This entails of a vacation that would be happening and veterans of the challenges and problems.

This explains how the oet veiws the cat as a extesion of his own soul, how they are alike yet simmilar all the same. They both have their challenges hey both overcome, taking resolve in ones company.

This saying that just them together is all he needs and feels like a reward to him

Being together with his cat is a reward to him, he loves his cat.

For the most part though the missing type annotations won't change the behavior of the program. I guess unless you're using numeric literals with type annotations to change what the type of the number is. Othe wise it normally will just not type check.

Violation of the gradual guarantee. I'm coming around to this being fine but I think that it needs to be declared loudly in a languages design description and possibly alternative syntax for type declarations should be used so it's more obvious it's not just for clarity.

Trying a second annotation here

My first annotation

for - to - essay - The Gospel of Wealth - Andrew Carnegie

article details\ - title: How a poor boy from Scotland became the richest man on Earth - author: Daniel Coughlin - publication: https://www.msn.com/en-za/news/other/how-a-poor-boy-from-scotland-became-the-richest-man-on-earth/ss-AA1snaUl?ocid=widgetonlockscreen&cvid=7d9709f2a3784d09b14e49f448a1a4cc&ei=12#image=25 - date - 2024, Oct 18

top-down process

= bottom-up process

Provide an example of such a campaign from each of the three countries. Cite your sources.

What are Illinois laws on gaining access (being included) on a presidential ballot? Cite your source(s).

Explore ballot laws in another Western democracy and find either support for or against Nader's claim. Cite your source(s).

How many electoral votes does Illinois have? Cite yourr source(s).

By what method are ballots counted in Illinois?

Two parts: 1) Does Illinois use a primary or a caucus system? 2) Does Illinois have an "open" or "closed" primary vote, and what does that mean? Cite your source(s)

Under the US Constitution, what are the rules for becoming a Presidential candidate? Cite your source(s}

We conjure up the emotion in order to fulfill the goal

Pro instructors

Tom Kurkwood

Interesting because I've heard the exact opposite statement

belangrijke term

Choosing the next president by strictly majority vote would essentially make the whole process into a popularity contest. If this stayed in affect, a president with little to no real solutions regarding the improvement of the United States could be elected, setting back the nation by at least four years. However, this notion of selecting the five highest numbers and choosing the president based on their quality is how Hamilton ensures that corruption would not surface.

If less than half of the voting population is not in agreement on the election, it could be unsafe. Therefore, the House of Representatives will choose, in their opinion, who is the best fit. This could get out of hand as the HOR could be corrupt and may not represent the people accurately.

If all electors congregated in one place, there might be arguments and disagreements leading to the disruption of the task at hand.

Truy xuất: các phương pháp truy xuất dựa trên embedding của câu được sử dụng cùng với điểm tương đồng cosine làm thước đo đánh giá. K câu tương đồng gần nhất sẽ được sử dụng làm ngữ cảnh.

RA-IT: Một cách khác để thực hiện instruction-tuning trong việc chiết xuất có mục tiêu, được thể hiện khái quát ở hình 1. Trong cách tiếp cận này, mỗi mẫu sẽ được tăng cường với một đoạn ngữ cảnh được truy xuất. Đoạn ngữ cảnh này bao gồm k mẫu dữ liệu tương đồng về mặt ngữ cảnh được trích xuất từ bộ dữ liệu huấn luyện. Đoạn ngữ cảnh này được thêm vào đoạn hội thoại gốc. từ đó tạo ra mẫu instruction được tăng cường truy xuất. Bằng cách tinh chỉnh các mô hình ngôn ngữ (LMs) theo hướng này, LMs sẽ được trang bị khả năng tạo sinh câu trả lời NER cùng với RAG. Điều này có nghĩa là LMs có thể được cài đặt dễ dàng để thích ứng với nhiều kịch bản khác nhau bằng cách xác định xem có cần sử udngj RAG trong quá trình infer ko dựa trên các đặc tính cụ thể của kịch bản

IT đơn thuần: Mẫu instruction tuning ban đầu được sử dụng trong quá trình chiết xuất có mục tiêu được thể hiện ở bên dưới của hình 1, còn được gọi là mẫu IT đơn thuần. Trong mẫu này, mỗi câu đầu vào thô và các thực thể liên quan sẽ được hoán đổi thành một đoạn hội thoại có nhiều lượt.

Bước chuẩn bị: Chiết xuất có mục tiêu: Được làm dựa trên nghiên cứu UniNER, vốn đã thành công trong việc chiết xuất khả năng mạnh mẽ của chatGPT và truyền nó vào các mô hình nhỏ hơn mà không cần dữ liệu do con người gán nhãn. Qúa trình này bao gồm các bước: - Xây dựng dữ liệu: Các mẫu đầu vào thô được chọn từ nhiều domain đa dạng khác nhau và sử dụng chatGPT để gán nhãn. - Chiết xuất: Sau khi có được dữ liệu gán nhãn tự động, Instruction tuning được áp dụng để chiết xuất khả năng của chatGPT và truyền vào các mô hình nhỏ hơn.

Đóng góp chính của nhóm tác giả: - Tiến hành nghiên cứu RA-IT cho bài toán open NER. Các dữ liệu tăng cường truy xuất được chuẩn bị với các mẫu tương đồng về mặt ngữ nghĩa. Các thực nghiệm được tiến hành để đánh giá mức độ ảnh hưởng của các chiến lược truy xuất. - Xây dựng bộ dữ liệu IT cho bài toán open NER bằng tiếng Trung và tiến hành thực nghiệm đánh giá với cả tiếng Anh và tiềng Trung.

Các kết luận thu được: - RA-IT đạt được độ cải thiện bền vững với dữ liệu ở nhiều phạm vi khác nhau, từ đó thể hiện sự cần thiết của việc fine-tuning với ngữ cảnh được mở rộng. - Việc truy xuất các mẫu tương đồng về mặt ngữ cảnh làm cải thiện đáng kể chất lượng huấn luyện, tùy thuộc vào chiến lược chọn mẫu trích xuất. - Việc truy xuất các mẫu out-domain trong quá trình infer yêu cầu các chiến lược lọc mẫu để đạt được hiệu quả mong muốn. Ngược lại, việc cung cấp các mẫu in-domain sẽ làm gia tăng hiệu quả của quá trình infer.

Bằng hướng tiếp cận RA-IT, đối với mỗi mẫu huấn luyện, các mẫu tương đồng về mặt ngữ nghĩa sẽ được trích xuất từ bộ dữ liệu huấn luyện và được thêm vào mẫu dữ liệu huấn luyện gốc, tạo thành mẫu huấn luyện được tăng cường ngữ cảnh. Ngoài ra , một bộ dữ liệu huấn luyện bằng tiếng Trung cho bài toán openNER cũng được xây dựng và được dùng để đánh giá hiệu quả của mô hình trên cả tiếng Anh và tiếng Trung.

UniNER đã chắt lọc khả năng mạnh mẽ của ChatGPT trong bài toán open NER và truyền các mô hình bé hơn thông qua instruction tuning mà không cần dữ liệu do con người xây dựng. Nghiên cứu này được tiến hành theo hướng tương tự và RA-IT được điều tra theo hướng chắt lọc có mục tiêu (targeted distillation)

Ý tưởng chính: Sử dụng phương pháp instruction tuning có tăng cường truy hồi (RA-IT) cho bài toán IE, tập trung vào bài toán Open NER. Cụ thể, với mỗi mẫu huấn luyện, các mẫu khác có tính tương đồng về mặt ngữ nghĩa sẽ được truy xuất ra từ bộ dữ liệu và thêm các mẫu này vào đầu vào của mẫu huấn luyện ban đầu.

A covalent link (or covalent bond) occurs when two atoms share electrons, forming a stable connection between them. In DNA, covalent bonds link the deoxyribose sugar of one nucleotide to the phosphate group of the next,

Down-market: lower price (AH basic) Up-market: increase price, more luxurious (AH bio)

Formatting is similar to Q1 a

Truth table formatting remark?

From what I know iOS 17.0-17.3 is supported on RHEL. should we mention it?

When you are programming, you are writing a control flow diagrams embedded with state transition methods through code.

Acc to structured program theorem, You essentially need three control structures to write any computable program. 1. Sequence <code> <code> <code> ... 2. Selection if <condition> | <code_this> # if true else <code_that> # if false 3. Iteration <loop> | <code> # exists for do..while | if <condition> | escape # if true | <code> # exists for while repeat <loop>

Due to the reliable ubiquity of the first structure, any set of sequenced program statements could be deemed as a code block. Then, control flow structures command the order in which these various code blocks are executed.

There are various control flow methods. * Conditionals for selection (like if, switch...) * Loops for iteration (like for, while..) * Routines for abstraction (like function, class...)

Résumé de la vidéo [00:00:00][^1^][1] - [00:20:43][^2^][2]:

La vidéo examine le rapport de la Cour des comptes sur les dépenses des collectivités territoriales en France. Elle critique certaines conclusions du rapport et explore les réalités financières des collectivités.

Moments forts: + [00:00:00][^3^][3] Introduction et contexte * Pierre Moscovici propose de supprimer 100,000 postes * Économies potentielles de 4 milliards d'euros par an * Présentation du rapport de la Cour des comptes + [00:01:59][^4^][4] État des lieux financiers * Croissance du déficit global en 2024 * Différences entre les catégories de collectivités * Augmentation des dépenses de fonctionnement et d'investissement + [00:06:39][^5^][5] Rôle des collectivités dans le redressement * Dépenses locales représentent 18% des dépenses publiques * Marges d'amélioration sur les dépenses de personnel * Critiques sur le temps de travail et l'absentéisme + [00:11:01][^6^][6] Propositions de la Cour des comptes * Réduction de 100,000 emplois d'ici 2030 * Économies de 4,1 milliards d'euros par an * Non-remplacement des départs en retraite + [00:17:25][^7^][7] Critiques du rapport * Transferts de charges non compensés * Augmentation des dépenses sociales et des normes * Besoin de données chiffrées précises

Résumé de la vidéo [00:20:44][^1^][1] - [00:24:19][^2^][2]:

Cette vidéo discute des dépenses des collectivités territoriales et des critiques de la Cour des comptes. Elle met en lumière les problèmes de gestion des effectifs et des compétences, ainsi que les défis liés à la sécurité et aux normes.

Moments forts: + [00:20:44][^3^][3] Critiques des dépenses * Dépenses jugées inutiles ou exagérées * Manque de données tangibles * Raccourcis de la Cour des comptes + [00:21:18][^4^][4] Problèmes d'effectifs * Enchevêtrement des compétences * Recrutement des communes * Nouvelles charges à assumer + [00:21:50][^5^][5] Sécurité et effectifs * Augmentation des effectifs de police municipale * Pression de la population pour plus de sécurité * Désengagement de l'État + [00:22:05][^6^][6] Dépenses et services publics * Effet de vase communicant entre l'État et les collectivités * Nécessité de couper dans certains services publics * Importance de la clarté dans les choix de coupes + [00:22:50][^7^][7] Pistes d'économies * Chasse aux doublons et compétences entremêlées * Développement de l'intelligence artificielle * Réduction des normes et ressources nécessaires

Je ne comprends pas la solution donnée, n'est-elle pas incomplète ? le titre, le paragraphe et le lien sont tous entassés et je n'arrive pas à les espacer même en mettant des marges définies ou un padding. La photo ne se positionne pas à droite avec la solution, il a fallu que je rajoute un div sur la page html index autour de l'image pour que cela fonctionne. Est-ce la bonne façon de faire ?

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

PPARgamma is a nuclear receptor that binds to orthosteric ligands to coordinate transcriptional programs that are critical for adipocyte biogenesis and insulin sensitivity. Consequently, it is a critical therapeutic target for many diseases, but especially diabetes. The malleable nature and promiscuity of the PPARgamma orthosteric ligand binding pocket have confounded the development of improved therapeutic modulators. Covalent inhibitors have been developed but they show unanticipated mechanisms of action depending on which orthosteric ligands are present. In this work, Shang and Kojetin present a compelling and comprehensive structural, biochemical, and biophysical analysis that shows how covalent and noncovalent ligands can co-occupy the PPARgamma ligand binding pocket to elicit distinctive preferences of coactivator and corepressor proteins. Importantly, this work shows how the covalent inhibitors GW9662 and T0070907 may be unreliable tools as pan-PPARgamma inhibitors despite their widespread use.

Strengths:

- Highly detailed structure and functional analyses provide a comprehensive structure-based hypothesis for the relationship between PPARgamma ligand binding domain co-occupancy and allosteric mechanisms of action. - Multiple orthogonal approaches are used to provide high-resolution information on ligand binding poses and protein dynamics.

- The large number of x-ray crystal structures solved for this manuscript should be applauded along with their rigorous validation and interpretation.

Weaknesses

- Inclusion of statistical analysis is missing in several places in the text. - Functional analysis beyond coregulator binding is needed.

We added additional statistical analyses as recommended (Source Data 1, a Microsoft Excel spreadsheet).

Related to functional analysis, we cite and studies from our previous publication (Hughes et al. Nature Communications 2014 5:3571) where we demonstrated that the covalent inhibitor ligands (GW9662 and T0070907) do not block the activity of other ligands using a PPARγ transcriptional reporter assay and gene expression analysis in 3T3-L1 preadipocytes. Our study here expands on this finding and other published studies showing the structural mechanism for the lack of blocking activity by the covalent inhibitors.

Reviewer #2 (Public Review):

Summary:

The flexibility of the ligand binding domain (LBD) of NRs allows various modes of ligand binding leading to various cellular outcomes. In the case of PPARγ, it's known that two ligands can co-bind to the receptor. However, whether a covalent inhibitor functions by blocking the binding of a non-covalent ligand, or co-bind in a manner that weakens the binding of a non-covalent ligand remains unclear. In this study, the authors first used TR-FRET and NMR to demonstrate that covalent inhibitors (such as GW9662 and T0070907) weaken but do not prevent non-covalent synthetic ligands from binding, likely via an allosteric mechanism. The AF-2 helix can exchange between active and repressive conformations, and covalent inhibitors shift the conformation toward a transcriptionally repressive one to reduce the orthosteric binding of the non-covalent ligands. By co-crystal studies, the authors further reveal the structural details of various non-covalent ligand binding mechanisms in a ligand-specific manner (e.g., an alternate binding site, or a new orthosteric binding mode by alerting covalent ligand binding pose).

Strengths:

The biochemical and biophysical evidence presented is strong and convincing.

Weaknesses:

However, the co-crystal studies were performed by soaking non-covalent ligands to LBD pre-crystalized with a covalent inhibitor. Since the covalent inhibitors would shift the LBD toward transcriptionally repressive conformation which reduces orthosteric binding of non-covalent ligands, if the sequence was reversed (i.e., soaking a covalent inhibitor to LBD pre-crystalized with a non-covalent ligand), would a similar conclusion be drawn? Additional discussion will broaden the implications of the conclusion.

This is an interesting point, which we now expand upon in a new (third) paragraph of the discussion in our revised manuscript:

“In our previous study, we observed synthetic and natural/endogenous ligand co-binding via co-crystallography where preformed crystals of PPARγ LBD bound to unsaturated fatty acids (UFAs) were soaked with a synthetic ligand, which pushed the bound UFA to an alternate site within the orthosteric ligand-binding pocket 8. In the scenario of synthetic ligand cobinding with a covalent inhibitor, it is possible that soaking a covalent inhibitor into preformed crystals where the PPARγ LBD is already bound to a non-covalent ligand may prove to be difficult. The covalent inhibitor would need to flow through solvent channels within the crystal lattice, which may not be a problem. However, upon reaching the entrance surface to the orthosteric ligand-binding pocket, it may be difficult for the covalent inhibitor to gain access to the region of the orthosteric pocket required for covalent modification as the larger non-covalent ligand could block access. This potential order of addition problem may not be a problem for studies in solution or in cells, where the non-covalent ligand can more freely exchange in and out of the orthosteric pocket and over time the covalent reaction would reach full occupancy.”

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

- IC50 or EC50 values are not reported for the coregulator interaction assays, R2 for fit should also be reported where Ki and IC50s are disclosed.

We now report fitting statistics and IC50/EC50 values when possible in Figure 2B and Source Data 1 along with R2 values for the fit. We note that some data do not show complete or robust enough binding curves to faithfully fit to a dose response equation.

-  Reporter gene or qPCR should be performed for the combinations of covalent and noncovalent ligands to show how these molecules impact transcriptional activities rather than just coregulator binding profiles.

We previously performed PPARγ transcriptional reporter assay and gene expression analysis in 3T3-L1 preadipocytes to demonstrate that cotreatment of a covalent inhibitor (GW9662 or T0070907) with a non-covalent ligand does not block activity of the non-covalent ligand and showed cobinding-induced activation relative to DMSO control (Hughes et al., 2024 Nature Communications). We did not specifically mention this in our original manuscript, but we now call this out in the first paragraph of the results section.

- Inclusion of a structure figure to show the different helix 12 orientations should be included in the introduction. Likewise, how the overall structure of the LBD changes as a result of the cobinding in the discussion or a summary model would be helpful.

Our revised manuscript includes a structure figure called out in the introduction describing the active and repressive helix 12 PPARγ LBD conformations (new Figure 1). There are no major changes to the overall structure of the LBD compared to the active conformation that crystallized, so we did not include a summary model figure but we do refer readers to our previous paper (Shang and Kojetin, Structure 2021 29(9):940-950) in the penultimate paragraph of the discussion. We also added the following sentence to the crystallography results section related to the overall LBD changes:

“The structures show high structural similarity to the transcriptionally active LBD conformation with rmsd values ranging from 0.77–1.03Å (Supplementary Table S2)”

A typo in paragraph 3 of the discussion says "long-live" when it should probably say "long-lived."

We corrected this typo.

Reviewer #2 (Recommendations For The Authors):

It's interesting that ligand-specific binding mode of non-covalent ligands was observed. Would modifications of the chemical structure of a covalent inhibitor alter the allosteric binding behavior of non-covalent ligands in a predictive manner? If so, how can such SAR be used to guide the design of covalent inhibitors to more broadly and effectively inhibit agonists of various chemical structures? Discussion on this topic could be valuable.

This is an interesting point, which we now discuss in the penultimate and last paragraphs of the discussion:

“Another way to test this structural model could be through the use of covalent PPARγ inverse agonist analogs with graded activity 23, where one might posit that covalent inverse agonist analogs that shift the LBD conformational ensemble towards a fully repressive LBD conformation may better inhibit synthetic ligand cobinding.”

“It may be possible to use the crystal structures we obtained to guide structure-informed design of covalent inhibitors that would physically block cobinding of a synthetic ligand. This could be the potential mechanism of a newer generation covalent antagonist inhibitor we developed, SR16832, that more completely inhibit alternate site ligand binding of an analog of MRL20, rosiglitazone and the UFA docosahexaenoic acid (DHA)

21 and thus may be a better choice for the field to use as a covalent ligand inhibitor of PPARγ.”

eLife Assessment

This landmark study elucidates the intricate structural mechanisms by which both covalent and non-covalent synthetic ligands can co-occupy the binding pocket of the nuclear receptor transcription factor PPARγ. Through a compelling integration of structural, biochemical, and biophysical evidence, the authors challenge the reliability of two commonly used covalent inhibitors. These findings have far-reaching implications for the broader field of nuclear receptor research. This work will be of high interest to structural biologists and biochemists exploring ligand interactions within the nuclear receptor superfamily.

Reviewer #1 (Public review):

Summary:

PPARgamma is a nuclear receptor that binds to orthosteric ligands to coordinate transcriptional programs that are critical for adipocyte biogenesis and insulin sensitivity. Consequently, it is a critical therapeutic target for many diseases but especially diabetes. The malleable nature and promiscuity of the PPARgamma orthosteric ligand binding pocket has confounded the development of improved therapeutic modulators. Covalent inhibitors have been developed but they show unanticipated mechanisms of action depending on which orthosteric ligands are present. In this work, Shang and Kojetin present a compelling and comprehensive structural, biochemical, and biophysical analysis that shows how covalent and noncovalent ligands can co-occupy the PPARgamma ligand binding pocket to elicit distinctive preferences of coactivator and corepressor proteins. Importantly, this work shows how the covalent inhibitors GW9662 and T0070907 may be unreliable tools as pan-PPARgamma inhibitors despite their wide-spread use.

Strengths:

- Highly detailed structure and functional analyses provide a comprehensive structure-based hypothesis for the relationship between PPARgamma ligand binding domain co-occupancy and allosteric mechanisms of action.<br /> - Multiple orthogonal approaches are used to provide high resolution information on ligand binding poses and protein dynamics.<br /> - The large number of x-ray crystal structures solved for this manuscript should be applauded along with their rigorous validation and interpretation.

Reviewer #2 (Public review):

Summary:

The flexibility of the ligand binding domain (LBD) of NRs allows various modes of ligand binding leading to various cellular outcomes. In the case of PPARγ, it's known that two ligands can cobind to the receptor. However, whether a covalent inhibitor functions by blocking the binding of a non-covalent ligand, or cobind in a manner that weakens the binding of a non-covalent ligand remains unclear. In this study, the authors first used TR-FRET and NMR to demonstrate that covalent inhibitors (such as GW9662 and T0070907) weaken but do not prevent non-covalent synthetic ligands from binding, likely via an allosteric mechanism. The AF-2 helix can exchange between active and repressive conformations, and covalent inhibitors shift the conformation toward a transcriptionally repressive one to reduce orthosteric binding of the non-covalent ligands. By co-crystal studies, the authors further reveal the structural details of various non-covalent ligand binding mechanisms in a ligand specific manner (e.g., an alternate binding site, or a new orthosteric binding mode by alerting covalent ligand binding pose).

Strengths:

The biochemical and biophysical evidence as presented is strong and convincing.

Additional comments:

The co-crystal studies were performed by soaking a non-covalent ligand to LBD pre-crystalized with a covalent inhibitor. Since the covalent inhibitors would shift the LBD toward transcriptionally repressive conformation which reduces orthosteric binding of non-covalent ligands, one might ask if the sequence was reversed (i.e., soaking a covalent inhibitor to LBD pre-crystalized with a non-covalent ligand), would similar conclusion be drawn? The authors have reasonably speculated that it might be difficult to soak a covalent inhibitor into preformed crystals where the PPARγ LBD is already bound to a non-covalent ligand, because the larger non-covalent ligand could block the covalent inhibitor to gain access to the region of the orthosteric pocket required for covalent modification.

relational: duurzaam, samen, etc transactional: korte-termijn, individueel

bovenstaand figuur: consistency of social expectations = verwachten alle stakeholders hetzelfde?

eLife Assessment

The manuscript presents an important model for the field of endosome maturation, providing perspective on the role of the deubiquitinating enzyme UPS-50/USP8 in the process. The evidence presented in the paper is clear, incorporating well-designed experiments that suggest the dual actions of UPS-50 and USP8 in the conversion of early endosomes into late endosomes. Overall, the work is convincing and centers on an intriguing subject.

Reviewer #1 (Public review):

Summary:

The manuscript focuses on the role of the deubiquitinating enzyme UPS-50/USP8 in endosome maturation. The authors aimed to clarify how this enzyme drives the conversion of early endosomes into late endosomes. Overall, they did achieve their aims in shedding light on the precise mechanisms by which UPS-50/USP8 regulates endosome maturation. The results support their conclusions that UPS-50 acts by disassociating RABX-5 from early endosomes to deactivate RAB-5 and by recruiting SAND-1/Mon1 to activate RAB-7. This work is commendable and will have a significant impact on the field. The methods and data presented here will be useful to the community in advancing our understanding of endosome maturation and identifying potential therapeutic targets for diseases related to endosomal dysfunction. It is worth noting that further investigation is required to fully understand the complexities of endosome maturation. However, the findings presented in this manuscript provide a solid foundation for future studies.

Strengths:

The major strengths of this work lie in the well-designed experiments used to examine the effects of UPS-50 loss. The authors employed confocal imaging to obtain a picture of the aftermath of USP-50 loss. Their findings indicated enlarged early endosomes and MVB-like structures in cells deficient in USP-50/USP8.

Weaknesses:

Specifically, there is a need for further investigation to accurately characterize the anomalous structures detected in the ups-50 mutant. Also, the correlation between the presence of these abnormal structures and ESCRT-0 is yet to be addressed, and the current working model needs to be revised to prevent any confusion between enlarged early endosomes and MVBs.

Reviewer #2 (Public review):

Summary:

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that localization of USP8 to early endosomes are disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although that Rabex5 are accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

Strengths:

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidences to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members in the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

Weaknesses:

- The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell that whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion. Note that the authors have provided convincing evidence about the effects on Rab proteins in the revised manuscript.<br /> - The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient of both factors displayed similar defects in late endosomes/lysosomes. But the authors didn't confirm whether and/or to which extent that USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model. Note that the authors have provided convincing evidence about the role of USP8-Rabx5 axis in the revised manuscript.

Reviewer #3 (Public review):

Summary:

The authors elucidated the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of abnormal enlarged vesicles (aberrant early endosomes) when USP8 function was lost. They showed that USP8 interacts with Rabx5 to dissociate it from early endosomes promoting the recruitment of the Rab7 GEF SAND-1/Mon1 and the maturation of the endosomes. The authors provided evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells.

Strengths:

The use of two models, C. elegans and a mammalian cell line to describe a similar mechanism.

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #1 (Public Review): 

Summary: 

The manuscript focuses on the role of the deubiquitinating enzyme UPS-50/USP8 in endosome maturation. The authors aimed to clarify how this enzyme drives the conversion of early endosomes into late endosomes. Overall, they did achieve their aims in shedding light on the precise mechanisms by which UPS-50/USP8 regulates endosome maturation. The results support their conclusions that UPS-50 acts by disassociating RABX-5 from early endosomes to deactivate RAB-5 and by recruiting SAND-1/Mon1 to activate RAB-7. This work is commendable and will have a significant impact on the field. The methods and data presented here will be useful to the community in advancing our understanding of endosome maturation and identifying potential therapeutic targets for diseases related to endosomal dysfunction. It is worth noting that further investigation is required to fully understand the complexities of endosome maturation. However, the findings presented in this manuscript provide a solid foundation for future studies. 

We thank this reviewer for the instructive suggestions and encouragement.

Strengths: 

The major strengths of this work lie in the well-designed experiments used to examine the effects of UPS-50 loss. The authors employed confocal imaging to obtain a picture of the aftermath of the USP-50 loss. Their findings indicated enlarged early endosomes and MVB-like structures in cells deficient in USP-50/USP8. 

We thank this reviewer for the instructive suggestions and encouragement.

Weaknesses: 

Specifically, there is a need for further investigation to accurately characterize the anomalous structures detected in the usp-50 mutant. Also, the correlation between the presence of these abnormal structures and ESCRT-0 is yet to be addressed, and the current working model needs to be revised to prevent any confusion between enlarged early endosomes and MVBs. 

Excellent suggestions. USP8 has been identified as a protein associated with ESCRT components, which are crucial for endosomal membrane deformation and scission, leading to the formation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). In usp-50 mutants, we observed a significant reduction in the punctate signals of HGRS-1::GFP and STAM-1 (Figure 1G and H; and Figure1-figure supplement 1B), indicating a disruption in ESCRT-0 complex localization (Author response image 1). Additionally, lysosomal structures are markedly reduced in these mutants. In contrast, we found that early endosomes, as marked by FYVE, RAB-5, RABEX5, and EEA1, are significantly enlarged in usp-50 mutants. Electron microscopy (EM) imaging further revealed an increase in large cellular vesicles containing various intraluminal structures. Given the reduction in lysosomal structures and the enlargement of early endosomes in usp-50 mutants, these enlarged vesicles are likely aberrant early endosomes rather than late endosomal or lysosomal structures. To address potential confusion, we have revised the manuscript according to the reviewer's comments and updated the model to accurately reflect these observations.

Reviewer #2 (Public Review): 

Summary: 

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that the localization of USP8 to early endosomes is disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although Rabex5 is accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation. 

We thank this reviewer for the instructive suggestions and encouragement.

Strengths: 

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidence to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members of the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled. 

We thank this reviewer for the instructive suggestions and encouragement.

Weaknesses: 

- The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion. 

Good suggestion. Indeed, to test whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion as fluorescent protein reporters, we supplemented our approach with the utilization of endogenous markers. These markers, including Rab5, RAB-5, Rabex5, RABX-5, and EEA1 for early endosomes, as well as RAB-7, Mon1a, and Mon1b for late endosomes, were instrumental in our investigations (refer to Figure 3, Figure 6, Figure 5-figure supplement 1, Figure 5-figure supplement 2, and Figure 6-figure supplement 1). Our comprehensive analysis, employing various methodologies such as tissue-specific fused proteins, CRISPR/Cas9 knock-in, and antibody staining, consistently highlights the critical role of USP8 in early-to-late endosome conversion.

- The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient in both factors displayed similar defects in late endosomes/lysosomes. However, the authors didn't confirm whether and/or to which extent USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model. 

Excellent point. To test whether USP-50 regulates endosome maturation through RABX-5, we performed additional genetic analyses. In rabx-5(null) mutant animals, the morphology of 2xFYVE-labeled early endosomes is comparable to that of wild-type controls (Figure 4H and I). Introducing the rabx-5(null) mutation into usp-50(xd413) backgrounds resulted in a significant suppression of the enlarged early endosome phenotype characteristic of usp-50(xd413) mutants (Figure 4H and I). These findings suggest that USP-50 may modulate the size of early endosomes through its interaction with RABX-5.

Reviewer #3 (Public Review): 

Summary: 

The authors were trying to elucidate the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of "abnormal" enlarged vesicles when USP8 function was lost. At this specific point, it's not clear what the objective of the authors was. What would have been their hypothesis addressing whether the reduced lysosomal structures in USP8 (-) animals were linked to MVB formation? Then they observed that the abnormally enlarged vesicles, marked by the PI3P biosensor YFP-2xFYVE, are bigger but in the same number in USP8 (-) compared to wild-type animals, suggesting homotypic fusion. They confirmed this result by knocking down USP8 in a human cell line, and they observed enlarged vesicles marked by YFP-2xFYVE as well. At this point, there is quite an important issue. The use of YFP-2xFYVE to detect early endosomes requires the transfection of the cells, which has already been demonstrated to produce differences in the distribution, number, and size of PI3P-positive vesicles (doi.org/10.1080/15548627.2017.1341465). The enlarged vesicles marked by YFP-2xFYVE would not necessarily be due to the loss of UPS8. In any case, it appears relatively clear that USP8 localizes to early endosomes, and the authors claim that this localization is mediated by Rabex-5 (or Rabx-5). They finally propose that USP8 dissociates Rabx-5 from early endosomes facilitating endosome maturation. 

Weaknesses: 

The weaknesses of this study are, on one side, that the results are almost exclusively dependent on the overexpression of fusion proteins. While useful in the field, this strategy does not represent the optimal way to dissect a cell biology issue. On the other side, the way the authors construct the rationale for each approximation is somehow difficult to follow. Finally, the use of two models, C. elegans and a mammalian cell line, which would strengthen the observations, contributes to the difficulty in reading the manuscript. 

The findings are useful but do not clearly support the idea that USP8 mediates Rab5-Rab7 exchange and endosome maturation, In contrast, they appear to be incomplete and open new questions regarding the complexity of this process and the precise role of USP8 within it. 

We thank this reviewer for the insightful comments. Fluorescence-fused proteins serve as potent tools for visualizing subcellular organelles both in vivo and in live settings. Specifically, in epidermal cells of worms, the tissue-specific expression of these fused proteins is indispensable for studying organelle dynamics within living organisms. This approach is necessitated by the inherent limitations of endogenously tagged proteins, whose fluorescence signals are often weak and unsuitable for live imaging or genetic screening purposes. Acknowledging concerns raised by the reviewer regarding potential alterations in organelle morphology due to overexpression of certain fused proteins, we supplemented our approach with the utilization of endogenous markers. These markers, including Rab5, RAB-5, Rabex5, RABX-5, and EEA1 for early endosomes, as well as RAB-7, Mon1a, and Mon1b for late endosomes, were instrumental in our investigations (refer to Figure 3, Figure 6, Figure 5-figure supplement 1, Figure 5-figure supplement 2, and Figure 6-figure supplement 1). Our comprehensive analysis, employing various methodologies such as tissue-specific fused proteins, CRISPR/Cas9 knock-in, and antibody staining, consistently highlights the critical role of USP8 in early-to-late endosome conversion. Specifically, we discovered that the recruitment of USP-50/USP8 to early endosomes is depending on Rabex5. However, instead of stabilizing Rabex5, the recruitment of USP-50/USP8 leads to its dissociation from endosomes, concomitantly facilitating the recruitment of the Rab7 GEF SAND-1/Mon1. In cells with loss-of-function mutations in usp-50/usp8, we observed enhanced RABX-5/Rabex5 signaling and mis-localization of SAND-1/Mon1 proteins from endosomes. Consequently, this disruption impairs endolysosomal trafficking, resulting in the accumulation of enlarged vesicles containing various intraluminal contents and rudimentary lysosomal structures.

Through an unbiased genetic screen, verified by cultured mammalian cell studies, we observed that loss-of-function mutations in usp-50/usp8 result in diminished lysosome/late endosomes. Electron microscopy (EM) analysis indicated that usp-50 mutation leads to abnormally enlarged vesicles containing various intraluminal structures in worm epidermal cells. USP8 is known to regulate the endocytic trafficking and stability of numerous transmembrane proteins. Given that lysosomes receive and degrade materials generated by endocytic pathways, we hypothesized that the abnormally enlarged vesicular structures observed in usp-50 or usp8 mutant cells correspond to the enlarged vesicles coated by early endosome markers. Indeed, in the absence of usp8/usp-50, the endosomal Rab5 signal is enhanced, while early endosomes are significantly enlarged. Given that Rab5 guanine nucleotide exchange factor (GEF), Rabex5, is essential for Rab5 activation, we further investigated its dynamics. Additional analyses conducted in both worm hypodermal cells and cultured mammalian cells revealed an increase of endosomal Rabex5 in response to usp8/usp-50 loss-of-function. Live imaging studies further demonstrated active recruitment of USP8 to newly formed Rab5-positive vesicles, aligning spatiotemporally with Rabex5 regulation. Through systematic exploration of putative USP-50 binding partners on early endosomes, we identified its interaction with Rabex5. Comprehensive genetics and biochemistry experiments demonstrated that USP8 acts through K323 site de-ubiquitination to dissociate Rabex5 from early endosomes and promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In summary, our study began with an unbiased genetic screen and subsequent examination of established theories, leading to the formulation of our own hypothesis. Through multifaceted approaches, we unveiled a novel function of USP8 in early-to-late endosome conversion.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) Within Figures 1K-N, diverse anomalous structures were detected in the usp-50 mutant. Further scrutiny is needed to definitively characterize these structures, particularly as the images in Figures 1M and 1L exhibit notable similarities to lamellar bodies.

We thank the reviewer for the insightful question regarding the resemblance between the vesicles observed in our study and lamellar bodies (LBs). Lamellar bodies are specialized organelles involved in lipid storage and secretion1, prominently studied in keratinocytes of the skin and alveolar type II (ATII) epithelial cells in the lung2. These organelles contain not only lipids but also cell-type specific proteins and lytic enzymes. Due to their acidic pH and functional similarities, LBs are classified as lysosome-related organelles (LROs) or secretory lysosomes3,4. In usp-50 mutants, we observed a considerable number of abnormal vesicles, some of which contain threadlike membrane structures and exhibit morphological similarities to LBs (Figure 2O). However, further analysis with a comprehensive panel of lysosome-related markers demonstrated a significant reduction in lysosomal structures within these mutants. In contrast, vesicles marked by early endosome markers, such as FYVE, RAB-5, RABX-5, and EEA1, were notably enlarged. These results suggest that the enlarged vesicles observed in usp-50 mutants are more likely aberrant early endosomes rather than true lamellar bodies. We have revised the manuscript to reflect these findings and to clearly differentiate between these structures and lysosome-related organelles.

(2) The correlation between the presence of these abnormal structures and ESCRT-0 remains unaddressed, thus the assertion that UPS-50 regulates endolysosome trafficking in conjunction with ESCRT-0 lacks empirical support.

We thank the reviewer for the valuable suggestions. We apologize for any confusion and appreciate the opportunity to clarify our findings. The ESCRT machinery is essential for driving endosomal membrane deformation and scission, which leads to the formation of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs). Recent research has shown that the absence of ESCRT components results in a reduction of ILVs in worm gut cells5. In wild type animals, the ESCRT-0 components HGRS-1 and STAM-1 display a distinct punctate distribution (Figure 1G and H). However, in usp-50 mutants, the punctate signals of HGRS-1::GFP and STAM-1::GFP are significantly reduced (Figure 1G and H; and Figure 1-figure supplement 1B), indicating a role for USP-50 in stabilizing the ESCRT-0 complex. Our TEM analysis revealed an accumulation of abnormally enlarged vesicles containing intraluminal structures in usp-50 mutants. When we examined a panel of early endosome and late endosome/lysosome markers, we found that early endosomes are significantly enlarged, while late endosomal/lysosomal structures are markedly reduced in these mutants. This suggests that the abnormal structures observed in usp-50 mutants are likely enlarged early endosomes rather than classical MVBs. To further investigate whether the reduction in ESCRT components contributes to the late endosome/lysosome defects, we analyzed stam-1 mutants. In these mutants, the size of RAB-7-coated vesicles was reduced (Author response image 1C), and the lysosomal marker LAAT-1 indicated a reduction in lysosomal structures (Author response image 1B). These results highlight the importance of the ESCRT complex in late endosome/lysosome formation. However, the morphology of early endosomes, as marked by 2xFYVE, remained similar to that of wild type in stam-1 mutants (Author response image 1A). Therefore, while reduced ESCRT-0 components may contribute to the late endosome/lysosome defects observed in usp-50 mutants, the enlargement of early endosomes in these mutants may involve additional mechanisms. We have revised the manuscript to incorporate these insights and to address the reviewer's comments more comprehensively.

Author response image 1.

(A) Confocal fluorescence images of hypodermis expressing YFP::2xFYVE to detect EEs in L4 stage animals in wild type and stam-1(ok406) mutants. Scale bar: 5 μm. (B) Confocal fluorescence images of hypodermal cell 7 (hyp7) expressing the LAAT-1::GFP marker to highlight lysosome structures in 3-day-old adult animals. Compared to wild type, LAAT-1::GFP signal is reduced in stam-1(ok406) animals. Scale bar, 5 μm. (C) The reduction of punctate endogenous GFP::RAB-7 signals in stam-1(ok406) animals. Scale bar: 10 μm.

(3) Endosomal dysfunction typically leads to significant alterations in the spatial arrangement of marker proteins across distinct endosomes. In the manuscript, the authors examined the distribution and morphology of early endosomes, multivesicular bodies (MVBs), late endosomes, and lysosomes in a usp-50 deficient background primarily through single-channel confocal imaging. By employing two color images showing RAB-5 and RAB-7, in conjunction with HGRS-1, a more comprehensive picture of the aftermath of USP-50 loss can be obtained.

Good suggestions. We have conducted a double-labeling analysis to examine the distribution of RAB-5 and RAB-7 in conjunction with HGRS-1. In wild type animals, HGRS-1 exhibits a punctate distribution that is partially co-localized with both RAB-5 and RAB-7. In contrast, in usp-50 mutants, the punctate signal of HGRS-1 is significantly reduced, along with its co-localization with RAB-5 and RAB-7 (Author response image 2). These results suggest that, in the absence of USP-50, the stabilization of ESCRT-0 components on endosomes is compromised.

Author response image 2.

ESCRT-0 is adjacent to both early endosomes and late endosomes. (A) Confocal fluorescence images of wild-type and usp-50(xd413) hypodermis at L4 stage co-expressing HGRS-1::GFP (hgrs-1 promoter) and endogenous wrmScarlet::RAB-5. (B) HGRS-1 and RAB-5 puncta were analyzed to produce Manders overlap coefficient M1 (HGRS-1/RAB-5) and M2 (RAB-5/HGRS-1) (N=10). (C) Confocal fluorescence images of wild-type and usp-50(xd413) hypodermis at L4 stage co-expressing HGRS-1::GFP (hgrs-1 promoter) and endogenous wrmScarlet::RAB-7. (D) HGRS-1 and RAB-7 puncta were analyzed to produce Manders overlap coefficient M1 (HGRS-1/RAB-7) and M2 (RAB-7/HGRS-1) (N=10). Scale bar: 10 μm for (A) and (C).

(4) The authors observed enlarged early endosomes in cells depleted of usp-50/usp8, along with enlarged MVB-like structures identified through TEM. The potential identity of these structures as the same organelle could be determined using CLEM.

We thank the reviewer for the valuable suggestion. Our TEM analysis identified a large number of abnormally enlarged vesicles with various intraluminal structures accumulated in usp-50 mutants. As the reviewer correctly noted, CLEM (correlative light and electron microscopy) would be an ideal approach to further characterize these structures. We have been attempting to implement CLEM in C. elegans for a few years. Given that CLEM relies on fluorescence markers, in this study we focused on two tagged proteins, RAB-5 and RABX-5, which show enlargement in their vesicles in usp-50 mutants. Unfortunately, we encountered significant challenges with this approach, as the GFP-tagged RAB-5 and RABX-5 signals did not survive the electron microscopy procedure. Attempts to align EM sections with residual GFP signaling yielded results that were not convincing. Consequently, we concentrated our analysis on a panel of molecular markers, including 2xFYVE, RAB-5, RABX-5, RAB-7, and LAAT-1. These markers consistently indicated that early endosomes are specifically enlarged in usp-50 mutants, while late endosomal/lysosomal structures are notably reduced. Thus, the abnormal structures identified in usp-50 mutants via TEM are likely to be enlarged early endosomes rather than the classical view of MVBs. We have revised the manuscript to reflect these findings and to clarify this point.

(5) The working model depicted in Figure 6 Y (right) requires revision, as it has the potential to mislead authors into mistaking enlarged early endosomes for multivesicular bodies (MVBs).

We thank the reviewer for the excellent suggestion. We have revised the model to clarify that it is the enlarged early endosomes, rather than MVBs, that are observed in usp-50 mutants.

Reviewer #2 (Recommendations For The Authors):

(1) Is there any change of Rabx5 protein level in USP8/USP50 mutant cells?

Good question. In the absence of usp-50/usp8, we indeed observed a noticeable increase in the signal of Rabex5 on endosomes. To determine whether usp-50/usp8 affects the protein level of Rabex5, we investigated the endogenous levels of RABX-5 using the RABX-5::GFP knock-in line. Compared to wild-type controls, we found an elevated protein level of RABX-5::GFP in the knock-in line (Author response image 3). This suggests that USP-50 may play a role in the destabilization of RABX-5/Rabex5 in vivo.

Author response image 3.

The endogenous RABX-5 protein level is increased in usp-50 mutants. (A) The RABX-5::GFP KI protein level is increased in usp-50(xd413). (B) Quantification of endogenous RABX-5::GFP protein level in wild type and usp-50(xd413) mutant animals.

(2) It is interesting that "The rabx-5(null) animals are healthy and fertile and do not display obvious morphological or behavioral defects.", which seems contrary to its role in regulating USP8 localization and endosome maturation.

It has been previously documented that rabx-5 functions redundantly with rme-6, another RAB-5 GEF in C. elegans, to regulate RAB-5 localization in oocytes6. RNA interference (RNAi) targeting rabx-5 in a rme-6 mutant background results in synthetic lethality, whereas neither rabx-5 nor rme-6 single mutants are essential for worm viability. RME-6 co-localizes with clathrin-coated pits, while Rabex-5 is localized to early endosomes. Rabex-5 forms a stable complex with Rabaptin-5 and is part of a large EEA1-positive complex on early endosomes, whereas RME-6 does not interact with Rabaptin-5 (RABN-5) or EEA-1. These findings suggest that while RME-6 and RABX-5 may function redundantly, they likely play distinct roles in regulating intracellular trafficking processes. In the absence of RABX-5, USP-50 appears to lose its endosomal localization, although the size of the early endosome remains comparable to that of wild type. This observation contrasts with the phenotype associated with USP-50 loss-of-function, in which the early endosome is notably enlarged. These results suggest that residual USP-50 present in the endosomes is sufficient to maintain its role in the endocytic pathway. Conversely, the complete absence of USP-50 likely disrupts the transition of early endosomes to late endosomes, indicating a crucial role of USP-50 in this conversion process. It is also noteworthy that, although loss-of-function of rabx-5 does not result in obvious changes to early endosomes, increasing the gene expression level of rabx-5/Rabex-5 alone is sufficient to cause enlargement of early endosomes (Author response image 4) . Indeed, we observed that loss-of-function mutations in u_sp-50/usp_8 lead to abnormally enlarged early endosomes, accompanied by an enhanced signal of endosomal RABX-5. When the rabx-5(null) mutation was introduced into usp-50 mutant animals, the enlarged early endosome phenotype seen in usp-50 mutants was significantly suppressed (Figure 4H and I). This implies that maintaining a lower level of Rab5 GEF may be crucial for endolysosomal trafficking.

(3) Does Rabx5 mutation has any impact on early endosomes?

To address the question, we utilized the CRISPR/Cas9 technique to create a molecular null for rabx-5 (Figure 4E). In the rabx-5(null) mutant animals, we found that the 2xFYVE-labeled early endosomes are indistinguishable from wild type (Figure 4H and 4I). Given that r_abx-5_ functions redundantly with rme-6, another RAB-5 GEF in C. elegans, it is likely that the regulation of early endosome size involves a cooperative interaction between RABX-5 and RME-6.

(4) The authors observed a reduction of ESCRT-0 components in USP8 mutant cells, could this contribute to the late endosome/lysosome defects?

Good suggestion. In wild-type animals, the two ESCRT-0 components, HGRS-1 and STAM-1, exhibit a distinct punctate distribution (Figure 1G and H). However, in usp-50 mutants, the punctate signals of HGRS-1::GFP and STAM-1::GFP are significantly diminished (Figure 1G and H; and Figure 1-figure supplement 1B), which aligns with the role of USP-50 in stabilizing the ESCRT-0 complex. To investigate whether the reduction in ESCRT components might contribute to defects in late endosome/lysosome formation, we examined stam-1 mutants. In stam-1 mutants, we observed a reduction in the size of RAB-7-coated vesicles (Author response image 1). Further, when we introduced the lysosomal marker LAAT-1::GFP into stam-1 mutants, we found a substantial decrease in lysosomal structures compared to wild-type animals (Author response image 1). This suggests that the ESCRT complex is essential for proper late endosome/lysosome formation. In contrast, the morphology of early endosomes, as indicated by the 2xFYVE marker, appeared normal in stam-1 mutants, similar to wild-type animals (Author response image 1). This implies that while a reduction in ESCRT-0 components may contribute to the late endosome/lysosome defects observed in usp-50 mutants, the early endosome enlargement phenotype in _usp-5_0 mutants may involve additional mechanisms.

(5) Rabx5 is accumulated in USP8 mutant cells, I am very curious about the phenotype of USP8-Rabx5 double mutants. Could over-expression of Rabx5 (wild type or mutant forms) cause any defects?

Excellent suggestions. To address the question, we employed the CRISPR/Cas9 technique to create a molecular null for rabx-5 (Figure 4E). In the rabx-5(null) mutant animals, we observed that the punctate USP-50::GFP signal became diffusely distributed (Figure 4F and G). This suggests that rabx-5 is necessary for the endosomal localization of USP-50. Interestingly, in rabx-5(null) mutant animals, the 2xFYVE-labeled early endosomes appeared similar to those in wild-type animals (Figure 4H and I). When rabx-5(null) was introduced into usp-50 mutant animals, the enlarged early endosome phenotype observed in usp-50 was significantly suppressed (Figure 4H and I). This finding indicates that usp-50 indeed functions through rabx-5 to regulate early endosome size. Additionally, we constructed strains overexpressing either wild-type or K323R mutant RABX-5. Our results showed that overexpression of wild-type RABX-5 led to early endosome enlargement (as indicated by YFP::2xFYVE labeling) (Author response image 4A, B and D). In contrast, overexpression of the K323R mutant RABX-5 did not result in noticeable early endosome enlargement (Author response image 4A, C and D). Together, these data are in consistent with our model that USP-50 may regulate RABX-5 by deubiquitinating the K323 site.

Author response image 4.

(A-C) Over-expression wild type RABX-5 causes enlarged EEs (labeled by YFP::2xFYVE) while RABX-5(K323R) mutant form does not. (D) Quantification of the volume of individual YFP::2xFYVE vesicles. Data are presented as mean ± SEM. ****P<0.0001. ns, not significant. One-way ANOVA with Tukey’s test.

(6) Rabx5 could be ubiquitinated at K88 and K323, and Rabx5-K323R showed different activity when compared with the wild-type protein in USP8 mutant cells. Could the authors provide evidence that USP8 could remove the ubiquitin modification from K323 in Rabx5 protein?

We appreciate the reviewer's insightful suggestions. To explore the potential of USP-50 in removing ubiquitin modifications from lysine 323 on the RABX-5 protein, we undertook a series of experiments. Initially, we sought to determine whether USP-50 influences the ubiquitination level of RABX-5 in vivo. However, due to the low expression levels of USP-50, we encountered challenges in obtaining adequate amounts of USP-50 protein from worm lysates. To overcome this, we expressed USP-50::4xFLAG in HEK293 cells for subsequent affinity purification. Concurrently, we utilized anti-GFP agarose beads to purify RABX-5::GFP from worms expressing the rabx-5::gfp construct. We then incubated RABX-5::GFP with USP-50::4xFLAG for varying durations and performed immunoblotting with an anti-ubiquitin antibody. As shown in Author response image 5A, our results revealed a decrease in the ubiquitination level of RABX-5 in the presence of USP-50, suggesting that USP-50 directly deubiquitinates RABX-5. Previous studies have indicated that only a minor fraction of recombinant RABX-5 undergoes ubiquitination in HeLa cells, which is believed to have functional significance7. Our findings are consistent with this observation, as only a small fraction of RABX-5 in worms is ubiquitinated. Rabex-5 is known to interact with both K63- and K48-linked poly-ubiquitin chains. To further elucidate whether USP-50 specifically targets K48 or K63-linked ubiquitination at the K323 site of RABX-5, we incubated various HA-tagged ubiquitin mutants with either wild-type or K323R mutant RABX-5 protein. Our results indicated that the K323R mutation reduces K63-linked ubiquitination of RABX-5 (Author response image 5). This experiment was repeated multiple times with consistent results. Additionally, while overexpression of wild-type RABX-5 led to an enlargement of early endosomes, as evidenced by YFP::2xFYVE labeling, overexpression of the K323R mutant did not produce a noticeable effect on endosome size (Author response image 4). Collectively, this finding indicates that RABX-5 is subject to ubiquitin modification in vivo and that USP-50 plays a significant role in regulating this modification at the K323 site.

Author response image 5.

(A) RABX-5::GFP protein was purified from worm lysates using anti-GFP antibody. FLAG-tagged USP-50 was purified from HEK293T cells using anti-FLAG antibody. Purified RABX-5::GFP was incubated with USP-50::4FLAG for indicated times (0, 15, 30, 60 mins), followed by immunoblotting using antibody against ubiquitin, FLAG or GFP. In the presence of USP-50::4xFLAG, the ubiquitination level of RABX-5::GFP is decreased. (B) Quantification of RABX-5::GFP ubiquitination level from three independent experiments. (C) HEK293T cells were transfected with HA-Ub or indicated mutants and 4xFLAG tagged RABX-5 or RABX-5 K323R mutant for 48h. The cells were subjected to pull down using the FLAG beads, followed by immunoblotting using antibody against HA or Flag.

(7) The authors described "the almost identical phenotype of usp-50/usp8 and sand-1/Mon1 mutants", found protein-protein interaction between USP8 and sand-1, and showed that sand1-GFP signal is diminished in USP8 mutant cells. These observations fit with the possibility that USP8 regulates the stability of sand-1 to promote endosomal maturation. Could this be tested and integrated into the current model?

are grateful for the insightful comments provided by the reviewer. Rab5, known to be activated by Rabex-5, plays a crucial role in the homotypic fusion of early endosomes. Rab5 effectors also include the Rab7 GEF SAND-1/Mon1–Ccz1 complex. Rab7 activation by SAND-1/Mon1-Ccz1 complex is essential for the biogenesis and positioning of late endosomes (LEs) and lysosomes, and for the fusion of endosomes and autophagosomes with lysosomes. The Mon1-Ccz1 complex is able to interact with Rabex5, causing dissociation of Rabex5 from the membrane, which probably terminates the positive feedback loop of Rab5 activation and then promotes the recruitment and activation of Rab7 on endosomes. In our study, we identified an interaction between USP-50 and the Rab5 GEF, RABX-5. In the absence of USP-50, we observed an increased endosomal localization of RABX-5 and the formation of abnormally enlarged early endosomes. This phenotype is reminiscent of that seen in sand-1 loss-of-function mutants, which also exhibit enlarged early endosomes and a concomitant reduction in late endosomes/lysosomes. Notably, USP-50 also interacts with SAND-1, suggesting a potential role in regulating its localization. We could propose several models to elucidate how USP-50 might influence SAND-1 localization, including:

(1) USP-50 may stabilize SAND-1 through direct de-ubiquitination.

(2) In the absence of USP-50, the sustained presence of RABX-5 could lead to continuous Rab5 activation, which might hinder or delay the recruitment of SAND-1.

(3) USP-50 could facilitate SAND-1 recruitment by promoting the dissociation of RABX-5.

We are actively investigating these models in our laboratory. Due to space constraints, a more detailed exploration of how USP-50 regulates SAND-1 stability will be presented in a separate publication.

References:

(1) Schmitz, G., and Müller, G. (1991). Structure and function of lamellar bodies, lipid-protein complexes involved in storage and secretion of cellular lipids. J Lipid Res 32, 1539-1570.

(2) Dietl, P., and Frick, M. (2021). Channels and Transporters of the Pulmonary Lamellar Body in Health and Disease. Cells-Basel 11. https://doi.org/10.3390/cells11010045.

(3) Raposo, G., Marks, M.S., and Cutler, D.F. (2007). Lysosome-related organelles: driving post-Golgi compartments into specialisation. Current opinion in cell biology 19, 394-401. https://doi.org/10.1016/j.ceb.2007.05.001.

(4) Weaver, T.E., Na, C.L., and Stahlman, M. (2002). Biogenesis of lamellar bodies, lysosome-related organelles involved in storage and secretion of pulmonary surfactant. Semin Cell Dev Biol 13, 263-270. https://doi.org/10.1016/s1084952102000551.

(5) Ott, D.P., Desai, S., Solinger, J.A., Kaech, A., and Spang, A. (2024). Coordination between ESCRT function and Rab conversion during endosome maturation. bioRxiv, 2024.2005.2014.594104. https://doi.org/10.1101/2024.05.14.594104.

(6) Sato, M., Sato, K., Fonarev, P., Huang, C.J., Liou, W., and Grant, B.D. (2005). Caenorhabditis elegans RME-6 is a novel regulator of RAB-5 at the clathrin-coated pit. Nature cell biology 7, 559-569. https://doi.org/10.1038/ncb1261.

(7) Mattera, R., Tsai, Y.C., Weissman, A.M., and Bonifacino, J.S. (2006). The Rab5 guanine nucleotide exchange factor Rabex-5 binds ubiquitin (Ub) and functions as a Ub ligase through an atypical Ub-interacting motif and a zinc finger domain. The Journal of biological chemistry 281, 6874-6883. https://doi.org/10.1074/jbc.M509939200.

eLife Assessment

This study demonstrates mRNA-specific regulation of translation by subunits of the eukaryotic initiation factor complex 3 (eIF3) using convincing methods, data, and analyses. The investigations have generated important information that will be of interest to biologists studying translation regulation. However, the physiological significance of the gene expression changes that were observed is not clear.

Reviewer #1 (Public review):

Summary:<br /> In this manuscript, Herrmannova et al explore changes in translation upon individual depletion of three subunits of the eIF3 complex (d, e and h) in mammalian cells. The authors provide a detailed analysis of regulated transcripts, followed by validation by RT-qPCR and/or Western blot of targets of interest, as well as GO and KKEG pathway analysis. The authors confirm prior observations that eIF3, despite being a general translation initiation factor, functions in mRNA-specific regulation, and that eIF3 is important for translation re-initiation. They show that global effects of eIF3e and eIF3d depletion on translation and cell growth are concordant. Their results support and extend previous reports suggesting that both factors control translation of 5'TOP mRNAs. Interestingly, they identify MAPK pathway components as a group of targets coordinately regulated by eIF3 d/e. The authors also discuss discrepancies with other reports analyzing eIF3e function.

Strengths:<br /> Altogether, a solid analysis of eIF3 d/e/h-mediated translation regulation of specific transcripts. The data will be useful for scientists working in the Translation field.

Weaknesses:<br /> The authors could have explored in more detail some of their novel observations, as well as their impact on cell behavior.

The manuscript has improved with the new corrections. I appreciate the authors' attention to the minor comments, which have been fully solved. The authors have not, however, provided additional experimental evidence that uORF-mediated translation of Raf-1 mRNA depends on an intact eIF3 complex, nor have they addressed the consequences of such regulation for cell physiology. While I understand that this is a subject of follow-up research, the authors could have at least included their explanations/ speculations regarding major comments 2-4, which in my opinion could have been useful for the reader.

Reviewer #2 (Public review):

Summary:<br /> mRNA translation regulation permits cells to rapidly adapt to diverse stimuli by fine tuning gene expression. Specifically, the 13-subunit eukaryotic initiation factor 3 (eIF3) complex is critical for translation initiation as it aids in 48S PIC assembly to allow for ribosome scanning. In addition, eIF3 has been shown to drive transcript-specific translation by binding mRNA 5' cap structures through the eIF3d subunit. Dysregulation of eIF3 has been implicated in oncogenesis, however the precise eIF3 subunit contributions are unclear. Here, Herrmannová et al. aim to investigate how eIF3 subcomplexes, generated by knock down (KD) of either eIF3e, eIF3d or eIF3h, affect the global translatome. Using Ribo-seq and RNA-seq, the authors identified a large number of genes that exhibit altered translation efficiency upon eIF3d/e KD, while translation defects upon eIF3h KD were mild. eIF3d/eKD share multiple dysregulated transcripts, perhaps due to both subcomplexes lacking eIF3d. Both eIF3d/e KD increase translation efficiency (TE) of transcripts encoding lysosomal, ER and ribosomal proteins, suggesting a role of eIF3 in ribosome biogenesis and protein quality control. Many transcripts encoding ribosomal proteins harbor a TOP motif, and eIF3d KD and eIF3e KD cells exhibit a striking induction of these TOP-modified transcripts. On the other hand, eIF3d KD and eIF3e KD leads to a reduction of MAPK/ERK pathway proteins. Despite this downregulation, eIF3d KD and eIF3e KD activates MAPK/ERK signaling as ERK1/2 and c-Jun phosphorylation was induced. Finally, in all three knockdowns, MDM2 and ATF4 protein levels are reduced. This is notable because MDM2 and ATF4 both contain short uORFs upstream of the start codon, and further supports a role of eIF3 in reinitiation. Altogether, Herrmannová et al. have gained key insights to precise eIF3-mediated translational control as it relates to key signaling pathways implicated in cancer.

Strengths:<br /> The authors have provided a comprehensive set of data to analyze RNA and ribosome footprinting upon perturbation of eIF3d, eIF3e, and eIF3h. As described above in the summary, these data present many interesting starting points to understand additional roles of the eIF3 complex and specific subunits in translational control.

Weaknesses:<br /> - The differences between eIF3e and eIF3d knockdown are difficult to reconcile, especially since eIF3e knockdown leads to reduction in eIF3d levels.<br /> - The paper would be strengthened by experiments directly testing what RNA determinants allow for transcript-specific translation regulation by the eIF3 complex. This would allow the paper to be less descriptive.<br /> - The paper would have more biological relevance if eIF3 subunits were perturbed to mimic naturally occurring situations where eIF3 is dysregulated. For example, eIF3e is aberrantly upregulated in certain cancers, and therefore an overexpression and profiling experiment would have been more relevant than a knockdown experiment.

The first review is unchanged as no additional experiments were provided to address the first review.

Reviewer #3 (Public review):

Summary:<br /> In this article, Hermannova et al catalog the changes in ribosome association with mRNAs when the multisubunit eukaryotic translation initiation factor 3 is disrupted by knocking down individual subunits. They find that RNAs relying on TOP motifs for translation, such as ribosomal protein RNAs, and RNAs encoding modification enzymes in the ER and components of the lysosome are upregulated. In contrast, proteins encoding components of MAP kinase cascades are downregulated when subunits of eIF3 are knocked down, but retain elevated levels of activity.

Strengths:<br /> The authors use ribosome profiling of well-characterized mutants lacking subunits of eIF3 and assess the changes in translation that take place. They supplement the ribosome association studies with western blotting to determine protein level changes of affected transcripts. They analyze what transcripts undergo translation changes, which is important for understanding more broadly how translation initiation factor levels affect cancer cell translatomes. Changes observed by both ribosome profiling and western blotting supports their claims that eIF3 functions in mRNA-specific control of translation.

Weaknesses:<br /> (1) The paper would be strengthened if there were a clear model tying the various effects together or linking individual subunit knockdown to cancerous phenotypes. It is noted that the authors plan to address such outcomes of eIF3 dysregulation in future work, which will be of interest.

(2) The paper could also be strengthened if some of the experiments were performed in at least one other cell type to determine whether changes observed are general or cell-type specific. The authors discuss this issue and provide a literature citation to support a more general mechanism.

If you came from programming background; Each year divisible by 4 is a leap year. EXPECT Each year divisible by 100 is not. BUT Each year, that is divisible by 100, which is also divisible by 400, is a leap year. div 4 to div 100 to div 400

Ontstopping Antwerpen

• 期望值计算的基本原理：事件发生的概率乘以事件发生时的"代价"（在这里是检查次数）

我们利用期望来计算平均时间复杂度

如果元素出现在第二个位置，我们需要检查两次，这里权重就是2

• 期望的计算方法： 期望是该变量输出值的加权平均

. From this study, we concluded that over the course of a whole small-sided game, the forward-backward motion of the centroids is most strongly linearly related. Furthermore, goals show a specific pattern in the forward-backward motion of the centroid. Therefore, surface area and particularly centroid position may provide a sound basis for a collective variable that captures the dynamics of attacking and defending in soccer at team level. Future research should develop these ideas further

eLife Assessment

This study presents an important finding on durotaxis in various amoeboid cells that is independent of focal adhesions. The evidence supporting the authors' claims is compelling. The work will be of interest to cell biologists and biophysicists working on rigidity sensing, the cytoskeleton, and cell migration.

Reviewer #1 (Public review):

In their paper, Kang et al. investigate rigidity sensing in amoeboid cells, showing that, despite their lack of proper focal adhesions, amoeboid migration of single cells is impacted by substrate rigidity. In fact, many different amoeboid cell types can durotax, meaning that they preferentially move towards the stiffer side of a rigidity gradient.

The authors observed that NMIIA is required for durotaxis and, buiding on this observation, they generated a model to explain how durotaxis could be achieved in the absence of strong adhesions. According to the model, substrate stiffness alters the diffusion rate of NMAII, with softer substrates allowing for faster diffusion. This allows for NMAII accumulation at the back, which, in turn, results in durotaxis.

The evidence provided for durotaxis of non adherent (or low-adhering) cells is strong. I am particularly impressed by the fact that amoeboid cells can durotax even when not confined. I wish to congratulate the authors for the excellent work, which will fuel discussion in the field of cell adhesion and migration.

Reviewer #2 (Public review):

Summary:

The authors developed an imaging-based device, that provides both spatial confinement and stiffness gradient, to investigate if and how amoeboid cells, including T cells, neutrophils and Dictyostelium can durotax. Furthermore, the authors showed that the mechanism for the directional migration of T cells and neutrophils depends on non-muscle myosin IIA (NMIIA) polarized towards the soft-matrix-side. Finally, they developed a mathematical model of an active gel that captures the behavior of the cells described in vitro.

Strengths:

The topic is intriguing as durotaxis is essentially thought to be a direct consequence of mechanosensing at focal adhesions. To the best of my knowledge, this is the first report on amoeboid cells that are not dependent on FAs to exert durotaxis. The authors developed an imaging-based durotaxis device that provides both spatial confinement and stiffness gradient and they also utilized several techniques such as quantitative fluorescent speckle microscopy and expansion microscopy. The results of this study have well-designed control experiments and are therefore convincing.

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #1 (Public Review):

In their paper, Kang et al. investigate rigidity sensing in amoeboid cells, showing that, despite their lack of proper focal adhesions, amoeboid migration of single cells is impacted by substrate rigidity. In fact, many different amoeboid cell types can durotax, meaning that they preferentially move towards the stiffer side of a rigidity gradient. 

The authors observed that NMIIA is required for durotaxis and, buiding on this observation, they generated a model to explain how durotaxis could be achieved in the absence of strong adhesions. According to the model, substrate stiffness alters the diffusion rate of NMAII, with softer substrates allowing for faster diffusion. This allows for NMAII accumulation at the back, which, in turn, results in durotaxis. 

The authors responded to all my comments and I have nothing to add. The evidence provided for durotaxis of non adherent (or low-adhering) cells is strong. I am particularly impressed by the fact that amoeboid cells can durotax even when not confined. I wish to congratulate the authors for the excellent work, which will fuel discussion in the field of cell adhesion and migration.

We thank the reviewer for critically evaluating our work and giving kind suggestions. We are glad that the reviewer found our work to be of potential interest to the broad scientific community.

Reviewer #2 (Public Review):

Summary:

The authors developed an imaging-based device that provides both spatialconfinement and stiffness gradient to investigate if and how amoeboid cells, including T cells, neutrophils, and Dictyostelium, can durotax. Furthermore, the authors showed that the mechanism for the directional migration of T cells and neutrophils depends on non-muscle myosin IIA (NMIIA) polarized towards the soft-matrix-side. Finally, they developed a mathematical model of an active gel that captures the behavior of the cells described in vitro.

Strengths:

The topic is intriguing as durotaxis is essentially thought to be a direct consequence of mechanosensing at focal adhesions. To the best of my knowledge, this is the first report on amoeboid cells that do not depend on FAs to exert durotaxis. The authors developed an imaging-based durotaxis device that provides both spatial confinement and stiffness gradient and they also utilized several techniques such as quantitative fluorescent speckle microscopy and expansion microscopy. The results of this study have well-designed control experiments and are therefore convincing.

Weaknesses:

Overall this study is well performed but there are still some minor issues I recommend the authors address:

(1) When using NMIIA/NMIIB knockdown cell lines to distinguish the role of NMIIA and NMIIB in amoeboid durotaxis, it would be better if the authors took compensatory effects into account.

We thank the reviewer for this suggestion. We have investigated the compensation of myosin in NMIIA and NMIIB KD HL-60 cells using Western blot and added this result in our updated manuscript (Fig. S4B, C). The results showed that the level of NMIIB protein in NMIIA KD cells doubled while there was no compensatory upregulation of NMIIA in NMIIB KD cells. This is consistent with our conclusion that NMIIA rather than NMIIB is responsible for amoeboid durotaxis since in NMIIA KD cells, compensatory upregulation of NMIIB did not rescue the durotaxis-deficient phenotype. 

(2) The expansion microscopy assay is not clearly described and some details are missed such as how the assay is performed on cells under confinement.

We thank the reviewer for this comment. We have updated details of the expansion microscopy assay in our revised manuscript in line 481-485 including how the assay is performed on cells under confinement:

Briefly, CD4+ Naïve T cells were seeded on a gradient PA gel with another upper gel providing confinement. 4% PFA was used to fix cells for 15 min at room temperature. After fixation, the upper gradient PA gel is carefully removed and the bottom gradient PA gel with seeded cells were immersed in an anchoring solution containing 1% acrylamide and 0.7% formaldehyde (Sigma, F8775) for 5 h at 37 °C.

(3) In this study, an active gel model was employed to capture experimental observations. Previously, some active nematic models were also considered to describe cell migration, which is controlled by filament contraction. I suggest the authors provide a short discussion on the comparison between the present theory and those prior models.

We thank the reviewer for this suggestion. Active nematic models have been employed to recapitulate many phenomena during cell migration (Nat Commun., 2018, doi: 10.1038/s41467-018-05666-8.). The active nematic model describes the motion of cells using the orientation field, Q, and the velocity field, u. The director field n with (n = −n) is employed to represent the nematic state, which has head-tail symmetry. However, in our experiments, actin filaments are obviously polarized, which polymerize and flow towards the direction of cell migration. Therefore, we choose active gel model which describes polarized actin field during cell migration. In the discussion part, we have provided the comparison between active gel model and motor-clutch model. We have also supplemented a short discussion between the present model and active nematic model in the main text of line 345-347:

The active nematic model employs active extensile or contractile agents to push or pull the fluid along their elongation axis to simulate cells flowing (61). 

(4) In the present model, actin flow contributes to cell migration while myosin distribution determines cell polarity. How does this model couple actin and myosin together?

We thank the reviewer for this question. In our model, the polarization field is employed to couple actin and myosin together. It is obvious that actin accumulate at the front while myosin diffuses in the opposite direction. Therefore, we propose that actin and myosin flow towards the opposite direction, which is captured in the convection term of actin ) and myosin () density field.

ir is not just onenes

Para criar um subclasse de uma classe principal utiliza-se a seguinte sintaxe java public class PlaceManager extends UnicastRemoteObject para implementar a interface java public class PlaceManager extends UnicastRemoteObject implements PlacesListInterface

eLife Assessment

The study is considered important with solid evidence that demonstrates the impact of plasma membrane nano-domains and protein interactions in the plant defence response to viruses. It includes a molecular understanding of the role of a calcium dependent kinase (CPK3) and a remorin protein in the cell-to-cell spread of viruses and cytoskeletal dynamics demonstrating, conclusively, the role of CPK3 with multiple lines of evidence. The work opens avenues to investigate different viruses and other plasma membrane proteins to gain a fuller picture of the involvement of plasmodesmata and other nanodomains in virus spreading.

Reviewer #1 (Public review):

Summary:

How plants perceive their environment and signal during growth and development is of fundamental importance for plant biology. Over the last few decades, nano domain organisation of proteins localised within the plasma-membrane has emerged as a way of organising proteins involved in signal pathways. Here, the authors addressed how a non-surface localised signal (viral infection) was resisted by PM localised signalling proteins and the effect of nano domain organisation during this process. This is valuable work as it describes how an intracellular process affects signalling at the PM where most previous work has focused on the other way round, PM signalling effecting downstream responses in the plant. They identify CPK3 as a specific calcium dependent protein kinase which is important for inhibiting viral spread. The authors then go on to show that CPK3 diffusion in the membrane is reduced after viral infection and study the interaction between CPK3 and the remorins, which are a group of scaffold proteins important in nano domain organisation. The authors conclude that there is an interdependence between CPK3 and remorins to control their dynamics during viral infection in plants.

Strengths:

The dissection of which CPK was involved in the viral propagation was masterful and very conclusive. Identifying CPK3 through knockout time course monitoring of viral movement was very convincing. The inclusion of overexpression, constitutively active and point mutation non-functioning lines further added to that.

Weaknesses:

I would like to thank the researchers for including some additional work suggested in the previous round of peer review. However, I still have concerns over this work which are two fold.

(1) Firstly, the imaging described and shown is not sufficient to support the claims made. The PM localisation and its non-PM localised form look similar and with no PM stain or marker construct used to support this. In addition, the quality of lots of the confocal based imaging (including new figure on colocalisation) is simply not sufficient. The images are too noisy and no clear conclusions can be made. The point made previously, the system this data was collected on has an Airyscan detector capable of 120nm resolution and as such NDs can be resolved. The sptPALM data conclusions are nice and fit the narrative. The inclusion of sptPALM movies is useful for the reader and tracks numbers is highly beneficial. But they do not show a high signal to noise ratio compared to other work in the field (see work from Alex Martineire) and the mEOS prticles are only just observable over the detector noise in some videos. As such, I worry about the data quality on which the analysis is based on. In addition, in some of the videos the conversion laser seems too high as it is difficult to separate some of the single particles as they emerge which would again, hinder the analysis.

(2) Secondly, remorins are involved in a lot of nano domain controlled processes at the PM. The authors have not conclusively demonstrated that during viral infection the remorin effects seen are solely due to its interaction with CPK3. The sptPALM imaging of REM1.2 in a cpk3 knockout line goes part way to solve this and the inclusion of CPK3-CA also strengthens the authors claims. But to propose a kiss and go model bearing in mind the differences in diffusion between CPK3 and REM3 and differential changes to diffusion between the two proteins after PIAMV infection without two colour imaging of both proteins at the same time, the claims are much stronger than the evidence. Negative control experiments are required here utilising other PM localised proteins which have no role during viral infection (such as Lti6B).

Overall, I think this work has the potential to be a very strong manuscript but additional evidence supporting interaction claims would significantly strengthen the work and make it exceptional.

Reviewer #3 (Public review):

Summary:

This study examined the role that the activation and plasma membrane localisation of a calcium dependent protein kinase (CPK3) plays in plant defence against viruses.<br /> The authors clearly demonstrate that the ability to hamper the cell-to-cell spread of the virus P1AMV is not common to other CPKs which have roles in defence against different types of pathogens, but appears to be specific to CPK3 in Arabidopsis. Further, they show that lateral diffusion of CPK3 in the plasma membrane is reduced upon P1AMV infection, with CPK3 likely present in nano-domains. This stabilisation however, depends on one of its phosphorylation substrates a Remorin scaffold protein REM1-2. However, when REM1-2 lateral diffusion was tracked, it showed an increase in movement in response to P1AMV infection. These contrary responses to P1AMV infection were further demonstrated to be interdependent, which led the authors to propose a model in which activated CPK3 is stabilised in nano-domains in part by its interaction with REM1.2, which it binds and phosphorylates, allowing REM1-2 to diffuse more dynamically within the membrane.

The likely impact of this work is that it will lead to closer examination of the formation of nano-domains in the plasma membrane and dissection of their role in immunity to viruses, as well as further investigation into the specific mechanisms by which CPK3 and REM1-2 inhibit the cell-to-cell spread of viruses, including examination of their roles in cytoskeletal dynamics.

Strengths:

The paper provided compelling evidence about the roles of CPK3 and REM1-2 through a combination of logical reverse genetics experiments and advanced microscopy techniques, particularly in single particle tracking.

Weaknesses:

There is limited discussion or exploration of the role that CPK3 has in cytoskeletal organisation and whether this may play a role in the plant's defence against viral propagation. Further. although the authors show that there is no accumulation of CPK3/Rem1.2 at plasmodesmata, it would be interesting to investigate whether the demonstrated reduction of viral propagation is due to changes in PD permeability.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

How plants perceive their environment and signal during growth and development is of fundamental importance for plant biology. Over the last few decades, nano domain organisation of proteins localised within the plasma-membrane has emerged as a way of organising proteins involved in signal pathways. Here, the authors addressed how a non-surface localised signal (viral infection) was resisted by PM localised signalling proteins and the effect of nano domain organisation during this process. This is valuable work as it describes how an intracellular process affects signalling at the PM where most previous work has focused on the other way round, PM signalling effecting downstream responses in the plant. They identify CPK3 as a specific calcium dependent protein kinase which is important for inhibiting viral spread. The authors then go on to show that CPK3 diffusion in the membrane is reduced after viral infection and study the interaction between CPK3 and the remorins, which are a group of scaffold proteins important in nano domain organisation. The authors conclude that there is an interdependence between CPK3 and remorins to control their dynamics during viral infection in plants.

Strengths:

The dissection of which CPK was involved in the viral propagation was masterful and very conclusive. Identifying CPK3 through knockout time course monitoring of viral movement was very convincing. The inclusion of overexpression, constitutively active and point mutation non functioning lines further added to that.

Weaknesses:

My main concerns with the work are twofold.

(1) Firstly, the imaging described and shown is not sufficient to support the claims made. The PM localisation and its non-PM localised form look similar and with no PM stain or marker construct used to support this. The sptPALM data conclusions are nice and fit the narrative. However, no raw data or movie is shown, only representative tracks. Therefore, the data quality cannot be verified and in addition, the reporting of number of single particle events visualised per experiment is absent, only number of cells imaged is reported. Therefore, it is impossible for the reader to appreciate the number of single molecule behaviours obtained and hence the quality of the data.

(2) Secondly, remorins are involved in a lot of nanodomain controlled processes at the PM. The authors have not conclusively demonstrated that during viral infection the remorin effects seen are solely due to its interaction with CPK3. The sptPALM imaging of REM1.2 in a cpk3 knockout line goes part way to solve this but more evidence would strengthen it in my opinion. How do we not know that during viral infection the entire PM protein dynamics and organisation are altered? Or that CPK3 and REM are at very distant ends of a signalling cascade. Negative control experiments are required here utilising other PM localised proteins which have no role during viral infection. In addition, if the interaction is specific, the transiently expressed CPK3-CA construct (shown to from nano domains) should be expressed with REM1.2-mEOS to show the alterations in single particle behaviour occur due to specific activations of CPK3 and REM1.2 in the absence of PIAMV viral infection and it is not an artefact of whole PM changes in dynamics during viral infection.

In addition, displaying more information throughout the manuscript (such as raw particle tracking movies and numbers of tracks measured) on the already generated data would strengthen the manuscript further.

Overall, I think this work has the potential to be a very strong manuscript but additional reporting of methods and data are required and additional lines of evidence supporting interaction claims would significantly strengthen the work and make it exceptional.

Reviewer #2 (Public Review):

Summary:

The paper provides evidence that CPK3 plays a role in plant virus infection, and reports that viral infection is accompanied by changes in the dynamics of CPK3 and REM1.2, the phosphorylation substrate of CPK3, in the plasma membrane. In addition, the dynamics of the two proteins in the PM are shown to be interdependent.

Strengths:

The paper contains novel, important information.

Weaknesses:

The interpretation of some experimental data is not justified, and the proposed model is not fully based on the available data.

Reviewer #3 (Public Review):

Summary:

This study examined the role that the activation and plasma membrane localisation of a calcium dependent protein kinase (CPK3) plays in plant defence against viruses.<br /> The authors clearly demonstrate that the ability to hamper the cell-to-cell spread of the virus P1AMV is not common to other CPKs which have roles in defence against different types of pathogens, but appears to be specific to CPK3 in Arabidopsis. Further they show that lateral diffusion of CPK3 in the plasma membrane is reduced upon P1AMV infection, with CPK3 likely present in nano-domains. This stabilisation however, depends on one of its phosphorylation substrates a Remorin scaffold protein REM1-2. However, when REM1-2 lateral diffusion was tracked, it showed an increase in movement in response to P1AMV infection. These contrary responses to P1AMV infection were further demonstrated to be interdependent, which led the authors to propose a model in which activated CPK3 is stabilised in nano-domains in part by its interaction with REM1.2, which it binds and phosphorylates, allowing REM1-2 to diffuse more dynamically within the membrane.

The likely impact of this work is that it will lead to closer examination of the formation of nano-domains in the plasma membrane and dissection of their role in immunity to viruses, as well as further investigation into the specific mechanisms by which CPK3 and REM1-2 inhibit the cell-to-cell spread of viruses.

Strengths:

The paper provided compelling evidence about the roles of CPK3 and REM1-2 through a combination of logical reverse genetics experiments and advanced microscopy techniques, particularly in single particle tracking.

Weaknesses:

There is a lack of evidence for the downstream pathways, specifically whether the role that CPK3 has in cytoskeletal organisation may play a role in the plant's defence against viral propagation. Also, there is limited discussion about the localisation of the nano-domains and whether there is any overlap with plasmodesmata, which as plant viruses utilise PD to move from cell to cell seems an obvious avenue to investigate.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Viral spread work in CPK mutants with time courses is beautiful!

Regarding my public points on my issues with the imaging:

- Figure 2A shows 'PM' localisation of CPK3 and 'non-PM' imaging of CPK3-G2A. The images are near identical both showing cell outlines and cytoplasmic strands. Here a PM marker (such as Lti6B) tagged with a different fluorophore or PM stain should be used in conjunction with surface views (such as in Figure 2C) to show it really is at the PM and the G2A line is not.

Impaired membrane localization of CPK3-G2A is documented in Mehlmer et al., 2010 using microsomal fractionation. Although Figure 2A main purpose is to show correct expression of the constructs in the lines used for PlAMV propagation (Figure 2B), we replaced the images with wider view pictures to be more representative of the subcellular localization of CPK3 and CPK3-G2A.

- Regarding Figure 2C, this is extremely noisy and PM heterogeneity is barely observable over the noise from the system (looking at the edges of surface imaged). You mention low resolution was an issue. I notice from the methods you have taken confocal images on an Zeiss 880 with Airyscan. These images must be confocal but If imaged with Airyscan the PM heterogeneity would be much clearer (see work from John Runions lab).

Indeed, these are tangential views images obtained by Zeiss 880 with Airyscan. Based on tessellation analysis (Figure 2H-J), CPK3 is rather homogeneously distributed and forms ND of around 70nm of diameter. Objects of such size cannot be resolved using pixel reassignment methods such as Airyscan. Note also that AtREM in our study are less heterogeneously distributed than what was described in the literature for StREM1.3.

- Regarding all sptPALM data. At least an example real data image and video is required otherwise the data can’t be assessed. The work of Alex Martiniere (sptPALM) or Alex Jonson (TIRF) all show raw data so the reader can appreciate the quality of the data. In addition, number of events (particles tracked) has to be shown in the figure legend, not just number of cells otherwise was one track performed per cell? Or 10,000? Obviously where the N sits in this range gives the reader more or less confidence of the data.

We agree and we added example videos of sptPALM experiments in the supplementary data, we also indicated the number of tracked particles in the figure legends.

- On a slight technical aside, how do you know the cells being imaged for sptPALM with PIAMV are actually infected with the virus? In Fig 2C you use a GFP tagged version but in sptPALM you use none tagged. I think a sentence in methods on this would help clarify.

PlAMV-GFP was used for spt-PALM experiment and cell infection was assessed during PALM experiment. This is now precised in the corresponding figures and methods.

- I also have a concern over some of the representative images showing the same things between different figures. Your clustering data in 3F looks very convincing. However, in Figure 2H the mock and PIAMV-GFP look very similar. How is Figure 3F so different for the same experiment? Especially considering the scale bars are the same for both figures. Same for CPK3-mRFP1.2 in Fig 2C and 3A, the same thing is being imaged, at the same scale (scale bars same size) but the images are extremely different.

Figure 2 data were generated using CPK3 stably expressed in A. thaliana while Figure 3 data were obtained upon transient over-expression of CPK3 in N. benthamiana. We do not have a clear explanation for such a difference in CPK3 PM behavior, it could lie on a different PM composition or actin organization between those two species, this point is now addressed in the discussion.

- Line 193&194 - you state that the CA CPK3 is reminiscent of the CPK3 upon PIAMV expression. I don't agree, while CPK3CA is less mobile (2D), the MSD shows it is in-between CPK3 and CPK3 + PIAMV. Therefore, can’t the opposite also be true? That overall the behaviour of CPK3-CA is reminiscent of WT CPK. I think this needs rewording.

We agree and we reworded that part

- Line 651 - what numerical aperture are you using for the lens during confocal microscopy. This is fundamentally important information directly related to the reproducibility of the work. You report it for the sptPALM.

The numerical aperture is now indicated in the methods.

Regarding my bigger point about specific interactions between CPK3 and remorin during viral infection to strengthen your claim the following need doing. I am not suggesting you do all of these but at least two would significantly enhance the paper.

(1) Image a none related PM protein during viral infection using sptPALM and demonstrate that its behaviour is not altered (such as lti6b). This would show the affects on remorin behaviour are specific to CPK3 and not a whole scale PM alteration in dynamics due to viral infection.

(2) Two colour SPT imaging of CPK3 and REM1.2. You show in absence of proteins (knockouts effect on each other) but your only interaction data is from a kinase assay (where CPK1 and 2 also interact, even though they are not localised at the same place) and colocalisation data (see below). A two colour SPT imaging experiment showing interaction and clustering of CPK3 and REM1.2 with each other and the change in their behaviours when viral infected and simultaneously imaged would address all of my concerns.

- On another note, the co-localisation data (fig 5 sup 4) needs additional analysis. I would expect most PM proteins to show the results you show as the data is very noisy. In order to improve I would zoom in to fill the field of view and then determine correlation and also when one image is rotated 90 degrees (as described in Jarsch et al., plant cell) to enhance this work.

(3) In the absence of viral infection, but presence of CPK3-CA, is sptPALM REM1.2 behaviour in the PM altered, if so then the interaction is specific and changes in remorin dynamics are not due to whole scale PM changes during viral infection and the manuscript substantially strengthened.

(4) Building on from 3), if you have a CPK3 mutated with both CPK3-CA and G2A this would be constitutively active and non-PM localised and as such should not affect Remorin behaviour if your model is true, this would strengthen the case significantly but I appreciate is highly artificial and would need to be done transiently.

Regarding the first point, since the role of PM proteins involved in potexvirus infection is barely assessed, picking a non-related PM protein might be tricky. The data obtained with mEOS3.2-REM1.2 expressed in cpk3 null-mutant point towards a specific role of CPK3 in PlAMV-induced REM1.2 diffusion and not a general alteration of PM protein behavior.

Regarding the second point, we already reported the in vivo interaction between AtCPK3CA and AtREM1.2/AtREM1.3 by BiFC in N.benthamiana (Perraki et al 2018) and AtCPK3 was shown to co-IP with AtREM1.2 (Abel et al, 2021). While we agree on the relevance of doing dual color sptPALM with CPK3 and REM1.2, it is so far technically challenging and we would not be able to implement this in a timely manner. For the colocalization, although the whole cell is displayed in the figure, the analysis was performed on ROI to fill the field of analysis.

We agree with the relevance of adding the colocalization analysis of randomized images (mTagBFP2 channel rotated 90 degrees), this is now added to Figure 5 – supplement figure 5.

Finally, for the third and fourth points, spt-PALM analysis of REM1.2 in presence of CPK3-CA and CPK3-CA-G2A was performed (Figure 5 - figure supplement 4). The results suggest a specific role of CPK3-CA in REM1.2 diffusion.

Minor points:

Line 59 - from, I think you mean from.

Line 63 - Reference needed after latter.

Line 68 - Reference required after viral infection.

Line 85 - Propose not proposed.

Line 156 - Allowed us to not allows to.

Line 204 - add we previously 'demonstrated'

Line 622 and 623 - You say lines obtained from Thomas Ott. This is very odd phrasing considering he is an author. I appreciate citing the work producing the lines but maybe reword this

These points were corrected, thank you.

Reviewer #2 (Recommendations For The Authors):

The paper provides evidence that CPK3 plays a role in plant virus infection, and reports that viral infection is accompanied by changes in the dynamics of CPK3 and REM1.2, the phosphorylation substrate of CPK3, in the plasma membrane. In addition, the dynamics of the two proteins in the PM are shown to be interdependent. The paper contains novel, important information that can undoubtedly be published in eLife. However, I have some concerns that should be addressed before it can be accepted for publication.

Major concerns

When the authors say that CPK3 plays a role in viral propagation, it should be clarified what is meant by 'propagation', - replication of the viral genome, its cell-to-cell transport, or long-distance transport via the phloem. By default the readers will tend to assume the former meaning. In my opinion, the term 'propagation' is misleading and should be avoided.

We purposely chose the term “propagation” because it sums replication and cell-to-cell movement. Nevertheless, we previously showed that group 1 StREM1.3 doesn’t alter PVX replication (Raffaele et al., 2009 The Plant Cell). In this paper, as we do not investigate the role of AtREM1.2 or AtCPK3 in the replication of the viral PlAMV genome, we cannot state that these proteins are strictly involved in cell-to-cell movement of the virus.

The authors show that viral infection is associated with decreased diffusion of CPK3 and increased diffusion of REM1.2 in the PM. However, it remains unclear whether these changes are related to partial resistance to viral infection involving CPK3 and REM1.2, or whether they are simply a consequence of viral infection that may lead to altered PM properties and altered dynamics of PM-associated proteins. Therefore, the model presented in Fig. 6 appears to be entirely speculative, as it postulates that changes in CPK3 and REM1.2 dynamics are the cause of suppressed virus 'propagation'. In addition, the model implies that a decrease in CPK3 mobility leads to activation of its kinase activity. This view is not supported by experimental data (see my next comment). The model should be deleted (both as the figure and its description in the Discussion) or substantially reworked so that it finally relies on existing data.

For the first point, the results obtained from the additional experiments proposed by reviewer #1 supports the hypothesis of a direct impact of CPK3 on REM1.2 diffusion (Figure 5 - figure supplement 4).

We agree with the second point and reworked the model to remove the link between CPK3 activation and its increased diffusion.

The statement that 'changes in CPK3 dynamics upon PlAMV infection are linked to its activation' (line 194) is based on a flawed logic, and the conclusion in this section of Results ('changes in CPK3 dynamics upon PlAMV infection are linked to its activation') is incorrect, as it is not supported by experimental data. In fact, the authors show that CPK3 dynamics and clustering upon viral infection is somewhat reminiscent of the behavior of a CPK3 deletion mutant, which is a constitutively active protein kinase. However, this partial similarity cannot be taken as evidence that CPK3 dynamics upon PlAMV infection are related to its activation. Furthermore, the authors emphasize the similarity of the mutant and CPK3 in infected cells without taking into account a drastic difference in their localization (Fig. 3A, middle and right panels) showing that the reduced dynamics or the compared proteins may have different causes. I suggest the removal of the section 'CPK3 activation leads to its confinement in PM ND' from the paper, as the results included in this section are not directly related to other data presented.

The PM lateral organization of PM-bound CPKs in their native or constitutively active form as well as the role of lipid in such phenomenon was never shown before. We believe that this section contains relevant information for the community. We kept the section but reworded it to tamper the correlation made between CPK3 PM organization upon viral infection and its activation.

Line 270 - 'group 1 REMs might play a role in CPK3 domain stabilization upon viral infection'. This is an overstatement. The size of the CPK3-containing NDs may have no correlation with their stability.

We reworded the sentence.

Minor points

Line 204 - we previously that Line 234 and hereafter - "the D" sounds strange. Suggest using "the diffusion coefficient".

This was reworded.

Reviewer #3 (Recommendations For The Authors):

The authors have previously demonstrated that there was an increase in REM1.2 localisation to plasmodesmata under viral challenge. It would be useful to see if there was any co-localisation of REM1.2 and CPK3 with plasmodesmata in response to PlAMV and how this is affected in the mutants. This could be carried out relatively simply using aniline blue.

These experiments were added to the supplementary data of Figure 2 – figure supplement 2.  and Figure 4 – figure supplement 4. , no enrichment of CPK3 or REM1.2 at plasmodesmata could be observed upon PlAMV infection.

Fig 3 supplementary figure 2 would be better incorporated into the main body of Figure 3 as this underpins discussion on the involvement of lipids such as sterols in the formation of nanodomains.

We moved Figure 3 – Supplementary figure 2 to the main body of Figure 3.

Minor corrections:

Whilst the paper is generally well written there are a number of grammatical errors:

Line 1 & 2: Title doesn't quite read correctly, suggest a rewording for clarity.

L31: Insert "a"after only

L33: Replace "are playing" with "play"

L34: Begin sentence "Viruses are intracellular pathogens and as such the role..."59: replace "form" with "from"

L63: Insert "was demonstrated" after REM1.2)

L85: Replace "proposed" with "propose"

L86: replace "encouraging to explore" with "which will encourage further exploration of "

L129: replace "we'll focus on" with "we concentrated on"

L131: insert "an" before ATP

L138: change "among" to "amongst"

L156: change "allows to analyse" to "allows the analysis of"

L204: Insert "showed" after previously.

L232: "root seedlings" should this be the roots of seedlings?

L235: insert "to" after "as"

L280: insert "a" after "only"

L281: change " to play" with "as playing": change CA to superscript

L307: Insert "was" after "transcription"

L320: change "display" to "displaying"

L321: change "form" to forms"

L340: "hampering" should come before viral

L365: insert"us' after "allow"

Thank you, these were corrected

this is the basic building block of the model

very dubious claim

problematic methods

Il y a une petite erreur avec l'article défini, ils l'ont utilisé au singulier, alors que son adjectif qualificatif est au feminin.

apparent dissolution between the subject and the object

Ideal Gas is a model! It is incorrect based on the fact that gases never have 100% the assumptions it states but it proves to be very useful!

we all need a place to play.

eLife Assessment

This valuable work describes results from a set of simulation and empirical studies of a set-up assessing exploratory behavior in a potentially rewarding environment that contains danger. The core idea is that an instrumental agent can be helped to be both effective and safe, thus avoiding excessive danger, during exploratory behavior, if its influence is flexibly gated by an independent Pavlovian fear learning system. The conclusion that safe, but effective exploration can be achieved based on a flexibly weighted combination of a Pavlovian and an instrumental agent is solid.

Reviewer #1 (Public review):

Summary:

This paper provides a computational model of a synthetic task in which an agent needs to find a trajectory to a rewarding goal in a 2D-grid world, in which certain grid blocks incur a punishment. In a completely unrelated setup without explicit rewards, they then provide a model that explains data from an approach-avoidance experiment in which an agent needs to decide whether to approach or withdraw from, a jellyfish, in order to avoid a pain stimulus, with no explicit rewards. Both models include components that are labelled as Pavlovian; hence the authors argue that their data show that the brain uses a Pavlovian fear system in complex navigational and approach-avoid decisions.

In the first setup, they simulate a model in which a component they label as Pavlovian learns about punishment in each grid block, whereas a Q-learner learns about the optimal path to the goal, using a scalar loss function for rewards and punishments. Pavlovian and Q-learning components are then weighed at each step to produce an action. Unsurprisingly, the authors find that including the Pavlovian component in the model reduces the cumulative punishment incurred, and this increases as the weight of the Pavlovian system increases. The paper does not explore to what extent increasing the punishment loss (while keeping reward loss constant) would lead to the same outcomes with a simpler model architecture, so any claim that the Pavlovian component is required for such a result is not justified by the modelling.

In the second setup, an agent learns about punishments alone. "Pavlovian biases" have previously been demonstrated in this task (i.e. an overavoidance when the correct decision is to approach). The authors explore several models (all of which are dissimilar to the ones used in the first setup) to account for the Pavlovian biases.

Strengths:

Overall, the modelling exercises are interesting and relevant and incrementally expand the space of existing models.

Weaknesses:

I find the conclusions misleading, as they are not supported by the data.

First, the similarity between the models used in the two setups appears to be more semantic than computational or biological. So it is unclear to me how the results can be integrated.

Secondly, the authors do not show "a computational advantage to maintaining a specific fear memory during exploratory decision-making" (as they claim in the abstract). Making such a claim would require showing an advantage in the first place. For the first setup, the simulation results will likely be replicated by a simple Q-learning model when scaling up the loss incurred for punishments, in which case the more complex model architecture would not confer an advantage. The second setup, in contrast, is so excessively artificial that even if a particular model conferred an advantage here, this is highly unlikely to translate into any real-world advantage for a biological agent. The experimental setup was developed to demonstrate the existence of Pavlovian biases, but it is not designed to conclusively investigate how they come about. In a nutshell, who in their right mind would touch a stinging jellyfish 88 times in a short period of time, as the subjects do on average in this task? Furthermore, in which real-life environment does withdrawal from a jellyfish lead to a sting, as in this task?

Crucially, simplistic models such as the present ones can easily solve specifically designed lab tasks with low dimensionality but they will fail in higher-dimensional settings. Biological behaviour in the face of threat is utterly complex and goes far beyond simplistic fight-flight-freeze distinctions (Evans et al., 2019). It would take a leap of faith to assume that human decision-making can be broken down into oversimplified sub-tasks of this sort (and if that were the case, this would require a meta-controller arbitrating the systems for all the sub-tasks, and this meta-controller would then struggle with the dimensionality j).

On the face of it, the VR task provides higher "ecological validity" than previous screen-based tasks. However, in fact, it is only the visual stimulation that differs from a standard screen-based task, whereas the action space is exactly the same. As such, the benefit of VR does not become apparent, and its full potential is foregone.

If the authors are convinced that their model can - then data from naturalistic approach-avoidance VR tasks is publicly available, e.g. (Sporrer et al., 2023), so this should be rather easy to prove or disprove. In summary, I am doubtful that the models have any relevance for real-life human decision-making.

Finally, the authors seem to make much broader claims that their models can solve safety-efficiency dilemmas. However, a combination of a Pavlovian bias and an instrumental learner (study 1) via a fixed linear weighting does not seem to be "safe" in any strict sense. This will lead to the agent making decisions leading to death when the promised reward is large enough (outside perhaps a very specific region of the parameter space). Would it not be more helpful to prune the decision tree according to a fixed threshold (Huys et al., 2012)? So, in a way, the model is useful for avoiding cumulatively excessive pain but not instantaneous destruction. As such, it is not clear what real-life situation is modelled here.

A final caveat regarding Study 1 is the use of a PH associability term as a surrogate for uncertainty. The authors argue that this term provides a good fit to fear-conditioned SCR but that is only true in comparison to simpler RW-type models. Literature using a broader model space suggests that a formal account of uncertainty could fit this conditioned response even better (Tzovara et al., 2018).

Reviewer #2 (Public review):

Summary:

The authors tested the efficiency of a model combining Pavlovian fear valuation and instrumental valuation. This model is amenable to many behavioral decision and learning setups - some of which have been or will be designed to test differences in patients with mental disorders (e.g., anxiety disorder, OCD, etc.).

Strengths:

(1) Simplicity of the model which can at the same time model rather complex environments.

(2) Introduction of a flexible omega parameter.

(3) Direct application to a rather advanced VR task.

(4) The paper is extremely well written. It was a joy to read.

Weaknesses:

Almost none! In very few cases, the explanations could be a bit better.

Reviewer #3 (Public review):

Summary:

This paper aims to address the problem of exploring potentially rewarding environments that contain the danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so.

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards.

Strengths:

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally, there is little to criticise about the study.

Weaknesses:

The extent of the testing in human participants is fairly limited but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later). The description of the models is also a little difficult to parse.

Author response:

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

This paper provides a computational model of a synthetic task in which an agent needs to find a trajectory to a rewarding goal in a 2D-grid world, in which certain grid blocks incur a punishment. In a completely unrelated setup without explicit rewards, they then provide a model that explains data from an approach-avoidance experiment in which an agent needs to decide whether to approach or withdraw from, a jellyfish, in order to avoid a pain stimulus, with no explicit rewards. Both models include components that are labelled as Pavlovian; hence the authors argue that their data show that the brain uses a Pavlovian fear system in complex navigational and approach-avoid decisions. 

We thank the reviewer for their thoughtful comments. To clarify, the grid-world setup was used as a didactic tool/testbed to understand the interaction between Pavlovian and instrumental systems (lines 80-81) [Dayan et al., 2006], specifically in the context of safe exploration and learning. It helps us delineate the Pavlovian contributions during learning, which is key to understanding the safety-efficiency dilemma we highlight. This approach generates a hypothesis about outcome uncertainty-based arbitration between these systems, which we then test in the approach-withdrawal VR experiment based on foundational studies studying Pavlovian biases [Guitart-Masip et al., 2012, Cavanagh et al., 2013].

Although the VR task does not explicitly involve rewards, it provides a specific test of our hypothesis regarding flexible Pavlovian fear bias, similar to how others have tested flexible Pavlovian reward bias without involving punishments (e.g., Dorfman & Gershman, 2019). Both the simulation and VR experiment models are derived from the same theoretical framework and maintain algebraic mapping, differing only in task-specific adaptations (e.g., differing in action sets and temporal difference learning for multi-step decisions in the grid world vs. Rescorla-Wagner rule for single-step decisions in the VR task). This is also true for Dayan et al. [2006] who bridge Pavlovian bias in a Go-No Go task (negative auto-maintenance pecking task) and a grid world task. Therefore, we respectfully disagree that the two setups are completely unrelated and that both models include components merely labelled as Pavlovian.

We will rephrase parts of the manuscript to prevent the main message of our manuscript from being misconveyed. Particularly in the Methods and Discussion, to clarify that our main focus is on Pavlovian fear bias in safe exploration and learning (as also summarised by reviewers #2 and #3), rather than on its role in complex navigational decisions. We also acknowledge the need for future work to capture more sophisticated safe behaviours, such as escapes and sophisticated planning which span different aspects of the threat-imminence continuum [Mobbs et al., 2020], and we will highlight these as avenues for future research.

In the first setup, they simulate a model in which a component they label as Pavlovian learns about punishment in each grid block, whereas a Q-learner learns about the optimal path to the goal, using a scalar loss function for rewards and punishments. Pavlovian and Q-learning components are then weighed at each step to produce an action. Unsurprisingly, the authors find that including the Pavlovian component in the model reduces the cumulative punishment incurred, and this increases as the weight of the Pavlovian system increases. The paper does not explore to what extent increasing the punishment loss (while keeping reward loss constant) would lead to the same outcomes with a simpler model architecture, so any claim that the Pavlovian component is required for such a result is not justified by the modelling. 

Thank you for this comment. We acknowledge that our paper does not compare the Pavlovian fear system to a purely instrumental system with varying punishment sensitivity. Instead, our model assumes the coexistence of these two systems and demonstrates the emergent safety-efficiency trade-off from their interaction. It is possible that similar behaviours could be modelled using an instrumental system alone. In light of the reviewer’s comment, we will soften our claims regarding the necessity of the Pavlovian system, despite its known existence.

We also encourage the reviewer to consider the Pavlovian system as a biologically plausible implementation of punishment sensitivity. Unlike punishment sensitivity (scaling of the punishments), which has not been robustly mapped to neural substrates in fMRI studies, the neural substrates for the Pavlovian fear system (e.g., the limbic loop) are well known (see Supplementary Fig. 16).

Additionally, we point out that varying reward sensitivities while keeping punishment sensitivity constant allows our PAL agent to differentiate from an instrumental agent that combines reward and punishment into a single feedback signal. As highlighted in lines 136-140 and the T-maze experiment (Fig. 3 A, B, C), the Pavlovian system maintains fear responses even under high reward conditions, guiding withdrawal behaviour when necessary (e.g., ω = 0.9 or 1), which is not possible with a purely instrumental model if the punishment sensitivities are fixed. This is a fundamental point.

We will revise our discussion and results sections to reflect these clarifications.

In the second setup, an agent learns about punishments alone. "Pavlovian biases" have previously been demonstrated in this task (i.e. an overavoidance when the correct decision is to approach). The authors explore several models (all of which are dissimilar to the ones used in the first setup) to account for the Pavlovian biases. 

Thank you, we respectfully disagree with the statement that our models used in the experimental setup are dissimilar to the ones used in the first setup. Due to differences in the nature of the task setup, the action set differs, but the model equations and the theory are the same and align closely, as described in our response above. The only additional difference is the use of a baseline bias in human experiments and the RLDDM model, where we also model reaction times with drift rates which is not a behaviour often simulated in grid world simulations. We will improve our Methods section to ensure that model similarity is highlighted.

Strengths: 

Overall, the modelling exercises are interesting and relevant and incrementally expand the space of existing models. 

We thank reviewer #1 for acknowledging the relevance of our models in advancing the field. We would like to further highlight that, to the best of our knowledge, this is the first time reaction times in Pavlovian-Instrumental arbitration tasks have been modelled using RLDDM, which adds a novel dimension to our approach.

Weaknesses: 

I find the conclusions misleading, as they are not supported by the data. 

First, the similarity between the models used in the two setups appears to be more semantic than computational or biological. So it is unclear to me how the results can be integrated. 

We acknowledge the dissimilarity between the task setups (grid-world vs. approach-withdrawal). However, we believe these setups are computationally similar and may be biologically related, as suggested by prior work like Dayan et al. [2006], which integrates Go-No Go and grid-world tasks. Just as that work bridged findings in the appetitive domain, we aim to integrate our findings in the aversive domain. We will provide a more integrated interpretation in the discussion section of the revised manuscript.

Dayan, P., Niv, Y., Seymour, B., and Daw, N. D. (2006). The misbehavior of value and the discipline of the will. Neural networks, 19(8):1153–1160.

Secondly, the authors do not show "a computational advantage to maintaining a specific fear memory during exploratory decision-making" (as they claim in the abstract). Making such a claim would require showing an advantage in the first place. For the first setup, the simulation results will likely be replicated by a simple Q-learning model when scaling up the loss incurred for punishments, in which case the more complex model architecture would not confer an advantage. The second setup, in contrast, is so excessively artificial that even if a particular model conferred an advantage here, this is highly unlikely to translate into any real-world advantage for a biological agent. The experimental setup was developed to demonstrate the existence of Pavlovian biases, but it is not designed to conclusively investigate how they come about. In a nutshell, who in their right mind would touch a stinging jellyfish 88 times in a short period of time, as the subjects do on average in this task? Furthermore, in which real-life environment does withdrawal from a jellyfish lead to a sting, as in this task? 

Thank you for your feedback. As mentioned above, we invite the reviewer to potentially think of Pavlovian fear systems as a way how the brain might implement punishment sensitivity. Secondly, it provides a separate punishment memory that cannot be overwritten with higher rewards (see also Elfwing and Seymour 2017, and Wang et al, 2021)

Elfwing, S., & Seymour, B. (2017, September). Parallel reward and punishment control in humans and robots: Safe reinforcement learning using the MaxPain algorithm. In 2017 Joint IEEE International Conference on Development and Learning and Epigenetic Robotics (ICDL-EpiRob) (pp. 140-147). IEEE. 

Wang, J., Elfwing, S., & Uchibe, E. (2021). Modular deep reinforcement learning from reward and punishment for robot navigation. Neural Networks, 135, 115-126.

The simulation setups such as the following grid-worlds are common test-beds for algorithms in reinforcement learning [Sutton and Barto, 2018].

Any experimental setup faces the problem of having a constrained experiment designed to test and model a specific effect versus designing a lesser constrained exploratory experiment which is more difficult to model. Here we chose the former, building upon previous foundational experiments on Pavlovian bias in humans [Guitart-Masip et al., 2012, Cavanagh et al., 2013].  The condition where withdrawal from a jellyfish leads to a sting, though less realistic, was included for balancing the four cue-outcome conditions. Overall the task was designed to isolate the effect we wanted to test - Pavlovian fear bias in choices and reaction times, to the best of our ability. In a free operant task, it is very well likely that other components not included in our model could compete for control.

Crucially, simplistic models such as the present ones can easily solve specifically designed lab tasks with low dimensionality but they will fail in higher-dimensional settings. Biological behaviour in the face of threat is utterly complex and goes far beyond simplistic fight-flight-freeze distinctions (Evans et al., 2019). It would take a leap of faith to assume that human decision-making can be broken down into oversimplified sub-tasks of this sort (and if that were the case, this would require a meta-controller arbitrating the systems for all the sub-tasks, and this meta-controller would then struggle with the dimensionality j). 

We agree that safe behaviours, such as escapes, involve more sophisticated computations. We do not propose Pavlovian fear bias as the sole computation for safe behavior, but rather as one of many possible contributors. Knowing about the existence about the Pavlovian withdrawal bias, we simply study its possible contribution. We will include in our discussion that such behaviours likely occupy different parts of the threat-imminence continuum [Mobbs et al., 2020].

Dean Mobbs, Drew B Headley, Weilun Ding, and Peter Dayan. Space, time, and fear: survival computations along defensive circuits. Trends in cognitive sciences, 24(3):228–241, 2020.

On the face of it, the VR task provides higher "ecological validity" than previous screen-based tasks. However, in fact, it is only the visual stimulation that differs from a standard screen-based task, whereas the action space is exactly the same. As such, the benefit of VR does not become apparent, and its full potential is foregone. 

We thank the reviewer for their comment. We selected the action space to build on existing models [Guitart-Masip et al., 2012, Cavanagh et al., 2013] that capture Pavlovian biases and we also wanted to minimize participant movement for EEG data collection. Unfortunately, despite restricting movement to just the arm, the EEG data was still too noisy to lead to any substantial results. We will explore more free-operant paradigms in future works.

On the issue of the difference between VR and lab-based tasks, we note the reviewer's point. Note however that desktop monitor-based tasks lack the sensorimotor congruency between the action and the outcome. Second, it is also arguable, that the background context is important in fear conditioning, as it may help set the tone of the fear system to make aversive components easier to distinguish.

If the authors are convinced that their model can - then data from naturalistic approach-avoidance VR tasks is publicly available, e.g. (Sporrer et al., 2023), so this should be rather easy to prove or disprove. In summary, I am doubtful that the models have any relevance for real-life human decision-making. 

We thank the reviewers for their thoughtful inputs. We do not claim our model is the best fit for all naturalistic VR tasks, as they require multiple systems across the threat-imminence continuum [Mobbs et al., 2020] and are currently beyond the scope of the current work. However, we believe our findings on outcome-uncertainty-based arbitration of Pavlovian bias could inform future studies and may be relevant for testing differences in patients with mental disorders, as noted by reviewer #2. At a general level, it can be said that most well-controlled laboratory-based tasks need to bridge a sizeable gap to applicabilty in real-life naturalistic behaviour; although the principle of using carefully designed tasks to isolate individual factors is well established

Finally, the authors seem to make much broader claims that their models can solve safety-efficiency dilemmas. However, a combination of a Pavlovian bias and an instrumental learner (study 1) via a fixed linear weighting does not seem to be "safe" in any strict sense. This will lead to the agent making decisions leading to death when the promised reward is large enough (outside perhaps a very specific region of the parameter space). Would it not be more helpful to prune the decision tree according to a fixed threshold (Huys et al., 2012)? So, in a way, the model is useful for avoiding cumulatively excessive pain but not instantaneous destruction. As such, it is not clear what real-life situation is modelled here. 

We thank the reviewer for their comments and ideas. In our discussion lines 257-264, we discuss other works which identify similar safety-efficiency dilemmas, in different models. Here, we simply focus on the safety-efficiency trade-off arising from the interactions between Pavlovian and instrumental systems. It is important to note that the computational argument for the modular system with separate rewards and punishments explicitly protects (up to a point, of course) against large rewards leading to death because the Pavlovian fear response is not over-written by successful avoidance in recent experience. Note also that in animals, reward utility curves are typically convex. We will clarify this in the discussion section.

We completely agree that in certain scenarios, pruning decision trees could be more effective, especially with a model-based instrumental agent. Here we utilise a model-free instrumental agent, which leads to a simpler model - which is appreciated by some readers such as reviewer #2. Future work can incorporate model-based methods.

A final caveat regarding Study 1 is the use of a PH associability term as a surrogate for uncertainty. The authors argue that this term provides a good fit to fear-conditioned SCR but that is only true in comparison to simpler RW-type models. Literature using a broader model space suggests that a formal account of uncertainty could fit this conditioned response even better (Tzovara et al., 2018). 

We thank the reviewer for bringing this to our notice. We will discuss Tzovara et al., 2018 in our discussion in our revised manuscript.

Reviewer #2 (Public review): 

Summary: 

The authors tested the efficiency of a model combining Pavlovian fear valuation and instrumental valuation. This model is amenable to many behavioral decision and learning setups - some of which have been or will be designed to test differences in patients with mental disorders (e.g., anxiety disorder, OCD, etc.). 

Strengths: 

(1) Simplicity of the model which can at the same time model rather complex environments. 

(2) Introduction of a flexible omega parameter. 

(3) Direct application to a rather advanced VR task. 

(4) The paper is extremely well written. It was a joy to read. 

Weaknesses: 

Almost none! In very few cases, the explanations could be a bit better. 

We thank reviewer #2 for their positive feedback and thoughtful recommendations. We will ensure that, in our revision, we clarify the explanations in the few instances where they may not be sufficiently detailed, as noted.

Reviewer #3 (Public review): 

Summary: 

This paper aims to address the problem of exploring potentially rewarding environments that contain the danger, based on the assumption that an independent Pavlovian fear learning system can help guide an agent during exploratory behaviour such that it avoids severe danger. This is important given that otherwise later gains seem to outweigh early threats, and agents may end up putting themselves in danger when it is advisable not to do so. 

The authors develop a computational model of exploratory behaviour that accounts for both instrumental and Pavlovian influences, combining the two according to uncertainty in the rewards. The result is that Pavlovian avoidance has a greater influence when the agent is uncertain about rewards. 

Strengths: 

The study does a thorough job of testing this model using both simulations and data from human participants performing an avoidance task. Simulations demonstrate that the model can produce "safe" behaviour, where the agent may not necessarily achieve the highest possible reward but ensures that losses are limited. Interestingly, the model appears to describe human avoidance behaviour in a task that tests for Pavlovian avoidance influences better than a model that doesn't adapt the balance between Pavlovian and instrumental based on uncertainty. The methods are robust, and generally, there is little to criticise about the study. 

Weaknesses: 

The extent of the testing in human participants is fairly limited but goes far enough to demonstrate that the model can account for human behaviour in an exemplar task. There are, however, some elements of the model that are unrealistic (for example, the fact that pre-training is required to select actions with a Pavlovian bias would require the agent to explore the environment initially and encounter a vast amount of danger in order to learn how to avoid the danger later). The description of the models is also a little difficult to parse. 

We thank reviewer #3 for their thoughtful feedback and useful recommendations, which we will take into account while revising the manuscript.

We acknowledge the complexity of specifying Pavlovian bias in the grid world and appreciate the opportunity to elaborate on how this bias is modelled. In the human experiment, the withdrawal action is straightforwardly biased, as noted, while in the grid world, we assume a hardwired encoding of withdrawal actions for each state/grid. This innate encoding of withdrawal actions could be represented in the dPAG [Kim et. al., 2013]. We implement this bias using pre-training, which we assume would be a product of evolution. Alternatively, this could be interpreted as deriving from an appropriate value initialization where the gradient over initialized values determines the action bias. Such aversive value initialization, driving avoidance of novel and threatening stimuli, has been observed in the tail of the striatum in mice, which is hypothesized to function as a Pavlovian fear/threat learning system [Menegas et. al., 2018].

Additionally, we explored the possibility of learning the action bias on the fly by tracking additional punishment Q-values instead of pre-training, which produced similar cumulative pain and step plots. While this approach is redundant, and likely not how the brain operates, it demonstrates an alternative algorithm.

We thank the reviewer for pointing out these potentially unrealistic elements, and we will revise the manuscript to clarify and incorporate these explanations and improve the model descriptions.

Eun Joo Kim, Omer Horovitz, Blake A Pellman, Lancy Mimi Tan, Qiuling Li, Gal Richter-Levin, and Jeansok J Kim. Dorsal periaqueductal gray-amygdala pathway conveys both innate and learned fear responses in rats. Proceedings of the National Academy of Sciences, 110(36):14795–14800, 2013

William Menegas, Korleki Akiti, Ryunosuke Amo, Naoshige Uchida, and Mitsuko Watabe-Uchida. Dopamine neurons projecting to the posterior striatum reinforce avoidance of threatening stimuli. Nature neuroscience, 21(10): 1421–1430, 2018

eLife Assessment

The authors identify a novel relationship between exosome secretion and filopodia formation that has implications for cancer cell metastasis and neuronal synapse formation. Further, they identify the exosomal cargo, THSD7A, as a regulator of this process. The data presented is convincing, and represents an important advancement in our understanding of how these two biological processes are linked and play roles in regulating cell migration and cell-cell communication.

Reviewer #1 (Public review):

Summary:

The study significantly advances our understanding of how exosomes regulate filopodia formation. Filopodia play crucial roles in cell movement, polarization, directional sensing, and neuronal synapse formation. McAtee et al. demonstrated that exosomes, particularly those enriched with the protein THSD7A, play a pivotal role in promoting filopodia formation through Cdc42 in cancer cells and neurons. This discovery unveils a new extracellular mechanism through which cells can control their cytoskeletal dynamics and interaction with their surroundings. The study employs a combination of rescue experiments, live-cell imaging, cell culture, and proteomic analyses to thoroughly investigate the role of exosomes and THSD7A in filopodia formation in cancer cells and neurons. These findings offer valuable insights into fundamental biological processes of cell movement and communication and have potential implications for understanding cancer metastasis and neuronal development.

Weaknesses:

The conclusions of this study are in most cases supported by data, but some aspects of data analysis need to be better clarified and elaborated. Some conclusions need to be better stated and according to the data observed.

Reviewer #2 (Public review):

Summary:

The authors show that small EVs trigger the formation of filopodia in both cancer cells and neurons. They go on to show that two cargo proteins, endoglin, and THSD7A, are important for this process. This possibly occurs by activating the Rho-family GTPase CDC42.

Strengths:

The EV work is quite strong and convincing. The proteomics work is well executed and carefully analyzed. I was particularly impressed with the chick metastasis assay that added strong evidence of in vivo relevance.

Weaknesses:

The weakest part of the paper is the Cdc42 work at the end of the paper. It is incomplete and not terribly convincing. This part of the paper needs to be improved significantly

Reviewer #3 (Public review):

Summary:

The authors identify a novel relationship between exosome secretion and filopodia formation in cancer cells and neurons. They observe that multivesicular endosomes (MVE)-plasma membrane (PM) fusion is associated with filopodia formation in HT1080 cells and that MVEs are present in filopodia in primary neurons. Using overexpression and knockdown (KD) of Rab27/HRS in HT1080 cells, melanoma cells, and/or primary rat neurons, they found that decreasing exosome secretion reduces filopodia formation, while Rab27 overexpression leads to the opposite result. Furthermore, the decreased filopodia formation is rescued in the Rab27a/HRS KD melanoma cells by the addition of small extracellular vesicles (EVs) but not large EVs purified from control cells. The authors identify endoglin as a protein unique to small EVs secreted by cancer cells when compared to large EVs. KD of endoglin reduces filopodia formation and this is rescued by the addition of small EVs from control cells and not by small EVs from endoglin KD cells. Based on the role of filopodia in cancer metastasis, the authors then investigate the role of endoglin in cancer cell metastasis using a chick embryo model. They find that injection of endoglin KD HT1080 cells into chick embryos gives rise to less metastasis compared to control cells - a phenotype that is rescued by the co-injection of small EVs from control cells. Using quantitative mass spectrometry analysis, they find that thrombospondin type 1 domain containing 7a protein (THSD7A) is downregulated in small EVs from endoglin KD melanoma cells compared to those from control cells. They also report that THSD7A is more abundant in endoglin KD cell lysate compared to control HT1080 cells and less abundant in small EVs from endoglin KD cells compared to control cells, indicating a trafficking defect. Indeed, using immunofluorescence microscopy, the authors observe THSD7A-mScarlet accumulation in CD63-positive structures in endoglin KD HT1080 cells, compared to control cells. Finally, the authors determine that exosome-secreted THSD7A induces filopodia formation in a Cdc42-dependent mechanism.

Strengths:

(1) While exosomes are known to play a role in cell migration and autocrine signaling, the relationship between exosome secretion and the formation of filopodia is novel.

(2) The authors identify an exosomal cargo protein, THSD7A, which is essential for regulating this function.

(3) The data presented provide strong evidence of a role for endoglin in the trafficking of THSD7A in exosomes.

(4) The authors associate this process with functional significance in cancer cell metastasis and neurological synapse formation, both of which involve the formation of filopodia.

(5) The data are presented clearly, and their interpretation appropriately explains the context and significance of the findings.

Weaknesses:

(1) A better characterization of the nature of the small EV population is missing:

It is unclear why the authors chose to proceed to quantitative mass spectrometry with the bands in the Coomassie from size-separated EV samples, as there are other bands present in the small EV lane but not the large EV lane. This is important to clarify because it underlies how they were able to identify THSD7A as a unique regulator of exosome-mediated filopodia formation. Is there a reason why the total sample fractions were not compared? This would provide valuable information on the nature of the small and large EV populations.

(2) Data analysis and quantification should be performed with increased rigor:

a) Figure 1C - The optical and temporal resolution are insufficient to conclusively characterize the association between exosome secretion and filopodia. Specifically, the 10-second interval used in the image acquisitions is too close to the reported 20-second median time between exosome secretion and filopodia formation. Two-5 sec intervals should be used to validate this. It would also be important to correlate the percentage of filopodia events that co-occur with exosome secretion. Is this a phenomenon that occurs with most or only a small number of filopodia? Additionally, resolution with typical confocal microscopy is subpar for these analyses. TIRF microscopy would offer increased resolution to parse out secretion events. As the TIRF objective is listed in the Methods section, figure legends should mention which images were acquired using TIRF microscopy.

b) Figure 2 - It would be important to perform further analysis to concretely determine the relationship between exosome secretion and filopodia stability. Are secretion events correlated with the stability of filopodia? Is there a positive feedback loop that causes further filopodia stability and length with increased secretion? Furthermore, is there an association between the proximity of secretion with stability? Quantification of filopodia more objectively (# of filopodia/cell) would be helpful.

c) Figure 6 - Why use different gel conditions to detect THSD7A in small EVs from B16F1 cells vs HT1080 and neurons? Why are there two bands for THSD7A in panels C and E? It is difficult to appreciate the KD efficiency in E. The absence of a signal for THSD7A in the HT1080 shEng small EVs that show a signal for endoglin is surprising. The authors should provide rigorous quantification of the westerns from several independent experimental repeats.

(3) The study lacks data on the cellular distribution of endoglin and THSD7A:

a) Figure 6 - Is THSD7A expected to be present in the nucleus as shown in panel D (label D is missing in the Figure). It is not clear if this is observed in neurons. a Western of endogenous THSD7A on cell fractions would clarify this. The authors should further characterize the cellular distribution of THSD7A in both cell types. Similarly, the cellular distribution of endoglin in the cancer cells should be provided. This would help validate the proposed model in Figure 8.

b) Figure 7 - Although the western blot provides convincing evidence for the role of endoglin in THSD7A trafficking, the microscopy data lack resolution as well as key analyses. While differences between shSCR and shEng cells are clear visually, the insets appear to be zoomed digitally which decreases resolution and interferes with interpretation. It would be crucial to show the colocalization of endoglin and THSD7A within CD63-postive MVE structures. What are the structures in Figure 7E shSCR zoom1? It would be important to rule out that these are migrasomes using TSPAN4 staining. More information on how the analysis was conducted is needed (i.e. how extracellular areas were chosen and whether the images are representative of the larger population). A widefield image of shSCR and shEng cells and DAPI or HOECHST staining in the higher magnification images should be provided. Additionally, the authors should quantify the colocalization of external CD63 and mScarlet signals from many independently acquired images (as they did for the internal signals in panel F). Is there no external THSD7A signal in the shEng cells?

Author response:

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

The study significantly advances our understanding of how exosomes regulate filopodia formation. Filopodia play crucial roles in cell movement, polarization, directional sensing, and neuronal synapse formation. McAtee et al. demonstrated that exosomes, particularly those enriched with the protein THSD7A, play a pivotal role in promoting filopodia formation through Cdc42 in cancer cells and neurons. This discovery unveils a new extracellular mechanism through which cells can control their cytoskeletal dynamics and interaction with their surroundings. The study employs a combination of rescue experiments, live-cell imaging, cell culture, and proteomic analyses to thoroughly investigate the role of exosomes and THSD7A in filopodia formation in cancer cells and neurons. These findings offer valuable insights into fundamental biological processes of cell movement and communication and have potential implications for understanding cancer metastasis and neuronal development. 

Weaknesses: 

The conclusions of this study are in most cases supported by data, but some aspects of data analysis need to be better clarified and elaborated. Some conclusions need to be better stated and according to the data observed. 

We appreciate the reviewer's recognition of the impact of our study.  We will address the concerns about data analysis and statement of our conclusions in our full response to reviewers.

Reviewer #2 (Public review): 

Summary: 

The authors show that small EVs trigger the formation of filopodia in both cancer cells and neurons. They go on to show that two cargo proteins, endoglin, and THSD7A, are important for this process. This possibly occurs by activating the Rho-family GTPase CDC42. 

Strengths: 

The EV work is quite strong and convincing. The proteomics work is well executed and carefully analyzed. I was particularly impressed with the chick metastasis assay that added strong evidence of in vivo relevance. 

Weaknesses: 

The weakest part of the paper is the Cdc42 work at the end of the paper. It is incomplete and not terribly convincing. This part of the paper needs to be improved significantly.

We appreciate the reviewer's recognition of the impact of our study.  Indeed, more work needs to be done to clarify the role of Cdc42 in the induction of filopodia by exosome-associated THSD7A.  We anticipate that this will be a separate manuscript, delving in-depth into how exosome-associated THSD7A interacts with recipient cells to activate Cdc42 and carrying out a variety of assays for Cdc42 activation.

Reviewer #3 (Public review): 

Summary: 

The authors identify a novel relationship between exosome secretion and filopodia formation in cancer cells and neurons. They observe that multivesicular endosomes (MVE)-plasma membrane (PM) fusion is associated with filopodia formation in HT1080 cells and that MVEs are present in filopodia in primary neurons. Using overexpression and knockdown (KD) of Rab27/HRS in HT1080 cells, melanoma cells, and/or primary rat neurons, they found that decreasing exosome secretion reduces filopodia formation, while Rab27 overexpression leads to the opposite result. Furthermore, the decreased filopodia formation is rescued in the Rab27a/HRS KD melanoma cells by the addition of small extracellular vesicles (EVs) but not large EVs purified from control cells. The authors identify endoglin as a protein unique to small EVs secreted by cancer cells when compared to large EVs. KD of endoglin reduces filopodia formation and this is rescued by the addition of small EVs from control cells and not by small EVs from endoglin KD cells. Based on the role of filopodia in cancer metastasis, the authors then investigate the role of endoglin in cancer cell metastasis using a chick embryo model. They find that injection of endoglin KD HT1080 cells into chick embryos gives rise to less metastasis compared to control cells - a phenotype that is rescued by the co-injection of small EVs from control cells. Using quantitative mass spectrometry analysis, they find that thrombospondin type 1 domain containing 7a protein (THSD7A) is downregulated in small EVs from endoglin KD melanoma cells compared to those from control cells. They also report that THSD7A is more abundant in endoglin KD cell lysate compared to control HT1080 cells and less abundant in small EVs from endoglin KD cells compared to control cells, indicating a trafficking defect. Indeed, using immunofluorescence microscopy, the authors observe THSD7A-mScarlet accumulation in CD63-positive structures in endoglin KD HT1080 cells, compared to control cells. Finally, the authors determine that exosome-secreted THSD7A induces filopodia formation in a Cdc42-dependent mechanism. 

Strengths: 

(1) While exosomes are known to play a role in cell migration and autocrine signaling, the relationship between exosome secretion and the formation of filopodia is novel. 

(2) The authors identify an exosomal cargo protein, THSD7A, which is essential for regulating this function. 

(3) The data presented provide strong evidence of a role for endoglin in the trafficking of THSD7A in exosomes. 

(4) The authors associate this process with functional significance in cancer cell metastasis and neurological synapse formation, both of which involve the formation of filopodia. 

(5) The data are presented clearly, and their interpretation appropriately explains the context and significance of the findings. 

Weaknesses: 

(1) A better characterization of the nature of the small EV population is missing: 

It is unclear why the authors chose to proceed to quantitative mass spectrometry with the bands in the Coomassie from size-separated EV samples, as there are other bands present in the small EV lane but not the large EV lane. This is important to clarify because it underlies how they were able to identify THSD7A as a unique regulator of exosome-mediated filopodia formation. Is there a reason why the total sample fractions were not compared? This would provide valuable information on the nature of the small and large EV populations. 

We would like to clarify that there are two sets of proteomics data in the manuscript. The first was comparing bands from a Coomassie gel from two samples: small EVs and large EVs from B16F1 cells. In this proteomics experiment, we identified endoglin as present in small EVs, but not large EVs. For this experiment, we only sent 4 bands from the small EV lane, chosen based on their obvious banding pattern difference on the Coomassie gel.

In the second proteomics experiment, we used quantitative iTRAQ proteomics to compare small EVs purified from B16F1 control (shScr) and endoglin KD (shEng1 and shEng2) cell lines. In this experiment, we sent total protein extracted from small EV samples for analysis. So, these samples included the entire EV content, not just selected bands from a gel. In this experiment, we identified THSD7A as reduced in the shEng small EVs.

(2) Data analysis and quantification should be performed with increased rigor: 

a) Figure 1C - The optical and temporal resolution are insufficient to conclusively characterize the association between exosome secretion and filopodia. Specifically, the 10-second interval used in the image acquisitions is too close to the reported 20-second median time between exosome secretion and filopodia formation. Two-5 sec intervals should be used to validate this. It would also be important to correlate the percentage of filopodia events that co-occur with exosome secretion. Is this a phenomenon that occurs with most or only a small number of filopodia? Additionally, resolution with typical confocal microscopy is subpar for these analyses. TIRF microscopy would offer increased resolution to parse out secretion events. As the TIRF objective is listed in the Methods section, figure legends should mention which images were acquired using TIRF microscopy. 

We acknowledge that the frame rate naturally limits our estimates of the timing of filopodia formation after exosome secretion. We set out to show a relationship between exosome secretion and filopodia formation, based on their proximity in timing. While our data set shows a median time interval of 20 seconds, the true median could be between 10-30 seconds, based on our frame rate.  Regardless of the exact timing, our data show that exosome secretion is rapidly followed by filopodia formation events.

To address the question of the percentage of filopodia events that are preceded by exosome secretion, the reviewer is correct in stating that we might need TIRF microscopy to get an accurate calculation of this number.  Nonetheless, we will review our live imaging data for this experiment to determine if this calculation is possible. Again, we will be limited by the frame rate we used to capture the images, so we could possibly be missing secretion events taking place between the 10 second time intervals.  Regardless, for the secretion events that we visualized, we always observed subsequent filopodia formation.

No TIRF imaging was used in this manuscript.  A TIRF objective was used for selected neuron imaging (see methods); however, it was used for spinning disk confocal microscopy, not for TIRF imaging.  We will clarify this in the methods.

b) Figure 2 - It would be important to perform further analysis to concretely determine the relationship between exosome secretion and filopodia stability. Are secretion events correlated with the stability of filopodia? Is there a positive feedback loop that causes further filopodia stability and length with increased secretion? Furthermore, is there an association between the proximity of secretion with stability? Quantification of filopodia more objectively (# of filopodia/cell) would be helpful. 

Our data shows that manipulation of general exosome secretion, via Hrs knockdown, affects both de novo filopodia formation and filopodia stability (Fig 2g,h). Interestingly, knockdown of endoglin only affects de novo filopodia formation, while filopodia stability is unaffected (Fig 4g,h). These results suggest that filopodia stability is dependent upon exosome cargoes besides endoglin/THSD7A.  Such cargoes might include other extracellular matrix molecules, such as fibronectin. We previously showed that exosomes promote nascent cell adhesion and rapid cell migration, through exosome-bound fibronectin (Sung et al., Nature Communications, 6:7164, 2015). We also previously found that inhibition of exosome secretion affects the persistence of invadopodia, which are filopodia-dependent structures (Hoshino et al., Cell Reports, 5:1159-1168, 2013).  We agree that this is an interesting research direction, and perhaps future work could focus on exosomal factors that are responsible for filopodia persistence.

With regard to the way we plotted the filopodia data, we plotted the cancer cell data as filopodia per cell area so that it matched the neuron data, which was plotted as filopodia per 100 mm of dendrite distance. Since the neurons cannot be imaged as a whole cell, the quantification is based on the length of the dendrite in the image. We found that graphing the cancer cell data as filopodia per cell gave similar results as filopodia per cell area, as there were no significant differences in cell area between conditions and experiments. We plan to include a new supplementary figure showing the data in Figure 2 plotted as filopodia per cell to show that this quantification gives the same results.

c) Figure 6 - Why use different gel conditions to detect THSD7A in small EVs from B16F1 cells vs HT1080 and neurons? Why are there two bands for THSD7A in panels C and E? It is difficult to appreciate the KD efficiency in E. The absence of a signal for THSD7A in the HT1080 shEng small EVs that show a signal for endoglin is surprising. The authors should provide rigorous quantification of the westerns from several independent experimental repeats. 

Detection of THSD7A via Western blot was, unfortunately, not straightforward and simple. Due to the large size (~260 kDa) of THSD7A, its low level of expression in cancer cells, as well as the inconsistency of commercially available THSD7A antibodies, we had to troubleshoot multiple conditions.  We found that it was much easier to detect THSD7A in the human fibrosarcoma cell line HT1080 than in the mouse B16F1 cells, both in the cell lysates and in the small EVs. We were usually unable to detect THSD7A using these same conditions for the mouse melanoma B16F1 samples, but were successful using native gel conditions. We also detected THSD7A in rat primary neuron samples. All these samples were from different source organisms (human, mouse, rat) and from either cell lysates or extracellular vesicles, further complicating the analyses. Expression and maturation of THSD7A in these different cell types and compartments could involve different post-translational modifications, such as glycosylation, thus requiring different methods needed to detect THSD7A on Western blots and leading to different banding patterns. Based on our THSD7A trafficking data, we believe that in control cells, most of the THSD7A is getting trafficked and secreted via small EVs. As you can see in Figure 7A, the band for THSD7A in the shScr cell lysate is relatively light and also shows a double band similar to Figure 6E (both HT1080 samples).

With regard to the level of knockdown of THSD7A in the Western blot shown in Figure 6E, the normalized level is quantitated below the bands.  If you compare that quantitation to the filopodia phenotypes in the same panel, they are quite concordant.  Figures 7B and 7C show quantification of triplicate Western blots, highlighting the significant accumulation of THSD7A in shEng cell lysates, as well as significant small EV secretion of THSD7A in control and WT rescued conditions.

(3) The study lacks data on the cellular distribution of endoglin and THSD7A: 

a) Figure 6 - Is THSD7A expected to be present in the nucleus as shown in panel D (label D is missing in the Figure). It is not clear if this is observed in neurons. a Western of endogenous THSD7A on cell fractions would clarify this. The authors should further characterize the cellular distribution of THSD7A in both cell types. Similarly, the cellular distribution of endoglin in the cancer cells should be provided. This would help validate the proposed model in Figure 8. 

The image in figure 6D shows an HT1080 cell stained with phalloidin-Alexa Fluor 488 to visualize F-actin with or without expression of THSD7A-mScarlet.  In order to fully visualize the thin filopodia protrusions, the cellular plane of focus of the images for this panel was purposely taken at the bottom of the cell, where the cell is attached to the coverslip glass. Thus, we interpret the red signal across the cell body as THSD7A-mScarlet expression on the plasma membrane underneath the cell, not in the nucleus. The neuron images only include the dendrite portion of the neurons; therefore, there is no nucleus present in the neuronal images.

b) Figure 7 - Although the western blot provides convincing evidence for the role of endoglin in THSD7A trafficking, the microscopy data lack resolution as well as key analyses. While differences between shSCR and shEng cells are clear visually, the insets appear to be zoomed digitally which decreases resolution and interferes with interpretation. It would be crucial to show the colocalization of endoglin and THSD7A within CD63-postive MVE structures. What are the structures in Figure 7E shSCR zoom1? It would be important to rule out that these are migrasomes using TSPAN4 staining. More information on how the analysis was conducted is needed (i.e. how extracellular areas were chosen and whether the images are representative of the larger population). A widefield image of shSCR and shEng cells and DAPI or HOECHST staining in the higher magnification images should be provided. Additionally, the authors should quantify the colocalization of external CD63 and mScarlet signals from many independently acquired images (as they did for the internal signals in panel F). Is there no external THSD7A signal in the shEng cells? 

The images for Figure 7E were taken with high resolution on a confocal microscope.  Insets for Figure 7E were zoomed in so that readers could see the tiny structures.  Zoom 1 in Figure 7E shows areas of extracellular deposition. In these areas, we can see small punctate depositions that are positive for CD63 and/or THSD7A-mScarlet. Our interpretation of this staining is that the cells are secreting heterogeneous small EVs that are then attached to the glass coverslip. The images and zooms in Fig 7E were chosen to be representative and indeed reveal that there is more extracellular deposition of THSD7A-mScarlet outside the control shScr cells compared to the shEng cells, consistent with more export of THSD7A into small EVs from shScr cells when compared to those of shEng cells (Fig 7A,B). However, we did not quantify this difference, as these experiments were conducted with transient transfection of THSD7A-mScarlet and it is challenging to determine which cell the extracellular THSD7A-mScarlet came from, complicating any quantitative analysis on a per-cell basis.  Quantification of internal THSD7A localization is much more straightforward in this experimental regime.  Indeed, in Figure 7F we assessed internal colocalization of THSD7A-mScarlet and CD63, which we obtained by choosing only cells that were visually positive for THSD7A-mScarlet in each transient transfection and omitting all extracellular signals. Quantifying the extracellular colocalization of THSD7A and CD63 could certainly be a future direction for this project and would require establishing cells that stably express THSD7A-mScarlet.

Reporter John Dickerson talking about his notebook.

While he doesn't mention it, he's capturing the spirit of the commonplace book and the zettelkasten.

[...] I see my job as basically helping people see and to grab ahold of what's going on.

You can decide to do that the minute you sit down to start writing or you can just do it all the time. And by the time you get to writing you have a notebook full of stuff that can be used.

And it's not just about the thing you're writing about at that moment or the question you're going to ask that has to do with that week's event on Face the Nation on Sunday.

If you've been collecting all week long and wondering why a thing happens or making an observation about something and using that as a piece of color to explain the political process to somebody, then you've been doing your work before you ever sat down to do your work.

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Field Notes: Reporter's Notebook from Coudal Partners on Vimeo.

eLife Assessment

This valuable study investigates how the size of an LLM may influence its ability to model the human neural response to language recorded by ECoG. Overall, solid evidence is provided that larger language models can better predict the human ECoG response. Further discussion would be beneficial as to how the results can inform us about the brain or LLMs, especially about the new message that can be learned from this ECoG study beyond previous fMRI studies on the same topic. This study will be of interest to both neuroscientists and psychologists who work on language comprehension and computer scientists working on LLMs.

Reviewer #1 (Public review):

Summary:

The authors perform an analysis of the relationship between the size of an LMM and the predictive performance of an ECoG encoding model made using the representations from that LMM. They find a logarithmic relationship between model size and prediction performance, consistent with previous findings in fMRI. They additionally observe that as the model size increases, the location of the "peak" encoding performance typically moves further back into the model in terms of percent layer depth, an interesting result worthy of further analysis into these representations.

Strengths:

The evidence is quite convincing, consistent across model families, and complementary to other work in this field. This sort of analysis for ECoG is needed and supports the decade-long enduring trend of the "virtuous cycle" between neuroscience and AI research, where more powerful AI models have consistently yielded more effective predictions of responses in the brain. The lag analysis showing that optimal lags do not change with model size is a nice result using the higher temporal resolution of ECoG compared to other methods like fMRI.

Weaknesses:

I would have liked to have seen the data scaling trends explored a bit too, as this is somewhat analogous to the main scaling results. While better performance with more data might be unsurprising, showing good data scaling would be a strong and useful justification for additional data collection in the field, especially given the extremely limited amount of existing language ECoG data. I realize that the data here is somewhat limited (only 30 minutes per subject), but authors could still in principle train models on subsets of this data.

Separately, it would be nice to have better justification of some of these trends, in particular the peak layerwise encoding performance trend and the overall upside-down U-trend of encoding performance across layers more generally. There is clearly something very fundamental going on here, about the nature of abstraction patterns in LLMs and in the brain, and this result points to that. I don't see the lack of justification here as a critical issue, but the paper would certainly be better with some theoretical explanation for why this might be the case.

Lastly, I would have wanted to see a similar analysis here done for audio encoding models using Whisper or WavLM as this is the modality where you might see real differences between ECoG and other slower scanning approaches. Again, I do not see this omission as a fundamental issue, but it does seem like the sort of analysis for which the higher temporal resolution of ECoG might grant some deeper insight.

Reviewer #2 (Public review):

Summary:

This paper investigates whether large language models (LLMs) of increasing size more accurately align with brain activity during naturalistic language comprehension. The authors extracted word embeddings from LLMs for each word in a 30-minute story and regressed them against electrocorticography (ECoG) activity time-locked to each word as participants listened to the story. The findings reveal that larger LLMs more effectively predict ECoG activity, reflecting the scaling laws observed in other natural language processing tasks.

Strengths:

(1) The study compared model activity with ECoG recordings, which offer much better temporal resolution than other neuroimaging methods, allowing for the examination of model encoding performance across various lags relative to word onset.

(2) The range of LLMs tested is comprehensive, spanning from 82 million to 70 billion parameters. This serves as a valuable reference for researchers selecting LLMs for brain encoding and decoding studies.

(3) The regression methods used are well-established in prior research, and the results demonstrate a convincing scaling law for the brain encoding ability of LLMs. The consistency of these results after PCA dimensionality reduction further supports the claim.

Weaknesses:

(1) Some claims of the paper are less convincing. The authors suggested that "scaling could be a property that the human brain, similar to LLMs, can utilize to enhance performance", however, many other animals have brains with more neurons than the human brain, making it unlikely that simple scaling alone leads to better language performance. Additionally, the authors claim that their results show 'larger models better predict the structure of natural language.' However, it remains unclear to what extent the embeddings of LLMs capture the "structure" of language better than the lexical semantics of language.

(2) The study lacks control LLMs with randomly initialized weights and control regressors, such as word frequency and phonetic features of speech, making it unclear what the baseline is for the model-brain correlation.

(3) The finding that peak encoding performance tends to occur in relatively earlier layers in larger models is somewhat surprising and requires further explanation. Since more layers mean more parameters, if the later layers diverge from language processing in the brain, it raises the question of what aspects of the larger models make them more brain-like.

Reviewer #3 (Public review):

This manuscript studies the connection between neural activity collected through electrocorticography and hidden vector representations from autoregressive language models, with the specific aim of studying the influence of language model size on this connection. Neural activity was measured from subjects who listened to a segment from a podcast, and the representations from language models were calculated using the written transcription as the input text. The ability of vector representations to predict neural activity was evaluated using 10-fold cross-validation with ridge regression models.

The main results are that (as well summarized in section headings):

(1) Larger models predict neural activity better.

(2) The ability of language model representations to predict neural activity differs across electrodes and brain regions.

(3) The layer that best predicts neural activity differs according to model size, with the "SMALL" model showing a correspondence between layer number and the language processing hierarchy.

(4) There seems to be a similar relationship between the time lag and the ability of language model representations to predict neural activity across models.

Strengths:

(1) The experimental and modeling protocols generally seem solid, which yielded results that answer the authors' primary research question.

(2) Electrocorticography data is especially hard to collect, so these results make a nice addition to recent functional magnetic resonance imaging studies.

Weaknesses:

(1) The interpretation of some results seems unjustified, although this may just be a presentational issue.

a) Figure 2B: The authors interpret the results as "a plateau in the maximal encoding performance," when some readers might interpret this rather as a decline after 13 billion parameters. Can this be further supported by a significance test like that shown in Figure 4B?

b) Figure S1A: It looks like the drop in PCA max correlation is larger for larger models, which may suggest to some readers that the same trend observed for ridge max correlation may not hold, contra the authors' claim that all results replicate. Why not include a similar figure as Figure 2B as part of Figure S1?

(2) Discussion of what might be driving the main result about the influence of model size appears to be missing (cf. the authors aim to provide an explanation of what seems to drive the influence of the layer location in Paragraph 3 of the Discussion section). What explanations have been proposed in the previous functional magnetic resonance imaging studies? Do those explanations also hold in the context of this study?

(3) The GloVe-based selection of language-sensitive electrodes (at least to me) isn't explained/motivated clearly enough (I think a more detailed explanation should be included in the Materials and Methods section). If the electrodes are selected based on GloVe embeddings, then isn't the main experiment just showing that representations from larger language models track more closely with GloVe embeddings? What justifies this methodology?

(4) (Minor weakness) The main experiments are largely replications of previous functional magnetic resonance imaging studies, with the exception of the one lag-based analysis. Is there anything else that the electrocorticography data can reveal that functional magnetic resonance imaging data can't?

Author response:

We thank the reviewers for their thoughtful feedback and valuable comments. We plan to fully address their concerns by including the following experiments and analyses:

Reviewer 1 suggested exploring data scaling trends for encoding models, as successful scaling would justify larger datasets for language ECoG studies. To estimate scaling effects, we will develop encoding models on subsets of our data.

Reviewer 2 expressed uncertainty about the baseline for model-brain correlation and recommended adding control LLMs with randomly initialized weights. In response, we will generate embeddings using untrained LLMs to establish a more robust baseline for encoding results.

Reviewer 2 also proposed incorporating control regressors such as word frequency and phonetic features of speech. We will re-run our modeling analysis using control regressors for word frequency, 8 syntactic features (e.g., part of speech, dependency, prefix/suffix), and 3 phonetic features (e.g., phonemes, place/manner of articulation) to assess how much these features contribute to encoding performance.

Reviewer 3 raised concerns that the “plateau in maximal encoding performance” was actually a decline for the largest models. We will add significance tests in Figure 2B to clarify this issue.

Reviewer 3 also noted that in Supplementary Figure 1A, the decline in encoding performance was more pronounced when using PCA to reduce embedding dimensionality, in contrast to the trend observed when using ridge regression. To address this, we will attempt to replicate the observed scaling trends in Figure 2B using PCA combined with OLS.

Additionally, we will provide a point-by-point response and revise the manuscript with updated analyses and figures in the near future.

Paul Pham shares an interesting take on how expectations play out in schools. He points out that if a student from a different background, like a Latina/o, joins a mostly Asian class or activity, they often get a lot of positive attention just for being there. While Paul sees this as a good thing, there’s a deeper issue tied to Claude Steele’s idea of disidentification. In this view, if a non-Asian student joins an AP class or a tough extracurricular, they might be greeted with surprise and praise for doing something unexpected. This reinforces the stereotype that they’re exceeding low expectations. This stigma can make them feel disengaged from academics unless they have mentors or support that recognize and encourage their abilities. It really shows how crucial it is to challenge these stereotypes and create a more welcoming environment for everyone.

While many schools are starting to focus more on students' mental health and offering counseling services, the counselors often don’t really know how to help students of color. Violet, who is multiracial Asian American and part Latino, shares how she feels invisible in her mostly white community and often gets lumped together with Mexican Americans. It shows a real lack of understanding about her identity. When a white counselor tried to help by taking students on just one tour of a local community college, it felt pretty shallow and didn’t tackle the real issues they face. It really emphasizes how important it is for counselors to understand and be sensitive to the unique experiences of diverse students.

When Asian American students try to speak up about bullying, they often get told to “thicken their skin” and not take it seriously, while the kids making fun of them face little to no consequences. Parents worry about complaining because they’re afraid it’ll just cause more trouble or make things worse. Even though schools are pushing for multiculturalism and celebrating diversity, sometimes it feels pretty surface-level. This “happy” approach can blind teachers and administrators to the real effects of racism on students of color and the bigger problem of institutional racism in schools. It’s frustrating to see how these attitudes can really undermine the experiences of students who are struggling.

I think it highlights the mixed messages about how Asian Americans are seen. On one side, they’re often labeled as the “model minority,” seen as super successful and even “whiter than whites.” But at the same time, they deal with serious racism and discrimination, facing barriers in education and jobs, plus real threats of violence—similar to what Black communities experience. Gary Okihiro points out that this puts Asians in a tricky spot within the larger black-and-white racial landscape, where both groups face their own kinds of oppression. It really shows how complex these issues are and how important it is to understand the different ways racial struggles affect Asian Americans.

It really shows how harmful stereotypes about Asian Americans are still everywhere in media. Cartoons like "Mr. Wong" feature ridiculous, over-the-top characters that just feed into negative views, and some people think it’s just a joke, which is super frustrating. Movies like "Team America: World Police" do the same thing, piling on racial mockery and telling anyone who’s offended to lighten up. Even shows like "Desperate Housewives" join in with lines that reinforce stereotypes, like questioning a doctor’s background just because of where they’re from. It’s disappointing that this kind of stuff is still considered okay in mainstream media, and it’s awesome to see Asian and Pacific Islander communities standing up against it.

This paragraph emphasizes the stress and pain that come from dealing with racism and stereotypes. The "model minority" label, often used by those in power, creates a lot of pressure to fit into a white-dominated culture, which can be really tough. It highlights how these expectations can lead to mental health struggles, making it clear that it’s more about the external pressures than anything inherent in the community.

very informative Thanks atman for such Information

这不是很显然的吗？

Parth’s annotation on this line introduces an interesting interpretation of its origins in Antony and Cleopatra. He identifies that “this line is an imitation of a line we see early on in Antony and Cleopatra—with one change: the word "barge" is changed to a capitalized "Chair" here…Chair might not be referring to a literal chair, which isn't a proper noun—but an organizational position. A "Chair" in a company is an executive position; likewise, a metaphorical Chair in a kingdom may refer to one's supreme status” (Jain). I’d like to expand the scope of the Antony and Cleopatra reference beyond specific lines that Eliot incorporated into “The Waste Land,” now through Parth’s lens of Cleopatra’s status and power within the play, her “Chair” within Antony and Cleopatra, and therefore “The Waste Land” as well. When reading Antony and Cleopatra, I couldn’t help but take note of the many ways in which Cleopatra both adopts the role of the queen in a literal game of chess. In a very broad sense, Cleopatra’s decision to fake her own suicide, to temp Antony with her death, is quite analogous to the physical and strategic features of the queen on a chess board. For example, while Cleopatra plots to win the attention of Antony, she says, “I have nothing Of woman in me: now from head to foot // I am marble-constant” (Shakespeare, V.II). In portraying herself as hard as marble, she renders herself part of a chess set, as they are often made of marble. The image of a chess board is then transferred to the marble as it appears here in “The Waste Land,” further convincing me that Cleopatra is the queen of Eliot’s chess set as it is understood in “A Game of Chess.” Additionally, Cleopatra embodies the characteristics of a chess piece as a powerful strategic asset when used correctly. She even says, “come hither, come! come, come, and take a queen worth many babes and beggars” (Shakespeare, V.II). “Take” is the terminology in a game of chess for the capture of a piece. I believe it’s possible that both Shakespeare and Eliot recognized the parallels between Cleopatra’s actions and the act of sacrificing a piece in the game of chess. However, Cleopatra’s move was in vain, and ultimately, when she sacrifices her life, amounts to nothing. In other words, the power she possessed through her “Chair” is only valuable insofar as it is spent to win the Game of Chess. Meanwhile, the woman in “The Waste Land” also sits dead on her throne, her status reduced, presumably having met a similarly wasteful fate.

I would like to elaborate on Quisha’s point in her annotation for this section. She investigates the similarities between the original epigraph from Heart of Darkness, and the one from Ovid in the final draft, stating that the Sybil “deems her body as if it is a piece of waste. Kurtz is similar – he is ill; his health in a wasted state. The moment before his death he, too, is filled with superfluous knowledge; yet he cannot make use of it for death soon transcends him. The two epigraphs do resemble each other in many ways, and I'm curious about the exact reason as to why Eliot chose the one of Sybil over Kurtz” (Lee). I think she does something really interesting here by connecting the title of the poem to the epigraph, which is further supported by Weston’s way of connecting the physical condition of a person to the state of the land itself. This suggests that Eliot intended for Kurtz, and later the Sybil, to embody the Waste Land, the poem itself. That said, I believe the Sybil epigraph actually accomplishes this in a more complete way, which is why Eliot chose it.

To begin, Eliot’s original title for “The Waste Land” was “He Do the Police in Different Voices,” from Dickens’ Our Mutual Friend. We see here the origins of the polyphony of different voices throughout the poem–in the final draft, the multitude of voices essentially constitutes the poem itself. When Eliot swapped this title for something that didn’t directly relate to the “Different Voices,” He likely sought to compensate using the epigraph. Like the Sybil, Kurtz wishes to die. He says, “I am lying here in the dark waiting for death” (Conrad, 3). However, unlike the Sybil, when he is on the brink of death, he actually loses his voice: “he cried out twice, a cry that was no more than a whisper” (Conrad, 3). Considering the time Eliot spends alluding to various hellscapes and states of death throughout the poem, it simply does not suit the form of the poem if the establishing character were to lose their voice in death, or, as Quisa pointed out, when their body becomes a waste land.

The Sybil, on the other hand, serves Eliot’s purpose beautifully. She, too, wishes for death, perpetually suffering as she wastes away in this in-between state. However, significantly she retains her voice. As she leans closer and closer to true death, more and more of her physical being deteriorates. Not only is her voice one of her defining characteristics, but if her body is “The Waste Land,” is Eliot’s poem itself, then it too assumes an perpetually shrinking quality. Her physical form grows smaller and smaller, approaching a state of nothingness, but (for the purposes of this investigation) never reaching it. Mathematically, an infinitely diminishing quantity is considered to approach zero. Zero is a very difficult number to define in the realm of reality. It is nothing, yet it is nothing in a pluralistic sense–it is “not any,” but that is not the same as “not one” or “not 10.” If the Sybil and her voice, and therefore “The Waste Land” and its voice, can be defined as zero, we see that The Sybil successfully represents the indefinite cacophony of the voices of the poem, as well as its tendency towards a state of nothingness.

While the “Death by Water” section of “The Waste Land” is seemingly short, Eliot’s original draft was actually quite long. In this draft, Eliot evokes multiple concepts that I understand as foundational to understanding the published version. Ultimately, however, I want to explore a sense of penetrability and the impenetrability throughout “Death By Water”. One of the first points in which the notion of impenetrability appears is in Eliot’s original draft when the narrator speaks of the horizon. They describe the horizon as, “a long white line, a wall, a barrier”, which suggests a sense of the horizon as infinite and impenetrable, especially in the sense that it is unconquerable for sailors. Another aspect of impenetrability in sailing is the ship itself, which floats on water that constantly tries to get in. Additionally, the sails, inferred by the mentions of wind, are also impenetrable to the forces of nature, such as the wind that is actually harnessed to achieve human goals. Thus, we can understand this impenetrability as representative of humanities disconnect from the wider world itself, which constantly seeks to enter and conquer “humanity”. Another important mention of penetration from the draft can be seen with Eliot’s multiple references to the sailors drinking alcohol. Through drinking, the sailors try to forget their anguish at sea by thinking about “home and dollars and the pleasant violin//At Arm Brown’s joint, and the girls and the gin”. Thus, the sailors are allowing their bodies (which are generally impenetrable by liquid), to consume alcohol, a poison, in order to escape their pain. And so, we are met with a paradox as Sailors become more vulnerable and penetrable to liquid in order to escape pain. When we arrive at the published version of Eliot’s poem (which appears at the very end of his draft), Phlebas the sailor is described as drowned. On line 317, Eliot describes how “a current under sea // Picked his bones in whispers”. Again, we return to the impenetrable human body, which when drowning, becomes penetrated by water, and is reduced to nothing but bones, which are not only penetrable but completely submerged in water themselves. Regarding the previous mentioning of drinking, we can interpret drowning as an ultimate form of becoming penetrated as a method of escapism. Interestingly, this escapism is an allowing of the previously disconnect nature to penetrate the human self, or “humanity”.

These lines of “The Waste Land” can be traced back to Dante’s Inferno, specifically Purgatorio, or Purgatory. Translated to English, Dante’s text reads “please remember me, who am La Pia. Siena made me, in Maremma I was undone.” The mention of Italian cities such as Siena and Maremma and the state of being undone clearly connects with Eliot’s mentioning of Richmond and Kew, also cities, undoing the narrator. Thus, we can infer that Eliot intended to connect the “trams and dusty trees” to Purgatory, a realm essentially between hell and heaven. The use of trams, interestingly, expanded from carrying passengers to carrying war cargo and industrial workers during the Great War and industrialism. Alongside these roles of trams, the “dusty trees” seem to suggest ash or dust from factories or explosions, also tying into industrialism and war. Thus, we can understand the once innocent trams and trees as tainted by a world of destructive progress and violence, forcing them into a middle state similar to purgatory. Additionally, the notion of the narrator lying supine, or back against the floor, in a narrow canoe, introduces another tension between the narrator and the water around him. While he is unable to move the canoe, and the water essentially guides it, he is still in this impenetrable man-made vessel, able to be on the level with the water, but not in it. Thus, the reader can interpret a sense of anxiety, which is exacerbated by the notion of being “undone” by the cities of Richmond and Kew. Interestingly, the river Thames, which is previously mentioned in this poem, is the only river that connects Richmond and Kew. As for the “undoing” of the narrator by the cities, one can interpret the narrator’s frustration and imprisonment in the canoe as spiritually degrading, especially as they pass cities bustling with culture and people who are able to move about. The narrator’s inescapable isolation from these cities portrays a contrast between nature and humanity, in which the narrator finds themselves in a middle-ground as well.

Eliot’s interest in death by suicide (as we see in the Sybil epigraph) is brought to life here in an overlap of Shakesphere references. Of course, the first line here is an echo of the Tempest reference in Burial of the Dead. Notably, it is a reference to Alonso, who tried to drown himself upon hearing false news of his son’s death. Then, the second line is quite feasibly a reworking of the famous, “To be, or not to be,” given that they are syntactically analogous with the placement of commas. Actually, they are structurally inverted, with the Hamlet comma placed in between the second and third syllable (x x, x x x x), while Eliot’s comma is placed in between the fourth and fifth syllable (x x x x, x x). Hamlet’s line is most popularly interpreted as a contemplation of suicide. Together, these two references seem to point to Ophelia’s death, and suggest Eliot decides that hers was a death by suicide as well.

The way Eliot inverts the structure of Hamlet’s line brings to mind the concept of a reflection, such as the “glassy stream” in which Ophelia drowns, as if the Eliot line floats on the surface of a body of water. If we were to indulge that imagery, it certainly would reaffirm the ideas of death and corpses, seeing as corpses float in water. This calls to mind the line “fear death by water” from Burial of the Dead in the tarot section. Perhaps Eliot means to prophesize suicidal drowning, which adds an interesting wrinkle to the grave motif that we’ve seen so often up to this point: does Eliot’s focus shift from the ground as a resting place to that of a water-submerged grave in A Game of Chess?

I think there is a strong relationship between poverty and education. Poverty can impact child’s ability to succeed in school. The educators and policymakers should design strategies to help economically disadvantaged students to ensure all students, regardless of their socioeconomic background, receive support in various aspects. There is no excuse for policymaker not to provides sufficient needs for all students.

Poverty, in my opinion, is the struggle of those who lack the means to improve their lot in life. Even though many people struggle, it might be difficult to succeed and get by. Fortunately, those living in poverty have access to opportunities provided by schools and scholarships, enabling them to improve their lives and attend prestigious universities across the globe.

While the father is clearly moving in a positive direction in terms of acceptance of his son's uniqueness, the fact that it took this long for him to appreciate his son is concerning, and I struggle to believe that there aren't any more underlying issues. It's a very significant change going from occasionally wishing your son would disappear to being amazed by him, and while it's positive, the fact that those initial thoughts even existed in the first place is troubling.

This passage stuck out to me because I had to reread it a few times before realizing it was referring to a blind girl, and I thought the way in which Kaden perceived this girl and her cane as a wand was beautiful

I understand where he is coming from, but I still struggle to get behind the idea that he has wished that his child would disappear.

I find this so disturbing. I can't believe a father would ever feel this way about his own son.

I still struggle to understand why Martin was so unhappy about Sebastian being able to stand. I don't see why it would be such a problem.

The fact that the father wasn't immediately happy upon seeing his child standing shows that he may have an underlying ableist attitude toward his son.

I am not sure why, but this line seems dehumanizing to me

The fact that Martin didn't call the police and get as much help as possible because he wasn't sure how to describe his son struck me because I would expect a father to do whatever he could possible to find his missing child

This fear of the mother's may reflect the fears that parents of children with disabilities may often face in regards to others seeing them as different

The playpen is described as a "hurdle" suggesting that the baby likely felt the need to escape the parents' constant arguments

The image of a baby jumping over the rail of a playpen and running away surpasses typical infant abilities as most babies aren't even capable of walking until they are one, suggesting that the child’s unique body makes him extraordinary

When trolling is used to illustrate fraud, it can be beneficial and promote critical thinking. However, when used to harass or bully someone, it can also be harmful and cause mental pain. The purpose of trolling and how it affects the targeted people or communities determine the result.

Stereotypes can be a hard thing to ethically judge. One should always leave room for humility in their understandings of other people. So much wrong about this world is the stubbornness and unwillingness to learn about others and see where an individual is coming from. At the same time, generalizations (like the one I just made) can be useful tools that at the very least hold partial/likely truths. In this way, they are like risky shortcuts; they can be useful if you are willing to take a few steps backwards should you find yourself in the wrong.

Problem #2の変数可視化表

To handle trolling, it's best to ignore and avoid engaging, as trolls thrive on attention. Users can use platform tools to block or report trolls, keeping the online environment controlled and safe.

I think the real issue here is treating "don't feed the trolls" as a maxim. Of course it is wrong to fault victims, but there is a big difference between assigning fault and offering a strategy to be employed. It won't always work, but it is an action one can take to attempt to minimize their personal suffering while waiting for a more ideal change in the system.

Problem #4の変数可視化表

I think this is what AI should be. I think that it makes most sense for humans to live in a world where AI exists this way. What is wrong with AI remaining this way? If it pretends to reciprocate why is real consciousness needed?

I don't think that these robots should deserve rights. They are real humans or Americans that are protected by the U.S. Constitution. Again, I think if Americans had to treat Ai as if it were a real U.S. citizen it may bring more harm than good.

I think that abiding ethical morals, is more important than having AI that has sentience. With it's advancements I think it imposes more on real humans' lives and it would probably do more harm than good if it had genuine emotions and consciousness.

If the current AI realm is what used to see as science-fiction, and consciousness is within the near future, then is what is "out of reach" going to become a reality in the future?

the freedom came with something she was not free of controlling

For an inexpensive starter machine ($5-25) that's easy to find, easy to get parts for and has a reasonable chance of working when in "unknown" or "untested" condition, I'd recommend one of the following ubiquitous, but solid machines which show up almost daily on ShopGoodwill.com:

• Royal Quiet De Luxe https://typewriterdatabase.com/Royal.Quiet+De+Luxe.72.bmys
• Remington Quiet Riter https://typewriterdatabase.com/Remington.Quiet-Riter.42.bmys
• Smith-Corona Clipper (their Sterling, Silent, and Silent Super are very similar, but can sometimes be more expensive; they often have a few more features like tabs, but are otherwise the same machine) https://typewriterdatabase.com/Smith+Corona.Clipper.86.bmys

They'd all make excellent starter machines for a younger kid. The black models with glass keys from the 1940s will look a bit more old school/classic while the more industrial browns and grays with plastic keys from the 1950s are still solid choices. You might also find some later 60s/70s versions of these machines (or variations), and while they may be a bit more colorful, they'll usually have a lot more cheap plastic and can potentially have cheaper builds. (My parents got me my first typewriter, a 1948 Smith-Corona Clipper, in the mid-1980s when I was 10—I have it today and it still works as well as it did then; I still also love the airplane on the hood.)

If you want something simple with a bit of color you can also look at the 70s/80s Brother Charger 11 which is pretty ubiquitious and inexpensive as well.

Since you have some time, you can wait for one in better looking cosmetic condition (and with a case) which means it was probably better taken care of, and less likely to need aggressive cleaning, and more likely to work without needing any repairs. You can also wait to find one local that you can pick up in person (to save shipping cost and/or potential damage) or which will be cheaper to ship from nearby.

Without any experience, you might try looking at Just My Typewriter's Typewriter 101 series on YouTube: https://www.youtube.com/playlist?list=PLJtHauPh529XYHI5QNj5w9PUdi89pOXsS She covers most of the basics there.

Cleaning a machine isn't horribly difficult and can be done pretty cheaply ($20 or less for some paint thinner/isopropyl and a small toothbrush), but if you need it or get a machine that needs some repair work, try https://site.xavier.edu/polt/typewriters/tw-repair.html.

If you're in an area with lots of yard sales, try shopping around and see if you find something interesting. It's at these that you'll have a potential chance of finding more collectible machines for pennies on the dollar and it'll also give you the chance to put your hands on machines to test them out to make sure they work.

Good luck! 🎄

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reply to u/strawberystegosaurus at https://old.reddit.com/r/typewriters/comments/1g5rgi4/typewriter_for_christmas_please_help/

He is the only noble gas with 2 valence e and not 8

It’s easier for Non metals to gain electrons (become anions) to make the shell full aka octet rule

anyone knows what this means？

no entiendo por que Esposito hace eso, el hecho que en el personalismo no se tenga en cuenta el cuerpo, la verdad derrumba esta crítica de Esposito, la persona es cuerpo, pero no se reduce al cuerpo únicamente

I've actually seen lots of videos where the AI is biased towards one political candidate versus another, you can ask specific questions like "Why should I vote for Trump/Kamala" and you may receive biased answers

This would be super interesting, AI would be able to pry into what they may have believed about certain topics just solely based on their works and then be the person that wrote them.

I've only ever really used AI for research, this is an incredible approach to AI. Asking for help with specific problems or for help when improving an essay is a great idea.

In my experience with AI, I have never encountered an error.

It's interesting that they knock out the pros and cons of AI immediately in the beginning of the article. An approach I have not seen before.

This is incredibly interesting! I have never heard of Theseus before. It is super interesting to learn that AI has been here since before the internet.

AI-native social media doesn't seem that far away tho

how human-in-the-loop a tool is will also be a factor. IC humans will do more QA; higher-level humans will do more orchestration

bearish on prosumer AI?

should generational products always aim for this (i.e. FB and Google approach of doing work for people) instead of augmenting people's abilities?

improve the backend tech instead of simplifying the UX

key to young people utilizing tech in more interesting and innovative ways, I think, is that they simply have more free time to explore the product. adults are always busy and in a rush

they just needed access to a bicycle

sort of like a tour guide for translating biz reqs -> tech

the equivalent for Copilot now would be that's for people who don't currently code

VisiCalc gives people who couldn't do something before a step function change bc now they CAN do it; Copilot is a marginal improvement that relies on there being an existing behavior

sweet spot of bicycle-riding: it's what one augmented person can produce

what 10x more ambitious thing does the technological shift enable?

This is very surprising to hear. I don't think any of my teachers from HIgh School ever used AI and if they did, they didn't tell us or show us in any way.

While this is helpful, AI doesn't really think for itself, it sounds like thinking. I think AI is better used as a tool for understanding other topics better.

why not?

This shows that the creators of AI, OpenAI in particular is taking special attention to how the education industry has been affected and is offering a tool to support Higher Education.

I also appreciate the origin and definition of AI being present before we get into the meat of the article.

I like that they give us a clear reason to trust them and it really builds their ethos and credibility.

I would move this section up much higher on the page right underneath the kind words section.

I would also revamp this section entirely because the lessons are already released (I assume) and ready to watch as soon as they join. So I would say something like: Access additional short sessons from each of the guest artists as well as the othe amazing benefits waiting for you inside the membership.

And then I'd list the sessions as well as the other top-line inclusions of your membership in bullet point format.

Keep growing with your FREE month inside the Roben's Nest

and receive ONE MONTH FREE inside the Roben's Nest, the membership .... (valued at $24).

This exlusive launch offer and pricetag disappear when the timer strikes zero

and ongoing

I would make this the heading

virtual summit

I'd change the CTA here too... DOWNLOAD THE DIGITAL VERSION

I'm curious... are you not able to link to the print version or do you ask them to sign up so you capture their email addresses? If it's a conscious decision to make them sign up, I'd reword the button CTA slightly. GET YOUR EXCLUSIVE LINK TO ORDER... and then the CTA in the pop-up box would be SEND ME THE EXCLUSIVE LINK TO ORDER

I wonder whether you can include a photo of yourself with the print version in the header so there's a warm smile as soon as they land on the page.

Les quartiers sont rénovés. Ils ont une nouvelle image grâce à l'art de rue.*

L'art de rue attire les touristes. L'art de rue a une bonne position à Paris.

Cet article est sur l'art de rue à Paris. Il parle aux parisiens curieux. Il parle aussi aux visiteurs d'autres pays. Cet art aide le tourisme à Paris.

J'aime le titre de cet article

Can you add a little more detail around how many hours it includes and the outcome of the day?

I would move this up so it's in line with the inclusions in the column on the left. And bold the VIP day inclusion on the left...

I would centre this

This font seems different to the headings above?

Can you centre this section on the page and bold the first sentence?

Can you move this text next to the photo of your with your medal at all?

Could you structure this section as 4 columns at all? I think it would make it look cleaner.

I feel like there's something missing over the header image?

!!!!

Content specific NCC are neuronal populations which are active when specific "contents of experience" are perceived or thought about, and are inactive otherwise. For example, if the NCC for experiencing the contents of a face are stimulated

This is a continuation of Elliot showing his sympathy for Bonnie after realizing she was a victim of sexual assault. A great way of showing sympathy is to simply be in a space that brings the other comfort and just sitting with them in it.

I think this is too much in the header section. I would remove this section entirely.

As for the button copy, I'd remove Register Here!

I would beef these up in a separate 'this is what you can expect' section underneath your about section.

Some of these ideas would be great to the 'right now' section further up on the page

I'd try to condense your about section to one visual section on the page. So possibly see how you can move this bit into your 'main' about section

BB ARt Journal Masterclass will help you...

I'd then follow it with a paragraph that addresses the right now and how they're currently approaching their art. The frustration of doubting themselves, not knowing where to start, lacking feedback so they know how they can improve, etc...

And then I would introduce your BB Art Journals Masterclass as the solution in the next section.

I would lead with what's possibe here... the dream future!

What if you activated your full creative potential and said goodbye to overwhelm?

Imagine what it would feel like to eliminate the sheer impossible obsticles that have been holidng you back from creating? And you not only started creating consistently, but you could watch your skills grow week by week?

And you could trust your artistic intuition?

Someting like this...