interesting story and also lysa connection with he dying in water and the tears of lys thing

yay more tullys

lady stoneheart

last words last chapter being catelyn and now its her chapter

he is one unfortuntly

jon....

YEAH

very polite way of saying he's horny

PLEASE

ooo them i know theyd get the job done

and he sees him again and his head is spiked

yess

interesting interesting

i wish she got to meet him

oh not HIM

liarrr

which it does but dany is gonna have 3 dragon children in place of it

cut their heads off....

she's so cute

wrong one but dw i thought this too

:(

he really does know everythingg

varys..

who?

vhagar is here somewhere

the dragonss

NAHH THATS CRAZY

oo cool

theyre kinda silly

OMG YESS

i like this cat

why fire?

it being full of them because no more ratcatchers so otto brought in more cats lol

ohh when he WON it nvm

wait a minute i thought he betted FOR him??

nvm i thought this was harrenhall

that would be an interesting couple ngl kinda like dany x robb enemies to lovers lol

oh yeah!!

aw nooo

thats so mean but he did get his first revenge

lol

i love herr

UFINW LITTLEFINGER WHEN I CATCH YOU

lmao

interesting

yess loyalty but maybe it shows cat's death as well since harrenhall IDK

i love this sm

see she is smart at times

actually i think you opened your mouth

ohh ok so he's a random guy

eww

strange name

Este comando de upower fue útil para revisar que la batería externa estaba cargando. La interna aún no carga.

Estos fueron los métodos que usé para resolver el problema de la batería en mi portátil, que se manifestó una vez le actualicé la RAM de 8 a 24 GB (agregué 16 GB).

Finalmente removí la batería externa, apliqué el reset de hardware y dejé el portátil con el cargador aplicado a la batería interna. Finalmente volvió a cargar.

Résumé de la vidéo [00:00:00][^1^][1] - [00:29:54][^2^][2]:

Cette vidéo présente une conférence d'Esther Duflo sur les approches expérimentales en éducation, mettant en lumière les résultats des élèves en CP et CE1 en France, les dispositifs d'évaluation, et l'impact des vacances scolaires sur les inégalités de réussite.

Points forts: + [00:00:23][^3^][3] Introduction de la conférence * Présentation par Sandra Andreux, chef du bureau de la conception et du pilotage des évaluations des élèves * Focus sur les résultats des élèves en CP et CE1 + [00:01:01][^4^][4] Statistiques de scolarisation * 6,4 millions d'élèves dans le premier degré en France * 1,1 million d'élèves dans des territoires défavorisés + [00:02:15][^5^][5] Dispositifs d'évaluation * Évaluations sur échantillon et exhaustives * Participation à des études internationales comme PISA + [00:06:54][^6^][6] Objectifs des évaluations nationales * Fournir des outils standardisés aux enseignants * Doter les acteurs locaux d'indicateurs de référence * Contribuer au débat public avec des indicateurs nationaux + [00:15:01][^7^][7] Résultats des évaluations nationales * Analyse des inégalités de réussite selon le secteur de scolarisation et le genre * Impact de la crise sanitaire sur les performances + [00:30:00][^8^][8] Impact des vacances scolaires * Étude sur l'évolution des performances pendant les vacances * Augmentation des inégalités de réussite pendant l'été

Résumé de la vidéo [00:00:00][^1^][1] - [00:08:04][^2^][2]:

Cette vidéo présente une conférence sur les approches expérimentales en éducation, animée par Esther Duflo. Elle aborde l'importance de la libre diffusion des connaissances, la collaboration interdisciplinaire, et les efforts pour améliorer l'éducation grâce à des données et des expériences en milieu scolaire.

Points forts: + [00:00:23][^3^][3] Introduction de la conférence * Esther Duflo accueille les participants * Mention de la nature bilingue de l'événement * Présentation du Collège de France et de son rôle + [00:02:04][^4^][4] Objectifs de la conférence * Collaboration entre scientifiques et praticiens * Présentation du réseau ID et de la Fondation pour l'Initiative d'Apprentissage * Discussion sur l'enseignement et l'apprentissage des enfants + [00:03:11][^5^][5] Efforts en matière de données et d'expérimentation * Création de données et partenariats pour des expériences en milieu scolaire * Projets interdisciplinaires entre scientifiques cognitifs et économistes * Évaluation de l'impact des programmes éducatifs + [00:04:33][^6^][6] Engagement du Collège de France en éducation * Présentation de l'action "Agir pour l'éducation" * Discussion sur les défis du système éducatif français * Développement d'outils basés sur les sciences cognitives pour l'enseignement

? what does this say and why dooes it matter

What is paul celans significance

Yet another definition of Elio, in three contrasting words to the top (oliver)

Does this show maturity? Independence from external influences on his identity?

Is he looking towards others to figure out how he feels, what is real and what is not, what is right or wrong? In some way he is also trying to see himself in her, just as narcissus does. And he does this to know himself, which was his main goal anyway

Is this trying to see another part of Oliver, to know another side of him? What does this have to do with identity/bodily continuity?

Why does he always need to bear witness? Why does he need to define? Why does he need to know everything? That is also his love for oliver, the knowing of the intimate sides of him, of his underskin

How tie this to the thing about sexuality? And bodily continuity?

What does this say about his maturity? age 6?

?

?

?

contradiction

another side

Speaks to the communal and collaborative nature of whaling, present throughout these chapters

Recalls "Young Goodman-Brown." Does it put a question to Hawthorne in some sense--have you, lubber, any notion of the true terrors of the world?

A line which recalls the unabating liminality of this tale, a within-and-without quality which lends to it the overawing character of the sublime.

The analysis of the men into their constituent parts magnifies the kind of horror of the scene, the many-armed hauling of a corpse across the dark water.

Likewise Melville's story, which is composed of so many yarns spun together. Can they bear the monstrous weight he puts on them? Can they bear us, who are likewise fast and loose fish?

The combination of magic and terror hints at that characteristic of the sublime already alluded to by several others here--that experience of being brought before the aesthetically terrible object which keeps the soul in tension between the sense of being overwhelmed and the sense of the self expanding to the bounds of that overwhelming thing.

This is an example

直接设备会干扰受试者的活动。

面部特征容易收到外界因素的干扰，个体差异大。

Test notes

Résumé de la vidéo [00:00:05][^1^][1] - [00:16:32][^2^][2]:

Cette vidéo présente une étude sur l'efficacité de la méthode DR-All (Differential Reinforcement for All) pour gérer les comportements perturbateurs en classe. L'étude a été réalisée dans le cadre d'une thèse dirigée par Pascal Pensu et vise à réduire les comportements qui nuisent au déroulement des cours, augmentent le stress des enseignants et diminuent les performances scolaires des élèves.

Points forts: + [00:00:18][^3^][3] Présentation de l'étude * Introduction de l'étude sur la méthode DR-All * Objectif de réduire les comportements perturbateurs en classe * Importance de la gestion des comportements pour le bien-être des élèves et des enseignants + [00:03:46][^4^][4] Stratégie DR-All * Explication de la stratégie de renforcement différentiel pour tous * Transfert de l'attention des comportements perturbateurs vers les comportements appropriés * Application de la stratégie de manière équitable pour favoriser l'apprentissage par observation + [00:05:45][^5^][5] Résultats de l'étude * Présentation des participants et de la méthodologie de l'étude * Comparaison des comportements perturbateurs avant et après la formation des enseignants à la méthode DR-All * Réduction significative des comportements perturbateurs dans le groupe expérimental + [00:14:00][^6^][6] Conclusion et implications * Efficacité de la méthode DR-All pour résoudre les problèmes de discipline en classe * Importance de la formation et du suivi des enseignants pour un changement de pratique * Suggestions pour des études futures et l'application de la stratégie à plus grande échelle

Résumé de la vidéo [00:00:00][^1^][1] - [00:07:22][^2^][2]:

Cette vidéo présente une discussion avec Pier, directeur d'école depuis 17 ans, qui partage ses méthodes pour accueillir les familles dans son établissement. Il souligne l'importance des entretiens préalables avec les familles pour comprendre leur relation avec l'école et leurs enfants, et comment cela peut influencer l'apprentissage. Pier discute également de la diversité des structures familiales en 2023 et de l'utilisation d'un guide d'entretien non institutionnel pour faciliter la communication.

Points forts: + [00:00:20][^3^][3] L'accueil des familles * Entretiens préalables pour comprendre le rapport à l'école * Importance de connaître les liens avec les enfants * Vigilance sur les problématiques d'apprentissage + [00:01:02][^4^][4] La diversité des familles * Reconnaissance de différentes structures familiales * Adaptation de l'accueil à chaque situation unique * Sensibilité aux besoins spécifiques des enfants et parents + [00:01:23][^5^][5] L'utilisation d'un guide d'entretien * Guide conçu par l'école pour faciliter les discussions * Questions sur la relation avec l'enfant et les attentes envers l'école * Nécessité d'aller au-delà du guide pour une communication approfondie + [00:03:04][^6^][6] Observations lors des entretiens * Exemple d'une interaction touchante entre une mère et sa fille * Importance de la question de la séparation pour certains enfants * Attention particulière aux liens parents-enfants et au passé des parents avec l'école + [00:04:44][^7^][7] Transmission d'informations aux enseignants * Partage sélectif des informations recueillies avec les collègues * Réunions avec les enseignants et les adsem pour discuter des besoins des élèves * Suivi tout au long de l'année scolaire + [00:05:11][^8^][8] Le rôle du directeur d'école * Réflexion sur l'équilibre entre accompagnement et autorité * Changement de pratique professionnelle pour inclure davantage les familles * Importance de créer des relations de confiance avec les familles

Résumé de la vidéo [00:00:05][^1^][1] - [00:15:23][^2^][2]:

Cette vidéo est une rediffusion d'une émission sur l'accueil des émotions des élèves face à l'actualité, initialement diffusée le 27 avril 2022 pendant la guerre en Ukraine. Elle aborde l'importance de reconnaître et de gérer les émotions des enfants à l'école, en particulier lorsqu'ils sont confrontés à des événements mondiaux majeurs ou à des crises. Les invités discutent des stratégies pour aider les enfants à exprimer leurs émotions et à les comprendre, ainsi que des outils pédagogiques qui peuvent être utilisés pour faciliter ce processus.

Points forts: + [00:01:00][^3^][3] L'importance de l'accueil des émotions * L'impact des actualités sur les enfants * La nécessité de paroles rassurantes d'adultes * Le rôle des enseignants dans la structuration des émotions + [00:02:36][^4^][4] Stratégies pour accueillir les émotions * L'importance de nommer et d'exprimer les émotions * L'utilisation de jeux et d'activités pour explorer les émotions * La création d'un espace pour que les enfants puissent partager leurs sentiments + [00:07:00][^5^][5] Outils pédagogiques pour gérer les émotions * Les petits laboratoires d'empathie et le jeu des trois figures * L'engagement des adolescents dans des actions et des projets * L'adaptation des approches en fonction de l'âge des enfants

Résumé de la vidéo [00:08:00][^1^][1] - [00:15:23][^2^][2]:

Cette vidéo est une rediffusion d'une émission sur la manière d'accueillir les émotions des élèves face à l'actualité, initialement diffusée le 27 avril 2022 pendant la guerre en Ukraine. Les invités discutent de l'importance de reconnaître et de gérer les émotions des enfants à l'école, en particulier lorsqu'ils sont confrontés à des événements mondiaux majeurs ou à des crises.

Points forts: + [00:08:00][^3^][3] Gestion des émotions à l'école * Importance de reconnaître les émotions des enfants * Utilisation de jeux et d'activités pour exprimer les émotions * Identification des besoins sous-jacents aux émotions + [00:10:01][^4^][4] Théâtre et expression corporelle * Le théâtre comme moyen d'expression des émotions * L'importance du mouvement et de l'incarnation pour les enfants * Utilisation de l'activité théâtrale pour aider les enfants à gérer leurs émotions + [00:12:22][^5^][5] Adaptation aux différents âges * Différences dans la gestion des émotions selon l'âge * Approches spécifiques pour les tout-petits, les enfants plus âgés et les adolescents * Importance de l'engagement et de l'action chez les adolescents

Résumé de la vidéo [00:00:06][^1^][1] - [01:01:06][^2^][2]:

Cette vidéo est une table ronde intitulée "Développer les compétences psychosociales à l'école - Osons la Communication NonViolente", organisée par le réseau Canopé. Elle aborde l'importance de la communication non violente (CNV) et des compétences psychosociales dans le contexte éducatif, en particulier face aux défis sociétaux actuels tels que le harcèlement et les tensions géopolitiques. Les intervenants discutent des stratégies pour promouvoir un climat scolaire apaisé et la formation des enseignants à ces compétences essentielles.

Points forts: + [00:00:06][^3^][3] Introduction et contexte * Présentation de la table ronde et de son importance * Mention de la semaine de lutte contre le harcèlement * Réflexion sur le rôle de l'école face aux tensions sociétales + [00:03:50][^4^][4] L'importance de travailler en amont * Discussion sur l'investissement dans l'éducation dès l'école maternelle * L'importance de l'équilibre entre le cognitif et l'affectif * La nécessité de la sécurité émotionnelle pour les enfants + [00:24:23][^5^][5] Expériences personnelles avec la CNV * Témoignages sur l'utilisation de la CNV dans l'enseignement * L'impact de la CNV sur la gestion des émotions et des comportements * La valeur de l'empathie et de l'autoempathie dans l'éducation + [00:37:45][^6^][6] Formations en présentiel et leur importance * Annonce des ateliers à venir sur la CNV et les compétences psychosociales * Remerciements aux intervenants pour leur contribution * Discussion sur la professionnalisation des métiers de l'éducation + [00:49:34][^7^][7] La CNV comme outil parmi d'autres * Débat sur l'intégration de la CNV dans la formation initiale * L'importance de former les formateurs à adopter une posture adéquate * La CNV comme moyen de créer une dynamique positive dans les établissements + [00:59:07][^8^][8] Les défis de l'adoption de la CNV * Reconnaissance du processus difficile de changement des habitudes * La CNV n'est pas une solution magique, mais un outil parmi d'autres * L'importance de la formation continue pour le bien-être et la santé mentale

Résumé de la vidéo [00:00:00][^1^][1] - [00:08:45][^2^][2]:

La vidéo présente l'application de la Communication Non Violente (CNV) dans une école primaire en France, où les enseignants et les élèves pratiquent l'empathie, l'écoute active et l'expression des sentiments et des besoins pour améliorer l'interaction et le bien-être à l'école.

Points forts: + [00:00:00][^3^][3] Introduction à la CNV à l'école * Nadia Amrani, directrice, explique le projet d'école girafe basé sur la CNV * Formation de l'équipe pédagogique par une formatrice certifiée du CNVC + [00:01:01][^4^][4] Principes de base de la CNV * Importance de lâcher prise, écouter les autres, et reconnaître sentiments et besoins * Droit des élèves et des adultes à s'exprimer et à s'écouter mutuellement + [00:02:03][^5^][5] Exercice de gratitude 'l'ami secret' * Chaque enfant observe positivement un camarade et exprime sa gratitude * Renforcement de l'estime de soi et du bien-être + [00:02:20][^6^][6] Application de la CNV en classe * Observation, sentiment, besoin, et demande comme fondements de la CNV * Approche empathique face aux élèves qui ne travaillent pas + [00:04:08][^7^][7] Témoignages de solidarité entre élèves * Augmentation de la solidarité et de l'entraide observée en classe et en récréation * Exemple de l'entraide spontanée des élèves de CM2 envers les CP + [00:06:21][^8^][8] Outil 'la marelle de la colère' * Aide les élèves à exprimer leur colère de manière constructive * Accompagnement par un adulte pour résoudre les conflits

This is another example

This is an example comment for renaming this section

https://www.theguardian.com/world/article/2024/jun/26/german-activists-take-government-to-court-over-climate-policy

如何理解？

没看懂，财务知识还是没学好

劳动法与工会

tech for tech's sake; need for context

challenging authority of museum as established preserver of cultural history/heritage

removing context

BANGER!!!

为什么options对象只在runtime执行

这就是通过constructor在注入依赖吗

这里的providers？

这个是什么

自定义的provider就要在inject的时候传入token吗

这个就是token

所以这个注册的过程是

这是什么意思

https://www.theguardian.com/environment/article/2024/jun/26/voluntary-carbon-market-offsetting-industry-reforms-cccg-climate-crisis-advisory-group-aoe

Résumé de la vidéo [00:00:03][^1^][1] - [00:38:57][^2^][2]:

Cette vidéo est un débat sur la transgression et la sanction dans le contexte éducatif, animé par Hélène et l'équipe d'Extra Classe. Les invités, Éric Prera, philosophe, et Aurélien Becker, conseiller principal d'éducation, discutent de la distinction entre punition et sanction, l'importance de la transgression pour le développement des adolescents, et l'évolution des pratiques disciplinaires dans les écoles.

Points forts: + [00:01:15][^3^][3] Éric Prera, philosophe de l'éducation * Professeur à l'Université de Laoren * Auteur de l'ouvrage "Le que sais-je la sanction en éducation" * Discute de la légitimité de l'école et de la réflexion sur la sanction + [00:02:21][^4^][4] Aurélien Becker, conseiller principal d'éducation * Expérience de 15 ans dans l'éducation * Formateur académique et conseiller en ingénierie de formation * Partage une expérience récente de gestion de punition + [00:15:45][^5^][5] Histoire des sanctions éducatives * Évolution des pratiques de sanction depuis 1800 * Introduction de nouvelles formes de sanctions comme le renvoi et les arrêts * Influence des courants hygiénistes et des pédagogies nouvelles + [00:21:00][^6^][6] Rôle des parents dans l'éducation * Changement du rôle des parents de relais à avocats de leurs enfants * Nécessité de redonner crédit aux pratiques de sanction dans les écoles + [00:33:53][^7^][7] Prévention de la violence et formation des élèves * Importance de la prévention et de la compréhension des règles par les élèves * Formation des élèves à des techniques de défense pacifique + [00:37:20][^8^][8] Inspirations et lectures recommandées * Éric Prera recommande Howard Zehr, père de la justice restaurative * Aurélien Becker suggère le livre de Daniel Marcelli, pédopsychiatre

https://www.theguardian.com/environment/article/2024/jun/26/most-of-it-was-dead-scientists-discovers-one-of-great-barrier-reefs-worst-coral-bleaching-events

https://www.theguardian.com/environment/article/2024/jun/25/us-climate-finance-gas-expansion-john-podesta-biden

https://www.theguardian.com/environment/article/2024/jun/25/rising-sea-levels-flooding

https://www.theguardian.com/world/article/2024/jun/25/eu-regulated-sustainable-funds-invest-14bn-in-biggest-polluters

Résumé de la vidéo [00:00:05][^1^][1] - [00:47:50][^2^][2]:

Cette vidéo intitulée "Croire en chaque élève - Parlons pratiques ! #32" sur YouTube explore les approches éducatives positives dans les établissements situés dans des quartiers populaires, en mettant l'accent sur l'éducabilité et l'émancipation des élèves. Elle souligne l'importance de la maîtrise de la langue et présente des témoignages d'enseignants et de coordinateurs qui croient en le potentiel de chaque élève, en particulier ceux scolarisés dans les réseaux d'éducation prioritaire (REP et REP+).

Points forts: + [00:00:05][^3^][3] Introduction au sujet * Présentation de l'épisode dédié aux pratiques éducatives * Focus sur les établissements de quartiers populaires * Importance de croire en chaque élève + [00:00:54][^4^][4] Invités et leurs contributions * Marguerite, enseignante et membre d'un collectif éducatif * Discussion sur le collectif et ses publications * Engagement envers l'éducation dans les quartiers populaires + [00:02:38][^5^][5] Rôle des coordinateurs REP+ * Myriam, coordinatrice REP+, explique son rôle * Soutien aux enseignants et collaboration avec les partenaires * Importance de l'accompagnement dans les projets éducatifs + [00:03:26][^6^][6] Expériences d'une principale adjointe * Émilie partage son parcours et son choix de travailler en REP+ * L'impact de l'approche empathique sur les élèves * La nécessité de comprendre les élèves au-delà de leur rôle scolaire + [00:18:10][^7^][7] Initiatives pour engager les élèves * Projets pour encourager les activités hors écran * Implication des enseignants dans la vie du quartier * Création d'un environnement d'apprentissage dynamique + [00:27:17][^8^][8] Équité et aspirations des élèves * Discussion sur l'équité et la lutte contre le déterminisme social * Encouragement des élèves à poursuivre des études supérieures * Importance des modèles et de la représentation pour les élèves

Résumé de la vidéo [00:00:05][^1^][1] - [00:47:50][^2^][2]:

Cette vidéo intitulée "Croire en chaque élève - Parlons pratiques ! #32" explore les pratiques éducatives dans les quartiers populaires, en mettant l'accent sur l'importance de croire en chaque élève, en particulier ceux scolarisés dans les réseaux d'éducation prioritaire (REP). Les invités discutent de l'éducabilité, de l'émancipation et du rôle fondamental de la maîtrise de la langue française.

Points forts: + [00:00:05][^3^][3] Introduction au sujet * Présentation des approches éducatives positives dans les établissements de quartiers populaires * Discussion sur l'éducabilité et l'émancipation des élèves + [00:30:00][^4^][4] L'importance de la langue française * La maîtrise de la langue française est cruciale pour l'émancipation et l'intégration des élèves * Les enseignants partagent des stratégies pour aider les élèves à améliorer leurs compétences linguistiques + [00:33:39][^5^][5] Déconstruire le déterminisme social * Les intervenants abordent les défis liés au déterminisme social et comment ils encouragent les élèves à viser haut * Importance de l'orientation et de l'accompagnement des élèves dans leurs choix d'études supérieures + [00:47:03][^6^][6] Conclusion et inspirations * Les invités partagent des références culturelles et des expériences personnelles qui inspirent leur pratique éducative * L'émission se termine sur une note positive, soulignant la capacité de chaque élève à réussir

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ok

eLife assessment

This manuscript aims to unravel the contribution of cholesterol to aquaporin-0 (AQP0) tetramer array formation within lens membranes. Compelling electron crystallography data are combined with solid molecular dynamics experiments to identify a specific cholesterol binding site of significance to protein clustering within lipid rafts. The important work advances our understanding of membrane biology and will be of broad interest to membrane transport biologists, biochemists, and structural biologists.

Reviewer #1 (Public Review):

Aquaporin-0 forms 2D crystals in the lens of the eye. This propensity to form 2D crystals was originally exploited to solve the structure of aquaporin-0 reconstituted in membranes. Existing structures do not explain why the proteins spontaneously form these arrays, however. In this work the authors investigate the hypothesis that the main lipids in the native membranes, sphingomyelin and cholesterol, contribute to lattice formation. By titrating the cholesterol: sphingomyelin ratio, the authors identify cholesterol binding sites of increasing stability. The authors identify a cholesterol that interacts with adjacent tetramers and is bound at an unusual membrane depth. Computational simulations suggest that this cholesterol is only stable in the context of adjacent tetramers (ie lattice formation) and that the presence of the cholesterol increases the stability of that interface. The exact mechanism is not clear, but the authors propose that the so-called "deep cholesterol" improves shape complementarity between adjacent tetramers and modulates the kinetics of protein-protein interactions. Finally, the authors provide a reasonable model for the role of cholesterol in

Strengths of this manuscript include the analysis of multiple structures determined with different lipid compositions and lipid:cholesterol ratios. For each of these, multiple lipids can be modelled, giving a good sense of the lipid specificity at various favorable lipid binding positions. In addition, multiple hypotheses are tested in a very thorough computational analysis that provides the framework for interpreting the structural observations. The authors also provide a thorough scholarly discussion that connects their work with other studies of membrane protein-cholesterol interactions.

The model presented by the authors is consistent with the data described.

Reviewer #2 (Public Review):

Summary:

In the manuscript by Chiu et al., "Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts," the authors address the effect of cholesterol on array formation by AQP0. Using a combination of electron crystallography and molecular dynamics simulations, the authors show binding of a "deep" cholesterol molecule between AQP0 tetramers. Each AQP0 tetramer binds four deep cholesterols to form a crystallographic array of AQP0.

Strengths:

The combined approaches of electron crystallography and MD simulations under different lipid conditions (different sphingomyelin and cholesterol concentrations) are a strength of the study. The authors provide a thorough and convincing assessment of cholesterol binding, protein-protein interactions, and array formation by AQP0. The MD simulations allow the authors to consider the propensity of cholesterol to occupy the observed binding sites in the absence of crystal contacts. The combined methods and the breadth of analyses set a high standard in the field of membrane protein structural biology.

The findings of the authors fit nicely into a growing body of literature on cholesterol binding sites that mediate membrane protein-protein interactions. Cholesterol interacts with a variety of membrane proteins via its smooth alpha face of rough beta face. AQP0 is somewhat unique in that it binds the rough face of cholesterol in a "deep" binding site that places cholesterol in the middle of the membrane bilayer. So-called "deep" cholesterol binding sites have been described for GPCRs and docking studies suggest they may exist on other ion channels and transporters. In the case of AQP0, the deep cholesterol acts as a glue that holds two tetramers together. Since each tetramer has four binding sites for deep cholesterol, the assembly and mechanical stability of an extended two-dimensional array of AQP0 tetramers is a natural consequence in lens membranes.

Weaknesses:

The authors report that the findings generally apply to raft formation in membranes. However, this point is less clear as the lens membrane in which AQP0 resides is rather unique in lipid and protein content and density. Nonetheless, the authors achieve the overall goal of evaluating cholesterol binding to AQP0, and there are many valuable and informative figures in the main manuscript and supplement that provide convincing results and interpretations.

Reviewer #3 (Public Review):

Summary:

This manuscript aims to unravel the mechanisms behind Aquaporin-0 (AQP0) tetramer array formation within lens membranes. The authors utilized electron crystallography and molecular dynamics (MD) simulations to shed light on the role of cholesterol in shaping the structural organization of AQP0. The evidence suggests that cholesterol not only defines the positions and orientations of associated molecules but also plays a crucial role in stabilizing AQP0 tetramer arrays. This study provides valuable insights into the potential principles driving protein clustering within lipid rafts, advancing our understanding of membrane biology.

In this review, I will focus on the MD simulations part, since this is my area of expertise. The authors conducted an impressive set of MD simulations aiming at understanding the role of cholesterol in structural organization of AQP0 arrays. These simulations clearly demonstrate the well-defined localization of cholesterol molecules around a single AQP0 tetramer, aligning with previous computational studies and the crystallographic structures presented in this manuscript. Interestingly, the authors identified an unusual position for one cholesterol molecule, located near the center of the lipid bilayer, which was stabilized by the adjacent AQP0 tetramers. The authors showed that these adjacent tetramers can withstand a larger lateral detachment force when deep cholesterol molecules are present at the interface compared to scenarios with sphingomyelin (SM) molecules at the interface between two AQP0 tetramers. Authors interpret that result as evidence that deep cholesterol molecules mechanically stabilize the interface of the AQP0 tetramers.

The simple steered MD simulations are typically employed to either identify pathways for subsequent free energy calculations, such as umbrella sampling or perform numerous non-equilibrium simulations, utilizing the Jarzynski equation to extract free energy. In this paper, the authors conducted steered MD simulations to examine the maximum force required to separate tetramers, and they did not carry out the more rigorous but challenging free energy calculations. The observation that the maximum force needed to separate tetramers in the presence of cholesterol (compared to the SM case) suggests a positive direction in the authors' work, however, free energy calculations would be needed to fully support the cholesterol stabilization effect.

Author response:

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

Reviewer #1 (Public Review):

The model presented by the authors is consistent with the data described. Further testing of this model, for example by mutating the deep cholesterol binding site, would strengthen the model. However, such experiments might be challenging due to the relatively non-specific/hydrophobic nature of the deep cholesterol binding site.

We completely agree that testing of the deep cholesterol-binding site by mutagenesis would be ideal. However, as the reviewer points out, such experiments would be challenging, not only because of the non-specific/hydrophobic nature of the deep cholesterol-binding site but also because we have been purifying AQP0 from natural sources (sheep eyes) and because it would be very difficult to secure the substantial amount of cryo-EM time needed to generate an electron crystallographic structure.

Reviewer #2 (Public Review):

The authors report that the findings generally apply to raft formation in membranes. However, this point is less clear as the lens membrane in which AQP0 resides is rather unique in lipid and protein content and density.

We agree that the lens membrane is quite unique in its lipid and protein content and density, but rafts are also characterized by the same lipids and high protein density. Nonetheless, we do agree that our suggested implications for lipid rafts are speculative and so we emphasize this more in the revised version of the manuscript by writing: “This model is specific for the formation of AQP0 arrays in lens membranes, but we speculate that similar principles may underlie the organization of lipid rafts”.

Reviewer #3 (Public Review):

The authors showed that these adjacent tetramers can withstand a larger lateral detachment force when deep cholesterol molecules are present at the interface compared to scenarios with sphingomyelin (SM) molecules at the interface between two AQP0 tetramers. Authors interpret that result as evidence that deep cholesterol molecules mechanically stabilize the interface of the AQP0 tetramers. This conclusion has minor weaknesses, and the rigor of the lateral detachment simulations could be increased by establishing a reference point for the detachment force needed to separate AQP0 tetramers in a scenario without lipids at the interface between tetramers, and by increasing the number of repeats for the non-equilibrium steered MD simulations. Thermodynamic integration might be a better approach to compute the stabilization energy in the presence of cholesterol compared to the SM case.

In all electron crystallographic structures of AQP0 determined to date, lipids have always been observed sandwiched in between the AQP0 tetramers (see, for example, Gonen et al., Nature, 2005 and Hite et al., EMBO J., 2010). Therefore, considering a scenario without lipids at the interface would be unnatural and the AQP0 array would likely not be stable. Such a scenario would thus not be the most appropriate reference point for the lateral detachment simulations. In our view, comparison of a scenario with the deep cholesterol at the interface versus a scenario without it appeared a more realistic setup to investigate the stabilizing role the deep cholesterol has on the association of AQP0 tetramers. In the Results subsection regarding these simulations, we added the following sentence to further stress the rationale of our experimental setup: “Comparison of these two cases should allow us to assess the effect of the deep-binding Chol3 molecules on the mechanical stability of the associated AQP0 tetramers.”

Concerning the second suggestion of the reviewer of increasing the number of repeats, we doubled the number of simulation replicas: now it is n=20 for each pulling velocity and lipid interface. The trend of higher detachment forces for the interface containing cholesterol prevailed in a statistically significant, robust fashion (see Figure 7 of the revised manuscript and the main text referring to it). In consequence, as the reviewer suggested, extension of the dataset increased the rigor of the lateral detachment simulations. In addition to Figure 7 and the Results section, the Methods section and Table 4 have been updated to reflect the expanded dataset. 

Finally, concerning the usage of thermodynamic integration to compute the stabilization energy, we agree with the reviewer that calculation of the free energy would be better to determine the thermodynamic stabilization imparted by the cholesterol molecules. At an earlier stage of the project, we did indeed consider carrying out this type of simulations, but we decided against it because of the complexity and poor convergence of such calculations. Our choice is also based on a previous attempt in which it proved very challenging to use free energy calculations to assess the binding of lipids to a flippase (see Wang et al. BioRxiv, https://doi.org/10.1101/ 2020.06.24.169771, 2021). We now included this consideration in the revised manuscript by adding the following sentence in the Discussion: “Although we provide solid evidence here that deep cholesterol impart mechanical stabilization, free energy calculations would be required to obtain the full picture of thermodynamic stabilization. Such free energy calculations are challenging for lipids, due to the chemical complexity and poor convergence involved (Wang et al., 2021), and are thus beyond the scope of the current work.”

Reviewer #1 (Recommendations For The Authors):

Reorganizing a few concepts would make the story easier to follow. For example, the analysis of the bilayer thickness seems disjointed. Although Figure 4 shows measurements, it is not clear that the measurements represent bilayer thickness until the last paragraph of page 21 in the discussion, where "Hydrophobic thickness" is first introduced. Moving that first paragraph of page 22 that refers to Fig. 4A to the results would be helpful to understand the figure, and would prepare the reader for this part of the discussion.

In response to the reviewer, we moved the description of the measurements of the hydrophobic thickness to the Results section (Page 12) and adjusted the Discussion to minimize repetition (page 22).

Likewise, Figure 4E shows measurements of something, but it is not clear that these are the dimensions of a protein pocket until well into the discussion.

In response to the reviewer’s comment, we added a sentence both in the Results section [It sits in a pocket between the two adjacent AQP0 tetramers that is wider in the extracellular leaflet than the cytoplasmic leaflet (Figure 4E)] as well as to the caption of Figure 4E [The dotted lines indicate the distance between the two adjacent AQP0 tetramers at the positions of the ring system (~8.5 Å) and the acyl chain (~2.5 Å)].

Figure 2 - a comment for the non-specialists on what this region of the protein is would be helpful context. Is this the pore with part of the NPA motif?

We agree with the referee and added the following sentence to the caption of Figure 2: “A region of the water-conducting pathway close to the NPA (asparagine-proline-alanine), the AQP signature motif, is shown”.

Reviewer #2 (Recommendations For The Authors):

There is only one recommendation: In the results section entitled "Cholesterol positions observed in the electron crystallographic structures are representative of those around single AQP0 tetramers" the authors do not describe their approach. They refer to a reference (AponteSantamaria et al., 2012). The authors state the problem (investigate cholesterol positions), but it would be helpful to the readers if they also described the experimental approach.

We agree with the reviewer and made the following addition to the sentence “we performed MD simulations and calculated time-averaged densities to investigate ...”

Reviewer #3 (Recommendations For The Authors):

Technical comments:

(1) Authors stated: "Equilibration simulations were then performed until bulk membrane properties, such as thickness and deuterium order parameters, became stable and congruent with previous reports such as those by (Doktorova et al., 2020) and others (Figure 5-figure supplement 2 and Figure 5-figure supplement 3)." However, bilayer thickness is not represented in these figures. Additionally, I observed that the area per lipid (APL) appeared to be somewhat variable. This variation was particularly noticeable in systems where SM:CHOL=2:1, which seem to be not fully equilibrated. Is the figure displaying APL data for only one repetition? Could you please include plots for the other repetitions?

We thank the reviewer for pointing this out. We would like to clarify that we used CHARMMGUI to generate one lipid bilayer configuration for each mixture and system size. These configurations (one per system) were extensively simulated to generate stable initial configurations of the lipid bilayers. Figure 5 – supplements 2 and 3 refer to this pre-equilibration step. The final pre-equilibrated configurations were then used in the subsequent multiple equilibrium MD runs that we performed, either with a single cholesterol molecule or with the AQP0 tetramer(s) inserted. We have clarified this procedure in the revised manuscript (see changes in the Methods section for the MD equilibrium simulations).  

Concerning this pre-equilibration step, we have chosen the area per lipid, not thickness, to characterize the equilibration of the pure lipid bilayers. Accordingly, the area per lipid is the quantity shown in Figure 5 – figure supplement 3. We no longer refer to the membrane thickness in the revised manuscript.

Concerning the variability in the area per lipid, we note that the large changes occur within the first few tens of nanoseconds of the pre-equilibration step, after which the area per lipid stabilizes. We would like to also point out that in Figure 5 – figure supplement 3, we chose a logarithmic scale for the time axis to actually make it possible for the reader to see the major changes that occur at the beginning of the pre-equilibration step (which would otherwise be difficult to see). In the particular case of the SM:CHOL=2:1 mixture_,_ the 64 lipids/leaflet system converged to a stable area per lipid value in the last 70 ns and the 244 lipids/leaflet system approached the same value in approximately the last 30 ns. This was a good indication that the large system had also converged. After equilibration of the membranes, a single cholesterol or AQP0 tetramer(s) were inserted and equilibrium simulations were initiated. However, the first 100 ns (or 300 ns in the case of the double tetramer system) were considered as a further equilibration time and were not included in the analysis. This is now explicitly stated in the revised manuscript: “The first 100 ns of each simulation replica (the first 300 ns for the two tetramer simulations) were considered as additional equilibration time and were not included in further analysis.”

(2) Could you clarify the reasoning behind conducting the simulations at 323 K?

We conducted the simulations at 323 K to ensure that the lipid bilayers were in the liquid phase.

SM:CHOL mixtures have been reported to be in the liquid phase above 314 K (Keyvanloo et al. Biophys. J. 114: 1344, 2018). 323 K was thus chosen to be well above this value. Note that this temperature was also chosen in a previous MD simulation study of pure sphyngomyelin bilayers (Niemelä et al. Biophys. J. 87: 2976, 2004). This reasoning, as well as the two references, have been added to the Methods section in the revised manuscript.  

(3) There appears to be a discrepancy in Figure 7. Panel F does not align with the provided caption. 

We apologize for this mistake. The captions for panels E and F were switched. We corrected this mistake.

(4) Likewise, in Figure 8, there is a mismatch between the caption and the figures. Furthermore, in the text, the authors assert, "In the absence of cholesterol, the AQP0 surface is completely covered by sphingomyelin in the hydrophobic region of the membrane and by water outside this region (Figure 8A, left column). As noted before, there are essentially no direct protein-protein interactions between the adjacent tetramers. When cholesterol was present at the interface, it interacted with AQP0 at the center of the membrane and remained mostly in place (Figure 8A, right column)." However, the left column shows cholesterol density. Could you please clarify this inconsistency, especially regarding the absence of cholesterol?

We apologize for this mistake. The panels in Figure 8A showing the AQP0 surfaces in the absence and presence of cholesterol were switched. We corrected this mistake.

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Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

This manuscript by Estevam et al. reports new insights into the regulation of the receptor tyrosine kinase MET gained from two deep mutational scanning (DMS) datasets. In this paper, the authors use a classic selection system for oncogenic kinase signaling, the murine Ba/F3 cell line, to assess the functional effects of thousands of mutations in the kinase domains of MET in two contexts: (1) fusion of the whole MET intracellular region to the dimerization domain TPR, and (2) the same fusion protein, but with exon 14, which encodes part of the juxtamembrane region of MET, skipped. Critically, exon 14 skipping yields a version of MET that is found in many cancers and has higher signaling activity than the canonical MET isoform. The authors extensively analyze their DMS data to very convincingly show that their selection assay reports on kinase activity, by illustrating that many functionally important structural components of the kinase domain are not tolerant of mutation. Then, they turn their attention to a helical region of the juxtamembrane region (αJM), immediately after exon 14, which is posited to play a regulatory role in MET. Their DMS data illustrate that the strength and mutational tolerance of interactions between αJM and the key αC helix in the kinase domain depends on the presence or absence of exon 14. They also identify residues in the N-lobe of the kinase, such as P1153, which are not conserved across tyrosine kinases but appear to be essential for MET and MET-like kinases. Finally, the authors analyze their DMS data in the context of clinically-observed mutations and drug-resistance mutations.

Overall, this manuscript is exciting because it provides new insights into MET regulation in general, as well as the role of exon 14. It also reveals ways in which the JM region of MET is different from that of many other receptor tyrosinekinases. The exon 14-skipped fusion protein DMS data is somewhat underexplored and could be discussed in greater detail, which would elevate excitement about the work. Furthermore, some of the cell biological validation experiments and the juxtaposition with clinical data are perhaps not assessed/interpreted as clearly they could be. Some constructive suggestions are given below to enhance the impact of the manuscript.

Strengths:

The main strengths of this paper, also summarized above in the summary, are as follows:

(1) The authors very convincingly show that Ba/F3 cells can be coupled with deep mutational scanning to examine MET mutational effects. This is most clearly shown by highlighting how all of the known kinase structure and regulatory elements are highly sensitive to mutations, in accordance with a few other DMS datasets on other kinases.

(2) A highlight of this paper is the juxtaposition of two DMS datasets for two different isoforms of the MET receptor. Very few comparisons like this exist in the literature, and they show how small changes to the overall architecture of a protein can impact its regulation and mutational sensitivity.

(3) Another exciting advance in this manuscript is the deep structural analysis of the MET juxtamembrane region with respect to that of other tyrosine kinases - guided by the striking effect of mutations in the juxtamembrane helical region. The authors illustrate how the JM region of MET differs from that of other tyrosine kinases.

(4) Overall, this manuscript will provide a resource for interpreting clinically relevant MET mutations.

Weaknesses:

(1) The manuscript is front-loaded with extensive analysis of the first DMS dataset, in which exon 14 is present, however, the discussion and analysis of the exon 14-skipped dataset is somewhat limited. In particular, a deeper discussion of the differences between the two datasets is warranted, to lay out the full landscape of mutations that have different functional consequences in the two isoforms. Rather, the authors only focus on differences in the JM region. What are the broader structural effects of exon 14 skipping across the whole kinase domain?

Thank you for your feedback on our manuscript and our analysis of the exon 14 skipped mutational scanning data. The lack of a robust growth differential  between the wild type MET intracellular domain and the exon 14 skipped isoform within the Ba/F3 system suggests that there is not a significant growth advantage related to exon 14 skipping, likely due to the constitutive activation of both constructs by the TPR domain, which also suggests that the assay is potentially less sensitive to nuanced JM-driven effects between these two isoforms, aside from the highly sensitive ⍺JM-helix. We also lose insight on membrane-related interactions imposed on the juxtamembrane that may be important to fully understand the differences between these two isoforms in the cytoplasmically-expressed context. Therefore, we can at most speculate exon 14 skipped related differences between these two datasets.

With these caveats in mind, to further address exon 14 and juxtamembrane-driven differences between these two mutational landscapes, we calculated the absolute score difference between TPR-METΔEx14 and TPR-MET (|METΔEx14 - MET|) and plotted the |ΔScore| in a heatmap. Overall, the two landscapes, as noted in the text, are largely similar with differences emerging mostly for specific mutations. Where we see the largest secondary structural difference continues to be the ⍺JM-helix, where MET is more sensitive to helix-breaking mutations such as proline. Again L1062 has the greatest difference in sensitivity between these two datasets for the ⍺JM-helix, with the introduction of negative charge resulting in loss-of-function for the TPR-MET kinase domain but having a null effect in the TPR-METΔEx14 kinase domain. Other positions with strong differences include the ⍺G and APE motif.

We have incorporated more detailed discussion in text. 

(2) It is unclear if gain-of-function mutations can actually be detected robustly in this specific system. This isn't a problem at face value, as different selection assays have different dynamic ranges. However, the authors don't discuss the statistical significance and reproducibility of gain- vs loss-of-function mutations, and none of the gain-of-function mutations are experimentally validated (some appear to show loss-of-function in their cellular validation assay with full-length MET). The manuscript would benefit from deeper statistical analysis (and discussion in the text) of gain-of-function mutations, as well as further validation of a broad range of activity scores in a functional assay. For the latter point, one option would be to express individual clones from their library in Ba/F3 cells and blot for MET activation loop phosphorylation (which is probably a reasonable proxy for activity/activation).

Thank you for your comment on the statistical interpretations of gain-of-function (GOF) and loss-of-function (LOF) mutations. In this study we classify GOF and LOF based on the following metrics:

(1) The difference between the missense mutation score and the wild type synonymous score for a given position must be smaller than the calculated propagated error, for both IL-3 withdrawal and IL-3 conditions

(2) Missense mutations must be ≥ ±2 standard deviations (SD) from the mean of wild type synonymous mutations

Given that our assay was conducted in a constitutively active kinase in the TPR-fusion context, gain-of-function mutations are expected to not only be rare, but also supersede baseline fitness. Within the IL-3 conditions, we expect that cells are not reliant or “addicted” to MET for growth proliferation. Nevertheless, due to the parallel nature of the screen, we can compare scores for variants in the IL-3 control and IL-3 withdrawal conditions to filter mutations that are solely exhibiting high fitness under selective pressure.

To identify these mutations we 1) calculated the propagation of error between IL-3 and IL-3 withdrawal scores for the same variant 2) calculated the absolute difference between IL-3 and IL-3 withdrawal scores for the same variant 3) filtered variants if the IL-3 withdrawal score was ≥ +2 SDs, the IL-3 score was ≤ 0, and the absolute score difference between IL-3 and withdrawal conditions was larger than the propagated error.

In analyzing mutations within the IL-3 withdrawal conditions, applying our statistical metrics, we find 33 mutations within the MET library, and 30 in the METΔEx14 library, that have a score of ≥ +2 SD and low propagated error. By increasing our boundary to ≥+2.5 SD, we can classify mutations with even higher confidence, identifying 10 mutations within the MET library, and 9 in the METΔEx14 library (Supplemental Data Figure 7).

(3) In light of point 2, above, much of the discussion about clinically-relevant gain-of-function mutations feels a bit stretched - although this section is definitely very interesting in premise. A clearer delineation of gain-of-function, with further statistical support and ideally also some validation, would greatly strengthen the claims in this section.

To address this concern, we have provided additional analysis and details on gain-of-function (GOF) classification in Supplemental Data Figure 5 and the overlap between GOF and clinically associated mutations in Supplemental Data Figure 8. Within our gain-of-function classifications, we pick up on several mutations at positions that have been clinically detected and experimentally validated in previous studies in both libraries (D1228, G1163, L1195), and show that GOF mutations also have low variance.

Reviewer #2 (Public Review):

Summary:

The authors describe a deep mutational scanning (DMS) study of the kinase domain of the c-MET receptor tyrosine kinase. The screen is conducted with a highly activated fusion oncoprotein - Tpr-MET - in which the MET kinase domain is fused to the Tpr dimerization element. The mutagenized region includes the entire kinase domain and an alpha-helix in the juxtamembrane region that is essentially part of the MET kinase domain. The DMS screen is carried out in two contexts, one containing the entire cytoplasmic region of MET, and the other with an "exon 14 deletion" which removes a large portion of the juxtamembrane region (but retains the aforementioned alpha-helix). The work provides a robust and essentially exhaustive catalog of the effect of mutations (within the kinase domain) on the ability of the Tpr-MET fusion oncoproteins to drive IL3-independent growth of Ba/F3 cells. Every residue in the kinase is mutated to every natural amino acid. Given the design of the screen, one would expect it to be a powerful tool for identifying mutations that impair catalytic activity and therefore impair IL3-independent proliferation, but not the right tool for identifying gain-of-function mutations that operate by shifting the kinase from an inactive to active state (because the Tpr-Met fusion construct is already very highly activated). This is borne out by the data, which reveal many many deleterious mutations and few "gain-of-function" mutations (which are of uncertain significance, as discussed below).

Strengths:

The authors take a very scholarly and thorough approach to interpreting the effect of mutations in light of available information for the structure and regulation of MET and other kinases. They examine the effect of mutations in the so-called catalytic (C) and regulatory (R) spines, the interface between the JM alpha-helix and the C-helix, the glycine-rich loop, and other key elements of the kinase, providing a structural rationale for the deleterious effect of mutations. Comparison of the panoply of deleterious mutations in the TPR-met versus TPR- exon14del-MET DMS screens reveals an interesting difference - the exon14 deletion MET is much more tolerant of mutations in the JM alpha-helix/C-helix interface. The reason for this is unclear, however.

Weaknesses:

Because the screens were conducted with highly active Tpr-MET fusions, they have limited power to reveal gain-of-function mutations. Indeed, to the extent that Tpr-MET is as active or even more active than ligand-activated WT MET, one could argue that it is "fully" activated and that any additional gain of fitness would be "super-physiologic". I would expect such mutations to be rare (assuming that they could be detected at all in the Ba/F3 proliferation assay). Consistent with this, the authors note that gain-of-function mutations are rare in their screen (as judged by being more fit than the average of synonymous mutations). In their discussion of cancer-associated mutations, they highlight several "strong GOF variants in the DMS". It is unclear what the authors mean by "strong GOF", indeed it is unclear to this reviewer whether the screen has revealed any true gain of function mutations at all. A few points in this regard:

(1) More active than the average of synonymous mutations (nucleotide changes that have no effect on the sequence of the expressed protein) seems to be an awfully low bar for GOF - by that measure, several synonymous mutations would presumably be classified as GOF.

We completely agree that any mutation above the average synonymous would not be a robust assessment and thus why we statically filtered mutations in our entire analysis. To this point, and that of  Reviewer 1, we have further outlined our statistical definitions. In classifying mutations as GOF or LOF, the following parameters were used:

(1) The difference between the missense mutation score and the wild type synonymous score for a given position must be smaller than the calculated propagated error, for both IL-3 withdrawal and IL-3 conditions

(2) Missense mutations must be ≥ ±2 standard deviations (SD) from the mean of wild type synonymous mutations

Therefore, only variants at the tail-ends of the mutational distribution were assessed, and further filtered based on propagation of error. For this reason, a “strong GOF” mutation as noted in this study is one that improves the fitness of an already active kinase. As pointed out, within our analysis, these are very rare occurrences, and in focusing on cancer-associated mutations we find that the variants that meet these statistical parameters require a larger genetic “leap” in the codon space. Overall, we have also changed our language in reference to GOF mutations in text.

We hope this concern has been addressed in the new Supplemental Data Figures.

(2) In the +IL3 heatmap in supplemental Figure 1A, there is as much or more "blue" indicating GOF as in the -IL3 heatmap, which could suggest that the observed level of gain in fitness is noise, not signal.

We hope this concern has been addressed in the previous responses and new Supplemental Data Figures.

(3) And finally, consistent with this interpretation, in Supplemental Figure 1C, comparing the synonymous and missense panels in the IL3 withdrawal condition suggests that the most active missense mutations (characterized here as strong GOF) are no more active than the most active synonymous mutations.

We hope this concern has been addressed in the previous responses and figures above.

My other major concern with the work as presented is that the authors conflate "activity" and "activation" in discussing the effects of mutations. "Activation" implies a role in regulation - affecting a switch between inactive and active conformations or states - at least in this reviewer's mind. As discussed above, the screen per se does not probe activation, only activity. To the extent that the residues discussed are important for activation/regulation of the kinase, that information is coming from prior structural/functional studies of MET and other kinases, not from the DMS screen conducted here. Of course, it is appropriate and interesting for the authors to consider residues that are known to form important structural/regulatory elements, but they should be careful with the use of activity vs. activation and make it clear to the reader that the screen probes the former. One example - in the abstract, the authors rightly note that their approach has revealed a critical hydrophobic interaction between the JM segment and the C-helix, but then they go on to assert that this points to differences in the regulation of MET and other RTKs. There is no evidence that this is a regulatory interaction, as opposed to simply a structural element present in MET (and indeed the authors' examination of prior crystal structures shows that the interaction is present in both active and inactive states.

Thank you, and we completely agree that the distinction between “activity” and “activation” is important and that we can at most speculate and propose models for effects related to activation from this screen. We have edited the text to reflect these distinctions. In respect to activation and the second point, we believe the screen highlights the ⍺JM-C interface as a critical structural region, which may have a role in regulation based on the paradigm of juxtamembrane regulation in RTKs, the presence of a similar interface in TAM family kinases, the co-movement of the ⍺JM-helix and ⍺C-helix between active and inactive conformations in the structural ensemble, and the observation that within the TPR-METΔEx14 library there is a greater tolerance for mutations at interface positions than TPR-MET. We hope that are follow-up studies that directly probe the ⍺JM-C interface in respect to the entire juxtamembrane to truly say if/ what role this conserved motif plays in regard to MET function. We have changed the language of the text to reflect how these differences contribute to our proposed model, rather than any unintended assertion on direct regulatory effects.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Suggested major points to address:

(1) Although the authors show that several key functional residues in the kinase domain are highly sensitive to mutation, it would be nice if the authors further established a clear connection between kinase activity and enrichment in the Ba/F3 assay. Specifically, it is unclear to what extent there is a correlation between the extent of enrichment/depletion and kinase activity - is a larger activity score necessarily indicative of higher kinase activity? This is partly validated by the P1153L mutation autophosphorylation western blots in Figure 4B, but this correlation is somewhat undermined by the data in 5F. Autophosphorylation data (or phosphorylation data on a direct downstream substrate) for a few mutants would really solidify what the activity score is truly reporting. This might also clarify the extent to which the difference between the two screens can be interpreted, and the extent to which gain-of-function can be interpreted.

The Ba/F3 assay was carefully chosen for its addiction to exogenous IL-3, which serves as a permissive signaling switch. Any mutation that prevents TPR-MET/ΔEx14 from properly functioning is therefore dampening its signaling ability. Nevertheless, it is possible that some mutations with high scores are truly improving activity and others are sustaining activity through more stable interactions than the wild type kinase domain or with downstream signaling partners, which would require careful biochemical dissection outside the scope of this study. To address these points, we now refer to the mutation score simply as “score” rather than “activity score” and further discuss these caveats in text.

(2) Overall, the exon 14-skipped dataset is under-discussed in the paper. The comparison of the two datasets is where most deep insights are likely to be found, and so a more thorough analysis/discussion of this dataset would really elevate the significance of the paper. For example, there appear to be a very large number of mutations that have divergent effects in the two screens (everything along the dashed lines in Figure 5D), but it's unclear where most of these mutations lie on the structure. It would be helpful if the residues with divergent mutational effects between the two screens (Supplementary Figure 5E) were mapped onto a structure of the JM-KD construct.

To address this concern, new analysis has been added to the supplement, showing the score differences between MET and METΔEx14 mutations as a heatmap (Supplemental Data Figure 7A). Within this analysis we further applied our statistical filtering methods and structurally mapped positions with the greatest differential scores to show where divergent effects cluster (Supplemental Data Figure 7D). Consistent with our previous reports, the ⍺JM-helix and ⍺C-helix show the largest cluster of divergent effects, in addition to sites such as the ⍺G and APE motif. Further discussion of these points have been added to the text.

(3) Based on the observations that αJM-αC interactions seem to be less strictly required in the exon 14 mutant, the hypothesis that exon 14 skipping merely removes a Cbl docking site seems largely unsatisfactory. There seems to be more direct structural alterations that could explain this change, but these are not really discussed or speculated on. Related to this, while L1062 mutations are more tolerated, as the authors showed in both the mutational heatmap and the cellular experiments, its binding counterpart L1125 still seems to be somewhat immutable based on the heatmaps. So, more hypothesis/exploration of how exon 14 skipping affects MET KD structure would be a nice addition to the paper.

We agree that loss of the Cbl docking site is an insufficient model to capture the full nature of JM regulation and exon 14 skipping effects, which was a major incentive for this study. The outstanding ⍺JM-⍺C-helix sensitivity also excites us because it points to a potential regions of the JM that potentially is involved in kinase activity through ⍺C-helix interactions, much like the CDK models and other RTK-JM interactions. We observed that the ⍺JM-⍺C helix retain contact, and propose that the ⍺JM-⍺C helix move in unison between active and inactive conformations. However, it is possible that a more complicated mechanism might also exist, where there is a larger degree of maintenance of these contacts in a homodimer. For instance, in Figure 3G, if you compare the ⍺JM-helix conformations, in both RON and AXL there is more distance and a pivot away from the ⍺C-helix. It’s is possible that there are shared mechanisms between the MET and TAM families that could further elucidate exactly how these ⍺JM-helices interact with the kinase domain during the activity transitions and what biophysical role JM truncations play.

(4) The discussion about mutations S1122Q and L1062D is a bit confusing and incomplete. From the DMS data, it appears that L1062D should be mildly gain-of-function for the exon 14 deletion variant and very loss of function for wild-type MET. In the validation HeLa cell experiments L1062D is loss-of-function in both contexts, but a mention of this discrepancy is omitted. Then, when the discordance between DMS and HeLa cell experiments is observed again for S1122Q, it is explicitly called out for activation-loop phosphorylation, but then there is no mention of the fact that HGF stimulation leads to greater pERK levels for S1122Q in the exon 14 deletion context (the opposite of the DMS result). The Erk phosphorylation discrepancy should be mentioned. It is entirely reasonable, as the authors suggest, that there are differences between full-length MET and the TPR fusions, but the enhanced Erk phosphorylation by the S1122Q mutation is surprising (and intriguing!). This section could use some re-analysis/re-writing and further discussion.

Thank you for this comment. As noted L1062D shows slight GOF in METΔEx14 but LOF in MET. The blots show expression of L1062D and S1122Q in the full length receptor in the absence and presence of HGF stimulation. L1062D is loss of function for both contexts only in -HGF conditions, but shows expression in phosphorylated METΔEx14, but not MET. For S1122Q, indeed there is a stronger pERK signal in the METΔEx14, which highlights how probing all regions of phosphorylation (A-loop and C-tail) and many MET-associate pathways (ERK, AKT) may be important to understand in what way these mutations are affect MET phosphorylation and proliferation. We have included this point in the text.

(5) Related to the previous point, one other thing to consider here is that perhaps gain-of-function mutations are simply not detectable in this particular DMS assay. The authors state that GOF and LOF are defined as 2 standard deviations from the mean of the WT-synonymous distribution. How many mutations are actually designated to be GOF based on this criterion? Are those GOF mutations as reproducible as the LOF mutations? It would be worthwhile to separately analyze the variance in activity scores for every loss-of-function mutation and gain-of-function mutation. It seems likely that loss-of-function scores are a lot more reproducible than gain-of-function ones, suggesting that the most apparent gain-of-function signal is just noise in the assay. The few outliers to this point (true gain-of-function mutations) may be some of the ones discussed in Figure 6. If this is true, it would lend confidence to the claims associated with Figure 6.

In analyzing and classifying both GOF and LOF mutations, error was a main filtering parameter. Each fitness score, calculated by Enrich2, is representative of the slope across time points  and biological replicates for the read frequency of the mutation. The associated standard error (SE) reflects the variance for each mutation within the scoring framework (Rubin et al., 2017). Mutations were then further filtered based on low propagated error, calculated by comparing the standard error (SE) of each missense mutation to the SE of the respective wild type synonymous mutation. Therefore, mutations were only classified as GOF or LOF if there was low error, in addition to the other score filters previously described. We have plotted the classified GOF mutations with their respective SE in the newly incorporated Supplemental Data Figure 8C.

(6) In the discussion of panels 6C and 6D, the assertion is that the "clinical, not validated" category has more mutations that are low-fitness outliers than the "clinical, validated" category. From the graphs, it's actually hard to tell if this is the case for two reasons: (1) the way the graphs are normalized, (to the largest value in each histogram), you cannot compare bar heights (and thus number of mutations) between two histograms on the same graph. (2) Just looking at the shapes of the distributions, or considering maybe the mean or median values, it's unclear whether the "validated" and "not validated" populations are actually different from one another.

This is an important indication, and we have added analysis showing the distribution and number of clinically-associated mutations within our libraries without normalization in the main text and in Supplemental Data Figure 8A-B.

(7) This sentence in the last results section is somewhat unclear: "GOF resistance mutations may indicate an effect on the equilibrium of kinase activation, whereas LOF resistance mutations likely affect inhibitor-protein interactions directly." The first part makes sense, but it is not totally obvious how one can infer anything about inhibitor-protein interactions from mutations that are LOF with respect to kinase activity. Related to this, how are LOF mutations selected in the presence of an inhibitor? Is the assumption here that the mutation might totally abrogate inhibitor binding but only slightly impair the kinase? Perhaps this could be explained a bit more.

Here, the idea we wanted to get across is that there are two models  that can explain how a mutation can contribute to resistance: shift the activity equilibrium at baseline or directly impair drug effects and restore baseline activity. Mutations that are labeled resistant and GOF, favor the first model. Mutations that are labeled resistant and LOF, favor the second model. In the presence of an inhibitor, which is in the scope outside of this study, LOF mutations would be sensitive to the inhibitor (ie WT-like and sensitive).

(8) Some additional details of the library preparation and sequencing should be given in the methods section. It appears that the variable region of the library is roughly 275 amino acid residues long, which means >800 bases. How was this sequenced? From the methods, it sounds like all of the variants were pooled into a single library, but then sequencing was done using a 300x300 paired-end Illumina kit, which would not cover the length of the whole variable region. Was the library actually screened in segments as sub-libraries and then separately sequenced? Alternatively, was the whole library screened at once, and then different segments were amplified out for sequencing? If the latter approach is used, this could yield confounding results for counting wild-type variants that have the parent wild-type coding sequence. For example, if you amplify your kinase library in three segments after a single selection on the whole library, and you sequence those three segments separately, you might find a read that appears as wild-type in the part you amplified/sequenced but has a mutation in a region that you did not sequence. If this approach is taken, the counts for the wild-type sequence would be inaccurate, in which case, how is the data normalized with WT as a reference? Regardless of the method used, some more details should be provided in the methods section.

In this study, we used the Nextera XT DNA Library Preparation Kit (Illumina), which uses a tagmentanation approach that randomly fragments our 861 bp amplicon into ~300 bp fragments with a transposase, resulting in a Poisson distribution of fragment sizes. This allows for direct sequencing of all amplicons and libraries with an SP300 paired-end run, which we ran on two lanes of a NovaSeq6000. Samples are demultiplexed  and processed by our analysis pipeline with a lookup table that associates the unique dual index to the specific sample (library, time point, biological replicate, IL-3 condition).

The TPR-MET and TPR-METΔEx14 libraries were prepared in parallel throughout the entire experiment, from cloning to virus generation to transductions, screening, cell harvesting, sequencing prep, and sequencing. In other words, the TPR-MET and TPR-METΔEx14 were transduced into their own, respective batch of cells for each biological replicate, then selected and screened on the same day for each replicate and time point. Each library and condition (time point, biological replicate, IL-3 condition) was prepared in parallel but still an independent sample. At the stage of tagmentation, each sample was arrayed, where each well corresponds to a library, biological replicate, and time point. At the stage of sequencing, samples across the two libraries were normalized to 10mM (library, biological replicate, time point, IL-3 condition) then pooled together and all run on two lanes of the same NovaSeq6000 flow cell.

PCR and sequencing bias was one of the most important parameters for us, which is why we performed tagmentation in parallel and sequenced everything on the same run. We have added extra details to the methods and hope that we have clarified your questions on this matter.

Suggested minor points to address:

(1) TPR (as in TPR-MET fusion) is not defined in the text when it is first mentioned. And it wasn't immediately clear that this is not a membrane-associated domain (Figure 5E makes this way more obvious than Figure 1B does). Perhaps this could be made more explicit in the text or in Figure 1.

We have incorporated a new schematic in Figure 1B to better illustrate the TPR-fusion constructs used within this study. The usage of the TPR-fusion is first mentioned in the introduction, paragraph 4, and revised the main-text to delineate the usage of the TPR-fusion more clearly.

(2) In Figure 2G, it would be helpful if the wild-type amino acid residue was listed underneath the position number in the two graphs (even though those residues are also highlighted in 2H).

Thank you for this recommendation, we have added the wild type amino acid next to the position number in the x-axis label.

(3) For Supplementary Data Figure 2, is it possible to calculate conservation scores at each position using some kind of evolutionary model, rather than relying on visual inspection of the sequence logo? Can one quantitatively assert that the C-spine is less conserved than the R-spine overall, or can this only be said for certain positions? Related to this, in comparing Figure 2G to Supplementary Data Figure 2, it is interesting that there isn't any obvious correspondence between mutational tolerance and conservation within the C-spine. For example, 1165 seems to be the most conserved position in the C-spine, but several substitutions are tolerated at this position, just like 1210, which is one of the least conserved positions in the C-spine. Finally, it's very likely that positions 1165, 1210, 1272, and 1276 co-vary, given that they all pack into the same hydrophobic cluster. This might be why they appear less conserved. These last few points might be worth discussing briefly if the authors want to relate mutational tolerance to evolutionary conservation.

Thank you for this recommendation. To better quantitatively determine C-spine versus R-spine conservation, we performed a multiple sequence alignment of all RTK kinase domain sequences to properly identify corresponding R- and C-spine locations, as previously done in generating the spine logos, then used the bio3D structural bioinformatics package in R to calculate the conservation score of each residue position by amino acid “similarity” with a blosum62 matrix (Supplemental Data Figure 2B). In concordance with the logos, we find that C-spine positions 1092, 1108, 1165 have the highest conservation scores, even compared to some R-spine mutations. We also see across the alignment that indeed, C-spine positions 1165 1210,1211,1212, and 1272, and 1276 co-vary within RTK families. We have revised the text to reflect these points, and more specifically discuss position-level conservation rather than generalizing conservation for the C- and R-spines.

(4) On Page 7 of the merged document, there appear to be some figure labeling errors. In the first and second paragraphs of the "Critical contacts between..." section, Figure 3B is referenced multiple times as a structural alignment/ensemble, but this is a heatmap.

Thank you for catching this! The correct figure panels are now referenced.

(5) In the text describing Figure 3A, it is stated that the structures were aligned to the N-lobe, but the figure legend says that all structures were aligned to alpha-C and alpha-JM.

Thank you - a local alignment to the ⍺JM-helix and ⍺C-helix is correct, the idea here being that if the ⍺JM-helix and ⍺C-helix are linked to an active/inactive conformation like in the case of the insulin receptor, these two clusters could be revealed through the structural ensemble. However, we discovered this was not the case, combined with the DMS sensitivity to mutations at the packing interface leads us to believe that the MET JM has a distinctive regulatory mechanism that relies on this ⍺C-helix interface. We have made this correction to the text.

(6) It would be helpful if the alpha-C and alpha-JM helices in Figure 3D were labeled on the MET structures.

The ⍺C-helix and ⍺JM-helix are now labeled in Figure 3D.

(7) It appears that Figure 4E is never explicitly referenced in the text.

Thank you, Figure 4E is now appropriately referenced in the text.

(8) Throughout the Figure 6 legend, for the histograms, it is stated that "Counts are normalized to the total mutations in each screen dataset." This might not be the correct description of normalization, as this would mean that the sum of all of the bins should equal 1. Rather, the normalization appears to be to the bin with the largest number of mutants in it, which is given a value of 1. This difference is really critical to how one visually inspects the overlaid histograms.

Thank you for this comment. Here, the intention was to aid in the visualization of the distribution of cancer-associated and resistance associated mutations, which is a much smaller population compared to the whole library and becomes easily masked. We originally applied a “stat(ncount)” function in R, which as noted scales the data and sets the peak to 1, which only applied to the clinical and cancer-associated mutations plotted. Now, to better compare distributions, normalization has been removed, instead opting to overlay the distributions of all missense mutations and the subset of clinical mutations directly with their own y-axis scale. This modification has been made throughout Figure 6 panels, hopefully improving interpretability.

Reviewer #2 (Recommendations For The Authors):

A few thoughts/suggestions:

(1) Regarding kinase regulation, the "closing of N- and C-lobe" upon activation is an often mentioned component of activation, and I'm sure is true in many cases, but it is not a general feature of kinase activation.

The text has been updated - we removed the description of N- and C-lobe closure. 

(2) With respect to the inactive state of MEK, the DFG-flipped structure discussed here is almost certainly an inhibitor-induced conformation. Again, DFG-flip is often discussed as a mechanism of kinase regulation, and while in some kinases this might be the case, more often it is a drug-induced or drug-stabilized inactive conformation. The SRC/CDK-like inactive conformation in 2G15 is more likely a physiologically relevant inactive state. (or even better, the ATP-bound inactive state structure 3DKC, which exhibits a somewhat different SRC/CDK-like inactive conformation).

The PDB 3R7O structure was chosen as the main representation because it was the clearest representation of a wild type structure with an aligned R- and C- spine, solvent-exposed, phosphorylated activation loop. Although 3DKC is bound to ATP, this structure is still in an inactive conformation and has stabilizing mutations (Y1234/F, Y1235D) and an atypical alpha helix structure in the activation loop. However, we agree the SRC/CDK-like inactive conformation is an important representation and we have incorporated our structural mapping on 2G15 in the new supplemental figures with further details on statistical analysis and comparison of libraries.

(3) Following the comments above, I would describe the process of activation in a simpler way (in any case, it is peripheral to the work described here). Something along the lines of "phosphorylation on tyrosines XX and XX induces rearrangement of the activation segment and promotes and stabilizes the inward active position of the C-helix." Can go on to mention that this forms the E1127/K1110 salt bridge. (The DFG is already "in" in the SRc/CDK-like inactive state).

We have changed the language to more simply describe activation. Thank you!

(4) Would be great to see DMS with the intact receptor done in a way that could identify mutations that lead to activation in a ligand-independent manner. (but obviously beyond the scope of this paper).

Agreed! This would be an excellent follow up for the future, especially to elucidate juxtamembrane regulation, as the membrane context is likely required.

A typo or two:

Boarded instead of bordered/outlined in legend to Fig. 1.

P11553L in the 2nd line of the 2nd paragraph in that section.

Thank you, we have addressed these typos!

eLife assessment

This manuscript describes a deep mutational scanning study of the kinase domain of the MET receptor tyrosine kinase. The study yields an important catalog of essentially all possible deleterious mutations in this portion of the receptor., with convincing evidence. The manuscript will be of interest to researchers working in the field of receptor tyrosine kinases.

Reviewer #2 (Public Review):

Summary:

The authors describe a deep mutational scanning (DMS) study of the kinase domain of the c-MET receptor tyrosine kinase. The screen is conducted with a highly activated fusion oncoprotein - Tpr-MET - in which the MET kinase domain is fused to the Tpr dimerization element. The mutagenized region includes the entire kinase domain and an alpha-helix in the juxtamembrane region that is essentially part of the MET kinase domain. The DMS screen is carried out in two contexts, one containing the entire cytoplasmic region of MET, and the other with an "exon 14 deletion" which removes a large portion of the juxtamembrane region (but retains the aforementioned alpha-helix). The work provides a robust and essentially exhaustive catalog of the effect of mutations (within the kinase domain) on the ability the Tpr-MET fusion oncoproteins to drive IL3-independent growth of Ba/F3 cells. Every residue in the kinase is mutated to every natural amino acid. Given the design of the screen, one would expect it to be a powerful tool for identifying mutations that impair catalytic activity and therefore impair IL3-independent proliferation. This is borne out by the data, which reveal many many deleterious mutations. The study reveals relatively few "gain-of-fitness" mutations, but this is not unexpected because it is carried out with an already-activated form of the MET kinase (the oncogenic Tpr-met fusion).

Strengths:

The authors take a very scholarly and thorough approach in interpreting the effect of mutations in light of available information for the structure and regulation of MET and other kinases. They examine the effect of mutations in the so-called catalytic (C) and regulatory (R) spines, the interface between the JM alpha-helix and the C-helix, the glycine-rich loop and other key elements of the kinase, providing a structural rationale for the deleterious effect of mutations. Comparison of the panoply of deleterious mutations in the TPR-met versus TPR- exon14del-MET DMS screens reveals an interesting difference - the exon14 deletion MET is much more tolerant of mutations in the JM alpha-helix/C-helix interface. The reason for this is unclear, however.

An important qualification of the study is that it was carried out with the already highly activated Tpr-Met fusion. As a consequence, it is not expected to reveal mutations that activate the kinase -- activate in the sense of promoting a switch between physiologically-relevant inactive and active states. Consistent with this, the authors note that gain-of-fitness mutations are rare in their screen, and those that are identified induce modest but significant increases in fitness.

Reviewer #2 (Public Review):

Summary:

The study focuses on the vomeronasal organ, the peripheral chemosensory organ of the accessory olfactory system, by employing single-cell transcriptomics. The author analyzed the mouse vomeronasal organ, identifying diverse cell types through their unique gene expression patterns. Developmental gene expression analysis revealed that two classes of sensory neurons diverge in their maturation from common progenitors, marked by specific transient and persistent transcription factors. A comparative study between major neuronal subtypes, which differ in their G-protein sensory receptor families and G-protein subunits (Gnai2 and Gnao1, respectively), highlighted a higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons. Moreover, distinct differences in ER content and ultrastructure suggest some intriguing roles of ER in Gnao1-positive vomeronasal neurons. This work is likely to provide useful data for the community and is conceptually novel with the unique role of ER in a subset of vomeronasal neurons. This reviewer has some minor concerns and some suggestions to improve the manuscript.

Strengths:

(1) The study identified diverse cell types based on unique gene expression patterns, using single-cell transcriptomic.

(2) The analysis suggests that two classes of sensory neurons diverge during maturation from common progenitors, characterized by specific transient and persistent transcription factors.

(3) A comparative study highlighted differences in Gnai2- and Gnao1-positive sensory neurons.

(4) Higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons.

(5) Distinct differences in ER content and ultrastructure suggest unique roles of ER in Gnao1-positive vomeronasal neurons.

(6) The research provides conceptually novel on the unique role of ER in a subset of vomeronasal neurons, offering valuable insights to the community.

Weaknesses:

(1) The connection between observations from sc RNA-seq and EM is unclear.

(2) The lack of quantification for the ER phenotype is a concern.

eLife assessment

This valuable study uses single-cell transcriptomics to explore the mouse vomeronasal organ and represents an advance that enhances our understanding of neural diversity within this sensory system. Findings suggest a unique endoplasmic reticulum (ER) structure in Gnao1 neurons and allow for the synthesis of a developmental trajectory from stem cells to mature vomeronasal sensory neurons. Convincing methods, data, and analyses broadly support the claims, although experiments supporting the main ER-related claim require additional quantification of co-expression and statistics on labeling intensity or coverage. Adding these data would greatly strengthen the conclusions of the paper.

Reviewer #1 (Public Review):

Devakinandan and colleagues present a manuscript analyzing single-cell RNA-sequencing data from the mouse vomeronasal organ. The main advances in this manuscript are to identify and verify the differential expression of genes that distinguish apical and basal vomeronasal neurons. The authors also identify the enriched expression of ER-related genes in Gnao1 neurons, which they verify with in situ hybridizations and immunostaining, and also explore via electron microscopy. Finally, the results of this manuscript are presented in an online R shiny app. Overall, these data are a useful resource to the community. I have a few concerns about the manuscript, which I've listed below.

General Concerns:

(1) The authors mention that they were unable to identify the cells in cluster 13. This cluster looks similar to the "secretory VSN" subtype described in a recent preprint from C. Ron Yu's lab (10.1101/2024.02.22.581574). The authors could try comparing or integrating their data with this dataset (or that in Katreddi et al. 2022) to see if this is a common cell type across datasets (or arises from a specific type of cell doublets). In situ hybridizations for some of the marker genes for this cluster could also highlight where in the VNO these cells reside.

(2) I found the UMAPs for the neurons somewhat difficult to interpret. Unlike Katreddi et al. 2022 or Hills et al. 2024, it's tricky to follow the developmental trajectories of the cells in the UMAP space. Perhaps the authors could try re-embedding the data using gene sets that don't include the receptors? It would also be interesting to see if the neuron clusters still cluster by receptor-type even when the receptors are excluded from the gene sets used for clustering. Plots relating the original clusters to the neuronal clusters, or dot plots showing marker gene expression for the neuronal clusters might both be useful. For example, right now it's difficult to interpret clusters like n8-13.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Devakinandan and colleagues have undertaken a thorough characterization of the cell types of the mouse vomeronasal organ, focusing on the vomeronasal sensory neurons (VSNs). VSNs are known to arise from a common pool of progenitors that differentiate into two distinct populations characterized by the expression of either the G protein subunit Gnao1 or Gnai2. Using single-cell RNA sequencing followed by unsupervised clustering of the transcriptome data, the authors identified three Gnai2+ VSN subtypes and a single Gnao1+ VSN type. To study VSN developmental trajectories, Devakinandan and colleagues took advantage of the constant renewal of the neuronal VSN pool, which allowed them to harvest all maturation states. All neurons were re-clustered and a pseudotime analysis was performed. The analysis revealed the emergence of two pools of Gap43+ clusters from a common lineage, which differentiate into many subclusters of mature Gnao1+ and Gnai2+ VSNs. By comparing the transcriptomes of these two pools of immature VSNs, the authors identified a number of differentially expressed transcription factors in addition to known markers. Next, by comparing the transcriptomes of mature Gnao1+ and Gnai2+ VSNs, the authors report the enrichment of ER-related genes in Gnao1+ VSNs. Using electron microscopy, they found that this enrichment was associated with specific ER morphology in Gnao1+ neurons. Finally, the authors characterized chemosensory receptor expression and co-expression (as well as H2-Mv proteins) in mature VSNs, which recapitulated known patterns.

Strengths:

The data presented here provide new and interesting perspectives on the distinguishing features between Gnao1+ and Gnai2+ VSNs. These features include newly identified markers, such as transcription factors, as well as an unsuspected ER-related peculiarity in Gnao1+ neurons, consisting of a hypertrophic ER and an enrichment in ER-related genes. In addition, the authors provide a comprehensive picture of specific co-expression patterns of V2R chemoreceptors and H2-Mv genes.

Importantly, the authors provide a browser (scVNOexplorer) for anyone to explore the data, including gene expression and co-expression, number and proportion of cells, with a variety of graphical tools (violin plots, feature plots, dot plots, ...).

Weaknesses:

The study still requires refined analyses of the data and rigorous quantification to support the main claims.

The method description for filtering and clustering single-cell RNA-sequencing data is incomplete. The Seurat package has many available pipelines for single-cell RNA-seq analysis, with a significant impact on the output data. How did the authors pre-process and normalize the data? Was the pipeline used with default settings? What batch correction method was applied to the data to mitigate possible sampling or technical effects? Moreover, the authors do not describe how cell and gene filtering was performed. The data in Figure 7-Supplement 3 show that one-sixth of the V1Rs do not express any chemoreceptor, while over a hundred cells express more than one chemoreceptor. Do these cells have unusually high or low numbers of genes or counts? To exclude the possibility of a technical artifact in these observations, the authors should describe how they dealt with putative doublet cells or debris. Surprisingly, some clusters are characterized by the expression of specific chemoreceptors (VRs). Have these been used for clustering? If so, clustering should be repeated after excluding these receptors.

The identification of the VSN types should be consistent across the different analyses and validated. The data presented in Figure 1 lists four mature VSN types, whereas the re-clustering of neurons presented in Figure 3 leads to a different subdivision. At present, it remains unclear whether these clusters reflect the biology of the system or are due to over-clustering of the data, and therefore correspond to either noise or arbitrary splitting of continua. Clusters should be merged if they do not correspond to discrete categories of cells, and correspondence should be established between the different clustering analyses. To validate the detected clusters as cell types, markers characteristic of each of these populations can be evaluated by ISH or IHC.

There is a lack of quantification of imaging data, which provides little support for the ER-related main claim. Quantification of co-expression and statistics on labeling intensity or coverage would greatly strengthen the conclusions and the title of the paper.

Author response:

eLife assessment

This valuable study uses single-cell transcriptomics to explore the mouse vomeronasal organ and represents an advance that enhances our understanding of neural diversity within this sensory system. Findings suggest a unique endoplasmic reticulum (ER) structure in Gnao1 neurons and allow for the synthesis of a developmental trajectory from stem cells to mature vomeronasal sensory neurons. Convincing methods, data, and analyses broadly support the claims, although experiments supporting the main ER-related claim are incomplete and lack quantification of co-expression and statistics on labeling intensity or coverage. Adding these data would greatly strengthen the conclusions of the paper.

Public Reviews:

Reviewer #1 (Public Review):

Devakinandan and colleagues present a manuscript analyzing single-cell RNA-sequencing data from the mouse vomeronasal organ. The main advances in this manuscript are to identify and verify the differential expression of genes that distinguish apical and basal vomeronasal neurons. The authors also identify the enriched expression of ER-related genes in Gnao1 neurons, which they verify with in situ hybridizations and immunostaining, and also explore via electron microscopy. Finally, the results of this manuscript are presented in an online R shiny app. Overall, these data are a useful resource to the community. I have a few concerns about the manuscript, which I've listed below.

General Concerns:

(1) The authors mention that they were unable to identify the cells in cluster 13. This cluster looks similar to the "secretory VSN" subtype described in a recent preprint from C. Ron Yu's lab (10.1101/2024.02.22.581574). The authors could try comparing or integrating their data with this dataset (or that in Katreddi et al. 2022) to see if this is a common cell type across datasets (or arises from a specific type of cell doublets). In situ hybridizations for some of the marker genes for this cluster could also highlight where in the VNO these cells reside.

Cluster13 (Obp2a+) cells identified in our study have similar gene expression markers to those identified with the “putative secretory” cells in Hills et al. manuscript. At the time this manuscript was available publicly, our publication was already finalized and communicated. We welcome the suggestion to integrate data, which we will attempt and address in our revision.      

(2) I found the UMAPs for the neurons somewhat difficult to interpret. Unlike Katreddi et al. 2022 or Hills et al. 2024, it's tricky to follow the developmental trajectories of the cells in the UMAP space. Perhaps the authors could try re-embedding the data using gene sets that don't include the receptors? It would also be interesting to see if the neuron clusters still cluster by receptor-type even when the receptors are excluded from the gene sets used for clustering. Plots relating the original clusters to the neuronal clusters, or dot plots showing marker gene expression for the neuronal clusters might both be useful. For example, right now it's difficult to interpret clusters like n8-13.

We will represent the UMAPs to make the developmental trajectory clearer. How neuron clusters are affected by the presence or exclusion of receptors is an interesting question that we will address in our revision, along with showing markers of each neuronal cluster, as suggested by the reviewer.  

Reviewer #2 (Public Review):

Summary:

The study focuses on the vomeronasal organ, the peripheral chemosensory organ of the accessory olfactory system, by employing single-cell transcriptomics. The author analyzed the mouse vomeronasal organ, identifying diverse cell types through their unique gene expression patterns. Developmental gene expression analysis revealed that two classes of sensory neurons diverge in their maturation from common progenitors, marked by specific transient and persistent transcription factors. A comparative study between major neuronal subtypes, which differ in their G-protein sensory receptor families and G-protein subunits (Gnai2 and Gnao1, respectively), highlighted a higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons. Moreover, distinct differences in ER content and ultrastructure suggest some intriguing roles of ER in Gnao1-positive vomeronasal neurons. This work is likely to provide useful data for the community and is conceptually novel with the unique role of ER in a subset of vomeronasal neurons. This reviewer has some minor concerns and some suggestions to improve the manuscript.

Strengths:

(1) The study identified diverse cell types based on unique gene expression patterns, using single-cell transcriptomic.

(2) The analysis suggests that two classes of sensory neurons diverge during maturation from common progenitors, characterized by specific transient and persistent transcription factors.

(3) A comparative study highlighted differences in Gnai2- and Gnao1-positive sensory neurons.

(4) Higher expression of endoplasmic reticulum (ER) associated genes in Gnao1 neurons.

(5) Distinct differences in ER content and ultrastructure suggest unique roles of ER in Gnao1-positive vomeronasal neurons.

(6) The research provides conceptually novel on the unique role of ER in a subset of vomeronasal neurons, offering valuable insights to the community.

Weaknesses:

(1) The connection between observations from sc RNA-seq and EM is unclear.

(2) The lack of quantification for the ER phenotype is a concern.

We would like to point out that the connection between scRNA-seq and EM was made in our experiments that investigated the localization of ER proteins via IHC (in Figure 5). The intriguing observation that the levels of a number of ER luminal and membrane proteins were higher in Gnao1 compared to Gnai2 neurons, led us to hypothesize a differential ER content or ultrastructure, which was verified by EM. The quantification of ER phenotype would definitely strengthen our observations, which we will add in our revised manuscript.       

Reviewer #3 (Public Review):

Summary:

In this manuscript, Devakinandan and colleagues have undertaken a thorough characterization of the cell types of the mouse vomeronasal organ, focusing on the vomeronasal sensory neurons (VSNs). VSNs are known to arise from a common pool of progenitors that differentiate into two distinct populations characterized by the expression of either the G protein subunit Gnao1 or Gnai2. Using single-cell RNA sequencing followed by unsupervised clustering of the transcriptome data, the authors identified three Gnai2+ VSN subtypes and a single Gnao1+ VSN type. To study VSN developmental trajectories, Devakinandan and colleagues took advantage of the constant renewal of the neuronal VSN pool, which allowed them to harvest all maturation states. All neurons were re-clustered and a pseudotime analysis was performed. The analysis revealed the emergence of two pools of Gap43+ clusters from a common lineage, which differentiate into many subclusters of mature Gnao1+ and Gnai2+ VSNs. By comparing the transcriptomes of these two pools of immature VSNs, the authors identified a number of differentially expressed transcription factors in addition to known markers. Next, by comparing the transcriptomes of mature Gnao1+ and Gnai2+ VSNs, the authors report the enrichment of ER-related genes in Gnao1+ VSNs. Using electron microscopy, they found that this enrichment was associated with specific ER morphology in Gnao1+ neurons. Finally, the authors characterized chemosensory receptor expression and co-expression (as well as H2-Mv proteins) in mature VSNs, which recapitulated known patterns.

Strengths:

The data presented here provide new and interesting perspectives on the distinguishing features between Gnao1+ and Gnai2+ VSNs. These features include newly identified markers, such as transcription factors, as well as an unsuspected ER-related peculiarity in Gnao1+ neurons, consisting of a hypertrophic ER and an enrichment in ER-related genes. In addition, the authors provide a comprehensive picture of specific co-expression patterns of V2R chemoreceptors and H2-Mv genes.

Importantly, the authors provide a browser (scVNOexplorer) for anyone to explore the data, including gene expression and co-expression, number and proportion of cells, with a variety of graphical tools (violin plots, feature plots, dot plots, ...).

Weaknesses:

The study still requires refined analyses of the data and rigorous quantification to support the main claims.

The method description for filtering and clustering single-cell RNA-sequencing data is incomplete. The Seurat package has many available pipelines for single-cell RNA-seq analysis, with a significant impact on the output data. How did the authors pre-process and normalize the data? Was the pipeline used with default settings? What batch correction method was applied to the data to mitigate possible sampling or technical effects? Moreover, the authors do not describe how cell and gene filtering was performed.

The data in Figure 7-Supplement 3 show that one-sixth of the V1Rs do not express any chemoreceptor, while over a hundred cells express more than one chemoreceptor. Do these cells have unusually high or low numbers of genes or counts? To exclude the possibility of a technical artifact in these observations, the authors should describe how they dealt with putative doublet cells or debris.

Surprisingly, some clusters are characterized by the expression of specific chemoreceptors (VRs). Have these been used for clustering? If so, clustering should be repeated after excluding these receptors.

The identification of the VSN types should be consistent across the different analyses and validated. The data presented in Figure 1 lists four mature VSN types, whereas the re-clustering of neurons presented in Figure 3 leads to a different subdivision. At present, it remains unclear whether these clusters reflect the biology of the system or are due to over-clustering of the data, and therefore correspond to either noise or arbitrary splitting of continua. Clusters should be merged if they do not correspond to discrete categories of cells, and correspondence should be established between the different clustering analyses. To validate the detected clusters as cell types, markers characteristic of each of these populations can be evaluated by ISH or IHC.

There is a lack of quantification of imaging data, which provides little support for the ER-related main claim. Quantification of co-expression and statistics on labeling intensity or coverage would greatly strengthen the conclusions and the title of the paper.

scRNA-seq data analysis methods: We agree with the reviewer and will elaborate on the various criterion, parameters and methods in our revision. As described above, our revised manuscript will include analysis of how inclusion / exclusion of VRs affects cell clusters, as well as quantification of the ER phenotype. We will address the reviewer’s concern of over-clustering.

We think that the cells expressing zero as well as two V1Rs are real and cannot be attributed to debris or doublets for the following reasons:

a) Cells expressing no V1Rs are not necessarily debris because they express other neuronal markers at the same level as cells that express one or two V1Rs. Higher expression threshold values used in our analysis may have somewhat increased the proportion of cells with zero V1Rs. We will modify figure 7-supplement 3c to add another group showing Gnai2 level in cells expressing zero V1Rs.

b) Cells co-expressing V1R genes: We listed the frequency of cells co-expressing V1R gene combinations in Supplementary table - 8. Among 134 cells that express two V1Rs, 44 cells express Vmn1r85+Vmn1r86, 21 express Vmn1r184+Vmn1r185, 13 express Vmn1r56+Vmn1r57, 6 express Vmn1r168+Vmn1r177, and so on. Doublets generally are a random combination of two cells. Here, each specific co-expression combination represents multiple cells and is highly unlikely by random chance. Some of the co-expression combinations were identified earlier and verified experimentally in Lee et al., 2019 and Hills et. al. Furthermore, Figure-7 supplement 3c shows that the level of Gnai2 expression is comparable across cells expressing one or two V1Rs. If the V1R expressing cells are doublets, we expect the level of Gnai2 to be higher, as compared to cells expressing single V1R. We will elaborate on this in our revised manuscript.

eLife assessment

This study compiles a wide range of results on the connectivity, stimulus selectivity, and potential role of the claustrum in sensory behavior. While most of the connectivity results confirm earlier studies, this valuable work provides incomplete evidence that the claustrum responds to multimodal stimuli and that local connectivity is reduced across cells that have similar long-range connectivity. The conclusions drawn from the behavioral results are weakened by the animals' poor performance on the designed task.This study has the potential to be of interest to neuroscientists.

Reviewer #1 (Public Review):

Summary:

The paper by Shelton et al investigates some of the anatomical and physiological properties of the mouse claustrum. First, they characterize the intrinsic properties of claustrum excitatory and inhibitory neurons and determine how these different claustrum neurons receive input from different cortical regions. Next, they perform in vitro patch clamp recordings to determine the extent of intraclaustrum connectivity between excitatory neurons. Following these experiments, in vivo axon imaging was performed to determine how claustrum-retrosplenial cortex neurons are modulated by different combinations of auditory, visual, and somatosensory input. Finally, the authors perform claustrum lesions to determine if claustrum neurons are required for performance on a multisensory discrimination task

Strengths:

An important potential contribution the authors provide is the demonstration of intra-claustrum excitation. In addition, this paper provides the first experimental data where two cortical inputs are independently stimulated in the same experiment (using 2 different opsins). Overall, the in vitro patch clamp experiments and anatomical data provide confirmation that claustrum neurons receive convergent inputs from areas of the frontal cortex. These experiments were conducted with rigor and are of high quality.

Weaknesses:

The title of the paper states that claustrum neurons integrate information from different cortical sources. However, the authors did not actually test or measure integration in the manuscript. They do show physiological convergence of inputs on claustrum neurons in the slice work. Testing integration through simultaneous activation of inputs was not performed. The convergence of cortical input has been recently shown by several other papers (Chia et al), and the current paper largely supports these previous conclusions. The in vivo work did test for integration because simultaneous sensory stimulations were performed. However, integration was not measured at the single cell (axon) level because it was unclear how activity in a single claustrum ROI changes in response to (for example) visual, tactile, and visual-tactile stimulations. Reading the discussion, I also see the authors speculate that the sensory responses in the claustrum could arise from attentional or salience-related inputs from an upstream source such as the PFC. In this case, claustrum cells would not integrate anything (but instead respond to PFC inputs).

The different experiments in different figures often do not inform each other. For example, the authors show in Figure 3 that claustrum-RSP cells (CTB cells) do not receive input from the auditory cortex. But then, in Figure 6 auditory stimuli are used. Not surprisingly, claustrum ROIs respond very little to auditory stimuli (the weakest of all sensory modalities). Then, in Figure 7 the authors use auditory stimuli in the multisensory task. It seems that these experiments were done independently and were not used to inform each other.

One novel aspect of the manuscript is the focus on intraclaustrum connectivity between excitatory cells (Figure 2). The authors used wide-field optogenetics to investigate connectivity. However, the use of paired patch-clamp recordings remains the ground truth technique for determining the rate of connectivity between cell types, and paired recordings were not performed here. It is difficult to understand and gain appreciation for intraclaustrum connectivity when only wide-field optogenetics is used.

In Figure 2, CLA-rsp cells express Chrimson, and the authors removed cells from the analysis with short latency responses (which reflect opsin expression). But wouldn't this also remove cells that express opsin and receive monosynaptic inputs from other opsin-expressing cells, therefore underestimating the connectivity between these CLA-rsp neurons? I think this needs to be addressed.

In Figure 5J the lack of difference in the EPSC-IPSC timing in the RSP is likely due to 1 outlier EPSC at 30ms which is most likely reflecting polysynaptic communication. Therefore, I do not feel the argument being made here with differences in physiology is particularly striking.

In the text describing Figure 5, the authors state "These experiments point to a complex interaction ....likely influenced by cell type of CLA projection and intraclaustral modules in which they participate". How does this slice experiment stimulating axons from one input relate to different CLA cell types or intra-claustrum circuits? I don't follow this argument.

In Figure 6G and H, the blank condition yields a result similar to many of the sensory stimulus conditions. This blank condition (when no stimulus was presented) serves as a nice reference to compare the rest of the conditions. However, the remainder of the stimulation conditions were not adjusted relative to what would be expected by chance. For example, the response of each cell could be compared to a distribution of shuffled data, where time-series data are shuffled in time by randomly assigned intervals and a surrogate distribution of responses generated. This procedure is repeated 200-1000x to generate a distribution of shuffled responses. Then the original stimulus-triggered response (1s post) could be compared to shuffled data. Currently, the authors just compare pre/post-mean data using a Mann-Whitney test from the mean overall response, which could be biased by a small number of trials. Therefore, I think a more conservative and statistically rigorous approach is warranted here, before making the claim of a 20% response probability or 50% overall response rate.

Regarding Figure 6, a more conventional way to show sensory responses is to display a heatmap of the z-scored responses across all ROIs, sorted by their post-stimulus response. This enables the reader to better visualize and understand the claims being made here, rather than relying on the overall mean which could be influenced by a few highly responsive ROIs.

For Figure 6, it would also help to display some raw data showing responses at the single ROI level and the population level. If these sensory stimulations are modulating claustrum neurons, then this will be observable on the mean population vector (averaged df/f across all ROIs as a function of time) within a given experiment and would add support to the conclusions being made.

As noted by the authors, there is substantial evidence in the literature showing that motor activity arises in mice during these types of sensory stimulation experiments. It is foreseeable that at least some of the responses measured here arise from motor activity. It would be important to identify to what extent this is the case.

All claims in the results for Figure 6 such as "the proportion of responsive axons tended to be highest when stimuli were combined" should be supported by statistics.

In Figure 7, the authors state that mice learned the structure of the task. How is this the case, when the number of misses is 5-6x greater than the number of hits on audiovisual trials (S Figure 19). I don't get the impression that mice perform this task correctly. As shown in Figure 7I, the hit rate is exceptionally low on the audiovisual port in controls. I just can't see how control and lesion mice can have the same hit rate and false alarm rate yet have different d'. Indeed, I might be missing something in the analysis. However, given that both groups of mice are not performing the task as designed, I fail to see how the authors' claim regarding multisensory integration by the claustrum is supported. Even if there is some difference in the d' measure, what does that matter when the hits are the least likely trial outcome here for both groups.

In the discussion, it is stated that "While axons responded inconsistently to individual stimulus presentations, their responsivity remained consistent between stimuli and through time on average...". I do not understand this part of the sentence. Does this mean axons are consistently inconsistent?

In the discussion, the authors state their axon imaging results contrast with recent studies in mice. Why not actually do the same analysis that Ollerenshaw did, so this statement is supported by fact? As pointed out above, the criteria used to classify an axon as responsive to stimuli were very liberal in this current manuscript.

I find the discussion wildly speculative and broad. For example, "the integrative properties of the CLA could act as a substrate for transforming the information content of its inputs (e.g. reducing trial-to-trial variability of responses to conjunctive stimuli...)". How would a claustrum neuron responding with a 10% reliability to a stimuli (or set of stimuli) provide any role in reducing trial-to-trial variability of sensory activity in the cortex?

Reviewer #2 (Public Review):

Summary:

In this manuscript, Shelton et al. explore the organization of the Claustrum. To do so, they focus on a specific claustrum population, the one projecting to the retrosplenial cortex (CLA-RSP neurons). Using an elegant technical approach, they first described electrophysiological properties of claustrum neurons, including the CLA-RSP ones. Further, they showed that CLA-RSP neurons (1) directly excite other CLA neurons, in a 'projection-specific' pattern, i.e. CLA-RSP neurons mainly excite claustrum neurons not projecting to the RSP and (2) received excitatory inputs from multiple cortical territories (mainly frontal ones). To confirm the 'integrative' property of claustrum networks, they then imaged claustrum axons in the cortex during single- or multi-sensory stimulations. Finally, they investigated the effect of CLA-RSP lesion on performance in a sensory detection task.

Strengths:<br /> Overall, this is a really good study, using state-of-the-art technical approaches to probe the local/global organization of the Claustrum. The in-vitro part is impressive, and the results are compelling.

Weaknesses:<br /> One noteworthy concern arises from the terminology used throughout the study. The authors claimed that the claustrum is an integrative structure. Yet, integration has a specific meaning, i.e. the production of a specific response by a single neuron (or network) in response to a specific combination of several input signals. In this study, the authors showed compelling results in favor of convergence rather than integration. On a lighter note, the in-vivo data are less convincing, and do not entirely support the claim of "integration" made by the authors.

Reviewer #3 (Public Review):

The claustrum is one of the most enigmatic regions of the cerebral cortex, with a potential role in consciousness and integrating multisensory information. Despite extensive connections with almost all cortical areas, its functions and mechanisms are not well understood. In an attempt to unravel these complexities, Shelton et al. employed advanced circuit mapping technologies to examine specific neurons within the claustrum. They focused on how these neurons integrate incoming information and manage the output. Their findings suggest that claustrum neurons selectively communicate based on cortical projection targets and that their responsiveness to cortical inputs varies by cell type.

Imaging studies demonstrated that claustrum axons respond to both single and multiple sensory stimuli. Extended inhibition of the claustrum significantly reduced animals' responsiveness to multisensory stimuli, highlighting its critical role as an integrative hub in the cortex.

However, the study's conclusions at times rely on assumptions that may undermine their validity. For instance, the comparison between RSC-projecting and non-RSC-projecting neurons is problematic due to potential false negatives in the cell labeling process, which might not capture the entire neuron population projecting to a brain area. This issue casts doubt on the findings related to neuron interconnectivity and projections, suggesting that the results should be interpreted with caution. The study's approach to defining neuron types based on projection could benefit from a more critical evaluation or a broader methodological perspective.

Nevertheless, the study sets the stage for many promising future research directions. Future work could particularly focus on exploring the functional and molecular differences between E1 and E2 neurons and further assess the implications of the distinct responses of excitatory and inhibitory claustrum neurons for internal computations. Additionally, adopting a different behavioral paradigm that more directly tes2ts the integration of sensory information for purposeful behavior could also prove valuable.

guaranteed permanent employment, especially as a teacher or lecturer, after a probationary period; security of tenure

making a certain situation or outcome likely or possible

demand or specify (a requirement), typically as part of an agreement: he stipulated certain conditions before their marriage.

noun

1 a comfortable or suitable position in life or employment: he is now a partner at a leading law firm and feels he has found his niche | he is carving out a niche for himself as one of the most talented and reliable character actors around.

2 a specialized segment of the market for a particular kind of product or service: he believes he has found a niche in the market

adjective

denoting products, services, or interests that appeal to a small, specialized section of the population: other companies in this space had to adapt to being niche players

Marktforschung konkret

Résumé vidéo [00:00:00][^1^][1] - [00:27:44][^2^][2]:

Ce webinaire présente la campagne de vaccination HPV dans les collèges, expliquant l'importance de la vaccination pour prévenir les cancers liés au papillomavirus et les modalités de mise en œuvre dans les établissements scolaires.

Points forts: + [00:00:00][^3^][3] Objectif du webinaire * Présentation de la campagne de vaccination en milieu scolaire + [00:01:03][^4^][4] Avancement de la campagne * Nouvelle-Aquitaine en avance sur l'organisation + [00:05:28][^5^][5] Épidémiologie du HPV * Explication des risques et de la transmission du virus + [00:08:56][^6^][6] Recommandations de vaccination * Vaccination recommandée pour les jeunes filles depuis 2007 et pour les garçons depuis 2021 + [00:13:08][^7^][7] Vaccination dans les collèges * Discussion sur l'efficacité de la vaccination en milieu scolaire + [00:22:26][^8^][8] Communication avec les parents * Importance de l'engagement des parents et des solutions pour les zones défavorisées Résumé vidéo [00:27:47][^1^][1] - [00:56:19][^2^][2]: Le webinaire aborde la campagne de vaccination dans les collèges, mettant l'accent sur la communication et l'organisation. Il souligne l'importance de la collaboration avec les professionnels de santé et l'accessibilité des informations pour les parents.

Points clés: + [00:27:47][^3^][3] Communication de la campagne * Messages pré-rédigés par l'ARS * Disponibilité des informations en ligne + [00:30:34][^4^][4] Calendrier de la campagne * Informations initiales en juin * Évaluation de la couverture vaccinale en juillet 2024 + [00:31:45][^5^][5] Plan de communication * Sensibilisation sur les réseaux sociaux * Ciblage des parents et adolescents + [00:37:12][^6^][6] Engagement des établissements * Planification de la vaccination * Mise à disposition des ressources pour les familles + [00:44:21][^7^][7] Organisation des centres de vaccination * Déplacement vers les établissements * Collaboration avec les acteurs locaux + [00:52:58][^8^][8] Rôle des chefs d'établissement * Facilitation de la vaccination * Support dans l'utilisation des outils numériques Résumé de la vidéo [00:56:21][^1^][1] - [00:57:31][^2^][2]: La partie 3 de la vidéo aborde la communication avec les familles concernant la campagne de vaccination dans les collèges, les difficultés rencontrées et les solutions proposées.

Points forts: + [00:56:21][^3^][3] Communication avec les familles * Utilisation de papier pour les autorisations + [00:57:00][^4^][4] Lien avec centre de vaccination * Mise à disposition de documents nécessaires + [00:57:15][^5^][5] Conclusion de la réunion * Envoi des documents promis * Interruption pour une autre réunion

a district of a city or town as defined for policing purposes.

| snatʃ |

verb [with object]

quickly seize (something) in a rude or eager way: she snatched a biscuit from the plate |

To have a broad, enthusiastic smile.

take (someone) away by force or deception; kidnap: the millionaire who disappeared may have been abducted.

The SM3 and SM4

Subtle differences between the two models.

铁扇公主 Princess Iron Fan

大闹天宫 Havoc in Heaven

骆驼献舞 The Dance of Camel

小蝌蚪找妈妈 Where is Mama

哪吒闹海 Nezha Conquers the Dragon King

天书奇谭 The Legend of Sealed Book

三个和尚 Three Monks

九色鹿 A Deer of Nine Colors

中国奇谭 Yao-Chinese Folktales

没头脑和不高兴 Mei Tounao and Bu Gaoxing

giving pleasure or satisfaction

| ˈrɛpətwɑː |

noun

a stock of plays, dances, or items that a company or a performer knows or is prepared to perform.

| ˈɑːtɪstri |

noun [mass noun]

creative skill or ability: the artistry of the pianist.

| ɪˈnɔːɡjʊrəl |

adjective [attributive]

marking the beginning of an institution, activity, or period of office: his inaugural concert as Music Director.

something that serves as a sign or evidence of a specified fact, event, or quality: growing attendance figures are a testament to the event's popularity.

put an end to (a rebellion or other disorder), typically by the use of force: extra police were called to quell the disturbance

eLife assessment

This valuable study uses dynamic metabolic models to compare perturbation responses in a bacterial system, analyzing whether they return to their steady state or amplify beyond the initial perturbation. The evidence supporting the emergent properties of perturbed metabolic systems to network topology and sensitivity to specific metabolites is compelling. However, the mathematical explanation of the perturbation response is incomplete, and a more comprehensive metabolic and biosynthesis model would be beneficial.

Reviewer #1 (Public Review):

Summary

The author studied metabolic networks for central metabolism, focusing on how system trajectories returned to their steady state. To quantify the response, systematic perturbation was performed in simulation and the maximal destabilization away from the steady state (compared with the initial perturbation distance) was characterized. The author analyzed the perturbation response and found that sparse networks and networks with more cofactors are more "stable", in the sense that the perturbed trajectories have smaller deviations along the path back to the steady state.

Strengths and major contributions

The author compared three metabolic models and performed systematic perturbation analysis in simulation. This is the first work to characterize how perturbed trajectories deviate from equilibrium in large biochemical systems and illustrated interesting findings about the difference between sparse biological systems and randomly simulated reaction networks.

Weaknesses

There are two main weaknesses in this study:

First, the metabolic network in this study is incomplete. For example, amino acid synthesis and lipid synthesis are important for biomass and growth, but they are not included in the three models used in this study. NADH and NADPH are as important as ATP/ADP/AMP, but they are not included in the models. In the future, a more comprehensive metabolic and biosynthesis model is required.

Second, this work does not provide a mathematical explanation of the perturbation response χ. Since the perturbation analysis is performed close to the steady state (or at least belongs to the attractor of single-steady-state), local linear analysis would provide useful information. By complementing with other analysis in dynamical systems (described below) we can gain more logical insights about perturbation response.

Discussion and impact for the field

Metabolic perturbation is an important topic in cell biology and has important clinical implications in pharmacodynamics. The computational analysis in this study provides an initiative for future quantitative analysis on metabolism and homeostasis.

Reviewer #2 (Public Review):

Summary:

The authors have conducted a valuable comparative analysis of perturbation responses in three nonlinear kinetic models of E. coli central carbon metabolism found in the literature. They aimed to uncover commonalities and emergent properties in the perturbation responses of bacterial metabolism. They discovered that perturbations in the initial concentrations of specific metabolites, such as adenylate cofactors and pyruvate, significantly affect the maximal deviation of the responses from steady-state values. Furthermore, they explored whether the network connectivity (sparse versus dense connections) influences these perturbation responses. The manuscript is reasonably well written.

Strengths:

Well-defined and valuable research questions.

Weaknesses:

(1) In the study on determining key metabolites affecting responses to perturbations (starting from line 171), the authors fix the values of individual concentrations to their steady-state values and observe the responses. Such a procedure adds artificial constraints to the network because, in the natural responses of cells (and models) to perturbations, it is highly unlikely that metabolites will not evolve in time. By fixing the values of specific metabolites, the authors prohibit the metabolic network from evolving in the most optimal way to compensate for the perturbation. Instead of this procedure, have the authors considered for this task applying techniques from variance-based sensitivity analysis (Sobol, global sensitivity analysis), where they can calculate the first-order sensitivity index and total effect index? Using this technique, the authors would be able to determine the key metabolites while allowing for metabolic responses to perturbations without unnatural constraints.

(2) To follow up on the previous remark, the authors state that the metabolites that augment the response coefficient when their concentration is fixed tend to be allosteric regulators. The authors should report which allosteric regulations are implemented in each of the models so that one can compare against Figure 2. Again, the effect of allosteric regulation by a specific metabolite that is quantified the way the authors did is biased by fixing the concentration value - it is true that negative feedback is broken when the metabolite concentration is fixed, however, in the rate law, there is still the fixed inhibition term with its value corresponding to the inhibition at the steady state. To see the effect of allosteric regulation by a metabolite, one can change the inhibition constants instead of constraining the responses with fixed concentrations.

(3) Given the role of ATP in metabolic processes, the authors' finding of the sensitivity of the three networks' responses to perturbations in the AXP concentrations seems reasonable. However, drawing such firm conclusions from only three models, with each of them built around one steady state and having one kinetic parameter set despite that they were built for different physiologies, raises some questions. It is well-known in studies related to basins of attraction of the steady states that the nonlinear responses also depend on the actual steady states, the values of kinetic parameters, and implemented kinetic rate law, i.e., not only on the topology of the underlying systems. In the population of only three models, we cannot exclude the possibility of overlaps and strong similarities in the values of kinetic parameters, steady states, and enzyme saturations that all affect and might bias the observed responses. Ideally, to eliminate the possibility of such biases, one should simulate responses of a large population of models for multiple physiologies (and the corresponding steady states) and multiple parameter sets per physiology. This can be a difficult task, but having more kinetic models in this work would go a long way toward more convincing results. Recently, E. coli nonlinear kinetic models from several groups appeared that might help in this task, e.g., Haiman et al., PLoS Comput Biol, 17(1): e1008208, (2021), Choudhury et al., Nat Mach Intell, 4, 710-719, (2022); Hu et al., Metab Eng, 82, 123-133 (2024), Narayanan et al., Nat Commun, 15:723, (2024).

(4) Can the authors share their insights on what could be the underlying reasons for the bimodal distribution in Figure 1E? Even after adding random reactions, the distribution still has two modes - why is that?

(5) Considering the effects of the sparsity of the networks on the perturbation responses (from line 223 onwards), when we compare the three analyzed models, it is clear that the Khodayari et al. model is a superset of the other two models. Therefore, this model can be considered as, e.g., Chassagnole model with Nadd reactions (though not randomly added). Based on Figures 1b and S2, one can observe that the responses of the Khodayari models have stronger responses, which is exactly opposite to the authors' conclusion that adding the reactions weakens the responses. The authors should comment on this.

А есть возможность изменить написание слова на рисунке? В словаре закрепили форму с е.

В моём варианте в строке всего три этих слова и чудовищно большие пробелы между ними. Не знаю, можно ли с этим что-то сделать.

Тут что-то сбилось, не понимаю что.

У меня в конце строки.

Résumé de la vidéo [00:00:02][^1^][1] - [00:52:12][^2^][2]:

Cette vidéo, intitulée "EVARS, on se lance ? - Parlons pratiques ! #39", aborde l'éducation à la vie affective, relationnelle et à la sexualité (EVARS) dans le contexte scolaire français. Elle souligne l'importance de cette éducation pour la santé publique, l'égalité entre les sexes, la lutte contre les violences et discriminations, et le bien-être des élèves. Des experts discutent des défis et des ressources disponibles pour les enseignants, ainsi que des stratégies pour intégrer l'EVARS dans les programmes scolaires.

Points forts: + [00:00:06][^3^][3] Introduction à l'EVARS * Importance pour la santé publique et l'égalité * Sensibilité du sujet et intégration dans les programmes * Difficultés rencontrées dans les écoles + [00:01:26][^4^][4] Rôle de l'école et obstacles * Définition de la santé sexuelle * Besoins des enseignants en outils et connaissances * Importance de commencer l'éducation dès le plus jeune âge + [00:20:39][^5^][5] Mise en œuvre de l'EVARS * Freins institutionnels et manque de temps * Nécessité de coordination et de ressources * Importance de la formation des enseignants + [00:36:28][^6^][6] Ressources et coanimation * Utilisation de partenaires éducatifs * Ressources en ligne et formations gratuites * Importance de la coanimation avec les intervenants extérieurs

Résumé de la vidéo [00:37:00][^1^][1] - [00:52:12][^2^][2]:

Cette vidéo aborde l'éducation à la vie affective, relationnelle et sexuelle (EVARS) dans le contexte scolaire français. Elle souligne l'importance de l'EVARS pour la santé publique, l'égalité entre les sexes et le bien-être des élèves. Les intervenants discutent des défis et des ressources disponibles pour mettre en œuvre l'EVARS dans les écoles.

Points forts: + [00:37:01][^3^][3] L'éducation sexuelle dans les écoles * Importance de commencer tôt * Nécessité d'utiliser un vocabulaire clair et partagé * L'éducation sexuelle est une mission de l'école et de santé publique + [00:42:35][^4^][4] Ressources et partenaires * Le Planning Familial et les Crips comme ressources * Sites web et brochures pour adolescents et éducateurs * Importance de la coanimation et de la formation des enseignants + [00:48:07][^5^][5] La prévention des violences sexuelles * Urgence de former les enseignants à repérer et protéger les enfants * Nécessité de s'entourer de ressources et de soutien * Importance de ne pas rester seul face à ces enjeux

below screenshot should show customer contact with country code

S$

"Cards"

this is not working

not needed for SG

CPython

cPython 是个什么？

我公司交的是7%

days of AR AP INVENTORY can flux - to rationale decisions.

IMPORTANT NOTE: when you are projecting your estimated AR (for the WC schedule); you use your PROJECTED REVENUE (from what you did above) AND PROJECTED AR DAYS (based on your past history)

this is similar to AP and inventory.

sales volume / plant capacity

should always have some past data for you to establish a stand

an example that this venom was not originally in the animal but was developed over a long period of time

Being too early is the same as being wrong :'(

at LEADS ... "give me a good old one"

the farmer in the dell, the farmer in the dell"

HI HO, IVES DAIRY, THE FARMER IN THE DELL ..

BIG IDEA OF THIS SECTION

School "improvement requires fundamental changes in the way public schools and school systems are designed, and in the ways they are led," which is what "the existing institutional structure of public schooling is specifically designed not to do."

BIG IDEA OF THIS SECTION

Every major education reform of this century has become weakened, adulterated, and unrecognizable by the time it arrives in the classroom.

BIG IDEA OF THIS SECTION

Schools with weak collective values and internal accountability will fail to significantly alter educator behaviors and classroom practice, ensuring that loosely-coupled 'improvement' efforts will be unsuccessful.

BIG IDEA OF THIS SECTION

Organizations improve when they have strong 'internal accountability' but most school systems instead have weak internal accountability mechanisms that are 'long on pressure and short on support.'

BIG IDEA OF THIS SECTION

District Two in New York City is an example of a school system that has strong internal accountability and thus is seeing concurrent improvement in learning outcomes.

BIG IDEA OF THIS SECTION

Public school educators should be grateful for standards-based reform because the alternative is an inequitable 'open market' solution that results in public schools serving the students that no one else wants to serve.

BIG IDEA OF THIS SECTION

Utilizing distributed leadership principles and practices, educators in schools must recognize that 1) what occurs in classrooms is a 'collective good,' and 2) isolated practice results in inequities and lack of schoolwide improvement.

BIG IDEA OF THIS SECTION

School leaders are those who can actually guide and implement 'improvement,' not just 'activity.'

BIG IDEA OF THIS SECTION

Like previous reforms, standards-based reform probably will fail to change learning and teaching because most schools will bend the new policy requirements to the existing structure rather than altering core functions of schooling.

BIG IDEA OF THIS ARTICLE

School improvement efforts rarely improve actual learning and teaching because they do not fundamentally alter how educators think about and do their work.

BIG IDEA OF THIS SECTION

"Loose coupling" explains why educational reforms rarely result in significant impacts on leaders' and teachers' practice... and student learning.

BIG IDEA OF THIS SECTION

Implementation of the SAM model not only can positively impact the frequency and quality of principals' instructional leadership activities, it also can provide SAMs with valuable opportunities to gain leadership experience.

BIG IDEA OF THIS SECTION

Some limitations of this study are that: * it was conducted in one district * that district had support from the Wallace Foundation * it didn't make any connections between SAMs and student learning outcomes

BIG IDEA OF THIS SECTION

Effective uses of SAMs frees up principals to spend more time being instructional leaders and also can foster greater teacher leadership.

BIG IDEA OF THIS SECTION

Principals and SAMs agreed that regular (even if brief) meetings were important for communication and collaboration.

BIG IDEA OF THIS SECTION

The success of SAMs may vary by school setting (e.g., elementary v. middle school) and also by implementation model.

BIG IDEA OF THIS SECTION

SAMs are a promising strategy for clawing back instructional leadership time for principals that has been eroded by "growing job responsibilities and unrealistic expectations."

BIG IDEA OF THIS SECTION

Both middle school and elementary school teachers agreed that SAMs positively impacted school management and instructional leadership responsibilities, with greater perceived impacts by middle school teachers.

BIG IDEA OF THIS SECTION

Principals have a difficult time handing over managerial responsibilities to SAMs.

BIG IDEA OF THIS SECTION

The surveys exhibited high degrees of internal consistency as assessed by Cronbach's alpha.

BIG IDEA OF THIS SECTION

Principals take on the roles of mentors, coaches, and trainers of SAMs, which requires strong levels of trust between them.

BIG IDEA OF THIS SECTION

5 focus groups (2 with SAMs; 3 with principals), ranging from 6 to 12 participants each.

BIG IDEA OF THIS SECTION

Each of the 5 focus groups was recorded, transcribed, and coded.

BIG IDEA OF THIS SECTION

Middle school teachers perceived greater impacts of SAMs on principals' instructional leadership than elementary school teachers.

BIG IDEA OF THIS SECTION

311 usable teacher surveys from 4 middle schools and 11 elementary schools.

BIG IDEA OF THIS SECTION

SAMs take managerial tasks away from principals and can either be new hires or existing employees who are given new responsibilities.