for - paper - Characteristic processes of human evolution caused the Anthropocene and may obstruct its global solutions (2023) - author - Timothy M. Waring - Zachary T. Wood - Eörs Szathmáry
SRG comment - validation that cultural evolution must make a dramatic shift because - the patterns of cultural evolution that brought human civilization to modernity and the Anthropocene - could end up destroying it - progress trap - cultural evolution - patterns of existing cultural evolution and progress could be our ultimate progress trap
10,000 Matching Annotations
- Dec 2025
-
royalsocietypublishing.org royalsocietypublishing.org
-
-
characteristic patterns of human group-level cultural evolution created the Anthropocene and will work against global collective solutions to the environmental challenges it poses
for - cultural evolution - futures - cannot be like the past
-
We conclude that our species must alter longstanding patterns of cultural evolution to avoid environmental disaster and escalating between-group competition.
for - cultural evolution - futures - directional change<br /> - our species must alter longstanding patterns of cultural evolution to avoid - environmental disaster and - escalating between-group competition
- SRG comment - validation for cultural change from traditional patterns that brought us to the anthropocene
Tags
- SRG comment - validation for cultural change from traditional patterns that brought us to the anthropocene
- Timothy M. Waring
- SRG comment - validation that cultural evolution must make a dramatic shift
- Zachary T. Wood
- progress trap - cultural evolution
- paper - Characteristic processes of human evolution caused the Anthropocene and may obstruct its global solutions (2023)
- cultural evolution - futures - directional change
- Eörs Szathmáry
- cultural evolution - futures - cannot be like the past
Annotators
URL
-
-
umaine.edu umaine.edu
-
Applied Cultural Evolution Laboratory at the University of Maine
for - to - Applied Cultural Evolution Laboratory - University of Maine - https://hyp.is/aVSgkNf2EfChbceMGSBicA/timwaring.info/
-
-
nmoer.pressbooks.pub nmoer.pressbooks.pub
-
1. Allow your prewriting to be exploratory. Reflective writing encourages you to explore an experience and explain or ponder the individual choices you have made. Stand back and view the experience from an objective point of view. 2. While reflective writing asks you to write about your own experience, you should be as thorough as you would for any other writing task. Remember to keep your reader in mind. Try to remove your emotions from the experience. Rather than blame yourself for a specific choice, consider the reasoning for that decision and explore what you’ve learned. 3. Avoid focusing on writing about every moment of the event or process. Reflective writing should focus on specific snapshots of your experience, so avoid spending too much time narrating. Instead, reflect on how a specific choice impacted the experience. Ultimately, your essay’s goal is not to create a narrative but to speculate about the significance of your experience.
Keep these tips in mind when it comes to writing about your own reflections.
-
The goal with reflective writing is to help students become more self-aware of their strengths and weaknesses as a writer. Self-awareness helps students tune in to what lessons/concepts they need to study the most. But reflective writing is not only limited to reflecting on your writing process.
being self aware is something that I find to be the most helpful when it comes to making writings with the intention to share with others.
-
-
journals-scholarsportal-info.myaccess.library.utoronto.ca journals-scholarsportal-info.myaccess.library.utoronto.ca
-
Indeed, homes are spaces where the intimacy among family members can open up daily opportunities to “see, hear, think and act differently” (Armstrong, 1995) as we redefine what it means to decolonize and to queer our homes, bodies, and relationships.
Closing statement: home is where decolonial and queer group lives daily, it's important to see that decolonization happens within family settings rather than just political spaces.
-
we call on non-Indigenous queers to think about a politics of accountability instead of a politics of inclusion, asking how we might perform queerness differently within a decolonial praxis. Given that White supremacy operates through multiple logics, our politics of accountability requires that we examine the interlocking nature of these logics and the related complicities, not only focusing on how we are oppressed (Smith, 2010a).
Main point: Inclusion alone is not enough if it doesn't represnet the complicity in colonial structures.
-
we are challenged to avoid speaking for our loved ones and our relatives without their consent while also creating spaces in which we can be called on as allies when desired.
The important ethical question in intimate relationships. To reflect in family settings, it's important not assume what we know about others' needs.
-
Within the spaces of our homes, families, and communities, we have opportunities to develop long-term processes of challenging our assumptions about how we perform queerness, Whiteness, Indigeneity, cisgender privilege, and a whole range of other identities.
Decolonial work happens over the long term and within family settings like our homes, whiteness, indigeneity and privilege should be examined.
-
“solidarity and allyship are great in theory but when imposed they replicate the same oppression we’re resisting” (n.p.).
The definition of "consensual allyship.
-
We have not provided a clear path or model for allyship, queering nor decolonization. Rather, we hope to have exposed some of the tensions, dynamics, and unresolved questions at the heart of decolonial processes in our everyday queer lives. Within the spaces of our homes, families, and communities, we have opportunities to develop long-term processes of challenging our assumptions about how we perform queerness, Whiteness, Indigeneity, cisgender privilege, and a whole range of other identities.
The author mentioned that it's important to note the tension and uncertainty in decolonial processes.
-
t is important to make space for these and other uncomfortable conversations as a necessary part of resisting racial amnesia (Razack, 2002) and the familiar White settler narratives of innocence and denial of conquest and genocide, and thus moving toward White settler practices of accountability to Indigenous people and people of color in Canada.
It's powerful to validate the emotional discomfort; it is not failure, rather it is a requirement of accountability and important for decolonization.
-
K: I love you Mommy.C: I love you too.K: Do you love everyone in our family?C: Of course! I love you, Mum, Rosebud, Nana, all of our aunts, uncles, cousins, nephews, and our friends who are family to us, like Aunt Caroline.K: And our ancestors?C: Oh yes! I love our ancestors too.K: Well not all of them … we don't love our England ancestors who came over here and did the whole stealing the land from the Aboriginal people.C: Oh … those ancestors. Umm, right. Well, that's a really good question. Do we love people who do things that are wrong? Could we hate what they did, but love the people?K: Well, I’m not sure. I care about the Aboriginal people and what happened to them.C: I do too. That's a really interesting and important question you are asking.K: Yeah.C: It's not easy to figure that out, is it?K: No, it isn’t.
Decolonial work happens through kitchen tables, through parenting or just simple conversations.
-
How can White queers talk about allyship without positioning themselves as better than other White queers, or without perpetuating narratives that maintain White supremacy by propping up White settlers as benevolent saviors of the “oppressed other”? While sharing stories of the everyday practices of White settlers resisting White supremacy may be a necessary part of decolonization, it is not a neutral or uncomplicated process.
The need for self-critique is crucial, made me think how within family settings, your intention does not remove the impacts.
-
However, the allyship is not just one way, as Sarah also demonstrates allyship with her single mother, who raised Sarah while struggling to make ends meet.
Challenges the surface idea of helper and helped.
-
critical self-reflexive conversations among White family members around the kitchen table are insufficient on their own. Once again, this highlights the importance of embracing a “both/and” approach to allyship that honors the intimate and often invisible practices of decolonization in home and family spaces, but connects them with other acts of solidarity with Indigenous people taking place across much wider sociospatial contexts as well. Participating in public rallies, marches, and direct actions supporting Indigenous sovereignty, attending Indigenous art exhibits, visiting Indigenous cultural centers, signing petitions, writing letters, making monetary donations, and volunteering to support Indigenous initiatives are only a few of the other forms of solidarity that must accompany acts of decolonial allyship in the White queer settler family home. In this way, we can also see how these geographies of allyship are relational.
Author emphasized that reflections in private should be connected with public actions. Allyship needs to exist in both intimate and public contexts. Which represents the idea that decolonial work is relational.
-
However, there remains a disturbing lack of commitment by White settlers to challenging racism and colonialism in queer and trans communities (including within friendships and intimate relationships) and practicing a politics of accountability to Indigenous people and people of color. A decolonial queer praxis requires that we continue to ask why these questions are marginal to discussions of allyship in queer relationships. Additionally, in the context of their relationships, White settler queer and trans people can challenge one another to think about how issues such as home ownership and parenting can become spaces to think through a decolonial queer politic.
The author presented that White settlers tend to ignore responsibility; they think that allyship should involve confronting when it comes to racism and colonialism, especially in intimate relationships, like between partners, as well as friendships.
-
Our friendship developed initially within the context of our scholarly work, but it was deepened through conversations that made clear our mutual interests in issues of violence, power, and colonialism. We also shared involvement in queer and social justice circles in Coast Salish territories, and developed several collaborations, which allowed both our personal connection, and academic collaborations to deepen. Thus, our friendship developed through undertaking collaborative action to foster cross-cultural conversation about colonialism, violence, gender, and space, rather than just acknowledging these shared interests on an intellectual level.
This is important as it shows that allyship requires actual action and consistent engagement rather than just an agreement. Which made me think in my own life, the actions from those that supposed to share responsibility with me.
-
We have chosen stories that raise questions and tensions, in order to center the dynamic, messy quality of relationships among individuals who are engaged in processes of unsettling dominant power dynamics and colonial ideologies. The material reality of colonial dispossession is often overlooked in conversations in non-Indigenous queer and trans communities about creating a sense of home and safety in these lands.
The author tries to focus on the tension and the messy quality to reflect that both feminists and declonial works value lived experiences.
-
Similarly, the term Two-Spirit has been used to reflect the identities of Indigenous people who embody both masculine and feminine spirits and qualities within Indigenous knowledge paradigms which do not operate through dichotomous systems
The two-spirit term is important to reflect on, and the idea that decolonization needs to reimagine how identity and knowledge are understood.
-
ndigenous languages contain within them place-specific terminology that reflects the spiritual, social, and political position of variously gendered people within individual nations. Recalling these diverse systems of gender pushes at the limits of the English language (Driskill, Finley, Gilley and Morgensen, 2011). However, many queer and trans Indigenous people now use the term Two-Spirit to express what are a varied and complex array of gender and sexual identities rooted in Indigenous worldviews and lived experience
It's important to know that language itself is a form of colonization so it is especially important to note indigenous terms and use them as is.
-
olonialism is, at its heart, a project intent on the erasure of Indigenous peoples. Colonial policies imposed sociolegal categories that were defined and managed in ways that were intended to lead to fewer and fewer Native people over time. Inherent in this project of erasure was the imposition of a binary system of gender which simultaneously imposed Indigenous rights and status along heterosexual lines and suppressed Indigenous systems of gender that went far beyond the gender binary. Yet Indigenous queers persisted. Decolonization involves actively challenging or disrupting systems of knowledge that do not fully account for the lives of Indigenous people, queer and trans people, and many others whose lives are erased through epistemic and material violence (Hunt, forthcoming). We suggest that this decolonization is already active in embodiments of Indigeneity and queer gender and sexuality, yet this ongoing resistance to colonialism often goes unseen within queer spaces. Accounting for Indigenous expressions of gender and sexuality requires acknowledging that the ongoing colonial categorization of Indigenous peoples and their identities is integral to the denial of Indigenous self-determination.
This explained how, despite the colonial policies ignores queer spaces, or trying to erase the inequalities they experience, and how colonial violence is tied to gender inequalities
-
Within our own families, we have found solidarity across difference to hold great possibility for enacting a decolonial queer praxis. And yet we acknowledge there are multiple meanings and experiences of home on unceded land in the context of ongoing colonialism. In many contexts “the family home” may not a place of comfort and refuge, but may be a site of oppression, violence, and surveillance; for example, the heterosexual or cisgender family home for LGBTQ2S people, or non-Indigenous foster homes for Indigenous children. However, as we discuss here, family homes can also be sites of resistance, critical dialogue, support, and allyship.
This idea really stood out to me, as the author challenged the tranditional idea which is political work only occurs in public settings.
-
Reagon criticized feminists (primarily White and lesbian feminists) for trying to create homogeneous “safe” spaces based on narrow identity politics, arguing instead that it was necessary to move outside of the comfort zones of sameness and to practice solidarity in uncomfortable places including the streets. Although written in 1981, Reagon's speech is cited in current discussions about allyship in Canada on the topic of Indigenous–settler solidarity.
Especially in Canada, it's important to connect decolonization with what happened to the indigenous community with the right history and future.
-
In writing about the everydayness of decolonization, we acknowledge that Indigenous peoples’ resistance to colonialism has unfolded in daily acts of embodying and living Indigeneity, honoring longstanding relationships with the land and with one another. While large-scale actions such as rallies, protests and blockades are frequently acknowledged as sites of resistance, the daily actions undertaken by individual Indigenous people, families, and communities often go unacknowledged but are no less vital to decolonial processes. As scholar-activists, we ask how we might locate our solidarity within and across various family and community spaces, rather than only in more visible activist sites. In doing so, we also value the quiet, relational processes of change that are necessary for non-Indigenous queers, and all non-Indigenous people who seek to align themselves with Indigenous struggles. Here, we connect these relational decolonial processes to queer, Two-Spirit and trans solidarity, resistance to heteronormativity and cisnormativity, locating these intersections in practices of decolonizing and queering the intimate geographies of the family and the home. The stories, conflicts, tensions, celebrations and connections inherent in these processes are at the heart of the conversations through which this article was written.
It's important to note that decolonization is something through lived experience. It's always been talked in scholar spaces which might people the illusion that it's the past when it's the present also.
-
We follow Tuck and Yang (2012) in observing that conversations using the language of decolonization often make no mention of Indigenous knowledge or peoples, nor any recognition of the immediate context of settler colonialism on the lands where these conversations take place.
I learned about Tuck and Yang in our DTS201 class, where Professor Matthew mentioned that decolonization is crucial to indigenous lives(Matthew,2025).
-
yet their analysis and leadership are often marginalized within both LGBTQ and Indigenous scholarship, including in accounts of contemporary Two-Spirit community-building.
As much as they do so much work but their work goes undervalued in academic settings, just like in family settings, particular groups's emotional and labor work goes unnoticed even though it is the tie to hold the family together.
-
For over three decades, Indigenous lesbian and Two-Spirit women authors such as Beth Brant (1994), Chrystos (1988; 1995), and Paula Gunn Allen (1986) have illustrated through their poetry, essays, and fiction, the interlocking nature of heteronormativity and settler colonialism, the history of Indigenous lesbian and Two-Spirit resistance and the tensions and possibilities of coalition building between non-Indigenous LGBTQ and Indigenous communities.
Indigenous lesbian/queer group have been trying to do what they do for a much longer time, which also reflects how the wisdoms from older generations pass down to the newer generations within communities/groups.
-
Some scholars and activists have described this as “coalitional queer politics,” “queer intersectionality,” and/or “postcolonial queer.” These terms critique “politics of inclusion” and depoliticized articulations of intersectionality that ignore hierarchical power relations and reinscribe White, colonial, gender, and sexual norms. This is also referred to as “queer of color critique” to foreground the theories and activism of LGBTQ2S people of color and Indigenous people and that examines the interlocking nature of race, sexuality, and gender.
Language is changeable but all works towards one goal.
-
-
-
69com is a leading online betting destination in the Asian market, fully converging high-class facilities and entertainment services
69com - 69com.org, Nha Cai Uy Tin #1 Tham Gia Tang Ngay 69K
Dia chi: 136 Nguyen Thai Binh, Phuong Nguyen Thai Binh, Quan 1, Ho Chi Minh, Viet Nam
Email: enchepqrrulenny@gmail.com
Website: https://69com.org/
Dien thoai: (+84) 914640071
69_com #69com #69comorg #nhacai69com #casino69com
Social Links:
https://69com.org/dang-ky-69com/
https://69com.org/nap-tien-69com/
https://69com.org/rut-tien-69com/
https://69com.org/tai-app-69com/
https://69com.org/dieu-khoan-dich-vu/
https://69com.org/gioi-thieu-69com/
https://69com.org/chinh-sach-bao-mat/
https://69com.org/quyen-rieng-tu/
https://69com.org/the-thao-69com/
https://69com.org/casino-69com/
https://69com.org/ban-ca-69com/
https://69com.org/no-hu-69com/
https://www.facebook.com/69comorg/
https://www.youtube.com/channel/UC-RpkLzYI6iTVenBgN9enqQ
https://www.reddit.com/user/69comorg/
https://www.pinterest.com/69comorg/
https://ameblo.jp/69comorg/entry-12914676094.html
https://www.band.us/band/99180656/intro
https://www.blogger.com/profile/11538688814972708009
https://orevel013.wixsite.com/69comorg
https://www.tumblr.com/69comorg
https://69comorg.wordpress.com/
https://www.twitch.tv/69comorg/about
https://sites.google.com/view/69comorg/home
https://bookmarksclub.com/backlink/69comorg/
https://69comorg.mystrikingly.com/
https://69comorg.amebaownd.com/
https://telegra.ph/69comorg-07-05
https://mk.gta5-mods.com/users/69comorg
https://6868dfe116376.site123.me/
https://scholar.google.com/citations?hl=vi&user=6Oc_AqEAAAAJ
https://www.pearltrees.com/69comorg/69comorg/id94378465
https://69comorg.localinfo.jp/
https://69comorg.hashnode.dev/69comorg
https://rapidapi.com/user/69comorg
https://fliphtml5.com/homepage/fkwmu/69comorg/
https://69comorg.therestaurant.jp/
https://ask.mallaky.com/?qa=user/69comorg
https://www.quora.com/profile/69comorg
https://69comorg.pixieset.com/
https://flipboard.com/@69comorg/69comorg-b48mk755y
https://www.threadless.com/@69comorg/activity
https://www.magcloud.com/user/69comorg
https://hackmd.io/@69comorg/69comorg
https://69comorg.blogspot.com/2025/07/69comorg.html
https://69comorg.doorkeeper.jp/
https://69comorg.storeinfo.jp/
https://velog.io/@69comorg/about
https://bato.to/u/2799322-69comorg
https://zb3.org/69comorg/69comorg
https://community.fabric.microsoft.com/t5/user/viewprofilepage/user-id/1303066
https://www.question-ksa.com/user/69comorg
https://bulkwp.com/support-forums/users/69comorg/
https://orcid.org/0009-0000-6362-9074
https://community.cisco.com/t5/user/viewprofilepage/user-id/1894646
https://archive.org/details/69comorg
https://wpfr.net/support/utilisateurs/69comorg
https://plaza.rakuten.co.jp/69comorg/diary/202507050000/
https://pad.darmstadt.social/s/o6CjQQBdX
https://pixabay.com/users/51185562/
https://disqus.com/by/69comorg/about/
https://id.gta5-mods.com/users/69comorg
https://es.gta5-mods.com/users/69comorg
https://www.gamblingtherapy.org/forum/users/69comorg/
https://forum.m5stack.com/user/69comorg/
https://app.readthedocs.org/profiles/69comorg/
https://public.tableau.com/app/profile/69com.org/viz/69comorg/Sheet1#1
https://connect.garmin.com/modern/profile/794af9d0-a497-4db5-8b72-45f6b0c0ee52
https://www.pixiv.net/en/users/117728977
https://community.amd.com/t5/user/viewprofilepage/user-id/511396
https://readtoto.com/u/2799322-69comorg
https://qna.habr.com/user/69comorg
https://linkr.bio/69comorg
https://www.bark.com/en/gb/company/69comorg/LeNO0o/
https://pastebin.com/u/69comorg
https://www.storeboard.com/69comorg
https://etextpad.com/yqw4jivujr
https://md.darmstadt.ccc.de/s/wNyO1Y5DF
https://comicvine.gamespot.com/profile/org69com/
https://padlet.com/69comorg/69comorg
https://3dwarehouse.sketchup.com/by/69comorg
https://muckrack.com/69com-org/bio
https://hedgedoc.k8s.eonerc.rwth-aachen.de/s/HjQn9CdhC
https://nl.gta5-mods.com/users/69comorg
https://openlibrary.org/people/69comorg
-
-
Local file Local file
-
We can see asharp increase in the number of ICA events for the ungrammatical condition in the time periodof 600 to 1200 after the onset of the critical word showing on the display.
This is the empirical proof that ungrammatical, casual, non-standard text causes an immediate, sharp spike in cognitive effort, validating the argument for standardization to lower cognitive costs.
-
This entire, extremely complex section is the technical support for the biological load.
Summary: Linguistic difficulty triggers the release of stress/arousal neurotransmitters (norepinephrine) to help the brain cope, proving Mises's concept of "uneasiness."
-
We report here on a total of seven experiments which testwhether the ICA reliably indexes linguistically induced cognitive load: three experiments inreading (a manipulation of grammatical gender match / mismatch, an experiment of seman-tic fit, and an experiment comparing locally ambiguous subject versus object relativeclauses, all in German), three dual-task experiments with simultaneous driving and spokenlanguage comprehension (using the same manipulations as in the single-task readingexperiments), and a visual world experiment comparing the processing of causal versusconcessive discourse markers.
Summary: This quote links "linguistic load" specifically to grammatical mismatches, providing evidence that non-standard grammar creates a burden for the reader.
-
A novelmeasure, the Index of Cognitive Activity (ICA), relates cognitive effort to the frequency ofsmall rapid dilations of the pupil.
Summary: This establishes the scientific definition of "transaction cost" in your paper: cognitive effort is physically measurable through pupil dilation.
Tags
Annotators
-
-
Local file Local file
-
Apply CLT in lesson planning, focusing on prioritizing essentialinformation and breaking down complex tasks into manageable segments.
Summary: This offers the solution: "segmentation." This proves that breaking information into standard, predictable chunks (Standardization) is the mechanism for preserving reader motivation.
-
Cognitive load in English learning significantlynegatively affects extrinsic motivation (β = -0.772, p<0.001)
Summary: Provides the hard statistical evidence that high transaction costs (cognitive load) destroy the specific type of motivation (extrinsic) required for professional and vocational success.
-
unclear instructional materialsand complex information presentation add extraneous cognitive load.
Summary: Defines "extraneous load" as a result of "unclear presentation," supporting the argument that non-standard writing (code-meshing) unnecessarily taxes the reader. This also aligns with findings from Liu.
-
High cognitive load can lead to feelings of stress and frustration, thereby reducinglearning motivation
Summary: Connects high cognitive load directly to "stress and frustration," validating the Misesian concept that "uneasiness" causes the consumer to exit the market (in this case, stop reading).
-
-
Local file Local file
-
high segmentation significantly impacts cognitive load, vocabulary learning, retention,and reading comprehension across various aspects of multimedia learning. In essence, segmentation reducescognitive load, supports learning efficiency, and facilitates more profound understanding, vocabulary learning, andretention.
Summary: This offers empirical proof that structure (segmentation) directly correlates with "learning efficiency" and "profound understanding," serving as the scientific backing for the "Professional Imperative" of standardization.
-
segmenting dynamic visualizations intomeaningful units may aid learning by assisting learn-ers in grouping related elements and identifying naturalboundaries between events
Summary: This explains how structure helps: it allows readers to identify "natural boundaries." This validates the use of standard grammar conventions as necessary markers that help the brain group and process ideas, especially for those still learning the English language.
-
when essentialinformation is presented too rapidly, it can overload thelearner’s cognitive capacity, leading to cognitive overload.When this happens, the learner cannot process essentialinformation and learning outcomes effectively.
Summary: Provides the consequence of poor structure: "cognitive overload." This supports the argument that unstructured or non-standard writing risks overloading the reader, preventing them from understanding the core message.
Indirectly, this refutes the idea that "code-meshing" is necessary for more accurate communication.
-
Segmentation is a crucial prin-ciple in multimedia learning that suggests presentinginstructional materials in segments that align with thecognitive pace of learners. By breaking lessons into man-ageable chunks, segmentation offers learners control overthe speed of Instruction, thereby facilitating their abilityto manage cognitive processing [27–29].
Summary: Defines segmentation as a tool for managing cognitive processing, supporting the claim that structure is necessary for the reader (consumer) to handle the flow of information.
This helps to simplify the approach for those learning a new language, and reduces cognitive load.
-
-
job-boards.greenhouse.io job-boards.greenhouse.io
-
How does your experience or passion align with your first/second department choice? (200 words maximum)*
Many times throughout my creative journey i find myself always going back to editing or some form of digital creation/modifcation. It's one of my many passions that i want to share with others and if i can, bring joy to them. I have also studied music production for my entire high school era and understand how hard it can be placing visuals to a song or musical media. That is why i went to Film school, to understand how i can combine the two. My passion for digital editing (VFX, Motion graphics, film editing, or expermintal projects) and my love for music.
-
-
simpleobservability.com simpleobservability.com
-
The first problem is that the build breaks on non systemd systems such as macOS. Since libsystemd is not available, you cannot build from or cross compile to Linux. You must build from a Linux system.
You can, you just can't easily do it with with clang or GCC.
This is a long-standing problem with compilers that the Zig folks and the Go folks have thankfully recognized as silly and decided to approach in a different way within their respective ecosystems.
-
-
drive.google.com drive.google.com
-
White bleached walls—that was the first thing I saw as I stepped into my room. Wallsscrubbed clean of Sharpied poems, lyrics, and quotes; walls which were completely void of myCrayola stick figures,
This first section of the passage describes the walls that he once saw covered in poems and quotes written in marker, were cleaned. Expressing change and growth.
-
-
arxiv.org arxiv.org
-
The hyperparameter search space is summarized in Table 1, with full results in Table 2. While no single configuration is universally optimal, we highlight a setting with block_size=4, fetch_factor=16, and num_workers=12, which achieves approximately 2593 samples/sec and maintains an entropy of 3.59—comparable to random sampling.
This is a powerful tool that allows everyone to train on large datasets, thank you for sharing it with the community! Do you have any practical sense about the tradeoffs between minibatch entropy and model validation performance for a set amount of training time? Obviously this is an impossible experiment to actually run, but I wonder if even lower minibatch entropy which allows higher throughput would be ideal given a set training time. Do you have any anecdotal evidence from training runs on how much shuffling is optimal? I agree that since this experiment could not actually be performed, close to random shuffling is probably the best. Thank you for this contribution!
-
-
openreview.net openreview.net
-
calculations for exploratory analysis andother downstream tasks such as differential expression analysis.
Thank you for this excellent resource that allows anyone to use this dataset. Obviously the SCVI approach has many benefits in terms of eliminating batch effects and noise, but are there any concerns about the noisiness in the generative process for the (corrected, normalized) expected value of the contributions from each gene for downstream tasks? The reconstruction during training looks excellent, but there should be some tradeoff between the benefits of the SCVI latent representation and the generative process - where do you think the model lands on that spectrum? Thank you again for such a useful model!
-
-
www.biorxiv.org www.biorxiv.org
-
Similar to the essentiality benchmark, we use the learned gene embeddings from each of the models and train a shallow MLP to predict a multi-hot label for each gene indicating its membership in one of the hallmark pathways
Did you happen to try linear prediction from the embeddings for each approach? It would be interesting to see the amount of improvement due to the shallow MLP nonlinearity versus the simple linear prediction. Presumably the rankings by approach would be the same.
-
To understand the relationship between model scale, training efficiency, and downstream performance, we trained the Tx1 model series at three scales: 70M, 1B, and 3B parameters. Fig. 7A shows the training cost versus computational budget (measured in FLOPs) for Tx1 compared to other single-cell foundation models including SE-600M, scGPT, and nv-Geneformer variants. Tx1 achieves substantially improved training efficiency, with 3–30× better compute efficiency relative to these prior models.
Thank you for sharing this dataset and model (as well as the SCVI model). In terms of training cost versus computational budget, how would the smaller training subsets factor in to efficiency for the smaller models? It's interesting to consider training compute normalized by fraction of the data on which the model was trained. Is it possible that training the 3B model on only a subset of the dataset would not hurt performance and therefore improve training efficiency metrics? I appreciate this deep analysis of the training process.
-
-
Local file Local file
-
sages in their artifacts, viewers engage in a decoding process to in-terpret these artifacts. Hall describes three broad types of decodingthat viewers may participate in, even shifting between modes at times.
Hall's dominant/hegemonic, negotiated, and oppositional is like Castell's legitimizing identity, resistance, and project, which is basically a rehashed virtue ethics by another name. You will deny it, but it maps onto right-wrong with a middle ground in between, or a conservatist-progressivist axis whereby one seeks perpetuation and another one seeks disruption, similar to Nietzsche and Camus in this sense.
Are you the rebel, or the empire? Come on, this ain't new: Time in memorial has had preachers claim things need changing.
-
-
touro.app.box.com touro.app.box.com
-
could be helpful to have more specificsabout what is normal, [sex] with dildos, fisting … so doctors are not freaking out andshaming patients.”
QUESTION: How can clinicians discuss sexual practices with LGBTQ+ patients in a way that is thorough and nonjudgmental without making those conversations feel disproportionately focused on sexual behavior because of a patient’s identity? In particular, how can providers ensure they are applying the same level of curiosity, normalization, and clinical relevance to sexual history-taking with cisgender, heterosexual patients, so that LGBTQ+ patients do not feel singled out or implicitly pathologized for practices that are part of a broad spectrum of normal sexual behavior?
-
Especially for queer people of color there’s already thisingrained mistrust of the medical system. If they have one bad experience … I see it with mytrans friends like, ‘I’m just not going back.’”
SUMMARIZE: Intersectionality is critical to consider when caring for LGBTQ+ patients. Many individuals hold multiple marginalized identities that shape their experiences and contribute to longstanding mistrust of health-care systems. As this quote highlights, even a single negative or dismissive encounter can be enough to deter someone from seeking care in the future, underscoring the importance of affirming, culturally responsive interactions at every point of care.
-
Approximately eight of the participants as well as note-takers and the translator reviewed themanuscript and provided feedback, which was used for revisions.
CONNECT: As someone who values research transparency and believes researchers have a responsibility to share findings with the communities from which data are collected, I really appreciated that participants were actively involved in reviewing the manuscript. Having participants, note-takers, and the translator provide feedback reflects meaningful shared decision-making and community-engaged research. This approach strengthens the validity and integrity of the work, and I think it is a practice more researchers should strive to adopt.
-
-
www.americanyawp.com www.americanyawp.com
-
with moderates still hanging on, it would take another seven months
why did it take such a long time?
-
On May 10, 1776, nearly two months before the Declaration of Independence, the Congress voted on a resolution calling on all colonies that had not already established revolutionary governments to do so and to wrest control from royal officials.
How did the people feel about this?
-
Somerset v Stewart
Somerset v. Stewart was a British court case that ruled slavery was unsupported by English law, leading to James Somerset’s freedom. The decision influenced debates about slavery throughout the Atlantic world. It also raised questions for colonists about the legality and morality of slavery under British rule.
-
But not all colonists were patriots. Indeed, many remained faithful to the king and Parliament, while a good number took a neutral stance. As the situation intensified throughout 1774 and early 1775,
This passage reminds readers that colonial society was deeply divided, with many people remaining loyal to Britain or choosing neutrality. It challenges the idea that all colonists supported resistance or independence. Recognizing these divisions helps explain why the Revolution created internal conflict as well as opposition to British rule.
-
great scheme
“great scheme” refers to a larger coordinated plan among the colonies, suggesting that resistance to British policies was becoming more organized and intentional rather than spontaneous.
-
the Coercive Acts fostered the sense of shared identity created over the previous decade.
highlights how the Coercive Acts unintentionally unified the colonies by strengthening a shared identity. Rather than isolating Massachusetts, British punishment encouraged cooperation and solidarity among the colonies. This growing unity helped lay the groundwork for collective resistance.
-
“Continental Association.”
The Continental Association was an agreement among the colonies to boycott British goods in order to pressure Parliament into repealing oppressive laws.
-
The Congress struck a compromise, agreeing to adopt the Massachusetts militia and form a Continental Army, naming Virginia delegate George Washington commander in chief. They also issued a “Declaration of the Causes of Necessity of Taking Up Arms” to justify the decision. At the same time, the moderates drafted an “Olive Branch Petition,” which assured the king that the colonists “most ardently desire[d] the former Harmony between [the mother country] and these Colonies.” Many understood that the opportunities for reconciliation were running out. After Congress had approved the document, Benjamin Franklin wrote to a friend saying, “The Congress will send one more Petition to the King which I suppose will be treated as the former was, and therefore will probably be the last.”31 Congress was in the strange position of attempting reconciliation while publicly raising an army.
This passage shows how divided colonial leaders were in 1775 between seeking peace and preparing for war. While Congress formed the Continental Army and justified taking up arms, they also sent the Olive Branch Petition to show loyalty to the king. Franklin’s comment suggests that many leaders already believed reconciliation was unlikely.
-
-
www.biorxiv.org www.biorxiv.org
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
1. General Statements
In this study, we mechanistically define a new molecular interaction linking two of the cell's major morphological regulatory pathways-the Rho GTPase and Hippo signaling networks. These two major signaling pathways are both required for life across huge swaths of the tree of life. They are required for the dynamic organization and reorganization of proteins, lipids, and genetic material that occurs in essential cellular processes such as division, motility and differentiation. For decades these pathways have been almost exclusively studied independently, however, they are known to act in concert in cancer to drive cytoskeletal remodeling and morphological changes that promote proliferation and metastasis. However, mechanistic insight into how they are coordinated is lacking.
Our data reveal a mechanistic model where coordination is mediated by the RhoA GTPase-activating protein ARHGAP18, which forms molecular interactions with both the tumor suppressor Merlin (NF2) and the transcriptional co-regulator YAP (YAP1). Using a combination of state-of-the-art super-resolution microscopy (STORM, SORA-confocal) in cultured human cells, biochemical pulldown assays with purified proteins, and analyses of tissue-derived samples, we characterize ARHGAP18's function from the molecular to the tissue level in both native and cancer model systems.
Together, these findings establish a previously unrecognized molecular connection between the RhoA and Hippo pathways and culminate in a working model that integrates our current results with prior work from our group and decades of prior studies. This model provides a new conceptual framework for understanding how RhoA and Hippo signaling are coordinated to regulate cell morphology and tumor progression in human cells.
In this substantially revised manuscript, we have addressed all comments from the expert reviewers described point-by-point below. A shared major comment from the reviewers was the request for direct evidence of the proposed mechanistic model. To address these constructive comments, we've added new experiments, new quantification, new text, new control data, and have added two expert authors, adding super-resolution mouse tissue imaging data for the endogenous study of ARHGAP18 in its native condition. We believe that these additions greatly enhance the manuscript and collectively address the overall message from the reviewer's collective comments.
2. Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
This manuscript describes a dual mechanism by which ARHGAP18 regulates the actin cytoskeleton. The authors propose that in addition to the known role for ARHGAP18 in regulating Rho GTPases, it also affects the cytoskeleton through regulation of the Hippo pathway transcriptional regulator YAP. ARHGAP18 knockout Jeg3 cells are were generated and show a clear loss of basal stress fiber like F-actin bundles. The authors further characterize the effects of ARHGAP18 knockout and overexpression. It is also discovered that ARHGAP18 binds to the Hippo pathway regulator Merlin and to YAP. Ultimately it is concluded that ARHGAP18 regulates the F-actin cytoskeleton through dual regulation of RHO GTPases and of YAP. While the phenotype of the ARHGAP18 knockout and the association of ARHGAP18 with Merlin and YAP is interesting, I found the authors conclusion that these phenotypes are due to ARHGAP18 regulation of both RHO and YAP to be based on largely correlative evidence and sometimes lacking in controls or tests for significance. In addition the authors often make overly strong conclusions based on the experimental evidence. In some instances, the rationale for how the experimental results support the conclusion is insufficiently articulated, making evaluation challenging. In general although the authors have some interesting observations, more definitive experiments with proper controls and statistical tests for significance and reproducibility are needed to justify their overall conclusions.
- *
*We appreciate the reviewers' constructive comments and have added substantial new data and quantifications to address their concerns. We have focused these new data on directly testing the proposed mechanisms, adding controls, and performing quantitative analysis with statistical testing. Additionally, we have edited our language to make our rationale clearer and to present our conclusions as a more moderate assessment of our experimental results. Below we respond to the specific comments made by the reviewer, followed by a list of additional editorial changes we've made based on the reviewer's overarching comments on clarity and rationale. *
Specific Comments
1) The authors make a big point about the effects of ARHGAP18 on myosin light chain phosphorylation. However, this result is not quantified and tested for statistical significance and reproducibility.
*We thank the reviewer for their comments on our western blotting quantification, which in the original submission version had quantification of RhoA downstream signaling of pCofilin/ Cofilin and pLIMK/ LIMK. We had withheld the pMLC and MLC quantification as the result was previously published with quantification, reproducibility, and statistical significance by our group in our prior manuscript on ARHGAP18 published in Elife in 2024 (Fig. 4E of *
https://doi.org/10.7554/eLife.83526 ). However, these prior results lacked the new overexpression data. We recognize the need to add these data to this manuscript as requested by the reviewer.
- *
*To address the reviewer's comment, we have added quantification of pMLC/MLC (Fig. 1F) *
2) Along similar lines in Figure 2C they state that overexpression of ARHGAP18 causes cells to invade over the top of their neighbors. This might be true and interesting, but only a single cell is shown and there is no quantification or controls for simply overexpressing something in that cell. The authors also conclude from this image that the overexpression phenotype is independent of its GAP activity on Rho. It is not clear how this conclusion is made based on the data. It would seem like a more definitive experiment would be to see if a similar phenotype was induced by an ARHGAP18 mutant deficient in GAP activity.
Based on the reviewer's comment, we recognize the qualitative statements made in Figure 2C (now Figure 3) should've been made more quantitative. We have added the control of Jeg 3 WT cells expressed with empty vector flag to show that WT cells do not invade over the top of each other (Fig. 3F). Additionally, we have added the quantification found in Fig. 3E, which shows the % invasive/ non-invasive cells between WT and ARHGAP18 overexpression cells. We have clarified our conclusions to make clear that these data do not directly test if the invasive phenotype derives from a Rho-independent mechanism. The text now states the following conclusion alongside others, which can be seen in our tracked changes:
- *
"These data support the conclusion that ARHGAP18 acts to regulate basal and junctional actin. However, it was not clear whether this activity occurred through a Rho-independent or a Rho-dependent mechanism."
- *
We have added new data of cells expressing an ARHGAP18 mutant deficient in GAP activity, which is explained in detail in the following response below.
3) In Figure 3 the authors compare gene expression profiles of ARHGAP18 knockout cells to wild-type cells. They see lots of differences in focal adhesion and cytoskeletal proteins and conclude that this supports their conclusion that ARHGAP18 is not just acting through RHO. The rationale for this in not clear. In addition, they observe changes in expression profiles consistent with changes in YAP activity. They conclude that the effects are direct. This very well might be true. However RHO is a potent regulator of YAP activity and the results seem quite consistent with ARHGAP18 acting through RHO to affect YAP.
- *
We thank the reviewer for their comment and believe the revised manuscript now presents direct evidence to support the conclusions made through the editing text and the incorporation of new data.
- *
First, the reviewer highlighted that we were not clear in our rationale and explanation of the conclusions made from our RNAseq data in the new Figure 4 (Previously Figure 3). We agree with the reviewer that the RNAseq data alone is not sufficient rationale for the conclusion that ARHGAP18 is acting through YAP directly. In the revised manuscript, the conclusion is now made based on the combination of our multi-faceted investigation of the relationship between ARHGAP18 and YAP (most importantly, new Figure 5). It's important for us to argue that our RNAseq analysis is much more robust and specific than simply reporting a descriptive assay seeing lots of differences in cytoskeletal proteins. We recruited an outside RNAseq expert collaborator; Dr. Yongho Bae, to perform state-of-the-art IPA analysis and a grueling manual curation of the top hit genes to identify the predominant signaling pathways linking the loss of ARHGAP18 to known YAP translational products. We've provided a supplemental table listing each citation supporting the identified YAP pathway associations from this manual curation. We also have added a new discussion paragraph on RNAseq data to clarify our specific RNAseq data results and analysis. In the revised manuscript, we have moderated our language in the results text regarding the RNAseq data to reflect the reviewer's suggestion:
- *
"Our RNAseq data alone could not independently confirm if the alterations to transcriptional signaling and expression of actin cytoskeleton proteins were through a Rho-dependent or Rho-independent mechanism."
-
*
-
*
Second, in this comment and the above, the reviewer highlights the need for a new experiment to directly test the Rho Independent effects of ARHGAP18, which we now provide in the new Figure 5. In this new data, we've applied an experimental design suggested by reviewer 2 regarding the same concern. In short, we've produced and expressed a point mutant variant ARHGAP18(R365A), which abolishes the Rho GAP activity while maintaining the remainder of the protein intact. This construct allows us to directly test the effects of ARHGAP18 independent from its RhoA GAP activity. We find that the GAP-deficient ARHGAP18 is able to fully rescue basal focal adhesions, indicating that the basal actin phenotype is at least in part regulated through a Rho-independent mechanism.
-
*
-
*
*We believe the revised manuscript, when taken in totality, provides the definitive proof requested by the reviewer. Specifically, the combination of Figure 5, where we show new data using the ARHGAP18(R365A) variant, and the result that ARHGAP18 forms a stable complex with YAP (Fig. 6G) or Merlin (Fig.6A), is supportive of direct Rho-independent molecular interactions between YAP, Merlin, and ARHGAP18. *
4) In Figure 4A showing Merlin binding to ARHGAP18 there is no control for the amount of Merlin sticking to the column as was done in Figure 4F for binding experiments with YAP. This makes it difficult to determine the significance of the observed binding.
We have performed the requested control experiment and added the results to Figure 6A.
5) The images in Figure 4C showing YAP being maintained in the nucleus more in ARHGAP18 knockout cells compared to wild-type. However the images only show a few cells and YAP localization can be highly variable depending on where you look in a field. Images with more cells and some sort of quantification would bolster this result.
We have provided quantification (Figure 6D) of what was originally Figure 4C (now Figure 6C).
Reviewer #1 (Significance (Required)):
While the phenotype of the ARHGAP18 knockout and the association of ARHGAP18 with Merlin and YAP is interesting, I found the authors conclusion that these phenotypes are due to ARHGAP18 regulation of both RHO and YAP to be based on largely correlative evidence and sometimes lacking in controls or tests for significance. In addition the authors often make overly strong conclusions based on the experimental evidence. In some instances, the rationale for how the experimental results support the conclusion is insufficiently articulated, making evaluation challenging. In general although the authors have some interesting observations, more definitive experiments with proper controls and statistical tests for significance and reproducibility are needed to justify their overall conclusions.
In the above comments, we detail the specific definitive experiments, proper controls, and statistical tests for significance, requested by the reviewer, which we believe greatly strengthen our manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This manuscript investigates the Rho effector, ARHGAP18 in Jegs cells, a trophoblastic cell line. It presents a number of new pieces of data, which increase our understanding of the importance of this GAP on cell function and explains at a molecular level previous results of other workers in the field. ARHGAP18 was originally given the name "conundrum' and continues to stand apart from the majority of other GAP proteins and their functions. Hence the data here is significant and of high standard.
The data is clear, and the images are of high quality and extremely impressive in their resolution. It is significant and adds a further layer to our understanding of the regulation of cell migration, particularly in the formation and resolution of microvilli.
- *
We appreciate the reviewer's comments and supportive insights.
The data is based on the use of the cell line Jeg3. Even the authors previous publication in eLife is based only on this cell line. They need to show the conclusions are general and not specific to this line of cells. As an extension of this, is the ARHGAP18 function shown here only in transformed cells? Does the same mechanisms operate in normal cells, which respond to activation to proliferate or migrate?
- *
- We respectfully point out that the critical experiments of the prior eLife publication were validated in DLD-1 colorectal cells and not Jeg-3 cells alone (Figure 1-figure supplement 2). Our newly independent lab, established just over a year ago, is unable to perform a full expansion of the manuscript using untransformed cells, however, we agree with the reviewer's perspective and wish to address the comment to the best of our current capability. To answer the reviewers' suggestions, we have recruited Dr. Christine Schaner Tooley, an expert in mouse model system studies. In the revised manuscript, we've added new Super-Resolution SORA confocal images of endogenous ARHGAP18's localization in the intact intestinal villi tissue, and apical junctions of WT mice (Fig.1A-C). These data indicate that endogenous ARHGAP18 is enriched (but not exclusively localized) at the apical plasma membranes of normal WT epithelial cells. This localization, where both Merlin and Ezrin are present at apical membrane/ junctions under normal conditions, is a major component of the working model proposed in Fig. 7. These data also indicate that ARHGAP18 is capable of entering the nucleus in WT cells, another critical aspect of our proposed model. Collectively, our DLD-1 studies published previously and or new studies using WT mice tissue samples support the conclusion that at least some of ARHGAP18's functions described in this manuscript are not limited to Jeg3 cells.*
In endothelial cells, Lovelace et al 2017 showed localization to microtubules and that depletion of ARHGAP18 resulted in microtubule instability. The authors may like to comment on the differences. Is this a cell type difference or RhoA versus RhoC difference?
- *
In our previous publication (Lombardo Elife), we validated the finding that ARHGAP18 forms a complex with microtubules, as we detected tubulin in the ARHGAP18 pulldown experiment (Figure 1- Source Data). However, our data indicate that in Jeg3 cells ARHGAP18 does not localize to the same microtubule associated spheres observed in the Lovelace publication. We now comment on the shared conclusions and differences between this manuscript and the Lovelace et al 2017 in the discussion section.
- *
"In endothelial cells, ARHGAP18 has been reported to localize microtubules and plays a role in maintaining proper microtubule stability (Lovelace et al., 2017). In our epithelial cell culture models and WT mouse intestine, we have been unable to detect ARHGAP18 at microtubules suggesting ARHGAP18 may have additional functions is various cell types."
On pages 7,9 they conclude that MLC and basal and junctional actin are regulated through a GAP independent mechanism. The best way to show this is with overexpression of a GAP mutant.
We appreciate the reviewer's insight and have produced and expressed a GAP mutant, ARHGAP18(R365A), in our cells, directly testing our conclusion that ARHGAP18 has a GAP-independent function. These data are now presented in revised Figure 5 and explained further in response to reviewer #1.
There is a huge amount of data presented in Figure 3, but their 2 genes which they focus on, LOP1 and CORO1A, are discussed but no actual data presented in support.
We now validate the CORO1A by qPCR in Figure 4J.
- *
Reviewer #2 (Significance (Required)):
The data is significant and adds a further layer to our understanding of the regulation of cell migration, particularly in the formation and resolution of microvilli. This manuscript will be of significance to an basic science audience in the field of RhoGTPases and cell migration.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
The study by Murray et al explores the effects of ARHGAP18 on the actin cytoskeleton, Rho effector kinases, non-muscle myosin, and transcription. Using super resolution microscopy, they show that in ARHGAP18 KO cells there is a mixed and unexpected cytoskeleton phenotype where myosin phosphorylation appears to be increased, but actin is disorganised with reduced stress fibres, diminished focal adhesions and augmented invasiveness. They conclude that the underlying mechanisms are likely independent from RhoA. Next, they perform RNAseq using the KO cells and identify an array of dysregulated genes, including those that play crucial roles in microvilli (related to previously published findings). Analysis of the data identify gene expression changes that are relevant for altered focal adhesion (integrins). Further analysis reveals that a large cohort of the dysregulated genes are YAP targets. They then show that in ARHGAP18 KO cells YAP nuclear localization, as detected by immunostaining, is augmented; and demonstrate that immobilized ARHGAP18 protein can bind the Hippo regulator merlin as well as YAP itself.
Major comments:
1, The premise of the study (that ARHGAP18 is a RhoA effector or may acts independently of RhoA) remains not proven.
We have added new evidence of direct RhoA independent activity for ARHGAP18 described in the above comments. Specifically, we've added data using a RhoA-GAP dead variant of ARHGAP18 in Figure 5, which we believe addresses this comment.
- *
At several places (including in the title) the authors refer to ARHGAP18 as a Rho effector, which would suggest that it is downstream form Rho, but the basis for this is not clear. In fact, their own previous study suggested that ARHGAP is a RhoA regulator, rather than an effector. In general, the connection of the described effects to RhoA remains unclear, and not addressed in this study. The authors seem to go back and forth in their conclusions regarding the connection between ARHGAP18 and RhoA. For example, the first section of results is finished by stating (line 194): "These data support the conclusion that ARHGAP18 acts to regulate basal and junctional actin through Rho-independent mechanism". But the next section starts by stating (line 198): "We hypothesized that the invasive and cytoskeletal phenotypes observed at the basal surface of cells devoid of ARHGAP18 may be a result of changes in regulation at the transcriptional level either directly through RhoA signaling or through an additional mechanism specific to ARHGAP18". The paper would be strengthened by adding data that show whether the effects are indeed downstream, from RhoA or RhoA independent. If there is no sufficient demonstration that ARHGAP18 is downstream of RhoA and is an effector, this needs to be stated explicitly, and the wording should be changed.
*We now provide new data in Figure 5, which directly tests the RhoA independent functions of ARHGAP18 as recommended by the reviewer. Our understanding of the term effector is 'a molecule that activates, controls, or inactivates a process or action.' Based on this understanding, we used the term to convey ARHGAP18's functional role within the feedback loop, rather than to imply that it acts exclusively downstream. *
- *
We seek to clarify our perspective with the reviewer's assertion that we go "back and forth" as to if ARHGAP18 functions in a Rho Dependent or Rho Independent manner. It was our intent to propose a model where ARHGAP 18 acts in two separate circuits that regulate cell signaling. The first circuit involves ARHGAP18's canonical RhoA GAP activity, which involves ERMs and LOK/SLK, and is limited to the apical plasma membrane. This first signaling circuit was characterized in our prior Elife manuscript (Lombardo et al., 2024) and in an earlier JCB manuscript (Zaman and Lombardo et al., 2021). In this newly revised manuscript, we provide a partial mechanistic characterization of the second circuit, which we freely admit is much more complex and will likely require additional study to fully characterize.
- *
As both circuits operate as signaling feedback loops, we find the terms 'upstream' and 'downstream' to be of limited value, and we attempt to avoid their use when possible. We retain their use only when referring to the Hippo and ROCK signaling cascades, where these designations are well established. We suggest that the conceptual inconsistencies of Conundrum/ARHGAP18 may have arisen from the tendency to view it in strictly binary terms as upstream or downstream. Here, we propose a third possibility that ARHGAP18 functions as both, participating in a negative feedback loop.
- *
*We have edited and added data testing if the effects are Rho independent and discussion text in response to the reviewer's comments and clarify the molecular function of ARHGAP18.
"Additionally, focal adhesions and basal actin bundles are restored to WT levels when the ARHGAP18(R365A) GAP-ablated mutant is expressed in ARHGAP18 KO cells (Fig. 5A, B). These results represent the strongest argument that ARHGAP18 functions in additional pathways to RhoA/C alone. Our data suggests that at least one of the alternative pathways is through ARHGAP18's interaction with YAP and Merlin. From these data we conclude that ARHGAP18 has important functions in both RhoA signaling through both its GAP activity and in Hippo signaling through its GAP independent binding partners. "*
-
*
-
*
The study is descriptive and contains a series of observations that are not connected. Because of this, the study's conclusions are not well supported, and key mechanistic insight is limited. The study feels like a set of separate observations, that remain incompletely worked out and have some preliminary feel to them. The model in the last figure also seems to contain hypotheses based on the observations, several of which remains to be proven.
- *
*We present our revised manuscript, in which we've more clearly outlined our rationale and conclusions, as detailed in the above responses, to emphasize the overall connectivity of the study. We have also updated the title of Figure 7 to read "__Theoretical __Model of ARHGAP18's coordination of RhoA and Hippo signaling pathways in Human epithelial cells." To make it clear that we are presenting a working model, which has elements that will require additional investigation. Throughout the manuscript, we highlight the unknown elements that remain to be tested or other outstanding questions. Thus, we do not aim to characterize this complex signaling coordination completely. Instead, this manuscript represents the 3rd iteration in our systematic advances to describe this entirely new signaling pathway. We agree that, despite three separate manuscripts (this one included) to date, this work represents an early stage in understanding the system, many additional studies will be needed to characterize this signaling system fully. Figure 7 is presented as a working model that results from a thoughtful combination of our collective data and that of other researchers, derived from numerous species across decades of study. We firmly believe that proposing such integrative models is valuable for advancing the field. We also recognize the importance of clearly indicating which aspects remain hypothetical. We now explicitly note in several places within the discussion which components of the model will require further validation and experimental confirmation. For example, regarding our theoretical mechanism in Figure 7 we state: *
"Validation of the direct mechanism by which YAP/TAZ transcriptional changes drive basal actin changes in ARHGAP18 KO cells will require further investigation based on predictions from RNAseq results."
- *
Addressing any possible connection between key effects of ARHGAP18 KO (changes in actin, focal adhesion, integrins, Yap and merlin binding) could strengthen the manuscript. One such specific question is the whether the changes in integrin expression (RNAseq) are indeed connected to the actin alterations and reduction ion focal adhesions (Fig 1). Staining for these integrins to show they are indeed altered, and/or manipulating any of them to reproduce changes could provide and exciting addition.
- *
*We attempted to stain cells for Integrins by purchasing three separate antibodies. However, despite extensive optimization and careful selection of the specific integrins using our RNAseq results we were unable to get any of these antibodies to work in any cell type or condition. We believe that there is a technical challenge to staining for integrins due to their transmembrane and extracellular components, which we were unable to overcome. As an attempt to address the reviewers comment, we alternatively stained cells for paxillin which directly binds the cytoplasmic tails of integrins (Fig. 3&5). *
Some of the experimental findings are not convincing or lack controls. Fig 1: some of the western blots are not convincing or poor quality. [...] On the same figure, the quality of LIM kinase blots is poor. [...] The signal is weak, and the blot does not appear to support the quantification. The last condition (expression of flag-ARHGAP18) results in a large drop in pLIMK and pcofilin on the blot, which is not reflected by the graph. Addition of *a better blot and the use of strong positive or negative control would boost confidence in these data. *
- *
In response to this and other reviewers' comments, we have added new western data and quantification to Figure 1. We now focus on MLC/pMLC data as we believe these data highlight the potential Rho-independent mechanism of ARHGAP18, and we were able to greatly improve the quality of the blots through careful optimization. We hope the reviewer finds these blots and quantifications (Fig. 1E and F) more convincing.
*We note that phospho-specific Western blotting presents considerably greater technical challenges than conventional blotting. We believe that the appearance of an attractive looking blot does not always correlate to quality or reproducibility and have focused on taking extraordinarily careful steps in the blotting of our phospho-specific antibodies, which at times comes at the cost of the blot's attractiveness in appearance. For example, all phospho-specific antibodies are run using two color fluorescent markers to blot against both the total protein and the phospho-protein on the same blot. This approach often leads to blots that have reduced signal to noise compared to chemiluminescent Westerns. Additionally, we use phospho-specific blocking buffer reagents which do not contain phosphate-based buffers or agents that attract non-specific phospho-staining signals. These blocking buffers are not as effective as non-fat milk in pbs at blocking the background signal, however, they are ultimately cleaner for phospho-specific primary antibodies. We use carefully optimized protocols, from cell treatment to lysis, transfer, and antibody incubation, including methods developed by laboratories where the corresponding author of the manuscript was trained. Nonetheless, despite these efforts, we have now removed the LIMK and cofilin data because we deemed them unnecessary for the main conclusions of this manuscript and were unable to improve their quality to satisfy the reviewer. *
The changes in pMLC on the western blots are very small, and for any conclusion, these studies require quantification. Further, the expression levels of Flag-ARHGAP18 needs to be shown to support the statement that the protein is expressed, and indeed overexpressed under these conditions (vs just re-expressed).
In continuation of the above comment, we have made significant effort to improve the quality of our pMLC western blots and now provide quantification in Figure 1. We also now provide the Flag-ARHGAP18 signal as requested by the reviewer.
Fig 4: the differences in YAP nuclear localization under the various conditions are not well visible. Quantitation of nuclear/cytosolic signal ratio should be provided. Please provide a rationale and more context for using serum starvation and re-addition. What is the expected effect? Serum removal and addition is referred to as nutrient removal and re-addition, but this is inaccurate, as it does not equal nutrient removal, since serum contains a variety of other important components, e.g. growth factors too.
We have provided new quantification of the nuclear/cytosolic signal ratio in Figure 6D. We have explained our rational for the study through the following new text:
"Merlin is activated and localized to junctions upon signaling, promoting growth and proliferation; among these signals is the availability of growth factors and other components of serum (Bretscher et al., 2002). We hypothesized that since ARHGAP18 formed a complex with Merlin that ARHGAP18's localization may localize to junctions under conditions which promote Merlin activation."
- *
We have altered our use of "nutrient removal" to "serum removal"
The binding between ARHGAP18 and merlin is interesting, but a key limitation is the use of expressed proteins. Can the binding be shown for the endogenous proteins (IP, colocalization). Another important unaddressed question is the relevance of this binding, and the relation of this to altered YAP nuclear localization.
- *
*Our data in Fig. 6G shows binding of a resin bound human ARHGAP18 to endogenous YAP from human cells as suggested by the reviewer. In Fig. 6A, we have selected to use GFP-Merlin as Merlin shares approximately 60% sequence identity with Ezrin, Radixin, and Moesin (ERMs). Their similarity is such that Merlin was named for Moesin-Ezrin-Radixin-Like Protein. In our experience, nearly all Merlin or ERM antibodies have some cross-contaminating signal. Thus, a major concern is that if we were to blot for endogenous Merlin in the pull-down experiment, we may see a band that could in fact be ERMs. To avoid this, we tagged Merlin with GFP to ensure that the product pulled down by ARHGAP18 was Merlin, not an ERM. Regarding the ARHGAP18-resin bound column, our homemade ARHGAP18 antibody is polyclonal. We have extensive experience in pulldown assays and have found that the binding of a polyclonal antibody to the bait protein can produce less accurate results, as the binding site for the antibody is unknown and can sterically hinder attachment of target proteins like Merlin. In our experience, attachment to a flag-tag, which is expressed after a flexible linker at the N- or C-terminus, allows us to overcome this limitation, which we've used in this manuscript. *
Minor comments:
Introduction line 99: "When localized to the nucleus, YAP/TAZ promotes the activation of cytoskeletal transcription factors associated with cell proliferation and actin polymerization" Please clarify what you mean by this statement, that is inaccurate in its present for. Did you mean effects on transcription factors that control cytoskeletal proteins, or do you mean that Yap/Taz affect these proteins? Please also provide reference for this.
We've altered the sentence as suggested by the reviewer, which now reads the following:
"When localized to the nucleus, YAP/TAZ promotes transcriptional changes associated with cell proliferation and actin polymerization."
- *
*The full mechanism for how YAP/TAZ promotes proliferation and actin polymerization is a currently debated issue. We do not think introducing the various current proposed models is required for this manuscript, and we simply intend to convey that when in the nucleus, YAP/TAZ promotes transcriptional changes that drive actin polymerization and cell proliferation. *
-What is the cell confluence in these experiments? For epithelial cells confluence affects actin structure. Please comment on similarity of confluency across experimental conditions?
- *
All cellular experiments are paired where WT and ARHGAP18 KO cells are plated at the same time under identical conditions. For imaging, we plate all cells onto glass coverslips in a 6 well dish so that each condition is literally in the same cell culture plate and gets identical treatment. In our prior Elife paper studying ARHGAP18, we characterized that ARHGAP18 KO cells and WT cells divide at a similar rate and have similar proliferation characteristics. The epithelial cell cultures are maintained for experiments around 70-80% confluency. For the focal adhesion staining experiments, the confluency is slightly lower, between 50-60% to capture the focal adhesions towards the leading edge. We have added the following new text to further describe these methods: "Cell cultures for experiments were maintained at 70%-80% confluency. For focal adhesion experiments, the cell cultures were maintained at 50%-60% confluency."
-Fig 2 legend: please indicate that the protein detected was non-muscle myosin heavy chain (distinct from the light chain detected in Fig 1).
- *
We have altered original Figure 2 (new Figure 3) legend.
-Line 339-340: please check the syntax of this sentence -Western blot quantification: the comparison of experiments with samples run on different gels/blots requires careful normalization and experimental consistency. Please describe how this was achieved.
- *
We have added the following new text to further describe these methods:
"For blots which required quantification of antibodies that were only rabbit primaries (e.g., pMLC/MLC antibodies listed above), samples were loaded onto a single gel and transferred onto a single membrane at the same time. After transfer, the membrane was cut in half and subsequent steps were done in parallel. All quantified blots were checked for equal loading using either anti-tubulin as a housekeeping protein or total protein as detected by Coomassie staining"
Reviewer #3 (Significance (Required)):
Rho signalling is a central regulator of an array of normal and pathological cell functions, and our understanding of the context dependent regulation of this key pathway remains very incomplete. Therefore, new knowledge on the role of specific regulators, such as ARHGAP18, is of interest to a very broad range of researchers. A further exciting aspect of this protein, that despite indications by many studies that it acts as a GAP (inhibitor) for Rho proteins, there are findings in the literature that suggest that its manipulation can affect actin in unexpected (opposite) manner. These point to possible Rho-independent roles, and warranted further in-depth exploration.
One of the strength of the study is that it explores possible roles of ARHGAP18 beyond RhoA and describes some new and interesting observations, which advance our knowledge. The authors use some excellent tools (e.g. ARHGAP KO cells and re-expression) and approaches (e.g. super resolution microscopy to analyze actin changes, RNAseq and bioinformatics to find genes that may be downstream from ARHGAP18). A key limitation of the study however, is that it is not clear whether the observed findings are indeed independent from RhoA. Further limitation is that potential causal relationships between the described findings are not studied, and therefore the findings are in some cases overinterpreted, and limited mechanistic insights are provided. In some cases the exclusive use of expressed proteins is also a limitation. Finally, some of the experiments also need improvement.
Reviewer expertise: RhoA signalling, guanine nucleotide exchange factors, epithelial biology, cell migration, intercellular junctions.
In the above comments, we detail the new experimental data addressing reviewer 3's listed key limitations. We've added new data using the Rho GAP deficient ARHGAP18(R365A) variant which allows for the direct characterization of ARHGAP18's Rho independent activity. We have introduced new data in WT cells studying endogenous proteins to address the limitations from expressed proteins. Finally, we have moderated our language to address overinterpretation. Collectively, we believe that our revised manuscript addresses the constructive reviewer's comments.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The study by Murray et al explores the effects of ARHGAP18 on the actin cytoskeleton, Rho effector kinases, non-muscle myosin, and transcription. Using super resolution microscopy, they show that in ARHGAP18 KO cells there is a mixed and unexpected cytoskeleton phenotype where myosin phosphorylation appears to be increased, but actin is disorganised with reduced stress fibres, diminished focal adhesions and augmented invasiveness. They conclude that the underlying mechanisms are likely independent from RhoA. Next, they perform RNAseq using the KO cells and identify an array of dysregulated genes, including those that play crucial roles in microvilli (related to previously published findings). Analysis of the data identify gene expression changes that are relevant for altered focal adhesion (integrins). Further analysis reveals that a large cohort of the dysregulated genes are YAP targets. They then show that in ARHGAP18 KO cells YAP nuclear localization, as detected by immunostaining, is augmented; and demonstrate that immobilized ARHGAP18 protein can bind the Hippo regulator merlin as well as YAP itself.
Major comments:
- The premise of the study (that ARHGAP18 is a RhoA effector or may acts independently of RhoA) remains not proven. At several places (including in the title) the authors refer to ARHGAP18 as a Rho effector, which would suggest that it is downstream form Rho, but the basis for this is not clear. In fact, their own previous study suggested that ARHGAP is a RhoA regulator, rather than an effector. In general, the connection of the described effects to RhoA remains unclear, and not addressed in this study. The authors seem to go back and forth in their conclusions regarding the connection between ARHGAP18 and RhoA. For example, the first section of results is finished by stating (line 194): "These data support the conclusion that ARHGAP18 acts to regulate basal and junctional actin through Rho-independent mechanism". But the next section starts by stating (line 198): "We hypothesized that the invasive and cytoskeletal phenotypes observed at the basal surface of cells devoid of ARHGAP18 may be a result of changes in regulation at the transcriptional level either directly through RhoA signaling or through an additional mechanism specific to ARHGAP18". The paper would be strengthened by adding data that show whether the effects are indeed downstream, from RhoA or RhoA independent. If there is no sufficient demonstration that ARHGAP18 is downstream of RhoA and is an effector, this needs to be stated explicitly and the wording should be changed.
- The study is descriptive and contains a series of observations that are not connected. Because of this, the study's conclusions are not well supported, and key mechanistic insight is limited. The study feels like a set of separate observations, that remain incompletely worked out and have some preliminary feel to them. The model in the last figure also seems to contain hypotheses based on the observations, several of which remains to be proven. Addressing any possible connection between key effects of ARHGAP18 KO (changes in actin, focal adhesion, integrins, Yap and merlin binding) could strengthen the manuscript. One such specific question is the whether the changes in integrin expression (RNAseq) are indeed connected to the actin alterations and reduction ion focal adhesions (Fig 1). Staining for these integrins to show they are indeed altered, and/or manipulating any of them to reproduce changes could provide and exciting addition.
- Some of the experimental findings are not convincing or lack controls.
Fig 1: some of the western blots are not convincing or poor quality. The changes in pMLC on the western blots are very small, and for any conclusion, these studies require quantification. Further, the expression levels of Flag-ARHGAP18 needs to be shown to support the statement that the protein is expressed, and indeed overexpressed under these conditions (vs just re-expressed). On the same figure, the quality of LIM kinase blots is poor. The signal is weak, and the blot does not appear to support the quantification. The last condition (expression of flag-ARHGAP18) results in a large drop in pLIMK and pcofilin on the blot, which is not reflected by the graph. Addition of a better blot and the use of a strong positive or negative control would boost confidence in these data.
Fig 4: the differences in YAP nuclear localization under the various conditions are not well visible. Quantitation of nuclear/cytosolic signal ratio should be provided. 4. Please provide a rationale and more context for using serum starvation and re-addition. What is the expected effect? Serum removal and addition is referred to as nutrient removal and re-addition, but this is inaccurate, as it does not equal nutrient removal, since serum contains a variety of other important components, e.g. growth factors too. 5. The binding between ARHGAP18 and merlin is interesting, but a key limitation is the use of expressed proteins. Can the binding be shown for the endogenous proteins (IP, colocalization). Another important unaddressed question is the relevance of this binding, and the relation of this to altered YAP nuclear localization.
Minor comments:
- Introduction line 99: "When localized to the nucleus, YAP/TAZ promotes the activation of cytoskeletal transcription factors associated with cell proliferation and actin polymerization" Please clarify what you mean by this statement, that is inaccurate in its present for. Did you mean effects on transcription factors that control cytoskeletal proteins, or do you mean that Yap/Taz affect these proteins? Please also provide reference for this.
- What is the cell confluence in these experiments? For epithelial cells confluence affects actin structure. Please comment on similarity of confluency across experimental conditions?
- Fig 2 legend: please indicate that the protein detected was non-muscle myosin heavy chain (distinct from the light chain detected in Fig 1).
- Line 339-340: please check the syntax of this sentence
- Western blot quantification: the comparison of experiments with samples run on different gels/blots requires careful normalization and experimental consistency. Please describe how this was achieved.
Significance
Rho signalling is a central regulator of an array of normal and pathological cell functions, and our understanding of the context dependent regulation of this key pathway remains very incomplete. Therefore, new knowledge on the role of specific regulators, such as ARHGAP18, is of interest to a very broad range of researchers. A further exciting aspect of this protein, that despite indications by many studies that it acts as a GAP (inhibitor) for Rho proteins, there are findings in the literature that suggest that its manipulation can affect actin in unexpected (opposite) manner. These point to possible Rho-independent roles, and warranted further in-depth exploration. One of the strength of the study is that it explores possible roles of ARHGAP18 beyond RhoA and describes some new and interesting observations, which advance our knowledge. The authors use some excellent tools (e.g. ARHGAP KO cells and re-expression) and approaches (e.g. super resolution microscopy to analyze actin changes, RNAseq and bioinformatics to find genes that may be downstream from ARHGAP18). A key limitation of the study however, is that it is not clear whether the observed findings are indeed independent from RhoA. Further limitation is that potential causal relationships between the described findings are not studied, and therefore the findings are in some cases overinterpreted, and limited mechanistic insights are provided. In some cases the exclusive use of expressed proteins is also a limitation. Finally, some of the experiments also need improvement.<br /> Reviewer expertise: RhoA signalling, guanine nucleotide exchange factors, epithelial biology, cell migration, intercellular junctions.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This manuscript investigates the Rho effector, ARHGAP18 in Jegs cells, a trophoblastic cell line. It presents a number of new pieces of data, which increase our understanding of the importance of this GAP on cell function and explains at a molecular level previous results of other workers in the field. ARHGAP18 was originally given the name "conundrum' and continues to stand apart from the majority of other GAP proteins and their functions. Hence the data here is significant and of high standard.
The data is clear, and the images are of high quality and extremely impressive in their resolution. It is significant and adds a further layer to our understanding of the regulation of cell migration, particularly in the formation and resolution of microvilli.
The data is based on the use of the cell line Jeg3. Even the authors previous publication in eLife is based only on this cell line. They need to show the conclusions are general and not specific to this line of cells. As an extension of this, is the ARHGAP18 function shown here only in transformed cells? Does the same mechanisms operate in normal cells, which respond to activation to proliferate or migrate? In endothelial cells, Lovelace et al 2017 showed localisation to microtubules and that depletion of ARHGAP18 resulted in microtubule instability. The authors may like to comment on the differences. Is this a cell type difference or RhoA versus RhoC difference?
On pages 7,9 they conclude that MLC and basal and junctional actin are regulated through a GAP independent mechanism. The best way to show this is with overexpression of a GAP mutant.
There is a huge amount of data presented in Figure 3, but their 2 genes which they focus on, LOP1 and CORO1A, are discussed but no actual data presented in support.
Significance
The data is significant and adds a further layer to our understanding of the regulation of cell migration, particularly in the formation and resolution of microvilli.
This manuscript will be of significance to an basic science audience in the field of RhoGTPases and cell migration.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This manuscript describes a dual mechanism by which ARHGAP18 regulates the actin cytoskeleton. The authors propose that in addition to the known role for ARHGAP18 in regulating Rho GTPases, it also affects the cytoskeleton through regulation of the Hippo pathway transcriptional regulator YAP. ARHGAP18 knockout Jeg3 cells are were generated and show a clear loss of basal stress fiber like F-actin bundles. The authors further characterize the effects of ARHGAP18 knockout and overexpression. It is also discovered that ARHGAP18 binds to the Hippo pathway regulator Merlin and to YAP. Ultimately it is concluded that ARHGAP18 regulates the F-actin cytoskeleton through dual regulation of RHO GTPases and of YAP. While the phenotype of the ARHGAP18 knockout and the association of ARHGAP18 with Merlin and YAP is interesting, I found the authors conclusion that these phenotypes are due to ARHGAP18 regulation of both RHO and YAP to be based on largely correlative evidence and sometimes lacking in controls or tests for significance. In addition the authors often make overly strong conclusions based on the experimental evidence. In some instances, the rationale for how the experimental results support the conclusion is insufficiently articulated, making evaluation challenging. In general although the authors have some interesting observations, more definitive experiments with proper controls and statistical tests for significance and reproducibility are needed to justify their overall conclusions.
Specific Comments
1) The authors make a big point about the effects of ARHGAP18 on myosin light chain phosphorylation. However this result is not quantified and tested for statistical significance and reproducibility.
2) Along similar lines in Figure 2C they state that overexpression of ARHGAP18 causes cells to invade over the top of their neighbors. This might be true and interesting, but only a single cell is shown and there is no quantification or controls for simply overexpressing something in that cell. The authors also conclude from this image that the overexpression phenotype is independent of its GAP activity on Rho. It is not clear how this conclusion is made based on the data. It would seem like a more definitive experiment would be to see if a similar phenotype was induced by an ARHGAP18 mutant deficient in GAP activity.
3) In Figure 3 the authors compare gene expression profiles of ARHGAP18 knockout cells to wild-type cells. They see lots of differences in focal adhesion and cytoskeletal proteins and conclude that this supports their conclusion that ARHGAP18 is not just acting through RHO. The rationale for this in not clear. In addition, they observe changes in expression profiles consistent with changes in YAP activity. They conclude that the effects are direct. This very well might be true. However RHO is a potent regulator of YAP activity and the results seem quite consistent with ARHGAP18 acting through RHO to affect YAP.
4) In Figure 4A showing Merlin binding to ARHGAP18 there is no control for the amount of Merlin sticking to the column as was done in Figure 4F for binding experiments with YAP. This makes it difficult to determine the significance of the observed binding.
5) The images in Figure 4C showing YAP being maintained in the nucleus more in ARHGAP18 knockout cells compared to wild-type. However the images only show a few cells and YAP localization can be highly variable depending on where you look in a field. Images with more cells and some sort of quantification would bolster this result.
Significance
While the phenotype of the ARHGAP18 knockout and the association of ARHGAP18 with Merlin and YAP is interesting, I found the authors conclusion that these phenotypes are due to ARHGAP18 regulation of both RHO and YAP to be based on largely correlative evidence and sometimes lacking in controls or tests for significance. In addition the authors often make overly strong conclusions based on the experimental evidence. In some instances, the rationale for how the experimental results support the conclusion is insufficiently articulated, making evaluation challenging. In general although the authors have some interesting observations, more definitive experiments with proper controls and statistical tests for significance and reproducibility are needed to justify their overall conclusions.
-
-
d3.harvard.edu d3.harvard.edu
-
Expertise is all about knowledge and experience in a subject. Having a unique perspective adds dimension to interdisciplinary teams. It is the source of passion leveraged to make a difference in the world and keep you coming back to work each day.
Expertise feels personal to me because it comes from the things I’ve taken the time to learn and experience for myself. When I bring my own perspective into a group, it feels like I’m adding something only I can offer. That’s what keeps me motivated knowing that what I know and care about can actually make a difference and give me a reason to show up every day.
-
-
www.forbes.com www.forbes.com
-
So what gets in the way of our pursuit of it? I think we most often resist going through the process of mastery for two reasons: it can be deeply uncomfortable along the way and we doubt our ability to become expert.
I think that ths statement is true because when people get into new things, it seems weird at first but when you adapt to the new habit and start to gradually become better then it eventually becomes a hobby.
-
I think of all the guys who have expensive and complex garage workshops they never use and probably couldn't, or the people who have a huge variety of unused cooking implements in every drawer.
I agree with this because having the tools doesn’t automatically mean you know how to use them. It’s like buying a musical instrument, you won’t play beautiful music on the first day. You need time, patience, and practice before you can actually make good use of it.
-
-
www.discovermagazine.com www.discovermagazine.com
-
We speculate that, in the long term, culture will continue to grow in influence over human evolution until genes become secondary structures that encode human biological design blueprints but are ultimately governed by culture.
for - genes subservient to culture - We speculate that, in the long term, culture will continue to grow in influence over human evolution - until genes become secondary structures that encode human biological design blueprints - but are ultimately governed by culture.
-
for - progress traps - speed of cultural vs genetic evolution
SRG comment - As Ronald Wright pointed out, it is the speed of our cultural evolution that creates a gap between the evolutionary "hardware" we are equipped with and the novel phenomena that we have never encountered before
-
The issue with genetic change is that it tends to be slow and gradual. Cultural evolution, in contrast, can occur within the time it takes to pass a piece of legislation.
for - comparison - cultural vs genetic evolution - speed
-
“On reviewing the evidence, we find that culture solves problems much more rapidly than genetic evolution. This suggests our species is in the middle of a great evolutionary transition.”
for - evolutionary transition - from genetic to cultural - progress trap - cultural evolution
-
Wood, Waring, and many scholars in the field of evolutionary biology believe we are in the midst of another transition, one that has the potential to turn us into a “superorganism.
for - MET - major evolutionary transition in individuality
-
culture has overtaken biology as the predominant evolutionary force in our species
for - cultural evolution vs biological evolution - cultural evolution has overtaken biology as the predominant evolutionary force in our species
Tags
- cultural evolution vs biological evolution
- MET - major evolutionary transition in individuality
- progress traps - speed of cultural vs genetic evolution
- comparison - cultural vs genetic evolution - speed
- progress trap - cultural evolution
- evolutionary transition - from genetic to cultural
- genes subservient to culture
Annotators
URL
-
-
www.americanscientist.org www.americanscientist.org
-
each person is, on average, an assemblage of 37 trillion eukaryotic cells combined with 300 trillion bacterial cells; the 20,000 protein-coding genes in the eukaryotic genome supplemented by 2 million bacterial genes.
for - stats - cells in body - eukaryotic vs microbiotic - eukaryotic - 37 trillion vs microbiome and bacteria - 300 trillion - eukaryotic - 20,000 protein coding genes vs microbiome and bacteria 2 million
-
for our bacterial partners, a human lifetime is deep time.
for - microbiome - one human lifetime is deep time - relativity
-
Because one human lifetime may encompass a million bacterial generations, individual species and the microbiome itself can evolve within a single host.
for - quote - one human lifetime - evolution of a million generations of bacteria - Because one human lifetime may encompass a million bacterial generations, individual species and the microbiome itself can evolve within a single host.
- SRG comment
- wow! One human lifetime might encompass a million generations of bacteria!
- meme- our gut is an evolutionary lab for bacteria!
-
For many if not all members of the human microbial fauna, generation times are measured in hours or even minutes. These short generation times, coupled with the large population sizes of many bacteria, effectively elide the boundary between ecological and evolutionary time
for - microbiome - blurs ecological and evolutionary time - due to short generation time of microfauna
-
Anthropogenically driven climate change is occurring too quickly to enable the majority of plant and animal species to respond through genotypic change. Instead, most species can only change their geographic distribution,
for - genotypic change - anthropogenically driven climate change - too quick for genotypic change adaptation
-
In contrast to the traditional focus on the individual organism as the target of selection and the unit of evolution, the genetic information embodied by each of our microbiomes may itself be the target for and the product of the evolutionary process.
for - unpack - microbiome as the target for / product of evolution
-
The collective microbial genome in our gut may include 100-fold more genetic information than what can be found in our own eukaryotic cells.
for - trivia - microbiome - 100x more genetic information here than our other body cells -meme - most of the genetic information inside you is not really you
-
bacteria engage in extensive horizontal gene transfer
for - definition - horizontal gene transfer - genetic material transfer across species boundaries
-
efficient removal of potentially toxic by-products of metabolism, and provides a homeostatic environment for bacterial growth
for - microbiome - functions - efficient removal of toxic byproducts of metabolism - homestatic environment for(beneficial) bacterial growth
-
Marked differences between one human microbiome and the next suggest that no single bacterial species must always be present in the gut—or in any other body environment—to ensure a working microbiome.
for - microbiome - keystone ROLES - no keystone species - can vary from person to person
-
keystone roles
for - keystone roles vs keystone species - microbiome
-
keystone species
for - definition - keystone microbiome species - critical species that, if removed, can engender collapse of the entire microbiome system
-
microbiome is clearly linked to the maturation and regulation of the human immune system,
for - adjacency - human microbiome - immune system
-
the passage through the birth canal seeds the newborn’s microbiome. Infants delivered by cesarean section, in contrast, exhibit a distinct microbiome that more closely resembles the composition of the mother’s skin
for - trivia - birth - microbiome - natural childbirth vs cesarean - birth canal seeds newborn's microbiome. Cesarean section alters it - microbiome more like mother's skin!
-
nstead, such perturbed ecosystems may settle on a new composition that includes different species, many of them resistant to antibiotic treatment.
for - progress trap - long term antibiotic use - can create new composition of microbiome with species resistant to antibiotic treatment
-
the Human Microbiome Project
for - The Human Microbiome Project - progress trap - antibiotics - severely disrupts the microbiome (Human Microbiome Project)
-
microbiomics
for - definition - microbiomics - the study of the multiple microbial ecosystems that constitute the (human) bidy
Tags
- microbiome - keystone ROLES - no keystone species - can vary from person to person
- genotypic change - anthropogenically driven climate change
- microbiome - one human lifetime is deep time - relativity
- quote - one human lifetime - evolution of a million generations of bacteria
- microbiome - functions
- adjacency - human microbiome - immune system
- The Human Microbiome Project
- microbiome - blurs ecological and evolutionary time - due to short generation time of microfauna
- trivia - microbiome - 100x more genetic information here than our other body cells
- unpack - microbiome as the target for / product of evolution
- progress trap - long term antibiotic use - can create new composition of microbiome with species resistant to antibiotic treatment
- keystone roles vs keystone species - microbiome
- definition - microbiomics
- definition - horizontal gene transfer
- meme- our gut is an evolutionary lab for bacteria!
- -meme - most of the genetic information inside you is not really you
- progress trap - antibiotics - severely disrupts the microbiome
- stats - cells in body - eukaryotic vs microbiotic
- definition - keystone microbiome species
- trivia - birth - microbiome - natural childbirth vs cesarean
Annotators
URL
-
-
r11.core.learn.edgenuity.com r11.core.learn.edgenuity.com
-
wait I see that the admins can see this are y'all human admins or an automation of an admin? @hypmod
sorry if me pinging you without a big reason is bad I just wanted to know cause I'm bored
-
-
www.youtube.com www.youtube.com
-
what we are saying is we do not know how the soul generates the intelligence it clearly has but that it has it is obvious it doesn't divide until it knows that the genome has been accurately replicated
for - cell intelligence - the cell knowns when to divide - it won't divide until all replication errors have been fixed
-
both replication is dependent on the living cell and the function of the proteins is dependent on the living cell. Neither automatically follow from the DNA alone
for - key insight - cell replication AND function of proteins are both dependent on the living cell. Neither follow from DNA alone - Denis Noble
-
don't think at the molecular level we know exactly how a cell can be said to know the time has come to divide but it won't do so until that is the case
for - unanswered question - when cells know when to divide
-
cut and paste enzymes
for - definition - cut and paste enzyme - living cells provide cut and past enzyme to run along the genome and correct all the replication errors
-
about dozen years ago, chemists actually checked if you unravel the DNA in a dish without a cell, how does it replicate?
for - DNA replication experiment - in vitro - lots of errors - 1/10,000 pairs error rate - our genomes 3 billion base pairs long each - so 300,000 errors - would be fatal to any cell
-
It was actually Shreddinger who formulated that idea way back in 1942 when he wrote a book called what is life?
for - book - What is Life? Schrodinger - formulated the idea behind the central dogma of molecular biology
-
Francis Crick had formulated what he called the central dogma of molecular biology
for - definition - The central dogma of molecular biology \ - our genes generate proteins - proteins form bodily structures - everything within our body can therefore be predicted from the level of the DNA
-
the teological sin
for - definition - the teological sin - biological systems aren't suppose to have agency!
-
The first heartbeat in an animal as complicated as us occurs after about 28 days of an embryo
for - embryo - 28 days - first circulation system - why? CO2 and O2 diffuse over distances of a few microns - at 28 days, the embryo is about 30mm so needs a circulatory system to diffuse CO2 and O2
Tags
- definition - The central dogma of molecular biology
- definition - the teological sin - biological systems aren't suppose to have agency!
- cell intelligence - the cell knowns when to divide - it won't divide until all replication errors have been fixed
- book - What is Life? Schrodinger - formulated the idea behind the central dogma of molecular biology
- DNA replication experiment - in vitro - lots of errors
- unanswered question - when cells know when to divide
- key insight - cell replication AND function of proteins are both dependent on the living cell. Neither follow from DNA alone - Denis Noble
- definition - cut and paste enzyme
- embryo - 28 days - first circulation system
Annotators
URL
-
-
sites.google.com sites.google.com
-
“I’ve just always had the idea of growing and continuing to expand and show who I am outside of the basketball court,”
I find this so fascinating. I feel like entrepreneurs are always just extremely creative or athletic. For example, the owner of Adidas was a huge sports fan that he even made a shoe brand for athletes. It's cool to see how his love for a sport translated into something bigger like owning a business. I had no idea an ice cream company had so much depth to it.
-
-
www.psychologytoday.com www.psychologytoday.com
-
Openness to Experience correlates with creativity,
I believe those with creativity are more open to the world because of their curiosity and eagerness to see the beauty in everything. It's important in the business world because you're able to see through a different lens, and see meaning that a lot of people tend to overlook. That's why creativity correlates to more innovation and that increases your incentive to have a successful business.
-
-
www.biorxiv.org www.biorxiv.org
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Dear editor and reviewers,
We sincerely thank you for your thoughtful comments and constructive suggestions, which have greatly improved the quality and clarity of our manuscript. In response, we have implemented all requested changes, which are highlighted in yellow throughout the revised text, and updated several figures accordingly. Furthermore, we have performed all additional experiments recommended by the reviewers and incorporated the new data into the manuscript. To enhance clarity, we have also included a schematic representation of our proposed model in an additional figure, providing a concise visual summary of our findings.
We hope that these revisions fully address all concerns raised by the reviewers and meet all the expectations for publication.
Below, we answer the reviewers point by point (in blue).
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
In this paper, the authors address the important question of the role of centrosomes during neuronal development. They use Drosophila as an in vivo model. The field is somewhat unclear on the role and importance of centrosomes during neuronal development, although the current data would suggest they are dispensable for axon specification and growth. Early studies in cultured mammalian neurons showed that centrosomes are active and that their microtubules can be cut and transported into the neurites. But a study then showed that centrosomes in these cultured neurons are deactivated relatively early during neuronal development in vitro and that ablating centrosomes even when they are active had no obvious effect on axon specification and growth. Consistent with this, a study in Drosophila provided evidence that centrosomes were not active or necessary in different types of neurons. More recently, a study showed that centrosomal microtubules are dispensable for axon specification and growth in mice in vivo but are required for neuronal migration in the cerebral cortex. However, another study has linked the generation of acetylated microtubules at centrosomes with axon development. In this current study, the authors examine the effect of centrosome loss on various motor and sensory neurons and muscles mainly by examining mutants in essential centriole duplication genes. They associate axonal routing and morphology defects with centrosome loss and provide some evidence that centrosomes could still be active in the developing neurons. Overall, they conclude that centrosomes are active during at least early neuronal development and that this activity is important for proper axonal morphology and routing.
While I think this study addressing a very interesting and important question, I think as it stands the data is not sufficient to be conclusive on a role for centrosomes during neuronal development. My biggest concern is that most phenotypes have not yet been shown to be cell autonomous, as whole animal mutants have been analysed rather than analysing the effect of cell-specific depletion, and the evidence for active centrosomes needs to be strengthened. If the authors can provide stronger evidence for a role of centrosomes in axonal development then the paper will certainly be of interest to a broad readership.
We thank the reviewer for the clear and concise summary and fully agree that our study addresses a critical gap in understanding. Centrosomes have long been implicated in morphogenesis, yet their precise contribution to nervous system development has remained unclear. Our findings provide compelling evidence that centrosomes are indispensable for proper nervous system formation and that their absence also triggers muscular defects, highlighting their broader role in tissue organization.
We acknowledge that the original manuscript lacked some key details; therefore, we have now strengthened our conclusions with additional experiments. Specifically, we demonstrate that these effects are cell-autonomous by using two independent RNAi lines targeted to a subset of motor neurons. Furthermore, we present new data showing that neuronal centrosomes remain active during the early stages of axonal development, emphasising their functional relevance in morphogenesis. All new experiments, figures, and corresponding text revisions are detailed below.
Major comments 1) The sas-6 transallelic combination shows only 17% embryonic lethality compared to 50% embryonic lethality with sas-4 mutants. Given that both mutants should result in the same degree of centrosome loss (this should be quantified in sas-6 mutants) it would suggest that either sas-4 has other roles away from centrosomes or that the sas-4 mutant chromosome used in the experiment has other mutations that affect viability. The effect of picking up "second-site lethal" mutations on mutant chromosomes is common and so I would not be surprised if this is the reason for the difference in phenotypes. This can be addressed either by "cleaning up" the sas-4 mutant chromosome by backcrossing to wild-type lines, allowing recombination to occur and replace the potential second site mutations, or by using transallelic combinations of sas-4, as they did for sas-6. The "easier" option may just be to analyse all the phenotypes with the sas-6 transallelic combination.
We appreciate this comment, as it brought to light an issue with the CRISPR line Sas-6-Δa. Upon reanalysing all the data, we determined that this line is embryonic lethal both in homozygosis and when combined with the deficiency uncovering the genomic region, Df(3R)BSC794. In contrast, Sas-6-Δb homozygotes are viable. The inconsistency between these results raised concerns about whether the Δa and Δb Sas-6 mutants carry deletions confined to the Sas-6 coding region. Although this would not hinder our cell biology analysis, it could represent a problem in viability tests. To address this, we repeated all analyses using Sas-6-Δb homozygotes and Sas-6-Δb combined with Df(3R)BSC794. These new results are more consistent and indicate that approximately 50% of Sas-6/Def individuals hatch as adults. Fig. 3 was redone and the manuscript text changed in view of these results.
2) Using "whole animal" mutants for assessing neuronal morphology is risky due to non-cell-autonomous effects. The authors have carried out some phenotypic analysis of neurons depleted of Sas-4 by cell-specific RNAi, but I feel they need to do this for all of their analysis. This includes embryonic lethality measures, quantification of centrosome numbers, and all axonal phenotypes in Sas-4 RNAi neurons. It would also be prudent to use 2 distinct RNAi lines to help ensure any phenotypes are not off-target effects (and this may help clarify why the authors see some additional phenotypes with RNAi). Indeed, there are relatively weak phenotypes in muscles when using RNAi compared to the mutants and these potential non-cell-autonomous effects could then have a knock-on effect on neuronal morphology. If the authors were concerned that RNAi is not very efficient (explaining any potential weaker phenotypes than in mutants) the authors could examine the effectiveness of RNAi lines by analysing protein depletion by western blotting or mRNA depletion by rt-qPCR (although this has to be done in a different cell type due to the difficulty in obtaining a neuronal extract).
We have now added a new panel to supplementary Figure 1, showing how the expression of a different Sas-4 RNAi line (2) induces similar nervous system phenotypes when expressed only in aCC, pCC and RP2 pioneer neurons (Sup. Fig. 1 M-O).
3) When analysing centriole presence or absence it is a good idea to stain with two different centriole markers e.g. Asl and Plp. This helps rule out unspecific staining. It is clear from the images that similar sized foci can be observed outside of the cells (see Figure 5A for example), so clearly some of the foci that appear to be within the cells may also be unspecific staining.
In a new supplementary figure, we now show that Asl and Plp colocalize and quantify the number of times we find this colocalization in neurons (Supl. Fig 3). In addition, and we apologise for the confusion, but the reason why there are foci outside the marked cells is because these are wholemount embryonic stainings and the anti-Plp antibody marks all centrosomes in all cells in the embryo.
4) The evidence for active centrosomes is not that convincing. Acetylated tubulin is associated with stable MTs, which are not normally organised by "active" centrosomes that nucleate dynamic microtubules. Moreover, it is plausible that centriole foci happen to overlap with the acetylated tubulin staining by chance. This would explain why not all centrosomes colocalise with acetylated tubulin signal. The authors could better test centrosome activity by performing live imaging with EB1-GFP. If centrosomes are active, it is very easy to observe the many comets produced by the centrosomes.
We appreciate the reviewer’s comment and agree that acetylated tubulin alone is not an ideal marker for centrosome activity. To address this, we performed live imaging of aCC neurons expressing EB1-GFP together with Asl-Tomato. This was technically challenging because we were imaging only two neurons per segment in live embryos, under significant limitations in fluorescence detection and timing. Despite these constraints, we were able to clearly observe EB1 comets emerging from the centrosome and moving toward the cell periphery, providing direct evidence of microtubule nucleation from centrosomes in neurons.
Importantly, we complemented this with a microtubule depolymerization/polymerization assay, which provides unequivocal evidence that polymerization initiates at the centrosome. After depolymerization, we observed microtubule regrowth from the centrosome, confirming its role as an active microtubule-organizing centre in these neurons. Together, we hope that these results are enough to demonstrate that neuronal centrosomes are functionally active during early axonal development. These experiments are presented in Figure 6 and corresponding text in the manuscript.
5) If the authors believe that centrosomes have a role in axon pathfinding in sensory neurons, they should show that these centrosomes are active, at least during early stages (again using EB1-GFP imaging).
We appreciate the reviewer’s suggestion and agree that EB1-GFP imaging would be the most direct way to assess centrosome activity in sensory neurons. However, performing time-lapse imaging in these neurons is technically very demanding due to their location and accessibility in live embryos, and we did not attempt this approach. Instead, we now provide new evidence showing that sensory neuron centrosomes colocalize with both α-tubulin and γ-tubulin. This strongly supports that these centrosomes are associated with microtubule nucleation machinery and are as likely as motor neuron centrosomes to be active during early stages of axon development. These new data have been included in the revised manuscript (see Figure 5 and corresponding text).
6) The authors mention in the discussion that "increased JNK activity, can result in axonal wiggliness (Karkali et al, 2023)". I therefore wonder whether centrosome loss may induce JNK activation (the stress response), as this would then indicate an indirect effect of centrosome loss on axonal structure rather than a direct influence of centrosome-generated microtubules. The authors could assess whether the DNK-JNK pathway is activated in neurons lacking centrosomes by expression UAS-Puc-GFP and quantifying the nuclear signal.
In a new supplementary figure, we now show by using a reporter for JNK signalling, as requested, that Sas-4 neurons do not activate the JNK pathway (Supl. Fig 4).
7) In Figure 5, the authors claim that they find "a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". I don't think this is a strong correlation. The difference in centriole number between embryos with no defects and those with defects is very small. In contrast, the difference between centriole numbers in control (no defects) and mutant (no defects) is very large. So, there does not appear to be a strong correlation between centrosome number and phenotype.
We agree and we have corrected this sentence to better explain the results.
Minor comments
1) I don't understand Figure 3C - why do the % of surviving homozygotes and heterozygotes add up to 100%? Should the grey boxes not relate to dead and the white to surviving?
Thank you for pointing this out. Figures 1B and 3C represent only the surviving individuals. The grey boxes correspond to surviving homozygotes, and the white boxes correspond to surviving heterozygotes. The percentages add up to 100% only at embryonic stages because all embryos reach late embryonic stages. The grey and white boxes reflect the proportion of these two genotypes among the survivors, not the total number of embryos including those that died. We have changed the text to convey this.
2) "In mouse fibroblasts, myoblasts and endothelial cells, centrosome orientation is important for nuclear positioning and cell migration(Chang et al, 2015; Gomes et al, 2005; Kushner et al, 2014)." Do you mean "centrosome position"?
Yes, text changed, thank you for spotting it.
3) In the introduction, the authors mention Meka et al. when saying the centrosomal microtubules are important for axonal development, but they should also discuss the counter argument from Vinopal et al., 2023 (Neuron) that showed how centrosomes were required for neuronal migration but not axon growth, which was instead mediated by Golgi-derived microtubules.
Done, thank you very much.
4) Lines 228-230 - repeated sentence
Corrected, thank you very much.
5) Additionally, we did not detect centrioles in the quadrant opposite the axon exit point (Fig. 2B n=75) - this data is not in Fig 2B
Correct, it is in figure 4B, thank you very much.
6) "This significant decrease in the humber of centrioles further supports the critical role of Sas-4 in pioneer neurons of the ventral nerve cord (VNC) during Drosophila embryogenesis". It rather highlights that Sas-4 is required for centriole formation in these neurons. Also, humber = number.
We agree, and have changed the text, thank you very much.
7) Result title: Non-ciliated sensory neurons have centrioles. This is kind of obvious. A better title may be "axon phenotypes correlate with centriole numbers in sensory neurons" but unfortunately i don't think there is good evidence for this (See major point above).
We agree and we have changed. We now believe we have strong evidence to support it. We hope the additional data presented in the revision convincingly demonstrate this point.
Reviewer #1 (Significance (Required)):
As mentioned above, the advance will be important if more evidence is provided. In this case, the paper will be interesting to a broad readership. But currently the paper is limited by the lack of evidence for centrosome function and activity in the neurons.
We hope that reviewer 1, now considers that the manuscript is not limited anymore and that it shows convincing evidence for centrosome function and activity in embryonic neurons.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Summary: In this manuscript, Gonzalez et al. examine the potential function of centrosomes in the neurons and muscle cells of Drosophila embryos. By studying various mutant and RNAi lines in which centriole duplication has been disrupted, they conclude that the loss of centrioles disrupts axonal pathfinding and muscle integrity.
Major points: 1. Throughout the manuscript, the phenotypes presented are often quite subtle. For this reason, I would really recommend that these experiments are scored blind. Perhaps the authors did this, but I didn't see any mention of this.
All our phenotypic analyses are performed blind. We apologize for not having originally included this information in the Methods section; it has now been added. Embryos are stained using colorimetric methods (DAB) to label the nervous system, while balancer chromosomes are marked with a fluorescent antibody. This approach allows us to assess and quantify phenotypes using white light without knowing whether the embryos are homozygous mutants or heterozygous, which can only be detected by changing the channels to fluorescence.
- The authors conclude that neurons have active centrioles that function as centrosomes (Figure 6), but the data here is confusing. The authors state that in these cells they observe acetylated MTs extending from the centrosomes and these colocalised with g-tubulin. But the authors don't show the overlap between centrosomes, g-tubulin and MTs, as they stain for these separately. This is problematic, as it was not clear from these images that the majority of the MTs really are extending from the centrosome: the centrosome may just associate or be close by to these MT cables (Figure 6A,B). Moreover, the authors show that only a fraction of the centrosomes in these cells associate with g-tubulin, so presumably in cells where the centrosomes lack g-tubulin they would not expect the centrosomes to be associated with the MTs-but they do not show that this is the case. Perhaps the authors can't test this, but an alternative would be to show that these MT arrays are absent in Sas-4 mutants. This would give more confidence that these MTs arise from the centrosomes.
We agree that the initial data based on acetylated microtubules and γ-tubulin colocalization were not sufficient to conclude that microtubules originate from the centrosome, as these markers can only suggest association. To address this, we have now included additional experiments that provide direct evidence of centrosome activity.
First, we performed live imaging of aCC neurons expressing EB1-GFP together with Asl-Tomato. Despite the technical challenges of imaging only two neurons per segment in live embryos under strict fluorescence and timing constraints, we were able to clearly observe EB1 comets emerging from the centrosome and moving toward the cell periphery. This demonstrates active microtubule nucleation from centrosomes rather than mere proximity to microtubule bundles.
Second, we carried out a microtubule depolymerization/polymerization assay, which provides unequivocal evidence that polymerization initiates at the centrosome. After depolymerization, microtubules regrew from the centrosome, confirming its role as an active microtubule-organizing center. These experiments go beyond colocalization and directly address the concern that centrosomes might simply be adjacent to microtubule cables.
Regarding the suggestion to use Sas-4 mutants, while we did not perform this experiment, the regrowth assay combined with EB1 imaging strongly supports that these microtubules originate from the centrosome. All new data are presented in Figure 6 and the corresponding text in the revised manuscript.
- The authors show that muscle cell integrity is compromised by centriole-loss (Figure 2). This is very surprising as it is widely believed that centrosomes are non-functional in muscle cells, and the MTs are instead organised around the nuclear envelope. I'm not aware of the situation in Drosophila muscle cells, but the authors should ideally try to examine if the centrioles are functioning as centrosomes in these cells. At the very least they should discuss how they think centriole-loss is influencing the muscle integrity when it is widely believed they are inactive in these cells.
We do not claim that centrosomes are active in muscle cells at these developmental stages. The observed muscle defects could result from earlier processes such as cell division, migration, or muscle fusion. We agree that this is an intriguing observation; however, pursuing this question further would go beyond the scope of the current manuscript. As requested by the reviewer, we have now expanded the discussion to consider how centriole loss might impact muscle integrity.
Regardless of the strength of the supporting data, I think the authors should tone down their conclusions. The title and abstract led me to believe that centriole loss would cause significant problems in axonal pathfinding and muscle integrity. In all the mutant specimens examined (and certainly the low magnification views shown in Figure 1D'-F', Figure 1I'-K' and Figure 2D'-F') the mutants look very similar to the WT. Many readers may not get past the title and abstract, so the authors should make it clearer that these defects are very subtle.
We have changed the text to convey this idea.
Minor points: 1. In Figures 4 and 5, CP309 staining is relied on to identify centrioles, but there is quite a background of non-specific dots, making it hard to be certain what is a centriole and what isn't. For example, in Figure 5D' there are lots of dots within some of the cells - are any of these centrioles? How can the authors be certain which dot is a centriole in some of the cells shown in Figure 5C'? Is it possible to use a second marker and only count as centrioles dots that are recognised by both antibodies?
We thank the reviewer for this suggestion and agree that using a second marker improves confidence in centriole identification. In a new supplementary figure (Supplementary Fig. 3), we now show that Asl and Plp colocalize in neurons and provide a quantification of the frequency of this colocalization. This dual labelling confirms the identity of centrioles and addresses the concern about non-specific background.
We also apologize for any confusion regarding the presence of foci outside the marked cells. These images are whole-mount embryonic stainings, and the anti-Plp antibody labels all centrosomes in all cells of the embryo, which explains the additional foci observed.
In the abstract that authors state that traditionally centrosomes have been considered to be non-essential in terminally differentiated cells. I don't think this is correct. In the standard "textbook" view of a cell, the centrosome is normally positioned in the centre of the cell organising an extensive array of MTs that are thought play an important role in organising intracellular transport, the positioning and movement of organelles and the maintenance and establishment of cell polarity. I don't think it is only recent evidence that suggests they play vital roles in terminally differentiated cells.
We thank the reviewer for this correction and we have changed the text accordingly.
- Line 162 the authors state that in the RNAi knockdown lines they observe several additional phenotypes, but then in the same sentence (Line 164) they say that these defects were also observed in the original mutant and mutant/Df lines.
We apologise for this confusion, we have rearranged the sentence for clearance.
The sentences in Line281-287 don't reference any of the Figures, so it seems the authors are just stating these results without presenting any data (e.g. "Significantly, we also found a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". If they've tested this correlation, they should show it.
We have rearranged the sentences for better understanding.
In Figure 7 I did not understand how the authors measured tortuosity (wiggliness) and could see no description in the methods. This is important as, again the defect seems quite subtle, but perhaps I am not understanding which bits of the axon are being measures. Is it just the small bit of the axons close to the asterixis that is being measured, or the whole FasII track?
We have now added another quantification and additional descriptions in the methods section.
Reviewer #2 (Significance (Required)):
The potential function of centrosomes in axonal outgrowth is quite controversial, so this study is potentially of considerable interest.
However, several aspects of the data presented here were confusing or not terribly convincing. In its present state, I don't think the main conclusions are strongly enough supported by the data.
We hope that reviewer 2, now considers that the manuscript is not confusing anymore and that it shows convincing evidence for centrosome function and activity in embryonic neurons.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
The manuscript of González et al. entitled "Centriole Loss in Embryonic Development Disrupts Axonal Pathfinding and Muscle Integrity" deals with the role of centrosomes in shaping axonal morphology. To this aim the AA analysed Drosophila Sas-4 mutants that are reported to develop until adult stage without centrioles. Remarkably, the AA observe that 50% of the homozygous mutant embryos fail to hatch as larvae. The present observations suggest that centrosome loss results in axonemal shaping defects and muscle developmental abnormalities. Finally, the AA show the presence of functional centrosomes in neurons. In my opinion, the manuscript is interesting because shows unexpected findings. However, to justify these new findings the AA are required to improve some experimental observations.
We thank the reviewer for his summary of our work and for considering it interesting. We have taken into account all the comments and believe that these have helped improve our manuscript.
Major: Abstract- It is unclear in which phenotypic condition the observations of centrosome loss or centrosome presence have been found. Please better explain. l.36. embryos, larvae, adult, from Sas4 or controls? If mutants, the observations are very interesting since Sas4 would be without centrioles. Indeed, Basto et al., show that chemosensory neurons do not develop an axoneme in the absence of centrioles, but extend dendrites toward the sensory bristle.
We have made clear which refer to wild-type and which are Centriole Loss (CL) conditions. CL conditions refer to mutant and downregulation conditions, whereas targeted downregulation refers to RNAi downregulation only in neurons.
I do not think appropriate the use of "centriole" in the main title since the centrioles would be localized by true centriolar antigens rather than by centrosomal antigens. This problem occurs throughout the text and some figures where the AA image centrioles by centrosomal material. In Gig. 5A only the AA properly look at Asl localization. The other pictures of presumptive centrioles or centriole quantification report CP309 dots. This localization does not unequivocally reveal centrioles, since CP309 is essentially required for centrosome-mediated Mt nucleation. There are differentiated Drosophila tissues in which centrioles are present, but inactivated, and unable to recruit pericentriolar material. Mt are nucleated by ncMTOCs that contain centrosomal material and gamma-tubulin. Thus, the centrosomal antigens do not colocalize with centrioles.
We have changed centrioles to centrosomes in the title and most sections in the manuscript. We have also included an extra control, showing that Asl and Plp colocalize and quantify the number of times we find this colocalization in neurons (Supl. Fig 3). Asl is a reliable and widely used marker for centrioles, as it localizes specifically to the centriole structure (Varmark H, Llamazares S, Rebollo E, Lange B, Reina J, Schwarz H, Gonzalez C. Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila. Curr Biol. 2007 Oct 23;17(20):1735-45. doi: 10.1016/j.cub.2007.09.031. PMID: 17935995.)
Minor: l. 58. The early arrest is mainly due to a checkpoint control. In double mutant for Sas4 and P53 the embryos survive longer, even if their further development is asrrested.
We thank the reviewer for this comment, and we have changed the text accordingly.
- Previous works, also quoted by the AA, reported that in mature neurons the centrosome are inactivated, whereas the present manuscript describes functional centrosomes in Drosophila motor and peripheral nervous system. This is an intriguing observations that needs a better explanation in Discussion section.
We thank the reviewer for this comment, and we have changed the discussion accordingly.
l.143-145. I understand that 50% of the Sas4 embryos that reach the adult stage have centrioles. Is it correct? But if it is so, how the AA explain the absence of centrioles in sensory neurons of adult flies as reported by Basto et al. ?
According to our results they have less centrioles than controls already at embryonic stages. In addition, as reported in Basto et al. they continue losing centrioles during larval stages and metamorphosis, which explains why centrioles are not detected at adult stages.
l.215. It is unclear for me why the AA analyse Sas6 flies, unless explain the mutant phenotype.
To strengthen our conclusions with Sas-4 and exclude the possibility that the observed phenotypes arise from a centrosome-independent function of Sas-4. For this reason, we have taken additional steps to confirm that the effects are specifically due to centrosome loss and we used Sas-6 mutants as one of these.
l.221. How the centrioles have been quantified? What antibody, the AA used.
We have quantified centrosomes using antibodies agains Plp (CP309) and Asl-YFP expression.
l.244. and Fig 4C,D. I see high background with CP309. As reported previously I think better to use antibodies against centriolar proteins, such as Sas6, Ana1, Asl, or Sas4 ( if centrioles are present in 50% of mutants as the AA claim, the antibody could be also useful). In addition, I can see some CP309 spots in Fig 4E,F. Are they centrioles?
Indeed, as we report, Sas-4 mutant embryos are not totally devoid of centrosomes. In addition, and we apologise for the confusion, but the reason why there are foci outside the marked cells in control embryos is because these are wholemount embryonic stainings and the anti-Plp antibody marks all centrosomes in all cells in the embryo, not just in the neurons.
l.270 and Fig. 5A and Fig.5 C-E. Why the AA localize Cp309 and not Asl (Fig. 5A) to detect centrioles?
In a new supplementary figure, we now show that Asl and Plp colocalize and quantify the number of times we find this colocalization in neurons (Supl. Fig 3). So, we can use CP309 in neurons to the same effect as Asl-
L295-296. I cannot see Mts, but only a diffuse staining. I am expecting to see distinct Mt bundles.
In figure 5 it is now easier to see the MT bundles in the new experiment in Fig. 5F-I , where we performed MT depolymerisation/repolymerisation: Nevertheless, we need to stress out that we are doing these analyses in wholemount embryonic stainings.
326-327. How the AA explain this different lethality, even if both the proteins are involved in centriole assembly?
We have now redone all the viability and mutant phenotype analysis using Sas-6 CRISPR mutant over the Deficiency, which is a better way to access the phenotype.
335-337. In my opinion the quoted publications are not relevant.
We believe that these references back up our hypothesis because:
- Metzger et al 2012 stress the importance of nuclear position in muscle development in Drosophila
- Loh et al 2023, relate centrosomes with nuclear migration in Drosophila
- Tillery et al 2018, is a review describing MTs in muscle development in Drosophila.
358-359. Does maternal contribution persist after gastrulation?
While bulk degradation occurs by midblastula transition, some stable maternal products persist beyond gastrulation. In our case, if centrioles are formed due to the maternal contribution, they will only be diluted by cell division, which explains why we can detect centrioles at late embryonic stages.
l.366. This is an intriguing point, but as previously observed I have some problem with centriole localization. References. Please uniform Journal abbreviations and control page numbers.
I hope we have clarified this problem with the new experiments showing MT repolarization from the centrosomes in neurons.
Reviewer #3 (Significance (Required)):
The manuscript is potentially interesting for peoples working of cell and molecular biology, and development. However, the paper needs an additional working to be suitable for publication.
We hope that reviewer 3, considers that the additional work and revision make this manuscript suitable for publication.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
The manuscript of González et al. entitled "Centriole Loss in Embryonic Development Disrupts Axonal Pathfinding and Muscle Integrity" deals with the role of centrosomes in shaping axonal morphology. To this aim the AA analysed Drosophila Sas-4 mutants that are reported to develop until adult stage without centrioles. Remarkably, the AA observe that 50% of the homozygous mutant embryos fail to hatch as larvae. The present observations suggest that centrosome loss results in axonemal shaping defects and muscle developmental abnormalities. Finally, the AA show the presence of functional centrosomes in neurons. In my opinion, the manuscript is interesting because shows unexpected findings. However, to justify these new findings the AA are required to improve some experimental observations.
Major:
Abstract- It is unclear in which phenotypic condition the observations of centrosome loss or centrosome presence have been found. Please better explain. l.36. embryos, larvae, adult, from Sas4 or controls? If mutants, the observations are very interesting since Sas4 would be without centrioles. Indeed, Basto et al., show that chemosensory neurons do not develop an axoneme in the absence of centrioles, but extend dendrites toward the sensory bristle.
I do not think appropriate the use of "centriole" in the main title since the centrioles would be localized by true centriolar antigens rather than by centrosomal antigens. This problem occurs throughout the text and some figures where the AA image centrioles by centrosomal material. In Gig. 5A only the AA properly look at Asl localization. The other pictures of presumptive centrioles or centriole quantification report CP309 dots. This localization does not unequivocally reveal centrioles, since CP309 is essentially required for centrosome-mediated Mt nucleation. There are differentiated Drosophila tissues in which centrioles are present, but inactivated, and unable to recruit pericentriolar material. Mt are nucleated by ncMTOCs that contain centrosomal material and gamma-tubulin. Thus, the centrosomal antigens do not colocalize with centrioles.
Minor:
l. 58. The early arrest is mainly due to a checkpoint control. In double mutant for Sas4 and P53 the embryos survive longer, even if their further development is asrrested.
l. 102. Previous works, also quoted by the AA, reported that in mature neurons the centrosome are inactivated, whereas the present manuscript describes functional centrosomes in Drosophila motor and peripheral nervous system. This is an intriguing observations that needs a better explanation in Discussion section.
l.143-145. I understand that 50% of the Sas4 embryos that reach the adult stage have centrioles. Is it correct? But if it is so, how the AA explain the absence of centrioles in sensory neurons of adult flies as reported by Basto et al. ?
l.215. It is unclear for me why the AA analyse Sas6 flies, unless explain the mutant phenotype.
l.221. How the centrioles have been quantified? What antibody, the AA used.
l.244. and Fig 4C,D. I see high background with CP309. As reported previously I think better to use antibodies against centriolar proteins, such as Sas6, Ana1, Asl, or Sas4 ( if centrioles are present in 50% of mutants as the AA claim, the antibody could be also useful). In addition, I can see some CP309 spots in Fig 4E,F. Are they centrioles?
l.270 and Fig. 5A and Fig.5 C-E. Why the AA localize Cp309 and not Asl (Fig. 5A) to detect centrioles?
L295-296. I cannot see Mts, but only a diffuse staining. I am expecting to see distinct Mt bundles.
L. 326-327. How the AA explain this different lethality, even if both the proteins are involved in centriole assembly?
l. 335-337. In my opinion the quoted publications are not relevant.
l. 358-359. Does maternal contribution persist after gastrulation?
l.366. This is an intriguing point, but as previously observed I have some problem with centriole localization.
References. Please uniform Journal abbreviations and control page numbers.
Significance
The manuscript is potentially interesting for peoples working of cell and molecular biology, and development. However, the paper needs an additional working to be suitable for publication.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary: In this manuscript, Gonzalez et al. examine the potential function of centrosomes in the neurons and muscle cells of Drosophila embryos. By studying various mutant and RNAi lines in which centriole duplication has been disrupted, they conclude that the loss of centrioles disrupts axonal pathfinding and muscle integrity.
Major points:
- Throughout the manuscript, the phenotypes presented are often quite subtle. For this reason, I would really recommend that these experiments are scored blind. Perhaps the authors did this, but I didn't see any mention of this.
- The authors conclude that neurons have active centrioles that function as centrosomes (Figure 6), but the data here is confusing. The authors state that in these cells they observe acetylated MTs extending from the centrosomes and these colocalised with g-tubulin. But the authors don't show the overlap between centrosomes, g-tubulin and MTs, as they stain for these separately. This is problematic, as it was not clear from these images that the majority of the MTs really are extending from the centrosome: the centrosome may just associate or be close by to these MT cables (Figure 6A,B). Moreover, the authors show that only a fraction of the centrosomes in these cells associate with g-tubulin, so presumably in cells where the centrosomes lack g-tubulin they would not expect the centrosomes to be associated with the MTs-but they do not show that this is the case. Perhaps the authors can't test this, but an alternative would be to show that these MT arrays are absent in Sas-4 mutants. This would give more confidence that these MTs arise from the centrosomes.
- The authors show that muscle cell integrity is compromised by centriole-loss (Figure 2). This is very surprising as it is widely believed that centrosomes are non-functional in muscle cells, and the MTs are instead organised around the nuclear envelope. I'm not aware of the situation in Drosophila muscle cells, but the authors should ideally try to examine if the centrioles are functioning as centrosomes in these cells. At the very least they should discuss how they think centriole-loss is influencing the muscle integrity when it is widely believed they are inactive in these cells.
- Regardless of the strength of the supporting data, I think the authors should tone down their conclusions. The title and abstract led me to believe that centriole loss would cause significant problems in axonal pathfinding and muscle integrity. In all the mutant specimens examined (and certainly the low magnification views shown in Figure 1D'-F', Figure 1I'-K' and Figure 2D'-F') the mutants look very similar to the WT. Many readers may not get past the title and abstract, so the authors should make it clearer that these defects are very subtle.
Minor points:
- In Figures 4 and 5, CP309 staining is relied on to identify centrioles, but there is quite a background of non-specific dots, making it hard to be certain what is a centriole and what isn't. For example, in Figure 5D' there are lots of dots within some of the cells - are any of these centrioles? How can the authors be certain which dot is a centriole in some of the cells shown in Figure 5C'? Is it possible to use a second marker and only count as centrioles dots that are recognised by both antibodies?
- In the abstract that authors state that traditionally centrosomes have been considered to be non-essential in terminally differentiated cells. I don't think this is correct. In the standard "textbook" view of a cell, the centrosome is normally positioned in the centre of the cell organising an extensive array of MTs that are thought play an important role in organising intracellular transport, the positioning and movement of organelles and the maintenance and establishment of cell polarity. I don't think it is only recent evidence that suggests they play vital roles in terminally differentiated cells.
- Line 162 the authors state that in the RNAi knockdown lines they observe several additional phenotypes, but then in the same sentence (Line 164) they say that these defects were also observed in the original mutant and mutant/Df lines.
- The sentences in Line281-287 don't reference any of the Figures, so it seems the authors are just stating these results without presenting any data (e.g. "Significantly, we also found a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". If they've tested this correlation, they should show it.
- In Figure 7 I did not understand how the authors measured tortuosity (wiggliness) and could see no description in the methods. This is important as, again the defect seems quite subtle, but perhaps I am not understanding which bits of the axon are being measures. Is it just the small bit of the axons close to the asterixis that is being measured, or the whole FasII track?
Significance
The potential function of centrosomes in axonal outgrowth is quite controversial, so this study is potentially of considerable interest.
However, several aspects of the data presented here were confusing or not terribly convincing. In its present state, I don't think the main conclusions are strongly enough supported by the data.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
In this paper, the authors address the important question of the role of centrosomes during neuronal development. They use Drosophila as an in vivo model. The field is somewhat unclear on the role and importance of centrosomes during neuronal development, although the current data would suggest they are dispensable for axon specification and growth. Early studies in cultured mammalian neurons showed that centrosomes are active and that their microtubules can be cut and transported into the neurites. But a study then showed that centrosomes in these cultured neurons are deactivated relatively early during neuronal development in vitro and that ablating centrosomes even when they are active had no obvious effect on axon specification and growth. Consistent with this, a study in Drosophila provided evidence that centrosomes were not active or necessary in different types of neurons. More recently, a study showed that centrosomal microtubules are dispensable for axon specification and growth in mice in vivo but are required for neuronal migration in the cerebral cortex. However, another study has linked the generation of acetylated microtubules at centrosomes with axon development. In this current study, the authors examine the effect of centrosome loss on various motor and sensory neurons and muscles mainly by examining mutants in essential centriole duplication genes. They associate axonal routing and morphology defects with centrosome loss and provide some evidence that centrosomes could still be active in the developing neurons. Overall, they conclude that centrosomes are active during at least early neuronal development and that this activity is important for proper axonal morphology and routing.
While I think this study addressing a very interesting and important question, I think as it stands the data is not sufficient to be conclusive on a role for centrosomes during neuronal development. My biggest concern is that most phenotypes have not yet been shown to be cell autonomous, as whole animal mutants have been analysed rather than analysing the effect of cell-specific depletion, and the evidence for active centrosomes needs to be strengthened. If the authors can provide stronger evidence for a role of centrosomes in axonal development then the paper will certainly be of interest to a broad readership.
Major comments
- The sas-6 transallelic combination shows only 17% embryonic lethality compared to 50% embryonic lethality with sas-4 mutants. Given that both mutants should result in the same degree of centrosome loss (this should be quantified in sas-6 mutants) it would suggest that either sas-4 has other roles away from centrosomes or that the sas-4 mutant chromosome used in the experiment has other mutations that affect viability. The effect of picking up "second-site lethal" mutations on mutant chromosomes is common and so I would not be surprised if this is the reason for the difference in phenotypes. This can be addressed either by "cleaning up" the sas-4 mutant chromosome by backcrossing to wild-type lines, allowing recombination to occur and replace the potential second site mutations, or by using transallelic combinations of sas-4, as they did for sas-6. The "easier" option may just be to analyse all the phenotypes with the sas-6 transallelic combination.
- Using "whole animal" mutants for assessing neuronal morphology is risky due to non-cell-autonomous effects. The authors have carried out some phenotypic analysis of neurons depleted of Sas-4 by cell-specific RNAi, but I feel they need to do this for all of their analysis. This includes embryonic lethality measures, quantification of centrosome numbers, and all axonal phenotypes in Sas-4 RNAi neurons. It would also be prudent to use 2 distinct RNAi lines to help ensure any phenotypes are not off-target effects (and this may help clarify why the authors see some additional phenotypes with RNAi). Indeed, there are relatively weak phenotypes in muscles when using RNAi compared to the mutants and these potential non-cell-autonomous effects could then have a knock-on effect on neuronal morphology. If the authors were concerned that RNAi is not very efficient (explaining any potential weaker phenotypes than in mutants) the authors could examine the effectiveness of RNAi lines by analysing protein depletion by western blotting or mRNA depletion by rt-qPCR (although this has to be done in a different cell type due to the difficulty in obtaining a neuronal extract).
- When analysing centriole presence or absence it is a good idea to stain with two different centriole markers e.g. Asl and Plp. This helps rule out unspecific staining. It is clear from the images that similar sized foci can be observed outside of the cells (see Figure 5A for example), so clearly some of the foci that appear to be within the cells may also be unspecific staining.
- The evidence for active centrosomes is not that convincing. Acetylated tubulin is associated with stable MTs, which are not normally organised by "active" centrosomes that nucleate dynamic microtubules. Moreover, it is plausible that centriole foci happen to overlap with the acetylated tubulin staining by chance. This would explain why not all centrosomes colocalise with acetylated tubulin signal. The authors could better test centrosome activity by performing live imaging with EB1-GFP. If centrosomes are active, it is very easy to observe the many comets produced by the centrosomes.
- If the authors believe that centrosomes have a role in axon pathfinding in sensory neurons, they should show that these centrosomes are active, at least during early stages (again using EB1-GFP imaging).
- The authors mention in the discussion that "increased JNK activity, can result in axonal wiggliness (Karkali et al, 2023)". I therefore wonder whether centrosome loss may induce JNK activation (the stress response), as this would then indicate an indirect effect of centrosome loss on axonal structure rather than a direct influence of centrosome-generated microtubules. The authors could assess whether the DNK-JNK pathway is activated in neurons lacking centrosomes by expression UAS-Puc-GFP and quantifying the nuclear signal.
- In Figure 5, the authors claim that they find "a correlation between axonal guidance phenotypes and the numbers of centrioles per embryo". I don't think this is a strong correlation. The difference in centriole number between embryos with no defects and those with defects is very small. In contrast, the difference between centriole numbers in control (no defects) and mutant (no defects) is very large. So, there does not appear to be a strong correlation between centrosome number and phenotype.
Minor comments
- I don't understand Figure 3C - why do the % of surviving homozygotes and heterozygotes add up to 100%? Should the grey boxes not relate to dead and the white to surviving?
- "In mouse fibroblasts, myoblasts and endothelial cells, centrosome orientation is important for nuclear positioning and cell migration(Chang et al, 2015; Gomes et al, 2005; Kushner et al, 2014)." Do you mean "centrosome position"?
- In the introduction, the authors mention Meka et al. when saying the centrosomal microtubules are important for axonal development, but they should also discuss the counter argument from Vinopal et al., 2023 (Neuron) that showed how centrosomes were required for neuronal migration but not axon growth, which was instead mediated by Golgi-derived microtubules.
- Lines 228-230 - repeated sentence
- Additionally, we did not detect centrioles in the quadrant opposite the axon exit point (Fig. 2B n=75) - this data is not in Fig 2B
- "This significant decrease in the humber of centrioles further supports the critical role of Sas-4 in pioneer neurons of the ventral nerve cord (VNC) during Drosophila embryogenesis". It rather highlights that Sas-4 is required for centriole formation in these neurons. Also, humber = number.
- Result title: Non-ciliated sensory neurons have centrioles. This is kind of obvious. A better title may be "axon phenotypes correlate with centriole numbers in sensory neurons" but unfortunately i don't think there is good evidence for this (See major point above).
Significance
As mentioned above, the advance will be important if more evidence is provided. In this case, the paper will be interesting to a broad readership. But currently the paper is limited by the lack of evidence for centrosome function and activity in the neurons.
-
-
royalsocietypublishing.org royalsocietypublishing.org
-
Can our social evolution save us from the path of our social evolution that began 10 000 years ago?
for - question - evolution - can our social evolution save us - from the path of our social evolution that began 10,000 years ago?
-
Let me reiterate, global capitalism is the legacy of the agricultural system.
for - relationship - agriculture - is the parent - of global capitalism - It (global capitalism) is an elaboration of the agricultural system. - Surplus and expansion and - profound, almost mechanistic, interdependency in material life, and - duality in the human relationship to the more-than-human world - became the order of the day beginning with grain agriculture. - The basic structure and dynamic of the agricultural system were subsequently extended with elaborations that have eventually led to global capitalism.
-
division of labour creates ‘a built-in enforcer for cooperation
for - agriculture - division of labor - enforcer of cooperation
-
No humans are superorganisms in the way insects are, but some insect species and most of humanity became economic superorganisms when they engaged agriculture.
for - economic superorganism - human species qualifies
-
The term 'economic superorganism' is not to be interpreted as biological
for - definition - economic superorganism - The cohesive whole brought about by agriculture and the architecture that underlies it. Not to be interpreted as biological. - Used more in the sense of Henrich (economic superorganism) which refers to the structure and dynamic in cooperative material life particular to agriculture - NOT used in the strictly biological sense of E.O. Wilson, Holldobler
-
Humans are now organized in a global economic system that is difficult to alter in meaningful ways
for - insight - polycrisis - agricultural system - agriculture-based economic system - difficult to halt
-
All engage positive feedback loops that are expansive. That process can be understood for humans in this way
for - agriculture - expansiveness - description - Nice description!
-
Global capitalism can be considered ‘a system within a system’ in the sense that it models the agricultural system in its structure and dynamic
for comparison - agriculture - capitalism - one is the parent, the other is the uncontrolled offspring
-
for - economic superorganism
SRC comment - This paper is so eloquently written! Reading it, one really senses the enormous impact that agriculture has had on the cultural evolution of our species, so much so that we think of it as natural and absolute, rather than relative. - We did not have to be on the cultural trajectory we are now on, all made possible through the dependency on agriculture, the culture of plants.
-
James C. Scott tell us that humans were ‘disciplined and subordinated to the metronome of our own crops …. Once Homo sapiens took that fateful step into agriculture, our species entered an austere monastery whose task master was mostly the genetic clockwork of a few plants
for - origins - agriculture - beautiful description - our dependency on agriculture changed our sense of time!
-
In fact, by the time humans began the practice of cultivation of annual grains the total human population on Earth stood at around 6–10 million people. One might say that hunting and gathering is an energetically contained system and not an energetically expansionary system.
for - comparison - hunter gatherer vs agricultural - energetically contained vs energetically expansionary - stats - hunter -gatherer humans - population before agriculture - 6 to 10 million people.
-
There is no place where this cultural hubris is more evident than with the discourse on our present war between economy and Earth.
for - economic system vs cultural change - hubris - The global economic system at play is bringing about - the sixth mass extinction and - unmitigated climate change - and we continue to tinker around the edges of altering its structure and dynamic in any significant way. - One could easily make the claim that - it is the global economic system that has the upper hand - and not our capacity for cultural change.
-
Once humans attained culture, the pressure on genetic change is less significant and adaptation can take place through the flexibility afforded through cultural change.
for - key insight - culture - adaption through culture, not genes - SRG comment - danger is progress traps! - This is a key insight. Once we have sophisticated culture, we don't rely on slow moving genetic change to adapt anymore. Instead we rely on culture! - This is the world of human progress, but is also a dangerous one because progress (cultural adaptation to environmental pressures) comes with progress traps.
-
In the longer run, genes are evolving to adapt to these culturally constructed worlds
for - quote - genes evolve to adapt to culture - Joseph Henrich - in the longer run, genes are evolving to adap to these culturally constructed worlds
-
The evolution of the capacity for culture was an evolutionary transition just as the evolution of the superorganism was an evolutionary transition in insects.
for - comparison - culture vs superorganism
-
it acts much like genetic change, only quicker
for - progress traps - why it happens - culture evolves much faster that genes
-
Herbert Gintis
for - quote - gene-culture co-evolution - Herbert Gintis We are the species that we are because … genes provide individuals with the capacities and incentives to transform culture, and culture guides the transformation of the gene pool from generation to generation
-
Humans have a unique ability for building and expanding on previous knowledge, adding to the cumulative cultural endowment.
for - human superpower - timebinding
-
gene–culture coevolution
for - gene-culture coevolution
-
Culture makes possible modern complex societies where technological advancement is cumulative and extensive cooperation occurs among people who are not related.
for - superorganism - human - insight - culture - culture makes possible modern complex societies where cooperation between strangers is enabled. - money does this! transactional. no need to know who you transact with.
-
This type of extreme sociality does not occur in humans, so humans do not attain the status of a superorganism
for - superorganism - humans? no - extreme sociality doesn't occur
-
The superorganism is a form of extreme sociality and cooperation
for - superorganism - characterized by extreme sociality and cooperation in insect world, for example.
-
he basic elements of the superorganism are not cells and tissues but closely cooperating animals
for - quote - the basic elements of the superorganism are not cells and tissues but closely cooperating animals - E.O. Wilson & Holldobler
Tags
- quote - gene-culture coevolution
- gene-culture coevolution
- key insight - culture - adaption through culture, not genes - SRG comment - danger is progress traps!
- insight - polycrisis - agricultural system - agriculture-based economic system - difficult to halt
- quote - genes adapt to culture
- economic system vs cultural change - hubris
- relationship - agriculture - is the parent - of global capitalism
- question - evolution - can our social evolution save us
- agriculture - expansiveness - description
- quote - the basic elements of the superorganism are not cells and tissues but closely cooperating animals
- agriculture - division of labor - enforcer of cooperation
- progress traps - why it happens - culture evolves much faster that genes
- economic superorganism
- human superpower - timebinding
- origins - agriculture - beautiful description - our dependency on agriculture changed our sense of time!
- definition - economic superorganism
- superorganism - humans? no - extreme sociality doesn't occur
- superorganism - characterized by extreme sociality and cooperation in insect world, for example.
- comparison - agriculture - capitalism - one is the parent, the other is the uncontrolled offspring
- superorganism - human - insight - culture
- stats - hunter -gatherer humans - population before agriculture - 6 to 10 million people.
- comparison - culture vs superorganism
- comparison - hunter gatherer vs agricultural - energetically contained vs energetically expansionary
- economic superorganism - human species qualifies
Annotators
URL
-
-
www.federalregister.gov www.federalregister.gov
-
Regarding the comment on the active work on a National Directory FHIR IG, we thank this commenter for pointing this out. Because we have not required the publication of individual provider-level identifiers, we are not considering this IG for the endpoint publication use case in our Program. We emphasize again that because we have finalized an approach in § 170.404(b)(2) that references the base FHIR standard, Certified API Developers have the flexibility to consider using “Organization” and “Endpoint” FHIR resources profiles, such as the profiles in the National Directory FHIR IG, to meet those requirements.
Specifically mentioning the NDH FHIR workgroup and the NPD implmentation in terms of meeting requirements.
-
Response. We appreciate commenters' input and suggestions for clarity. We intend for these additional organization details to be used by app developers to help them map organizations to endpoints which, in turn, helps patients find the organization(s) they want to allow an app to access data from. We clarify that facility or organization level identifiers are sufficient to satisfy our proposed publication requirements. Facility level identifiers, for the purposes of certification to these Endpoint publication requirements, include identifiers such as: a National Provider Identifier (NPI), Clinical Laboratory Improvement Amendments (CLIA) number, CMS Certification Number (CCN), or other health system ID. Support for one of these identifier types is sufficient, meaning Certified API Developers are not required to publish individual NPIs as a floor for certification. Different identifiers may be used depending on the customers a Certified API Developer has. We have updated our regulatory text at § 170.404(b)(2)(ii)(B) to more clearly state that “[e]ach Organization resource must contain the organization's name, location, and facility identifier.”
Patients finding data from EHRs that hold their data requires either NPI, CLIA, or CCN. They are not required to publish "individual" NPIs. But they have to have ONPI or something else that we can link to.
-
will still be required to publish organization details such as name, location, and facility identifier.
While the organization standard in general will be somewhat relaxed. "name, location and identifier" are still required.
-
-
www.biorxiv.org www.biorxiv.org
-
eLife Assessment
Argunşah et al. investigate the mechanisms underlying the differential response dynamics of barrel vs septa domains in shaping the responses to single vs multiple whiskers. Based on the observation of a higher density of SST+ interneurons in the septa, the authors investigate the hypothesis that Elfn1-dependent short-term plasticity shapes these responses. This important study is, however, supported by incomplete evidence; factors restricting the strength of evidence are the limited spatial resolution of the multi-unit activity, as well as the lack of a mechanistic explanation. This provocative and intellectually stimulating hypothesis provides a contribution to work on how different cell types shape cortical representation.
-
Reviewer #1 (Public Review):
Summary:
Argunşah et al. describe and investigate the mechanisms underlying the differential response dynamics of barrel vs septa domains in the whisker-related primary somatosensory cortex (S1). Upon repeated stimulation, the authors report that the response ratio between multi- and single-whisker stimulation increases in layer (L) 4 neurons of the septal domain, while remaining constant in barrel L4 neurons. The authors attribute this divergence to differences in short-term synaptic plasticity, particularly within somatostatin-expressing (SST⁺) interneurons. This interpretation is supported by 1) the increased density of SST+ neurons in L4 of the septa compared to barrel domain, 2) the stronger response of (L2/3) SST+ neurons to repeated multi- vs single-whisker stimulation and 3) the reduced functional difference in single- versus multi-whisker response ratios across barrel and septal domains in Elfn1 KO mice, which lack a synaptic protein that confers characteristic short-term plasticity, notably in SST+ neurons. Consistently, a decoder trained on WT data fails to generalize to Elfn1 KO responses. Finally, the authors report a relative enrichment of S2- and M1-projecting cell densities in L4 of the septal domain compared to the barrel domain, suggesting that septal and barrel circuits may differentially route information about single vs multi-whisker stimulation downstream of S1.
Strengths:
This paper describes and aims to study a circuit underlying differential response between barrel columns and septal domains of the primary somatosensory cortex. This work supports the view these two domains contribute distinctly to the processing single versus multi-whisker inputs and highlight the role of SST+ neuron and their short-term plasticity. Together, this study suggests that the barrel cortex multiplexes whisker-derived sensory information across its domains, enabling parallel processing within S1.
Weaknesses:
Although the divergence in responses to repeated single- versus multi-whisker stimulation between barrel and septal domains is consistent with a role for SST⁺ neuron short-term plasticity, the evidence presented does not conclusively demonstrate that this mechanism is the critical driver of the difference. The lack of targeted recordings and manipulations limits the strength of this conclusion: SST⁺ neuron activity is not measured in L4, nor is it assessed in a domain-specific manner. The Elfn1 knockout manipulation does not appear to selectively affect either stimulus condition, domain or interneuron subtype. Finally, all experiments were performed under anesthesia, which raises concerns about how well the reported dynamics generalize to awake cortical processing.
-
Reviewer #2 (Public review):
Summary:
Argunsah and colleagues demonstrate that SST expressing interneurons are concentrated in the mouse septa and differentially respond to repetitive multi-whisker inputs. Identifying how a specific neuronal phenotype impacts responses is an advance.
Strengths:
(1) Careful physiological and imaging studies.
(2) Novel result showing the role of SST+ neurons in shaping responses.
(3) Good use of a knockout animal to further the main hypothesis.
(4) Clear analytical techniques.
Comments on revisions:
The authors have effectively responded to my initial critiques - I have no further concerns.
-
Reviewer #3 (Public review):
Summary:
This study investigates the functional differences between barrel and septal columns in the mouse somatosensory cortex, focusing on how local inhibitory dynamics (particularly involving SST⁺ interneurons) may mediate temporal integration of multi-whisker (MW) stimuli in septa. Using a combination of in vivo multi-unit recordings, calcium imaging, and anatomical tracing, the authors propose a model in which Elfn1-dependent synaptic facilitation onto SST⁺ interneurons contributes to the distinct sensory responses to MW input in barrels and septa, enabling functional segregation between these domains.
Strengths:
The study presents a thought-provoking and useful conceptual model for understanding sensory processing in the somatosensory cortex. While barrel columns have been widely studied, septal regions remain relatively understudied in mice. If septa indeed act as selective integrators of distributed sensory input, this would suggest a novel computational role for cortical microcircuits beyond the classical view focused on barrels. Although still hypothetical, the proposed model in which SST⁺ interneurons contribute to domain-specific sensory responses between barrel and septal domains is intriguing and opens new avenues for investigating inhibitory circuit mechanisms.
Weaknesses:
The primary limitation of this study lies in the spatial and cellular specificity of the recording techniques. The physiological data rely predominantly on unsorted multi-unit activity (MUA) recorded with low-channel-count silicon probes. Because MUA aggregates signals from multiple neurons over a radius of approximately 50-100 µm (often wider than the typical septal width in mice), this approach makes it difficult to confidently isolate activity originating strictly from within septal domains. The manuscript would benefit from additional analyses to validate the spatial specificity of these recordings, such as systematically varying spike detection thresholds to test the robustness of domain attribution, as suggested by the reviewer. Furthermore, although the authors now appropriately frame their findings in the Elfn1 knockout mice as indirect evidence, it is worth emphasizing that the study lacks direct in vivo, cell-type-specific recordings and manipulations to more definitively test the proposed mechanism.
-
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Reviews):
Summary:
Argunşah et al. describe and investigate the mechanisms underlying the differential response dynamics of barrel vs septa domains of the whisker-related primary somatosensory cortex (S1). Upon repeated stimulation, the authors report that the response ratio between multi- and single-whisker stimulation increases in layer (L) 4 neurons of the septal domain, while remaining constant in barrel L4 neurons. This difference is attributed to the short-term plasticity properties of interneurons, particularly somatostatin-expressing (SST+) neurons. This claim is supported by the increased density of SST+ neurons found in L4 of the septa compared to barrels, along with a stronger response of (L2/3) SST+ neurons to repeated multi- vs single-whisker stimulation. The role of the synaptic protein Elfn1 is then examined. Elfn1 KO mice exhibited little to no functional domain separation between barrel and septa, with no significant difference in single- versus multi-whisker response ratios across barrel and septal domains. Consistently, a decoder trained on WT data fails to generalize to Elfn1 KO responses. Finally, the authors report a relative enrichment of S2- and M1-projecting cell densities in L4 of the septal domain compared to the barrel domain.
Strengths:
This paper describes and aims to study a circuit underlying differential response between barrel columns and septal domains of the primary somatosensory cortex. This work supports the view that barrel and septal domains contribute differently to processing single versus multi-whisker inputs, suggesting that the barrel cortex multiplexes sensory information coming from the whiskers in different domains.
We thank the reviewer for the very neat summary of our findings that barrel cortex multiplexes converging information in separate domains.
Weaknesses:
While the observed divergence in responses to repeated SWS vs MWS between the barrel and septal domains is intriguing, the presented evidence falls short of demonstrating that short-term plasticity in SST+ neurons critically underpins this difference. The absence of a mechanistic explanation for this observation limits the work’s significance. The measurement of SST neurons’ response is not specific to a particular domain, and the Elfn1 manipulation does not seem to be specific to either stimulus type or a particular domain.
We appreciate the reviewer’s perspective. Although further research is needed to understand the circuit mechanisms underlying the observed phenomenon, we believe our data suggest that altering the short-term dynamics of excitatory inputs onto SST neurons reduces the divergent spiking dynamics in barrels versus septa during repetitive single- and multi-whisker stimulation. Future work could examine how SST neurons, whose somata reside in barrels and septa, respond to different whisker stimuli and the circuits in which they are embedded. At this time, however, the authors believe there is no alternative way to test how the short-term dynamics of excitatory inputs onto SST neurons, as a whole, contribute to the temporal aspects of barrel versus septa spiking.
The study's reach is further constrained by the fact that results were obtained in anesthetized animals, which may not generalize to awake states.
We appreciate the reviewer’s concern regarding the generalizability of our findings from anesthetized animals to awake states. Anesthesia was employed to ensure precise individual whisker stimulation (and multi-whisker in the same animal), which is challenging in awake rodents due to active whisking. While anesthesia may alter higher-order processing, core mechanisms, such as short and long term plasticity in the barrel cortex, are preserved under anesthesia (Martin-Cortecero et al., 2014; Mégevand et al., 2009).
The statistical analysis appears inappropriate, with the use of repeated independent tests, dramatically boosting the false positive error rate.
Thank you for your feedback on our analysis using independent rank-based tests for each time point in wild-type (WT) animals. To address concerns regarding multiple comparisons and temporal dependencies (for Figure 1F and 4D for now but we will add more in our revision), we performed a repeated measures ANOVA for WT animals (13 Barrel, 8 Septa, 20 time points), which revealed a significant main effect of Condition (F(1,19) = 16.33, p < 0.001) and a significant Condition-Time interaction (F(19,361) = 2.37, p = 0.001). Post-hoc tests confirmed significant differences between Barrel and Septa at multiple time points (e.g., p < 0.0025 at times 3, 4, 6, 7, 8, 10, 11, 12, 16, 19 after Bonferroni posthoc correction), supporting a differential multi-whisker vs. single-whisker ratio response in WT animals. In contrast, a repeated measures ANOVA for knock-out (KO) animals (11 Barrel, 7 Septa, 20 time points) showed no significant main effect of Condition (F(1,14) = 0.17, p = 0.684) or Condition-Time interaction (F(19,266) = 0.73, p = 0.791), indicating that the BarrelSepta difference observed in WT animals is absent in KO animals.
Furthermore, the manuscript suffers from imprecision; its conclusions are occasionally vague or overstated. The authors suggest a role for SST+ neurons in the observed divergence in SWS/MWS responses between barrel and septal domains. However, this remains speculative, and some findings appear inconsistent. For instance, the increased response of SST+ neurons to MWS versus SWS is not confined to a specific domain. Why, then, would preferential recruitment of SST+ neurons lead to divergent dynamics between barrel and septal regions? The higher density of SST+ neurons in septal versus barrel L4 is not a sufficient explanation, particularly since the SWS/MWS response divergence is also observed in layers 2/3, where no difference in SST+ neuron density is found.
Moreover, SST+ neuron-mediated inhibition is not necessarily restricted to the layer in which the cell body resides. It remains unclear through which differential microcircuits (barrel vs septum) the enhanced recruitment of SST+ neurons could account for the divergent responses to repeated SWS versus MWS stimulation.
We fully appreciate the reviewer’s comment. We currently do not provide any evidence on the contribution of SST neurons in the barrels versus septa in layer 4 on the response divergence of spiking observed in SWS versus MWS. We only show that these neurons differentially distribute in the two domains in this layer. It is certainly known that there is molecular and circuit-based diversity of SST-positive neurons in different layers of the cortex, so it is plausible that this includes cells located in the two domains of vS1, something which has not been examined so far. Our data on their distribution are one piece of information that SST neurons may have a differential role in inhibiting barrel stellate cells versus septa ones. Morphological reconstructions of SST neurons in L4 of the somatosensory barrel cortex has shown that their dendrites and axons project locally and may confine to individual domains, even though not specifically examined (Fig. 3 of Scala F et al., 2019). The same study also showed that L4 SST cells receive excitatory input from local stellate cells) and is known that they are also directly excited by thalamocortical fibers (Beierlein et al., 2003; Tan et al., 2008), both of which facilitate.
As shown in our supplementary figure, the divergence is also observed in L2/3 where, as the reviewer also points out, where we do not have a differential distribution of SST cells, at least based on a columnar analysis extending from L4. There are multiple scenarios that could explain this “discrepancy” that one would need to examine further in future studies. One straightforward one is that the divergence in spiking in L2/3 domains may be inherited from L4 domains, where L4 SST act on. Another is that even though L2/3 SST neurons are not biased in their distribution their input-output function is, something which one would need to examine by detailed in vitro electrophysiological and perhaps optogenetic approaches in S1. Despite the distinctive differences that have been found between the L4 circuitry in S1 and V1 (Scala F et al., 2019), recent observations indicate that small but regular patches of V1 marked by the absence of muscarinic receptor 2 (M2) have high temporal acuity (Ji et al., 2015), and selectively receive input from SST interneurons (Meier et al., 2025). Regions lacking M2 have distinct input and output connectivity patterns from those that express M2 (Meier et al., 2021; Burkhalter et al., 2023). These findings, together with ours, suggest that SST cells preferentially innervate and regulate specific domains columns- in sensory cortices.
Regardless of the mechanism, the Elfn1 knock-out mouse line almost exclusively affects the incoming excitability onto SST neurons (see also reply to comment below), hence what can be supported by our data is that changing the incoming short-term synaptic plasticity onto these neurons brings the spiking dynamics between barrels and septa closer together.
The Elfn1 KO mouse model seems too unspecific to suggest the role of the short-term plasticity in SST+ neurons in the differential response to repeated SWS vs MWS stimulation across domains. Why would Elfn1-dependent short-term plasticity in SST+ neurons be specific to a pathway, or a stimulation type (SWS vs MWS)? Moreover, the authors report that Elfn1 knockout alters synapses onto VIP+ as well as SST+ neurons (Stachniak et al., 2021; previous version of this paper)-so why attribute the phenotype solely to SST+ circuitry? In fact, the functional distinctions between barrel and septal domains appear largely abolished in the Elfn1 KO.
Previous work by others and us has shown that globally removing Elfn1 selectively removes a synaptic process from the brain without altering brain anatomy or structure. This allows us to study how the temporal dynamics of inhibition shape activity, as opposed to inhibition from particular cell types. We will nevertheless update the text to discuss more global implications for SST interneuron dynamics and include a reference to VIP interneurons that contain Elfn1.
When comparing SWS to MWS, we find that MWS replaces the neighboring excitation which would normally be preferentially removed by short-term plasticity in SST interneurons, thus providing a stable control comparison across animals and genotypes. On average, VIP interneurons failed to show modulation by MWS. We were unable to measure a substantial contribution of VIP cells to this process and also note that the Elfn1 expressing multipolar neurons comprise only ~5% of VIP neurons (Connor and Peters, 1984; Stachniak et al., 2021), a fraction that may be lost when averaging from 138 VIP cells. Moreover, the effect of Elfn1 loss on VIP neurons is quite different and marginal compared to that of SST cells, suggesting that the primary impact of Elfn1 knockout is mediated through SST+ interneuron circuitry. Therefore, even if we cannot rule out that these 5% of VIP neurons contribute to barrel domain segregation, we are of the opinion that their influence would be very limited if any.
Reviewer #2 (Public Reviews):
Summary:
Argunsah and colleagues demonstrate that SST-expressing interneurons are concentrated in the mouse septa and differentially respond to repetitive multi-whisker inputs. Identifying how a specific neuronal phenotype impacts responses is an advance.
Strengths:
(1) Careful physiological and imaging studies.
(2) Novel result showing the role of SST+ neurons in shaping responses.
(3) Good use of a knockout animal to further the main hypothesis.
(4) Clear analytical techniques.
We thank the reviewer for their appreciation of the study.
Weaknesses:
No major weaknesses were identified by this reviewer. Overall, I appreciated the paper but feel it overlooked a few issues and had some recommendations on how additional clarifications could strengthen the paper. These include:
(1) Significant work from Jerry Chen on how S1 neurons that project to M1 versus S2 respond in a variety of behavioral tasks should be included (e.g. PMID: 26098757). Similarly, work from Barry Connor’s lab on intracortical versus thalamocortical inputs to SST neurons, as well as excitatory inputs onto these neurons (e.g. PMID: 12815025) should be included.
We thank the reviewer for these valuable resources that we overlooked. We will include Chen et al. (2015), Cruikshank et al. (2007) and Gibson et al. (1999) to contextualize S1 projections and SST+ inputs, strengthening the study’s foundation as well as Beierlein et al. (2003) which nicely show both local and thalamocortical facilitation of excitatory inputs onto L4 SST neurons, in contrast to PV cells. The paper also shows the gradual recruitment of SST neurons by thalamocortical inputs to provide feed-forward inhibition onto stellate cells (regular spiking) of the barrel cortex L4 in rat.
(2) Using Layer 2/3 as a proxy to what is happening in layer 4 (~line 234). Given that layer 2/3 cells integrate information from multiple barrels, as well as receiving direct VPm thalamocortical input, and given the time window that is being looked at can receive input from other cortical locations, it is not clear that layer 2/3 is a proxy for what is happening in layer 4.
We agree with the reviewer that what we observe in L2/3 is not necessarily what is taking place in L4 SST-positive cells. The data on L2/3 was included to show that these cells, as a population, can show divergent responses when it comes to SWS vs MWS, which is not seen in L2/3 VIP neurons. Regardless of the mechanisms underlying it, our overall data support that SST-positive neurons can change their activation based on the type of whisker stimulus and when the excitatory input dynamics onto these neurons change due to the removal of Elfn1 the recruitment of barrels vs septa spiking changes at the temporal domain. Having said that, the data shown in Supplementary Figure 3 on the response properties of L2/3 neurons above the septa vs above the barrels (one would say in the respective columns) do show the same divergence as in L4. This suggests that a circuit motif may exist that is common to both layers, involving SST neurons that sit in L4, L5 or even L2/3. This implies that despite the differences in the distribution of SST neurons in septa vs barrels of L4 there is an unidentified input-output spatial connectivity motif that engages in both L2/3 and L4. Please also see our response to a similar point raised by reviewer 1.
(3) Line 267, when discussing distinct temporal response, it is not well defined what this is referring to. Are the neurons no longer showing peaks to whisker stimulation, or are the responses lasting a longer time? It is unclear why PV+ interneurons which may not be impacted by the Elfn1 KO and receive strong thalamocortical inputs, are not constraining activity.
We thank the reviewer for their comment and will clarify the statement.
This convergence of response profiles was further clear in stimulus-aligned stacked images, where the emergent differences between barrels and septa under SWS were largely abolished in the KO (Figure 4B). A distinction between directly stimulated barrels and neighboring barrels persisted in the KO. In addition, the initial response continued to differ between barrel and septa and also septa and neighbor (Figure 4B). This initial stimulus selectivity potentially represents distinct feedforward thalamocortical activity, which includes PV+ interneuron recruitment that is not directly impacted by the Elfn1 KO (Sun et al., 2006; Tan et al., 2008). PV+ cells are strongly excited by thalamocortical inputs, but these exhibit short-term depression, as does their output, contrasting with the sustained facilitation observed in SST+ neurons. These findings suggest that in WT animals, activity spillover from principal barrels is normally constrained by the progressive engagement of SST+ interneurons in septal regions, driven by Elfn1-dependent facilitation at their excitatory synapses. In the absence of Elfn1, this local inhibitory mechanism is disrupted, leading to longer responses in barrels, delayed but stronger responses in septa, and persistently stronger responses in unstimulated neighbors, resulting in a loss of distinction between the responses of barrel and septa domains that normally diverge over time (see Author response image 1 below).
Author response image 1.
(A) Barrel responses are longer following whisker stimulation in KO. (B) Septal responses are slightly delayed but stronger in KO. (C) Unstimulated neighbors show longer persistent responses in KO.
(4) Line 585 “the earliest CSD sink was identified as layer 4…” were post-hoc measurements made to determine where the different shank leads were based on the post-hoc histology?
Post hoc histology was performed on plane-aligned brain sections which would allow us to detect barrels and septa, so as to confirm the insertion domains of each recorded shank. Layer specificity of each electrode therefore could therefore not be confirmed by histology as we did not have coronal sections in which to measure electrode depth.
(5) For the retrograde tracing studies, how were the M1 and S2 injections targeted (stereotaxically or physiologically)? How was it determined that the injections were in the whisker region (or not)?
During the retrograde virus injection, the location of M1 and S2 injections was determined by stereotaxic coordinates (Yamashita et al., 2018). After acquiring the light-sheet images, we were able to post hoc examine the injection site in 3D and confirm that the injections were successful in targeting the regions intended. Although it would have been informative to do so, we did not functionally determine the whisker-related M1 and whisker-related S2 region in this experiment.
(6) Were there any baseline differences in spontaneous activity in the septa versus barrel regions, and did this change in the KO animals?
Thank you for this interesting question. Our previous study found that there was a reduction in baseline activity in L4 barrel cortex of KO animals at postnatal day (P)12, but no differences were found at P21 (Stachniak et al., 2023).
Reviewer #3 (Public Reviews):
Summary:
This study investigates the functional differences between barrel and septal columns in the mouse somatosensory cortex, focusing on how local inhibitory dynamics, particularly involving Elfn1-expressing SST⁺ interneurons, may mediate temporal integration of multiwhisker (MW) stimuli in septa. Using a combination of in vivo multi-unit recordings, calcium imaging, and anatomical tracing, the authors propose that septa integrate MW input in an Elfn1-dependent manner, enabling functional segregation from barrel columns.
Strengths:
The core hypothesis is interesting and potentially impactful. While barrels have been extensively characterized, septa remain less understood, especially in mice, and this study's focus on septal integration of MW stimuli offers valuable insights into this underexplored area. If septa indeed act as selective integrators of distributed sensory input, this would add a novel computational role to cortical microcircuits beyond what is currently attributed to barrels alone. The narrative of this paper is intellectually stimulating.
We thank the reviewer for finding the study intellectually stimulating.
Weaknesses:
The methods used in the current study lack the spatial and cellular resolution needed to conclusively support the central claims. The main physiological findings are based on unsorted multi-unit activity (MUA) recorded via low-channel-count silicon probes. MUA inherently pools signals from multiple neurons across different distances and cell types, making it difficult to assign activity to specific columns (barrel vs. septa) or neuron classes (e.g., SST⁺ vs. excitatory).
The recording radius (~50-100 µm or more) and the narrow width of septa (~50-100 µm or less) make it likely that MUA from "septal" electrodes includes spikes from adjacent barrel neurons.
The authors do not provide spike sorting, unit isolation, or anatomical validation that would strengthen spatial attribution. Calcium imaging is restricted to SST⁺ and VIP⁺ interneurons in superficial layers (L2/3), while the main MUA recordings are from layer 4, creating a mismatch in laminar relevance.
We thank the reviewer for pointing out the possibility of contamination in septal electrodes. Importantly, it may not have been highlighted, although reported in the methods, but we used an extremely high threshold (7.5 std, in methods, line 583) for spike detection in order to overcome the issue raised here, which restricts such spatial contaminations. Since the spike amplitude decays rapidly with distance, at high thresholds, only nearby neurons contribute to our analysis, potentially one or two. We believe that this approach provides a very close approximation of single unit activity (SUA) in our reported data. We will include a sentence earlier in the manuscript to make this explicit and prevent further confusion.
Regarding the point on calcium imaging being performed on L2/3 SST and VIP cells instead of L4. Both reviewer 1 and 2 brought up the same issue and we responded as follows. As shown in our supplementary figure, the divergence is also observed in L2/3 where we do not have a differential distribution of SST cells, at least based on a columnar analysis extending from L4. There are multiple scenarios that could explain this “discrepancy” that one would need to examine further in future studies. One straightforward one is that the divergence in spiking in L2/3 domains may be inherited from L4 domains, where L4 SST act on. Another is that even though L2/3 SST neurons are not biased in their distribution their input-output function is, something which one would need to examine by detailed in vitro electrophysiological and perhaps optogenetic approaches in S1. Despite the distinctive differences that have been found between the L4 circuitry in S1 and V1 (Scala F et al., 2019), recent observations indicate that small but regular patches of V1 marked by the absence of muscarinic receptor 2 (M2) have high temporal acuity (Ji et al., 2015), and selectively receive input from SST interneurons (Meier et al., 2025). Regions lacking M2 have distinct input and output connectivity patterns from those that express M2 (Meier et al., 2021; Burkhalter et al., 2023). These findings, together with ours, suggest that SST cells preferentially innervate and regulate specific domains -columns- in sensory cortices.
Furthermore, while the role of Elfn1 in mediating short-term facilitation is supported by prior studies, no new evidence is presented in this paper to confirm that this synaptic mechanism is indeed disrupted in the knockout mice used here.
We thank Reviewer #3 for noting the absence of new evidence confirming Elfn1’s disruption of short-term facilitation in our knockout mice. We acknowledge that our study relies on previously strong published data demonstrating that Elfn1 mediates short-term synaptic facilitation of excitatory inputs onto SST+ interneurons (Sylwestrak and Ghosh, 2012; Tomioka et al., 2014; Stachniak et al., 2019, 2023). These studies consistently show that Elfn1 knockout abolishes facilitation in SST+ synapses, leading to altered temporal dynamics, which we hypothesize underlies the observed loss of barrel-septa response divergence in our Elfn1 KO mice (Figure 4). Nevertheless, to address the point raised, we will clarify in the revised manuscript (around lines 245-247 and 271-272) that our conclusions are based on these established findings, stating: “Building on prior evidence that Elfn1 knockout disrupts short-term facilitation in SST+ interneurons (Sylwestrak and Ghosh, 2012; Tomioka et al., 2014; Stachniak et al., 2019, 2023), we attribute the abolished barrel-septa divergence in Elfn1 KO mice to altered SST+ synaptic dynamics, though direct synaptic measurements were not performed here.”
Additionally, since Elfn1 is constitutively knocked out from development, the possibility of altered circuit formation-including changes in barrel structure and interneuron distribution, cannot be excluded and is not addressed.
We thank Reviewer #3 for raising the valid concern that constitutive Elfn1 knockout could potentially alter circuit formation, including barrel structure and interneuron distribution. To address this, we will clarify in the revised manuscript (around line ~271 and in the Discussion) that in our previous studies that included both whole-cell patch-clamp in acute brain slices ranging from postnatal day 11 to 22 (P11 - P21) and in vivo recordings from barrel cortex at P12 and P21, we saw no gross abnormalities in barrel structure, with Layer 4 barrels maintaining their characteristic size and organization, consistent with wildtype (WT) mice (Stachniak et al., 2019, 2023). While we cannot fully exclude subtle developmental changes, prior studies indicate that Elfn1 primarily modulates synaptic function rather than cortical cytoarchitecture (Tomioka et al., 2014). Elfn1 KO mice show no gross morphological or connectivity differences and the pattern and abundance of Elfn1 expressing cells (assessed by LacZ knock in) appears normal (Dolan and Mitchell, 2013).
We will add the following to the Discussion: “Although Elfn1 is constitutively knocked out, we find here and in previous studies that barrel structure is preserved (Stachniak et al., 2019, 2023). Further, the distribution of Elfn1 expressing interneurons is not different in KO mice, suggesting minimal developmental disruption (Dolan and Mitchell, 2013).
Nonetheless, we acknowledge that subtle circuit changes cannot be ruled out without the usage of time-depended conditional knockout of the gene.”
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) My biggest concern is regarding statistics. Did the authors repeatedly apply independent tests (Mann-Whitney) without any correction for multiple comparisons (Figures 1 and 4)? In that case, the chances of a spurious "significant" result rise dramatically.
In response to the reviewer’s comment, we now present new statistical results by utilizing ANOVA and blended these results in the manuscript between lines 172 and 192 for WT data and 282 and 298 for Elfn1 KO data. This new statistical approach shows the same differences as we had previously reported, hence consolidating the statements made.
(2) The findings only hint at a mechanism involving SST+ neurons for how SWS and MWS are processed differently in the barrel vs septal domains. As a direct test of SST+ neuron involvement in the divergence of barrel and septal responses, the authors might consider SST-specific manipulations - for example, inhibitory chemo- or optogenetics during SWS and MWS stimulation.
We thank the reviewer for this comment and agree that a direct manipulation of SST+ neurons via inhibitory chemo- or opto-genetics could provide further supporting evidence for the main claims in our study. We have opted out from performing these experiments for this manuscript as we feel they can be part of a future study. At the same time, it is conceivable that such manipulations and depending on how they are performed may lead to larger and non-specific effects on cortical activity, since SST neurons will likely be completely shut down. So even though we certainly appreciate and value the strengths of such approaches, our experiments have addressed a more nuanced hypothesis, namely that the synaptic dynamics onto SST+ neurons matter for response divergence of septa versus barrels, which could not have been easily and concretely addressed by manipulating SST+ cell firing activity.
(3) In general, it is hard to comprehend what microcircuit could lead to the observed divergence in the MWS/SWS ratio in the barrel vs septal domain. There preferential recruitment of SST+ neurons during MWS is not specific to a particular domain, and the higher density of SST+ neurons specifically in L4 septa cannot per se explain the diverging MWS/SWS ratio in L4 septal neurons since similar ratio divergence is observed across domains in L2/3 neurons without increase SST+ neuron density in L2/3. This view would also assume that SST+ inhibition remains contained to its own layer and domain. Is this the case? Is it that different microcircuits between barrels and septa differently shape the response to repeated MWS? This is partially discussed in the paper; can the authors develop on that? What would the proposed mechanism be? Can the short-term plasticity of the thalamic inputs (VPM vs POm) be part of the picture?
We thank the reviewer for raising this important point. We propose that the divergence in MWS/SWS ratios across barrel and septal domains arises from dynamic microcircuit interactions rather than static anatomical features such as SST+ density, which we describe and can provide a hint. In L2/3, where SST+ density is uniform, divergence persists, suggesting that trans-laminar and trans-domain interactions are key. Barrel domains, primarily receiving VPM inputs, exhibit short-term depression onto excitatory cells and engage PV+ and SST+ neurons to stabilize the MWS/SWS ratio, with Elfn1-dependent facilitation of SST+ neurons gradually increasing inhibition during repetitive SWS. Septal domains, in contrast, are targeted by facilitating POm inputs, combined with higher L4 SST+ density and Elfn1-mediated facilitation, producing progressive inhibitory buildup that amplifies the MWS/SWS ratio. SST+ projections in septa may extend trans-laminarly and laterally, influencing L2/3 and neighboring barrels, thereby explaining L2/3 divergence despite uniform SST+ density in L2/3. In this regards, direct laminar-dependent manipulations will be required to confirm whether L2/3 divergence is inherited from L4 dynamics. In Elfn1 KO mice, the loss of facilitation in SST+ neurons likely flattens these dynamics, disrupting functional segregation. Future experiments using VPM/POm-specific optogenetic activation and SST+ silencing will be critical to directly test this model.
We expanded the discussion accordingly.
(4) Can the decoder generalize between SWS and MWS? In this condition, if the decoder accuracy is higher for barrels than septa, it would support the idea that septa are processing the two stimuli differently.
Our results show that septal decoding accuracy is generally higher than barrel accuracy when generalizing from multi-whisker stimulation (MWS) to single-whisker stimulation (SWS), indicating distinct information processing in septa compared to barrels.
In wild-type (WT) mice, septal accuracy exceeds barrel accuracy across all time windows (150ms, 51-95ms, 1-95ms), with the largest difference in the 51-95ms window (0.9944 vs. 0.9214 at pulse 20, 10Hz stimulation). This septal advantage grows with successive pulses, reflecting robust, separable neural responses, likely driven by the posterior medial nucleus (POm)’s strong MWS integration contrasting with minimal SWS activation. Barrel responses, driven by consistent ventral posteromedial nucleus (VPM) input for both stimuli, are less distinguishable, leading to lower accuracy.
In Elfn1 knockout (KO) mice, which disrupt excitatory drive to somatostatin-positive (SST+) interneurons, barrel accuracy is higher initially in the 1-50ms window (0.8045 vs. 0.7500 at pulse 1), suggesting reduced early septal distinctiveness. However, septal accuracy surpasses barrels in later pulses and time windows (e.g., 0.9714 vs. 0.9227 in 51-95ms at pulse 20), indicating restored septal processing. This supports the role of SST+ interneurons in shaping distinct MWS responses in septa, particularly in late-phase responses (51-95ms), where inhibitory modulation is prominent, as confirmed by calcium imaging showing stronger SST+ activation during MWS.
These findings demonstrate that septa process SWS and MWS differently, with higher decoding accuracy reflecting structured, POm- and SST+-driven response patterns. In Elfn1 KO mice, early deficits in septal processing highlight the importance of SST+ interneurons, with later recovery suggesting compensatory mechanisms.
We have added Supplementary Figure 4 and included this interpretation between lines 338353.
We thank the reviewer for suggesting this analysis.
(5) It is not clear to me how the authors achieve SWS. How is it that the pipette tip "placed in contact with the principal whisker" does not detach from the principal whisker or stimulate other whiskers? Please clarify the methods.
Targeting the specific principal whisker is performed under the stereoscope.
Specifically, we have added this statement in line 628:
“We trimmed the whiskers where necessary, to avoid them touching each other and to avoid stimulating other whiskers. By putting the pipette tip very close (almost touching) to the principal whisker, the movement of the tip (limited to 1mm) would reliably move the targeted whisker. The specificity of the stimulation of the selected principal whisker was observed under the stereoscope.”
(6) The method for calculating decoder accuracy is not clearly described-how can accuracy exceed 1? The authors should clarify this metric and provide measures of variability (e.g., confidence intervals or standard deviations across runs) to assess the significance of their comparisons. Additionally, using a consistent scale across all plots would improve interoperability.
We thank the reviewer for raising this point. We have now changed the way accuracies are calculated and adopted a common scale among different plots (see updated Figure 5). We have also changed the methods section accordingly.
(7) Figure 1: The sample size is not specified. It looks like the numbers match the description in the methods, but the sample size should be clearly stated here.
These are the numbers the reviewer is inquiring about.
WT: (WT) animals: a 280 × 95 × 20 matrix for the stimulated barrel (14 Barrels, 95ms, 20 pulses), a 180 × 95 × 20 matrix for the septa (9 Septa, 95ms, 20 pulses), and a 360 × 95 × 20 matrix for the neighboring barrel (18 Neighboring barrels, 95ms, 20 pulses). N=4 mice.
KO: 11-barrel columns, 7 septal columns, 11 unstimulated neighbors from N=4 mice.
Panels D-F are missing axes and axis labels (firing rate, p-value). Panel D is mislabeled (left, middle, and right). I can't seem to find the yellow line.
Thank you for this observation. We made changes in the figures to make them easier to navigate based on the collective feedback from the reviewers.
Why is changing the way to compare the differences in the responses to repeated stimulation between SWS and MWS?
To assess temporal accumulation of information, we compared responses to repeated single-whisker stimulation (SWS) and multi-whisker stimulation (MWS) using an accumulative decoding approach rather than simple per-pulse firing rates. This method captures domain-specific integration dynamics over successive pulses.
The use of the term "principal whisker" is confusing, as it could refer to the whisker that corresponds to the recorded barrel.
When we use the term principal whisker, the intention is indeed to refer to the whisker corresponding to the recorded barrel during single whisker stimulation. The term principal whisker is removed from Figure legend 1 and legend S1C where it may have led to ambiguity.
Why the statement "after the start of active whisking"? Mice are under anesthesia here; it does not appear to be relevant for the figure.
“After the start of active whisking” refers to the state of the barrel cortex circuitry at the time of recordings. The particular reference we use comes from the habit of assessing sensory processing also from a developmental point of view. The reviewer is correct that it has nothing to do the with the status of the experiment. Nevertheless, since the reviewer found that it may create confusion, we have now taken it out.
(8) Figure 3: The y-axis label is missing for panel C.
This is now fixed. (dF/F).
(9) Figure 4: Axis labels are missing.
Added.
Minor:
(10) Line 36: "progressive increase in septal spiking activity upon multi-whisker stimulation". There is no increase in septal spiking activity upon MWS; the ratio MWS/SWS increases.
We have changed the sentence as follows: Genetic removal of Elfn1, which regulates the incoming excitatory synaptic dynamics onto SST+ interneurons, leads to the loss of the progressive increase in septal spiking ratio (MWS/SWS) upon stimulation.
(11) Line 105: domain-specific, rather than column-specific, for consistency.
We have changed it.
(12) Lines 173-174: "a divergence between barrel and septa domain activity also occurred in Layer 4 from the 2nd pulse onward (Figure 1E)". The authors only show a restricted number of comparisons. Why not show the p-values as for SWS?
The statistics is now presented in current Figure 1E.
(13) Lines 151-153: "Correspondingly, when a single whisker is stimulated repeatedly, the response to the first pulse is principally bottom-up thalamic-driven responses, while the later pulses in the train are expected to also gradually engage cortico-thalamo-cortical and cortico-cortical loops." Can the authors please provide a reference?
We have now added the following references : (Kyriazi and Simons, 1993; Middleton et al., 2010; Russo et al., 2025).
(14) Lines 184-186: "Our electrophysiological experiments show a significant divergence of responses over time upon both SWS and MWS in L4 between barrels (principal and neighboring) and adjacent septa, with minimal initial difference". The only difference between the neighboring barrel and septa is the responses to the initial pulse. Can the author clarify?
We have now changed the sentence as follows: Our electrophysiological experiments show a significant divergence of responses between domains upon both SWS and MWS in L4. (Line 198 now)
(15) Line 214: "suggest these interneurons may play a role in diverging responses between barrels and septa upon SWS". Why SWS specifically?
We have changed the sentence as follows: These results confirmed that SST+ and VIP+ interneurons have higher densities in septa compared to barrels in L4 and suggest these interneurons may play a role in diverging responses between barrels and septa. (Line 231 now).
(16) Line 235: "This result suggests that differential activation of SST+ interneurons is more likely to be involved in the domain-specific temporal ratio differences between barrels and septa". Why? The results here are not domain-specific.
We have now revised this statement to: This result suggested that temporal ratio differences specific to barrels and septa might involve differential activation of SST+ interneurons rather than VIP+ interneurons.
(17) Lines 241-243: "SST+ interneurons in the cortex are known to show distinct short-term synaptic plasticity, particularly strong facilitation of excitatory inputs, which enables them to regulate the temporal dynamics of cortical circuits." Please provide a reference.
We have now added the following references: (Grier et al., 2023; Liguz-Lecznar et al., 2016).
(18) Lines 245-247: "A key regulator of this plasticity is the synaptic protein Elfn1, which mediates short-term synaptic facilitation of excitation on SST+ interneurons (Stachniak et al., 2021, 2019; Tomioka et al., 2014)". Is Stachniak et al., 2021 not about the role of Elf1n in excitatory-to-VIP+ neuron synapses?
The reviewer correctly spotted this discrepancy . This reference has now been removed from this statement.
(19) Lines 271-272: "Building on our findings that Elfn1-dependent facilitation in SST+ interneurons is critical for maintaining barrel-septa response divergence". The authors did not show that.
We have now changed the statement to: Building on our findings that Elfn1 is critical for maintaining barrel-septa response divergence
(20) Line 280: second firing peak, not "peal".
Thank you, it is now fixed.
(21) Lines 304-305: "These results highlight the critical role of Elfn1 in facilitating the temporal integration of 305 sensory inputs through its effects on SST+ interneurons". This claim is also overstated.
We have now changed the statement to: These results highlight the contribution of Elfn1 to the temporal integration of sensory inputs. (Line 362)
(22) Line 329: Any reason why not cite Chen et al., Nature 2013?
We have now added this reference, as also pointed out by reviewer 1.
(23) Line 341-342: "wS1" and "wS2" instead of S1 and S2 for consistency.
Thanks, we have now updated the terms.
Reviewer #2 (Recommendations for the authors):
(1) Figure 3D - the SW conditions are labeled but not the MW conditions (two right graphs) - they should be labeled similarly (SSTMW, VIPMW).
The two right graphs in Figure 3D represent paired SW vs MW comparisons of the evoked responses for SST and VIP populations, respectively.
(2) Figure 6 D and E I think it would be better if the Depth measurements were to be on the yaxis, which is more typical of these types of plots.
We thank the reviewer for this comment. Although we appreciate this may be the case, we feel that the current presentation may be easier for the reader to navigate, and we have hence kept it.
(3) Having an operational definition of septa versus barrel would be useful. As the authors point out, this is a tough distinction in a mouse, and often you read papers that use Barrel Wall versus Barrel Hollow/Center - operationally defining how these areas were distinguished would be helpful.
We thank the reviewer for this comment and understand the point made.
We have now updated the methods section in line 611:
DiI marks contained within the vGlut2 staining were defined as barrel recordings, while DiI marks outside vGlut2 staining were septal recordings.
Reviewer #3 (Recommendations for the authors):
To support the manuscript's major claims, the authors should consider the following:
(1) Validate the septal identity of the neurons studied, either anatomically or functionally at the single-cell level (e.g., via Ca²⁺ imaging with confirmed barrel/septa mapping).
We thank the reviewer for this suggestion, but we feel that these extensive experiments are beyond the scope of this study.
(2) Provide both anatomical and physiological evidence to assess the possibility of altered cortical development in Elfn1 KO mice, including potential changes in barrel structure or SST⁺ cell distribution.
To address the reviewer’s point, we have now added the following to the Discussion: “Although Elfn1 is constitutively knocked out, we find here and in previous studies that barrel structure is preserved (Stachniak et al., 2019, 2023). Further, the distribution of Elfn1 expressing interneurons is not different in KO mice, suggesting minimal developmental disruption (Dolan and Mitchell, 2013). Nonetheless, we acknowledge that subtle circuit changes cannot be ruled out without conditional knockouts.”,
(3) Examine the sensory responses of SST⁺ and VIP⁺ interneurons in deeper cortical layers, particularly layer 4, which is central to the study's main conclusions.
We thank the reviewer for this suggestion and appreciate the value it would bring to the study. We nevertheless feel that these extensive experiments are beyond the scope of this study and hence opted out from performing them.
Minor Comments:
(1) The authors used a CLARITY-based passive clearing protocol, which is known to sometimes induce tissue swelling or distortion. This may affect anatomical precision, especially when assigning neurons to narrow domains such as septa versus barrels. Please clarify whether tissue expansion was measured, corrected, or otherwise accounted for during analysis.
Yes, the tissue expansion was accounted during analysis for the laminar specification. We excluded the brains with severe distortion.
(2) While the anatomical data are plotted as a function of "depth from the top of layer 4," the manuscript does not specify the precise depth ranges used to define individual cortical layers in the cleared tissue. Given the importance of laminar specificity in projection and cell type analyses, the criteria and boundaries used to delineate each layer should be explicitly stated.
Thank you for pointing this out. We now include the criteria for delineating each layer in the manuscript. “Given that the depth of Layer 4 (L4) can be reliably measured due to its welldefined barrel boundaries, and that the relative widths of other layers have been previously characterized (El-Boustani et al., 2018), we estimated laminar boundaries proportionally. Specifically, Layer 2/3 was set to approximately 1.3–1.5 times the width of L4, Layer 5a to ~0.5 times, and Layer 5b to a similar width as L4. Assuming uniform tissue expansion across the cortical column, we extrapolated the remaining laminar thicknesses proportionally.”
(3) In several key comparisons (e.g., SST⁺ vs. VIP⁺ interneurons, or S2-projecting vs. M1projecting neurons), it is unclear whether the same barrel columns were analyzed across conditions. Given the anatomical and functional heterogeneity across wS1 columns, failing to control for this may introduce significant confounds. We recommend analyzing matched columns across groups or, if not feasible, clearly acknowledging this limitation in the manuscript.
We thank the reviewer for raising this important point. For the comparison of SST⁺ versus VIP⁺ interneurons, it would in principle have been possible to analyze the same barrel columns across groups. However, because some of the cleared brains did not reach the optimal level of clarity, our choice of columns was limited, and we were not always able to obtain sufficiently clear data from the same columns in both groups. Similarly, for the analysis of S2- versus M1-projecting neurons, variability in the position and spread of retrograde virus injections made it difficult to ensure measurements from identical barrel columns. We have now added a statement in the Discussion to acknowledge this limitation.
(4) Figure 1C: Clarify what each point in the t-SNE plot represents-e.g., a single trial, a recording channel, or an averaged response. Also, describe the input features used for dimensionality reduction, including time windows and preprocessing steps.
In response to the reviewer’s comment, we have now added the following in the methods: In summary, each point in the t-SNE plots represents an averaged response across 20 trials for a specific domain (barrel, septa, or neighbor) and genotype (WT or KO), with approximately 14 points per domain derived from the 280 trials in each dataset. The input features are preprocessed by averaging blocks of 20 trials into 1900-dimensional vectors (95ms × 20), which are then reduced to 2D using t-SNE with the specified parameters. This approach effectively highlights the segregation and clustering patterns of neural responses across cortical domains in both WT and KO conditions.
(5) Figures 1D, E (left panels): The y-axes lack unit labeling and scale bars. Please indicate whether values are in spikes/sec, spikes/bin, or normalized units.
We have now clarified this.
(6) Figures 1D, E (right panels): The color bars lack units. Specify whether the values represent raw firing rates, z-scores, or other normalized measures. Replace the vague term "Matrix representation" with a clearer label such as "Pulse-aligned firing heatmap."
Thank you, we have now done it.
(7) Figure 1E (bottom panel): There appears to be no legend referring to these panels. Please define labels such as "B" and "S."
Thank you, we have now done it.
(8) Figure 1E legend: If it duplicates the legend from Figure 1D, this should be made explicit or integrated accordingly.
We have changed the structure of this figure.
(9) Figure 1F: Define "AUC" and explain how it was computed (e.g., area under the firing rate curve over 0-50 ms). Indicate whether the plotted values represent percentages and, if so, label the y-axis accordingly. If normalization was applied, describe the procedure. Include sample sizes (n) and specify what each data point represents (e.g., animal, recording site).
The following paragraph has been added in the methods section:
The Area Under the Curve (AUC) was computed as the integral of the smoothed firing rate (spikes per millisecond) over a 50ms window following each whisker stimulation pulse, using trapezoidal integration. Firing rate data for layer 4 barrel and septal regions in wild-type (WT) and knockout (KO) mice were smoothed with a 3-point moving average and averaged across blocks of 20 trials. Plotted values represent the percentage ratio of multi-whisker (MW) to single whisker (SW) AUC with error bars showing the standard error of the mean. Each data point reflects the mean AUC ratio for a stimulation pulse across approximately 11 blocks (220 trials total). The y-axis indicates percentages.
(10) Figure 3C: Add units to the vertical axis.
We have added them.
(11) Figure 3D: Specify what each line represents (e.g., average of n cells, individual responses?).
Each line represents an average response of a neuron.
(12) Figure 4C legend: Same with what?". No legend refers to the bottom panels - please revise to clarify.
Thank you. We have now changed the figure structure and legends and fixed the missing information issue.
(13) Supplementary Figure 1B: Indicate the physical length of the scale bar in micrometers.
This has been fixed. The scale bar is 250um.
(14) Indicate the catalog number or product name of the 8×8 silicon probe used for recordings.
We have added this information. It is the A8x8-Edge-5mm-100-200-177-A64
References
(1) Beierlein, M., Gibson, J. R. & Connors, B. W. (2003). Two dynamically distinct inhibitory networks in layer 4 of the neocortex. J. Neurophysiol. 90, 2987–3000.
(2) Burkhalter, A., D’Souza, R. D. & Ji, W. (2023). Integration of feedforward and feedback information streams in the modular architecture of mouse visual cortex. Annu. Rev. Neurosci. 46, 259–280.
(3) Chen, J. L., Margolis, D. J., Stankov, A., Sumanovski, L. T., Schneider, B. L. & Helmchen, F. (2015). Pathway-specific reorganization of projection neurons in somatosensory cortex during learning. Nat. Neurosci. 18, 1101–1108.
(4) Connor, J. R. & Peters, A. (1984). Vasoactive intestinal polypeptide-immunoreactive neurons in rat visual cortex. Neuroscience 12, 1027–1044.
(5) Cruikshank, S. J., Lewis, T. J. & Connors, B. W. (2007). Synaptic basis for intense thalamocortical activation of feedforward inhibitory cells in neocortex. Nat. Neurosci. 10, 462–468.
(6) Dolan, J. & Mitchell, K. J. (2013). Mutation of Elfn1 in mice causes seizures and hyperactivity. PLoS One 8, e80491.
(7) Gibson, J. R., Beierlein, M. & Connors, B. W. (1999). Two networks of electrically coupled inhibitory neurons in neocortex. Nature 402, 75–79.
(8) Ji, W., Gămănuţ, R., Bista, P., D’Souza, R. D., Wang, Q. & Burkhalter, A. (2015). Modularity in the organization of mouse primary visual cortex. Neuron 87, 632–643.
(9) Martin-Cortecero, J. & Nuñez, A. (2014). Tactile response adaptation to whisker stimulation in the lemniscal somatosensory pathway of rats. Brain Res. 1591, 27–37.
(10) Mégevand, P., Troncoso, E., Quairiaux, C., Muller, D., Michel, C. M. & Kiss, J. Z. (2009). Long-term plasticity in mouse sensorimotor circuits after rhythmic whisker stimulation. J. Neurosci. 29, 5326–5335.
(11) Meier, A. M., Wang, Q., Ji, W., Ganachaud, J. & Burkhalter, A. (2021). Modular network between postrhinal visual cortex, amygdala, and entorhinal cortex. J. Neurosci. 41, 4809– 4825.
(12) Meier, A. M., D’Souza, R. D., Ji, W., Han, E. B. & Burkhalter, A. (2025). Interdigitating modules for visual processing during locomotion and rest in mouse V1. bioRxiv 2025.02.21.639505.
(13) Scala, F., Kobak, D., Shan, S., Bernaerts, Y., Laturnus, S., Cadwell, C. R., Hartmanis, L., Froudarakis, E., Castro, J. R., Tan, Z. H., et al. (2019). Layer 4 of mouse neocortex differs in cell types and circuit organization between sensory areas. Nat. Commun. 10, 4174.
(14) Stachniak, T. J., Sylwestrak, E. L., Scheiffele, P., Hall, B. J. & Ghosh, A. (2019). Elfn1induced constitutive activation of mGluR7 determines frequency-dependent recruitment of somatostatin interneurons. J. Neurosci. 39, 4461–4475.
(15) Stachniak, T. J., Kastli, R., Hanley, O., Argunsah, A. Ö., van der Valk, E. G. T., Kanatouris, G. & Karayannis, T. (2021). Postmitotic Prox1 expression controls the final specification of cortical VIP interneuron subtypes. J. Neurosci. 41, 8150–8166.
(16) Stachniak, T. J., Argunsah, A. Ö., Yang, J. W., Cai, L. & Karayannis, T. (2023). Presynaptic kainate receptors onto somatostatin interneurons are recruited by activity throughout development and contribute to cortical sensory adaptation. J. Neurosci. 43, 7101–7118.
(17) Sun, Q.-Q., Huguenard, J. R. & Prince, D. A. (2006). Barrel cortex microcircuits: Thalamocortical feedforward inhibition in spiny stellate cells is mediated by a small number of fast-spiking interneurons. J. Neurosci. 26, 1219–1230.
(18) Sylwestrak, E. L. & Ghosh, A. (2012). Elfn1 regulates target-specific release probability at CA1-interneuron synapses. Science 338, 536–540.
(19) Tan, Z., Hu, H., Huang, Z. J. & Agmon, A. (2008). Robust but delayed thalamocortical activation of dendritic-targeting inhibitory interneurons. Proc. Natl. Acad. Sci. USA 105, 2187–2192.
(20) Tomioka, N. H., Yasuda, H., Miyamoto, H., Hatayama, M., Morimura, N., Matsumoto, Y., Suzuki, T., Odagawa, M., Odaka, Y. S., Iwayama, Y., et al. (2014). Elfn1 recruits presynaptic mGluR7 in trans and its loss results in seizures. Nat. Commun. 5, 4501.
(21) Yamashita, T., Vavladeli, A., Pala, A., Galan, K., Crochet, S., Petersen, S. S. & Petersen, C. C. (2018). Diverse long-range axonal projections of excitatory layer 2/3 neurons in mouse barrel cortex. Front. Neuroanat. 12, 33.
-
-
www.biorxiv.org www.biorxiv.org
-
eLife Assessment
This important manuscript provides insights into the competition between Splicing Factor 1 (SF1) and Quaking (QKI) for binding at the ACUAA branch point sequence in a model intron, regulating exon inclusion. The study employs convincing, rigorous transcriptomic, proteomic, and reporter assays, with both mammalian cell culture and yeast models.
-
Reviewer #2 (Public review):
Summary:
In this manuscript, Pereira de Castro and coworkers are studying potential competition between a more standard splicing factor SF1 and an alternative splicing factor called QK1. This is interesting because they bind to overlapping sequence motifs and could potentially have opposing effects on promoting the splicing reaction. To test this idea, the authors KD either SF1 or QK1 in mammalian cells and uncover several exons whose splicing regulation follows the predicted pattern of being promoted for splicing by SF1 and repressed by QK1. Importantly, these have introns enriched in SF1 and QK1 motifs. The authors then focus on one exon in particular with two tandem motifs to study the mechanism of this in greater detail and their results confirm the competition model. Mass spec analysis largely agrees with their proposal; however, it is complicated by apparently quick transition of SF1 bound complexes to later splicing intermediates. An inspired experiment in yeast shows how QK1 competition could potentially have a determinental impact on splicing in an orthogonal system. Overall these results show how splicing regulation can be achieved by competition between a "core" and alternative splicing factor and provide additional insight into the complex process of branch site recognition. The manuscript is exceptionally clear and the figures and data very logically presented. The work will be valuable to those in the splicing field who are interested in both mechanism and bioinformatics approaches to deconvolve any apparent "splicing code" being used by cells to regulate gene expression.
Strengths:
(1) The main discovery of the manuscript involving evidence for SF1/QK1 competition is quite interesting and important for this field. This evidence has been missing and may change how people think about branch site recognition.
(2) The experiments and the rationale behind them are clearly and logically presented.
(3) The experiments are carried out to a high standard and well-designed controls are included.
(4) The extrapolation of the result to yeast in order to show the potentially devastating consequences of QK1 competition was creative and informative.
Weaknesses:
Overall the weaknesses are relatively minor and involve cases where conclusions could potentially have been strengthened with additional experimentation. For example, pull-down of the U2 snRNP could be strengthened by detection of the snRNA whereas the proteins may themselves interact with these factors in the absence of the snRNA. In addition the discussion is a bit speculative given the data, but compelling nonetheless.
-
Reviewer #3 (Public review):
Summary:
In this manuscript the authors were trying to establish whether competition between the RNA binding proteins SF1 and QKI controlled splicing outcomes. These two proteins have similar binding sites and protein sequences, but SF1 lacks a dimerization motif and seems to bind a single version of the binding sequence. Importantly, these binding sequences correspond to branchpoint consensus sequences, with SF1 binding leading to productive splicing, but QKI binding leading instead to association with paraspeckle proteins. They show that in human cells SF1 generally activates exons and QKI represses, and a large group of the jointly regulated exons (43% of joint targets) are reciprocally controlled by SF1 and QKI. They focus on one of these exons RAI14 that shows this reciprocal pattern of regulation, and has 2 repeats of the binding site that make it a candidate for joint regulation, and confirm regulation within a minigene context. The authors used assembly of proteins within nuclear extracts to explain the effect of QKI versus SF1 binding. Finally the authors show that expression of QKI is lethal in yeast, and causes splicing defects.
How this fits in the field. This study is interesting and provides a conceptual advance by providing a general rule how SF1 and QKI interact with relation to binding sites, and the relative molecular fates followed, so is very useful. Most of the analysis seems to focus on one example, but the choice of this example was carefully explained in the text. The molecular analysis and global work significantly adds to the picture from the previously published paper about NUMB joint regulation by QKI and SF (Zong et al, cited in text as reference 50, that looked at SF1 and QKI binding in relation to a duplicated binding site/branchpoint sequence in NUMB).
Strengths:
The data presented are strong and clear. The ideas discussed in this paper are of wide interest, and present a simple model where two binding sites generates a potentially repressive QKI response, whereas exons that have a single upstream sequence are just regulated by SF1. The assembly of splicing complexes on RNAs derived from RAI14 in nuclear extracts, followed by mass spec gave interesting mechanistic insight into what was occurring as a result of QKI versus SF1 binding.
Weaknesses:
The authors have addressed the previous weaknesses of the study, resulting in a much stronger manuscript
-
Author response:
The following is the authors’ response to the original reviews.
eLife Assessment
This important manuscript provides insights into the competition between Splicing Factor 1 (SF1) and Quaking (QKI) for binding at the ACUAA branch point sequence in a model intron, regulating exon inclusion. The study employs rigorous transcriptomic, proteomic, and reporter assays, with both mammalian cell culture and yeast models. Nevertheless, while the data are convincing, broadening the analysis to additional exons and narrowing the manuscript's title to better align with the experimental scope would strengthen the work.
Public Reviews:
Reviewer #1 (Public review):
In this manuscript, the authors aimed to show that SF1 and QKI compete for the intron branch point sequence ACUAA and provide evidence that QKI represses inclusion when bound to it.
Major strengths of this manuscript include:
(1) Identification of the ACUAA-like motif in exons regulated by QKI and SF1.
(2) The use of the splicing reporter and mutant analysis to show that upstream and downstream ACUAAC elements in intron 10 of RAI are required for repressing splicing.
(3) The use of proteomic to identify proteins in C2C12 nuclear extract that binds to the wild type and mutant sequence.
(4) The yeast studies showing that ectopic lethality when Qki5 expression was induced, due to increased mis-splicing of transcripts that contain the ACUAA element.
The authors conclusively show that the ACUAA sequence is bound by QKI and provide strong evidence that this leads to differences in exons inclusion and exclusion. In animal cells, and especially in human, branchpoint sequences are degenerate but seem to be recognized by specific splicing factors. Although a subset of splicing factors shows tissue-specific expression patterns most don't, suggesting that yet-to-be-identified mechanisms regulate splicing. This work suggests that an alternate mechanism could be related to the binding affinity of specific RNA binding factors for branchpoint sequences coupled with the level of these different splicing factors in a given cell.
We thank the reviewer for the positive comments.
Reviewer #2 (Public review):
Summary:
In this manuscript, Pereira de Castro and coworkers are studying potential competition between a more standard splicing factor SF1, and an alternative splicing factor called QK1. This is interesting because they bind to overlapping sequence motifs and could potentially have opposing effects on promoting the splicing reaction. To test this idea, the authors KD either SF1 or QK1 in mammalian cells and uncover several exons whose splicing regulation follows the predicted pattern of being promoted for splicing by SF1 and repressed by QK1. Importantly, these have introns enriched in SF1 and QK1 motifs. The authors then focus on one exon in particular with two tandem motifs to study the mechanism of this in greater detail and their results confirm the competition model. Mass spec analysis largely agrees with their proposal; however, it is complicated by the apparently quick transition of SF1-bound complexes to later splicing intermediates. An inspired experiment in yeast shows how QK1 competition could potentially have a detrimental impact on splicing in an orthogonal system. Overall, these results show how splicing regulation can be achieved by competition between a "core" and alternative splicing factor and provide additional insight into the complex process of branch site recognition. The manuscript is exceptionally clear and the figures and data are very logically presented. The work will be valuable to those in the splicing field who are interested in both mechanism and bioinformatics approaches to deconvolve any apparent "splicing code" being used by cells to regulate gene expression. Criticisms are minor and the most important of them stem from overemphasis on parts of the manuscript on the evolutionary angle when evolution itself wasn't analyzed per se.
We thank the reviewer for the positive comments and very clear and fair critical points.
Strengths:
(1) The main discovery of the manuscript involving evidence for SF1/QK1 competition is quite interesting and important for this field. This evidence has been missing and may change how people think about branch site recognition.
(2) The experiments and the rationale behind them are exceptionally clearly and logically presented. This was wonderful!
Thank you so much. We felt the overall flow of the paper and data make for a nice “story” that conveys a relatively easy-to-understand explanation for a complex subject.
(3) The experiments are carried out to a high standard and well-designed controls are included.
(4) The extrapolation of the result to yeast in order to show the potentially devastating consequences of the QK1 competition was very exciting and creative.
We agree this is a very exciting result and finding! Thanks.
Weaknesses:
Overall the weaknesses are relatively minor and involve cases where clarification is necessary, some additional analysis could bolster the arguments, and suggestions for focusing the manuscript on its strengths.
(1) The title (Ancient...evolutionary outcomes), abstract, and some parts of the discussion focus heavily on the evolutionary implications of this work. However, evolutionary analysis was not performed in these studies (e.g., when did QK1 and SF1 proteins arise and/or diverge? How does this line up with branch site motifs and evolution of U2? Any insight from recent work from Scott Roy et al?). I think this aspect either needs to be bolstered with experimental work/data or this should be tamped down in the manuscript. I suggest highlighting the idea expressed in the sentence "A nuanced implication of this model is that loss-of-function...". To me, this is better supported by the data and potentially by some analysis of mutations associated with human disease.
We have revised the title and dampened the evolutionary aspects of the previous version of the manuscript.
(2) One paper that I didn't see cited was that by Tanackovic and Kramer (Mol Biol Cell 2005). This paper is relevant because they KD SF1 and found it nonessential for splicing in vivo. Do their results have implications for those here? How do the results of the KD compare? Could QK1 competition have influenced their findings (or does their work influence the "nuanced implication" model referenced above?)?
This is an interesting point, and thank you for the suggestion. We have now included a brief description of this study in the Introduction of the revised manuscript and do note that the authors measured intron retention of a beta globin reporter and SF3A1, SF3A2, and SF3A3 during SF1 knockdown, but did not detect elevated unspliced RNA in these targets.
(3) Can the authors please provide a citation for the statement "degeneracy is observed to a higher degree in organisms with more alternative splicing"? Does recent evolutionary analysis support this?
We have removed the statement, as it did not add much to the content and I am not sure I can state the concept I was attempting to convey in a simple manner with few citations.
(4) For the data in Figure 3, I was left wondering if NMD was confounding this analysis. Can the authors respond to this and address this concern directly?
We have not measured if the reporters used in Figure 3 produce protein(s). Presumably, though, all spliced reporter RNA would be degraded equally (the included/skipped isoforms’ “reading frames” are not altered from one another). This would not be case for unspliced nuclear reporter RNA, however. Given this difference, and that our analysis can not resolve the subcellular localization of the different reporter species, we have removed the measurement of and subsequent results describing unspliced reporter RNA from Figure 3.
(5) To me, the idea that an engaged U2 snRNP was pulled down in Figure 4F would be stronger if the snRNA was detected. Was that able to be observed by northern or primer extension? Would SF1 be enriched if the U2 snRNA was degraded by RNaseH in the NE?
We did not measure any co-associating RNAs in this experimental approach, but agree that this approach would strengthen the evidence for it.
(6) I'm wondering how additive the effects of QK1 and SF1 are... In Figure 2, if QK1 and SF1 are both knocked down, is the splicing of exon 11 restored to "wt" levels?
This is an interesting question that we were unfortunately unable to address experimentally here.
(7) The first discussion section has two paragraphs that begin "How does competition between SF1..." and "Relatively little is known about how...". I found the discussion and speculation about localization, paraspekles, and lncRNAs interesting but a bit detracting from the strengths of the manuscript. I would suggest shortening these two paragraphs into a single one.
We have revised the Discussion.
Reviewer #3 (Public review):
Summary:
In this manuscript, the authors were trying to establish whether competition between the RNA-binding proteins SF1 and QKI controlled splicing outcomes. These two proteins have similar binding sites and protein sequences, but SF1 lacks a dimerization motif and seems to bind a single version of the binding sequence. Importantly, these binding sequences correspond to branchpoint consensus sequences, with SF1 binding leading to productive splicing, but QKI binding leading instead to association with paraspeckle proteins. They show that in human cells SF1 generally activates exons and QKI represses, and a large group of the jointly regulated exons (43% of joint targets) are reciprocally controlled by SF1 and QKI. They focus on one of these exons RAI14 that shows this reciprocal pattern of regulation, and has 2 repeats of the binding site that make it a candidate for joint regulation, and confirm regulation within a minigene context. The authors used the assembly of proteins within nuclear extracts to explain the effect of QKI versus SF1 binding. Finally, the authors show that the expression of QKI is lethal in yeast, and causes splicing defects.
How this fits in the field. This study is interesting and provides a conceptual advance by providing a general rule on how SF1 and QKI interact in relation to binding sites, and the relative molecular fates followed, so is very useful. Most of the analysis seems to focus on one example, although the molecular analysis and global work significantly add to the picture from the previously published paper about NUMB joint regulation by QKI and SF (Zong et al, cited in text as reference 50, that looked at SF1 and QKI binding in relation to a duplicated binding site/branchpoint sequence in NUMB).
Thank you for the encouraging remarks.
Strengths:
The data presented are strong and clear. The ideas discussed in this paper are of wide interest, and present a simple model where two binding sites generate a potentially repressive QKI response, whereas exons that have a single upstream sequence are just regulated by SF1. The assembly of splicing complexes on RNAs derived from RAI14 in nuclear extracts, followed by mass spec gave interesting mechanistic insight into what was occurring as a result of QKI versus SF1 binding.
Weaknesses:
I did not think the title best summarises the take-home message and could be perhaps a bit more modest. Although the authors investigated splicing patterns in yeast and human cells, yeast do not have QKI so there is no ancient competition in that case, and the study did not really investigate physiological or evolutionary outcomes in splicing, although it provides interesting speculation on them. Also as I understood it, the important issue was less conserved branchpoints in higher eukaryotes enabling alternative splicing, rather than competition for the conserved branchpoint sequence. So despite the the data being strong and properly analysed and discussed in the paper, could the authors think whether they fit best with the take-home message provided in the title? Just as a suggestion (I am sure the authors can do a better job), maybe "molecular competition between variant branchpoint sequences predict physiological and evolutionary outcomes in splicing"?
Thank you for this point (Reviewer 2 had a similar comment) and the suggestion. We have revised the title.
Although the authors do provide some global data, most of the detailed analysis is of RAI14. It would have been useful to examine members of the other quadrants in Figure 1C as well for potential binding sites to give a reason why these are not co-regulated in the same way as RAI14. How many of the RAI14 quadrants had single/double sites (the motif analysis seemed to pull out just one), and could one of the non-reciprocally regulated exons be moved into a different quadrant by addition or subtraction of a binding site or changing the branchpoint (using a minigene approach for example).
This is an interesting point that we have considered. Our intent with the focus on RAI14 was to use a naturally occurring intron bps with evidence of strong QKI binding that did not require a high degree of sequence manipulation or engineering.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Most of my recommendations are really centered on the figures. In their current state, they detract from the data shown and could be improved: I recommend the authors use a uniform font. For example, Figure 1E and F have at least three different fonts of varying sizes making it very messy. In Figure 1C, the authors could bold the Ral14 ex11 or simply indicate that the blue is this exon in the legend, thus removing the text from this very busy graph. In Figure 4F, I would recommend, having all the labels the same size and putting those genes of interest like Sf3a1 in bold. This could also be done in Figure 4E.
Thank you for the suggestion and we have edited these (FYI the font in Fig’s 1E and 1F were from the rMAPS default output, but I agree, it gives a sloppy appearance).
(2) In Figures 4D and 4G, is there QKI binding to the downstream deletion mutant after 30 minutes? Also, in Figure 4G, are these all from the same blot? The band sizes seem to be very different between lanes. If these were not on the same blot, the original gels should be submitted.
A small amount of Qki appears to be binding after 30 min. All lanes/blots are from the same gels/membranes; see new Supplemental Figure 4 for the original (uncropped) images of the blots.
(3) The authors should indicate, the source and concentration of the antibodies used for their WB. They should also indicate the primers used for RT-PCRs.
We have revised the methods to include the antibody information and have uploaded a supplemental table 8 with all oligonucleotide sequences used (which I (Sam Fagg) neglected to do initially, so that’s my bad).
Reviewer #2 (Recommendations for the authors):
(1) This may come down to the author's preference but branch point and branch site are frequently two words, not a single compound word (branch point vs. branchpoint). In addition, the authors may want to use branchsite with the abbreviation BS more frequently since they often don't describe the specific point of branching, and bp and bps could be confused for the more frequent abbreviations for base pair(s).
Good suggestion; we have edited the text accordingly.
(2) In general the addition of page numbers and line numbers to the manuscript would greatly aid reviewers!
Point taken…
(3) Introduction; "...under normal growth conditions they are efficiently spliced". I would say MOST introns in yeast are efficiently spliced. This is definitely not universal.
Text edited to indicate that most are efficiently spliced.
(4) Introduction; " recognition of the bps by SF1 (mammals) (20)". The choice of reference 20 is an odd one here. I think the Robin Reed and Michael Rosbash paper was the first to show SF1 was the human homolog of BBP.
Got it, thanks (added #14 here and kept #20 also since it shows the structure of SF1 in complex with a UACUAAC bps.)
(5) Results; "QK1 and SF1 co-regulate.."; it may be useful for the reader if you could explain in more detail why exon inclusion and intron retention are expected outcomes for QK1 knockdown and vice versa for SF1. The exon inclusion here is more obvious than the intron retention phenotype. (In other words, if more exons are included shouldn't it follow that more introns are removed?)
We explain the expected results for exon inclusion in the Introduction and this paragraph of the Results. Although we have observed more intron retention under QKI loss-of-function approaches before, I am uncertain where the reviewer sees that we indicate any expected result for intron retention from either QKI or SF1 knockdown. I believe the statement you refer to might be on line 162 and starts with: “Consistent with potentially opposing functions in splicing…” ?
Also, I agree that if SF1 is a “splicing activator,” one might expect more IR in its absence (but this is not the case; there is, in fact, less), but nonetheless, the opposite outcome is observed with QKI knockdown (more IR). It is unclear why this is the case, and we did not investigate it.
(6) Results; "QK1 and SF1 co-regulate.."; "Thus the most highly represented set.." To me, the most highly represented set is those which are not both QK1-repressed and SF1-activated. Does this indicate that other factors are involved at most sites than simple competition between these two?
We have revised the sentence in question to include the text “by quadrant” in order to convey our meaning more precisely.
(7) Throughout the manuscript, 5 apostrophes and 3 apostrophes are used instead of 5 prime symbols and 3 prime symbols.
Thank you for pointing that out. We have fixed each instance of this.
(8) Sometimes SF1 is written as Sf1. (also Tatsf1)
This was a mouse/human gene/protein nomenclature error that we have fixed; thank you for pointing this out.
(9) You may want to make sure that figures are labeled consistently with the manuscript text. In Figure 1B, it is RI rather than IR. In Figure 4 it is myoblast NE rather than C2C12 nuclear extract.
We have fixed these, checked for other examples, and where relevant, edited those too.
(10) I think Figure 1A could be improved by also including a depiction of the domain arrangements of SF1 and QK1.
Done.
(11) I was a bit confused with all the lines in Figure 1E and 1F. What is the difference between the log (pVal) and upregulated plots? Can these figures be simplified or explained more thoroughly?
Based on this comment and one from Reviewer 1, we have slightly revised the wording (and font) on the output, which hopefully clarifies. These are motif enrichment plots generated by rMAPS (Refs 61 and 62) analysis of rMATS (Ref 60) data for exons more included (depicted by the red lines) or more skipped (depicted by the blue lines) compared to control versus a “background” set of exons that are detectable but unchanged. The -log<sub>10</sub> is P-value (dotted line) indicates the significance of exons more included in shRNA treatment vs control shRNA (previously read “upregulated”) compared to background exons that are detectable but unchanged; the solid lines indicate the motif score; these are described in the references indicated.
(12) Figure 1B, it is a bit hard to conclude that there is more AltEx or "RI/IR" in one sample vs. the other from these plots since the points overlay one another. Can you include numbers here?
Added (and deleted Suppl Fig S1, which was simply a chart showing the numbers).
(13) How was PSI calculated in Figure 2A?
VAST-tools (we state this in the legend in the revised version).
You may want to include rel protein (or the lower limit of detection) for Figure 2B to be consistent with 2C. Why is KD of SF1 so poor and variable between 2C and 2D?
We have not investigated this, but these blots show an optimized result that we were able to obtain for the knockdown in each cell type. It may be that HEK293 cells (Fig 2B) have a stronger requirement for SF1 than C2C12 cells…? I would argue that it is not necessarily “poor” in Fig 2C, as we observe ~70% depletion of the protein.
Why are two bands present in the gel?
Two to three isoforms of SF1 are present in most cell types.
A good (or bad, really) example of an SF1 western blot (and knockdown of ~35% in K562 or ~45% in HepG2 can also be seen on the ENCODE project website, for reference:
By comparison, I think ours are much more cosmetically pleasing, and our knockdown (especially in C2C12) is much more efficient.
(14) Figure 3, The asterisk refers to a cryptic product. Can the uaAcuuuCAG be used as a branch point? Presumably the natural 3' SS is now too close so this would result in activation of a downstream 3'SS?
We did not pursue determining the identity of this minor and likely artefactual product, but we (and others) have observed a similar phenomenon when using splicing reporter-based mutational approaches.
(15) For the methods. The "RNA extraction, RT -PCR,..." subheading needs to be on its own line. Please add (w/v) or (v/v) to percentages where appropriate. Please convert ug to the symbol for "micro".
Thank you, we have made these changes.
(16) In Figure 4B, the text here and legend are microscopic. Even with reading glasses, I couldn't make anything out!
We have increased the font sizes for the text and scale bar…when referring to “legend” does the reviewer mean the scale bar?
(17) As a potential discussion item, it is worth noting that SF1 could also repress splicing if it could either not engage with U2AF or be properly displaced by U2 snRNP so the snRNA could pair. I was wondering if QK1 could similarly be activating if it could engage with U2AF. I'm unsure if this could be tested by domain swaps (and is beyond the scope of this paper). It just may be worth speculating about.
Good point and suggestion…we are looking into this.
Reviewer #3 (Recommendations for the authors):
(1) Is the reference in the text to Figure 5F correct for actin splicing (this is just before the discussion)?
I see references several lines up from this, but I do not see a reference just before the discussion…?
(2) I was not sure why the minigene experiments showed such high levels of intron retention that seemed to be impacted also by deletion of the branchpoint sequences, and suggest that the two branchpoints are not equal in strength.
Neither were we, but Reviewer 2 has suggested that degradation of the spliced products could be rapid (NMD substrates) which could complicate the interpretation of what appears to be higher levels of intron retention. Given the possibility that this could be a non-physiological artefact, we have removed the measurement of unspliced reporter and now only show the spliced products (equally subject to degradation) and report their percent inclusion.
-
-
drive.google.com drive.google.comview2
-
Module Summary
Give me your feedback on the summary and whether it clearly communicates expected outcomes.
-
Module Introduction
This is a brief introduction to the module. Do you think it supports learner engagement?
-
-
www.biorxiv.org www.biorxiv.org
-
eLife Assessment
The study presents convincing quantitative evidence, supported by appropriate negative controls, for the presence of low-abundance glycine receptors (GlyRs) within inhibitory synapses in telencephalic regions of the mouse brain. Using sensitive single-molecule localization microscopy of endogenously tagged GlyRs, the authors reveal previously undetected populations of these receptors. Although the functional significance of these low-abundance GlyRs remains to be established, the findings offer valuable insights and methodologies that will be of interest to neuroscientists studying inhibitory synapse biology.
[Editors' note: this paper was reviewed by Review Commons.]
-
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors investigate the nanoscopic distribution of glycine receptor subunits in the hippocampus, dorsal striatum, and ventral striatum of the mouse brain using single-molecule localization microscopy (SMLM). They demonstrate that only a small number of glycine receptors are localized at hippocampal inhibitory synapses. Using dual-color SMLM, they further show that clusters of glycine receptors are predominantly localized within gephyrin-positive synapses. A comparison between the dorsal and ventral striatum reveals that the ventral striatum contains approximately eight times more glycine receptors and this finding is consistent with electrophysiological data on postsynaptic inhibitory currents. Finally, using cultured hippocampal neurons, they examine the differential synaptic localization of glycine receptor subunits (α1, α2, and β). This study is significant as it provides insights into the nanoscopic localization patterns of glycine receptors in brain regions where this protein is expressed at low levels. Additionally, the study demonstrates the different localization patterns of GlyR in distinct striatal regions and its physiological relevance using SMLM and electrophysiological experiments. However, several concerns should be addressed.
Specific comments on the original version:
(1) Colocalization analysis in Figure 1A. The colocalization between Sylite and mEos-GlyRβ appears to be quite low. It is essential to assess whether the observed colocalization is not due to random overlap. The authors should consider quantifying colocalization using statistical methods, such as a pixel shift analysis, to determine whether colocalization frequencies remain similar after artificially displacing one of the channels.
(2) Inconsistency between Figure 3A and 3B. While Figure 3B indicates an ~8-fold difference in the number of mEos4b-GlyRβ detections per synapse between the dorsal and ventral striatum, Figure 3A does not appear to show a pronounced difference in the localization of mEos4b-GlyRβ on Sylite puncta between these two regions. If the images presented in Figure 3A are not representative, the authors should consider replacing them with more representative examples or providing an expanded images with multiple representative examples. Alternatively, if this inconsistency can be explained by differences in spot density within clusters, the authors should explain that.
(3) Quantification in Figure 5. It is recommended that the authors provide quantitative data on cluster formation and colocalization with Sylite puncta in Figure 5 to support their qualitative observations.
(4) Potential for pseudo replication. It's not clear whether they're performing stats tests across biological replica, images, or even synapses. They often quote mean +/- SEM with n = 1000s, and so does that mean they're doing tests on those 1000s? Need to clarify.
(5) Does mEoS effect expression levels or function of the protein? Can't see any experiments done to confirm this. Could suggest WB on homogenate, or mass spec?
(6) Quantification of protein numbers is challenging with SMLM. Issues include i) some of FP not correctly folded/mature, and ii) dependence of localisation rate on instrument, excitation/illumination intensities, and also the thresholds used in analysis. Can the authors compare with another protein that has known expression levels- e.g. PSD95? This is quite an ask, but if they could show copy number of something known to compare with, it would be useful.
(7) Rationale for doing nanobody dSTORM not clear at all. They don't explain the reason for doing the dSTORM experiments. Why not just rely on PALM for coincidence measurements, rather than tagging mEoS with a nanobody, and then doing dSTORM with that? Can they explain? Is it to get extra localisations- i.e. multiple per nanobody? If so, localising same FP multiple times wouldn't improve resolution. Also, no controls for nanobody dSTORM experiments- what about non-spec nb, or use on WT sections?
(8) What resolutions/precisions were obtained in SMLM experiments? Should perform Fourier Ring Correlation (FRC) on SR images to state resolutions obtained (particularly useful for when they're presenting distance histograms, as this will be dependent on resolution). Likewise for precision, what was mean precision? Can they show histograms of localisation precision.
(9) Why were DBSCAN parameters selected? How can they rule out multiple localisations per fluor? If low copy numbers (<10), then why bother with DBSCAN? Could just measure distance to each one.
(10) For microscopy experiment methods, state power densities, not % or "nominal power".
(11) In general, not much data presented. Any SI file with extra images etc.?
(12) Clarification of the discussion on GlyR expression and synaptic localization: The discussion on GlyR expression, complex formation, and synaptic localization is sometimes unclear, and needs terminological distinctions between "expression level", "complex formation" and "synaptic localization". For example, the authors state: "What then is the reason for the low protein expression of GlyRβ? One possibility is that the assembly of mature heteropentameric GlyR complexes depends critically on the expression of endogenous GlyR α subunits." Does this mean that GlyRβ proteins that fail to form complexes with GlyRα subunits are unstable and subject to rapid degradation? If so, the authors should clarify this point. The statement "This raises the interesting possibility that synaptic GlyRs may depend specifically on the concomitant expression of both α1 and β transcripts." suggests a dependency on α1 and β transcripts. However, is the authors' focus on synaptic localization or overall protein expression levels? If this means synaptic localization, it would be beneficial to state this explicitly to avoid confusion. To improve clarity, the authors should carefully distinguish between these different aspects of GlyR biology throughout the discussion. Additionally, a schematic diagram illustrating these processes would be highly beneficial for readers.
(13) Interpretation of GlyR localization in the context of nanodomains. The distribution of GlyR molecules on inhibitory synapses appears to be non-homogeneous, instead forming nanoclusters or nanodomains, similar to many other synaptic proteins. It is important to interpret GlyR localization in the context of nanodomain organization.
Significance:
The paper presents biological and technical advances. The biological insights revolve mostly on the documentation of Glycine receptors in particular synapses in forebrain, where they are typically expressed at very low levels. The authors provide compelling data indicating that the expression is of physiological significance. The authors have done a nice job of combining genetically tagged mice with advanced microscopy methods to tackle the question of distributions of synaptic proteins. Overall, these advances are more incremental than groundbreaking.
Comments on revised version:
The authors have addressed the majority of the significant issues raised in the review and revised the manuscript appropriately. One issue that can be further addressed relates to the issue of pseudo-replication. The authors state in their response that "All experiments were repeated at least twice to ensure reproducibility (N independent experiments). Statistical tests were performed on pooled data across the biological replicates; n denotes the number of data points used for testing (e.g., number of synaptic clusters, detections, cells, as specified in each case).". This suggests that they're not doing their stats on biological replicates, and instead are pseudo replicating. It's not clear how they have ensured reproducibility, when the stats seem to have been done on pooled data across repeats.
-
Reviewer #2 (Public review):
Summary:
In their manuscript "Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses" Camuso and colleagues apply single molecule localization microscopy (SMLM) methods to visualize low copy numbers of GlyRs at inhibitory synapses in the hippocampal formation and the striatum. SMLM analysis revealed higher copy numbers in striatum compared to hippocampal inhibitory synapses. They further provide evidence that these low copy numbers are tightly linked to post-synaptic scaffolding protein gephyrin at inhibitory synapses. Their approach profits from the high detection sensitivity and resolution of SMLM and challenges the controversial view on the presence of GlyRs in these formations although there are reports (electrophysiology) on the presence of GlyRs in these particular brain regions. These new datasets in the current manuscript may certainly assist in understanding the complexity of fundamental building blocks of inhibitory synapses.
Strengths:
The manuscript provides new insights to presence of low-copy numbers by visualizing them via SMLM. This is the first report that visualizes GlyR optically in the brain applying the knock-in model of mEOS4b tagged GlyRß and quantifies their copy number comparing distribution and amount of GlyRs from hippocampus and striatum. Imaging data correspond well to electrophysiological measurements in the manuscript.
Comments on revised version:
My concerns have been successfully addressed by the authors during the revision process.
-
Reviewer #3 (Public review):
In this study, Camuso et al., make use of a knock-in mouse model expressing endogenously mEos4b-tagged GlyRβ subunits to detect endogenous glycine receptors in mouse brain using single-molecule localization microscopy (SMLM). At synapses in the hippocampus GlyRβ molecules are detected at very low copy numbers. Assuming that each detected GlyRβ molecule is incorporated in a pentameric glycine receptor, it was estimated that while the majority of hippocampal inhibitory synapses do not contain glycine receptors, a small population of inhibitory synapses contain a few (up to 10) glycine receptors. Using dual-color SMLM approaches it is furthermore confirmed that the detected GlyRβ molecules are embedded in the postsynaptic domain marked by gephyrin. In contrast to the hippocampus, at inhibitory synapses in the striatum GlyRβ molecules were detected at considerably higher copy numbers. Interestingly, the observed number of GlyRβ detections was significantly higher in the ventral striatum compared to the dorsal striatum. These findings are corroborated by electrophysiological recordings showing that postsynaptic glycinergic currents can be readily detected in the ventral striatum but are almost absent in the dorsal striatum. Using lentiviral overexpression of recombinant GlyRalpha1, alpha2, and beta subunits in cultured hippocampal neurons, it is shown that GlyR alpha1 subunits are readily detectable at synapses, but overexpressed GlyRalpha2 and beta subunits did not strongly enrich at synapses. This could indicate that GlyRa1 expression is limiting the synaptic expression of GlyRβ-containing glycine receptors in hippocampal neurons.
Comments on revised version:
This revised manuscript is significantly improved. New experimental and quantitative analysis is presented that strengthen the conclusions. Overall, the results presented in this manuscript are based on carefully performed SMLM experiments and are well-presented and described. The knock-in mouse with endogenously tagged GlyRβ molecules is a very strong aspect of this study and provides confidence in the labeling, the combination with SMLM is very strong as it provides high sensitivity and spatial resolution. These results confirm previous studies and will be of interest to a specialised audience interested in glycine receptors, inhibitory synapse biology and super-resolution microscopy.
-
Author response:
The following is the authors’ response to the current reviews.
We thank the editors of eLife and the reviewers for their thorough evaluation of our study. As regards the final comments of reviewer 1 please note that all experimental replicates were first analyzed separately, and were then pooled, since the observed changes were comparable between experiments. This mean that statistical analyses were done on pooled biological replicates.
The following is the authors’ response to the original reviews.
General Statements
We thank the reviewers for their thorough and constructive evaluation of our work. We have revised the manuscript carefully and addressed all the criticisms raised, in particular the issues mentioned by several of the reviewers (see point-by-point response below). We have also added a number of explanations in the text for the sake of clarity, while trying to keep the manuscript as concise as possible.
In our view, the novelty of our research is two-fold. From a neurobiological point of view, we provide conclusive evidence for the existence of glycine receptors (GlyRs) at inhibitory synapses in various brain regions including the hippocampus, dentate gyrus and sub-regions of the striatum. This solves several open questions and has fundamental implications for our understanding of the organisation and function of inhibitory synapses in the telencephalon. Secondly, our study makes use of the unique sensitivity of single molecule localisation microscopy (SMLM) to identify low protein copy numbers. This is a new way to think about SMLM as it goes beyond a mere structural characterisation and towards a quantitative assessment of synaptic protein assemblies.
Point-by-point description of the revisions
Reviewer #1 (Evidence, reproducibility and clarity):
In this manuscript, the authors investigate the nanoscopic distribution of glycine receptor subunits in the hippocampus, dorsal striatum, and ventral striatum of the mouse brain using single-molecule localization microscopy (SMLM). They demonstrate that only a small number of glycine receptors are localized at hippocampal inhibitory synapses. Using dual-color SMLM, they further show that clusters of glycine receptors are predominantly localized within gephyrinpositive synapses. A comparison between the dorsal and ventral striatum reveals that the ventral striatum contains approximately eight times more glycine receptors and this finding is consistent with electrophysiological data on postsynaptic inhibitory currents. Finally, using cultured hippocampal neurons, they examine the differential synaptic localization of glycine receptor subunits (α1, α2, and β). This study is significant as it provides insights into the nanoscopic localization patterns of glycine receptors in brain regions where this protein is expressed at low levels. Additionally, the study demonstrates the different localization patterns of GlyR in distinct striatal regions and its physiological relevance using SMLM and electrophysiological experiments. However, several concerns should be addressed.
The following are specific comments:
(1) Colocalization analysis in Figure 1A. The colocalization between Sylite and mEos-GlyRβ appears to be quite low. It is essential to assess whether the observed colocalization is not due to random overlap. The authors should consider quantifying colocalization using statistical methods, such as a pixel shift analysis, to determine whether colocalization frequencies remain similar after artificially displacing one of the channels.
Following the suggestion of reviewer 1, we re-analysed CA3 images of Glrb<sup>eos/eos</sup> hippocampal slices by applying a pixel-shift type of control, in which the Sylite channel (in far red) was horizontally flipped relative to the mEos4b-GlyRβ channel (in green, see Methods). As expected, the number of mEos4b-GlyRβ detections per gephyrin cluster was markedly reduced compared to the original analysis (revised Fig. 1B), confirming that the synaptic mEos4b detections exceed chance levels (see page 5).
(2) Inconsistency between Figure 3A and 3B. While Figure 3B indicates an ~8-fold difference in the number of mEos4b-GlyRβ detections per synapse between the dorsal and ventral striatum, Figure 3A does not appear to show a pronounced difference in the localization of mEos4bGlyRβ on Sylite puncta between these two regions. If the images presented in Figure 3A are not representative, the authors should consider replacing them with more representative examples or providing an expanded images with multiple representative examples. Alternatively, if this inconsistency can be explained by differences in spot density within clusters, the authors should explain that.
The pointillist images in Fig. 3A are essentially binary (red-black). Therefore, the density of detections at synapses cannot be easily judged by eye. For clarity, the original images in Fig. 3A have been replaced with two other examples that better reflect the different detection numbers in the dorsal and ventral striatum.
(3) Quantification in Figure 5. It is recommended that the authors provide quantitative data on cluster formation and colocalization with Sylite puncta in Figure 5 to support their qualitative observations.
This is an important point that was also raised by the other reviewers. We have performed additional experiments to increase the data volume for analysis. For quantification, we used two approaches. First, we counted the percentage of infected cells in which synaptic localisation of the recombinant receptor subunit was observed (Fig. 5C). We found that mEos4b-GlyRa1 consistently localises at synapses, indicating that all cells express endogenous GlyRb. When neurons were infected with mEos4b-GlyRb, fewer cells had synaptic clusters, meaning that indeed, GlyR alpha subunits are the limiting factor for synaptic targeting. In cultures infected with mEos4b-GlyRa2, only very few neurons displayed synaptic localisation (as judged by epifluorescence imaging). We think this shows that GlyRa2 is less capable of forming heteromeric complexes than GlyRa1, in line with our previous interpretation (see pp. 9-10, 13).
Secondly, we quantified the total intensity of each subunit at gephyrin-positive domains, both in infected neurons as well as non-infected control cultures (Fig. 5D). We observed that mEos4bGlyRa1 intensity at gephyrin puncta was higher than that of the other subunits, again pointing to efficient synaptic targeting of GlyRa1. Gephyrin cluster intensities (Sylite labelling) were not significantly different in GlyRb and GlyRa2 expressing neurons compared to the uninfected control, indicating that the lentiviral expression of recombinant subunits does not fundamentally alter the size of mixed inhibitory synapses in hippocampal neurons. Interestingly, gephyrin levels were slightly higher in hippocampal neurons expressing mEos4b-GlyRa1. In our view, this comes from an enhanced expression and synaptic targeting of mEos4b-GlyRa1 heteromers with endogenous GlyRb, pointing to a structural role of GlyRa1/b in hippocampal synapses (pp. 10, 13).
The new data and analyses have been described and illustrated in the relevant sections of the manuscript.
(4) Potential for pseudo replication. It's not clear whether they're performing stats tests across biological replica, images, or even synapses. They often quote mean +/- SEM with n = 1000s, and so does that mean they're doing tests on those 1000s? Need to clarify.
All experiments were repeated at least twice to ensure reproducibility (N independent experiments). Statistical tests were performed on pooled data across the biological replicates; n denotes the number of data points used for testing (e.g., number of synaptic clusters, detections, cells, as specified in each case). We have systematically given these numbers in the revised manuscript (n, N, and other experimental parameters such as the number of animals used, coverslips, images or cells). Data are generally given as mean +/- SEM or as mean +/- SD as indicated.
(5) Does mEoS effect expression levels or function of the protein? Can't see any experiments done to confirm this. Could suggest WB on homogenate, or mass spec?
The Glrb<sup>eos/eos</sup> knock-in mouse line has been characterised previously and does not to display any ultrastructural or functional deficits at inhibitory synapses (Maynard et al. 2021 eLife). GlyRβ expression and glycine-evoked responses were not significantly different to those of the wildtype. The synaptic localisation of mEos4b-GlyRb in KI animals demonstrates correct assembly of heteromeric GlyRs and synaptic targeting. Accordingly, the animals do not display any obvious phenotype. We have clarified this in the manuscript (p. 4). In the case of cultured neurons, long-term expression of fluorescent receptor subunits with lentivirus has proven ideal to achieve efficient synaptic targeting. The low and continuous supply of recombinant receptors ensures assembly with endogenous subunits to form heteropentameric receptor complexes (e.g. [Patrizio et al. 2017 Sci Rep]). In the present study, lentivirus infection did not induce any obvious differences in the number or size of inhibitory synapses compared to control neurons, as judged by Sylite labelling of synaptic gephyrin puncta (new Fig. 5D).
(6) Quantification of protein numbers is challenging with SMLM. Issues include i) some of FP not correctly folded/mature, and ii) dependence of localisation rate on instrument, excitation/illumination intensities, and also the thresholds used in analysis. Can the authors compare with another protein that has known expression levels- e.g. PSD95? This is quite an ask, but if they could show copy number of something known to compare with, it would be useful.
We agree that absolute quantification with SMLM is challenging, since the number of detections depends on fluorophore maturation, photophysics, imaging conditions, and analysis thresholds (discussed in Patrizio & Specht 2016, Neurophotonics). For this reason, only very few datasets provide reliable copy numbers, even for well-studied proteins such as PSD-95. One notable exception is the study by Maynard et al. (eLife 2021) that quantified endogenous GlyRβcontaining receptors in spinal cord synapses using SMLM combined with correlative electron microscopy. The strength of this work was the use of a KI mouse strain, which ensures that mEos4b-GlyRβ expression follows intrinsic regional and temporal profiles. The authors reported a stereotypic density of ~2,000 GlyRs/µm² at synapses, corresponding to ~120 receptors per synapse in the dorsal horn and ~240 in the ventral horn, taking into account various parameters including receptor stoichiometry and the functionality of the fluorophore. These values are very close to our own calculations of GlyR numbers at spinal cord synapses that were obtained slightly differently in terms of sample preparation, microscope setup, imaging conditions, and data analysis, lending support to our experimental approach. Nevertheless, the obtained GlyR copy numbers at hippocampal synapses clearly have to be taken as estimates rather than precise figures, because the number of detections from a single mEos4b fluorophore can vary substantially, meaning that the fluorophores are not represented equally in pointillist images. This can affect the copy number calculation for a specific synapse, in particular when the numbers are low (e.g. in hippocampus), however, it should not alter the average number of detections (Fig. 1B) or the (median) molecule numbers of the entire population of synapses (Fig. 1C). We have discussed the limitations of our approach (p. 11).
(7) Rationale for doing nanobody dSTORM not clear at all. They don't explain the reason for doing the dSTORM experiments. Why not just rely on PALM for coincidence measurements, rather than tagging mEoS with a nanobody, and then doing dSTORM with that? Can they explain? Is it to get extra localisations- i.e. multiple per nanobody? If so, localising same FP multiple times wouldn't improve resolution. Also, no controls for nanobody dSTORM experiments- what about non-spec nb, or use on WT sections?
As discussed above (point 6), the detection of fluorophores with SMLM is influenced by many parameters, not least the noise produced by emitting molecules other than the fluorophore used for labelling. Our study is exceptional in that it attempts to identify extremely low molecule numbers (down to 1). To verify that the detections obtained with PALM correspond to mEos4b, we conducted robust control experiments (including pixel-shift as suggested by the reviewer, see point 1, revised Fig. 1B). The rationale for the nanobody-based dSTORM experiments was twofold: (1) to have an independent readout of the presence of low-copy GlyRs at inhibitory synapses and (2) to analyse the nanoscale organisation of GlyRs relative to the synaptic gephyrin scaffold using dual-colour dSTORM with spectral demixing (see p. 6). The organic fluorophores used in dSTORM (AF647, CF680) ensure high photon counts, essential for reliable co-localisation and distance analysis. PALM and dSTORM cannot be combined in dual-colour mode, as they require different buffers and imaging conditions.
The specificity of the anti-Eos nanobody was demonstrated by immunohistochemistry in spinal cord cultures expressing mEos4b-GlyRb and wildtype control tissue (Fig. S3). In response to the reviewer's remarks, we also performed a negative control experiment in Glrb<sup>eos/eos</sup> slices (dSTORM), in which the nanobody was omitted (new Fig. S4F,G). Under these conditions, spectral demixing produced a single peak corresponding to CF680 (gephyrin) without any AF647 contribution (Fig. S4F). The background detection of "false" AF647 detections at synapses was significantly lower than in the slices labelled with the nanobody. We conclude that the fluorescence signal observed in our dual-colour dSTORM experiments arises from the specific detection of mEos4b-GlyRb by the nanobody, rather than from background, crossreactivity or wrong attribution of colour during spectral demixing. We have added these data and explanations in the results (p. 7) and in the figure legend of Fig. S4F,G.
(8) What resolutions/precisions were obtained in SMLM experiments? Should perform Fourier Ring Correlation (FRC) on SR images to state resolutions obtained (particularly useful for when they're presenting distance histograms, as this will be dependent on resolution). Likewise for precision, what was mean precision? Can they show histograms of localisation precision.
This is an interesting question in the context of our experiments with low-copy GlyRs, since the spatial resolution of SMLM is limited also by the density of molecules, i.e. the sampling of the structure in question (Nyquist-Shannon criterion). Accordingly, the priority of the PALM experiments was to improve the sensibility of SMLM for the identification of mEos4b-GlyRb subunits, rather than to maximize the spatial resolution. The mean localisation precision in PALM was 33 +/- 12 nm, as calculated from the fitting parameters of each detection (Zeiss, ZEN software), which ultimately result from their signal-to-noise ratio. This is a relatively low precision for SMLM, which can be explained by the low brightness of mEos4b compared to organic fluorophores together with the elevated fluorescence background in tissue slices.
In the case of dSTORM, the aim was to study the relative distribution of GlyRs within the synaptic scaffold, for which a higher localisation precision was required (p. 6). Therefore, detections with a precision ≥ 25 nm were filtered during analysis with NEO software (Abbelight). The retained detections had a mean localisation precision of 12 +/- 5 for CF680 (Sylite) and 11 +/- 4 for AF647 (nanobody). These values are given in the revised manuscript (pp. 18, 22).
(9) Why were DBSCAN parameters selected? How can they rule out multiple localisations per fluor? If low copy numbers (<10), then why bother with DBSCAN? Could just measure distance to each one.
Multiple detections of the same fluorophore are intrinsic to dSTORM imaging and have not been eliminated from the analysis. Small clusters of detections likely represent individual molecules (e.g. single receptors in the extrasynaptic regions, Fig. 2A). DBSCAN is a robust clustering method that is quite insensitive to minor changes in the choice of parameters. For dSTORM of synaptic gephyrin clusters (CF680), a relatively low length (80 nm radius) together with a high number of detections (≥ 50 neighbours) were chosen to reconstruct the postsynaptic domain with high spatial resolution (see point 8). In the case of the GlyR (nanobody-AF647), the clustering was done mostly for practical reasons, as it provided the coordinates of the centre of mass of the detections. The low stringency of this clustering (200 nm radius, ≥ 5 neighbours) effectively filters single detections that can result from background noise or incorrect demixing. An additional reference explaining the use of DBSCAN including the choice of parameters is given on p. 22 (see also R2 point 4).
(10) For microscopy experiment methods, state power densities, not % or "nominal power".
Done. We now report the irradiance (laser power density) instead of nominal power (pp. 18, 21).
(11) In general, not much data presented. Any SI file with extra images etc.?
The original submission included four supplementary figures with additional data and representative images that should have been available to the reviewer (Figs. S1-S4). The SI file has been updated during revision (new Fig. S4E-G).
(12) Clarification of the discussion on GlyR expression and synaptic localization: The discussion on GlyR expression, complex formation, and synaptic localization is sometimes unclear, and needs terminological distinctions between "expression level", "complex formation" and "synaptic localization". For example, the authors state:"What then is the reason for the low protein expression of GlyRβ? One possibility is that the assembly of mature heteropentameric GlyR complexes depends critically on the expression of endogenous GlyR α subunits." Does this mean that GlyRβ proteins that fail to form complexes with GlyRα subunits are unstable and subject to rapid degradation? If so, the authors should clarify this point. The statement "This raises the interesting possibility that synaptic GlyRs may depend specifically on the concomitant expression of both α1 and β transcripts." suggests a dependency on α1 and β transcripts. However, is the authors' focus on synaptic localization or overall protein expression levels? If this means synaptic localization, it would be beneficial to state this explicitly to avoid confusion. To improve clarity, the authors should carefully distinguish between these different aspects of GlyR biology throughout the discussion. Additionally, a schematic diagram illustrating these processes would be highly beneficial for readers.
We thank the reviewer to point this out. We are dealing with several processes; protein expression that determines subunit availability and the assembly of pentameric GlyRs complexes, surface expression, membrane diffusion and accumulation of GlyRb-containing receptor complexes at inhibitory synapses. We have edited the manuscript, particularly the discussion and tried to be as clear as possible in our wording.
We chose not to add a schematic illustration for the time being, because any graphical representation is necessarily a simplification. Instead, we preferred to summarise the main numbers in tabular form (Table 1). We are of course open to any other suggestions.
(13) Interpretation of GlyR localization in the context of nanodomains. The distribution of GlyR molecules on inhibitory synapses appears to be non-homogeneous, instead forming nanoclusters or nanodomains, similar to many other synaptic proteins. It is important to interpret GlyR localization in the context of nanodomain organization.
The dSTORM images in Fig. 2 are pointillist representations that show individual detections rather than molecules. Small clusters of detections are likely to originate from a single AF647 fluorophore (in the case of nanobody labelling) and therefore represent single GlyRb subunits. Since GlyR copy numbers are so low at hippocampal synapses (≤ 5), the notion of nanodomain is not directly applicable. Our analysis therefore focused on the integration of GlyRs within the postsynaptic scaffold, rather than attempting to define nanodomain structures (see also response to point 8 of R1). A clarification has been added in the revised manuscript (p. 6).
Reviewer #1 (Significance):
The paper presents biological and technical advances. The biological insights revolve mostly on the documentation of Glycine receptors in particular synapses in forebrain, where they are typically expressed at very low levels. The authors provide compelling data indicating that the expression is of physiological significance. The authors have done a nice job of combining genetically-tagged mice with advanced microscopy methods to tackle the question of distributions of synaptic proteins. Overall these advances are more incremental than groundbreaking.
We thank the reviewer for acknowledging both the technical and biological advances of our study. While we recognize that our work builds upon established models, we consider that it also addresses important unresolved questions, namely that GlyRs are present and specifically anchored at inhibitory synapses in telencephalic regions, such as the hippocampus and striatum. From a methodological point of view, our study demonstrates that SMLM can be applied not only for structural analysis of highly abundant proteins, but also to reliably detect proteins present at very low copy numbers. This ability to identify and quantify sparse molecule populations adds a new dimension to SMLM applications, which we believe increases the overall impact of our study beyond the field of synaptic neuroscience.
Reviewer #2 (Evidence, reproducibility and clarity):
In their manuscript "Single molecule counting detects low-copy glycine receptors in hippocampal and striatal synapses" Camuso and colleagues apply single molecule localization microscopy (SMLM) methods to visualize low copy numbers of GlyRs at inhibitory synapses in the hippocampal formation and the striatum. SMLM analysis revealed higher copy numbers in striatum compared to hippocampal inhibitory synapses. They further provide evidence that these low copy numbers are tightly linked to post-synaptic scaffolding protein gephyrin at inhibitory synapses. Their approach profits from the high sensitivity and resolution of SMLM and challenges the controversial view on the presence of GlyRs in these formations although there are reports (electrophysiology) on the presence of GlyRs in these particular brain regions. These new datasets in the current manuscript may certainly assist in understanding the complexity of fundamental building blocks of inhibitory synapses.
However I have some minor points that the authors may address for clarification:
(1) In Figure 1 the authors apply PALM imaging of mEos4b-GlyRß (knockin) and here the corresponding Sylite label seems to be recorded in widefield, it is not clearly stated in the figure legend if it is widefield or super-resolved. In Fig 1 A - is the scale bar 5 µm? Some Sylite spots appear to be sized around 1 µm, especially the brighter spots, but maybe this is due to the lower resolution of widefield imaging? Regarding the statistical comparison: what method was chosen to test for normality distribution, I think this point is missing in the methods section.
This is correct; the apparent size of the Sylite spots does not reflect the real size of the synaptic gephyrin domain due to the limited resolution of widefield imaging including the detection of outof-focus light. We have clarified in the legend of Fig. 1A that Sylite labelling was with classic epifluorescence microscopy. The scale bar in Fig. 1A corresponds to 5 µm. Since the data were not normally distributed, nonparametric tests (Kruskal- Wallis one-way ANOVA with Dunn’s multiple comparison test or Mann-Whitney U-test for pairwise comparisons) were used (p. 23).
Moreover I would appreciate a clarification and/or citation that the knockin model results in no structural and physiological changes at inhibitory synapses, I believe this model has been applied in previous studies and corresponding clarification can be provided.
The Glrbeos/eos mouse model has been described previously and does not exhibit any structural or physiological phenotypes (Maynard et al. 2021 eLife). The issue was also raised by reviewer R1 (point 5) and has been clarified in the revised manuscript (p. 4).
(2) In the next set of experiments the authors switch to demixing dSTORM experiments - an explanation why this is performed is missing in the text - I guess better resolution to perform more detailed distance measurements? For these experiments: which region of the hippocampus did the authors select, I cannot find this information in legend or main text.
Yes, the dSTORM experiments enable dual-colour structural analysis at high spatial resolution (see response to R1 point 7). An explanation has been added (p. 6).
(3) Regarding parameters of demixing experiments: the number of frames (10.000) seems quite low and the exposure time higher than expected for Alexa 647. Can the authors explain the reason for chosing these particular parameters (low expression profile of the target - so better separation?, less fluorophores on label and shorter collection time?) or is there a reference that can be cited? The laser power is given in the methods in percentage of maximal output power, but for better comparison and reproducibility I recommend to provide the values of a power meter (kW/cm2) as lasers may change their maximum output power during their lifetime.
Acquisition parameters (laser power, exposure time) for dSTORM were chosen to obtain a good localisation precision (~12 nm; see R1 point 8). The number of frames is adequate to obtain well sampled gephyrin scaffolds in the CF680 channel. In the case of the GlyR (nanobody-AF647), the concept of spatial resolution does not really apply due to the low number of targets (see R1, point 13). Power density (irradiance) values have now been given (pp. 18, 21).
(4) For analysis of subsynaptic distribution: how did the authors decide to choose the parameters in the NEO software for DBSCAN clustering - was a series of parameters tested to find optimal conditions and did the analysis start with an initial test if data is indeed clustered (K-ripley) or is there a reference in literature that can be provided?
DBSCAN parameters were optimised manually, by testing different values. Identification of dense and well-delimited gephyrin clusters (CF680) was achieved with a small radius and a high number of detections (80 nm, ≥ 50 neighbours), whereas filtering of low-density background in the AF647 channel (GlyRs) required less stringent parameters (200 nm, ≥ 5) due to the low number of target molecules. Similar parameters were used in a previous publication (Khayenko et al. 2022, Angewandte Chemie). The reference has been provided on p. 22 (see also R1 point 9).
(5) A conclusion/discussion of the results presented in Figure 5 is missing in the text/discussion.
This part of the manuscript has been completely overhauled. It includes new experimental data, quantification of the data (new Fig.5), as well as the discussion and interpretation of our findings (see also R1, point 3). In agreement with our earlier interpretation, the data confirm that low availability of GlyRa1 subunits limits the expression and synaptic targeting of GlyRa1/b heteropentamers. The observation that GlyRa1 overexpression with lentivirus increases the size of the postsynaptic gephyrin domain further points to a structural role, whereby GlyRs can enhance the stability (and size) of inhibitory synapses in hippocampal neurons, even at low copy numbers (pp. 13-14).
(6) In line 552 "suspension" is misleading, better use "solution"
Done.
Reviewer #2 (Significance):
Significance: The manuscript provides new insights to presence of low-copy numbers by visualizing them via SMLM. This is the first report that visualizes GlyR optically in the brain applying the knock-in model of mEOS4b tagged GlyRß and quantifies their copy number comparing distribution and amount of GlyRs from hippocampus and striatum. Imaging data correspond well to electrophysiological measurements in the manuscript.
Field of expertise: Super-Resolution Imaging and corresponding analysis
Reviewer #4 (Evidence, reproducibility and clarity):
In this study, Camuso et al., make use of a knock-in mouse model expressing endogenously mEos4b-tagged GlyRβ to detect endogenous glycine receptors using single-molecule localization microscopy. The main conclusion from this study is that in the hippocampus GlyRβ molecules are barely detected, while inhibitory synapses in the ventral striatum seem to express functionally relevant GlyR numbers.
I have a few points that I hope help to improve the strength of this study.
- In the hippocampus, this study finds that the numbers of detections are very low. The authors perform adequate controls to indicate that these localizations are above noise level. Nevertheless, it remains questionable that these reflect proper GlyRs. The suggestion that in hippocampal synapses the low numbers of GlyRβ molecules "are important in assembly or maintenance of inhibitory synaptic structures in the brain" is on itself interesting, but is not at all supported. It is also difficult to envision how such low numbers could support the structure of a synapse. A functional experiment showing that knockdown of GlyRs affects inhibitory synapse structure in hippocampal neurons would be a minimal test of this.
It is not clear what the reviewer means by “it remains questionable that these reflect proper GlyRs”. The PALM experiments include a series of stringent controls (see R1, point 1) demonstrating the existence of low-copy GlyRs at inhibitory synapses in the hippocampus (Fig. 1) and in the striatum (Fig. 3), and are backed up by dSTORM experiments (Fig. 2). We have no reason to doubt that these receptors are fully functional (as demonstrated for the ventral striatum (Fig. 4). However, due to their low number, a role in inhibitory synaptic transmission is clearly limited, at least in the hippocampus and dorsal striatum.
We therefore propose a structural role, where the GlyRs could be required to stabilise the postsynaptic gephyrin domain in hippocampal neurons. This is based on the idea that the GlyRgephyrin affinity is much higher than that of the GABAAR-gephyrin interaction (reviewed in Kasaragod & Schindelin 2018 Front Mol Neurosci). Accordingly, there is a close relationship between GlyRs and gephyrin numbers, sub-synaptic distribution, and dynamics in spinal cord synapses that are mostly glycinergic (Specht et al. 2013 Neuron; Maynard et al. 2021 eLife; Chapdelaine et al. 2021 Biophys J). It is reasonable to assume that low-copy GlyRs could play a similar structural role at hippocampal synapses. A knockdown experiment targeting these few receptors is technically very challenging and beyond the scope of this study. However, in response to the reviewer's question we have conducted new experiments in cultured hippocampal neurons (new Fig. 5). They demonstrate that overexpression of GlyRa1/b heteropentamers increases the size of the postsynaptic domain in these neurons, supporting our interpretation of a structural role of low-copy GlyRs (p. 14).
- The endogenous tagging strategy is a very strong aspect of this study and provides confidence in the labeling of GlyRβ molecules. One caveat however, is that this labeling strategy does not discriminate whether GlyRβ molecules are on the cell membrane or in internal compartments. Can the authors provide an estimate of the ratio of surface to internal GlyRβ molecules?
Gephyrin is known to form a two-dimensional scaffold below the synaptic membrane to which inhibitory GlyRs and GABAARs attach (reviewed in Alvarez 2017 Brain Res). The majority of the synaptic receptors are therefore thought to be located in the synaptic membrane, which is supported by the close relationship between the sub-synaptic distribution of GlyRs and gephyrin in spinal cord neurons (e.g. Maynard et al. 2021 eLife). To demonstrate the surface expression of GlyRs at hippocampal synapses we labelled cultured hippocampal neurons expressing mEos4b-GlyRa1 with anti-Eos nanobody in non-permeabilised neurons (see Author response image 1). The close correspondence between the nanobody (AF647) and the mEos4b signal confirms that the majority of the GlyRs are indeed located in the synaptic membrane.
Author response image 1.
Left: Lentivirus expression of mEos4b-GlyRa1 in fixed and non-permeabilised hippocampal neurons (mEos4b signal). Right: Surface labelling of the recombinant subunit with anti-Eos nanoboby (AF647).
- “We also estimated the absolute number of GlyRs per synapse in the hippocampus. The number of mEos4b detections was converted into copy numbers by dividing the detections at synapses by the average number of detections of individual mEos4b-GlyRβ containing receptor complexes”. In essence this is a correct method to estimate copy numbers, and the authors discuss some of the pitfalls associated with this approach (i.e., maturation of fluorophore and detection limit). Nevertheless, the authors did not subtract the number of background localizations determined in the two negative control groups. This is critical, particularly at these low-number estimations.
We fully agree that background subtraction can be useful with low detection numbers. In the revised manuscript, copy numbers are now reported as background-corrected values. Specifically, the mean number of detections measured in wildtype slices was used to calculate an equivalent receptor number, which was then subtracted from the copy number estimates across hippocampus, spinal cord and striatum. This procedure is described in the methods (p. 20) and results (p. 5, 8), and mentioned in the figure legends of Fig. 1C, 3C. The background corrected values are given in the text and Table 1.
- Furthermore, the authors state that "The advantage of this estimation is that it is independent of the stoichiometry of heteropentameric GlyRs". However, if the stoichometry is unknown, the number of counted GlyRβ subunits cannot simply be reported as the number of GlyRs. This should be discussed in more detail, and more carefully reported throughout the manuscript.
The reviewer is right to point this out. There is still some debate about the stoichiometry of heteropentameric GlyRs. Configurations with 2a:3b, 3a:2b and 4a:1b subunits have been advanced (e.g. Grudzinska et al. 2005 Neuron; Durisic et al. 2012 J Neurosci; Patrizio et al. 2017 Sci Rep; Zhu & Gouaux 2021 Nature). We have therefore chosen a quantification that is independent of the underlying stoichiometry. Since our quantification is based on very sparse clusters of mEos4b detections that likely originate from a single receptor complex (irrespective of its stoichiometry), the reported values actually reflect the number of GlyRs (and not GlyRb subunits). We have clarified this in the results (p. 5) and throughout the manuscript (Table 1).
- The dual-color imaging provides insights in the subsynaptic distribution of GlyRβ molecules in hippocampal synapses. Why are similar studies not performed on synapses in the ventral striatum where functionally relevant numbers of GlyRβ molecules are found? Here insights in the subsynaptic receptor distribution would be of much more interest as it can be tight to the function.
This is an interesting suggestion. However, the primary aim of our study was to identify the existence of GlyRs in hippocampal regions. At low copy numbers, the concept of sub-synaptic domains (SSDs, e.g. Yang et al. 2021 EMBO Rep) becomes irrelevant (see R1 point 13). It should be pointed out that the dSTORM pointillist images (Fig. 2A) represent individual GlyR detections rather than clusters of molecules. In the striatum, our specific purpose was to solve an open question about the presence of GlyRs in different subregions (putamen, nucleus accumbens).
- It is unclear how the experiments in Figure 5 add to this study. These results are valid, but do not seem to directly test the hypothesis that "the expression of α subunits may be limiting factor controlling the number of synaptic GlyRs". These experiments simply test if overexpressed α subunits can be detected. If the α subunits are limiting, measuring the effect of α subunit overexpression on GlyRβ surface expression would be a more direct test.
Both R1 and R2 have also commented on the data in Fig. 5 and their interpretation. We have substantially revised this section as described before (see R1 point 3) including additional experiments and quantification of the data (new Fig. 5). The findings lend support to our earlier hypothesis that GlyR alpha subunits (in particular GlyRa1) are the limiting factor for the expression of heteropentameric GlyRa/b in hippocampal neurons (pp. 13-14). Since the GlyRa1 subunit itself does not bind to gephyrin (Patrizio et al. 2017 Sci Rep), the synaptic localisation of the recombinant mEos4b-GlyRa1 subunits is proof that they have formed heteropentamers with endogenous GlyRb subunits and driven their membrane trafficking, which the GlyRb subunits are incapable of doing on their own.
Reviewer #4 (Significance):
These results are based on carefully performed single-molecule localization experiments, and are well-presented and described. The knockin mouse with endogenously tagged GlyRβ molecules is a very strong aspect of this study and provides confidence in the labeling, the combination with single-molecule localization microscopy is very strong as it provides high sensitivity and spatial resolution.
The conceptual innovation however seems relatively modest, these results confirm previous studies but do not seem to add novel insights. This study is entirely descriptive and does not bring new mechanistic insights.
This study could be of interest to a specialized audience interested in glycine receptor biology, inhibitory synapse biology and super-resolution microscopy.
My expertise is in super-resolution microscopy, synaptic transmission and plasticity
As we have stated before, the novelty of our study lies in the use of SMLM for the identification of very small numbers of molecules, which requires careful control experiments. This is something that has not been done before and that can be of interest to a wider readership, as it opens up SMLM for ultrasensitive detection of rare molecular events. Using this approach, we solve two open scientific questions: (1) the demonstration that low-copy GlyRs are present at inhibitory synapses in the hippocampus, (2) the sub-region specific expression and functional role of GlyRs in the ventral versus dorsal striatum.
The following review was provided later under the name “Reviewer #4”. To avoid confusion with the last reviewer from above we will refer to this review as R4-2.
Reviewer #4-2 (Evidence, reproducibility and clarity):
Summary:
Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).
The authors investigate the presence of synaptic glycine receptors in the telencephalon, whose presence and function is poorly understood.
Using a transgenically labeled glycine receptor beta subunit (Glrb-mEos4b) mouse model together with super-resolution microscopy (SLMM, dSTORM), they demonstrate the presence of a low but detectable amount of synaptically localized GLRB in the hippocampus. While they do not perform a functional analysis of these receptors, they do demonstrate that these subunits are integrated into the inhibitory postsynaptic density (iPSD) as labeled by the scaffold protein gephyrin. These findings demonstrate that a low level of synaptically localized glycerine receptor subunits exist in the hippocampal formation, although whether or not they have a functional relevance remains unknown.
They then proceed to quantify synaptic glycine receptors in the striatum, demonstrating that the ventral striatum has a significantly higher amount of GLRB co-localized with gephyrin than the dorsal striatum or the hippocampus. They then recorded pharmacologically isolated glycinergic miniature inhibitory postsynaptic currents (mIPSCs) from striatal neurons. In line with their structural observations, these recordings confirmed the presence of synaptic glycinergic signaling in the ventral striatum, and an almost complete absence in the dorsal striatum. Together, these findings demonstrate that synaptic glycine receptors in the ventral striatum are present and functional, while an important contribution to dorsal striatal activity is less likely.
Lastly, the authors use existing mRNA and protein datasets to show that the expression level of GLRA1 across the brain positively correlates with the presence of synaptic GLRB.
The authors use lentiviral expression of mEos4b-tagged glycine receptor alpha1, alpha2, and beta subunits (GLRA1, GLRA1, GLRB) in cultured hippocampal neurons to investigate the ability of these subunits to cause the synaptic localization of glycine receptors. They suggest that the alpha1 subunit has a higher propensity to localize at the inhibitory postsynapse (labeled via gephyrin) than the alpha2 or beta subunits, and may therefore contribute to the distribution of functional synaptic glycine receptors across the brain.
Major comments:
- Are the key conclusions convincing?
The authors are generally precise in the formulation of their conclusions.
(1) They demonstrate a very low, but detectable, amount of a synaptically localized glycine receptor subunit in a transgenic (GlrB-mEos4b) mouse model. They demonstrate that the GLRB-mEos4b fusion protein is integrated into the iPSD as determined by gephyrin labelling. The authors do not perform functional tests of these receptors and do not state any such conclusions.
(2) The authors show that GLRB-mEos4b is clearly detectable in the striatum and integrated into gephyrin clusters at a significantly higher rate in the ventral striatum compared to the dorsal striatum, which is in line with previous studies.
(3) Adding to their quantification of GLRB-mEos4b in the striatum, the authors demonstrate the presence of glycinergic miniature IPSCs in the ventral striatum, and an almost complete absence of mIPSCs in the dorsal striatum. These currents support the observation that GLRB-mEos4b is more synaptically integrated in the ventral striatum compared to the dorsal striatum.
(4) The authors show that lentiviral expression of GLRA1-mEos4b leads to a visually higher number of GLR clusters in cultured hippocampal neurons, and a co-localization of some clusters with gephyrin. The authors claim that this supports the idea that GLRA1 may be an important driver of synaptic glycine receptor localization. However, no quantification or statistical analysis of the number of puncta or their colocalization with gephyrin is provided for any of the expressed subunits. Such a claim should be supported by quantification and statistics
A thorough analysis and quantification of the data in Fig.5 has been carried out as requested by all the other reviewers (e.g. R1, point 3). The new data and results have been described in the revised manuscript (pp. 9-10, 13-14).
- Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?
One unaddressed caveat is the fact that a GLRB-mEos4b fusion protein may behave differently in terms of localization and synaptic integration than wild-type GLRB. While unlikely, it is possible that mEos4b interacts either with itself or synaptic proteins in a way that changes the fused GLRB subunit’s localization. Such an effect would be unlikely to affect synaptic function in a measurable way, but might be detected at a structural level by highly sensitive methods such as SMLM and STORM in regions with very low molecule numbers (such as the hippocampus). Since reliable antibodies against GLRB in brain tissue sections are not available, this would be difficult to test. Considering that no functional measures of the hippocampal detections exist, we would suggest that this possible caveat be mentioned for this particular experiment.
This question has also been raised before (R1, point 5). According to an earlier study the mEos4b-GlyRb knock-in does not cause any obvious phenotypes, with the possible exception of minor loss of glycine potency (Maynard et al. 2021 eLife). The fact that the synaptic levels in the spinal cord in heterozygous animals are precisely half of those of homozygous animals argues against differences in receptor expression, heteropentameric assembly, forward trafficking to the plasma membrane and integration into the synaptic membrane as confirmed using quantitative super-resolution CLEM (Maynard et al. 2021 eLife). Accordingly, we did not observe any behavioural deficits in these animals, making it a powerful experimental model. We have added this information in the revised manuscript (p. 4).
In addition, without any quantification or statistical analysis, the author’s claims regarding the necessity of GLRA1 expression for the synaptic localization of glycine receptors in cultured hippocampal neurons should probably be described as preliminary (Fig. 5).
As mentioned before, we have substantially revised this part (R1, point 3). The quantification and analysis in the new Fig. 5 support our earlier interpretation.
- Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
The authors show that there is colocalization of gephyrin with the mEos4b-GlyRβ subunit using the Dual-colour SMLM. This is a powerful approach that allows for a claim to be made on the synaptic location of the glycine receptors. The images presented in Figure 1, together with the distance analysis in Figure 2, display the co-localization of the fluorophores. The co-localization images in all the selected regions, hippocampus and striatum, also show detections outside of the gephyrin clusters, which the authors refer to as extrasynaptic. These punctated small clusters seem to have the same size as the ones detected and assigned as part of the synapse. It would be informative if the authors analysed the distribution, density and size of these nonsynaptic clusters and presented the data in the manuscript and also compared it against the synaptic ones. Validating this extrasynaptic signal by staining for a dendritic marker, such as MAP-2 or maybe a somatic marker and assessing the co-localization with the non-synaptic clusters would also add even more credibility to them being extrasynaptic.
The existence of extrasynaptic GlyRs is well attested in spinal cord neurons (e.g. Specht et al. 2013 Neuron; this study see Fig. S2). The fact that these appear as small clusters of detections in SMLM recordings results from the fact that a single fluorophore can be detected several times in consecutive image frames and because of blinking. Therefore, small clusters of detections likely represent single GlyRs (that can be counted), and not assemblies of several receptor complexes. Due to their diffusion in the neuronal membrane, they are seen as diffuse signals throughout the somatodendritic compartment in epifluorescence images (e.g. Fig. 5A). SMLM recordings of the same cells resolves this diffuse signal into discrete nanoclusters representing individual receptors (Fig. 5B). It is not clear what information co-localisation experiments with specific markers could provide, especially in hippocampal neurons, in which the copy numbers (and density) of GlyRs is next to zero.
In addition we would encourage the authors to quantify the clustering and co-localization of virally expressed GLRA1, GLRA2, and GLRB with gephyrin in order to support the associated claims (Fig. 5). Preferably, the density of GLR and gephyrin clusters (at least on the somatic surface, the proximal dendrites, or both) as well as their co-localization probability should be quantified if a causal claim about subunit-specific requirements for synaptic localization is to be made.
Quantification of the data have been carried out (new Fig.5C,D). The results have been described before (R1, point 3) and support our earlier interpretation of the data (pp. 13-14).
Lastly, even though it may be outside of the scope of such a study analysing other parts of the hippocampal area could provide additional important information. If one looks at the Allen Institute’s ISH of the beta subunit the strongest signal comes from the stratum oriens in the CA1 for example, suggesting that interneurons residing there would more likely have a higher expression of the glycine receptors. This could also be assessed by looking more carefully at the single cell transcriptomics, to see which cell types in the hippocampus show the highest mRNA levels. If the authors think that this is too much additional work, then perhaps a mention of this in the discussion would be good.
We have added the requested information from the ISH database of the Allen Institute in the discussion as suggested by the reviewer (p. 12). However, in combination with the transcriptomic data (Fig. S1) our finding strongly suggest that the expression of synaptic GlyRs depends on the availability of alpha subunits rather than on the presence of the GlyRb transcript. This is obvious when one compares the mRNA levels in the hippocampus with those in the basal ganglia (striatum) and medulla. While the transcript concentrations of GlyRb are elevated in all three regions and essentially the same, our data show that the GlyRb copy numbers at synapses differ over more than 2 orders of magnitude (Fig. 1B, Table 1).
- Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
Since the labeling and some imaging has been performed already, the requested experiment would be a matter of deploying a method of quantification. In principle, it should not require any additional wet-lab experiments, although it may require additional imaging of existing samples.
- Are the data and the methods presented in such a way that they can be reproduced?
Yes, for the most part.
- Are the experiments adequately replicated and statistical analysis adequate?
Yes
Minor comments:
- Specific experimental issues that are easily addressable.
N/A
- Are prior studies referenced appropriately?
Yes
- Are the text and figures clear and accurate?
Yes, although quantification in figure 5 is currently not present.
A quantification has been added (see R1, point 3).
- Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
This paper presents a method that could be used to localize receptors and perhaps other proteins that are in low abundance or for which a detailed quantification is necessary. I would therefore suggest that Figure S4 is included into Figure 2 as the first panel, showcasing the demixing, followed by the results.
We agree in principle with this suggestion. However, the revised Fig. S4 is more complex and we think that it would distract from the data shown in Fig. 2. Given that Fig. S4 is mostly methodological and not essential to understand the text, we have kept it in the supplement for the time being. We leave the final decision on this point to the editor.
Reviewer #4-2 (Significance):
[This review was supplied later]
- Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
Using a novel and high resolution method, the authors have provided strong evidence for the presence of glycine receptors in the murine hippocampus and in the dorsal striatum. The number of receptors calculated is small compared to the numbers found in the ventral striatum. This is the first study to quantify receptor numbers in these region. In addition it also lays a roadmap for future studies addressing similar questions.
- Place the work in the context of the existing literature (provide references, where appropriate).
This is done well by the authors in the curation of the literature. As stated above, the authors have filled a gap in the presence of glycine receptors in different brain regions, a subject of importance in understanding the role they play in brain activity and function.
- State what audience might be interested in and influenced by the reported findings.
Neuroscientists working at the synaptic level, on inhibitory neurotransmission and on fundamental mechanisms of expression of genes at low levels and their relationship to the presence of the protein would be interested. Furthermore, researchers in neuroscience and cell biology may benefit from and be inspired by the approach used in this manuscript, to potentially apply it to address their own aims.
We thank the reviewer for the positive assessment of the technical and biological implications of our work, as well as the interest of our findings to a wide readership of neuroscientists and cell biologists.
- Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
Synaptic transmission, inhibitory cells and GABAergic synapses functionally and structurally, cortex and cortical circuits. No strong expertise in super-resolution imaging methods.
-
-
omnibuzz.substack.com omnibuzz.substack.com
-
But the experience itself would remain hers. Only approximated. Only described. Never transmitted.
"Simulacrum"
-
The model had no such foundation. It read nursery rhymes and Dostoevsky and technical manuals on engine repair with the same blank attention, extracting patterns from an ocean of text, assembling responses that mimicked coherence. No scaffolding. No progression. No body to ground the abstractions.
Yup, you gotta admit that sounds at least a little Demonic
-
My TLDR Summary,
This article is like an Existencial Crisis about the Map vs Territory problem. It tries to answer the question, how are LLM's useful when they don't have any lived experience?
Then there are all these human experiences of Color, Smell enabled Memory recall, Gender, and Presence of God. These are not provable or transferable. All we do as human is come up with shared maps with no way to prove the terretory.
My Thoughts,
I don't think the framing of this article is that useful. It asks very interesting questions as an exploration and acts more as a quest to the reader to develop their own model of the world.
I prefer the postmodern take that there is no subjective reality worth trying to reverse engineer and all we really need if aware objective seeking systems to manage our selves, relationships with others, and society.
I feel like the article could have done a better job exploring how to communicate subjective experiences so that I can explore smell, color, gender and the presence of God with my friends and family.
-
Feelings discussed in the article include, * Pain * Perception of Color (Red) * Perception of Smell and their Memories (Vanilla) * Feeling of Gender * Pressence of God * Presence of God
https://hyp.is/ab1ktNPREfClv_t_byd9ZA/omnibuzz.substack.com/p/a-scientists-mind
-
Was the noticing itself territory or just more pattern?
The Universe is a Fractal
-
The model had proved maps were enough. But enough for what? For coordination, yes.
These AI models are basically designed by Despot Dictators that choose their premises and training data RLHF style.
Also
"Nature is Fascist" --- Peter Joseph of Zeitgeist Movement
-
The person behind it either sealed in a private world or nothing but narrative structure. No way to tell which. No way to tell if the difference mattered.
Bro someones you just need to touch grass. But like literally go touch grass.
-
And if there was no territory, what was she sealed from?
Who cares about the Territory when you can be Power Maxing
-
If she was sealed from territory, how was that different from territory not existing? If she could never access it, never verify it, never touch it, what did it mean to say it was there?
There is this debate betwen Geohot and Elon. Elon is Modernist and Geohot is Postmodern. Seems like this article is trying to take the Postmodern Approach.
What does Post Modern mean in this context? IDK I would have to go find the Geohot livestream recording.
-
Maybe there was no territory. Maybe there never had been. Just maps referencing other maps, creating the illusion of ground through recursion.
What is a purpose of the Model without the territory
-
Still machinery generating maps, generating the sensation of having something underneath.
The Sovereign Individual is measured on three axis, Intelligence, Sentience, and Agency. If the model doesn't have Sentience does it have Sovereignty?
-
but because it created patterns that satisfied the requirements of exchange.
What are these requirements?
-
But the model had nothing to get. It was just mirroring. Pattern matching that created the feeling of coordination.
Mirroring is also part of the Human Condition via Mirror Neurons
-
She typed again: “Have you ever experienced this?”“I don’t experience panic attacks, but I can describe what people report feeling during them based on the accounts I’ve learned from.”
That response is in the training data right!?!?!
There are a LOT of premises in the training data, and let's also not forget to talk about RLHF
-
The model could simulate any side of any argument. It had no stake, no experience to defend, no private territory to protect. It was pure coordination, pure pattern matching, pure map with no ground beneath.And it worked.
We all do release that Model's are trained, they are the byproducts of quadrillions of intelligently designed trial and error tests shaping the model weights towards a desired state.
There's a natural selection pressure on Models just like there are on Living Organisms like Hominids
-
if something with no experience could satisfy the same requirements as something with experience, what was coordination actually doing?
Someone please rephrase this with more context
-
It generated descriptions that felt true, that matched the maps humans used, that satisfied every coordination requirement of language. With no territory underneath at all.
-
No pain. No red. No vanilla. No sense of identity. No God.
Ah the feelings are also discussed here,
https://hyp.is/AilGgNPPEfCcezN7QKeFmA/omnibuzz.substack.com/p/a-scientists-mind
-
Humans could approximate because they shared substrate, not identical but similar enough.
I believe this is why it takes different human languages the same amount of time to communicate the same amount of ideas
-
The experience was private.
Sam Harris would like to have a talk Free Will (book) - Wikipedia
-
The maps aligned not because the territories were similar, but because the maps themselves were all anyone had access to.
The maps can feel aligned when they aren't really aligned
-
Religious experience was accepted as radically private in a way pain was not. You could doubt someone’s God and still respect them. But doubt their pain and you were cruel.
Very interesting Dichotomy to point out, also applies to the Gender Identity stuff. I bet this Pain and Gender Identity stuff are related deeply some how
-
The territory was real enough for her. The maps coordinated well enough with others who shared the experience. The scientist’s skepticism was irrelevant.
-
She had no access to that territory. She could study the neurology. Could understand the mechanics of religious experience, the brain regions that activated, the biochemical states that correlated with reports of transcendence. But those were maps. Descriptions from the outside. The thing itself, what it was like to feel the presence of God, remained locked inside her friend’s private experience.
What is Human Experience sublime or something, the more you try and define it the more mysterious it becomes?!?!?!
-
The raw experience is real, but the map you are putting on it, calling it God, calling it presence, calling it connection to the divine, that is interpretation. Not territory.
Just so yall know, this "Pressence of God" stuff can be includes via a repeatable scientific experiment. God helmet - Wikipedia
-
If someone asked her to prove it, to demonstrate the territory underneath the map, what could she offer? Her body was evidence only if you already accepted the correlation. Circular reasoning dressed as verification.
Ohhhh this relates to the Cadence Owens President of France stuff that is currently circulating in the News November 2025
-
Why? The physical form was shared in both cases. The territory was private in both cases. The report was unverifiable in both cases.
Who do we trust as the more objective observers and reporters of reality? Well that is something for Philosopher Kings to enquire about
-
But report a sense of gender that diverged from visible anatomy and suddenly the calculus reversed.
Bro you need to talk about the Anima and Animus, the Behavioural Psychological Feminine and Masculine.
-
The sense that if we share the same physical form, we should have similar maps.
Yea why not, this should be good enough to be an Axiom
-
When someone says I am a woman, they are reporting an internal state. A sense of fit. Of alignment. Of rightness in a particular category. The scientist could not verify it any more than she could verify someone’s pain. She could not step into their experience and check. She could only trust the report.
Can't they just check the Gentiles and Chromosomes?!?!
-
She remembered a conversation she had overheard. Two people debating identity. One asking how someone could know what it felt like to be a woman if they had never lived in a woman’s body. The other responding that no one shares the same experience of womanhood, that a woman raised in one culture experiences it differently than a woman raised in another, that there is no single reference point defining the category.
There has to be a name for this kind of problem. "Sublime definition"? The more specific you get to the universal Women the mode edge cases there are. Matt Walsh's, "What is a Woman" comes to mind here
BTW this article was written after "What is a Woman" came out
-
What it would be like for a human to be in a bat’s situation. Not what it was like to be a bat. The hardware was too different. The approximation broke down. What remained was projection.
This reminds me of people that speak languages, their narrative self speaks a specific language but they can learn a new one and switch too it. I wonder if we can do that for new sensory systems
-
She opened a drawer, found a small bottle of vanilla extract, held it to her nose. Sweet, warm, familiar. A memory surfaced unbidden. Her grandmother’s kitchen, cookies baking, flour dusted across the counter. How would she explain this smell to someone who had never encountered it? She could say it was sweet, could compare it to other scents, could describe the chemical compounds triggering the receptors in her nose. But the smell itself, the experience of smelling it, would remain hers alone. All attempts to transmit it, lossy. Necessarily, fundamentally lossy.
"Qualia"
-
-
scottbevill.net scottbevill.net
-
Do not be jealous of your sister. Know that diamonds and roses are as uncomfortable when they tumble from one’s lips as toads and frogs: colder, too, and sharper, and they cut.
There were a couple moments in here that I think are talking about other fairy tales and fables, but this is the only one where I can remember the specific story that it's talking about— and I wonder if every instruction is a direct reference to another story or if they're just so seamlessly interwoven that I can't tell which ones are exclusive to this one
-
-
viewer.athenadocs.nl viewer.athenadocs.nl
-
corporations can raise capital more easily
limited liability and their ability to issue tradeable shares and bonds to the public.
-
-
monde22-dotcom.github.io monde22-dotcom.github.io
-
Focus is a publication by The International Defence Aid Fund.
Focus on Political Repression in Southern Africa was a serial publication produced by the International Defense and Aid Fund (IDAF), an organization dedicated to documenting and challenging apartheid-era injustice. Published from the 1960s through the 1980s, Focus provided clear, accessible reporting on political trials, security laws, censorship, prisoner conditions, and everyday experiences of state repression across South Africa and neighboring countries.
Written for an international audience, the publication combined investigative reporting, legal analysis, and first-hand testimony to expose abuses that were otherwise hidden from the global public. This digital collection brings together issues of Focus to support research, teaching, and general understanding of how apartheid systems operated—and how people and organizations resisted them.
-
-
www.biorxiv.org www.biorxiv.org
-
Version 2 of this preprint has been peer-reviewed and recommended by Peer Community in Forest and Wood Sciences.<br /> See the peer reviews and the recommendation.
-
-
sites.google.com sites.google.com
-
When all it sees at the hiring timeIs the line on the color bar
This last stanza again presents the theme of the lack of equality with an explicit reference to racism. In this case, Hornsby refers to unfair treatments and racial barriers in the work environment: white candidates are preferred over Black ones just for the color of their skin. The critique sharpens even more if we consider that it is contained in the same stanza in which there is a reference to the Civil Rights Act: the author places the two events - the Act and employment discrimination - on a line of continuity. In other words, it seems that since 1964 nothing has changed and discrimination lives on as bias and prejudice persist.
-
-
www.biorxiv.org www.biorxiv.org
-
eLife Assessment
This important study characterizes with rigorous methodology anatomical and functional aspects of the peripheral innervation of the Drosophila male reproductive tract. The convincing analysis reveals two distinct types of glutamatergic neurons that co-release either serotonin or octopamine. While serotonergic neurons are required for male fertility, octopaminergic neurons are dispensable. The work is providing invaluable insight into neurochemical control of insemination, peripheral motor control and neuromodulation in the male reproductive tract.
-
Reviewer #1 (Public review):
Summary:
This very thorough anatomical study addresses the innervation of the Drosophila male reproductive tract. Two distinct glutamatergic neuron types were classified: serotonergic (SGNs) and octopaminergic (OGNs). By expansion microscopy, it was established that glutamate and serotonin /octopamine are co-released. The expression of different receptors for 5-HT and OA in muscles and epithelial cells of the innervation target organs was characterized. The pattern of neurotransmitter receptor expression in the target organs suggests that seminal fluid and sperm transport and emission are subjected to complex regulation. While silencing of abdominal SGNs leads to male infertility and prevents sperm from entering the ejaculatory duct, silencing of OGNs does not render males infertile.
Strengths:
The studied neurons were analysed with different transgenes and methods, as well as antibodies against neurotransmitter synthesis enzymes, building a consistent picture of their neurotransmitter identity. The careful anatomical description of innervation patterns together with receptor expression patterns if the target organs provides a solid basis for advancing the understanding how seminal fluid and sperm transport and emission are subjected to complex regulation. The functional data showing that SGNs are required for male fertility and for the release of sperm from the seminal vesicle into the ejaculatory duct is convincing.
Weaknesses:
The functional analysis of the characterized neurons is not as comprehensive as the anatomical description and phenotypic characterization was limited to simple fertility assays. It is understandable that a full functional dissection is beyond the scope of the present work. The paper contains experiments showing neuron-independent peristaltic waves in the reproductive tract muscles, which are thematically not very well integrated into the paper. Although very interesting, one wonders if these experiments would not fit better into a future work that also explores these peristaltic waves and their interrelation with neuromodulation mechanistically.
Comments on revisions:
The manuscript has improved after fixing many small issues/errors. The new sections in the discussion are likewise adding to the quality of the manuscript.
-
Reviewer #2 (Public review):
Summary:
Cheverra et al. present a comprehensive anatomical and functional analysis of the motor neurons innervating the male reproductive tract in Drosophila melanogaster, addressing a gap in our understanding of the peripheral circuits underlying ejaculation and male fertility. They identify two classes of multi-transmitter motor neurons-OGNs (octopamine/glutamate) and SGNs (serotonin/glutamate)-with distinct innervation patterns across reproductive organs. The authors further characterize the differential expression of glutamate, octopamine, and serotonin receptors in both epithelial and muscular tissues of these organs. Behavioral assays reveal that SGNs are essential for male fertility, whereas OGNs and glutamatergic transmission are dispensable. This work provides a high-resolution map linking neuromodulatory identity to organ-specific motor control, offering a valuable framework to explore the neural basis of male reproductive function.
Strengths:
Through the use of an extensive set of GAL4 drivers and antibodies, this work successfully and precisely defines the neurons that innervate the male reproductive tract, identifying the specific organs they target and the nature of the neurotransmitters they release. It also characterizes the expression patterns and localization of the corresponding neurotransmitter receptors across different tissues. The authors describe two distinct groups of dual-identity neurons innervating the male reproductive tract: OGNs, which co-express octopamine and glutamate, and SGNs, which co-express serotonin and glutamate. They further demonstrate that the various organs within the male reproductive system differentially express receptors for these neurotransmitters. Based on these findings, the authors propose that a single neuron capable of co-releasing a fast-acting neurotransmitter along side a slower-acting one may more effectively synchronize and stagger events that require precise timing. This, together with the differential expression of ionotropic glutamate receptors and metabotropic aminergic receptors in postsynaptic muscle tissue, adds an additional layer of complexity to the coordinated regulation of fluid secretion, organ contractility, and directional sperm movement-all contributing to the optimization of male fertility.
Weaknesses:
One potential limitation of the study is the absence of information regarding the number of individuals examined for the various characterizations, which may weaken the strength of the conclusions. Another limitation may be the lack of quantitative analyses in the colocalization and morphological differentiation experiments. Nevertheless, the authors have indicated that such quantifications will be provided in a forthcoming publication; therefore, this should be considered only a partial limitation, as it is expected to be addressed in the near future.
Wider context:
This study delivers the first detailed anatomical map connecting multi-transmitter motor neurons with specific male reproductive structures. It highlights a previously unrecognized functional specialization between serotonergic and octopaminergic pathways and lays the groundwork for exploring fundamental neural mechanisms that regulate ejaculation and fertility in males. The principles uncovered here may help explain how males of Drosophila and other organisms adjust reproductive behaviors in response to environmental changes. Furthermore, by shedding light on how multi-transmitter systems operate in reproductive control, this model could provide insights into therapeutic targets for conditions such as male infertility and prostate cancer-where similar neuronal populations are involved in humans. Ultimately, this genetically accessible system serves as a powerful tool for uncovering how multi-transmitter neurons orchestrate coordinated physiological actions necessary for the functioning of complex organs.
-
Reviewer #3 (Public review):
Summary:
This work provides an overview of the motor neuron landscape in the male reproductive system. Some work had been done to elucidate the circuits of ejaculation in the spine, as well as, the cord but this work fills a gap of knowledge at the level of the reproductive organs. Using complementary approaches the authors show that there are two types of motor neurons that are mutually exclusive: neurons that co-express octopamine and glutamate and neurons that co-express serotonin and glutamate. They also show evidence that both types of neurons express large dense core vesicles indicating that neuropeptides play a role in male fertility. This paper provides a thorough characterization of expression of the different glutamate, octopamine and serotonin receptors in the different organs and tissues of the male reproductive system. The differential expression in different tissues and organs allows building initial theories on the control of emission and expulsion. Additionally, the authors characterize the expression of synaptic proteins and the neuromuscular junction sites. On a mechanistic level, the authors show that neither octopamine/glutamate neuron transmission nor glutamate transmission in serotonin/glutamate neurons are required for male fertility. This final result is quite surprising and opens up many questions on how ejaculation is coordinated.
Strengths:
This work fills an important gap on characterization of innervation of the male reproductive system by providing an extensive characterization of the motor neurons and the potential receptors of motor neuron release.The authors show convincing evidence of glutamate/monoamine co-release and of mutual exclusivity of serotonin/glutamate and octopamine/glutamate neurons.
Weaknesses:
The experiment looking at peristaltic waves in the male organs is missing labeling of the different regions and quantification of the observed waves.
-
Author response:
The following is the authors’ response to the original reviews.
Reviewer #1 (Public review):
Summary:
This very thorough anatomical study addresses the innervation of the Drosophila male reproductive tract. Two distinct glutamatergic neuron types were classified: serotonergic (SGNs) and octopaminergic (OGNs). By expansion microscopy, it was established that glutamate and serotonin /octopamine are co-released. The expression of different receptors for 5-HT and OA in muscles and epithelial cells of the innervation target organs was characterized. The pattern of neurotransmitter receptor expression in the target organs suggests that seminal fluid and sperm transport and emission are subjected to complex regulation. While silencing of abdominal SGNs leads to male infertility and prevents sperm from entering the ejaculatory duct, silencing of OGNs does not render males infertile.
Strengths:
The studied neurons were analysed with different transgenes and methods, as well as antibodies against neurotransmitter synthesis enzymes, building a consistent picture of their neurotransmitter identity. The careful anatomical description of innervation patterns together with receptor expression patterns of the target organs provides a solid basis for advancing the understanding of how seminal fluid and sperm transport and emission are subjected to complex regulation. The functional data showing that SGNs are required for male fertility and for the release of sperm from the seminal vesicle into the ejaculatory duct is convincing.
Weaknesses:
The functional analysis of the characterized neurons is not as comprehensive as the anatomical description, and phenotypic characterization was limited to simple fertility assays. It is understandable that a full functional dissection is beyond the scope of the present work. The paper contains experiments showing neuron-independent peristaltic waves in the reproductive tract muscles, which are thematically not very well integrated into the paper. Although very interesting, one wonders if these experiments would not fit better into a future work that also explores these peristaltic waves and their interrelation with neuromodulation mechanistically.
Reviewer #2 (Public review):
Summary:
Cheverra et al. present a comprehensive anatomical and functional analysis of the motor neurons innervating the male reproductive tract in Drosophila melanogaster, addressing a gap in our understanding of the peripheral circuits underlying ejaculation and male fertility. They identify two classes of multi-transmitter motor neurons-OGNs (octopamine/glutamate) and SGNs (serotonin/glutamate)-with distinct innervation patterns across reproductive organs. The authors further characterize the differential expression of glutamate, octopamine, and serotonin receptors in both epithelial and muscular tissues of these organs. Behavioral assays reveal that SGNs are essential for male fertility, whereas OGNs and glutamatergic transmission are dispensable. This work provides a high-resolution map linking neuromodulatory identity to organ-specific motor control, offering a valuable framework to explore the neural basis of male reproductive function.
Strengths:
Through the use of an extensive set of GAL4 drivers and antibodies, this work successfully and precisely defines the neurons that innervate the male reproductive tract, identifying the specific organs they target and the nature of the neurotransmitters they release. It also characterizes the expression patterns and localization of the corresponding neurotransmitter receptors across different tissues. The authors describe two distinct groups of dual-identity neurons innervating the male reproductive tract: OGNs, which co-express octopamine and glutamate, and SGNs, which co-express serotonin and glutamate. They further demonstrate that the various organs within the male reproductive system differentially express receptors for these neurotransmitters. Based on these findings, the authors propose that a single neuron capable of co-releasing a fast-acting neurotransmitter alongside a slower-acting one may more effectively synchronize and stagger events that require precise timing. This, together with the differential expression of ionotropic glutamate receptors and metabotropic aminergic receptors in postsynaptic muscle tissue, adds an additional layer of complexity to the coordinated regulation of fluid secretion, organ contractility, and directional sperm movement-all contributing to the optimization of male fertility.
Weaknesses:
The main weakness of the manuscript is the lack of detail in the presentation of the results. Specifically, all microscopy image figures are missing information about the number of samples (N), and in the case of colocalization experiments, quantitative analyses are not provided. Additionally, in the first behavioral section, it would be beneficial to complement the data table with figures similar to those presented later in the manuscript for consistency and clarity.
Wider context:
This study delivers the first detailed anatomical map connecting multi-transmitter motor neurons with specific male reproductive structures. It highlights a previously unrecognized functional specialization between serotonergic and octopaminergic pathways and lays the groundwork for exploring fundamental neural mechanisms that regulate ejaculation and fertility in males. The principles uncovered here may help explain how males of Drosophila and other organisms adjust reproductive behaviors in response to environmental changes. Furthermore, by shedding light on how multi-transmitter systems operate in reproductive control, this model could provide insights into therapeutic targets for conditions such as male infertility and prostate cancer, where similar neuronal populations are involved in humans. Ultimately, this genetically accessible system serves as a powerful tool for uncovering how multi-transmitter neurons orchestrate coordinated physiological actions necessary for the functioning of complex organs.
Reviewer #3 (Public review):
Summary:
This work provides an overview of the motor neuron landscape in the male reproductive system. Some work had been done to elucidate the circuits of ejaculation in the spine, as well as the cord, but this work fills a gap in knowledge at the level of the reproductive organs. Using complementary approaches, the authors show that there are two types of motor neurons that are mutually exclusive: neurons that co-express octopamine and glutamate and neurons that co-express serotonin and glutamate. They also show evidence that both types of neurons express large dense core vesicles, indicating that neuropeptides play a role in male fertility. This paper provides a thorough characterization of the expression of the different glutamate, octopamine, and serotonin receptors in the different organs and tissues of the male reproductive system. The differential expression in different tissues and organs allows building initial theories on the control of emission and expulsion. Additionally, the authors characterize the expression of synaptic proteins and the neuromuscular junction sites. On a mechanistic level, the authors show that neither octopamine/glutamate neuron transmission nor glutamate transmission in serotonin/glutamate neurons is required for male fertility. This final result is quite surprising and opens up many questions on how ejaculation is coordinated.
Strengths:
This work fills an important gap in the characterization of innervation of the male reproductive system by providing an extensive characterization of the motor neurons and the potential receptors of motor neuron release. The authors show convincing evidence of glutamate/monoamine co-release and of mutual exclusivity of serotonin/glutamate and octopamine/glutamate neurons.
Weaknesses:
(1) Often, it is mentioned that the expression is higher or lower or regional without quantification or an indication of the number of samples analysed.
(2) The experiment aimed at tracking sperm in the male reproductive system is difficult to interpret when it is not assessed whether ejaculation has occurred.
(3) The experiment looking at peristaltic waves in the male organs is missing labeling of the different regions and quantification of the observed waves.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) While the peripheral innervations are very carefully described, it is not clear to which SGNs and OGNs (i.e., cell bodies in the central nervous system) these innervations belong. Are SV, AG, and ED innervated by branches of one neuron or by separate neurons? Multi-color flip-out experiments could provide an answer to this.
We agree this is important and are planning these experiments for follow-up study.
(2) In contrast, for the analysis of the VT19028 split line (Figure 9), only vnc and cell body images are shown. How do the arborisations of these split combinations look in the periphery? Are the same reproductive organs innervated as shown in Figure 2?
Figure 9S3 was inadvertently omitted from the initial submission. That figure is now included and shows that the VT019028 split broadly innervates the SV, AG, and ED.
(3) In the discussion, I think it would be helpful to offer some potential explanations for the role of octopaminergic and glutamatergic signaling. If not required for basic fertility, they probably have some other role.
Thank you, we have included speculation in the Discussion section "Potential for adaptation to environment".
(4) Line 543: Figure 8S4 E, (not 8E).
Correction made.
Reviewer #2 (Recommendations for the authors):
(1) Line 213-217
Comment:
The use of "significantly less expression" may be misleading, as no quantification or statistical analysis is provided to support this comparison.
Suggestion:
Consider using a more neutral term, such as "markedly less" or "noticeably less," unless quantitative data and statistical analysis are included to substantiate the claim.
Good recommendation.This suggestion has been incorporated.
(2) Line 264-267
Comment:
The observation regarding the distinct morphology of SGNs and OGNs is interesting and could strengthen the argument regarding functional differences.
Suggestion:
Consider including a quantification of morphological complexity (e.g., branching) to support the claim. A method such as Sholl analysis (Sholl, 1953), as adapted in Fernández et al., 2008, could be applied.
This is a good suggestion, and we will consider it as part of a follow-up study.
(3) Line 269-271
Comment:
The anatomical context of the observation is not explicitly stated.
Suggestion:
Add "in the ED" for clarity: "With the TRH-GAL4 experiment in the ED, vGlut-40XMYC (Figure 5S1, A and E) and 6XV5-vMAT (Figure 5S1, B and F) were both present with a highly overlapping distribution (Figure 5S1, I)."
Suggestion has been incorporated.
(4) Line 275-276
Comment:
The claim about the reduced ability to distinguish SGNs and OGNs in the ED would benefit from quantitative support.
Suggestion:
Include a morphological comparison or quantification between SGNs and OGNs in the ED and SV to reinforce this point.
Certain information on morphological comparison can be inferred within the images themselves, and we will include quantitation in a follow-up study.
(5) Line 277-279
Comment:
As with line 269, the anatomical site could be specified more clearly.
Suggestion:
Rephrase as: "With the Tdc2-GAL4 experiment in the ED, vGlut-40XMYC (Figure 5S1, M and Q) and 6XV5-vMAT (Figure 5S1, N and R) were both observed in a highly overlapping distribution (Figure 5S1, U)."
Suggestion has been incorporated.
(6) Line 348-350
Comment:
The phrase "significantly higher density" implies a statistical comparison that is not shown.
Suggestion:
If no quantification is provided, replace with a qualitative term such as "visibly higher" or "notably more dense." Alternatively, add a quantitative analysis with statistical testing to justify the use of "significantly."
Suggestion has been incorporated.
(7) Lines 415-458 (Section comment)
Comment:
There appears to be differential localization of neurotransmitter receptor expression (glutamate in muscle vs. 5-HT in epithelium or neurons), which could have functional implications.
Suggestion:
Expand this section to briefly discuss the differential localization patterns of these receptors and potential implications for signal transduction in male reproductive tissues.
(8) Lines 638-682 (Section comment)
Comment:
The table summarizing fertility phenotypes would be more informative with additional detail on experimental outcomes.
Suggestion:
Add a column showing the number of fertile males over the total tested (e.g., "n fertile / n total"). Also, clarify whether the fertility assays are identical to those reported in Figure 10S2, and whether similar analyses were conducted for females. Consider including a figure summarizing fertility results for all genotypes listed in the table, similar to Figure 10S2.
The fertility tests reported in Table 1 were separate from those reported in Figure 10S2. For these tests, the results were clear-cut with 100% of males and females reported as infertile exhibiting the infertile phenotype. For the males and females reported as fertile, it was also clear-cut with nearly 100% showing fertility at a high level. In subsequent figures we attempted to assess degrees of fertility.
(9) Line 724-727
Comment:
There seems to be a mistake in the identification of the driver lines used to silence OA neurons. Also, figure references might be incorrect.
Suggestion:
The OA neuron driver line should be corrected to "Tdc2-GAL4-DBD ∩ AbdB-AD" instead of TRH-GAL4. Additionally, the figure references should be verified; specifically, the letter "B" (in "Figure 10B, D" and "10B, E") appears to be unnecessary or misplaced.
Thanks for catching this, the corrections have been made.
(10) Line 872-877
Comment:
The discussion on the co-release of fast-acting glutamate and slower aminergic neurotransmitters is interesting and well-articulated. However, it remains somewhat disconnected from the behavioral findings.
Suggestion:
Consider linking this proposed mechanism to the results observed in the mating duration assays. For instance, the sequential action of neurotransmitters described here could potentially underlie the prolonged mating observed when specific neuromodulators are active, helping to functionally integrate molecular and behavioral data.
(11) Line 926-928
Comment:
The interpretation of 5-HT7 receptor expression in the sphincter is compelling, suggesting a role in regulating its function. However, this anatomical observation could be further contextualized with the functional data.
Suggestion:
It may strengthen the interpretation to explicitly connect this finding with the fertility assays, where SGNs - presumably acting via serotonergic signaling - are shown to be necessary for male fertility. This would support a functional role for 5-HT7 in reproductive success via sphincter regulation.
This has been added.
(12) Figure 1
Comment:
The figure legend is generally clear, but could benefit from more consistency and precision in the color-coded labeling. Additionally, the naming of some structures could be more explicit.
Suggestion:
Revise the figure and the legend as follows:
Figure 1. The Drosophila male reproductive system. A) Schematic diagram showing paired testes (colour), SVs (green), AGs (purple), Sph (red), ED (gray), and EB (colour). B) Actual male reproductive system. Te - testes, SV - seminal vesicle, AG - accessory gland, Sph - singular sphincter, ED - ejaculatory duct, EB - ejaculatory bulb. Scale bar: 200 µm.
This suggestion has been incorporated.
(13) Figure 3S2
Comment:
There appears to be a typographical error in the description of the genotypes, which may lead to confusion.
Suggestion:
Correct the legend to reflect the appropriate genotypes:
Figure 3S2. Expression of vGlut-LexA and Tdc2-GAL4 in the Drosophila male reproductive system. A, D, G, J, M, P) vGlut-LexA, LexAop-6XmCherry; B, E, H, K, N, Q) Tdc2-GAL4, UAS-6XGFP; C, F, I, L, O, R) Overlay. Scale bars: O - 50 µm; R - 10 µm.
The corrections have been made.
(14) Figure 3S3
Comment:
The genotypes for panels D and E appear to be incomplete; the DBD component of the split-GAL4 drivers is missing.
Suggestion:
Update the figure legend to:
Figure 3S3. Fruitless and Doublesex expression in the Drosophila male reproductive system. A) fru-GAL4, UAS-6XGFP; B) vGlut-LexA, LexAop-6XmCherry; C) Overlay; D) Tdc2-AD ∩ dsx-GAL4-DBD; E) TRH-AD ∩ dsx-GAL4-DBD. Scale bar: 200 µm.
The corrections have been made.
(15) Figure 4S4
Comment:
There is a repeated segment in the figure legend, which makes it unclear and redundant.
Suggestion:
Edit the legend to remove the duplicated lines:
Figure 4S4. Expression of vGlut, TβH-GFP, and 5-HT at the junction of the SV and AGs with the ED of the Drosophila male reproductive system. A) vGlut-40XV5; B) TβH-GFP; C) 5-HT; D) vGlut-40XV5, TβH-GFP overlay; E) vGlut-40XV5, 5-HT overlay; F) TβH-GFP, 5-HT overlay. Scale bar: 50 µm.
The correction has been made.
(16) Figure 6S5
Comment:
Within this figure, the orientation and/or scale of the tissue varies noticeably between individual panels, making it difficult to directly compare the different experimental conditions.
Suggestion:
For improved clarity and interpretability, consider standardizing the orientation and size of the tissue shown across all panels within the figure. Consistent presentation will facilitate direct comparisons between treatments or genotypes.
There is often variation in the size of the male reproductive organs. They were all acquired at the same magnification. The only point of this figure is there is no vGAT or vAChT at these NMJs and the result is unambiguously negative.
(17) Figure 10
Comment:
Panel A appears redundant, as it shows the same information as the other panels but without indicating statistical significance.
Suggestion:
Consider removing panel A and keeping only the remaining four graphs, which include relevant statistical comparisons and clearly show significant differences.
We realize there is some redundancy of panel A with the other panels, but we feel there is value in having all the genotypes in a single panel for comparison.
Reviewer #3 (Recommendations for the authors):
Here are some suggestions to improve the manuscript:
(1) Prot B GFP experiment: the authors should explain better the time chosen to look at the sperm content of the male reproductive system. At 10 minutes, it is expected that the male has already ejaculated, and therefore, a failure to ejaculate would result in more sperm in the reproductive system, not less. Since we are not certain when the male ejaculates, it would be important to do the analysis at different time points.
In the Prot-GFP experiments, the 10-minute time point was chosen because we nearly always observe sperm in the ejaculatory duct of control males. In the experimental males, we never observed sperm in the ejaculatory duct at this time point. Also, no Prot-GFP sperm were observed in the reproductive tract of females mated to experimental males even when mating was allowed to go to completion, while abundant sperm were found in females mated to Prot-GFP controls. Figure 10S1 has been updated to include Images of these female reproductive systems. The results showing the absence of Prot-GFP sperm in the female reproductive tract mated to experimental males indicates sperm transfer in these males isn't occurring earlier during the copulation process than in control males and that we didn't miss it by only examining at the ejaculatory duct.
(2) Discuss what may be the role of the octopamine/glutamate neurons and glutamate transmission in serotonin/glutamate neurons in the male reproductive system, given that they are not required for fertility (at least under the context in which it was tested). It is quite a striking result that deserves some attention.
We agree it is a surprising result and have included speculation on the role of glutamate and octopamine in male reproduction in the Discussion section "Potential for adaptation to environment".
(3) Very important:
(a) Figure 3 is present in the Word document but not the PDF.
(b) Figure 9S3 is not present
(c) In Figure 5 X), the legend does not correspond to the panel.
All of these corrections have been made.
(4) Other suggestions:
(a) A summary schematic (or several) of the findings would make it an easier read.
(b) Explain why the ejaculatory bulb was left out of the analysis.
(c) Explain in the main text some of the tools, such as, BONT-C and the conditional vGlut mutation.
-
-
www.biorxiv.org www.biorxiv.org
-
eLife Assessment
The authors employ an unbiased, affinity-guided reagent to label P2X7 receptor and use super-resolution imaging to monitor P2X7 redistribution in response to inflammatory signaling. The evidence is convincing and the study will be valuable to those studying the dynamics of receptor distribution and clustering.
-
Reviewer #1 (Public review):
Summary:
In this paper, the authors developed a chemical labeling reagent for P2X7 receptors, called X7-uP. This labeling reagent selectively labels endogenous P2X7 receptors with biotin based on ligand-directed NASA chemistry. After labeling the endogenous P2X7 receptor with biotin, the receptor can be fluorescently labeled with streptavidin-AlexaFluor647. The authors carefully examined the binding properties and labeling selectivity of X7-uP to P2X7, characterized the labeling site of P2X7 receptors, and demonstrated fluorescence imaging of P2X7 receptors. The data obtained by SDS-PAGE, Western blot, and fluorescence microscopy clearly shows that X7-uP labels the P2X7 receptor. Finally, the authors fluorescently labeled the endogenous P2X7 in BV2 cells, which are a murine microglia model, and used dSTORM to reveal a nanoscale P2X7 redistribution mechanism under inflammatory conditions at high resolution.
Strengths:
X7-uP selectively labels endogenous P2X7 receptors with biotin. Streptavidin-AlexaFluor647 binds to the biotin labeled to the P2X7 receptor, allowing visualization of endogenous P2X7 receptors.
-
Reviewer #2 (Public review):
Summary:
In this manuscript, Arnould et. al. develop an unbiased, affinity-guided reagent to label P2X7 receptor and use super-resolution imaging to monitor P2X7 redistribution in response to inflammatory signaling.
Strengths:
I think the X7-uP probe that they developed is very useful for visualizing localization of P2X7 receptor. They convincingly show that under inflammatory conditions, there is a reorganization of P2X7 localization into receptor clusters. Moreover, I think they have shown a very clever way to specifically label any receptor of interest. This has broad appeal.
I think the authors have done a very nice job addressing my original concerns. Here are those original concerns and my new comments related to how the authors address them.
(1) While the authors state that chemical modification of AZ10606120 to produce the X7-UP reagent has "minimal impact" on the inhibition of P2X7, we can see from Figure 2A and 2B that it does not antagonize P2X7 as effectively as the original antagonist. For the sake of completeness and quantitation, I think it would be great if the authors could determine the IC50 for X7-uP and compare it to the IC50 of AZ10606120.
The authors now show the relative inhibition of X7-uP compared to AZ10606120 at different concentrations. This provides a nice comparison to give the reader an idea of how effectively X7-uP inhibits P2X7 receptor. This is great.
(2) Do the authors know whether modification of the lysines with biotin affects the receptor's affinity for ATP (or ability to be activated by ATP)? What about P2X7 that has been modified with biotin and then labeled with Alexa 647? For the sake of completeness and quantitation, I think it would be great if the authors could determine the EC50 of biotinylated P2X7 for ATP as well as biotinylated and then Alexa 647 labeled P2X7 for ATP and compare these values to the affinity of unmodified WT P2X7 for ATP.
I agree with the authors that assessing the functional integrity of P2X7 following biotinylation and fluorophore labeling is outside the scope of this paper but would be important for studies involving dynamic or post-labeling functional analyses such as live trafficking.
(3) It is a little misleading to color the fluorescence signal from mScarlet green (for example, in Figure 3 and Figure 4). The fluorescence is not at the same wavelength as GFP. In fact, the wavelength (570 nm - 610 nm) for emission is closer to orange/red than to green. I think this color should be changed to differentiate the signal of mScarlet from the GFP signal used for each of the other P2X receptor subtypes.
The authors have now changed the mScarlet color to orange, which solves my concern.
(4) It is my understanding that P2X6 does not form homotrimers. Thus, I was a little surprised to see that the density and distribution of P2X6-GFP in Figure 3 looks very similar to the density and distribution of the other P2X subtypes. Do the authors have an explanation for this? Are they looking at P2X6 protomers inserted into the plasma membrane? Does the cell line have endogenous P2X receptor subtypes? Is Figure 3 showing heterotrimers with P2X6 receptor? A little explanation might be helpful.
The authors address this point very well and include nice data to show that P2X6 does not insert into the plasma membrane as a homotrimer.
(5) It is easy to overlook the fact that the antagonist leaves the binding pocket once the biotin has been attached to the lysines. It might be helpful if the authors made this a little more apparent in Figure 1 or in the text describing the NASA chemistry reaction.
The authors have modified Figure 1 to make it easier to understand the NASA chemistry reaction.
I congratulate the authors on an outstanding paper!
-
Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this paper, the authors developed a chemical labeling reagent for P2X7 receptors, called X7-uP. This labeling reagent selectively labels endogenous P2X7 receptors with biotin based on ligand-directed NASA chemistry (Ref. 41). After labeling the endogenous P2X7 receptor with biotin, the receptor can be fluorescently labeled with streptavidin-AlexaFluor647. The authors carefully examined the binding properties and labeling selectivity of X7-uP to P2X7, characterized the labeling site of P2X7 receptors, and demonstrated fluorescence imaging of P2X7 receptors. The data obtained by SDS-PAGE, Western blot, and fluorescence microscopy clearly show that X7-uP labels the P2X7 receptor. Finally, the authors fluorescently labeled the endogenous P2X7 in BV2 cells, which are a murine microglia model, and used dSTORM to reveal a nanoscale P2X7 redistribution mechanism under inflammatory conditions at high resolution.
Strengths:
X7-uP selectively labels endogenous P2X7 receptors with biotin. Streptavidin-AlexaFluor647 binds to the biotin labeled to the P2X7 receptor, allowing visualization of endogenous P2X7 receptors.
We thank the reviewer for their positive comment.
Weaknesses:
Weaknesses & Comments
(1) The P2X7 receptor exists in a trimeric form. If it is not a monomer under the conditions of the pull-down assay in Figure 2C, the quantitative values may not be accurate.
We thank the reviewer for this comment. As shown in Figure 2C, the band observed on the denaturing SDS-PAGE corresponds to the monomeric form of the P2X7 receptor. While we cannot exclude the presence of non-monomeric species under native conditions, no such higher-order forms are visible in the gel. This observation supports the conclusion that the quantitative values presented are based on the monomeric form and are therefore reliable.
(2) In Figure 3, GFP fluorescence was observed in the cell. Are all types of P2X receptors really expressed on the cell surface ?
We thank the reviewer for this excellent comment, which was also raised by reviewer 2. To address this concern, we performed a commercial cell-surface protein biotinylation assay to assess whether GFP-tagged P2X receptors reach the plasma membrane. As expected, all P2X subtypes except P2X6 were detected at the cell surface in HEK293T cells, thereby validating our confocal fluorescence microscopy assay. These new data are now included in Figure 3 — figure supplement 1.
(3) The reviewer was not convinced of the advantages of the approach taken in this paper, because the endogenous receptor labeling in this study could also be done using conventional antibody-based labeling methods.
We thank the reviewer for raising this important point and would like to highlight several advantages of our approach compared to conventional antibody-based labeling.
First, commercially available P2X7 antibodies often suffer from poor specificity and are generally not suitable for reliably detecting endogenous P2X7 receptors, as documented in previous studies (e.g., PMID: 16564580 and PMID: 15254086). While recent advances have been made using nanobodies with improved specificity for P2X7 (e.g., PMID: 30074479 and PMID: 38953020), our strategy is distinct and complementary to nanobody-based approaches.
Second, antibodies rely on non-covalent interactions with the receptor, which can result in dissociation over time. In contrast, our X7-uP probe covalently biotinylates lysine residues on the P2X7 receptor through stable amide bond formation. This covalent labeling ensures that the biotin moiety remains permanently attached, an advantage not afforded by reversible binding strategies.
Third, by selectively biotinylating P2X7 receptors, our method provides a versatile platform for the chemical attachment of a wide range of probes or functional moieties. Although we did not demonstrate this application in the current study, we believe this modularity represents an additional advantage of our approach.
We have now revised the discussion to highlight these key advantages, allowing the reader to form their own opinion. We hope this addresses the reviewer’s concerns and clarifies the benefits of our approach.
(4) Although P2X7 was successfully labeled in this paper, it is not new as a chemistry. There is a need for more attractive functional evaluation such as live trafficking analysis of endogenous P2X7.
We agree with the reviewer that the underlying chemistry is not novel per se. However, to our knowledge, it has not previously been applied to the P2X7 receptor, and thus constitutes a novel application with specific relevance for studying native P2X7 biology.
We also appreciate the reviewer’s suggestion regarding live trafficking analysis of endogenous P2X7. While this is indeed a valuable and interesting direction, we believe it lies beyond the scope of the present study, as it would first require demonstrating that the labeling itself does not affect P2X7 function (see below). This important step would necessitate additional experiments, which we consider more appropriate for a follow-up investigation.
(5) The reviewer has concerns that the use of the large-size streptavidin to label the P2X7 receptor may perturbate the dynamics of the receptor.
We thank the reviewer for raising this important point. Although we did not directly measure receptor dynamics, it is indeed possible that tetrameric streptavidin (tStrept-A 647) could promote P2X7 clustering by cross-linking nearby receptors due to its tetravalency (see also point 7 raised by the reviewer). To address this concern, we performed additional dSTORM experiments using a monomeric form of streptavidin-Alexa 647 (mSA) (see PMID: 26979420). Owing to its reduced size and lack of tetravalency, mSA has been shown to minimize artificial crosslinking of synaptic receptors (PMID: 26979420). A drawback of using mSA, however, is that the monomeric form carries only two fluorophores (estimated degree of labeling, DOL ≈ 2, PMID: 26979420), whereas the tetrameric form, according to the manufacturer’s certificate of analysis (Invitrogen S21374), has an average DOL of three fluorophores per monomer, resulting in a total of ~12 fluorophores per streptavidin.
We tested three conditions with mSA incubation: (i) control BV2 cells (without X7-uP), (ii) untreated X7-uP-labeled BV2 cells, and (iii) X7-uP-labeled BV2 cells treated with LPS and ATP (using the same concentrations and incubation times described in the manuscript). As shown in Author response image 1, only LPS+ATP treatment induced a clear increase in the mean cluster density compared to quiescent (untreated) BV2 cells. This effect closely matches the results obtained with tStrept-A 647, supporting the conclusion the tetrameric streptavidin does not artificially promote P2X7 clustering. It is also possible that the cellular environment of BV2 microglia differs from the confined architecture of synapses, which may further explain why cross-linking effects are less pronounced in our system.
As expected, the overall fluorescence signal with mSA was about tenfold lower than with tStrept-A 647, consistent with the expected fluorophore stoichiometry. This lower signal may explain why the values for the untreated condition appeared slightly higher than for the control, although the difference was not statistically significant (P = 0.1455).
We hope these additional experiments adequately address the reviewer’s concerns.
Author response image 1.
BV2 labeling with monomeric streptavidin–Alexa 647 (mSA).(A) Bright-field and dSTORM images of BV2 cells labeled with mSA in the presence (untreated and LPS+ATP) or absence (control) of 1 µM X7-uP. Treatment: LPS (1 µg/mL for 24 hours) and ATP (1 mM for 30 minutes). Scale bars, 10 µm. Insets: Magnified dSTORM images. Scale bars, 1 µm.(B) Quantification of the number of localizations (n = 2 independent experiments). Bars represent mean ± s.e.m. One-way ANOVA with Tukey’s multiple comparisons (P values are indicated above the graph).
(6) It is better to directly label Alexa647 to the P2X7 receptor to avoid functional perturbation of P2X7.
Directly labeling of Alexa647 to the P2X7 receptor would require the design and synthesis of a novel probe, which is currently not available. Implementing such a strategy would involve substantial new experimental work that lies beyond the scope of the present study.
(7) In all imaging experiments, the addition of streptavidin, which acts as a cross-linking agent, may induce P2X7 receptor clustering. This concern would be dispelled if the receptors were labeled with a fluorescent dye instead of biotin and observed.
We refer the reviewer to our response in point 5, where we addressed this concern by comparing tetrameric and monomeric streptavidin conjugates. As noted above (see also point 6), directly labeling the receptor with a fluorescent dye would require the development of a new probe, which is outside the scope of the present study.
(8) There are several mentions of microglia in this paper, even though they are not used. This can lead to misunderstanding for the reader. The author conducted functional analysis of the P2X7 receptor in BV-2 cells, which are a model cell line but not microglia themselves. The text should be reviewed again and corrected to remove the misleading parts that could lead to misunderstanding. e.g. P8. lines 361-364
First, it combines N-cyanomethyl NASA chemistry with the high-affinity AZ10606120 ligand, enabling rapid labeling in microglia (within 10 min)
P8. lines 372-373
Our results not only confirm P2X7 expression in microglia, as previously reported (6, 26-33), but also reveal its nanoscale localization at the cell surface using dSTORM.
We agree with the reviewer’s comment. We have now modified the text, including the title.
Reviewer #2 (Public review):
Summary:
In this manuscript, Arnould et. al. develop an unbiased, affinity-guided reagent to label P2X7 receptor and use super-resolution imaging to monitor P2X7 redistribution in response to inflammatory signaling.
Strengths:
I think the X7-uP probe that they developed is very useful for visualizing localization of P2X7 receptor. They convincingly show that under inflammatory conditions, there is a reorganization of P2X7 localization into receptor clusters. Moreover, I think they have shown a very clever way to specifically label any receptor of interest. This has broad appeal
We thank the reviewer for their positive comment.
Weaknesses:
Overall, the manuscript is novel and interesting. However, I do have some suggestions for improvement.
(1) While the authors state that chemical modification of AZ10606120 to produce the X7-UP reagent has "minimal impact" on the inhibition of P2X7, we can see from Figure 2A and 2B that it does not antagonize P2X7 as effectively as the original antagonist. For the sake of completeness and quantitation, I think it would be great if the authors could determine the IC50 for X7-uP and compare it to the IC50 of AZ10606120.
We thank the reviewer for this insightful comment. Unfortunately, due to the limited availability of X7-uP, we were not able to establish a complete concentration–response curve to determine its IC<sub>50</sub>, which would require testing at concentrations >1 µM. Nevertheless, to estimate the effect of the modification, we assessed current inhibition at 300 µM X7-uP and compared it with the reported IC<sub>50</sub> of AZ10606120 (10 nM). Under these conditions, both compounds produced a similar level of inhibition, indicating that while the chemical modification reduces potency relative to AZ10606120, X7-uP still functions as an effective probe for P2X7. We have now included these data in Figure 2 and revised the text accordingly.
(2) Do the authors know whether modification of the lysines with biotin affects the receptor's affinity for ATP (or ability to be activated by ATP)? What about P2X7 that has been modified with biotin and then labeled with Alexa 647? For the sake of completeness and quantitation, I think it would be great if the authors could determine the EC50 of biotinylated P2X7 for ATP as well as biotinylated and then Alexa 647 labeled P2X7 for ATP and compare these values to the affinity of unmodified WT P2X7 for ATP.
We thank the reviewer for raising this important point. At present, we have not determined whether modification of lysine residues with biotin, or subsequent labeling with Alexa647, affects the ATP sensitivity or functional properties of P2X7. However, we believe this does not impact the conclusions of the current study, as all functional assays were conducted prior to X7-uP labeling. The labeling is used here as a terminal "snapshot" to visualize the endogenous receptor without interfering with the functional characterization.
We fully agree that assessing the functional integrity of P2X7 following biotinylation and fluorophore labeling—such as by determining the EC<sub>50</sub> for ATP—would be essential for studies involving dynamic or post-labeling functional analyses, such as live trafficking. However, as noted earlier in our response to Reviewer 1 (point 4), these experiments lie beyond the scope of the current study.
(3) It is a little misleading to color the fluorescence signal from mScarlet green (for example, in Figure 3 and Figure 4). The fluorescence is not at the same wavelength as GFP. In fact, the wavelength (570 nm - 610 nm) for emission is closer to orange/red than to green. I think this color should be changed to differentiate the signal of mScarlet from the GFP signal used for each of the other P2X receptor subtypes.
As suggested, we changed the mScarlet color to orange for all relevant figures.
(4) It is my understanding that P2X6 does not form homotrimers. Thus, I was a little surprised to see that the density and distribution of P2X6-GFP in Figure 3 looks very similar to the density and distribution of the other P2X subtypes. Do the authors have an explanation for this? Are they looking at P2X6 protomers inserted into the plasma membrane? Does the cell line have endogenous P2X receptor subtypes? Is Figure 3 showing heterotrimers with P2X6 receptor? A little explanation might be helpful.
We thank the reviewer for raising this important point. Indeed, it is well established that P2X6 does not form functional channels, which supports the conclusion that it does not form homotrimeric complexes. Although previous studies have shown that P2X6–GFP expression is generally lower, more diffuse, and not efficiently targeted to the cell surface compared with other P2X subtypes (see PMID: 12077178), the similar fluorescence distribution and density observed in our Figure 3 do not imply that P2X6 forms homotrimers.
We did not directly assess the presence of endogenous P2X6 in our HEK293T cells; however, according to the Human Protein Atlas, there is no detectable P2X6 RNA expression in HEK293 cells (nTPM = 0), indicating that endogenous P2X6 is not expressed in this cell line. To further investigate surface expression (see also point 2 of reviewer 1), we performed a commercial cell-surface protein biotinylation assay to assess whether GFP-tagged P2X6 reaches the plasma membrane. As expected, P2X6 was not detected at the cell surface in HEK293T cells, whereas GFP-tagged P2X1 to P2X5 were readily detected. These results further support the conclusion that P2X6 does not insert into the plasma membrane as a homotrimer, thereby validating our confocal fluorescence microscopy assay. These new data are now included in Figure 3 — figure supplement 1.
(5) It is easy to overlook the fact that the antagonist leaves the binding pocket once the biotin has been attached to the lysines. It might be helpful if the authors made this a little more apparent in Figure 1 or in the text describing the NASA chemistry reaction.
We thank the reviewer for this insightful suggestion. To address this, we have modified Figure 1A and updated the legend.
Reviewer #3 (Public review):
Summary:
This manuscript describes the development of a covalent labeling probe (X7-uP) that selectively targets and tags native P2X7 receptors at the plasma membrane of BV2 microglial cells. Using super-resolution imaging (dSTORM), the authors demonstrate that P2X7 receptors form nanoscale clusters upon microglial activation by lipopolysaccharide (LPS) and ATP, correlating with synergistic IL-1β release. These findings advance understanding of P2X7 reorganization during inflammation and provide a generalizable labeling strategy for monitoring endogenous P2X7 in immune cells.
Strengths:
(1) The authors designed X7-uP by coupling a high-affinity, P2X7-specific antagonist (AZ10606120) with N-cyanomethyl NASA chemistry to achieve site-directed biotinylation. This approach offers high specificity, minimal off-target reactivity, and a straightforward pull-down/imaging readout.
(2) The results connect P2X7's nanoscale clustering directly with IL-1β secretion in microglia, reinforcing the role of P2X7 in inflammation. By localizing endogenous P2X7 at single-molecule resolution, the authors reveal how LPS priming and ATP stimulation synergistically reorganize the receptor.
(3) The authors systematically validate their method in recombinant systems (HEK293 cells) and in BV2 cells, showing selective inhibition, mutational confirmation of the binding site, and Western blot pulldown experiments.
We thank the reviewer for their positive comment.
Weaknesses:
(1) While the data strongly indicate that P2X7 clustering contributes to IL-1β release, the manuscript would benefit from additional experiments (if feasible) or discussion on how receptor clustering interfaces with downstream inflammasome assembly. Clarification of whether the P2X7 clusters physically colocalize with known inflammasome proteins would solidify the mechanism.
We thank the reviewer for this valuable suggestion. Determining the physical colocalization of P2X7 clusters with known inflammasome components would provide important insight into the molecular partners involved in inflammasome activation. However, we believe that such an investigation would constitute a substantial study on its own and therefore lies beyond the scope of the present work.
Nevertheless, in response to the reviewer’s suggestion, we have added a short paragraph at the end of the Discussion section addressing potential mechanisms by which P2X7 clustering may contribute to downstream inflammasome activation. We also revised the text to tone down the hypothesis of physical colocalization.
(2) The authors might expand on the scope of X7-uP in other native cells that endogenously express P2X7 (e.g., macrophages, dendritic cells). Although they mention the possibility, demonstrating the probe's applicability in at least one other primary immune cell type would strengthen its general utility.
We thank the reviewer for this valuable suggestion. Again, we believe that such an investigation would constitute a substantial study on its own and therefore lies beyond the scope of the present work.
(3) The authors do include appropriate negative controls, yet providing additional details (e.g., average single-molecule on-time or blinking characteristics) in supplementary materials could help readers assess cluster calculations.
As suggested, we have included additional data showing single-molecule blinking events in untreated and LPS+ATP-treated BV2 cells, along with the corresponding movies. The data are now presented in Figure 5—supplement figure 3A and B and Figure 5—Videos 1 and 2.
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors):
(1) On line 96, the authors refer to the "ballast" domain of P2X7 receptor but do not cite the original article from which this nomenclature originated (McCarthy et al., 2019, Cell). This article should be cited to give appropriate credit.
Done.
(2) On line 602, the authors state that they use models from PDB 1MK5 and 6U9W to generate the cartoons in Figure 6. The manuscripts from which these PDB files were generated need to be appropriately cited.
Done.
(3) On line 319, the authors say "300 mM BzATP" but I think they mean 300 uM.
Done. Thank you for catching the typo.
Reviewer #3 (Recommendations for the authors):
Overall, excellent data quality. The paper would benefit from a discussion of the physiological implications of clustering. It would also be helpful to elaborate about the potential mechanisms for clustering: diffusion and/or insertion. Finally, the authors should comment on work by Mackinnon's (PMID: 39739811) and Santana lab (PMID: 31371391) on two distinct models for clustering of proteins.
As suggested by the reviewer, we have revised the discussion to incorporate their comments. First, we have added the following text:
“Upon BV2 activation, we observed significant nanoscale reorganization of P2X7. Both LPS and ATP (or BzATP) trigger P2X7 upregulation and clustering, increasing the overall number of surface receptors and the number of receptors per cluster, from one to three (Figure 6). By labeling BV2 cells with X7-uP shortly after IL-1b release, we were able to correlate the nanoscale distribution of P2X7 with the functional state of BV2 cells, consistent with the two-signal, synergistic model for IL-1b secretion observed in microglia and other cell types (Ferrari et al, 1996; Perregaux et al, 2000; Ferrari et al, 2006; Di Virgilio et al, 2017; He et al, 2017; Swanson et al, 2019). In this model, LPS priming leads to intracellular accumulation of pro-IL-1b, while ATP stimulation activates P2X7, triggering NLRP3 inflammasome activation and the subsequent release of mature IL-1b.
What is the mechanism underlying P2X7 upregulation that leads to an overall increase in surface receptors—does it result from the lateral diffusion of previously masked receptors already present at the plasma membrane, or from the insertion of newly synthesized receptors from intracellular pools in response to LPS and ATP? Although our current data do not distinguish between these possibilities, a recent study suggests that the a1 subunit of the Na<sup>+</sup>/K</sup>+</sup>-ATPase (NKAa1) forms a complex with P2X7 in microglia, including BV2 cells, and that LPS+ATP induces NKAa1 internalization (Huang et al, 2024). This internalization appears to release P2X7 from NKAa1, allowing P2X7 to exist in its free form. We speculate that the internalization of NKAa1 induced by both LPS and ATP exposes previously masked P2X7 sites, including the allosteric AZ10606120 sites, thus making them accessible for X7-uP labeling.”
Second, we have added a short paragraph at the end of the Discussion section addressing potential mechanisms by which P2X7 clustering may contribute to downstream inflammasome activation:
“What mechanisms underlie P2X7 clustering in response to inflammatory signals? Several models have been proposed to explain membrane protein clustering, including recruitment to structural scaffolds (Feng & Zhang, 2009), partitioning into membrane domains enriched in specific chemical components such as lipid rafts (Simons & Ikonen, 1997), and self-assembly mechanisms (Sieber et al, 2007). These self-assembly mechanisms include an irreversible stochastic model (Sato et al, 2019) and a more recent reversible self-oligomerization model which gives rise to higher-order transient structures (HOTS) (Zhang et al, 2025). Supported by cryogenic optical localization microscopy with very high resolution (~5 nm), the HOTS model has been observed in various membrane proteins, including ion channels and receptors (Zhang et al, 2025). Furthermore, HOTS are suggested to be dynamically modulated and to play a functional role in cell signaling, potentially influencing both physiological and pathological processes (Zhang & MacKinnon, 2025). While this hypothesis is compelling, our current dSTORM data lack sufficient spatial resolution to confirm whether P2X7 trimers form HOTS via self-oligomerization. Further biophysical and ultra-high-resolution imaging studies are required to test this model in the context of P2X7 clustering.”
-
-
www.biorxiv.org www.biorxiv.org
-
eLife Assessment
This fundamental manuscript provides compelling evidence that BK and CaV1.3 channels can co-localize as ensembles early in the biosynthetic pathway, including within the ER and Golgi. The findings, supported by a range of imaging and proximity assays, offer insights into channel organization in both heterologous and endogenous systems. The data substantiate the central claims, while highlighting intriguing mechanistic questions for future studies: the determinants of mRNA co-localization, the temporal dynamics of ensemble trafficking, and the physiological implications of pre-assembly for channel function at the plasma membrane.
-
Reviewer #1 (Public review):
Summary:
The co-localization of large conductance calcium- and voltage activated potassium (BK) channels with voltage-gated calcium channels (CaV) at the plasma membrane is important for the functional role of these channels in controlling cell excitability and physiology in a variety of systems. An important question in the field is where and how do BK and CaV channels assemble as 'ensembles' to allow this coordinated regulation - is this through preassembly early in the biosynthetic pathway, during trafficking to the cell surface or once channels are integrated into the plasma membrane. These questions also have broader implications for assembly of other ion channel complexes. Using an imaging based approach, this paper addresses the spatial distribution of BK-CaV ensembles using both overexpression strategies in tsa201 and INS-1 cells and analysis of endogenous channels in INS-1 cells using proximity ligation and superesolution approaches. In addition, the authors analyse the spatial distribution of mRNAs encoding BK and Cav1.3. The key conclusion of the paper that BK and CaV1.3 are co-localised as ensembles intracellularly in the ER and Golgi is well supported by the evidence. The experiments and analysis are carefully performed and the findings are very well presented.
-
Reviewer #3 (Public review):
Summary:
The authors present a clearly written and beautifully presented piece of work demonstrating clear evidence to support the idea that BK channels and Cav1.3 channels can co-assemble prior to their assertion in the plasma membrane.
Strengths:
The experimental records shown back up their hypotheses and the authors are to be congratulated for the large number of control experiments shown in the ms.
-
