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Reviewer #2 (Public Review):

In this study, Pinatel et al. address the role of interneuron myelination in the hippocampus using a 4.1B protein mouse knockout model. They show that deficiency in 4.1B significantly reduces myelin in CA1 stratum radiatum, specifically myelin along axons of parvalbumin and somatostatin hippocampal interneurons. In addition, there are striking defects in the distribution of ion channels along myelinated axons, with misplacement of Na channel clusters along the nodes of Ranvier and the heminodes, and a pronounced decrease in potassium channels (Kv1) at juxtaparanodes. The axon initial segments of SST are also shorter. Because the majority of myelinated axons in the stratum radiatum of the hippocampus belong to PV and SST interneurons such profound changes in myelination are expected to affect interneuronal function. Interestingly, the authors show that PV basket cells' properties appear largely unaffected, while there are substantial changes in stratum oriens O-LM cells. Inhibitory inputs to pyramidal neurons are also changed. Behaviorally, the 4.1B KO mice exhibit deficits in spatial working memory, supporting the role of interneuronal myelination in hippocampal function. This study provides important insights into the role of myelination for the function of inhibitory interneurons, as well as in the mechanisms of axonal node development and ion channel clustering, and thus will be of interest to a broad audience of circuit and cellular neuroscientists. However, the claims of the specificity of the reported changes in myelination need to be better supported by evidence.

Strengths:<br /> The authors combine a wide array of genetic, immunolabeling, optical, electrophysiological, and behavioral tools to address a still unresolved complex problem of the role of myelination of locally projecting inhibitory interneurons in the hippocampus. They convincingly show that changing myelination and ion channel distribution along nodes and heminodes significantly impairs the function of at least some interneuron types in the hippocampus and that this is accompanied by behavioral deficits in spatial memory.

Regarding the organization of myelinated axons, the lack of 4.1B causes striking changes at the nodes of Ranvier that are convincingly and beautifully presented in the Figures. While the reduction in Kv1 in 4.1B KO mice has been previously reported, the mislocalization of sodium channels at the nodes and heminodes had only been observed in developing but not adult spinal cords. This difference in the dependence of the sodium channel distribution on 4.1B in adult hippocampus vs spinal cord may hold important clues for the varying role of myelin along axons of different neuronal types.

The manuscript is very well written, the discussion is comprehensive, and provides detailed background and analysis of the current findings and their implications.

Weaknesses:<br /> Because of the wide diversity of interneuron types in the hippocampus, and also the presence of myelinated axons from other neuron types as well, including pyramidal neurons, it is very difficult to disentangle the effects of the observed changes in the 4.1 B KO mouse model. While the authors have been careful to explore different possibilities, some of the claims of the specificity of the reported changes in myelination are not completely founded. For example, there is no compelling evidence that the myelination of axons other than the local interneurons is unchanged. The evidence strongly supports the claims of changes in interneuronal myelination, but it leaves open the question of whether 4.1B lack affects the myelination of hippocampal pyramidal neurons or of long-range projections.

To be able to better interpret the changes in the 4.1B KO mice, knowledge of the distribution of 4.1B in the hippocampus of control mice will be very helpful. The authors state that 4.1B is observed in PV neurons but not in pyramidal neurons, however, the evidence is not convincing. Thus, the lack of immunolabeling at the pyramidal neuron cell bodies does not indicate that 4.1B is missing at the axonal level. The analysis also leaves out the question of whether 4.1 B is seen in the axons of somatostatin neurons.

Art. 5º O advogado postula, em juízo ou fora dele, fazendo prova do mandato.

§ 1º O advogado, afirmando urgência, pode atuar sem procuração, obrigando-se a apresentá-la no prazo de quinze dias, prorrogável por igual período.

§ 2º A procuração para o foro em geral habilita o advogado a praticar todos os atos judiciais, em qualquer juízo ou instância, salvo os que exijam poderes especiais.

§ 3º O advogado que renunciar ao mandato continuará, durante os dez dias seguintes à notificação da renúncia, a representar o mandante, salvo se for substituído antes do término desse prazo.

§ 4º As atividades de consultoria e assessoria jurídicas podem ser exercidas de modo verbal ou por escrito, a critério do advogado e do cliente, e independem de outorga de mandato ou de formalização por contrato de honorários.

• for: awe experiment
• experimental results
  • awe upregulates
    • Default Mode Network (DMN) which controls interaction to multiple brain regions, increasing:
      • creative thinking
      • daydreaming
  • awe downregulates executive control

Importante para recursos interactivos.

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Reply to the reviewers

Manuscript number: RC-2023-01991

Corresponding author(s): Chaitanya A. Athale

1. General Statements [optional]

*We are grateful to the editors sending our manuscript out to review, and the reviewers for the careful reading and critical comments. In the following sections we describe our plan for revisions that will address the comments of the reviewers. We have added these in a point-wise manner. In summary most of the comments are addressable with additional experiments, simulations and data analysis. These will indeed serve to strengthen the findings without altering the fundamental findings. However, we would require upto 90 days to make these changes. *

Description of the planned revisions

Reviewer #1 Evidence, reproducibility and clarity

Summary: This work combines in-vitro experiments and numerical modeling to study the dynamics ofmicrotubules, driven by molecular motors. In this bottom-up approach, molecular motors areimmobilized on the surface and microtubule filaments are anchored to the surface from one end. The dynamics results in "beating like" motion of the anchored microtubules. The authors establish aphase diagram of the different dynamical patterns of "beating like" motions by varying the molecular motor density and the length of the microtubule anchored to the surface. They use a numerical framework that captures the observed patterns.

Our response: We are grateful to the reviewer for the careful reading and agree with the summary of our work. In the following sections we detail how we plan to address the specific comments.

Major comments:

1. Overall the experiments and results are well described and claims are supported by the data.

Both experimental and numerical methods are presented in a way that they can be reproduced.

Our response: We are grateful for the reviewer’s assessment about our findings and presentation of the results.

Minor comments:

1. A key feature of beating cilia is the asymmetry of the beat pattern (fast stroke and slow recovery). It might be interesting to use the kymographs or the Phy vs time analysis to see whether or not this feature exists in this simplified experimental model.

Our response: We agree with the reviewer that it could be of interest to examine whether the dynamics of the tip-angle phi (φ) shows a difference between the strokes at onset and return, to compare to the fast-slow asymmetry observed in cilia. This will be approached in two ways:

1) We will obtain more data from more fields of view

2) Use the time-derivative of the tip-angle, phi (φ) dynamics to examine whether the onset and return strokes are asymmetric and how this compares to ciliary dynamics.

3) We will also analyze the tangent angle to the contour, psi (ψ) plots with time (y-axis) and MT length (x-axis).

A qualitative analysis of a few time-series suggests indeed that the onset v/s return stroke of the ‘beating’ is likely to be asymmetric in the manner qualitatively distinct from cilia and flagella, that appear to be symmetric. This would suggest we avoid the term flagella-like to describe the dynamics.

2. Also, the beating frequency is very low (mHz) compared to real cilia/flagella (~Hz). Would it be possible to use the model to predict which parameter would need to be tuned to reach more

physiologically relevant beating frequencies?

Our response: We agree that the oscillations we observe have a frequency thousand fold lower as compared toflagella and cilia and have highlighted this in our discussion. When we modified motor velocity and stall forces, we found only a marginal increase in frequency of oscillations by a factor of 2-5, but not 10-fold or more. We also attempted in simulations to mimic kinesin-like properties. However we do not see a dramatic improvement. This suggests an involvement of higher-order organization of the filament. Indeed we plan to perform simulations that test the following scenarios not already tested:

1) the role of microtubule bundling factors resulting in 2-, 3-, and higher order complexes of MTs

2) varying the bending rigidity of the microtubules within ranges of what may be experimentally feasible with differences reported for taxol and GMPCPP filaments

3) altering the duty ratio of the motor

These will be in the nature of “what if” simulations that could provide the basis of future experimental design to test such predictions. This comment is similar to one by the other reviewers.

Significance

This study is part of the field of in-vitro reconstitution, from a minimal set of components, that aims to reproduce a biological function to identify and understand the minimal physical/biophysical mechanisms underlying a function. This study might be of interest for the people who address questions of the self-organization of cytoskeletal elements in minimal systems.

Our response: We agree with the assessment of the reviewer of the significance of the study and the readership that might be most interested in this work.

*The main limitation of this study relies on the claim of reproducing a flagella-like motion. Indeed, the frequency of the described oscillations is in the mHz range while the frequency of cilia is in the range of few Hz to tens of Hz. This suggests that the mechanism at play in such a reconstituted system is not the one that drives beating in real cilia/flagella. Yet, this limitation also applies to other studies in the field (Vilfan et al. 1999, Guido et al. 2022 ...). *

Our response: We agree with the reviewer that the 10^3 to 10^4 difference in oscillation frequency with that observed in cilia is striking. Indeed our claim was limited to the wavelike nature of the oscillation of the free end of a clamped microtubule driven by molecular motors producing a buckling instability, release and re-engagement of motors. Therefore it is evident we are missing many components in our minimal system as compared to cilia. However, we would like to emphasize for now that the beating is only qualitatively comparable to cilia and flagella. So far we have not compared the two waveforms. As a part of our revision plan, we aim to objectively describe the quantitativeaspects that could strengthen our claim of a similarity or lack thereof in wave-forms.

Indeed this limitation is also observed in the work of Vilfan et al. (2019) and Guido et al. (2022). However, we believe with changes to the experimental setup and a robust and tractable model we have improved on these studies.

References:

Vilfan A, Subramani S, Bodenschatz E, Golestanian R, Guido I (2019). Flagella- like Beating of a Single Microtubule. Nano Lett 19(5), 3359–3363.

Guido I, Vilfan A, Ishibashi K, Sakakibara H, Shiraga M, Bodenschatz E, Golestanian R, Oiwa K (2022). A synthetic minimal beating axoneme. Small e2107854.

My second concern is that the added value with regards to state of art is not clearly explicit. I'm thinking about the work of the Isabelle Guido's team where they have more complex reconstituted systems (a pair of 2 microtubules); or the work of Pascal Martin's lab where the design of the system allows to capture more complex mechanisms such as myosin density waves, which result infrequency beat of 0.1Hz.

Our response: We agree that the advances of our study can be highlighted. In the following points we highlight the value added to prior art:

1. In previous work, MT bundles have been shown to produce synchronized base-to-tip oscillations in vitro driven by kinesin in presence of crowdants (Sanchez et al., 2011??). However, the study lacked control over MT length,, something we have addressed in our study.

2. Cilia reconstitution with MT length and motor density control (Sasaki et al., 2018) are closer to control of the system but because of the complexity it is hard to distinguish what effect emerged from which componen.

3. The generation of a bending wave driven by outer dynein arm (ODA) combined with pairs of MTs nucleated from Chlamydomonas axonemal fragments (Guido et al., 2022) was probably a close mimic of a minimal system; it not only lacked ??lacked variation in motor density and length but failed to show oscillations, with S-shaped buckling patterns observed.

As a result it is reasonable to state that this work is a distinct improvement on previous work. In some senses it provides a consistency check on the previous results and at the other with a model and novel order-parameter an opportunity to improve our understanding.

References:

1. Vilfan A, Subramani S, Bodenschatz E, Golestanian R, Guido I (2019). Flagella-like Beating of a Single Microtubule. Nano Lett 19(5), 3359–3363.

2. Sanchez T, Welch D, Nicastro D, Dogic Z (2011). Cilia-like beating of active microtubule bundles. Science 333(6041), 456–9.

3. Sasaki R, Kabir AMR, Inoue D, Anan S, Kimura AP, Konagaya A, Sada K, Kakugo A (2018). Construction of artificial cilia from microtubules and kinesins through a well-designed bottom-up approach. Nanoscale 10(14), 63236332.

4. Guido I, Vilfan A, Ishibashi K, Sakakibara H, Shiraga M, Bodenschatz E, Golestanian R, Oiwa K (2022). A synthetic minimal beating axoneme. Small e2107854.

Reviewer #2 Evidence, reproducibility and clarity Summary:

The authors use a modified version of conventional gliding assays to induce microtubule bending, buckling, looping and cyclic beating (which they term "flagella-like") via clamping the plus ends of gliding microtubules to the surface. They find that the pattern of motion depends on different factors such as microtubule length and motor density. They build a simple computational model that predicts transitions between microtubule motion patterns depending on these parameters.

Our response: We agree with the assessment of the reviewer summarizing our work in terms of the approach taken and the inferences.

Major comments:

- Overall, the experimental data is extremely sparse. As far as I can see, there are only two replicas for the lower motor density. It is not clear to me how the authors define the boundaries in the

experimental phase diagram in Fig. 7. To build a phase diagram - where one axis corresponds to the motor density - on just two experiments is not convincing. I would need to see more experiments covering a larger range of motor densities and at least three replica per condition.

Our response: The comment refers to Fig. 7, whose purpose was to answer the question- can we test the phase diagram predicted in simulations by comparing to experiment? The answer was provided with representative data, in order to demonstrate that the model is qualitatively validated.

The reviewer is asking for a systematic experimental test that rigorously demonstrates such a match between simulation and experiment. To this end, the phase diagram may not be the ideal form for such a test. We will attempt to examine the beating frequency and wave-transition in line with a comment by reviewer #3, as a measure of experiment-theory validation.

We agree with the reviewer that our data could be enriched with replicates, with more densities of the motor. We will then analyze all the experimental data using common metrics to compare to simulations.

- It is not clear to me why the proportion of pinned vs. free microtubule segments should affect the beating pattern. I would expect that the free microtubule segment does not "feel" the length of the clamped segment, if it is indeed fixed all along its length and unable to move / bend. The simulations use only two anchor points at the pinned tip. The segment in between the anchor points bends, which could affect how the free microtubule segment behaves. To support the claim that it is indeed the proportion of the lengths of the pinned vs. free segments and not simply the length of the free segment alone that influence the beating pattern, I would expect to

(1) see the corresponding and thoroughly quantified experimental data that verifies this simulation-based prediction. Fig. 5C is based on only three microtubules and it is not clear how long the segments are.

(2) the entire pinned segments in the simulation should be fixed. This should also be compared to experimental data, where the lengths of the free segments are the same and only the lengths of the pinned segments

vary.

Our response: Originally the intention of comparing pinned length changes was based on experimental design, in which we incubated biotinylated tubulin to obtain longer or shorter clamped plus-ends. The contrast between a point-pinning and a longer segment is based on beam bending and buckling theory, corresponding to the difference between a swivelling point of immobilization (pinning) and a clamped end (clamped). However, we agree with the reviewer that beyond the pining scenario, once a segment is pinned the only thing really driving the beating is the free length. To address the specific comment we aim to add simulation calculations that will include a fixed clamp and increasing free length demonstrating that the primary driver in changing dynamics (so long as a segment is clamped) is the free length.

(1) To address the question of experimental comparison we will examine more data with increasing free segment lengths for the same density of motors and plot the dynamics, as well as characterize the oscillations with frequency estimation.

(2) This relates to the earlier part of this comment and we aim to re-run the filament clamped segment simulations to make it consistent with expectation and theory from related papers in the field, with only the free-segment length varied.

- In relation to my previous comments: I would expect a direct comparison between the simulation-based prediction that the beating pattern changes with microtubule length and motor density in a quantitative manner, where all pinned microtubules observed experimentally are analyzed. The figures are often based on single observations.

Our response: The experimental phase diagram had representative beating MTs, as compared to simulations. We agree that showing more statistics on these patterns could help. We aim to perform more experiments and analyze more data, which will be systematically plotted to make statistically relevant inferences of patterns as a function of density and length of MT.

- The authors report that the pinned microtubules typically undergo 2-3 cycles of beating. This

number is very low, and I am hesitant to call it "flagella-like" cyclic beating. Is this due to the dynein motors being much slower than e.g. kinesis? To confirm this and support the generality claimed by the authors, I would like to see experiments with a different, faster motor. If other motors are not readily available to the authors, this would imply a substantial amount of time and effort though.

Our response: The slower velocity of yeast cytoplasmic dynein is indeed one the contributing factors for the slow oscillations seen. In preliminary experiments with kinesin we indeed see a faster oscillation, but still in the 10 mHz range. These experiments will be added to the revised manuscript.

- Please perform statistical analysis of the experimental data.

Our response: Most of the data, while statistical, is not being compared for means (e.g. simulation v/s experiment). However we will analyze the frequency as a function of length and density and examine differences based on standard statistical tests.

Minor comments:

- Number of replicates and samples should be indicated in the figures.

Our response: With additional analyzed data and new experiments we will have more datasets and

Significance

- The approach to clamp the plus ends of gliding microtubules in order to induce buckling, bending and beating is elegant and should be easily transferable to other groups who may be interested in this method, since it is straightforward to adapt conventional gliding assays to induce pinning.

Our response: We agree with this assessment of the reviewer.

- The study could potentially be interesting to an audience studying flagella-like systems. Since the system is simple and based on in vitro components with defined parameters, it could serve as a basis for studying more complex systems or testing the influence of particular proteins associated with flagella. However, I do not see a major advance regarding our understanding of flagella or similar structures based on the manuscript. In combination with the model, I see it majorly as a useful tool, providing methodological advance. It would be desirably to make the computational model available to the public.

Our response: We agree that this system of minimal in vitro components could in future be made more complex in a step-wise manner. Once the manuscript is accepted after review, we have intended to make the code available in OpenSource. The source code of Cytosim already is OpenSource and can be downloaded here: https://gitlab.com/f-nedelec/cytosim.

- The computational model seems useful and straightforward to me, yet my background is purely experimental and I cannot judge the model in detail.

Our response: The computational model is indeed straightforward, and is based on a set of C++ codes that are OpenSource and those with a computational training have tested it in multiple studies both by us and other labs.

- In my view, the most important limitation of the manuscript is its lack of thorough experimental data to support the claims made by the authors. In its current state, the manuscript seems rather preliminary and I see the need for significant additional experimental evidence.

Our response: We plan to take the reviewers criticism on board and perform new experiments, analyses and simulations to address this gap of additional experiments. These experiments we believe will go to strengthen the manuscript, but not fundamentally alter the result.

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

-Summary:

*This manuscript reports experimental in vitro gliding assays demonstrating bending oscillations when single microtubules are anchored at their plus end and compressed beyond a buckling threshold by dynein molecular motors immobilized on a solid substrate. Together with numerical simulations based on the well-established Cytosim software, the authors identify three main classes of motile behavior under the control of microtubule length and motor density: aperiodic fluctuations (flapping), periodic beating with bending traveling waves over at least part of the filament length, and looping behaviors where the microtubule can curl on itself near its free tip. The authors claim that these movements are reminiscent of the beating movements of eukaryotic cilia and flagella and may provide useful information of the mechanism underlying the oscillatory instability. *

Our response: We are grateful to the reviewer for a careful reading and have in the following sections outlined our plan for revision in response to the specific comments.

*- Major comments: *

1. The observed oscillations show only a few cycles (up to only 4, but often 2-3 (Fig. 1-2)) and are in addition very noisy. Oscillations thus appear to happen only transiently, i.e. do not show a dynamic steady state on timescale much larger than the oscillation period. Demonstrating the emergence of true (and stable) regular oscillations thus remains a challenge, in contrast to the authors' claim. The large variability of behavior from filament to filament (as seen in SV3), as well as in a single filament over time, also makes it difficult to achieve a robust quantitative description of these movements (see below).

Our response: We have observed at times 4 and at times more cycles but we believe this is limited by the fact that mechanical pulling on the streptavidin-biotin linkage could result in occasional detachment of the filament from the surface. Stable oscillations of the form that the reviewer is pointing to may not emerge due to practical challenges and may require an alternative experiment such as optical tweezer to clamp the filament for a longer period. This is currently beyond the scope of the study, but could be attempted in future.

Regarding the variability, we are aiming to analyze more data that has already been recorded and is also being acquired. These additional datapoints will allow for more representative statistics. The variability should tell us more about the nature of the system. We will estimate frequency of oscillations as a parameter for comparison along with our order parameter (span). This is similar to the comment by reviewer #2.

2. Overall, the amount of experimental control seems relatively limited, for there is systematic variation of microtubule length (free or pinned) and only two motor densities have been explored.

Our response: We will address this shortcoming by performing more experiments, with a few more motor densities of intermediate value. This will be supplemented with additional data analysis.

• *One wonders why the motor density has not been more extensively varied and what determines the range of densities that can be achieved. What happens if the density gets larger than 50/µm^2? Do the filaments fail to remain anchored? Is buckling still permitted at high motor density? *

Our response: The range of densities are obtained after the experiment, since this is not a patterning system. At times the density is either too low, and the filaments do not beat, or too high and they detach. This results in only two reported densities, less than perhaps desirable as pointed out by the reviewer. Now that we know what densities work, we aim for a fine-grained scan in the same range expected to produce regular oscillations.

We will titrate the motors to obtain intermediate densities in the range that we have already found to result in stable oscillations with between 4-5 periods and hope to address this question.

• *Important fundamental issues remain here unfortunately untouched in experiments and are also only qualitatively discussed in simulations (bottom of p11 and Fig. 4), namely the dependence of the frequency and wavelength of wave-like beating as a function of motor density and microtubule length. These limitations result from a lack of control over the microtubule lengths and that only two motor densities have been tested. Using the natural variability in length of the anchored filaments may be potentially used to study length effects but then a relatively large amount of data will be required to reliably conclude that filaments ensembles of different mean lengths reliably show different behaviors. Similarly, I do not see where in the data one can see that increasing motor density actually controls the oscillation frequency, as concluded from simulation data (but not analyzed quantitatively). *

Our response: We plan to systematically analyze the frequency, which we have already demonstrated we can measure. The dependence on MT length and density will be tested and added as additional data. We will perform experiments with more motor densities to address that aspect too. We will also run additional simulations and compare outputs. This will help to address the comment and is in line with suggestions by the other reviewers too.

3. The authors repeatedly claim that the movements they observe are "flagella-like". However, the comparison remains vague as there is no quantitative assessment of the similarity or dissimilarity between the movements observed here and biological beating of flagella or cilia (e.g. using data in Riedel-Kruse et al HSFP Journal 2007. DOI:10.2976/1.2773861 as a reference).

Our response: We have compared frequency of oscillations from previous literature but find them to be extremely disparate – by a factor of 1,000. We will use the suggested references to find geometric properties that could test our claim of flagella-like in terms either of waveforms, symmetry of beating or the dynamics or tip-behavior.

• *What does it mean to resemble flagellar beating? It would be desirable to be more explicit/quantitative and not be ashamed to point to differences (could be event more instructive) as well as to similarities. Note that oscillations of the tangent angle in flagella of the bull sperm are nearly sinusoidal, and are thus smooth, with no snaps (Riedel-Kruse et al HSFP 2007), thus challenging the claimed resemblance between bending oscillations in this work and the flagellar beat. *

Our response: This is similar to the previous point. We agree that a quantitative comparison between the dynamics we observe of single filaments and of bonafide flagella, could strengthen the findings of this manuscript. We will use multiple metrics such as the tangent angle-with time of the free end, and the average angle along the flagella (as reported by Riedel-Kruse et al.) to make a more concrete comparison.

• *In my opinion, the authors should tone done the resemblance of their system with cilia and flagella and be much more quantitative about the detailed features of the observed movements in their in-vitro assay. *

Our response: We will take the reviewers comment on board and discuss the work in the absence of the flagellar connection since indeed there is no direct link so far- our comparison with flagella-like systems will be moved to the discussion section with a qualitative comparison of waveforms as this reviewer and others have suggested.

• *In the present gliding assay, motors produce compressive tangential forces on the microtubule, which can result in buckling and thus in an elastic load applied by the filament to the motor with a component perpendicular to the filament. Instead, flagellar motors produce force dipoles that result in neighboring-filament sliding which is then converted in bending of the filament bundle as a result of elastic constrains. Symmetries of the problem thus seem very different. It is also worth noting that many (but not all) models of the flagellar beat actually assume a constant inter-filament distance so that there is no effective normal force acting on the motors to detach them, yet faithfully reproduce beating waveforms (e.g. Camalet and Jülicher New J of Phys (2000) DOI: 10.1088/1367-2630/2/1/324; Riedel-Kruse et al HSFP J (2010)). More generally, whether the present study provides any useful information to inform our current understanding of the flagellar beat is not clear to me and the authors' claim that it may be the case is not motivated enough. Accordingly, the statement (P19) "qualitative transitions (...) expected from not just the minimal but even the potentially complex flagellum" is not justified. *

Our response: This distinction will be more elaborately discussed in the revised discussions section and similar to the previous point, we will avoid reference to flagella-like behavior.

4. I could not find a detailed statistical account of the total number of filaments that was used for the paper, how many fell in the four classes of movement (swiveling, fluctuations, beating, and looping) identified by the authors, and whether the population in each class could actually be controlled experimentally, e.g. by varying motor density or microtubule length. This gave the unfortunate impression that the conclusions were based on cherry picking, which is troublesome considering the large variability in behavior between filaments and the ambition of the authors to provide a state diagram of the dynamics (Fig. 7). To reach clear conclusions, one parameter must be changed while the others remain fixed. For instance, to discuss the effects of the pinned length, one would like to fix the total microtubule length (but then the free length varies) or vary the pinned length with constant free length (thus changing the total microtubule length). I understand that this might be difficult (in experiments), but the authors should then acknowledge these limitations and mitigate their conclusions. In principle, if the yield of the experiment (number of anchored filaments per slide) were sufficient, one could to address these issues by classifying the filaments in ensembles of a given properties (e.g. same total length by variable pinned length). To reach this goal, there is a need to obtain a sufficiently large quantity of data. The reader gets an estimate on the order of 10 usable filaments per slide (video SV3 and inset in Fig. 2D), with only a few replicates (4 experiments at 46/µm^2 and 2 experiments at 27/µm^2). The authors talk about "representative filaments" throughout the text but there is no detail about the ensemble of filaments that show a given behavior and the number of filaments that are used to reach a given conclusion is not given. Length distributions for the free and pined ends of the microtubules, for the maximal amplitude of tangent-angle oscillations, and other measures that characterize the microtubule movements (curvature, wave speed) ought to be given, provided that enough data has been collected to compute reliable ensemble averages.

Our response: For now we have only considered the average behavior with the dynamics observed from multiple fields of view, combined in terms of MT lengths and motor densities. Since Fig. 7 was meant to be representative and therefore a qualitative comparison with simulation predictions, replicates were not added. However, in response to reviewer’s question, we will analyze more data and add it in the supplementary material, in order to support the statistical validity of our claims- that are not based on purely selective evidence.

*5. The effect of motor density on beating properties, in particular frequency, is discussed in simulations but not clearly demonstrated in experiments. One cannot conclude that experiments confirm the prediction of the theory in this respect. *

Our response: Currently we have used a novel metric for the type of oscillation and pattern, the span-parameter (S). However, this was meant to capture large qualitative changes observed in experiment and simulation in terms of patterns.

In response to this comment, we will also analyze the dominant frequency of filaments using FFT on the tip-angles from multiple conditions of MT length and motor density. The scaling of frequency with length and motor density will be compared to simulation predictions. The comparison will then allow an additional quantitative comparison between experiment and simulation.

*- Minor comments: *

6. More extensive quantitative analysis of the waveform of oscillation (noisy sinusoid vs. sawtooth or relaxation oscillations?) and bending wave propagation (speed and curvature vs position along the filament) is needed. In particular, it is claimed that the filaments "snap" and thus evince a "recovery stroke" (e.g. p7). I agree that snaps are evident in some of the videos, and are expected at low motor density. However, I would expect the movements to get smoother at higher motor density, as shown in simulations (looping regime). In any case, one could use the analysis of the tip or (better) tangent angle as a function of time to assess whether 'snaps' indeed occur; due to noise, snapping behavior is not so clear in the data provided in Fig. 1D-E.

Our response: We agree with the reviewer that “snap-back” movement arising from potentially low motor density scenarios changes when the motor density is increased to a more smooth motion. We have observed this, and will characterize it quantitatively to make this point more clear. The tip-dynamics will be analyzed for velocity and symmetry to make this point more apparent.

*7. Because the tips of the microtubules are "sticky" due to their biotinylated tips, I wonder whether the histogram of gliding velocity of the microtubules that are not anchored is modified, i.e shifted toward lower velocities, as compared to that of bare gliding microtubules. This is assuming that a majority of the microtubules are equipped with biotinylated "heads"; this information ought to be provided in the Methods if, as the author claim, the biotinylated tips can be visualized. Analysis of gliding velocities (e.g. in video SV3) could potentially reveal the enhanced interaction between the microtubules and the surface. *

Our response: We will analyze the instantaneous gliding velocity and test the hypothesis that some filaments may be transiently immobile, while others may move unhindered at typical gliding assay velocities (50 to 80 nm/s).

*8. Demonstrating that the anchoring strategy has actually improved the chance to anchor a microtubule, as compared to random anchoring to surface defects that occur naturally in gliding assays, would be welcome. *

Our response: We will analyze the frequency histogram of gliding assay velocities and compare them to the filament-oscillation scenario with biotinylated filaments. We expect to see a zero-velocity mode in the clamped filament scenario and only transient (and therefore less frequent) pinned or clamped filaments. This is already our qualitative observation but we will seek to quantify it.

*9. The simulations should be analyzed more quantitatively and extensively to study how motor density and microtubule length affect the wavelength and frequency of oscillations in the wave-like beating regime, going beyond what can be achieved experimentally. In particular, one could compute the speed of the bending waves, asses how it varies during wave progression from base to tip of the microtubule, describe the increase in the magnitude of tangent-angle and curvature oscillation as a function of curvilinear abscissa. *

Our response: We have now analyzed the frequency and amplitude of filament oscillations in simulations. This will in the next step be used to look for trends as a function of MT length and motor density. We hope indeed to look beyond what experimentally achievable ranges might be, including measuring the propagation of the bending wave along the contour as suggested by the reviewer.

*- Suggestions to help improving the presentation: *

*1. First section of the results (p5-7): this section is full of methological details that get in the way of the description of the actual result (Fig. 1). I would suggest moving these details (e.g. there is not need here to explain how the motors are attached to the substrate, which you use cytoplasmic yeast dynein, and other details). *

Our response: We will rewrite the manuscript to improve the clarity and move the methods to the section dedicated to the methodology.

*2. The top of P9 could also be moved to Discussion section. *

Our response: We will move the page 9 text referred to into the discussion.

*3. P12-13: I also find that the Results section mixes results with discussion, which is not very effective. I would again move elements of discussion (here associated with bending energies) to the Discussion section and focus on results only. *

Our response: We have done so due perhaps to a requirement from an earlier round of reviews. However, we will be happy to separate results from discussion- for example the reference to bending energies.

*4. Throughout the result section: Move any comparison to actual flagellar dynamics to a dedicated section in Discussion. *

Our response: The flagellar discussion will be moved out of the results section entirely unless we invoke the analysis of bonafide flagella.

5. P12: doesn't the increase of the clamped length reduce the length of the free length, moving in the state diagram toward regions of shorter filaments. One wonders whether the clamped length really matter as long as the filament is clamped near the plus end. I would naively expect that it is the free-filament length that maters rather than the total length or the faction of the filament that is clamped.

Our response: We agree with the reviewer with one caveat- filaments pinned at one end (point pinning) are distinct from those with long segments clamped. However, the reviewer is correct in pointing out cases where filaments have a substantial clamp, the free length is more important. We will revise our figures and results section to clarify this.

*6. Figure 4: this figure shows very interesting simulation data that, in my opinion could be much more extensively studied. In particular, one could plot the oscillation frequency, the bending-wave speed, and wavelength as a function of the filament length and the motor density. Also, to characterize the beating waveform more in detail, it would be worth computing how the magnitude of tangent-angle oscillation increases with the curvilinear abscissa for representative examples of waveforms in the three regimes (see again in Riedel-Kruse et al HSFP 2007. DOI: 10.2976/1.2773861 or Pochitaloff et al Nat Phys 2022 DOI: 10.1038/s41567-022-01688-8). *

Our response: We have performed fourier series analysis to obtain dominant frequencies. This will indeed be applied to the simulations in Fig. 4 in order to examine the rich dynamics, as well as provide a point of quantitative comparison to exepriments.

*7. Figure 5: the way to display the data in A-B (simulations) and C-D (experiments) does not allow for an easy comparison between simulations and experiments. I would use beating patterns and kymographs of the tangent angle for both. *

Our response: We are in the process of revising Fig. 5 in order to examine the effect of free MT length on oscillations and will put experimental and simulation analyses that match each other in the nature of the analysis. The analysis itself will be elaborated to include aspects such as average tangent angle as a function of arc-length (Riedel-Kruse et al., 2007) along with frequency.

*8. Figure 6: the way to present the experimental beating patterns is no so clear (thick colored lines). I would recommend showing black lines resulting from automatic tracking of the microtubule. *

Our response: The data in Fig. 6 is raw data projected in order to provide a picture within the limits of magnification. In order to address this comment we will project the tracked contour of the filament and that will result in a finer and better resolved image.

*9. Legend of Fig. S1: the panels (D) and (E) of the figure are not called properly. *

Our response: We will rectify the issue of sub-figure callouts.

*10. Fig. S3: use the same scale in the different panels of (a) and (b) to allow for an easier comparison. It would also be nice to show videos of the simulated motion. *

Our response: The current differences were in order for visual clarity and the modified axis values are mentioned. We will revise Fig. S3 simulation outputs where filaments are projected on the same axis for consistency.

*11. Fig. S4: Hard to read, in particular the motors are not visible. Would be better to have the patterns in black on a white background. The panels look like screen shots. *

Our response: The unbound motors have been deliberately made invisible for clarity. We can provide a figure update with the motors made visible again.

*12. Fig.S5: indicate in the title that this figure deals with results of simulations. The legend refers to color bars but the figure is in grey scale. *

Our response: Colorbar is indeed in grayscale. The legend entry will be modified to read “grayscale bar”.

*Reviewer #3 (Significance (Required)): *

*The motile phenomena reported here are qualitatively already well known in the field. Indeed, anyone who has performed a gliding assay, with microtubules or actin filaments has probably seen undulating or spiraling filaments accidentally anchored on surface defects. Accordingly, the topic has already been somewhat adressed in previous publications (e.g. Bourdieu et al Phys Rev Lett 1995; Sekimoto et al Phys rev Lett 1995; Vilfan et al Nanoletter 2019). As a matter of fact, microtubules anchored on defects in standard gliding assay can show oscillations very similar to those shown here. However, the lack of control over filament anchoring has precluded a systematic experimental study of the oscillatory filament dynamics. It is worth noting that ther bottom-up approaches have used filament bundles instead of single filaments, either with microtubules and kinesin motors (Sanchez et al Science 2011) or actin filaments and myosin motors (Pochitaloff et al Nature Phys 2022). These assays evince more regular oscillations (over tens of cycles) and waveforms that more closely resemble those of eukaryotic flagella than reported here. *

Our response: We agree with this summary of our work, and will highlight the possible reasons why it differs from the work of Pochitaloff et al.

*Here, the authors have developed an experimental strategy to increase the chance of anchoring single filaments' plus end to the substrate, potentially allowing for more control of the experimental conditions that lead to the emergence of oscillations (but see my criticisms above). Anchoring is made more likely, because short segments of biotinylated tubulin are added to the end of bare microtubules to make them stick to the substrate, which has been functionalized with streptavidin. A similar protocol had been reported before in the literature to study buckling of single microtubules by single kinesin motors (Gittes et al Biophys J 1996), but is here used at larger motor densities on the substrate. There is unfortunately no quantification of the success of the approach. *

Our response: We propose to perform more experiments and analyze the data more quantitatively using multiple measures described in the literature and cited by this reviewer. We believe these changes will adequately address the concerns.

*The comparison of the experimental data to Cytosim simulations is, to my knowledge, novel and a clear asset of the work, although this comparison could be more effective, as detailed above. *

Our response: We will add a more complete quantitative comparison to supplement the already provided qualitative comparison to address the comments.

*The emergence of periodic wave-like beating oscillations in motor-filament systems is a classical problem in biophysics. This problem is particularly relevant in the context of eukaryotic cilia and flagellar beating in biology. The audience for the present work is thus potentially broad, although the simplistic and artificial nature of the in-vitro system, with only one microtubule, will probably appeal more to biophysicists and theoretical physicists than biologists. *

Our response: We appreciate the effort of this reviewer to evaluate our work. We however believe that the relevance of this work could extend beyond purely biophysics and theoretical physics as claimed by the reviewer.

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Temperatura

Reviewer #2 (Public Review):

The authors phototag DA and GABA neurons in the VTA in mice performing a t-maze task, and report choice-specific responses in the delay period of a memory-guided task, more so than in a variant task w/o a memory component. Overall, I found the results convincing. While showing responses that are choice selective in DA neurons is not entirely novel (e.g. Morris et al NN 2006, Parker et al NN 2016), the fact that this feature is stronger when there is a memory requirement is an interesting and novel observation.

I found the plots in 3B misleading because it looks like the main result is the sequential firing of DA neurons during the Tmaze. However, many of the neurons aren't significant by their permutation test. Often people either only plot the neurons that are significant, or plot with cross-validation (ie sort by half of the trials, and plot the other half).

Relatedly, the cross-task comparisons of sequences (Fig, 4,5) are hampered by the fact that they sort in one task, then plot in the other, which will make the sequences look less robust even if they were equally strong. What happens if they swap which task's sequences they use to order the neurons? I do realize they also show statistical comparisons of modulated units across tasks, which is helpful.

Overall, the introduction was scholarly and did a good job covering a vast literature. But the explanation of t-maze data towards the end of the introduction was confusing. In Line 87, I would not say "in the same task" but "in a similar task" because there are many differences between the tasks in question. And not clear what is meant by "by averaging neuronal population activities, none of these computational schemes would have been revealed. " There was trial averaging, at least in Harvey et al. I thought the main result of that paper related to coding schemes was that neural activity was sequential, not persistent. I think it would help the paper to say that clearly. Also, I'm not aware it was shown that choice selectivity diminishes when the memory demand of the task is removed - please clarify if that is true in both referenced papers. If so, an interpretation of this present data could be found in Lee et al biorxiv 2022, which presents a computational model that implies that the heterogeneity in the VTA DA system is a reflection of the heterogeneity found in upstream regions (the state representation), based on the idea that different subsets of DA neurons calculate prediction errors with respect to different subsets of the state representation.

I am surprised only 28% of DA neurons responded to reward - the reward is not completely certain in this task. This seems lower than other papers in mice (even Pavlovian conditioning, when the reward is entirely certain). It would be helpful if the authors comment on how this number compares to other papers.

DOI: 10.1016/j.celrep.2023.112884

Resource: (Cell Signaling Technology Cat# 9661, RRID:AB_2341188)

Curator: @scibot

SciCrunch record: RRID:AB_2341188

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DOI: 10.1016/j.celrep.2023.112927

Resource: RRID:Addgene_12259

Curator: @scibot

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prueba II

Nad touto sekcí je opravdu velký margin a pod touto sekcí zase skoro žádný není

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Reply to the reviewers

We appreciate the positive feedback from both reviewers and their critical comments, which will help us to improve the manuscript. Below, we provide a point-by-point response and how we propose to address their queries and comments.

As the laboratory is currently undergoing a major transition, we propose essential experiments that are realistic to perform under these circumstances. We are positive that we can address all the most critical points identified by the reviewers.

Suggestions for minor changes to the figures are already included.

We also include responses to questions the reviewers raise.

Reviewer #1 (Evidence, reproducibility and clarity):

Major comments:

1 - The authors assessed acridine orange incorporation in BECs upon LiverZap and concluded that LiverZap triggers hepatocyte-specific cell death without a bystander effect in adjacent cells (Figure 1 D-E). What happened to endothelial cells, which could also be affected either directly by ROS production in hepatocytes or indirectly by gross morphological changes in tissue organization?

Response:

The reviewer raises two excellent points:

(i) Bystander effect of hepatocyte-produced ROS on endothelial cells: the cell death analysis included in the manuscript, shows that Acridine Orange staining overlaps with hepatocyte _tg(fabp10a:dsRed) expression, but not with biliary tg(tp1:H2B-mCherry) indicating cell death is specific to hepatocytes. Moreover, singlet oxygen species as produced by LiverZap have been shown to have a very short half-life and short range of action, suggesting that neighbouring cells are unlikely affected (Liang et al., 2020).

PLAN: Investigate potential bystander effect in endothelial cells, by activating the LiverZap tool in livers expressing transgenic tg(kdrl:mcherry) marking the vascular network followed by live staining with Acridine Orange at 8 hours post illumination.

(ii) indirect effects of morphological tissue changes on endothelial cells: studying the tissue response of the vascular network to hepatocyte ablation would be very interesting. A separate and detailed study would be required to generate meaningful data and insights into said process. This could encompass for instance the use of the transgenic endothelial _tg(kdrl:mcherry) line for LiverZap experiments and parallel those in Figure 4A-S. Thus, it seems beyond the scope of this work.

NO CHANGES PROPOSED.

2 - The evaluation criteria for distinguishing mCherry and cells in imaging experiments should be clearly described in the methods section. The authors should also provide some quantitative data regarding the level of correlation between the mCherry hepatocytes and the BEC-derived hepatocytes strictly defined based on the TP1-H2B-EGFP lineage tracing, as the former was used as a surrogate marker for the latter in some experiments.

Response:

Here, we believe the reviewer refers to the Tp1:H2B-mCherry-based lineage tracing, since the tg(tp1:egfp) line has not been used for this purpose. Similar to previous studies in the regeneration field (e.g. Choi et al., 2014; He at al., 2014), we have used histone inheritance of Tp1:H2B-mCherry for short-term lineage tracing. Tp1:H2B-mCherry-based lineage tracing was assessed on the whole organ level, for which we will describe the quantification pipeline. Tp1:H2B-mCherrylow cells were identified as BEC-derived hepatocytes after severe hepatocyte ablation, as shown in Fig. 2A,C, correlating with hepatocyte marker tg(fabp10a:GFP) expression. Tp1high and Tp1low cell numbers were quantified for 12, 24, 48 and 72 hpi and can be added as supplementary information.

PLAN: Update the material and methods section and produce a more detailed description. This would include the following information: Whole-mounted livers of tg(tp1:H2B-mCherry) fish were stained for mCherry and imaged using an Leica SP8 confocal microscope. Image processing was carried out using the Imaris software. All mCherry-expressing cells in the liver were masked using the “spots” function, which allows quantification of signal intensity of all cells, represented by a sphere. Tp1high and Tp1low cells were identified using an automatically generated intensity threshold. Due to intensity differences with increasing imaging depth/z-position, segmented Tp1high cells were manually curated.

To showcase the analysis strategy, we propose to include an example showing original image data, semi-automated quantification at the surface and deep tissue levels, as well as the overall Tp1:H2B-mCherry intensities for all positive cells and specifically Tp1high cells for all z-positions of an entire liver (see data figure below). This example could be included as supplementary data. Likewise, cell number quantification for Tp1high and Tp1low across regeneration can be added to Fig. S2.

Fig. Quantification of Tp1:H2B-mCherryhigh cells. (A,B) 10 µm maximum intensity projections from whole mount stained tg(LiverZap);tg(tp1:H2B-mCherry) livers at 48 hpi: at the surface (A-A’) and deep in the liver (B-B’). Tp1high cells are identified by fluorescence intensity of segmented nuclei, outlined in yellow (A’ and B’). Graphs showing distribution of all Tp1:H2B-mCherry nuclei (C) and Tp1high nuclei (D) by fluorescence intensity and z-position (C). The intensity of all mCherry+ nuclei decreases with increasing z-position (C-D). The dotted line outlines the liver in A-B’.

3 - OPTIONAL: In the locally restricted ablation model, do hepatocytes located adjacent to the ROI proliferate and/or contribute to the regeneration of the injured region?

Response:

An important consideration, as highlighted by the reviewer, is whether neighbouring hepatocytes also contribute to regeneration following ROI ablation.

PLAN: To address this point, LiverZap ROI ablation will be followed by cell proliferation analysis using an EdU incorporation assay at 24 and 72 hpi. These time points are selected based on the proliferation results following global LiverZap ablation; see Fig. 2D-F. The experiment will be performed in a tg(tp1:H2B-mcherry); _tg(fabp10a:gfp)_background to distinguish proliferating GFP-positive hepatocytes, which are H2B-mCherry-negative, from LPC-derived hepatocytes that have inherited H2B-mCherry (Tp1low). The resulting insights may help to refine hypotheses regarding the process(es) stimulating the formation of new hepatocytes adjacent to the ablated region.

4 - OPTIONAL: Figure 4, A-S. It should be of significant interest if the authors could also analyze the BEC dynamics using the locally restricted hepatocyte ablation model, comparing those in the injured region (ROI) and the outside of the ROI.

Response:

We agree with the reviewer that this is the exciting next question, as it likely would provide insights into the cellular mechanism by which the biliary network is de- and re-constructed, as well as the mechanism by which BECs outside the ROI may initiate the LPC response to give rise to hepatocytes in a semi-systemic response. For this, the experimental set-up introduced in Fig.4J-P, in which BECs in the ROI are distinguished from adjacent ones by photoconversion, would be followed by extended live light-sheet microscopy of the regenerating liver. Due to the complexity, extent of the experiments and current unavailability of a light-sheet microscope, we would address this optional comment in future investigations.

NO CHANGES PROPOSED.

5A- Figure 4, T-V'. The data shown here for the changes in E-cadherin distribution is difficult to understand and interpret. The authors should provide magnified images and better description on how to distinguish the membranous (spotted signals?) and intracellular localization. Quantitative assessment should certainly be a plus, if possible.

Response:

We appreciate that it may be difficult to recognize the changes in E-Cadherin localisation, in particular at BEC membranes, given that there are intracellular puncta, and that E-Cadherin is expressed both in BECs and hepatocytes. We are convinced of the related data described in Figures 4 and S4, because the first experiment allowed quantification of the staining using both Tp1:H2B-mCherry to identify BECs and intestinal E-Cadherin for normalisation, which revealed a 51% E-Cadherin reduction at BEC cell membranes following injury. Unfortunately, the signal-to-noise ratio declined in consecutive experiments precluding further quantification although we could still observe a change in localisation. We tested alternative antibodies against E-Cadherin as well as optimized staining protocols, yet without success.

5B - OPTIONAL: In relation to the above point, it is this reviewer's candid impression that the very last part regarding the possible role of E-cadherin dynamics in regulating the biliary network remodeling is still preliminary compared to the remaining parts, thereby rather depreciating the value of the entire manuscript. Perhaps this part could be published separately, together with more functional evidence regarding the causal relationship between them (e.g., showing the effect of Ecadherin knockdown in hepatocytes on the biliary remodeling and the induction of the BECdependent regeneration program)

Response:

PLAN: Following this reviewer’s and reviewer 2’s comments and suggestions, we agree to remove the data on E-Cadherin. Loss of adhesion as a mechanism for adopting an LPC-state remains very exciting, future investigations with novel tools to monitor and modulate E‑Cadherin expression in BECs would thus be needed.

6 - Do zebrafish livers possess lobular structures with the portal-to-central vein axis and the metabolic zonation as typically observed in mammalian livers? As has been described in the manuscript, the "localized" injury patters in the mammalian livers usually occur at the sub-lobular structure levels (i.e., peri-portal region-restricted vs. peri-central region-restricted). Although the "localized" injury model described in this study using the zebrafish livers was indeed localized from the viewpoint of the entire organ (or the lobe), it still seemed much more "global" when considering those situations in the mammalian livers, so that the authors' claim that the former recapitulating the latter might be too exaggerated and somehow misleading. The authors should clarify and discuss this point in the manuscript.

Response:

The reviewer raises an important point, and it seems that our wording might not have been clear. In mammals, boundaries between injured and healthy tissue arise, because liver injuries frequently occur at the sub-lobular level. Although zebrafish livers are composed of metabolically diverse hepatocytes, a spatial arrangement comparable to mammalian zonation has so far not been identified (Morrison et al. 2022; Oderberg and Goessling, 2023). Yet, the liver lobes in the adult zebrafish have a central vein and periportal veins at the periphery of the organ, similar to the mammalian lobular organisation (Ota et al. 2022). Therefore, the scale of injury in the mammalian setting and the ROI-ablation model introduced in the current work differs. It, nevertheless, creates boundaries of healthy and injured liver tissue relevant for uncovering dynamic cellular processes mediating tissue repair in chronic liver disease. Importantly, with its suitability for advanced live imaging and optogenetic methods (e.g. photoconversion), LiverZap, complements mammalian models, in which this is still challenging. This offers therefore the powerful opportunity to employ LiverZap to screen for dynamic repair behaviours, which subsequently can be validated in a target approach in mammalian injury models.

PLAN: To describe the relevance of our ROI ablation paradigm for elucidating repair processes at the interface of injured and healthy tissue more precisely. We will further edit and clarify text to place the ROI ablation into the context of hepatic injuries at the sub-lobular level throughout the mammalian liver.

Minor comments:

7 - Figure 4. Panels D and G should correspond to the same one image and the way of labeling be changed (as in Figure 1G). Likewise, in panel J, the bars shown separately as "M" and "S" at 12 dpi should correspond to the same data, so that they should be unified as one bar.

Response:

Thank you for pointing this out, this is changed in the updated figures; panels Fig. 4D and I.

8 - Figure S3L. How was the ROI border defined? Perhaps the shape of the ROI should change significantly during regeneration due to dynamic tissue remodeling processes, thereby moving the position of the border as well.

Response:

The ROI border was defined as the interface between photoconverted and non-converted BECs. We concur with the reviewer’s notion that cell movement and rearrangement may occur during the regeneration process (see Fig. 4A-J), and the initially straight ROI border could consequently change during the regeneration process. Nevertheless, the border between photoconverted and non-converted BECs persists, serving as a landmark for the measurements shown in Figure S3L.

Fig.: Quantification strategy for determining the region exhibiting an LPC-response outside the ROI ablation region. The dashed line of the ROI indicates morphogenetic changes of the interface between photoconverted and nonconverted cells over time due to repair-related cell rearrangement.

PLAN: In the revised manuscript, we propose to include the below schematic as panel J to Figure S3. Moreover, we also suggest to change the solid line of the squares indicating the ROI area in figure panels 3C,G,O,P and S3D,H,K into a dashed line at the interface between photoconverted and non-converted tissue (see below figure as an example).

9 - The authors should comment in the manuscript as to whether the system can be applicable for induction of more restricted areas (e.g., at a single hepatocyte level; in particular metabolic zones, if existing), as well as for ablation of other hepatic cell types such as BECs and endothelial cells.

Response:

Indeed, the optogenetic nature of the LiverZap system allows to induce hepatocyte death at the single cell level, as well as any defined region of interest that can be generated by the light source (e.g. confocal microscope software).

Likewise, the FAP-TAP system can be easily applied to BECs or endothelial cells, or any cell type for which a specific promoter has been identified to drive the genetic FAP component fluorogen-activating protein dL5**.

Response:

PLAN: Both points will be included in the discussion section of the manuscript.

Reviewer #2 (Evidence, reproducibility and clarity):

MAJOR COMMENTS:

1 - The LiverZap is an elegant new tool to induce localized ablation of hepatocytes. It is not as claimed by the authors a real breakthrough: (1) While localized ablation is nice compared to NTR-MTZ model in zebrafish, mice model such as CCl4 chronic injury can also study the interaction between healthy and injured tissue. (2) Although not using MTZ, the system still requires injection or exposure to malachite green derivate dye MG-2I. A few searches suggest that this compound could induce toxicity. Can the authors study and compare the toxicity of malachite green derivate dye MG-2I to the toxicity of MTZ? This is important as this would be indeed a strong argument in favor of the presented tool.

Response:

Point 1 – studying interactions between healthy and injured liver tissue: The reviewer is of course correct that interactions between healthy and injured tissue can also be studied in the mouse. However, ROI ablation with the LiverZap system can be combined with live imaging, thereby enabling the observation of cellular responses of the same sample over time, at a resolution currently difficult to achieve in mammals. Moreover, the possibility to induce cell death in a defined ROI, also allows to simultaneously employ other genetic tools, including cell-type specific lineage tracing by photoconversion, which is difficult to achieve in mammalian systems. The finding that BECs beyond the ROI of hepatocyte ablation produce new hepatocytes by a LPC response, illustrates the power of this approach. The optogenetic LiverZap ablation system would therefore complement existing mammalian and zebrafish liver regeneration models.

PLAN: to include a more detailed discussion of this point and the complementary knowledge that can be gained in the discussion section.

Point 2 – MG-2I toxicity__: Indeed, as described in the manuscript, the FAP-TAP system, underlying LiverZap hepatocyte ablation, requires MG-2I incubation for the formation of the photosensitiser. Compared to the NTR/MTZ system, incubation with MG2I is short, requiring <3 hours in contrast to more than 24hours MTZ incubation. The system, including MG-2I has also been employed in cells, as well as in the zebrafish heart and nervous system without reported adverse effects (He et al., 2016; Xie et al., 2020). Consistently, we have not observed any apparent adverse effects between 0-72 hpi following 3-18 hour MG-2I incubation (unpublished). Nevertheless, toxicity studies evaluating survival upon MG-2I incubation have not yet been carried out and may be required for comparison with MTZ.

PLAN: To perform toxicity studies for MG-2I, similar to those previously performed for MTZ (e.g. Mathias et al. 2014), in which larval survival after 3, 24 and 48 hour MG-2I exposure starting at 4 dpf will be assessed daily until 8 days post fertilisation.

2 -The term ablation is choose because it is anticipated that it induces heaptospecific death. However, the consequences of cell death is not shown. In particular, the inflammatory immune response is not shown nor discussed.

Response:

The reviewer raises an interesting point, namely the inflammatory immune response, which is not the focus of this manuscript. Acridine Orange- and TUNEL-positive cells during the ablation process indicate that the reactive oxygen species produced by the FAP-TAP system cause hepatocyte apoptosis. We predict that this would recruit and be cleared by macrophages with little or no inflammatory response, like findings for the NTR-MTZ system (Stoddard et al., 2019). However, the role of neutrophils is unclear due to a possible direct effect of MTZ on this cell type.

PLAN: We will include this point in the discussion.

Future in-depth live imaging of transgenic reporters will be required for detailed studies of macrophage and neutrophil recruitment and their role in efferocytosis, including transcriptome analysis of specific gene signatures to detect an inflammatory response.

3 - The difference between mild and severe ablation is hard to grasp. Can the authors explain more clearly the differences between mild and severe: what are the criteria as there is no difference in liver volume between mild and severe ablation? How do you achieve mild or severe ablation? It appears that the severity of the ablation is judged a posteriori and not decided per the experiment.

Response:

Concerning the first point, there must be a misunderstanding. Mild and severe hepatocyte ablation result in clearly different liver sizes, for instance at 30 hpi, the end of ablation, liver volumes are reduced by 23 % for mild or 64 % for severe cases (Fig. 1Q). This is supported by representative image data in Figs. 1F-P and S1A-C. Nevertheless, for consistency, we had represented the 12 hpi volume data as the same two data bars, although we cannot distinguish them yet at that timepoint of the experiment, as shown by images in Fig. 1F-G.

PLAN: Adjust Fig.1Q and represent the 12 hpi liver volume data as a shared graph for mild/severe ablation, see included figure 1Q. We propose to similarly represent all 12 hpi quantifications, as represented in Figs. S1F, 2D-F and S2A.

For the second point, the reviewer is correct that ablation severity is evaluated and determined between 24-30 hpi, at the end of hepatocyte ablation, given there is some variability in the response. Nonetheless, both length of 660nm illumination and oxygen availability can be used to shift the proportion of mild and severe ablation, depending on the desired outcome (Figs. 1Q, S1G-H).

NO CHANGES PLANNED.

4 - The work supports that biliary-driven regeneration also occurs when hepatocyte ablation happens in a small area of interest. Our knowledge is that you need a large defect in hepatocyte or a chronic liver injury ro activate the BDC-driven auxiliary process for regeneration. Could this be a specificity of the fish model?

Response:

Like the reviewer, our understanding is that severe hepatocyte loss, senescence or chronic liver injury activate BEC-derived regeneration in mammals and in zebrafish. All these cases are characterised by substantial reduction of local hepatocyte density or loss of function (in senescence). Given the overall hepatocyte loss is only 10-20% in the ROI model, the induction of the local LPC response was very surprising, on the other hand it corresponds to a near complete local hepatocyte depletion. The hepatobiliary architecture in zebrafish is similar to that of the mammalian ductular reaction, an adaptation of the biliary network to severe hepatocyte loss. In both cases, the majority of hepatocytes connect directly via their apical canaliculi to biliary ductules to ensure physiologic transport of hepatocyte products, often preceding the LPC response (Sato et al., 2019; Caviglia et al., 2022). Therefore, we propose that the LPC response following ROI hepatocyte ablation is not specific to the zebrafish model, but a common mechanism elicited across species and related to the severity of the injury and the configuration of the hepatobiliary network at the time of injury, such as the ductular reaction.

PLAN: To edit the text and discuss this point clearly.

5 - Pathways revealed to control liver regeneration or BEC-driven regeneration in fish have not be found to have a similar drastic predominance in rodents. This mitigate perhaps the use of fish for this type of research?

Response:

On the contrary, zebrafish has been established and validated as a model to investigate and elucidate developmental hepatic programs as well as regeneration (Goessling and Sadler, 2015; Wang et al., 2017). However, we acknowledge that more comparative studies are needed to understand the molecular pathways driving regeneration both in zebrafish and mammals and their similarity.

Specifically, zebrafish and mammals display high conservation in the parenchymal and non-parenchymal cell types of the liver as well as their developmental programs (Goessling and Sadler, 2015; Wang et al., 2017). Using different injury paradigms in zebrafish, including ethanol, acetaminophen toxicity and the pharmacogenetic NTR-MTZ model, it has been shown that cellular responses to liver injury are also remarkably conserved with mammals where hepatocyte proliferation governs repair after mild injury while severe injury repair is driven by conversion of BECs into LPCs (So, et al., 2020; Forbes and Newsome, 2019). Major pathways, such as Wnt, FGF and BMP signaling show conserved functions in restorative hepatocyte proliferation (Goessling et al., 2008; Kan et al. 2009, Böhm et al 2010). At present, only very little is known about the molecular mechanisms controlling the BEC/LPC to hepatocyte conversion particularly in rodent models (Kim et al., 2023), while a number of zebrafish studies have started to elucidate the signals governing the different steps of this process (Kim et al., 2023), due to the relative ease of using the larval zebrafish model for this work. Notably, the Notch pathway plays multiple roles in both mouse and zebrafish LPC-mediated repair (Minnis-Lyons et al., 2021; Huang et al 2014; Russel et al.,2019), however further work will be necessary to determine the detailed corresponding functions. Therefore, future work in both rodents and zebrafish will be essential to uncover the molecular mechanisms of this repair process relevant for chronic injury. Given the large conservation of developmental and repair mechanism between mammals and zebrafish observed so far, it is highly likely that this will also apply to LPC-mediated repair. Studies promise to uncover even greater similarity between zebrafish and human (e.g. Fang et al 2011), underscoring the power of using complementary vertebrate models.

PLAN: To edit the text in the introduction and discussion to clarify and highlight the similarities, differences, and opportunities the zebrafish model offers for understanding the mechanisms of vertebrate liver regeneration in general and in particular by using the LiverZap system.

6 - The authors show that in the case of mild ablation, hepatocytes are responsible for replenishment of the parenchyma, but in the context of severe ablation, LPC-mediated regeneration takes control. However, when the authors perform localized and controlled ablation, which is small (around 10-20%) and, to my understanding, a mild / local ablation, however the authors show that LPC mediates the regeneration. Can the authors explain the discrepancy between their results?

Response:

We agree with the reviewer that the LPC response in the smaller, local ROI ablation was unexpected. However, it could be explained by the following: while such ROI hepatocyte ablation represents only a 10-20% ablation of the total hepatocyte population, by sheer numbers comparable to a mild global ablation, the near-complete local hepatocyte loss however makes it more similar to a severe or chronic global injury. Notably, the zebrafish hepatobiliary architecture in zebrafish is similar to that of the mammalian ductular reaction, an adaptation of the biliary network to severe hepatocyte loss. In both cases, the majority of hepatocytes connect directly via their apical canaliculi to biliary ductules to ensure physiologic transport of hepatocyte products, often preceding the LPC response (Sato et al., 2019; Caviglia et al., 2022). We hypothesize that if a similar local, near complete hepatocyte loss would be induced in a mammalian liver exhibiting a ductular reaction, it would similarly induce local LPC-mediated repair. Since this is, to our knowledge not possible, the LiverZap model represents a unique opportunity to induce the LPC-response in a controlled manner and in addition investigate the underlying cellular and molecular processes of injured and adjacent healthy tissues at high resolution in an in vivo context.

PLAN: We will edit the discussion to clarify this important point.

7 - The last part of the paper about E-Cadherin expression is not convincing. I am not sure about the quality of the IF stainings of E-Cadherin, and it is not helping proving the point of the authors. Can the authors provide better stainings for this figure?

Response:

(Same response as to point 5A+B of reviewer 1). We appreciate that it may be difficult to recognize the changes in E-Cadherin localisation, in particular at BEC membranes, given that there are intracellular puncta and that E-Cadherin is expressed both in BECs and hepatocytes. We are convinced of the related data described in Figures 4 and S4, because the first experiment allowed quantification of the staining using both Tp1:H2B-mCherry to identify BECs and intestinal E-Cadherin for normalisation, which revealed a 51 % E-Cadherin reduction at BEC cell membranes following injury. Unfortunately, the signal to noise ratio declined in consecutive experiments, while we could still observe a change in localisation, it challenged a meaningful quantification. We tested alternative antibodies against E-Cadherin, yet without success.

PLAN: Following both reviewers’ comments and suggestions, we agree to remove the data on E-Cadherin.

8 - Could the authors provide a bit more information on the live imaging. Exactly how do they achieve imaging for such a long time?

Response:

Thank you for pointing this out, the information was not very detailed. We used relatively standard mounting conditions (low-melting point agarose and Tricaine anaesthesia, see below for details), combined with light-sheet microscopy, which was the key to achieving the long imaging. We believe that in addition to the known gentle imaging condition, the mounting set-up is critical as the fish is completely suspended in a very low-percentage, low melting point agarose within a large volume of embryo medium.

PLAN: Update the material and methods section with the following details: Long-term live imaging was performed using a LS1 Live light sheet microscopy system (Viventis Microscopy Sàrl). Larvae were with anesthetized with 0.4% Tricaine and mounted ventrally in 0.8% low melting point agarose in E3/PTU media supplemented with 0.16% tricaine. Once the agarose solidified, the chamber was filled with E3/PTU with 0.16% Tricaine to maintain anaesthesia. A 25X objective was used and acquisition was performed every 20 minutes.

MINOR COMMENTS:

9 - It is hard to imagine the full-size liver in Figure 1, bad contrast. Can the authors manually delineate it?

Response:

The livers in this figure are now outlined in the updated figures, see new Figure 1.

10 - "This finding is very surprising, since current understanding in the field links the generation of new hepatocytes from BECs/LPCs with global hepatocyte death." This statement lacks references.

Response:

PLAN: To add the following primary references to the above sentence: (Choi et al., 2014; He et al., 2014; Manco et al., 2019; Raven et al., 2017) and recent review (Kim et al_._, 2023).

REFERENCES

Böhm F, Köhler UA, Speicher T, Werner S. Regulation of liver regeneration by growth factors and cytokines. EMBO Mol Med. 2010 doi:10.1002/emmm.201000085.

Caviglia S, Unterweger IA, Gasiūnaitė A, Vanoosthuyse AE, Cutrale F, Trinh LA, Fraser SE, Neuhauss SCF, Ober EA. Fraeppli: a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish. Development. 2022 doi:10.1242/dev.199615.

Choi TY, Ninov N, Stainier DY, Shin D. Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish. Gastroenterology. 2014 doi:10.1053/j.gastro.2013.10.019.

Fang L, Green SR, Baek JS, Lee SH, Ellett F, Deer E, Lieschke GJ, Witztum JL, Tsimikas S, Miller YI. In vivo visualization and attenuation of oxidized lipid accumulation in hypercholesterolemic zebrafish. J Clin Invest. 2011 doi: 10.1172/JCI57755.

Forbes SJ, Newsome PN. Liver regeneration - mechanisms and models to clinical application. Nat Rev Gastroenterol Hepatol. 2016 doi:10.1038/nrgastro.2016.97

Goessling W, North TE, Lord AM, Ceol C, Lee S, Weidinger G, Bourque C, Strijbosch R, Haramis AP, Puder M, Clevers H, Moon RT, Zon LI. APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development. Dev Biol. 2008 doi:10.1016/j.ydbio.2008.05.526.

Goessling W, Sadler KC. Zebrafish: an important tool for liver disease research. Gastroenterology. 2015 doi:10.1053/j.gastro.2015.08.034.

He J, Lu H, Zou Q, Luo L. Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish. Gastroenterology. 2014 doi:10.1053/j.gastro.2013.11.045.

He J, Wang Y, Missinato MA, Onuoha E, Perkins LA, Watkins SC, St Croix CM, Tsang M, Bruchez MP. A genetically targetable near-infrared photosensitizer. NatMethods. 2016 doi:10.1038/nmeth.3735.

Huang M, Chang A, Choi M, Zhou D, Anania FA, Shin CH. Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model. Hepatology. 2014 doi:10.1002/hep.27285.

Kan NG, Junghans D, Izpisua Belmonte JC. Compensatory growth mechanisms regulated by BMP and FGF signaling mediate liver regeneration in zebrafish after partial hepatectomy. FASEB J. 2009 doi:10.1096/fj.09-131730.

Kim M, Rizvi F, Shin D, Gouon-Evans V. Update on Hepatobiliary Plasticity. Semin Liver Dis. 2023 doi: 10.1055/s-0042-1760306.

Liang P, Kolodieznyi D, Creeger Y, Ballou B, Bruchez MP. Subcellular Singlet Oxygen and Cell Death: Location Matters. Front Chem. 2020. doi:10.3389/fchem.2020.592941.

Manco R, Clerbaux LA, Verhulst S, Bou Nader M, Sempoux C, Ambroise J, Bearzatto B, Gala JL, Horsmans Y, van Grunsven L, Desdouets C, Leclercq I. Reactive cholangiocytes differentiate into proliferative hepatocytes with efficient DNA repair in mice with chronic liver injury. J Hepatol. 2019

doi: 10.1016/j.jhep.2019.02.003.

Mathias JR, Zhang Z, Saxena MT, Mumm JS. Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase. Zebrafish. 2014 doi: 10.1089/zeb.2013.0937.

Minnis-Lyons SE, Ferreira-González S, Aleksieva N, Man TY, Gadd VL, Williams MJ, Guest RV, Lu WY, Dwyer BJ, Jamieson T, Nixon C, Van Hul N, Lemaigre FP, McCafferty J, Leclercq IA, Sansom OJ, Boulter L, Forbes SJ. Notch-IGF1 signaling during liver regeneration drives biliary epithelial cell expansion and inhibits hepatocyte differentiation. Sci Signal. 2021 doi:10.1126/scisignal.aay9185.

Morrison JK, DeRossi C, Alter IL, Nayar S, Giri M, Zhang C, Cho JH, Chu J. (2022) Single-cell transcriptomics reveals conserved cell identities and fibrogenic phenotypes in zebrafish and human liver. Hepatol Commun. doi: 10.1002/hep4.1930.

Oderberg IM, Goessling W. (2023) Biliary epithelial cells are facultative liver stem cells during liver regeneration in adult zebrafish. JCI Insight. doi: 10.1172/jci.insight.163929.

Ota N, Shiojiri N. Comparative study on a novel lobule structure of the zebrafish liver and that of the mammalian liver. Cell Tissue Res. 2022 doi:10.1007/s00441-022-03607-y.

Raven A, Lu WY, Man TY, Ferreira-Gonzalez S, O'Duibhir E, Dwyer BJ, Thomson JP, Meehan RR, Bogorad R, Koteliansky V, Kotelevtsev Y, Ffrench-Constant C, Boulter L, Forbes SJ. Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration. Nature. 2017 doi:10.1038/nature23015.

Russell JO, Ko S, Monga SP, Shin D. Notch Inhibition Promotes Differentiation of Liver Progenitor Cells into Hepatocytes via _sox9b_Repression in Zebrafish. Stem Cells Int. 2019 doi:10.1155/2019/8451282.

Sato K, Marzioni M, Meng F, Francis H, Glaser S, Alpini G. Ductular Reaction in Liver Diseases: Pathological Mechanisms and Translational Significances. Hepatology. 2019 doi: 10.1002/hep.30150.

So J, Kim A, Lee SH, Shin D. Liver progenitor cell-driven liver regeneration. Exp Mol Med. 2020 doi: 10.1038/s12276-020-0483-0.

Stoddard M, Huang C, Enyedi B, Niethammer P. Live imaging of leukocyte recruitment in a zebrafish model of chemical liver injury. Sci Rep. 2019 doi: 10.1038/s41598-018-36771-9.

Wang S, Miller SR, Ober EA, Sadler KC. Making It New Again: Insight Into Liver Development, Regeneration, and Disease From Zebrafish Research. Curr Top Dev Biol. 2017 doi: 10.1016/bs.ctdb.2016.11.012.

Xie W, Jiao B, Bai Q, Ilin VA, Sun M, Burton CE, Kolodieznyi D, Calderon MJ, Stolz DB, Opresko PL, St Croix CM, Watkins S, Van Houten B, Bruchez MP, Burton EA. Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity. Elife. 2020 doi: 10.7554/eLife.51845.

Compartir y saber diferentes tipos de vista, es una manera de interactuar con personas de manera virtual o remota. Cuando alguien pueda que no entienda otra persona te lo va replicando explicando de una manera adecuada. Por eso es interesante este párrafo ya que se puede construir textos en formato digital.

Esta parte es importante tenerla en cuenta, ya que va dejando claro el uso de procesos de aprendizaje entre pares y el porque debería usarse de ese modo.

¿Y si quien o quienes escriben se contradicen?¿Y sí la información que se escribe no es la correcta? ¿Cómo identificar?

Factores de riesgo, relacionados al síndrome metabólico.

subrayando las frases me parece interesante porque es una guía para uno no perderse del tema central.

cuando hayas ingresado a tu cuenta con tu login, (usuario y contraseña), te sale un cuadro con las cosas o anotaciones que has hecho en esta página, al lado derecho suyo, aparece un enlace que dice Visit Anotation in context. Ahi te aparece nuevamente la pagina de Hypotesis ya con los cuadritos que describe para uno poder realizar la lectura anotada

Sin duda, actualmente nuestras labores están ligadas a las redes sociales de alguna manera, así que, el poder compartir las lecturas por medio de estas es una apuesta interesante para la academía; por un lado, porque el acceso puede ser más "rápido" y por otro lado, por la apuesta a cambiar estos hábitos de lectura sobre todo en Colombia que el indice de lectura es bastante bajo.

es importante aprender nuevas tecnicas y herramientas para replicar a nuestros compañeros familiares o amigos

Que interesante que este tipo de herramientas se puedan compartir por redes sociales y mejorar esta practica de lectura y escritura.

• for: progress trap, unintended consequence, ecological realization, ecological awakening
  • claim
    • the idea that we need a profound, sudden and massive change in ourselves in a dangerous notion
    • comment
      • why?
      • it presumes we have a deficit as an ecological being
      • when in actual fact, we cannot be otherwise
      • so instead, our job is to awaken our already ecological nature
      • by this, we mean our deep, intrinsic ecological nature as ecological (interdependent) beings
      • we humans have a strange and very limited kind of interdependence, which is exploitative to other people and other species
      • we have to become aware of that culturally conditioned limitation

This doesn't really make sense when some of the bonuses are specifically there because there is infrastructure. It changes the requirement from just having infrastructure to needing infrastructure and FLU updates

This also perpetuates historical inequities between neighborhoods.

Okay this analysis is good. We should lean on this when talking about maximizing existing FLU.

Jedná se o Rekreační a školící středisko Loutí?

COmment

để fashion (fashion ở đây là động từ) thế giới theo hướng care nhiều hơn, ta cần thay đổi cách hiểu kn passion.

cuốn sách này muốn làm điều đó

Sin determinar con precisión (o por alguna definición) la frontera entre ambos términos...

o it is not necessary to use force to constrain the convict to good behaviour, the madman to calm, the worker to work, the schoolboy to application, the patient to the observation of the regulations

This is a ridiculous request from men and white women to be asking of black women and black queer women and tracks back to slavery in it's way of giving power to oppressors

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

Please find our point-to-point response to the reviewer’s comments below, where we marked all changes implemented in the manuscript in italics.

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

With the emergence and spread of resistance to Artemisinin (ART), a key component of current frontline malaria combination therapies, there is a growing effort to understand the mechanisms that lead to ART resistance. Previous work has shown that ART resistant parasites harbour mutations in the Kelch13 protein, which in turn leads to reduced endocytosis of host haemoglobin. The digestion of haemoglobin is thought to be critical for the activation of the artemisinin endoperoxide bridge, leading to the production of free radicals and parasite death. However, the mechanisms by which the parasites endocytose host cell haemoglobin remain poorly understood.

Previous work by the authors identified several proteins in the proximity of K13 using proximity-based labelling (BioID) (Birnbaum et al. 2020). The authors then went on to characterise several of these proteins, showing that when proteins including EPS15, AP2mu, UBP1 and KIC7 are disrupted, this leads to ART resistance and defects in endocytosis leading to the hypothesis that these two processes are inextricably linked.

In this manuscript, Schmidt et al. set themselves the task of characterising more K13 component candidates identified in their previous work (Birnbaum et al. 2020) that were not previously validated or characterised. They chose 10 candidates and investigated their localisations, and colocalisation with K13, and their involvement in endocytosis and in vitro ART resistance, 2 processes mediated by K13 and some members of the K13 compartments

The authors show that of their 10 candidates, only 4 can be co-localised with K13. Then, using a combination of targeted gene disruption (TGD) as well as knock sideways (KS), they characterised these 4 proteins found in the K13 compartment. They show that MyoF and KIC12 are involved in endocytosis and are important for parasite growth, however their disruption does not lead to a change in ART sensitivity. The authors also confirm the findings of their previous publication (Birnbaum et al. 2020), using a slightly different TGD

(note from the authors: we apologise if this has not properly transpired from the manuscript but the difference between the TGDs is substantial and relevant: one has less than 3% of the protein left and hence can be considered to fully inactivate MCA2 and has a growth defect whereas the other contains about two thirds of the protein (1344 amino acids/~66% are left), has no growth defect, although it lacks the MCA2 domain (hence that domain can not be critical for the growth defect)),

that MCA2 is involved in ART resistance, however they did not check whether its disruption impacts haemoglobin uptake. They also show that KIC11 is not involved in mediating haemoglobin uptake or ART resistance. To finish, the authors used AlphaFold to identify new domains in the proteins of the K13 compartment. This led them to the conclusion that vesicle trafficking domains are enriched in proteins of the K13 compartment involved in endocytosis and in vitro ART resistance.

The majority of the experiments conducted by the authors are performed to a good standard in biological and technical replicates, with the correct controls. Their findings provide confirmation that their 4 candidate genes seem to be important for parasite growth, and show that some of their candidates are involved in endocytosis. While the KD and KS approaches employed by the authors to study their candidate genes each have their own advantages and can be excellent tools for studying a large sets or genes, this manuscript highlights the many limitations of these approaches. For example, the large tag used for the KS approach can mislocalise proteins or disrupt their function (as is the case for MyoF), resulting in spurious results, or indeed the inability to generate the tagged line (as is the case for MCA2). The KS approach also makes the results of a protein with a dual localisation, like KIC12, extremely difficult to interpret.

We thank the reviewer for this thorough and insightful review.

The limitations mentioned above were addressed in the response to the main points and a general detailed response in regards to the systems used for this research are added at the end of this rebuttal. Briefly summarised here: while we agree that there are limitations of the system used, we are convinced that

• the advantages of using a large tag in most cases outweighs the drawbacks as it permits to track the inactivation of the target, if need be on the individual cell level

• while not optimal for MyoF, the partial inactivation actually helps in its functional study as detailed in major point 23&28 or reviewer#3 major point 11: it shows a consistent correlation of the phenotype with different causes and degrees of inactivation (this is now better illustrated in Figure 1L1M). Further, regarding the concern of the large tag: the effect of the tag based on localisation was overestimated in the review by what seems to have been a mix up comparing numbers from MyoF with a number from MCA2 (there is a difference, but it is only small) (see reviewer#1 major point #23).

• KS is the optimal method for most of the assays in this work (e.g. bloated food vacuole assays and RSAs); these assays would be impossible or difficult to use with other inactivation systems currently used in P. falciparum research (see details in the response to the specific points and after the rebuttal)

In regards to the difficulty to interpret KIC12 data: this is only true for measuring absolute essentiality, everything else we believe we actually have the optimal method. If not KS, which method targets a specific pool of a protein with a dual localisastion? Again, our assays targeting the K13 pool and revealing the specific function would have been difficult or impossible with any other system.

Ultimately the question is whether any other system would have resulted in a different conclusion on the function of the proteins studied. At present we are confident this would not be the case and other systems probably would not have delivered the specific functional data shown in this work. Clearly, more in depth work will provide more nuanced and detailed insights into the proteins analysed in this work and this likely will also include the use of other systems for specific aspects they are most suitable for. However, this (e.g. different complementations in a diCre cKO) is complex and therefore beyond what fits into this work which had the goal to assess which proteins are true positives for the K13 compartment and to place them into functional groups in regards to endocytosis.

Moreover, the manuscript is disjointed at times, with the authors choosing to conduct certain experiments for only a subset of genes, but not for others. For example, considering that the aim of this paper was to identify more proteins involved in ART resistance and endocytosis, it is confusing why the authors do not perform the endocytosis assays for all their selected proteins, and why they do not do this for the proteins they identify in their domain search. There is significant room for improvement for this manuscript, and a generally interesting question.

The reviewer remarks that not every experiment was done for every target. Based on the rebuttal we tried to amend this but also note that there was some sentiment by the reviewers to better stick to the point and not make the manuscript more disjointed. We attempted to balance that as much as possible and hope we were able to honour both aspects (amendments were done as detailed in the point by point response below).

In regards to endocytosis and choice of targets: We did do endocytosis assays for all proteins that showed a growth phenotype upon inactivation in this work. We therefore assume the reviewer here refers to major point #40 asking for endocytosis assays with KIC4 and KIC5 (which were not studied in this manuscript) as well as MCA2 (point 17). We fully agree with the reviewer that this would fill a gap in the work on K13 compartment proteins but such assays are difficult with TGDs (there are issues with non-comparable samples and compensatory effects) and proteins that are not essential (and hence likely have a smaller impact on endocytosis when truncated). We nevertheless now carried them out, but due to the limitations to do this with these lines would be hesitant to draw definite conclusions (see major point 17 and 40 for details and outcomes).

But in it's current format, other than confirming that MCA2 is involved in ART resistance (which was already known from the Birnbaum paper), the authors do not further expand our understanding of the link between ART resistance and endocytosis in this manuscript.

We would like to point out that the importance of the K13 compartment and endocytosis goes beyond ART resistance (see e.g. also newly published papers on the K13 compartment in Toxoplasma, (Wan et al., 2023; Koreny et al., 2023)). Endocytosis is an essential and prominent process in blood stages. However, in contrast to processes such as invasion, our understanding about endocytosis is only rudimentary. Hence, this manuscript provides important insights on an emerging topic that in our opinion deserves more attention:

• it identifies novel proteins at the K13 compartment and provides 2 new proteins in endocytosis (MyoF and KIC12); getting an as complete as possible list of proteins involved in the process will be critical to study and understand it

• it leads to the realisation that not all growth-relevant proteins detected at the K13 compartment are needed for endocytosis

• it provides domains and stage specificity of function for several K13 compartment proteins, overall bolstering the model of endocytosis in ART resistance and providing a framework critical to direct future studies on endocytosis and their detailed mechanistic function at the cytostome

• the identified vesicle trafficking domains (for instance now also found in UBP1) are expected to strengthen the support for the role of endocytosis of the K13 compartment; this and also the above points are important as (based on the current literature) there still seems to be prominent sentiment in the field that (in part due to the involvement of UBP1 and K13) the cause of ART resistance is due to various unclearly defined stress response pathways

• with MyoF it also shows the first protein in connection with the K13 compartment that acts downstream of the generation of hemoglobin-filled containers in the parasite and provides the first protein that explains the suspected involvement of actin in endocytosis (so far this was only based on CytD studies)

Overall we therefore believe this manuscript contains critical information and a framework for future studies on endocytosis and the K13 compartment. We hope the relevance of endocytosis as one of the most prominent and essential processes in the parasites and the connection to various aspects linked with many commercial drugs (in addition to the role of endocytosis in ART resistance), is adequately explained in the introduction. We also would like to mention that the main focus of the work is reflected in the title of the manuscript which does not mention ART susceptibility.

Major Comments

1) line 31: please change defined to characterised - defined suggests that novel proteins were identified in this study, which is not the case.

We apologise, but we do not fully understand this comment. We did identify novel proteins not before known to be at the K13 compartment (MCA2 (admittedly this one was likely but had not previously been verified), MyoF, KIC11 and KIC12). In our view "further defining the composition of the K13 compartment" therefore is an accurate statement. Additionally, the identification of previously not-discovered domains, the stage-specificity and function of these proteins helped to further define the K13 compartment.

If the reviewer is referring to the fact that the proteins analysed in this study were taken from a previously generated list of hits, we would like to stress that the presence in such a list (obtained from a BioID, but also if from an IP etc) can not be equalled for them to be true positives, they are merely candidates that still need to be experimentally validated. This is what we did in this work to find out which further proteins from the list can be classified as K13 compartment proteins (for hits with lower FDRs this is even more relevant as illustrated by the fact that 6 of the here analysed hits were not at the K13 compartment). In an attempt to address this comment in the manuscript, we changed the wording of this sentence to (line 31): "Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site."

2) line 37: please change 'second' to "another". As explained further below, the authors identified 3 classes of proteins (confer ART resistance + involved in HCCU, involved in HCCU only, or involved in neither).

We realized that the groups description wasn’t clear in the abstract. Please see response to major comment #41 for a detailed answer to this (endocytosis is an overarching criterion, ART resistance is a subgroup and applies only to those proteins with a function in endocytosis in ring stages). To clarify this (see also major point #8) we added an explanation on the influence of stage-specificity of endocytosis on ART susceptibility to the introduction (line 76): “In contrast to K13 which is only needed for endocytosis in ring stages (the stage relevant for in vitro ART resistance), some of these proteins (AP2µ and UBP1) are also needed for endocytosis in later stage parasites (Birnbaum et al., 2020). At least in the case of UBP1, this is associated with a higher fitness cost but lower resistance compared to K13 mutations (Behrens et al., 2021; Behrens et al., 2023). Hence, the stage-specificity of endocytosis functions is relevant for in vitro ART resistance: proteins influencing endocytosis in trophozoites are expected to have a high fitness cost whereas proteins not needed for endocytosis in rings would not be expected to influence resistance.” The abstract was changed in response to this and other comments and hope it is now clearer in regards to the groups.

3) Line 40: You define KIC11 as essential but according to your data some parasites are still alive and replicating 2 cycles after induction of the knock sideways. Please consider changing "essential" to "important for asexual parasite growth".

We fully agree with the reviewer, we reworded the sentence as suggested.

4) Line 40: please change 'second group' to 'this group'

We reworded this part of the abstract and it know reads: (line 38): “While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process.”

5) line 41: state here that despite it being essential, it is unknown what it is involved in.

With the newly added data we show that this protein either has a function in invasion or very early ring development although we did not see any evidence for the latter. We therefore changed the sentence to (line 43): “We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion*..” *

6) Line 50: the authors should state here that there is actually a reversal in this trend over the last few years.

Done as suggested.

7) Line 54: please separate out the references for each of the two statements made in this line (a: that ART resistance is widespread in SEA, and b: that ART resistance is now in Africa) Reference 14 also seems to reference ART resistance in Amazonia - which is not covered by the statement made by the authors (in which case the authors should state ART is now present in Africa and South America). The authors should also reference PMID: 34279219 for their statement that ART resistance is now found in Africa (albeit a different mutation to the one found in SEA).

Done as suggested.

8) Line 65: it is also worth mentioning here that there are other mutations in proteins other than K13, such as AP2mu and UBP1 (PMID: 24994911;24270944) that can lead to ART resistance.

As suggested by the reviewer, we included a sentence about non-K13 mutations linked with reduced ART susceptibility in the introduction (line 74): “Beside K13 mutations in other genes, such as Coronin (Demas et al., 2018) UBP1 (Borrmann et al., 2013; Henrici et al., 2020b; Birnbaum et al., 2020; Simwela et al., 2020) or AP2µ (Henriques et al., 2014; Henrici et al., 2020b)* have also been linked with reduced ART susceptibility." *

We here also added data on fitness cost that is related to this and is also relevant for the issue of proteins with a stage-specific function in endocytosis, making a transition for this statement which might help clarifying the grouping of K13 compartment proteins (see also major point #2).

9) Line 80, 86: ref 43 is misused. Reference 43 refers to Maurer's clefts trafficking which takes place in the erythrocyte cytosol and is not involved in haemoglobin uptake as far as I know. Please replace ref 43 with one showing the role of actin in haemoglobin uptake.

We thank the reviewer for pointing this out, Ref 43 was removed from the manuscript.

10) Line 98: the authors state here that they 'identified' further candidates from the K13 proxiome. This suggests that they identified new proteins in this paper, when in fact the list was already generated in ref 26. All they did was characterise proteins from that list that were not previously characterised. The authors should therefore remove identified from this statement.

We agree with the reviewer that we did not identify further candidates, we identified new K13 compartment proteins from the list of potential K13 compartment proteins. We therefore changed “identified further candidates” into “identified further K13 compartment proteins” (line 116). Please see also response to major comment #1.

11) Line 107-108: it is not clear from this sentence why these proteins were left out of the initial analysis in Ref 26. A sentence here explaining this would be valuable for the reader.

This is a good point. One reason why we did not analyse more in our previous publication was that we had to stop somewhere and adding more would have been very difficult to fit into what was already a packed paper. However, as shown in this work, the list does contain further interesting candidates (e.g. K13 compartment proteins that are involved in endocytosis).

We altered the relevant part of the introduction to highlight that we previously analysed the top hits, clarifying that the 'remaining' hits analysed in this work were further down in the list. This now reads: (line 113)“We reasoned that due to the high number of proteins that turned out to belong to the K13 compartment when validating the top hits of the K13 BioID (Birnbaum et al., 2020), the remaining hits of these experiments might contain further proteins belonging to the K13 compartment.” We hope this clarifies that we simply moved further down in the candidate list.

12) Line 117-123: The authors say that PF3D7_0204300, PF3D7_1117900 and PF3D7_1016200 were not studied because they were not in the top 10 hits. However, the current organisation of Supplementary Table 1 shows all 3 proteins among the top 10 hits (MyoF, KIC12, UIS14 and 0907200 being after them). I think the authors should reorganise their table. It is also unclear according to what the proteins in the table are ranked. Could the authors indicate the metric used for the ranking?

We thank the reviewer for alerting us to this. The issue here is that the 3 non-analysed proteins belong to a 'lower stringency' group comprising hits significant with FDRThe information about ranking is now also included as “Table legend” in the revised manuscript and the Table heading has been changed to: “List of putative K13 compartment proteins, proteins selected for further characterization in this manuscript are highlighted.”

13) Line 129-141: Can the authors be clearer with their explanations of the identification of mutation Y1344Stop? One dataset (ref 61) shows that 52% of African parasites have a mutation in MCA2 in position 1344 leading to a STOP codon. But another dataset (ref 62) shows that the next base is also mutated, reverting the stop codon. That should have been seen in the first dataset as well. Could the authors please clarify.

This mutation was first spotted in the MalariaGEN database (https://www.malariagen.net) (MalariaGEN et al., 2021), which allows online accessing of the data by using the “variant catalogue” tool, which is in a table format of frequency rather than in a sequence context. Hence, only after further research later on it became evident to us, that this mutation does not occur alone when looking at individual MCA2 sequences from patient samples in (Wichers et al., 2021b). We hope this is accurately reflected in our results section.

14) Line 147: the authors say that MCA2 is expressed throughout the intraerythrocytic cycle as shown by live cell imaging. In Birnbaum et al 2020 fig 4I, the authors show that MCA2 is mainly expressed between 4 and 16hpi. But in Figure 1B of this manuscript there is a clear multiplication of MCA2 signal between trophozoite and schizont. How do the authors explain this discrepancy? Could expression of the truncated MCA2 be different than the full length? This cannot be assessed as expression and localisation of the full-length HA tag MCA2 is not shown in Schizonts.

The key difference lies in transcription vs protein expression (usually protein levels peak after mRNA levels peak and - depending on turnover - protein levels can stay high even after mRNA levels have declined). Figure 4 of the Birnbaum et al paper presents transcriptomic data, but with a peak in trophozoites (The axis label in Fig. 4l of that publication is a bit confusing, as hour 0 is at the top, 48 h at the bottom; it is clearer in Fig. S13 of that paper) which would fit very well with the multiplication of the signal between trophozoites and schizonts mentioned by the reviewer. So, overall, the temporal peaks of transcripts and protein of that protein fit well.

For the signal in rings: Likely the protein has a turnover rate that is sufficiently low for some protein to be taken into the new cycle after re-invasion. Also different transcriptomic datasets e.g. (Otto et al., 2010; Wichers et al., 2019; Subudhi et al., 2020) available on plasmoDB show some mRNA present across the complete asexual development cycle, with each dataset showing maximum peak at a slightly different stage.

Even when located in foci and hence aiding detection of small amounts of protein (as is the case for MCA2-Y1344-GFP), the MCA2 signal in rings is not strong. For MCA2-TGD, the GFP signal is dispersed and therefore likely below our detection limit, while the same amount of protein concentrated at the K13 compartment is visible as foci in the MCA2-Y1344 cell line. Please note that MCA2-TGD has only 2.8% of the protein left whereas MCA2-Y1344 has 66.5% left and based on our manuscript is almost fully functional, hence fitting the different locations between the two versions.

Overall we believe this shows that there are actually no significant discrepancies of the expression of the different MCA2 versions.

15) Line 158: would it not have been more useful for the authors to have episomally expressed MCA2-3xHA in their MCA2Y1344STOP-GFPENDO line to make sure that the truncated protein is indeed going to the correct compartment? The experiments done by the authors suggests that the MCA2Y1344STOP goes to the right location but does not really confirm it.

We appreciate the reviewers caution here. However, considering that MCA2Y1344STOP-GFPendo co-locates with mCherryK13 and endogenously HA-tagged full length MCA2 does the same to a similar extent, there is in our opinion little doubt that MCA2 is found at the K13 compartment and that this is similar with both constructs. If there are minor differences, these might as well occur if MCA2 is episomally (as suggested in the comment) instead of endogenously expressed. Given the limited insight, we therefore decided against the episomal overexpression (which due to its size of > 6000bp may also be somewhat less straight forward than it may sound).

16) Line 191: it is stated that MCA2 confers resistance independently of the MCA domain, however in both the MCA2-TGD and MCA2Y1344STOP-GFPENDO parasites, the MCA domain is deleted, and for both parasites, there is resistance (albeit to a lower level in the MCA2Y1344STOP-GFPENDO line). Therefore, how can the authors state that the ART resistance is independent of the MCA domain? This statement should be that resistance is dependent on the loss of the MCA domain.

We agree that this can’t be categorically excluded. However, a ~5 fold difference in ART sensitivity was observed between the parasites with MCA2 truncated at amino acid 57 compared to those with MCA at amino acid 1344 even though both do not contain the MCA2 domain. Hence, at least this difference is not dependent on the MCA2 domain. The larger construct missing the MCA domain shows only a very moderate reduction in RSA survival, again suggesting the MCA domain is not the main factor. We amended our statement in an attempt to more accurately reflect the data (line 487): “This considerable reduction in ART susceptibility in the parasites with the truncation at MCA2 position 57 compared to the parasites still expressing 1344 amino acids of MCA2, despite both versions of the protein lacking the MCA domain, indicates that the influence on ART resistance is not, or only partially due to the MCA domain.” We would be hesitant to state the reviewer's conclusion that “resistance is dependent on the loss of the MCA domain”, as the larger construct missing the MCA2 domain has a milder RSA effect compared to MCA2-TGD, which suggests the reduction in ART susceptibility is independent of the MCA domain. These considerations also agree with the fact that the parasites with the longer MCA2 version (in contrast to the MCA2-TGD) do not have any detectable growth defect which indicates that the protein can fulfil its function without the MCA2 domain.

17) Line 192: Why did the authors not check if MCA2 is involved in endocytosis? They state later on in the manuscript that they did not do endocytosis assays with TGD lines, however if the authors include the correct controls, this could be easily done. It would also be really interesting to see whether endocytosis gets progressively worse going from WT to MCA2Y1344STOP to MAC2TGD. This experiment (as well as doing endocytosis assays for KIC4 and KIC5 TGD lines) would drastically increase the impact of this study. These experiments would not take more than 3 weeks to perform, and would not require the generation of new lines.

So far were very hesitant to do bloated FV assays with TGDs (even though TGDs were available for the genes encoding MCA2 and KIC4 and KIC5). The reason for this was:

1. the fact that these proteins could be disrupted indicated either redundancy or only a partial effect on endocytosis which might lead to only small effects that likely are difficult to pick up in an assay scoring for the rather absolute phenotype of bloated vs non-bloated. Using the refined assay measuring FV size could partly amend this but we note that also FV without hemoglobin have a certain size, reducing the relative effect if there are smaller differences.
2. a TGD line does not permit tightly controlled inactivation of the target which makes comparing the outcome of bloated food vacuole assays difficult if there are smaller growth and stage differences to the 3D7 control.
3. in contrast to conditional inactivation parasites, the TGD lines had ample times to adapt to loss of the target protein (compensatory mechanisms are well known for endocytosis, for instance in clathrin mediated endocytosis loss of individual components can be compensated (Chen and Schmid, 2020)). We nevertheless see the reviewer's point that this should at least be attempted and now conducted these assays (see also major point 40). For MCA2 (as requested in this point), the data is shown in Figure S5C-E. This assay showed that in MCA2-TGD, MCA2Y1344STOP-GFPendo (similar to the 3D7 control) >95% of parasites developed bloated food vacuoles. Additionally, we also measured the parasite and food vacuole size of individual cells in an attempt to solve some of the problems with TGDs with such assays. In order to specifically solve problem 2 mentioned above, we analysed the food vacuoles of similarly sized parasites, however, they were non-distinguishable between the three lines. Of note, in agreement with the reduced parasite proliferation rate (Birnbaum et al., 2020) a general effect on parasite and food vacuole size was observed for MCA2-TGD parasites, indicating reduced development speed in these parasites. Hence, it is possible that a potential endocytosis reduction was accompanied by a slowed growth, and the comparison of similarly sized parasites may have obscured the effect. It is therefore not sure if there indeed is no endocytosis phenotype, although we can exclude a strong effect in trophozoites.

Based on the RSA results at least rings can be expected to have a reduced endocytosis in the MCA2-TGD. Apart from options 1-3 mentioned above, it is therefore possible there is an effect restricted to rings, although in that case the reduced growth in trophozoites would be due to other functions of MCA2. Overall, we can conclude that the MCA2-TGD parasites do not have a strongly reduced endocytosis, but given the fact that the parasites are viable, this is not surprising. Whether the MCA2-TGD has no effect at all on endocytosis we would be very hesitant to postulate based on these results.

18) The authors should consider re-organising the MCA2 section, first showing that the 3xHA tagged line colocalises with K13, then performing the new truncation.

We attempted to re-organise as suggested but because we now included additional fluorescence microscopy images of schizont and merozoites (in response to reviewer 2 major comment 3) the main figure would become even larger. To prevent this, we kept the 3xHA data in the supplement.

19) Line 197: Once again ref 43 is not correct to illustrate that actin/myosin is involved in endocytosis

We thank the reviewer for pointing this out – we removed Ref 43.

20) Line 202: the authors state that MyoF localises near the food vacuole from ring stage/trophs onwards. However, how can this statement be made in schizonts based on these images (Fig. 2A), where it doesn't look like MyoF is anywhere near the FV? This statement can only be made for schizonts if co-localised with a FV marker (which is done in Fig. 2B), however, based on the number of MyoF foci, it appears that this was not done for schizonts. Please either remove the statement that MyoF is near the food vacuole from trophs onwards (because it is only seen near the FV up until trophs) or show the data in Fig. 2B of schizonts to substantiate these claims.

This is a valid point. We originally did not focus on schizonts because most markers end up in some focal area in the forming merozoite but other proteins (such as e.g. K13) also have one or more additional foci at the FV, making interpretation unclear, particularly if the schizont is still organizing to become fully segmented. This is why we generally focused the K13 co-localisations on the trophozoite stage to obtain the clearest information on endocytosis. However, given the fact that this manuscript gives the first localization of MyoF in P. falciparum parasites, we now provide a comprehensive time course (Figure 1C, S1A) including schizonts, which show quite a complex pattern: while the MyoF-GFP localization in trophozoites appeared as multiple foci close to K13 and also the FV, the MyoF-GFP pattern changes in late schizonts (fully segmented) and merozoites, appearing as elongated foci no longer close to K13 or the FV. Of note, this pattern has been previously reported for MyoE in P. berghei (Wall et al., 2019).

We therefore revised the statement about MyoF localization in schizont to better reflect the observed localization: (line 175): “In late schizonts and merozoite the MyoF-GFP signal was not associated with K13, but showed elongated GFP foci (Figure 1C, S2A) reminiscent of the MyoE signal previously reported in P. berghei schizonts (Wall et al., 2019).”

21) Line 204-206: what does this statement bring to the paper? Is it to show that it is the real localisation of MyoF because 2 tag cell line show the same localisation? I don't think this is needed, especially as later in the manuscript an HA-tag MyoF line is used and show similar localisation.

We see the reviewers point, but prefer to keep this data included in the supplement, particularly because potential differences in the location of tagged MyoF were a major concern.

Related to the tag issue: in order to get a better understanding of the effect of C-terminally tagging with different sized tags we now performed a more detailed analysis of the MyoF-3xHA cell line (Figure S2F-G), showing that this cell line shows a growth rate similar to the 3D7 wild type parasites, and has less vesicles than the 2x-FKBP-GFP-2xFKBP cell line, but still slightly, but significantly more than 3D7 parasites. Overall, this indicates that the smaller 3xHA tag has less effect on the parasite, than the larger 2x-FKBP-GFP-2xFKBP tag (see also new Figure 1L, showing a correlation of level of inactivation and the endocytosis phenotype for MyoF).

22) Line 212: The overlap of K13 with MyoF in Figure 2C 3rd panel (1st trophozoite panel) is not obvious, especially as the MyoF signal seems inexistant. I would advise the authors to replace with a better image. Also, why are there no images of schizonts shown in Figure 2C?

As suggested we exchanged the trophozoite image of panel Figure 2 C (now Figure 1C) and expanded this panel with images covering the complete asexual development cycle including schizonts in response to this and the previous points. As indicated above (point 20), schizont stages are complex to interpret. While late schizonts likely are not very relevant for endocytosis this is the first description of the location of the protein in this parasite and we therefore now provide a more thorough representation of the MyoF location across asexual stages in Figure1C and S2A.

23) Line 217: the spatial association of MyoF with K13 is very different when it is tagged with GFP and when it is tagged with 3xHA. The way the authors word it here, it seems that there is agreement with the two datasets, when this is not in fact the case (59% overlap for MyoF-GFP and only 16% overlap with MyoF-3xHA). These data suggest that the GFP and the multiple FKBP tags are doing something to the protein and therefore maybe the ensuing results using this line should not be trusted or be taken with a pinch of salt.

We agree with the reviewer that the location of this MyoF-GFP in the cell might differ due to the partial inactivation but in contrast to this comment, the data does not indicate any large differences. It seems the reviewer mixed something up (the 59% mentioned might come from the MCA2 figure?). The data with the two lines with differently tagged MyoF co-localised with K13 are actually quite comparable: GFP-tagged vs HA-tagged MyoF overlapping with K13 was 8% vs 16% full overlap, 12% vs 19% partially overlapping foci, 36% vs 63% foci that were touching but not overlapping (compare what now is Figure 1D and Figure S2C). Only in the 'no overlap' there is a much smaller proportion in the HA-tagged line. However, given that these are IFAs which on the one hand are more sensitive to see small protein pools but on the other hand also have pitfalls due to fixing of the cells (e.g. tiny increase in focus size due to fixing could increase the number of touching foci that in live cells might be close but did not touch), some variation can be expected to the live cells. We agree though that the partly reduced functionality of MyoF might be the reason for the consistent tendency of a lower overlap even though the difference is much less than indicated in the comment. We added "with a tendency for higher overlap with K13 which might be due to the partial inactivation of the GFP-tagged MyoF" to the sentence "IFA confirmed the focal localisation of MyoF and its spatial association with mCherry-K13 foci"

While we expect the fact that the difference between these parasites is only small somewhat reduces the "pinch of salt" with the MyoF line, we do agree that the partial functional inactivation of the GFP-tagged MyoF line may have some impact. However, we do not think that this means the results with the MyoF-GFP line are untrustworthy. On the contrary, it provides insights into its function that in some ways is equivalent to a knock down or TGD. Overall all the MyoF lines show: few vesicles occur in the MyoF-HA-line, more in the MyoF-GFP line and even more after knock sideways of MyoF-GFP. Importantly the severity of this phenotype correlates with the growth rates in these lines. Hence, together with the bloated food vacuole assays, this provides consistent data indicating that MyoF has a role in the transport of HCC to the FV and its level of activity correlates with the number of vesicles and growth. To better highlight this, it is now summarised in Figure 1M.

24) Line 219: the authors state here that they could not detect MyoF-GFP in rings, when in Figure 2C they show MyoF-GFP in rings, and also show that they could detect MyoF in Sup Fig. 3B with the 3xHA tagged line. Is this a labelling mistake in Figure 2C? If the authors could indeed not see MoyF-GFP in rings, this statement should have been made when Figure 2A was presented, and not so late in the manuscript, which causes confusion.

We thank the reviewer for pointing this out. We now provide a detailed time course (see also previous points) which shows that there is no detectable MyoF-GFP signal during ring stage development until the stage where the parasites starts the transition to trophozoites (i.e. MyoF-GFP signal could only be observed in parasites already containing hemozoin). In addition to the extended time course in Figure 1C (previously 2C) we included a panel of example ring stage images below to further highlight this. We also changed the labelling of the parasite with MyoF-GFP signal the reviewer mentions in Figure 1C to “late ring stage” (it already contains hemozoin) to clarify this.

The description of Figure 1A is now changed to: (line 153) *“The tagged MyoF was detectable as foci close to the food vacuole from the stage parasites turned from late rings to young trophozoite stage onwards, while in schizonts multiple MyoF foci were visible (Figure 1A, S2A).” *

Please see our answer to major comment #45 where we provide an explanation for the difference between MyoF-3xHA and MyoF-GFP signal in ring stage parasites.

[Figure MyoF]

25) Line 237: Showing a DNA marker (DAPI, Hoecht) for Figure 2E, and subsequent figures using mislocalisation to the nucleus, would help the reader assess efficiency of the mislocalisation.

Please see response to major comment #64 for a detailed answer on why we did not include DNA staining in the imaging used to assess mislocalization upon knock-sideways.

26) Line 254-256: authors should show the results of the bloating assay for parental 3D7 parasites (+ and - rapalog) to see whether the MyoF line - rapalog has increased baseline bloating. This applies to all subsequent FV bloating assays.

We did do several controls for bloated assays (including +/- rapalog of an irrelevant knock sideways line as well as using a chemical insult for which the control was 3D7 without treatment) in previous work (Birnbaum et al., 2020), which indicated that there is no effect of rapalog to reduce bloating. Although these controls are more stringent, we nevertheless did a 3D7 +/- rapalog control and added this to the manuscript (Figure S2I). As it is not possible to do this side by side with the assays that are already in the manuscript and the +/- rapalog 3D7 cells consistently showed no or very low numbers of cells without bloating (and stringent controls in the past equally did not show an effect), we believe adding this control once suffices.

27) Line 254-257: The authors say that because fewer parasites show a bloated food vacuole upon inactivation of MyoF it means that less hemoglobin reached the food vacuole. I understand the authors statement, however, shouldn't they look at the size of the food vacuole, instead of the number of parasites with bloated FV, to make such a statement? This has been done for KIC12 so why not doing it for MyoF?

This was now done and is provided as Figure 1J-K, S2J. The results confirm the assessment scoring bloated vs non-boated food vacuoles.

28) Line 259-261: these results would be difficult to interpret namely because the authors have dying parasites, which is exacerbated with the protein being knocked sideways. The authors should mention the pitfalls their knock sideways and tagging design here. Line 260-261: RSA is an assay relying on measuring parasite growth 1 cycle after a challenge with ART for 6 hours.

Fortunately, this concern is unfounded, as the survival (measured by parasitemia after one cycle) of the same sample + and - DHA is assessed, isolating the DHA effect independent of potential growth defects which are cancelled out. Hence, if there were parasites dying in the MyoF line (please note that they might not actually die, but simply grow more slowly), this factor applies for both the + and - ART condition. As we are testing for a decreased susceptibility to ART which would manifest as an increased survival in RSA surfacing above 1%, antagonistic effects of reduced MyoF function and ART treatment would not result in detectable differences as without effect, the RSA survival is always close to zero.

The same applies for the knock sideways where we assess the survival of +rapalog between +ART and -ART. If the reduced MyoF activity of the knock sideways leads to a decreased survival, this applies to both +ART and -ART. Please also note that rapalog was lifted after the DHA pulse (see e.g. Figure S2K).

That effects on growth are cancelled out is nicely illustrated for proteins where there is a stronger and more rapid effect on growth upon their conditional inactivation. For instance when KIC7 is knocked aside, there is a considerable increased of RSA survival, even though continued inactivation of KIC7 would have a severe growth defect (Birnbaum et al., 2020). Vice versa, a growth defect alone does not result in reduced RSA susceptibility as evident from knock sideways of an unrelated protein or using a chemical insult (Figure 4H in (Birnbaum et al., 2020) or simply slowing the ring stage by e.g. reducing EXP1 levels (Mesén-Ramírez et al., 2019). Hence, a growth reduction is not expected to alter the RSA outcome. And even if it did, it would only lead to an underestimation of the readout if growth is too severely affected (which would be obvious in the + rapalog without DHA sample, which was not the case).

In that respect it is valuable to have the rapid kinetics of knock sideways which permit inactivation of a protein before severe growth defects occur (although the only partial responsiveness of MyoF clearly is not the most optimal). In contrast, the absolute loss of a gene (as is the case if diCre is used) prevents (or at least makes it extremely difficult as the timing would need to exactly hit sufficient protein reduction without killing the parasite until the end of the RSA) using this system in these experiments (again see (Mesén-Ramírez et al., 2021) where in a EXP1 diCre based knock out RSA was only possible because we complemented with a lowly, episomally expressed EXP1 copy to have parasites with only a partial phenotype to do this assay).

29) Line 261-263: the authors sate that MyoF has a function in endocytosis but at a different step compared to K13 compartment proteins. I am not sure what they mean here. Can this be clarified?

The different steps in endocytosis are explained in the introduction and we now tried to further clarify this (line 98). “So far VPS45 (Jonscher et al., 2019), Rbsn5 (Sabitzki et al., 2023), Rab5b (Sabitzki et al., 2023), the phosphoinositide-binding protein PX1 (Mukherjee et al., 2022), the host enzyme peroxiredoxin 6 (Wagner et al., 2022) and K13 and some of its compartment proteins (Eps15, AP2µ, KIC7, UBP1) (Birnbaum et al., 2020) have been reported to act at different steps in the endocytic uptake pathway of hemoglobin. While inactivation of VPS45, Rbsn5, Rab5b, PX1 or actin resulted in an accumulation of hemoglobin filled vesicles (Lazarus et al., 2008; Jonscher et al., 2019; Mukherjee et al., 2022; Sabitzki et al., 2023), indicative of a block during endosomal transport (late steps in endocytosis), no such vesicles were observed upon inactivation of K13 and its compartment proteins (Birnbaum et al., 2020), suggesting a role of these proteins during initiation of endocytosis (early steps in endocytosis).“

VPS45 has not apparent spatial connection to the K13 compartment but the fact that MyoF does - and its inactivation also results in vesicle accumulation - indicates that it is downstream of vesicle initiation, providing the first connection from the initiation phase to the transport phase. More evidence for these different steps of endocytosis has been published in a recent preprint from our lab, where we simultaneously inactivated a protein of both “endocytosis steps” (Sabitzki et al., 2023).

To clarify this in the results as requested, we changed the statement to: (line 256) “Overall, our results indicate a close association of MyoF foci with the K13 compartment and a role of MyoF in endocytosis albeit not in rings and at a step in the endocytosis pathway when hemoglobin-filled vesicles had already formed and hence is subsequent to the function of the other so far known K13 compartment proteins.”

30) Do the authors mean that it is involved in endocytosis but not in ART resistance? If so, this is a very difficult statement to make since the parasites are dying. Is there any evidence of point mutations in MyoF in the field?

We split this point to address all issues raised here. Please see response to point 29 which clarifies that this was meant in a different way and our response to point 28 which explains why the dying parasite issue is not expected to affect the RSA (please also note that we do not have evidence of actually dying parasites in the MyoF-2xFKBP-GFP-2xFKBP line, most likely the growth is slowed).

The mutation issue is interesting. In fact evidence exists that MyoF mutations may be associated with resistance (Cerqueira et al., 2017) (please note that there it is still called MyoC) but in a recent preprint from our lab we did not find any evidence for a significantly changed RSA survival in 12 tested mutations in the corresponding gene (Behrens et al., 2023).

To clarify this we added the following statement to the discussion (line 709): "Of note, mutations in myoF have previously been found to be associated with reduced ART susceptibility (Cerqueira et al., 2017), but 12 mutations tested in the laboratory strain 3D7 did not result in increased RSA survival (Behrens et al., 2023)*. *

31) Line 298: the authors state that there is no growth defect in the first cycle when rapalog is added to the KIC11 line, however based on Figure 3D, there is evidently a 25% reduction in growth compared to - rapalog at day 1 post treatment, and a 60% reduction by day 2, which is still within the 1st growth cycle. The authors should either revise their statement or provide an explanation for these findings. The authors should also explain why their Giemsa data in Fig. 3E is not in accordance with their FACS data.

We think there is a misunderstanding here, as our figure legend was not detailed enough and we apologise if this had been misleading. The growth effect is restricted to invasion or possibly the first hours of ring stage development (see point 4&5, reviewer 2), which in asynchronous cultures more rapidly takes effect as the culture also contains schizonts that immediately generate cells that re-invade but can't due to inactivation of KIC11 (due to the rapid action of the knock sideways, KIC11 is already inactivated). In contrast, in highly synchronous cultures, this effect can only be evident once the parasites reached the schizont stage (starting with rings this takes close to 2 days). We now clarify that Figure 2E (previously Figure 3D) shows growth data obtained with an asynchronous parasite culture, while in Figure 2F the growth assay is performed with tightly synchronized (4h window) parasites as stated in the Figure legend.

We now explicitly state in each Figure legend and for each growth experiment throughout the manuscript whether we used asynchronous or synchronized parasites for growth assays.

Related to this, the incorrect y-axis label of what is now Figure 2E mentioned in major comment #58 is now corrected.

32) Line 301: KIC11 could also be important very early for establishment of the ring stage for example for establishment of the PV. Also, was mislocalisation assessed in rapalog-treated parasites at 72 hours or in cycle 3?

This is a valid point and this has now been addressed. We performed an invasion/egress assay revealing similar schizont rupture rates, but significantly reduced numbers of newly formed ring stage parasites (Figure 2H, S3G), indicating an effect of KIC11 inactivation either on invasion or possibly the first hours of ring stage development. A very similar point was raised by Reviewer 2, please see reviewer 2; major comment #4. This is now also reflected in line 302, which now reads: ”… indicating an invasion defect or an effect on parasite viability in merozoites or early rings but no effect on other parasite stages (Figure 2F-H, Figure S3F-G).”

We further included an assessment of mislocalization 80 hours after the induction of knock-sideways by addition of rapalog in Figure S3E which showed mislocalization of KIC11 to the nucleus.

33) Line 311: the authors should change the sentence from 'not related to endocytosis' to 'not related to endocytosis or ART resistance'.

Done as suggested.

34) Line 323-325: Authors say that a nuclear GFP signal can be observed in early schizonts for KIC12. According to the pictures provided in Figure 4A and Figure S5A it is not very obvious. Also faint cytoplasmic GFP signal could only be background as we can see that exposure is higher for schizont pictures

We changed the sentence (line 339) to: “…nuclear signal and a faint uniform cytoplasmic GFP signal was detected in late trophozoites and early schizonts and these signals were absent in later schizonts and merozoites (Figure 3A, Figure S4A,B).” in order to emphasize that the nuclear signal disappears early during schizont development.

35) Line 326-328: The authors say that kic12 transcriptional profile indicate mRNA levels peak (no s at peak) in merozoites. Should they show live cell imaging of merozoites then? Because from the Figure 4A schizont pictures where schizonts are almost fully segmented no signal can be observed.

The observation that mRNA levels of early ring stage expressed proteins tend to increase already in mature schizonts and merozoites is well established (e.g. (Bozdech et al., 2003)). A very good example for this are exported proteins of which most show a transcription peak in schizonts but the proteins are only detected in rings see e.g. (Marti et al., 2004). Hence, our observation for KIC12 is quite typical.

We originally did not include merozoites, as in the last row of Figure 3B fully developed merozoites within a schizont with already ruptured PVM are shown and no GFP signal can be detected in these parasites. We now provide images of free merozoites in Figure S4A-B showing again no detectable GFP signal.

We thank the reviewer for pointing out the typo, "peak" has been corrected.

36) Line 347: The authors state that using the Lyn mislocaliser the nuclear pool of KIC12 is inactivated by mislocalisation to the PPM. This tends to suggest that only the nuclear pool of KIC12 is mislocalised. How is it possible that only the nuclear pool is mislocalised?

The Lyn mislocaliser is at the PPM which is continuous with the cytostomal neck where the K13 compartment likely is found. The effect of the Lyn mislocalizer on the KIC12 protein pool localizing at the K13 compartment is therefore somewhat unclear. For this reason we already had the following statement in the original submission (line 400): “Foci were still detected in the parasite periphery and it is unclear whether these remained with the K13 compartment or were also in some way affected by the Lyn-mislocaliser.” We would like to stress here that the same does not apply to the nuclear mislocaliser, which is only a trafficking signal delivering KIC12 to the nucleus and hence likely does not affect the nuclear pool of KIC12, only the K13 compartment pool (the main interest of this manuscript).

We realised that the statement towards the end of this paragraph was unnecessarily ambiguous in regards to the K13 compartment pool of KIC12 which might have caused some confusion about the function of this pool of KIC12 and therefore modified it to (line 374): "Due to the possible influence on the K13 compartment located foci of KIC12 with the Lyn mislocaliser, a clear interpretation in regard to the functional importance of the nuclear pool of KIC12 other than that it confirms the importance of this protein for asexual blood stages is not possible. In contrast, the results with the nuclear mislocaliser indicate that the K13 located pool of KIC12 is important for efficient parasite growth.". It is also important to note that this limitation does not apply to the NLS knock sideways in regard to the K13 compartment and that the endocytosis function of this pool of KIC12 seems solid which with this statement is enforced.

37) Line 368-369: Effect was also only partial for MyoF. Why didn't you measure the same metrics for MyoF?

This was now done and is provided as Figure 1J-K, S2J, confirming our previous interpretation, see also point #27 which raises the same point.

38) Line 379: you don't know if all proteins acting later in endocytosis will have an increased number of vesicles as a phenotype

This is based on our current definition as stated in the introduction. It assumes a directional vesicular transport of hemoglobin to the food vacuole where inhibition of early stages will prevent transport before HCC-filled autonomous vesicular containers have formed and entered the cell. In contrast later inhibition stops such containers from further transport, leading to their accumulation. Such an accumulation is visible after VPS45-inactivation and other proteins (Jonscher et al., 2019; Mukherjee et al., 2022; Sabitzki et al., 2023) or treatment with cytochalasin D (Lazarus et al., 2008). While it is possible that there may be smaller intermediates formed at the K13 compartment that later on unite or fuse with the compartment evident after VPS45 inactivation and these might be missed due to small size (i.e. inhibition of a step between K13 compartment and an early endosome or equivalent), this would still be upstream of the VPS45 induced containers and hence would be earlier. We therefore believe that based on the framework given in the introduction (see also (Spielmann et al., 2020)) to assume that a phenotype manifesting as reduced food vacuole bloating without formation of detectable vesicles likely signifies inhibition of the process early whereas reduced bloating but with vesicles signifies inhibition later in the process.

39) Line 413-414: The authors state that no growth defect was observed upon KS of 1365800. Is growth alone enough to say that there is no impact on endocytosis?

This is an interesting point. The endocytosis proteins we studied so far indicate that efficient impairment of endocytosis manifests as a severe growth defect. Hence, lack of a growth defect can be assumed to be an indicator for absence of an important role for endocytosis (or any other growth relevant process). Clearly there is a gradual response, such as seen in the different MyoF versions resulting in proportional growth and vesicle appearance phenotypes. Hence, a protein with a minor role might have slipped our attention but then it probably is also not a very important protein in endocytosis.

To further strengthen our assessment of PF3D7_1365800 importance for asexual blood stage development, we now also generated a cell line expressing the PPM Mislocalizer, enabling knock sideways to the PPM. This was done because this protein consistently has a focus at the nucleus that may be within the nucleus. Again this revealed no growth defect upon inactivation (Figure S7D).

40) Line 432: in this section, the authors state that KIC4 and KIC5 seem to have domains that may suggest these proteins are involved in endocytosis, based on the alpha fold data that is publicly available. Considering the authors have TGD-SLI versions of these lines (Birnbaum et al. 2020) and have already confirmed in this previous publication that they confer resistance to ART; it would make sense to look at endocytosis for these genes. This would be a relatively simple and straightforward experiment, taking no longer than two to three weeks, and would require no additional reagents or line generation. Doing these experiments would add a lot more weight to this final section. The authors later state that KIC4 and 5 are TGD lines, so not the best for endocytosis assays. It is unclear why this would be difficult to do if an adequate control is contained in the experiment (such as parental 3D7). It explains why they did not perform the MCA2 endocytosis assays further up, but in my opinion, an attempt at doing these assays is important and would significantly increase the impact of this paper. Identical as major comment #17.

As stated in the manuscript and above, we were originally hesitant to do these assays due to the fact that we can't induce inactivation which is less ideal than comparing the identical parasite population split into plus and minus and is further complicated by the likely smaller effect as the TGDs still permitted growth. However, we see the point of the reviewer and now performed these assays using 3D7 as controls and taking extra care to account for stage differences between the TGD lines and 3D7. However, there was no significant difference in the bloated food vacuole assays with these cell lines. Due to the reasons mentioned in major point 17, we are not sure this indeed means these proteins have no role in endocytosis. One possible reason why we were able to obtain these TGDs may have been because the effect on endocytosis is less than in the essential proteins (or is ring stage specific) and in a TGD an endocytosis defect may therefore not be detectable with our assays (see details and further possible explanations in response to point 17).

In an attempt to address the TGD issue, we generated knock sideways cell lines for KIC4 and KIC5. Unfortunately, the mislocalization of KIC5 to the nucleus was inefficient (see figure below). As this did not result in a growth defect (in contrast to the clear KIC5-TGD growth defect (Birnbaum et al., 2020)), this line is not suitable to study a potential role of this protein in endocytosis. Therefore, we performed the bloated food vacuole assay only with KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser parasites. However, this revealed no effect on HHC uptake, which is in line with the normal growth of KIC4-TGD parasites (Birnbaum et al., 2020) and suggests that this protein could only have a minor or redundant role in endocytosis (it is the line that shows the smallest effect in RSA). As the KIC4 and KIC5 knock sideway lines did not permit any conclusions, we did not include them into the revised manuscript but they can be found here:

[Figure KIC4 knock sideways & KIC5 knocksideways]

Figure legend: (A) Live-cell microscopy of knock sideways (+ rapalog) and control (without rapalog) KIC4-2xFKBP-GFP-2xFKBPendo+ 1xNLS mislocaliser parasites 4 and 20 hours after the induction of knock-sideways by addition of rapalog. Scale bar, 5 µm. Relative growth of asynchronous KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser plus rapalog compared with control parasites over five days. Three independent experiments were performed. Growth of knock sideways (+ rapalog) compared to control (without rapalog) KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser (blue) or KIC5-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser (red) parasites over five days. Mean relative parasitemia ± SD is shown. (B) Live-cell microscopy of knock sideways (+ rapalog) and control (without rapalog) KIC5-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser parasites 4 and 20 hours after the induction of knock-sideways by addition of rapalog. Scale bar, 5 µm. Growth of asynchronous KIC5-2xFKBP-GFP-2xFKBPendo+ 1xNLSmislocaliser plus rapalog compared with control parasites over five days. Four independent experiments were performed. __(C) __Bloated food vacuole assay with KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser parasites 8 hours after inactivation of KIC4 (+rapalog). Cells were categorized as with ‘bloated FV’ or ‘non-bloated FV’ and percentage of cells with bloated FV is displayed; n = 3 independent experiments with each n=19-30 (mean 21.4) parasites analysed per condition. Representative DIC are displayed. Area of the FV, area of the parasite and area of FV divided by area of the corresponding parasites were determined. Mean of each independent experiment indicated by coloured symbols, individual datapoints by grey dots. Data presented according to SuperPlot guidelines (Lord et al., 2020); Error bars represent mean ± SD. P-value determined by paired t-test. Area of FV of individual cells plotted versus the area of the corresponding parasite. Line represents linear regression with error indicated by dashed line.

41) Line 490-493: the authors state that the K13 compartment proteins fall in two groups, some that are involved in ART resistance AND endocytosis, and some that have different functions. However, in this manuscript the authors have demonstrated 3 flavours that K13 compartment proteins can come in: • Some that confer ART resistance and are involved in HCCU (MCA2) • Some that are involved in HCCU but not ART resistance (MyoF & KIC12) • Some that are involved in neither (KIC11) The authors should therefore revise this statement.

We agree that this was not well phrased. To account for the fact that not all endocytosis proteins confer increased RSA survival to the parasites when inactivated we changed this statement (line 604): "This analysis suggests that proteins detected at the K13 compartment can be classified into at least two groups of which one comprises proteins involved in endocytosis or in vitro ART resistance whereas the other group might have different functions yet to be discovered.“

Generally, we believe that endocytosis is the overarching criterion and we therefore would like to keep the definitions of the main groups (endocytosis or not). As indicated by the title, the focus of the manuscript is on the K13 compartment for which so far endocytosis is the only experimentally associated function. That this group contains proteins that do not confer reduced ART susceptibility when conditionally inactivated (KIC12 and MyoF) is explained by their stage-specificity, making this a subgroup of the overarching endocytosis group.

We realise that with the endocytosis data on the KIC4, KIC5 and MCA2 TGD there is now also a subgroup we were unable to demonstrate an endocytosis effect in trophozoites although they show changes in RSA survival. However, as indicated above, we would be hesitant to fully exclude some role of these proteins in endocytosis in rings. Particularly as a comparably small reduction in endocytosis protein activity or abundance is sufficient to increase RSA survival (Behrens et al., 2023). A principal classification of "endocytosis or ART resistance" or "neither endocytosis nor ART resistance" still accounts for this and therefore seems to us to be the most useful, particularly also in light of our domain identification that then can be linked with one or the other group.

42) Line 508: the authors state that they expanded the repertoire of K13 compartments, when in fact they functionally analysed them - they did not do another BioID to identify more candidates.

We respectfully disagree with the reviewer in this point, we did expand the repertoire of known K13 compartment proteins. Only independently experimentally validated proteins from proximity biotinylation experiments can be considered part of the K13 compartment (or any other cellular site or complex). Without validation of the location, the identified proteins can only be considered candidates. This is highlighted in this manuscript by the finding that several proteins of the list did not localize at the K13 compartment.

43) Line 570-572: has anyone ever tested whether CytoD or JAS treatment in rings, is sufficient to mediate ART resistance? Something similar to what was done in PMID 21709259 with protease inhibitors. If not this would be a pretty interesting experiment for the authors to do that could shed more light on the MyoF data. It would take maybe 2 weeks to do and not require the generation of any new lines. This would clarify whether other Myosins other than MyoF are involved in endocytosis, as is suggested by previous publications (PMID: 17944961).

We now included this experiment. In agreement with a lacking need of MyoF in rings and no effect on RSA survival, there was no increased survival of the parasites in RSA (neither on 3D7 nor on K13 C580Y parasites) after cytD treatment (new part in Figure 1M). We thank the reviewer for pointing out that this experiment might also inform on whether other myosins influence endocytosis in ring stages. We added (line 250): “Similarly, also incubation with the actin destabilising agent Cytochalasin D (Casella et al., 1981), had no effect on RSA survival in 3D7 or K13C580Y (Birnbaum et al., 2020) parasites, indicating an actin/myosin independent endocytosis pathway in ring stage parasites (Figure 1M) and speaking against other myosins taking over the MyoF endocytosis function in rings.”

44) Line 608: inhibitors targeting the metacaspase domain of MCA2 may inadvertently inactivate other essential parts of the protein. They authors should acknowledge this possibility in the text.

The inhibitors used in the cited studies (Kumari et al., 2018) are validated metacaspase inhibitors, such as Z-FA-FMK (Lopez-Hernandez et al., 2003). Activity against the other parts of PfMCA2 - which apart from the MCA domain shows no homology to other proteins - is therefore unlikely.

45) Line 624-625: the authors state that MyoF is 'lowly expressed in rings' - indeed this is the case in their MyoF-2xFKBP-GFP-2xFKBP line which the authors established has defects due to the tag, but it appears from their MyoF-3xHA tagged line that it is expressed in rings. The authors should therefore revise their statement, and be careful of making claims based on their defective line and using fluorescence imaging as their only metric. If they do want to make the statement that it is not there in rings, they should also do a western blot, which is much more sensitive since it amplifies the signal compared to an image of one parasite.

This comment is related to major point #24. We also would like to stress that while the MyoF-GFP line already shows a phenotype, the impression of defectiveness based on its location is due to a mix up (see major point #23).

We now provide a comprehensive time course of the MyoF-GFP signal (Figure 1C, S2A) showing that there is no detectable MyoF-GFP signal until the transition from ring to trophozoite stage. As this is all under the endogenous promoter, we do not think the partial functional inactivation of the tagging is the reason for the absence of the signal. If anything, we would have expected adding a stably folded structure such as GFP to increase the stability of the protein. The main reason for the discrepancy of MyoF signal in rings between the GFP-tagged line (of note there is also no detectable MyoF-GFP signal in MyoF-2xFKBP-GFP ring stage parasites (Figure S2B)) and the HA-tagged line likely is that IFA is much more sensitive than live GFP detection (similar to the high sensitivity the reviewer mentions in regards to WB). This discrepancy therefore is likely due to the fact that the lowly expressed MyoF only become apparent with the HA-tagged line due to the IFA. We therefore believe that MyoF is 'lowly expressed in rings' is an appropriate description of our results obtained with three different cell lines (MyoF-2xFKBP-GFP-2xFKBP, MyoF-2xFKBP-GFP and MyoF-3xHA). We hope this is sufficiently well reflected in the manuscript where we write ‘a low level of expression of MyoF in ring stage parasites.’ not that it is ‘not there in rings’ (line 174).

46) Line 635: arguably this is the 3rd variety and not the 2nd (the authors already mentioned 2 types - ones that are involved in HCCU AND ART and those involved in HCCU only). See comment for line 490-493 above.

See response for major comment #41, we now consistently used "or" instead of "and". See line 490-493 how this was resolved for what previously was line 635.

47) Line 785: Bloated food vacuole assay/E64 hemoglobin uptake assay method specify that a concentration of 33mM E64protease inhibitor was used. However, in reference 44, cited in the manuscript, a concentration of 33µM E64 was used. Please confirmed if this is just a typo or if 1000x E64 concentration was used which renders the experiment invalid.

We thank the reviewer for pointing this out, we corrected this typo and will look out for symbol font conversion errors for the resubmission.

48) Line 788: it is unclear from this section what is considered a bloated food vacuole - is there an area above which the FV is considered bloated? Do the authors do these measurements manually or use an addon in FIJI/ImageJ? What is the cutoff for if a FV is bloated? Please clarify. Additionally, for the representative images + rapalog for Figures 2H and 4H, it would be useful to see where the authors delineate the FV (add a white circle showing what is actually measured).

The bloated FV assay is well established (Jonscher et al., 2019; Birnbaum et al., 2020; Sabitzki et al., 2023). Although the bloating of the FV is a human judgment call, it is actually quite obvious: bloating appears as an easily spotted bulging of the FV in DIC. As also minor bloating is scored as 'bloated', it is a very conservative assay. Using an-add on to measure this is not straight forward. It is unclear how this bulging effect of the FV in DIC could be spotted by a software and due to the obviousness to human operators, potentially lengthy and complicated efforts to design appropriate machine learning options were not undertaken. The situation faced by the scorer of the assay is evident from Figure S4F-G which contains close to 50 "on rapalog" cells and close to 50 control cells, giving representative cells from all replicas of bloated FV assays with KIC12. Please note that these images shows the most complicated situation as far as bloated assays go, because the phenotype is not 100% (see Figure 3F) compared to e.g. KIC7 inactivation which leads to lack of bloating in almost all cells (see (Birnbaum et al., 2020) Figure 3E) but nevertheless the difference is still obvious. We are aware that in such situations (less than absolute inhibition) this assay scoring of "yes" or "no" is a surrogate for the actual level of inhibition and may be more subjective. This is why in this case we also did the FV size measurements (which are less dependent on human judgment) to further support this and give a better quantifiable measure. Of note, the bloated food vacuole judgments are done "blinded", i.e. the examiner does not know which sample they are looking at.

In response to this reviewer's point we now also added the FV size refinement of the assay for MyoF inactivation which is one of the cases where inhibition of bloating is not in 100% of the cells (see major comment #27). Please also note here the advantage of the rapidly acting knock sideways technique for these assays which shows the sum of effect 8 h after initiating inactivation and for which we carefully control size of the cells which shows that there is no significant growth reduction over the assay time, excluding secondary effects due to a generally reduced viability. Compared to slower acting systems suggested to have been used instead (see introductory part and significance of this review), the rapid speed of knock sideways reduces the risk of potential pleiotropic or compensatory effects due to the time needed for proteins to be depleted if the gene or mRNA is targeted instead.

The suggestion to include a ‘white circle’ (raised also as minor comment#27) is useful as an aid to see the food vacuole. However, in contrast to the Figures in (Birnbaum et al., 2020) (where we did add such a circle), we here included the DHE staining images in the figure, labelling the parasite cytosol which readily shows the FV (the FV corresponds to the region where there is no DHE staining). As this shows the position of the FV we would prefer to not obscure the DIC images with additional features to permit the reader to see the difference between bloated or non-bloated food vacuoles and keeping the image as natural as possible.

49) Line 863-864: this sentence seems to be out of place.

We thank the reviewer for pointing this out, the details of nucleus staining were moved to the correct part.

50) Line 875: the authors state that there is a light blue wedge, when the circle consists of grey and black wedges. Please revise this.

This has been corrected.

51) Line 1059-1061: it is unclear whether the individual growth curves are different clones or whether they are just the same experiment repeated? If it is the latter, then why are they not combined, as is traditionally done?

These are the individual replicates of the growth curves shown in Figure 1G of the same cell lines done on a different occasion. We always try to show as much of the primary data as possible and believe that showing individual data points from the different experiments is better than only the combined values which obscure the actual course of each experiment.

52) Line 919-924: the authors mention a blue and red line, but there is only a black line in figure 3D. Moreover, the experiment of using the LYN mislocaliser was only done for KIC12 according to the manuscript. Additionally, the y axis of the figure states relative growth day 4[%] compared to rapalog, but then on the x axis there are several days. In the text it says there is no growth defect until the second cycle, but from this graph it appears the growth defect is evident as early as 1 day post rapalog treatment. Can the authors please clarify and correct the issues pointed out.

We thank the reviewer for pointing this out, this was due to a copy & paste error in the figure legend that was now amended. We also fixed the incorrect axis label. For the last part (growth defect) please see detailed answer to Major comment#31 raising the same concern for KIC11 (in synchronous parasites the defect only takes effect once the cells reached the relevant stage whereas in asynchronous cultures there are always cells in the relevant stage that due to the rapid effect of the knock sideways already have a growth phenotype).

53) Figure 1 panel B & C: the label of the figure where the signal from MCA2Y1344STOP-GFP is shown with the DAPI signal overlayed is deceptive since it suggests that this is the signal of full length MCA2. Please change the label of this panel from MAC2/DAPI to MCA2Y1344STOP/DAPI. The same is true for Panel C for the image labeled MCA2/K13 - please change this to MCA2Y1344STOP/K13.

Done as requested.

54) Figure 2B: what stages are these parasites? Please state this in the figure. Based on the MyoF pattern, it looks like rings in the upper panel and trophs in the bottom pannel. Why were schizonts not shown?

Both are trophozoites (early trophozoite in top panel and late trophozoite in bottom panel). This is now labelled in what now is figure 1B. As stated above, schizont stages are less relevant for the topic of this manuscript and in order to prevent the manuscript from getting more disjointed and keeping it more focussed on the main topic, we decided to not include a schizont in the manuscript. Nevertheless, we included an example image below.

[Figure MyoF_p40px schizont]

55) Figure 2D&F: it is not very meaningful when growth assays are shown as a final bar after 4 days of growth. It is much more useful and informative to see a growth curve instead (as is shown in the supplementary), since it shows if the defect is apparent in the first growth cycle or later. With the way the data is currently shown, this is not apparent. I would advise the authors to switch the graph in 2F out of a combined graph of all the biological replicates growth curves for S3D - showing error bars.

While we in principle fully agree with the reviewer in showing the course of the full experiment (which is available in Figure S2E), the key here is to show the overall difference. Hence, we would like to keep this comparison of the overall effect on growth in what now is Figure 1E and G. It is part of the argument to the doubts this reviewer raises to the function of MyoF (mainly in the overall assessment and the significance statement) to show that the phenotype is actually very consistent (partial inactivation through tagging or further inactivation using knock sideways increases endocytosis phenotypes, correlating with parasite viability).

Please also note, that the growth curves upon knock sideways shown in Figure 1G, S2E are performed with asynchronous parasite cultures, which doesn’t allow us to draw direct conclusions about growth cycle effects.

Nevertheless, we now also included the suggested combined data representation in Figure S2E.

56) Figure 3: why were the calculation of FV area, parasite area and FV/parasite area only done for KIC12 and not done for MyoF? It would be interesting to see if any of these values are different for MyoF - whether the parasites are smaller in area and therefore FV smaller. Please present them Figure 2. Images should be already available and would not require further experiments to be done, only the analysis.

This now has been done (confirming our results) and is included as Figure 1J-K, S2J. This point was also raised as major comment #37, please also see detailed answer there.

57) Figure 3B: why is there no spatial association assessment for KIC11 and K13 as was done for the MCA2 and MyoF? The authors should show a pie chart showing the degree of association here as was done for the other proteins.

This is now included in Figure 2C.

58) Figure 3D: The y axis of the figure states relative growth day 4[%] compared to rapalog, but then on the x axis the experiment takes place over several days. Is this a typo in the y axis? Additionally, the authors state in line 287-290 that the growth defect upon addition of rapalog is only seen in the second cycle, but from this graph it appears the growth defect is already evident 1 day post rapalog addition. The figure legend also does not make sense for this figure since it mentions a blue and a red line, when there is only a black line present. The legend also mentions the LYN mislocaliser which was used for KIC12 not KIC 11 (see above).

We apologise for the inadequate legend and colour issues, this was amended. This point was also raised in major comment #31 and #52, please find detailed answer there.

59) Figure 3E: the colour for Control and Rapalog 4 hpi are very similar and very hard to discern. Please choose an alternative colour or add a pattern to one of the samples. The y axis is also missing a label. Is this supposed to be parasitemia (%)?

We thank the reviewer for pointing this out, the missing label is now included and the colour has been adapted to make them better distinguishable.

60) Figure 4A: the ring shown in this figure does not appear to be a ring (it is far too large and appears to have multiple nuclei?). Do the authors have any other representative images to show instead?

This is in fact a ring, but we realize that we accidentally included an incorrect size bar in the ring image of Figure 4A (now Figure 3A) (size bar for 63x objective instead of the correct one for the 100x objective), we apologise for this oversight. We don’t think this parasite has multiple nuclei, instead the Hoechst signal shows the often elongated nucleus seen in rings that can appear as two foci in Giemsa stained smears which leads to the typical diagnostic feature of P. falciparum rings in diagnostics. In order to exclude any doubts about the nuclear localization of KIC12 in rings, we here attached a panel with more examples of KIC12-2xFKBP-GFP-2xFKBP ring stage parasites.

[Figure KIC12]

61) Figure 4B: why is there no spatial association assessment for KIC12 and K13 as was done for the MCA2 and MyoF? The authors should show a pie chart showing the degree of association here as was done for the other proteins. This should be done for the different life cycle stages considering the changing localisation of KIC12.

This is now provided in Figure S4A. As suggested by the reviewer, we independently quantified the association for ring stage, early trophozoite and late trophozoites stage. As there is no KI12 signal in schizonts, we did not include a quantification for this stage.

62) Figures 4C&E: it is extremely important to show the DNA stain in both these samples considering that a portion of KIC12 is in the nucleus! Please add the DAPI signal for these figures (as for all other figures!).

Please see major comment #64 for a detailed answer why we did not include DNA staining in the imaging used to assess mislocalization upon knock-sideways.

63) Figure 4E: this figure should be presented before 4D (considering the line being presented in 4E is used in an experiment in 4D). The authors should switch the order of these two.

We see the point the reviewer is raising here, Figure 4D (now Figure 3D) also contains the data with the Lyn mislocaliser while we first talk about the NLS mislocaliser. This permits a better comparison between the two mislocaliser lines. However, first explaining the Lyn-mislocaliser and then going back to the NLS would make it rather complicated for the reader to follow the storyline and therefore we would like to keep the order as it is. We realise that this means the reader has to go back one figure part for seeing the Lyn growth data, but believe this is worth the benefit that the data is there compared to the NLS result.

64) It is unclear why in many of the fluorescence images the authors do not show the DAPI signal - particularly when colocalising with K13 and when doing the knock sideways experiments. Please add these images to the figures - I would assume they have already been taken, so would simply involved adding the images to the panel.

We did not include DNA staining (DAPI or Hoechst) for any of the images used to assess the efficacy of mislocalization, as we would prefer to keep the parasites as representative of a viable parasites in culture as possible. Hence they were imaged without DNA stain (these stains are toxic). We would like to point out that a DNA stain is not necessary, as the mislocaliser already marks the nucleus (in the case of the NLS mislocaliser), actually even somewhat more accurately, as it fills the entire nuclear space rather than only the DNA which is marked by DAPI or Hoechst.

For LYN this admittedly is not the case, there the mislocaliser marks the plasma membrane. However, we think the proper control for efficient mislocalisation is the comparison between the GFP-tagged protein of interest and the mCherry mislocaliser to show mislocalisation, as previously done in our lab (e.g. (Birnbaum et al., 2017; Jonscher et al., 2019; Birnbaum et al., 2020)).

Due to their toxicity, we also avoided nuclear staining in some other parts of the manuscript when we were of the opinion that a nucleus signal was not necessary.

65) Throughout the manuscript, there is no western blot confirming the correct size of their modified proteins. This should be provided.

We did perform Western blot analysis for both MCA2 cell lines. MCA2 is the only gene-product for which we generated a disruption for this work, and together with the severe truncation from previous work, we provided a Western blot-based confirmation of the correct size.

The MCA2 disruptions are at least partially dispensable for in vitro parasite growth, hence if degradation occurred, this might not have been noticed. In that case we considered it relevant to show that the truncations were of the expected size. The other proteins in the main figures are essential for growth. Hence, if the tagging approach would lead to unexpected changes in protein integrity (which we assume is what was intended by this concern to be assessed with a Western blot), the parasites expressing the tagged MyoF, KIC11 and KIC12 would - due to their importance for asexual blood stage development - not have been obtained. Hence, we can assume the integrity of the tagged protein is very unlikely to have been affected in a functionally relevant way.

66) None of the figures are appropriate for individuals with colour blindness, limiting their accessibility to the paper. Please change the colour schemes for all fluorescent images using magenta/green or an alternative colour combination appropriate for colourblind individuals.

We thank the reviewer for this comment. This has now been amended, individual channels of fluorescence microscopy images are now shown in greyscale, while the overlay was changed to green/magenta.

Minor Comments

1) line 29: remove 'are'.

Done.

2) Line 29: the text says "HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins are among the few proteins so far functionally linked to this process." The sentence should be: 'HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins among the few proteins so far functionally linked to this process."

Done.

3) line 44: remove 'the'

Done.

4) Line 48: consider mentioning here that malaria is caused by the parasite Plasmodium - otherwise the first mention of parasite in line 52 is confusing for the non-specialist reader.

Done.

5) Line 49: estimated malaria-related death and case numbers are from the 2021 WHO World malaria report. You cite the 2020 WHO World malaria report.

We now cite the newest WHO report.

6) Line 53: please insert the word 'have' between now and also.

Done.

7) Line 54: please change 'was linked' to is linked

Done

8) Line 72: I would specify that free heme is toxic to the parasite. Especially as you mention that hemozoin is nontoxic.

Sentence would be "where digestion results in the generation of free heme, toxic to the parasite, which is further converted into nontoxic hemozoin"

Done.

9) Line 90: authors should either say "in previous works" or "in a previous work"

The text has been altered to say: “ in a previous work”.

10) Line 91: "We designated these proteins as K13 interaction candidates (KICs)"

Done.

11) Line 95: please change 'rate' to number

Done.

12) Line 109: Please include a coma before (ii).

Done.

13) Line 112: as shown by Rudlaff et al in the paper you are citing, PPP8 is actually associated with the basal complex. You can say that "(ii) were either linked or had been shown to localise to the inner membrane complex (IMC) or the basal complex (PF3D7...).

Done.

14) Line 114: Protein PF3D7_1141300 is called APR1 in the manuscript but ARP1 in Supplementary Table 1. Please correct.

Done.

15) Line 131: please define SNP - this is the first use of the acronym.

Done.

16) Line 133-134: South-East Asia instead of "South Asia"

Done.

17) Line 135: please explain what TGD is - it is referred to over and over again in the manuscript without ever being explained.

We apologise for this oversight. We now explain what is meant with TGD at the suggested point of the manuscript.

18) Line 145: change 'Western blot' to western blot - only Southern blot is capitalised since it is named after an individual, while the other techniques are not.

To the best of our knowledge this issue has not been resolved, some Journals capitalize the “W” (e.g. Science), while others don’t (e.g. Nature). We would prefer to continue to capitalize the “W”, as this is consistent with the original publication from (Burnette, 1981), but if there are strong objections, we would be happy to change this____.

19) Line 152: add "the" between 'and spatial'

Done.

20) Line 158: please define SLI as selected linked integration, since it is the first use of the acronym.

Done.

21) Line 178: introduce a coma after protein. Sentence should be "Proliferation assays with the MCAY1344STOP-GFPendo parasites which express a larger portion of this protein, yet still lacking the MCA domain (Figure 1), indicated no growth ...

Done.

22) Line 195: the authors could mention that MyoF was previously called MyoC in the Birnbaum 2020 paper. I wanted to check back in the Birnbaum 2020 paper and could not find MyoF

Good point, this was done.

23) Line 200: "Expression and localisation of the fusion protein was analysed by fluorescent microscopy". Why expression was not analysed also by western Blot same as for MCA2?

Please see major comment #64 for a detailed answer.

24) Line 204: I could not find any mention of MyoF (Pf3D7_1329100) in reference 65. Please remove reference 65 if not correct. Also reference 66 looks at Plasmodium chabaudii transcriptomes so I would specify that "This expression pattern is in agreement with the transcriptional profile of its Plasmodium chabaudii orthologue"

Reference 65 (Wichers et al., 2019) provides an RNAseq transcriptome dataset for asexual blood stage development of 3D7 (originating from the same source as the 3D7 used in this study). While Ref 66 (Subudhi et al., 2020) indeed contain transcriptomic data from P. chabaudi, the authors also provide a nice 2h window RNAseq transcriptome dataset for asexual blood stage development of Plasmodium falciparum. Both datasets are therefore suitable as reference for the statement about myoF transcription pattern. Both datasets are also easily accessible and show the pattern in a graph in PlasmoDB.

25) Line 208: Please indicate a reference for P40 being a marker of the food vacuole

Done.

26) Line 220-224: The authors should consider changing to " Taken together these results show that MyoF is in foci that are mainly close to K13 and, at times, overlapping, indicating that MyoF is found in a regular close spatial association with the K13 compartment."

The suggested wording introduces "mainly" for "frequently" and likely was in part motivated by the discrepancy in location between cell lines that we hope we now could clarify to be only minor (see major point #23). We therefore think the original wording appropriately summarises the findings (line 178): “*Taken together these results show that MyoF is in foci that are frequently close or overlapping with K13, indicating that MyoF is found in a regular close spatial association with the K13 compartment and at times overlaps with that compartment.” *

27) Line 255: In Figure 2H, and subsequent figures showing bloated FV assay, I would delineate the food vacuole with dashed line as in Birnbaum et al. 2020 to help the reader understanding where the food vacuole is.

In contrast to the Figures in Birnbaum et al. 2020, we here included the DHE staining (parasite cytosol) in images of bloated FV assays which visualizes the FV. We therefore decided to avoid any further marking, to keep the image as unprocessed as possible (see also major point 48).

28) Line 265-266: Here the title says that KIC11 is a K13 compartment associated protein, but the title of Figure 3 says KIC11 is a K13 compartment protein. I noticed that you make the difference between K13 compartment protein et K13 compartment associated protein for MyoF for example which is not clearly associated with the K13 compartment. Which one is it for KIC11?

The interpretation of the reviewer is correct, we indeed graded this subconsciously based on level of overlap. Based on the newly added quantification shown in Figure 2C, we describe KIC11 now as K13 compartment protein.

29) Line 309-310: indicate a reference for your statement "which is in contrast to previously characterised essential K13 compartment proteins".

Done, we now included Birnbaum et al. 2020 as reference for this.

30) Line 377: Figure 4I, please correct 1st panel Y axis legend

Done.

31) Line 404: replace "dispensability" with dispensable

Done.

32) Line 416: can the authors provide any speculation as to why they observed these proteins as hits in the BioID experiments?

As some of these proteins were less well or less consistently enriched, they could be background of the experiment. Alternatively, some could be proteins that only transiently interact with the K13 compartment.

33) Line 451: Where the "97% of proteins containing these domains also contain an Adaptin_N domain and function in vesicle adaptor complexes as subunit a" come from. Do you have a reference?

The statement now includes references and reads (with small changes to original submission): "More than 97% of proteins containing these domains also contain an Adaptin_N (IPR002553) domain (Blum et al., 2021) and in this combination typically function in vesicle adaptor complexes as subunit α (Hirst and Robinson, 1998; Traub et al., 1999) (Figure 5D) but no such domain was detectable in KIC5."

34) Line 465-467: the same could be said for KIC4 as it also has a VHS domain.

The critical issue is the combination of domains and their position within the protein. While KIC4 also contains a VHS domain, the VHS domain in KIC4 is N-terminal, not in a central position and it is also not the first structural domain to be identified in KIC4. The similarity to adaptin domains was already described ((Birnbaum et al., 2020) and annotated in PlasmoDB) and these domains are also involved in vesicle formation and trafficking. These aspects of the statement can therefore not be extended to KIC4. With regards to VHS domains being involved in vesicle trafficking, this is already stated in line 538: «KIC4 contained an N-terminal VHS domain (IPR002014), followed by a GAT domain (IPR004152) and an Ig-like clathrin adaptor α/β/γ adaptin appendage domain (IPR008152) (Figure 5A-C, Figure S8). This is an arrangement typical for GGAs (Golgi-localised gamma ear-containing Arf-binding proteins) which are vesicle adaptors first found to function at the trans-Golgi (Dell’Angelica et al., 2000; Hirst et al., 2000).»

35) Line 477-479: Can be rephrased to "However, we found this protein as being likely dispensable for intra-erythrocytic parasite development and no colocalisation with K13 could be demonstrated, suggesting a limited role for PF3D7_1365800 in endocytosis. Or something like that. Makes it clearer.

We rephrased this sentence and it now reads (line 592): “However, we found this protein as being likely dispensable for intra-erythrocytic parasite development and no colocalisation with K13 was observed, suggesting PF3D7_1365800 is not needed for endocytosis“.

36) Line 535: Have AP-2a or AP-2b been shown to be at the K13 compartment?

AP2m is at the K13 compartment (Birnbaum et al., 2020). Adaptor complexes are heterotetramers and their subunits do not typically function on their own and this is conserved across evolutionarily distant organisms. In agreement that this is also the case in P. falciparum, Henrici et al. (Henrici et al., 2020a) showed that both, AP-2a and AP-2b, were present in an AP2µ Co-IP, indicating that the AP2 complex consist of the ‘classical’ subunits in P. falciparum. Therefore, the presence of all subunits at the K13 compartment is very likely, although this has only been experimentally confirmed for AP2µ. Of note, for Toxoplasma gondii the presence of AP-2a and AP-2b at the micropore has been experimentally confirmed (Wan et al., 2023; Koreny et al., 2023) and interaction suggested by presence in the same IP as DRPC (Heredero-Bermejo et al., 2019).

37) Line 569: reference 43 is wrong

We thanks the reviewer for pointing this out – we removed Ref 43.

38) Line 746: typo "ot" instead of or.

Changed.

39) Line 801: method for Domain Identification using AlphaFold specify that RMSDs of under 5Å over more than 60 amino acids are listed in the results. However, there is a typo in Figure 5B for KIC5 where it says "RMSD 4.0 Å over 8 aa". Please correct.

Done. In addition, we have now applied a more stringent cut-off of 4Å over more than 60 amino acids to ensure a higher reliability of our hits. This decision was based on results from our preprint (Behrens and Spielmann, 2023). Because of this the phosphatase domain in KIC12 is no longer included in this manuscript and accordingly the following sentence has been deleted. “In KIC12 we identified a potential purple acid phosphatase (PAP) domain. However, with the high RMSD of 4.9 Å, the domain might also be a divergent similar fold, such as a C2 domain, which targets proteins to membranes.”

40) Line 856: In Figure 1E, please use the same Y axis legend as in Figure 2D "relative growth at day 4 [%] compared with 3D7"

Done.

41) Figure S1: Some PCR gels check for integration are presented as 5', 3' and ori whereas other gels are presented as ori, 5' and 3'. This is confusing.

We agree that ideally the order of sample loading should be consistent and we apologise for this. The explanation for this is that these gels were run by different people at different times before we were able to better standardize the loading scheme. However, in the interest of not unnecessarily using resources for something that has a similar meaning, we would prefer not to repeat these PCRs and re-run them only for consistency reasons (as the conclusion is not affected by the different loading schemes).

42) Figure S1: Why was the expression of only MCA2 was verified by Western blot? What about the other proteins?

See response to major comment 56.

43) Line 493: Considering KIC11 was not involved in HCCU or ART resistance it might be worth mentioning in this section that it is of note that there are no domains detected that would be involved in endocytosis.

We agree that this is the case, however it is also the case for all other proteins that either are not involved in endocytosis and/or lowered susceptibility to ART. We therefore now added a summary statement addressing this in line 602: “In contrast, the K13 compartment proteins where no role in ART resistance (based on RSA) or endocytosis was detected, KIC1, KIC2, KIC6, KIC8, KIC9 and KIC11, do not contain such domains (Figure 5E).” We did not add this at the suggested part of the manuscript as at that point the domain search results are not yet introduced and doing this each time for all the individual proteins would disconnect the flow of the manuscript.

44) Line 503-506: is it wise to generate more drugs that target a pathway that is already highly susceptible to mutations? The authors should add a statement explaining how this might be avoided.

The only protein for which mutations do not have a large fitness cost is K13 (see also our preprint on fitness cost of ubp1 mutation (Behrens et al., 2023) and even with K13 the level of resistance seems to be limited by amino acid deprivation when endocytosis is reduced (Mesén-Ramírez et al., 2021). We therefore do not think that this pathway is particularly prone for mutations. Further, the number of commercial drugs targeting the "endproduct" of endocytosis (hemoglobin digestion and detoxification of heme) highlight it as the most prominent vulnerability for drug-based intervention if we go by number of commercially available drugs acting on things associated with a single process.

45) Throughout, scale bars are stated in the figure legends at the end of the legend. This is a slightly confusing format. The authors should consider stating the scale bar for each sub-legend where a fluorescence image is taken.

Done.

** Referees cross-commenting**

After reading reviewer 2 and 3's comments, I think there are significant overlaps in the key points raised in terms of questions about fusion proteins and their potential partial mis-localisation, better descripton of results and target selection. Overall I think we agree that the work has potential, but in its current form does not represent a major advance. It would be immensely helpful if the manuscript would be carefully edited for a better flow and linear description of results.

We now rearranged the manuscript for better flow but would like to highlight that the many requests for smaller experimental issues (and "better description of results") worked somewhat in the opposite way of a more linear description. We hope the rearranged version acceptably balances these two issues. The issues raised in regards to target selection and potential partial mis-localisation are addressed in our responses mainly to this reviewer. Please also see comments on systems used at the end of the rebuttal.

Reviewer #1 (Significance (Required)):

The authors set out to test whether other proteins that are in the vicinity of K13 are involved in mediating ART resistance and endocytosis. This is an interesting question. However, other than MCA2 which was already known to be involved in mediating ART resistance (and was not tested for its involvement in endocytosis), none of their candidate proteins seem to be involved in mediating both these functions. The authors show that the other proteins tested appear important for parasite growth, with KIC12 and MyoF involved in mediating endocytosis. While these findings are novel, the KS approach used by the authors casts some doubt over the findings, and would mean that these findings would have to be re-tested with a more reliable approach, such as the GlmS system or generating a conditional knockout using the DiCre system. Despite not advancing our understanding of ART resistance, or identifying further players involved in this process, this manuscripts provides two candidates that are involved in mediating endocytosis and a further candidate that appears to be important for parasite growth. Further work on these proteins will be required to understand their exact roles. As stated above, there is currently limited interest for these results (limited to researchers working on endocytosis in apicomplexan parasites and possibly the wider endocytosis field from an evolutionary perspective), however with further work, this could increase the impact and interest of this work substantially.

The authors do not describe any novel methods/approaches within this work.

In the significance statement the reviewer indicates that other systems would have been more reliable for the work here. This is addressed in our response above and in a detailed considerations on the properties of conditional inactivation systems at the end of the rebuttal. The systems used in this work were not only chosen because they permit rapid targeting of many different proteins, but because they have merits that are beneficial for our assays. In fact many of the functional assays in this manuscript are difficult or impossible to carry with the suggested conditional inactivation systems (please note that we have extensive experience with the systems considered preferable:

• DiCre (Birnbaum et al., 2017; Mesén-Ramírez et al., 2019; Mesén-Ramírez et al., 2021; Wichers et al., 2022; Kimmel et al., 2023)

• glmS (Wichers et al., 2021c; Wichers et al., 2021a; Wichers et al., 2022; Wichers-Misterek et al., 2023)).

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

In a previous publication the Spielmann lab identified the molecular mechanism of ART resistance in P. falciparum by connecting reduced levels of the protein K13 to decreased endocytosis (uptake of hemoglobin from the RBC cytosol), which results in reduced ART susceptibility. Using quantitative BioID the authors further identified proteins belonging to a K13 compartment, highlighting an unusual endocytosis mechanism.

In the present manuscript the authors follow up on this work and closely examine ten more proteins of the K13/Eps15-related "proxiome". They successfully link MCA2 to ART resistance in vitro, while the proteins MyoF and KIC12 are involved in endocytosis but do not confer in vitro ART resistance when impaired. They further characterize one candidate (KIC11) that partially colocalizes with K13 in trophozoites but to a lesser degree in schizonts. Growth assays suggest an important function for KIC11 in late stages of the intraerythrocytic developmental cycle. Five analyzed proteins however do not colocalize with the K13 compartment, while a sixth was refractory to endogenous tagging.

Using AlphaFold predictions of the KIC protein structures the author identify domains in most constituents of the K13 compartment, highlighting vesicle trafficking-related features that were not identified on primary sequence level before.

The combination of functional data together with structure predictions leads them to propose a refinement of the K13 compartment as being divided into proteins participating in endocytosis and proteins that have an unknown function.

We thank the reviewer for the assessment of the manuscript and the constructive comments.

Major comments:

1) -Table 1 is missing

We apologise for this mistake; Table 1 is now included.

2) -Lines 117-123: Given the total list of uncharacterized candidates encompasses 13 proteins, can the author gives the reason why only the top 10 and not all 13 were characterized in this study?

A similar point has been raised by Reviewer 1 in major comment #12, please see our response there for an explanation why we chose which targets.

3) -Line 174: 20% of observed MCA2 foci show no overlap with K13 and 21% only partially overlap, can the author confirm that the observed MCA2 foci in schizonts are the ones that co-localize with K13. (Addition of a schizont stage image in Fig 1C would be sufficient).

We now extended Figure 4C with images of MCA2-Y1344STOP-GFP+mCherryK13 parasites covering the schizont and merozoite stage, showing that the majority of the MCA2 foci in schizonts are also mCherry-K13 positive.

4) -The localization and observed phenotype of KIC11 is interesting but unfortunately the authors do not explore it further. Does KIC11 localize with markers of e.g. the secretory organelles (micronemes or rhoptries) in schizonts and could therefore be involved in RBC invasion?

While we intended to focus mainly on the endocytosis aspect of these proteins, we see the reviewer's point and now generated new cell lines enabling assessment of spatial association of KIC11 with markers for rhoptry (ARO), micronemes (AMA1), and inner membrane complex (IMC1c). This revealed that the KIC11-GFP signal in schizonts does not overlap with apical organelle markers and the signal does not resemble a typical apical localization. In addition, we assessed all three organelle markers after inactivating KIC11 by knock sideways which showed that KIC11 inactivation has no apparent effect on the appearance of these markers, suggesting no major alterations in schizont morphology in respect to apical markers. These results are now presented as Figure S3A and in line 304 of the results.

5) Can the author distinguish if KIC11 is involved in RBC invasion or in establishment of the ring-stage parasite?

In order to look into this, we performed egress/invasion assays, quantifying schizont and ring stage parasites in tightly synchronized parasites at two different time points (pre-egress: 38-42 hpi & post-egress: 46-50 hpi). This revealed a significant decrease in newly formed ring stage parasite per ruptured schizont in parasites with inactivated KIC11, while the egress efficacy remained unaffected. This indicated an invasion or very early ring stage development defect (new Figure 2H, Figure S3G). To further determine at which point exactly the phenotype occurs (ie during invasion or early after invasion) would require extensive experimentation that goes beyond the scope of this study (e.g. invasion assays using video microscopy with a representative number of parasites or sophisticated flow based quantification assays). We hope by excluding egress and gross changes of apical organelles as well as no indication for similar number of early rings (indicating it is invasion or a very early ring-establishment phenotype) will sufficiently narrow down the phenotype for labs interested in invasion to more definitely answer this question.

Minor comments:

1) Table S1: Please add the criterion for the order of proteins (abundance in "proxiome"?) in the table as a separate column. I would also suggest adding a new column that highlights the 10 proteins investigated in this study as I found the color-coding slightly confusing.

Done as suggested: we now include the “average log2 Ratio normalized Kelch13” values from the four DiQ-BioID experiments performed with K13 in (Birnbaum et al., 2020), as well as the suggested column to highlight the investigated proteins. Please also see reviewer 1 major point # 12 for additional information on the selection criteria and how this was added to the manuscript.

2) -154-155: There is a discrepancy between the text and Fig1C regarding the % of partial overlapping and non-overlapping foci.

We thank the reviewer for pointing this out, this was corrected.

3) -The y-axis label is missing in Fig 3E

Done.

4) -Fig 4I left graph, the superscript 2 is missing in μm2

We thank the reviewer for pointing this out, this is now changed.

5) -Did the author colocalize KIC11 in schizonts with other proteins found in the K13 compartment group of proteins not involved in endocytosis/ART resistance? This may help to further subgroup these proteins.

This is an interesting point but would actually be technically challenging to do. For this we would need to generate a KIC11endo parasite line for each of these KICs and then do co-localisation in schizonts. However, the outcome of this likely would not be very clear. The reason for this is as follows. There are foci of KIC11 that do overlap with K13 in schizonts. One can expect that these foci show KIC11 at the K13 compartment and that the other KICs would overlap with KIC11 in these K13 foci in schizonts. Hence, we would also need to see K13 to find the non-K13 compartment KIC11 foci and see if these contained the KIC of interest. This is technically challenging because it would mean we would need a third fluorescent protein which is not that trivial to do. Due to the difficulty to do this and the large amount of work involved and the already considerable amount of data in this manuscript, we believe this will be better suited for a different study.

6) -As a general comment: to make the beautiful IFAs more accessible to a broader readership, I would encourage the authors to switch the color-coding to green/magenta/blue or an equivalent color system or add grayscale images.

This was done as suggested, all fluorescence images are now provided as greyscale images and the overlays are shown in magenta/green.

Reviewer #2 (Significance (Required)):

Characterizing the molecular components involved in Plasmodium endocytosis will not only reveal interesting biology in these highly adapted parasites, but will more importantly lead to a better understanding and potentially open new avenues for intervention of ART resistance. The here presented manuscript is a carefully executed follow-up on previous work done in Dr. Spielmann's lab focusing on the K13 compartment. The authors use established assays to characterize novel components and reveal three new players in endocytosis with one mediating ART resistance in vitro. The proposition that parts of the K13 compartment have a function other than endocytosis is interesting, but will have to await more data from future studies. Taken together this manuscript adds significantly to our understanding of endocytosis in P. falciparum.

This work is of interest for cell and molecular biologists working on Apicomplexa, but especially for the Plasmodium community.

We thank the reviewer for this positive assessment.

I am a cell and molecular biologist working on Toxoplasma gondii

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

Summary: The authors characterized 4 proteins from P. falciparum via cellular (co-)localization, endocytosis, parasite growth, and artemisinin resistance assays. These proteins have been identified as candidates for Kelch13 compartment and a possible role in endocytosis in their previously work with quantitative BioID for potential proximity to K13 and Eps15 (Birnbaum et al. 2020). In the current work, additional 6 proteins were not confirmed as being associated to the K13 compartment. This experimental work was complemented by an in-silico analysis of protein domains based on AlphaFold algorithm. For this protein structure evaluation all proteins were chosen, which were experimentally confirmed to be linked to the K13 compartment in the current publication and previous work. With the work 3 novel proteins linked to artemisinin resistance or endocytosis could be functionally described (KIC12, MCA2, and MyoF) and a number of hypotheses were generated.

We thank the reviewer for the assessment of the manuscript and the constructive comments.

Major comments:

The quality of the presented work is solid, the experimental design is adequate, and methods are presented clearly. The publication contains a lot of results both presented in text and in the figures and it is not always straight forward for the reader to follow the descriptions due to many details presented and a lack of context for some of these experiments.

We thank the reviewer for this overall positive assessment.

We now reordered the results section in an attempt to increase the flow of the manuscript. We also made changes to improve the context for the results. Given the further (very valid) requests for data on schizonts and invasion, there was an increased danger for a less linear manuscript that we hope to have acceptably managed with the re-arrange.

Specific suggestions for consideration by the authors to improve the manuscript. Abstract: 1) R 31: Mention how the 4 proteins were identified as candidates, you need to refer to previous work to clarify this

To clarify this the sentence was changed to (line 31): "Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site."

2) R38: "Second group of proteins" is confusing - different from the 4 mentioned above? Significance to endocytosis unclear. Please unify terminology in the manuscript, see also comment below on proxiome.

We changed the wording to clarify the group issue in the abstract as follows line 34: "Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these and previously identified proteins showed that canonical vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins, While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process. Another group of K13 compartment proteins did not influence endocytosis or ART susceptibility and lacked detectable vesicle trafficking domains. We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion.”

3) Abstract can only be understood after reading the full publication

We attempted to amend this by expanding the abstract, particularly the changes highlighted in the previous two points.

Results: 4) Table 1 is missing from the submitted materials

We apologise for this mistake. Table 1 is now included.

5) Consider to shorten and stratify the result section to focus on the significant data

We rearranged the results in an attempt to streamline this section and are now starting with MyoF in the revised manuscript. However, as highlighted by the requests from reviewer 1, many details need to be available to support our conclusions. For instance the fact that GFP-tagging partially inactivated MyoF asked for further data to support our conclusion (HA-tagged version, showing that the location of the GFP-tagged version was consistent with the HA-tagged version, showing to what extent the different constructs affected growth and correlated with number of vesicles and bloating, see new figure 1M) or that KIC12 has two locations. Overall, we are therefore hesitant to remove data or description from the result part.

6) Unclear how the localization and functionalization assays might be impaired by the fusion proteins Significance of ART resistance assay is not clear, in presence of strong growth effects due to inactivation or truncation of genes/proteins

As indicated also in the example given in the previous point (this reviewer #5), the use of different cell lines (GFP-tagged live cells and small epitope tag in IFA) for targets with an indication for an effect of the tagging confirm that the location we assigned is reasonable. In the case of MyoF, the HA-tagged line, the partial inactivation due to GFP and the further inactivation in the GFP-tagged line by knock sideways show plausible increase of phenotypes (vesicle accumulation and bloated FV assays). Thereby the GFP-tagged line can be seen as a partial inactivation line that further supports our conclusions and overall this paints a consistent picture of the function of this protein in endocytosis (see new Figure 1M better illustrating this). Please note that the difference in location shown by this line compared to the HA-tagged proteins is only small (see also reviewer 1 major point 23ff). See also general discussion on tags at the end of this rebuttal.

Significance of ART resistance assay: The ‘ART resistance assay’ is done comparing +/- ART (DHA) in identical parasites (originating from the same culture and the same condition). Hence, any growth effects are cancelled out and effects in reducing ART susceptibility would - if at all - be underestimated (see more detailed response to point 28, reviewer 1 and controls in Birnbaum et al., 2020 where we tested an unrelated essential protein, unrelated chemical insult and rapalog on 3D7 and did not detect any effect on RSA survival).

MCA 7) Stratify results, order by significance of findings, it appears to be described in chronological order, improve readability/flow, eg ART resistance if mentioned in r138, but only reported in r183ff

We attempted to stratify, but then the reason for generating the partial MCA2 disruption parasite line becomes very arbitrary and would leave the reader wondering why we at all truncated the protein at two thirds of the protein. Hence, we do not see a way around this chronological reporting. However, this part is now not at the start of the experimental results section anymore, possibly making it overall a bit more palatable.

MyoF 8) R195 to 197 - consider moving to discussion as it is distracting here

This was shortened and additional information (asked for by reviewer 1, major point 22) to clarify that MyoF was previously called MyoC, was added (line 147): “The presence of MyosinF (MyoF; PF3D7_1329100 previously also MyoC), in the K13 proxiome could indicate an involvement of actin/myosin in endocytosis in malaria parasites. "

9) Term proxiome is introduced above, but not used in result section - suggest to unify language, eg r195 uses "K13 compartment DiQ-BioIDs" instead, which is not very convenient for the reader

We carefully reviewed this and made this more consistent.

10) What is the enrichment factor? Please provide for this and the following proteins, eg in Table 1

The enrichment factor is log2 enrichment over control and this is now provided in table S1 (see also detailed answer for Reviewer 1 major point 12).

11) R225 to 243 - overall significance of the growth experiments with mislocaliser is not clear, consider removing from manuscript or explain relevance more clearly

See also point 28, reviewer 1: This experiment is actually quite important. It shows that if we conditionally inactivate the GFP-tagged MyoF, the growth is further reduced, as stated in line 208. It might have been confusing that the mislocalisation is only partial, but this is equivalent to a partial knock down and hence is useful. This becomes even more relevant with the specific assays following in the next paragraph: while the tagging of MyoF already resulted in vesicles, conditional inactivation with KS generated even more vesicles, showing that the same phenotype was rapidly increased when MyoF was further inactivated by a different means and this also correlated with growth. Hence, this is actually a very consistent phenotype that despite some shortcomings of the tools available to analyse this protein (due to the partial inactivation by the GFP tag) in our eyes looks very convincing. We now added a graph showing the correlation of growth and phenotypes to illustrate this (Figure 1L).

We also tried to make this clearer by changing line 200 to: “Hence, conditional inactivation of MyoF further reduced growth despite the fact that the tag on MyoF already led to a substantial growth defect, indicating an important role for MyoF during asexual blood stage development.” And line 208 to:“ This was even more pronounced upon conditional inactivation of MyoF by KS (Figure 1H), suggesting this is due to a reduced function of MyoF.”

12) KIC11/KIC12 Enrichment factor?

The enrichment (’average log2 Ratio normalized Kelch13 from Birnbaum et al. 2020’) is 1.65 for KIC11 and 1.32 for KIC12, which is now also explicitly shown in column D of Table S1.

** Referees cross-commenting**

I would like to applaud reviewer #1 for a great, very thorough review and lots of detailed suggestions. I agree with the conclusions mentioned in the significance evaluation from reviewer #1 and #2: the work presented does not contain novel methods and the scope is rather narrow with the current results. (I am working on clinical studies with novel antimalarial agents)

Reviewer #3 (Significance (Required)):

On the one hand side, the authors have wrapped up some of the remaining protein candidates of the K13 compartment and could verify 4 of 10 proteins. The work is of interest for the scientific community working on endocytosis and malaria drug resistance mechanisms. Overall, the conclusions and findings from the previous work, Birnbaum et al. 2020, could be confirmed and extended mainly using the methods previously described. On the other hand, the authors made use of progress in protein structure predictions and identified domains linking the K13 compartment proteins to putative functions. The overlaid protein folds of the newly identified domains in figure 5 look convincing, but I can't comment on the technical details or cut-off used for this in-silico analysis.

Extended general remarks on the systems used for this work:

Mainly reviewer 1 suggest (in the general comments and the significance statement) that other systems would have been better suited to use for this work, namely glmS and diCre and also has concerns about the large tag which is seconded by a comment of reviewer 3. In light of this we here provide some extended considerations on the properties for conditional systems and tagging in regards to the goals of this work.

We would like to point out that we do have experience with the systems considered better-suited by the reviewer (one of the first authors has extensively used glmS (Wichers et al., 2021c; Wichers et al., 2021a; Wichers et al., 2022; Wichers-Misterek et al., 2023) and our lab was one of the first to adopt the diCre system in P. falciparum parasites and we regularly us it (Birnbaum et al., 2017; Mesén-Ramírez et al., 2019; Kimmel et al., 2023)). Clearly, these methods have a lot of strengths but there are a number of issues to be considered for the assays we use in this work (see the next section on conditional inactivation systems). In a nutshell, we believe diCre would give a more reliable readout of the absolute level of "essentiality" (i.e. importance for growth) but is unsuitable or at least difficult to use for the assays that reveal the function of our interest in this work. GlmS basically combines the drawbacks of diCre and knock sideways and hence for most targets is not expected to give a better readout of level of "essentiality" but is similarly difficult to use for our specific assays. The fact that both of these systems are possible to use without adding a tag to the target may be an advantage but without tag one loses some very important features that can be critical to understand the outcome with a given system (see considerations on the tag further below).

Conditional inactivation systems:

1. __ speed of inactivation:__ glms acts on mRNA and diCre on the gene level, which makes them slower than techniques acting directly on the protein such as DD or KS. With diCre, mRNA and protein is still left, even if the gene is very rapidly excised. For instance for Kelch13 it takes 3-4 days after excising the gene until protein levels have waned enough that this manifests in a reduced growth (Birnbaum et al., 2017). While in some instances diCre permits same cycle analyses if the protein has a very rapid turn-over (e.g. Rab5a, (Birnbaum et al., 2017)), control in a few hours is still difficult. For vesicle accumulation and bloated food vacuole assays, which are done over comparably short time frames and with specific stages, it is rather challenging to hit the correct time of induction to have all the cells at the correct stage with suitably (and uniformly, ie all cells) sufficiently reduced target protein levels during the assay time. Slow acting systems are also more prone to secondary effects. The more immediate the inactivation, the closer it is to the core of the affected function. With vesicle trafficking processes this is particularly relevant as all vesicle trafficking in a cell is interconnected and there are always recycling pathways that maintain the membrane and protein homeostasis of individual compartments. Particularly for endocytosis there seem to be compensatory capacities at least in other organisms (see e.g. (Chen and Schmid, 2020)). One reason why knock sideways was developed is that it permitted to avoid compensatory changes when vesicle adaptors are inactivated (Robinson et al., 2010).

The comparably short time frame for malaria parasites to go through different stages during blood stage development also is an issue relevant for inactivation speed. The advantage of speed and the danger of obscured phenotypes is highlighted by our work on VPS45 which showed that in trophozoites this protein is involved in the transport of hemoglobin to the FV whereas in late stages it also has a role in secretory processes. Both of these functions we were able to specifically assess in the same growth cycle using KS to rapidly inactivate the protein (Bisio et al., 2020) but with a slower system would have been more complicated to dissect.

Speed of effect with glmS: unless the KS does not work well, glmS is slower acting than KS (it does not target the already synthesised protein which can remain in the cell) and also often suffers from only partial inactivation, hence the benefit of using it here is unclear. The option to have an untagged protein is a plus, however it also is a minus, as assessing efficiency (particularly in live cells e.g. for bloated assays etc a fluorescent tag is the only direct option to assess inactivation of target) is critical to ensure the phenotype manifests at the stage of interest.

lethality/absolute phenotypic effects are detrimental to some assays to study the functions we are interested in for this work: no RSA can be conducted, if the gene is lost and the parasites die. Again, with diCre, one could attempt to hit the point when the parasites have lost sufficient amounts of the target protein when they are placed under ART but then the parasites need to continue growing for ~3 days, which is not possible if the cKO is lethal except for very slowly turning over proteins. However, in that latter case, the parasites likely still had full functionality of the target protein at the beginning of the RSA, when the drug pulse happens and there would be no effect. Knock sideways solves these problems by permitting knock sideways inactivation only under ART (or with a few hours pre-incubation depending on the inactivation speed) to not yet affect growth in a severe manner but inhibiting the process the protein is involved in. It may be possible to use glmS for RSAs, but the slow speed would complicate it (it would not permit control of target protein levels in a matter of a few hours to inactivate the target protein and then re-install it).

None-absolute inactivation is also a strength for some functional assays. While we really like using diCre, in the case of EXP1 it made it necessary to complement the exp1 cKO parasites with low levels of EXP1 to be able to do functional assays without killing the parasites (Mesén-Ramírez et al., 2019; Mesén-Ramírez et al., 2021). While the lethality issue does not apply to glmS (like knock sideways, it also can be tuned), it is unclear what would be gained over knock sideways. Knockdown levels with glmS vary from gene to gene and cannot be predicted, it is in most cases considerably slower than KS, it requires glucosamine which becomes toxic at higher concentrations and might introduce off target effects and tracking protein levels during the assay would equally need GFP tagging.

Integration of properties of conditional systems

Given the above discussed properties, several factors have to be considered to be able to use a system for a given assay. Stage-specific transcription is one example. For diCre a protein not expressed in e.g. rings permits to remove the gene and the protein is never made in that parasite development cycle. We exploited this for instance for two proteins only expressed from the trophozoite stage onwards (Kimmel et al., 2023). However, if lethal (absolute effect problem), this also means one can also only see the phenotype on onset of expression of the target (e.g. if in mitosis, the first nuclear division in case the protein is absolutely essential for the process). This is just one example of such issues. Expression timing, turnover of the protein and homogeneity of stage-specific loss of protein will all influence how clearly the phenotype can be determined. All this will decide the exact time of loss/inactivation of the target protein to levels generating a phenotype and ideally therefore can be monitored during an assay (see considerations on tagging).

For these reasons vesicle accumulation or bloated food vacuole assays are difficult with slow systems as ideally the target should rapidly be inactivated at the trophozoite stage and the result monitored before the cells have moved to the schizont stage. For this a well responding knock sideways is ideal as the protein can be rapidly taken away (sometimes within seconds) to visualise the immediate, direct effect in the cell.

As shown for KIC11, there is also no disadvantage of using KS for proteins with other assays or proteins that result in different phenotypes. It permits stage-specific same cycle inactivation without having to worry about the turnover of mRNA and protein (Fig. 2F,G). Thus, besides the advantages of knock sideways for endocytosis related assays and RSAs, we also see no disadvantage of using knock sideways for the functional study of KIC11 which has a role other than endocytosis. KS also permits to specifically target the K13 pool of KIC12, something impossible or very difficult to do with other systems. Hence, we are of the opinion that the system for inactivation was adequate for most of the proteins analysed in this manuscript.

Large tag: we agree that GFP-tagging can be a disadvantage but in our opinion its benefits often outweigh the drawbacks because it permits easy and immediate (on individual cell level, if need be) monitoring of the presence/location of the target protein (e.g. after KS, but given the discrepancy of the timing between gene excision and protein loss, it might be even more important for techniques such as diCre). No fixing/permeabilisation (prone to artifacts, prevents immediate view of cells) to detect a target with specific antibodies or via a small tag is needed with GFP. Similarly, the use of Western blots to do this is time consuming and impractical if monitoring of left-over protein in the course of an assay such as a bloated food vacuole assay is needed.

In many cases, adding GFP has no negative effect. In addition, if the bulky folded structure of GFP is tolerated, it usually also tolerates the 2 to 4 12kDa FKBP domains in our standard tag. We also typically add a linker. This approach has worked for a large number of different proteins, including many essential ones for which we would not otherwise have obtained the integration cell lines (Birnbaum et al., 2017; Jonscher et al., 2019; Hoeijmakers et al., 2019; Birnbaum et al., 2020; Kimmel et al., 2023; Sabitzki et al., 2023). Hence, whenever a cell line is obtained with it, this tag in most cases is not a disadvantage. Admittedly an exception in this is MyoF and to some extent maybe MCA2 (we would like to stress that in the case of MCA2 the reason for not being able to obtain the full length tagged cell line is unclear: the protein can be severely truncated to less than 3% of its amino acid sequence and a GFP-tag is tolerated on the version with 2/3s of the protein left, which gives no good reason why the full length was not obtained; a potential reason could be a dominant negative effect). However, we obtained the full length with a small tag detected by IFA for both, MyoF and MCA2 and the location of these agreed well with the GFP tagged versions, indicating that the GFP-tagged versions are useful to show the location of these proteins in live cells.

There are also tricks to attempt monitoring the effect of e.g. diCre without tagging the target. For instance, if a fluorescent protein is connected to excision without actually being fused to the target (ie excision of the gene leads to its expression of e.g. GFP), which would avoid adding a tag to the target itself. However, the problem with this is that expression of GFP does only show excision, but mRNA producing the target protein and left over target protein may still be there in the cell. All in all, the GFP-tag on the target, while with some drawbacks, is still our preferred method to control to monitor the target protein in the cell (in principle permitting quantification of ablation efficiency on the individual cell level).

Conclusion on these considerations for this manuscript

Based on these considerations we do not see the immediate benefit of changing the system for the conclusions drawn from this study and are unsure if they are indeed better suited for this work as suggested. While a more exact readout of "essentiality" might be possible with the diCre system we are of the opinion this is less important than learning the function of a protein which - as outlined above - we believe to be considerably more difficult with diCre and even more so with glmS considering our target functions. The same applies to target specific cellular pools of a protein as done here for KIC12. Clearly MyoF is one example where the employed systems shows limitations, but with the new Figure part showing consistency in phenotype with degree of inactivation (importantly with two different forms of inactivation) and the clarification that the location of the GFP-tagged and HA-tagged versions are actually quite similar in location, we do not think employing an extra system is warranted for the conclusions of this work. Admittedly, the apparent lack of need in ring stags might give an opening to attack MyoF using diCre (by excision before its major expression peak), but depending on lethality this might preclude extended analyses (possibly vesicle assays, for sure not RSAs).

In the end the question is, if our approach provides the function of target analysed in this work and based on the data in our manuscript and the arguments in the rebuttal, we are reasonably confident that this is the case. It is not very likely the other mentioned techniques would result in a different conclusion on the function of the here studied proteins. In fact, we expect other commonly used techniques to be less suitable for the key assays in this work.

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Se pueden poner comentarios publicos o privados.

Tienen que crear una cuenta en Hypothesis.

En la proxima leccion vemos como.

O Python não arredonda números, ele apenas ignora o que está à direita do ponto flutuante.

Outra dica muito importante. Colocar linhas vazias, que são ignoradas pelo interpretador, e comentários ajudam qualquer pessoa a analisar o código. "Use os indiscriminadamente".

Una enfermedad insidiosa o gradual se refiere a cualquier enfermedad que comienza lentamente, y que no tiene síntomas obvios al principio. La persona no está consciente de que la enfermedad se está presentando.

Muito legal

foobar

se sopra i mutati erano arancioni dovrebbero esserlo anche qua, o viceversa :) Inoltre dovremmo mantenere l'ordine di raw e normalized, metterei sempre prima raw

• quote
  • power corrupts, absolute power corrupts absolutely
• Author
  • Lord Acton
• Context
  • Lord Acton coined the term to whiitewash the brutality o Spanish Inquisition

Before taking this assertion and then structuring ALL learning with this model, I'd want to explore who and why some people learn w/o examples or demonstration...

Author Response

Reviewer #1 (Public Review):

This study uses electrophysiological techniques in vitro to address the role of the Na+ leak channel NALCN in various physiological functions in cartwheel interneurons of the dorsal cochlear nucleus. Comparing wild type and glycinergic neuron-specific knockout mice for NALCN, the authors show that these channels 1) are required for spontaneous firing, 2) are modulated by noradrenaline (NA, via alpha2 receptors) and GABA (through GABAB receptors), 3) how the modulation by NA enhances IPSCs in these neurons.

This work builds on previous results from the Trussell's lab in terms of the physiology of cartwheel cells, and from other labs in terms of the role of NALCN channels, that have been characterized in more and more brain areas somewhat recently; for this reason, this study could be of interest for researchers that work in other preparations as well. The general conclusions are strongly supported by results that are clearly and elegantly presented.

I have a few comments that, in my opinion, might help clarify some aspects of the manuscript.

1) It is mentioned throughout the manuscript, including the abstract, that the results suggest a closed apposition of NALCN channels and alpha2 and GABAB receptors. From what I understand, this conclusion comes from the fact that GABAB receptors activate GIRK channels through a membrane-delimited mechanism. Is it possible that these receptors converge on other effectors, for example adenylate cyclase (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374141/).

It will be of interest to test the roles of adenylyl cyclase modulation in the control of NALCN, as a complement to the studies we have presented here.

2) In Figure 2G, the neurons from NALCN KO mice appear to reach a significantly higher frequency than those from WT (figure 2E, 110 vs. 70 spikes/s). Was this higher frequency a feature of all experiments? The results mention a rundown of peak firing rate due to whole-cell dialysis, but, from what I understand, the control conditions should be similar for all experiments.

The peak firing rates in control solutions for WT and KO CWC are not statistically different.

3) Also in Figure 2, the firing patterns for neurons from WT and NALCN KO mice appear to be quite different, with spikes appearing to be generated during the hyperpolarization of the bursts in the second half of the current step for WT neurons but always during the depolarization in KO neurons. Was this always the case? If so, could NALCN channels be involved in this type of firing? Along these lines, it would be interesting to show an example of a firing pattern of neurons from WT mice in the presence of NA, which inhibits NALCN channels.

The specific pattern of spikes in CWC is quite variable from trial-to-trial or cell-to-cell, as it is dependent on multiple CaV and calcium dependent K channels subtypes, and is not dependent on the genotypes used here. The primary effects observed in the KO are in background firing and sensitivity to NA, both reflected alterations in rheobase. The firing pattern example requested was shown in the raster plot of fig 2B2.

4) It might be interesting to discuss how the hyperpolarization induced by the activation of GIRK channels and inhibition of NALCN channels could have different consequences due to their opposite effect on the input resistance.

We considered this as a point of discussion, but decided that making sense of it would depend on assumptions about the location of the channels (dendritic vs somatic, distance to AIS) that we do not have data for. For example, a dendritic increase in resistance through NALCN block, leading to a hyperpolarization of the soma, might have actions similar to a somatic hyperpolarizing conductance increase by GIRK, as far as the voltage at the AIS is concerned.

Reviewer #3 (Public Review):

The study by Ngodup and colleagues describes the contribution of sodium leak NALCN conductance on the effects of noradrenaline on cartwheel interneurons of the DCN. The manuscript is very well-written and the experiments are well-controlled. The scope of the study is of high biological relevance and recapitulates a primary finding of the Khaliq lab (Philippart et al., eLife, 2018) in ventral midbrain dopamine neurons, that Gi/o-coupled receptors inhibit NALCN current to reduce neuronal excitability. Together these studies provide unequivocable evidence for NALCN as a downstream target of these receptors. There are no major concerns. I have only minor suggestions:

Minor

1) As introduced in the introduction, NALCN is inhibited by extracellular calcium which has led to some discourse of the relevance of NALCN when recorded in 0.1 mM calcium. A strength of this study is the effect of NA on NALCN is recorded in physiological levels of calcium (1.2 mM). I suggest including the concentration of extracellular calcium in the aCSF in the Results section instead of relying on the reader to look to the Methods.

Will do.

2) It would be interesting to include the basal membrane properties of the KO compared to wildtype, including membrane resistance and resting membrane potential. From the example recording in Figure 2, one might think that the KOs have lower membrane resistance, so it is interesting that the 2 mV hyperpolarization produced similar effects on rheobase. In addition, from the example in Figure 2G, it appears that NA has an effect on firing frequency with large current injection in the KO. Is this true in grouped data and if so, is there any speculation into how this occurs?

Will do.

3) Please expand on the rationale for why GABAB and alpha2 must be physically close to NALCN. To my knowledge, the mechanism by which these receptors inhibit NALCN is not known. Must it be membrane-delimited?

Given the known membrane delimited modulation of GIRK by GABAB, and that alpha2 and GABAB receptors appear to share the same population of NALCN channels, and that alpha2 receptors do not appear to target GIRK channels, we felt the simplest explanation would be coupling through G-proteins, with spatial segregation of different receptor/channel pools providing the means for separating GIRK and NALCN effects. However, the involvement of an additional second messenger is testable.

Reviewer #3 (Public Review):

The study by Ngodup and colleagues describes the contribution of sodium leak NALCN conductance on the effects of noradrenaline on cartwheel interneurons of the DCN. The manuscript is very well-written and the experiments are well-controlled. The scope of the study is of high biological relevance and recapitulates a primary finding of the Khaliq lab (Philippart et al., eLife, 2018) in ventral midbrain dopamine neurons, that Gi/o-coupled receptors inhibit NALCN current to reduce neuronal excitability. Together these studies provide unequivocable evidence for NALCN as a downstream target of these receptors. There are no major concerns. I have only minor suggestions:

Minor<br /> 1. As introduced in the introduction, NALCN is inhibited by extracellular calcium which has led to some discourse of the relevance of NALCN when recorded in 0.1 mM calcium. A strength of this study is the effect of NA on NALCN is recorded in physiological levels of calcium (1.2 mM). I suggest including the concentration of extracellular calcium in the aCSF in the Results section instead of relying on the reader to look to the Methods.

2. It would be interesting to include the basal membrane properties of the KO compared to wildtype, including membrane resistance and resting membrane potential. From the example recording in Figure 2, one might think that the KOs have lower membrane resistance, so it is interesting that the 2 mV hyperpolarization produced similar effects on rheobase. In addition, from the example in Figure 2G, it appears that NA has an effect on firing frequency with large current injection in the KO. Is this true in grouped data and if so, is there any speculation into how this occurs?

3. Please expand on the rationale for why GABAB and alpha2 must be physically close to NALCN. To my knowledge, the mechanism by which these receptors inhibit NALCN is not known. Must it be membrane-delimited?

es una teoría cientifíca que los mciroorganismos son causantes de una amaplia gama de enfermedes

O logo do Inspira está cortado?

DOI: 10.1016/j.xcrm.2023.101110

Resource: (Bethyl Cat# A304-328A, RRID:AB_2620524)

Curator: @olekpark

SciCrunch record: RRID:AB_2620524

What is this?

DOI: 10.1016/j.immuni.2023.06.020

Resource: (R and D Systems Cat# AF457, RRID:AB_2072083)

Curator: @scibot

SciCrunch record: RRID:AB_2072083

What is this?

first occurence : selection

This process is simple selection: i t proceeds by examining in turn every one of a large set of irems and by picking out those which have certain specified characterist~cs.Therc 1s another form of sclcction bcsr dlusrratrd by thc aururnaric relephone exchange. You dial a number and the machine selects and connects just one of a million poss~blesta'tions. I t does not run over them all. Ir pays attention only to a class givcn by a first digit, then only to a suhclars of this given by the second d i g ~ t ,and so on; and thus proceeds rapidly and almost ~lncrringlyto the selected station. I t requires a few seconds t o make the selection, although the process could hc speeded up if in- creased speed were economically warranred

O shell mode é uma forma onde o interpretador fornece rapidamente o resultado das expressões digitadas. Essa é uma forma de avaliar a qualidade e funcionalidade do código na medida em que se digita. É como um rascunho de teste. O shell mode pode ser feito com facilidade no colab sem a necesidade de ">>>".

O interpretador transforma o código de alto nível em código de baixo nível linha por linha na medida em que vai executando. Já o compilador primeiro lê todo o código de alto nível, para em seguida criar um código objeto (baixo nível) que será executado pela máquina.

"º" degrees

"º" missing :P

en fait, il n'existe que 2 type de 'channel' (des destination pour les fichiers de backup) : 1. disque 2. ruban magnétique

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

We would like to thank reviewers for their insightful comments.

Overall, there were two major concerns/suggestions:

• Applicability to humans of the increase of BTC in non-alcoholic steatohepatitis (NASH) and mechanisms of downregulation of BTC by omega-3. We now analyzed __3 __additional human gene expression datasets and show that BTC not only is increased in human NASH (as we have already shown for liver cancer meta-analysis), but is also decreased in livers of patients who received omega-3.

• One of the reviewers suggested investigating a potential mechanism of how BTC is regulated by omega3 fatty acids. Although a complete answer to this question would require entirely new studies to be done, we still performed additional investigation that was possible within a reasonable timeframe. We found that transcription factor FOXO3 (well-known inhibitor of carcinogenesis) is a highly probable mediator of the DHA inhibitory effect on BTC.

See all details of items 1 and 2 as well as answers to other (less critical concerns) below after each specific question.

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

This work by Padiadpu and colleagues investigate the mechanism by which pufa of the n-3 series (mostly DHA) may influence NAFLD progression using systems biology analysis and multiple omics analysis. The work is interesting and may provide a novel view of the topic. However, there are a number of issues the authors may wish to consider in order to improve their manuscript.

Major issues: Clarity: Since the authors refer to previously published experiments, they must refer to this work in the figure legends and improve the clarity of such legends. Here are a list of issues that must be fixed:

Fig.1: First panel is not clear. What does the table tell the reader? What are the effects of the different diets on NAFLD?

All the transcriptomic data are newly generated from the samples of previously published studies. The table shows the number of features changed by DHA and/or EPA in each of the -omics and phenotypic data used in the analysis.

I understand that the results are published elsewhere, but the authors must provide information regarding the NAFLD/ NASH scores.

We now added a supplementary table 1a showing the scores.

Fig.4: Why is there sometimes a DHA diet, sometimes DHA and EPA. Legend is not clear. What does WD + Mean? I guess it is olive oil... But the legend must be improved.

We added details in the legend for more clarity. Specifically, WD+O means WD + olive oil added as a control for WD+DHA, WD+EPA. As described in the 2nd paragraph of results, when both EPA and DHA had a similar and significant effects in reversing WD effect, it was defined as “EPA&DHA category” of parameters. When only WD+DHA or WD+EPA were significantly changed vs WD+O, those were assigned as “DHA category” or “EPA category”, respectively.

One issue the authors may consider trying to fix is the specificity of the effect of DHA on BTC.

Is it really specific? It seems to me that EPA has more or less the same effect. If the effect is DHA-specific, than make this clearer through the text.

Although BTC expression was reduced by both DHA and EPA comparing to WD, DHA had a statistically significant stronger effect than EPA (Fig. 3D).

Another issue the authors may wish to investigate is the relationship between W3 consumption and BTC expression in studies performed by other labs (if available on Gene expression omnibus?).

Thanks for the suggestion. We used publicly available data of human and mouse studies that showed significant increase in liver BTC gene expression in NASH in multiple datasets while a human trial with Omega 3 treatment for one year showed its significant reduction (Figures 3F - human data, S3G-mouse data).

Finally, a key issue would be to identify the mechanism by which DHA inhibits BTC expression? How does this happen? could such inhibition be induced by other fatty acids of the W3 series? I understand that this is not easy to address but it would significantly strengthen the manuscript.

Thanks to your question we investigated and found at least one of potential mechanisms contributing to how “DHA inhibits BTC expression”. See details in the answer to next question. As for “other fatty acids” while we agree this is important question, it is outside of the scope of the current study but will be investigated in future studies.

Moreover, it might be possible to identify the set of genes highly co-regulated with BTC expression and to investigate the possible transcription factors at play in the control of such gene set.

We really appreciate this question as our efforts in this direction provided one potential mechanism. A direct screen of transcription factor (TF) motifs in genes co-regulated with BTC did not provide any clear results. Therefore, we implemented a combination of network analysis and screen for motifs in BTC gene with the in vivo and in vitro treatment results and found FOXO3 as a candidate TF regulated by DHA upstream of BTC.

See details of the analysis and results in a new Supplementary Figure S6 and corresponding text located at the end of the results.

Minor: the authors use the term "beneficial" transcriptome alterations by DHA.

I do not think it is correct to use "beneficial".

We agree and removed the word "beneficial”.

Reviewer #1 (Significance (Required)):

Strength: This paper uses new approaches to investigate the relationship between W3 consumption and liver gene expression and its relevance to chronic metabolic liver diseases.

The experiments and data set used to perform systems biology are from an excellent lab (the authors lab) who has published a lot of important and reproducible discoveries in the field of regulation of gene expression by dietary fatty acids.

The work has high translational relevance in medicine / hepatology / metabolism.

I am not a qualified reviewer to assess the systems biology that has been done.

Limitation: The mechanistic link between DHA consumption and BTC expression is not very clear. The specificity of this effect could also be tested (DHA vs other W3 and/or W6).

Although BTC expression was reduced by both DHA and EPA comparing to WD, DHA had a significantly stronger effect than EPA (Fig. 3D). Other omega fatty acids were not tested but it can be done in future studies.

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

The authors files a manuscript describing the impact of the suppression of betacellulin as a key mechanism to counteract fibrosis and inflammation in NASH by modulating fatty acids in WD-fed mice.

Major Comments: (i) No histological analysis was presented and indeed this is of clinical relevance for NASH since diagnosis is still based on biopsy.

While histological evaluation was presented in the originally published papers (PMID: 28422962, 23303872), it is now provided in Supplementary Table S1a.

(ii) Human comparative analysis: is done with HCC not with NASH patients.

This cancer-related dataset is most likely obtained from different etiologies.

I would suggest comparing these mouse datasets with GSE48452 (human NAFLD-NASH spectra).

Thanks for this important question. We now analyzed available human data of NASH and show significant increase of BTC expression in two datasets while a human trial with omega-3 treatment for one year showed its significant reduction of BTC expression (Figure 3F) resembling our observations in mice.

(iii) to compare the inflammation and fibrosis (also lipid metabolism), one can compare these mouse datasets with GSE222576 and cite this preprint (https://doi.org/10.21203/rs.3.rs-2009380/v1)

Using the suggested dataset (of a chemically induced liver fibrosis), we first observed that Btc gene expression was significantly increased over 10 weeks of the model and now included this result in Fig. S3G.

We also queried the 66 genes from the network modules described by the authors to check their changes in our NASH model. We observed that 28 genes were differentially expressed in NASH with 14 of them belonging to the module that authors named as “Pathways in Cancer”. Other genes were from the lipid metabolism (4 genes), immunity (2) and inflammation (2 genes). In addition, we observed that several genes we found regulated by omega-3 and changed in this fibrosis model contained other inflammatory genes such as classical macrophage genes (Mmp12, Lgals3, Cd68, Trem2), fibrosis (Col4a1, Col27a1, Itga2b, Itga8) and lipid metabolism (Scd2, Lpl, Soat1). Of note, the preprint has been published and we now cite the corresponding article.

Minor comments:

(i) The heatmap in Figure 1B and another heatmap should show all mice not the average to see the variability

The supplementary figure with all the individual mouse data as another heatmap is added to show the variability and similarity (Figure S1D).

Reviewer #2 (Significance (Required)): The authors files a manuscript describing the impact of the suppression of betacellulin as a key mechanism to counteract fibrosis and inflammation in NASH by modulating fatty acids.

This is well designed experiment, and the results are of interest to hepatologists and should be indeed published after consideration of the following points

Strength is multiOMICs approach.

Weakness is human applicability.

We improved human applicability by investigating 3 additional human datasets of NASH (Fig. 3F) and finding consistent changes in BTC expression closely resembling our observations in mouse NASH model, including one trial with omega-3 treatment of patients for one year showing significant reduction in BTC gene expression.

1 u-turn, nhưng mà không được lâu

thế kỉ 13 để xuất phân đoạn thời gian cho quá trình phát triển phôi thai

thế kỉ 5 vẫn vậy

vẫn thế, không có gì khác biệt

S - A class should have a single responsibility

O - Classes should be open for extension, but closed for modification

L - Objects of a superclass should be replaceable with objects of its subclass without affecting the correctness of the program

I - Clients should not be forced to depend on methods that they do not use.

D -  High-level modules should not depend on low-level modules. Both should depend on the abstraction. Abstractions should not depend on details. Details should depend on abstractions.

Factor de preconcentración

Çoğu IP araması, tek noktaya yayın yönlendirme şemasını kullanır. DNS, bir URL'yi sizi belirli bir sunucuya götüren bir IP adresine çözümler. Ancak Deno Deploy ve Cloudflare , bir IP adresinin bir bilgisayar havuzuyla eşlendiği herhangi bir yayını kullanır. Ağ (en azından bir WAN'da, yani internette) daha sonra adresi en yakın bilgisayara çözer. Genel olarak, bir istemci bir uç çalışandan bir uç işlevi aracılığıyla veya konuşlandırılmış bir kod paketinde bir şey istediğinde, ince bir ters proxy sunucusuna ulaşır. Bu proxy onu istemciye yakın bir sunucuya yönlendirir (bu durumda yakın, o konum için en hızlı anlamına gelir) ve istenen işlevi yürütür. Kodun gerçekten yürütüldüğü sunucu, kaynak olarak bilinir. Orada tipik sunucu tarafı işlevleri sağlayabilir: veritabanlarından veri çekin, dinamik bilgileri doldurun ve istemciyi ağır JavaScript yükleriyle yormaktan kaçınmak için bölümleri statik HTML olarak işleyin. Ubl, "Yerel makinenizde çalışan şeyi çevirin ve altyapıya yerleştirdiğimizde tam olarak aynı şekilde davranacak şekilde sarın" dedi. "Bu, uç fonksiyonları ürünümüzü daha soyut bir kavram haline getiriyor çünkü onları doğrudan kullanmıyorsunuz.

DOI: 10.1158/1078-0432.CCR-23-0311

Resource: Samtools (RRID:SCR_002105)

Curator: @scibot

SciCrunch record: RRID:SCR_002105

What is this?

On average case is O(1), but in the worst case is O(n)

Author Response

Reviewer #1 (Public Review):

This paper investigates whether bistable rhodopsins can be used to manipulate GPCR signalling in zebrafish. As a first step, the authors compared the performance of bistable rhodopsins fused with a flag tag or with a fluorescent protein tag (TagCFP). Constructs were compared by expressing in HEK cells followed by calcium imaging with aequorin or cAMP monitoring with GloSensor. This showed that the protein with a smaller flag tag performed better. Then, a series of transgenic zebrafish lines were made, in which tagged rhodopsins were expressed in reticulospinal neurons or cardiomyocytes.

The data indicate that bistable rhodopsin can be used to manipulate Gq and Gi/o signalling in zebrafish. The Gq-coupled SpiRh1 was effective in manipulating reticulospinal neurons, as indicated by analysis of tail movements and calcium imaging of the neurons. Gi/o signalling could be manipulated by Opn3 from mosquitoes, TMT from pufferfish, and parapinopsin from lamprey, as shown by their effects on the heartbeat. Lamprey parapinopsin has the interesting property that it can be turned on and off by different wavelengths of light, and this was used to stop and restart the heart. Finally, the authors show that the cardiac effects are mediated by an inward-rectifier K+ channel, through the use of pharmacological inhibitors.

A strength of this paper is the testing of a range of bistable rhodopsins, with a total of 10 proteins tested. This provides a good resource for future experiments. A weakness is the failure to show that some experiments involved repeated sampling of the same animal. Figure 3 gives the impression that there are 48 independent datapoints. However, there are 8 animals, with 6 datapoints coming from each. Similarly, Figure 4 shows the data from 6 trials of 4 animals, not 24 independent animals. Repeated sampling should be reflected in the data presentation, and in the statistical analysis. Was there an effect of trial number, which is suggested in Figure 6?

In response to the reviewer’s comments, we modified the graph to show the average data for individual animals in Figure 3A-E, Figure 3-supplement 2, Figure 4D-F, H, and Figure 4-supplement 2B. We also showed the effect of trial number (difference between trials 1 and 6) in Figure 3-supplement 1 and Figure 4-supplement 1. In addition, we also showed all data as source data. We believe that more accurate statistical analyses were conducted using data from each individual animal.

Delta F/F refers to relative change, which should be (F-F0)/F0. This should be zero when t = 0. The values in Figure 3E, and 3F are ~ 1 when t = 0, however. Are these figures showing F/F0?

The reviewer is correct. It is indeed F-F0/F0 (ΔF/F0). In Figure 3F (3E in the original manuscript), t=0 was the time when 470-495 nm light (for both stimulation of SpiRh1 and detection of GCaMP6s fluorescence) started to be applied. In the experiment in Figure 3G (3F in the original manuscript), 405 nm light was applied to activate SpiRh1[S186F] for 2 s and then 470-495 nm light was applied to detect GCaMP6s fluorescence. In other words, t=0 is the time when 405 nm light started to be applied.

The authors' conclusions that the bistable rhodopsins are useful tools in the zebrafish system appear largely justified. This is consistent with findings from other organisms, including mouse (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097317/, https://www.sciencedirect.com/science/article/pii/S0896627321001616). The tools here are likely to find broad use by scientists who use the zebrafish as the experimental system for a variety of different areas.

For the studies on LamPP and MosOpn3, we cited the references mentioned by the reviewer. We believe that our study substantiates that LampPP and MosOpn3, as well as other bistable rhodopsins, are valuable tools for zebrafish research, as pointed out by the reviewer.

Reviewer #2 (Public Review):

The presented study aims at deciphering the physiological function of GPCR signaling in excitable cells. To this end, the authors developed transgenic zebrafish models expressing a selection of Gq- and Gi/o-coupled bistable rhodopsins in either reticulospinal neurons or cardiomyocytes and elucidated behavioral responses (tail movements) or physiological responses (heartbeat) as well as intracellular Ca2+ dynamics following optical stimulation of rhodopsins.

One of the major strengths of the presented study is the functional comparison of five Gq- and five Gi/o-coupled rhodopsins in two major classes of excitable cells, however; the selection of rhodopsins tested remains elusive. More importantly, it is not obvious why some of the effects of rhodopsin activation were assessed in both neurons and cardiomyocytes, while others were only tested in one of the two systems without further explanation. The main chosen experimental readouts (swimming/tail bending or cardiac contractions) have limited informative value regarding GPCR signaling, as they will only report the peak of the iceberg, namely whether movements are elicited or heartbeats inhibited. No analysis on subtle changes in heart rate and contraction force was included, but such modulation of cardiac activity (e.g. positive or negative chronotropic, inotropic, dromotropic, bathmotropic, and/or lusitropic responses) would represent better the physiological modulation of the heart via GPCR and down-stream signaling events. In line, the presented data only represents behavior at one light intensity tested, whereas a light titration of observed effects could provide more meaningful insight into both rhodopsin responses and signaling mechanisms. Also, the potential promiscuity of G protein activation of selected receptors has not been addressed, neither experimentally nor in the discussion part. As a result of the above-mentioned limitations, it is difficult to follow the logic of the study and especially to interconnect the data obtained in reticulospinal neurons (where activation of jumping spider rhodopsin elicited tail bending) to myocyte data (where three Gi-coupled rhodopsins suppressed cardiac activity). Moreover, as such, the study does not provide explanations on why a certain tool might evoke an effect in one system or the other, or not, which could be the main deliverable of such a comparative analysis.

We are grateful for helpful and insightful comments from the reviewer. We believe that the presentation of experimental findings in the original manuscript may have led to a misunderstanding. We examined the effects of Gq and Gi/o-coupled bistable rhodopsins on both reticulospinal V2a neurons and cardiomyocytes. We observed noticeable effects of Gq rhodopsins on reticulospinal V2a neurons, but no significant effects on cardiomyocytes. Similarly, we found effects of Gi/o-coupled rhodopsins on cardiomyocytes, but no significant effects on reticulospinal V2a neurons. These discrepancies could be attributed to differences in the target cells and experimental conditions, suggesting the need for further optimization. We described the data on page 13, lines 16-22 and page 16, lines 9-10 in the Result section and Table 1, and discussed the relationship between the activity of bistable rhodopsins and their effects on target cells on page 21, lines 6-15 and page 24, line 19-page 25, line 2 in the Discussion section of the revised manuscript.

In order to clarify the function of Gi/o-coupled rhodopsins on the heart in more detail, we conducted experiments in which we activated cardiomyocytes expressing bistable rhodopsins at various light intensities to observe the effects on heartbeats. We analyzed cardiac arrest rate, latency to cardiac arrest, and time to resumption of heartbeat. The results of these experiments are shown in Figure 4 and Figure 4-supplement 2, 3 in the revised manuscript. We described the data on page 15, line 16-page 16, line 1 in the revised manuscript, as follows.

To analyze the photosensitivity of Gi/o-coupled rhodopsins, we applied light of various intensities for 1 s and examine their effect on HBs (Figure 4-supplement 2). Cardiac arrest was induced and sustained for over 20 s after stimulation of MosOpn3 with 0.05 mW/mm2 light for 1 s. Photoactivation of PufTMT and LamPP at lower light intensities (0.2 or 0.05 mW/mm2) resulted in cardiac arrest, but faster HB recovery than stimulation with 0.5 mW/mm2 light (Figure 4-supplement 2). The data indicate that the ability of MosOpn3 to suppress HBs is more photosensitive than PufTMT and LamPP in the zebrafish heart. We further examined atrial-ventricular (AV) conductivity by measuring the time difference between atrial and ventricular contractions before and after light stimulation when HBs had slightly recovered. There was no significant difference in AV conductivity before and after light stimulation (Figure 4-supplement 3).

We performed experiments to the best of our ability with current technology regarding cardiac function. However, we hope that the reviewer is willing to acknowledge that there are certain limitations in conducting a detailed analysis of the zebrafish larval heart, since many experimental techniques, such as electrophysiological analysis, have not yet been fully or effectively established for this animal model.

While the presented data is interesting, the graphical presentation and description of the data are insufficient. Most importantly, the current version of the text does not include a quantitative description of effects and statistical analyses (which are found in the figures and legends!). The lack of quantitative description also extends to both the introduction and discussion, which remain general without a specific dissection of observed effects.

We have described quantitative data in the Result section.

One major concern is the selective citation of own work. While single statements in both the introduction and discussion are supported by up to ten own papers, recent studies using rhodopsins for dissecting GPCR signaling in neurons are not sufficiently discussed and new data is not compared to published results by other teams. Moreover, relevant papers on cardiomyocytes (e.g. PMID: 35579776, 35365606, 34987414, 30894542) are not cited at all, despite the use of similar rhodopsins and/or optogenetic activation of the same signaling pathways. Taking into account these published studies may help to better understand the observed responses.

We apologize for not citing important relevant papers in the original manuscript. We have now cited all four papers (Dai et la., 2022; Wagdi et al., 2022; Cokic et al., 2021; Makowka et al., 2019) mentioned by the reviewer, as well as a new paper describing the use of MosOpn3 and LamPP in C. elegans neurons (Koyanagi et al., 2022) in the Introduction section. We also discussed the differences between our findings and previously published data in the Discussion section.

Additional comment: Data were obtained from larvae zebrafish. It would be useful to include a discussion on how GPCR signaling might be different in adult fish compared to larvae, and how to test whether the observed effects are more generally applicable.

We discussed the differences between the hearts of zebrafish larvae and adults, and the differences in GPCR signaling, on page 27, lines 10-16, as follows. In this study, we used zebrafish larvae to study the role of GPCR signaling in cardiac function, and there are differences in heart structure and function between larvae and adult zebrafish. As a zebrafish grows, blood pressure increases and the heart becomes more complex with the development of valves and ventricular trabeculae. Therefore, GPCR signaling, which regulates heart structure and function, may differ between juvenile and adult fish. Optogenetic manipulation of the heart’s function in adult zebrafish using bistable opsins should clarify this issue.

Reviewer #2 (Public Review):

Jarysta and colleagues set out to define how similar GNAI/O family members contribute to the shape and orientation of stereocilia bundles on auditory hair cells. Previous work demonstrated that loss of particular GNAI proteins, or inhibition of GNAIs by pertussis toxin, caused several defects in hair bundle morphogenesis, but open questions remained which the authors sought to address. Some of these questions include whether all phenotypes resulting from expression of pertussis toxin stemmed from GNAI inhibition; which GNAI family members are most critical for directing bundle development; whether GNAI proteins are needed for basal body movements that contribute to bundle patterning. These questions are important for understanding how tissue is patterned in response to planar cell polarity cues.

To address questions related to the GNAI family in auditory hair cell development, the authors assembled an impressive and nearly comprehensive collection of mouse models. This approach allowed for each Gnai and Gnao gene to be knocked out individually or in combination with each other. Notably, a new floxed allele was generated for Gnai3 because loss of this gene in combination with Gnai2 deletion was known to be embryonic lethal. Besides these lines, a new knockin mouse was made to conditionally express untagged pertussis toxin following cre induction from a strong promoter. The breadth and complexity involved in generating and collecting these strains makes this study unique, and likely the authoritative last word on which GNAI proteins are needed for which aspect of auditory hair bundle development.

Appropriate methods were employed by the authors to characterize auditory hair bundle morphology in each mouse line. Conclusions were carefully drawn from the data and largely based on excellent quantitative analysis. The main conclusions are that GNAI3 has the largest effect on hair bundle development. GNAI2 can compensate for GNAI3 loss in early development but incompletely in late development. The Gnai2 Gnai3 double mutant recapitulates nearly all the phenotypic effects associated with pertussis toxin expression and also reveals a role for GNAIs in early movement of the basal body. Although these results are not entirely unexpected based on earlier reports, the current results both uncover new functions and put putative functions on more solid ground.

Based on this study, loss of GNAI1 and GNAO show a slight shortening of the tallest row of stereocilia but no other significant changes to bundle shape. Antibody staining shows no change in GNAI localization in the Gnai1 knockout, suggesting that little to no protein is found in hair cells. One caveat to this interpretation is that the antibody, while proposed to cross-react with GNAI1, is not clearly shown to immunolabel GNAI1. More than anything, this reservation mostly serves to illustrate how challenging it is to nail down every last detail. In turn, the comprehensive nature of the current study seems all the more impressive.

I believe the connection between mental deficiency and delinquency is a serious issue. I think a lot of people associate mental delinquency with being "slow" but according to the true crime shows I watch, a lot o criminals actually have high IQs. To me, mental delinquency can be a defect in a certain part of the brain that alters decision making. I believe the fetus experiences some sort of alteration when they are in utero from a mothers intake of drugs or alcohol or any other harmful substance. Brian damage after birth can also cause a mental defect that can set the tone for later delinquent behavior.

Sendo conhecedor da reflexão desenvolvida no seio do neo-humanismo alemão sobre o conceito de Bildung ou formação (por autores como Wlhelm von Humboldt, Hegel ou Herder) esta preocupação com a aplicação prática do saber suscita-me reservas. A Bildung é, acima de tudo, um processo de autodeterminação e de desenvolvimento pessoal não subordinado a fins práticos, baseado na multiplicação de experiências e na abertura a outras perspetivas. A Bildung pode coabitar com um ensino profissionalizante, mas não se confunde com ele. Importa não perder de vista esta distinção, que tem claras implicações práticas. Uma referência: Forming Humanity – Redeeming the German Bildung Tradition. Chicago University Press, 2019. Rui Silva

Na nossa última sessão síncrona, levantaram-se questões muito interessantes a partir das tensões entre estes dois verbos: avaliar e medir. Ainda que não queiramos reduzir o primeiro ao segundo e procuremos diferentes formas de monotorização das aprendizagens, a verdade é que no final do semestre temos sempre necessidade de apresentar um número que traduz o percurso dos nossos estudantes.

Abordagem da avaliação como uma ferramenta de aprendizagem ativa e contínua, em vez de ser apenas um momento final de verificação do aprendente, com vista à atribuição de uma classificação. Nessa perspetiva, a avaliação é vista como um processo integrante do ensino, fornecendo feedback relevante e permitindo identificar as lacunas com o objetivo de melhorar o desempenho ao longo do tempo. "O papel da avaliação enquanto promotora e reguladora da aprendizagem." [Maria do Carmo Martins e Helena Melo, Universidade dos Açores]

Todos os exercícios práticos, quer durante as aulas quer de avaliação são casos reais, com resultados reais. No entanto os alunos não têm a capacidade de verificar nem a noção se o seu resultado final é possível (real). (José Fontes UAç)

Isto me termos de cursos por exemplo na área de agronomia e nas outras de uma forma geral é de extrema importância para o estudante sentir a importância e aplicabilidade real do que apreendeu na UC

Este é um problema e um dilema real de todos os docentes para o qual urge encontrar forma de o resolver para despertar o interesse do estudante pela UC em causa.

A incorporação de "atitudes, emoções e valores" no conceito de competência surpreende-me, porque o meu termo de referência aqui é o programa do pensamento crítico que, desde as suas origens por volta dos anos 70/80 do séc. XX, defendeu a existência de uma dualidade competência/disposição (exemplo disso é Robert Ennis). Posso ter uma competência e não ter a disposição intelectual para a utilizar. Por isso é tão importante cultivar a dimensão disposicional do pensamento (virtudes intelectuais, disposições do pensamento crítico), um ponto que fica obscurecido diluindo-a no conceito de competência. Rui Silva

O tempo, especificamente na área da Matemática, é fundamental para consolidar conteúdos visando adquirir competências necessárias e preparação para obter conceitos mais avançados que necessitam de outros anteriores.

[Helena Melo e Maria do Carmo Martins, Universidade dos Açores]

Ao longo dos capítulos em que são apresentados exemplos práticos relativos a UCs do 1.º ciclo e do 2.º ciclo, referentes à área das Ciências da Educação, também poderiam exemplificar, de modo concreto, exemplos relativo às áreas das Ciências Exatas, atendendo à sua especificidade.

Salientamos que se torna difícil implementar tais ideologias formativas da área das Ciências da Educação diretamente, ou adaptada, às Ciência Exatas.

Observamos que a parte científica de uma UC na área das Ciências da Educação é muito similar às práticas pedagógicas utilizadas nesta área, enquanto que na área das Ciências Exatas a parte científica é distinta da prática pedagógica.

[Helena Melo e Maria do Carmo Martins, Universidade dos Açores]

Contudo, o avanço tecnológico é rápido e o estudante, no momento da sua utilização, pode estar desfasado, se não estiver constantemente a atualizar-se. Atendendo ao paradigma atual exige-se, do estudante e do formador, uma atitude reflexiva e interativa, um trabalho colaborativo incessante e um recurso permanente aos meios digitais, tendo em vista a uma preparação consciente para o amanhã e para lidar com o inesperado.

[Helena Melo e Maria do Carmo Martins, Universidade dos Açores]

A palavra falência é demasido forte. Talvez fizessse mais sentido usar "artificialidade" ou "crescente inedequabilidade". De facto, o sistema de ensino tem estado ligado as formas tradicionais psicométricas, mas se ele existe é sinal do seu sucesso, dai não se poder falar de falência.

Também há autores que questionam isto que parece ser uma sugestão de que a avaliação incide apenas sobre objetivos previamente definidos (o que foi planeado antes do ato de ensino), desvalorizando os imprevistos acontecendo ao longo do processo educativo e que podem ter grande potencial educativo, razão pela qual não deveriam ficar fora da avaliação. Como afirma Kliebard (1991), essa visão estreita da avaliação "não leva em consideração resultados latentes que talvez sejam os mais significativos" (p.126). Referência Kliebard (1991). Crítica aos princípios de Tyler. In F. A. Machado e M. F. Gonçalves (Orgs.), Currículo e Desenvolvimento Curricular: problemas e perspectivas (pp. 125-127). ASA. [Francisco Sousa, Universidade dos Açores]

Novo mesmo? Há décadas que quem estuda aprofundadamente a avaliação defende a ideia de que avaliação não é sinónimo de classificação, pois, como se afirma e desenvolve mais adiante nesta publicação, inclui uma dimensão de avaliação formativa. Por exemplo, Ribeiro e Ribeiro (1989) afirmam que "a função de avaliar corresponde a uma análise cuidada das aprendizagens planeadas, o que se vai traduzir numa descrição que informa professores e alunos sobre os objetivos atingidos e aqueles onde se levantam dificuldades", permitindo assim que se ofereça aos alunos "informação que lhes permite orientar os seus esforços, com o apoio do professor, no sentido de ultrapassar dificuldades" (p. 337). Referência Ribeiro, A. C. e Ribeiro, L. (1989). Planificação e avaliação do ensino-aprendizagem. Universidade Aberta. [Francisco Sousa, Universidade dos Açores]

Como sublinha Fino (2017), as expectativas de que o desenvolvimento tecnológico revolucionaria a educação e teria um forte impacto positivo no sucesso educativo têm sido cada vez mais reconhecidas como mitos. Mas as Tecnologias da Informação e da Comunicação (TIC) podem ser muito úteis na educação e têm características que lhes podem conferir o papel de peças importantes da inovação educacional, se integradas em estratégias de ensino transformadoras. Refiro-me ao ensino em geral e à avaliação em particular. Referência Fino, C. (2017). Um século de máquinas de ensinar, 50 anos de máquinas de aprender. Revista Hipótese, 3 (3), 58-74.Description [Francisco Sousa, Universidade dos Açores]

Car@s cibernautas, o desafio desta semana da nossa sala de aula virtual é comentar alguns dos desafios da avaliação digital no ES que encontrarem neste texto. Divirtam-se, façam anotações e highlights!! E podem interagir com as anotações já existentes.... saudações académicas António Moreira

Reviewer #1 (Public Review):

This is my first review of the article entitled "The canonical stopping network: Revisiting the role of the subcortex in response inhibition" by Isherwood and colleagues. This study is one in a series of excellent papers by the Forstmann group focusing on the ability of fMRI to reliably detect activity in small subcortical nuclei - in this case, specifically those purportedly involved in the hyper- and indirect inhibitory basal ganglia pathways. I have been very fond of this work for a long time, beginning with the demonstration of De Hollander, Forstmann et al. (HBM 2017) of the fact that 3T fMRI imaging (as well as many 7T imaging sequences) do not afford sufficient signal to noise ratio to reliably image these small subcortical nuclei. This work has done a lot to reshape my view of seminal past studies of subcortical activity during inhibitory control, including some that have several thousand citations.

In the current study, the authors compiled five datasets that aimed to investigate neural activity associated with stopping an already initiated action, as operationalized in the classic stop-signal paradigm. Three of these datasets are taken from their own 7T investigations, and two are datasets from the Poldrack group, which used 3T fMRI.

The authors make six chief points:<br /> 1. There does not seem to be a measurable BOLD response in the purportedly critical subcortical areas in contrasts of successful stopping (SS) vs. going (GO), neither across datasets nor within each individual dataset. This includes the STN but also any other areas of the indirect and hyperdirect pathways.<br /> 2. The failed-stop (FS) vs. GO contrast is the only contrast showing substantial differences in those nodes.<br /> 3. The positive findings of STN (and other subcortical) activation during the SS vs. GO contrast could be due to the usage of inappropriate smoothing kernels.<br /> 4. The study demonstrates the utility of aggregating publicly available fMRI data from similar cognitive tasks.<br /> 5. From the abstract: "The findings challenge previous functional magnetic resonance (fMRI) of the stop-signal task"<br /> 6. and further: "suggest the need to ascribe a separate function to these networks."

I strongly and emphatically agree with points 1-5. However, I vehemently disagree with point 6, which appears to be the main thrust of the current paper, based on the discussion, abstract, and - not least - the title.

To me, this paper essentially shows that fMRI is ill-suited to study the subcortex in the specific context of the stop-signal task. That is not just because of the issues of subcortical small-volume SNR (the main topic of this and related works by this outstanding group), but also because of its limited temporal resolution (which is unacknowledged, but especially impactful in the context of the stop-signal task). I'll expand on what I mean in the following.

First, the authors are underrepresenting the non-fMRI evidence in favor of the involvement of the subthalamic nucleus (STN) and the basal ganglia more generally in stopping actions.<br /> - There are many more intracranial local field potential recording studies that show increased STN LFP (or even single-unit) activity in the SS vs. FS and SS vs. GO contrast than listed, which come from at least seven different labs. Here's a (likely non-exhaustive) list of studies that come to mind:<br /> o Ray et al., NeuroImage 2012<br /> o Alegre et al., Experimental Brain Research 2013<br /> o Benis et al., NeuroImage 2014<br /> o Wessel et al., Movement Disorders 2016<br /> o Benis et al., Cortex 2016<br /> o Fischer et al., eLife 2017<br /> o Ghahremani et al., Brain and Language 2018<br /> o Chen et al., Neuron 2020<br /> o Mosher et al., Neuron 2021<br /> o Diesburg et al., eLife 2021<br /> - Similarly, there is much more evidence than cited that causally influencing STN via deep-brain stimulation also influences action-stopping. Again, the following list is probably incomplete:<br /> o Van den Wildenberg et al., JoCN 2006<br /> o Ray et al., Neuropsychologia 2009<br /> o Hershey et al., Brain 2010<br /> o Swann et al., JNeuro 2011<br /> o Mirabella et al., Cerebral Cortex 2012<br /> o Obeso et al., Exp. Brain Res. 2013<br /> o Georgiev et al., Exp Br Res 2016<br /> o Lofredi et al., Brain 2021<br /> o van den Wildenberg et al, Behav Brain Res 2021<br /> o Wessel et al., Current Biology 2022<br /> - Moreover, evidence from non-human animals similarly suggests critical STN involvement in action stopping, e.g.:<br /> o Eagle et al., Cerebral Cortex 2008<br /> o Schmidt et al., Nature Neuroscience 2013<br /> o Fife et al., eLife 2017<br /> o Anderson et al., Brain Res 2020

Together, studies like these provide either causal evidence for STN involvement via direct electrical stimulation of the nucleus or provide direct recordings of its local field potential activity during stopping. This is not to mention the extensive evidence for the involvement of the STN - and the indirect and hyperdirect pathways in general - in motor inhibition more broadly, perhaps best illustrated by their damage leading to (hemi)ballism.

Hence, I cannot agree with the idea that the current set of findings "suggest the need to ascribe a separate function to these networks", as suggested in the abstract and further explicated in the discussion of the current paper. For this to be the case, we would need to disregard more than a decade's worth of direct recording studies of the STN in favor of a remote measurement of the BOLD response using (provably) sub ideal imaging parameters. There are myriads of explanations of why fMRI may not be able to reveal a potential ground-truth difference in STN activity between the SS and FS/GO conditions, beginning with the simple proposition that it may not afford sufficient SNR, or that perhaps subcortical BOLD is not tightly related to the type of neurophysiological activity that distinguishes these conditions (in the purported case of the stop-signal task, specifically the beta band). But essentially, this paper shows that a specific lens into subcortical activity is likely broken, but then also suggests dismissing existing evidence from superior lenses in favor of the findings from the 'broken' lens. That doesn't make much sense to me.

Second, there is actually another substantial reason why fMRI may indeed be unsuitable to study STN activity, specifically in the stop-signal paradigm: its limited time resolution. The sequence of subcortical processes on each specific trial type in the stop-signal task is purportedly as follows: at baseline, the basal ganglia exert inhibition on the motor system. During motor initiation, this inhibition is lifted via direct pathway innervation. This is when the three trial types start diverging. When actions then have to be rapidly cancelled (SS and FS), cortical regions signal to STN via the hyperdirect pathway that inhibition has to be rapidly reinstated (see Chen, Starr et al., Neuron 2020 for direct evidence for such a monosynaptic hyperdirect pathway, the speed of which directly predicts SSRT). Hence, inhibition is reinstated (too late in the case of FS trials, but early enough in SS trials, see recordings from the BG in Schmidt, Berke et al., Nature Neuroscience 2013; and Diesburg, Wessel et al., eLife 2021).<br /> Hence, according to this prevailing model, all three trial types involve a sequence of STN activation (initial inhibition), STN deactivation (disinhibition during GO), and STN reactivation (reinstantiation of inhibition during the response via the hyperdirect pathway on SS/FS trials, reinstantiation of inhibition via the indirect pathway after the response on GO trials). What distinguishes the trial types during this period is chiefly the relative timing of the inhibitory process (earliest on SS trials, slightly later on FS trials, latest on GO trials). However, these temporal differences play out on a level of hundreds of milliseconds, and in all three cases, processing concludes well under a second overall. To fMRI, given its limited time resolution, these activations are bound to look quite similar.

Lastly, further building on this logic, it's not surprising that FS trials yield increased activity compared to SS and GO trials. That's because FS trials are errors, which are known to activate the STN (Cavanagh et al., JoCN 2014; Siegert et al. Cortex 2014) and afford additional inhibition of the motor system after their occurrence (Guan et al., JNeuro 2022). Again, fMRI will likely conflate this activity with the abovementioned sequence, resulting in a summation of activity and the highest level of BOLD for FS trials.

In sum, I believe this study has a lot of merit in demonstrating that fMRI is ill-suited to study the subcortex during the SST, but I cannot agree that it warrants any reappreciation of the subcortex's role in stopping, which are not chiefly based on fMRI evidence.

A few other points:<br /> - As I said before, this team's previous work has done a lot to convince me that 3T fMRI is unsuitable to study the STN. As such, it would have been nice to see a combination of the subsamples of the study that DID use imaging protocols and field strengths suitable to actually study this node. This is especially true since the second 3T sample (and arguably, the Isherwood_7T sample) does not afford a lot of trials per subject, to begin with.<br /> - What was the GLM analysis time-locked to on SS and FS trials? The stop-signal or the GO-signal?<br /> - Why was SSRT calculated using the outdated mean method?<br /> - The authors chose 3.1 as a z-score to "ensure conservatism", but since they are essentially trying to prove the null hypothesis that there is no increased STN activity on SS trials, I would suggest erring on the side of a more lenient threshold to avoid type-2 error.<br /> - The authors state that "The results presented here add to a growing literature exposing inconsistencies in our understanding of the networks underlying successful response inhibition". It would be helpful if the authors cited these studies and what those inconsistencies are.

! images - search - xanadu ted nelson

In the future will we have to specify time complexity like we did in python (ie big O notation) or is descriptors like quadratic enough?

Author Response:

We would like to thank the eLife reviewers for the considerable time and effort they have invested to review these manuscripts. We have also benefited from a previous round of review of the manuscript describing the proposed burial features, which underwent two rounds of revisions in a high-impact journal over a period of approximately 8 months during 2022 and early 2023. Both sets of reviews have reflected mixed responses to the evidence we have presented, with one reviewer recommending acceptance with minor editorial revisions, two recommending acceptance with minor revisions and the fourth recommending rejection based upon similar arguments to those reflected by some of the reviewers in this current round of reviews in eLife. Ultimately the managing editor of this first journal took the decision that the review process could not be completed in a timely manner and rejected the manuscript although the submission here reflected our consideration of these reviewers suggestions.

We have chosen in this initial response to the eLife reviews to include some references to the previous anonymous reviews in order to illustrate differences of opinion and differences in revision suggestions within the review process. Our goal is to offer maximal insight into our decision-making process and to acknowledge the considerable time and effort put into the assessment of these manuscripts by reviewers (for eLife and in the case of the earlier review process). We hope that this approach will assist the readers, and reviewers, of our manuscripts in understanding why we are proceeding with certain decisions during the revision process.

This is a new process for us and the reviewers, and one way in which it significantly differs from more traditional review is that both the reviews and our reply will be public well in advance of our revisions to the manuscript. Indeed, considering the scope of the reviews, some of those revisions may take considerable time, although many can be accomplished fairly easily. Thus, we are not in a position to say that we have solved every issue raised by the reviewers. Instead, we will examine what appear to be the key critical issues raised regarding the data and the analyses and how we propose to address these as we revise the papers. We will also address several philosophical and ethical issues raised by the reviews and our proposal for dealing with these. More specific editorial and citational recommendations will be dealt with on a case-by-case basis, and we do not address these point-by-point in this reply. Please note, this response to the reviewers is not the revision of the manuscript and is only the initial opinion of the corresponding authors with some guidance from the larger group of authors of all three papers. Our final submitted revision will reflect the input of all authors included on those submissions.

We took the decision to submit three separate papers consciously. The two different categories of evidence, burials and engravings, involve different kinds of analysis and different (although overlapping) teams of researchers, and we recognized that each deserved their own presentation and assessment. Meanwhile, together they inform the context of H. naledi in a way that requires some synthetic discussion, in which both kinds of evidence are relevant, leading to a third paper. But the mutual relevance of these different kinds of evidence and their review by a common set of reviewers naturally raises cross-cutting issues, and the reviewers have cross-referenced the three articles. This has sometimes led to suggestions about one manuscript based on the contents of another. Considering the situation, we accepted the recommendation that it would be clearer to consider all three articles in a single reply. Thus, while each of the three papers will proceed separately during the revision process, it will be necessary to highlight across all three papers occasionally in our responses.

Scientific Issues:

In reading the reviews, we feel there are 9 critical points/assertions raised by one or more of the reviewers that present a problem for, or challenge to, our hypothesis that the observed evidence (bone accumulations and engravings) described in the Dinaledi subsystem are of intentional naledigenic origin. These are:

1. The evidence presented does not demonstrate a clear interruption of the floor sediments, thus failing to demonstrate excavated holes.

2. The sediments infilling the holes where the skeletal remains are found have not been demonstrated to originate from the disruption of the floor sediments and thus could be part of a natural geological process (e.g. water movement, slumping) or carnivore accumulations.

3. Previous geological interpretations by our research group have given alternative geological explanations for formation of the bony accumulations that contradict the present evidence presented here and result in alternative origins hypotheses.

4. Burial cannot be effectively assessed without complete excavation of the features and site.

5. The skeletal remains as presented do not conform clearly to typical body arrangement/positions associated with human (Homo sapiens) burials.

6. There is no evidence of grave goods or lithic scatters that are typically associated with human burials.

7. Humans may have been involved with the creation of either the Homo naledi bone accumulations, the engravings, or both.

8. Without a date of the engravings, the null hypothesis should be the engravings were created by Homo sapiens.

9. The null hypothesis for explanation of the skeletal remains in this situation should be “natural accumulation”.

Our analysis of the Dinaledi Feature 1 leads us to accept that the laminated orange-red mudstone (LORM) sedimentary layer is interrupted, indicating a non-natural intervention, and that the hole created by the interruption was then filled by both a fleshed body (and perhaps parts of other bodies) which were then covered by sediment that originated from the hole that was dug. We recognize that the four eLife reviewers are not convinced that our presentation is sufficient to establish this. Interestingly, this was not the universal opinion of earlier reviewers of the initial manuscript several of whom felt we had adequately supported this hypothesis. The lack of clarity in this current version of the burial manuscript is our responsibility. In the upcoming revision of this paper to be submitted, we will take the reviewers’ critiques to heart and add additional figures that illustrate better the disruption of the LORM and clarify the sedimentological data showing the material covering the skeletal remains in the hole are the disrupted sediments excavated from the same hole. We are proposing to isolate this most critical evidence for burial into a separate section in the revised submission based on the reviewers’ comments. The fact that the LORM layer is disrupted, a fleshed body was placed in the hole created by this disruption, and the body (and perhaps parts of other bodies) was/were then covered by the same sediments from the hole is the central feature of our hypothesis that the bone accumulations observed reflect a burial and not a natural process.

The possibility of fluvial transport or involvement in the subsystem is a topic that we have addressed extensively in past work, and it is clear from these reviews that we must enhance our current manuscript to discuss this issue at greater length. Our previous work (Dirks et al. 2015; Dirks et al. 2017) emphasized that fluvial transport of whole bodies into the subsystem was precluded by several lines of sedimentological evidence. We excavated a rich accumulation of skeletal remains, including articulated limbs and other elements in subvertical orientations inconsistent with slow sedimentary infill, which were difficult to explain without positing either a large and dense pile of bodies and/or sediment movement. We encountered fractured chunks of laminated orange-red mudstone (LORM) in random orientations within our excavation area, within and among skeletal remains, which directly refuted that the remains were inundated with water at the time of burial, and this limited the possibility of fluvial transport. Water flow sufficient to displace bodies or complete skeletal evidence would also transport large and course sediment, which is absent from the subsystem, and would sort the commingled skeletal material that we found by size, which we do not observe. But our excavation only covered less than a square meter at very limited depth, and this was the limit to our knowledge of subsurface sediment. We thus were left with uncertainty that led us to suggest the possibility of sediment slumping or movement into subsurface drains, although these were not observed near our excavation. Our current work expands our knowledge of the subsurface and presents an alternative explanation for the disposition of skeletal remains from our earlier excavation. But we acknowledge that this new explanation is vulnerable to our own previous published proposals, and we must do a better job of explaining how the new information addresses our previous suggestions. By not clearly creating a section where we explained how these previous hypotheses were now nullified by new evidence, we clearly confused the reviewers with our own previous work. We will revise the manuscript by enhancing the review of the significant geological evidence demonstrating that there is no significant fluvial action in the system and making it clear how the burial hypothesis provides a clearer explanation for the situation of skeletal remains from our previous excavation work.

One of the central issues raised by reviewers has been a perceived need to excavate these features completely, totally exhuming all skeletal remains from them. Reviewers have written that it is necessary to identify every skeletal element that is present and account for any missing elements. On this point, we have both ethical and scientific differences from these reviewers. We express our ethical concerns first. Many of the best-preserved possible burials ever discovered by archaeologists were subjected to total excavation and exhumation. Cases like La Chapelle-aux-Saints, La Ferrassie, and Skhūl were fully excavated at a time when data recording and excavation methods did not include the range of spatial and geomorphological approaches that later became routine. The judgment of early investigators that these situations were intentional burials was challenged by later workers, and the kind of information that might enable better tests had been irrevocably lost (Gargett 1999; Dibble et al. 2015; Rendu et al. 2014).

Later, improved excavation standards have not sufficed to remove uncertainty or debate about possible burials. For example, it was long presumed that well-preserved remains of young children were by themselves diagnostic of intentional burial, such as those from Dederiyeh, Border Cave, or Roc de Marsal. Such cases were also fully excavated, with adequate documentation of the positioning of skeletal remains and their surrounding stratigraphic situation, but such cases were later challenged on several bases and the complete exhumation of material has confused or precluded testing of new hypotheses (e.g. Gargett 1999). The case of Roc de Marsal is one in which data from the initial excavation combined with data from the initial excavation combined with re-excavation and geoarchaeological analysis led to a naturalistic interpretation of the skeletal material (Sandgathe et al. 2011; Goldberg et al. 2017). But even in this case, the researchers erred in their interpretation of the skeleton’s situation due to a lack of identification of parts of the infant’s skeleton (Gómez-Olivencia and García-Martinez 2019). That is to say, it is not only the burial hypothesis but other hypotheses that suffer from complete excavation. Researchers concerned with preserving all possible information have sometimes taken extraordinary measures to remove and study possible burials at high-resolution in the laboratory. Such was the case of the Shanidar IV burial removed from the site and transported in plaster jacket by Solecki, which led to the disruption and loss of internal stratigraphic information (Pomeroy et al. 2020). Arguably, the current state of the art is full excavation with partial preparation, such as that undertaken at Panga ya Saidi (Martinón-Torres et al. 2021). But again, any future attempt to reinterpret or test the hypothesis of burial must rely on the adequacy of documentation as the original context has been removed.

In our decision to leave material in place as much as possible, we are expanding upon standard practice to leave witness sections and unexcavated areas for future research. The situation is novel, representing possible burials by a nonhuman species, and that makes it doubly important in our opinion to be conservative in not fully exhuming the skeletal material from its context. We anticipate that many other researchers, including future investigators, will suggest additional methods to further test the hypothesis of burial, something that would be impossible if we had excavated the features in their entirety prior to publishing a description of our work. We believe strongly that our ethical responsibility is to publish the work and the most likely interpretation while leaving as much evidence in place as possible to enable further testing and replication. We welcome the suggestions of additional methods/analyses to test the H. naledi burial hypothesis.

This being said, we also observe that total exhumation would not resolve the concerns raised by the reviewers. The recommendation of total exhumation is in pursuit of a full account of all skeletal material present and its preservation and spatial situation, in order to demonstrate that they conform to body positions comparable to human burials. As has been highlighted in forensic casework, the excavation of an inhumation feature does not necessarily provide an accurate spatial or anatomical manifest of the stratigraphical relationships between the body, encapsulating matrix, and any cut present due to preservational, taphonomic and operational factors (Dirkmaat and Cabo, 2016; Hunter, 2014). In particular, in cases where skeletal elements are highly fragmented, friable, or degraded (such as through bioerosion) then complete excavation—even under controlled laboratory conditions—may destroy bone and severely limit skeletal identification (Henderson, 1997; Hochrein, 2002; Owsley and Compton, 1997), particularly in elements where the ratio of trabecular to cortical bone is high (Darwent and Lyman, 2002; Lyman, 1994). As such, non-invasive methods of 3D and 4D modelling (preservation in situ) are often considered preferable to complete necropsy or excavation (preservation by record) where appropriate (Bolliger and Thali, 2009; Dell’Unto and Landeschi, 2022; Randolph-Quinney et al., 2018; Silver, 2016). 

The test of burial is not primarily positional, but taphonomic and geological. The position and number of bones can elaborate on process-driven questions of decay and destruction in the burial environment, or post-mortem modification, but are not singularly indicative of whether the remains were intentionally buried – the post-mortem narrative of all the processes affecting the cadaveric island is required (Knüsel and Robb, 2016). In previous cases, researchers have disputed or accepted the hypothesis of intentional hominin burial based upon assumptions about how modern humans or Neandertals would have positioned bodies, with the idea that some positions reflect ritual intent while others do not. But applying such assumptions is unjustifiable, particularly for a species like H. naledi, whose culture may have differed fundamentally from our own. Our work acknowledges that the present evidence does not enable a full reconstruction of the burial positions, but it does show that fleshed remains were encased in sediment prior to decomposition of soft tissue, and that subsequent spatial changes can be most parsimoniously explained by natural decomposition within sedimentary matrix contained within a burial feature (after Green, 2022; Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022). If the argument is that extraordinary claims require extraordinary evidence, we feel that the evidence documents excavation and interment (and will do so more clearly in the revision) and the fact of the remains do not match a “typical” human burial in body positioning is not in itself evidence that these are not H. naledi burials.

We feel that the reviewers (in keeping with many palaeoanthropologists) have a clear idea of what they “think” a burial should look like in an idealised sense, but this platonic ideal of burial form is not matched by the extensive literature in archaeothanatology, funerary archaeology and forensic science which indicates enormous variability in the activity, morphology and post-mortem system experienced by the human body in cases of interment and body disposal (e.g. Aspöck, 2008; Boulestin and Duday, 2005 and 2006; Connelly et al., 2005; Channing and Randolph-Quinney, 2006; Cherryson, 2008; Donnelly et al., 1995; Finley, 2000; Hunter, 2014; Parker Pearson, 1999; Randolph-Quinney, 2013). Decades of experience in the identification, recovery and interpretation of clandestine, deviant, and non-formal burials indicates the platonic ideal is rare, and in many contexts, the exception (Cherryson, 2008; Parker Pearson, 1999). This variability is particularly relevant to morphological traits in burial context, such as the informal nature of the grave cut in plan and section, shallow burial depth, and initial disposition of body (placement) during the early post-mortem period. These might run counter to the expectations of reviewers or others referencing the fossil hominin record, but are well accepted within the communities of researchers investigating Holocene archaeological sites and forensic contexts.

It is encouraging to see reviewers beginning to incorporate the extensive (often experimentally derived) literature from archaeothanatology and forensic taphonomy in their deliberations, and we will be taking these comments on board going forward. In particular, we acknowledge reviewers’ comments and the need to construct a more detailed post-mortem narrative, accounting for joint disarticulation (labile versus persistent joints etc), displacement, and final disposition of elements within the burial space. As such we will incorporate the hierarchy of decomposition (rank order disarticulation), associations between regions of anatomical association, areas of disassociation, and the voids produced during decomposition (after Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022) into our narrative. In doing so we acknowledge the tensions between the inductive archaeolothanatological narrative-driven approach (e.g. Duday, 2005 & 2009) versus robust decomposition data derived from human forensic taphonomic experimentation recently articulated by Schotsmans and colleagues (2022) - noting that we will highlight comparative data based on forensic experimental casework and actualistic modelling over inductive intuitive approaches which come with significant evidential shortcomings (Bristow et al. 2011).

Finally, from a taphonomic perspective it is worth pointing out to reviewers that we have already addressed the issue of lack of taphonomic evidence for carnivore involvement in the formation of the Dinaledi assemblage (Dirks, et al., 2016). Absence of any carnivore-induced bone surface modifications, patterns of skeletal part representation, and a total absence of any carnivore remains found within the Dinaledi chamber (following Kuhn and colleagues, 2010) lead us to reject carnivores as possible vectors of body accumulation within the Dinaledi Chamber and Hill Antechamber.

Reviewers suggest that without a date derived from geochronological methods, the engravings cannot be associated with H. naledi, and that it is possible (or probable) that the engravings were done in the recent past by H. sapiens. This suggestion neglects the context of the site. We have previously documented the structure and extremely limited accessibility of the Dinaledi subsystem. This subsystem was not recorded on maps of the documented Rising Star Cave system prior to our work and its discovery by our teams. Furthermore, there is no evidence of prehistoric human activity in the areas of the cave related to possible subterranean entrances There is no evidence that humans in the past typically ventured into such extreme spaces like those of Rising Star. It is clear from the presence of the remains of many individuals that H. naledi ventured into these spaces again and again. It is likely that H. naledi moved through these spaces more easily than humans do based on their physique. We show that the engravings overlay each other suggesting multiple engraving events.  These engravings took time and effort and the only evidence for use of the Dinaledi subsystem by any hominin is by H. naledi. The context leads to the null hypothesis that H. naledi made the marks. In our revision, we will elaborate on this argument to clarify the evidence for our stance on this hypothesis. Several reviewers took issue with the title of the engraving paper as we did not insert a qualifier in front of the suggested date range for the engravings. We deliberately left out qualifying language so that the title took the form of a testable hypothesis rather than a weak assertation. Should future work find the engravings were not produced within this time range, then we will restate this hypothesis.

Finally, with regards to the engravings we have chosen to report them because they exist. Not reporting the presence of engraved marks on the walls of a cave above hypothesized burials would be tantamount to leaving relevant evidence out of the description of an archeological context. We recognize and state in our manuscript that these markings require substantial further study, including attempts at geochronological dating. But the current evidence is clearly relevant to the archaeological context of the subsystem. We take a similar stance with reporting the presence of the tool shaped artefact near the hand of the H. naledi skeleton in the Hill Antechamber. It is evident that this object requires further study, as we stated in our manuscript, but again omitting it from our study would be leaving out relevant evidence.

Some have suggested that the null hypothesis should be that all of these observed circumstances are of natural origin. Our team took this approach in our early investigation of the Dinaledi subsystem (Dirks et al. 2015). We adopted the null hypothesis that the geological processes involved in the accumulation of H. naledi skeletal remains were “natural” (e.g., non-naledigenic involvement), and we were able to reject many alternative explanations for the assemblage, including carnivore accumulation, “death trap” accumulation, and fluvial transport of bodies or bones (Dirks et al. 2015). This led us to the hypothesis that H. naledi were involved in bringing the bodies into the spaces where they were found. But we did not hypothesize their involvement in the formation of the deposit itself beyond bringing the bodies to the location.

This approach seems conservative. It followed the traditional view that small-brained hominins do not engage in cultural practices. But we recognize in hindsight that this null hypothesis approach did harm to our analyses. It impeded us from recognizing within our initial excavations of the puzzle box area and other excavations between 2014 – 2017 that we might be encountering remains that were intrusive in the sedimentary floor of the chamber. If we had approached the accumulation of a large number of hominins from the perspective of the null hypothesis being that the situation was likely cultural, we perhaps would have collected evidence in a slightly different manner. We certainly note that if the Dinaledi system had been full of the remains of modern humans, there would have been little doubt that the null hypothesis would have been that this was a cultural space and not a “natural space”.  We therefore respectfully disagree with the reviewers who continue to support the idea that we should approach hominin excavations with the null hypothesis that they will be natural (specifically non-cultural) in origins. If excavations continue with this mindset we believe that potential cultural evidence is almost certain to be lost.

There has been a gradient across paleoanthropological excavations, archaeological work, and forensic investigation, with increasing precision of context. The reality is that the recording precision and frame of approach is typically different in most paleontological excavations than in those related to contemporary human remains. If anything comes from the present discussion of whether the Dinaledi system is a burial site for H. naledi or not, we hope that by taking seriously the possibility of deep cultural dynamics of hominins, we will encourage other teams to meet the highest standards of excavation in order to preserve potential cultural evidence. Given H. naledi’s cranial capacity we suggest that even very early hominin skeletal assemblages should be re-examined, if there is sufficient evidence or records available.  These would include examples such as the A.L. 333 Au. afarensis site (the so called First Family site in Hadar Ethiopia), the Dikika infant skeleton, WT 15000 (Turkana Boy) and even A.L. 288 (Lucy) as such unusual taphonomic situations where skeletons are preserved cannot be simply explained away as “natural” in origin, based solely on the cranial capacity and assumed lack of cognitive and cultural complexity of the hominins as emphasized by us in Fuentes et al. (2023). We are not the first to observe that some very early hominin situations may represent early mortuary activity (Pettitt 2013), but we would advocate a step further. We suggest it may be damaging to take “natural accumulation” as the standard null hypothesis for hominin paleoanthropology, and that it is more conservative in practice to engage remains with the null hypothesis of possible cultural formation.

We are deeply grateful for the time and effort all of the 8 reviewers (across three reviews) have taken with this work.  We also acknowledge the anonymous reviewers from previous submissions who’s opinions and comments will have made the final iterations of these manuscripts better for their efforts. As this process is rather public and includes commentary outside of the eLife forum, we ask that the efforts of all 37 authors and 8 reviewers involved be respected and that the discourse remain professional in all venues as we study this fascinating and quite complex occurrence. We appreciate also the efforts of members of the public who have engaged with this relatively new process where preprints are posted prior to the reviews allowing comments and interactions from colleagues and the public who are normally not part of the internal peer review process.  We believe these interactions will make for better final papers. We feel we have met the standards of demonstrating burials in H. naledi and that the engraving are most likely associated with H. naledi. However, given the reviews we see many areas where our clarity and context, and analyses, were less strong than they can be. With the clarifications and additions taken on board through these review processes the final papers will be stronger and clearer. We, recognize that this is an ongoing process of scientific investigation and further work will allow continued, and possibly better, evaluation of these hypothesis and others.

Lee R Berger, Agustín Fuentes, John Hawks, Tebogo Makhubela

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Author Response:

We would like to thank the eLife reviewers for the considerable time and effort they have invested to review these manuscripts. We have also benefited from a previous round of review of the manuscript describing the proposed burial features, which underwent two rounds of revisions in a high-impact journal over a period of approximately 8 months during 2022 and early 2023. Both sets of reviews have reflected mixed responses to the evidence we have presented, with one reviewer recommending acceptance with minor editorial revisions, two recommending acceptance with minor revisions and the fourth recommending rejection based upon similar arguments to those reflected by some of the reviewers in this current round of reviews in eLife. Ultimately the managing editor of this first journal took the decision that the review process could not be completed in a timely manner and rejected the manuscript although the submission here reflected our consideration of these reviewers suggestions.

We have chosen in this initial response to the eLife reviews to include some references to the previous anonymous reviews in order to illustrate differences of opinion and differences in revision suggestions within the review process. Our goal is to offer maximal insight into our decision-making process and to acknowledge the considerable time and effort put into the assessment of these manuscripts by reviewers (for eLife and in the case of the earlier review process). We hope that this approach will assist the readers, and reviewers, of our manuscripts in understanding why we are proceeding with certain decisions during the revision process.

This is a new process for us and the reviewers, and one way in which it significantly differs from more traditional review is that both the reviews and our reply will be public well in advance of our revisions to the manuscript. Indeed, considering the scope of the reviews, some of those revisions may take considerable time, although many can be accomplished fairly easily. Thus, we are not in a position to say that we have solved every issue raised by the reviewers. Instead, we will examine what appear to be the key critical issues raised regarding the data and the analyses and how we propose to address these as we revise the papers. We will also address several philosophical and ethical issues raised by the reviews and our proposal for dealing with these. More specific editorial and citational recommendations will be dealt with on a case-by-case basis, and we do not address these point-by-point in this reply. Please note, this response to the reviewers is not the revision of the manuscript and is only the initial opinion of the corresponding authors with some guidance from the larger group of authors of all three papers. Our final submitted revision will reflect the input of all authors included on those submissions.

We took the decision to submit three separate papers consciously. The two different categories of evidence, burials and engravings, involve different kinds of analysis and different (although overlapping) teams of researchers, and we recognized that each deserved their own presentation and assessment. Meanwhile, together they inform the context of H. naledi in a way that requires some synthetic discussion, in which both kinds of evidence are relevant, leading to a third paper. But the mutual relevance of these different kinds of evidence and their review by a common set of reviewers naturally raises cross-cutting issues, and the reviewers have cross-referenced the three articles. This has sometimes led to suggestions about one manuscript based on the contents of another. Considering the situation, we accepted the recommendation that it would be clearer to consider all three articles in a single reply. Thus, while each of the three papers will proceed separately during the revision process, it will be necessary to highlight across all three papers occasionally in our responses.

Scientific Issues:

In reading the reviews, we feel there are 9 critical points/assertions raised by one or more of the reviewers that present a problem for, or challenge to, our hypothesis that the observed evidence (bone accumulations and engravings) described in the Dinaledi subsystem are of intentional naledigenic origin. These are:

1. The evidence presented does not demonstrate a clear interruption of the floor sediments, thus failing to demonstrate excavated holes.

2. The sediments infilling the holes where the skeletal remains are found have not been demonstrated to originate from the disruption of the floor sediments and thus could be part of a natural geological process (e.g. water movement, slumping) or carnivore accumulations.

3. Previous geological interpretations by our research group have given alternative geological explanations for formation of the bony accumulations that contradict the present evidence presented here and result in alternative origins hypotheses.

4. Burial cannot be effectively assessed without complete excavation of the features and site.

5. The skeletal remains as presented do not conform clearly to typical body arrangement/positions associated with human (Homo sapiens) burials.

6. There is no evidence of grave goods or lithic scatters that are typically associated with human burials.

7. Humans may have been involved with the creation of either the Homo naledi bone accumulations, the engravings, or both.

8. Without a date of the engravings, the null hypothesis should be the engravings were created by Homo sapiens.

9. The null hypothesis for explanation of the skeletal remains in this situation should be “natural accumulation”.

Our analysis of the Dinaledi Feature 1 leads us to accept that the laminated orange-red mudstone (LORM) sedimentary layer is interrupted, indicating a non-natural intervention, and that the hole created by the interruption was then filled by both a fleshed body (and perhaps parts of other bodies) which were then covered by sediment that originated from the hole that was dug. We recognize that the four eLife reviewers are not convinced that our presentation is sufficient to establish this. Interestingly, this was not the universal opinion of earlier reviewers of the initial manuscript several of whom felt we had adequately supported this hypothesis. The lack of clarity in this current version of the burial manuscript is our responsibility. In the upcoming revision of this paper to be submitted, we will take the reviewers’ critiques to heart and add additional figures that illustrate better the disruption of the LORM and clarify the sedimentological data showing the material covering the skeletal remains in the hole are the disrupted sediments excavated from the same hole. We are proposing to isolate this most critical evidence for burial into a separate section in the revised submission based on the reviewers’ comments. The fact that the LORM layer is disrupted, a fleshed body was placed in the hole created by this disruption, and the body (and perhaps parts of other bodies) was/were then covered by the same sediments from the hole is the central feature of our hypothesis that the bone accumulations observed reflect a burial and not a natural process.

The possibility of fluvial transport or involvement in the subsystem is a topic that we have addressed extensively in past work, and it is clear from these reviews that we must enhance our current manuscript to discuss this issue at greater length. Our previous work (Dirks et al. 2015; Dirks et al. 2017) emphasized that fluvial transport of whole bodies into the subsystem was precluded by several lines of sedimentological evidence. We excavated a rich accumulation of skeletal remains, including articulated limbs and other elements in subvertical orientations inconsistent with slow sedimentary infill, which were difficult to explain without positing either a large and dense pile of bodies and/or sediment movement. We encountered fractured chunks of laminated orange-red mudstone (LORM) in random orientations within our excavation area, within and among skeletal remains, which directly refuted that the remains were inundated with water at the time of burial, and this limited the possibility of fluvial transport. Water flow sufficient to displace bodies or complete skeletal evidence would also transport large and course sediment, which is absent from the subsystem, and would sort the commingled skeletal material that we found by size, which we do not observe. But our excavation only covered less than a square meter at very limited depth, and this was the limit to our knowledge of subsurface sediment. We thus were left with uncertainty that led us to suggest the possibility of sediment slumping or movement into subsurface drains, although these were not observed near our excavation. Our current work expands our knowledge of the subsurface and presents an alternative explanation for the disposition of skeletal remains from our earlier excavation. But we acknowledge that this new explanation is vulnerable to our own previous published proposals, and we must do a better job of explaining how the new information addresses our previous suggestions. By not clearly creating a section where we explained how these previous hypotheses were now nullified by new evidence, we clearly confused the reviewers with our own previous work. We will revise the manuscript by enhancing the review of the significant geological evidence demonstrating that there is no significant fluvial action in the system and making it clear how the burial hypothesis provides a clearer explanation for the situation of skeletal remains from our previous excavation work.

One of the central issues raised by reviewers has been a perceived need to excavate these features completely, totally exhuming all skeletal remains from them. Reviewers have written that it is necessary to identify every skeletal element that is present and account for any missing elements. On this point, we have both ethical and scientific differences from these reviewers. We express our ethical concerns first. Many of the best-preserved possible burials ever discovered by archaeologists were subjected to total excavation and exhumation. Cases like La Chapelle-aux-Saints, La Ferrassie, and Skhūl were fully excavated at a time when data recording and excavation methods did not include the range of spatial and geomorphological approaches that later became routine. The judgment of early investigators that these situations were intentional burials was challenged by later workers, and the kind of information that might enable better tests had been irrevocably lost (Gargett 1999; Dibble et al. 2015; Rendu et al. 2014).

Later, improved excavation standards have not sufficed to remove uncertainty or debate about possible burials. For example, it was long presumed that well-preserved remains of young children were by themselves diagnostic of intentional burial, such as those from Dederiyeh, Border Cave, or Roc de Marsal. Such cases were also fully excavated, with adequate documentation of the positioning of skeletal remains and their surrounding stratigraphic situation, but such cases were later challenged on several bases and the complete exhumation of material has confused or precluded testing of new hypotheses (e.g. Gargett 1999). The case of Roc de Marsal is one in which data from the initial excavation combined with data from the initial excavation combined with re-excavation and geoarchaeological analysis led to a naturalistic interpretation of the skeletal material (Sandgathe et al. 2011; Goldberg et al. 2017). But even in this case, the researchers erred in their interpretation of the skeleton’s situation due to a lack of identification of parts of the infant’s skeleton (Gómez-Olivencia and García-Martinez 2019). That is to say, it is not only the burial hypothesis but other hypotheses that suffer from complete excavation. Researchers concerned with preserving all possible information have sometimes taken extraordinary measures to remove and study possible burials at high-resolution in the laboratory. Such was the case of the Shanidar IV burial removed from the site and transported in plaster jacket by Solecki, which led to the disruption and loss of internal stratigraphic information (Pomeroy et al. 2020). Arguably, the current state of the art is full excavation with partial preparation, such as that undertaken at Panga ya Saidi (Martinón-Torres et al. 2021). But again, any future attempt to reinterpret or test the hypothesis of burial must rely on the adequacy of documentation as the original context has been removed.

In our decision to leave material in place as much as possible, we are expanding upon standard practice to leave witness sections and unexcavated areas for future research. The situation is novel, representing possible burials by a nonhuman species, and that makes it doubly important in our opinion to be conservative in not fully exhuming the skeletal material from its context. We anticipate that many other researchers, including future investigators, will suggest additional methods to further test the hypothesis of burial, something that would be impossible if we had excavated the features in their entirety prior to publishing a description of our work. We believe strongly that our ethical responsibility is to publish the work and the most likely interpretation while leaving as much evidence in place as possible to enable further testing and replication. We welcome the suggestions of additional methods/analyses to test the H. naledi burial hypothesis.

This being said, we also observe that total exhumation would not resolve the concerns raised by the reviewers. The recommendation of total exhumation is in pursuit of a full account of all skeletal material present and its preservation and spatial situation, in order to demonstrate that they conform to body positions comparable to human burials. As has been highlighted in forensic casework, the excavation of an inhumation feature does not necessarily provide an accurate spatial or anatomical manifest of the stratigraphical relationships between the body, encapsulating matrix, and any cut present due to preservational, taphonomic and operational factors (Dirkmaat and Cabo, 2016; Hunter, 2014). In particular, in cases where skeletal elements are highly fragmented, friable, or degraded (such as through bioerosion) then complete excavation—even under controlled laboratory conditions—may destroy bone and severely limit skeletal identification (Henderson, 1997; Hochrein, 2002; Owsley and Compton, 1997), particularly in elements where the ratio of trabecular to cortical bone is high (Darwent and Lyman, 2002; Lyman, 1994). As such, non-invasive methods of 3D and 4D modelling (preservation in situ) are often considered preferable to complete necropsy or excavation (preservation by record) where appropriate (Bolliger and Thali, 2009; Dell’Unto and Landeschi, 2022; Randolph-Quinney et al., 2018; Silver, 2016). 

The test of burial is not primarily positional, but taphonomic and geological. The position and number of bones can elaborate on process-driven questions of decay and destruction in the burial environment, or post-mortem modification, but are not singularly indicative of whether the remains were intentionally buried – the post-mortem narrative of all the processes affecting the cadaveric island is required (Knüsel and Robb, 2016). In previous cases, researchers have disputed or accepted the hypothesis of intentional hominin burial based upon assumptions about how modern humans or Neandertals would have positioned bodies, with the idea that some positions reflect ritual intent while others do not. But applying such assumptions is unjustifiable, particularly for a species like H. naledi, whose culture may have differed fundamentally from our own. Our work acknowledges that the present evidence does not enable a full reconstruction of the burial positions, but it does show that fleshed remains were encased in sediment prior to decomposition of soft tissue, and that subsequent spatial changes can be most parsimoniously explained by natural decomposition within sedimentary matrix contained within a burial feature (after Green, 2022; Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022). If the argument is that extraordinary claims require extraordinary evidence, we feel that the evidence documents excavation and interment (and will do so more clearly in the revision) and the fact of the remains do not match a “typical” human burial in body positioning is not in itself evidence that these are not H. naledi burials.

We feel that the reviewers (in keeping with many palaeoanthropologists) have a clear idea of what they “think” a burial should look like in an idealised sense, but this platonic ideal of burial form is not matched by the extensive literature in archaeothanatology, funerary archaeology and forensic science which indicates enormous variability in the activity, morphology and post-mortem system experienced by the human body in cases of interment and body disposal (e.g. Aspöck, 2008; Boulestin and Duday, 2005 and 2006; Connelly et al., 2005; Channing and Randolph-Quinney, 2006; Cherryson, 2008; Donnelly et al., 1995; Finley, 2000; Hunter, 2014; Parker Pearson, 1999; Randolph-Quinney, 2013). Decades of experience in the identification, recovery and interpretation of clandestine, deviant, and non-formal burials indicates the platonic ideal is rare, and in many contexts, the exception (Cherryson, 2008; Parker Pearson, 1999). This variability is particularly relevant to morphological traits in burial context, such as the informal nature of the grave cut in plan and section, shallow burial depth, and initial disposition of body (placement) during the early post-mortem period. These might run counter to the expectations of reviewers or others referencing the fossil hominin record, but are well accepted within the communities of researchers investigating Holocene archaeological sites and forensic contexts.

It is encouraging to see reviewers beginning to incorporate the extensive (often experimentally derived) literature from archaeothanatology and forensic taphonomy in their deliberations, and we will be taking these comments on board going forward. In particular, we acknowledge reviewers’ comments and the need to construct a more detailed post-mortem narrative, accounting for joint disarticulation (labile versus persistent joints etc), displacement, and final disposition of elements within the burial space. As such we will incorporate the hierarchy of decomposition (rank order disarticulation), associations between regions of anatomical association, areas of disassociation, and the voids produced during decomposition (after Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022) into our narrative. In doing so we acknowledge the tensions between the inductive archaeolothanatological narrative-driven approach (e.g. Duday, 2005 & 2009) versus robust decomposition data derived from human forensic taphonomic experimentation recently articulated by Schotsmans and colleagues (2022) - noting that we will highlight comparative data based on forensic experimental casework and actualistic modelling over inductive intuitive approaches which come with significant evidential shortcomings (Bristow et al. 2011).

Finally, from a taphonomic perspective it is worth pointing out to reviewers that we have already addressed the issue of lack of taphonomic evidence for carnivore involvement in the formation of the Dinaledi assemblage (Dirks, et al., 2016). Absence of any carnivore-induced bone surface modifications, patterns of skeletal part representation, and a total absence of any carnivore remains found within the Dinaledi chamber (following Kuhn and colleagues, 2010) lead us to reject carnivores as possible vectors of body accumulation within the Dinaledi Chamber and Hill Antechamber.

Reviewers suggest that without a date derived from geochronological methods, the engravings cannot be associated with H. naledi, and that it is possible (or probable) that the engravings were done in the recent past by H. sapiens. This suggestion neglects the context of the site. We have previously documented the structure and extremely limited accessibility of the Dinaledi subsystem. This subsystem was not recorded on maps of the documented Rising Star Cave system prior to our work and its discovery by our teams. Furthermore, there is no evidence of prehistoric human activity in the areas of the cave related to possible subterranean entrances There is no evidence that humans in the past typically ventured into such extreme spaces like those of Rising Star. It is clear from the presence of the remains of many individuals that H. naledi ventured into these spaces again and again. It is likely that H. naledi moved through these spaces more easily than humans do based on their physique. We show that the engravings overlay each other suggesting multiple engraving events.  These engravings took time and effort and the only evidence for use of the Dinaledi subsystem by any hominin is by H. naledi. The context leads to the null hypothesis that H. naledi made the marks. In our revision, we will elaborate on this argument to clarify the evidence for our stance on this hypothesis. Several reviewers took issue with the title of the engraving paper as we did not insert a qualifier in front of the suggested date range for the engravings. We deliberately left out qualifying language so that the title took the form of a testable hypothesis rather than a weak assertation. Should future work find the engravings were not produced within this time range, then we will restate this hypothesis.

Finally, with regards to the engravings we have chosen to report them because they exist. Not reporting the presence of engraved marks on the walls of a cave above hypothesized burials would be tantamount to leaving relevant evidence out of the description of an archeological context. We recognize and state in our manuscript that these markings require substantial further study, including attempts at geochronological dating. But the current evidence is clearly relevant to the archaeological context of the subsystem. We take a similar stance with reporting the presence of the tool shaped artefact near the hand of the H. naledi skeleton in the Hill Antechamber. It is evident that this object requires further study, as we stated in our manuscript, but again omitting it from our study would be leaving out relevant evidence.

Some have suggested that the null hypothesis should be that all of these observed circumstances are of natural origin. Our team took this approach in our early investigation of the Dinaledi subsystem (Dirks et al. 2015). We adopted the null hypothesis that the geological processes involved in the accumulation of H. naledi skeletal remains were “natural” (e.g., non-naledigenic involvement), and we were able to reject many alternative explanations for the assemblage, including carnivore accumulation, “death trap” accumulation, and fluvial transport of bodies or bones (Dirks et al. 2015). This led us to the hypothesis that H. naledi were involved in bringing the bodies into the spaces where they were found. But we did not hypothesize their involvement in the formation of the deposit itself beyond bringing the bodies to the location.

This approach seems conservative. It followed the traditional view that small-brained hominins do not engage in cultural practices. But we recognize in hindsight that this null hypothesis approach did harm to our analyses. It impeded us from recognizing within our initial excavations of the puzzle box area and other excavations between 2014 – 2017 that we might be encountering remains that were intrusive in the sedimentary floor of the chamber. If we had approached the accumulation of a large number of hominins from the perspective of the null hypothesis being that the situation was likely cultural, we perhaps would have collected evidence in a slightly different manner. We certainly note that if the Dinaledi system had been full of the remains of modern humans, there would have been little doubt that the null hypothesis would have been that this was a cultural space and not a “natural space”.  We therefore respectfully disagree with the reviewers who continue to support the idea that we should approach hominin excavations with the null hypothesis that they will be natural (specifically non-cultural) in origins. If excavations continue with this mindset we believe that potential cultural evidence is almost certain to be lost.

There has been a gradient across paleoanthropological excavations, archaeological work, and forensic investigation, with increasing precision of context. The reality is that the recording precision and frame of approach is typically different in most paleontological excavations than in those related to contemporary human remains. If anything comes from the present discussion of whether the Dinaledi system is a burial site for H. naledi or not, we hope that by taking seriously the possibility of deep cultural dynamics of hominins, we will encourage other teams to meet the highest standards of excavation in order to preserve potential cultural evidence. Given H. naledi’s cranial capacity we suggest that even very early hominin skeletal assemblages should be re-examined, if there is sufficient evidence or records available.  These would include examples such as the A.L. 333 Au. afarensis site (the so called First Family site in Hadar Ethiopia), the Dikika infant skeleton, WT 15000 (Turkana Boy) and even A.L. 288 (Lucy) as such unusual taphonomic situations where skeletons are preserved cannot be simply explained away as “natural” in origin, based solely on the cranial capacity and assumed lack of cognitive and cultural complexity of the hominins as emphasized by us in Fuentes et al. (2023). We are not the first to observe that some very early hominin situations may represent early mortuary activity (Pettitt 2013), but we would advocate a step further. We suggest it may be damaging to take “natural accumulation” as the standard null hypothesis for hominin paleoanthropology, and that it is more conservative in practice to engage remains with the null hypothesis of possible cultural formation.

We are deeply grateful for the time and effort all of the 8 reviewers (across three reviews) have taken with this work.  We also acknowledge the anonymous reviewers from previous submissions who’s opinions and comments will have made the final iterations of these manuscripts better for their efforts. As this process is rather public and includes commentary outside of the eLife forum, we ask that the efforts of all 37 authors and 8 reviewers involved be respected and that the discourse remain professional in all venues as we study this fascinating and quite complex occurrence. We appreciate also the efforts of members of the public who have engaged with this relatively new process where preprints are posted prior to the reviews allowing comments and interactions from colleagues and the public who are normally not part of the internal peer review process.  We believe these interactions will make for better final papers. We feel we have met the standards of demonstrating burials in H. naledi and that the engraving are most likely associated with H. naledi. However, given the reviews we see many areas where our clarity and context, and analyses, were less strong than they can be. With the clarifications and additions taken on board through these review processes the final papers will be stronger and clearer. We, recognize that this is an ongoing process of scientific investigation and further work will allow continued, and possibly better, evaluation of these hypothesis and others.

Lee R Berger, Agustín Fuentes, John Hawks, Tebogo Makhubela

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Author Response:

We would like to thank the eLife reviewers for the considerable time and effort they have invested to review these manuscripts. We have also benefited from a previous round of review of the manuscript describing the proposed burial features, which underwent two rounds of revisions in a high-impact journal over a period of approximately 8 months during 2022 and early 2023. Both sets of reviews have reflected mixed responses to the evidence we have presented, with one reviewer recommending acceptance with minor editorial revisions, two recommending acceptance with minor revisions and the fourth recommending rejection based upon similar arguments to those reflected by some of the reviewers in this current round of reviews in eLife. Ultimately the managing editor of this first journal took the decision that the review process could not be completed in a timely manner and rejected the manuscript although the submission here reflected our consideration of these reviewers suggestions.

We have chosen in this initial response to the eLife reviews to include some references to the previous anonymous reviews in order to illustrate differences of opinion and differences in revision suggestions within the review process. Our goal is to offer maximal insight into our decision-making process and to acknowledge the considerable time and effort put into the assessment of these manuscripts by reviewers (for eLife and in the case of the earlier review process). We hope that this approach will assist the readers, and reviewers, of our manuscripts in understanding why we are proceeding with certain decisions during the revision process.

This is a new process for us and the reviewers, and one way in which it significantly differs from more traditional review is that both the reviews and our reply will be public well in advance of our revisions to the manuscript. Indeed, considering the scope of the reviews, some of those revisions may take considerable time, although many can be accomplished fairly easily. Thus, we are not in a position to say that we have solved every issue raised by the reviewers. Instead, we will examine what appear to be the key critical issues raised regarding the data and the analyses and how we propose to address these as we revise the papers. We will also address several philosophical and ethical issues raised by the reviews and our proposal for dealing with these. More specific editorial and citational recommendations will be dealt with on a case-by-case basis, and we do not address these point-by-point in this reply. Please note, this response to the reviewers is not the revision of the manuscript and is only the initial opinion of the corresponding authors with some guidance from the larger group of authors of all three papers. Our final submitted revision will reflect the input of all authors included on those submissions.

We took the decision to submit three separate papers consciously. The two different categories of evidence, burials and engravings, involve different kinds of analysis and different (although overlapping) teams of researchers, and we recognized that each deserved their own presentation and assessment. Meanwhile, together they inform the context of H. naledi in a way that requires some synthetic discussion, in which both kinds of evidence are relevant, leading to a third paper. But the mutual relevance of these different kinds of evidence and their review by a common set of reviewers naturally raises cross-cutting issues, and the reviewers have cross-referenced the three articles. This has sometimes led to suggestions about one manuscript based on the contents of another. Considering the situation, we accepted the recommendation that it would be clearer to consider all three articles in a single reply. Thus, while each of the three papers will proceed separately during the revision process, it will be necessary to highlight across all three papers occasionally in our responses.

Scientific Issues:

In reading the reviews, we feel there are 9 critical points/assertions raised by one or more of the reviewers that present a problem for, or challenge to, our hypothesis that the observed evidence (bone accumulations and engravings) described in the Dinaledi subsystem are of intentional naledigenic origin. These are:

1. The evidence presented does not demonstrate a clear interruption of the floor sediments, thus failing to demonstrate excavated holes.

2. The sediments infilling the holes where the skeletal remains are found have not been demonstrated to originate from the disruption of the floor sediments and thus could be part of a natural geological process (e.g. water movement, slumping) or carnivore accumulations.

3. Previous geological interpretations by our research group have given alternative geological explanations for formation of the bony accumulations that contradict the present evidence presented here and result in alternative origins hypotheses.

4. Burial cannot be effectively assessed without complete excavation of the features and site.

5. The skeletal remains as presented do not conform clearly to typical body arrangement/positions associated with human (Homo sapiens) burials.

6. There is no evidence of grave goods or lithic scatters that are typically associated with human burials.

7. Humans may have been involved with the creation of either the Homo naledi bone accumulations, the engravings, or both.

8. Without a date of the engravings, the null hypothesis should be the engravings were created by Homo sapiens.

9. The null hypothesis for explanation of the skeletal remains in this situation should be “natural accumulation”.

Our analysis of the Dinaledi Feature 1 leads us to accept that the laminated orange-red mudstone (LORM) sedimentary layer is interrupted, indicating a non-natural intervention, and that the hole created by the interruption was then filled by both a fleshed body (and perhaps parts of other bodies) which were then covered by sediment that originated from the hole that was dug. We recognize that the four eLife reviewers are not convinced that our presentation is sufficient to establish this. Interestingly, this was not the universal opinion of earlier reviewers of the initial manuscript several of whom felt we had adequately supported this hypothesis. The lack of clarity in this current version of the burial manuscript is our responsibility. In the upcoming revision of this paper to be submitted, we will take the reviewers’ critiques to heart and add additional figures that illustrate better the disruption of the LORM and clarify the sedimentological data showing the material covering the skeletal remains in the hole are the disrupted sediments excavated from the same hole. We are proposing to isolate this most critical evidence for burial into a separate section in the revised submission based on the reviewers’ comments. The fact that the LORM layer is disrupted, a fleshed body was placed in the hole created by this disruption, and the body (and perhaps parts of other bodies) was/were then covered by the same sediments from the hole is the central feature of our hypothesis that the bone accumulations observed reflect a burial and not a natural process.

The possibility of fluvial transport or involvement in the subsystem is a topic that we have addressed extensively in past work, and it is clear from these reviews that we must enhance our current manuscript to discuss this issue at greater length. Our previous work (Dirks et al. 2015; Dirks et al. 2017) emphasized that fluvial transport of whole bodies into the subsystem was precluded by several lines of sedimentological evidence. We excavated a rich accumulation of skeletal remains, including articulated limbs and other elements in subvertical orientations inconsistent with slow sedimentary infill, which were difficult to explain without positing either a large and dense pile of bodies and/or sediment movement. We encountered fractured chunks of laminated orange-red mudstone (LORM) in random orientations within our excavation area, within and among skeletal remains, which directly refuted that the remains were inundated with water at the time of burial, and this limited the possibility of fluvial transport. Water flow sufficient to displace bodies or complete skeletal evidence would also transport large and course sediment, which is absent from the subsystem, and would sort the commingled skeletal material that we found by size, which we do not observe. But our excavation only covered less than a square meter at very limited depth, and this was the limit to our knowledge of subsurface sediment. We thus were left with uncertainty that led us to suggest the possibility of sediment slumping or movement into subsurface drains, although these were not observed near our excavation. Our current work expands our knowledge of the subsurface and presents an alternative explanation for the disposition of skeletal remains from our earlier excavation. But we acknowledge that this new explanation is vulnerable to our own previous published proposals, and we must do a better job of explaining how the new information addresses our previous suggestions. By not clearly creating a section where we explained how these previous hypotheses were now nullified by new evidence, we clearly confused the reviewers with our own previous work. We will revise the manuscript by enhancing the review of the significant geological evidence demonstrating that there is no significant fluvial action in the system and making it clear how the burial hypothesis provides a clearer explanation for the situation of skeletal remains from our previous excavation work.

One of the central issues raised by reviewers has been a perceived need to excavate these features completely, totally exhuming all skeletal remains from them. Reviewers have written that it is necessary to identify every skeletal element that is present and account for any missing elements. On this point, we have both ethical and scientific differences from these reviewers. We express our ethical concerns first. Many of the best-preserved possible burials ever discovered by archaeologists were subjected to total excavation and exhumation. Cases like La Chapelle-aux-Saints, La Ferrassie, and Skhūl were fully excavated at a time when data recording and excavation methods did not include the range of spatial and geomorphological approaches that later became routine. The judgment of early investigators that these situations were intentional burials was challenged by later workers, and the kind of information that might enable better tests had been irrevocably lost (Gargett 1999; Dibble et al. 2015; Rendu et al. 2014).

Later, improved excavation standards have not sufficed to remove uncertainty or debate about possible burials. For example, it was long presumed that well-preserved remains of young children were by themselves diagnostic of intentional burial, such as those from Dederiyeh, Border Cave, or Roc de Marsal. Such cases were also fully excavated, with adequate documentation of the positioning of skeletal remains and their surrounding stratigraphic situation, but such cases were later challenged on several bases and the complete exhumation of material has confused or precluded testing of new hypotheses (e.g. Gargett 1999). The case of Roc de Marsal is one in which data from the initial excavation combined with data from the initial excavation combined with re-excavation and geoarchaeological analysis led to a naturalistic interpretation of the skeletal material (Sandgathe et al. 2011; Goldberg et al. 2017). But even in this case, the researchers erred in their interpretation of the skeleton’s situation due to a lack of identification of parts of the infant’s skeleton (Gómez-Olivencia and García-Martinez 2019). That is to say, it is not only the burial hypothesis but other hypotheses that suffer from complete excavation. Researchers concerned with preserving all possible information have sometimes taken extraordinary measures to remove and study possible burials at high-resolution in the laboratory. Such was the case of the Shanidar IV burial removed from the site and transported in plaster jacket by Solecki, which led to the disruption and loss of internal stratigraphic information (Pomeroy et al. 2020). Arguably, the current state of the art is full excavation with partial preparation, such as that undertaken at Panga ya Saidi (Martinón-Torres et al. 2021). But again, any future attempt to reinterpret or test the hypothesis of burial must rely on the adequacy of documentation as the original context has been removed.

In our decision to leave material in place as much as possible, we are expanding upon standard practice to leave witness sections and unexcavated areas for future research. The situation is novel, representing possible burials by a nonhuman species, and that makes it doubly important in our opinion to be conservative in not fully exhuming the skeletal material from its context. We anticipate that many other researchers, including future investigators, will suggest additional methods to further test the hypothesis of burial, something that would be impossible if we had excavated the features in their entirety prior to publishing a description of our work. We believe strongly that our ethical responsibility is to publish the work and the most likely interpretation while leaving as much evidence in place as possible to enable further testing and replication. We welcome the suggestions of additional methods/analyses to test the H. naledi burial hypothesis.

This being said, we also observe that total exhumation would not resolve the concerns raised by the reviewers. The recommendation of total exhumation is in pursuit of a full account of all skeletal material present and its preservation and spatial situation, in order to demonstrate that they conform to body positions comparable to human burials. As has been highlighted in forensic casework, the excavation of an inhumation feature does not necessarily provide an accurate spatial or anatomical manifest of the stratigraphical relationships between the body, encapsulating matrix, and any cut present due to preservational, taphonomic and operational factors (Dirkmaat and Cabo, 2016; Hunter, 2014). In particular, in cases where skeletal elements are highly fragmented, friable, or degraded (such as through bioerosion) then complete excavation—even under controlled laboratory conditions—may destroy bone and severely limit skeletal identification (Henderson, 1997; Hochrein, 2002; Owsley and Compton, 1997), particularly in elements where the ratio of trabecular to cortical bone is high (Darwent and Lyman, 2002; Lyman, 1994). As such, non-invasive methods of 3D and 4D modelling (preservation in situ) are often considered preferable to complete necropsy or excavation (preservation by record) where appropriate (Bolliger and Thali, 2009; Dell’Unto and Landeschi, 2022; Randolph-Quinney et al., 2018; Silver, 2016). 

The test of burial is not primarily positional, but taphonomic and geological. The position and number of bones can elaborate on process-driven questions of decay and destruction in the burial environment, or post-mortem modification, but are not singularly indicative of whether the remains were intentionally buried – the post-mortem narrative of all the processes affecting the cadaveric island is required (Knüsel and Robb, 2016). In previous cases, researchers have disputed or accepted the hypothesis of intentional hominin burial based upon assumptions about how modern humans or Neandertals would have positioned bodies, with the idea that some positions reflect ritual intent while others do not. But applying such assumptions is unjustifiable, particularly for a species like H. naledi, whose culture may have differed fundamentally from our own. Our work acknowledges that the present evidence does not enable a full reconstruction of the burial positions, but it does show that fleshed remains were encased in sediment prior to decomposition of soft tissue, and that subsequent spatial changes can be most parsimoniously explained by natural decomposition within sedimentary matrix contained within a burial feature (after Green, 2022; Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022). If the argument is that extraordinary claims require extraordinary evidence, we feel that the evidence documents excavation and interment (and will do so more clearly in the revision) and the fact of the remains do not match a “typical” human burial in body positioning is not in itself evidence that these are not H. naledi burials.

We feel that the reviewers (in keeping with many palaeoanthropologists) have a clear idea of what they “think” a burial should look like in an idealised sense, but this platonic ideal of burial form is not matched by the extensive literature in archaeothanatology, funerary archaeology and forensic science which indicates enormous variability in the activity, morphology and post-mortem system experienced by the human body in cases of interment and body disposal (e.g. Aspöck, 2008; Boulestin and Duday, 2005 and 2006; Connelly et al., 2005; Channing and Randolph-Quinney, 2006; Cherryson, 2008; Donnelly et al., 1995; Finley, 2000; Hunter, 2014; Parker Pearson, 1999; Randolph-Quinney, 2013). Decades of experience in the identification, recovery and interpretation of clandestine, deviant, and non-formal burials indicates the platonic ideal is rare, and in many contexts, the exception (Cherryson, 2008; Parker Pearson, 1999). This variability is particularly relevant to morphological traits in burial context, such as the informal nature of the grave cut in plan and section, shallow burial depth, and initial disposition of body (placement) during the early post-mortem period. These might run counter to the expectations of reviewers or others referencing the fossil hominin record, but are well accepted within the communities of researchers investigating Holocene archaeological sites and forensic contexts.

It is encouraging to see reviewers beginning to incorporate the extensive (often experimentally derived) literature from archaeothanatology and forensic taphonomy in their deliberations, and we will be taking these comments on board going forward. In particular, we acknowledge reviewers’ comments and the need to construct a more detailed post-mortem narrative, accounting for joint disarticulation (labile versus persistent joints etc), displacement, and final disposition of elements within the burial space. As such we will incorporate the hierarchy of decomposition (rank order disarticulation), associations between regions of anatomical association, areas of disassociation, and the voids produced during decomposition (after Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022) into our narrative. In doing so we acknowledge the tensions between the inductive archaeolothanatological narrative-driven approach (e.g. Duday, 2005 & 2009) versus robust decomposition data derived from human forensic taphonomic experimentation recently articulated by Schotsmans and colleagues (2022) - noting that we will highlight comparative data based on forensic experimental casework and actualistic modelling over inductive intuitive approaches which come with significant evidential shortcomings (Bristow et al. 2011).

Finally, from a taphonomic perspective it is worth pointing out to reviewers that we have already addressed the issue of lack of taphonomic evidence for carnivore involvement in the formation of the Dinaledi assemblage (Dirks, et al., 2016). Absence of any carnivore-induced bone surface modifications, patterns of skeletal part representation, and a total absence of any carnivore remains found within the Dinaledi chamber (following Kuhn and colleagues, 2010) lead us to reject carnivores as possible vectors of body accumulation within the Dinaledi Chamber and Hill Antechamber.

Reviewers suggest that without a date derived from geochronological methods, the engravings cannot be associated with H. naledi, and that it is possible (or probable) that the engravings were done in the recent past by H. sapiens. This suggestion neglects the context of the site. We have previously documented the structure and extremely limited accessibility of the Dinaledi subsystem. This subsystem was not recorded on maps of the documented Rising Star Cave system prior to our work and its discovery by our teams. Furthermore, there is no evidence of prehistoric human activity in the areas of the cave related to possible subterranean entrances There is no evidence that humans in the past typically ventured into such extreme spaces like those of Rising Star. It is clear from the presence of the remains of many individuals that H. naledi ventured into these spaces again and again. It is likely that H. naledi moved through these spaces more easily than humans do based on their physique. We show that the engravings overlay each other suggesting multiple engraving events.  These engravings took time and effort and the only evidence for use of the Dinaledi subsystem by any hominin is by H. naledi. The context leads to the null hypothesis that H. naledi made the marks. In our revision, we will elaborate on this argument to clarify the evidence for our stance on this hypothesis. Several reviewers took issue with the title of the engraving paper as we did not insert a qualifier in front of the suggested date range for the engravings. We deliberately left out qualifying language so that the title took the form of a testable hypothesis rather than a weak assertation. Should future work find the engravings were not produced within this time range, then we will restate this hypothesis.

Finally, with regards to the engravings we have chosen to report them because they exist. Not reporting the presence of engraved marks on the walls of a cave above hypothesized burials would be tantamount to leaving relevant evidence out of the description of an archeological context. We recognize and state in our manuscript that these markings require substantial further study, including attempts at geochronological dating. But the current evidence is clearly relevant to the archaeological context of the subsystem. We take a similar stance with reporting the presence of the tool shaped artefact near the hand of the H. naledi skeleton in the Hill Antechamber. It is evident that this object requires further study, as we stated in our manuscript, but again omitting it from our study would be leaving out relevant evidence.

Some have suggested that the null hypothesis should be that all of these observed circumstances are of natural origin. Our team took this approach in our early investigation of the Dinaledi subsystem (Dirks et al. 2015). We adopted the null hypothesis that the geological processes involved in the accumulation of H. naledi skeletal remains were “natural” (e.g., non-naledigenic involvement), and we were able to reject many alternative explanations for the assemblage, including carnivore accumulation, “death trap” accumulation, and fluvial transport of bodies or bones (Dirks et al. 2015). This led us to the hypothesis that H. naledi were involved in bringing the bodies into the spaces where they were found. But we did not hypothesize their involvement in the formation of the deposit itself beyond bringing the bodies to the location.

This approach seems conservative. It followed the traditional view that small-brained hominins do not engage in cultural practices. But we recognize in hindsight that this null hypothesis approach did harm to our analyses. It impeded us from recognizing within our initial excavations of the puzzle box area and other excavations between 2014 – 2017 that we might be encountering remains that were intrusive in the sedimentary floor of the chamber. If we had approached the accumulation of a large number of hominins from the perspective of the null hypothesis being that the situation was likely cultural, we perhaps would have collected evidence in a slightly different manner. We certainly note that if the Dinaledi system had been full of the remains of modern humans, there would have been little doubt that the null hypothesis would have been that this was a cultural space and not a “natural space”.  We therefore respectfully disagree with the reviewers who continue to support the idea that we should approach hominin excavations with the null hypothesis that they will be natural (specifically non-cultural) in origins. If excavations continue with this mindset we believe that potential cultural evidence is almost certain to be lost.

There has been a gradient across paleoanthropological excavations, archaeological work, and forensic investigation, with increasing precision of context. The reality is that the recording precision and frame of approach is typically different in most paleontological excavations than in those related to contemporary human remains. If anything comes from the present discussion of whether the Dinaledi system is a burial site for H. naledi or not, we hope that by taking seriously the possibility of deep cultural dynamics of hominins, we will encourage other teams to meet the highest standards of excavation in order to preserve potential cultural evidence. Given H. naledi’s cranial capacity we suggest that even very early hominin skeletal assemblages should be re-examined, if there is sufficient evidence or records available.  These would include examples such as the A.L. 333 Au. afarensis site (the so called First Family site in Hadar Ethiopia), the Dikika infant skeleton, WT 15000 (Turkana Boy) and even A.L. 288 (Lucy) as such unusual taphonomic situations where skeletons are preserved cannot be simply explained away as “natural” in origin, based solely on the cranial capacity and assumed lack of cognitive and cultural complexity of the hominins as emphasized by us in Fuentes et al. (2023). We are not the first to observe that some very early hominin situations may represent early mortuary activity (Pettitt 2013), but we would advocate a step further. We suggest it may be damaging to take “natural accumulation” as the standard null hypothesis for hominin paleoanthropology, and that it is more conservative in practice to engage remains with the null hypothesis of possible cultural formation.

We are deeply grateful for the time and effort all of the 8 reviewers (across three reviews) have taken with this work.  We also acknowledge the anonymous reviewers from previous submissions who’s opinions and comments will have made the final iterations of these manuscripts better for their efforts. As this process is rather public and includes commentary outside of the eLife forum, we ask that the efforts of all 37 authors and 8 reviewers involved be respected and that the discourse remain professional in all venues as we study this fascinating and quite complex occurrence. We appreciate also the efforts of members of the public who have engaged with this relatively new process where preprints are posted prior to the reviews allowing comments and interactions from colleagues and the public who are normally not part of the internal peer review process.  We believe these interactions will make for better final papers. We feel we have met the standards of demonstrating burials in H. naledi and that the engraving are most likely associated with H. naledi. However, given the reviews we see many areas where our clarity and context, and analyses, were less strong than they can be. With the clarifications and additions taken on board through these review processes the final papers will be stronger and clearer. We, recognize that this is an ongoing process of scientific investigation and further work will allow continued, and possibly better, evaluation of these hypothesis and others.

Lee R Berger, Agustín Fuentes, John Hawks, Tebogo Makhubela

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Un derecho implica necesariamente un deber para otro. Que un grupo social "A" tenga un derecho significa que el individuo u otro grupo social "B" tenga un deber respecto al primer grupo social "A". Si se otorgan derechos a grupos sociales se limita la libertad de otros, imponiendo su opinión o criterio de unos sobre otros. Si los derechos se aplican por igual a todos los individuos nadie se ve limitado ni se impone ninguna preferencia personal sobre nadie.

Porque eso depende de lo que quiera hacer cada individuo. Y en esto, no debe meterse el Estado.

El liberalismo considera que núcleo básico y esencial de todo es el individuo. Según esto es la sociedad. Pero un individuo o una pequeña familia puede vivir sin una sociedad alrededor. De forma aislada. Por lo tanto esta premisa es falsa.

Isn't that what would now be known as therapy?

La concentración de PSA tiene una sensibilidad muy superior, pero la especificidad es baja porque trastornos como la hipertrofia benigna de próstata o la prostatitis pueden producir elevaciones falsamente positivas, usando un umbral para el PSA de 4 ng/ml se pueden detectar el 70-80 % de los tumores

very nice

Esta la sacaría, se cruza con las respuestas de la primera pregunta

batería de preguntas ...

Tamaño letra

Creo que esta no va acá

Estas preguntas se pueden presentar en formato grilla o cuadro? para no repetir los encabezados.

O trabalho em parceria e o suporte com foco na solução estão intimamente ligados, e é durante as fases de definição de metas e estratégias que a assistência com foco na solução se evidencia.

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1. Onde se encontra neste momento em relação a este tema? 2

2. Onde gostaria de estar? 7

3. Como se sentirá quando lá chegar? Seria fantástico, sentir-se-ia orgulhosa, diminuiria o stress do bebé. Vai notar uma evolução no filho, são novos hábitos e conhecimentos.

4. O que já fez para chegar onde está? (...e o que mais?...) Tenta perceber quais os alimentos que o bebé gosta e procura oferecer esses alimentos em vez daqueles que ele rejeita. A novidade leva muitas vezes à rejeição, é necessário tempo, persistência e paciência para a construção de novos hábitos alimentares.

5. Qual seria um número realista para alcançar como próximo passo na escala? 4

6. Quando lá chegar, o que estará a fazer de diferente…? A Clara poderá sentar-se à mesa e comer algo com ele, assim estará a fazer aquilo que gostaria que ele fizesse. As crianças são o reflexo dos pais, se os pais se juntarem a estas novas aventuras, todos vão evoluir.

7. … e o que o(a) ajudará a fazer isso? (…e o que mais?...) Participar nas refeições será bom para os dois. Se a Clara estiver a comer, não vai querer apressá-lo tanto, o que também será melhor. E se comerem todos ao mesmo tempo, deixa de estar tão preocupada com os horários dos mais velhos. Criando uma harmonia em casa.

8. O que irá notar que mudou? (…e o que mais?...) A aprendizagem. Vai ficar menos impaciente e frustrada e o bebé não vai chorar tanto. Vai ser mais fácil a introdução de certos alimentos, porque com o stress o bebé também fica stressado e acaba por dispersar do foco no momento, a alimentação.

9. O que sentirá em relação a estas mudanças? Ele irá aceitar melhor os novos alimentos, o que acaba por trazer menos preocupação, será um alívio. Novos hábitos, mais conhecimento, melhor crescimento e evolução.

Author Response

Reviewer #1 (Public Review):

In this study, Shin and colleagues investigate the role of the posttranslational modification of the DNA methyltransferase by covalent linkage of the N-Acetylglucosamine (O-GlcNAc).

The authors present compelling evidence showing that a prolonged high fat/sucrose diet causes global protein O-GlcNAcylation in the liver and DNMT1 is among the proteins that increase their O-GlcNAc level. This result is significant because of the paucity of in vivo data addressing the interplay between metabolism and protein O-GlcNAcylation. The paper also shows that DNMT1's O-GlcNAcylation level correlated to the extracellular glucose levels in other cell types.

Using mass spectrometry, the authors identify S878 as the main site for O-GlcNAcylation. It is noteworthy that the mapping was performed with hyper-O-GlcNAcylated cells and may be different in a physiological situation. To investigate how O-GlcNAcylation of S878 of DNMT1 impacts its activity and ultimately DNA methylation patterns, Shin and colleagues mostly use a cellular model of hyper O-GlcNAcylation induced by the combination of high glucose and a chemical inhibitor of OGA (the only enzyme responsible for O-GlcNAc removal). The data shows that increased O-GlcNAcylation resulting from the combination of high glucose and OGA inhibition causes a reduction of DNMT1 activity and local loss of DNA methylation specifically at partially methylated domains.

This study brings completely new knowledge on the regulatory function of glycosylation of DNMT1 and its impact on its methyl-transferase activity and downstream genomic methylation. Furthermore, the manuscript introduces new data on the interplay between cellular metabolism and O-GlcNAcylation on DNMT1 and other proteins. The experiments are well-controlled, and their interpretation is sound. This study should be of special interest to the fields of fundamental and environmental epigenetics, as well as metabolism.

The main limitation of the study is the convolution of the functional experiments where the perturbation is a combination of high glucose and chemical inhibition of OGA. The relative contribution of the two variables is partially addressed in Figure 3-figure supplement 1B which shows that high glucose increases DNMT1 activity (Hep3B cells) while Figure 3D shows that high glucose when combined with OGA inhibitor decreases DNMT1 activity (Hep3B cells). As discussed, the data suggest that high-glucose and OGA inhibition may have an antagonistic effect on DNMT1 activity. An experiment of treatment of the cells with the OGA inhibitor in physiological glucose conditions would address this gap of knowledge.

We thank the reviewer for the suggestion. The physiological glucose levels are between 5 to 7 mM, and 25mM is in hyperglycemic range, which corresponds to severe diabetes. The new Figure 1A shows TMG treatment with physiological glucose conditions. We have included new WB data of 5mM glucose, 5mM glucose + TMG, 25mM glucose, and 25mM glucose + TMG (Figure 1A).

To understand the impact of the environment (in this study: extracellular glucose level) on the epigenome, one should keep in mind the variation of cytosine methylation patterns between individuals and over time. A recent large-scale profiling of DNA methylation of 137 individuals shows a near absence of individual variation between replicates of the same cell type, suggesting that genomic methylation patterns are largely insensitive to the environment (https://doi.org/10.1038/s41586-022-05580-6).

Comparative methylomes of healthy and diabetic individuals are needed to examine the medical significance of the findings presented here. It is possible that the modulation of DNMT1 activity by O-GlcNAc modification is relevant for a specific cell type or developmental stage that remains to be discovered.

We thank the reviewer for the suggestion. While the present study is focused on the functional impact of glucose concentrations on O-GlcNAcylation of DNMT1, the extension of this work to diabetic individuals is a goal for a follow up project.

Reviewer #2 (Public Review):

I've read the manuscript by Shin et al with great interest. The authors describe the identification of O-GlcNAcylation of DNMT1 and the impact this modification has on the maintenance activity of DNMT1 genome-wide and that modification of S878 leads to enzyme inhibition. The manuscript is written in a clear and understandable way making it easy for the reader to understand the logic as well as the steps of the experimental approach.

The authors identify O-GlcNAcylation of DNMT1 in a number of different cell lines by combining inhibition studies and WB and further on they identify the modification sites with LC/MS, predictions, and mutational studies. I really like the experimental approach, which while being straightforward (albeit technically challenging), is powerful and well-controlled in this case to unequivocally prove the modification of DNMT1 and identify the site. However, mutation of the two identified modification sites does not remove all the O-GlcNAcylation signal associated with DNMT1, thus possibly not all the possible sites were identified. While this is not a criticism of this manuscript, it would be interesting to know what other sites are modified and the enzymatic/biological effects associated.

We completely agree with the reviewer. As the O-GlcNAc band was also detected in double mutated DNMT1 (Figure 2D), it is expected that undetected O-GlcNAcylated sites will exist. This is a limitation of current MS analysis and is known to be difficult to detect in the case of modified sites located at both 5’- and 3’- ends of the protein or around the site cut by endoprotease such as trypsin. In follow up work we plan to detect more diverse O-GlcNAc modified sites using more types of endoproteases and observe changes in the phenotype of various cells accordingly.

Also, the authors isolate the modified DNMT1 from cells using immunoprecipitation, which is indeed useful to study the changes in catalytic activity but does not provide any information if the cellular localisation of modified DNMT1 changes.

We apologize for this oversight. We have added a DNMT1 localization assay via immunofluorescence (IF) in the revised manuscript (Figure 3—figure supplement 3). We found no difference in DNMT1 localization between wild type and S878A mutants.

Subsequently, the authors checked the impact of high glucose diet on the genome-wide DNA methylation patterns. The observed effects (Fig 4A) are very strong, almost as strong as observed with Aza treatment and therefore I wonder if LINE/IAP or other elements are getting activated (as observed with genome-wide demethylation with Aza).

We thank the reviewer for the suggestion. Changes in methylation of LINE-1 by hyperglycemia condition are displayed in Figure 4—figure supplement 4. In the case of LINE-1, DNA methylation is lost globally in hyperglycemia conditions. While beyond the scope of this study, a more thorough examination of the impact of the observed loss of methylation under high glucose conditions is of interest.

Do the authors see any changes in cell phenotype, slower/faster proliferation, or increased apoptosis due to the activation of mobile elements (not only ROS)?

This is also a very interesting idea. We plan on further investigating this as part of a follow up study.

Another point is that the S878A mutant seems not to be able to fully maintain the DNA methylation (Fig 4A). Does O-GlcNAcylation recruit any additional interactors? Given that the authors immunoprecipitated DNMT1 and use it for activity assay, it is possible, that the modification attracts an additional protein factor that could in turn inhibit DNMT1 activity (as observed). Therefore, the observed kinetic effect could be indirect, while still interesting and important, the mechanism of inhibition would be different.

We thank the reviewer for the great suggestions. According to Figure 4A, in the case of mutated DNMT1, a slight methylation loss appears to occur in both conditions. There could be for a number of reasons. It may be due to interacting proteins or it may be caused by some damage of DNMT1 itself. A further investigation of this is planned as a follow up project.

DNA methylation clock can be used to estimate the biological age of a tissue/cells. While not directly in the line of the manuscript, I was wondering if the DNA methylation changes in the high glucose diet would affect the methylation sites used for the DNAme clock. Meaning, would the cells/tissue epigenetically age faster when in high glucose media, and if the Ala mutant could provide resistance to that?

We thank the reviewer for the interesting suggestion. We believe this is beyond the scope of this manuscript, but we'll consider this with interest in the future.

In discussion, the authors write that this is the first investigation of O-GlcNAcylation in relation to DNA methylation, while this is true for DNMTs, TET enzymes, that oxidise 5mC and trigger active DNA demethylation have been shown before to also be modified.

We have toned down the language throughout the revised manuscript. This is the first investigation into the maintenance of DNA methylation. Although there is a great deal of evidence regarding the important regulatory role of O-GlcNAcylation in gene regulation, a direct link with maintenance of DNA methylation has not previously been established.

A nice and rigorous study, with important observations and connections to biological effects. It would be nice to prove that the effects are direct and not associated with other factors that could be recruited by the modification and impact the activity of DNMT1. I find it a bit surprising that phosphorylation of the target serine does not impact DNMT1 activity as well.

We thank the reviewer for the positive comments and agree that there are many interesting avenues to follow up on this.

Reviewer #3 (Public Review):

The authors investigate the potential effect of OGlcNacylation on the activity of the DNA methyltransferase DNMT1.

Some results that are convincingly obtained include:

• There is more overall OGlcNacylation when Glucose concentration in the culture medium or the feed is high;

• DNMT1 is OGlcNacylated, and more so in high glucose or on rich chow;

• The position S878 can be OGlcNacylated;

• The activity of transfected DNMT1 is decreased in high glucose conditions. This effect is lessened when S878 is mutated to A or D.

Some results that are suggested but not fully backed by experimental data include:

• This process happens to the endogenous protein under physiologically relevant conditions;

We agree that we could not completely rule out endogenous DNMT1 in our experiments. We have adjusted the language in the revised manuscript to acknowledge this. However, we confirmed the change in activity of recombinant DNMT1 (Figure 3D), and also demonstrated the change in activity under physiological conditions (normal physiological glucose level vs hyperglycemic range) in Figure 3—figure supplement 1B. This is a result that directly shows that the activity of DNMT1 changes under physiological conditions. In addition, DNA hypomethylation due to high glucose has been previously reported, already (Kandilya et al., 2020; Lan et al., 2016). Our results suggest a possible mechanism for this.

Kandilya, D., Shyamasundar, S., Singh, D.K., Banik, A., Hande, M.P., Stunkel, W., Chong, Y.S., and Dheen, S.T. (2020). High glucose alters the DNA methylation pattern of neurodevelopment associated genes in human neural progenitor cells in vitro. Sci Rep 10, 15676.

Lan, C.C., Huang, S.M., Wu, C.S., Wu, C.H., and Chen, G.S. (2016). High-glucose environment increased thrombospondin-1 expression in keratinocytes via DNA hypomethylation. Transl Res 169, 91-101 e101-103.

• This process is responsible for changes in DNA methylation, leading to changes in gene expression, leading to increased ROS and increased apoptosis.

We confirmed that ROS levels increased under high glucose conditions through DCFH-DA fluorescence experiments (Figure 5A). In addition, γH2A.X fluorescence experiments showed that DNA damage was increased under high glucose conditions (Fig. 5B). On the other hand, in the case of the S878A mutant, DNA damage was reduced under hyperglycemic conditions compared to wild type DNMT1 despite an increase in ROS levels (Fig. 5B). Moreover, we verified that the DNA damage did not come from oxidative stress through 8-OHdG analysis (Figure 5—figure supplement 4). Therefore, DNA oxidative stress is suppressed by DNMT1 due to the increase of ROS under high glucose conditions. However, the reduction of DNA methylation by O-GlcNAcylation of DNMT1 induces apoptosis due to oxidative stress.

Studying the connection between cellular metabolism and epigenetic phenomena is interesting. However, I feel that the article falls short of its aims because of the limits of the experimental system, some missing controls, and some data overinterpretation.

We hope the reviewer finds our revised manuscript more suitable.

eLife assessment

This study explores the regulatory function of O-GlcNAcylation on DNA methyltransferase 1 and identifies serine 878 as the main target. This study is of interest to those in epigenetics and metabolism. The significance is important and the strength of the evidence is convincing.

Reviewer #1 (Public Review):

In this study, Shin and colleagues investigate the role of the posttranslational modification of the DNA methyltransferase by covalent linkage of the N-Acetylglucosamine (O-GlcNAc).

The authors present compelling evidence showing that a prolonged high fat/sucrose diet causes global protein O-GlcNAcylation in the liver and DNMT1 is among the proteins that increase their O-GlcNAc level. This result is significant because of the paucity of in vivo data addressing the interplay between metabolism and protein O-GlcNAcylation. The paper also shows that DNMT1's O-GlcNAcylation level correlated to the extracellular glucose levels in other cell types.

Using mass spectrometry, the authors identify S878 as the main site for O-GlcNAcylation. It is noteworthy that the mapping was performed with hyper-O-GlcNAcylated cells and may be different in a physiological situation. To investigate how O-GlcNAcylation of S878 of DNMT1 impacts its activity and ultimately DNA methylation patterns, Shin and colleagues mostly use a cellular model of hyper O-GlcNAcylation induced by the combination of high glucose and a chemical inhibitor of OGA (the only enzyme responsible for O-GlcNAc removal). The data shows that increased O-GlcNAcylation resulting from the combination of high glucose and OGA inhibition causes a reduction of DNMT1 activity and local loss of DNA methylation specifically at partially methylated domains.

This study brings completely new knowledge on the regulatory function of glycosylation of DNMT1 and its impact on its methyl-transferase activity and downstream genomic methylation. Furthermore, the manuscript introduces new data on the interplay between cellular metabolism and O-GlcNAcylation on DNMT1 and other proteins. The experiments are well-controlled, and their interpretation is sound. This study should be of special interest to the fields of fundamental and environmental epigenetics, as well as metabolism.

The main limitation of the study is the convolution of the functional experiments where the perturbation is a combination of high glucose and chemical inhibition of OGA. The relative contribution of the two variables is partially addressed in Figure 3-figure supplement 1B which shows that high glucose increases DNMT1 activity (Hep3B cells) while Figure 3D shows that high glucose when combined with OGA inhibitor decreases DNMT1 activity (Hep3B cells). As discussed, the data suggest that high-glucose and OGA inhibition may have an antagonistic effect on DNMT1 activity. An experiment of treatment of the cells with the OGA inhibitor in physiological glucose conditions would address this gap of knowledge.

To understand the impact of the environment (in this study: extracellular glucose level) on the epigenome, one should keep in mind the variation of cytosine methylation patterns between individuals and over time. A recent large-scale profiling of DNA methylation of 137 individuals shows a near absence of individual variation between replicates of the same cell type, suggesting that genomic methylation patterns are largely insensitive to the environment (https://doi.org/10.1038/s41586-022-05580-6).

Comparative methylomes of healthy and diabetic individuals are needed to examine the medical significance of the findings presented here. It is possible that the modulation of DNMT1 activity by O-GlcNAc modification is relevant for a specific cell type or developmental stage that remains to be discovered.

Reviewer #2 (Public Review):

I've read the manuscript by Shin et al with great interest. The authors describe the identification of O-GlcNAcylation of DNMT1 and the impact this modification has on the maintenance activity of DNMT1 genome-wide and that modification of S878 leads to enzyme inhibition.<br /> The manuscript is written in a clear and understandable way making it easy for the reader to understand the logic as well as the steps of the experimental approach.

The authors identify O-GlcNAcylation of DNMT1 in a number of different cell lines by combining inhibition studies and WB and further on they identify the modification sites with LC/MS, predictions, and mutational studies. I really like the experimental approach, which while being straightforward (albeit technically challenging), is powerful and well-controlled in this case to unequivocally prove the modification of DNMT1 and identify the site. However, mutation of the two identified modification sites does not remove all the O-GlcNAcylation signal associated with DNMT1, thus possibly not all the possible sites were identified. While this is not a criticism of this manuscript, it would be interesting to know what other sites are modified and the enzymatic/biological effects associated.

Also, the authors isolate the modified DNMT1 from cells using immunoprecipitation, which is indeed useful to study the changes in catalytic activity but does not provide any information if the cellular localisation of modified DNMT1 changes. Subsequently, the authors checked the impact of high glucose diet on the genome-wide DNA methylation patterns. The observed effects (Fig 4A) are very strong, almost as strong as observed with Aza treatment and therefore I wonder if LINE/IAP or other elements are getting activated (as observed with genome-wide demethylation with Aza). Do the authors see any changes in cell phenotype, slower/faster proliferation, or increased apoptosis due to the activation of mobile elements (not only ROS)? Another point is that the S878A mutant seems not to be able to fully maintain the DNA methylation (Fig 4A). Does O-GlcNAcylation recruit any additional interactors? Given that the authors immunoprecipitated DNMT1 and use it for activity assay, it is possible, that the modification attracts an additional protein factor that could in turn inhibit DNMT1 activity (as observed). Therefore, the observed kinetic effect could be indirect, while still interesting and important, the mechanism of inhibition would be different.

DNA methylation clock can be used to estimate the biological age of a tissue/cells. While not directly in the line of the manuscript, I was wondering if the DNA methylation changes in the high glucose diet would affect the methylation sites used for the DNAme clock. Meaning, would the cells/tissue epigenetically age faster when in high glucose media, and if the Ala mutant could provide resistance to that?

In discussion, the authors write that this is the first investigation of O-GlcNAcylation in relation to DNA methylation, while this is true for DNMTs, TET enzymes, that oxidise 5mC and trigger active DNA demethylation have been shown before to also be modified.

A nice and rigorous study, with important observations and connections to biological effects. It would be nice to prove that the effects are direct and not associated with other factors that could be recruited by the modification and impact the activity of DNMT1. I find it a bit surprising that phosphorylation of the target serine does not impact DNMT1 activity as well.