1, there are no glioma cells or GSC that express DRAIC to levels comparable to LnCaP. We have shown that GLUT1 mRNA decreases in the glioma cells when DRAIC is overexpressed (Supp. Fig. S4. We also show in Fig. 7G that AMP levels increase when DRAIC is overexpressed in glioma cells.__

__

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In this manuscript, the authors describe DRAIC as a lncRNA downregulated in prostate cancer. They postulate that DRAIC expression surpasses invasion, migration and growth. Mechanistically, the authors show that DRAIC activates AMPK by suppressing NFkB target gene TOR and indirectly impacting translation and autophagy. Collectively the observation is interesting and robust. However, I have several technical requests, particularly regarding the mechanistic part of the paper.

Answer: We appreciate the positive feedback. We have addressed the reviewer’s concerns in our modified manuscript.

Major Comments:

1) The authors should rescue Ko phenotypes by over expressing DRAIC to consider potential off target effects.

Answer: DRAIC OE alone is sufficient to have exactly the opposite effect as DRAIC KO in protein translation (Fig. 3C-F), so DRAIC OE will rescue the effect of DRAIC KO. We make a similar argument for all the phenotypes, including mTOR, S6K and ULK1(S757) phosphorylation (Fig. 3G-J), AMPK and FoxO3a phosphorylation (Fig. 4B-C; J-L), autophagic flux (Fig. 5B, C) and effects on LC3B and p62 mRNAs (Fig. 5D, E). The same is true for our published phenotypes of DRAIC KO on invasion, migration and NF-kB activity (Saha, Cancer Research, 2020)

2) The blots showing TOR and ULK1 phosphorylation need to be repeated. This is an important part of the paper and I feel that these blots are hard to interpret. p-S6K typically run a bit higher in gels. there may be a technical problem.

Answer: We are not sure which specific blots the reviewer is referring to, and it is possible that the blots the other reviewers pointed to are the ones under question. We have changed those blots so that the results are clear.

3) GLUT1-related results are interesting, but the authors should provide genetic evidence that the effects are mediated by GLUT1. How do we know that glucose uptake is indeed upregulated upon knockout?

Answer: In Fig. 7 C-F we show that the effects of DRAIC KO on invasion, protein translation, AMP levels and AMPK activity are reversed by the GLUT1 inhibitor Bay-876. This is a cleaner result than using siRNA to knockdown GLUT1. siRNAs can have off-target activity and sometimes cannot decrease a protein sufficiently below the threshold necessary to see reversal of action.

Minor Comments:

4) The figures need to be updated. FOnts are all different, lots of unaligned graphs, quality of the blots are poor.

Answer: We have updated the Figures and changed fonts as reviewer mentioned.

Reviewer #2 (Significance (Required)):

The observation is interesting, but the mechanism is incompletely understood. This is a nice addition to the literature, even without the mechanism.

Answer: We want to thank the reviewer for the constructive criticism.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

**Summary:**

Shaha and colleagues present a study demonstrating the tumor suppressive role of DRAIC, a long non-coding RNA transcript, through transmission of the signal from IKK/NF-kB to the AMPK/mTOR pathway via regulation of GLUT1 expression. The inhibition of mTOR by this pathway results in the reduction of protein translation, cellular invasion and activation of autophagy. Several diseases and models as well as multiple genetic and pharmacological manipulations were used to investigate the mechanisms at play. The manuscript is well written and the experiments are well designed. The conclusions are supported by the results. The following major and minor comments should be addressed:

Answer: We appreciate the reviewer for the positive comments on our study.

Major Comments:

1) In addition to reporting the effect of DRAIC overexpression on tumor volume, the authors should present survival studies with one or more models.__

Answer: __We thought of doing the survival study in our glioblastoma model but unfortunately, the tumor growth is very rapid (exceeding the size permitted by our IACUC in 2-3 weeks). The animal ethics welfare committee did not allow us to keep the mice for a longer time to perform the survival study.

2) Since the authors study metabolic energy sensor pathways, related to glycolysis, it would be important to perform some of the key experiments in physiological level of glucose: e.g., pmTOR, pAMPK, LC3-II expression level in DRAIC overexpressing and deficient cells.

Answer: The concentration of glucose in plasma is 1G/L, while that of the RPMI medium is 2G/L. We do not think we are too far from the physiological levels of glucose.

3) In addition to RT-PCR data, GLUT1 protein levels should be investigated in the different DRAIC expressing cells.

Answer: We have incorporated the GLUT1 protein expression data from DRAIC KO cells in Figure 7B and DRAIC overexpressing cells in supplementary Figure 4G-H. The blots from the same gels were split into different panels, the loading control GAPDH remain same in Figure 4K and Supplementary Figure S4H.

4) The effect of DRAIC on GLUT1 expression is also measured in condition of glucose saturation, which does not reflect disease state. The decrease of GLUT1 in response to DRAIC overexpression and the increased GLUT1 level in DRAIC deficient cells should be investigated in physiological levels of glucose.

Answer Same as above. We are near physiological levels of glucose.

__Minor Comments:

__

5) All the data are generated with established cell lines (e.g., U87) but more clinically relevant models, such as patient-derived primary cells like the ones used in Fig. S1, could be used to replicate some of the key findings.

Answer: As we showed in Fig. S1 that DRAIC is not expressed in glioma stem cells, and so knockout experiments are not possible. We believe that the knockout experiments are the most relevant to this paper because they do not run the risk of artefacts from overexpression of an RNA far beyond physiological levels.

6) Also please provide further details about the patient-derived cells from Fig. S1.

Answer: We have mentioned the details of the cell lines in our modified manuscript.

7) The statistical analysis section states that the number of measurements is indicated however I don't see the sample size of the experiments.

Answer: We have now incorporated the number the experiments in our modified text.

Reviewer #3 (Significance (Required)): The study reports a new model of regulation of tumor via long non-coding RNA. This article adds to the growing literature The topic and content of the article is relevant and significant to the field of tumor research but the significance and impact could be enhanced with the use of more physiologically relevant models and conditions as pointed in the major comments.

Answer: We want to thank the reviewer for the positive feedback on our study.

This is something that I've been thinking about a lot lately, particularly as a white academic. Students have things to say, but if we require them to conform to "Standard American English" (I'm in the US, so I'm using SAE as an example, but this would presumably apply elsewhere) and scholarly/disciplinary academic writing expectations/form, then a teacher's fixation on "proper grammar" or "academic voice" or "scholarly form" likely DO stifle the ideas and knowledges that students may have to share ("How many points off for not using an Oxford comma?" "What if I use 'informal language' or 'slang'?") As one student explained just before they were about to graduate, they had spent most of their academic career "whitewashing" their ideas and language, often writing multiple drafts, first in their own voice, then progressively more "academic," so they were never actually able to express what they really had to say...until a two faculty said "just write it the way you want. Here are some models to consider, but you don't have to conform to these either."

Author Response:

Reviewer #1 (Public Review):

[...] The authors do a great job of listing and evaluating possible explanations, one of which is simply that the strains carry multiple mutations of small effect. All but one of the successfully mapped variants consists of missense and nonsense mutations. I think it's important to note that this represents a particular range of the effect-size distribution of mutations affecting the YFP phenotype. We know from the authors' earlier work that there are lots of mutations that can affect gene expression in cis, and so the absence of trans-acting cis-regulatory variants here is parsimoniously interpreted as due to their small effects. In general, work in other systems (particularly human genetics) has shown that even molecular traits are often hugely polygenic, affected by thousands of variants of tiny but non-zero magnitude. With a forward screen of the sort performed here, it's difficult to know how much of the phenotypic variance is due to unmapped small-effect variants, but two lines of evidence suggest it may be a lot: first, the absence of mappable causal mutations in 36/82 mutants, and second, the differences between EMS mutant strains and their matched single-site mutants. The authors commendably report and discuss these issues but to my mind they neglect them in drawing inferences and generalizations from their findings.

We thank the reviewer for these encouraging comments and also appreciate the reviewer pointing out these concerns.

With respect to the overlap of the trans-regulatory mutations we mapped and previously identified eQTL, we agree the possibility of similar mapping biases in the two BSA-seq studies contributing to the overlap of trans-regulatory mutations and eQTL warrants further exploration. We interpret the reviewer’s comment to suggest that if some regions of the genome systematically showed lower sequencing read coverage (because of poor read mappability, PCR biases or any other reason), the power to detect trans-regulatory mutations and eQTL in these regions would be decreased compared to regions of the genome with higher coverage because the G-tests used to identify significant associations with expression in both studies are based on read counts. Consequently, variation in sequencing read coverage across the genome shared in this study and the prior study identifying eQTL, both of which used BSA-seq, could lead to the enrichment of transregulatory mutations in eQTL regions. Indeed, consistent patterns of read coverage across the S. cerevisiae genome have been observed in prior work.

To determine whether trans-regulatory mutations were enriched in regions of the genome with higher sequence read coverage, we compared read coverage between regions of the genome identified as having trans-regulatory mutation or non-regulatory mutations. The identification of variants classified as non-regulatory is expected to be less dependent on the depth of sequencing read coverage because this designation does not require a statistically significant G-test. We found that the mutations identified as trans-regulatory showed 120x coverage whereas mutations identified as non-regulatory showed only 100x coverage, consistent with greater power to detect associations with expression in regions of the genome with higher sequencing read coverage. However, eliminating this difference in read coverage by excluding non-regulatory mutations with lower sequence read coverage did not eliminate the observed enrichment of trans-regulatory mutations in regions previously shown to contain eQTL. Non-regulatory mutations with higher and lower sequencing read coverage were also equally likely to be found within eQTL regions, suggesting that similar variation in sequence read coverage across the genome between the two studies is unlikely to explain the observed overlap of trans-regulatory mutations and eQTL. These analyses are now included in a new Figure 7-figure supplement 1.

With respect to better incorporating biases in what we were able to map and considerations for extending findings from this work to other systems, we have tried to better address these issues in the revised discussion.

Reviewer #2 (Public Review):

Fabien Duveau et al. tried to characterize mutations in trans-regulation effects on expression of the TDH3 by using EMS mutants with TDH3 reporter in Saccharomyces cerevisiae. This work is an extension of works of Gruber et al. (2012) and Metzger et al. (2016) with specific mutation effect on TDH3 expression. They found that these trans-regulatory mutations that have effects on expression of TDH3 reporter were enriched in coding sequences of transcription factors. They found that the trans regulatory mutations with effect are associated with natural variants of trans within S. cerevisiae. In summary, the data is well described and supports their claims. The method of study could be used for study the mechanism how regulatory network works.

[...] Although the paper does have strengths in principle, some weaknesses of the paper would cause the quality of data presented. [...]

We thank the reviewer for taking the time to evaluate this work and have the following responses to the weaknesses noted:

1) The reviewer is correct that we focused this paper on trans-regulatory mutations because cis-regulatory mutations affecting TDH3 expression were previously characterized. Furthermore, long distance enhancers with cis-acting effects on expression have not been described in S. cerevisiae and the term promoter is commonly used to encompass both the basal (core) promoter (including a TATA box for some genes) as well as other upstream activating sequences (UAS) and upstream repressing sequences (URS). In other words, the cis-acting sequences for S. cerevisiae genes are confined to a particular region much more than in multicellular eukarlyotes. In fact, our prior work with TDH3 (Metzger et al. 2015) showed that 97% of cis-acting variation affecting TDH3 expression could be explained by sequence variation in the 678 bp region we define as its promoter. Consequently, all mutations outside of this region were considered to have transregulatory effects on TDH3 expression. In the revised version, we extended the discussion to specifically compare the structure of regulatory sequences in S. cerevisiae to other eukaryotic model systems.

2) In this study, a mutation is defined as trans-regulatory if it affects TDH3 expression and is not located in the TDH3 promoter, regardless of whether or not it also affects growth rate. In fact, mutations in RAP1 and GCR1 affect growth rate (Figure 5), but are clearly trans-regulators of TDH3 with well-established binding sites in the TDH3 promoter. In other words, we do not think that mutations should be discounted as having trans-regulatory effects because they also impact growth rate.

3) (A) Prior work examining the statistics of BSA-seq has shown that G-tests are most appropriate because they take into account the independent sampling from two bulk populations inherent to bulk-segregant analysis (Magwene et al. 2011 PLOS Computational Biology). (B) We are guessing that the reviewer is asking about multiple testing corrections rather than post-hoc tests, as we used a false discovery rate correction for multiple tests in Figure 2-supplement 5A. Although we did not use a multiple test correction for the BSA-seq data, we used a conservative significance threshold of 0.001 that was expected to result in a 3.5% false positive rate. Perhaps more importantly, we functionally validated the effects of 40 of the 41 associated mutations tested. (C) We may indeed have been overly optimistic about mapping power when choosing mutants to analyze with BSA-seq given that the 36 EMS mutants for which we failed to find a significant association between a mutation and fluorescence tended to have smaller effects on PTDH3-YFP expression than the EMS mutants for which we observed one or more associated sites (Figure 3-figure supplement 3). The reviewer’s comment also made us realize that our original sentence referring to mapping power had reported the effect size for estimated RNA levels rather than fluorescence. To avoid confusion, and because our anticipated mapping power does not affect the results of the study, we deleted this statement from the revised manuscript. Regardless of our anticipated mapping power, we were ultimately able to map mutations that affected fluorescence by as little as 1.6% relative to the wildtype strain.

4) The GO enrichment analysis was performed with widely used tools on www.pantherdb.org. The statistical significance of enrichment for each GO term was computed using Fisher’s exact tests that compared 1) the proportion of genes with non-regulatory mutations and 2) the proportion of genes with trans-regulatory mutations that corresponded to the tested GO term. Because the total number of genes identified in our study with trans-regulatory mutations (42 genes) was much lower than the total number of genes with non-regulatory mutations (1043 genes), it was possible to obtain strong and statistically significant enrichment (P < 0.05 in Fisher’s exact test) even if only a small number of genes corresponded to the GO term in both categories. Although we found a large number of enriched GO terms, these GO terms were not always independent from each other. For instance, GO:0009168 (purine ribonucleoside monophosphate biosynthetic process) and GO:0009167 (purine ribonucleoside monophosphate metabolic process) refers to the same biological process and contains the same genes. For this reason, even though we reported all enriched GO terms in Supplementary File 8, we only showed GO terms that were at the tips of different branches in the GO hierarchy on Figure 6 and we grouped GO terms in four main categories that together encompassed most genes with trans-regulatory mutations.

5) We agree with the reviewer that trans-regulatory mutations can affect either the function of a gene product (including the ability of a transcription factor to bind to DNA) as well as the abundance of that gene product, but we do not think this is a weakness of the study. In fact, we think one of the strengths of the study is that we have empirical data testing the relative frequency of these two types of possible changes, finding that mutations in coding regions (presumably more likely to affect the function of the gene product than its expression) are the primary source of changes in TDH3 expression greater than 1%.

6) The goal of the study was to characterize the effects of individual trans-regulatory mutations, thus we did not look at the combined effects of mutations in proteins that might work in a complex. We do, however, mention transcription factors working in a complex: "Transcription factors encoded by the TYE7 and GCR2 genes found to harbor trans-regulatory mutations affecting expression of PTDH3-YFP are known to regulate the expression of glycolytic genes (including TDH3) by forming a complex with transcription factors encoded by the RAP1 and GCR1 genes” (line 461). We think that looking at the combined effects of mutations that all impact the same complex of regulatory proteins is an interesting direction for future work.

Finally, we’d like to point out that the reviewer’s statement in their opening summary about mutations being enriched in the coding sequence of transcription factors is not quite correct: the mutants we mapped were enriched in coding sequences, and we found more mutations in transcription factors previously shown to regulate (directly or indirectly) expression of TDH3 than expected by chance, but trans-regulatory mutations were not significantly enriched in genes encoding transcription factors relative to non-regulatory mutations (as described in the manuscript).

Reviewer #3 (Public Review):

[...] The mutagenesis approach in yeast the authors used is very powerful, but it naturally has drawbacks. The regulatory landscape in yeast is arguably simpler compared to e.g. metazoa or plants, in that the cis-regulatory regions are predominantly closely linked to target genes, the genes in majority do not have introns and post-transcriptional regulation of mRNA through e.g. splicing is rare. These features distinguish the systems, as in animals and plants introns are a very prominent source of regulatory elements (close to half of all enhancers are intronic in many animals), and alternative splicing of e.g. transcription factors are known to play major roles in transcriptional regulation. Further, chromatin is a very important layer in metazoan and plant gene regulation. To benefit the general readership, it would be informative to further elaborate on the significance of the findings for researchers studying other organisms. In addition, it would help to clarify what aspects of the differences in the regulatory landscape the authors think are important to distinguish.

We thank the reviewer for their kind words and recognition of the novelty of this work. We have modified the introduction to try to clarify the relationship of this work to eQTL studies, which we hope addresses the reviewer’s first concern. Specifically, we’ve tried to clarify that the complex, polygenic nature of trans-regulatory variation segregating within a species is well established by prior eQTL studies. We also sought to clarify that our work (which maps single mutations from mutagenized strains rather than natural variation) provides complementary insight into the distribution of regulatory mutations within the genome and within a gene’s regulatory network. Revisions have also been made to try to clarify that the single mutations we mapped were from EMS-induced mutants containing only ~24 mutations per genome, which is more than 1000-fold less than the number of single nucleotide polymorphisms between two strains of S. cerevisiae. That is, this study was designed to identify single trans-regulatory mutations rather than to characterize the genetic architecture of naturally occurring trans-regulatory variation. Although we intentionally focused on characterizing properties of single mutations here, we agree with the reviewer that testing for epistatic interactions among trans-regulatory mutations will be an interesting avenue for future work, and have added this point to the revised discussion. We have also added text to the discussion describing some similarities and differences in gene regulation as among eukaryotes that should be considered when trying to generalize from this work.

Reviewer #1 (Public Review):

This paper aims to characterize mutations that act in trans to affect a single gene's expression in yeast. Trans-acting mutations potentially play an important role in variation and disease within species and in phenotypic evolution. The authors have previously described the mutational architecture and natural variation in the gene's cis-regulatory activity, creating a powerful experimental model for the causes of phenotypic variation. Trans-acting variation is much more challenging, because the mutational target space is the whole genome. The authors use a forward-genetic screen and bulked segregant analysis to identify 52 point mutations that affect their focal transgene's activity, and they identify an additional 17 by directly searching within a handful of candidate genes. The paper includes elegant validation using genome engineering to confirm the mapped variants are causal.

With this collection of trans-acting mutations in hand, the authors can compare their characteristics to a set of mutations that do not have detectable effects on the transgene. Overall, they conclude that trans-acting mutations are enriched for genes that are known to sit upstream of the focal gene in transcriptional cascades, but the majority of mutations are in other kinds of genes, outside the network of transcription factors.

This work is a valuable contribution to the authors' important experimental assault on the genetics of regulatory variation, a useful complement to their previous work on cis-regulatory mutations and polymorphisms. They provide evidence that experimentally defined regulatory networks have predictive value for the location of trans-acting mutations, and they reinforce the result (well established and widely accepted, but important to show in this kind of rigorous way) that trans-acting variation is distributed across a wide range of cellular and molecular functions. There are also some useful fine-grained results, such as the absence of mutations in a known regulator, RAP1, probably due to pleiotropic constraints, and an excess of mutations in iron homeostasis.

Because the dataset of trans-acting mutations is relatively modest in size (necessarily- it's a heroic effort to identify this many), many of the enrichments are also modest. In particular, the finding that mutations are enriched in eQTL regions holds for only two of three previous eQTL studies, and involves a slight elevation over the baseline that 66% of the genome is in eQTL regions. Because both the eQTL and the mutations were discovered by bulked-segregant analysis, biases in mappability will affect both similarly, and so I do not find the enrichment for overlapping hits to be completely persuasive.

This work is important in substantial measure because of its contribution to the larger yeast TDH3 model trait project, which is a landmark research program for understanding phenotypic variation and evolution. On its own, the results in this manuscript would be difficult to generalize to regulatory variation more broadly. There are narrow reasons for this (yeast has a distinctive compact CDS-dense genome; the focal transcript is YFP and so has no endogenous post-transcriptional regulation; only one class of mutations assayed), but the bigger reason is that the researchers are only able to discover mutations with effects above a particular size. Even among the 82 mutant strains they start with, some 36 strains have altered YFP levels but no successfully mapped causal variants. The authors do a great job of listing and evaluating possible explanations, one of which is simply that the strains carry multiple mutations of small effect. All but one of the successfully mapped variants consists of missense and nonsense mutations. I think it's important to note that this represents a particular range of the effect-size distribution of mutations affecting the YFP phenotype. We know from the authors' earlier work that there are lots of mutations that can affect gene expression in cis, and so the absence of trans-acting cis-regulatory variants here is parsimoniously interpreted as due to their small effects. In general, work in other systems (particularly human genetics) has shown that even molecular traits are often hugely polygenic, affected by thousands of variants of tiny but non-zero magnitude. With a forward screen of the sort performed here, it's difficult to know how much of the phenotypic variance is due to unmapped small-effect variants, but two lines of evidence suggest it may be a lot: first, the absence of mappable causal mutations in 36/82 mutants, and second, the differences between EMS mutant strains and their matched single-site mutants. The authors commendably report and discuss these issues but to my mind they neglect them in drawing inferences and generalizations from their findings.

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Referee #3

Evidence, reproducibility and clarity

Summary

Most laboratory research using T. b. brucei has made use of two strains, the monomorphic Lister 427 strain and the pleomorphic EATRO1125 strain. These strains have been passaged in vitro for decades in separate laboratories, thus selecting for populations that differ from both the original isolate and between laboratories.

In this study, Mulindwa et al. describe the isolation of two new T. b. brucei strains from cattle in Uganda, MAK65 and MAK98, which show differences in virulence and propensity for stumpy form differentiation in rodents. To assess the effect of culture adaptation on trypanosomes, the researchers compared the gene copy number of the MAK65 and MAK98 isolates before and after culture adaptation. The study found that isolates cultured for as little as a week already demonstrated a broader gene copy number distribution than those that were not culture adapted. Broader gene copy number distributions, compared to the non-culture-adapted isolates, were also observed for a number of routinely-passaged Lister 427 and EATRO1125 cultures. The researchers observed reproducible increases in copy number for certain genes, such as those encoding histones, HSP70 and PFR proteins. The study postulated that changes in gene copy number observed across the genome increased bias towards rapid proliferation and stress tolerance upon culture adaptation.

Major comments

I have some concerns about the method that was used for the copy-number calculations. Firstly, I can imagine that a non-uniform distribution of the reads across the genome for any of the datasets could influence the results. Was this checked? Secondly, in the methods section lines 394/395 it is said that the modal RPKM for each dataset was 'adjusted slightly' to get a symmetrical distribution. Upon checking the modal RPKMs and the adjusted values used for the calculations, the adjusted values appear to have been adjusted to different extents between the different datasets to fit the authors assumptions. Do the authors believe that this could perhaps account for some of the subtle gene copy number changes observed (as is discussed in lines 287-290)? Next, why were the reads aligned 20 times? I think in general the method needs to be explained far more clearly so that the audience can understand what you did.

Minor comments

Additional experiments:

Would it be possible to generate a phylogenetic tree comparing these new isolates with the Lister 427, TREU927 and EATRO1125 isolates in circulation to get an idea of how these strains may have evolved? I this could add to the strength of the manuscript.

Title: Since no other unicellular eukaryotes are mentioned throughout the text, I think a more appropriate title for this study might be 'Adaptation of Trypanosoma brucei brucei bloodstream forms to in vitro culture results in gene copy-number changes.'

Line 59: There have been more recent studies than the referenced paper (Cross et al., 2014) that quote far higher numbers of alternative VSG genes or pseudogenes in the Lister 427 strain. In Müller et al (2018, doi: 10.1038/s41586-018-0619-8) over 2500 VSGs are quoted and in Cosentino et al (2021, doi: 10.1101/2021.04.13.439624) 2872 VSGs are identified.

Line 109/110: The dates of isolation written here, Feb 1st and July 30th 2016, are different to what is written under the Date of Isolation column in Supplementary text 1, table 1- 25/5/2016 and 30/7/2017. Do these two dates represent different things?

Line 233: Should Figure 5 A, B, C and E (rather than A, B, D and E) be referenced here to show all the initial, not cultured results?

Line 270/271: PIP39 does not promote differentiation to stumpy forms, but instead contributes towards the efficient differentiation of stumpies to procyclic forms (Szoor et al, 2010, doi: 10.1101/gad.570310).

Discussion:

It should be discussed in the text that changes in gene copy number have also been observed upon Leishmania culture adaptation (see, for e.g., Gerald Späth's work). This will strengthen the authors' conclusions. Furthermore, similar observations of aneuploidy and triploidy in some T. brucei Lister 427 strains have recently been reported in Cosentino et al (2021, doi: 10.1101/2021.04.13.439624). This could also be referenced somewhere in the text.

Line 364: I think the Nijuru et al (2005, doi: 10.1007/s00436-004-1267-5) paper would be the correct reference here since it describes the use of the ITS-1 PCR. Reference 13 is actually referring to another paper, I cannot find Nijuru et al in the references list.

Line 379: PAD staining should be referenced to be more informative and allow reproducibility.

Figure 1, Line 580: How many cells were counted for determining the % PAD positivity?

Figure 1, Line 581: Scale bar should be included in D.

Supplement Text 1:

I found Supplement Text 1 a little confusing for two reasons. Firstly, I was never sure if the tables or references being referenced were from the main text or from the supplement text 1, perhaps this could be made a little clearer to aid the reader. Secondly, the names of the isolates switched between, for e.g., MAK 65 and Tb065. For simplicity it could help to try and stick to one naming system.

It might be worth adding a sentence about why Tb236B was not followed up. Was this because it could not easily be distinguished from MAK65 by microsatellite analysis?

S3 Figure: Legend describes red bars but there are none in the figure.

Table 2: In the legend for Sheet 2, the cut-off for the increase is missing.

Significance

Though the T. b. brucei Lister 427 and EATRO1125 strains are used most commonly for laboratory-based research, they have been extensively passaged in vitro without characterisation of the changes that have occurred between them and the original isolate, or indeed, between laboratories.

Mulindwa et al. demonstrated that changes to gene copy number occur rapidly upon adaptation to culture of field isolates, and that different cultures of the same isolate can furthermore have different ploidies. This is an important advance and raises awareness a) that trypanosomes undergo changes upon laboratory adaptation, b) of the nature of some of these changes and c) that the changes can occur rapidly. Changes to gene copy number have also been shown to effect Leishmania donovani upon culture adaptation (Prieto Barja et al, 2017, doi: 10.1038/s41559-017-0361-x).

This study will be of interest to the trypanosome community in general, but particularly those who work on biology that we already know is impacted by prolonged in vitro passage-differentiation, virulence and antigenic variation.

Reviewer expertise: BSF differentiation, antigenic variation

Author Response:

Reviewer #1 (Public Review):

[...] Authors' rigorous experimental design (based on bacterial genetics and structural biology), solid biochemical assays (including photo-crosslinking, cysteine crosslinking, and Western blotting), and carefully drawn interpretation and conclusions are impressive. Finally, authors delineate the mechanisms of BepA activation and LptD biogenesis, which are supported by the current and previous studies by the authors and other research groups.

Thank you for the nice summarization and the positive evaluation of our study.

While this is overall a wonderful piece of work, this manuscript would be further improved by clarifying the following points:

1. Authors examined how mutations (Pro and Cys scanning) on the edge-strand of BepA affected degradation and maturation of LptD.

It was assumed that these mutations impact the structure of BepA only locally. However, a mutational effect can be propagated in an unexpected way affecting the structural integrity of other regions. Although authors tested that A106P retains proteolytic activity as shown by self-cleavage, a similar test (for example, in vitro experiments using a structureless substrate) may need to be extended to other mutations to support the conclusions.

Thank you for the suggestion. Unfortunately, we have not succeeded in reproducibly detecting the proteolytic activity of BepA with purified BepA even when an unstructured substrate (-casein) is used and the assay was conducted at an elevated temperature, possibly because the protases activity of isolated BepA is tightly repressed by the mechanism that included His-246-mediated regulation as described in our paper (please see Introduction). Although BepA mutants with a mutation of His-246 or a deletion of H9 loop (these mutations release the His-246-mediated repression) significantly degrade -casein, a combination of these mutations with the edge-strand mutations should make the interpretation of the results complicated. We thus think that the suggested experiments cannot be conducted soon.

Instead, we described the following points in the revised manuscript. Although we mentioned the self-cleavage activity of only the A106P mutant in the original manuscript, our results showed that the other edge-strand Pro mutants (other than F107P) exhibited significant self-cleavage activities as well (Figure 1-figure supplement 2B). In addition, the Pro mutants other than the A106P mutant degraded mis- or un-folded BamA at a detectable level (Figure 1-figure supplement 2A). Furthermore, all the Pro mutants accumulated at a level comparable to that of wild-type BepA. These observations together indicate that most of the Pro mutations specifically affected the edge-strand structure, but not drastically altered the active site or the protein's overall structures. We described the above points in the revised text (line 174 in p7 to line 181 in p8).

1. In the result (Line 159), authors report chaperone-like activity of BepA. Here, the term "chaperone-like" is rather obscure regarding whether this activity facilitates LptD maturation without proteolysis (i.e., via holdase activity), or involves proteolysis as a part of quality control mechanisms. In another experiment, authors show that the chaperone-like activity may not necessarily involve proteolysis. It would be good to describe a possible molecular principle of how the edge-stand binding to the substrate can lead to chaperone activity.

We suppose that the interaction of BepA (via the edge-strand) with an assembly intermediate of LptD on the BAM complex stabilizes the partially unfolded assembly intermediate of LptD on the BAM complex to help the association of LptE with LptD. This was explained in Discussion (lines 388–392 in p16) and the legend to Figure 5.

Reviewer #2 (Public Review):

The authors found that a conserved β-strand (edge-strand β2 of BepA) directly contacts with the N-terminal half of the β-barrel-forming domain of an immature LptD; the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacts with a "seam" strand of BamA in the BAM complex. By combining crosslinking and mutational studies, they showed that the edge-strand of BepA may have both the proteolytic and the chaperone-like functions. Based on the authors' previous studies of BepA, they proposed a model that the edge-strand and His switch of BepA regulate BepA in LptD assembly and degradation.

Thank you for the nice summarization of our study.

Reviewer #3 (Public Review):

[...] By performing an impressive systematic cross linking analysis, combined with previous known findings, the authors are able to dissect the general architecture of how BepA interacts with beta-barrel substrates as they are being assembled by the Bam complex. The experiments presented are nicely executed and the data are of high quality. I am convinced that the edge strand of BepA interacts with LptD, likely as it is assembling on the Bam complex. It is also clear that this interaction is functionally important because mutations in this region that disrupt the BepA-LptD interaction interfere with LptD maturation and degradation. This suggests that the substrate binding to the protease domain of BepA is important for both its chaperone and proteolytic activity. The work is well executed and will be of value to others interested in the regulation of membrane protein folding and, more broadly, in the biogenesis of the bacterial cell envelope.

Thank you for the nice summarization and the positive evaluation of our study.

While the authors conclusively establish a link between this region of BepA and its function, the data do not explain the underlying mechanism of how BepA discriminates between substrates targeted for integration into the membrane and those targeted for destruction. The model proposed at the end incorporates the presence of the edge strand of BepA, but its role in the process remains vague. As mentioned in the discussion, the mechanisms that control the switch from chaperone to protease function in BepA is likely governed by the loops that gate access to the catalytic residues proximal to the edge strand. It is possible that the edge strand may just be reporting on substrate binding to the protease domain active site. While this may be important for substrate recognition, it does not mean that the edge strand-substrate interaction plays a deterministic role in subsequent protein triage during LptD assembly.

Our data demonstrated that the edge-strand of BepA directly binds a substrate. As pointed out by the reviewer, the involvement of the edge-strand in substrate binding has been known for other proteases. However, it was not known whether the substrate binding at the edge-strand contributes to the chaperone-like function; it was possible that the binding sites of a substrate on BepA during its proteolysis and its maturation are totally different as the chaperone-like activity of BepA is independent of its protease activity (it was conceivable, for example, that substrate binding during it maturation occurs on the surface of the C-terminal TPR domain that has been shown to interact with LptD）. Our results showed that the defective binding of a substrate (LptD) at the edge-strand impairs not only its proteolysis but also its normal maturation (assembly). Because the edge-strand-bound substrate would be directly presented to the proteolytic active site for its degradation, this binding step should be important for the determination of the fates of the bound substrate. Our results strongly suggest that the substrate binding by the edge-strand is a crucial common step required for the subsequent protein triage during the LptD assembly.

I think this is a super key consideration, and I think we all have to acknowledge the possibility that our findings for this project are only a part of the story, and may not be representative of the entire campus community. I'm really looking forward to seeing the final project and learning about your interpretation of the results.

Reviewer #3 (Public Review): 

Pancreatic beta cells in each islet of Langerhans act cooperatively to produce a coordinated response to blood glucose, which takes the form of roughly synchronous electrical oscillations that result in coherent pulses of insulin secretion. However, it has become more appreciated recently that the cells are heterogeneous and that there may subgroups of cells within the islet that contribute in different ways or control different aspects of the collective behavior. 

This paper addresses a subset of cells, termed first responders, that are the earliest to transition into activity when glucose is stepped up from a sub-stimulatory level. It is shown that the first responders determine the first transient phase of electrical activity, and implicitly secretion, that precedes the start of steady-state oscillations of the second phase. The first phase is of interest for the pathogenesis of diabetes because it is (or claimed to be) one of the first indications of the disease. The clinical data on this are actually ambiguous, but nonetheless the first phase is an important aspect of how secretion from islets is organized. This is clear from the existence of a subset of readily releasable insulin vesicles, but the electrical activity correlates that synergize with vesicle availability are less well understood. As such, the paper is an important contribution to both islet biology and potentially diabetology. 

The approach is to use a genetically encoded calcium indicator, GCamps, and confocal imaging to identify cells that show the earliest rise in calcium and then to verify that this property remains consistent when the glucose stimulus is repeated about an hour later. Further testing over 48 h shows the first responder slowly fading. This thus appears to be a matter of continuous variation of cell properties within the islet and moreover one that is persistent but decaying over long-enough periods of time, rather than a discrete sub-type of beta cell. This is confirmed by simulations using an islet model in which first responders emerge from random variation of properties. The text is a bit ambiguous about whether we should think of the glass as half full or half empty and could be clarified in this regard. 

The properties that matter are the density of KATP channels and gap junctional coupling which are both lower. This is also confirmed by simulations. The intriguing suggestion is made that there may be a reciprocal negative feedback relationship between these quantities that regulates their variation over time. This would be rather different from a persistent genetic difference and would be a good subject for future investigation. 

Interestingly and perhaps surprisingly, increased sensitivity to glucose is not a feature of the first responders. In contrast, other putative "leader" or "hub" cells that have been identified for the second phase of electrical activity are proposed to have increased glucose sensitivity. This and other features lead to the conclusion that the two types of leader cells are probably distinct. The much-discussed topic of hub cells provides interesting context and relevance to the paper, but its results stand by themselves and can be judged independent of hubs.

Author Response:

Reviewer #1 (Public Review):

Sokolsky et al. propose a new statistical model class for descriptive modeling of stimulus encoding in the spiking activity of neural populations. The main goals are to provide a model family that (G1) captures key activity statistics, such as spike count (noise) correlations, and their stimulus dependence, in potentially large neural populations, (G2) is relatively easy to fit, and (G3) when used as a forward encoder model for Bayesian decoders leads to efficient and accurate decoding. There are also three additional goals or claims: (C1) that this descriptive model family can serve to quantitatively test computational theories of probabilistic population coding against data, (C2) that the model can offer interpretable representations of information-limiting noise correlations, (C3) that the model can be extended to the case of temporal coding with dynamic stimuli and history dependence.

The starting point of their model is a finite mixture of independent Poisson distributions, which is then generalized and extended in two ways. Due to the "mixture", the model can account for correlations between neurons (see G1). As any mixture model, the model can be viewed in the language of latent variables, which (in this case) are discrete categorical variables corresponding to different mixture components. The two extensions of the model are based on realizing that the joint distribution (of the observed spike counts and the latent variables) is in the exponential family (EF), which opens the door to powerful classical results to be applied (e.g. towards G2-G3), and allows for the two extensions by: (E1) generalizing Poisson distributions in mixture components to Conway-Maxwell-Poisson distributions, and (E2) introducing stimulus dependence by allowing the natural parameters of the EF to depend on stimulus conditions. They call the resulting model a Conditional Poisson Mixture or CPM (although the "Poisson" in CPM really means Conway-Maxwell-Poisson). E1 is key for capturing under-dispersion, i.e. Fano Factors below 1. For the case of discrete set of stimulus conditions, they propose minimal, maximal versions of E2; depending on which natural parameters are stimulus dependent. In the case of a continuum of stimuli (they only consider 1D continuum of stimulus orientations, e.g. in V1 encoding) they also consider a model-based parametric version of the minimal E2 which gives rise to Von Mises orientation tuning curves.

Strengths:

-Proposing a new descriptive encoding model of spike responses that can account for sub-poissonian and correlated noise structure, and yet can be tractably fit and accurately decoded.

-Their experiments with simulated and real (macaque V1) data presented in Figs. 2-5 and Tables 1-2 provide good evidence that the model supports G1-3.

-Working out a concrete Expectation Maximization algorithm that allows efficient fits of the model to data.

-Exploiting the EP framework to provide a closed form expression for the model's Fisher Information for the minimal model class, a measure that plays a key role in theoretical studies of probabilistic population coding.

As such, the papers makes a valuable contribution to the arsenal of descriptive models used to describe stimulus encoding in neural population, including the structure and stimulus dependence of their higher-order statistics.

Thank you very much for your thorough, exact, and positive evaluation of our manuscript!

Weaknesses:

1) I found the title and abstract too vague, and not informative enough as to the concrete contributions of this paper. These parts should more concretely and clearly describe the proposed/developed model family and the particular contributions listed above.

We found your summary of the paper and model to be highly accurate, and we rewrote the abstract to summarize the key strengths as you’ve listed them. We found it difficult to develop a more exact title which wasn’t overlong, so we left it as is.

2) I was not convinced about claims C1 and C2 (which also contribute to the vagueness of abstract), but I think even without establishing these claims the more solid contributions of the paper are valuable. And while I can see how the model can be extended towards C3, there are no results pertaining to this in the current paper, nor even a concrete discussion of how the model may be extended in this direction.

2.1) Regarding C1, the claim is supposed to follow from the fact that the model's joint distribution is in the exponential family (EF), and that they have reasonably shown G1-G3 (in particular, that it captures noise correlations and its Bayesian inversion provides an accurate decoder). While I agree with the latter part, what puzzles me is that in the probabilistic population coding (PPC) theoretical models that claim can be quantitatively tested using their descriptive model are, as far as I remember/understand, the encoder itself is in EF. By contrast here the encoder is a mixture of EF's and as such is not itself in EF. Perhaps this distinction is not key to the claim - but if so, this has to be clearly explained, and more generally the exact connection between the descriptive encoder model here and the models used in the PPC literature should be better elaborated.

This claim was indeed poorly explained in our manuscript, and not self-evident. There is a deeper connection between our conditional models and PPCs, which we now make explicit in a new section of the manuscript (Constrained conditional mixtures support linear probabilistic population coding, line 364), which includes an equation (Equation 4) that shows their exact relationship.

2.2) Regarding C2, I do not see how their results in Fig 5 (and corresponding section) provide any evidence for this claim. As a theoretical neuroscientist, I take "interpretable" to mean with a mechanistic or computational (theoretical) interpretation. But, if anything, I think the example studied in Fig 5 provides a great example of the general point: that even when successful descriptive models accurately capture the statistics of data, they may nevertheless not reveal (or even hide or mis-identify) the mechanisms underlying the data. In this example's ground-truth model, the stimulus (orientation) is first corrupted by input noise and then an independent population of neurons with homogeneous tuning curves (and orientation-independent average population rate) responds to this corrupted version of the stimulus. That is a very simple AND mechanistic interpretation (which of course is not manifest to someonw only observing the raw stimulus and spiking data). The fit CPM, on the other hand, does not reveal the continuous input noise mechanism (and homogeneous population response) directly, but instead captures the resulting noise correlation structure by inferring a large (~20) number of mixture components, in each of which population response prefers a certain orientation. For a given stimulus orientation, the fluctuations between (3-4 relevant) mixture components then approximate the effect of input noise. This captures the generated data well, but misses the true mechanism and its simpler interpretation. Let me be clear that I don't take this as a fault of their descriptive model. This is a general phenomenon, despite which their descriptive model, like any expressive and tractible descriptive model, still can be a powerful tool for neural data analysis. I'm just not convinced about the claim.

This is a very fair point, and we’ve reformulated a few passages to emphasize that the model is primarily descriptive, at least in our applications in the paper (see new section title at like 393, the first corresponding paragraph).

2.3) Regarding C3, I think the authors can at least add a discussion of how the model can be extended in this direction (and as I'm sure they are aware, this can be done by generalizing the Von Mises version of the model, whereby the model I believe can be more generally thought of as a finite mixture of GLMs).

In Appendix 4 we detail the relationship between CPMs and GLMs. We also note here that, at least as far as we understand, CPMs are formally distinct from finite mixtures of GLMs — the easiest way to see this distinction is to note that the index probabilities of a CPM depend on the stimulus, whereas the equivalent index probabilities in a finite mixture of GLMs would not. We have also explained this in Appendix 4.

Reviewer #2 (Public Review):

Sokoloski, Aschner, and Coen-Cagli present a modeling approach for the joint activity of groups of neurons using a family of exponential models. The Conway-Maxwell (CoM) Poisson models extend the "standard" Poisson models, by incorporating dependencies between neurons.

They show the CoM models and their ability to capture mixture of Poisson distributions. Applied to V1 data from awake and anesthetized monkeys, they show it captures the Fano Factor values better than simple Poisson models, compare spike count variability and co-variability. Log-likelihood ratios in Table 1 show on-par or better performance of different variant of the CoM models, and the optimal number of parameters to use for maximizing the likelihood [balancing accuracy and overfitting] and are useful for decoding. Finally, they show how the latent variables of the model can help interpret the structure of population codes using simple simulated Poisson models over 200 neurons.

In summary, this new family of models offer a more accurate approach to the modeling and study of large populations, and so reflects the limited value of simple Poisson based models. Under some conditions it gives has higher likelihood than Poisson models and uses fewer parameters than ANN model.

However, the approach, presentation, and conclusions fall short on several issues that prevents a clear evaluation of the accuracy or benefits of this family of models. Key of them is the missing comparison to other statistical models.

1) Critically, the model is not evaluated against other commonly used models of the joint spiking patterns of large populations of neurons. For example: GLMs (e.g. Pillow et al Nature 2008), latent Gaussian models (e.g. Macke et al Neural Comp 2009), Restricted Boltzmann Machines (e.g. Gardella et al PNAS 2018), Ising models for large groups of neurons (e.g. Tkacik etal PNAS 2015, Meshulam et al Neuron 2017), and extensions to higher order terms (Tkacik et al J Stat Mech 2013), coarse grained versions (Meshulam et al Phys Rev Lett 2019), or Random Projections models (Maoz et al biorxiv 2018).

. Most of these models have been used to model comparable or even larger populations than the ones studied here, often with very high accuracy, measured by different statistics of the populations and detailed spiking patterns (see more below). Much of the benefit or usefulness of the new family of models hinges on its performance compared to these other models.

We agree very much with this point, and have done our best to address it by thoroughly comparing our model with a factor analysis encoding model in Appendices 1 and 2, and summarizing these results at appropriate points in the manuscript (lines 196–199 and 325–328). In particular, we visualized and compared the performance of factor analysis with our mixture models, and found that (i) factor analysis is better at capturing the first and second order statistics of the data, but (ii) when evaluated on held-out data, the performance gap more-or-less vanishes. Moreover, we found that an encoding model based on FA performs poorly as a Bayesian decoder, and we provided preliminary evidence that this is because our mixture models can capture higher-order statistics that FA cannot. We believe that these results have been very valuable to conveying the strengths and weaknesses of the mixture model approach.

We have also extended the introduction to explain the differences between other model families suggested by the reviewer and our approach, to explain how the different assumptions about the form of data make it difficult to compare them quantitatively (see lines 42–63). To wit, GLMs and latent Gaussian models are both models that critically depend on modelling spike trains, and not spike counts. On the other hand, Restricted Boltzmann machines, Ising models, and random projection models all assume binary, rather than counting spiking data. As such, any comparison would depend on coming up with methods for either (i) reshaping our datasets and comparing spike- train/binary spike-count likelihoods to trial-to-trial likelihoods, or (ii) extending our conditional mixture approach to temporal/binary data, both of which are beyond the scope of our paper. We instead used factor analysis because it has been applied widely to modelling trial-to-trial spike counts, and thus avoid further transformations that might reduce the validity of our comparisons.

2) As some of these models are exponential models, their relations to the family of the models suggested by the authors is relevant also in terms of the learned latent variables. Moreover, the number of parameters that are needed for these different models should be compared to the CoM and its variants.

In our comparisons with factor analysis we also compared number of latent states/dimensions required to achieve maximum performance. Overall FA was consistently the most efficient, at least when evaluated on the ability to capture second-order statistics, although our mixture models also performed quite well with modest numbers of parameters.

3) The analysis focuses on simple statistics of neural activity, like Fano Factors (Fig. 2) and visual comparisons rather than clear quantitative ones. More direct assessments of performance in terms of other spiking statistics for single neurons and small groups (e.g., correlations of different orders ) and direct comparison to individual spiking patterns (which would be practical for groups of up to 20 neurons) would be valuable

In the Appendix 2 we evaluated the ability of our mixtures to capture the empirical skewness and kurtosis of recorded neurons, and found that the CoM-based mixture performs quite well (r2 for the CoM-Based mixture was between 0.6 and 0.9). Because FA cannot capture these higher-order moments, we speculate that modelling these higher-order moments is critical for maximizing decoding performance. This adds another perspective on the strengths of our approach, and we appreciate the suggestion.

Reviewer #3 (Public Review):

The authors use multivariate mixtures of Poisson or Conway-Maxwell-Poisson distributions to model neural population activity. They derive an EM algorithm, a formula for Fisher information, and a Bayesian decoder for such models, and show it is competitive with other methods such as ANNs. The paper is clear and didactically written, and I learned a lot from reading it. Other than a few typos the math and analyses appear to be correct.

Thank you for the positive evaluation!

Nevertheless there are some ways the study could be further improved.

Most important, code for performing these analyses needs to be publicly released. The EM algorithm is complicated, involving a gradient optimization on each iteration - it is very unlikely people will rewrite this themselves, so unless the authors release well-packaged and well-documented code, their impact will be limited.

We very much agree, and we have done this. We provide a link to our gitlab page, where all relevant code can be downloaded, and installation instructions are provided (we indicate this in the manuscript at lines 799–803).

Second, it would be nice to extend the model to continuous latent factors. It seems likely that one or two latent factors could do the work of many mixture components, as well as increasing the interpretability of the models.

We certainly agree that in some cases continuous latent variables could be much more parsi- monious. However, to the best of our knowledge most of the expressions that we rely on would no longer be closed-form, and so the machinery of the model would require suitable approximations. Nevertheless, it’s an interesting possibility that we now address in the Discussion (lines 482–491).

Third, it would be interesting to see the models applied to more diverse types of population data (for example hippocampal place field recordings).

We certainly agree with the importance of applying our model to other datasets, and indeed the purpose of our manuscript is to offer a method that can be applied broadly, and our goal in making the code available publicly is to facilitate that. However, we have decided to maintain the focus of this manuscript on the method itself, and limit the application to one kind of data (V1), for which we also now provide more extensive analysis and quantification of the response statistics (Figure 2 C-D, Figure 3 G-H, Appendix 2), a study of the sample sizes required to fit the model (Appendix 3), and model-comparison (Appendix 1–2). Overall we feel that the paper is already quite long and dense even when limited to a single kind of data. We believe applications to multiple kinds of data would perhaps be better suited for a different study, focusing on the comparisons between them. In that regard, we are certainly open to future collaborations on large-scale recordings from various stimulus-driven brain areas.

Fourth, how does a user choose how many mixture components to add?

To clarify this, we’ve added a section in the methods (Strategies for choosing the CM form and latent structure), and in particular the number of mixture components.

Author Response:

Reviewer #2 (Public Review):

[...] 1) A weakness of the paper is the disruption of the complex during cryoEM grid preparation resulting in about half of the observed particles missing the membrane arm and likely also contributing to the disorder and biased orientation seen in the intact complexes. This leads to poor density in the membrane arm for all of the intact complex I structures presented and large variations in the local resolution of the membrane arm focused refinement.

Purified E. coli complex I has always been known to be labile in particularly at the junction of peripheral and membrane arms (https://pubmed.ncbi.nlm.nih.gov/12637579/).

Air-water interface likely plays a role in disrupting the complex in addition to other possible causes. Indeed, the dissociated arms, preferred particle orientation, and low protein concentration (~0.1 mg/ml) used to produce grids with high particle density all indicate that reconstituted complex I does interact with air-water interface. While disruption and denaturation of protein complexes on air-water interface has been well documented, (https://pubmed.ncbi.nlm.nih.gov/3043536/, https://pubmed.ncbi.nlm.nih.gov/30932812/ ), we are not aware of examples where air-water interfaces caused higher mobility of a complex or induced a stable conformation, different from the one in bulk solution. Therefore, we think that air-ware interface is neither the cause of the observed high arms mobility nor of their relative rotation.

Preferential orientation was observed in the cryo-EM studies of most complex I homologs (Gutiérrez-Fernández et al., 2020; Parey et al., 2019; Zhu et al., 2016) as well as of other proteins, suggesting that adsorption of complex I on air water interface is a common phenomenon. In this case it is not clear why relative movement of the arms observed in all the structurally characterized complex I homologs is not due to the air-water interface, but in the case of E. coli complex it is.

To provide additional support to our interpretation of the structural data we purified complex I in detergent LMNG, showed that it catalyzes redox reactions and solved its structure to resolution of 6.7 Å (Figure 6 and corresponding figure supplements). Because cryo-EM grids had to be prepared at a protein concentration of 2-3 mg/ml and the particles displayed nearly homogeneous distribution of orientations, we conclude that the interaction with the air-water interface was reduced. Still, the complex assumes a very similar, or even somewhat more uncoupled conformation and the relative mobility of the arms remained comparable to that in the nanodisc-reconstituted complex reconstructions. These data allow us to rule out the air-water interface and reconstitution of the protein into lipid nanodiscs as the possible causes of the high mobility and the unusual relative position of the arms.

The corresponding modifications were added to the manuscript on lines 372-382:

“To better understand the reasons for the observed uncoupled conformation and the missing density for HTMH1, we purified E. coli complex I in detergent LMNG, showed that it can catalyze redox reactions (Figure 6 - figure supplement 1) and solved its structure to resolution of 6.7 Å (Figure 6 - figure supplement 2). The detergent-solubilized complex also displays high relative mobility of the arms (Figure 6 - figure supplement 3) and has uncoupled conformation (Figure 6). Its peripheral arm is rotated even further away from the expected coupled state position than in the nanodisc-reconstituted structures. Both the cryo-EM sample preparation conditions and more homogeneous distribution of particle orientations indicate that interaction of the complex with air-water interface was significantly reduced when compared with the complex in nanodiscs. This allows us to conclude that neither air-water interface nor reconstitution into nanodiscs cause the uncoupled conformations.”

It is not very clear what referee means by “poor”, when referring to the focused density of the membrane arm. The density corresponds well to the reported resolution of 3.7 Å. Indeed, it is in a stark contrast with the quality of the density obtained for the peripheral arm at 2.1 Å resolution. Given high mobility of the membrane arm it had to be refined essentially independently of the peripheral arm which remains still challenging for a ~200 kDa membrane protein without water-soluble domains in lipid nanodiscs. The density is heterogeneous as clearly stated at the beginning of the section “Structure of membrane arm” from line 264:

“The model of complete membrane arm, including the previously missing subunit NuoH (Efremov and Sazanov, 2011), was built into the density map with local resolution better than 3.5 Å at the arm center and approximately 4.0 Å at its periphery (Figure 1A, Figure 1 - figure supplement 4).”

Finally, for most complex I homologs the resolution was gradually improved over several years, as reflected in multiple publications of essentially the same structures. In contrast, no high-resolution structure information was available for the intact E. coli complex I until now. Therefore, it would be unreasonable to expect the complete structure to be solved at resolution of 2 Å at once.

The resolution of the membrane domain in reconstructions of complete complex I is indeed lower due to high flexibility of the complex and the fact that refinement naturally focuses on more stable peripheral arm that does not have heterogeneous nanodisc around and that contains Fe-S clusters enhancing particle alignment power. Still, these conformations clearly resolve the interface between subunits albeit at lower resolution.

This fact was also clearly stated at the beginning of results section lines 102-106:

“Three conformations of the entire complex were reconstructed to average resolutions between 3.3 and 3.7 Å (Figure 1 - figure supplement 4) resolving the interface between the arms; however, due to high-residual mobility of the arms, the antiporter-like subunits were resolved at below 8 Å (Figure 1 - figure supplement 4).”

2) A weakness of the paper is the disorder of important functional regions of the complex, namely the NuoH TMH1, whose disorder is unique to these nanodisc E. coli structures, and the NuoA TMH1-TMH2 loop. As the NuoH TMH1 forms part of the entry to the quinone tunnel of the complex, its absence in the structure leads to concerns regarding the function of the nanodisc preparation. Its absence it curious as this suggests flexibility of the helix, as pointed out by the authors, but the authors also state that there is not enough room in the nanodisc to accommodate this helix (given the visible density for the lipid and membrane scaffold protein). These observations suggest denaturation or unfolding in this region of the complex as opposed to simple flexibility.

According to the usual definition of complex I activity our preparation in nanodiscs is active. We complemented our data with additional measurements and included NADH:DQ assays (see next point) that also indicate that our preparation is active. Additional 3D reconstruction of E. coli complex I that we obtained for protein solubilized in LMNG does resolve HTMH1 and its environment appears to be more similar to other detergent-solubilized structures of complex I homologues. At the same time, the helices around HTMH1 appear to be more tightly packed and more curved than in the nanodiscs which may reflect suppressed dynamics and distorted protein conformation. Most importantly, the overall conformation of the complex remains nearly the same and still corresponds to what we call the uncoupled conformation. That of course does not allow us to say where HTMH1 is positioned within the nanodisc, but it does enable us to conclude that the local changes in the vicinity of HTMH1 do not influence the global conformation of the complex.

The additional structure is not described on lines 383-388:

“The HTMH1 helix is resolved in the detergent-solubilized complex (Figure 6A). Its density is weaker than that of the surrounding helices and it is strongly bent (Figure 6B). Simultaneously, HAH1 takes the conformation resembling other complex I homologs while ATMH1 bends towards the arm core. The arrangement of helices in detergent-solubilized reconstruction appears to be more compact and more bent than in the lipid environment which may restrain the otherwise more flexible HTMH1.”

In the revised discussion the environment of HTMH1 is described more clearly on lines 426-433:

“The absence of HTMH1 density in nanodiscs, but not in detergent, is another unique feature of E. coli complex I. HTMH1 is exposed to the lipid environment and the width of the nanodisc next to HTMH1 is similar to other regions around the membrane arm (Movie 1). Moreover, homology modelled HHTM1 fits the empty space without steric clashes suggesting that HHTM1 is dynamic rather than displaced or unfolded. By comparing the detergent-solubilized and reconstituted complexes we can conclude that position and dynamics of this helix is neither the cause of the uncoupled conformation nor of the high relative mobility of the arms.”

Disorder of ATMH1-TMH2 loop is not unique to E. coli complex I but also observed in some conformations of ovine complex I PDB 6zkd, 6zke, 6zkf.

3) Unfortunately, the NADH:Q1 functional data do not fully address these concerns at Q1 is far more soluble that the native Q8 substrate of the complex. Although the Q1 activity is sensitive to the inhibitor Piericidin A, which clearly demonstrates that the Q1 reduction is occurring in the native quinone binding site as Piericidin A binds specifically at that site, this does not preclude the possibility of Q1 accessing this binding site via a different path. In fact, the structures indicate that given the flexibility in the connection between peripheral and membrane arms of the complex, the quinone binding site is likely open to the cytoplasm. This leads the authors themselves to conclude that the structures presented are likely disrupted/uncoupled states in which the energy converting mechanism of the complex is not likely possible.

To address the raised concern, we have measured the activity of complex I in nanodiscs with less soluble decylubiquinone (DQ) as well as its inhibition. Small amounts of LMNG was used to increase the DQ solubility. Our results have confirmed that E. coli complex I in nanodiscs is active and the NADH:DQ activity is sensitive to piericidin A (see the modified Figure 1-figure supplement 2). We have also remeasured the Q1 activity and its inhibition which showed lower values than previously, due to a flaw in the activity measurements reported in the original submission (qualitatively, the results remained unchanged). Moreover, we have observed a similar activity results with somewhat higher values for E. coli complex I in LMNG (Figure 6-figure supplement 1). These data demonstrate that in the reconstituted complex I quinone analogues can enter the Q-site through the membrane. It is worth noting that due to extremely low solubility of longer quinones, including native ones, they are not used for activity measurements in purified preparations.

Regarding the complex I conformation, we do think our reconstruction represents uncoupled state which is not able to pump protons (as states in the title). We have improved the clarity of this point throughout the manuscript including the discussion lines starting from the line 412.

“The high mobility of the interfacial regions and the relative rotation of the arms disrupts conserved interfacial interactions and exposes Q-cavity to the solvent (Figure 5A). This differentiates E. coli complex I from its structurally characterized homologs in which the Q-cavity is sealed from the solvent. Thus, we interpret the observed conformation as an uncoupled state.”

And from line 469: “We also observed the relative rotation of the membrane and peripheral arms disrupting the conserved interface and trapping the complex in an uncoupled conformation. Whether this conformation is biologically relevant or is a result of protein purification is to be clarified by further research.”

4) A weakness of the paper is the building of atomic models into regions of the map which do not contain sufficient detail to warrant atomic models. This is particularly the case for the intact models of complex I as well as the membrane arm focused maps and results in low map-model correlations (0.58-0.71). The models were clearly highly restrained during refinement, resulting in good geometry, as is necessary for low resolution regions. But being able to restrain the geometry is not sufficient for placing atoms into regions where the density is weak or absent. If additional information was used in building/constraining the model, such as the X-ray structure, the regions of the model that are biased towards the X-ray structure model needs to be made clearer. Also, in several places in the membrane arm map residues bulge out of the density (side chain and main chain) leading to possible frame shifts with respect to the match between subsequent residues in the model and the map (see NuoM Ile168 for example).

A large part of the membrane domain has been solved using X-ray crystallography to resolution of 3.0 Å which was used as a starting model for model building, therefore we don’t think there are register shifts in our model. We used standard setting for model refinement in phenix_refine. Our building and refinement procedure has been described in fine details in the original submission, see from line 674:

“For the membrane domain, the previously obtained E. coli model (PDB ID: 3RKO) was real-space-refined in PHENIX. The missing NuoH subunit was homology-modelled using the T. thermophilus structure (PDB ID: 4HEA) in Coot 0.9. The final model was obtained after several rounds of manual rebuilding and real-space refinement using standard parameters with Ramachandran restrains, secondary-structure restrains applied to the NuoL TMH9-13, without ADP restrains, and with the optimized nonbonded_weight parameter. To generate the model of the complete complex I, the separate peripheral and membrane arm structures were combined and the missing parts at the interface (Table 2) were built manually. As the density of NuoL and NuoM was very poor in all the resolved full conformations, these subunits were subjected to rigid-body refinement in PHENIX, whereas the others were subjected to real-space refinement with minimization_global, local_grid_search, morphing, and ADP refinement. Ramachandran, ADP, and secondary-structure restrains were used. After manual rebuilding in Coot, real-space refinement of the full complex was performed with standard parameters and restrains.”

To improve clarity, we added a following sentence to the Results section from line 116:

“Using the resulting maps, atomic models of the peripheral and membrane arms have been built. The entire E. coli complex I was modelled by fitting models of the arms and extending additionally resolved loops and termini. Due to limited resolution, the antiporter-like subunits were refined as rigid bodies.”

The model has been improved and side chains with absent density were truncated to C position.

The density for focused refinement density of the membrane fragment is relatively week, but of sufficient quality to allow building side chains for most of the map. It even visualizes lipid densities (not described in the manuscript). Such weaker densities are common for small membrane proteins. While fully usable for model building, they naturally result in lower model map FSC and consequently, in lower real-space correlation. In addition, real-space correlation is lower when the map is heterogeneous, and it strongly depends on the way the heterogeneous map has been filtered. Therefore, lower cross correlations do not necessarily mean that the model fit is poor. In our case they reflect weaker signal to noise of the density. Model-map FSCs (Figure 1 figure supplement 4) are more informative than a single number and show that model-map cross correlations remain above 0.5 for the complete resolution range for all models.

5) A weakness of the paper is that several specific claims are made about the positions of side chains but, when investigated, the density for those side chains is poorly resolved. An example of this is NuoH Lys274, which is in a low-resolution region of the map and although is fit as well as possible must be considered low confidence given the local resolution (nearby residues Phe277 and Phe282 have almost no side chain density for example).

At lower resolution, a presence of residues density strongly depends on their mobility. Well-ordered residues may have well-defined densities while others, even in the proximity, may have a poor density. In the case of Lys274, there is a clear density for the side chain, its position makes chemical sense, and it is hydrogen-bonded to the backbone oxygen of Gly258. In fact, if examined closely, this is also the only meaningful position for Lys274 side chain. At the same time, the conformations of Phe277 and Phe282 are not restrained by interactions with other residues in their vicinity which is likely why their densities are weaker.

6) A weakness of the paper is that the conformational changes seen between the membrane and peripheral arm of the complex in the different 3D classes are difficult to interpret. It is unclear if they are mechanistically significant or, perhaps more likely given the amount of broken complex observed, due to partial disruption of the complex before it completely breaks apart.

As we discussed above, the observed multiple conformations are not due to the complex disruption. It is not very clear what the reviewer means by ‘difficult to interpret’. Many conformations of the peripheral and membrane arms observed for the complex I homologues are likely not mechanistically meaningful per see, but rather reflect overall flexibility of such a large complex. Here, our goal was to describe our structural data as accurately as possible which resulted in several resolved conformations.

We do think they all represent the uncoupled complex I, in this respect they do not have different mechanistic meanings. However, they do permit us to understand how the arms move relative to each other and what degree of freedom exists between them.

7) A strength of the paper is the interesting and original mechanistic proposal put forward by the authors. But a weakness is that it is unclear how this proposal stems from the structural data presented. Also, the arguments presented are difficult to follow in their current form and warrant a more detailed discussion with the requisite thermodynamic treatment. This may warrant a more complete discussion in an appendix or unless the authors can more convincingly show how the data presented in the paper suggests their proposed mechanism perhaps a separate review article. Furthermore, the proposed mechanism, as presented would make a simple prediction that in the absence of NuoM and NuoL (or equivalent subunits in other species) complex I would not pump any net protons. Experiments that are relevant to this prediction have been done in E. coli (NuoL deletion) and Y. lipolytica (nb8m deletion that results in loss of both NuoM and NuoL subunits). See https://pubmed.ncbi.nlm.nih.gov/21417432/ and https://pubmed.ncbi.nlm.nih.gov/21886480/. In both cases the complex is still able to pump protons. The behavior of the NuoL deletion in E. coli is reconcilable with their proposed mechanism as NuoM is still present, however, the case of the nb8m deletion in Y. lipolytica is more difficult to reconcile with their proposed mechanism. The authors would need to address these experiments in order to include their proposed mechanism.

The description of the mechanism has been modified. It is very briefly outlined in the main text along with the Figure 7 and more detailed description, including thermodynamic considerations, is moved to the supplementary text. We have also explained more clearly how the model stems from the experimental data on line 435:

“The absence of a continuous proton-translocation pathway between the Q-site and subunit NuoN, as well as high flexibility of the peripheral arm interface are not consistent with the recently proposed coupling mechanisms relying on specific movements of the interfacial loops (Cabrera-Orefice et al., 2018; Kampjut and Sazanov, 2020). This led us to ask whether a coupling mechanism consistent with known complex I properties, but without the movements of interfacial loops is conceivable.”

Furthermore, we state that at this point this is a hypothetical mechanism.

Supplementary data describing mechanism in more details now also includes the discussion of both papers mentioned by the reviewer from line 1368.

“Experiments with engineering E. coli complex I lacking subunit NuoL and Y. lipolytica complex I lacking homologs of subunits NuoM and NuoL (Dröse et al., 2011; Steimle et al., 2011)(Dröse et al., 2011; Steimle et al., 2011) (correspond to n=2 and 1, respectively) both suggested that the engineered complexes were active and for both constructs stoichiometry was estimated as 2H+/2e-. While NuoL deletion experiments support our model, the NuoL/M deletion clearly contradicts it. Both experiments should be interpreted cautiously, however. Results of NuoL deletion for E. coli complex I were not reproducible (Verkhovskaya and Bloch, 2012). In the case of Y. lipolytica, the homologs of NuoL/M dissociated from the complex along with another 11 subunits upon deletion of supernumerary subunit NB8M located at the tip of NuoL (Zickermann et al., 2015). Since the proton-translocating modules were not deleted per se, the presence of contaminating amounts of assembled complex I in the preparations that generated observed proton pumping cannot be completely excluded. It is important to note that mutation of the conserved ionizable residues on the interface between NuoN and NuoM, i.e. ME144 (Torres-Bacete et al., 2007) or its counter ion NK395 (Amarneh and Vik, 2003), result in a completely inactive complex I suggesting that dissociation of subunits NuoL/M also should render complex I inactive (Verkhovskaya and Bloch, 2012).”

The main problem with these experiments that that they have never been reproduced by other laboratories and are not completely consistent with the mutagenesis data. Deletion of subunits may also result in distinct pumping behavior of the remaining subcomplex. For example, it was shown for the bovine complex I that it can translocate Na+ ions in the deactive state (https://pubmed.ncbi.nlm.nih.gov/22854968/).

Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

8) Overall, despite the many strengths of this paper detailed above it is unclear whether the authors achieved their goal of a structure of functional E. coli respiratory complex I reconstituted in lipid nano-discs. It appears that under the current grid preparation conditions that the complex is under excessive stress resulting in partial denaturation and partial-to-complete dissociation. Given the clear biophysical data presented on the intactness of the complex in solution, this disruption likely occurs during grid preparation and further optimization of grid conditions may resolve this issue. With the current maps more work needs to be done to improve the map-to-model correlation and to clearly indicate the regions in the models where this correlation is low.

Additional reconstruction of complex I solubilized in LMNG help us to exclude the interaction of the complex with water-air interface and its reconstitution into lipid nanodiscs as the causes of the relative subunit rotation and high flexibility between the arms. At this moment, whether the structure represents an artifact of purification or is a biologically-relevant state remains an open question. However, answering it goes beyond the current study and will require additional research. This is now explained in the discussion section.

Reviewer #2 (Public Review):

The authors performed six experiments examining the influence of beliefs regarding pain experience on behavioral and neural indices of empathy for pain and altruistic behavior. They demonstrate that manipulations that to reduce beliefs that individuals making painful expressions are actually in pain (e.g., revealing them to be actors, indicating that their treatment has been successful, etc.) attenuates subjective judgments of pain intensity, real monetary donations to these targets, and P2 amplitudes, and further, that regions involved in perspective-taking and emotion regulation are sensitive to representations of pain beliefs. While I think that the authors have done an admirable job in laying out the evidence for their argument across six well-devised experiments, I do think that the manuscript has some room for improvement. In particular, I hope that the authors can offer stronger grounding in the background literature and clarify some task and stimulus details.

1. In laying out their hypotheses, the authors write, "The current work tested the hypothesis that BOP provides a fundamental cognitive basis of empathy and altruistic behavior by modulating brain activity in response to others' pain. Specifically, we tested predictions that weakening BOP inhibits altruistic behavior by decreasing empathy and its underlying brain activity whereas enhancing BOP may produce opposite effects on empathy and altruistic behavior." While I'm a little dubious regarding the enhancement effects (see below), a supporting assumption here seems to be that at baseline, we expect that painful expressions reflect real pain experience. To that end, it might be helpful to ground some of the introduction in what we know about the perception of painful expressions (e.g., how rapidly/automatically is pain detected, do we preferentially attend to pain vs. other emotions, etc.).

2. For me, the key takeaway from this manuscript was that our assessment of and response to painful expressions is contextually-sensitive - specifically, to information reflecting whether or not targets are actually in pain. As the authors state it, "Our behavioral and neuroimaging results revealed critical functional roles of BOP in modulations of the perception-emotion-behavior reactivity by showing how BOP predicted and affected empathy/empathic brain activity and monetary donations. Our findings provide evidence that BOP constitutes a fundamental cognitive basis for empathy and altruistic behavior in humans." In other words, pain might be an incredibly socially salient signal, but it's still easily overridden from the top down provided relevant contextual information - you won't empathize with something that isn't there. While I think this hypothesis is well-supported by the data, it's also backed by a pretty healthy literature on contextual influences on pain judgments (including in clinical contexts) that I think the authors might want to consider referencing (here are just a few that come to mind: Craig et al., 2010; Twigg et al., 2015; Nicolardi et al., 2020; Martel et al., 2008; Riva et al., 2015; Hampton et al., 2018; Prkachin & Rocha, 2010; Cui et al., 2016).

3. I had a few questions regarding the stimuli the authors used across these experiments. First, just to confirm, these targets were posing (e.g., not experiencing) pain, correct? Second, the authors refer to counterbalancing assignment of these stimuli to condition within the various experiments. Was target gender balanced across groups in this counterbalancing scheme? (e.g., in Experiment 1, if 8 targets were revealed to be actors/actresses in Round 2, were 4 female and 4 male?) Third, were these stimuli selected at random from a larger set, or based on specific criteria (e.g., normed ratings of intensity, believability, specificity of expression, etc.?) If so, it would be helpful to provide these details for each experiment.

4. The nature of the charitable donation (particularly in Experiment 1) could be clarified. I couldn't tell if the same charity was being referenced in Rounds 1 and 2, and if there were multiple charities in Round 2 (one for the patients and one for the actors).

5. I'm also having a hard time understanding the authors' prediction that targets revealed to truly be patients in the 2nd round will be associated with enhanced BOP/altruism/etc. (as they state it: "By contrast, reconfirming patient identities enhanced the coupling between perceived pain expressions of faces and the painful emotional states of face owners and thus increased BOP.") They aren't in any additional pain than they were before, and at the outset of the task, there was no reason to believe that they weren't suffering from this painful condition - therefore I don't see why a second mention of their pain status should *increase* empathy/giving/etc. It seems likely that this is a contrast effect driven by the actor/actress targets. See the Recommendations for the Authors for specific suggestions regarding potential control experiments. (I'll note that the enhancement effect in Experiment 2 seems more sensible - here, the participant learns that treatment was ineffective, which may be painful in and of itself.)

6. I noted that in the Methods for Experiment 3, the authors stated "We recruited only male participants to exclude potential effects of gender difference in empathic neural responses." This approach continues through the rest of the studies. This raises a few questions. Are there gender differences in the first two studies (which recruited both male and female participants)? Moreover, are the authors not concerned about *target* gender effects? (Since, as far as I can tell, all studies use both male and female targets, which would mean that in Experiments 3 and on, half the targets are same-gender as the participants and the other half are other-gender.) Other work suggests that there are indeed effects of target gender on the recognition of painful expressions (Riva et al., 2011).

7. I was a little unclear on the motivation for Experiment 4. The authors state "If BOP rather than other processes was necessary for the modulation of empathic neural responses in Experiment 3, the same manipulation procedure to assign different face identities that do not change BOP should change the P2 amplitudes in response to pain expressions." What "other processes" are they referring to? As far as I could tell, the upshot of this study was just to demonstrate that differences in empathy for pain were not a mere consequence of assignment to social groups (e.g., the groups must have some relevance for pain experience). While the data are clear and as predicted, I'm not sure this was an alternate hypothesis that I would have suggested or that needs disconfirming.

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Reviewer 1

This paper proposes a noise-aware approach SCRaPL for modelling the associations of single cell multi-omic data. For gene expression, it uses Poisson-lognormal model. For DNAm data, it uses Binomial noise model which explicitly takes into account the average within the region. The Bayesian hierarchical framework employed by SCRaPL could achieve higher sensitivity and better robustness in identifying correlations, and also offer a template for the application of more complex analysis techniques to multi-omics data. The symbols of this paper are a little bit confusing, and I suggest authors to carefully check them.

We thank the reviewer for his/ her appreciation, and apologise for the confusion arising from the dense notation, which we will thoroughly revise.

1. The symbols used in this paper are messy. For example, "1" and "2" are subscripts in Eq.(2) but become superscripts in Figure 5. Besides, there are many symbols not explained such as mj, Hj, Ψ0, etc. Also, I don't know if x{j,i}^{(1)} , x{j,i}^{(2)} in Figure 5 are same with x{ij1} and x{ij2} in Eq.(3). There are many places mismatch, authors should check carefully.

1. Why the equations in Fig.5 are totally different with Section 4.2? For example, pj ∼Beta(αj ,βj ) in Fig.5 but ρj ∼ Beta−1,1 in Eq.(8).

We apologise for the notational confusion, this will be fully revised.

The paper involves a lot of hyper-parameters which doesn't demonstrate their selection. For example, c1, c2, d1, d2.

This is a good point. We will include a sensitivity analysis on the hyperparameters, justifying the choices on both simulated and real data.

In section4.8, I am confused about $ρ_j$ the experiment 2, 5, 8, 11. Why $ρ_j$ both represents ZI rate and correlation?

We apologise for the notational oversight, which will be rectified.

In Section 4.5, it is difficult to understand the sentence "for me threshold u". Besides, what is $r$ represent in Section 4.5?

We apologise for the confusing sentence. $r$ is the Pearson correlation coefficient, as explained at the start of 4.5

Why there is "(6a)Agreement between SCRaPL and Pearson" in Fig. 4?

This simply means that the panel shows a methylation/ expression scatterplot for a gene where estimation by SCRaPL and Pearson return both a significant association. We will expand the caption to explain further.

For Fig.1, I cannot see the text in the rectangle.

Apologies, we will improve the readability of the figures

I would like to see the efficiency analysis for SCRaPL.

As part of part of re-implementation in a more accessible programming language, we have preformed preliminary efficiency analysis for MCMC , demonstrating linear scalability. Results will appear in the revised manuscript.

Reviewer 2

The authors present a Bayesian model to determine noise-corrected correlation coefficients for gene expression (RNA) and DNA-methylation data at single-cell resolution. The authors present a series of simulation data and an example of matched multi-omics data, and compare their results with Pearson correlation. Noise modelling allows the model to determine gene-methylation correlation patterns more accurately. While the authors demonstrate a neat application on accurate quantification of correlation coefficients, I see a limited use of the model for the broader single-cell community. The authors may therefore improve their manuscript on several aspects.

We thank the reviewer for the encouraging words, and thank him/ her for the critical observations, which we have taken at heart, considerably broadening the scope of our paper to make it more attractive to a larger community.

- Abstract: please specify the omics layers that you are analyzing (RNA + DNA methylation) in the abstract

We acknowledge that, while SCRaPL is potentially general, in the first submission we focused only on RNA and DNA methylation. We have now decided to expand our analyses to include 10X data of simultaneous chromatin accessibility (ATAC-seq) and RNA.

- What is the benefit of using a Bayesian model formulation in this setting?

The benefit is twofold: a principled treatment of noise, and a quantification of the resulting uncertainty which allows for a meaningful way to compute Bayesian significance levels. We will expand the discussion of the relative merits of a Bayesian vs frequentist approach.

- Does it also apply to unmatched data?

In principle, given measurements with the same number of cells in all modalities, it is possible to apply SCRaPL. However, unless there is a natural pairing between different cells, the scaling of this approach will be quadratic in the number of cells, hence potentially expensive (although largely parallelizable). We will discuss this now, particularly in the light of applying SCRaPL in conjunction with other suites such as Seurat.

• Would SCRaPL allow for differential correlation testing?

At the moment, SCRaPL does not allow for differential correlation testing. Of course, one may run SCRaPL separately on two groups of cells and compare the resulting estimates, which would be informative. Nevertheless, extending SCRaPL to perform differential correlation testing (e.g. using Bayesian model selection) would be a non-trivial effort. We will add a comment on this issue to the discussion section.

• Figure 1: The graphical description of the model is rudimentary. I believe that the model description could profit from a graphical model representation of SCRaPL (as presented in figure 5).

We will redraw Fig. 1 and incorporate the graphical model from Fig 5.

- Simulated data: all experiments seem to have rather low cell numbers (max. 200) and genes (max. 300). Given that 10X Genomics is the most widely-used sequencing platform with approx. 10,000 cells and 3,000 (highly variable) genes per experiment, and given that the authors show a use-case with 9480 genes in 487 cells, it seems appropriate to extend the simulations and runtime estimates of the presented model to several thousands of cells and genes, respectively.

Thank you for this comment. The original simulation settings were designed with scMT data in mind, where indeed only a few hundred cells can be assayed at most. Partly because of this feedback, and also because of the request of implementing SCRaPL in a different language, we are working on a more scalable Tensorflow implementation which will be able to handle thousands of cells and genes in a matter of tens of minutes . The new simulated data will therefore extend into this regime with larger data sets.

- Figure 4: Please revise the figure legend as I did not understand the plotted results based on the description.

We will do so.

- Results section 2.5: Please formulate your whole argument about epigenetic regulators. I do not think that "For further information please refer to supplementary figure XYZ." Is an appropriate closing statement for a paragraph, nor does it motivate the reader to look at the supplementary figures (I did look at them and I do not see how they support the point made in the paragraph). Please elaborate and consider a "take home message" for the paragraph such that the reader is able to understand the benefit of SCRaPL without revisiting the original data publication.

Thank you for this pointer, we will take it on board in the full revision.

- Conclusion: The authors mention that SCRaPL would further offer a "template for the application of more complex analysis techniques (such as clustering, dimensionality reduction and network inference)". If that was the case, the authors should consider a comparison to other tools, which offer exactly that (e.g. Seurat's CCA or non-negative matrix factorization in LIGER). Further, the authors should set their work into context with tools like bindSC.

Thank you for the suggestion. As far as we can tell, all of these methods are thought for unmatched data, rather than multi-omics assays performed in the same cells. Having said that, it is in principle possible to “preprocess” data with SCRaPL and then feed to Seurat or other tools the latent means computed by SCRaPL. We will include an example of how this may be done in the revision.

- Implementation: Matlab is used in about 6% of the single-cell RNAseq tools (according to scrna-tools.org). To reach a larger scientific community, do the authors plan to provide an R or Python implementation of their model?

We are now implementing SCRaPL in Python using Tensorflow probability, hoping to achieve substantial speedups (see response to previous point).

Additional minor points about formatting by Reviewer 2 will all be addressed.

Reviewer 3

Maniatis et al propose a sound strategy to analyse single-cell multi-comic data sets. A key advance is to use bespoke error models for each of the omics data. These are integrated into a multivariate gaussian model. This method is a novel and, in my opinion, a valuable addition to the analyses of the growing multi-omics single-cell data sets.

We thank this reviewer for his/ her appreciation of our work.

- Authors make a convincing argument of the importance of principle methods and in particular to use noise models that tailored to the data at hand. To further support this, can authors elaborate on how results would be different from using commonly applied methods ? Eg those embedded in the Seurat, OSCA, and scanpy 'suites'? Authors compare to Pearson correlation-based methods but is not clear if that is the true state-of-the-art on those methods

As far as we know, volcano plots of p-value versus Pearson correlation are the most commonly employed approaches to assess correlations amongst different molecular modalities in single-cell multi-omics (see e.g. Argelaguet et al, Nature 2020). Seurat and other methods normally do not deal with single-cell multi-omics (i.e., multiple omics measured in the same cell), rather with multiple single-cell omics (different molecular modalities assayed in different cells). Nevertheless, it is possible to pre-apply SCRaPL to non-matched data and then use another suite; as an illustration, we will perform an analysis on scMT data using SCRaPL followed by Seurat.

- In the case study on mouse embryonic stem cells, authors excluded the chromatic accessibilty. Why not using it to more clearly show the value of the method?

We did use SCRaPL also on chromatin accessibility, however the signal was weaker and we did not include it in the manuscript, we will now present these results as supplementary material.

- It would also be great if authors would use a different single-cell multi-comic data sets, using other dat modalities, e.g. CITE-Seq data. If this not possible, at least they should elaborate on which omics SCRAPL can handle, what would be the noise models for different data types, etc.

We have started analysing a joint scATAC-scRNA- seq data set generated using the new 10X commercial platform, and will add the results of this analysis to the revised manuscript. We will also expand the description of the suitability for different data types.

*- As the authors acknowledge, computational burden is high, which presumably limits scalability. Are authors able to further explore this (scalability on Insilico data)? Or how complex is adopting the variational inference method suggested? I appreciate that the variational inference implementation might be out of the scope of this paper, though.

• It is a pity that the method is in Matlab. Nearly no-one in single-cell omics use Matlab. Our own lab is largely invested in this topic and we do not even have Matlab licenses. I strongly encourage authors to implement their method in e.g. R or python, ideally compatible with the broadly used 'suites' (Seurat, OSCA, and scanpy,...).*

We are addressed these two comments jointly by re-implementing SCRaPL in Tensorflow probability (Python based), which allow us to leverage powerful libraries for variational inference. We hope that this will lead to a substantial increase of scalability, providing the possibility of running on thousands of cells / genes in under one hour (results will appear in ).

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Reply to the reviewers

First of all, we would like to thank the editor and all reviewers for the effort to evaluate our paper in this difficult era of COVID-19.

Reviewer #1

(Significance): Overall, this manuscript is very clear and easy to follow. The manuscript could be improved by making the following changes:

We thank the reviewer for the favorable comment and will revise the manuscript according to the suggestions.

Reviewer #2

(Evidence, reproducibility and clarity): The use of genetics is particularly impressive but the lack of major discoveries dampens the enthusiasm. Additional efforts to mechanistically define wave initiation and wave propagation would significantly improve the impact of the manuscript. Moreover, some of the conclusions are not fully supported by the data and require further experimentation and/or analysis.

We admit that marked redundancy of function among the EGFR ligands and their essential roles in cell growth prevent us from obtaining very clear results. Considering the importance of EGFR ligands in biology, we believe, our observation will give invaluable suggestions to whom wishes to clarify the roles played by EGFR-family protein in other biological contexts.

(Significance): While it is known that ADAM17 is critical to process EGFR ligands, the specific or redundant roles of different ligands remains an open question. The authors find that all ADAM17 ligands contribute to ERK signaling waves but may have specific contributions to other phenotypes. This work would be of interest to the signaling dynamics, epithelial and developmental biology communities.

We thank the reviewer for the favorable comment.

Reviewer #3

(Evidence, reproducibility and clarity): Overall, this study is carried out with a high degree of rigor and technical excellence, with clear reporting of experimental details and replication. The writing and figures are very clear, and there are no obvious technical problems. However, there are some areas in which the strength and clarity of the conclusions could be strengthened by relatively simple experiments.

We thank the reviewer for the favorable comment. We have already performed some of the experiments suggested by the reviewer. As the reviewer might have anticipated, co-culture with the wild type MDCK cells helps mutant cells to survive. We believe we could propose a clearer model in the revised paper.

(Significance): This study definitively establishes the role of 4 EGFR ligands in the generation of ERK activity waves in MDCK cells. While other studies, including some from the senior author's lab, have strongly indicated that EGFR autocrine signaling is important for these waves, this study goes further in comparing the roles of these ligands using knockouts to unambiguously establish the autocrine factors involved. Others who use this common experimental system (MDCK) to study epithelial dynamics will find this study of great interest. A wider audience of those who work on EGFR-mediated signaling will also find the data quite fascinating as an example of the complex relationship between ERK activation and its downstream effects. The technical excellence of the paper will make it a must-read for those in these fields. However, there are some factors that limit the scope of the significance. MDCK cells are an important experimental model system but differ in substantial ways from other epithelial cells, particularly in the expression of EGFR ligands. Because different ligands such as amphiregulin dominate in other systems (as noted by the authors, and PMID 27405981), the ability to extrapolate from these findings to other cell types is somewhat limited. Also, the paper avoids addressing the major question of how ERK waves relate to collective migration rate. From the data presented it is clear that this relationship is complex; for example, bath application of the ligands restores a high migration rate but not ERK waves. Given this lack of a clear relationship it is an understandable decision to leave this question for future work; however this does limit the conclusions that can be drawn from the study.

We completely agree with the reviewer’s view. It is uncertain to what extent the observation with MDCK cells can be generalized to other cell types. We also admit that the conclusion is not very simple because EGFR signaling is required for various cellular functions including cell survival and migration. Even though the gene editing becomes so easy, it is still labor consuming work to knock out many genes in a single cell line with extensive characterization. We believe the data shown in our work will provide a basis for the understanding of EGFR ligands.

Reviewer #1

For Fig 1F, 3 individual experiments should be conducted to confirm results.

We will follow the reviewer’s suggestion and repeat the experiment.

For Fig 1G, could the authors please show the original western blot data in full rather than just the densitometry graphs?

We did not show just for the sake of brevity. We are happy to will include the images as a supplementary data.

The authors should explain the origin/phenotype of MDCK cells for those who are not familiar with the cell line.

We will modify the text according to the reviewer’s suggestion.

The authors should give a future outlook/direction for future experimentation to further confirm redundancy in EGF ligands in the propagation of ERK activation waves.

We will discuss on the redundancy in other cell types based on available NGS data.

Some mention of the use of biosensors in the abstract and introduction is recommended as this is a major part of the experimental work.

We will refer to the biosensors in the abstract and introduction.

Reviewer #2

There are conflicts with some of the conclusions made about ligands. dEGFR cells have basal ERK activity as high as WT which argues against EGF being responsible for basal ERK activity. Further, basal ERK activity was not rescued by restoration of EGF in the 4KO-EGF cells. The authors should address this discrepancy.

We agree that some new questions have arisen from our observations. The discrepancy of the phenotypes between dEGFR cells and dEGF cells is an example. We are currently establishing dEGF cell lines, in which different genomic sequences of the EGF gene were targeted. We have already started to develop these cell lines and will obtain them within a month. The result will provide some clues to answer the questions. However, even if we could not solve the question, we believe, it is worth reporting observations that could not be easily understood, because such questions are often leading to another discovery.

Besides the ones genetically disrupted in this work, other EGFR ligands seem to play functional roles given that dEGFR cells less migration and fewer ERK waves than 4KO cells. The authors could test if other ligands are upregulated in 4KO cells to compensate. On a similar note determining whether ADAM17 deficient cells are more similar to 4KO cells or dEGFR cells could provide some insight.

According to the reviewer’s suggestion, we will conduct qPCR of growth factors in mutant cell lines to see the expression levels of seven EGFR ligands might have changed significantly. At the same time, as the reviewer suggested, we will establish ADAM17 knockout cell lines and compare the phenotype with those of cell lines deficient from EGFR ligand genes.

• The authors propose that Nrg1 is responsible for ERK waves in QKO, 4KO, dEGFR, and 4KO-EGF cells but are limited in testing this due to Nrg1 being essential in 4KO cells. First, Nrg1 should have been deleted in TKO cells to confirm that it is only essential in the absence of the four EGFR ligands. Additionally, Nrg1 could be knocked out in 4KO-EGF cells to demonstrate the claim that EGF-induced ADAM17 cleavage of Nrg1 is responsible for ERK waves.*

We do not think the deletion of Nrg1 in the TKO cells will abolish the ERK activation waves because EREG in TKO cells could transmit the waves. To overcome the problem of cell growth, we will try to obtain 5KO cells by Cre-induced deletion of NRG1 in 5KO-loxP-NRG1 cells, wherein EGF is supplied exogenously. We already had preliminary data suggesting that co-culture with wild type MDCK cells helps 5KO cells grow.

The authors state that ERK activation waves are important for collective migration and seek to understand the roles of each EGFR ligand, but despite measuring migration and properties of ERK activity, there is very little analysis or commentary on the relationship between the two. The ability of HB-EGF to restore migration without ERK waves suggests that waves are not required per se. It is interesting to note that with restoration of ligands, migration is higher than WT but ERK activity is lower.

We refrained from spending much space about the essential role of ERK activation waves in collective cell migration, because several papers have already described this issue.

Probably, we should have spent more space to emphasize that the collective cell migration is comprised of at least two different phenomena. The migration of leader cells and the follower cells. The ERK activation waves are essential for the follower cells but not the leader cells. In 4KO cells, both the leader cell and follower cell migrations are impaired. We showed that GFs expression restore the leader cell migration, but not the follower cells. We will revise the text to include this issue.

It is suggested that the total amount of EGFR ligands may be the primary determinant of migration, but deletion of TGFα alone causes a significant decrease in migration comparable to the DKO cells. TGFα has the lowest expression of the four ligands studied but is the only ligand to have a significant impact on migration in the single knockout context, which disagrees with that conclusion.

Each EGFR ligand has different affinity to EGFR, which makes it difficult to link the mRNA levels directly to the effect of each EGFR ligand. We will modify the discussion to include this argument.

Other:

Fig. S3B needs clarification that the WT (black) and 4KO (green) did not receive a stimulus.

We will follow the reviewer’s advice.

Reviewer #3:

The experiments in Fig. 5 are undertaken with the purpose of assessing whether NRG acts as an additional ligand that mediates the residual ERK waves in 4KO/QKO cells. However, this question is never addressed in the NRG/4KO cells. While it might be challenging due to the proliferative defect, it seems important to attempt this experiment in some way; measuring the ERK waves for these cells would establish whether all of the critical autocrine factors have been identified. Can the proliferation be rescued by application of high amounts of growth factors?

This question is similar to a question raised by reviewer #2.

To overcome the problem of cell growth, we will try to obtain 5KO cells by Cre-induced deletion of NRG1 in 5KO-loxP-NRG1 cells, wherein EGF is supplied exogenously.

The bath exposure to EGFR ligands shown in Fig. S3A is an important experiment, but it is surprising that ERK signaling is not maintained under these conditions. Is this due to depletion of the added ligands, perhaps locally? Or is the intermittent nature of paracrine signaling needed to maintain ERK activity? These possibilities could be distinguished by checking whether the added EGF or the other ligands are depleted after several hours, or by restimulating with a new bolus of ligand after several hours.

We thank the reviewer for this invaluable suggestion. We will conduct the experiments suggested by the reviewer.

The connection between ERK activity and migration is somewhat confusing. It would be helpful to show the dose sensitivity of migration to a MEK or ERK inhibitor. Are other pathways downstream of EGFR such as PI3K involved in the autocrine-mediated migration? This could also be established with the appropriate inhibitors.

We should have spent more space to emphasize that the collective cell migration is comprised of at least two different phenomena. The migration of leader cells and the follower cells. The ERK activation waves are essential for the follower cells but not the leader cells. In 4KO cells, both the leader cell and follower cell migrations are impaired. We showed that GFs expression restore the leader cell migration, but not the follower cells. We will emphasize this issue in the revised manuscript.

Reviewer #1:

Line 47 in Abstract should read "Aiming for" not "Aiming at".

We have corrected the mistake as suggested.

Some in the field call fluorescence lifetime microscopy "FLIM", you could adopt the same wording in your manuscript to attract more readers.

We have included FLIM according to the reviewer’s suggestion.

Reviewer #1 :

Figure 1D, the images should be presented using the same scale for both the EKAREV and EKARrEV constructs so that they can be directly compared.

Because the basal FRET/CFP ratio is significantly different between EKAREV-NLS and EKARrEV-NLS, the changes during mitosis become unclear if we applied the same scale. This figure is prepared to show the reactivity to Cdk1 during mitosis; therefore, we believe the current scale is better for presentation.

The names QKO and 4KO are a bit confusing. Could the authors please change the naming of the knockout cells so that readers understand that QKO and 4KO are two separate cell types? Perhaps instead of 4KO use FKO for "full knockout" or something similar. The 5KO line might also need to be named something else if you change to FKO.

We have discussed this issue with the co-authors, but could not reach a better idea. Instead of changing the names, we will include a detailed explanation for each cell line.

Reviewer #2:

The interpretation of the RA-SOS coculture experiments is confusing. Based on the author's reasoning, I would expect ADAM17 shedding in the RA-SOS cells to trigger signaling at the interface of both WT and 4KO cells but the 4KO should be unable to propagate the wave farther away from the interface. This does not seem to be the case. Do RA-SOS ADAM17KO cells still trigger waves of ERK signaling in the WT cells? Do ADAM17KO cells behave as the 4KO cells in this coculture system?

Probably, the reviewer misunderstood the method. The GF-less 4KO cells were co-cultured with wild type cells harboring the RA-SOS system. We will describe more in detail to avoid misunderstanding.

Finally, the growth curve in Fig. 5B indicates that 5KO-loxP-NRG1-CreERT2 cells are viable for about two days after Cre induction. The authors could perform a confinement release assay of these cells 1-1.5 days after Cre induction to look for further reduction of ERK waves and migration to demonstrate the role of Nrg1.

This experiment may not be necessary. It is clear that NRG1 is required for the survival of 4KO cells. The reason why cells are still alive 1 to 2 days after 4-OHT application is simply because NRG1 protein is remaining. The interpretation of the results would be difficult during the phase of NRG1 reduction.

In Fig. 1G, the normalization of all WT pERK samples to 1 artificially lowers the variance to zero when performing the T-test.

For the comparison of immunoblotting data derived from independent experiments, the signals must be normalized to the control. We believe the use of pERK/ERK of the wild type cells as the control is reasonable for this experiment.

Author Response:

Reviewer #1 (Public Review):

I think that it is important for the authors to consider that for most (if not all) SARS-CoV-2 variants, increased transmissibility of the virus has not been directly demonstrated. While it is clear that numerous variants have emerged and will continue to emerge, the rapid upsurge of cases with a variant may be related to many factors (e.g. host susceptibility due to immunity or genetic factors, virus seeding events, predominant replication in particular age cohorts, ...) that cannot simply be captured as "transmissibility of the virus". Even for B.1.1.7 and D614G mutants, the direct evidence of increased transmissibility in humans is extremely limited if available at all. Most studies erroneously simply take the increasing occurrence of particular lineages or mutations in sequence databases as a measure of increased "transmissibility", which should be avoided, also in the present manuscript. Increased transmissibility can only be derived from field studies where transmission is measured directly.

We thank the reviewer for pointing out that this is a controversial area. We have adjusted the text throughout to accommodate the fact that the published evidence of increased transmissibility/infectivity is not definitive.

On several occasions in the manuscript (e.g. page 3, page 4 L58-59, page 9 in submitted version), the authors seem to suggest that changes that lead to increased "transmission" or binding affinity and changes that lead to immune escape are mutually exclusive. But the opposite might be true. Viruses may escape from antibody-mediated immunity by amino acid substitutions in linear or structural antibody-binding epitopes. However, viruses may also escape from antibody-mediated immunity through altered protein density on virion surfaces (e.g. less Spike) and/or altered affinity, making it harder for antibody to inhibit virus attachment. As an example, increased affinity may facilitate virus replication with less dense Spike protein, allowing more effective antibody escape. Lower affinity but more dense coverage of Spike may reduce accessibility of critical virus parts by antibodies. Several viruses are known to escape from antibody-mediated neutralization through changes in affinity/avidity.

We agree with this point and have modified the text to avoid implying that increased transmissibility and antibody escape are mutually exclusive.

In relation to the previous point, it is important that authors mention some limitations of the present work in the discussion. SARS-CoV-2 virion attachment to cells is not just a matter of spike protein binding and certainly not of a monomeric RBD. Escape from antibodies and effects on affinity are heavily influenced by the entire (trimeric) spike protein, including its N-terminal domains. Such components are not taken into account in the present experimental designs, and this should be discussed, as e.g. the NTD can be important in attachment and antibody-mediated neutralization.

We thank the reviewer for this suggestion. We have added an appropriate caveat to the Discussion.

The authors suggest that the pandemic virus as it spread across the globe initially did not have "optimized" affinity. However, in the first months of the pandemic, there was relatively limited variation in spike protein sequences. The major variants emerged only later and mostly in areas where population immunity was building up. Again, this begs the question whether natural selection is occurring as a consequence of receptor affinity or immune escape?

We thank the reviewer for making this point. However, we do not think it is that surprising that it took a few months for the first Spike variants to be detected, for the following reasons. Firstly, the number of infections would have been relatively low early in the pandemic and SARS-CoV-2 replicates with a comparatively low error rate for an RNA virus. Secondly, the introduction of strict non-pharmacological measures (social-distancing etc), which would have increased the selective pressure on the virus, was somewhat delayed. Thirdly, it would take some time for any variant that emerged by chance to expand sufficiently to be detected by sequencing. While there is evidence suggestive of broader immunity in populations were the Beta and Gamma variants emerged, which we cite, we are not aware of evidence of widespread immunity in populations where the D614G, S477N and Alpha variants first emerged.

Reviewer #2 (Public Review):

Barton and colleagues investigated the effect of common SARS-CoV-2 RBD mutations and two ACE2 mutations on the RBD/ACE2 interaction. They concluded that the N501Y, E484K and S477N increased receptor binding while the K417N/T had the opposite effect. Double and triple mutants were also included. The ACE2 mutations (that are rare in the human population) also increased binding to most RBD mutants. The study is well-performed and written clearly.

The primary conclusions of the manuscript were supported by the results. However, the interpretation was too speculative. In the abstract (lines 14-17), the authors suggest that the 501 and 477 mutations enhance transmission solely based on data on the RBD-ACE2 interaction. It is unknown whether increased affinity to ACE2 is beneficial for transmission. In addition, increased RBD affinity to ACE2 does not mean that the whole spike or virus particle also binds stronger to ACE2. Lastly, increasing ACE2 affinity does not necessarily increase binding to cells (for example S1A binding to sugars or spike abundance can also influence this).

We agree that it would be inappropriate to assume, based on our affinity/kinetic studies alone, that 501 and 477 enhance transmission. That is why the relevant sentence in the abstract starts with the phrase, “Taken together with other studies”. We summarises the evidence from these other studies in the Discussion. We acknowledge that we have not examined the effects of the mutations on binding of the whole Spike protein to ACE2 or viruses to cells, and have added a suitable caveats to the Discussion.

The overall impact on the field will be limited as there is substantial overlap with already published studies. The observation that the N501Y and E484K increase receptor binding while the K417N/T mutations decrease binding was already made prior by Laffeber et al (2021; J Mol Biol). Laffeber et al also investigated double and triple mutants and came to similar conclusions. Liu et al (2021) confirmed that the N501Y increases binding whereas the K417N/T have opposing effects (Liu et al., 2021 mAbs). The observation that the Y501N increases ACE2 affinity has been made by several groups (e.g. Liu et al 2021 Cell research; Starr et al 2020 Cell).

We thank the reviewer for highlighting these addition studies, two of which are very recent. We have now cited these studies.

Starr et all 2020 was a high throughput study in which the affinity measurements were semi-quantitative, and no kinetic analysis was performed. Liu et al (2021) and Laffeber et al (2021) were performed at 25 C and without rigorous controls for mass-transport and protein aggregation. Liu et al (2021) did not report kinetic measurements. Their results are broadly consistent with ours but their affinity and kinetic measurments are ~ 10 fold different. While we accept that some of the measurements of the effects of mutations have been made before, our measurements of affinity and especially kinetics are performed more rigorously than in previous studies and, for the first time, at a physiological temperature (37 C). Thus, the affinity and kinetic data that we have obtained for single and combinations variants are more definitive. As noted in our Discussion there is a wide variation in reported binding affinities and kinetics in previously published studies. We think the comprehensive data that we report here, the same robust method to measure binding properties of all these variants, adds significant value.

Reviewer #3 (Public Review):

[...] 1) The ACE2 receptor exists naturally as a dimeric form and the RBD is a component of the SARS-CoV-2 spike trimer. The assay format here was monomeric RBD binding against monomeric ACE2 throughout this study. While the measurements are indeed carefully executed and under more physiological conditions than many other reported studies, the authors should discuss potential avidity effects, the consequences of mutations on the accessibility of the RBD in VOC versus wildtype, and impact of other domains such as the NTD, in the context of their monomeric ACE2 measurements with isolated RBD here.

We thank the reviewer for raising this issue. We have added a section to the Discussion addressing these important points.

2) As shown in Figure S2, RBD WT, K417N, K417T, KN/EK, KT/EK, and S477N, the ~30kDa monomeric proteins were flanked by additional ~60kDa bands (which correspond to the smaller peaks to the left of the main peaks) some of which bleed through to the main fraction to different extents, whereas RBDs SA, UK1, UK2, BR, and E484K, do not seem to have as much or any of these extra species. Can the authors comment on whether these contaminants are RBD-dimers as observed before (Dai et al. 2020)? If yes, would such dimers affect the affinity and kinetics?

We thank the reviewer for pointing out these larger ~60 kDa bands in some RBD preps. We think that it is unlikely that these are RBD dimers as these are reducing gels. The strictly monophasic kinetics of all RBD preps, also argues against this being an RBD dimer. We have confirmed by densitometry that the larger band comprises less that 5% of the protein in all the preparations. This will have only a minor effect on estimated of RBD concentration. We have added this information to the Figure S2 legend.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

**Summary:**

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2.

In this paper the authors used a targeted approach to identify rare mutations in a cohort of glioma patients. Using this approach they identified a recurrent mutation in the TOP2A gene encoding for Topoisomerase 2A, and suggest that this mutation creates a more effective protein, binding DNA strongly and maybe more enzymatically active. RNAseq analysis of TOP2A WT and TOP2A mut tumor samples suggest different transcription patterns and points to possible splicing defects. The most recurrent variant (E9448Q) is described in depth and some experimental information shows this variant might be a gain-of-function mutation.

**Major comments:**

• Are the key conclusions convincing? The validation of both the methodology and the presence of never described TOP2A variations in HGG is done quite successfully. Interesting evidence about relevance of the most frequent mutation is provided. However, besides having computational and biochemistry assays performed, lack of details about in vitro experiment statistics (no p-values are provided in figures 4 and 5, neither sample size, repetitions) weakens the conclusions claimed by the authors about the properties of the mutated topoisomerase. Ad. In the revised version we provided more details about in vitro experiments, including statistics when is applicable, sample size and a number of repetitions. In the fig. 4 we show the results of two repetitions (so we can’t calculate statistics) but I would like to stress that we tested independently two fragments of the protein and the results were similar, so our conclusion was justified. However, we do agree with the reviewer that a statistical analysis of those biochemical tests is required. We already started to produce a new batch of recombinant proteins and we will add repetitions to reinforce our claims. We will provide statistical analysis details once all experiments are performed. __

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Claims about E948Q variant function should be revised. Data is not presented in a convincing way, plus there is ambiguous language used from the results ("We conclude that the E9448Q TOP2A protein is functional, and MIGHT have a higher activity than the WT protein") to the rest of the paper where they strongly support the claims about the TOP2A activity. Ad. We will provide more data on biochemical features of the TOP2A variant to confirm the impact of the E948Q substitution on enzyme activities, which would allow more strong conclusions. This will present our results in more convincing way. A language of the manuscript has been critically revised and modified (see a version with tracked changes).

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. In line with the presented data in the paper, additional experiments that show catalytic changes of the E9448Q variation must be added. It is shown that there are differences in the DNA binding capacity by EMSA compared to the WT form, however, the DNA supercoil relaxation activities is not that different, at least the way the results are presented. The authors suggest that TOP2A mutation is a driver mutation but no validation in vitro of this claim is shown. Can this mutation alone or in combination with e.g. tumor suppressors transform normal cells to cancer cells? Do cell lines expressing this mutation (compared to parental TOP2A wt expressing cells) display increased transcription? Increased invasion? Ad. In the revised version we moderated our conclusions and we do not state that the mutated TOP2A is an oncogenic driver. We suggest this mutation (and possibly other TOP2A mutations, as we analyzed the impact of other variants on the TOP2A protein function) contribute to gliomagenesis. This conclusion is based not only on the changes in biochemical properties, but also on the observation of the impact of the mutation of transcription and patient survival. We expanded the analysis of TOP2A mutations and expression levels on TCGA datasets and those new results support our conclusions about a pathogenic nature of TOP2A overexpression and mutations (the supplementary fig.4). We believe in such situation, there is no rationale to make a classical oncogenic driver experiment.

Due to a rarity of the TOP2A mutations it is impossible to find a patient derived cell line with such defect. We attempted to overexpress TOP2A in glioma cells but apparently there is some autoregulation preventing overexpression of this protein is cells with endogenous TOP2A expression. Therefore, we can’t verify if cell lines expressing this variant (compared to parental TOP2A wt expressing cells) have increased transcription. Moreover, such experiments are costly and require more time investment for substantial experiments

I would like to stress that modeling some events in cell cultures is difficult and we found in GBMs the link between the mutated TOP2A and increased transcription along with decrease of splicing factors expression.

We have attempted to make CRISPR/Cas9 mediated knock-in in glioma cells but without success. This is a difficult and time consuming procedure. Although in principle, we agree on the rationale for such experiment, we think that the current data are consistent and convincing. If reviewers find it necessary we may attempt to create glioma cell lines with TOP2A knock-out and overexpression of the mutated TOP2A gene and study it functionally, but it would require more time.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. If the authors can complement the already presented in vitro experiments with additional ones supporting their hypothesis, this should be feasible. The authors can use patient derived glioma cells or glioma cell lines manipulated to express either the parental TOP2A wt enzyme or the identified mutated form. __Ad. Due to a rarity of the TOP2A mutations it is impossible to find a patient derived cell line with such defect. Our findings partly relied on frozen historical samples, so it is not possible to develop patient-derived cell lines. As mentioned above, we can create a TOP2A knock-out cell line and overexpress a wild type or mutated version but there is no certainty that TOP2A deficient cells would survive (this is an essential enzyme) and such manipulation would be feasible.__

• Are the data and the methods presented in such a way that they can be reproduced? Yes, the authors provide a quite detailed explanation of the methods implemented to reach each one of the results they are presenting.

• Are the experiments adequately replicated and statistical analysis adequate? No, there is no information about the statistical analysis or number of replicates in any of the in vitro experiments performed. This information should be added to the manuscript.

Ad. In the revised version the requested information was added where was possible and additional repetitions for biochemical experiments are currently in progress.

**Minor comments:**

• Specific experimental issues that are easily addressable.
• Are prior studies referenced appropriately? Yes, authors clearly address the state of the art regarding previous NGS methodologies and let us know the advantages and novelty of their approach.

• Are the text and figures clear and accurate? There are some discrepancies between the strength of the language used in different sections of the paper to refer the conclusions they can infer from the results they are showing. While they are all valid, authors should revise it. Ad. The text of the manuscript has been unified and revised.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? First of all, describe the statistical analysis used in every figure, include number of biological and technical replicates. I would also suggest to change the title or the scope of the discussion, there is too much focus on the TOP2A in the introduction, neglecting all the technical NGS work that actually lead to several new variants being described. This may be confusing when it collides with a conclusion that is heavily focused on the first half describing potential implications of at least another 3 proteins where genetic alterations were described. Given the fact there is not much experimental work that shows TOP2A mutations relevance in HGG or strong enough evidence of the variant's function I would suggest to change a bit the scope of the title. Ad. The description of the results and discussion have been revised to include additional data/discussion on technicalities and other finding not related to TOP2A. We performed additional computational analyses of TOP2A expression/mutations in the TCGA datasets. We believe that the planned experiments on genetically modified cell lines would provide additional support for our claims. We think that in the revised version a balance between landscape/NGS content and TOP2A content is well balanced.

Reviewer #1 (Significance (Required)):

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. The authors describe a methodology that proved to be sensitive and specific enough in order to allow them to detect rare genetic alterations in patient glioma samples. This information could be valuable to describe new driver mutations or infer in genetic pathway alterations that could be potential therapeutic targets. As the authors state at the beginning of the paper, given the poor therapeutical approaches existing for HGG currently, information of this kind could still be highly useful and provide a better outcome to a specific cohort of patients.

On a personal note, I think there is too much speculation about how TOP2A mutations could be interesting from a biological point of view (authors referred to evidence about implications of this mutation in other forms of cancer) but since no experimental validation is provided in glioma cells, it is difficult to conclude that this enzyme gain-of-function mutation could have a relevant role in HGG and thus make these variants a potential therapeutic target. There are no experiments conducted in glioma cells that express TOP2A variants, it would be interesting to see if it has an effect in the migratory/invasive phenotype like described in other cancer types or like it is suggested by analysis of the genetic pathways activated in the HGG patients samples harboring TOP2A mutation. In addition, there is no evidence of the TOP2A mutations possible role as a driver mutation, which is an interesting aspect that could be further explored from both a computational and an experimental approach.

Ad. As mentioned above, there is no glioma cells that express TOP2A variants and we are not convinced that such experiment will be feasible taking into account an essential role of TOP2A. We will attempt to perform experiments with CRISP/Cas9 knock-in cell lines and functional validation, but until now we did not accomplish knock-in in glioma cells. We will try to knock-out the endogenous TOP2A using CRISPR and express a TOP2A WT or E948Q variant from plasmids encoding these proteins, but we can’t predict if TOP2A KO cell would survive. If we manage to produce such cells, then we will investigate proliferation, migration and invasion of cells expressing TOP2A WT or mutated variant.

We do agree with the reviewer that our previous conclusions were too strong, and in the revised version we moderated our claims. We do not say that the mutated TOP2A is an oncogenic driver. We suggest this mutation (and possibly other TOP2A mutations, as we analyzed the impact of other variants on the TOP2A protein structure) contribute to gliomagenesis.

__Data on the Fig. 1A suggests that TOP2A has a mutational hotspot in the position E948Q in our dataset. In the revised version of the manuscript we have extended RNA-seq analysis of our datasets and TCGA PanCancer datasets to search for TOP2A mutations/ overexpression. We found that another computational prediction using CADD algorithm strongly confirms that TOP2A E948Q is in the top 1% of most deleterious variants in the human genome (CADD score >20). This results was added to Supplementary Table 2.__

• Place the work in the context of the existing literature (provide references, where appropriate). The quality of the paper is high and in line with other studies in the literature that perform genome and transcriptome analysis of tumor samples. It is only the experimental validation that is lacking data supporting the "in silico" findings. Ad. We would like to point that we provided the results of experimental, biochemical validation (2 assays) showing that the variant TOP2A proteins have different properties. The associations of transcriptional dysregulation in variant TOP2A bearing gliomas was not a in silico prediction but the result of the analysis of real tumor samples.

As stated above, we are ready to perform further biological validation if the editors find it necessary.

• State what audience might be interested in and influenced by the reported findings. Computational biologists are the right audience to target this paper. If additional experimental work further validating their initial bioinformatic findings is added to the manuscript then probably a wider population could be targeted.

Ad. As stated above, we are working now on providing more replicates of biochemical assays and we are ready to perform further biological validation if the editors find it necessary. I would like to stress that genome editing by knock-in is not always possible/feasible, and these type of experiments is time and money consuming.

• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Brain tumors, immunotherapy, cancer stem cells, tumor microenvironment, tumor heterogeneity. I do not have sufficient expertise to evaluate the bioinformatic analysis and software/programs used to analyze the NGS data.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

By exon targeted resequencing of 664 genes frequently mutated in cancer, authors identify novel mutations associated to Glioma in a cohort of 182 Polish and Canadian samples. Most of these novel mutations have been identified as potential rare germline mutations, somatic mosaicism or loss-of-heterozygosity variants. Among them, authors focus on mutations associated to the TOP2A gene, which encodes one of the two Type II topoisomerases paralogs present in humans. By a limited number of in vitro experiments, authors conclude that TOP2A recurrent variant E948Q, displays increased binding to DNA and topoisomerase activity. Therefore, authors suggest that the TOP2A E948Q variant is a gain-of-function mutation.

**Major comments:**

• Authors show an interesting plethora of new exon mutations associated with High Grade Glioma. Nevertheless, the characterization of TOP2A E948Q variant, which is the main focus of the study, although very interesting and potentially clinically relevant, remains incomplete. Association of the TOP2A E948Q glioma variant with a gain-of-function mutation would require to improve the statistical power of the presented experiments (increase number of replicates). With the existing experimental evidence, the increased DNA binding and activity of the TOP2A E948Q variant should be considered as preliminary, especially in the case of 431-1193 aa fragment. I would consider mandatory to increase experimental replicates and to analyse statistical significance in the case of DNA binding experiments and DNA relaxation assays with the TOP2A 431-1193 aa fragment. A more detailed biochemical characterization should be performed. A titration of different amounts of protein should be included in these experiments, and at least two batches of purified proteins should be analysed. Decatenation assays should also be performed to characterize the activity of the mutant protein in more detail. Recapitulation of DNA binding and activity results with other TOP2A variants obtained in this study will significantly reinforce authors claims too. This improved biochemical characterization should not take longer than two months.

Ad. We would like to stress that while two replicates are presented, we were testing two forms of TOP2A proteins and the results were similar, confirming our conclusions. But we agree that additional replicates would strengthen our claims. Therefore, we are in the process of producing another batch of recombinant proteins to increase a number of replicates and calculate statistics for the biochemical assays (binding and relaxation assay). We will perform titration of different amounts of the protein using two batches of purified proteins.

The occurrence of other TOP2A variants is low (identified in only a single patient sample), therefore we will perform experimental validation only for E948Q. However, we performed additional computational analysis for other TOP2A variants showing the influence of the substitution on DNA binding by docking the DNA fragment into TOP2A binding pocket (Supplementary table 4).

• To increase the significance of the results, I would encourage authors to include experiments showing the functional impact of this TOP2A mutation in cells. The connection with transcriptomic alterations is merely correlative, and would be greatly strengthened by functional experiments in cellular models. To draw definitive conclusions regarding the changes in transcription, I would encourage authors to complement the results with experiments that point to the physiological impact of TOP2A variants within the cell. Overexpression of WT and E948Q variants in a cell model and transcriptomic analysis would be desirable, but validation in these experimental models of some of the target genes identified as deregulated in patients could suffice. These experiments could be accomplished in no more than 3-4 months.

Ad. We agree that the connection of the TOP2A mutation with transcriptomic alterations is correlative, and would be greatly strengthened by functional experiments in cellular models. If we develop a TOP2A E948Q knock-in cell line or TOP2A KO cell line with E948Q over-expression, we are planning to evaluate transcriptomic changes on selected genes by qPCR or whole transcriptome by RNAseq. We estimate that developing a stable CRISPR/Cas9 cell line may take up to 6 months.

We provided additional results showing that the connection of the TOP2A mutation with transcriptomic alterations may be due to different expression of splicing factors (Supplementary Fig. 6).

• Some of the methods are not presented with sufficient detail. Regarding the DNA and RNA sequencing experiments, I consider necessary to specify the DNA fragmentation method, reference for the indexed adapters and ligation and amplification procedures (ligase reference, number of PCR cycles, etc). It would be helpful to clarify or reference which are the "special oligonucleotide probes" that are mentioned. Finally, a reference for the "special beads" and final amplification number of cycles is needed. The sequence of primers used for TOP2A cloning and mutagenesis should be included. The reference for the "site mutagenesis kit" used is missing. When studying the survival rate of glioma patients depending of TOP2A expression levels, it should be clarified what is considered HIGH or LOW expression (i.e: which percentiles are used).

Ad. We expanded the description of methodological aspects of DNA and RNA sequencing experiments. This description was revised and more details are provided in the revised version. Regarding cloning and mutagenesis, we added a table with primer sequences (Supplementary Table 5). We did not use any kit for cloning and mutagenesis. Standard methods and primers with modified nucleotides were used.

__We have included information about the partitioned groups in the survival analyses in the figure 2 caption. “D - Kaplan-Meier overall survival curve for patients with high (> TOP2A mRNA median expression x 1.25) or low (- There is a major concern about how the experiments are replicated and about the statistical analysis, which is inexistent in some cases. Indeed, Figures 4 and 5 do not present any statistical analysis, it is therefore hard to draw any conclusion. In Figure 4b, the results for the 890-996 aa fragment looks qualitatively clear, but this is not the case for the 431-1193 aa fragment. More replicates and statistical analysis are mandatory, together with a protein titration. The replicates should be performed with at least two independent batches of protein purifications. The individual values of each experiment should be included in the graph to provide a better understanding of experimental variability. All this also applies to Figure 5.

Ad. We will increase a number of replicates for the binding and relaxation assay. We will perform a titration of different amounts of protein in these experiments using two batches of purified proteins.

**Minor comments:**

• The effect on transcription of co-occurrence of TOP2A mutations with other mutations could also be analysed with the already available data. Also, a more detailed analysis of genome-wide transcription could also be used to at least partially address the proposed hypotheses of increased transcriptional rate or splicing aberrations.

Ad. We don’t have enough samples with the TOP2A mutation to analyze the effect on transcription of co-occurrence of TOP2A with other mutations.

We addressed the hypothesis of increased transcriptional rate or splicing aberrations by performed additional analyses of RNA-seq data to confirm splicing aberrations. Indeed we found splicing machinery genes down-regulated in the E948Q TOP2A glioma samples (Supplementary Fig.6).

• There is no reference for the following argument "As the identified germline variants were exceptionally rare in the general population ... it is likely that these variants are pathogenic". I also find low number of references to support the suggested high frequency of altered genes in gliomas compare to other cancer types. I miss specific works relating TOP2 activity with transcriptional regulation.

Ad. The appropriate references are provided to back-up these statements.

• At several points in the text there are quantitative and comparative statements that should be backed up by the actual numbers (e.g. "The results of the targeted sequencing indicate a high frequency of altered genes", "The most altered gene was TP53, followed by IDH1...", "Other genes that were found to be frequently altered included KDM6B...", "These partial results combined with a low frequency of this variant in the Polish population suggest a somatic mutation"). The same thing applies to the co-occurrence of mutations, in which the percentage of co-occurrence and significance is not indicated. This lack of detail in the description is also observed in the description of the transcriptomic alterations in which no detail is provided regarding how many of the 105 analyzed samples correspond to low or high gliomas.

Ad. We apologize that the frequencies of mutated genes were not specified. This information is included in the main text of the revised version. We now provide a gnomAD frequency for all variants of interest, confirming the low frequency in the population (AF__ __

Regarding the total number of samples in the transcriptomic analysis, we provided an updated supplementary table covering also samples that were used for transcriptomic analyses (Supplementary Table 1).

• For TOP2A mutation analysis, sometimes is not clear when the analysis is done with the 9 mutated samples and when with the 4 recurrent TOP2A E948Q variants. For example, in figure 2b and 2c analysis are done with 9 samples while the figure 2e is based on the 4 E948Q variants. At least this is what I have deduced from the main text, it should be clarified in the figure legend).

Ad. This information has been included in the captions of Figure 2B, 2C and 2E and now we specify how many samples were used in each analysis.

• Fig1. In figure 1b it would be interesting to color-code patients by glioma grade. This would also apply to Figure S1a, S1c, 2a, S3 and S4. In figure 1D it would be very informative to distinguish mutations that passed the quality control or not with different colors.

Ad. Following reviewer’s suggestions, we have added this information, and oncoplot figures derived from the germline analysis have a distinct color for each glioma grade. In the figure 1D, all of the presented mutations have passed a quality control in terms of quality of sequencing. One additional criterion that was used for all genomic results (except some of the TOP2A variants) was a criterion of 20% variant penetration (20% of reads in the position had to come from the alternative allele). We corrected the description in the Supplementary Table to “passed 20% penetration criterion”. The rationale behind this criterion for TOP2A variants was a fact that for one of the E948Q samples it was ~13% and we didn’t want to lose this sample from the analysis due to rarity of the mutation.

• Fig2. In figure 2b and 2c the statistical significance of differences between TOP2A and the rest of genotypes should be included. Looking at Figures 2d and 2e it looks surprising how similar is the overall survival of HIGH TOP2A mRNA expression (500 days, fig 2d) with the overall survival of the TOP2A WT samples (400 days, fig 2e). Here a I would include a graph that summarizes the TOP2A mRNA expression levels of each group in fig 2d and 2e.

Ad. We agree that median overall survival is similar comparing patients with high TOP2A mRNA expression to TOP2A WT patients in our cohort. It is worth noting, however, that both datasets were produced using different library protocols, and the methodology is different, so it can’t be expected the levels to be equal. We think that adding two more graphs, as suggested, would add another layer of information to this section of the analysis. We have included two boxplots depicting TOP2A mRNA RPKMs, and it is clear now that the medians of High TOP2 mRNA and TOP2A mutant (E948Q) are more closely related, despite the fact that we only have a few patients with the mutation.

• Fig3. It would be interesting to include the same simulation for the rest of TOP2A mutations as supplementary figure.

Ad. We agree that the other TOP2A SNPs could potentially affect DNA binding. We focused on the recurrent mutation and did not analyze those occurring in a single patient. In the revised version we included predictions whether these variants could affect TOP2A DNA binding. For WT TOP2A and variants, we calculated the Gibbs free energy (ΔG). This information can be found in Supplementary Table 4. We have extended description in the Results section: “The TOP2A E948Q substitution may affect protein-DNA interactions”

• Fig4 and Fig5. Include statistical analysis and dots representing individual replicates.

Ad. For Fig 4 we have two replicates for two protein fragments, so we can’t present statistics now. As mentioned above we are preparing a new batch of proteins and will make more repetitions of EMSA and relaxation assays. For Fig 5. we have 3 replicates but despite a trend there is no statistical significance. We intent to make more replicates and a separate protein preparation. After including additional repetitions we will present the results as dots representing individual replicates.

• Fig6. In Figure 6d I would increase the size differences in the dots representing the gene counts, as it is not easily perceived with current parameters.

Ad. The dot size in Fig 6d did not reflect the true meaning. To make it easier to understand, we changed a plot type to a barplot, which now represents the number of differentially expressed genes involved in each pathway.

• FigS2. In figure S2B, it would be informative to establish which dots are significatively above or below the diagonal.

Ad. The purpose of this figure was to show which oncogenic signaling pathways from TCGA cohorts were affected in our cohort. The pathway's size is a variable that is used to normalize the calculation (shown in abscissa axis in S2B). RTK-RAS and NOTCH pathways contain hundreds of genes, whereas other pathways, such as the NRF2 oncogenic pathway, contains only a few. On the other hand, we counted how many genes in each pathway in our cohort were mutated (shown in ordinate axis, S2B). We used logarithms in both axes for visualization purposes, but this has no effect on the enrichment of these pathways, which is shown in the color-coded legend.

• FigS3. How were the samples shown selected from the total?

Ad. In this plot we show only somatic variants that were found in at least two different patients. We apologize that this information was missing, and we have added it to the figure's caption.

• FigS4. I would include a line with the TOP2A mutation to have an idea of how these mutations are distributed between groups.

Ad. Based on the feedback of the reviewer, this figure has been modified and improved. A new row has been added to the figure, displaying TOP2A mutations alongside other highly frequent mutations in other genes.

Reviewer #2 (Significance (Required)):

In this work authors have identified new mutations associated to gliomas by targeted exome sequencing using an important cohort of 182 samples. Among these new mutations epigenetic enzymes and modifiers are found. These results potentially increase the repertoire of putative molecular targets for future cancer therapies. Authors focus in mutations associated to TOP2A gene, that provides stronger DNA binding and DNA relaxation capacity in vitro. Although further characterization is needed, tumours harbouring this kind of mutations could show higher level of sensitivity to TOP2 drugs, providing potentially interesting clinical implications. Although the link between TOP2A expression and cancer prognosis is well established, the relevance of specific mutations in still largely unexplored.

On one hand this work brings novelties in the field of Glioma providing a series of putative new players in the development of this type of cancer. Audience interested in basic or clinical aspects of these tumours would be a good target for this work. On the other hand, this putative gain-of-function mutation of TOP2A represent an interesting aspect for the DNA topology and topoisomerases field. Although, as stated above a more detailed biochemical and functional characterization would be required to draw the attention of this audience-

Scientifically, I have experience in the DNA topology and topoisomerases field, 3D genome organization and gene regulation. I have no experience in Gliomas or any other clinical aspect of cancer, so it is difficult for me to properly establish the potential impact of the newly discovered mutations. Technically I have no capacity to critically evaluate the aspects related to the targeted exome sequencing and the suitability of the analysis performed for mutation identification.

**Referee Cross-commenting**

I fully agree with the comments of the other reviewer, which are perfectly aligned with my own regarding the preliminary nature of the conclusions about the biochemical and functional characterization of the TOP2A mutations.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate). Please place your comments about significance in section 2. In this paper the authors used a targeted approach to identify rare mutations in a cohort of glioma patients. Using this approach they identified a recurrent mutation in the TOP2A gene encoding for Topoisomerase 2A, and suggest that this mutation creates a more effective protein, binding DNA strongly and maybe more enzymatically active. RNAseq analysis of TOP2Awt and TOP2Amut tumor samples suggest different transcription patterns and points to possible splicing defects. The most recurrent variant (E9448Q) is described in depth and some experimental information shows this variant might be a gain-of-function mutation.

Major comments:

• Are the key conclusions convincing? The validation of both the methodology and the presence of never described TOP2A variations in HGG is done quite successfully. Interesting evidence about relevance of the most frequent mutation is provided. However, besides having computational and biochemistry assays performed, lack of details about in vitro experiment statistics (no p-values are provided in figures 4 and 5, neither sample size, repetitions) weakens the conclusions claimed by the authors about the properties of the mutated topoisomerase.

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Claims about E948Q variant function should be revised. Data is not presented in a convincing way, plus there is ambiguous language used from the results ("We conclude that the E9448Q TOP2A protein is functional, and MIGHT have a higher activity than the WT protein") to the rest of the paper where they strongly support the claims about the TOP2A activity.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. In line with the presented data in the paper, additional experiments that show catalytic changes of the E9448Q variation must be added. It is shown that there are differences in the DNA binding capacity by EMSA compared to the WT form, however, the DNA supercoil relaxation activities is not that different, at least the way the results are presented. The authors suggest that TOP2A mutation is a driver mutation but no validation in vitro of this claim is shown. Can this mutation alone or in combination with e.g. tumor suppressors transform normal cells to cancer cells? Do cell lines expressing this mutation (compared to parental TOP2A wt expressing cells) display increased transcription? Increased invasion?

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. If the authors can complement the already presented in vitro experiments with additional ones supporting their hypothesis, this should be feasible. The authors can use patient derived glioma cells or glioma cell lines manipulated to express either the parental TOP2A wt enzyme or the identified mutated form.

• Are the data and the methods presented in such a way that they can be reproduced? Yes, the authors provide a quite detailed explanation of the methods implemented to reach each one of the results they are presenting.

• Are the experiments adequately replicated and statistical analysis adequate? No, there is no information about the statistical analysis or number of replicates in any of the in vitro experiments performed. This information should be added to the manuscript.

Minor comments:

• Specific experimental issues that are easily addressable.

• Are prior studies referenced appropriately? Yes, authors clearly address the state of the art regarding previous NGS methodologies and let us know the advantages and novelty of their approach.

• Are the text and figures clear and accurate? There are some discrepancies between the strength of the language used in different sections of the paper to refer the conclusions they can infer from the results they are showing. While they are all valid, authors should revise it.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions? First of all, describe the statistical analysis used in every figure, include number of biological and technical replicates. I would also suggest to change the title or the scope of the discussion, there is too much focus on the TOP2A in the introduction, neglecting all the technical NGS work that actually lead to several new variants being described. This may be confusing when it collides with a conclusion that is heavily focused on the first half describing potential implications of at least another 3 proteins where genetic alterations were described. Given the fact there is not much experimental work that shows TOP2A mutations relevance in HGG or strong enough evidence of the variant's function I would suggest to change a bit the scope of the title.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. The authors describe a methodology that proved to be sensitive and specific enough in order to allow them to detect rare genetic alterations in patient glioma samples. This information could be valuable to describe new driver mutations or infer in genetic pathway alterations that could be potential therapeutic targets. As the authors state at the beginning of the paper, given the poor therapeutical approaches existing for HGG currently, information of this kind could still be highly useful and provide a better outcome to a specific cohort of patients.

On a personal note, I think there is too much speculation about how TOP2A mutations could be interesting from a biological point of view (authors referred to evidence about implications of this mutation in other forms of cancer) but since no experimental validation is provided in glioma cells, it is difficult to conclude that this enzyme gain-of-function mutation could have a relevant role in HGG and thus make these variants a potential therapeutic target. There are no experiments conducted in glioma cells that express TOP2A variants, it would be interesting to see if it has an effect in the migratory/invasive phenotype like described in other cancer types or like it is suggested by analysis of the genetic pathways activated in the HGG patients samples harboring TOP2A mutation. In addition, there is no evidence of the TOP2A mutations possible role as a driver mutation, which is an interesting aspect that could be further explored from both a computational and an experimental approach.

• Place the work in the context of the existing literature (provide references, where appropriate). The quality of the paper is high and in line with other studies in the literature that perform genome and transcriptome analysis of tumor samples. It is only the experimental validation that is lacking data supporting the "in silico" findings.

• State what audience might be interested in and influenced by the reported findings. Computational biologists are the right audience to target this paper. If additional experimental work further validating their initial bioinformatic findings is added to the manuscript then probably a wider population could be targeted.

• Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Brain tumors, immunotherapy, cancer stem cells, tumor microenvironment, tumor heterogeneity. I do not have sufficient expertise to evaluate the bioinformatic analysis and software/programs used to analyze the NGS data.

SciScore for 10.1101/2021.07.21.21260691: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

However, this study also had several limitations. First, because we conducted a cross-sectional study, causality could not be determined. However, since it is theoretically unlikely that treatment interruption experienced by an individual will increase the COVID-19 infection rate in a region, we think it is likely that high regional infection rates cause treatment interruption. Second, we did not identify workers’ reasons for discontinuing treatment in this study. As discussed above, there are various possible causes of treatment interruption, which may vary by region. Third, we did not inquire about the diseases being treated. Treatment interruption may vary depending on the presence or absence of symptoms and the potential disadvantages of discontinuing treatment for a particular disease.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Reply to the reviewers

We are grateful for the constructive and highly supportive reviews provided by our Reviewers. We especially appreciate the efforts they have made to provide suggestions on how to make our revised manuscript even more robust. We have incorporated many of these suggestions into the revised manuscript that will post to Biorxiv and will be submitted to an affiliate journal. We have provided point-by-point responses to each Reviewer below each item (starting with Response: …), along with any changes made in response to that comment/suggestion (starting with In our revised manuscript, …).

Finally, we agree with all Reviewers that this work should be of broad interest to the molecular biology, cell biology, and parasitology communities. Our discovery that Plasmodium and two related genera have taken the unorthodox approach of duplicating their NOT1 protein, and that Plasmodium has dedicated it for its unique transmission strategy, is a fascinating adaptation of the use of this core eukaryotic complex. We believe that those that focus on diverse aspects of RNA biology, including RNA preservation/decay, the maternal to zygotic transition, translational repression, and beyond will find this work to be of interest and relevant to their own research questions.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

The manuscript „The Plasmodium NOT1-G paralogue acts as an essential nexus for sexual stage maturation and parasite transmission" investigates the two forms of NOT1 in rodent malaria parasites. The authors found out that the original NOT1 is crucial for gametocyte induction as well as transmission to the mosquito, they therefore renamed it NOT1-G. The paralogous proteins, on the other hand, appears to be crucial for intraerythrocytic growth, since it cannot be knocked out. The authors then investigated NOT1-G in more detail, using standard phenotyping assays. They found a slightly increased gametocytemia and a minor effect on transmission to the mosquito.

Response: In our submitted manuscript, we do focus on PyNOT1-G because of the exciting role it has for both sexes of gametocytes, which results in a complete defect in transmission to mosquitoes. Our investigations of what domains of PyNOT1-G focused on the most likely suspect: the putative tristetraprolin-binding domain (TTPbd). It was through deletion of this domain that we observed only a minor defect in the prevalence of infection of mosquitoes, indicating that the portion of PyNOT1-G that is required for transmission lies elsewhere (in part or in total). It is also important to correct Reviewer 1’s statement regarding the other (perhaps canonical) PyNOT1. To our surprise, PyNOT1 could be deleted, but resulted in a parasite that has an extreme fitness cost and a very slow growth phenotype. This is in stark contrast to other eukaryotes, where NOT1 is essential.

Reviewer #1 (Significance (Required)):

If the authors are able to provide convincing data that NOT1-G is indeed important for gametocyte induction and transmission to the mosquito, then the report would be of high significance for the malaria and molecular cell biology fields.

Response**: We have in fact shown this and more in the originally submitted manuscript, and thus we are grateful that Reviewer 1 considers this work to be of high significance in a broad readership (molecular and cell biology, parasitology). In our revised manuscript, we have added text throughout to make these results even more apparent and clear for the reader.

My expertise: molecular cell biology of gametocytes, translational regulation, parasite transmission

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

**Summary**

The manuscript by Hart et al. builds upon a fascinating finding presented in a previous manuscript by the same authors, in which they show that CCR4 seems to be able to associate with two members of the NOT1 family. In this work, the authors first re-annotate the two NOT1 paralogs in Plasmodium yoelii and then perform an in depth characterization of the role of NOT1-G during gametocytogenesis and early mosquito development. Using gene knockout and different genetic crosses, the authors show that NOT1-G is essential for male gametocyte development and leads to an arrest of development in zygotes arising from female gametocytes. Using RNA-seq the authors show that NOT1-G leads to lower transcript abundances, leading to the hypothesis that NOT1-G might be involved in preserving mRNAs in a larger RNA-binding complex. Lastly, the authors characterize a NOT1-G defining TPP domain and find that it is not essential for either male/female phenotype observed for the whole gene KO.

Response**: We appreciate the concise and accurate summary of these findings.

**Major comments:**

• Are the key conclusions convincing?

  The phenotypic characterization of NOT1-G during gametocytogenesis / early mosquito development is nicely presented and the experiments are well performed. Because a duplication of NOT1 with possibly opposing roles of the paralogs is a very unique feature with broad implication on RNA metabolism, it would have been great to see two select experiments on the molecular level adding evidence that 1) NOT1/NOT1-G are mutually exclusive in a complex with CCR4/CAF1 and 2) NOT1-G acts post-transcriptionally in an antagonistic way to NOT1 (i.e. as a mRNA 'stabilizer' as proposed by the authors).

Response**: We agree that inclusion of those two aspects would make for a more complete story about these two NOT1 paralogues.

First, we also think that it is highly likely that NOT1 and NOT1-G are mutually exclusive, as in other eukaryotes NOT1 acts as a scaffold protein upon which effector proteins bind and bridging interactions are made. In our original manuscript, we did not include a mention of our previous attempts to address this question through colocalization and proteomic approaches, as they were largely unsuccessful. Specifically, we generated rabbit polyclonal antisera to PyNOT1-G’s tristetraprolin-binding domain but it did not pass our rigorous quality control (e.g. too much staining persisted in pynot1-g- parasites). Using both asexual and sexual blood stage parasites, we also attempted immunoprecipitation (with and without chemical crosslinking) and proximal labeling approaches via BioID and TurboID but all approaches did not produce rigorous results and thus we did not report them in our original manuscript. However, this question of whether the two NOT1 paralogues were mutually exclusive in complexes was also taken up by the Bozdech Laboratory in their 2020 preprint (Liu et al.) where they were able to capture the P. falciparum NOT1-G and NOT1 proteins (called Not1.1 and Not1.2 in that work). While their proteomic evidence showed that they could capture these bait proteins and that the NOT1 paralogues were not in the same complex, these results should be taken with a grain of salt: all mass spectrometry-based proteomic approaches are limited in that an absence of evidence does not mean that the protein is not present/interacting. Moreover, these efforts only identified a few other proteins that were already known to interact with the CAF1/CCR4/NOT complex, but even so, they did not use statistically rigorous methods in an attempt to quantify these results. In our revised our manuscript, we have included additional text to describe our unsuccessful efforts to do these capture proteomics experiments, and we have expanded our discussion of the Liu et al findings that provide some evidence in support of a mutually exclusive complex.

Second, we also hypothesize that PyNOT1-G acts post-transcriptionally to affect mRNA abundance and translation. However, it is important to emphasize that NOT1 proteins typically act as scaffolds, with the recruited effector proteins acting to hasten the degradation and/or to preserve associated transcripts. We believe that studying these effector proteins is the next important effort to undertake. In fact, we hypothesized that these antagonistic effector proteins would be analogous to TTP and ELAV/HuR-family proteins as are found in other eukaryotes, and that the critical interaction with PyNOT1-G would be via its putative TTP-binding domain. It was for that reason that we interrogated the TTP-binding domain itself, and were surprised that its deletion did not phenocopy the complete gene deletion. Ongoing work will be focused on identifying these antagonistic effector proteins that likely are expressed in a stage-enriched manner, and to define how they interact with PyNOT1-G in order to direct specific mRNAs to their fates. Additionally, it would be very important and exciting to directly test if PyNOT1 and PyNOT1-G are functionally opposed. However, this would be exceptionally challenging to study from a technical standpoint. While we were able to delete the pynot1 gene after many repeated attempts, these parasites are very sickly and grow very slowly. Because of this, we believe that assessing direct versus indirect effects of PyNOT1 in these cells would not be feasible or robust. Given this, comparing functions between PyNOT1 and PyNOT1-G could not be done in a conclusive manner.** In our revised manuscript, we have expanded our descriptions of the mechanisms by which we believe PyNOT1-G and its complex affects mRNA fates. In particular, we have expanded our Discussion section to incorporate the results that indicate that the TTP-binding domain is not required for the essential functions of PyNOT1-G.

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

  The authors describe the role of NOT1-G as 'preserving' mRNA. The lower abundance of many transcripts in the NOT1-G knockout suggest this, but experimental proof is not provided (see suggestions below). Maybe rephrase to 'putatively preserved/stabilized' or 'has a potentially stabilizing function'. The same is true for the mutually exclusive association of the two paralogs with CCR4/CAF1. The authors refer to a protein co-IP of CCR4 showing that CCR4 can interact with both NOT1 and NOT1-G, but a reciprocal experiment is lacking.

Response**: In our first publication on the deadenylase members of this complex, we also saw a similar effect on specific mRNAs when pyccr4-1 was deleted: the abundance of specific mRNAs went up in pyccr4-1- parasites. In that work and here in this manuscript, we have carefully decided to apply the word “preserved” to the fate of these mRNAs as it describes in a general way what is happening. In order to robustly state that mRNAs are stabilized by PyNOT1-G (directly or indirectly) would require additional experiments designed to test this (more description on this is provided on a response below). Second, as described above, we agree that doing a reciprocal IP for mass spectrometry-based proteomics would be ideal, we attempted four different approaches to do this to no avail. However, the composite proteomics data that is already available in the literature and via the Liu et al. preprint from the Bozdech Lab all indicate that these interactions occur, and perhaps that NOT1 and NOT1-G are mutually exclusive as expected. In our revised manuscript, we have provided further explanation in the Discussion for our use of the descriptor “preserve” instead of “stabilize”, and as noted above, and we have expanded our Discussion to more comprehensively define the interaction network depicted in Figure 7.

In both cases, the conclusions of the authors are very likely (e.g. downregulation of many genes as seen by RNA-seq), but the final experimental evidence is not provided and a network such as in Figure 7 is not fully supported. If the authors would like to maintain these statements, then they should be rephrased and made clear or the additional experimental evidence suggested below is necessary.

Response**: We hold that the published proteomic datasets do support such a network, with further support offered from the preliminary proteomic evidence from the Liu et al preprint. Therefore, we have not modified our manuscript beyond the additional text now provided in the Discussion as noted above.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

  The essential claim that NOT1-G is important for gametocytogenesis and early mosquito development is well presented and fully supported by the experiments. As for the role of NOT1-G in 'preserving' mRNA, an mRNA half-life experiment would be necessary (or the text should be adjusted as mentioned above). In a short-term in vitro culture, pynot1-g- and WT parasites could be treated with ActD and abundances of select transcripts are measured by RT-qPCR.

Response**: We appreciate that Reviewer 2 considers the rigor of our experiments to be high. Regarding the use of the term “preserve” vs “stabilize”, we agree that to shift from our more general descriptor (preserve) to one that has specific connotations (stabilize) would require additional experimentation. To correctly and most robustly make the claim of stabilization would require work on par with that done by Painter et al. (PMID: 29985403) that uses a thiol-containing nucleotide (4-TU) along with a yeast-derived fusion enzyme (yFCU) to convert it for use by Plasmodium. Previously we have shown that an associated deadenylase (PyCCR4-1) also acted to preserve mRNAs, and moreover that deletion of its gene resulted in no discernable effect upon the poly(A) tail or 3’ UTR of an mRNA that is bound by this complex (p28).

While understanding mRNA stability is an exciting area of study, this 4-TU labeling experiment alone warranted a standalone, high impact publication for Painter et al. As this has not been adapted for any rodent-infectious Plasmodium species to date, and as adaptation of this labeling approach took several years for Dr. Painter while in the Llinas Laboratory (personal communication), we believe this work is beyond the scope of this study. Moreover, the additional information that it would provide to understand NOT1-g functions (preserve vs stabilize) would be incremental beyond the major storyline presented in this manuscript. In our revised manuscript, we have added text to ensure that our choice of “preserve” is well defined and explained.

To support the idea that NOT-1 and NOT1-G associate in a mutually exclusive way or to just show that they act in distinct complexes despite their similar expression patterns, an IFA with a double stained NOT1/NOT-1G cell line could be performed. Alternatively, the authors could perform a protein co-IP using the already existing NOT1/NOT1-G-GFP cell line and show that the proteins don't interact with each other or even have certain distinct interaction partners.

Response**: We agree, and these studies were attempted but were unsuccessful (described in our responses above). In our revised manuscript, we have included this information as noted above.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

  All necessary cell lines for a NOT1/NOT1-G co-IP and the ActD experiment are already present. The authors already present a ring to schizont in vitro culture (for ActD) and also have substantial experience in protein co-IP and proteomics.

  I am not sure about the cost for a proteomics experiment at the author's institute and I don't want to make a guess on time investment given the still on-going COVID situation.

Response**: We agree that these experiments would be interesting, and would be costly to do at a transcriptome-wide scale and would require substantial time to conduct. We believe that the 4-TU approach noted above is the most rigorous, but is well beyond the scope of this study as it has not yet been adapted to rodent-infectious malaria parasites. As noted above, we have attempted four different proteomics approaches to provide reciprocal evidence for the complex composition which were unsuccessful. In our revised manuscript, we have added text to ensure that our choice of “preserve” is well defined and explained, and have noted the unsuccessful reciprocal proteomics approaches.

• Are the data and the methods presented in such a way that they can be reproduced?

  The MM section is well structured and presented and the supplemental material includes all data.

Response**: Thank you. We want to ensure that our work is clearly described and can be reproduced with the information reported.

• Are the experiments adequately replicated and statistical analysis adequate?

  There is hardly any test of significance presented in the main text of the manuscript (e.g. Figure 3B and 4A). Please show the individual data points for these graphs and make sure the n= and the statistical test is described in the figure legend. If you use the term significant in the text, then just add the p-value behind it. This is also true for the RNA-seq data: Genes are sorted by fold-changes, leaving it unclear if these changes are significant. These data are however presented in Table S1 and could be incorporated in the main text.

Response**: We agree. In our revised manuscript, we have incorporated additional details about the statistical tests used, p-values for noteworthy comparisons, and have included more panels for our comparative RNA-seq datasets (heatmap, PCA, MA plots). We have also made adjustments to our plots to make individual data points more readily observed, especially when error bars may block them (e.g. Figure 3B). And as in the original submission, all of the pertinent values, including fold changes, statistics and more are provided in our comprehensive supplementary files. We have structured the Supplementary Tables to flow from one tab to the next with the filtering/threshold applied noted both in the tab name and in the README tab that is found first among the tabs.

**Minor comments:**

• Specific experimental issues that are easily addressable.

  One idea that is also not discussed but could be added is for example that NOT1-G itself doesn't even have a stabilizing effect itself, but act as a decoy for other components of the CCR4/Caf1 complex, keeping them from associating with NOT1. In the NOT1-G knockout, the decrease in RNA abundance might then be just a result of an 'overactivity' of CCR4/Caf1/NOT1.

Response**: This hypothesis proposed by Reviewer 2, that PyNOT1-G is acting as a decoy or a binding partner sponge, is certainly feasible. For this scenario to be effective, PyNOT1-G would need to be in excess of PyNOT1 and/or would need to be able to bind to the critical effector protein(s) better than does PyNOT1. However, our microscopy data, along with the transcriptomic data presented here and previously published proteomic data would indicate that these two gene products are in approximately balanced proportions and are similarly localized. This does not exclude the possibility that PyNOT1-G could act as a sponge for relevant binding partners. In our revised manuscript, we have raised this possibility as an alternate explanation for the phenotype in the Discussion section.

• Are prior studies referenced appropriately?

  Throughout the manuscript, the authors should make clear what results come from which organism. Just as an example, the genome wide KO screens were performed in P. berghei and P. falciparum, CCR4/CAF1 experiments were performed in P. yoelii, whereas the original DDX6 work was done in P. berghei.

Response**: We agree. In our revised manuscript, we have added additional text to further clarify what data comes from which Plasmodium species.

• Are the text and figures clear and accurate?

  The Introduction is a bit long and partially turns into a minireview of eukaryotic RNA degradation. In the main text on page 13, the authors introduce a model for proteins involved in translational repression. This in not fully accurate, since for many of the proteins in this network, an effect on translation has actually not been shown. This includes NOT1-G characterized in the present work that most likely has an effect on mRNA stability, but for which a role in regulating translation is not presented.

Response**: We believe the length and content of this Introduction is appropriate to provide the context that some readers outside of the parasitology field will need to appreciate these findings. Regarding designations for these proteins as being related to translational repression, we think that the ample proteomic evidence tying them to translationally repressive complexes warrants this. In our revised manuscript, we have made it more clear that these proteins themselves have not been directly implicated in translational repression.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

  Overall the RNA-seq is underrepresented and Figure 5 could easily be expanded by adding several panels that would help the future reader getting a better idea of the data:

1. Summary graphs such as PCA/MDS plots of the different replicates and MA-plots (all of which can be easily generated in DESeq2)
2. Heatmaps comparing the expression patterns of pynot1-g-, pbdozi-, pbcith-, pyalba4- highlighting some key gametocyte genes mentioned in the text

3. Alternatively to 2., a simple Venn Diagram would already be very informative

   An informative representation might also be to sort the differentially expressed genes as predominant male and/or female. The P. berghei data by Yeoh et al (PMID: 28923023) could be a starting point.

Response**: We agree. In our revised manuscript, we have expanded Figure 5 to include additional plots that speak the rigor of these datasets. Specifically, we have added a comprehensive heatmap and PCA plots, as well as MA plots as recommended. We have chosen not to include a Venn diagram for the overlap of affected mRNAs across these transgenic parasite lines, as we hold that this information is best provided in the text (high level observations) and the Supplement (details).

Reviewer #2 (Significance (Required)):

**Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.**

Technically this manuscript builds on standard methods of the field that are well executed. There is no direct clinical advancement, although one might argue that a unique adaptation of the parasite could always be a novel therapeutic target. Conceptually this is great advancement for the parasitology field as it is, providing additional evidence for the importance of post-transcriptional regulation for parasite transmission. With the two experiments suggested above and the additional evidence gained from it, this manuscript could also gain great interest to readers outside the field by clearly showing how alternative ways to regulate RNA stability evolved.

Response**: We are grateful for your careful review of our work and for the recommendations that you provided. We have incorporated many of them into the revised manuscript to make it even more rigorous and comprehensive. We also appreciate hearing that this work would be of great interest to a broader community. We feel that this is already the case, as the duplication of NOT1 and the dedication of one paralogue to an essential function is exciting and novel among eukaryotes.

**Place the work in the context of the existing literature (provide references, where appropriate)**

The work builds on the early reports of the particular RNA metabolism in gametocytes performed in the groups of Andy Waters. Since then, the authors themselves have published a great set of manuscripts extending our knowledge of the proteins involved in gametocytogenesis and nicely place the current work into this framework.

Response**: We appreciate this positive feedback. This is a fascinating topic to study.

**State what audience might be interested in and influenced by the reported findings.**

The manuscript as it stands is particularly interesting for the parasitology and potentially the evolutionary biology field. For a broader readership for example in the RNA field, the possibly antagonistic roles and mutually exclusive association with CAF1/CCR4 are likely most interesting.

Response**: We agree that this should be interesting to readers beyond our own field, as the duplication and specialization of NOT1, and the finding that the “canonical” PyNOT1 can be deleted, are both of general interest to how eukaryotes have adapted and deployed a highly conserved and essential RNA metabolic complex.

**Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.**

**Expertise:**

RNA biology, Plasmodium falciparum, Bioinformatics

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

In this manuscript, the authors investigate the requirement of two possible Not1 paralogs for the development of asexual blood stages and for the sexual transmission stages of Plasmodium yoelii. While Not1 is critical for asexual blood stages, its putative paralog, Not1G is important for the development of sexual transmission stages. In the absence of Not1G, male gametes are not formed while female gametes are formed and can be fertilised by wt male gametes. However, the resulting zygote cannot develop further into ookinete. The in vitro genetic cross assay to show this is elegant! A transcriptomic analysis further indicates that the transcriptomes of Not1G deficient parasites are significantly different from their WT counterpart.

Response**: We are thrilled that you found our evidence and approaches to be rigorous and compelling. Thank you.

**Major comments:**

The discussion section is very nice and the authors describe well what is speculative and should be further confirmed by additional experiments. However, I did find this was not the case in the results section where the authors are proposing conclusions that are not supported by the results. I think the reading of this manuscript would be much more enjoyable if the authors only describe the results shown and move all the discussions to the dedicated section. Below are some examples. The data presented in this manuscript is not showing a nexus, this is a suggestion based on the results of other articles, the word should thus be removed from the title (and kept for a future review!). The last two sentences of the localisation section should be moved to the discussion because they do refer to results not shown in this manuscript. The last sentence of the second paragraph of the zygote development section should also be moved to the discussion. For the transcriptomic analysis there is also no formal comparison with transcriptomes of other previously analysed mutants: the results of the comparisons should either be shown or not discussed in the result section. Finally, the discussions mentioning interactors of the complex should be removed from the result section and moved to the discussion unless the results are formally analysed.

Response**: We again thank you for the complement. In our original manuscript, we opted to provide some limited interpretations and context within the Results section in order to help guide readers along our train-of-thought and line-of-experimentation. While a more traditional split of keeping essentially all discussion and interpretation for the Discussion is a tried-and-true approach, we prefer this more narrative method and have opted to keep these short sections in the Results section.

I would strongly suggest the author the better present and describe their transcriptomic results. There is only one volcano plot indicating the overall defect in mixed gametocytes in the main figure. Apart from this, the results are only described in the main text or in supplementary tables. It is therefore difficult to understand the subtilities of the analysis. For example, the authors frequently mention dysregulated genes, but without specifying whether it is up or down-regulated in the mutant. To address this issue, I would suggest the authors to better describe their results in the figures. They could show the GO term enrichment analysis they mention and show how they assign GO term or transcripts to male and female parasites. It would also be nice to discuss some of the results a bit more in details. For example, it is not surprising to see a reduction in transcripts that are under the control of AP2-O in retort-arrested ookinetes as the parasite do not reach this stage. It is thus highly speculative to specifically link this observation with ALBA4 without further detailed analysis. On the other hand, it is more surprising to see a decrease in ap2g transcripts, while the authors observe an increased gametocytaemia. Could the authors comment this observation? It may also be nice to better present the comparison between gametocytes and schizonts to possibly speculate on the early requirement of Not1G in committed schizonts.

Response**: We (and Reviewer 2) agree. In our revised manuscript, we have expanded Figure 5 to include additional plots that speak the rigor of these datasets. Specifically, we have added a heatmap, and PCA and MA plots as recommended. We have chosen not to include a Venn diagrams for the overlap of affected mRNAs across these transgenic parasite lines for the reasons stated above in our response to Reviewer 2. Similarly, we have opted to keep the specifics of the GO Term analyses in the Supplement as we believe these should always be taken with a grain of salt (especially high level GO Terms, as many choose to report). Finally, we have expanded our discussion on our observation that pyapiap2-g transcript levels are lower in the pynot1-g- line, despite seeing a slight increase in gametocytemia.

The conclusion regarding the similar localisation of Not1 and Not1G with other members of the CAF1/CCR4/NOT complex is not really convincing for two reasons. First, there is not colocalization shown and, second, the distribution is not very peculiar so it is difficult to draw any conclusion with this level of resolution. The presence of alpha-tubulin in the nucleus of male gametocytes is also very surprising as it is rather nucleus-excluded in both P. falciparum and P. berghei, could the authors comment this peculiar localisation?

Response**: We agree and disagree here. First, we agree that no colocalization data is presented here to place NOT1-G within the limit of resolution of fluorescence microscopy. What we can (and do) state is that these proteins are all localized to cytosolic puncta, which matches what is observed for essentially all other studied eukaryotes. In further support of this, our published, quantitative proteomic data indicates that the bioinformatically predictable members of the CAF1/CCR4/NOT complex do associate as anticipated. In the same vein, the micrographs presented were not captured by confocal microscopy, and thus the apparent localization of alpha tubulin “in” the nucleus is most likely attributed to being above and/or below the nucleus. Taken together, we do feel that the combined evidence is convincing. As we have already made all of these points in the original manuscript, we have not adjusted the revised manuscript further.

One of my major frustration when reading this manuscript was that the authors are not trying to discriminate between an early role of Not1G during gametocytogenesis or later in gametogenesis. The fact that the transcriptomes of gametocytes and schizonts seem to show similarities suggests that the phenotype observed during both male gametogenesis or ookinete development are probably linked to early knock-on defects during gametocytogenesis. Could the authors test whether male gametocytes replicate DNA or female activate translation? These are of course non-essential experiments as the authors are careful with their conclusions and mention possible defects during both gametocytogenesis or gametogenesis. Addressing this question may however add significant insights into the requirement for Not1G.

Response**: We are sorry for the frustration. We wrote the manuscript so as to state what we feel we could robustly say, and where we are drawn to speculate, we made that speculation clear. As Reviewer 3 notes, we have not attempted to discriminate between functions that PyNOT1-G may be playing in different stages or substages of development because we do not believe the experiments allow that discrimination. While we could investigate finer and finer aspects of possible defects in both male and female gametocyte development, the most impactful take home messages remain the same. We continue to address questions related to translational repression and its release, and anticipate that PyNOT1-G will play a substantial and essential role in this. As Reviewer 3 noted, we have already discussed these possibilities in the original manuscript, and thus have not added anything further about this in our revised manuscript.

**Minor comments:**

Please use page and line numbering for your next submissions! Please describe what "bioinformatics" was used. I would show the nice localisation in oocyst and sporozoite in the main section. The conclusions drawn from the genetic cross seem to come from a single biological replicate, if this is the case please indicate it clearly.

Response**: We apologize for these oversights. In our revised manuscript, we have provided page and line numbering, have expanded on what bioinformatic processes were done in the manuscript, and have made it more clear that the genetic crosses come from multiple biological replicates (biological triplicate for the transmission-based genetic cross, biological duplicate for the in vitro culture genetic cross). However, we have opted to retain the oocyst and sporozoite IFA data in the Supplement, as the rest of the story is focused on blood stage and early mosquito stage.

Reviewer #3 (Significance (Required)):

This manuscript highlights the requirement of a Not1 paralog in the transmission stages of a Plasmodium parasite. More specifically it describes a new player in the control of RNA biology during this process where our knowledge is scarce. It will be a valuable manuscript for molecular parasitologists interested in transmission or RNA biology.

Response**: We agree and are grateful that our colleagues find this study to be a valuable addition in our efforts to understand how malaria parasites have adapted classic eukaryotic mechanisms to suit their purposes.

Our expertise is largely in molecular and cellular parasitology.

I think this is a framework that could use more emphasis. It’s one I am cueing into more after Caviola et al. (2021) included it in their review, “the psychology of (in)effective giving.”

People have a strong aversion to prioritizing some lives over others (see Tetlock et al., 2003, "Thinking the unthinkable: sacred values and taboo cognitions"). With limited resources, we of course do this all the time. But CBA makes it uncomfortably explicit. To prioritize some recipients as a result of CBA means to deprioritize others, which feels unfair. This is one explanation for why people prefer “distributed helping” when there are multiple possible recipients, even at the expense of helping more, since then at least no one is fully deprioritized (Caviola et al. 2020a, obstacle 5; Sharps & Schroder, 2019, “The Preference for Distributed Helping”). This could also be an explanation for Berman et al., 2018 finding that people prefer to prioritize investments rather than charities, since deprioritizing an investment isn’t nearly as aversive.

A moral aversion to (de)prioritization may also explain social judgments of people who donate effectively seeming “cold” (section 7.1). This is evidenced by the differences in instinctive judgments of “coldness” based on what is deprioritized. For example, deprioritizing investing in textbooks because it isn’t an effective intervention feels much different than deprioritizing investment in childhood cancer treatment because one could help more kids dying of malaria. People would likely make harsher judgments about someone doing the latter even though the reasoning is the same – it’s what is deprioritized that is different.

There might also be something else at play related to ‘CBA’ discomfort: choosing whom to help makes it clear to individuals that they can’t help everyone. It reminds people of all the suffering in the world that they can’t alleviate, whereas just choosing a neat charity only introduces one cause of suffering and then gives the donor the satisfaction that they have done something to alleviate it. I can imagine that CBAs role in revealing the reality of triage (1) makes people less inclined to engage in CBA and (2) less likely to donate a lot in accordance with CBA because there is less warm glow/ that one cause just isn’t as sexy anymore. (2) is related to the idea of Pseudoineffecay developed in (Slovic, 2007; Västfjäll et al., 2015). People are less inclined to help when they learn about others they can’t help.

A key idea that I think is relevant here is the “affect heuristic,” the importance of instinctive emotional cues of “goodness” or “badness” informing decisions (LINK). Deprioritization of emotional cause → instinctively violates moral value → aversion → less likely to engage with, worse social . Similarly, reminder of all the suffering in the world → feeling of sadness + helplessness → avoidant behavior. These oversimplified decision pathways can be overruled by rational, deliberate processing (see Tetlock, 2003 for discussion specific to sacred values) , but charitable giving is largely a system 1/emotional arena.

I feel this is one of the most important parts of documentation and observation. I feel we should give children the space to think about what they make to give them a sense of ownership and accomplishment. If we constantly share our ideas, the child may not see theirs as valid.

I think that awareness is key for being able to teach within a context we as adults may be familiar with. In this particular experience there was almost a system of checks and balances to make sure the students hypotheses and ideas stayed at the forefront of their research.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

We thank all four reviewers for their positive and constructive comments! We have carefully considered these comments and provided a point-by-point response below.

Reviewer #1 (Evidence, reproducibility and clarity):

This paper explores an interesting problem of SHP1/SHP2 preferences of inhibitory immunoreceptors. The author are quick to point out that many of their individual data points confirm published results at some level, but the power of the paper is in the parallel analysis of both PD1, which is strongly biased towards SHP2 and BTLA, which is biased towards SHP1. This gives them the opportunity to test the predictions of descriptive experiment by making simple mutated receptors with swapped ITIM or ITSM domains.

The work is very well done and generally the authors are quite careful and precise about the language used to describe results, in general.

The results are quite striking in that the find plenty of evidence for transient interaction of SHP1 with PD1 based on the biophysical measurements, but don't detect the interactions in pull down or in "in cell" microcluster recruitment experiments. In describing the pull-downs they discuss the issue of dissociation during washing potentially missing interactions that are taking place. I would prefer that the pull down is fine evidence for binding, but lack of pull down is not evidence for lack of binding. They should double check that this language is consistent. Also, unless something has changed in the microcluster binding experiments, this in situ recruitment of SHP2 to PD1 is only observed or a 2-3 minutes and then can't be detected, the situation for SHP2 becoming the same as it is for SHP1. If the kinetics are different in the cleaner systems that have now developed they should show this in a primary figure as this would be then different when what is reported previously.

We agree with the reviewer that pull down is evidence for binding. Indeed, in most, if not all of our assays, our results with pull down were consistent with those in the microcluster imaging. As suggested by the reviewer, we will check through the manuscript and ensure the language is accurate and consistent. In our recent study (Xu et al., JCB, 2020, PMID: 32437509), we conducted a side-by-side comparison of SHP2 and SHP1 recruitment kinetics to PD-1 in a similar system as the current study. Both microcluster imaging and co-IP assays showed that PD-1:SHP2 association lasted at least 10 minutes, whereas PD-1:SHP1 recruitment was nearly undetectable. The duration of PD- 1:SHP2 association was in good agreement with Takashi Saito’s finding in CD4+ mouse T cells (Yokosuka et al., JEM, 2012, PMID: 22641383). Regardless the somewhat different kinetics in different studies, SHP2 recruitment was transient, as pointed out by the reviewer. We believe that some other effectors contribute to PD-1 inhibitory signaling. In supportive of this notion, we recently found that PD-1 remains partially inhibitory in CD8+ T cells deficient in both SHP1 and SHP2 (Xu et al., JCB, 2020).

The gap in this study is lack of any functional analysis. The Jurkat model could be quite useful as they have a relatively clean system for asking if the transient binding of SHP1 to PD1 has any functional impact, which they have not yet followed through on. Does PD-1 recruited SHP2 have any impact on function after the 5 minutes? Furthermore, the authors need to keep in mind that mice deficient in SHP2 respond to anti-PD1 checkpoint therapies (Rota, G., Niogret, C., Dang, A. T., Barros, C. R., Fonta, N. P., Alfei, F., Morgado, L., Zehn, D., Birchmeier, W., Vivier, E., & Guarda, G. (2018). Shp-2 Is Dispensable for Establishing T Cell Exhaustion and for PD-1 Signaling In Vivo. Cell Rep, 23(1), 39-49. https://doi.org/10.1016/j.celrep.2018.03.026). This is an important issue to discuss in light the the very interesting binding analysis the authors have performed. But I think the functional analysis can be part of a future paper.

In our recent publication (Xu et al. JCB, 2020, PMID: 32437509), we found that deletion of SHP1 from Jurkat cells had little, if any effect on PD-1 mediated suppression of IL-2 production. As the reviewer alluded to, we did observe SHP2 dissociation from PD-1 after 10 minutes, so the question of whether and how PD-1:SHP2 complex influence T cell function in a longer term is a great one. We currently are pursuing a hypothesis that there is a SHP2-independent mechanism of PD-1 inhibitory function, and indeed, in our recent study (Xu et al. JCB, 2020, PMID: 32437509), we found that PD-1 retains its partial inhibitory function in SHP1/SHP2 double knockout murine primary T cells. These results are consistent with the in vivo data by Rota et al. cited by the reviewer. We will also briefly discuss this point in a revised manuscript.

I would suggest that the title be modified slightly from "SHP1/SHP2 discrimination" to "differential SHP1/SHP2 interaction" and leave discussion of discrimination until they have the functional data integrated over times that are relevant to T cell transcriptional regulation (1-2 hrs). The functional analysis can be in another paper, but it would be interesting to have a paragraph in the discussion raising the outstanding issues beyond stable binding detected by the pull-down and microcluster recruitment experiments- what are the implications for function. Could the transient interactions in the noise of the steady state and equilibrium measurements be functional?

We thank the reviewer for the suggestion, even though reviewer #3 felt that our current title is appropriate. We will be happy to change the title at the editors’ discretion.

I would summarise that the work is outstanding as biochemistry and biophysics and it should be published nearly as is. I'm suggesting minor revisions in that the changes are just to text, but I think this is important and somewhat nuanced aspect of the paper that will make it even more helpful to readers.

We appreciate the positive and insightful comments!

Reviewer #1 (Significance):

The authors generate a detailed descriptive data set about the component interaction of SHP1 and SHP2 SH2 domains with PD1 and BTLA intracellular domains. They then test hypotheses generated from the descriptive data set to better define the nature of the interactions and why PD1 recruits primarily SHP2, while BTLA mainly recruits SHP1. PD1 is a major driver or the cancer immunotherapy revolution and SHP2 is the major candidate for a signalling effector of PD1. This paper can become the reference paper for the specificity and engineering of this interaction, which will make it highly significant in a very active and still expanding field.

Referee Cross-commenting

I still feel that "discrimination" has a functional/activity connotation that is not addressed at all in this paper, but can be addressed. I'm happy to have the suggestion stand and let the authors decide. They need to live with it once its published. Another suggestion- the citations on regulation are mostly old. A good recent paper is Pádua, R. A. P., Sun, Y., Marko, I., Pitsawong, W., Stiller, J. B., Otten, R., & Kern, D. (2018). Mechanism of activating mutations and allosteric drug inhibition of the phosphatase SHP2. Nature Communications, 9(1),

1. https://doi.org/10.1038/s41467-018-06814-w .

We believe that some of the functional questions raised by this reviewer, including the SHP1 and SHP2 contribution in PD-1 signaling, was addressed in our recent publication (Xu et al., JCB, 2020). Using SHP1 KO and SHP2 KO T cells, we showed that PD-1 inhibitory function is contributed by SHP2, but very little if any by SHP1. Thus in the current study, we focus on the mechanism behind the striking SHP2 preference by PD-1. We thank this reviewer for suggesting this excellent reference. We will cite this reference in the revised manuscript.

Reviewer #2 (Evidence, reproducibility and clarity):

In this study, Xu and co-workers investigate the biophysical nature of the interaction between the structurally-related non-transmembrane PTPs Shp1 and Shp2 with the ITIM/ITSM-containing inhibitory receptors PD-1 and BTLA using cell-based, biochemical, biophysical and domain swapping assays. The primary aim being to better understand how these receptors discriminate between binding Shp1 and/or Shp2, and the orientation of Shp1 and Shp2 engagement. These are major unresolved questions in the field that the authors go some way to addressing in a methodical, rigorous, clear and concise manner. Findings are convincing, correlate well with previous findings and internally, and are complemented with excellent schematics, making it easy to comprehend.

Major comments

The authors focus primarily on binding affinities to explain differential binding of Shp1 and Shp2 by PD-1 and BTLA ITIMs and ITSMs, but this is only part of the story. Avidity, compartmentalization, stoichiometry of kinases, and relative abundance of Shp1 and Shp2 are also important aspects of the discriminatory mechanism that are not addressed. Competition assays would go some way to addressing the latter point and should be at least be considered and discussed.

We agree that various parameters mentioned by this reviewers, such as compartmentalization and relative expression levels would be a concern for purely cell-free assays such as SPR, however, we feel that our cell-based assays already integrate these parameters. This is also precisely the reason why we chose to examine the recruitments of Shp1/2 in a cellular context instead of a purely cell-free system.

Regarding the competition, we have confirmed our key results in both WT and SHP2 KO background, with or without the potential competition from endogenous SHP2, suggesting that competition might not be a dominant mechanism for the recruitment specificity we observed.

Similarly, authors do not address how distortion of the pY binding pocket of Shp1 and Shp2 nSH2 domains in the auto-inhibited conformation is released, allowing the domain to engage with phopho-ITIM/ITSM. Again, this should be at least discussed. Current binding studies do not address this issue.

We feel that the overall recruitment to the PD-1 microclusters as we observed in cells already integrate this auto-inhibition mechanism of Shp1 and Shp2, because we used full length proteins. We do agree with the reviewer that future studies are warranted to address the contributions of each mechanism, including auto-inhibition, concentration, competition, etc., to the overall recruitment. This might require careful and extensive biophysical analyses coupled with mathematical modeling.

Minor comments:

Phosphorylation should be indicated in schematic representations in Figures 3, 6 b, c.

We thank the reviewer for this advice, we will indicate phosphorylation in the revised figure 3.

Cellular and physiological significance should be further discussed, as well as broader implications of findings to other ITIM/ITSM-containing receptors in other lineages.

We will further discuss this as suggested.

Reviewer #2 (Significance)

Findings from this study advance our knowledge of how inhibitory checkpoint regulatory receptors discriminate between Shp1 and Shp2, which has important implications for understanding how the unique biochemical, cellular and physiological functions of these receptors and phosphatases are dictated. Indeed, findings lay the foundation for a universal mechanism, that may apply to all ITIM/ITSM receptors in other cell lineages, and perhaps novel ways of targeting these interactions therapeutically.

Compare to existing published knowledge

Although largely correlative with previous studies, findings from this study start to fill major gaps in our knowledge of these biochemical processes, in a highly rigorous, concise and clear manner. Findings from previous studies were more 'piecemeal', whereas this study consolidates and advances important nuances of these interactions. Moreover, it lays the foundation for further structural, physiological and therapeutic studies.

Audience

The immune receptor signaling community and beyond, including any lineage in which ITIM/ITSM-containing receptors play a major role in regulating cellular responses.

Your expertise

ITIM/ITSM-containing receptors, kinase-phosphatase molecular switches, cellular reactivity to extracellular matrix proteins

Referee Cross-commenting

Generally agree with reviewer's comments. Constructive overall and fair. Although I was thinking additional competition experiments, I do not think necessary. Over the top for this study. Hence, 1 month should suffice to revise accordingly.

We thank this reviewer for the excellent comments and understanding!

Reviewer #3 (Evidence, reproducibility and clarity):

Summary:

Inhibitory immune receptors containing ITIMs function through recruiting the phosphatases SHP-1 and SHP-2. SHP-1 and SHP-2 are remarkably similar yet have different roles in vivo. How can ITIM-containing immune receptors specifically recruit SHP-1 or SHP-2? In this paper, Xu et al ask how SHP-1 vs SHP-2 specificity is achieved. They use very thorough biochemical assays to measure the affinity of SHP-1 and SHP-2 for various ITIM/ITSMs and finally pin point some key amino acids that switch an ITIM/ITSM from SHP-2 to SHP-1 specificity. The in vitro biochemical assays are augmented by in cell assays that support their conclusions. Overall, this paper is an incredibly elegant and straight forward paper addressing how SHP-1/SHP-2 specificity is achieved.

Major Comments: none

Minor Comments:

• Could the western blots in Figure 1 be quantified as the western blots in other figures?

We will quantify the western blots in Figure 1 as suggested in the revised manuscript.

• The data that the y+1 reside is essential for SHP-1/2 specificity is very convincing. We are curious if the other residues of the ITIM/ITSM also contribute to this specificity, albeit less potently. The PD-1 G224A mutant is still less potent than the PD-1 BTLA ITIM swap, suggesting that while the y+1 position is most important, the other residues contribute some specificity. The authors also included data on a PD-1 variant with the BTLA ITIM A224G mutation (8f), which is slightly better at recruiting SHP-1 than the PD-1 ITIM. It may be worth mentioning this data in the text of the paper as well as displaying it in the figure.

The reviewer raised an excellent point, yes, our data does suggest that other pY-flanking residues within the ITIM also contribute to SHP1 binding. However, the pY+1 residue replacement produced the strongest effect as the reviewer noted. In the revised manuscript, we will acknowledge the potential contributions of other residues.

• A brief introduction to ITIM vs ITSM in the introduction of the paper may be helpful background for readers. For example, ITIM receptors are reasonably well known but how ITSM functionally differs is probably less well known.

We will rewrite the introduction about ITIM and ITSM for better clarity.

• Although not the major focus of the paper, broadening out this SHP-1/2 specificity to other immune receptors in the discussion is fascinating. (a) The authors find that a Valine, Leucine, or Isoleucine in place of the Alanine in y+1 is very close to equivalent, yet the A is highly conserved. The authors speculate that there may be an advantage to sub-maximal SHP-1 affinity because it is more easy to regulate. I think this is reasonable speculation but a little unsatisfying given the very small observed difference in SHP-1 binding. If the authors have additional thoughts, I would be interested to hear them. (b) The authors note that PD-1 is the only ITIM with a glycine in the Y+1 position. Are there other receptors that function primarily through SHP-2, and how might they achieve this specificity?

Response to a: Even though valine, leucine or isoleucine did not produce a striking enhancement in Shp1 recruitment over alanine, the differences were statistically significant. In fact, when we performed these point mutations at a BTLA ITIM background, valine, leucine or isoleucine markedly enhanced the SHP1 recruitment (see unpublished data below). We speculate that other pY-flanking residues in BTLA, as this reviewer alluded to above, creates an environment that amplifies the differences. The strong sensitivity on pY+1 residue, as observed in BTLA, might be true for other SHP1-recruiting receptors too. If they were to have leucine or isoleucine at the pY+1 position of ITIM, they may recruit too much SHP1 that presumably decreases the fitness/growth of the cells. We propose to show this unpublished data as a supplemental figure in the revised manuscript. We will also discuss the potential contributions of other pY-flanking residues as this reviewer suggested.

{{images cannot be rendered at this time in reply letters}}

Response to b: Among the several receptors that we tested, PD-1 is the only receptor that exhibited no recruitment of SHP1. The lack of SHP1 recruitment is also true for murine PD-1, which has a glutamate residue (charged) at Y+1 position. In addition, earlier work reported that PECAM1 also selectively recruits SHP2, but not SHP1. We have noted that PECAM1 contain a threonine (polar) at the pY+1 position of their ITIMs. Thus, their inability to recruit SHP1 is consistent with our model that a nonpolar residue at Y+1 position is required for strong SHP1 recruitment. We will discuss these points in the revised manuscript.

• Figure 9 b Val not Vla, Figure 3a - a legend for the color code may be nice (ie, 20-1000 nM) Thanks for catching this, we will fix the error in Figure 9b and provide the color code in Figure 3a in the revised manuscript.

Reviewer #3 (Significance):

Significance:

SHP-1 and SHP-2 play a critical role in regulating immune system function. In addition, the receptors recruiting these phosphatases (like PD-1) are important immunotherapy targets. Previously, the question of SHP-1/SHP-2 specificity has been primarily described for ITIM bearing receptors individually. Other studies have predicted consensus sequences for the tSH2 domains of SHP-1 or SHP-2, but not addressed the defining molecular characteristics of these consensus sites or how these could be combined on ITIM receptors to generate selectivity between these related phosphatases. This paper represents a significant step forward because it provides a unifying mechanism explaining how ITIM-bearing immune receptors specifically recruit SHP-1 or SHP-2. I expect this paper will be broadly interesting to biochemists, immunologists and cancer biologists.

Referee Cross-commenting

I generally think the other reviewers comments are reasonable and insightful. Together, they suggest no new experiments are necessary. As for the proposed title change, I prefer the authors title and find it to be justified given their data.

Reviewer #4 (Evidence, reproducibility and clarity):

In this manuscript, Xu and college performed an elaborate study to investigate the molecular basis of Shp1 and Shp2 discrimination by immune checkpoints PD-1 and BTLA. The paper is original, clear, and well written. I only have a few minor comments:

1. Please label the molecular weights to all the western blots/IPs results.

We will label the molecular weights to all the blots in the revised manuscript.

1. Please add scale bars to all the microscopy pictures.

We will add scale bars to all the microcopy images in the revised manuscript.

1. For the SPR data, please add the fitting curves.

We thank the reviewer for the suggestion. However, we did not use the fitting curve to calculate the Kd, we plotted the maximum response as a function of concentration to determine the Kd. This is another well accepted method for Kd calculation. In fact, some of the SPR curves fit poorly with the existing algorithm. Thus, showing the fitting curve might distract the readers.

Reviewer #4 (Significance):

The strength of this paper relies on the details they dissected by using a series of mutagenesis screening experiments, which should be interesting to cell biologists and cancer immunologists.

Referee Cross-commenting

I think the other reviewer's comments are insightful and constructive, the suggested experiments are necessary and will improve the paper.

We thank this reviewer for the positive comments!

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Referee #3

Evidence, reproducibility and clarity

Summary:

Inhibitory immune receptors containing ITIMs function through recruiting the phosphatases SHP-1 and SHP-2. SHP-1 and SHP-2 are remarkably similar yet have different roles in vivo. How can ITIM-containing immune receptors specifically recruit SHP-1 or SHP-2? In this paper, Xu et al ask how SHP-1 vs SHP-2 specificity is achieved. They use very thorough biochemical assays to measure the affinity of SHP-1 and SHP-2 for various ITIM/ITSMs and finally pin point some key amino acids that switch an ITIM/ITSM from SHP-2 to SHP-1 specificity. The in vitro biochemical assays are augmented by in cell assays that support their conclusions. Overall, this paper is an incredibly elegant and straight forward paper addressing how SHP-1/SHP-2 specificity is achieved.

Major Comments:

none

Minor Comments:

• Could the western blots in Figure 1 be quantified as the western blots in other figures?
• The data that the y+1 reside is essential for SHP-1/2 specificity is very convincing. We are curious if the other residues of the ITIM/ITSM also contribute to this specificity, albeit less potently. The PD-1 G224A mutant is still less potent than the PD-1 BTLA ITIM swap, suggesting that while the y+1 position is most important, the other residues contribute some specificity. The authors also included data on a PD-1 variant with the BTLA ITIM A224G mutation (8f), which is slightly better at recruiting SHP-1 than the PD-1 ITIM. It may be worth mentioning this data in the text of the paper as well as displaying it in the figure.
• A brief introduction to ITIM vs ITSM in the introduction of the paper may be helpful background for readers. For example, ITIM receptors are reasonably well known but how ITSM functionally differs is probably less well known.
• Although not the major focus of the paper, broadening out this SHP-1/2 specificity to other immune receptors in the discussion is fascinating. (a) The authors find that a Valine, Leucine, or Isoleucine in place of the Alanine in y+1 is very close to equivalent, yet the A is highly conserved. The authors speculate that there may be an advantage to sub-maximal SHP-1 affinity because it is more easy to regulate. I think this is reasonable speculation but a little unsatisfying given the very small observed difference in SHP-1 binding. If the authors have additional thoughts, I would be interested to hear them. (b) The authors note that PD-1 is the only ITIM with a glycine in the Y+1 position. Are there other receptors that function primarily through SHP-2, and how might they achieve this specificity?
• Figure 9 b Val not Vla, Figure 3a - a legend for the color code may be nice (ie, 20-1000 nM)

Significance

SHP-1 and SHP-2 play a critical role in regulating immune system function. In addition, the receptors recruiting these phosphatases (like PD-1) are important immunotherapy targets. Previously, the question of SHP-1/SHP-2 specificity has been primarily described for ITIM bearing receptors individually. Other studies have predicted consensus sequences for the tSH2 domains of SHP-1 or SHP-2, but not addressed the defining molecular characteristics of these consensus sites or how these could be combined on ITIM receptors to generate selectivity between these related phosphatases. This paper represents a significant step forward because it provides a unifying mechanism explaining how ITIM-bearing immune receptors specifically recruit SHP-1 or SHP-2. I expect this paper will be broadly interesting to biochemists, immunologists and cancer biologists.

Referee Cross-commenting

I generally think the other reviewers comments are reasonable and insightful. Together, they suggest no new experiments are necessary. As for the proposed title change, I prefer the authors title and find it to be justified given their data.

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Reply to the reviewers

Editor comments:

Thank you for sending your manuscript entitled "In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography" to Review Commons. We have now completed the peer review of the manuscript. Please find the full set of reports below.

We thank the editors of Review Commons and all the reviewers for their insightful comments which helped us to improve our manuscript. We have now modified our manuscript based on the Reviewers’ comments and would like to ask you to consider our revised manuscript for publication.

Reviewer #1:

This manuscript by the Jensen lab surveys a plethora of bacterial outer-membrane projections captured over the years by in situ cryo-tomography under near-native conditions. The authors classify the different visualized structures, highlighting both similarities and differences among them. They further describe molecular complexes that are associated with these projections. The manuscript highlights the abundance of such understudied structures in nature, indicating the need to deepen our exploration into their biological functions and mechanisms of action.

We thank the reviewer for her/his insightful comments that allowed us to improve our manuscript.

The authors should state in the Abstract and Introduction that only diderm bacteria and outer- membrane extensions are included in the study.

Done. We have modified the title, the abstract and the introduction to explicitly highlight this point.

In the Introduction or Discussion the authors should mention the limits of the in situ cryo-tomography, such as the difficulty to observe regions in between neigbouring bacterial cells, and into the thick bacterial cell body.

Done. We have added the following to our revised manuscript:

“Currently, only electron cryo-tomography (cryo-ET) allows visualization of structures in a near-native state inside intact (frozen-hydrated) cells with macromolecular (~5 nm) resolution. However, this capability is limited to thin samples (few hundred nanometers thick, like individual bacterial cells of many species) while thicker samples like the central part of eukaryotic cells, thick bacterial cells, or clusters of bacterial cells are not amenable for direct cryo-ET imaging. Such thick samples can be rendered suitable for cryo-ET experiments by thinning them first using different methods including focused ion beam milling and cryosectioning [30]. Cryo-ET has already been invaluable in revealing the structures of several membrane extensions, including Shewanella oneidensis nanowires [6], Helicobacter pylori tubes [15], Delftia acidovorans nanopods [25], Vibrio vulnificus OMV chains [16], and more recently cell-cell bridges in the archaeon Haloferax volcanii [31].” (Lines 108-118)

Please provide a legend to Table S1 explaining the numbers (organelles?), how many cells were viewed? I think that at least part of it should be included in the main text. Also, there are examples of vesicles emanating from H. pylori. This information is missing from Table S1.

Done. We added a column to the table indicating the number of cells available for each species. We also added the information about the vesicles in H. pylori to the table. This table is now incorporated into the main text of the manuscript as Table 1.

Please provide an ordered list including all the strains (and IDs of the specific isolates) used in this study and their genotypes.

Done. We added Table S1 to the revised manuscript that contains this information. This table also includes relevant references to all the published papers where these strains were previously used.

The authors describe in detail the H. pylori tubes that seem to be flagellum-core independent. However, the authors found previously (ref 15) that during infection, these structures are dependent on CagA T4SS, and they visualized T4SS sub-complexes in proximity to the point of tube emanation. This should be described and discussed in the text. Also, please indicate if the "host-independent" tubes are similarly dependent on T4SS.

Done. We added the following to the revised manuscript:

“The scaffolded uniform tubes of H. pylori that we observed were formed in samples not incubated with eukaryotic cells, indicating that they can also form in their absence. However, the tubes we found had closed ends and no clear lateral ports, while some of the previously-reported tubes (formed in the presence of eukaryotic host cells) had open ends and prominent ports [15]. It is possible that such features are formed only when H. pylori are in the vicinity of host cells. Moreover, while it was previously hypothesized that the formation of membrane tubes in H. pylori (when they are in the vicinity of eukaryotic cells) is dependent on the cag T4SS [15], we could not identify any clear correlation between the emanation of membrane tubes and cag T4SS particles in our samples where H. pylori was not incubated with host cells. We also show that the tubes of H. pylori are CORE-independent, indicating that they are different from the CORE-dependent nanotubes described in other species.” (Lines 303-313)

Is there any difference in the frequency or length of the tubes in the mutants presented in Figure 4? The flgS mutant in the image exhibits a very short filament; is that typical?

We did not see any significant statistical difference in the number or lengths of the tubes in these different mutants. We added Table S2 to the revised manuscript which details the number of cells we visualized for each mutant and the number of the tubes seen there. In all these mutants the lengths of the tubes ranged between few tens to hundreds of nanometers. In addition, we added Fig. S2 to show more examples of these tubes in each of these mutants.

Minor points:

-Please check full bacterial names that are sometimes missing (e.g., lines 110-112).

Done.

-There is no reference to panel 2G. Please check the references to all panels.

Done. Please see lines 154 and 183 in the main text.

-Lines 181-184: There is no figure related to the formation of teardrop-like extensions from C. pinensis. Please review the text accordingly.

Done. Corrected.

-Line 235, not clear to what "as these" refers to.

Done. We modified the text as the following:

“As these MEs/MVs from S. oneidensis were purified” (Lines 246-247)

-Line 241, not clear what "a secretin-like complex" is, and no reference is provided.

Done. We modified the text as the following:

“In the third category, we observed a secretin-like complex in many tubes and vesicles of F. johnsoniae. Secretins are proteins that form a pore in the outer membrane and are associated with many secretion systems like type IV pili and type II secretion systems (T2SS) [39–41]” (Lines 252-254)

Reviewer #1 (Significance)

As described in this manuscript, even in model bacteria these structures are generated (e.g., Caulobacter forms the hardly studied nanopod extensions). The manuscript also provides visual categories of these structures, defining "extension types" that are likely to be used by the scientific community for years to come, similar to the initial pili classification during the 1960s-70s. It is a "descriptive study," in the positive sense of the term, as it significantly contributes to the field of bacteriology.

We thank the reviewer for her/his kind words and enthusiasm about our work. It is an honor to have our work compared to the seminal pili classification work done in the 1960s-70s by pioneers in the field of bacteriology.

Reviewer #2:

The manuscript "In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography" by Kaplan et al., identifies and catalogues membrane extensions (MEs) and membrane vesicles (MVs) from 13 different species using cryo-electron tomography. Furthermore, they identify and discuss several protein complexes observed in these membrane projections.

The manuscript is beautifully written, interesting, and genuinely got this reviewer excited about the biology. I applaud the authors on their manuscript and have only minor comments and a few thoughts that the authors may wish to think on and discuss.

We thank the reviewer for her/his kind words and insightful comments that allowed us to improve our manuscript.

Some schematics throughout the introduction would be useful to readers new to the field/ outside the field who are not used to these different membrane structure features.

We thank the Reviewer for this suggestion. First, we made an extra figure with schematics showing the cell body and membrane tubes but that was rather redundant with Figure 8. For this reason, we added explicit labels to figure 1 highlighting the cell body and the tubes in these examples to help the reader following that figure and the subsequent ones. However, if the Reviewer has an explicit suggestion/view about the schematics then we would be very happy to do that.

The size of scale bars should be indicated on the figure panels themselves rather than in the figure legend to assist the reader.

Done.

In reference to lines 193-196 - what was the extracellular environment like in these micrographs? Were other cells present? Could it be the extracellular environment/surrounding cells that stimulate pearling? Have the authors considered this? Please discuss if relevant/insightful.

This is a good point. The cells were usually plunge-frozen in their standard growth media (except in H. pylori where the cells were resuspended in PBS and subsequently plunge-frozen). Yes, there are other cells present in the sample, however, usually, only one cell is present in the field of view of the tomogram as areas with multiple cells have thick ice and therefore not amenable for cryo-ET imaging. We added the following to the revised manuscript:

“As usually only one (or part of a) cell is present in the cryo-tomogram, we can’t exclude that differences in the extracellular environments, like the presence of a cluster of cells in the vicinity of the individual cells with pearling tubes, might play a role in this observation” (Lines 198-201).

"Randomly-located complexes" in this reviewers opinion should actually be described "seemingly randomly-located complexes" given there may be an organization present that is beyond the resolution limit of this study.

The is a good point. Indeed, we can’t exclude that these complexes have a preferred localization in specific lipid patches that we can’t detect in our cryo-tomograms. We added the following statement to the revised manuscript:

“These complexes, which were also found in the OM of intact cells, did not exhibit a preferred localization or regular arrangement within the tube at least within the fields of view provided by our cryo- tomograms (Fig. 5a & b).” (lines 227-230).

In reference to lines 287-292 - is it possible this has to do with lipid composition? Have the authors considered this? Please discuss if relevant/insightful.

Done. We added the following to the revised manuscript:

“In addition, differences in the lipid compositions among the various species investigated here might also play a role in the formation of these different forms of projections” (Lines 299-301).

Reviewer #2 (Significance ):

These results advance the field by shedding new light on bacterial membrane extension morphologies. The authors use a cryo-ET to catalogues membrane extensions and membrane vesicles which has not been done before.

This paper is likely to be of interest to structural biologists, biophysicist, membrane protein biologists, virologists and microbiologists.

This reviewer is a single-particle cryo-EM structural biologist with interest in membrane proteins._

We thank the reviewer for her/his enthusiasm about our work described here.