Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor/a #2

La temática propuesta por los autores surte gran efecto en las dinámicas actuales de ciencia ciudadana y alfabetización científica, en la cual, el biohacking social plantea una ruta disruptiva en el escenario colombiano de los jóvenes y la ciencia. Algunas de las observaciones respecto al documento son las siguientes:

• En el resumen, es necesario complementar este tópico con datos generales y concretos de los resultados y las conclusiones finales a las que llevó la pesquisa en cuestión.
• En la introducción, se hace una alusión a las recomendaciones ONU de educación en Colombia, algunas acciones de ciencia ciudadana, y casos específicos sobre “Do It Yourself”, pero es necesario dar mayor profundidad teórica en el despliegue de acciones globales y locales de alfabetización científica, la misma ciencia ciudadana, dando margen no solo a las referencias experienciales, sino a conceptualización teórica sobre los tópicos grandes que aborda el documento: biohacking y alfabetización científica.
• En los Resultados, los enunciados “Implementación del modelo”, “Divulgación científica”, “Intervención de comunidades”, y los siguientes son expuestos de manera directa, sin siquiera ser detallados en la metodología. Por ejemplo, en lo descrito en Metodología, “Divulgación científica”, los autores plantean la realización de una serie televisiva, un programa radial y un taller. En la misma metodología, no se describen los métodos para llevar a cabo estos o por qué son pertinentes estas acciones en la propuesta. Ahora bien, al revisar los resultados, los autores envían enlaces del programa televisivo, radial y enlaces de Tweets de los talleres, sin explicar, describir o detallar el impacto como tal de estas acciones. Esto sugiero abordarlo más, dar mayor profundidad y asegurarse que los enlaces puedan tener una permanencia en el tiempo.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor/a #1

El artículo trata de un tema muy interesante que es la posibilidad de crear espacios que articulan la producción de conocimiento con la innovación ciudadana a partir de una metodología basada en el DIY que despierta la curiosidad, la creatividad y el ánimo de los ciudadanos por temas científicos e intervención directa en su realidad. Creo que el texto es un relato de experiencia y me parece que, al intentar encajar en un formato más convencional de artículo, con secciones predeterminadas - como metodología, resultados, debate y conclusión - su escrita se hace menos directa, menos objetiva y se pierde la riqueza del relato de sus dos años de trabajo voluntario con comunidades. Siguen algunas sugerencias.

• El resumen podría ser reescrito. El trabajo es más interesante e importante de lo que indica.
• Sería interesante tener una presentación más ordenada de Biohacking porque se entiende, en parte lo que es, a lo largo del texto. Cuando el Biohacking es constituído? ¿Por qué? ¿Es una ONG, un grupo informal, un coletivo? ¿Quién hace parte? Cuales son sus principales actividades, donde se realizan, como se realizan, qué apoyos cuentan? Es una manera de enseñar su trayectoria y su importancia como proponiente de modelos de educación cientifica/intervención comunitária.
• En metodologia, sacar el título "descripción del proyecto", pues no es etapa metodológica.
• La "Análisis de necesidades" es en realidad, Identificación de necesidades y podría ser la etapa 1, siendo "Encuesta para la población de estudiantes de básica secundaria" la etapa 1.1 y la "Encuesta para la población de jóvenes egresados de educación básica secundaria" es 1.1.2.
• Numerar las preguntas del cuestionario.
• La primera parte de "Resultados”, llamada “Impactos negativos de la carencia de laboratorio” debería venir luego después, enseñando el contexto de la actuación.
• Se podría hacer un par de gráficos para hacer la lectura de los resultados más agradable.
• Seguiría con la numeración de las etapas. La siguiente es la etapa 2 y se llamaría "Elaboración de" Modelo de intervención. Aquí, cúal es la referencia de los principios de Formar, Articular, Investigar y Replicar? ¿Cómo dialogan/impulsan el desarrollo de la experiencia?
• La sección "Biohacking Colombia: Escuelas de Creación" debería estar abajo del iten (nuevo) "Elaboración de" Modelo de intervención.
• La sección "Implementación del modelo" se trata del desarrollo de lo que fue descrito en metodología como "Diseño y construcción de prototipos"; es la descripción de una etapa de construcción de la intervención.
• En "El modelo planteado se ha probado inicialmente con la participación de aproximadamente 100 estudiantes de pregrado de universidades e instituciones universitarias de la ciudad de Medellín" - se puede añadir los nombres de las instituciones como nota de pié de página. ¿Son las mismas que participaron de la encuesta o no? Decirlo.
• Las figuras deben estar junto al texto que las menciona para mejorar el entendimiento del lector.
• En divulgación científica, todos los enlaces deben ser transformados en nota de pie de página. Sería interesante añadir los títulos de los videos para que el lector sepa los temas tratados.
• Los videos son bien hechos, con calidad profesional, pero están poco valorados en el trabajo. Si se inserta un print screen, el lector ya ve que es material audiovisual profesional. Hay alguna información de su alcance? Audiencia, comentários de viewers, repercusión?
• El enlace de Facebook está privado, no se puede consultar.
• Las partes "Intervención de comunidades" y "Talleres a comunidades en el Exploratorio del Parque Explora Medellín", "Talleres a niños y adultos mayores en la Biblioteca EPM de Fundación EPM" y "Museo de Ciencias Naturales de la Salle del Instituto Tecnológico Metropolitano-ITM", "“Ciencia a la mano” del Parque Explora", "BioMAE", "Participación en el MinkaLab", "Con-ciencia, Ciencia para la Paz" y "Actividades de apropiación social del conocimiento" son consideradas parte de divulgación científica? Diferenciar con una enumeración o diferente tamaño de letra.
• En la parte del debate, hubo algún tipo de evaluación con participantes - alumnos de pregrado, universitarios o ciudadanos - que pueda dar base a alguna de las afirmaciones? ¿O son evaluaciones del grupo promotor? Se podría ejemplificar algunas de las afirmaciones para entender mejor a que nivel estaba "la resistencia de las comunidades a comprender ciencia y apropiarsela", "visiones deformadas de la ciencia", "sensación de impotencia".
• Es importante retomar, en la sección debate, los principios de “Formar, Articular, Investigar y Replicar” y hacer una pequeña reflexión a la luz de las acciones implementadas por el grupo.
• Sería interesante citar, a lo largo del texto, temas cotidianos que el grupo utilizó para atraer su atención ya que parten de la premisa que la educación científica les ayudaría a tomar decisiones más adecuadas. Es decir, como aprender a utilizar un equipo de laboratorio me hace reflexionar sobre mi realidad e intervenir. ¿Hay algún impacto más expresivo que se pueda citar?
• En conclusión, creo que es demasiado fuerte decir que se propone un modelo de trabajo; sería más prudente afirmar que el relato colabora para la construcción de un modelo de trabajo.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisora: Gimena del Rio Riande

El texto es de gran interés y puede transformarse en un caso de estudio a seguir por la comunidad interesada en la publicación científica y el acceso abierto en América Latina. Disfruté mucho la lectura.

Hago aquí algunas puntualizaciones con el objetivo de mejorar la claridad expositiva del trabajo y sus objetivos:

1. El título del trabajo debe mencionar que se trata de un corpus de revistas de Humanidades y Cs Sociales, de lo contrario, parece a simple vista que su reflexión es para todas las ciencias.
2. En el abstract en inglés sugiero cambiar “magazine” por “journal”.
3. La cita de Ramirez y Samoilovich es algo criticable. Brasil es América Latina, no tenemos por qué ponerlo aparte. Es hora de cambiar ese estigma: https://es.wikipedia.org/wiki/Am%C3%A9rica_Latina
4. No estoy de acuerdo con la visión sobre Wikipedia/Wikidata: por un lado, como ya señala el otro revisor, la participación colectiva y horizontal no es tan así, y ha sido ya objeto de críticas y nuevas proposiciones. También se registran críticas de editores cuyas entradas se han borrado de Wikipedia sin mediar un diálogo previo. Asimismo, no es tan sencillo saber editar Wikidata y comprender su funcionamiento con Wikipedia. Creo que aquí el trabajo necesita matices.
5. Es necesaria una reflexión a mayores sobre el acceso abierto radical, que es lo que se propone en el título. Justamente por eso, se hace necesario que defina el acceso abierto radical. Dónde nace esta rama del AA? dónde se registra hoy día? alguna disciplina específica? nombres? bibliografía?
6. Con respecto a la verticalidad/horizontalidad, creo que es necesario ver el equilibrio. Latinrev es una bbdd en la que cualquier persona puede proponer una revista. Aunque la BBDD se cura por especialistas, no se necesita más que buena voluntad para proponer una revista al directorio. Algo similar sucede con Dialnet. Y aún más, si unx encuentra un error solo tiene que abrir un cuadro de consulta. Se suelen contestar a las 48 horas. Latinrev, a diferencia de Dialnet no apunta a métricas o impacto.
7. Sugiero mejore la sección donde se explica el corpus; me costó entender cuál era el corpus final de análisis.
8. Es necesario que discuta la noción de AA frente a las licencias CC que levanta de su corpus. El alto uso de la licencia ND habla de este AA que no es tan abierto.
9. Por momentos habla de revistas de cs sociales pero estas son de humanidades y cs sociales.
10. Entiendo que esta investigación es cuantitativa y cualitativa; solo se la menciona como cualitativa en el trabajo. Sugiero revisar.
11. Creo que este trabajo amerita que los datos de su corpus estén en abierto para poder leerlo más cabalmente.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor: David Ramírez-Ordóñez

El trabajo es muy pertinente, por cómo el autor lo indica en la introducción, la incidencia que puede tener desde la ciencia abierta en Latinoamérica y el Caribe. La definición de herramientas como Wikidata, que incluyen una parte tecnológica, una ontología y una comunidad, hacen que se consideren diferentes aspectos del ecosistema de la cultura libre y la ciencia abierta.

Discrepo de afirmaciones relacionadas a que “cualquiera puede editar Wikipedia”, debido a que se ha demostrado que existen diferentes tipos de brechas, como por ejemplo la brecha de género que incluye brecha de contenido, de lectura y de editoras. La edición de Wikidata tiene brechas similares, por ejemplo de interfaz o de conocimiento técnico para realizar queries. El artículo de Heather Ford y Judy Wajcman ponen en duda que cualquiera puede editar, por mencionar un artículo que se titula de manera que riñe diametralmente con esta afirmación: “¿Cualquiera puede editar? ¡No todo el mundo lo hace!” se titula el artículo. El texto “Quantifying the Gap: A Case Study of Wikidata Gender Disparities” muestra la brecha en Wikidata, donde menos del 22% de los ítems que representan personas, son mujeres.

Creo que se debe hacer un análisis crítico al término “apertura radical” frente al uso de cualquier tipo de licencias Creative Commons. Me parece que el artículo toma por sentado que las licencias más restrictivas de Creative Commons permiten una “apertura radical” cuando no necesariamente sea así. Si tomamos la definición del artículo, la apertura radical incluye:

1. Publicación de revistas que permiten poseer un archivo personal en digital. Entiendo que permiten la descarga del archivo del artículo.
2. No incluye cobros de acceso.
3. No incluye cobros por el procesamiento de artículos.
4. Es una nueva forma de cooperar
5. Es una nueva forma de cuidarse (cuidarnos) en los procesos de investigación
6. Genera archivos y repositorios con estas características
7. Genera redes entre las autoras y lectoras.
8. Genera otras iniciativas que permiten ampliar y generar conocimiento libre y abierto.

Al comparar esta definición con la de Contenido Cultural Libre, lo que quiere decir que ponemos un estándar alto para entender lo que es libre de lo que no es, únicamente lo que se encuentra en dominio público, atribución y atribución - compartir igual pasaría este estándar. La Figura 7 nos muestra que 64.9% de las revistas no pasarían este estándar, lo que nos podría hacer dudar de que tan “radical” es esta apertura. Dichas restricciones podrían estar en contra de los puntos 4) Es una nueva forma de cooperar y 8) Genera otras iniciativas que permiten ampliar y generar conocimiento libre y abierto; principalmente y probablemente colateralmente otros de estos puntos.

Las obras que dan libertad a las autoras no sólo son las licencias Creative Commons. Si bien este trabajo incluye otro tipo de licencias, definidas en el trabajo como “acceso abierto”, resulta interesante este estándar de facto que se presenta. Sugeriría considerar definiciones como la de Contenido Cultural Libre para revisar éste análisis y posteriores.

Valoro mucho que este trabajo use herramientas abiertas. Resulta bastante coherente escribir sobre ciencia abierta presentando trabajos que usan herramientas abiertas.

Un paso adicional que el trabajo no consideró pero que puede ser interesante explorar, es la apertura en las revistas científicas más allá de las licencias. Quiero decir: de las revistas analizadas ¿cuántas, cuáles y cómo ofrecen acceso a los archivos fuente para permitir su modificación y re uso? ¿Se ofrece acceso a los datos de investigación? ¿Se puede acceder a las imágenes en alta resolución para promover su re uso? Y aunque no sé si ocurrió con este trabajo, el mismo podría pensarse en clave de apertura: ¿Este trabajo brinda acceso al archivo .bib generado en Zotero para hacerlo replicable?

Finalmente, no me queda más que felicitar a Luis por su trabajo. Fue muy interesante leerle, entender su propuesta y ver cómo ésta se enmarca en otras como la del listado de obras mexicanas en dominio público. Este tipo de trabajos ayudan a comprender y expandir la cultura libre y la ciencia abierta. Dejé mis comentarios al documento por si resultan de ayuda. Además, considero que este trabajo puede abrir puertas a diferentes colaboraciones en Latinoamérica, como el artículo lo menciona.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisora: Saray Córdoba

El artículo no es de tipo de investigación, es más bien descriptivo, le falta profundidad. Se recomienda extenderse mucho más para complementar varias ideas que quedan truncadas, poco específicas o rozadas superficialmente.

El enfoque que las autoras le dan al tema se podría interpretar como parcial, pues se enfatizan dos organizaciones, sin embargo hay otras más que sostienen infraestructuras de ciencia abierta. Las autoras las mencionan (por ejemplo, ARK y dARK) pero no dan detalles de esos PIDs y de su organización, tampoco justifican por qué no fueron incluidas.

También se recomienda completar más el último apartado sobre el desarrollo de estas herramientas en Iberoamérica, más allá de expresar que deben ser promovidos, se podrían presentar datos sobre cuáles países los utilizan y qué resultados se han obtenido.

Agregan la taxonomía de Foster para que el lector ubique a los PIDs dentro de las infraestructuras abiertas, pero estas no están explícitamente en el esquema que se incluye. Mayor contexto ayudaría a clarificar la pertinencia de esta taxonomía.

Se recomienda extender la bibliografía para incluir estudios realizados sobre la efectividad de las herramientas -por ejemplo- o del uso de estas en Iberoamérica.

En las figuras se usan nomenclaturas diferentes, aunque una enumeración continua. En el primer caso se usa “cuadro” y en el número dos, se usa “figura”. Todos deben denominarse “figuras”.

Conviene también una revisión respecto a la escritura.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor/a #1

El manuscrito se encuentra bien redactado y aborda una temática relevante para la interoperabilidad entre infraestructuras a través de PIDs. Las dos infraestructuras cuya colaboración se describe en el trabajo cuentan con una aportación e historia importantes para el campo.

Por otro lado, el trabajo no cuenta con una estructura de artículo de investigación científica y tecnológica, ya que narra de forma descriptiva la colaboración entre ambas infraestructuras. Los productos de la colaboración son tecnológicos y tienen relevancia. Sin embargo, el trabajo no presenta información técnica, metodológica y de resultados acorde al desarrollo tecnológico que se menciona. Es por ello que no se ajusta an artículo de tipo de investigación científica y tecnológica, a menos que se agregue lo necesario para detallar la aportación tecnológica con la estructura e información acorde a ello.

Si bien es un trabajo descriptivo de la colaboración entre infraestructuras, presenta reflexiones en algunos puntos como la importancia de los PIDs en la conectividad de contenido y su impacto en la trazabilidad de los procesos de investigación. Sin embargo, sería importante profundizar en la reflexión y el pensamiento crítico sobre el tema más allá de la descripción de la experiencia de la colaboración, así como mostrar un apartado de resultados alcanzados. Aunado a ello, sería muy relevante para el público lector y pares investigadores conocer la arquitectura, servicios, nivel de interoperabilidad de los desarrollos tecnológicos descritos así como explicar el beneficio e impacto, así como también proveer información de cómo se puede acceder a ellos para aplicar, extender o realizar nuevos desarrollos a partir de esta aportación.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisora: Carolina Botero

La ciencia abierta es un tema de moda que ha tenido un importante desarrollo en América Latina asociado con el movimiento de Acceso Abierto y más recientemente con la obligación también de publicar los datos abiertos. No sorprende por tanto que la investigación y los resultados que desarrolla el artículo muestre cómo que ese es el sentido que ha calado en la percepción de los científicos que se ocupan de ciencias básicas en el Perú sobre lo que es ciencia abierta. El texto hace un buen recuento de esta situación y lo contextualiza en forma clara, la confirmación y el entorno que describe -especialmente en lo relacionado con los problemas de financiación e incentivos- exponen una realidad y reflexión del campo de estudio que puede ser muy útil a la hora de analizar o desarrollar políticas públicas e institucionales con el propósito de incentivar y apoyar la ciencia abierta. De la lectura se extraña que la autora no vaya un poco más allá para aprovechar su reflexión, bien sea para proponer recomendaciones que movilicen esta modalidad de ciencia abierta en su entorno o en el sentido de arriesgarse a predecir la evolución de la ciencia abierta en uno u otro sentido.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor: Ignasi Labastida

El trabajo presentado ofrece los resultados de un estudio sobre las percepciones sobre la ciencia abierta de un grupo reducido de investigadores en Perú.

El título indica que se estudiarán las políticas de ciencia abierta pero en realidad el estudio nos ofrece la percepción de las prácticas de la ciencia abierta más que las políticas. De hecho no queda claro a qué políticas se hace referencia ni cuáles son los objetivos de estas políticas. Se indican algunas normativas de aplicación en Perú pero no se explican ni se informa de que implican estas normativas. En la parte descriptiva de los resultados obtenidos no se indica ninguna pregunta ni ninguna respuesta que se haga referencia a alguna de las políticas de ciencia abierta. Si que se hace referencia a una serie de prácticas.

Mi sugerencia sería cambiar el título para reflejar lo que realmente aporta el trabajo: la percepción de los investigadores respecto a las prácticas de ciencia abierta. El título también es ambicioso porque habla de comunidades de investigadores y finalmente el trabajo se limita a tres disciplinas: biología, física y química y a un número muy limitado de investigadores. Aquí también se tendría que modificar el título para clarificar que el estudio es muy limitado y no tan ambicioso como podría parecer.

También parece un poco contradictorio que se indique de manera reiterada que los campos científicos más productivos del país no son justamente los que se tratan en el trabajo sino el de ciencias de la salud y la agricultura. Parecería interesante conocer la opinión de los investigadores de estos campos.

Este sería mi principal punto a corregir de manera global en el trabajo que por su parte sí que ofrece una interesante información sobre lo que perciben estos investigadores respecto a la ciencia abierta. Muy relevante, por ejemplo, es cómo entiende cada investigador el concepto de ciencia abierta.

Me gustaría también hacer algunas sugerencias de más detalle al texto:

En el apartado de la introducción se utilizan unos términos que dudo que estén generalizados para un público general y quizás requieran alguna explicación suplementaria. Estos términos son: “el modo 2 de producción de conocimiento”, “paises centrales” o “paises hegemónicos”.

En este mismo apartado se indica que “Perú invierte poco en investigación y desarrollo” Esta afirmación, en mi opinión, requiere de una cita.

En el penúltimo párrafo de la introducción se afirma que “durante los últimos años, muchas instituciones han comenzado a diseñar, adoptar y aplicar políticas de ciencia abierta” No queda claro a qué tipo de instituciones se refiere la autora ni si son instituciones latinoamericanas o del ámbito internacional. A continuación se señalan algunas normativas y normativas peruanas que requerirían una descripción más amplia para entender que implican y cómo afectan a los investigadores que después son las personas a quien van a ser preguntadas al respecto. Se mezclan normativas, iniciativas, portales pero no queda claro si alguna puede tener la consideración de política.

En el apartado de resultados hay una frase que no acaba de entenderse: “el pago de costos de procesamiento de artículos se encuentra totalmente normalizado, pues entienden que hay que pagar para contar con material de calidad” Parece como si se mezclase el modelo de pagar por publicar con el modelo de pagar por leer.

En la sección de discusión se hacen una reflexiones sobre las diferencias entre las disciplinas, por ejemplo indicando que el área de química parece más conservadora pero no se infiere directamente esta afirmación con las respuestas aportadas en el apartado de resultados, quizás faltaría alguna evidencia más.

En esta misma sección se indica que en algunos países existe normalmente una oficina de apoyo a la ciencia abierta que realiza el trabajo de limpiar datos y códigos. Creo que sería necesario indicar algún ejemplo de estas oficinas.

Finalmente en las conclusiones se hace énfasis en el código abierto pero no aparece este tema en los resultados de forma clara y al final del apartado se mencionan las revistas predatorias que es un término que hasta entonces no ha aparecido en el texto lo que parece sorprendente. Quizás sería necesario aportar evidencias al respecto en la sección de resultados.

Sugiero a la autora que tenga en cuenta estos comentarios para poder mejorar el trabajo

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisora: Sheila Godínez Larios

El trabajo presentado por las autoras es una contribución sumamente valiosa desde dos aristas: aborda un ámbito de urgencia para la humanidad que es la conservación de la bibliodiversidad y la propuesta de investigación está orientada hacia la integración y participación ciudadana.

Acertadamente se considera en el eje de experiencias y metodologías de ciencia ciudadana y participativa, dado que aplica un ciclo de monitoreo participativo pero situando la complejidad del contexto de Barrancabermeja, por ejemplo, al recurrir a asociaciones de pescadores, al diseñar diversos formatos de participación como encuentros de trabajo y divulgación y al considerar las prácticas y saberes de los participantes en las estrategias de captura de datos e implementación del monitoreo.

Pese a lo anterior, la complejidad del proceso de diagnóstico, monitoreo, sistematización y análisis se vuelve confusa e inconexa en la presentación formal. Algunas decisiones y procedimientos se infieren, pero no se hacen explícitos, por ejemplo, la elección de un único punto de monitoreo para aguas de consumo; asimismo, estrategias valiosas que se llevaron a cabo de forma adicional aunque no eran centrales en el proyecto, por ejemplo, la realización del taller sobre tratamiento de aguas a nivel domiciliario. Una presentación más cuidadosa podría dar mayor claridad sobre el trabajo realizado.

A lo anterior podría contribuir también modificar algunos recursos de imagen, con la finalidad de ilustrar con claridad lo deseado; por ejemplo, los registros fotográficos, los cuales son pequeños y no permiten aprehender el trabajo de campo que se realizó. De igual forma, algo que podría contribuir a la claridad del trabajo es agregar hipervínculos a recursos como es la libreta de campo; sería enriquecedor conocer detalles como el instrumento mismo de monitoreo del que hicieron uso las vecinas y vecinos de la comunidad. En ambos casos, manejo de imagen y uso de hipervínculos, cabe considerar que puede no tratarse de una decisión de las autoras, sino de las posibilidades mismas del formato de exposición requerido (PDF).

Finalmente, se sugiere enfatizar los resultados obtenidos, justamente, por tratarse de un ejemplo de ciencia ciudadana que derive en un artículo que se oriente hacia la Ciencia Abierta y permita, entre otros aspectos, la replicabilidad. Se menciona muy brevemente, por ejemplo, que algunos datos generados se hicieron públicos en GBIF y SiB de Colombia.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor/a #1

El manuscrito es una contribución importante; no tengo duda. Sin embargo, su aportación no la sitúo en los “hallazgos” o “propuestas de política”, sino como una propuesta metodológica (con evidencias de pasos y procesos muy claros) para la investigación participativa con comunidades, en específico las cuestiones ambientales.

Me parece un acierto el uso de los elementos gráficos que organizan la información y reducen la complejidad. Cuando leía el documento, consideraba que no sólo era la narrativa de una investigación participativa con la comunidad local, sino que mostraba la experiencia de forma tal que se convertía en un insumo importante para hacer este tipo de investigación y, sobre todo, empezar a concretar eso llamado ciencia ciudadana o vinculación de la investigación con las necesidades locales. Un gran ejemplo de hacia dónde deben girar las investigaciones de la región.

Me hubiera gustado y no lo encontré, links a los documentos; por ejemplo, a la guía de campo -sería de gran ayuda-, así como ligas donde la experiencia se registró a mucho mayor profundidad de como se muestra, a los datos, la transcripción de las entrevistas semiestructuradas, etc.

Evidentemente, todo lo que comento no es parte de un artículo, pero los links o las ligas a los materiales (no sólo a los datos), permitirían enriquecer el trabajo.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisora:Karen Soacha

Como artículo de investigación principalmente descriptivo contribuye a documentar y visibilizar los impactos de las iniciativas de ciencia ciudadana. Está bien estructurado y redactado. Tiene relevancia los datos que comparte sobre las contribuciones al conocimiento de la biodiversidad urbana. Como artículo de investigación sería necesario profundizar en al menos uno de los aspectos que componen el desarrollo de la actividad. El reto ciudad naturaleza de La Paz se puede analizar y documentar en principio desde dos componentes. El componente de conocimiento de la biodiversidad (aporte a la ciencia) y el de participación e involucramiento de la ciudadanía (impactos en las personas que participaron tanto registrando, como organizando y validando información). Sería importante que se profundice en al menos uno de ellos dentro del artículo, incluyendo un análisis más detallado sobre los datos que se recolectaron como distribución por grupos biológicos, distribución geográfica, vacíos, niveles de identificación, para mencionar algunos aspectos. Si bien algunos de ellos se mencionan de forma general no se detallan en el documento.

El artículo tiene una metodología general bien definida, sin embargo, tienen un margen importante de mejora. Algunas de mis sugerencias a continuación:

1) Crear una introducción sobre la metodología que se sigue a nivel global y que es definida por los organizadores del CNC.

2) Incluir una sección sobre la fase de planeación del alcance del CNC en La Paz. En la que se incluya la parte del área de estudio. Sería ideal hacer una aclaración sobre lo que se entiende como territorio urbano para este ejercicio, dado que se menciona que es un concurso enfocado en territorios urbanos y se hace mención explícita a la inclusión de áreas protegidas y parques naturales. Que si bien hacen parte de lo que se entiende como ciudad de forma integral es necesario aclararlo.

3) Mencionaría también en la metodología que es la herramienta iNaturalist, aspectos básicos de la plataforma y la gestión de los datos y que al final los datos son abiertos y están disponibles para ser descargados y utilizados. Dado que es un artículo con un fuerte componente divulgativo es importante que se definan estos componentes.

4) Incluiría una sección de ética y buenas prácticas seguidas durante la implementación del CNC en La Paz, como por ejemplo que prácticas se implementaron o recomendaron para gestionar el impacto ambiental de la visitas a espacios naturales y especialmente dado que mencionan la visita de 100 personas en un día en un espacio natural ¿Se tuvo en cuenta este tema durante las capacitaciones?

5) ¿Se midió de alguna forma el impacto en las personas que participaron en el CNC? especialmente dado que la mayoría fue población estudiantil ¿se hizo alguna encuesta o se midió de alguna forma incrementos en conocimiento de biodiversidad o similar? Esto puede ser a futuro materia de otro articulo relacionado con la experiencia.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor/a #1

El artículo detalla la experiencia desarrollada con grupos de ciudadanos en una región específica de la Paz (Bolívia), de manera comparativa muestra el aumento de especies identificadas durante los años 2019 y 2022, el número de participantes involucrados en los procesos de participación y la contribución taxonómica en especies endémicas y en vía de extinción.

Es relevante dar a conocer el proceso de gestación, desarrollo y producción final de la experiencia, la cual contribuye a destacar el rol que puede aportar la participación ciudadana y su interacción con herramientas tecnológicas.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor/a #2

Es un documento muy interesante, claro y pertinente que aborda la Ciencia Abierta desde una perspectiva de materialización, describiendo la experiencia real de una Universidad que ha desarrollado diversos esfuerzos orientados a hacer realidad este tema dentro de sus dinámicas institucionales. El documento aborda la experiencia y el proceso de la institución de manera concreta, presentando sus avances, pero además como estos no han sido aislados, y han estado conectados con el camino de la Ciencia Abierta en el mundo y en su región. El contenido en general y sus conclusiones aportan al avance de la Ciencia Abierta, siendo de gran utilidad para instituciones que están recorriendo este camino, y en general para toda la comunidad interesada en poner en práctica estos principios en su entorno. ¡Muy buen trabajo!

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisora: Malgorzata Lisowska

El artículo presenta avances reales de una institución de educación superior, respecto a la implementación de la estrategia institucional de ciencia abierta, materializando de esta manera las recomendaciones de la UNESCO.

Es un trabajo completo, claro y conciso, de lectura obligatoria para aquellas instituciones que quieren empezar o ya están caminando por los senderos de innovación abierta. Felicitaciones a los autores!

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor: Gabriel Vélez Cuartas

El trabajo presenta un desarrollo de una plataforma para el procesamiento de datos y presentación de indicadores cienciométricos y altimétricos más comunes. Así mismo presenta una validación en la selección de bases de datos para integrar al sistema. Las tecnologías empleadas les permiten un desarrollo estandarizado de protocolos de comunicación que integran datos importantes para la plataforma de OJS, asunto necesario para apoyar los procesos de desarrollo editorial de las revistas científicas latinoamericanas.

Es un desarrollo muy potente que podría generar una herramienta muy útil para revistas que no necesariamente están indexadas en WoS y Scopus.

Es importante anotar algunos aspectos que podrían mejorarse:

1. La plataforma se propone como una herramienta editorial que seguramente enriquecerá de manera importante los metadatos de las publicaciones OJS. Sin embargo, es importante, con miras a un mejoramiento de la gestión editorial una reflexión más profunda sobre las métricas. Hay abundante literatura y discusión sobre el uso de métricas e indicadores para la evaluación científica, documentos críticos y propuestas que permitan discernir de mejor forma sobre los instrumentos métricos a emplear. En este sentido sería necesario hacer un breve repaso por los principales problemas contemporáneos de las métricas y hacer una relación con el trabajo de gestión editorial para poder hacer un discernimiento sobre lo que debería seleccionarse o no como indicador para estos procesos. No basta con mencionar que existen estos indicadores, habría que evaluarlos de alguna manera parecida como se hizo la selección de las bases de datos a considerar para este desarrollo tecnológico.

2. Tal vez podría ser importante anexar fichas de indicadores que permitan no sólo entender el uso de estos sino también las fuentes de las que se nutren de manera más precisa.

3. Sería importante citar algunas experiencias similares que presten servicios similares a la gestión editorial. Esto permitiría tener un contexto más amplio de los alcances de una herramienta como la propuesta en comparación con otras ofertas comerciales y no comerciales.

4. Debido a las características del OJS y su multidiversidad en formas de indexación, lo que lleva a que muchos artículos no tengan DOI y muchos de los autores no tengan ORCID, no hay expresado un estimativo de los datos no capturados o procesados en la identificación tanto de objetos como de autores? Se presentan los resultados positivos, pero no los negativos lo que permitiría establecer el sesgo de la información entregada. Esto es importante, pues siendo un principio estadístico bastante importante (el reconocimiento del error), en cienciometría se sigue trabajando con los datos posibles de encontrar y se sacan conclusiones importantes sobre su desempeño y no a partir del cálculo del error o el sesgo, que permitirían una información más confiable a los editores en sus procesos de gestión, de otra forma estarían caminando a ciegas en datos de los cuales no se conoce su sesgo.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor: Alejandro Uribe Tirado

Es una propuesta interesante, pero no es claro si por asuntos de forma (límite de extensión del texto o?) no se desarrollan aspectos claves del mismo en lo metodológico, los resultados y su aporte a la comunicación científica, las métricas y los distintos agentes que daría esta integración de herramientas-arquitectura, además del qué seguiría? para que así, quede claro para los lectores, se debe, por tanto, mejorar el texto para ser publicado.

Se hacen varios comentarios de forma-fondo a continuación:

• Unificar en forma o fondo el objetivo del trabajo, pues no se presenta en el texto de la misma manera en varios apartados (Resumen, Arquitectura propuesta)
• Se usa más la palabra altmetrics o altmetría que altimetría
• En las tablas 2 y 3, se hace la comparación y se elige una opción, en la 4 y 5, se presentan, luego se comenta, pero no se elige, y eso genera inquietud, considerando la lógica que lleva el texto al seleccionar ORCID y DOI en los puntos anteriores. ¿Por qué? faltó? o fue intencionado por...?
• No es clara del todo, o debería haber otra(s) razón(es) para esta elección?(Esto debería estar antes en el texto, por lo indicado en un punto anterior):: Analizando las alternativas que se tienen para la recolección de información por parte de las revistas para determinar indicadores bibliométricos, se optó por vincular las revistas disponibles en OJS, por la disponibilidad que tienen de los datos por medio del protocolo OAI-PMH. Dicho protocolo recibe peticiones por medio de la URL obteniendo los datos registrados por las revistas para cada artículo publicado. Por otra parte, como estándar de metadatos se seleccionó Dublin Core, al ser un estándar ampliamente reconocido por los elementos que lo componen, que son la instanciación del recurso con su identificador y la información del recurso

• En la parte explicativa (párrafos) tras la figura 1. Diseño de arquitectura propuesta, se identifica un problema significativo: Este apartado debe ser más amplio, más explicado, indicar el por qué y cómo de estos componentes de la arquitectura y cómo funcionan... hay una narrativa más paso a paso al inicio, pero luego hay un gran salto, y se elige el OJS y de allí se salta a este gráfico, y no se explica mucho ni el por qué y su utilidad (para editores o tomadores de decisión desde la integración de fuentes-métricas-indicadores que se pretende) y este sería el propósito del texto, su mayor aporte…

• Finalmente, en las conclusiones:

Ampliar la información de la aplicación de este piloto-prototipo para los 162 artículos indicados (el cómo, y su utilidad...)

No es claro el qué (qué incluye, qué suma y cómo se llegó a...) en lo que se indica de puntaje?

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisor/a #2

Sin dudas, el manuscrito aborda un tema de suma actualidad como es la promoción del acceso abierto y la ciencia abierta por parte de diversas instituciones. En este caso, el texto se centra en la descripción de las acciones llevadas a cabo por el Instituto Brasileño de Información en Ciencia y Tecnología (Ibict), para poner en marcha iniciativas relacionadas con el tema. Resulta interesante conocer estas iniciativas y observar el rol que el Ibict ha tenido en su promoción.

En mi opinión, más allá del interés por la temática, se trata de un texto informativo que describe una serie de proyectos en los que ha participado la institución, pero no se adecua a los requisitos de un artículo científico. En este sentido, se observa que la introducción resulta adecuada en cuanto a información histórica sobre la institución pero no se aporta -como es de esperar en un artículo de investigación- información sobre otros estudios similares ni se presenta un “estado de la cuestión” que permita conocer iniciativas similares. Por otro lado, se echa en falta una explicación metodológica amplia en la que se detalle cuáles son las fuentes seleccionadas para recoger información, con qué criterio se eligieron, cómo se ha delimitado el concepto de “ciencia abierta” y “acceso abierto” para la recogida de datos. Asimismo, sería fundamental explicar qué procedimiento se ha seguido para analizar los proyectos/acciones presentados. Al no contar con esta información, el texto pasa a convertirse en un “informe” descriptivo de una serie de acciones realizadas. En la misma línea, en el apartado de resultados se ofrece una descripción de las 14 acciones en las que participa el Ibict pero no aparece un análisis ni una interpretación al respecto. En la sección de conclusiones se hacen unos comentarios finales pero no se ponen en común los resultados obtenidos, ni se aporta información comparativa con la acciones realizadas en otros contextos.

Por todo lo expuesto considero que se trata de un texto de interés para conocer la actividad del Ibicit y que cumple su función como documento informativo o divulgativo. Sin embargo, en mi opinión, en su estado actual, el texto no cuenta con los requisitos necesarios para ser considerado como artículo científico. Para transformarlo en un artículo de investigación me permito hacer algunas sugerencia a los autores:

• plantear una hipótesis a contrastar o validar
• proponer objetivos no descriptivos
• definir una metodología que permita alcanzar los objetivos propuestos y que sea reproducible (indicando y justificando qué fuentes se utilizan, por qué se han seleccionado, qué aportan al estudio, con qué criterio se selecciona la información, etc).
• proponer métodos de análisis (cuantitativos y/o cualitativos) novedosos y exhaustivos.
• incluir interpretaciones profundas de los resultados obtenidos (comparar los resultados, ponerlos en contexto con otras acciones, comprobar si validan o refutan la hipótesis inicial, etc)
• plantear una discusión en la que se mencione cuál es la aportación del trabajo para otros autores de la comunidad académica (¿qué ofrece este trabajo metodológica o conceptualmente a otros investigadores del área?). Al tratarse de un estudio de caso, es importante tener en cuenta esta observación.

Revisión completada como parte de la evaluación de trabajos postulados al Congreso Iberoamericano de Ciencia Abierta

Revisora: Iratxe Puebla

El manuscrito proporciona una descripción concisa de las actividades del Instituto Brasileño de Información en Ciencia y Tecnología desde 2005 en apoyo del acceso abierto y la ciencia abierta. Para encajar como un manuscrito de investigación, creo que el artículo debe proporcionar más detalles sobre la metodología que se utilizó, reconsiderar cómo se presentan los resultados y ubicar mejor la información en el contexto del ecosistema de ciencia abierta en Brasil. Si se prefiere no elaborar en la metodología, recomiendo re-enfocar el manuscrito como un artículo de revisión y ampliar el contexto sobre dónde encaja este instituto en el ecosistema institucional en torno a la ciencia abierta en Brasil y en Iberoamérica en general.

Comentarios mayores

• Introducción - Recomiendo proporcionar más contexto acerca del instituto ¿dónde encaja en la infraestructura y los marcos nacionales de investigación o información?, ¿Qué actividades ha realizado desde su creación hasta 2005?
• Metodología - En esta sección se enumeran diferentes tipos de enfoques de investigación cualitativa, ¿podrían por favor clarificar qué metodología en particular se utilizó para este estudio? ¿Podrían indicar también la estrategia de búsqueda para identificar documentos, las bases de datos empleadas y qué palabras clave se emplearon para las búsquedas?
• Resultados - Aquí o en la sección Metodología, ¿podrían por favor indicar cuantos documentos se incluyeron/utilizaron (y cuantos se excluyeron), y de qué fuentes?
• Resultados - Para los diferentes repositorios y/o servidores, ¿podrían por favor indicar si están disponibles sólo para investigadore/as brasilen@s y si otr@s pueden también depositar sus trabajos? Hay requerimientos dentro de políticas institucionales o nacionales para que l@s investigadore/as brasileños depositen sus trabajos en estas plataformas?
• Resultados - Algunas de las plataformas descritas parecen tener enfoques superpuestos, por ejemplo, el Repositorio Institucional del Ibict (RIDI) también incluye tesis, que son el enfoque de la Biblioteca Digital Brasileña de Tesis y Disertaciones (BDTD). Recomiendo proporcionar contexto sobre la necesidad de las diferentes plataformas, cómo son complementarias y las necesidades específicas que atiende cada una, en particular aquellas creadas en los últimos años en relación a otras ya existentes.
• Resultados - Con respecto a las diferentes iniciativas, me pregunto si en lugar de proporcionar un resumen cronológico anclado en los manifiestos, sería más informativo enumerarlas según áreas específicas de ciencia abierta, es decir, acceso abierto, datos abiertos, políticas abiertas etc. Esto podría dar una visión general más clara de dónde encaja cada actividad dentro del ecosistema de ciencia abierta y cómo las diferentes actividades/plataformas se complementan dentro de esas áreas.
• Tabla 1- La tabla actual no me parece muy informativa, ya que solo resume la lista de plataformas o actividades ya cubiertas en el texto. En lugar de la tabla actual, recomiendo incluir una tabla que enumere las diferentes plataformas y actividades dando más detalles sobre cada una:
• Para las plataformas de datos/tesis/artículos: información en cuantos trabajos están depositados, si las plataformas asignan identificadores permanentes (por ejemplo DOIs), si están indexadas y dónde, qué licencia(s) se aplican a los trabajos depositados, cuantas instituciones participan (si aplicable).
• Para otras actividades como las conferencias se puede indicar cuantas organizaciones participantes y asistentes han tenido.
• Para todas las actividades descritas se podría mapear su enfoque a las recomendaciones de la UNESCO, esto es, indicar qué áreas de la ciencia abierta cubren.
• Resultados - No conocía el servidor Emerging Research Information (EmeRI), podrían dar más detalles sobre este servidor, ¿pueden los autores depositar sus manuscritos directamente o los preprints se canalizan a través de colaboraciones con revistas que envían manuscritos bajo consideración? ¿Cuántas preprints alberga actualmente?
• Conclusiones - Agradecería una ampliación de la sección de conclusiones (o incluir una sección de discusión) para cubrir brevemente cuál ha sido el impacto de las diferentes actividades, por ejemplo, ¿está aumentando la deposición de datos y tesis? ¿Cómo ha evolucionado el número de artículos disponibles en acceso abierto en Brasil desde 2005? Creo que también puede valer la pena agregar algo de discusión sobre cómo las iniciativas del Ibict se comparan con las de otros institutos en Brasil o Iberoamérica.

Sugerencias menores

• Introducción - recomiendo hacer mención en la introducción a las Recomendaciones sobre Ciencia Abierta de la UNESCO, sus líneas principales y qué pasos se están dando en Brasil, como país firmante, para implementar las recomendaciones.
• Metodologia - ‘Especificados os procedimentos metodológicos, na próxima seção se discorre sobre os resultados, com vistas ao cumprimento do objetivo estabelecido para a pesquisa.’ Este fragmento está en portugués y no en español.
• Las notas al pie del manuscrito también están en portugués.
• Conclusiones ‘resaltando a Brasil en la perspectiva internacional’ - pueden elaborar en como se ha resaltado el país a través de las actividades, dando ejemplos del impacto de las actividades y si es posible incluyendo referencias.

Avaliação em grupo ASAPbio-SciELO Preprints

Esta avaliação reflete contribuições de Kamyla Pedrosa, Iratxe Puebla, Mario Cézar de Oliveira. Síntese por Adeilton Brandão.

O artigo apresenta um trabalho que, através da identificação de podcasts educacionais abordando a pandemia da covid-19, teve como objetivo sugerir o uso do podcast como metodologia complementar no processo de ensino-aprendizagem. Os autores concluem que o uso educacional de podcasts amplia as características do processo de ensino-aprendizagem e torna o processo pedagógico plural.

Sugestões dos revisores:

1. [Resumo] Recomenda-se alterar o resumo para que o mesmo expresse de forma clara os resultados obtidos no desenvolvimento do trabalho.

2. [Introdução] "Ensino Remoto em tempos de Pandemia": Pode valer a pena mencionar se/como a educação se adaptou em outras partes do mundo (e citando referências relevantes sobre isso).

3. [Introdução] "Podcast como ferramenta de aprendizagem e ensino inclusivo":

"...o que é percebido nas escolas...": A frase soa subjetiva, a menos que seja baseado em uma pesquisa sobre visões e percepções. Sugere-se uma reformulação para indicar 'o que é implementado nas escolas' ou algo nesse sentido.

"...que apresentaram um bom aproveitamento diante da utilização de podcast como ferramenta de aprendizagem...": Esses resultados se referem ao acesso, a facilidade com a tecnologia ou estão relacionados a resultados de aprendizagem? Isso pode ser especificado?

1. [Aspectos Metodológicos] "...submetidos a uma análise duplo-cego...": Não ficou claro como se pode concluir uma análise duplo-cega do podcast: ao ouvi-lo é possivel saber qual pessoa ou organização o administra? Seria interessante fornecer mais esclarecimentos sobre o que se entende por uma análise duplo-cego.

2. [Aspectos Metodológicos] Tabela 2: no texto é indicado que podcasts em idiomas que não fosse português foram excluídos. Sugerimos um esclarecimento sobre a inclusão de 'Idioma' na taxonomia, algo relacionado a clareza da linguagem...

3. [Aspectos Metodológicos] "... fidedignos...": forneça mais informações sobre como isso foi verificado.

4. [Aspectos Metodológicos] "...não que contemplassem os critérios de inclusão...": Sugere-se que os critérios de inclusão sejam descritos mais claramente nesta seção. Por exemplo, o nível educacional que os podcasts deveriam ter como alvo - ensino médio, graduação, pós-graduação; Uso por professores ou alunos, ou ambos.

5. [Resultados] Tabela 3: Há informações sobre se e como esses podcasts foram usados como parte dos cursos durante o período de estudo? Estas informações podem ser relevantes para apoiar o uso destes podcasts como material educativo.

6. [Discussão] Nesta seção as limitações do estudo devem ser discutidas, por exemplo:

a) o estudo não coletou dados sobre o uso real dos podcasts por alunos ou professores e, portanto, não pode estabelecer se os podcasts foram realmente usados na educação, por quem ou para quais propósitos.

b) também não há informações sobre os resultados reais de aprendizagem do uso do podcast, portanto, sua eficácia como ferramenta de ensino não pode ser estabelecida. Se houver estudos em outros lugares que estudaram a eficácia dos podcasts para resultados de aprendizagem, inclua uma discussão desses estudos.

1. [Discussão] "...mostrando as aplicações de podcasts em salas de aula...": Não está claro se as informações fornecem evidências do uso dos podcasts em salas de aula. Há informações de que as contagens de acesso vêm de alunos/escolas que recomendaram os podcasts?

2. [Discussão] Tabela 4: Não está claro se existe ligação entre esta lista de podcasts e as principais questões de pesquisa, pois não estão relacionadas a doenças infecciosas ou COVID-19.

3. [Discussão] "fake news": apresentar o conceito deste termo.

4. [Discussão] "os mais engajados no consumo de podcasts são pessoas com idades entre 20 a 34 anos": Forneça uma referência para esta declaração.

5. [Discussão] "...Visto que os jovens apresentaram-se como os maiores consumidores de podcasts nos últimos anos, quando comparado com outros públicos...": A afirmação anterior diz que o grupo mais engajado com podcasts são aqueles de 20 a 34 anos, isso pode ser esclarecido em relação a este fragmento?

6. [Discussão] "Não obstante, há indicação positiva de que a utilização dos podcasts pode auxiliar na inclusão de alunos com diferentes singularidades, como alunos cegos e com Síndrome de Down." Forneça uma referência para esta.

7. [Conclusão]

8. Recomenda-se enfatizar o objetivo da pesquisa "identificar podcasts voltados para a área educacional, disponibilizados em plataformas digitais". E também, enfatizar os resultados com as inferências.
9. "Uma vez que o docente pode utilizar...": esta conclusão não é suportada pelos resultados. O estudo não reuniu a opinião dos alunos sobre os podcasts, ou como eles os avaliam em comparação com as metodologias tradicionais de ensino.
10. "Além disso, a utilização desta ferramenta possibilita o planejamento de um ensino mais inclusivo...": este fragmento repete o último trecho da Discussão, e não é uma das conclusões do estudo, pois o uso dos podcasts por essas populações não foi avaliado.
11. "Uma vez que o docente pode utilizar as informações..."; "O interesse que os jovens possuem por tecnologias reforçam as automotivações…”: O estudo não fornece dados sobre a utilização real por parte dos professores, ou interesse/motivação dos alunos. Estes fragmentos podem ser melhor colocados na secção de Discussão, e não apresentados como conclusão do estudo.

Avaliação em grupo ASAPbio-SciELO Preprints

Esta avaliação reflete contribuições de Helvecio Cardoso Correa Povoa, Mariana de Almeida Rosa Rezende, Iratxe Puebla e Luciane Ferreira do Val . Síntese por Vanessa Bortoluzzi.

Neste estudo, os autores avaliaram, através de questionário, o conhecimento acerca (e prescrição) da Profilaxia Pré-Exposição (PrEP) à Síndrome da Imunodeficiência Adquirida (SIDA) em uma população de médicos do Estado de Goiás com inscrição ativa no Conselho Regional de Medicina do Estado, concluindo que este grupo tem alto nível de conhecimento prévio acerca da PrEP. Este trabalho pode ser relevante na elaboração de formas de se avaliar o conhecimento de profissionais da saúde no que diz respeito à PrEP, visando a capacitação desses profissionais para aplicação dessa importante estratégia de prevenção da SIDA. A seguir, as sugestões dos revisores:

[GERAL] Ao longo do texto, os autores se referem à Síndrome da Imunodeficiência Adquirida (SIDA) e acquired immunodeficiency syndrome (AIDS). É necessária a padronização do termo e concisão no seu uso. Além disso, a "síndrome da imunodeficiência adquirida" hora é chamada de infecção, hora de doença. Sugere-se rever as definições e revisão geral do texto.

[RESUMO] Na passagem “os resultados dessa pesquisa não estão em consonância com a hipótese de que apenas 20% dos médicos/as teriam conhecimento suficiente,” deixe claro de onde essa taxa de conhecimento se origina. Esse resultado foi obtido em população comparável à abordada neste estudo?

[INTRODUÇÃO] Sugere-se manter o termo “populações mais vulneráveis” ao invés de “grupos de risco,” visto o conceito de vulnerabilidade ter nascido justamente da pandemia do HIV/AIDS. Na perspectiva da vulnerabilidade, a exposição a agravos de saúde resulta de aspectos individuais e de contextos ou condições coletivas que produzem maior suscetibilidade a estes agravos e morte e, simultaneamente, menor acesso aos recursos para o seu enfrentamento. Dessa forma, para a interpretação do processo saúde-doença, considera-se que o risco indica probabilidades e a vulnerabilidade é um indicador da iniquidade e desigualdade social. A vulnerabilidade antecede ao risco e determina os diferentes riscos de se infectar, adoecer e morrer.

Bertolozzi, Maria Rita et al. Os conceitos de vulnerabilidade e adesão na Saúde Coletiva. Revista da Escola de Enfermagem da USP [online]. 2009, v. 43, n. spe2 [Acessado 22 Junho 2022] , pp. 1326-1330. Disponível em: https://doi.org/10.1590/S0080-62342009000600031. Epub 07 Abr 2010. ISSN 1980-220X. https://doi.org/10.1590/S0080-62342009000600031.

[INTRODUÇÃO] Em relação ao estudo realizado por Lucas Cardoso da Silva, os profissionais que fizeram a autodeclaração de conhecimento foram os mesmos avaliados quanto ao conhecimento sobre a PrEP?

[INTRODUÇÃO] Seria relevante discutir na já na Introdução a razão para um nível de conhecimento básico de 20%. Por que esse corte? É baseado em relatos da literatura? Em caso afirmativo, eles devem ser citados e discutidos. Apesar de ser mencionado após Métodos, é necessário delinear estes aspectos antes de declarar a hipótese.

[MÉTODOS] O trabalho de Christopher Turndrup et al. foi conduzido em uma população dos Estados Unidos; podemos assumir o mesmo nível de conhecimento nos EUA e no Brasil? Alguma justificativa para isso pode ser fornecida?

[MÉTODOS] Em relação ao estudo de Lucas Cardoso da Silva, seria relevante discutir como a população deste estudo se relaciona/compara com a pesquisada. Existem outros estudos relevantes no Brasil? A taxa de conhecimento da linha de base pode precisar de mais justificativas além de um único estudo no Brasil.

[MÉTODOS] Quantos médicos foram abordados, e qual a taxa de resposta obtida a partir dos convites enviados? Além disso, informe as datas em que o questionário foi enviado, por quanto tempo ficou aberto para respostas e se lembretes foram enviados. Também seria interessante deixar mais claro os critérios de seleção dos médicos. Todos os médicos com inscrição ativa e informações no domínio público foram convidados?

[MÉTODOS] Quanto ao questionário empregado, o mesmo foi validado em português?

[RESULTADOS] Sobre os resultados apresentados na Tabela 1, há informações se essa distribuição demográfica se correlaciona ou não com as características dos médicos da região ou do Brasil? Em caso afirmativo, seria relevante abordar essa perspectiva na Discussão.

[RESULTADOS] Na Tabela 3, a última pergunta está enquadrada de forma negativa e pode ter sido confusa/incerta para os entrevistados. É relevante abordar esse viés na Discussão.

[DISCUSSÃO] O estudo de Malika Sharma et al., no qual “45,9% dos participantes se sentiram ‘muito familiarizados’ com a PrEP e 45,4% dos entrevistados estavam dispostos a prescrevê-la,” foi concluído em 2013; é possível que o conhecimento sobre esta opção de tratamento tenha aumentado ao longo do tempo. Por favor, inclua referências mais recentes.

[DISCUSSÃO] Considerando que os resultados obtidos não estão em consonância com o outro estudo mencionado (Lucas Cardoso da Silva), realizado em Porto Alegre, é relevante comentar brevemente as razões da discrepância.

[DISCUSSÃO] Por favor, elabore as limitações do trabalho:

• comente sobre a taxa de resposta à pesquisa e se isso pode ter tido um impacto nos dados obtidos;
• discorra acerca do risco de autosseleção entre os entrevistados, ou seja, aqueles mais familiarizados com a PrEP podem ter sido mais propensos a concordar em participar;
• se o questionário não foi validado em português, indique isso também como limitação.

Avaliação em grupo ASAPbio-SciELO Preprints

Esta avaliação reflete contribuições de Carla Maria de Jesus Silva, Helvecio Cardoso Correa Povoa, Iratxe Puebla, Kamyla de Arruda Pedrosa, Marcos Roberto Furlan e Mariana de Almeida Rosa Rezende. Síntese por João Victor Cabral-Costa.

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Neste trabalho, Custódio e Freitas realizaram uma análise retrospectiva baseada na extração de dados do Benefício de Prestação Continuada (BPC) para a verificação da possibilidade de uma influência da pandemia sobre a mortalidade de idosos entre os anos de 2020 e 2021. Em uma extensiva análise demográfica, as autoras expuseram seus resultados acompanhados de uma completa descrição metodológica do processo de extração e tratamento dos dados, contribuindo para a reprodutibilidade de estudos análogos. Abaixo, elencamos comentários dos revisores sobre o estudo que poderiam fortalecê-lo e enriquecer o trabalho.

1. Seria interessante a condução de uma revisão ortográfica e de escrita para enriquecer a coesão textual. Sugerimos, por exemplo, evitar o uso de “etc.” ao elencar exemplos e informações, a fim de se evitar a finalização de construções textuais de forma aberta.
2. É frequente, no decorrer de todo o texto, o apontamento de informações/conclusões/resultados alheios sem a referenciação a uma citação específica. Do ponto de vista metodológico, toda informação parafraseada de um artigo deve ser referenciada a fim de se evitar questionamentos acerca da autoria da ideia/conceito. Por exemplo, em:

a) “Esses números representam um elevado valor no orçamento público anual, da ordem de 62 bilhões de reais, em 2021”

b) “em cumprimento ao Decreto nº  3.266, de 29 de novembro de 1999”

c)  “Os dados demográficos têm demonstrado (...) causas naturais).”

d) “a probabilidade de morte de uma pessoa do BPC-Idoso com 92 anos deve necessariamente (...) da população nacional”

e) “por si só, pelo fator idade, independentemente da incidência ou não de uma pandemia”

f)  "A covid-19 deixa marcas e seus impactos no médio e no longo prazo ainda não são totalmente conhecidos”

1. [RESUMO] Sugerimos incluir a faixa etária descrita nos critérios de seleção da pesquisa (Metodologia).
2. [RESUMO] “Os resultados confirmam que houve um aumento significativo da taxa de cessação” Sugerimos indicar o aumento real da taxa de cessação por óbito neste ponto do resumo.
3. [RESUMO] “Destaca-se a relevância de se estudar um dos maiores programas assistenciais do país, que possui orçamento anual da ordem de R$ 62 bilhões” Considerando que esta informação, per se, não constituiu uma conclusão do estudo em si, sugerimos a remoção deste trecho.
4. [INTRODUÇÃO] “Em termos históricos, a Mantidos possui registros sobre 8,24 milhões de pessoas, considerando ambas as espécies do BPC, para pessoas com deficiência e idosos” Existem dados disponíveis sobre o número de idosos com deficiência ou todos os beneficiários ativos com deficiência aqui referidos são também parte do grupo dos idosos?
5. [INTRODUÇÃO] Seria útil fornecer um contexto na introdução, antes de introduzir o escopo da análise, sobre a justificativa da relevância do foco na taxa de mortalidade e os motivos pelos quais uma alteração seria esperada.
6. [INTRODUÇÃO] “Como objetivos específicos, são apresentados dados quantitativos históricos dos benefícios do BPC-Idoso” Seria interessante ampliar este trecho, apresentando a ligação entre esses objetivos específicos e o objetivo principal, sobre a taxa de mortalidade.
7. [REVISÃO BIBLIOGRÁFICA] “recorte parcial da população brasileira, como são os idosos que  recebem o BPC” Existe alguma informação/estudo sobre a cobertura populacional do BPC, ou seja, alguma referência sobre a representatividade dos inscritos no programa, incluindo possíveis indivíduos que deveriam ser cobertos porém que não se encontram inscritos (e.g., falta de contato, questões administrativas, etc.).
8. [REVISÃO BIBLIOGRÁFICA] “entre as quais estão a logística, a curva de Gompertz” Sugerimos a seguinte construção: “dentre as quais estão, por exemplo, a logística e a curva de Gompertz”.
9. [REVISÃO BIBLIOGRÁFICA] “Os dados demográficos têm demonstrado (...) causas naturais).” Sugerimos a reconsideração sobre a necessidade deste excerto, uma vez que discute tendências demográficas gerais, e não o recorte dos resultados específicos de interesse do estudo.
10. [METODOLOGIA] Em essência, o manuscrito procedeu com uma ampla comparação de efeitos sexo-específicos. Seria interessante, pois, incluir uma justificativa no texto (Introdução, Revisão Bibliográfica e/ou Metodologia) sobre as razões/importância em se proceder com a análise de tal maneira – pode-se, inclusive, adicionar exemplos de efeitos análogos descritos na literatura, como forma de ressaltar a relevância desta forma de análise.
11. [METODOLOGIA] “As sintaxes de geração das três tabelas utilizadas para os cálculos das taxas de mortalidade estão no anexo II” Ressaltamos aqui a importância destas informações para a reprodutibilidade do estudo – um excelente recurso disponibilizado pelas autoras deste estudo.
12. [RESULTADOS] Gráficos 9 e 10 – Talvez fosse interessante também apresentar um gráfico contendo a taxa bruta de mortalidade total, incluindo toda a população (masculino + feminino).
13. [RESULTADOS] Gráficos 9 e 10 – Por conter um compilado de 17 séries em um mesmo gráfico, a visualização plena dos resultados é limitada. Há alguma alternativa para este formato de apresentação de dados (seja um formato de gráfico alternativo ou alguma forma secundária de comparar as curvas)? Uma ampliação do tamanho do eixo Y poderia já contribuir para uma menor sobreposição de algumas das curvas, por exemplo.  
14. [RESULTADOS] Gráficos 11 e 12 – Há alguma justificativa disponível para explicar o motivo pelo qual as taxas de cessação por óbito do ano de 2016 (ou seja, um ponto prévio à pandemia) sejam bem maiores, destoando dos outros anos?
15. [RESULTADOS] “por si só, pelo fator idade, __independentemente__ da incidência ou não de uma pandemia”

a) Sugerimos a substituição do trecho sublinhado por “de forma independente”.

b) Sugerimos referenciar dados baseados na literatura que corroborem com esta ideia. Seria importante discutir como os dados deste manuscrito se relacionam ou diferem (e, consequentemente, o porquê) dos dados de outros países, uma vez que estes resultados apontam uma tendência possivelmente diferente – não seria esperado que o impacto da COVID-19 na mortalidade aumentasse com a idade? E, inclusive, não seria essa uma das principais justificativas para a seleção deste recorte da população como grupo prioritário das primeiras campanhas de vacinação?

Este talvez seja um ponto crítico que requeira uma especial atenção das autoras, pois pode constituir um possível indicador de viés metodológico (e.g., potencial impacto da representatividade do recorte populacional dos inscritos no BPC em relação à população idosa em geral? Potencial variação nos mecanismos de dispersão/contaminação em relação a outros países, devido a fatores estruturais e sociais, como proporção de ILPIs?). Embora este ponto tenha sido brevemente comentado nas considerações finais, recomendamos que estes fatores (bem como outras possíveis implicações) sejam incluídos como comentários/discussões no texto a fim de fortalecer o trabalho. 

1. [RESULTADOS] “Uma, dentre outras explicações plausíveis para a ocorrência desse fenômeno, e que poderia ser objeto de novas pesquisas, seria a maior exposição de mulheres idosas à vida social, por questões culturais, o que as levaria a uma maior propensão a se contaminar pelo vírus” Sugerimos ampliar este trecho, explicitando a relação da taxa de infecção com o recorte populacional deste estudo. Inclusive, é possível que haja alguns fatores adicionais aqui, uma vez que dados publicados já no primeiro ano da pandemia apontaram para uma maior mortalidade por COVID-19 em homens (e.g., Abate et al. 2020 http://dx.doi.org/10.1136/bmjopen-2020-040129 e Peckham et al. 2020 https://doi.org/10.1038/s41467-020-19741-6)
2. [CONCLUSÃO] Sugerimos mover esta seção para o final do texto, após a discussão, possivelmente fundindo-se com os excertos considerados como “considerações finais”.
3. [DISCUSSÃO] “Lei Geral de Proteção de Dados Pessoais (__LGPC__,” Correção: LGPD.
4. [DISCUSSÃO] “Dados preliminares (não divulgados) indicam que a primeira hipótese é a que prevalece” Se esses dados não puderem ser disponibilizados, há alguma referência que possa ser fornecida para apoiar essa afirmação?
5. [DISCUSSÃO] “Corroborando com a hipótese, é de  se esperar (...) o que daria ensejo a novos estudos para verificar essa crença/esperança” Sugerimos que este trecho seja reescrito, referenciando e incluindo a discussão de estudos que relatam sobre a manutenção de óbitos pela COVID-19, colocando-a como uma doença endêmica.
6. [DISCUSSÃO] “A covid-19 deixa marcas e seus impactos no médio e no longo prazo ainda não são totalmente conhecidos” Há algum trabalho recente que tenha levantado/discutido essa perspectiva? Seria interessante abordar/referenciar esses possíveis impactos, para auxiliar na clarificação desse panorama em potencial.
7. [DISCUSSÃO] “Acredita-se que o recorte (...) acerca dos efeitos da pandemia ao longo do tempo” Sugerimos a reconsideração deste trecho – talvez seja interessante revisitá-lo e cogitar reescrevê-lo a fim de tornar mais clara a ideia que se deseja transmitir com este excerto.
8. [DISCUSSÃO] “Por fim, e em sede de conclusão, (...) visando a aprimorar os direitos sociais previstos na Constituição” Considerando que este não foi o foco principal do estudo, seria relevante ter como texto conclusivo um fragmento que foquem nos principais achados/conclusões sobre a mortalidade no contexto da pandemia (vide comentário #20).
9. [DISCUSSÃO] “tempos de big data” O termo ‘big data’ não foi introduzido no decorrer do texto. Recomendamos que o mesmo seja introduzido no preâmbulo do trabalho e, caso seja pertinente, discutido em algum ponto da Discussão.
10. [DISCUSSÃO] Recomendamos a inclusão de possíveis limitações do estudo a fim de fortalecer o trabalho, bem como enriquecer sua transparência. Por exemplo, vide comentários #9 e # 17b.

Avaliação em grupo ASAPbio-SciELO Preprints

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Esta avaliação reflete contribuições de Carla Maria de Jesus Silva, Helvecio Cardoso Correa Povoa, Iratxe Puebla, Kamyla de Arruda Pedrosa, Luciane Ferreira do Val. Síntese por João Victor Cabral-Costa.

Neste trabalho, Guimarães et al. fazem uma análise descritiva do excesso de mortalidade no primeiro ano de pandemia por COVID-19 (2020) em comparação com os 5 anos prévios (2015-2019). Trata-se de uma breve análise de dados extraídos do Sistema de Informações sobre Mortalidade (SIM) do Datasus, agrupados em: doenças infecciosas e parasitárias; neoplasias; doenças endócrinas; transtornos mentais; doenças cardiovasculares; doenças do aparelho respiratório; doenças do trato geniturinário; gravidez, parto e puerpério; causas externas; e causas mal definidas.

A análise tem um objetivo relevante, porém o manuscrito muito se beneficiaria de algumas alterações conforme os comentários levantados abaixo:

1. Seria de grande valia a condução de uma revisão ortográfica e de escrita para melhor conectar os parágrafos ou frases, deixando assim a leitura mais fluida, em especial para melhorar a organicidade da discussão;
2. A fim de dar maior clareza ao resumo, acrescentar uma breve introdução ao tema e ressaltar a importância do estudo;
3. Os autores usaram os descritores baseados nos termos controlados e não controlados (ou alternativos) do https://decs.bvsalud.org/, sendo um ponto positivo.
4. [INTRODUÇÃO] Recomendamos citar quais foram as des assistências dentro da rede assistencial do SUS (e.g., desassistência na atenção básica, atenção especializada, cirurgias, reabilitação, etc.), inserindo trabalhos que corroborem com essa informação;
5. [INTRODUÇÃO] Inserir um comentário com a justificativa da escolha do período “primeiro ano da pandemia (2020)” como foco de estudo do trabalho;
6. [INTRODUÇÃO] Seria interessante expandir um pouco a introdução de forma a possibilitar a discussão de alguns itens adicionais: a) Outros estudos sobre excesso de mortalidade no contexto da COVID-19, globalmente ou, pelo menos, no Brasil e/ou América do Sul. A introdução poderia então articular as lacunas da literatura no contexto de estudos prévios (ou comentar sobre a ausência de tais estudos abordando determinado tema com foco na região). b) Comentar sobre a organização do sistema de saúde no Brasil. A análise realizada por este trabalho apresenta dados em nível regional, contudo não há justificativa para tal escolha - por quê seria relevante conduzir esta avaliação em nível regional? Seria interessante discutir brevemente sobre potenciais diferenças no gerenciamento do sistema de saúde entre diferentes regiões/estados durante o primeiro ano de pandemia, bem como possíveis diferenças que já seriam esperadas entre estados.
7. [MÉTODOS] Detalhar o que é o Datasus e especificar melhor que os dados obtidos são de domínio público. Adicionar uma referência da base de dados, incluindo seu link de acesso (https://dados.gov.br/dataset/sistema-de-informacao-sobre-mortalidade).
8. [MÉTODOS] Esclarecer o motivo da seleção dos últimos cinco anos anteriores (2015-2019) como limite para a comparação prévia e comparando com o primeiro ano da pandemia (2020). Há algum motivo para a seleção de um período de 5 anos para a avaliação da tendência de crescimento de mortalidade? Se sim, seria útil incluir uma referência para justificar a escolha do corte temporal.
9. [MÉTODOS] Seria interessante que mais detalhes sobre a extração de dados do SIM/Datasus pudessem ser providenciados, incluindo a adoção (ou ausência de) possíveis filtros, fatores de agrupamento, fatores de exclusão, etc.
10. [MÉTODOS] “Optamos por fazer a tendência linear pelo reduzido número de pontos” Seria interessante justificar os argumentos que levaram a esta escolha metodológica (i.e., por qual razão, referência vantagem, etc., este método foi escolhido). Esse método foi empregado anteriormente para estudos de excesso de mortalidade relacionados ao COVID-19? Se sim, é possível fornecer uma referência?
11. [MÉTODOS] Detalhar nos Métodos quais doenças estão incluídas nos respectivos grupos, principalmente o referente às “Doenças Infecciosas e Parasitárias”, apontando quais doenças infecciosas são as principais causas de mortalidade. Adicionalmente, seria interessante esclarecer a definição do grupo “Causas mal definidas” e quais doenças ele historicamente inclui.
12. [RESULTADOS] Recomendamos uma padronização da forma com a qual os resultados numéricos são apresentados no decorrer do texto, i.e., (SMR ###, IC 95% ##-##) ou (SMR = ###, IC 95% ##-##), para melhor normatização.
13. [RESULTADOS] O termo “RT” não foi definido/descrito por extenso em primeira aparição no texto e/ou nos Métodos.
14. [RESULTADOS] Seria possível comparar a SMR por região/estado aos números de casos de COVID-19, de forma a apontar como a mortalidade por diferentes causas está relacionada (ou não) à incidência de COVID-19 em determinada região? Em caso positivo, pode-se conectar tais achados com a discussão sugerida no item #23.
15. [RESULTADOS] Houve algum tipo de intervenção sanitária ou surtos (e.g. dengue, Zika) que possam ter impactado - positiva ou negativamente - a SMR basal no período 2015-2019? Talvez seja interessante discutir este ponto e comentar sobre como isso foi considerado durante a análise, além de como a comparação entre estados poderia ser afetada de maneira heterogênea.
16. [RESULTADOS] Não seria interessante, de maneira adicional aos resultados já apresentados, gerar uma tabela com os resultados aglutinados por região do país, de forma a reduzir a capilarização da análise e facilitar uma visualização dos efeitos regionais? Vide comentário #22.
17. [DISCUSSÃO] “A respeito das causas externas, os resultados são coerentes com a adoção de medidas de distanciamento físico” Adicionar que as causas externas foram reduzidas ou aumentadas com as medidas de biossegurança “no primeiro ano do período pandêmico da COVID-19 no Brasil”.
18. [DISCUSSÃO] “As quedas na mobilidade têm um impacto esperado nos acidentes de trânsito, uma vez que as pessoas que ficam em casa não correm risco para esses eventos” Recomendamos adicionar referência da literatura para fundamentar o efeito esperado.
19. [DISCUSSÃO] “e a heterogeneidade entre as UF é reflexo das desigualdades regionais, seja para a exposição a fatores de risco, seja para a oportunidade diagnóstica e terapêutica” Pode ser importante aprofundar este ponto. Existem diferenças em nível do sistema de saúde entre as regiões que podem explicar algumas das diferenças? Não seria interessante, portanto, agregar os dados detalhados por estados em regiões de forma a facilitar a visualização dessas diferenças?
20. [DISCUSSÃO] O manuscrito se beneficiaria de uma ampliação da discussão, contextualizando os resultados com outros estudos sobre excesso de mortalidade associada à COVID-19 no Brasil e na América do Sul e discutindo se os resultados corroboram ou vão de encontro aos dados destas publicações prévias - e o que poderia justificar tais diferenças/semelhanças. Por exemplo, seria interessante adicionar à discussão uma contextualização dos resultados apresentados com os achados discutidos por dos Santos et al. (2021) (https://doi.org/10.11606/s1518-8787.2021055004137), que realizaram uma análise semelhante, embora com outro tipo de aninhamento.
21. [DISCUSSÃO] Uma discussão sobre as limitações da análise fortaleceria o trabalho (e.g., apontando que se trata de um detalhamento da mortalidade por agrupamento de causas e de estados, portanto não é possível ainda estabelecer causalidade com alguns dos tópicos mencionados, como respostas mediadas pelo sistema de saúde, uma vez que mais informações - como ocupação/capacidade dos serviços de saúde, número de casos, etc. - seriam necessárias para proceder com este tipo de análise). Adicionalmente, há algum dado sobre a precisão dos dados reportados/consolidados no SIM/Datasus? Em caso positivo, seria interessante reportar possíveis vieses/subnotificação.

Avaliação em grupo ASAPbio-SciELO Preprints

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Esta avaliação reflete contribuições de Carla Silva, Helvécio Cardoso C. Povoa, Iratxe Puebla, Kamyla Pedrosa, Mariana Rezende, Vanner Boere. Síntese por Adeilton Brandão.

O artigo apresenta um trabalho que teve como objetivo avaliar a qualidade da transição do cuidado de pacientes pós-covid-19 que tiveram alta do serviço hospitalar para o domicílio. A metodologia foi baseada em estudo transversal, descritivo e analítico, realizado em um hospital universitário no Norte do Brasil, com a coleta de dados realizada através de questionário sociodemográfico com o Care Transitions Measure (CTM-15). Os autores concluem que a qualidade da transição do cuidado percebida pelo paciente recuperado de COVID-19, ou por seus cuidadores, foi considerada alta.

Os comentários elaborados pelos revisores são apresentados abaixo como sugestões de alteração no texto para que os autores avaliem a adequação e a inclusão dessas sugestões.

Sugestões dos Revisores

1. [Resumo] Recomenda-se reescrever a descrição dos resultados mencionando apenas que foram obtidos níveis de satisfação aceitáveis acima de 80, e que o padrão recomendável é acima de 50.

2. [Resumo] O resumo deve apresentar clara e inequivocamente os resultados obtidos. Recomenda-se que seja retirado da conclusão a informação "...reduzindo as taxas de re-internações e complicações pós alta hospitalar." Não foram identificados no artigo dados que justifiquem esta informação.

3. [Introdução] Rede de Atenção À Saúde (RAS): Considerando a importância de informar o leitor sobre o sistema de saúde, recomenda-se a descrição da RAS dentro do SUS, e seguidamente em um outro parágrafo, descrever com referências o SUS e RAS no Brasil, mencionando a Atenção Primária à Saúde (APS) e a rede de urgência e emergência.

4. [Introdução] "Diante da escassez de estudos...":

   a) Seria possível discutir evidências sobre a transição da qualidade da assistência no Brasil, antes/além do contexto da COVID-19? Isso forneceria uma medida de linha de base para a qualidade da transição de cuidados no país/região, para comparação.

   b) Recomenda-se não citar "escassez", pois não foi realizado (apontado/mencionado) estudo sobre "o estado da arte" relativo ao tema desta pesquisa que justificasse a utilização deste termo.

5. [Introdução] "Nessa direção, a questão norteadora desta pesquisa foi...": Não está claro como o desenho do estudo pode responder a esta questão, dado o desenho transversal. Pode ser útil reformular a pergunta/direção de pesquisa para torná-la mais alinhada ao trabalho concluído.

6. [Introdução] "Hipótese": Seria útil ter algum contexto adicional sobre a qualidade de base da transição do cuidado no Brasil. Se houver evidências de que isso era alto antes/além do COVID-19, que forneceria uma base para essa hipótese.

7. [Método] "projeto multicêntrico": Existe referência para o estudo multicêntrico?

8. [Método] "Critérios de Seleção":

   a) recomenda-se descrever se o paciente ficou internado em leito de UTI COVID, se foi intubado, se foi traqueostomizado...

   b) o período mínimo de 72 horas de internação foi um dos critérios de inclusão, no entanto, não há descrição do período máximo de internação. Recomenda-se inserir o período máximo de internação.

9. [Método] "Definição da amostra":

   a) a amostra final foi menor, portanto, o estudo é de baixa potência. Recomenda-se que este fato seja discutido em termos de interpretação dos resultados.

   b) sobre a frase "descredenciamento do hospital pela Secretaria Municipal de Saúde do município de Belém": não está clara a relação entre este evento e os cuidados com a COVID-19.

10. [Método] "Coleta de dados": "...período de maio a dezembro de 2021." Recomenda-se fornecer uma justificativa para este período de estudo.

11. [Resultados] "... (83,7%) foram do sexo masculino...": Recomenda-se esclarecer a que esse dado se refere, p. ex. seria o número de participantes que eram pacientes (vs cuidadores)? É importante separar bem os grupos para que os valores sejam claros.

12. [Resultados] No trecho que começa com "Na tabela 2...": a) remover "descrita a seguir"; b) incluir "e o desvio padrão".

13. [Discussão] Recomenda-se uma apresentação e contextualização das limitações do estudo considerando:

a) de acordo com o cálculo do tamanho da amostra, o estudo foi insuficiente (underpowered), isso precisa ser explicitamente declarado e discutido;

b) a possibilidade de seleção/viés do respondente - quem teve melhor recuperação de melhor atendimento/transição de atendimento tem mais chances de se dispor a participar;

c) os dados são auto-relatados e, portanto, não podem ser totalmente extrapolados para refletir a qualidade real do atendimento;

d) o estudo não relatou medidas de saúde/complicações pós-alta e, portanto, não pode fazer associações entre a qualidade percebida da transição do cuidado e os resultados de saúde.

1. [Discussão] "...apresentando resultado semelhante com esse estudo...": Este estudo é comparável? Concentrou-se em pacientes idosos e estudos intervencionistas.

15 [Discussão] "...corroborando com estudos (4,9)...": Indique aqui se os estudos anteriores analisaram a qualidade da transição do cuidado na região/Brasil ou em outros lugares.

1. [Discussão] "...pacientes e seus cuidadores não são orientados sobre efeitos colaterais...": Essa é a prática no contexto de pacientes em recuperação do COVID-19? Seria útil esclarecer quais medicamentos os pacientes foram aconselhados a tomar após a alta.

2. [Conclusão] "...a qualidade da transição do cuidado...": Recomenda-se informar que são dados auto-relatados.

3. [Conclusão] "...pacientes recuperados de COVID-19 e/ou cuidadores da alta hospitalar para o domicílio...": Recomenda-se a alterar para "pacientes recuperados de COVID-19 após a alta hospitalar para o domicilio, sendo a avaliação respondidas pelos pacientes ou pelos seus respectivos cuidadores".

4. [Conclusão] "...o instrumento foi útil e permite uma avaliação global da transição do cuidado.Os itens relacionados a “Preparação da Gestão a Saúde” e “Compreensão dos Medicamentos” mostraram-se favoráveis para qualificar a qualidade de transição do cuidado.": Recomenda-se reescrever esta parte, pois fica entendido como se o estudo estivesse fazendo uma validação do instrumento, e não avaliando a qualidade da transição no cuidado.

5. [Conclusão] "Houve associação entre o tempo de internação e a qualidade de transição do cuidado, entretanto, não houve associação com a faixa etária." - Explicar com mais detalhes essa associação, especificando que correlação "aumenta" ou "diminui".

Author response

• A comment on the overall organization of the paper. Figure 2 has a major location in the paper, but it seems that its main takeaway is that these MAPs aren't really involved in the main process this paper is probing. While these are important findings, it might be more satisfying to move some of the central results earlier.

We agree that this figure displays mostly negative results. However, most work on anaphase B microtubule dynamics from our group and others has focused on the effect that motors and MAPs may have on microtubule dynamics (EB1 and kinesin-8 in budding yeast, klp9 in fission yeast). Therefore, we consider it is important to clearly show that previously proposed candidates are not required for the observed decrease in microtubule growth speed, prior to introducing the unexpected effect of the membrane.

*A model schematic might drive home the main finding of the paper, and be particularly useful for readers who are not experts in microtubule or spindle dynamics. That said, the Discussion does an excellent job of summarizing the findings and explaining the takeaway message(s), even for the non-expert.

We have added a model schematic and we have referred to it in the main text.

Specific comments

• ‘In higher eukaryotes’ - Suggest avoiding the terms higher and lower when describing organisms, and instead, directly defining which organisms, for instance in animals/metazoans that would be a better description.

We have removed this terminology.

• Figure 1 E-F - It is hard to see the difference in the distribution, maybe a different color could be used instead of stars.

We have used a different color.

• Figure 1 Data shown in pink in G comes from 832 midzone length measurements during anaphase, from 60 cells in 10 independent experiments - The pink here does not correspond to the pink coding in D, consider colour choice for clarity across panels.

We have changed this.

• Finally, yeasts undergo closed mitosis - How does this relate to the findings in the Dey paper (cited here) which shows it was somewhat semi-closed or semi-open. According to the Dey paper, the membrane disassembles locally twice, at the SPB and the bridge.

Membrane disassembly at the nuclear membrane bridge occurs at late anaphase, and leads to the disassembly of the spindle, presumably by the action of cytoplasmic factors (Dey et al. 2020). We do not believe the membrane disassembly itself has a role in spindle elongation or microtubule dynamics, as when it happens the spindle is then disassembled. However, the fact that les1D reduces the decrease in microtubule growth speed associated with internalisation of microtubules in the nuclear membrane bridge suggest that the organisation of the nuclear membrane bridge required for its local disassembly at late anaphase might affect microtubule growth (see section “Formation of Les1 stalks […]”).

• ‘vertical comets in kymographs (Fig. 1C) do not correspond to non-growing microtubules, but rather microtubules that grow at a speed matching the sliding speed’- For clarity, it might be nice to add: "(as the SPB moves away from the plus end in the kymograph)".

We have included this useful clarification.

• ‘significantly shorter than in interphase, where growth events last more than 120 seconds on average [42, 43]. Microtubule shrinking speed did not change during anaphase either (Fig. 1-Supplement 1D), and was on average 3.56±1.75 μm/min, also lower than in interphase (~8 min/μm)’ - This comment concerns the comparison of growth and shrinking rate as well as growth duration. The authors did not measure microtubule dynamics in interphase in this manuscript but compared their numbers to literature values. The comparison raises some questions for three reasons: 1) the microscopy method used is different in this paper and the two references provided, 2) the sample is mounted differently compared to the two references provided - 1) and 2) combined could lead to different levels of stress on the cells which could affect MT dynamics-, 3) (probably the most important caveat) the experiments are done at different temperatures: 27C in this paper versus 25C in the references provided. Microtubule dynamics are sensitive to temperature so this could explain part of the differences observed. Also, there are multiple values published for MT dynamics in interphase depending on the strain used and the microscopy method used. Suggest that the authors measure microtubule dynamics in interphase cells at 27C in SIM to ensure that the differences are not due to the technical parameters employed. Small item - should ‘8 min/μm’ read “8 μm/min"?

We have measured microtubule growth speed and growth event duration using GFP-Mal3 during interphase and anaphase B in the same conditions as proposed (see Figure 1 – Supplement 2). Unfortunately, shrinkage speed cannot be measured using GFP-Mal3, so we cannot confirm that the difference between our measurements and the literature values would be observed.

• ‘we observed two populations of microtubules (fast and slow growing)’ - Does this statement about thistle fast and slow growing populations refer to the data in Fig. 1C and 2A?

Yes, we have added reference to this figures in the next sentence (mentioned below).

• ‘In some cells, all microtubules seemed to switch to the slow growing phase simultaneously (Fig. 1C), while in others fast and slow growing microtubules co-existed (Fig. 2A)’ - This is a very interesting observation, could we know how many cells (%) were detected in each case? Is it that in 90% of the cells the switch is simultaneous, and hence the microtubule growth is somehow synchronized? Or is it more random, e.g. around 50%?

This was just to point the reader to two kymographs and show that a clear point where all microtubules change speed is not present in all kymographs, as one may think from Fig. 1C. Later in the paper, we show that the change in growth depends on whether the microtubule rescue occurs inside or outside the nuclear membrane bridge, so it is a matter of where microtubules are rescued once the dumbbell transition occurs, which is a stochastic process. We have added another sentence pointing the reader to examples in the kymograph (see line 152, This representation captures…).

• ‘On such a plot, the data points visibly cluster in two separate clouds and the variation of growth speeds can be fitted by an error function (Fig. 1F)’ - It is unclear that there are two distinct clusters, maybe the assertion should be toned down, or some sort of cluster analysis provided.

We acknowledge that the data is widely spread across the y axis, and given that the magnitude “distance to the closest pole at rescue” is continuous the transition is not a clear cut. However, we consider the fact that the averaged curve closely matches the error function fit to be sufficient evidence for the existence of two populations of microtubule growth. Additionally, R2 of the fit is ~0.5 indicating that half of the variance is explained by this model. In any case, we show later that these two populations do exist (Fig. 3D), and why plotting microtubule growth against distance to the closest pole at rescue is a good way to segregate them (Fig. 3E).

• ‘speed of interphase microtubules (~2.3 μm/min)’ - It would be interesting to see the dynamics in a les1 mutant (Dey Nature 2020) paper. Just as a control for presence/absence of the bridge?

We thank the reviewers for kindly suggesting this interesting experiment. We have included it after the ase1 section. Les1 forms stalks at the edges of the nuclear membrane bridge that restrict nuclear membrane disassembly to the center of the bridge at the end of mitosis (Dey at al. 2020). While les1 deletion does not prevent the formation of the nuclear membrane bridge, it has been proposed that Les1 stalks may constitute sites of close interaction between the nuclear membrane and the spindle. Therefore, these sites may influence microtuble growth. Indeed, we have found that removing these Les1 stalks by either deleting les1 or nem1 leads to a smaller decrease in microtubule growth speed when plus ends enter the nuclear membrane bridge (see section “Formation of Les1 stalks […]”)

*‘Figure 2, Transition from fast to slow microtubule growth occurs in the absence of known anaphase MAPs’ - It looks like the overlap zone is larger on the mal3 kymograph. Is the size of the midzone changed in some of the mutants? It could be important to report. Related to it, is the spindle length changed in some of the mutants? (It does not look like it from the kymographs displayed).

The midzone is indeed longer in mal3D strains, now this can be seen in Fig. 2 – Supp. 2 and it is mentioned in the main text in line 272. As for the spindle length, diverse kinds of alterations in spindle length have been previously reported for the mutants that we used in this study. For instance, ase1D /cls1off cells have shorter spindles at anaphase onset (Loiodice et al. 2005 and data not shown), and klp5Dklp6D have longer spindles at anaphase onset (Syrivatkina et al. 2013). klp9D / clp1D / dis1D cells have lower spindle elongation velocity and may not reach the wild-type spindle length by the end of anaphase (Kruger et al. 2019). Despite these differences, the decrease in microtubule growth as a function of distance to the closest pole has a similar tendency across conditions, suggesting that the mentioned differences in spindle length are unlikely to have an important effect.

• Additionally, adding the data about rescue localization in the mutant (equivalent of Fig 1 G) would be interesting to better describe the role of these different proteins. Figure 2, Panel G to L - Could the authors indicate the value for the average +/- error in each bin for the WT and the mutants? Also, it is hard to say from the plots, but it looks like the WT average speed in the first bin is different in every panel, that would be good to know to have an idea of the reproducibility/variability.

We have added a figure with the rescue distribution (see Fig. 2 – Supp. 2). This apparent difference in the wt speed in different experiments might have come from looking at normalised data. The new way of representing the data in fig. 2H and J shows that the microtubule growth velocity in the wild-type is very consistent across experiments. We have added a table with microtubule growth velocity values (Table 1), and the source data is available.

• The dots making up the "thick lines" are centered on 1.5/2.5/etc.. in some panels (G and K) and centered on 1/2/3/etc.. the others (I,J,L). Could the authors provide some clarification?

We have fixed this inconsistency across the paper.

• Figure 3 - Can the authors indicate the average values +/- error for each of the distributions in Fig. 3D? Maybe on the plot itself, in the legend or as a table. This would make them easily available without having to infer them from the Y axis. This comment is also valid for Fig 4I and 4J.

We have added tables with average values and confidence intervals in the appendix.

• Figure 3E ‘Distance from the plus-end to the nuclear membrane bridge edge at rescue as a function of distance from the plus-end to the closest pole at rescue’ - The Y axis reads as "distance to the bridge edge" but it shows negative values, could this be "position to the bridge edge" instead? (same item throughout the text).

We have fixed this.

• Figure 3 ‘Number of events: 442 (30 cells) wt, 260 (27 cells) klp9OE, 401 (35 cells) cdc25-22, from 3 independent experiments’ - P values this small raise a concern. Presumably the number of degrees of freedom in the regression analysis should not exceed the number of independent experiments. Instead, the DoF listed under "error" in the analysis output is hundreds or thousands instead of 3. To address this, the regression analysis should use either the "Error" function in R or a linear mixed-effects model to account for the nesting of the repeated measurements within each independent experiment. Alternatively, it is also possible to just calculate summary means for each independent experiment, and calculate p values based on that N=3. See: Lazic. Experimental Design for Laboratory Biologists. p. 157. and the supplemental file of: https://doi.org/10.1371/journal.pbio.2005282 and the additional file 1 of: https://doi.org/10.1186/s12868-015-0228-5 and this for an alternative plotting approach: https://doi.org/10.1083/jcb.202001064 Recommend either recalculating the p values by one of the methods above or removing the reported p values from the paper. The large effects observed in many cases are self-evident without a significance metric, so eliminating the p values would be acceptable here. (This comment applies to other figures through the paper that report p values based on number of cells or number of measurements instead of number of independent samples/experiments.)

We thank the reviewers for suggesting the improvements to the statistical analysis, as well as for pointing us to useful resources that described the statistical methods and their implementation in detail. We have followed Aarts et al. 2015 and used a linear mixed effects model (see Methods>Statistical Analysis)

Due to the change in statistical analysis method, to show that some of the differences we had reported previously were significant, we included more cells in the analysis from our existing data. We did this for klp5Dklp6D kymographs (Fig. 2I and Fig.2 – Supp. 1). Spindle dynamics in ase1D (Fig. 5D and Fig. 5 – Supp. 1) and klp9D (Fig. 2 – Supp. 3 A, C). Cell length (Fig. 3 – Supp. 1A).

For the same reason, we measured anaphase spindle elongation velocity (Fig. 3 – Supp. 1C) from kymographs instead of measuring them from the 1 minute interval movies that we had used previously (from Fig. 3 – Supp 1B). We have reflected this in the methods (see added text in line 800 and deleted text in line 809 in the document with changes highlighted).

None of these changes has altered our conclusions.

• Figure 4 - Nice experiment. It brings the question of how cell-shape affects all these dynamics (probably out of the scope of this work). But a for3 mutant for example?

This is an interesting suggestion, to be tested in the future. Furthermore, we believe that nuclear shape should also have an important effect, since the spindle is confined inside the nuclear membrane. We would expect that mutants that perturb nuclear shape might have effects on microtubule growth. We have observed that the decrease in growth speed associated with internalisation of microtubules in the nuclear membrane bridge is reduced upon nem1 deletion, which increases nuclear membrane surface, and produces membrane ruffling (Fig. 4-Supplement 2). However, nem1 deletion also removes les1 stalks from the nuclear bridge (Dey et al. 2020). It would be interesting to find a perturbation of the nuclear membrane that does not remove the les1 stalks.

• ‘Ase1 is required for microtubule growth speed to decrease during anaphase B, this is unlikely to be a direct effect’ - If it is unlikely to be a direct Ase1 effect is the title of the section accurate? "Ase1 is required for normal rescue distribution and for microtubule growth speed to decrease in anaphase B"

Ase1 recruits multiple proteins to the spindle midzone, so the fact that ase1 deletion produces a given phenotype does not necessarily mean that this phenotype results from the absence of Ase1 protein activity. For instance, deleting ase1 perturbs rescue distribution, but it does not mean that Ase1 acts as a rescue factor itself, or at least to a relevant extent, given that deletion of cls1 completely prevents rescue, but ase1 deletion does not. In the discussion we propose some indirect effects of ase1 deletion that may produce this effect. In any case, upon more careful analysis we have found that ase1 deletion does not prevent the decrease in microtubule growth speed during anaphase B, but rather makes it smaller (see section “The decrease in growth speed associated with internalisation of microtubules in the nuclear membrane bridge is reduced upon ase1 deletion”).

• Figure 5 - What about an ase1 lem1 double mutant?

We suppose that the intended gene is les1. We have studied the effects of les1 deletion in the new version of the manuscript. However, we do not see the information we would obtain from a double deletion ase1D les1D.

• ‘In summary, Ase1 is required for rescue organisation and for microtubule growth speed to decrease during anaphase B ‘- In this context it could make sense to discuss the observations from this paper (doi:10.1371/journal.pone.0056808) about the role of Ase1 ortholog's MAP65-1 in coordinating MT dynamics within bundles.

In the mentioned paper, the authors showed that the presence of PRC1 (ase1 orthologue) in bundles increases microtubule rescue rate, and that it slightly reduces microtubule growth speed.

We observe a small increase in microtubule growth speed throughout anaphase upon ase1 deletion (Fig. 5), which is consistent with the in vitro observation that PRC1 decreases microtubule growth. However, once more this might not be a direct effect of Ase1, since less Cls1 is recruited if ase1 is deleted, and Cls1 reduces microtubule growth speed (Fig. 2). In addition, this can also be a result of higher concentration of tubulin / MAPs resulting from less polymerised tubulin in ase1 deleted cells, which have less spindle microtubules on average.

Regarding the increase in rescue rate produced by PRC1 in vitro, it is possible that Ase1 contributes to microtubule rescue in the spindle. However, given that no rescues occur upon inactivation of cls1 (Bratman et al. 2007), we believe Cls1 is the dominant factor, and Ase1 contribution is likely negligible.

• ‘We initially set the microtubule growth velocity to 1.6 μm/min (early anaphase speed, Fig. 1F), and aimed to reproduce the experimental distribution of positions of rescue and catastrophe at early anaphase (spindle length < 6 μm’ - Kudos to the authors for detailing the model and its parameters in a way that even non-modelling experts can understand.

Discussion - ‘Our data suggests that microtubule growth speed is mainly governed by spatial cues’ - Is it right to assume that in the cases where fast and slow growing microtubules were simultaneously observed, the fast microtubules were not/had not yet reached the midzone?

Our data suggests that it’s not about being inside the midzone, but rather inside the nuclear membrane bridge formed after the dumbbell transition. We have elaborated more on this in the main text, pointing the reader to examples in the kymograph, and giving a quantitative argument for distance to the closest pole being a better predictor than anaphase progression or position with respect to the center (which is equivalent to distance to the midzone), see line 152.

• Methods - ‘PIFOC module (perfect image focus), and sCMOS camera’ - Is this Nikon's "Perfect Focus" autofocus, or some other manufacturer's system? And back-thinned sCMOS.

We have clarified this in the Methods section.

This review reflects comments and contributions by Elnaz Fazeli, Rachel Lau, Gregory Redpath, Mugdha Sathe and Wasim Sayyad.

The preprint reports the use of two color-3D STORM imaging, quantitative live-cell TIRF microscopy, fluorescence lifetime, and atomic force microscopy to explore the load-adapted endocytic mechanism in actin networks of mammalian cells. The results show that the actin network forms at the base of the endocytic pit and grows around and over the tip of the clathrin coat. The branched actin network generator Arp2/3 is required for normal clathrin-mediated endocytosis (CME). CME is perturbed when the tension in the plasma membrane increases which also increases the actin coverage over the clathrin coat.

This is a solid study; the manuscript reports appropriate control experiments and statistical analysis.

Specific comments

‘skin-melanoma cell line (SK-MEL-2)’ - Was this cell line chosen due to its availability in the lab? It would be interesting to see whether the results would be similar in normal human cell lines and not a cancer-specific mechanism. Perhaps this could be mentioned in the discussion as future work?

‘​​The standard deviations of positions of single fluorophores were 10 nm in-plane for XY and 19 nm in-depth for the Z dimension’ - This is obviously great resolution for STORM, but the manuscript reports using primary and secondary antibodies to detect clathrin etc. This will add a reasonable distance between the fluorophore and clathrin (or whatever protein) which will be relevant at this resolution. This should perhaps be mentioned as a potential caveat, or why it does not matter.

Figures 1 and 4 - The schematics are great and make the interpretation of the images so much easier!

Figure 1 ‘shape index’ - How was the shape index measured? Would it be possible to expand on how the two right columns of Figure 1E were separated into three categories (either in the Results or the Methods section).

‘We detected actin associated with 74% of the clathrin coats, which is comparable to previous measurements for the same cell type that we made of endocytic traces in live cells of dynamin2-GFP events associated with actin-RFP, given that our super-resolved images are snapshots of a time-lapse event‘- What is the aspect ratio distribution of Clathrin coats that lacked actin associated with it?

‘Coat with height similar to its width) (Figs. 1D’ - By looking at the fraction of shallow, u-shaped and omega-shaped clathrin coat shapes under steady state vs when membrane tension is altered, is it possible to see whether some stages are getting enriched or depleted?

Figure 1F, ‘There was no significant correlation between actin/coat coverage and coat shape for any of the three geometries’ - What is the size/volume distributions of CCPs for a given geometry?

Figure 1H ‘Indeed, as a function of actin/coat coverage, Dz increased from negative values to near zero’ - Conversely, should Dz decrease with regard to width of the clathrin pits?

‘Consistent with our conclusions about where actin assembly occurs at CCPs, N-WASP localized to the base of both shallow and highly curved clathrin coats (Fig. S6A). More unexpectedly, at some CME sites, N-WASP covered the entire clathrin coat irrespective of coat geometry (Fig. S6B)’ - The N-WASP data is very interesting, recommend completing quantification similar to the actin data: if the N-WASP distribution come out the same as actin distribution, where it is clear it is "nucleated" at the bottom of a clathrin structure, that would be a very interesting result that supports the findings regarding actin.

‘the CLTA-TagRFP-TEN and DNM2-eGFPEN lifetimes began to recover, most likely reflecting cellular adaptation to the hypotonic treatment’ - The recovery is noticeable for clathrin and dynamin2 lifetime, but is not reflected in endocytosis initiation and completion. Could some comments be provided about this?

‘DNM2-eGFPEN-only events showed a moderate response to elevated membrane tension’ - Recommend referencing Fig S7 for this, and perhaps saying "moderate, statistically significant response to elevated membrane tension only with 75 mOsm hypotonic media" just to highlight the specificity of the response to clathrin-associated dynamin.

‘Figure 3 Clathrin coat images for quantitative analysis were collected from at least 3 cells for each condition’ - Please report across how many independent experiments.

‘2d-3D STORM’ - Should this read 2c-3D STORM?

‘the average clathrin coat height decreased to 96 nm ± 24 nm’ - It seems that hypotonic shock in the background of arp2/3 inhibition actually rescues the Clathrin coat height back to DMSO levels. What is the height distribution with just hypotonic shock? Moreover, when arp2/3 is inhibited do all the CCPs lose actin around them? If yes, then is there any difference in shape distribution? If no, how does the actin distribution fare with regard to control?

‘​​actin grows higher in the z-dimension around clathrin coats’ - How will the actin distribution look under dynamin k44a (ideally temperature sensitive) mutant where the CCPs are stalled? Will the cell infer this as 'high tension'?

‘We showed quantitatively that actin assembly and organization adapt to changes in membrane tension, which we measured by AFM membrane tether pulling’ - Will this actin recruitment change in hypertonic shock in a manner opposite to the hypotonic shock?

‘such differences might reflect subcellular, local load variation resulting from variance in such factors as membrane tension and cell adhesion’ - Would it be possible to use actin coverage around CCPs to predict local tension? Computational modelling might be helpful in future studies.

‘the U-shaped stage, consistent with the effects of actin assembly inhibition reported for other cell types’ - Does dynamic recruitment care if the CCPs have U vs omega shape? It might be interesting to see if dynamin localisation in altered in Z.

‘for example by increasing contact of filaments associated with the coated pit with membrane-associated N-WASP-Arp2/3 complex’ - Through live TIRF imaging, Taylor & Merrifield et al. showed that 30-40% of CCPs showed N-WASP recruitment, so the question arises as to whether there are other regulators involved. Another question to consider is enrichment of BAR domain proteins. Would it be possible to comment on whether at high tension, more BAR domain proteins could also be recruited to help with membrane bending?

Methods

• ‘Data analysis, statistical analysis and data plotting’ - The following could come in this section: fluorescence lifetime, fluorophores used, shape index measurements, software used for image handling and image preprocessing, kymograph generation, STORM image reconstruction process and quality control, how was the data plotted and what parameters were used. This article discusses reporting for reproducible microscopy workflows: https://www.nature.com/articles/s41592-021-01156-w. Suggest a mention to whether the code and the original images are available at a repository (e.g. Github, Zenodo).
• It may be worth adding some comments on why STORM was used in the study, the need for super-resolution imaging rather than confocal and why STORM in particular. Also some comments about the limitations of traction force microscopy over atomic force microscopy, owing to it being more suitable for measuring tension on the ventral surface where the endocytic events’ images were acquired.

This review reflects comments and contributions by Karen Lange, Rachel Lau, Claudia Molina, Mafalda Pimentel, Sree Rama Chaitanya Sridhara and Sagar Varankar. Review synthesized by Sree Rama Chaitanya Sridhara.

The 3’ end of pre-mRNA molecule in metazoan cells is processed to make functional mRNA molecules. This involves chopping the ends to make free 3’ ends that are polyadenylated (or poly-A) to form mature mRNA molecules with a poly-A tail. The 3’ end processing is important for the stability and transport of mature mRNA molecules, transcription, and translation. 3’ end processing is achieved by a 7-subunit cleavage and polyadenylation specificity factor (CPSF). Despite functional genomics and some in vitro reconstitution experiments, the full protein composition and detailed molecular mechanisms of CPSF are not clear.

The current study reconstitutes the CPSF complex and the preprint reports that an accessory protein called RBBP6 is required for the activation of 3’end cleavage in an RNA-dependent manner. Altogether, the manuscript and data are very clear, and the claims are supported by appropriate experiments and respective controls. The schematics are extremely helpful for the reader to grasp the interactions of different subunits in this complex and also to understand the experiments. These data (with the "back-to-back" preprint in yeast; Rodrigez-Molina et al. 2021) determine a conserved machinery that efficiently cleaves the 3' end of pre-mRNAs in vitro.

Results

Major comments

1. 'Addition of CStF and CFIIm, either individually or together, failed to activate CPSF. However, addition of RBBP6 activated CPSF in the presence of CStF and CFIIm, promoting efficient cleavage of the pre-mRNA substrate.' – There is a faint 5'-cleavage product in the CPSF/RBBP6 and CPSF/CFIIm/RBBP6 lanes. It would be helpful to provide some comments or discussion about this faint band. (Results – Fig. 1 and Fig. S2)
2. 'Time-course cleavage assays of SV40 pre-mRNA substrates containing either a canonical PAS (RNAAAUAAA) or a mutant PAS (RNAAACAAA) sequence.' – Were any other mutants (e.g., AAGAAA or AAAAAA) tested as part of the study? (Results)
3. 'Upon addition of ATP, the 5’ cleavage product band disappears and is replaced by a smear corresponding to polyadenylated 5’ products containing poly(A) tails of variable length.' – The "smear" is not clear. It seems like the addition of ATP made cleavage less efficient because the lower 3'cleavage product appears to be fainter than the no ATP lane as well. It might be important to include some clarification on these points. (Results, Fig. S1D)

Minor comments

1. 'likely that the role of RBBP6 is conserved from yeast to human' – It is advisable to include the context under which the role of RBBP6 is conserved (Abstract).
2. 'in insect cells' – It might be helpful to clarify or discuss the status of post-translational modifications of human proteins expressed in insect cells if any? (Results)
3. 'Unstructured regions were removed from the CPSF subunits WDR33 and hFip1, and the CFIIm subunit Pcf11 to facilitate purification' – Suggest including some comments on how these deletions don't affect the protein function (Results).
4. 'We hypothesized that the conserved region of the multi-domain protein RBBP6 (residues 1-335; Supplementary Information) might also be required for endonuclease activation' – Is there a reason for this hypothesis or can a relevant reference be included? (Results)
5. Kwon et al., 2020 – typo Kwon et al., 2021. (Results)
6. 'We tested various combinations of 3’-end processing factors in cleavage assays and analyzed the results by denaturing gel electrophoresis of RNA (Figure 1C).' – Is this experiment performed with the deletion constructs mentioned earlier? (Results)
7. 'Overall, we determined that activation of the CPSF endonuclease requires three additional protein factors: CStF, CFIIm and RBBP6' – Does this mean only in vitro or also in vivo? (Results)
8. 'Each dot represents a single measurement.' – It is not clear how many measurements were taken on a graph. It seems there are more measurements for certain concentrations compared to others. For example, 0.1uM has four compared to 1 at the penultimate highest concentration. Maybe the number of measurements and biological replicates can be described more clearly in the figure legend. (Results, Fig. 2D)
9. (Sun et al, 2018) – Although a reference is provided, the PDB ID code in the figure legend might be helpful. (Results, Fig. 4D)
10. 'The DNA sequences encoding fragments of SV40 pre-mRNA with either wild-type (AAUAAA) or mutant PAS (AACAAA)' – It might be helpful to provide the SV70 pre-mRNA sequence for future reference. (Methods).

This review reflects comments and contributions by Rachel Lau, Claudia Molina, Sónia Gomes Pereira, Pablo Ranea-Robles, Gregory Redpath, Mugda Sathe, Bathia Sonam and Sagar Varankar.

In this study, Nazemi et al. investigated the role of the extracellular matrix in the metabolic adaptations of breast cancer cells under amino acid starvation. They found that ECM uptake and its lysosomal degradation provide amino acids that support cell growth. They also point to phenylalanine and tyrosine metabolism as an important pathway in this process, leading to the generation of the TCA cycle intermediate fumarate. This study fits well in the context of the broader literature showing that cancer cells upregulate endocytosis to continue proliferating in starvation conditions, and extends it well by identifying catabolism of ECM components as a part of this response and identifying precise metabolic pathways involved. This is a very interesting study, the data are solid and supportive of the claims. The study is well-written and the experiments are well contextualized, making the paper easy to read and understand.

Some aspects in the preprint require further clarification, major and minor points are outlined below, followed by comments on specific parts of the paper.

Major points

• Experimentally, the endocytosis sections could be improved. Filipin is used as an endocytosis inhibitor, despite it being non-specific and not currently accepted as an endocytosis inhibitor. These experiments could be strengthened with well accepted endocytosis inhibitors, and especially macropinocytosis inhibitors to tie the study in with the current literature.
• The in vitro data is promising, however, the authors could acknowledge in the discussion the limitation of not having in vivo data. The title should specify that the data have been obtained in vitro only.
• It is not clear when the experiments are completed with Matrigel and Collagen I, or only one of them. Ideally, to make the work more consistent, the two of them could be used for every parameter measured.
• Some IF experiments lack representative images associated with the quantification.
• It is not clear why the inhibitor of focal adhesion is tested if there is no change in focal adhesion signaling in cells cultured with collagen-I/Matrigel/Cross-linked matrices.
• It is not clear that the isotope labelling experiment data support the conclusion that "tyrosine derived from collagen I degradation could contribute to fumarate production." Data show a higher proportion of C13-labelled fumarate on plastic than on Collagen I.
• Data with HPD and HPDL siRNA show a big effect on cell number and fumarate levels but only with a 10% decrease in protein level by IF. It's hard to reconcile the fact that a siRNA that only decreases 10% of the protein level causes such a big effect. The manuscript should introduce some nuance when describing these data. Experiments with HPD and/or HPDL KO cells would be more conclusive.

Minor points

• In the 6-8 days experiment, what was the signal intensity at day 1 when the experiment started? These baseline values may be important to understand this process.
• It would be informative to include in Figure 1 the values of cells cultured in complete media shown in Fig. S1A-B, but the authors may prefer the current presentation.
• Can some rationale be provided for using DRAQ5 in some assays in Fig. 1 and Hoescht3342 in others, given that the outcome measured is the same.
• It may be worth discussing the fact that CDM generated from normal fibroblasts had a remarkable effect on cell growth under Gln starvation, even more than CDM generated from cancer-cells.
• Colocalization of mTOR with a lysosomal marker would be a nice experiment to support the conclusions.
• What is the explanation for the seemingly decreased uptake of collagen I upon Aa starvation when compared with complete media?
• Effect of crosslinking treatment alone should be measured as a control. Does it affect cell number by itself?
• Fig. S5A: May be worth mentioning in the Results or Discussion the fact that ECM uptake increased when cultured with MMP inhibitor.
• Fig. 4C: The scale bars look different, the filipin image is lacking a scale bar.
• Total FAK levels missing in Fig. 5A.
• Fig. 6A - For each amino acid there is a fold change depending on whether cells were cultured on plastic or collagen I. Is the fold-change collagen I vs plastic? Then why display two fold change bars per amino acid? Are they being compared to the respective controls?
• It’d be interesting to see if ECM is also incorporated on the MCF10CA1 cell line, and if this leads to an increase in phenylalanine/tyrosine metabolism, similar to that shown for MDA-MB-231 cells.
• It might be interesting to explore the expression levels and profiles for HPD and HPDL in the MCF10 cell line, or discuss this in the context of publicly available data regarding any correlation with cancer progression.
• Please add to the reporting of the statistical analyses. Is two-way ANOVA significant per se? Which were the factors compared?
• Please report in the Methods section or figure legends how many cells were quantified in each experiment, and how many times the experiments were performed.
• For reproducibility, recommend providing more details about the metabolomic pathway analysis.
• There are some small spelling or grammar errors in the text.
• The discussion may benefit from considerations on how ECM internalisation and degradation might alter cellular microenvironment and tissue properties, which might also facilitate cellular migration and invasiveness.

Additional points

‘glutamine and full amino acid starvation' - It is possible to provide the rationale for selecting these conditions as opposed to essential amino acids which cannot be synthesized by human cells, hence need to be supplied to the cells externally.

‘the down-regulation of an enzyme in this pathway’- Recommend updating the fragment to clarify which pathway is mentioned and expand the HPDL abbreviation.

‘invasive breast cancer’- All the assays were done on MDA-MB-231 cells (with the exception of one assay that was done with MCF10 lines) and the results are occasionally extrapolated to breast cancer. Given the extent of heterogeneity within breast cancers, it is recommended to do a couple of experiments (such as in Fig 1) to ensure that similar ECM-supported growth is observed in more than one line. The results might be specific for this particular line, so accounting for subtype or genetic make up is necessary. Some of the lines to consider might be luminal (MCF7, T47D etc), additional TNBC lines (MDA-MB-468) etc.

Results

‘CAF-CDM provided a more favourable environment for cancer cells’ growth compared to NF-CDM under amino acid deprivation’ - This is outside the scope of this paper, but it would be interesting to see what the differences are in protein composition between CAF-CDM and normal fibroblast CDM and whether these differences have been seen in the different studies referenced.

Figure 1

• The figures in general are a bit small, making them difficult to read in the printed version.
• The colour code to differentiate amino acid starvation and glutamine starvation is good and the legend is in the same order as the line graphs to allow for quick comparison.
• It is clear in Figure 1 E-F that there is a difference between growing the breast cancer cells on normal fibroblast and CAF cell derived matrices compared to plastic with glutamine starvation whilst only CAF cell derived matrices increased cell growth under amino acid deprivation compared to plastic. What is the rationale for measuring the cell growth by determining cell number by Hoechst whilst in the comparative experiment when growing cells on plastic, collagen or matrigel (Figure 1 A-D, G), DRAQ5 was used instead? Is this due to a technical aspect of the experiments or the availability of the reagents?
• Figure 1A - Is there a difference between "AA starvation…" vs "AA free starvation…"? ‘2mg/ml collagen I (coll I) or (C-D) 3mg/ml Matrigel (Matr)'*- What is the rationale for using these concentrations? And why is it different for collagen I vs Matrigel? Is there optimization data available that informed the choice of concentrations?
• Matrigel is a complex mixture of ECMs; is any particular component (for instance, collagen vs laminin), the key player for the observed growth benefit? This could be discussed in the discussion section.
• ‘Signal intensity’ - As opposed to signal intensity, would it be possible to focus on a more physiological measure like cell growth or proliferation or viability? The intensity data could be part of the Supplementary figures.
• ‘black dots’ - What do the other lighter colored dots represent?

‘This includes normal mammary epithelial cells (MCF10A), non-invasive ductal carcinoma in-situ breast cancer cells (MCF10A-DCIS) and metastatic breast cancer cells (MCF10CA1)’- It would be nice to see if the three cell lines had any growth differences when starved and grown in plastic. Additionally, what was the effect of NF-CDM and CAF-CDM on the growth of these three cell lines which are at different stages of cancer progression. Could these growth curves upon starvation be used for diagnostic purposes for patients?

Figure 2

• Suggest annotating n.s. for non-significant results to aid the interpretation of the graphs.
• ‘The ability to use the ECM to promote cell growth was acquired during carcinoma progression’- Why is the starting intensity for Fig2A and C so different between different samples? If cell numbers had remained the same, would the results be the same or different?

‘which passed through DNA synthesis and mitosis’ - As supporting data, was cell cycle analysis performed for these samples? Examining whether starved cells are halted in a specific stage of the cell cycle would provide a great addition to the EdU data. ‘activated caspase-3/7’ - Was any other cell death assay tried?

‘Although the apoptosis rate increased between day three and day eight in both complete and amino acid-free media, and amino acid starvation resulted in increased cell death, there were no significant difference between the apoptosis rate on collagen I and Matrigel compared to plastic either at day 3 (Figure S3A) or day 8 (Figure S3B) in amino acid depleted media’- Recommend revising the sentence for length and clarity.

Figure 3

• Recommend showing immunofluorescence images, especially for panels C & D.
• As a suggestion, comparing Figures 1 and 3 would prove to be really helpful in arriving at a conclusion regarding the effects of the ECM on cell growth and proliferation.
• Figure 3B - The black dots represent the mean of individual experiments, but seem high compared to the other dots in the first two bars (complete media+ matrigel and complete media+ plastic). The black dots also appear slightly above 100% and it is not possible to have more than 100% EdU positive cells. Do the black dots show the means necessary? Do they hide some other dots?
• Figure 3C-D -The y-axis is integrated intensity instead of average intensity. This is acceptable only in the situations where one knows that cell size is the same between different conditions. Since we are starving the cells, it may not be the case. Additionally, p-S6 should be technically normalised to the total S6 to rule out differences in merely changes in total S6 expression.

‘Collagen I and Matrigel rescued mTORC1 activity in starved cells’ - It may make it easier to follow for readers if each result heading refers to one figure. It seems like Fig 3 corresponding to this heading should contain the current Fig 3C-D (and any visualization data).

‘MDA-MB-231 cells’ - It would be helpful to provide the rationale for using different cell types for different assays.

‘ECM endocytosis’ - At this stage it is still not known whether there is endocytosis or not.

‘lysosomal degradation following internalisation’ - Using E64D as a lysosome inhibitor is good, but given that imaging experiments are possible based on what is presented in Fig4/5, verification of this result with co-localisation of ECM components and lysotracker or some other lysosomal marker would be good. In the images presented in the supplementary figures, the ECM localisation appears lysosomal so this result would be easy to get and visually compelling. Also, did these experiments inhibit cell growth? This would further suggest the importance of lysosomal degradation of ECM for cancer cell growth.

‘We then wanted to investigate whether the ECM-dependent growth of cancer cells under nutrient deficiency relied on ECM internalisation’ - Suggest to place this first in Fig 4. If EMC components are being degraded in the lysosome, they must be internalised by some endocytic mechanism. Assessing endocytosis and then looking at lysosomal targeting seems like the natural flow of events.

‘Interestingly, while the growth of MDA-MB-231 cells was not affected by matrix cross-linking in complete media’ - Is the cell migration affected by cross linking ECM under cell starvation conditions?

‘MMP inhibition did not affect cell growth under complete media or amino acid starvation on both plastic and collagen I' - The reduction does not seem so big; there may be another mechanism that explains the reduction in growth?

‘lipid raft-mediated endocytosis inhibitor filipin ‘- Filipin is a broad, non-specific endocytosis inhibitor. Filipin is really a cholesterol extracting/disrupting molecule, and cholesterol extraction from the plasma membrane has flow on effects on endocytosis. Filipin is not currently widely used as a bona fide endocytosis inhibitor. The filipin treatment was done for 3 days, after initial cholesterol extraction, it is unclear that filipin can continue to extract cholesterol for over three days. Further, "lipid raft mediated endocytosis" is not an accepted, specific endocytic pathway. The effect of filipin is also modest, so even if it is inhibiting a specific endocytic pathway, it is unlikely to be the predominant one mediating ECM uptake. To draw in an endocytic mechanism here to explain how ECM components are internalised, specific, bona fide endocytic pathways should be investigated. For example, as evidenced in papers cited in the introduction, macropinocytosis is an important endocytic pathway in nutrient scavenging by cancer cells. It would fit perfectly here - cells upregulate fluid phase engulfment by macropinocytosis, which results in increased ECM internalisation and preserves cell proliferation in cell starvation conditions. Alternatively, the filipin results could be replaced by more representative images of the various ECM uptake experiments with crosslinking.

Some suggestions for studying bona fide endocytic components:

1. Clathrin pathway can be blocked using pitstop or dynamic shRNA/siRNA. Dynamin inhibitors have off-target effects on actin cytoskeleton, they are not recommended.
2. Caveolin: can be blocked using CSD peptide
3. Macropinocytosis: A macropinocytosis inhibitor such as EIPA, RAC1 inhibitor or ctBP1 siRNA,
4. CLIC/GEEC pathway: CDC42 inhibitor, GBF1 inhibitor or IRSp53 knockdown

Figure 4

• (A) 2mg/ml collagen I (coll I) or (B - Please show representative images for these experiments.
• Panels C-D, ‘​​ECM uptake index was calculated with’ - Please report what the uptake index is.
• Panels F-G, ‘pH-rodo labelled 0.5mg/ml Matrigel’ - Other panels in the figure use 3mg/ml of labelled Matrigel except for F-G where authors use 0.5mg/ml, can the context for this be outlined?

‘predominantly mediated by ECM internalisation’- To strengthen this point, stronger endocytic inhibition studies should be undertaken. As mentioned, filipin is not the most specific or widely accepted endocytosis inhibitor, and its inhibition of ECM uptake is modest in Fig 4F/G. Performing a broader inhibitor screen would support the conclusion that the effects seen in the study are predominantly mediated by ECM endocytosis. An excellent review to help select endocytosis inhibitors is here: https://www.nature.com/articles/s41565-021-00858-8/tables/1

‘Given the requirement for HPDL’- Does the MCF10 series of cell lines have variable HPDL expression which may correspond to its ability to grow on ECM under amino acid starvation? Maybe the expression of HPDL in MDA-MB-231 and the MCF10 series of cell lines can be assessed?

Figure 6

‘Working model: invasive breast cancer cells internalised and degraded ECM components. This upregulated phenylalanine/tyrosine catabolism, leading to an increased production of fumarate, supporting cell proliferation under amino acid starvation' - Nice diagram to summarise the proposed mechanism. It would be worth clarifying where integrins come into play, as their uptake was not specifically investigated and the FAK studies ruled out integrin signalling as a factor?

Discussion

‘We showed that the presence of ECM fully rescued [...] suggesting that the ECM supports cell growth via triggering distinct downstream signalling pathways in different cancer types.’ - Recommend revising the paragraph for clarity.

‘enodcytosis inhibitor filipin’ - As filipin is not an ideal endocytosis inhibitor, recommend revising the statement to "cholesterol extractor which can have inhibitory effects on endocytosis". Bona fide endocytosis inhibitors should be used to strengthen this part of the manuscript.

Methods

Please provide catalogue numbers for the reagents used.

‘Telomerase immortalised normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs)’- Is it correct that both the normal fibroblasts and cancer-associated fibroblasts are breast tissue derived as opposed to the normal fibroblasts being from another tissue type (e.g skin)? Are these fibroblasts paired and from the same patient? Can the origin of the normal fibroblasts and cancer-associated fibroblasts be clarified?

‘150nM ON TARGET plus Human HPDL and HPD smart pool were added on each well’- It might be useful to provide the siRNA sequences.

Author response

We thank the reviewers who provided feedback on our manuscript through ASAPbio Crowd Review. We have revised the preprint based on their thoughtful commentary. Specifically, reviewers asked whether in Figure 3 polar ejection force levels depend on the expression level of wild type or mutant KIF22-GFP. We measured KIF22-GFP expression level in these cells and now include plots indicating that polar ejection forces are not correlated with expression levels of KIF22-GFP (Figure 3E and 3F). Our manuscript also includes a plot demonstrating that anaphase recongression is correlated with KIF22-GFP expression level (Supplemental Figure 3A). In addition to adding data to address this question raised by the reviewers, we have revised the text to clarify several points based on reviewer comments, including the efficiency of siRNA knockdown of KIF22, the absence of identified pathogenic mutations in P148 or R149 in other members of the kinesin superfamily, and the potential role of mutations in altering phosphoregulation of T463. We appreciate the interest of the reviewers in the use of a chondrocyte cell line or mouse model to study how mutations in KIF22 result in tissue-specific pathology in patients. We agree that these questions are important for follow-up studies.

This review reflects comments and contributions by Rachel Lau, Claudia Molina, Arthur Molines, Anup Parchure, Daniel Rios, Rajan Thakur and Sagar Varankar.

This is a very interesting and thorough study using a novel optogenetic approach for Bicoid regulation and uncovers a novel repressive function of this protein. The experiments are very well designed and the figures are easy to follow.

General clarification - would it be possible to comment on whether nos-tub bnt + Bcd LEXY/iRFP-bCD LEXY embryos hatch? What is the consequence of perturbing the gene network on the overall fitness of the animal?

Abstract

‘Our results recapitulate known relationships’ - Is it possible to specify what the genes listed in this sentence represent? The genes referred to here are likely developmental regulators. For non-specialist readers, it would be helpful to include the term which encompasses these genes to specify that their 'results recapitulate known relationships between _'.

Introduction

Recommend adding additional references in this section, pointing out to the source for different statements. Citing the original research paper rather than a review can attribute credit to the original authors (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953266).

Figure 1

‘Studying Bicoid-dependent gap gene responses using a stimulus-response approach in single-input embryos.(a-b)’ - Figure 1a-b is good for readers not familiar/specialists in the topic to understand gene networks and how they are conventionally characterised.

‘Illumination also produced nuclear export of similar magnitude and spatial precision, with a 4-fold change in nuclear concentration between dark and light conditions and a spatial precision of ∼10-12 μm (1-2 cells) (Figure S1b-d)’ - This is an important point, may be worth showing it in the main figure.

Figure 2

Figure 2A ‘Bicoid was fused to various fluorescent proteins at its N terminus and to the LEXY optogenetic system at its C terminus. 450 nm light illumination exposes LEXY’s nuclear export sequence (NES), leading to an expected decrease in Bicoid transcriptional activity.’ - For clarity, it may be better to show Bcd inside or outside the nucleus in each condition?

Figure 2C ‘Larval cuticles for Bcd-LEXY variants in dark and light conditions. Anterior head and posterior tail structures are indicated with the outlined and shaded arrows. Illuminated embryos exhibit loss of anterior structures or duplication of posterior structures, indicating progressive loss of Bicoid activity.’- Suggest adding a line below each genotype "X-Bcd-LEXY, bcd[E1]/bcd[E1]" encompassing both 'dark' and 'light' panels; to make it clearer that for each genotype there is a control and a light-treated embryo.

Figure 2 f-g ‘EGFP-Bcd-LEXY time course during cycle of optogenetic activation and deactivation. Representative images are shown in f; quantification during a nuclear cycle 14 (NC14) time course of light and dark exposure shown in g. (h) Quantification of import and export kinetics for five LEXY constructs is shown in h; similar kinetics of translocation are observed for fluorescent Bicoid variants and non-Bicoid-containing LEXY constructs.’ - We understand that both panels report EGFP-Bcd-LEXY, with the left panel being after light stimulation (blue border) and the right panel after the light is removed (grey border). Could this be clarified in the figure legend? Also clarify whether time points are in minutes. Would it be possible to comment on the change observed with light in Figure 2f, is that significant?

‘We also measured expression of the canonical Bcd target gene Hunchback (Hb) in embryos harboring each of the three Bcd-LEXY variants (Figure 2d-e)’ - It may be useful to report the images depicting the staining pattern of Hunchback.

‘Nevertheless, all three variants were expressed at higher levels than wild-type Bcd, suggesting that fusion to LEXY and/or a fluorescent protein also partially interferes with Bcd function’ - mCherry-Bcd-LEXY expression seems to be similar to wild type levels, even though it does not rescue very well the bcd mutant phenotype or the Hb expression levels. mCherry tends to form aggregates, could this explain why this construct shows the weakest rescue? This could be discussed along with the interference by the LEXY domain.

‘nos-tub bnt, are devoid of all three A-P patterning cues and produce a posterior-like gene expression state throughout the embryo (Figure S2a).’ - The manuscript reports use of the triple mutant line bnt (mutant for bcd, nos and tsl) plus nos-tub, but in Figure S2a the genotype is stated as a double mutant, for nos and tsl but not for bcd. Is this correct?

Figure 3

‘Spatially-uniform, single-input embryos to enable optogenetic interrogation of specific gap genes.’- suggest adding a wt panel, showing what's on f but in the same way as b-e panels.

‘Mapping approximate embryonic positions represented by dark and light conditions in each genetic background. Bottom: diagram from Ref. 2 quantifying gap gene expression as a function of A-P position, with posterior = 100% EL. Top: the approximate position based on gap gene expression for each optogenetic variant under in illuminated (open circle) and dark conditions (blue circle).’ - Is panel f a schematic rather than a data figure? If so, could this be specified in the figure legend.

‘This pattern was altered dramatically in the presence of uniformly expressed Bcd-LEXY, which drove an anterior-like of gt and hb transcription in the dark, shifting to a mid-embryo-like state of hb and Kr transcription in the light (Figure 3c). Uniform iRFP-Bcd-LEXY embryos transcribed gt, hb, and Kr in the dark, shifting to Kr and kni expression in the light (Figure 3d).’ - From the characterization experiments in Fig1, the Bcd-LEXY is a stronger inducer of anterior-like pattern than iRFP-Bcd-LEXY showing a wider Hb pattern. However in the dark, Hb transcription seems more pronounced in iRFP-Bcd-LEXY than Bcd-LEXY, could this be commented on?

‘2P excitation is ideal because it can be used for simultaneous GFP and mCherry imaging without triggering AsLOV2 excitation (Figure S3b; Video S5-6)’ - This setup is a great approach to combine optogenetic stimulations while still allowing to use other fluorophores like GFP.

Figure 4

Figure 4b ‘Example of light stimulation and two-color imaging of NLS-mCherry-LEXY and MCP foci for a gt MS2 transcriptional reporter. Images show ventral regions of representative embryos in the absence or presence of a 450 nm light input delivered in a stripe in the middle of the embryo.’ - Is it possible to report the length of stimulation?

Figure c-e - The scheme of representation used to depict which regions of a wildtype embryo are recapitulated by each of the uniformly expressed embryos is really helpful.

Figure 4i - Really good way of summarising the data for individual figures, to help readers remember the dynamics of specific genes in response to their regulators.

Figure 5

‘​​constant-light and constant-dark stimuli were used as controls’- would it be possible to comment on the lack of difference here?

‘Conversely, a light-induced increase in nuclear Bcd triggered a similarly rapid but smaller-amplitude decrease in kni transcription (Figure 6b). Just as in the case of gt, the stability of the high-kni-expressing state may be indicative of positive autoregulation of kni expression by its own protein product. Together, these data suggest that Bcd can act as an apparent repressor of kni expression, an unexpected role for Bcd which is typically considered to perform only transcriptional activation functions. The initiation of kni transcription within 2 min after Bcd nuclear export is only compatible with a direct regulatory link, not Bcd-induced expression of an intermediate repressor.’ - Very interesting data. As a possible follow up, it may be worth testing the tagged Bcd-LEXY constructs since they have different levels of activation and will have a different potency as repressors.

Figure 6

Suggest to include images along with the quantification.

Discussion

The authors have done an amazing job of introducing and efficiently utilising an approach to study the regulation of gene networks in this study. While understanding the morphological outcomes of the embryos may not be a key result in the study, it may be relevant to include some discussion on how combining morphological data along with the gene expression networks and how different the uniformly expressing embryos are from wild type embryos, with respect to comprehending the physiological proximity of their system.

Methods

‘The construct was further recombined to the Sp marker on the same chromosome to mark the transgene’- Probably not an issue but since Sp is an allele of Wg, is it possible to clarify if this may have an influence on the interpretation of the results?

‘We created a custom MATLAB script to analyze and visualize time-lapse MS2 counts.’ - A great asset of the work is the use of MS2 foci for quantifications instead of simple fluorescence intensity or other more indirect assays. This provides a direct numeric approximation for the perturbations.

This review reflects comments and contributions by Madison Bolger-Munro, Rachel Lau, Arthur Molines, Sónia Gomes Pereira, Mafalda Pimentel, Mugdha Sathe, and Wasim Sayyad

In this paper the authors test how mitotic entry is controlled by mechanical confinement or mechanical stimulation. Using a set of quantitative fluorescence experiments, they conclude that confinement triggers the dissolution of the nuclear envelope and eventually allows the cyclin B1 to translocate into the nucleus, thus triggering the G2/M transition. Further, using a set of small molecule inhibitors, the authors demonstrate that this mechanochemical pathway involves the LINC complex, cPLA2 and actomyosin contractility, while microtubule-related signalling is dispensable. The study is well-controlled and convincing.

Regarding the terminology, in the text the terms 'confinement', 'mechanical stimulation', and 'mechanical force' are used interchangeably, but these are not always necessarily equal to each other. For instance, the majority of the experiments are performed using long-term confinement of cells in PDMS chambers which flatten the nucleus. Does the mechanical stimulation on the nucleus persist through this long-term confinement, instead of being gradually dissipated? In other words, is it possible to separate the effect of confinement from the effect of mechanical stimulation? Figure 5 partly addresses this issue while testing for mitotic defects after 4 minute-long confinement. If the import of cyclin B1 into the nucleus could be tested after a short confinement like this, it would strengthen the case for mechanical stimulation triggering the mitotic entry as opposed to long-term confinement itself.

‘This signal relies on nuclear unfolding during the G2-M transition, which activates the stretch-sensitive cPLA2 on the nuclear envelope. This activation upregulates actomyosin contractility, determining the spatiotemporal translocation of cyclin B1 in the nucleus.’ - There is a question as to whether the causality between these events has been shown. The study reports interesting discoveries: artificial confinement induces a faster rate of nuclear cyclinB1 import, dependent on importin, but independent of KASH, actomyosin, cPLA2 and Ca2+. Because cPLA2 is recruited to the "unfolding" NE in prophase and in confined cells, and because inhibition of KASH, actomyosin, and cPLA2 decreases the rate of cyclin B1 import in the absence of confinement, the paper extrapolates that this pathway is generally used as mechanosensing pathway to determine mitotic timing. If cPLA2 was indeed transducing nuclear deformability into lipid second messengers, the inhibition of cPLA2 should probably not be rescued by mechanical confinement. Moreover, AAOCF3 treatment should significantly impact cyclin B1 nuclear import, which does not seem to be the case (only relative fluorescence values are shown, but NEP does not seem to be affected).

In the Abstract or early in the main text when the concept and players are introduced it would be nice to say what organism or kind of cells are referred to as these proteins are conserved across eukaryotes.

‘its mainly cytoplasmic localization’ - could be written as "is mainly cytoplasmic" or is "mainly localized in the cytoplasm".

‘RPE-1 cells expressing H2B-GFP/tubulin-RFP’ - The experiments demonstrate how the mechanical signal may regulate cyclin B1 translocation to the nucleus via cPLA2 and actomyosin contractility. Would this mechanism be universal across human cell lines or is it just specific to RPE1? Could this be discussed as future work.

‘Under these conditions, cells failed to enter mitosis’ - This aspect could be documented more as it is the baseline for the rest of the experiments. It is unclear from the result or the methods how long the cells were in the PLL-g-PEG before they were imaged. Could it be that they take longer to enter mitosis after the transfer? Fig 1G suggests that cyclin B1 accumulates in the nucleus in CTRL cells at a similar rate to the confined cells. The confinement ‘boosts’ the accumulation at the beginning of the experiment (in the first minute or so) but after that rates look similar. Is there a possibility that CTRL cells eventually divide but after a longer time? If that is the case, then it challenges the statement ‘cells failed to enter mitosis’ and the idea that adhesion is necessary. Would it be possible to perform some cell counting experiments over multiple days to show that cell concentration does not change?

‘acute mechanical stimulation’ - ‘mechanical stimulation’ is conceptually different from ‘confinement’. Can more details be provided here to clarify? From the figure it seems that the "suction cup" method was used to squeeze the cells. What was the control parameter in the experiments? Cell height? Applied pressure?

'mechanical confinement is sufficient to overcome the lack of external stimuli’ - Here ‘external stimuli’ can be replaced by ‘the adhesion’ as mechanical confinement is also an external stimulus.

‘after stimulation’ - What happens if the stimulation is transient? Let's say squeeze for 5 minutes then release? Is this enough to induce mitosis?

‘normal and confined conditions (fig. 1d,e)’ - Would it be possible to clarify when dynamic or static confinement set ups are used? Based on the ‘confinement’ label on top of some time lapse panels, it seems like the dynamic set up is used most often. Please report the pillar height for the dynamic confinement set up. There is little difference in nucleus shape and size when confinement is applied vs before (or vs control cells in most cases), except for Fig1a-b and supp Fig1a. Is it certain that the confiner without vacuum is not exerting any effect on cells already? An ideal control should be in the confinement device without vacuum, but it seems cells in 2D FBN dishes are used. Can this comparison and set up be discussed?

‘We defined time zero as the lowest fluorescence intensity levels of nuclear cyclin B1 and quantified its increase as cyclin B1 translocated to the nucleus, up until NEP’ - This statement seems to refer to the graphs in Fig1g but in Fig1d and e, Time 0 is at NEP. It might be useful to align the time indicated on the images in d and e with the time used for quantification so that it is easier to connect the images to the graphs. Also, the experimental timings could be clarified: If fluorescence was quantified until NEP, it is NEP that sets the 300ms timing (instead of 0ms being set by ‘lowest fluorescence intensity levels of nuclear cyclin B1’). These data imply that control cells had a lower fold increase (slower) in terms of nuclear cyclinB1 in the 300ms preceding NEP. How different are the absolute levels of nuclear cyclinB1 in CT vs confined before NEP?

‘This was not due to a rupture of the nucleus, as we could not detect any obvious tears or gaps in the NE when RPE-1 cells expressing Lap2β-mRFP were mechanically stimulated’ - It is difficult to tell for sure that the nucleus does not rupture from the 2D image in Supplementary Fig 1a. Perhaps it may be possible to try expressing NLS-GFP to show there is no leakage of the GFP signal into the cytoplasm during confinement.

(Supplementary Fig. 1a) - Were these cells also in late G2? How long does it take to unfold after confinement? How do the timings relate with nuclear cyclinB1 increase? In the 300ms quantifications, the increase seems to happen >200ms before NEP. How long is that after the confinement?

‘and resulted in an increase in the distance between neighbouring NPCs (Supplementary Fig. 1b, c; \**p<0,001), when compared to unconfined cells*’ - Did the nuclear volume change? If so, it would also change the concentration of everything in the nucleus and may affect the signaling pathway?

Figure 1

• Figure 1a - it would help to include in Fig1a a schematic of the confinement chamber and the following treatment, to make the concept easier to grasp for non-specialists.
• Figure 1b - the middle panel seems to have a misplaced scale bar in yz.
• It is confusing to see the H2B-GFP shown in magenta and tubulin-mRFP in green. Please clearly mention in the legends, and also in the legend of subsequent figures.
• Figure 1h ‘RPE-1 cells expressing cyclinB1-Venus/tubulin-mRFP dividing on a FBN coated substrate, treated with DMSO (top panel; n=15), a CDK1 inhibitor (middle panel, CDK1i; RO-3306; n=23) or with CDK1i and confinement (bottom panel; n=17). Time is in min:sec and time zero corresponds to NEP. Images were acquired with a 20 sec interval. Scale bar corresponds to 10μm’- Figure 1h demonstrates that confinement reversed the effects of CDK1 inhibition as cyclin B1 translocated to the nucleus. It may be nice to allow for visual comparison if the timepoints for CDKi (middle panel) and CDKi and confinement (bottom panel) were the same. Similarly, it may be nice to have the same timepoints for Supplementary Figure 2 and other figures after time zero (e.g Figure 4) for comparison.
• Figure 1k - This Y axis starts on 0.5. Since intensities are normalized to time 0ms, graph visualization and comparison could be strengthened if all Y axis started closer to 1.
• How CDK1 is blocked by inhibitor RO3306 needs a brief explanation as given for Importazole treatment.

‘Interestingly, confinement was sufficient to overcome the inhibition of CDK1 and force translocation of cyclin B1 to the nucleus (Fig. 1h, j; p<0.001). However, these cells failed to enter mitosis, as nuclear envelope permeabilization (NEP) was blocked by CDK1 inhibition31,32.This observation further strengthens the idea that mechanical stimulation per se does not affect the barrier function of the nucleus’ - If we understood correctly, under confinement cyclin B1 translocates into the nucleus independently of CDK1 activity. However, without CDK1 activity NEP doesn't take place and cells don't undergo mitosis. So, although there might not be a general effect on nucleus permeabilization, there is clearly an effect on the entry of certain proteins (like cyclin B1). How is CDK1 getting translocated in the presence of the inhibitor + confinement? Especially so when the NEP is blocked by the inhibitor. Does this mean that CDK1 translocation to the nucleus doesn't depend on its activation? Could these aspects be discussed further, or perhaps a scheme added to the figure, to guide the readers through the mechanism of CKD1-Cyclin B1-NEP-mitosis, and how this is changed by CDK1 inhibition and confinement.

Supplementary Figure 2 - Panel b lacks a scale-bar. Additionally, tubulin is not as visible in panel b as in a, for comparison purposes it could be a good idea to apply the same threshold to both images.

‘​​Similar delays in cyclin B1 translocation’ - The control experiment (30 min DMSO) is not shown in the graphs. Nevertheless, it is understandable that the treatments slightly decrease the rate of cyclinB1 import compared to the controls in other panels. It may be useful to calculate the rate over time, for comparison across experiments.

Fig. 2d; supplementary Fig 3a.**p<0.01 - The p-values seem to compare with and without confinement, not with and without drug. They are mentioned again below (‘Importantly, mechanical stimulation not only reverted … but also accelerated both cyclin B1 accumulation and NEP in cells where actomyosin contractility was inhibited Fig. 2d-h; p<0.01; *p<0.001’).

Supplementary Figure 3 - Panel d lacks a scale-bar. In some of the conditions it is a bit hard to check exactly when CyclinB1 is considered to have been translocated to the nucleus. Would it be possible to add a small panel on the figures, as in Figure 1?

Figure 2

• Figure 2b ‘in control non-confined cells expressing the DN-KASH mutant (black) and confined cells expressing the DN-KASH mutant (green)’ - It is not clear that the colors from the legend and graph correspond, can this be clarified?
• Figure 2h ‘Time lag between cyclin B1 and tubulin nuclear translocation in DMSO-treated, non-confined cells (black), cells with different treatments (green), and confined cells in the same conditions (magenta).’ - It may be clearer to say confined cells with different treatments? Please also clarify what comparisons the statistical analyses correspond to. It is assumed that this is the non-confined vs. confined cells in each treatment, but it could be added to the figure legend.

‘and an actomyosin-dependent translocation of cyclin B1 into the nucleus’ - While in normal conditions there's a delay in cyclin B1 translocation to the nucleus upon actomyosin perturbation/inhibition, under confinement this translocation appears to go faster when actomyosin is perturbed/inhibited. So, it is expected that the confinement can somehow overcome the need for the actomyosin contractility and lead to independent translocation. In the absence of actomyosin, cyclinB1 import rate is lower, but it is not clear if there is a connection to mechanical cues for the translocation of cyclinB1. Can this fragment be clarified?

Figure 3 - What is C-Pla2 tagged to?

‘​​Indeed, inhibition of cPLA2 activity with AAOCF3 led to a significant decrease in cyclin B1 nucleoplasmic shuttling (Fig. 3f, h, i; p<0.01’ - This p value is for AAOCF3 with and without confinement, not for AAOCF3 vs DMSO.

‘This likely occurs due to confinement-induced unfolding of the NE (Supplementary Fig. 1), that is sufficient to bypass the pharmacological inhibition of contractility and still induce an increase in NPC distance’ - The data suggest that mechanically induced cyclin B1 import is dependent of importin and independent of actomyosin, cyclin B1 activation, Nesprin, Calcium and cPLA2. How could NE unfolding and NPC distance explain the increased rate of cyclin B1 import? Perhaps reference 23 should be cited here, instead of above regarding the cPLA2 hypothesis, which the authors proved to be uncoupled.

‘To confirm this hypothesis, we seeded cells in a soft hydrogel (5kPa) or in a rigid glass, inducing low or high nuclear tension, respectively’ - Is this known from the literature (if so could a reference be provided) or was it established in this study?

‘indicating that increased tension facilitates mitotic entry’ - The study used glass vs hydrogel, instead of hydrogels with different stiffness. This should be taken into consideration when stating claims that the difference here is due to the change in NE tension.

Figure 4 - Please add a scale-bar to the lower panel.

‘Fig. 5e; 24±7 min for controls vs. 36±20 min’ - should this be 5f? These values don't seem to match the graph in panel F.

‘promote cyclin B1 nuclear translocation by permeabilizing the NE with laser microsurgery, therefore anticipating mitotic entry’ -Does this relate to prophase cells? In this experiment, rupturing the nucleus gives the same result as the mechanical perturbations. Additional evidence may be valuable to show that the confinement/mechanical perturbations do not cause nuclear rupture.

Supplementary Figure 5 - The lower panels are missing scale-bars

Figure 5

• 'Centrosomes angle at the time of NEP' - angles between centrosomes and long nuclear axis at the moment of NEP.
• ‘​​laser surgery was performed on the cytoplasm’ - For clarity, the top panel could say "cytoplasm surgery" and the bottom panel "NE surgery".
• ‘mechanical pathway based on nuclear tension’- Showing that nuclear tension changes would strengthen the claims. Could this be done with a FRET sensor to show that nuclear tension occurs when cyclin B1 translocation occurs? It is important to show that all the mechanical perturbations tried directly change nuclear tension.
• It is not clear that the experiments performed directly test the role of the NE tension as most perturbations used are pleiotropic in nature and NE tension is not measured directly (admittedly very hard to do). Is there a way to alter NE tension more directly? Maybe a lamina mutant, or a mutant with excess NE, or a transient hypotonic shock?

‘cyclin B1 translocation’ - How could cyclin B1/CDK activity be altered by mechanical stimulations (other than with the inhibitor)? Would it be possible to show that cyclin B1 is activated across the different mechanical stimulations with a western blot? It would be nice to address if the mechanical perturbations activate cyclinB1/CDK1 to induce its translocation into the nucleus or if the mechanical perturbations cause the translocation of not active cyclinB1/CDK1. Some more discussion on this idea would be useful.

‘In agreement, we were able to rescue cyclin B1 shuttling to the nucleus by mechanical stimulation, even in the absence of cPLA2 activity or actomyosin contractility’ -According to refs 24 and 28, mechanical constriction is upstream of unfolding, NPC spacing, and cPLA2 increase, which will later result in cortical (not perinuclear) actomyosin activation. The authors show that cyclin B1 is translocated under confinement independently of every condition tested, except for importin. Can the order in which the described events take place, and their interdependence, be clarified?

‘Establishing mechanical forces as a determinant for cyclin B1 nuclear translocation and mitotic entry raises the interesting possibility that the nucleus might act as a sensor for external forces’ - Could the level of conservation of the proteins involved across eukaryotes and the generality of this mechanism be discussed? Could such a mechanism work in organisms with a cell-wall like plants or yeast or organisms that have no lamina?

Methods

• Recommend using concentrations (M, v/v, g/v) instead of dilutions and volumes; can the cell cycle stage of cells imaged, height of pillars, type of confinement and controls be reported?
• ‘RPE-1 parental and RPE-1 H2B-GFP and tubulin-mRFP were already available in our lab’ - Were these previously published? If so, please include the citation. If not, could you report where these were obtained or how they were generated.
• Cell confinement setup - a scheme of these manipulations would be very useful for non-experts to understand the experimental set-up and design.
• ‘correlate the angle between the centrosomes and the nucleus, at the moment of NEP.’ - the nucleus and long nuclear axis.

This review reflects comments and contributions by Joachim Goedhart, Arthur Molines, Attya Omer, Sónia Gomes Pereira, Bhatia Sonam, Rajan Thakur. Review synthesized by Sree Rama Chaitanya Sridhara.

NOTCH1-mediated cellular signaling determines the fate of developing cells during embryogenesis and also helps maintain tissue homeostasis (e.g., blood vessel formation, nervous system plasticity, and bone regulation). NOTCH1 performs a plethora of biological functions by maneuvering gene expression patterns. Furthermore, anomalies in NOTCH1 signaling cause pathological conditions in humans. For example, in breast cancer, NOTCH1 signaling regulates cancer stem cells, proliferation rates, metastasis, and chemosensitivity. Hence, it is important to understand the spatio-temporal regulation of NOTCH1 signaling in mammalian cells. Therefore, the authors of the current preprint engineered a light-sensitive NOTCH1 in mammalian cells to investigate the NOTCH1-mediated gene expression program and its functional impact on breast cancer cells.

The preprint reports the major findings clearly, we lay out some suggestions that can improve the clarity and robustness of the reported findings.

Major comments

1.‘To facilitate the expression of both proteins in cells, we joined them through a P2A (porcine teschovirus-1 2A) “self-cleaving” sequence so that both could be expressed within one reading frame (Results, Fig 1B).’ - An excess of ZDK-NICD could lead to a constant activation. It’s advisable to look at the expression level of each of these proteins (e.g., immunoblots).

2.‘The P2A is a self-cleaving peptide, which enables the formation of two separate proteins after translation (Fig 1C). In the dark, LOV2 and Zdk1-NICD are located at the cell membrane. Upon release from the cell membrane, the hN1ICD translocate to the nucleus where it acts as a transcriptional activator together with CSL and MAML (Fig 1A).’ - These findings may require further experimental evidence or additional discussion. Since this is a proof-of-concept study, there should be sufficient details that the system is working as planned. This includes: testing the membrane localization of the receptor and the levels of the two peptides separated by the P2A, and the translocation of the NCID domain. It may also be relevant to check if the two separate proteins are actually synthesized (using immunoblots), to test the optimal cleavage. While some functional activation of the NOTCH pathway is reported by over-expression of HES1, HEY1 (presuming that the NICD is translocated) it would be helpful for the readers to visualize it.

1. While the initial experiments were performed in HEK293 cells, the latter was done in breast cancer cells. It is relevant to repeat the same initial experiments in breast cancer cells to make sure the reporter system is working in breast cancer cells.

4.‘The γ-secretase inhibitor DAPT (10 μM) was used to rule out an effect on the growth of spheroids by endogenous Notch activity.’- Please report the data for this statement. The following conditions could be tested to gauge the full effect of the system: untreated control, DMSO control, 10uM DAPT, Light, and Dark.

5.Figure 3 -There does not seem to be a clear difference between light and dark exposed spheroids. Also, the Y-axis is different between the graphs. It might be helpful to keep the Y-axis constant. Additionally, performing more experiments and imaging at higher magnifications could increase the robustness of the data.

6.‘Notch activity was blocked by a γ-secretase inhibitor (DAPT). Using optoNotch we can modulate receptor activation to discriminate between different modes of NOTCH1 activation, as suggested by differential binding and force used by each of the ligands.’ - It is advisable to report data supporting this statement.

7.The authors have shown that 1hr is the timepoint that induces the highest changes to gene expression (Fig. 1). However, some of the experiments were performed with light pulses for 3hrs (Fig. 2). Please clarify if there is a reason for using a different duration across experiments.

1. ‘The total content of N1ICD in both of these stable cell lines was substantially higher than in WT cells (where there is some endogenous N1ICD) (Fig 2B-C).’ Please provide quantification for this data. Also, is there a way to differentiate between the WT N1ICD and the engineered one? If so, could an explanation be provided.

Minor comments

1.Abstract ‘Here, we circumvent this step by regulating Notch activity by light. To achieve this, we have engineered a membrane-bound optogenetic NOTCH1 receptor (optoNotch) to control the activation of NOTCH1 intracellular domain (N1ICD) and its downstream transcriptional activities’ - For clarity, this fragment could be rewritten as follows: ‘Here, we report on a system that allows the activation of Notch signaling by light. To this end, we used the heterodimerization between the photosensitive protein LOV and Zdk. The interaction between the membrane located LOV protein and the Zdk fused to N1ICD is lost upon illumination with blue light. As a consequence, the N1ICDactivates downstream transcriptional activities.’ Strictly speaking, the engineered optoNotch is not a membrane bound receptor. The intracellular activating domain has been tethered to the plasma membrane using a heterodimerizing system that is sensitive to light. Light-induced dissociation releases the intracellular domain and triggers downstream activities.

2.Recommend spelling out abbreviations e.g., ICD, CSL, MAML.

3.'avoids' instead of ‘allows avoiding’ (Introduction).

4.Results ’via the Myristoylation-targeting sequence (MTS)’ -The consensus sequence is MG. Is that used here, or is the Myristoylation-targeting sequence (MTS) a longer peptide (as it is usually used to increase membrane labeling efficiency)?

5.Recommend checking that the figures have sufficient resolution after conversion of the document to PDF.

6.Consider illustrating a cartoon of the natural NOTCH receptor domain structure in Fig. 1 (for readers unfamiliar with NOTCH signaling).

7.Results ‘This reaction resulted in a very low luciferase signal after photoactivation of the optoNotch system no matter the length of activation (0.05-second pulses for 1, 3, or 12 hours) (Fig. 1E).’ - Before moving to the results, the text could include an explanation for the engineered target gene CSL-LUC that is used as a read-out here. Also, consider illustrating it in Fig 1.

8.Include the remnant of the P2A peptide in Fig 1C. The P2A leaves a 'scar' and it is important to include this information.

9.Keep the axes and scale consistent in Fig 1 E-H.

10.Data related to Fig 1H – was this measured in cells expressing WT LOV2 or the LOV2 V416L?

11.‘All three genes showed increased expression in light-activated cells.’- Is it known, and can it be discussed how this compares to the natural activation of the NOTCH receptor. This would be useful to mention even if unknown.

12.‘To confirm the influence of the optoNotch system on breast cancer development under physiological-mimicking conditions.’ - As the tests measure growth properties rather than development, recommend reframing the sentence.

13.Please state what the error bars represent in Fig 3A-D?

14.Molecular cloning - could the full sequence of the optoNotch system be reported (for instance as supplemental).

References

Please kindly cite relevant references:

• ‘receptor upon mechanical pulling by any of the ligands’ (Introduction).
• ‘LOVTRAP’ (Results).
• pTriEx-NTOM20-LOV plasmid, Zdark-1 (Zdk1) sequence from pTriEX-mCherry-Zdk1 (Methods, Molecular cloning) - Please cite the sources or cat.no.
• ‘to the original protocol’ (Methods, Molecular cloning).
• ‘MCF7 and MDA-MB-468 stable cell lines’ (Methods, Immunostaining and Flow Cytometry) - Please cite sources if not generated by the lab.

This review reflects comments and contributions by Sónia Gomes Pereira, Julia Grzymkowski and Wasim Sayyad.

The authors reported an improved method called nano-PrOteomics sample Preparation (nPOP) to quantify single-cell proteomics and its use in cell cycle analysis. The high throughput sample preparation method uses CellenOne cell sorting and liquid handling system which overcome the limitations of their previously proposed method Minimal ProteOmics sample Preparation (mPOP method) by reducing the sample volume 100-fold to a few nanoliters. The nPOP Workflow is explained very clearly. The Cell Cycle Analysis data presented in Figure 4 is an interesting validation of the technique, which seems really useful for single cell-omics studies.

The text in the paper could be tidied up slightly, especially surrounding Figure 1. Some minor issues which can be easily addressed:

• Please provide the full forms of some of the abbreviations used (e.g. SILAC, SP3, iST, RI, or TMT).
• There are some missing labels of subpanels in the Figures or the text. E.g.

a) Subpanel Fig. 1c and 1d mentioned in the text are missing in Fig. 1 as well as in the legend.

b) On page 5 and line 7 (PDF file), Fig. 2b should be mentioned at the end of the sentence “After 20 minutes of cell lysis, …”.

c) On page 7 (PDF file), ‘The relatively low CV values indicate that protein quantification derived from different peptides is internally consistent, Fig. 3c.’ - Fig. 3c should read Fig. 3b.

‘The samples were then diluted and combined for digestion. These results suggest that DMSO allows for efficient cell lysis without detectable biases against proteins originating from different cell compartments, Fig. 1a’ - There may be a sentence missing here to explain the graph in Fig. 1a. The first sentence describes the last part of the method and the next sentence starts with "These results suggest…", though no results were discussed. Perhaps the reference to Fig. 1a should be updated to refer to other figure panels.

‘We lysed U-937 monocytes and Jurkat T-cells with both DMSO and urea and compared the protein ratios estimated from the cells lysed with DMSO and with urea, Fig. 1c. U-937 monocytes cultured in heavy SILAC media and Jurkat T-Cells cultured in standard media were combined in equal amounts and lysed with either 90 % DMSO or 6M urea as shown in Fig. 1c.’ - Consider condensing this fragment into a single sentence.

‘This gave us further confidence that DMSO lysis is well suited for miniaturizing sample preparation on an open surface without using MS-incompatible chemicals’ - The confidence in using DMSO for lysis and therefore avoiding the use of "MS-incompatible chemicals" is supported. However, based solely on the information given in the paragraph and in Figure 1a, it is unclear how conclusions can be made about "miniaturizing sample preparation on an open surface," because it looks like the method was done in centrifuge tubes. Can more detail be provided about steps taken to establish whether DMSO lysis is suited for this miniaturization?

‘The addition brings the total volume to 13.5 nl, Fig. 2a. Samples are digested by 75 ng/μl trypsin for 5 hours’ - In the Methods it is stated that the volume is 14 nl and the trypsin concentration is 100 ng/ul. Suggest checking these details for consistency and accuracy. As in Fig. 1a the trypsin is given in nl (10nl), maybe both values should be in the text (e.g. 10nl of 75 ng/ul).

‘room temperature’ - Room temperature can mean different values in different countries. Could the authors clarify or provide a range of temperatures? This might be particularly important when taking into consideration the drying of samples with such reduced volumes.

Figure 2

• ‘a) A schematic of the nPOP sample preparation method illustrates the steps of cell lysis, protein digestion, peptide labeling with TMT, and quenching with two additions of hydroxylamine’ - The drying of the droplets appears to be done without the use of the nozzle. If this is correct, suggest removing it from this particular stage on the scheme. Also, please note that each incubation with hydroxylamine was indicated as lasting 20min (so a total of 40min), but the scheme indicates 1h.
• A suggestion for the visualization of Figure 2a is to divide the sections using vertical dashed lines into the following sections: Cell isolation - Lysis – Digestion - Labeling – Quenching - Pooling
• ‘(c) Total ion current chromatograms from three runs demonstrate low contaminants and consistent chromatography’ - This could be discussed in the text.

‘To improve the recovery of labeled peptides, the footprint of each array is washed by 4 μl of acetonitrile, which is collected and added to the corresponding combined set. This wash is option and used to maximize the recovery of labeled peptides from the slide.’ - Suggest rephrasing this fragment to "To improve the recovery of labeled peptides, an optional step of washing the footprint of each array…"

Figure 3 ‘The consistency of protein quantification is estimated as the coefficient of variation (CV) of the relative levels of peptides originating from the same protein. The median CVs per cells are tightly distributed, suggesting high consistency of sample preparation’ - It is not immediately clear what the pink and black numbers of cells represent; this could be clarified in the legend or in the text.

‘Similar to previous analysis the protein ratios in bulk samples agreed well with ratios in the single cells single cells’ - ‘single cells’ is duplicated.

‘A Pearson correlation of 0.8 indicates that the single-cell protein quantification is consistent with the proteomic measurements of established bulk method’ - Fig. 3d should be mentioned at the end of this sentence.

Figure 4

• Figure 4b, 'The boxplots display distributions for correlations between the phase markers and proteins from the large ribosomal subunit assembly GO term'- In the image itself it's written "Microtubule-based process," so please clarify what is shown on the boxplots.
• Figure 4c - as in b, please clarify in the legend what the boxplots represent.
• Methods ‘for a final concentration of 100 ng/μl of trypsin’ - as mentioned above, in the main text the value indicated for trypsin concentration is 75ng/ul, can this be clarified?

This review reflects comments and contributions by Julia Grzymkowski, Jake Herman, Yogaspoorthi Subramaniam, and Vladimir Volkov.

Alex Thompson and colleagues characterize the molecular defects of four previously documented and one newly described disease-associated mutation in the human KIF22 chromokinesin. They describe the patient’s history and go on to study the function of this kinesin in mitosis by expressing known pathogenic mutations of KIF22 in HeLa and RPE-1 cells.

After rejecting several hypotheses, the authors conclude that the mutations affect the fidelity of anaphase by an excessive polar ejection force. This work provides compelling evidence that the mutations prevent the normal silencing of this chromosome-associated kinesin motor during the final step of chromosome segregation. This failure to silence KIF22 results in chromosome segregation failures, abnormal spindle morphology, and proliferation defects.

This study was executed with robust controls and the conclusions presented are clearly represented by the data. A majority of the phenotypes were assayed in multiple cell lines and useful 'negative' results were reported (e.g. mutations do not alter the dynamic nor steady state localization of the protein). The imaging data are beautiful and sufficiently explained, the data is striking. The paper is well written. Altogether the work sheds light on important fundamental and medical questions.

There is one conceptual question which may be relevant to address to strengthen the interpretations. Mutant KIF22 constructs are introduced via two inducible pathways in the background of siRNA depletion of the endogenous KIF22, and the resulting expression levels of the mutants exceed the endogenous KIF22 by 2-3 fold, as judged by immunofluorescence. The polar ejection forces are then estimated by treating cells with monastrol and quantifying the distance between the spindle monopole and the chromosome ring around it. However, even expression of wild-type KIF22 led to elevated polar ejection force (Fig 3D), raising a question about whether overexpression of KIF22, rather than mutations, produces this phenotype. Is it possible to modulate the expression levels to address this question? At least, can the levels of KIF22-GFP in individual cells be correlated to the pole-chromosome distance in this experiment? The same question applies to the description of chromosome recongression phenotypes.

General comments:

• How complete is siRNA depletion of endogenous KIF22? Can a western blot be provided to test this?
• In the FRAP experiments, which regions were photobleached at different cell cycle stages?

Introduction

The introduction could feature more about the molecular aspect of KIF22 mutations and shorten the paragraph(s) about the disease pathologies.

‘These mutations occur in adjacent residues P148 and R149 in the α2 helix of the KIF22 motor domain (Figure 1B). P148 and R149 are conserved in kinesin-10 family members across species (Figure 1C) and in many human members of the kinesin superfamily (Figure 1D).’ - In addition to the mention in Figure 1A, it would be useful to describe the specific four KIF22 mutations in the text. Have mutations to the paralogous Pro and Arg residues in other kinesin proteins been correlated with pathologies?

‘However, KIF22 knockout in mice did not affect skeletal development’ - Often, a complete KO of a gene is relatively less/non-detrimental than a dominant mutant ascribing to its (im)proper functions. Given that, comparison with mutant mice may be more relevant than comparisons with KIF22 KO mice.

Results

‘(RPE-1) cell lines expressing wild type and mutant KIF22-GFP to assess any differences between the consequences of expressing mutant KIF22 in aneuploid cancer-derived cells (HeLa-Kyoto) and genomically stable somatic cells.’ - Based on the introduction, mutations in the KIF22 motor domain affect skeletal development severely and selectively. Epithelial cells such as HeLa or RPE may not fully represent the outcomes of KIF22 mutations, would usage of a chondrocyte cell line be relevant?

‘KIF22-GFP with pathogenic mutations demonstrated the same localization pattern throughout the cell cycle as wild type motor’ - Over-expression of KIFF22-GFP is several folds higher compared to the nascent content of KIFF22 in cells and the localization pattern remains unaltered. What does this imply with respect to force generated by excessive motor loading on microtubules and associated chromosomes?

‘Relative polar ejection forces were compared by measuring the distance from the spindle pole to the maximum DAPI signal (Figure 3A). Expression of mutant motor did not reduce polar ejection forces (Figure 3B and 3C).’ - Figure 3A is incredibly helpful and clear for interpreting the data. In Figure 3B, it is difficult to see overlap between GFP and DAPI when using green and blue, since the DAPI is already shown to the left, it may be more helpful to show only Centrin and GFP. In Figure 3B, is GFP signal similar between different mutants?

‘Together, the localization of mutant KIF22 and the ability of mutant KIF22 to generate polar ejection forces indicate that pathogenic mutations P148L, P148S, R149L, R149Q, and V475G do not result in a loss of KIF22 function during early mitosis.’ - Quantifying rescue phenotypes in terms of either force generated by them (activity) or protein content may be relevant to support this conclusion.

‘This phenotype was dominant and occurred in the presence of endogenous KIF22’ - In the introduction KIF22 mutants are described as dominant because they cause disease phenotypes as heterozygotes. Then the anaphase recongression and nuclear morphology phenotypes are described as dominant because they are observable without siRNA treatment. Recommend not applying the same term to both conditions because they are not equivalent. The outstanding characterization in Supplement 1 shows that ectopic KIF22 is expressed in excess of endogenous KIF22. Ectopic copies may also silence or outcompete endogenous KIF22 because ectopic KIF22 shows no decrease in mitotic localization when endogenous KIF22 is depleted (S1C and S1E). This is not a comment on the data but rather how they are discussed.

‘Additionally, the distance between chromosome masses at the time of cleavage furrow ingression was reduced in cells expressing KIF22-GFP R149Q or V475G, suggesting that the position of the chromosome masses may be physically obstructing cytokinesis’ - Does this mean that karyokinesis failed too, as the chromosome masses fail to sufficiently segregate?

‘was imaged in HeLa-Kyoto cells expressing fluorescent markers for the poles (pericentrin-RFP) and centromeres (CENPB-mCh) (Figure 5A)’ - It is difficult to see the centromere staining, can it be false colored to a different color than the poles?

‘Since expression of KIF22-GFP T463A does not cause anaphase recongression (Figure 8E), the level of compaction of the segregating chromosome masses was explored as a possible explanation for this modest increase in the percentage of cells with nuclear morphology defects.’ - How does KIF22A protein conformation influence availability/accessibility of T463 to phosphorylation by CDK1/cyclin B? head-tail auto-inhibition, does it occur due to masking of the T463 site?

This review reflects comments and contributions by Pablo Ranea-Robles, Gregory Redpath, Venkat Krishnan Sundaram, Rajan Thakur.

Determining the neurotoxic factors secreted by astrocytes has been an important area of study in the glial research community. In this study, Rooney et al. describe a mechanism to explain how inflammatory reactive astrocytes drive neurotoxicity. This scientific question is relevant as this mechanism is not well known but has a major contribution to neuroinflammatory and neurodegenerative diseases.

The preprint reports that upon induction of inflammatory response in hiPSC derived astrocytes there is significant lysosomal remodeling. Inflammatory reactive astrocytes have decreased lysosome acidification and degradative capacity with increased lysosomal exocytosis. Via pharmacological and genetic approaches it is shown that the neurotoxic effects of inflammatory reactive astrocytes could be mediated by increased exocytosis in these cells. This is demonstrated in vitro using Caspase 3/7 as a marker for neurotoxicity in neurons cultured with astrocytes conditioned media. The CRISPRi screen is an excellent tool that is smartly used in this study, and the finding of a strong inverse correlation between the pH and exocytosis phenotypes for many hit genes sustains the conclusions. The paper reports a number of hits in the mTOR pathway including mTOR itself, with very remarkable effects of mTOR KO in iAstrocytes on lysosome exocytosis in astrocytes and neurotoxicity.

The paper is interesting, the experiments sound and the findings are supported by the data. The manuscript is very well written and the findings clearly explained. The inclusion of all the uncropped blots and the corresponding replicates for the immunoblots is a great addition.

Some comments as well as suggestions for polishing the manuscript are offered below.

• It is recommended to provide further data to show clear evidence that basal level of autophagy is unaltered upon ITC treatment.
• There are certain discrepancies on LAMP1 expression on the cell surface post exocytosis. LAMP1 blot data should be correlated with log2FC value of LAMP1 in the proteome dataset.
• For most of the repetition experiments the paper reports ‘X’ independent wells for each condition. Are these wells (representing independent experiments) done from the same activated astrocytes which were split into ‘X’ wells or these were totally independent experiments? Please clarify if these are technical replicates or biological replicates.
• Images of cells with Hoescht staining could be provided, as it was used for normalization. It would be great to know how many images/ wells or cells/wells were used for this analysis.
• There could be further discussion about the results of the study. For example, the fact that OXPHOS genes were the top pathway among the hits of the CRISPRi screen, and how this relates to lysosomal exocytosis.
• Knocking out genes involved in exocytosis in iAstrocytes leads to a partial reduction in neurotoxicity. It may be worth showing some data to prove that the sgRNA targeted those genes and there is no functional protein. The manuscript could discuss in more detail what may be responsible for the remaining neurotoxicity.
• Readers may appreciate some further explanation for the selection of Caspase3/7 as the marker for neurotoxicity. Can this be complemented with other markers to strengthen the message?
• There is inconsistency around the presentation of statistical tests - some figures have them, some don't. Recommend that all graphs include relevant statistical tests. The bar graphs could be presented as just dot-plots.
• The vehicle is always presented beside the treatment condition. This makes sense, but in some figures the comparison of interest is between the other intervention (e.g., sgRNA target), in which case the graph may be easier to interpret with the vehicle +/- intervention and ITC +/- intervention beside each other.
• It is unclear how the TIRF images of lysosomal exocytosis add to the study, as they do not show clear exocytosis events that are consistent with published literature. Different videos displaying the distinct exocytic burst could be presented, the images retaken, or this data removed. The flow cytometry data on exocytosis is convincing enough as it is. If the authors desired another visual demonstration of lysosomal exocytosis, cells could be treated +/- ITC, cooled on ice, cell surface LAMP1 detected with their antibody to the luminal epitope and these cells fixed and imaged.

Figure 1

• Figure 1D - Is it confirmed that GAPDH is not differentially expressed in RNAseq and proteomics upon ITC treatment? The Log2FC value of GAPDH should be noted (as done for ACTB) in the proteomic plots. This would support the use of the protein as a loading control. This comment is made in light of the implication of GAPDH in autophagy. Furthermore, subtle changes in expression levels picked up by proteomics are not necessarily recapitulated in western blots.
• Figure 1D - Please mention under the Methods section how background correction, band definition, detection linearity was performed and verified using the LICOR software.
• Figure 1G - Recommend adding a statistical analysis to this figure. The graph of the autophagic flux with LC3B construct would be more complete with corresponding representative pictures.
• Figure 1I - To allow the reader to ascertain the extent of differential expression in the volcano plot, please mention the log2FC & P values for the proteins taken into consideration from the proteomics data. This could either be incorporated into the legend or presented as a separate table in the supplementary data.

‘lysosomes of ITC-treated iAstrocytes were indeed less acidic than those of control iAstrocytes'- As this was measured by flow cytometry, the standard flow dotplots or histograms could be presented as well.

‘Furthermore, ITC-treated iAstrocytes accumulated puncta of LC3B, an autophagosome marker degraded in the lysosome by acid-activated hydrolases (Fig. 1e)’- Recommend providing Western blot data of LC3I and LC3II in addition to the LC3 ICC. This is because important conclusions are derived on steady state autophagy levels and lysosomal degradation of LC3 in the autolysosomes.

‘Using a GFP-LC3-RFP-LC3ΔG reporter’- A brief description of the reporter and how this measures autophagic flux would be helpful - the color that is quenched when the autophagosome fuses with the lysosome should be mentioned.

‘reflected an impairment in degradative autophagic flux rather than an increase in the steady-state level of autophagic components’ - The data on lysosome acidification and impaired lysosomal degradation of LC3 is convincing. However, the expression levels of autophagic components upon ITC treatment is not mentioned. Recommend presenting the following data with Figure 1:

1. Log2FC values of Atg genes and LC3 from the RNAseq data along with other genes that pop up in autophagy related GO terms, if any.
2. The log2FC values of autophagy related genes from the total proteomics data. Also please indicate where LAMP1 is situated in the Lyso-IP proteome volcano plot (Fig 1I).
3. Western blot data on LC3I and LC3II as mentioned earlier would also add to points 1 and 2 above.

‘To test this hypothesis, we used TIRF microscopy to visualize lysosomes in iAstrocytes loaded with LysoTracker Green and expressing a lysosome membrane-targeted mCherry construct (Fig. 2a)’ - Are these control or ITC-treated astrocytes? In Fig 1E, it was shown that lysotracker could not permeate lysosomes as much as the control condition owing to a change in pH. However, it has been used in TIRF microscopy. Would it be possible to clarify how lysotracker signal is detected in this method but not in ICC. Is it simply a question of resolution?

‘Supplemental Movies 1,2’ - At the framerate the movies are presented, the exocytic events are not clear. Signal disappears, but the characteristic burst of intensity that accompanies an exocytic event was not observed. I realise kiss-and-run exocytosis would not involve this full fusion, and has been reported not to occur in lysosomal exocytosis (https://www.jneurosci.org/content/28/30/7648). Different videos displaying these classical fusion events may be used, or the authors could rely on their luminal epitope exposure assay to demonstrate this point.

Figure 2

• Figures 2A&B - Images for vehicle-treated astrocytes could be provided. It would be also interesting to know if the rate of lysosomal exocytosis is different or similar in control and activated astrocytes.
• Figure 2D ‘Total LAMP1 protein in ITC and vehicle-treated iAstrocyte’ - From the immunoblots and the source data, it looks like the membrane was stripped between LAMP2 and LAMP1. However, GAPDH blots seem to be used from the membrane before stripping. If that is the case, GAPDH should be reprobed after stripping for robust normalization. LAMP1 immunoblot data could be verified with the proteomic data to check that it is not differentially expressed between conditions.
• Figure 2G,H - Recommend a statistical comparison between vehicle and ITC-treated cells.
• Figure 2K - When comparing Fig 2C right panel and Fig 2K DMSO, the effect of ITC on LAMP1 MIF seems to be lost upon DMSO addition. Interestingly there is significant neurotoxicity. Could some clarification be added for this discrepancy, along with the P value.
• Figure 2I - SYT11 knockdown leads to significant increase in surface levels of LAMP1 in control treated cells while there is slight decrease in the surface levels of LAMP1 in activated astrocytes. Could these differences be discussed in the text? Does this mean that SYT11 differentially regulates surface LAMP1 levels in control vs activated astrocytes?
• The 50% decrease in neurotoxicity is not easy to see by looking at the bars in Fig 2J. Some numbers will help visualize the exact effect on neurotoxicity measured by the apoptotic marker.

‘no substantial effect of vacuolin-1 on cell-surface TFRC levels’- There is a substantial increase in TFRC at the cell surface when comparing vehicle and ITC treated, with around a 4x increase in MFI. This is an important point, because independent of the effect of vacuolin-1, it strongly implies that general, non-lysosome related trafficking (specifically Rab11-related recycling) is massively upregulated. This is a good demonstration of the specificity of vacuolin-1, but the apparent effect of ITC on increasing exocytosis (or cell surface expression) of other cargoes could be mentioned.

‘While SYT11 and VAMP7 knockdown resulted in only a small decrease in cell-surface LAMP1, this was sufficient to reduce ITC-induced neurotoxicity by ∼50% relative to control iAstrocytes’ - The text could acknowledge that the reduction in LAMP1 is not statistically significant for a couple of these knockdowns, and also mention that this may imply the reduction in toxicity could be due to either a non-exocytic mechanism, or lysosomal exocytosis mediated by non-conventional pathways.

Figure 3

• Figure 3M,N&O - Multiple pairwise comparison should be done to see if the inhibitor treatment is any different in control vs activated astrocytes.
• Figure 3N - The effect of TOR inhibitor PP242 on the surface levels of LAMP1 seems to be alike in control and activated astrocytes. This appears contradictory with the findings from the genetic knockdown of TOR.
• Figure 3P - The model predicts the lysosomal content to be different in control vs activated astrocytes. The secretome of control vs activated astrocytes could be compared to check if there are any significant differences.

‘Mechanistically, we have not yet resolved if the relationship between lysosome exocytosis and astrocyte-mediated neurotoxicity stems from direct toxicity of astrocyte lysosome contents, or whether these contents contribute to autocrine-paracrine signaling in astrocytes to induce neurotoxicity through an indirect mechanism’ - More data on astrocyte-mediated neurotoxicity appeared in a recent publication https://www.nature.com/articles/s41586-021-03960-y#Sec1, it may be interesting to compare these data.

Author response

_______________________

We thank the researchers within the ASAPbio community for taking the time to provide valuable feedback on our manuscript and also Iratxe Puebla for both facilitating this review of our preprint and for consolidating the comments we received. Here we provide comments to some of the points raised by the reviewers.

In regards to the reviewers’ comment that our “work focuses on the nwk mutant”, we note that Figures 3 and 4 show the unexpected EV cargo depletion phenotype for mutants of numerous components of the clathrin-mediated endocytic machinery. We chose the nwk mutant for our in-depth analysis because it best shows separability of functions in synaptic vesicle (mild defect) vs extracellular vesicle traffic (severe defect), and also produces null mutant viable adult flies for our APP functional studies. However, our work indicates that EV cargo regulation is a broader function for the endocytic machinery and raises the possibility that previously identified neuronal phenotypes for many endocytic mutants could be due to loss of EV cargoes from synapses. Related to this, in reference to the comment that the nwk mutant “affects EV release” we also wanted to highlight that while the EV phenotype we observed for nwk and other endocytic mutants shows both pre- and postsynaptic depletion of EV cargoes, our retromer(vps35);nwk double mutant result suggests that endocytic machinery such as Nwk is not directly regulating release of EV cargoes. Instead, we conclude that the reduction of postsynaptic EV cargoes is a secondary consequence of presynaptic depletion due to defective intracellular traffic. Your helpful feedback has alerted us that we could make these points more clear in the writing and organization of our manuscript.

In response to the points that “...the question arises as to how specific this pathway is to EVs” we should clarify that our findings seem to be specific to cargoes for which sorting to extracellular vesicles is at least a major trajectory (ie, Syt4 and hAPP, of which 30-50% of the synaptic complement is in EVs). We agree that they both have an intracellular component (either en route to EVs or for intracellular signaling functions, which have been well-documented for APP). In response to the comment that “other cargoes that undergo clathrin-dependent endocytosis and are not packaged into EVs would need to be tested”, indeed both Syt1 or Tkv require CME machinery for their traffic (PMID12795692, PMID16459302, PMID18498733), but we find that they are not detectable in EVs and are not depleted at CME machinery mutant synapses. This indicates that local synaptic depletion is specific to cargoes for which least a significant portion of their total pool is normally packaged in EVs.

The reviewers commented that APP and perhaps Syt4 also have intracellular itineraries and functions that may be affected by their depletion at synapses - we agree that our results have implications for both extracellular vesicle and intracellular functions of these cargoes. We fully agree that “the [Figure 2] results might not be specific to EV functions of Syt4 or hAPP” and that a more general statement (such as was suggested in the comments) here would make this possibility more explicit. Our results at least indicate that reduction of these cargoes in presynaptic terminals (but not axons, cell bodies, or dendrites) is sufficient to abrogate their functions. It will be critical in the future to identify trafficking mutants that specifically disrupt EV release without impacting levels in the donor cell, in order to directly query the physiological functions of EV sorting.

To “provide some more information on how the [postsynaptic ɑ-HRP puncta intensity] quantification was done”, we selected an intensity threshold sufficient to distinguish postsynaptic puncta from background muscle fluorescence. We did not directly select puncta manually. Puncta with brightness above this intensity threshold were measured within a 3 μm region around the neuronal HRP. Puncta brightness was not normalized to neuronal HRP brightness, but instead was normalized to the neuronal HRP volume. This analysis was not blinded as many endocytic mutants exhibit synaptic overgrowth phenotypes that are easily visible, thus complicating the blinding process. Using a complementary automated analysis for presynaptic Syt4-GFP, we found very similar results to our manual thresholding analysis. We were however unable to successfully automate the postsynaptic signal measurements due to signal-to-noise-ratio heterogeneity, especially for HRP. Here we’d also like to clarify that in regards to “postsynaptic objects smaller than 0.015 μm were excluded”, we meant postsynaptic objects smaller than 0.015 μm3.

In response to the comment that “...saying this trafficking opposes retromer complex sorting appears to extend beyond the results” we would like to clarify that while direct opposition of endocytic machinery to retromer on endosomes is one possible interpretation, it is not the one we favor in the discussion. We agree that endocytosis and retromer are more likely to oppose each other more indirectly by regulating overall flux through the recycling pathway. We intended to convey opposition as a genetic rather than a mechanistic argument, and we think this conclusion is supported by our data. However based on this feedback we see that we could make this more clear in our manuscript.

We thank the reviewers for pointing out that “In Figure 4B clc depletion does not yield a significant difference in pre-synaptic Syt4 levels. However, Figure 4D the levels of Syt4 are significantly lower in clc both pre- and postsynaptically”. One possibility is that this just reflects variance in the assay, and that the subtle Syt4 phenotype in the clc mutant reached our arbitrary threshold of significance in one experiment but did not in another. There is however also a potentially interesting biological explanation. The 4B clc experiment was conducted at 25℃ while the 4D experiment was conducted at 20℃, since we found that at the lower temperature we were able to recover more clc; nwk double mutant third instar larvae. Endocytosis is well-known as a temperature-dependent process, and perhaps there is some residual endocytosis at this lower temperature in the clc mutant, making it more similar to slowed endocytosis in the endocytic accessory protein mutants (see PMID16269341), compared to a more complete block in the chc or clc 25º condition. This would suggest that slow endocytosis drives cargo into the degradative pathway, fast endocytosis into the rapidly recycling and EV pathway, while no endocytosis traps cargo in unproductive membrane cisternae. Proving this would likely require more quantitative endocytosis assays than are currently available.

We are also appreciative of reviewers comments that will help to make our manuscript more clear, such as suggestions to present the plots consistently, to mention that individual points represent individual NMJs, and to report that C155 is a neuron-specific driver, among other helpful points.

This review reflects comments and contributions by Ricardo Carvalho, Joachim Goedhart, Sónia Gomes Pereira, Pratima Gurung, Samuel Lord, Claudia Molina, Arthur Molines, Gregory Redpath, Mugdha Sathe, Sagar Varankar. Review synthesized by Ewa Sitarska.

This preprint introduces a recombinant profilin that has a flexible linker to a genetically encoded fluorescent tag (either mApple or Halo). Fluorescent protein tagging is a popular and accepted method to study the properties of a protein of interest in solution and in cells. A careful analysis of the tagged protein relative to the untagged, native protein is crucial to understand whether the tagged protein faithfully reflects the behavior of the native protein. Therefore, studies like these are very valuable and the current manuscript is a good example of how such a study should be performed. The flexible linker presented here overcomes challenges observed in previous papers that found that linking a fluorescent protein to profilin disrupted some of its actin-related functions in cells and in vitro.

This study is carefully conducted and nicely describes the properties of a fluorescent protein tagged profilin in a detailed manner. In particular, the authors use various in vitro assays as well as rescue experiments to demonstrate that their tagged version of profilin appears to behave similarly to wildtype profilin. The manuscript is written in a clear manner and was an enjoyable read. The comments below cover a couple of experiments, clarifications and questions for consideration to further add to the work. Regardless, this work is likely to contribute to the field, as anyone studying profilin is likely to try this construct in their future experiments.

General comments:

• Why was mApple chosen as a tag (as opposed to the popular and best known fluorescent protein mEGFP)?
• The mApple is prone to photochromicity/photoswitching (https://doi.org/10.1038/nmeth.1209, https://doi.org/10.1038/nmeth.4074). This should be mentioned to warn future users of this fusion protein.
• It would be advisable to be consistent with the naming of ‘untagged profilin’ throughout the manuscript. Currently unlabeled, untagged, wild-type or rescue are used interchangeably.
• In Figures 4B, 5A there appears to be differences between untagged and tagged profilin in the images. Maybe a more representative image would be beneficial, where applicable.
• Depositing the plasmids from this paper at addgene.org would be beneficial for the public (the plasmids can be deposited under condition that these will be released only after publication of this work in a peer-reviewed journal).

Title

‘Functional fluorescently-tagged human profilin’– suggest clarifying in the title and throughout the text that the fluorescent tags are genetically encoded.

Abstract

‘high cellular concentrations (121 µM)’– This is a very precise number for such a general statement. It seems that the number is derived from a specific cell line, so it would be beneficial to present it as a number from this cell line or change it to an approximation (~100 µM).

Introduction

'Some profilin outcompetes actin bound'– suggest some rewording to clarify the fragment, for example, it could be mentioned whether it refers to F-actin or G-actin.

Results- Design of tagged profilin

‘Profilin is considerably smaller than the smallest fluorescent tags’ – clarifying that it is a genetically encoded fluorescent tag would be of advantage. There are no smaller fluorescent proteins (FP) yet, but genetically encoded FP of a similar size exist, for example miRFP670nano is 17kDa. https://doi.org/10.1038/s41467-018-08050-8

‘Traditional direct labeling approaches are cytotoxic and disrupt actin-based functions’ – is there data showing the new fluorescent profilin side-by-side with one without a flexible linker (or other version used previously) to show that the latter disrupts profilin's functions? It’s not essential but it would strengthen this point and confirm the improvement over prior work.

‘Estimates of cellular profilin concentration are very high depending on the cell type’ – would be nice to provide a rough estimate at this point, similarly to the introduction part.

‘with an mApple fluorescent probe or as Halo-tagged single molecules’ - What is the meaning of 'single' here?

‘We cloned mApple- or Halo-tags fused to a ten amino acid flexible linker on the N-terminus of human profilin-1.’ – As in the introduction, it is stated that “Positioning a GFP-derived fluorescent tag on the C- or N-terminus disrupts PLP- and PIP-binding interactions, effectively rendering the fluorescent version flawed for critical measurements in cells”, it would be beneficial to state the rationale for tagging profillin at the N-terminus? Also, how was the linker composition and length determined and how is it related to other linkers used in compromised fusions of profilin?

Figure 1

• In panel A, it would be helpful to indicate the N-terminus, as this is the side where the fluorescent protein is attached.
• In the legend, PFN1 is introduced for the first time and thus, it could be replaced with ‘tagged-profilin (PFN1)’.

Results - mApple-profilin binds phosphoinositide lipids with similar affinity as untagged profilin

‘PIP’ - PIP is usually an abbreviation for Phosphatidyl Inositol Phosphate (which is a lipid). Phosphoinositide is the same thing, but is not abbreviated as PIP. Recommend using Phosphatidyl Inositol Phosphate = PIP. These types of lipids can be indicated as PIPs (without the addition of lipids).

‘Profilin also binds PI(3,5)P2 which regulates critical signal transduction events through intracellular vesicles to the early endosome’ - The prevailing consensus now is that PI(3,5)P2 is involved in late endosomal trafficking to the lysosome. This could be different for profilin specific purposes, but this statement could be updated with recent references to PI(3,5)P2.

‘Thus, mApple-tagged profilin retains functional interactions with two important PIP lipids.’ – Testing other phospholipids, including PIP3 (Lu et al., 1996 showed an interaction with profilin) and some negative controls would be beneficial. Covering most phosphoinositide species by using the phospholipid-binding dot blots would make this figure stronger.

Figure 2

• In panel B, it would be easier to compare profilin and mApple-profilin binding affinity to each of the PIP lipids. For that, profilin and mApple-profilin samples could be run side by side in the same blot. The suggestion being that panel B includes profilin and mApple-profilin incubated with PI(3,5)P2, and panel C profilin and mApple-profilin incubated with PI(4,5)P2. For clarity, can it be specified in the figure legend or in the figure that the profilin-1 lane is the negative control pellet lacking the liposomes as well as that S and P stands for supernatant.
• Also, in panel B, what are the loading controls? The quantification and western-blots are unclear. For example, it is indicated that 1µM PFN1was used in this experiment, but the pellet band for mAp-PFN1 is not comparable to PFN1 pellet band. Despite this, quantification in 2D shows that they are similar. Please clarify and add the corresponding loading controls.
• Panel D: Please mention what is quantified here (supernatant, pellet or overall level),
• Panel D and E: the shades to pink may be tricky to distinguish, more contrasting colors and/or shapes might be useful.
• It is highly appreciated that the antibodies and dilutions are mentioned in the figure legend.

Results - Direct visualization of fluorescent-profilin with polymerizing actin filaments

‘We used fluorescence anisotropy to measure the binding affinity between profilin and Oregon-Green (OG)-labeled monomeric actin (Figure S2).‘ - This appears confusing with the 3D results. If a change in fluorescence anisotropy with OG-actin is not detected, then is it okay to use OG-actin for bulk polymerisation assay. Maybe the OG-tag interferes only during fluorescence anisotropy, but not during fluorescence microscopy.

‘Several studies demonstrate that thymosin b4 (Tb4) competes with profilin to bind actin monomers‘ – It is worth mentioning that this refers to untagged/unlabeled actin monomers.

Figure 3

• Please consider if it is relevant to compare the competitive and non-competitive data on the same graph.
• In panel A description, ‘10 nM GFP-thymosin b4 (GFP-Tb4) mixed with increasing concentrations of unlabeled actin.’ – Would ‘10nM unlabelled actin monomers in presence of increasing concentration of Τβ4’ be more appropriate?
• In panel D, the curves would be better visible when the y-axis runs from 0-30.
• In panel E, the mApple-Profilin samples show longer filaments, maybe quantification of the filament length could be performed?
• In panel G, are the errors bars based on the means of the technical replicates or on all aggregated data? The first option is preferred, as this plotting strategy was also used in 3F.
• In the panel description, ‘Data were quantified from four separate reactions (FOV) each.’ – could clarify whether the data derive from 4 totally independent reactions, or from the analysis of 4 different fields of view (FOV) from the same experimental procedure?
• In the panel description, ‘ns, not significantly different from 1 µM actin alone control; a, compared with control (p <0.05)‘ – such labeling may be confusing for the reader, it would be beneficial to state that the first ‘ns/a’ are related to the actin alone, and the second ‘ns’ are comparing labelled and unlabeled profilin or omit showing the statistical test in the plot, and show it in a table instead.

Results - Fluorescent profilin stimulates formin-based actin filament assembly

General: Based on the competition and interaction experiments it seems important to generate a dose-dependent inducible construct for profilin to govern the stoichiometry of interactions and study their relevance in the cells.

‘Similar to experiments in assessing only profilin-actin interactions (Figure 3F), we counted significantly fewer actin filaments in reactions containing actin and either profilin (Figure 4D)’ - In Figure 3F the plot shows around 45 actin filaments per FOV with 1 uM actin (20% oregon green actin). In Figure 4D the plot shows more than 100 actin filaments per FOV with 1 uM actin (10% alexa 647 actin). Surprisingly, the elongation rate are similar in Fig 3F and Fig 4D. Is the Oregon-Green actin known to be less efficient at nucleating filaments while retaining the same polymerization ability? If it is the case, it would be worth making a mention.

‘Thus, fluorescent profilin stimulates formin-mediated actin filament nucleation similar to the untagged version.’ - The data seem to suggest that the presence of profilin inhibits actin filament nucleation and polymerization, a clarification would be appreciated.

‘The ac-celerated rate of actin filament’ – Please change to ‘accelerated’.

Results - Profilin directly binds tubulin dimers and enhances the growth rate of microtubules in vitro

‘microtubule stability index‘ – The values indicated on the y-axis for the plot in Fig 5E are confusing (0 / 25/ 50 / 75 / 1). Is the index expressed as a percentage and the max value supposed to be 100? Or is the index supposed to be the number of rescues per catastrophe?

’This suggests a mechanism where profilin accelerates microtubule polymerization by directly binding to tubulin dimers to promote microtubule assembly and then diffusing along the sides of the microtubule lattice to further stabilize microtubule growth.‘ - Microtubules are more stable the faster they grow (catastrophe frequency scales inversely with polymerization rate). In this condition where profilin increases polymerization rate by around 5x, it is unclear how much of the increased stability is due to the lattice binding. The fragment could be softened regarding the role of the transient lattice binding in microtubule stabilization.

Figure 5

• In panel B, intensities are quite different, would it be possible to comment on this?
• In panels H an I, a black/magenta merge is tricky to see. Although it breaks consistency, a green/magenta or cyan/magenta merge may be more informative visually.

Results - Profilin regulates the morphology of N2a cells through actin and microtubule crosstalk

‘We used quantitative western blots to determine the level of endogenous profilin as well as levels of profilin in CRISPR knockout cells following transfection with plasmids containing untagged profilin, mApple-profilin, or Halo-profilin.’ - The levels of profilin are quantified from a blot, which is a bulk measurement. The transfection of profilin will show substantial cell-to-cell variation (some cells may have much higher or lower levels than the measured average). Mentioning it and discussing its implications would be advisable.

‘We chose this parameter because N2a cells have unique actin filament and microtubule cytoskeletal features but do not efficiently perform other classic cell processes that require intact cytoskeletal crosstalk (i.e., migration or division).‘ – While looking at cell shape is one strategy, an additional experiment looking into a migration phenotype may strengthen this point. Another interesting experiment to strengthen this point and providing a direct measure of profilin function could be performing a pulse chase experiment using drugs to depolymerise actin/microtubules. In such an experiment, a distinct change in depolymerisation should be noted between WT, KO, and the profilin rescue cells. This could show that the mApple-profilin can substitute for WT profilin.

‘super resolution confocal microscopy to image fixed cells.‘ – how is super-resolution achieved here? Or is ‘super’ unnecessary here?

‘the ratio of endogenous cell area to other cell conditions’ - This metric is a ratio of areas and it is only valid as an assessment for shape if the cells from each condition cover similar areas. If it is not the case, then the two parameters (shape and area) are convoluted and the ratio measures both the difference in shape and in area covered. It would be good to provide the average area of the cells in each condition for clarification.

‘We also stained these cells for actin filaments (Figures 6H and 6I) and micro-tubules (Figure 6J and 6K) and used a similar morphology parameter to detect broad differences in cytoskeletal architecture.’ – Please clarify the reasons for using the cell area ratio metric for quantification of cell morphology. How is the quantification metric (area) used for sub-cellular network like actin and microtubule? F-actin stained with phalloidin looks different in endogenous PFN1 vs mApple-PFN1, but by using area metric there is no morphological difference. Microtubule, on the other hand, appear similar in all the cells.

Figure 6

• In panel A, it does not seem like mApple-PFN1 and Halo-PFN1 reach endogenous levels. Maybe a quantification would be beneficial.
• In panel B, please report the number of independent replicates. Also, may be worth commenting on why tagged PFN1 rescue cells were not included?
• For panel E, please provide the error bars and the legend that contains the information from where the data derived or from how many independent experiments.
• In panel F, it may be advantageous to use red green and blue as colors for the overlay. This will generate unique colors for the different combinations of the three images.
• For the context of panel F, unprocessed images of the tagged profilin in living cells could be presented somewhere in the main text. They could be larger than the small panels here, and not be segmented into binary images. The point of the fluorescent profilin is that it can be used for live-cell imaging without substantially disrupting the typical profilin interactions. This should be confirmed by presenting live-cell images of the profilin construct. To avoid problems with high cytoplasmic concentrations of profilin drowning out any localization signal, maybe the fluorescent version could be expressed at a very low level or the Halo version could be used with a low concentration of fluorophore.
• In panel G, it is clear that the dots are from different cover slips, how many cells were analyzed per coverslip? Data could be shown from individual cells (not just their average). Also, please clarify if this quantification was made 24 hours after transfection as well?
• For panel I, actin morphology was calculated from actin filament signals similar to the cell morphology index. These calculations could be explained further in the methods. Does this mean that the actin morphology index is the ratio of actin area between the two conditions? Is the actin image somehow thresholded before taking the ratio?
• Panel L, is really appreciated and helpful to understand the "competition" between actin and microtubules for profilin. It would be also nice to represent the plasma membrane and the lipid-binding activity of profilin as well as binding to nucleation promoting factors (the proline-rich motifs of VASP and WASP), as this is mentioned throughout the paper.

Discussion

The fact that mApple and HaloTag both are entirely different and non-disturbing gives confidence that profilin can be fused with other tags, without losing functionality. Mentioning this in the discussion could give new insights for the readers.

‘Our genetic analyses in mammalian cells indicate that mApple-profilin and Halo-profilin are fully inter-changeable with the endogenous version.‘ - Authors have given the field an excelled tool which will be quite useful to study cellular functions of PFN1, its interaction with its several binding partners. Currently, cell shape is the metric used to determine if the tagged versions are fully inter-changeable with the endogenous version. Whether the tagged PFN1 can replace untagged PFN1 for other cellular functions will require further exploration. Also, high concentration of PFN1 will remain an issue even with the mApple-PFN1 developed here. Do the authors suggest mild over-expression as a strategy to go around the high concentration issue?

‘Based on localization experiments using the pan-formin inhibitor, SMIFH2, some interactions between profilin and the sides of micro-tubules are thought to be indirect.’ – suggest clarifying how SMIFH2 treatment leads to conclude that interactions between PFN1 and microtubules is indirect.

Methods

Statistical significance tests do not demonstrate that conditions are identical. That is, when two conditions are not statistically different, it is not possible to say that these are equal (https://doi.org/10.1053/j.seminhematol.2008.04.003). Suggest avoiding the use of "n.s." in graphs to indicate that the data are similar. It is clear from the data that (in many cases) the tagged and untagged profilin show similar properties. If equality needs to be demonstrated, recommend carrying out an equivalence test.

‘Different shades of data points show technical replicates.’ – Please rephrase to clarify, for example “Different colors represent biological replicates. Similar colored dots reflect technical replicates“. Does "technical replicate," mean repeated measurements within each independent experimental run? Or different experimental runs? If the shading is supposed to denote paired experiments (e.g. darkest shading in different conditions are from the same experimental run), that can be stated in the caption.

This review reflects comments and contributions by Joachim Goedhart, Sónia Gomes Pereira, Karen Lange, Arthur Molines, Gregory Redpath, Zara Weinberg.

The authors studied the release of extracellular vesicles (EV) from nerve terminals. Drosophila is used as a model and the work focuses on the nwk mutant, which affects EV release. The extracellular intensity of GFP-tagged proteins was quantified as a proxy for secretion. Overall, the study is well performed, with the correct analysis and controls, and the images are great.

The interpretation of the data could be further clarified or toned down, especially in Figure 2, 4 and 5 and the statements around endocytic machinery opposing retromer complex sorting. The authors have performed a good job of characterising the role of Nwk in endocytosis of cargoes that can be incorporated in EVs. However, these proteins also have intracellular roles (at least for APP, not sure for Drosophila Syt4, although the human version has clear intracellular roles) meaning that the question arises as to how specific this pathway is to EVs as such. The study most definitely uncovered a protein important in SV endocytosis and recycling, which has an impact on EVs.

Minor comments:

• The paper would benefit from an expansion to the legends, to indicate the conditions tested. In Figures 3A&B, 4D it would be helpful to outline the abbreviations used for the graphs.
• It is unclear that the bars in Figure 1/3/4/5 add useful information; suggest showing the data + error bars, this would be more consistent with the plots shown in Figure 2.
• The authors may want to include this recent report in the introduction and/or discussion: Ganguly et al. (2021) Neuron.Clathrin packets move in slow axonal transport and deliver functional payloads to synapses

Abstract

'EV cargo Amyloid Precursor Protein' - It could be mentioned (perhaps in the intro) that APP also has well established intracellular roles in endocytosis, as well as being an EV cargo. If the same is true for Drosophila Syt-4 (as it is for human Syt-4), a similar reference could be made.

Results

‘During our studies of the endocytic F-BAR and SH3 domain-containing protein Nervous Wreck (Nwk), we found that nwk null mutants exhibited a strong reduction in the intensity of postsynaptic α-HRP puncta (within 3 μm of the boundary of motor neurons innervating muscles 6 and 7) (Fig. 1A) - Was this a hypothesis-driven exploration? It may be worth expanding on the groundwork for this initial key observation.

Figure 1

• ‘Quantification of postsynaptic α-HRP puncta intensity’ - Is it possible to provide some more information on how the quantification was done. Were the puncta manually selected? Was selection blinded to condition? Is puncta brightness normalized to neuronal HRP brightness?
• ‘Data is represented as mean +/− s.e.m.; n represents NMJs’ - n values are not stated, suggest saying "individual dots represent".
• ‘See Tables S1 and S3 for detailed genotypes and statistical analyses’ - The authors did a great job in providing clear details of what genotypes were used in each experiment as well as exactly which analysis and parameters were considered. This is great to increase transparency and reproducibility!

‘These phenotypes were rescued by neuronal re-expression of Nwk using the binary GAL4/UAS system (Fig 1B)’ - While the number of Syt4 puncta appears to be fully rescued, this is less clear for the pre- or postsynaptic intensities (only a partial rescued is observed). The text reads as both phenotypes are rescued, suggest clarifying that this rescue is not similar for both phenotypes, and if possible, to speculate on the reasons for this.

‘cell autonomously regulates’ - This fragrant may benefit from some clarification, the results indicate Nwk alters Syt4 endocytosis, perhaps it could be stated in that manner.

'The calcium sensor Synaptotagmin-1 (Syt1)’ - Are these cargoes internalised by comparable endocytic pathways to Syt4, i.e. clathrin-dependent endocytosis? If so, this could be stated. If not, cargoes undergoing endocytosis by a comparable pathway could be used.

‘reduction of Syt4, hAPP, Evi, and Nrg in nwk mutants is specific to the EV trafficking itinerary’ - Suggest replacing "is" with "may be." If an expanded range of cargoes was investigated this could change.

‘Together these results suggest that Nwk specifically and locally regulates the levels of EV cargo proteins at synaptic terminals’ - This fragment could be reworded to reflect that some of these cargoes (at least APP) have intracellular roles, e.g. "Together these results suggest that Nwk specifically and locally regulates the levels of cargo proteins that can be incorporated into EVs at synaptic terminals." I realise the APP expressed here is human, but in human cells it has intracellular endocytic roles, so it could also play a role when expressed in Drosophila.

Figure 2

• ‘Figure 2A ‘before (top trace) and after (bottom trace)’ - This information could be added to the figure.
• Figure 2B - Consider removing some of the labels on the x-axis, to avoid the 45 degrees rotation (to improve readability).
• ‘Figure 2C ‘C57-GAL4 and Vglut-GAL4 are muscle and neuron-specific drivers, respectively’ - For readers unfamiliar with Drosophila drivers, it would be useful if the figure was labelled with "muscle" and "neuron" and then the legend listed the specific driver.
• Figure 2F ‘GAL4C155-driven’ - Would it be possible to report where this driver is expressed?

‘Together these results show that the depletion of EV cargoes Syt4 and hAPP at nwk mutant synaptic terminals correlates with a loss of function for these EV cargos in the nervous system’ - The results might not be specific to EV functions of Syt-4 or hAPP, an alternative wording for the statement might be "Together these results show that the depletion of Syt4 and hAPP at nwk mutant synaptic terminals correlates with a loss of function for these cargos in the nervous system."

Figure 4 ‘all measurements were further normalized to the mean of their respective controls, which is indicated with a dashed line’ - In Figure 4A, the control data on the graph are unclear. It appears that the results were normalized to Post AP-2α, would it be possible to clarify why this would be the control? A similar question regarding Figure 4B where it is not clear where the control data appear on the graph.

‘We found that simultaneous loss of clc and nwk phenocopied loss of clc alone with regards to levels and localization of Syt4-GFP pre- and postsynaptically (Fig. 4D)’ - In Figure 4B clc depletion does not yield a significant difference in pre-synaptic Syt4 levels. However, Figure 4D the levels of Syt4 are significantly lower in clc both pre- and postsynaptically. This could be commented on further, with an explanation offered (if possible).

‘This suggests that the Syt4-GFP accumulations in clc mutant synapses are not accessible to the mechanism by which EV cargoes are depleted at nwk mutant synapses’ - This could be clarified, does it mean that Nwk cannot retrieve cargo from CLC accumulations, i.e. Nwk acts downstream of clathrin?

'Together these results highlight a novel clathrin-dependent mechanism that regulates the traffic and levels of EV cargoes at synapses' - The data suggests Nwk acts in clathrin-dependent endocytosis to regulate uptake of Syt-4 and some other EV cargoes, and when this is defective these cargoes don't get packaged into EVs. This appears to be expected, when endocytosis is defective, downstream sorting will be defective. To support the statement ‘levels of EV cargoes at synapses’, other cargoes that undergo clathrin-dependent endocytosis and are not packaged into EVs would need to be tested.

‘suggesting that Nwk is involved in loading EV precursors and maintaining presynaptic cargo levels’ - Recommend adding "endocytic" in relation to ‘in loading EV precursors’, based on the statement above "in endocytic loading of cargoes into the EV-permissive precursor compartment that populates MVBs destined for EV release".

Figure 5A ‘Cartoon depicting potential EV phenotype outcomes for nwk; Vps35 experiment, testing if Nwk regulates MVB-PM release or EV cargo sorting’ - The schematic is good, consider making the label a touch clearer, e.g. "release defect", as release is not happening.

Discussion

‘Syt4-dependent synaptic plasticity’ - As mentioned in Fig 2, the loss of synaptic plasticity is interesting in the Nwk mutant, but not directly shown to be dependent on Syt-4 here. Recommend revising to "Nwk-dependent" synaptic plasticity.

‘endocytic machinery opposes the retromer complex in sorting cargoes’ - Endocytosis is clearly perturbed in the Nwk mutants. This endocytosis is indeed clearly important in the synaptic region, before cargoes attach to dynein mediated retrograde transport. However, saying this trafficking opposes retromer complex sorting appears to extend beyond the results. Synaptic endocytic machinery will ensure a rapid endocytic/exocytic cycle occurs, with the occasional endosome/synaptic vesicle targeted for retrograde trafficking and degradation. This endocytic machinery is part of an overarching endocytic network that ensures cellular function. A possible explanation here is that with the Nwk mutant, the cell has aberrant EV cargo trafficking and must deal with this somehow - likely degradation. Recommend incorporating further interpretations in addition to the one stated.

‘endocytic machinery protects' - Endocytic machinery feeds cargo into the synaptic vesicle cycle, when this trafficking is perturbed, an alternate pathway is utilised by the cell. Alternate explanations for the results could be provided in addition to what is already suggested.

‘Finally, our observation that AP2’ - µ missing here.

Materials and methods

‘​​Postsynaptic objects smaller than 0.015 μm were excluded’ - Could this be clarified? A size of 15 nm cannot be measured with the kind of conventional microscopy (spinning disk) used here.

This review reflects comments and contributions by Ankita Jha, Zara Weinberg, Julia Grzymkowski, Julien Berro, Karen Lange, Sónia Gomes Pereira, Arthur Molines, Jacob Herman, and Manoj Yadav. Review synthesized by Jacob Herman.

The work by Joseph Varberg and colleagues uses super resolution microscopy to better characterize the non-random distribution of nuclear pore complexes within the nuclei of the fission yeast Schizosaccharomyces pombe. This work also confirms findings in other organisms that nuclear pore complexes exist in multiple compositions. In addition to better documenting this phenomenon, this work begins to characterize the mechanisms by which nuclear pore position is regulated. Specifically, the authors show that clustering centromeres at the spindle pole body excludes nuclear pore complexes from the spindle pole body, and when these two complexes are forcibly dimerized mitotic defects result in decreased fitness.

The commenters were overall quite impressed with the imaging technique. The major conclusions and message of the preprint were generally well received, the comments or questions below relate to very specific text and experiments.

A few key themes mentioned in specific comments were:

• A desire for more consistent statistical analysis of data.
• Suggestions for additional data for some statements or toning down of the claims. NPC clustering is commonly discussed but there were questions as to how this phenotype was being measured.

Specific comments

Introduction

“perhaps explaining links between changes in NPC density and cancer” - The statement could note whether the correlation between NPC density and cancer is positive or negative.

“for example, emerin is enriched at pore-free regions of the NE in cultured cells (44). In budding yeast, NPC density is increased in the region of the NE near the spindle pole body (SPB),” - Does the SPB contain LEM domain proteins or is this a different possible mechanism for the non-random regulation of NPC density?

“Using S. pombe as a model system”- Why not S. cerevisiae, which is discussed earlier to have significant prior art in this regard? I'm sure there is a good reason, I think it could be outlined a bit more in the intro.

“We quantify NPC number under a range of conditions” - It would be useful to mention briefly at this point what types of conditions this refers to.

“Additionally, NPCs are excluded from the NE region surrounding the SPBs by Lem2 and other factors.” - Could the authors clarify if it is also something that is conserved or it is a new finding?

Results

Subheader “3D-SIM image analysis pipeline for NPC quantitation” - Worth mentioning the conclusion that NPC density is independent of cell cycle stage since that is the major conclusion from this section.

“This approach provides a roughly two-fold increase in resolution as compared to conventional light microscopy” (Figure 1A) - For those who have never imaged NPCs it would be really informative to see a confocal or wide field image to better understand how SIM imaging made this project possible.

Figure 1 legend “E) Mean number of NPCs, nuclear surface area and NPC density measurements from four independent replicates. Significant differences (*) determined using Wilcoxon rank sum tests. ns, not significant.”

• Each one of the coloured dots on the graph appear to represent the mean NPC number for each replicate. If so, then this information should be added to the figure legend, as it is not immediately obvious for a broader audience. If not, please clarify what each dot represents. Same in Figure S1E.
• Were these tests conducted pairwise? Are the reported p values corrected for multiple hypothesis testing? Having a dedicated "Statistics" section in the methods would be helpful for reporting this.

“We observed that the number of NPCs also increases through interphase to maintain a constant NPC density (Table S1, Fig. 1D-E)”- There are no cell-cycle markers used to determine cell-cycle progression but only a visual assessment from cell size and nucleus shape. It would be good to show the three plots in figure 1E as scatter plots with the X axis being cell size for cells that are not yet in mitosis (1 nucleus). Then do a correlation analysis between cell size and NPC number, Surface Area, NPC density.

“We observed occasional differences in NPC density between mother and daughter nuclei produced by the symmetric mitotic division in S. pombe, reminiscent of the elevated NPC density observed for daughter nuclei produced by the asymmetric mitosis in S. cerevisiae”

• It is not clear what the authors mean by "occasional", is it 1%, 5%,10%? It would be better to replace this with a specific number/% of events. Additionally, the question arises as to what happens afterwards in the daughter cells, do they retain the NPC density asymmetry? Or do they eventually achieve similar densities?
• Some of these points are addressed later on in the paragraph and Fig S1A. Some edits to this sentence should address this and provide clarity.

“Despite the improved lateral resolution offered by SIM, clustering of NPCs and the comparatively reduced axial resolution likely leads to undercounting of NPCs using 3D-SIM” - This is useful context for Figure 1C - it would be worth mentioning in that section how the automated counting handles clustered NPCs, or placing the paragraph earlier with a short description of the methods.

“Similarly, a constant NPC density was maintained when nuclear size was reduced in mitotic cells using a temperature-sensitive allele of Wee1 kinase (wee1.50)” - The NPC density does not change with temperature in the wee1 mutant but the NPC density in Fig 2B is lower than in a WT in Fig 1. The Nup tagged in the two figures are different, so this could be an explanation (as shown in Fig S1 E) but it could be good to make sure that the wee1 background does not have a different NPC density. I don't see a quantification of the NPC density using Nup37 in the WT elsewhere. In fact, Nup37 seems to be used only in wee1 background and in Sup Fig. 1 B.

“The increase in NPC number was dependent on NE membrane expansion during arrest, as chemical inhibition of fatty acid synthesis by treatment with cerulenin blocked nuclear growth while NPC density was maintained (36°C + Cerulenin = 6.8 ± 1.5 NPCs/μm2, n=110) (Fig. 2C)” -The effect of the Cerulenin drug on a WT background is not shown, was that control experiment done? It would also be helpful to include statistics in this section.

“Yeast lacking core components of the autophagy machinery (atg8Δ or atg1Δ) (75) that targets NPCs for degradation during nutrient deprivation do not show increased NPC density compared to wild-type cells, suggesting that autophagy is not used to remove NPCs to maintain NPC density (Fig. 2D)” - It is unclear that this experiment alone tells us much about the regulation of NPC density. If atg8 and atg1 are known to regulate NPC removal only in response to nutrient deprivation then consider performing these experiments under that condition or revisiting whether they fit here.

“NPC density is maintained by a mechanism that restricts the assembly of new NPCs in the absence of increased available NE surface area.” - This conclusion is indicative of a mechanism where NPC assembly is maintained by restricting the assembly, however all the data above is indicative of the mechanism where NPC assembly is correlated with NE surface area, for increase there must be an additive mechanism and for a decrease in the NE, there must be a mechanism of removal. This suggests that the NE surface area regulation mechanism could be tied to NPC density. One way to clearly show that could be a correlation plot of NE surface area and NPCS density, color coded for all the different conditions tested.

Figure 2- It appears as though no comparative statistical analysis was done with the quantitative data displayed in Figure 2, yet it is stated that e.g., "treatment with cerulenin blocked nuclear growth while NPC density was maintained" or "yeast lacking the autophagy machinery do not show increased NPC density". These conclusions would be strengthened if statistics were run on the data similar to Figure 1.

“NPC clustering is common phenotype in different cell types and in mutants defective in NPC assembly.” - Does this mean that NPC clustering is higher in mutants defective in NPC assembly? Would suggest including references for this. Also, this paragraph needs an introduction to why NPC clustering matters? Does it have any connection with the NPC distribution?

“3D-SIM images revealed the presence of multiple smaller clusters distributed throughout the NE (Fig. 3A).” - It is unclear (also not mentioned in the Methods section) how clusters are identified. The images show rings but it is hard to tell how many clusters compose that ring structure. It will be beneficial to show how clusters are quantified. Can that be resolved with 3DSIM?

“We frequently observed NPC clusters organized in a ring-like structure with diameters ranging from 250-300 nm (Fig. 3B)” - Is it possible to report what was the frequency of the ring structures in nup132-deleted and wt cells?

“Clustering increased in aged nup132Δ cells grown on plates (Fig. 3C)”- The figure depicts the NPC ring like structure, does this mean that the ring increased or the clustering has increased. Does increase in clustering make the rings more continuous?

“NPC clusters were frequently enriched in the anaphase bridge, along with excess membrane (Fig. 3E)” - Providing a quantification of NPC cluster enrichment in the anaphase bridge would be helpful.

“Following completion of nuclear division, the resulting daughter nuclei had normal NE morphologies and NPC densities equivalent to wild-type nuclei (Fig. S2). This suggests that nem1Δ nuclei can remove excess NE membrane and NPCs during mitosis via the anaphase bridge.”

• This implies that prior to mitosis nem1∆ cells have abnormal morphology and NPC densities but the latter is not measured.
• The NPC density reported in Figure S2 for the WT and the Nem1 mutant are different from the NPC density reported for the WT in figure 1 and figure S1 yet it is done using the same tagged Nup, Nsp1. It would be helpful to have an explanation. If the NPC density is “a constant” in the WT it should not be different from one figure to another. If the nem1 mutant has a density of 4 NPC/micron^2 then it is different from the WT. Also, the NPC density in the nem1 mutant in Figure S2 seems almost bimodal. Increasing the number of nem1-delta cells analyzed could help identify if it is bimodal or if it is due to under-sampling.
• For the nem1 mutant the clustering is not quantified.

“In contrast, NPC clusters in nup132Δ nuclei coalesced into larger clusters that preferentially localized to the SPBs in mitosis (Fig. 3G)”

• An overlay image could be included to support this statement.
• Fig. 3F could be referenced here too because otherwise it is not referenced until the discussion; at which point it is used to reference the data that is referenced here as Fig. 3G.

“We observed a clear reduction in NPC density over the nucleolus” - Is this referring to where the yellow and magenta staining meet in Fig. 4? It is not immediately obvious as to where "over the nucleolus" is in those slices. Can the regions that are being compared (NPC staining at NE vs. NPC staining over nucleolus) be highlighted/specified in some way so as to better understand the quantification method?

Figure 4

• 4C is gorgeous - really conveys the point well!
• In this figure the authors at first show a 3D-SIM image, but perform the intensity analysis on the confocal slice. What is the reason for it? Analysis of the 3D-SIM data could provide more information on the characteristics (number, spatial distribution) of NPS density reduction.

Figure 5

• Very minor comment -- the scale bar is very hard to see in Fig 5A.
• Statistics for Fig. 5B would strengthen the conclusion that the exclusion was cell-cycle independent.

Figure 6

• Figure 6D - Looking at the insets, the exclusion area in the lem2(delta)C-off appears to be the smallest one and closer to the exclusion area shown for the lem2(delta) in panel B. However, this is not represented in the quantification/results. I wonder if there is another image that would more closely represent the quantification outcome? Or if the insets might have been mislabeled, for instance lem2(delta)C-off could indeed represent the lem(delta)N-on and vice-versa?
• Figure 6E - This is listed as F in the legend.

“Tethering did not affect microtubule nucleation at the SPB, including the formation of cytoplasmic microtubules.” - Please provide evidence supporting this statement.

Discussion

“In nem1Δ mutants, both excess nuclear membrane material and NPCs are segregated into the anaphase bridge region during nuclear division” - This would benefit from some analysis - are there too many NPCs? Is it specifically the clustered NPCs? Currently the data supporting this is snapshots from a single movie.

“The ability for 3D-SIM to resolve and quantify individual NPCs labelled with multiple fluorescent proteins at endogenous levels provides tools to begin to interrogate__ how altered NPC compositions may allow for functional specialization of NPC function at distinct regions of the NE.” - The high resolution images are really beautiful! Great job in showing the power of 3D-SIM to help answer these types of biological questions.

Methods

“Images were acquired overa6 μm volume with 0.3 μm z-spacing for 45 min at 2 min intervals.” - For dynamic measurements a 2-minute interval is big and it would be interesting to see a few time-series imaging with smaller intervals to capture the fast changes.

This review reflects comments and contributions by Vaishnavi Ananthanarayanan, Xianrui Cheng, Joachim Goedhart, Arthur Molines, Eric Peterman, Sanjana Pillay, Pablo Ranea-Robles, Mugdha Sathe and Zara Weinberg

The study by Kidwell et al. reports that lateral transfer of mitochondria from macrophages to breast cancer cells activates signaling pathways that promote cell proliferation. The study uses single-cell imaging strategies to show transfer of fluorescently labeled mitochondria; the transfer is rare, but it is convincingly demonstrated. The study reports that transferred mitochondria were depolarized and accumulate ROS, which signaled through ERK and promoted proliferation of the recipient cells as well as their daughter cells who also inherited the transferred mitochondria.

The report sheds light on the mechanism by which macrophages stimulate cancer cell proliferation in the tumor microenvironment, which has strong implications in the cancer field. One of the novelties of this study is the fact that the transferred mitochondria are rather dysfunctional (depolarized), in contrast with most of the previous reports on mitochondrial transfer, which was thought to be a way to "rejuvenate" the mitochondrial pool of a cell.

The authors make some interesting observations and the methods are described in detail. With the current data, there are questions as to whether all effects are due to biological changes, and there may be a need to re-evaluate some of the data and nuance the interpretation of the results.

General comments are outlined below followed by annotations/specific comments on manuscript content (in order of appearance).

• One of the most important claims is that mitochondria are the organelles responsible for the activation of the signals of cell proliferation. However, a previous report by the last author reported that macrophages transfer cytoplasm to recipient cells. It cannot be excluded that other organelles or cellular fragments are transferred as well and contribute to the observed effects (ERK activity). Perhaps a good way to solve this would be the use of macrophages that are devoid of mitochondria. At least, this aspect should be discussed in the manuscript.
• Most of the positive examples of transferred mitochondria discussed appeared in a small clump. However, there also appears to be another population that was more diffuse and co-localizes with host mitochondria (e.g., Fig2B, bottom right panels). It would be helpful to show results of these sibling mitochondria for assays performed on their clumpy siblings. If they behave differently, it would be helpful to provide some explanation.
• The effects that are attributed to the transferred mitochondria are highly variable (figures 1F, 3A,E) and often due to a subpopulation of samples that show a few extreme values (e.g. figures 2D, 3E, S4B, S4D). This might be expected from effects that are caused by a single mitochondria (which has a small volume) that is transferred to a complete cell. This complicates the study of the transfer process and effects and should be discussed. Also, do the authors have ideas how to improve the system, to make it more robust and easier to study the effects?
• The authors conclude that the transfer of dysfunctional mitochondria generated a signal mediated by ROS that activates cell proliferation signals. The statement that "transferred mitochondria act as a signaling source that promotes cancer cell proliferation" is too strong. There is increased ROS production from mitochondria, yes, but an experiment in which ROS are decreased would be needed to properly sustain that conclusion. The title and abstract could be changed to better reflect the data.
• The study may benefit from more direct evidence to support its conclusion of increased proliferation after mitochondrial transfer. While the RNA-seq, flow cytometry, counting of completion of cytokinesis and dry mass measurements provided in the present study do lend some support to the proliferation hypothesis, they all seem indirect. With the biomarkers labeling the mitochondria of donor and potential recipient cells, high content imaging and tracking of cells could be used to monitor cell division. A comparison of cell division rates of transfer-positive cells and transfer-negative cells will provide a more pertinent test of whether mitochondrial transfer promotes recipient cell proliferation.

The authors have used such a tracking-based approach on a very small scale (n=5) to measure daughter cell growth rate. However, the data do not show a statistically significant difference between the growth rates of daughters that inherited transferred mitochondria and those who did not (Fig S3). Increasing the case number via high content imaging would help obtain sufficient data points for a reliable statistical test. In addition, as suggested above, an accounting of the daughter cells' division rate for transfer positive and negative cells would provide another line of evidence to either prove or disprove the increased proliferation rate hypothesis. The same suggestion goes to the optically induced ERK activation experiments shown in Fig3F. It is also helpful to include references that studied how ERK signaling promotes proliferation and compare the evidence here with evidence or assays used in those studies as a benchmark.

Specific comments

Figure S1A - The authors could perhaps use a more aggressive gating strategy here, clipping closer to the 231 population described in Fig S1A - picking only the center of the cluster in the upper left of the RFP vs CD11b plot would likely not affect results but make them more convincing by unequivocally excluding macrophages.

Figure S1B - Could perhaps be an interesting follow-up question for future works re: differences between cell lines and propensities to transfer mitochondria. Did the authors attempt to use other cell lines (ie, MDCK, HeLa, iPSCs, etc)?

Figure S1B - Did the authors see an increase in growth rate in MCF10A line despite the lower growth rate?

‘physically separated from macrophages by a 0.4μM trans-well insert’ - should this read 0.4 micrometer?

Figure S1F - The authors wrote that they used a two-way ANOVA analysis, could you report the factors used for that analysis in the Figure legend.

Figure 1B - It is difficult to see the arrowheads in 1B, suggest moving them so they are not covering the magenta fluorescence, have them point from a different angle, and make them more brightly colored. Insets here would help the reader. A negative control image from a monoculture would also be helpful, to ensure the GFP signal is not an artifact of culture conditions. Figure 1D - Not sure about the 0.2% baseline assigned for the monoculture of cancer cells (that does not have the macrophages with the Emerald mitochondria). It is determined with cytometry - I am no expert on that topic, so maybe I missed something - but it looks weird to see some cells with transfer when there is a monoculture.

Figure 1F - For graphs that do not show zero (as in 1F), the bar should be omitted. In these cases the length of the bar does not reflect the average of the data (as it does in 1D).

Figure 1 - ​​Given that these data are fractions of a population (i.e. can be described via a contingency table), isn't something like a Fisher's exact test a better measure of significance here?

Single cell RNA- sequencing - In the methods section the authors mention doing a differential analysis between the cells that received the mitochondria and the cells that didn’t. It might be worth introducing a figure (a heatmap or a U-MAP) relating to this analysis. Single cell sequencing would not only affirm the heterogeneity between these two populations but also help in highlighting the novel cell surface markers associated with the two populations.

Figure S3 - There is no statistical test to check for ‘increase in their rate of change of dry mass over time versus sister cells that did not inherit macrophage mitochondria’. What are the colours indicative of in S3B? Can this be reported in the figure legend. ‘mito-mEm+ mitochondria remained distinct from the recipient host mitochondrial network, with no detectable loss of the fluorescent signal for over 15 hours’ - It is surprising that the transferred mitochondria do (or cannot) fuse with the host 231 mitochondria. It is unclear in these images, but the 231 mitochondria appear fragmented too. Is it possible that the mitochondrial fusion machinery (Opa1 or Mfn1/2) are inactive?

Figure 2B - What does the MTDR staining of the macrophage mitochondria prior to transfer look like? Important to check this to confirm that only the transferred mitochondria had lower membrane potential.

Figure 2A, 2BB and S1D - How were the colocalizations assessed? Was it just a visual assessment? Given the importance of these experiments for the whole story, having a quantification of the level of colocalization with each dye would be important.

Figure S1D - The paper makes an argument about mitochondria transferred from Macrophages (marked green) having positive DNA stain (gray), but appearing depolarized (negative TMRM stain). The image in FigS1D is peculiar, as the majority of the 231 cells' mitochondria appear to not have any DNA stain but maintain membrane potential (positive in TMRM), while some (just above the green macrophage mitochondria) do have both DNA stain and membrane potential. The authors might want to clarify whether this is a typical scenario, and if so perhaps offer an explanation as to why the 231 mitochondria exhibit such heterogeneity.

‘we confirmed that 91% of transferred mitochondria were not encapsulated by a membranous structure, thus excluding sequestration as a mechanism for explaining the lack of degradation or interaction with the endogenous mitochondrial network’ - This is based on co-staining with MemBrite 640/660, which is a dye that "covalently labels the surface of live cells", thus there is a concern as to whether this approach allows to study whether the mitochondrium is encapsulated by an endomembrane.

Figure 2 ‘Majority (57%) of donated mitochondria do not colocalize with LysoTracker signal (N=24 cells, 4 donors) - Here the paper implies that some transferred mitochondria do co-localize with lysoTracker signal. More importantly, they co-localize with host mitochondria. It raises the question of whether they signal through ROS and ERK like their clumpy siblings who are in the limelight of most figures.

‘macrophage mitochondria are depolarized but remain in the recipient cancer cell’ - Did the authors examine the extent of cancer cell death in their co-culture system (due to the activation of apoptosis by the depolarized mitochondria)?

‘significantly higher ratios of oxidized:reduced protein were associated with the transferred mitochondria versus the host network’ - Here too, it would be important to check the mito-Grx1-roGFP2 readout of macrophage mitochondria prior to transfer.

Figure 2C–D - Like in Fig 2B, in the bottom left of panel of Fig 2C there are a lot of donor mitochondria not in highly oxidized state and the growth/proliferation phenotypes apply mostly to donor mitochondria that appear 'clumpy'. Perhaps it is worth commenting on whether there is a link between donor mitochondrial morphology and the suspected proliferation-enhancing phenotype.

‘At 24 hours, we observed a similar trend, but no statistically significant difference (Fig. S4D). These results indicate ROS accumulates at the site of transferred mitochondria in recipient cancer cells’ - if a specific sensor fails to show a significant oxidation at 24 hours compared mito-Grx1-roGFP2 which reports on mitochondrial glutathione redox state, does that mean there are ROS independent ways to oxidize Glutathione? The authors did see cell growth phenotype both in 24 and 48 hours which suggests that something is happening in 24 hours despite no significant difference in ROS H2O2 sensor.

The differences in ratio for the two sensors used are not very convincing. In Fig 2D and Fig S4B and D the “host” and “transfer” populations are very similar. The difference seems only due to the presence of a few outliers in the “transfer” populations. More importantly, sometimes it seems that these outliers come mostly from one donor rather than being present in all 3 donors. It could be good to show histograms of the two populations for each replicate/donor and maybe redo the stats excluding these outliers.

Figure S5C - it seems like the percentage of cells that divided is the same for unstimulated cells and cells with stimulated mito-KillerRed. Isn't this contrary to the expectation? The figure shows that photobleaching cytoplasm decreased % cell division, which is puzzling.

‘ROS induces several downstream signaling pathways’ - We would not expect the authors to investigate every signaling pathway, but wonder if the PI3K pathway was explored? It seems to be the other major cancer/proliferative pathway induced by ROS.

‘Recipient 231 cells had significantly higher cytoplasmic to nuclear (C/N) ERK-KTR ratios compared to cells that did not receive transfer’ - Since two different quantification styles with opposite fraction values were used, is it possible to please specify which one was used here.

Figure 3A - ​​In the 'cyto' condition 6 out of 13 fields have no cells that divide. Is that expected? What is the percentage of dividing cells for cells that were not illuminated at all (a control that is lacking)? There is large variation, ranging from 0% to 22%. The evidence that illumination of KillerRed leads to increased proliferation is rather weak. Also, since Cyto and Mito are different cells, is a "paired" statistical test the right kind of test to use here?

Figure 3B - Please show the outlines of the nuclei and that of the cell.

Figure 3C - Please omit bar, see comment on panel 1F.

Figure 3D - it is peculiar that ERK-KTR in Fig 3D is so strongly cytosolic while in Fig 3B it is almost exclusively nuclear. If this sensor behaves differently in different situations, the authors may want to comment on how that would affect their conclusions.

Figure 3E - The effect of 'opto-induced' ERK activity is weak. The initial ERK-KTR is 1 at time point zero (as the data is normalized to this timepoint) and around 1 for both the cyto and mito condition. A statistical difference is observed, but the effect is minor and it is unclear whether it is biologically meaningful. The 'cyto' condition shows an average below 1 and the mito condition remains 1, suggesting that ERK activity remains constant when ROS are produced in the mitochondria. Also from S8C and 3E it appears cyto actually shows a decrease rather than mito showing an increase, could the authors comment on this?

‘Furthermore, treatment with an ERK inhibitor (ERKi) was sufficient to inhibit ERK activity ‘- curious as to whether antioxidant treatment would reverse any proliferative phenotypes?

‘patient-derived xenografts (PDxOs)’ - As a control it would be relevant to include a normal mammary organoid model perhaps from the same patient to demonstrate that the transfer of mitochondria specifically to the cancer cells is more beneficial.

‘macrophages to both HCI-037 and HCI-038 PDxO cells (Fig. 4G)’ - Why is M0 able to transfer efficiently to HCL-037 tumour when its mitochondrial network is less fragmented as M2? Are mito transfer from M0 depolarised and accumulate ROS or show increased ERK activity or increased cell proliferation?

‘M2-like macrophages preferentially transferred mitochondria to the bone metastasis PDxO cells (HCI-038) when compared to primary breast tumor PDxO cells (HCI-037)’ -The authors may want to check this statement here as it is in consistent with their data plot. In Fig. 4G, M2/PDxO transfer percentages for HCI-037 and HCI-038 are about the same, unless the authors provide statistical tests to prove otherwise. Instead, M0 appears to transfer mitochondria to HCI-037 much more efficiently than it does HCI-038.

‘M2-like macrophages exhibit mitochondrial fragmentation’ - Is there a correlation between the status of the mitochondrial network in the donor and the % of transfer to the recipient? If so, this would be a correlation that would support the conclusions.

‘accumulate ROS, leading to increased ERK activity’ - Did the authors obtain similar results with the PDXOs? It would be an interesting observation if the primary samples also exhibit a mechanism similar to established cell lines wherein there are more accumulated genetic changes. It would also be interesting to examine whether there is any difference in the ROS-ERK mechanism for primary and metastatic tumour.

‘in cancer cells that receive exogenous mitochondria’ - Since these macrophages also transfer mitochondria to non-malignant cells, such as MCF10A cells shown in Fig S1B, perhaps the authors could comment on whether this is part of a physiological process that would also promote normal cell growth?

Review by Alain Queffelec here again the value of 85%.

Review by Alain Queffelec here again the word accurate seems strange to me.

Review by Alain Queffelec why the 80th percentile? Why not 95th, or 50th? This should be stated somewhere, in the methods or here.

Review by Alain Queffelec Given the numbers of Table 1, I don’t get how the 85% has been calculated.

Review by Alain Queffelec I wouldn’t say that the LCP from the south-to-north is more “accurate”. The calculation is as accurate in both directions. It is the Roman that followed the LCP more accurately in one direction than the other. The LCP is calculated based on several parameters and assumptions, but it is a LCP. The Roman road is not presupposed to be perfectly a route of less effort, it can be chosen due to other parameters than efforts, or with other parameters than slope. The word “accurate” does not seem to be the best here.

Review by Alain Queffelec computing time: for the reader it could be useful to have a broad idea of the computing time on a standard desktop computer. Is it seconds, minutes, hours, days? Maybe after the line 234, as a last sentence of the paragraph?

here is the RMSE value, and there is a citation about it. As I mentioned earlier, it would be nice to have a short explanation of how this value is calculated.

Review by Alain Queffelec (Université de Bordeaux)

except for the abstract, it is the first mention of the fact that the LCP will be calculated both from north-to-south and south-to-north. This is not obvious for me that this is useful. I guess that, if you do that, it is because it is not the same path in either direction. Would it be interesting to insert a sentence to explain that the route is not supposed to be the same if you change the direction? Ah… saw that in the lines 229-230.

Review by Alain Queffelec (Université de Bordeaux)

how is the distance calculated? Is it calculated for distributed points of the real road, or for every cell of the raster? Is it calculated orthogonally to the route, or on an east-west distance every north-south kilometer? Maybe this is obvious for GIS researchers, but it is not to me.

Review by Alain Queffelec (Université de Bordeaux)

I don’t get why the citations of 1930 and 1861 works are brought in this paragraph since Whitehead and Elswroth mapped the road in 2008. So there are at least 3 sources for this road’s route. The author chose one, but it is not very clear why this one. It is written that this road by Whitehead and Elsworth includes only the road that can be identified in the field. Did Bishop 2014 or Tabert & Bagnall 2000 invent some parts of the road? Please explain a bit more the reasons for this choice.

Review by Alain Queffelec (Université de Bordeaux)

Figures 6, 7 & 8: it would be easier for the reader to simply add a red arrow in the direction of the road (north-to-south or south-to-north), just parallel to the road.

Review by Alain Queffelec (Université de Bordeaux)

A north arrow would be useful. Maybe put the scale on the map to earn space?

Review by Alain Queffelec (Université de Bordeaux)

Figure 4, 6 7 8. A scale and north arrow would make the maps more complete. As well as a scale of the grey levels for altitude so that the reader has an idea of the topography of the region.

Review by Alain Queffelec (Université de Bordeaux)

Why the values of the orange cells in Fig. 2left are not the same as the values in the table in fig.2right? If I understood well, it should be the case. And if it is normal that it is not the case, it should be explained since it is disturbing.

Review by Alain Queffelec (Université de Bordeaux)

gdistance is said “comparable to other software” but the only explained parameter in the paragraph is a difference between gdistance and all the other software apparently. This does not seem very “comparable”, on the contrary. Or if studies exist that this specific parameter gives “comparable” results between different software, it should be stated.

Review by Alain Queffelec (Université de Bordeaux)

Figure 1: E: given that the text mentions that “the LCP results from the Monte Carlo simulation can be visually communicated probabilistically” (lines 83-84), it would be preferable that figure 1:E represents this visual aid, like one can see on figure 7.

Review by Alain Queffelec (Université de Bordeaux)

If Figure 1: F is called in the text before Fig 1: E, please change the text or the figure so that the text calls the subfigures in the alphabetical order.

Review by Alain Queffelec (Université de Bordeaux)

Since you write that “The vertical error is often summarized by a single global statistic such as the root mean square error”, it would be interesting to cite the other ways of giving the vertical errors for a DEM even if it is not so common.

Review by Alain Queffelec (Université de Bordeaux)

The discussion is rather short, and does not incorporate any citation of previous works. Surely these results can be integrated in the literature, what is not the case from one can read here. For example, as listed by the author, probabilistic LCPs have been used in other domains than archaeology before (lines 75-77), and it would be interesting to know if the results of the author does compare or not with the results from the literature.

It could be interesting, even if not perfectly in the scope of the paper, to propose some hypothesis for the fact that the real road does not follow the LCP in the middle-north part.

It would also be interesting to consider whether this type of result can, or cannot, be used to support archaeological hypothesis on the main directions of use of the road: did Roman use more the road from south to north so that the road better follow the LCP calculated from one side than the other? Or maybe the road has been built beginning in the south, so that the path has been chosen to be the easiest in this direction?

It could be interesting to have a paragraph written to explain to the reader whether these results show, or not, that the error in the DEM can have a stronger, or not, impact than the other parameters used generally in LCP studies. Indeed, we can see here that using and modelling the error of the DEM can change the final LCP as compared with classic LCP modelling ; it would be interesting to know whether this can change it more than taking into account other parameters than permeability to compute the LCP, or taking into account other parameters than Tobler’s Hiking cost function. I guess the slope is not the only parameter one would look at before constructing a road.

Are there examples in the literature where using this modelling of the error could lead to really different LCP? For example, while taking into account the error, the LCP could pass north of a mountain instead of south, and could change drastically the path? Maybe for older LCP studies with worse DEMs than we have today?

The conclusion can be considered to be the last paragraph of the discussion. By making a longer discussion, the author could separate this paragraph and make it a proper conclusion.

Review by Alain Queffelec (Université de Bordeaux)

The second paragraph of the 2.1 could be in the introduction.

This same paragraph could incorporate one or two sentences about how the RMSE for a DEM is calculated. Is it calculated by checking the values on some random points on the field? Is it purely mathematical? Is it optical, given the characteristics of the LIDAR device or the satellite, the GPS correction of the plane, the weather at the time of the measurement? Is it a mix of several artefacts of measurements + calculations? Is it systematically given by the provider of the DEM?

Review by Alain Queffelec (Université de Bordeaux)

The introduction misses a bit of background about the Roman case study: what is the aim of a case study, why this period, the importance of road in the Roman empire. And what is the hypothesis that can be tested thanks to a case study? Later the author use the word “accuracy” to determine whether the modeled LCP is close or not to the real road, as if the holy graal is the real road and the perfect LCP simulation should copy exactly the real road. This word has a meaning, I think, that deserves an explanation about what a comparison between the real road and the LCP should bring to the understanding in both directions : the LCP method but also the Roman road engineering. Unless this is something usually used in this field of research that I am not aware of.

This review was completed by Alain Queffelec (Université de Bordeaux) as part of ASAPbio’s #PreprintReviewChallenge

This manuscript by Joseph Lewis proposes to integrate the error of the Digital Elevation Model (DEM) while calculating the Least Cost Path (LCP), by running multiple modelisations thanks to Monte-Carlo replications. The manuscript explains the methodology, which has been implemented in an R package, and applies it to a case study of a Roman road in the north of England. The results show that the mean LCP, integrating the error of the DEM, represented by the path the most used in the 1000 models, can be qui different from the LCP that would be calculated without taking into account the error of the DEM.

I think the paper is clearly written, the figures are nice and explain clearly the point of the author, the result is very interesting and could probably be integrated as a basic method in the future LCP analysis in archaeology. This manuscript and the methodology it proposes has probably the possibility to become used by everyone in this field.

Code is proposed as a new function in an R package already developed by the author. Data and code specific for this manuscript and this case study are accessible on Zenodo.