That's a con. There's no need to sync globe-wide, creating a giant ledger. You have a set of peers that you want to share your stuff with (friends), leave it at that.

Good stuff. I.e., associate existing accs.

Better: always log in with a server, unless you choose to migrate.

Better: let peers have personal profiles of their friends.

Like you do with your contacts on phone. You know their id (phon number), you give it a name, assign a pic. It's up to you.

Additionally: community-based management, ban polls.

Alternative: per-user configuration of access. Let rooms be topics on which peers discuss. A friend can see what he's friends and foafs are saying.

That's a standard - good for devs. Yet it's grotesque.

Meh. Verbose, bad types, needs parsing.

And be able to store it locally, on trusted devices, replicated.

Ideally support p2p. Servers/brokers are optional.

Better: text messages & structured stuff (data).

Better: across devices (p2p) and (optionally) their servers.

That's good. Matrix Event Graph is cool.

Talk a lot about it without yet defining it

grammar nitpick

IoT/non-smartphone edge devices (=embedded devices) are notoriously less secure than cloud providers (source?) --> is this really a claim that can be done?

tested different modalities?

Uses edge deviecs as motivation --> tested on them afterwards?

Terrible use of bolding font.

leeswijzer heel fijn

In einem Brief , dessen Text die New York Times erhalten hat, haben Nasawissenschaftler dagegen protestiert, dass drei für die Beobachtung der Erdatmosphäre und unter anderem die Analyse des Strahlungs-Ugleichgewichts essentielle Satelliten nach dem bevorstehenden Ende ihrer Lebenszeit nicht ersetzt werden sollen. https://www.nytimes.com/2024/05/03/climate/nasa-satellites-data.html

The poem is written in a free verse.

The blinding glare is truly extremely bright, which is highlighted in these lines. Then we get the allteration of b: "blaze of branch, or brazier" which is connecting the words describing a fire. No fire, natural (branch), nor humanmade "brazier" is more blinding then the light that we get from the sun. The sun's light may also be viewed as the holy light, bringing life and bigger then anything terrestrial.

The winter was cold and long, however finally, the sun is coming, bringing life and hope. Alliteration of the letter h, showing how the life and heat are forever intertwined.

Sun in the cold ice. Again the image of life/death.

Again there are more of those contrasting words: here there are "pond and ditches", together with the previously mentioned words and "pole and tropic" in l. 3, they are all creating this theme of the inbetween. In this line we are also introduced to an aspect that is coming in this season and that is the sun that "flames the ice". We get to witness the blinding light that is reflecting off of the ice. The following lines are all talking about this light.

There are several cases of alliteration of the "s" sound. There is spring, season, sempiternal, and finally sodden. This creates an audible pulse to the text which is might refer to the pulsing of the beating heart which would correlate to the rebirth theme of the poem. There are again interesting contrasting words put in a juxtaposition: sempiternal and sodden in line 2. This is to highlight the slow tempo of the incoming spring as the winter seemed eternal and so the change is very slow but highly anticipated. Then there is also frost and fire which are describing the two opposite seasons, which makes the poetic persona thinking that they are "suspended in time". The lines 2-5 have all the metre of 11 syllables, thus connecting them as a descripting part of this new season.

Introduction of a new type of season, called the "Midwinter spring". The juxtaposition of the two contrary season is capturing the rebirth of life that is coming with the finishing winter. The metre is 9.

andere verwoording

stukje

zinstructuur loopt niet

woord weg

Dit is te hard; meer focussen op leden motiveren en opleiden. Niet iedereen eruit knikkeren. alv gaat dit niet pikken

Misschien focussen op dat het een casual laagdrempelig moment is? Niet een 'presentatie'

willen we hier nog iets concreets mee? introcie heeft dit niet als 'doel'

te informeel?

Misschien iets specifieker? We gaan kijken naar 'stamkroeg'

Stukje bezuinigen / contributie?

was vgm ooit op alv gezegd, kon niet terugvinden in notulen: waarom niet Kasco voorstel in beleidsvisie? dan is iedereen toch meteen lekker geinformeerd

En este seminario observo que hay una apuesta fuerte por la reivindicación de la comunidad como un escenario de desarrollo de la vida, construcción de identidad y fortalecimiento de relaciones con el entorno natural y social.

Hablar de Digital Storytelling es referirse <br /> a un tema difuso, bajo el cual se ampara una gran variedad de narrativas posibles, tanto por el contenido formal de las historias que comparten (el qué se cuenta, con sus hechos, personajes, tiempos y escenarios), como por su la expresión formal del discurso (el cómo se cuenta, con diferentes modalidades de organización y expresión, con los medios de materialización y con una diversidad de plataformas tecnológicas que permiten su registro, difusión y consumo).

Lo importante es aprender a utilizar los diferentes lenguajes que permitan contar historias de ficción y no ficción significativas a través de la radio, la televisión, el podcast, el cine, la prensa, las publicaciones digitales o el performance, entre muchas otras posibilidades, con la finalidad de generar espacios de transformación ante realidades sociales poco visibles y complejas.

A propósito de esta técnica, comparto reseña del libro Humanizando la Deportación, un proyecto realizado en el año 2016 en Tijuana, con el método de producción audiovisual participativa (digital storytelling), el cual ofreció a los migrantes una plataforma en donde podían compartir sus experiencias.

El proyecto se conformó por un equipo transnacional que realizó trabajo de campo durante un mes en las calles de Tijuana, donde grabaron distintas historias junto a los migrantes deportados de Estados Unidos. A partir de esto se publicaron 50 narrativas digitales (cortometrajes testimoniales) en la página web de Humanizando la Deportación.

Esta publicación documenta algunas de las experiencias y los aprendizajes obtenidos en el transcurso del primer año del proyecto en Tijuana, ciudad de migrantes que –quizá por ser la más emblemática en cuanto a la volatilidad de las dinámicas fronterizas de nuestro tiempo– ha sido y sigue siendo el corazón de Humanizando la Deportación.

Esta pregunta me lleva al trabajo de investigación que adelanto, en donde hago énfasis en el desarrollo creativo y constante de métodos e investigaciones que visibilicen y den herramientas a comunidades vulneradas, en este caso, de los migrantes. Sería interesante conocer otras investigaciones sobre la temática a tratar.

Florida hat die Produktion und den Verkauf von synthetischem Fleisch untersagt. Damit kommt man dem Druck der Fleisch- und Geflügelindustrie nach. Gouverneur DeSantis sprach bei der Vorstellung der Maßnahme auch von einem Schlag gegen die globalen Eliten. https://www.nytimes.com/2024/05/03/climate/florida-lab-grown-meat-ban.html

BIG AGREE

Risks of fitting PAT into the rigid contours of EBM

!!!!!!!

Pressure from EBM shaping PAT into something it was not meant to be

These issues are amplified in PAT

How EBM limits inquiry regarding PAT

Name

Auflage

Beschreibung

Unstrukturierte Angaben über die Auflage des Werks.

Abdeckung

Enthalten in ca. 5,5 Millionen Einträgen.

Provenienz

Stammt aus den MARC-Feld 240 $a.

URI

http://purl.org/ontology/bibo/edition

Name

Erstellungsdatum

Beschreibung

Enthält das Datum an dem die Ressource erfasst wurde oder durch Fremddatenübernahme in den Katalog kam im Format jjjj-mm-tt.

Verwendungsbeispiele

• Einschränken einer Suche auf Titel, die nach der RDA-Einführung im hbz-Verbund (1.1.2016) ergänzt wurden: https://lobid.org/resources/search?q=bibliothek+AND+describedBy.resultOf.object.dateCreated%3A%3E2016-01-01

Abdeckung

Alle Resourcen haben ein Erstellungsdatum.

Provenienz

ALMA-MARC spezifisches Feld MNG $b

URI

http://purl.org/dc/terms/created

Name

Bearbeitungsdatum

Beschreibung

Enthält das Datum, an dem das Katalogisat zum letzten Mal bearbeitet wurde im Format jjjj-mm-tt.

Abdeckung

Ca. 2,1 Millionen Ressourcen haben kein Bearbeitungsdatum, das heißt die Katalogeinträge wurden nach ihrer Erstellung nicht mehr angefasst.

Verwendungsbeispiele

• Eine Liste aller Ressourcen, die am 1. April 2024 das letzte Mal verändert wurden: https://lobid.org/resources/search?q=*+AND+describedBy.resultOf.object.dateModified%3A%3E2024-01-01

Provenienz

ALMA-MARC spezifisches Feld MGN $d

URI

http://purl.org/dc/terms/modified

Name

Beschrieben durch

Beschreibung

Dieses Objekt enthält Informationen zur Beschreibung (den Datensatz/Katalogeintrag) selbst. Momentan sind dies insbesondere Erstellungs- und Modifikationsdatum(describedBy.resultOf.object.dateCreated und describedBy.resultOf.object.dateModified). Es handelt sich wohlgemerkt, um die Modifikation der zugrundeliegenden MARC-/ALMA-Quelldaten, die nicht notwendigerweise eine Änderung in den lobid-Daten nach sich ziehen.

Verwendungsbeispiel

Diese Angaben sind besonders relevant, um sich inkrementelle Updates zu ziehen. Eine Liste aller Ressourcen, die in seit dem 1. April 2024 geändert oder ergänzt wurden: https://lobid.org/resources/search?q=*+AND+describedBy.resultOf.object.dateCreated%3A%3E2024-04-01+OR+describedBy.resultOf.object.dateModified%3A%3E2024-04-01

URI

http://www.w3.org/2007/05/powder-s#describedby

Name

Alternativer Name / Bezeichnung

Beschreibung

Enthält Namensvarianten der Person oder Institution. Basis dieser Varianten sind die über ALMA bereitgestellten GND-Anreicherungen.

Verwendungsbeispiele

Prinzipiell sollte eine Suche nach Ressourcen anhand beteiligter Personen auch die Namensvarianten abfragen. Beispiel: Eine Suche über die Namen und variierende Namensformen aller beitragenden Personen: https://lobid.org/resources/search?q=contribution.agent.label:Charles+Dodgson+AND+contribution.agent.altLabel:Charles+Dodgson

URI

http://www.w3.org/2004/02/skos/core#altLabel

Name

Akteur

Beschreibung

Enthält den Typ/type (Person oder Körperschaft/CorporateBody oder Veranstaltung/Konferenz/ConferenceOrEvent) des beitragenden Akteurs/Veransaltung und den Namen (label) sowie – wenn vorhanden – Namensvarianten (altLabel) der Person oder Institution, die diesen Beitrag erbracht hat und in ca. 94% Prozent der Fälle eine URI (id) zu einem Normdatensatz der DNB.

URI

http://id.loc.gov/ontologies/bibframe/agent

Name

Beitrag zu einem Werk

Beschreibung

Zu einem Werk gehören ein oder mehrere Beiträge, die als Objekte in einer sortierten Liste angegeben werden. Ein Beitrag besteht aus der Angabe der beitragenden Institution oder Person und ihrer Rolle in bezug auf die beschriebene Ressource.

Neben Organisationen und Personen können auch zugehörige Veranstaltungen als contribution ausgewiesen sein.

Abdeckung

Vorhanden bei ca. 22,7 Millionen Einträgen, fehlt bei ca. 4,9 Millionen Treffern.

Verwendungsbeispiele

Eine Suche über die Namen und variierende Namensformen aller beitragenden Personen: https://lobid.org/resources/search?q=contribution.agent.label:Harry+Rowohlt+AND+contribution.agent.altLabel:Harry+Rowohlt

Provenienz

MARC-Felder 100,110,111, 700, 710, 711.

URI

http://id.loc.gov/ontologies/bibframe/contribution

Name

Akteur

Beschreibung

Enthält den Typ/type (Person oder Körperschaft/CorporateBody oder Veranstaltung/Konferenz/ConferenceOrEvent) des beitragenden Akteurs/Veransaltung und den Namen (label) sowie – wenn vorhanden – Namensvarianten (altLabel) der Person oder Institution, die diesen Beitrag erbracht hat und in ca. 94% Prozent der Fälle eine URI (id) zu einem Normdatensatz der DNB.

URI

http://id.loc.gov/ontologies/bibframe/agent

Name

Beitrag zu einem Werk

Beschreibung

Zu einem Werk gehören ein oder mehrere Beiträge, die als Objekte in einer sortierten Liste angegeben werden. Ein Beitrag besteht aus der Angabe der beitragenden Institution oder Person und ihrer Rolle in bezug auf die beschriebene Ressource.

Neben Organisationen und Personen können auch zugehörige Veranstaltungen als contribution ausgewiesen sein.

Abdeckung

Vorhanden bei ca. 22,7 Millionen Einträgen, fehlt bei ca. 4,9 Millionen Treffern.

Verwendungsbeispiele

Eine Suche über die Namen und variierende Namensformen aller beitragenden Personen: https://lobid.org/resources/search?q=contribution.agent.label:Harry+Rowohlt+AND+contribution.agent.altLabel:Harry+Rowohlt

Provenienz

MARC-Felder 100,110,111, 700, 710, 711.

URI

http://id.loc.gov/ontologies/bibframe/contribution

Many potential 'confounders'

Ways in which PAT challenges the conventional framework of EBM

Integration of qualitative and quantitative measures

Yes! Open-minded exploration utilising narrative data to determine new parameters of relevance

Also good!

Good: expanding the notion of evidence to include qualitative evidence

What kind of evidence?

Increasingly removing themselves from the horror of prosecuting a genocide -- aerial bombing vs. ground combat, ai-directed bombing vs. human-operated drone bombing. c.f. techniques used to shield the mental health of nazi concentration camp administrators.

https://www.repubblica.it/economia/2024/05/04/news/decreto_agricoltura_lollobrigida_cosa_prevede-422821679/?ref=drla-f-1

delete

see footnote 94 notes and format the same way

separate into own footnote

Separate into separate footnote

hyperlink

hyperlink

no italics

no italics

no italics

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replace with https://open.umn.edu/opentextbooks/textbooks/817

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hyperlink

replace link with https://psycnet.apa.org/doi/10.1037/0022-3514.85.5.894

no italics

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reply to reviewer comments

• *

We extend our gratitude to the reviewers for their time and valuable feedback on our manuscript. We especially appreciate the insightful suggestions that have significantly contributed to refining our work and elucidating our findings. With the revisions made to the text and the inclusion of new experimental data, we believe our manuscript now effectively addresses all reviewer comments. We eagerly await your evaluation of our revised submission.

Small ARF-like GTPases play fundamental roles in dynamic signaling processes linked with vesicular trafficking in eukaryotes. Despite of their evolutionary conservation, there is little known about the ARF-like GTPase functions in plants. Our manuscript reports the biochemical and cell biological characterization of the small ARF-like GTPase TTN5 from the model plant Arabidopsis thaliana*. Fundamental investigations like ours are mostly lacking for ARF and ARL GTPases in Arabidopsis. *

We employed fluorescence-based enzymatic assays suited to uncover different types of the very rapid GTPase activities for TTN5. The experimental findings are now illustrated in a more comprehensive modified Figure 2 and in the form of a summary of the GTPase activities for TTN5 and its mutant variants in the NEW Figure 7A in the Discussion part. Taken together, we found that TTN5 is a non-classical GTPase based on its enzymatic kinetics. The reviewers appreciated these findings and highlighted them as being „impressive in vitro biochemical characterization" and "major conceptual advance". Since such experiments are "uncommon" for being conducted with plant GTPases, reviewers regarded this analysis as "useful addition to the plant community in general". The significance of these findings is given by the circumstance that „the ARF-like proteins are poorly addressed in Arabidopsis while they could reveal completely different function than the canonical known ARF proteins". Reviewers saw here clearly a "strength" of the manuscript.

With regard to the cell biological investigation and initial assessment of cell physiological roles of TTN5, we now provide requested additional evidence. First of all, we provide NEW data on the localization of TTN5 by immunolocalization using a complementing HA3-TTN5 construct, supporting our initial suggestions that TTN5 may be associated with vesicles and processes of the endomembrane system. The previous preprint version had left the reviewers „less convinced" of cell biological data due to the lack of complementation of our YFP-TTN5 construct, lack of Western blot data and the low resolution of microscopic images. We fully agree that these points were of concern and needed to be addressed. We have therefore intensively worked on these „weaknesses" and present now a more detailed whole-mount immunostaining series with the complementing HA3-TTN5 transgenic line (NEW Figure 4, NEW Figure 3P), Western blot data (NEW Supplementary Figures S7C and D), and we will provide all original images upon publication of our manuscript at BioImage Archives which will provide the high quality for re-analysis. BioImage Archives is an online storage for biological image data associated with a peer-reviewed publication. This way, readers will be able to inspect each image in detail. The immunolocalization data are of particular importance as they indicate that HA3-TTN5 can be associated with punctate vesicle structures and BFA bodies as seen with YFP studies of YFP-TTN5 seedlings. We have re-phrased very carefully and emphasized those localization patterns which are backed up by immunostaining and YFP fluorescence detection of YFP-TTN5 signals. To improve the comprehension, the findings are summarized in a schematic overview in NEW Figure 7B of the Discussion. We have also addressed all other comments related to the cell biological experiments to "provide the substantial improvement" that had been requested. We emphasize that we found two cell physiological phenotypes for the TTN5T30N mutant. YFP-TTN5T30N confers phenotypes, which are differing mobility of the fluorescent vesicles in the epidermis of hypocotyls (see Video material and NEW Supplementary Video Material S1M-O), and a root growth phenotype of transgenic HA3-TTN5T30N seedlings (NEW Figure 3O). We explain the cell physiological phenotypes in relation to enzymatic GTPase data. These findings convince us of the validity of the YFP-TTN5 analysis indicative of TTN5 localization.

*We are deeply thankful to the reviewers for judging our manuscript as "generally well written", "important" and "of interest to a wide range of plant scientists" and "for scientists working in the trafficking field" as it "holds significance" and will form the basis for future functional studies of TTN5. *

We prepared very carefully our revised manuscript in which we address all reviewer comments one by one. Please find our revision and our detailed rebuttal to all reviewer comments below. Changes in the revised version are highlighted by yellow and green color. In the "revised version with highlighted changes".

With these adjustments, we hope that our peer-reviewed study will receive a positive response.

We are looking forward to your evaluation of our revised manuscript and thank you in advance,

Sincerely

Petra Bauer and Inga Mohr on behalf of all authors

*

• *

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

The manuscript from Mohr and collaborators reports the characterization of an ARF-like GTPase of Arabidopsis. Small GTPases of the ARF family play crucial role in intracellular trafficking and plant physiology. The ARF-like proteins are poorly addressed in Arabidopsis while they could reveal completely different function than the canonical known ARF proteins. Thus, the aim of the study is important and could be of interest to a wide range of plant scientists. I am impressed by the biochemical characterization of the TTN5 protein and its mutated versions, this is clearly a very nice point of the paper and allows for proper interpretations of the other results. However, I was much less convinced on the cell biology part of this manuscript and aside from the subcellular localization of the TTN5 I think the paper would benefit from a more functional angle. Below are my comments to improve the manuscript:

1- In the different pictures and movies, TTN5 is quite clearly appearing as a typical ER-like pattern. The pattern of localization further extends to dotty-like structures and structures labeled only at the periphery of the structure, with a depletion of fluorescence inside the structure. These observations raise several points. First, the ER pattern is never mentioned in the manuscript while I think it can be clearly observed. Given that the YFP-TTN5 construct is not functional (the mutant phenotype is not rescued) the ER-localization could be due to the retention at the ER due to quality control. The HA-TTN5 construct is functional but to me its localization shows a quite different pattern from the YFP version, I do not see the ER for example or the periphery-labeled structures. In this case, it will be a crucial point to perform co-localization experiments between HA-TTN5 and organelles markers to confirm that the functional TTN5 construct is labeling the Golgi and MVBs, as does the non-functional one. I am also quite sure that a co-localization between YFP-TTN5 and HA-TTN5 will not completely match... The ER is contacting so many organelles that the localization of YFP-TTN5 might not reflects the real location of the protein.

__Our response: __

At first, we like to state that specific detection of intracellular localization of plant proteins in plant cells is generally technically very difficult, when the protein abundance is not overly high. In this revised version, we extended immunostaining analysis to different membrane compartments, including now immunostaining of complementing HA3-TTN5 in the absence and presence of BFA, along with immunodetection of ARF1 and FM4-64 labeling in roots (NEW Figure 3P, NEW Figure 4A, B). In the revised version, we focus the analysis and conclusions on the fluorescence patterns that overlap between YFP-TTN5 detection and HA3-TTN5 immunodetection. With this, we can be most confident about subcellular TTN5 localization. Please find this NEW text in the Result section (starting Line 323):

„For a more detailed investigation of HA3-TTN5 subcellular localization, we then performed co-immunofluorescence staining with an Alexa 488-labeled antibody recognizing the Golgi and TGN marker ARF1, while detecting HA3-TTN5 with an Alexa 555-labeled antibody (Robinson et al. 2011, Singh et al. 2018) (Figure 4A). ARF1-Alexa 488 staining was clearly visible in punctate structures representing presumably Golgi stacks (Figure 4A, Alexa 488), as previously reported (Singh et al. 2018). Similar structures were obtained for HA3-TTN5-Alexa 555 staining (Figure 4A, Alexa 555). But surprisingly, colocalization analysis demonstrated that the HA3-TTN5-labeled structures were mostly not colocalizing and thus distinct from the ARF1-labeled ones (Figure 4A). Yet the HA3-TTN5- and ARF1-labeled structures were in close proximity to each other (Figure 4A). We hypothesized that the HA3-TTN5 structures can be connected to intracellular trafficking steps. To test this, we performed brefeldin A (BFA) treatment, a commonly used tool in cell biology for preventing dynamic membrane trafficking events and vesicle transport involving the Golgi. BFA is a fungal macrocyclic lactone that leads to a loss of cis-cisternae and accumulation of Golgi stacks, known as BFA-induced compartments, up to the fusion of the Golgi with the ER (Ritzenthaler et al. 2002, Wang et al. 2016). For a better identification of BFA bodies, we additionally used the dye FM4-64, which can emit fluorescence in a lipophilic membrane environment. FM4-64 marks the plasma membrane in the first minutes following application to the cell, then may be endocytosed and in the presence of BFA become accumulated in BFA bodies (Bolte et al. 2004). We observed BFA bodies positive for both, HA3-TTN5-Alexa 488 and FM4-64 signals (Figure 4B). Similar patterns were observed for YFP-TTN5-derived signals in YFP-TTN5-expressing roots (Figure 4C). Hence, HA3-TTN5 and YFP-TTN5 can be present in similar subcellular membrane compartments."

We did not find evidence that HA3-TTN5 can localize at the ER using whole-mount immunostaining (NEW Figure 3P; NEW Figure 4A, B). Hence, we are careful with describing that fluorescence at the ER, as seen in the YFP-TTN5 line (Figure 3M, N) reflects TTN5 localization. We therefore do not focus the text on the ER pattern in the Result section (starting Line 295):

„Additionally, YFP signals were also detected in a net-like pattern typical for ER localization (Figure 3M, N). (...) We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

*And we discuss in the Discussion section (starting Line 552): *

„We based the TTN5 localization data on tagging approaches with two different detection methods to enhance reliability of specific protein detection. Even though YFP-TTN5 did not complement the embryo-lethality of a ttn5 loss of function mutant, we made several observations that suggest YFP-TTN5 signals to be meaningful at various membrane sites. We do not know why YFP-TTN5 does not complement. There could be differences in TTN5 levels and interactions in some cell types, which were hindering specifically YFP-TTN5 but not HA3-TTN5. (...) Though constitutively driven, the YFP-TTN5 expression may be delayed or insufficient at the early embryonic stages resulting in the lack of embryo-lethal complementation. On the other hand, the very fast nucleotide exchange activity may be hindered by the presence of a large YFP-tag in comparison with the small HA3-tag which is able to rescue the embryo-lethality. The lack of complementation represents a challenge for the localization of small GTPases with rapid nucleotide exchange in plants. Despite of these limitations, we made relevant observations in our data that made us believe that YFP signals in YFP-TTN5-expressing cells at membrane sites can be meaningful."

2- What are the structures with TTN5 fluorescence depleted at the center that appear in control conditions? They look different from the Golgi labeled by Man1 but similar to MVBs upon wortmannin treatment, except that in control conditions MVBs never appear like this. Are they related to any kind of vacuolar structures that would be involved in quality control-induced degradation of non-functional proteins?

Our response:

The reviewer certainly refers to fluorescence images from N. benthamiana leaf epidermal cells where different circularly shaped structures are visible. In these respective structures, the fluorescent circles are depleted from fluorescence in the center, e.g. in Figure 5C, YFP- fluorescent signals in TTN5T30N transformed leaf discs. We suspect that these structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al., 2020 for ANNI-GFP (reference in manuscript). The reviewer certainly does not refer to swollen MVBs that are seen following wortmannin treatment, as in Figure 5N-P, which look similar in their shape but are larger in size. Please note that we always included the control conditions, namely the images recorded before the wortmannin treatment, so that we were able to investigate the changes induced by wortmannin. Hence, we can clearly say that the structures with depleted fluorescence in the center as in Figure 5C are not wortmannin-induced swollen MVBs.To make these points clear to the reader, we added an explanation into the text (Line 385-388):

„We also observed YFP fluorescence signals in the form of circularly shaped ring structures with a fluorescence-depleted center. These structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al. (2020) for ANNI-GFP."

3- The fluorescence at nucleus could be due to a proportion of YFP-TTN5 that is degraded and released free-GFP, a western-blot of the membrane fraction vs the cytosolic fraction could help solving this issue.

Our response:

In an α-GFP Western blot using YFP-TTN5 Arabidopsis seedlings, we detected besides the expected and strong 48 kDa YFP-TTN5 band, three additional weak bands ranging between 26 to 35 kDa (NEW Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins expressed from aberrant transcripts. α-HA Western blot controls performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size (Supplementary Figure S7D). We must therefore be cautious about nuclear TTN5 localization and we rephrased the text carefully (starting Line 300):

„We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

4- It is not so easy to conclude from the co-localization experiments. The confocal pictures are not always of high quality, some of them appear blurry. The Golgi localization looks convincing, but the BFA experiments are not that clear. The MVB localization is pretty convincing but the images are blurry. An issue is the quantification of the co-localizations. Several methods were employed but they do not provide consistent results. As for the object-based co-localization method, the authors employ in the text co-localization result either base on the % of YFP-labeled structures or the % of mCherry/mRFP-labeled structures, but the results are not going always in the same direction. For example, the proportion of YFP-TTN5 that co-localize with MVBs is not so different between WT and mutated version but the proportion of MVBs that co-localize with TTN5 is largely increased in the Q70L mutant. Thus it is quite difficult to interpret homogenously and in an unbiased way these results. Moreover, the results coming from the centroid-based method were presented in a table rather than a graph, I think here the authors wanted to hide the huge standard deviation of these results, what is the statistical meaning of these results?

Our response:

First of all, we like to point out that, as explained above, the BFA experiments are now more clear. We performed additional BFA treatment coupled with immunostaining using HA3-TTN5-expressing Arabidopsis seedlings and coupled with fluorescence analysis using YFP-TTN5-expressing Arabidopsis plants. In both experiments, we observed the typical BFA bodies very clearly (NEW Figure 4B, C).

Second, we like to insist that we performed colocalization very carefully and quantified the data in three different manners. We like to state that there is no general standardized procedure that best suits the idea of a colocalization pattern. Results of colocalization are represented in stem diagrams and table format, including statistical analysis. Colocalization was carried out with the ImageJ plugin JACoP for Pearson's and Overlap coefficients and based on the centroid method. The plotted Pearson's and Overlap coefficients are presented in bar diagrams in Supplementary Figure S8A and C, including statistics. The obtained values by the centroid method are represented in table format in Supplementary Figure S8B and D, which *can be considered a standard method (see Ivanov et al., 2014). *

Colocalization of two different fluorescence signals was performed for the two channels in a specific chosen region of interest (indicating in % the overlapping signal versus the sum of signal for each channel). The differences between the YFP/mRFP and mRFP/YFP ratios indicate that a higher percentage of ARA7-RFP signal is colocalizing with YFP-TTN5Q70L signal than with the TTN5WT or the TTN5T30N mutant form signals, while the YFP signals have a similar overlap with ARA7-positive structures. This is not a contradiction. Presumably this answers well the questions on colocalization.

Please note that upon acceptance for publication, we will upload all original colocalization data to BioImage Archive. Hence, the high-quality data can be reanalyzed by readers.

5- The use of FM4-64 to address the vacuolar trafficking is a hazardous, FM4-64 allows the tracking of endocytosis but does not say anything on vacuolar degradation targeting and even less on the potential function of TTN5 in endosomal vacuolar targeting. Similarly, TTN5, even if localized at the Golgi, is not necessarily function in Golgi-trafficking. __Our response: __

*Perhaps our previous description was misleading. Thank you for pointing this out. We reformulated the text and modified the schematic representation of FM4-64 in NEW Figure 6A: *

"(A), Schematic representation of progressive stages of FM4-64 localization and internalization in a cell. FM4-64 is a lipophilic substance. After infiltration, it first localizes in the plasma membrane, at later stages it localizes to intracellular vesicles and membrane compartments. This localization pattern reflects the endocytosis process (Bolte et al. 2004)."

6- The manuscript lacks in its present shape of functional evidences for a role of TTN5 in any trafficking steps. I understand that the KO mutant is lethal but what are the phenotypes of the Q70L and T30N mutant plants? What is the seedling phenotype, how are the Golgi and MVBs looking like in these mutants? Do the Q70L or T30N mutants perturbed the trafficking of any cargos?

__Our response: __

*We agree fully that functional evidences are interesting to assign roles for TTN5 in trafficking steps. A phenotype associated with TTN5T30N and TTN5Q70L is clearly meaningful. *

First of all, we like to emphasize that it is incorrect that the manuscript lacks functional evidences for a role of TTN5 and the two mutants. In fact, the manuscript even highlights several functional activities that are meaningful in a cellular context. These include different types of kinetic GTPase enzyme activities, subcellular localization in planta and association with different endomembrane compartments and subcellular processes such as endocytosis. We surely agree that future research can focus even more on cell physiological aspects and the physiological functions in plants to examine the proposed roles of TTN5 in intracellular trafficking steps. For such studies, our findings are the fundamental basis.

Concerning the aspect of colocalization of the mutants with the markers we show in Figure 5C, D and G, H that YFP-TTN5T30N- and YFP-TTN5Q70L-related signals colocalize with the Golgi marker GmMan1-mCherry. Figure 5K, L and O, P show that YFP-TTN5T30N and YFP-TTN5Q70L-related signals can colocalize with the MVB marker, and this may affect relevant vesicle trafficking processes and plasma membrane protein regulation involved in root cell elongation.

*At present, we have not yet investigated perturbed cargo trafficking. These aspects are certainly interesting but require extensive work and testing of appropriate physiological conditions and appropriate cargo targets. We discuss future perspectives in the Discussion. We agree that such functional information is of great importance, but needs to be clarified in future studies. *

__Reviewer #1 (Significance (Required)): __

In conclusion, I think this manuscript is a good biochemical description of an ARF-like protein but it would need to be strengthen on the cell biology and functional sides. Nonetheless, provided these limitations fixed, this manuscript would advance our knowledge of small GTPases in plants. The major conceptual advance of that study is to provide a non-canonical behavior of the active/inactive cycle dynamics for a small-GTPase. Of course this dynamic probably has an impact on TTN5 function and involvement in trafficking, although this remains to be fully demonstrated. Provided a substantial amount of additional experiments to support the claims of that study, this study could be of general interest for scientist working in the trafficking field.

__Our response: __

We thank reviewer 1 for the very fruitful comments. We hope that with the additional experiments, NEW Figures and NEW Supplementary Figures as well as our changes in the text, all comments by the reviewer have been addressed.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

The manuscript by Mohr and colleagues characterizes the Arabidopsis predicted small GTPase TITAN5 in both biochemical and cell biology contexts using in vitro and in planta techniques. In the first half of the manuscript, the authors use in vitro nucleotide exchange assays to characterise the GTPase activity and nucleotide binding properties of TITAN5 and two mutant variants of it. The in vitro data they produce indicates that TITAN5 does indeed have general GTPase and nucleotide binding capability that would be expected for a protein predicted to be a small GTPase. Interestingly, the authors show that TITAN5 favors a GTP-bound form, which is different to many other characterized GTPases that favor GDP-binding. The authors follow their biochemical characterisation of TITAN with in planta experiments characterizing TITAN5 and its mutant variants association with the plant endomembrane system, both by stable expression in Arabidopsis and transient expression in N.benthamiana.

The strength of this manuscript is in its in vitro biochemical characterisation of TITAN5 and variants. I am not an expert on in vitro GTPase characterisation and so cannot comment specifically on the assays they have used, but generally speaking this appears to have been well done, and the authors are to be commended for it. In vitro characterisation of plant small GTPases is uncommon, and much of our knowledge is inferred for work on animal or yeast GTPases, so this will be a useful addition to the plant community in general, especially as TITAN5 is an essential gene. The in planta data that follows is sadly not as compelling as the biochemical data, and suffers from several weaknesses. I would encourage the authors to consider trying to improve the quality of the in planta data in general. If improved and then combined with the biochemical aspects of the paper, this has the potential to make a nice addition to plant small GTPase and endomembrane literature.

The manuscript is generally well written and includes the relevant literature.

Major issues:

1. The authors make use of a p35s: YFP-TTN5 construct (and its mutant variants) both stably in Arabidopsis and transiently in N.benthamiana. I know from personal experience that expressing small GTPases from non-endogenous promoters and in transient expression systems can give very different results to when working from endogenous promoters/using immunolocalization in stable expression systems. Strong over-expression could for example explain why the authors see high 'cytosolic' levels of YFP-TTN5. It is therefore questionable how much of the in planta localisation data presented using p35S and expression in tobacco is of true relevance to the biological function of TITAN5. The authors do present some immunolocalization data of HA3-TTN5 in Arabidopsis, but this is fairly limited and it is very difficult in its current form to use this to identify whether the data from YFP-TTN5 in Arabidopsis and tobacco can be corroborated. I would encourage the authors to consider expanding the immunolocalization data they present to validate their findings in tobacco. __Our response: __

We are aware that endogenous promoters may be preferred over 35S promoter. However, the two types of lines we generated with endogenous promoter did both not show fluorescent signals so that we could unfortunately not use them (not shown). Besides 35S promoter-mediated expression we were also investigating inducible expression vectors for fluorescence imaging in N. benthamiana (not shown). Both inducible and constitutive expression showed very similar expression patterns so that we chose characterizing in detail the 35S::YFP-TTN5 fluorescence in both N. bethamiana*and Arabidopsis. *

We have expanded immunolocalization using the HA3-TTN5 line and compare it now along with YFP fluorescence signal in YFP-TTN5 seedlings (NEW Figure 3P; NEW Figure 4).

„For a more detailed investigation of HA3-TTN5 subcellular localization, we then performed co-immunofluorescence staining with an Alexa 488-labeled antibody recognizing the Golgi and TGN marker ARF1, while detecting HA3-TTN5 with an Alexa 555-labeled antibody (Robinson et al. 2011, Singh et al. 2018) (Figure 4A). ARF1-Alexa 488 staining was clearly visible in punctate structures representing presumably Golgi stacks (Figure 4A, Alexa 488), as previously reported (Singh et al. 2018). Similar structures were obtained for HA3-TTN5-Alexa 555 staining (Figure 4A, Alexa 555). But surprisingly, colocalization analysis demonstrated that the HA3-TTN5-labeled structures were mostly not colocalizing and thus distinct from the ARF1-labeled ones (Figure 4A). Yet the HA3-TTN5- and ARF1-labeled structures were in close proximity to each other (Figure 4A). We hypothesized that the HA3-TTN5 structures can be connected to intracellular trafficking steps. To test this, we performed brefeldin A (BFA) treatment, a commonly used tool in cell biology for preventing dynamic membrane trafficking events and vesicle transport involving the Golgi. BFA is a fungal macrocyclic lactone that leads to a loss of cis-cisternae and accumulation of Golgi stacks, known as BFA-induced compartments, up to the fusion of the Golgi with the ER (Ritzenthaler et al. 2002, Wang et al. 2016). For a better identification of BFA bodies, we additionally used the dye FM4-64, which can emit fluorescence in a lipophilic membrane environment. FM4-64 marks the plasma membrane in the first minutes following application to the cell, then may be endocytosed and in the presence of BFA become accumulated in BFA bodies (Bolte et al. 2004). We observed BFA bodies positive for both, HA3-TTN5-Alexa 488 and FM4-64 signals (Figure 4B). Similar patterns were observed for YFP-TTN5-derived signals in YFP-TTN5-expressing roots (Figure 4C). Hence, HA3-TTN5 and YFP-TTN5 can be present in similar subcellular membrane compartments."

• *

Many of the confocal images presented are of poor quality, particularly those from N.benthamiana.

Our response:

All confocal images are of high quality in their original format. To make them accessible, we will upload all raw data to BioImage Archive upon acceptance of the manuscript.

The authors in some places see YFP-TTN5 in cell nuclei. This could be a result of YFP-cleavage rather than genuine nuclear localisation of YFP-TTN5, but the authors do not present western blots to check for this.

__Our response: __

As described in our response to reviewer 1, comment 3, Fluorescence signals were detected within the nuclei of root cells of YFP-TTN5 plants, while immunostaining signals of HA3-TTN5 were not detected in the nucleus. In an α-GFP Western blot using YFP-TTN5 Arabidopsis seedlings, we detected besides the expected and strong 48 kDa YFP-TTN5 band, three additional weak bands ranging between 26 to 35 kDa (NEW Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins expressed from aberrant transcripts. α-HA Western blot controls performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size (Supplementary Figure S7D). We must therefore be cautious about nuclear TTN5 localization and we rephrased the text carefully (starting Line 300):

• *

„We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

That YFP-TTN5 fails to rescue the ttn5 mutant indicates that YFP-tagged TTN5 may not be functional. If the authors cannot corroborate the YFP-TTN5 localisation pattern with that of HA3-TTN5 via immunolocalization, then the fact that YFP-TTN5 may not be functional calls into question the biological relevance of YFP-TTN5's localisation pattern.

__Our response: __

This refers to your comment 1, please check this comment for a detailed response. Please also see our answer to reviewer 1, comment 1.

At first, we like to state that specific detection of intracellular localization of plant proteins in plant cells is generally technically very difficult, when the protein abundance is not overly high. In this revised version, we extended immunostaining analysis to different membrane compartments, including now immunostaining of complementing HA3-TTN5 in the absence and presence of BFA, along with immunodetection of ARF1 and FM4-64 labeling in roots (NEW Figure 3P, NEW Figure 4A, B). In the revised version, we focus the analysis and conclusions on the fluorescence patterns that overlap between YFP-TTN5 detection and HA3-TTN5 immunodetection. With this, we can be most confident about subcellular TTN5 localization. Please find this NEW text in the Result section (starting Line 323):

„For a more detailed investigation of HA3-TTN5 subcellular localization, we then performed co-immunofluorescence staining with an Alexa 488-labeled antibody recognizing the Golgi and TGN marker ARF1, while detecting HA3-TTN5 with an Alexa 555-labeled antibody (Robinson et al. 2011, Singh et al. 2018) (Figure 4A). ARF1-Alexa 488 staining was clearly visible in punctate structures representing presumably Golgi stacks (Figure 4A, Alexa 488), as previously reported (Singh et al. 2018). Similar structures were obtained for HA3-TTN5-Alexa 555 staining (Figure 4A, Alexa 555). But surprisingly, colocalization analysis demonstrated that the HA3-TTN5-labeled structures were mostly not colocalizing and thus distinct from the ARF1-labeled ones (Figure 4A). Yet the HA3-TTN5- and ARF1-labeled structures were in close proximity to each other (Figure 4A). We hypothesized that the HA3-TTN5 structures can be connected to intracellular trafficking steps. To test this, we performed brefeldin A (BFA) treatment, a commonly used tool in cell biology for preventing dynamic membrane trafficking events and vesicle transport involving the Golgi. BFA is a fungal macrocyclic lactone that leads to a loss of cis-cisternae and accumulation of Golgi stacks, known as BFA-induced compartments, up to the fusion of the Golgi with the ER (Ritzenthaler et al. 2002, Wang et al. 2016). For a better identification of BFA bodies, we additionally used the dye FM4-64, which can emit fluorescence in a lipophilic membrane environment. FM4-64 marks the plasma membrane in the first minutes following application to the cell, then may be endocytosed and in the presence of BFA become accumulated in BFA bodies (Bolte et al. 2004). We observed BFA bodies positive for both, HA3-TTN5-Alexa 488 and FM4-64 signals (Figure 4B). Similar patterns were observed for YFP-TTN5-derived signals in YFP-TTN5-expressing roots (Figure 4C). Hence, HA3-TTN5 and YFP-TTN5 can be present in similar subcellular membrane compartments."

We did not find evidence that HA3-TTN5 can localize at the ER using whole-mount immunostaining (NEW Figure 3P; NEW Figure 4A, B). Hence, we are careful with describing that fluorescence at the ER, as seen in the YFP-TTN5 line (Figure 3M, N) reflects TTN5 localization. We therefore do not focus the text on the ER pattern in the Result section (starting Line 295):

„Additionally, YFP signals were also detected in a net-like pattern typical for ER localization (Figure 3M, N). (...) We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

*And we discuss in the Discussion section (starting Line 552): *

„We based the TTN5 localization data on tagging approaches with two different detection methods to enhance reliability of specific protein detection. Even though YFP-TTN5 did not complement the embryo-lethality of a ttn5 loss of function mutant, we made several observations that suggest YFP-TTN5 signals to be meaningful at various membrane sites. We do not know why YFP-TTN5 does not complement. There could be differences in TTN5 levels and interactions in some cell types, which were hindering specifically YFP-TTN5 but not HA3-TTN5. (...) Though constitutively driven, the YFP-TTN5 expression may be delayed or insufficient at the early embryonic stages resulting in the lack of embryo-lethal complementation. On the other hand, the very fast nucleotide exchange activity may be hindered by the presence of a large YFP-tag in comparison with the small HA3-tag which is able to rescue the embryo-lethality. The lack of complementation represents a challenge for the localization of small GTPases with rapid nucleotide exchange in plants. Despite of these limitations, we made relevant observations in our data that made us believe that YFP signals in YFP-TTN5-expressing cells at membrane sites can be meaningful."

• *

Without a cell wall label/dye, the plasmolysis data presented in Figure 5 is hard to visualize.

__Our response: __

Figure 6E-G (previously Fig. 5) show the results of plasmolysis experiments with YFP-TTN5 and the two mutant variant constructs. It is clearly possible to observe plasmolysis when focusing on the Hechtian strands. Hechtian strands are formed due to the retraction of the protoplast as a result of the osmotic pressure by the added mannitol solution. Hechtian strands consist of PM which remained in contact with the cell wall, visible as thin filamental structures. We stained the PM and the Hechtian strands by the PM dye FM4-64. This is similary done in Yoneda et al., 2020. We could detect in the YFP-TTN5-transformed cells, colocalization with the YFP channels and the PM dye in filamental structures between two neighbouring FM4-64-labelled PMs. Although an additional labeling of the cell wall may further indicate plasmolysis, it is not needed here.

Please consider that we will upload all original image data to BioImage Archive so that a detailed re-investigation of the images can be done.

• *

__Minor issues: __

In some of the presented N.benthamiana images, it looks like YFP-TTN5 may be partially ER-localised. However, co-localisation with an ER marker is not presented.

Our response:

*Referring to our response to comments 1 and 3 of reviewer 2 and to comment 1 of reviewer 1: *

We did not find evidence that HA3-TTN5 can localize at the ER using whole-mount immunostaining (NEW Figure 3P; NEW Figure 4A, B). Hence, we are careful with describing that fluorescence at the ER, as seen in the YFP-TTN5 line (Figure 3M, N) reflects TTN5 localization. We therefore do not focus the text on the ER pattern in the Result section (starting Line 295):

„Additionally, YFP signals were also detected in a net-like pattern typical for ER localization (Figure 3M, N). (...) We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

*And we discuss in the Discussion section (starting Line 552): *

„We based the TTN5 localization data on tagging approaches with two different detection methods to enhance reliability of specific protein detection. Even though YFP-TTN5 did not complement the embryo-lethality of a ttn5 loss of function mutant, we made several observations that suggest YFP-TTN5 signals to be meaningful at various membrane sites. We do not know why YFP-TTN5 does not complement. There could be differences in TTN5 levels and interactions in some cell types, which were hindering specifically YFP-TTN5 but not HA3-TTN5. (...) Though constitutively driven, the YFP-TTN5 expression may be delayed or insufficient at the early embryonic stages resulting in the lack of embryo-lethal complementation. On the other hand, the very fast nucleotide exchange activity may be hindered by the presence of a large YFP-tag in comparison with the small HA3-tag which is able to rescue the embryo-lethality. The lack of complementation represents a challenge for the localization of small GTPases with rapid nucleotide exchange in plants. Despite of these limitations, we made relevant observations in our data that made us believe that YFP signals in YFP-TTN5-expressing cells at membrane sites can be meaningful."

• *

There is some inconsistency within the N.benthamiana images. For example, compare Figure 4C of YFP-TTN5T30N to Figure 4O of YFP-TTN5T30N. Figure 4O is presented as being significant because wortmannin-induced swollen ARA7 compartments are labelled by YFP-TTN5T30N. However, structures very similar to these can already been seen in Figure 4C, which is apparently an unrelated experiment. This, to my mind, is likely a result of the very different expression levels between different cells that can be produced by transient expression in N.benthamiana.

__Our response: __

Former Figure 4 is now Figure 5. As detailed in our response to comment 2 of reviewer 1:

The reviewer certainly refers to fluorescence images from N. benthamiana leaf epidermal cells where different circularly shaped structures are visible. In these respective structures, the fluorescent circles are depleted from fluorescence in the center, e.g. in Figure 5C, YFP- fluorescent signals in TTN5T30N transformed leaf discs. We suspect that these structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al., 2020 for ANNI-GFP (reference in manuscript). The reviewer certainly does not refer to swollen MVBs that are seen following wortmannin treatment, as in Figure 5N-P, which look similar in their shape but are larger in size. Please note that we always included the control conditions, namely the images recorded before the wortmannin treatment, so that we were able to investigate the changes induced by wortmannin. Hence, we can clearly say that the structures with depleted fluorescence in the center as in Figure 5C are not wortmannin-induced swollen MVBs.To make these points clear to the reader, we added an explanation into the text (Line 385-388):

„We also observed YFP fluorescence signals in the form of circularly shaped ring structures with a fluorescence-depleted center. These structures can be of vacuolar origin as described for similar fluorescent rings in Tichá et al. (2020) for ANNI-GFP."

**Referees cross-commenting**

It sems that all of the reviewers have converged on the conclusion that the in planta characterisation of TTN5 is insufficient to be of substantial interest to the field, highlighting the fact that major improvements are required to strengthen this part of the manuscript and increase its relevance.

__Reviewer #2 (Significance (Required)): __

General assessment: the strengths of this work are in its in vitro characterisation of TITAN5, however, the in planta characterisation lacks depth.

Significance: the in vitro characterisation of TITAN5 is commendable as such work is lacking for plant GTPases. However, the significance of the work would be boosted substantially by better in planta characterisation, which is where most the most broad interest will lie.

My expertise: my expertise is in in planta characterisation of small GTPases and their interactors.

__Our response: __

We thank the reviewer for the kind evaluation of our manuscript. We are confident that the changes in the text and NEW Figures and NEW Supplementary Figures will be convincing to consider our work.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

Summary: Cellular traffic is an important and well-studied biological process in animal and plant systems. While components involved in transport are known the mechanism by which these components control activity or destination remains to be studied. A critical step in regulating traffic is proper budding and tethering of vesicles. A critical component in determining this step is a family proteins with GTPase activity, which act as switches facilitating vesicle interaction between proteins, or cytoskeleton. The current manuscript by Mohr and colleagues have characterized a small GTPase TITAN5 (TTN5) and identified two residues Gln70 and Thr30 in the protein which they propose to have functional roles. The authors catalogue the localization, GTP hydrolytic activity, and discuss putative functions of TTN5 and the mutants.

__Major comments: __

The core of the manuscript, which is descriptive characterization of TTN5, lies in reliably demonstrating putative roles. While the GTP hydrolysis rates are well-quantified (though the claims need to be toned down), the microscopy data especially the association of TTN5 with different endomembrane compartments is not convincing due to the quality (low resolution) of the figures submitted. The manuscript text is difficult to navigate due to repetition and inconsistency in the order that the mutants are referred. I am requesting additional experiments which should be feasible considering the authors have all the materials required to perform the experiments and obtain high-quality images which support their claims.

In general the figure quality needs to be improved for all microscopy images. I would suggest that the authors highlight 1-2 individual cells to make their point and use the current images as supplementary to establish a broader spread. __Our response: __

*We have worked substantially on the text and figures to make the content well comprehensive. The mutants are referred to in a consistent manner in the text and figures. We have addressed requested experiments. *

As we pointed out in the cover letter and our responses to reviewers 1 and 2, we will upload all raw image data to BioImage Archive upon acceptance of the manuscript so that they can be re-examined without any reduction of resolution. Furthermore, we have conducted new experiments on immunolocalization of HA3-TTN5 (NEW Figure 3P, NEW Figure 4A, B). The text has been improved in several places (see highlighted changes in the manuscript and as detailed in the responses to reviewer 1. We think, this addresses well the reviewers' concerns.

Fig. S1 lacks clarity. __Our response: __

Supplementary Figure S1 shows TTN5 gene expression in different organs and growing stages as revealed by transcriptomic data, made available through the AtGenExpress eFB tool of the Bio-Analytic Resource for Plant Biology (BAR). The figure visualizes that TTN5 is ubiquitously expressed in different plant organs and tissues, e.g. the epidermis layers that we investigated here, and throughout development including embryo development. In accordance with the embryo-lethal phenotype, this highlights well that TTN5* is needed throughout for plant growth and it emphasizes that our investigation of TTN5 localization in epidermis cells is valid. *

We have added a better description to the figure legend. We now also mention the respective publications from which the transcriptome data-sets are derived. The modified figure legend is:

"Supplementary Figure S1. Visualization of TTN5 gene expression levels during plant development based on transcriptome data. Expression levels in (A), different types of aerial organs at different developmental stages; from left to right and bottom to top are represented different seed and plant growth stages, flower development stages, different leaves, vegetative to inflorescence shoot apex, embryo and silique development stages; (B), seedling root tissues based on single cell analysis represented in form of a uniform manifold approximation and projection plot; (C), successive stages of embryo development. As shown in (A) to (C), TTN5 is ubiquitously expressed in these different plant organs and tissues. In particular, it should be noted that TTN5 transcripts were detectable in the epidermis cell layer of roots that we used for localization of tagged TTN5 protein in this study. In accordance with the embryo-lethal phenotype, the ubiquitous expression of TTN5 highlights its importance for plant growth. Original data were derived from (Nakabayashi et al. 2005, Schmid et al. 2005) (A); (Ryu et al. 2019) (B); (Waese et al. 2017) (C). Gene expression levels are indicated by local maximum color code, ranging from the minimum (no expression) in yellow to the maximum (highest expression) in red."

For the supplementary videos, it is difficult to determine if punctate structures are moving or is it cytoplasmic streaming? Could this be done with a co-localized marker? Considering that such markers have been used later in Fig. 4? __Our response: __

We had detected movement of YFP fluorescent structures in all analyzed YFP-TTN5 plant parts except the root tip. Movement of fluorescence signals in YFP-TTN5T30N seedlings was slowed in hypocotyl epidermis cells. To answer the reviewer comment, we added three NEW supplemental videos (NEW Supplementary Video Material S1M-O) generated with all the three YFP-TTN5 constructs imaged over time in N. benthamiana leaf epidermal cells upon colocalization with the cis-Golgi marker GmMan1-mCherry as requested by the reviewer. In these NEW videos, some of *the YFP fluorescent spots seem to move together with the Golgi stacks. GmMan1 is described with a stop-and-go directed movement mediated by the actino-myosin system (Nebenführ 1999) and similarly it might be the case for YFP-TTN5 signals based on the colocalization. *

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It would be good if the speed of movement is quantified, if the authors want to retain the current claims in results and the discussion. __Our response: __

*We describe a difference in the movement of YFP fluorescent signal for the YFP-TTN5T30N variant in the hypocotyl compared to YFP-TTN5 and YFP-TTN5Q70L. In hypocotyl cells, we could observe a slowed down or arrested movement specifically of YFP-TTN5T30N fluorescent structures, and we describe this in the Results section (Line 278-291). *

"Interestingly, the mobility of these punctate structures differed within the cells when the mutant YFP-TTN5T30N was observed in hypocotyl epidermis cells, but not in the leaf epidermis cells (Supplementary Video Material S1E, compare with S1B) nor was it the case for the YFP-TTN5Q70L mutant (Supplementary Video Material S1F, compare with S1E)."

*The slowed movement in the YFP-TTN5T30N mutant is well visible even without quantification. We checked that the manuscript text does not contain overstatements in this regard. *

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Fig.2 I am not sure what the unit / scale is in Fig. 2D/E if each parameter (Kon, Koff, and Kd) are individually plotted? Could the authors please clarify/simplify this panel?

__Our response: __

We presented kinetics for nucleotide association (kon) and dissociation (koff) and the dissociation constant (Kd) in a bar diagram for each nucleotide, mdGDP (Figure 2D) and mGppNHp (Figure 2E). We modified and relabeled the bar diagram representation. It should be now very clear which are the parameters and units. Please see also the other modified figures (NEW modified Figure 2A-H). We also modified the legend of Figure 2D and E:

"(D-E), Kinetics of association and dissociation of fluorescent nucleotides mdGDP (D) or mGppNHp (E) with TTN5 proteins (WT, TTN5T30N, TTN5Q70L) are illustrated as bar charts. The association of mdGDP (0.1 µM) or mGppNHp (0.1 µM) with increasing concentration of TTN5WT, TTN5T30N and TTN5Q70L was measured using a stopped-flow device (see A, B; data see Supplementary Figure S3A-F, S4A-E). Association rate constants (kon in µM-1s-1) were determined from the plot of increasing observed rate constants (kobs in s-1) against the corresponding concentrations of the TTN5 proteins. Intrinsic dissociation rates (koff in s-1) were determined by rapidly mixing 0.1 µM mdGDP-bound or mGppNHp-bound TTN5 proteins with the excess amount of unlabeled GDP (see A, C, data see Supplementary Figure S3G-I, S4F-H). The nucleotide affinity (dissociation constant or Kd in µM) of the corresponding TTN5 proteins was calculated by dividing koff by kon. When mixing mGppNHp with nucleotide-free TTN5T30N, no binding was observed (n.b.o.) under these experimental conditions."

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Are panels D and E representing values for mdGDP and GppNHP? This is not very clear from the figure legend.

__Our response: __

Yes, Figure 2D and E represent the kon, koff and Kd values for mdGDP (Figure 2D) and mGppNHP (Figure 2E). As detailed in our previous response to comment 2a, we modified figure and figure legend to make the representation more clear.

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Fig. 3 Same comments as in para above - improve resolution fo images, concentrate on a few selected cells, if required use an inset figure to zoom-in to specific compartments. Our response:

As detailed in our responses to reviewers 1 and 2, we will upload all original image data to BioImage Archive upon acceptance of the manuscript, so that a detailed investigation of all our images is possible without any reduction of resolution.

Please provide the non-fluorescent channel images to understand cell topography __Our response: __

*We presented our microscopic images with the respective fluorescent channel and for colocalization with an additional merge. We did not present brightfield images as the cell topography was already well visible by fluorescent signal close to the PM. Therefore, brightfield images would not provide any benefit. Since we will upload all original data to BioImage Archive for a detailed investigation of all our images, the data can be obtained if needed. *

Is the nuclear localization seen in transient expression (panel L-N) an artefact? If so, this needs to be mentioned in the text. Our response:

As explained in our responses to reviewers 1 and 2, fluorescence signals were detected within the nuclei of root cells of YFP-TTN5 plants, while immunostaining signals of HA3-TTN5 were not detected in the nucleus.

In an α-GFP Western blot using YFP-TTN5 Arabidopsis seedlings, we detected besides the expected and strong 48 kDa YFP-TTN5 band, three additional weak bands ranging between 26 to 35 kDa (NEW Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins expressed from aberrant transcripts. α-HA Western blot controls performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size (Supplementary Figure S7D). We must therefore be cautious about nuclear TTN5 localization and we rephrased the text carefully (starting Line 300):

„We also found multiple YFP bands in α-GFP Western blot analysis using YFP-TTN5 Arabidopsis seedlings. Besides the expected and strong 48 kDa YFP-TTN5 band, we observed three weak bands ranging between 26 to 35 kDa (Supplementary Figure S7C). We cannot explain the presence of these small protein bands. They might correspond to free YFP, to proteolytic products or potentially to proteins produced from aberrant transcripts with perhaps alternative translation start or stop sites. On the other side, a triple hemagglutinin-tagged HA3-TTN5 driven by the 35S promoter did complement the embryo-lethal phenotype of ttn5-1 (Supplementary Figure S7D, E). α-HA Western blot control performed with plant material from HA3-TTN5 seedlings showed a single band at the correct size, but no band that was 13 to 18 kDa smaller (Supplementary Figure S7D). (...) We did not observe any staining in nuclei or ER when performing HA3-TTN5 immunostaining (Figure 3P; Figure 4A, B), as was the case for fluorescence signals in YFP-TTN5-expressing cells. Presumably, this can indicate that either the nuclear and ER signals seen with YFP-TTN5 correspond to the smaller proteins detected, as described above, or that immunostaining was not suited to detect them. Hence, we focused interpretation on patterns of localization overlapping between the fluorescence staining with YFP-labeled TTN5 and with HA3-TTN5 immunostaining, such as the particular signal patterns in the specific punctate membrane structures."

Fig. 4 - In addition to the points made for Fig. 3 The authors should consider reducing gain/exposure to improve image clarity. Especially for the punctate structures, which are difficult to observe in TTN5, likely because of the cytoplasmic localization as well.

__Our response: __

Thank you for this comment. We record image z-stacks and represent in single z-planes. Reducing the gain to decrease the cytoplasmic signal does not increase the clarity of the punctate structures as the signal strength will become weak.. As mentioned above, we will upload all original image data to BioImage Archive for a detailed investigation of all our images without any reduction of resolution.

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Reducing Agrobacterial load could be considered. OD of 0.4 is a bit much, 0.1 or even 0.05 could be tried. If available try expression in N. tabaccum, which is more amenable to microscopy. However, this is OPTIONAL, benthamiana should suffice. __Our response: __

Thank you for the suggestion. We are routinely using N. benthamiana leaf infiltration. When setting up this method at first, we did not observe different localization results by using different ODs of bacterial cultures. Hence, an OD600 of 0.4 is routinely used in our institute. This value is comparable with the literature although some literature reports even higher OD values for infiltration (Norkunas et al., 2018; Drapal et al., 2021; Zhang et al., 2020, Davis et al., 2020; Stephenson et al., 2018).

A standard norm now is to establish the level of colocalization is by quantifying a pearson's or Mander's correlation. Which I believe has been done in the text, I didn't find a plot representing the same? Could the data (which the authors already have) be plotted alongwith "n" as a table or graph? __Our response: __

*Please check our response to reviewer 1, comment 4. *

We like to insist that we performed colocalization very carefully and quantified the data in three different manners. We like to state that there is no general standardized procedure that best suits the idea of a colocalization pattern. Results of colocalization are represented in stem diagrams and table format, including statistical analysis. Colocalization was carried out with the ImageJ plugin JACoP for Pearson's and Overlap coefficients and based on the centroid method. The plotted Pearson's and Overlap coefficients are presented in bar diagrams in Supplementary Figure S8A and C, including statistics. The obtained values by the centroid method are represented in table format in Supplementary Figure S8B and D, which *can be considered a standard method (see Ivanov et al., 2014). *

Colocalization of two different fluorescence signals was performed for the two channels in a specific chosen region of interest (indicating in % the overlapping signal versus the sum of signal for each channel). The differences between the YFP/mRFP and mRFP/YFP ratios indicate that a higher percentage of ARA7-RFP signal is colocalizing with YFP-TTN5Q70L signal than with the TTN5WT or the TTN5T30N mutant form signals, while the YFP signals have a similar overlap with ARA7-positive structures. This is not a contradiction. Presumably this answers well the questions on colocalization.

Please note that upon acceptance for publication, we will upload all original colocalization data to BioImage Archive. Hence, the high-quality data can be reanalyzed by readers.

The cartoons for the action of chemicals are useful, but need a bit more clarity. Our response:

The schematic explanations of pharmacological treatments and expected outcomes are useful to readers. For a better understanding, we added additional explaining sentences to the figure legends (Figure 5E, M; Figure 6A). We also modified Figure 6A and the corresponding legend.

"(E), Schematic representation of GmMan1 localization at the ER upon brefeldin A (BFA) treatment. BFA blocks ARF-GEF proteins which leads to a loss of Golgi cis-cisternae and the formation of BFA-induced compartments due to an accumulation of Golgi stacks up to a redistribution of the Golgi to the ER by fusion of the Golgi with the ER (Renna and Brandizzi 2020)."

"(M), Schematic representation of ARA7 localization in swollen MVBs upon wortmannin treatment. Wortmannin inhibits phosphatidylinositol-3-kinase (PI3K) function leading to the fusion of TGN/EE to swollen MVBs (Renna and Brandizzi 2020)."

"(A), Schematic representation of progressive stages of FM4-64 localization and internalization in a cell. FM4-64 is a lipophilic substance. After infiltration, it first localizes in the plasma membrane, at later stages it localizes to intracellular vesicles and membrane compartments. This localization pattern reflects the endocytosis process (Bolte et al. 2004)."

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Fig. 5 does the Q70L mutant show reduced endocytosis ?

__Our response: __

We have not investigated this question. As detailed in our response to reviewer 1, *we like to emphasize that we agree fully that functional evidences are interesting to assign role for TTN5 in trafficking steps. A phenotype associated with TTN5T30N and TTN5Q70L would be clearly meaningful. *

Concerning the aspect of colocalization of the mutants with the markers we show in Figure 5C, D and G, H that YFP-TTN5T30N- and YFP-TTN5Q70L-related signals colocalize with the Golgi marker GmMan1-mCherry. Figure 5K, L and O, P show that YFP-TTN5T30N and YFP-TTN5Q70L-related signals can colocalize with the MVB marker, and this may affect relevant vesicle trafficking processes and plasma membrane protein regulation involved in root cell elongation.

*At present, we have not yet investigated perturbed cargo trafficking. These aspects are certainly interesting but require extensive work and testing of appropriate physiological conditions and appropriate cargo targets. We discuss future perspectives in the Discussion. We agree that such functional information is of great importance, but needs to be clarified in future studies. *

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The main text needs to be organized in a way that a reader can separate what is the hypothesis/assumption from actual results and conclusions (see lines #143-149).

Our response:

*Thank you for this comment. We reformulated text throughout the manuscript. *

The text is repeated in multiple places, while I understand that this is not plagiarism, the repetitiveness makes it difficult to read and understand the text. I highlight a couple of examples here, but please check the whole text thoroughly and edit/delete as necessary. a. Lines #124-125 with Lines #149-151 Lines #140-143

__Our response: __

*We checked the text and removed unnecessary repetitions. *

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• Could the authors elaborate on whether there are plan homologs of TTN5? Also, have other ARF/ARLs been compared to TTN5 beyond HsARF1? *

Our response:

Phylogenetic trees of the ARF family in Arabidopsis in comparison to human ARF family were already published by Vernoud et al. (2003). In this phylogenetic tree ARF, ARL and SAR proteins of Arabidopsis are compared with the members in humans and S. cervisiae. It is difficult to deduce whether the proteins are homologs or orthologs. In this setting, an ortholog of TTN5 may be HsARL2 followed by HsARL3. In Figure 1A we represented some human GTPases as closely related in sequence to TTN5, these are HsARL2, HsARF1 and AtARF1 since they are the best studied ARF GTPases. HRAS is a well-known member of the RAS superfamily which we used for kinetic comparison in Figure 2. We additionally compared published kinetics of RAC1, HsARF3, *CDC42, RHOA, ARF6, RAD, GEM, and RAS GTPases. *

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On a related note, a major problem I have with these kinetic values is the assumption of significance or not. For eg. Line#180 the values represent and 2 and 6-fold increase, if these numbers do not matter can a significance threshold be applied so as to understand how much fold-change is appreciable?

Our response:

The kinetics of TTN5 and its two mutant variants can be compared with those of other studied GTPases. To provide a basis for the statements about differences in GTPase activities, we modified the text and added respective references in the text for comparisons of fold changes.

The new text is now as follows Line 175-231):

„ We next measured the dissociation (koff) of mdGDP and mGppNHp from the TTN5 proteins in the presence of excess amounts of GDP and GppNHp, respectively (Figure 2C) and found interesting differences (Figure 2D, E; Supplementary Figures S3G-I, S4F-H). First, TTN5WT showed a koff value (0.012 s-1 for mGDP) (Figure 2D; Supplementary Figure S3G), which was 100-fold faster than those obtained for classical small GTPases, including RAC1 (Haeusler et al. 2006)and HRAS (Gremer et al. 2011), but very similar to the koff value of HsARF3 (Fasano et al. 2022). Second, the koffvalues for mGDP and mGppNHp, respectively, were in a similar range between TTN5WT (0.012 s-1 mGDP and 0.001 s-1mGppNHp) and TTN5Q70L (0.025 s-1 mGDP and 0.006 s-1 mGppNHp), respectively, but the koff values differed 10-fold between the two nucleotides mGDP and mGppNHp in TTN5WT (koff = 0.012 s-1 versus koff = 0.001 s-1; Figure 2D, E; Supplementary Figure S3G, I, S4F, H). Thus, mGDP dissociated from proteins 10-fold faster than mGppNHp. Third, the mGDP dissociation from TTN5T30N (koff = 0.149 s-1) was 12.5-fold faster than that of TTN5WT and 37-fold faster than the mGppNHp dissociation of TTN5T30N (koff = 0.004 s-1) (Figure 2D, E; Supplementary Figure S3H, S4G). Mutants of CDC42, RAC1, RHOA, ARF6, RAD, GEM and RAS GTPases, equivalent to TTN5T30N, display decreased nucleotide binding affinity and therefore tend to remain in a nucleotide-free state in a complex with their cognate GEFs (Erickson et al. 1997, Ghosh et al. 1999, Radhakrishna et al. 1999, Jung and Rösner 2002, Kuemmerle and Zhou 2002, Wittmann et al. 2003, Nassar et al. 2010, Huang et al. 2013, Chang and Colecraft 2015, Fisher et al. 2020, Shirazi et al. 2020). Since TTN5T30N exhibits fast guanine nucleotide dissociation, these results suggest that TTN5T30N may also act in either a dominant-negative or fast-cycling manner as reported for other GTPase mutants (Fiegen et al. 2004, Wang et al. 2005, Fidyk et al. 2006, Klein et al. 2006, Soh and Low 2008, Sugawara et al. 2019, Aspenström 2020).

The dissociation constant (Kd) is calculated from the ratio koff/kon, which inversely indicates the affinity of the interaction between proteins and nucleotides (the higher Kd, the lower affinity). Interestingly, TTN5WT binds mGppNHp (Kd = 0.029 µM) 10-fold tighter than mGDP (Kd = 0.267 µM), a difference, which was not observed for TTN5Q70L (Kd for mGppNHp = 0.026 µM, Kd for mGDP = 0.061 µM) (Figure 2D, E). The lower affinity of TTN5WT for mdGDP compared to mGppNHp brings us one step closer to the hypothesis that classifies TTN5 as a non-classical GTPase with a tendency to accumulate in the active (GTP-bound) state (Jaiswal et al. 2013). The Kd value for the mGDP interaction with TTN5T30N was 11.5-fold higher (3.091 µM) than for TTN5WT, suggesting that this mutant exhibited faster nucleotide exchange and lower affinity for nucleotides than TTN5WT. Similar as other GTPases with a T30N exchange, TTN5T30Nmay behave in a dominant-negative manner in signal transduction (Vanoni et al. 1999).

To get hints on the functionalities of TTN5 during the complete GTPase cycle, it was crucial to determine its ability to hydrolyze GTP. Accordingly, the catalytic rate of the intrinsic GTP hydrolysis reaction, defined as kcat, was determined by incubating 100 µM GTP-bound TTN5 proteins at 25{degree sign}C and analyzing the samples at various time points using a reversed-phase HPLC column (Figure 2F; Supplementary Figure S5). The determined kcat values were quite remarkable in two respects (Figure 2G). First, all three TTN5 proteins, TTN5WT, TTN5T30N and TTN5Q70L, showed quite similar kcatvalues (0.0015 s-1, 0.0012 s-1, 0.0007 s-1; Figure 2G; Supplementary Figure S5). The GTP hydrolysis activity of TTN5Q70L was quite high (0.0007 s-1). This was unexpected because, as with most other GTPases, the glutamine mutations at the corresponding position drastic impair hydrolysis, resulting in a constitutively active GTPase in cells (Hodge et al. 2020, Matsumoto et al. 2021). Second, the kcat value of TTN5WT (0.0015 s-1) although quite low as compared to other GTPases (Jian et al. 2012, Esposito et al. 2019), was 8-fold lower than the determined koff value for mGDP dissociation (0.012 s-1) (Figure 2E). This means that a fast intrinsic GDP/GTP exchange versus a slow GTP hydrolysis can have drastic effects on TTN5 activity in resting cells, since TTN5 can accumulate in its GTP-bound form, unlike the classical GTPase (Jaiswal et al. 2013). To investigate this scenario, we pulled down GST-TTN5 protein from bacterial lysates in the presence of an excess amount of GppNHp in the buffer using glutathione beads and measured the nucleotide-bound form of GST-TTN5 using HPLC. As shown in Figure 2H, isolated GST-TTN5 increasingly bonds GppNHp, indicating that the bound nucleotide is rapidly exchanged for free nucleotide (in this case GppNHp). This is not the case for classical GTPases, which remain in their inactive GDP-bound forms under the same experimental conditions (Walsh et al. 2019, Hodge et al. 2020)."

Another issue with the kinetic measurements is the significance levels. Line #198-201. The three proteins are claimed to have similar values and in the nnext line, the Q70L mutant is claimed to be high.

Our response:

Please see our response and changes in the text according in our response to the previous comment 9. We have provided extra explanations and references to clarify why the kinetic behavior of TTN5 is unusual in several respects (Line 215-220).

„First, all three TTN5 proteins, TTN5WT, TTN5T30N and TTN5Q70L, showed quite similar kcat values (0.0015 s-1, 0.0012 s-1, 0.0007 s-1; Figure 2G; Supplementary Figure S5). The GTP hydrolysis activity of TTN5Q70L was quite high (0.0007 s-1). This was unexpected because, as with most other GTPases, the glutamine mutations at the corresponding position drastic impair hydrolysis, resulting in a constitutively active GTPase in cells (Hodge et al. 2020, Matsumoto et al. 2021)."

Provide data for conclusion in line#214-215

Our response:

We agree that a reference should be added after this sentence to make this sentence clearer (Line 228-231).

"As shown in Figure 2H, isolated GST-TTN5 increasingly bonds GppNHp, indicating that the bound nucleotide is rapidly exchanged for free nucleotide (in this case GppNHp). This is not the case for classical GTPases, which remain in their inactive GDP-bound forms under the same experimental conditions (Walsh et al. 2019, Hodge et al. 2020)."

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How were the mutants studied here identified? random mutation or was it directed based on qualified assumptions?

__Our response: __

We used the T30N and the Q70L point mutations as such types of mutants had been reported to confer specific phenotypes in these well-conserved amino acid positions in multiple other small GTPases (Erickson et al. 1997, Ghosh et al. 1999, Radhakrishna et al. 1999, Jung and Rösner 2002, Kuemmerle and Zhou 2002, Wittmann et al. 2003, Nassar et al. 2010, Huang et al. 2013, Chang and Colecraft 2015, Fisher et al. 2020, Shirazi et al. 2020). In particular, these positions affect the interaction between small GTPases and their respective guanine nucleotide exchange factor (GEF; T30N) or on GTP hydrolysis (Q70L). We introduced the mutants and described their potential effect on the GTPase cycle in the introduction and cited exemplary literature. Please see also our response to comment 6 and the proposed text changes (Line 142-151).

Could more simplification be provided for deifitinition of Kon/Koff values. And can these values be compared between mutants directly?

__Our response: __

*We introduce kon and koff in the modified Figure 2D, E, and they are described in the figure legends. Moreover, we present the data for calculations in Supplementary Figures S3, 4, where again we define the values in the respective figure legends. *

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Data provided are not convincing to claim that both the mutant forms have lower association with the Golgi.

__Our response: __

*Our conclusion is that both YFP-TTN5 and YFP-TTN5Q70L fluorescence signals tend to colocalize more with the Golgi-marker signals compared to YFP-TTN5T30N signals as deduced from the centroid-based colocalization method (Line 404-405). *

"Hence, the GTPase-active TTN5 forms are likely more present at cis-Golgi stacks compared to TTN5T30N."

The Pearson coefficients of all three YFP-TTN5 constructs were nearly identical, but we could identify differences in overlapping centers between the YFP and mCherry channel. 48 % of the GmMan1-mCherry fluorescent cis-Golgi stacks were overlapping with signal of YFP-TTN5Q70L, while for YFP-TTN5T30N an overlap of only 31 % was detected. This means that less cis*-Golgi stacks colocalized with signals in the YFP-TTN5T30N mutant than in YFP-TTN5Q70L, which is the statement in our manuscript. *

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IN general the Authors should strongly consider the claims made in the manuscript. For eg. "This study lays the foundation for studying the functional relationships of this small GTPase" (line 125) is unqualified as this is true for every protein ever studied and published. Considering that TTN was not isolated/identified in this study for the first time this claim doesn't stand.

__Our response: __

*We reformulated the sentence (Line 123-124). *

"This study paves the way towards future investigation of the cellular and physiological contexts in which this small GTPase is functional."

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Line #185 - "characterestics of a dominant-negative...." What is this based on? From the text it is not clear what are the paremeters. Considering that no complementation phenotypes have been presented, this is a far-fetched claim Our response:

Small GTPases in general are a well studied protein family and the here used mutations T30N and Q70L are conserved amino acids and commonly used for the characterization of the Ras superfamily members. We added explaining sentences with references to the text. The characteristics referred to in the above paragraph is based on the kinetic study.

We modified the text as follows (Line 186-197 ):

„Third, the mGDP dissociation from TTN5T30N (koff = 0.149 s-1) was 12.5-fold faster than that of TTN5WT and 37-fold faster than the mGppNHp dissociation of TTN5T30N (koff = 0.004 s-1) (Figure 2D, E; Supplementary Figure S3H, S4G). Mutants of CDC42, RAC1, RHOA, ARF6, RAD, GEM and RAS GTPases, equivalent to TTN5T30N, display decreased nucleotide binding affinity and therefore tend to remain in a nucleotide-free state in a complex with their cognate GEFs (Erickson et al. 1997, Ghosh et al. 1999, Radhakrishna et al. 1999, Jung and Rösner 2002, Kuemmerle and Zhou 2002, Wittmann et al. 2003, Nassar et al. 2010, Huang et al. 2013, Chang and Colecraft 2015, Fisher et al. 2020, Shirazi et al. 2020). Since TTN5T30N exhibits fast guanine nucleotide dissociation, these results suggest that TTN5T30N may also act in either a dominant-negative or fast-cycling manner as reported for other GTPase mutants (Fiegen et al. 2004, Wang et al. 2005, Fidyk et al. 2006, Klein et al. 2006, Soh and Low 2008, Sugawara et al. 2019, Aspenström 2020)."

The claims in Line #224-227 are exaggerated. Please tone down or delete __Our response: __

*We rephrased the sentence (Line 240-243). *

"Therefore, we propose that TTN5 exhibits the typical functions of a small GTPase based on in vitro biochemical activity studies, including guanine nucleotide association and dissociation, but emphasizes its divergence among the ARF GTPases by its kinetics."

Line#488-489 - This conclusion is not really supported. At best Authors can claim that TTN5 is associated with trafficking components, but the functional relevance of this association is not determined. Our response:

*We toned down our statement (Line 604-608). *

„The colocalization of FM4-64-labeled endocytosed vesicles with fluorescence in YFP-TTN5-expressing cells may indicate that TTN5 is involved in endocytosis and the possible degradation pathway into the vacuole. Our data on colocalization with the different markers support the hypothesis that TTN5 may have functions in vesicle trafficking."

__Minor comments: __

Line #95 - " This rolein vesicle....." - please clarify which role? Our response:

We rephrased the sentence (Line 96-99).

„These roles of ARF1 and SAR1 in COPI and II vesicle formation within the endomembrane system are well conserved in eukaryotes which raises the question of whether other plant ARF members are also involved in functioning of the endomembrane system."

Line #168 - "we did not observed" please change to "not able to measure/quantify" __Our response: __

*We changed the text accordingly (Line 169-171). *

„A remarkable observation was that we were not able to monitor the kinetics of mGppNHp association with TTN5T30N but observed its dissociation (koff = 0.026 s-1; Figure 2E)."

Line#179 - ARF# is human for Arabidopsis?

Our response:

*The study of Fasano et al., 2022 is based on human ARF3 and we added the information to the text (Line 180-181) *

"(...) very similar to the koff value of HsARF3 (Fasano et al. 2022)."

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Line #181 - compared to what is the 10-fold difference?

__Our response: __

The 10-fold difference is between the nucleotides mGDP and mGppNHp, for both TTN5WT and TTN5Q70L. We added the information on specific nucleotides to this sentence for a better understanding (Line 181-185).

„Second, the koff values for mGDP and mGppNHp, respectively, were in a similar range between TTN5WT (0.012 s-1mGDP and 0.001 s-1 mGppNHp) and TTN5Q70L (0.025 s-1 mGDP and 0.006 s-1 mGppNHp), respectively, but the koffvalues differed 10-fold between the two nucleotides mGDP and mGppNHp in TTN5WT (koff = 0.012 s-1 versus koff = 0.001 s-1; Figure 2D, E; Supplementary Figure S3G, I, S4F, H)."

Lines #314-323 - are diffciult to understand, consider reframing. Same goes for the conclusion following these lines.

__Our response: __

We added an explanation to these sentences for a better understanding (Line 392-405).

„We performed an additional object-based analysis to compare overlapping YFP fluorescence signals in YFP-TTN5-expressing leaves with GmMan1-mCherry signals (YFP/mCherry ratio) and vice versa (mCherry/YFP ratio). We detected 24 % overlapping YFP- fluorescence signals for TTN5 with Golgi stacks, while in YFP-TTN5T30N and YFP-TTN5Q70L-expressing leaves, signals only shared 16 and 15 % overlap with GmMan1-mCherry-positive Golgi stacks (Supplementary Figure S8B). Some YFP-signals did not colocalize with the GmMan1 marker. This effect appeared more prominent in leaves expressing YFP-TTN5T30N and less for YFP-TTN5Q70L, compared to YFP-TTN5 (Figure 5B-D). Indeed, we identified 48 % GmMan1-mCherry signal overlapping with YFP-positive structures in YFP-TTN5Q70L leaves, whereas 43 and only 31 % were present with YFP fluorescence signals in YFP-TTN5 and YFP-TTN5T30N-expressing leaves, respectively (Supplementary Figure S8B), indicating a smaller amount of GmMan1-positive Golgi stacks colocalizing with YFP signals for YFP-TTN5T30N. Hence, the GTPase-active TTN5 forms are likely more present at cis-Golgi stacks compared to TTN5T30N."

Authors might consider a longer BFA treatment (3-4h) to see more clearer ER-Golgi fusion (BFA bodies)

__Our response: __

We perforned addtional BFA treatments for HA3-TTN5-expressing Arabidopsis seedlings followed by whole-mount immunostaining and for YFP-TTN5-expressing Arabidopsis lines. In both experiments we could obtain the typical BFA bodies. We included the NEW data in NEW Figure 4B, C

**Referees cross-commenting**

I agree with both my co-reviewers that the manuscript needs substantial improvement in its cell biology based experiments and conclusions thereof. I think the concensus of all reviewers points to weakness in the in-planta experiments which needs to be addressed to understand and characterize TTN5, which is the main goal of the manuscript.

Reviewer #3 (Significance (Required)):

Significance: The manuscript has general significance in understanding the role of small GTPases which are understudied. Although the manuscript does not advance the field of either intracellular trafficking or organization it holds significance in attempting to characterize proteins involved, which is a prerequisite for further functional studies.

__Our response: __

Thank you for your detailed analysis of our manuscript and positive assessment. Our study is an advance in the plant vesicle trafficking field.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Cellular traffic is an important and well-studied biological process in animal and plant systems. While components involved in transport are known the mechanism by which these components control activity or destination remains to be studied. A critical step in regulating traffic is proper budding and tethering of vesicles. A critical component in determining this step is a family proteins with GTPase activity, which act as switches facilitating vesicle interaction between proteins, or cytoskeleton. The current manuscript by Mohr and colleagues have characterized a small GTPase TITAN5 (TTN5) and identified two residues Gln70 and Thr30 in the protein which they propose to have functional roles. The authors catalogue the localization, GTP hydrolytic activity, and discuss putative functions of TTN5 and the mutants.

Major comments:

The core of the manuscript, which is descriptive characterization of TTN5, lies in reliably demonstrating putative roles. While the GTP hydrolysis rates are well-quantified (though the claims need to be toned down), the microscopy data especially the association of TTN5 with different endomembrane compartments is not convincing due to the quality (low resolution) of the figures submitted. The manuscript text is difficult to navigate due to repetition and inconsistency in the order that the mutants are referred. I am requesting additional experiments which should be feasible considering the authors have all the materials required to perform the experiments and obtain high-quality images which support their claims.

1. In general the figure quality needs to be improved for all microscopy images. I would suggest that the authors highlight 1-2 individual cells to make their point and use the current images as supplementary to establish a broader spread.
   • a. Fig. S1 lacks clarity.
   • b. For the supplementary videos, it is difficult to determine if punctate structures are moving or is it cytoplasmic streaming? Could this be done with a co-localized marker? Considering that such markers have been used later in Fig. 4?
   • c. It would be good if the speed of movement is quantified, if the authors want to retain the current claims in results and the discussion.
2. Fig.2
   • a. I am not sure what the unit / scale is in Fig. 2D/E if each parameter (Kon, Koff, and Kd) are individually plotted? Could the authors please clarify/simplify this panel?
   • b. Are panels D and E representing values for mdGDP and GppNHP? This is not very clear from the figure legend.
3. Fig. 3
   • a. Same comments as in para above - improve resolution fo images, concentrate on a few selected cells, if required use an inset figure to zoom-in to specific compartments.
   • b. Please provide the non-fluorescent channel images to understand cell topography
   • c. Is the nuclear localization seen in transient expression (panel L-N) an artefact? If so, this needs to be mentioned in the text.
4. Fig. 4 - In addition to the points made for Fig. 3
   • a. The authors should consider reducing gain/exposure to improve image clarity. Especially for the punctate structures, which are difficult to observe in TTN5, likely because of the cytoplasmic localization as well.
   • b. Reducing Agrobacterial load could be considered. OD of 0.4 is a bit much, 0.1 or even 0.05 could be tried. If available try expression in N. tabaccum, which is more amenable to microscopy. However, this is OPTIONAL, benthamiana should suffice.
   • c. A standard norm now is to establish the level of colocalization is by quantifying a pearson's or Mander's correlation. Which I believe has been done in the text, I didn't find a plot representing the same? Could the data (which the authors already have) be plotted alongwith "n" as a table or graph?
   • d. The cartoons for the action of chemicals are useful, but need a bit more clarity.
5. Fig. 5
   • a. does the Q70L mutant show reduced endocytosis ?
6. The main text needs to be organized in a way that a reader can separate what is the hypothesis/assumption from actual results and conclusions (see lines #143-149).
7. The text is repeated in multiple places, while I understand that this is not plagiarism, the repetitiveness makes it difficult to read and understand the text. I highlight a couple of examples here, but please check the whole text thoroughly and edit/delete as necessary.
   • a. Lines #124-125 with Lines #149-151
   • b. Lines #140-143
8. Could the authors elaborate on whether there are plan homologs of TTN5? Also, have other ARF/ARLs been compared to TTN5 beyond HsARF1?
9. On a related note, a major problem I have with these kinetic values is the assumption of significance or not. For eg. Line#180 the values represent and 2 and 6-fold increase, if these numbers do not matter can a significance threshold be applied so as to understand how much fold-change is appreciable?
10. Another issue with the kinetic measurements is the significance levels. Line #198-201. The three proteins are claimed to have similar values and in the nnext line, the Q70L mutant is claimed to be high.
11. Provide data for conclusion in line#214-215
12. How were the mutants studied here identified? random mutation or was it directed based on qualified assumptions?
13. Could more simplification be provided for deifitinition of Kon/Koff values. And can these values be compared between mutants directly?
14. Data provided are not convincing to claim that both the mutant forms have lower association with the Golgi.
15. IN general the Authors should strongly consider the claims made in the manuscript. For eg. "This study lays the foundation for studying the functional relationships of this small GTPase" (line 125) is unqualified as this is true for every protein ever studied and published. Considering that TTN was not isolated/identified in this study for the first time this claim doesn't stand.
    • a. Line #185 - "characterestics of a dominant-negative...." What is this based on? From the text it is not clear what are the paremeters. Considering that no complementation phenotypes have been presented, this is a far-fetched claim
    • b. The claims in Line #224-227 are exaggerated. Please tone down or delete
    • c. Line#488-489 - This conclusion is not really supported. At best Authors can claim that TTN5 is associated with trafficking components, but the functional relevance of this association is not determined.

Minor comments:

1. Line #95 - " This rolein vesicle....." - please clarify which role?
2. Line #168 - "we did not observed" please change to "not able to measure/quantify"
3. Line#179 - ARF# is human for Arabidopsis?
4. Line #181 - compared to what is the 10-fold difference?
5. Lines #314-323 - are diffciult to understand, consider reframing. Same goes for the conclusion following these lines.
6. Authors might consider a longer BFA treatment (3-4h) to see more clearer ER-Golgi fusion (BFA bodies)

Referees cross-commenting

I agree with both my co-reviewers that the manuscript needs substantial improvement in its cell biology based experiments and conclusions thereof. I think the concensus of all reviewers points to weakness in the in-planta experiments which needs to be addressed to understand and characterize TTN5, which is the main goal of the manuscript.

Significance

The manuscript has general significance in understanding the role of small GTPases which are understudied. Although the manuscript does not advance the field of either intracellular trafficking or organization it holds significance in attempting to characterize proteins involved, which is a prerequisite for further functional studies.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The manuscript by Mohr and colleagues characterizes the Arabidopsis predicted small GTPase TITAN5 in both biochemical and cell biology contexts using in vitro and in planta techniques. In the first half of the manuscript, the authors use in vitro nucleotide exchange assays to characterise the GTPase activity and nucleotide binding properties of TITAN5 and two mutant variants of it. The in vitro data they produce indicates that TITAN5 does indeed have general GTPase and nucleotide binding capability that would be expected for a protein predicted to be a small GTPase. Interestingly, the authors show that TITAN5 favors a GTP-bound form, which is different to many other characterized GTPases that favor GDP-binding. The authors follow their biochemical characterisation of TITAN with in planta experiments characterizing TITAN5 and its mutant variants association with the plant endomembrane system, both by stable expression in Arabidopsis and transient expression in N.benthamiana.

The strength of this manuscript is in its in vitro biochemical characterisation of TITAN5 and variants. I am not an expert on in vitro GTPase characterisation and so cannot comment specifically on the assays they have used, but generally speaking this appears to have been well done, and the authors are to be commended for it. In vitro characterisation of plant small GTPases is uncommon, and much of our knowledge is inferred for work on animal or yeast GTPases, so this will be a useful addition to the plant community in general, especially as TITAN5 is an essential gene. The in planta data that follows is sadly not as compelling as the biochemical data, and suffers from several weaknesses. I would encourage the authors to consider trying to improve the quality of the in planta data in general. If improved and then combined with the biochemical aspects of the paper, this has the potential to make a nice addition to plant small GTPase and endomembrane literature. The manuscript is generally well written and includes the relevant literature.

Major issues:

• The authors make use of a p35s: YFP-TTN5 construct (and its mutant variants) both stably in Arabidopsis and transiently in N.benthamiana. I know from personal experience that expressing small GTPases from non-endogenous promoters and in transient expression systems can give very different results to when working from endogenous promoters/using immunolocalization in stable expression systems. Strong over-expression could for example explain why the authors see high 'cytosolic' levels of YFP-TTN5. It is therefore questionable how much of the in planta localisation data presented using p35S and expression in tobacco is of true relevance to the biological function of TITAN5. The authors do present some immunolocalization data of HA3-TTN5 in Arabidopsis, but this is fairly limited and it is very difficult in its current form to use this to identify whether the data from YFP-TTN5 in Arabidopsis and tobacco can be corroborated. I would encourage the authors to consider expanding the immunolocalization data they present to validate their findings in tobacco.
• Many of the confocal images presented are of poor quality, particularly those from N.benthamiana.
• The authors in some places see YFP-TTN5 in cell nuclei. This could be a result of YFP-cleavage rather than genuine nuclear localisation of YFP-TTN5, but the authors do not present western blots to check for this.
• That YFP-TTN5 fails to rescue the ttn5 mutant indicates that YFP-tagged TTN5 may not be functional. If the authors cannot corroborate the YFP-TTN5 localisation pattern with that of HA3-TTN5 via immunolocalization, then the fact that YFP-TTN5 may not be functional calls into question the biological relevance of YFP-TTN5's localisation pattern.
• Without a cell wall label/dye, the plasmolysis data presented in Figure 5 is hard to visualize.

Minor issues:

• In some of the presented N.benthamiana images, it looks like YFP-TTN5 may be partially ER-localised. However, co-localisation with an ER marker is not presented.
• There is some inconsistency within the N.benthamiana images. For example, compare Figure 4C of YFP-TTN5T30N to Figure 4O of YFP-TTN5T30N. Figure 4O is presented as being significant because wortmannin-induced swollen ARA7 compartments are labelled by YFP-TTN5T30N. However, structures very similar to these can already been seen in Figure 4C, which is apparently an unrelated experiment. This, to my mind, is likely a result of the very different expression levels between different cells that can be produced by transient expression in N.benthamiana.

Referees cross-commenting

It seems that all of the reviewers have converged on the conclusion that the in planta characterisation of TTN5 is insufficient to be of substantial interest to the field, highlighting the fact that major improvements are required to strengthen this part of the manuscript and increase its relevance.

Significance

General assessment: the strengths of this work are in its in vitro characterisation of TITAN5, however, the in planta characterisation lacks depth.

Significance: the in vitro characterisation of TITAN5 is commendable as such work is lacking for plant GTPases. However, the significance of the work would be boosted substantially by better in planta characterisation, which is where most the most broad interest will lie.

My expertise: my expertise is in in planta characterisation of small GTPases and their interactors.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

The manuscript from Morh and collaborators reports the characterization of an ARF-like GTPase of Arabidopsis. Small GTPases of the ARF family play crucial role in intracellular trafficking and plant physiology. The ARF-like proteins are poorly addressed in Arabidopsis while they could reveal completely different function than the canonical known ARF proteins. Thus, the aim of the study is important and could be of interest to a wide range of plant scientists. I am impressed by the biochemical characterization of the TTN5 protein and its mutated versions, this is clearly a very nice point of the paper and allows for proper interpretations of the other results. However, I was much less convinced on the cell biology part of this manuscript and aside from the subcellular localization of the TTN5 I think the paper would benefit from a more functional angle. Below are my comments to improve the manuscript:

1. In the different pictures and movies, TTN5 is quite clearly appearing as a typical ER-like pattern. The pattern of localization further extends to dotty-like structures and structures labeled only at the periphery of the structure, with a depletion of fluorescence inside the structure. These observations raise several points. First, the ER pattern is never mentioned in the manuscript while I think it can be clearly observed. Given that the YFP-TTN5 construct is not functional (the mutant phenotype is not rescued) the ER-localization could be due to the retention at the ER due to quality control. The HA-TTN5 construct is functional but to me its localization shows a quite different pattern from the YFP version, I do not see the ER for example or the periphery-labeled structures. In this case, it will be a crucial point to perform co-localization experiments between HA-TTN5 and organelles markers to confirm that the functional TTN5 construct is labeling the Golgi and MVBs, as does the non-functional one. I am also quite sure that a co-localization between YFP-TTN5 and HA-TTN5 will not completely match... The ER is contacting so many organelles that the localization of YFP-TTN5 might not reflects the real location of the protein.
2. What are the structures with TTN5 fluorescence depleted at the center that appear in control conditions? They look different from the Golgi labeled by Man1 but similar to MVBs upon wortmannin treatment, except that in control conditions MVBs never appear like this. Are they related to any kind of vacuolar structures that would be involved in quality control-induced degradation of non-functional proteins?
3. The fluorescence at nucleus could be due to a proportion of YFP-TTN5 that is degraded and released free-GFP, a western-blot of the membrane fraction vs the cytosolic fraction could help solving this issue.
4. It is not so easy to conclude from the co-localization experiments. The confocal pictures are not always of high quality, some of them appear blurry. The Golgi localization looks convincing, but the BFA experiments are not that clear. The MVB localization is pretty convincing but the images are blurry. An issue is the quantification of the co-localizations. Several methods were employed but they do not provide consistent results. As for the object-based co-localization method, the authors employ in the text co-localization result either base on the % of YFP-labeled structures or the % of mCherry/mRFP-labeled structures, but the results are not going always in the same direction. For example, the proportion of YFP-TTN5 that co-localize with MVBs is not so different between WT and mutated version but the proportion of MVBs that co-localize with TTN5 is largely increased in the Q70L mutant. Thus it is quite difficult to interpret homogenously and in an unbiased way these results. Moreover, the results coming from the centroid-based method were presented in a table rather than a graph, I think here the authors wanted to hide the huge standard deviation of these results, what is the statistical meaning of these results?
5. The use of FM4-64 to address the vacuolar trafficking is a hazardous, FM4-64 allows the tracking of endocytosis but does not say anything on vacuolar degradation targeting and even less on the potential function of TTN5 in endosomal vacuolar targeting. Similarly, TTN5, even if localized at the Golgi, is not necessarily function in Golgi-trafficking.
6. The manuscript lacks in its present shape of functional evidences for a role of TTN5 in any trafficking steps. I understand that the KO mutant is lethal but what are the phenotypes of the Q70L and T30N mutant plants? What is the seedling phenotype, how are the Golgi and MVBs looking like in these mutants? Do the Q70L or T30N mutants perturbed the trafficking of any cargos?

Significance

In conclusion, I think this manuscript is a good biochemical description of an ARF-like protein but it would need to be strengthen on the cell biology and functional sides. Nonetheless, provided these limitations fixed, this manuscript would advance our knowledge of small GTPases in plants. The major conceptual advance of that study is to provide a non-canonical behavior of the active/inactive cycle dynamics for a small-GTPase. Of course this dynamic probably has an impact on TTN5 function and involvement in trafficking, although this remains to be fully demonstrated. Provided a substantial amount of additional experiments to support the claims of that study, this study could be of general interest for scientist working in the trafficking field.

Der italienische Landwirtschaftsminister Francesco Lollobrigida plant, die Nutzung von Agrarflächen für die Installation von Fotovoltaik zu untersagen. Mit dieser und mit weiteren Maßnahmen entspricht er nach den Bauernprotesten einem Wunsch der Agrarlobby. Politisch ist dieser Schritt in Italien noch ümstritten. https://www.repubblica.it/economia/2024/05/03/news/governo_regalo_agricoltori_mutui_pannelli-422808012/

Résumé de la Vidéo

La vidéo aborde la thématique de la bienveillance dans le milieu éducatif, en particulier dans les établissements scolaires. Elle met en lumière les défis et les résistances rencontrés par les professionnels de l'éducation lorsqu'ils tentent d'appliquer la bienveillance en tant que pratique pédagogique. Les intervenants discutent des différentes interprétations de la bienveillance, de son importance pour le développement et la réussite des élèves, et de la manière dont elle peut coexister avec l'exigence et l'autorité.

Points Forts: 1. Introduction à la bienveillance [00:00:00][^1^][1] * Importance de la bienveillance dans l'éducation * Résistances et malentendus autour du concept * Bienveillance versus exigence et autorité 2. Définition et clarification [00:03:58][^2^][2] * Nécessité de clarifier la bienveillance * Distinction entre bienveillance et complaisance * Bienveillance à long terme versus court terme 3. Bienveillance et exigences professionnelles [00:11:43][^3^][3] * Exemples de pratiques bienveillantes * Programme PHARE contre le harcèlement * Diagnostic partagé et actions ciblées 4. Impact de la bienveillance sur les élèves [00:15:08][^4^][4] * Besoins fondamentaux des élèves * Gestes professionnels et feedback positif * Communication authentique et respectueuse Résumé de la Vidéo

La deuxième partie de la vidéo aborde l'importance de la bienveillance et de l'éthique dans le système éducatif. Elle met en lumière les initiatives prises par un collège pour créer un environnement propice à l'apprentissage et au bien-être des élèves, telles que l'introduction d'un jardin zen et d'une salle calme. L'intervenant discute également de l'importance de l'évaluation claire, des retours constructifs et de la nécessité de réinventer les espaces éducatifs pour répondre aux besoins des élèves.

Points Forts: 1. Initiatives pour le bien-être des élèves [00:22:57][^1^][1] * Création d'un jardin zen * Mise en place d'une salle calme * Récompense pour les efforts des élèves 2. L'importance de l'évaluation et des retours [00:24:29][^2^][2] * Clarification de l'évaluation * Multiplication des retours constructifs * Réinvention de la relation éducative 3. Réinvention des espaces éducatifs [00:25:20][^3^][3] * Adaptation des salles d'études * Espaces de coworking pour les élèves * Discussion sur le sens de l'école avec les familles 4. Éthique et bienveillance dans l'éducation [00:31:01][^4^][4] * Réflexion sur les actions éthiques * Bienveillance compatible avec l'autorité * Importance de l'accueil et de la diversité des idées Résumé de la Vidéo

La partie 3 de la vidéo aborde l'importance de la lecture, de la créativité, de la coopération et de l'activité physique dans l'éducation. Elle souligne l'impact positif de ces pratiques sur l'engagement des élèves et leur bien-être. La discussion met également en lumière les défis institutionnels et les stratégies pour améliorer le bien-être des élèves et des enseignants.

Points Forts: 1. L'engagement des élèves dans la lecture [00:45:02][^1^][1] * Des moments de lecture collective * Observation d'une amélioration de l'attention * Certains élèves continuent de lire après le temps alloué 2. La créativité et la coopération en classe [00:45:25][^2^][2] * Encouragement de la créativité * Importance de la coopération pour le bien-être * Introduction de la possibilité pour les élèves de bouger 3. L'importance de l'activité physique [00:45:34][^3^][3] * Mise en place de 30 minutes d'activité physique quotidienne * Utilisation de ballons et de vélos pour permettre le mouvement * Recherche montrant les bénéfices pour les élèves hyperactifs 4. Les défis institutionnels et les solutions [00:50:34][^4^][4] * Discussion sur l'évaluation bienveillante * Gestion de la pression évaluative et de la résilience des élèves * Importance de la qualité de vie au travail pour les enseignants

Résumé de la vidéo [00:00:00][^1^][1] - [00:23:46][^2^][2]:

Cette vidéo présente une conférence de Didier Fassin sur la faculté de punir, explorant les nuances linguistiques et philosophiques du châtiment en anglais et en français. Fassin discute des définitions et des critères du châtiment, en soulignant la complexité de son application dans divers contextes sociaux et légaux.

Points forts: + [00:00:28][^3^][3] Nuances linguistiques du châtiment * * Différences entre les termes anglais et français * * Variété des significations et des contextes * * Importance de la clarté dans la définition des termes + [00:03:54][^4^][4] Critères du châtiment selon Anthony Flew * * Nécessité d'un mal ou désagrément * * Réponse à une infraction supposée * * Action volontaire d'une autorité légitime + [00:06:09][^5^][5] Exclusions du châtiment standard * * Situations ne relevant pas du cadre légal * * Relations familiales ou scolaires * * Sanctions affectant indirectement ou collectivement + [00:13:43][^6^][6] Examen empirique du châtiment * * Étude des situations réelles et de leurs écarts avec la définition idéale * * Impact du châtiment au-delà du cadre juridique * * Conséquences sur les individus et les institutions Résumé de la vidéo [00:23:47][^1^][1] - [00:45:05][^2^][2]:

La vidéo présente une conférence de Didier Fassin sur la faculté de punir, en se concentrant sur les interactions entre la police et les jeunes hommes des quartiers populaires, souvent issus de minorités ethniques ou religieuses. Fassin explore les pratiques punitives de la police, qui incluent des contrôles d'identité fréquents et des traitements humiliants, souvent sans justification légale. Il discute également de cas spécifiques de brutalité policière et de leurs conséquences physiques et psychologiques graves, ainsi que de la perception publique de ces actions comme des châtiments injustes.

Points forts: + [00:23:47][^3^][3] Interactions avec la police * * Les jeunes hommes sont souvent contrôlés et humiliés * * Les pratiques policières peuvent être abusives et discriminatoires * * Les réactions inappropriées peuvent entraîner des accusations supplémentaires + [00:27:00][^4^][4] Conséquences des pratiques punitives * * Les victimes subissent des dommages physiques et psychiques * * Les pratiques illégales sont soutenues par l'institution policière * * Les sanctions judiciaires contre les policiers sont souvent insuffisantes + [00:37:03][^5^][5] Cas spécifique de brutalité policière * * L'affaire de Théo et les séquelles permanentes qu'il a subies * * La violence policière est parfois extrême et intentionnelle * * Les agressions sexuelles font partie de l'arsenal punitif de la police + [00:44:02][^6^][6] Réponse judiciaire aux violences policières * * Les peines prononcées contre les policiers sont perçues comme clémentes * * Les pratiques discriminatoires et extrajudiciaires de la police sont illégales * * La justice est critiquée pour son traitement inégal des affaires de brutalité policière Résumé de la vidéo [00:45:07][^1^][1] - [00:58:25][^2^][2]:

La vidéo présente une conférence de Didier Fassin sur l'évolution des pratiques punitives en France, notamment l'introduction de l'amende forfaitaire délictuelle qui permet aux forces de l'ordre d'imposer des sanctions pénales sans procédure judiciaire. Fassin critique cette mesure pour son impact disproportionné sur les populations marginalisées et pour avoir élargi les pouvoirs punitifs de la police au-delà du système judiciaire.

Points forts: + [00:45:07][^3^][3] L'amende forfaitaire délictuelle * * Introduction par la loi de 2016 * * Sanction pénale sans procédure judiciaire * * Impact sur les personnes à faible revenu + [00:48:03][^4^][4] Usage de stupéfiants * * Augmentation des condamnations pour usage simple * * Politique répressive et pouvoir punitif de la police * * Disparités dans l'application des sanctions + [00:51:01][^5^][5] Critiques et recommandations * * Dénonciation par la Défenseur des droits * * Appel à la fin de ces sanctions pénales * * Description comme une justice inégalitaire + [00:52:36][^6^][6] Violences policières * * Durcissement du maintien de l'ordre * * Condamnations internationales pour usage excessif de la force * * Pratiques punitives lors de manifestations

Smith Corona Vintage Typewriter Straighten Chrome Ring Keytops Twisted Crooked Letters Repair by [[Phoenix Typewriter]]

What is important here is that you can access only the last x number character by using the length function and subtracting 1 from that.

@nick..."Capture, Process, Share"? Or "Capture. Produce. Share"?

Useful when you don't like to initialise the counter variable from zero but instead prefer to start with 1.

For Loop Syntax

update with link: https://www.natcom.org/sites/default/files/pages/2017_Review_Credo_for_Ethical_Communication.pdf

no italics

no italics

no italics

check other edition to see if a footnote is missing, or if this is extra and can be deleted: https://milnepublishing.geneseo.edu/interpersonalcommunication/chapter/2/

this looks correct, so you can unitalicize

for author: consider this source instead: https://www.nfl.com/news/report-engagement-ring-returned-to-cowboys-williams-09000d5d820a5541

no italics

СК -- Следственный комитет ("Investigative committee"). Эта организация, наказывающая людей за антивоенные выступления. Оксимирон насмехается над ними. Кроме того, хотя на Обводном могут быть девушки, там тоже находится здание другой правительственной следственной организации, Главное следственное управление Санкт-Петербурга.

Пока идёт война, жизнь в России идёт нормально, и многие из них получают южный загар на отдыхе.

Бульбик -- краткое название бульбулятора, устройства для курения марихуаны. Капли в бульбике быстро разрушаются и потребляются для удовольствия человека, который курит. Oxxxymiron сравнивает человеческие жизни с этими каплями, и в этом ситуации, люди, которые курят -- государственные чиновники.

Котомка -- это как рюкзак. Это относится к вопросу эмигрантов о том, что оставить в России и что взять с собой. Как сохранить русскую культуру при нынешнем положении вещей -- тоже головоломка.

Старики здесь - правительственные чиновники, которые убивают молодых людей от других семей, чтобы помочь этой войне.

Это относится к прозвищу Путина "бункерный дед", которое он получил из-за того, что он так параноидально относится к своей безопасности. Более того, Oxxxymiron называет его гномом, чтобы посмеяться над его невысоким ростом, которого он стыдится.

Это игра слов выражения "По ком звонит колокол". Соловки -- соловецкий лагерь особого назначения, который был создан в 1920-х годах для политических заключенных. Колокола здесь тоже уместны, потому что лагерь был создан на территории соловецкого монастыря. Это указывает на растущую репрессивность правительства.

На видео мы видим, как Оксимирон удаляет с пальцев татуировки с надписью "Imperium". Кроме того, он призывает слушателей отказаться от своих проправительственных и империалистических убеждений.

Это описание флага сопротивления, российского флага без красного цвета, который символизирует Россию без крови. Не можно полностью избавляться от этого сопротивления.

Это говорит о том, что вы можете причинить нам боль, но мы все равно все восстановим и заживем позже. это, конечно, актуально для украинцев, чьи дома разрушаются, но также и для россиян, которые чувствуют, что их правительство разрушает их страну.

Название трека и повторение этой фразы в припеве пародируют русский боевой клич "гойда". Этот клич традиционно использовался во время войны, но его выкрикивают и на пропутинских митингах.

Infinite Loop - Self explanatory... Off-by-one-error - Incorrect operator used, resulting in an incorrect number of iterations.

Looping trace table format.

The implementation is exactly the same as a regular trace table. :P

Difference between a Repeat Until loop and a While Loop:

Repeat Until Loop: Executes the code as long as the given condition is False, once the condition turns to True, it stops.

While Loop: Executes the code as long as the given condition is True, once the condition turns to False, it stops.

While loop Syntax

Meaning features of a language that are used to essentially control the flow of execution. (If-else conditions and loops).

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this can go to 'Measuring'

for the numerical solution

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Compared to our initial submission to Review Commons, we have addressed all the reviewers' comments. We have extensively re-written the manuscript to make it clearer to a larger audience. In particular, we have transferred Figure EV1 to Figure 1 with more complete panels and included a scheme (Figure EV3) on the steps of D2R internalization which we measure with live cell imaging. We have added a new paragraph to the start of the Discussion to summarize our main conclusions and reordered the discussion on the possible mechanisms of membrane PUFA enrichment on D2R endocytosis. All the changes in the text are in red for easier comparison with the previous version.

As suggested by reviewer 1, we have performed additional experiments to test the specificity of the effects of PUFA treatments on D2R endocytosis, reinforcing the results shown in Figure 4 using feeding assays. We show with live cell TIRF imaging and the ppH assay that TfR-SEP endocytosis is not affected (Figure EV5) and that SEP-β2AR endocytosis and βarr2-mCherry recruitment to the plasma membrane are not affected (Figure EV6).

Reviewer #1

Evidence, reproducibility and clarity

*The manuscript, using different live and fixed cell trafficking assays, demonstrates that incorporation of poly-unsaturated, but not saturated, free fatty acids in the membrane phospholipids reduce agonist induced internalization of the D2 dopamine receptor but not the adrenergic beta2 receptors or the transferrin receptor. Pulsed pH (ppH) live microscopy further demonstrated that the reduced internalization by incorporation of free fatty acid was accompanied by a blunted recruitment of Beta-arrestin for the D2R.

I believe said claims put forward in the manuscript are overall well supported by the data and as such I do not believe that further experiments are necessarily needed to uphold these key claims. Also, the methodology is satisfactorily reported, and statistics are robust, although two-way Anova like used in Fig 1 seems appropriate for Fig 2 and 3*

We thank the reviewer for his/her positive assessment of our work. We have checked the statistical tests used for all our measures. For Figure 2 and 3 (now 3 and 4) we test for only one factor (PUFA treatment or not) so we ran ordinary one-way ANOVA using Graphpad Prism.

That said, I suggest that the fixed cell internalization experiments (Fig 2 and 3), which relate the effect on the D2R to B2AR and transferrin are revised. This is important since this is relevant to judge whether the effect is a general or a selective molecular mechanism since this is the one of the three assay which this comparison relies on. Alternatively, I suggest omitting this data and include the B2AR in the Live DERET assay and both B2AR and TfR in the ppH assay. Specifically, my concerns with the fixed cell internalization are: • The analysis is based on counting the number of endosomes, which is not necessarily equivalent to the number of receptors internalized

The number of puncta, as well as their fluorescence, is reported by the analysis program (written in Matlab2021 and available upon request). We chose to show number of puncta because they reflect more directly the number of labelled endosomes (in Figures 3 and 4). As shown in the figure below, we found slight but significant differences between groups for FLAG-D2R (88.6 % and 87.6 % of average fluorescence in DHA and DPA treated cells compared to control cells), (panel A), and no differences for FLAG-β2AR (panel B). We find a significant decrease in puncta fluorescence for transferrin uptake in cells incubated with DHA (but not DPA) relative to control cells (panel C). However, because we did not detect differences in the number of puncta or in the frequency and amplitude of endocytic vesicle creation events (see below), we still conclude that enrichment with exogenous PUFAs does not affect clathrin mediated endocytosis.

In conclusion, the most robust measure of endocytosis for this assay is the number of detected puncta per cell rather than their fluorescence.

• The analysis relies on fully effective stripping of the surface pool of receptors - i.e clustered surface receptors not stripped by the protocol will be assessed as internalized. It is often very difficult to obtain full efficiency of the Flag-tag stripping and this is somewhat expression dependent. • The protocol for the constitutive and agonist induced internalization is different and yet shown on the same absolute graph. Although I take it the microscope gain setting are unaltered between the constitutive and agonist induced internalization I don't believe the quantification can be directly related. This is confusing at the very least. More critically however, the membrane signal from the non-stripped condition of constitutive internalization will likely fully shield internalized receptors in the Rab4 membrane proximal recycling pathway leading to under-estimation of the in the constitutive endocytosis. I believe this methodological limitation underlies the massive relative difference in the constitutive endocytosis between panel 2A,B and 2C,D. For comparison, by a quantitative dual color FACS endocytosis assay, we have previously demonstrated the ligand endocytosis a ~4 fold increased over constitutive (in concert with Fig 2A,B here) (Schmidt et al 20XX). Importantly, high relative variability by this methodology could well shield an actual effect of incorporation of FFAs on the constitutive endocytosis. We thank the reviewer for pointing this difference in the protocol. As a matter of fact, we have not used acid stripping in all the conditions used for the uptake assays (Figures 3 and 4). We apologize for the confusion and we have clarified this point in the Methods section. In early experiments we compared conditions with or without stripping but we concluded from these experiments that indeed, the stripping was not complete. Moreover, we noticed early on that many cells treated with DHA or DPA did not have any detectable cluster (13 cells out of 58 quantified cells treated with DHA after addition of QPL, 12/56 cells treated with DPA, 0/68 for cells treated with vehicle). Stripping the antibody would have made these cells undetectable, biasing the analysis. Therefore, to make our results more consistent we decided to use non-stripping conditions. To detect endosomes specifically, we used a segmentation tool developed earlier (see Rosendale et al.* 2019). This tool is based on wavelet transforms which recognizes dot-like structures. In addition, we excluded from the cell mask the labelled plasma membrane by a mask erosion.

We agree the design of experiments was not aimed at comparing the effect of PUFA treatment on low levels of constitutive D2R endocytosis. This would require more sensitive assays and be addressed in subsequent studies.

'Optional' Also, it would be informative to see the ppH Beta-arrestin experiments with the B2AR to assess, whether the putative discrepancy between D2R and B2AR is upstream or downstream of the blunted Beta-arrestin recruitment. To the same point, it would be very informative to assess how the incorporation of the free fatty acids affect receptor signalling, which would also help relate the effect of incorporation of the FFA's in the phospholipids to previous experiment using short term incubation with FFA's

We have now performed live imaging experiments in HEK293 cells expressing SEP-β2AR, GRK2 and βarr2-mCherry and stimulated with isoproterenol (Figure EV6). We show that the clustering of SEP-β2AR, of βarr2-mCherry, as well as endocytosis, are not affected by treatments with DHA or DPA. In this study, we focused on the early trafficking steps of D2R internalization. It will be interesting in a future study to address its consequences on G protein dependent and independent signaling. Moreover, and for good measure, we performed experiments to assess TfR-SEP endocytosis with the ppH assay. Again, we found no difference between cells treated or not with PUFAs (Figure EV5)

*References overall seem appropriate although Schmidt et al would be relevant for reference of the constitutive vs agonist induced endocytosis of D2R and B2AR. *

We have now cited Schmidt et al. 2020 doi 10.1111/bcpt.13274 in the discussion with the following sentences: "D2R also shows constitutive endocytosis (Schmidt et al, 2020) which may be modulated by PUFAs although we did not detect any significant difference in our measures (see Figure 3) which were aimed at detecting high levels of internalization induced by agonists. Further work will be required to specifically examine the effect of PUFAs on constitutive GPCR internalization."

Overall, the figures are well composed and convey the messages fairly well. Specific point that would strengthen the rigor include: • Chosing actual representative pictures of the quantitative data in Fig 2 and 3 (e.g. hard to see 25 endocytic events in Fig 2A constitutive endo, EtOH)

We apologize for the confusion. We employ a normalization procedure to account for cell size. In addition, all numbers have been normalized to the condition stimulated with agonist with no PUFA treatment). In fact, we detect in unstimulated cells very few puncta (on average 0.6, range 0-5) compared to 27.3 clusters (range 2-87) in cells stimulated with QPL.

• Showing actual p values for the statistical comparisons* For easier reading, we have kept the stars convention for the figures but added two tables with all statistical tests and the p values for both main figures and EV figures.

Moreover, for ease of reading the figures (without consulting the legend repeatedly) it would be very helpful to headline individual panel with what the experiments assesses. Figure 1a and 1b for example can't be distinguished at all before reading the figure legend. Also, y-axis could be more informative on what I measured rather than just giving the unit.

We have added titles to panels (in particular for Figure 2A,B which correspond to former Figure 1A,B) and we have given new titles to Y axes to make them clearer. We hope that the reading of our figures will now be easier.

Finally, the figure presentation and description of S1 is very hard to follow. I cannot really make out what is assessed in the different panels.

We have changed substantially Figure EV1 (now Figure 1) with new presentation of data: all 4 conditions (control, treated with DHA, DPA or BA) systematically presented in the same graph, and clearer titles for the parameter displayed on the Y axes. We hope that this figure is now easier to follow.

Significance

*The strength of the manuscript is the use and validation of incorporation of FFA's in the plasma membrane, which more closely mimics the physiological situation than brief application of FFAs as often done. Is addition, the blunted recruitment of beta-arrestin as assessed by the ppH protocol is quite intriguing mechanistically. The limitation are the relative narrow focus on the D2 receptor (and not multiple GPCRs) that does not really speak to as or assess the physiological, pathophysiological or therapeutic role of the observations (except from referring the relation between FFAs and disease). Also, despite the putative role of Beta-arrestin recruitment in the process, the actual causation in the process is not clear. This shortcoming is underscored by the putative effect on the constitutive internalization described above.

My specific expertise for assessing the paper is within general trafficking processes (including the trafficking methodology applied), trafficking of GPCRs and function of the dopamine system including the role of D2 receptors.*

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

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The only conclusion that I was able to understand from the study was that enrichment of cell membranes with polyunsaturated fatty acids specifically inhibited agonist-induced internalization of D2 receptors. However, I think that the experiments used to conclude that PUFAs do not alter D2R clustering but reduce the recruitment of β-arrestin2 and D2R endocytosis need some clarification (i.e. data depicted in Fig. 2-5). This lack of clarity might be due to the fact I am not familiar enough with the employed technologies or to the unclear writing style of the paper. There was an overuse of acronyms, initialisms and abbreviations, which are difficult to understand for researchers outside of the specific lipid field. I think that the manuscript should be written in a way to be legible also for researchers not working in the immediate filed.

The paper was not written in a manner that a general audience of cell biologists or those interested in GPCR biology could understand and judge. It is indeed interesting that polyunsaturated fatty acids specifically inhibit D2R internalization in HEK293 cells, and it could be significant. But, it is difficult to judge the significance of the observation without more in vivo data.

I would suggest the following. Remove all acronyms and abbreviations. Significantly, expand the Materials and Methods section, either in the manuscript or in the Supplemental section. I suggest clearly explaining each construct used, and the function of each module in the construct, with diagrams. In addition, provide a comprehensive step by step description of each experimental protocol, providing the reader with the rationale for each step in the protocol with explanatory diagrams. The authors should also more clearly explain the rationale and logic that was utilized to make the conclusions that they did from the depicted observations. Only then can a broader audience determine if the authors' conclusions are justified.

We thank the reviewer for his/her comments. Indeed, our main message was that two types of PUFAs (DHA and DPA) specifically alter D2R endocytosis by reducing the recruitment of β-arrestin2 without changing D2R clustering at the plasma membrane. We are sorry that our writing was not clear enough. We also found out that in the last steps of the submission to Review Commons, the first paragraph of the Discussion was inadvertently erased. This made our main conclusions, summarized in this first paragraph, less clear. We have now put back this important paragraph. Moreover, we have extensively rewritten the manuscript thriving to make it as clear as possible to a large audience. We have reduced the use of acronyms to keep only the most used ones [e.g. PUFA (used 99 times), DHA (37 times), GPCR (34 times), D2R (126 times), GRK (17 times)] and made them consistent throughout the manuscript. Following the reviewer's suggestion, we have also added a scheme of the steps following D2R activation by agonist leading to its internalization (Figure EV3).

We understand that the reviewer implies by "in vivo data" results obtained in the brain of animals. As written in the Introduction and in the Discussion, the current work follows up on a recently published manuscripts by a subset of the authors, namely (i) Ducrocq et al. 2020 (doi 10.1016/j.cmet.2020.02.012) in which we show that deficits in motivation in animals deprived in ω3-PUFAs can be restored specifically by conditional expression of a fatty acid desaturase from c. elegans (FAT1) that allows restoring PUFA levels specifically in D2R-expressing striatal projection neurons (which mediate the so-called indirect pathway), and (ii) Jobin et al. 2023 (doi: 10.1038/s41380-022-01928-6) which combines in cellulo (HEK 293 cells) and in vivo data to show that PUFAs affects the ligand binding of the dopamine D2 receptor and its signaling in a lipid context that reflects patient lipid profiles regarding poly-unsaturation levels.

Reviewer #2 (Significance (Required)):

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In summary, I will reiterate that the reported experiments need to be much better explained to make the study understandable to a broader audience and for that audience to determine whether the conclusions are justified.

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

• *

Summary:

The authors investigate the role of lipid polyunsaturation in endocytic uptake of the dopamine D2 receptor (D2R). To modulate the degree of unsaturation in live cell plasma membranes, the authors incubate cell lines with pure fatty acid that is metabolized and incorporated into the cellular membranes. To quantify the internalization of D2R in these live cells, the authors utilized quantitative fluorescence assays such as DERET and endosome analysis to determine the degree and rate of D2R internalization in the presence of two model agonists - dopamine and quinpirole. The authors conclude that when the PUFA content of the plasma membrane is increased (i.e., via ω3 or ω6 fatty acids), both the quantity and rate of D2R internalization decrease substantially. The authors confirmed that these phenomena are specific to D2R as caveolar endocytosis and clathrin-mediated endocytosis were unaffected when these same experimental techniques were utilized for β2 adrenergic receptor and transferrin. Additionally, the authors conclude that the clustering ability of D2R is unaffected by lipid unsaturation but that the ability of D2R clusters to interact with β-arrestin2 is inhibited in the presence of excess PUFA. Based on these findings, the authors propose several hypothetical mechanisms for lipid-D2R interactions on the plasma membrane, which will likely be the scope of future work.

Overall, this is a highly thorough and rigorous body of work that convincingly illustrates the connection between PUFA levels and D2R activity. However, I do not agree with the authors' conclusions pertaining to how their results should be interpreted in the context of fatty acid-related disorders. Additionally, this manuscript could benefit from some reorganization which would present the work more clearly. Please see the comments below.

We thank the reviewer for the positive appreciation of our work, qualified as a "thorough and rigorous body of work that convincingly illustrates the connection between PUFA levels and D2R activity". We will address the specific points raised by the reviewer with our answers below.

Comments:

1. • A recurring motivation for this study that is brought up by the authors is that dietary deficiency of ω3 fatty acids is tied to D2R dysfunction. This would indicate that PUFA reduction in the plasma membrane results in D2R dysfunction. However, the experiments emphasized in this manuscript investigate the condition where PUFA content is INCREASED in the plasma membrane and D2R function is compromised. It seems inappropriate for the authors to cite dietary deficiency of ω3 as a motivation when they experimentally test a condition that is tied to ω3 surplus.* Regarding the general comment of the reviewer, we agree that direct conclusion cannot be drawn on the etiology of psychiatric disorders by looking at the effect of membrane fatty acid levels on D2R in HEK 293 cells. Nevertheless, we mention in the Introduction the intriguing occurrence of low PUFA levels in psychiatric disorders as starting point to look at D2R as an important target for psychoactive drugs prescribed for these disorders. In the Discussion, we propose that manipulating fatty acid levels might potentiate the efficacy of D2R ligands used as treatments. We felt raising these aspects was not putting too much emphasis on psychiatric disorders. However, in accordance with the reviewer's comment, we toned down these descriptions in the revised manuscript.

The goal of increasing the levels of fatty acids at the membrane in HEK 293, the most widely used cellular system to study GPCR trafficking, was to try to emulate the levels of lipids in brain cells. Indeed, the levels of PUFAs in our culture conditions are much lower (~8 %, Figure 1B) than in brain extracts (~30 %). Therefore, the "control" condition in HEK 293 cells would correspond to PUFA deficiency while after our enrichment protocol these levels are closer to those found in brain cells. Our results could therefore be interpreted as endocytosis of D2R being augmented under membrane PUFA decrease. Importantly, increased receptor internalization often correlates with decreased signaling. Therefore, membrane PUFA enrichment in our conditions would rather potentiate D2R signaling.

• Following up on the first comment, the authors' results seem to indicate that excess ω3's are detrimental to D2R function. This result would be at odds with the conventional view that ω3's are essential and that excessive ω3 may not be harmful. The authors should rationalize their findings in the context of what is known about excess dietary ω3.*

The Reviewer is right that the conventional view is that excessive ω3 PUFA may not be harmful. However, this rather applies to dietary consumption, which might have limited effect to brain fatty acid contents since their accretion is highly regulated. Moreover, the majority of studies looking at ω3 supplementation have been performed in young adults and the effects on the developing brain - as it might be happening in pathological conditions in which D2R is involved - remain poorly understood. Furthermore, as mentioned above, blunted internalization of D2R under membrane PUFA enrichment is not an indication of "detrimental" to D2R function. Nor do we argue that membrane enrichment corresponds to excess PUFAs.

• I would argue that the control experiments with saturated fatty acids (i.e., Behenic Acid in figure 1), represent a scenario mimicking ω3 deficiency as the enrichment of Behenic Acid causes an overall reduction in PUFAs (Figure EV1C - an increase in SFA must correspond to a decrease in PUFA). These Behenic acid results are the only experiments presented by the authors that mimic a scenario resembling ω3 deficiency and the results show that the D2R internalization is unaffected (Figure 1G-H). Therefore, I would further argue that if anything, the authors results suggest that ω3 deficiency is NOT correlated to D2R internalization. Again, the authors must rationalize these findings in the context of what is known about dietary intake of ω3's.*

The Reviewer must refer to the fact that nutrients rich in SFAs are usually poor in PUFAs and vice-versa. Based on our lipidomic analysis, we now present in Figure 1B the effect of treatments (DHA, DPA, BA) on the levels of PUFAs (Figure 1B) and saturated fatty acids (Figure 1C). In cells treated with behenic acid (BA), PUFA levels are not significantly changed relative to control, untreated cells, while saturated fatty acid levels are increased. BA was used here to determine whether the effects observed with PUFAs was related to the enrichment in unsaturations or due to carbon chain length (C22). It is not the case because BA treatment, unlike DHA or DPA treatment, does not affect D2R endocytosis (Figure 2G,H).

• It's not clear why the authors decided to include an ω6 fatty acid in this study. The authors built up a detailed rationale for investigating ω3's as they are dietarily essential and tied to disease when deficient. To my knowledge, ω6's are considered much less beneficial than ω3's in a dietary sense. The inclusion of an ω6 almost seems coerced as the ω6-related results don't provide any interesting additional insights. It would benefit the manuscript if the authors provided some additional discussion explaining why ω6's are being investigated in addition to ω3's. *

We agree that we could have made the rationale clearer. The goal in comparing ω3-DHA and ω6-DPA was to assess whether the position of the first unsaturation (n-3 vs n-6), with the same carbon chain length (C22) might differentially impact D2R endocytosis.

• In Figure EV1D, the AHA and DPA percentages each increase by ~6%. The corresponding Figure EV1B indicates that the overall PUFA% in the plasma membrane also increases by 6%. This makes sense as the total change in PUFA content is consistent with the amount of AHA or DPA being internalized to cells. However, this consistency was not observed with BA and SFAs. In Figure EV1E, the BA percentage increases only ~1% while the total SFA percentage in Figure EV1C increases by ~6%. How can something undergoing a 1% change (relative to total lipid content) result in a 6% overall change in SFA content?*

The reviewer is correct: the level of SFAs is increased by 5.2% (34.5 % of total FAs in control cells to 39.7 % in BA treated cells), more than the increase in BA alone (1.18% from 0.35 % to 1.53 %). A close look at our lipidomics data showed that many of the 10 saturated fatty acids quantified are enhanced. In particular, the two most abundant ones, palmitic acid (16:0) and stearic acid (18:0) are increased, from 21.37 % to 22.28 % and 8.47 % to 11.17%, respectively. The reasons for these apparent discrepancies may involve lipid metabolic pathways which convert the rare and long BA into more common and shorter SFAs to preserve lipid contents and thus membrane properties.

• In Figure 4, the discussion of kinetics does not make sense. How exactly are kinetics being monitored in this figure? (Recruitment kinetics are discussed in panels D and G)*

We wanted to convey the impression that the time to reach the peak βarr2-mCherry recruitment was shorter in PUFA-treated cells than in control cells. However, after analyzing the kinetics in individual cells, we did not find a statistically significant difference in the time to maximum fluorescence. Therefore, we removed this reference to the kinetics of recruitment.

We now write: " However, treatment with DHA or DPA significantly decreased peak βarr2-mCherry fluorescence (Figure 5F-G).."

• In Figure 5, What is the purpose of panel D? Would it be more helpful to include additional, overlaid "cumulative N" plots for scenarios in which PUFAs were enriched? This would work well in conjunction with panel F.*

The purpose of this panel is to show the kinetics of increase in the frequency of endocytic vesicle formation upon agonist addition, and the decrease in frequency when the agonist is removed. We have now added examples of cells treated with DHA and DPA of similar surface for direct comparison with control (EtOH) cells.

• For the readers who are new to this area or unfamiliar with the assays used, Figure 1 is not intuitive and initially difficult to interpret. It would greatly benefit the flow of the manuscript if Figures EV1A-C and EV2A were included in the main text and "Normalized R" was clearly defined in the main text, prior to discussion of Figure 1.*

We have now transferred Figure EV1 as Figure 1. We have adapted the scheme of the DERET assay and its legend (now in Figure EV1A) to make it clearer. We did not put in Figure 2 because this figure is already very big. We have changed "Normalized R" to "Ratio 620/520) (% max)" to be clearer and more consistent with the scheme.

Reviewer #3 (Significance (Required)):

• *

General assessment: The work, for the most part, is rigorous and scientifically sound. The authors utilize impressive, quantitative assays to expand our understanding of protein-lipid interactions. However, the authors need to improve their discussion of the actual physiological conditions that correspond to their experimental results.

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Advance: This work may fill a gap in our understanding of disorders related to the dopamine D2 receptor. However, some of the results may be at odds with what is currently known/understood about dietary ω3 fatty acids.

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Audience: This work will be of broad interest to researchers in the biophysics field, with particular emphasis on researchers who study protein and membrane biophysics. This work will also be of interest to researchers who study membrane molecular biology.

• *

Reviewer Expertise: quantitative fluorescence spectroscopy and microscopy; membrane biophysics; protein-lipid interactions

• *

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #3

Evidence, reproducibility and clarity

Summary:

The authors investigate the role of lipid polyunsaturation in endocytic uptake of the dopamine D2 receptor (D2R). To modulate the degree of unsaturation in live cell plasma membranes, the authors incubate cell lines with pure fatty acid that is metabolized and incorporated into the cellular membranes. To quantify the internalization of D2R in these live cells, the authors utilized quantitative fluorescence assays such as DERET and endosome analysis to determine the degree and rate of D2R internalization in the presence of two model agonists - dopamine and quinpirole. The authors conclude that when the PUFA content of the plasma membrane is increased (i.e., via ω3 or ω6 fatty acids), both the quantity and rate of D2R internalization decrease substantially. The authors confirmed that these phenomena are specific to D2R as caveolar endocytosis and clathrin-mediated endocytosis were unaffected when these same experimental techniques were utilized for β2 adrenergic receptor and transferrin. Additionally, the authors conclude that the clustering ability of D2R is unaffected by lipid unsaturation but that the ability of D2R clusters to interact with β-arrestin2 is inhibited in the presence of excess PUFA. Based on these findings, the authors propose several hypothetical mechanisms for lipid-D2R interactions on the plasma membrane, which will likely be the scope of future work.

Overall, this is a highly thorough and rigorous body of work that convincingly illustrates the connection between PUFA levels and D2R activity. However, I do not agree with the authors' conclusions pertaining to how their results should be interpreted in the context of fatty acid-related disorders. Additionally, this manuscript could benefit from some reorganization which would present the work more clearly. Please see the comments below.

Comments:

1. A recurring motivation for this study that is brought up by the authors is that dietary deficiency of ω3 fatty acids is tied to D2R dysfunction. This would indicate that PUFA reduction in the plasma membrane results in D2R dysfunction. However, the experiments emphasized in this manuscript investigate the condition where PUFA content is INCREASED in the plasma membrane and D2R function is compromised. It seems inappropriate for the authors to cite dietary deficiency of ω3 as a motivation when they experimentally test a condition that is tied to ω3 surplus.
2. Following up on the first comment, the authors' results seem to indicate that excess ω3's are detrimental to D2R function. This result would be at odds with the conventional view that ω3's are essential and that excessive ω3 may not be harmful. The authors should rationalize their findings in the context of what is known about excess dietary ω3.
3. I would argue that the control experiments with saturated fatty acids (i.e., Behenic Acid in figure 1), represent a scenario mimicking ω3 deficiency as the enrichment of Behenic Acid causes an overall reduction in PUFAs (Figure EV1C - an increase in SFA must correspond to a decrease in PUFA). These Behenic acid results are the only experiments presented by the authors that mimic a scenario resembling ω3 deficiency and the results show that the D2R internalization is unaffected (Figure 1G-H). Therefore, I would further argue that if anything, the authors results suggest that ω3 deficiency is NOT correlated to D2R internalization. Again, the authors must rationalize these findings in the context of what is known about dietary intake of ω3's.
4. It's not clear why the authors decided to include an ω6 fatty acid in this study. The authors built up a detailed rationale for investigating ω3's as they are dietarily essential and tied to disease when deficient. To my knowledge, ω6's are considered much less beneficial than ω3's in a dietary sense. The inclusion of an ω6 almost seems coerced as the ω6-related results don't provide any interesting additional insights. It would benefit the manuscript if the authors provided some additional discussion explaining why ω6's are being investigated in addition to ω3's.
5. In Figure EV1D, the AHA and DPA percentages each increase by ~6%. The corresponding Figure EV1B indicates that the overall PUFA% in the plasma membrane also increases by 6%. This makes sense as the total change in PUFA content is consistent with the amount of AHA or DPA being internalized to cells. However, this consistency was not observed with BA and SFAs. In Figure EV1E, the BA percentage increases only ~1% while the total SFA percentage in Figure EV1C increases by ~6%. How can something undergoing a 1% change (relative to total lipid content) result in a 6% overall change in SFA content?
6. In Figure 4, the discussion of kinetics does not make sense. How exactly are kinetics being monitored in this figure? (Recruitment kinetics are discussed in panels D and G)
7. In Figure 5, What is the purpose of panel D? Would it be more helpful to include additional, overlaid "cumulative N" plots for scenarios in which PUFAs were enriched? This would work well in conjunction with panel F.
8. For the readers who are new to this area or unfamiliar with the assays used, Figure 1 is not intuitive and initially difficult to interpret. It would greatly benefit the flow of the manuscript if Figures EV1A-C and EV2A were included in the main text and "Normalized R" was clearly defined in the main text, prior to discussion of Figure 1.

Significance

General assessment: The work, for the most part, is rigorous and scientifically sound. The authors utilize impressive, quantitative assays to expand our understanding of protein-lipid interactions. However, the authors need to improve their discussion of the actual physiological conditions that correspond to their experimental results.

Advance: This work may fill a gap in our understanding of disorders related to the dopamine D2 receptor. However, some of the results may be at odds with what is currently known/understood about dietary ω3 fatty acids.

Audience: This work will be of broad interest to researchers in the biophysics field, with particular emphasis on researchers who study protein and membrane biophysics. This work will also be of interest to researchers who study membrane molecular biology.

Reviewer Expertise: quantitative fluorescence spectroscopy and microscopy; membrane biophysics; protein-lipid interactions

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Referee #2

Evidence, reproducibility and clarity

The only conclusion that I was able to understand from the study was that enrichment of cell membranes with polyunsaturated fatty acids specifically inhibited agonist-induced internalization of D2 receptors. However, I think that the experiments used to conclude that PUFAs do not alter D2R clustering but reduce the recruitment of β-arrestin2 and D2R endocytosis need some clarification (i.e. data depicted in Fig. 2-5). This lack of clarity might be due to the fact I am not familiar enough with the employed technologies or to the unclear writing style of the paper . There was an overuse of acronyms, initialisms and abbreviations, which are difficult to understand for researchers outside of the specific lipid field. I think that the manuscript should be written in a way to be legible also for researchers not working in the immediate filed.

The paper was not written in a manner that a general audience of cell biologists or those interested in GPCR biology could understand and judge. It is indeed interesting that polyunsaturated fatty acids specifically inhibit D2R internalization in HEK293 cells, and it could be significant. But, it is difficult to judge the significance of the observation without more in vivo data.

I would suggest the following. Remove all acronyms and abbreviations. Significantly, expand the Materials and Methods section, either in the manuscript or in the Supplemental section. I suggest clearly explaining each construct used, and the function of each module in the construct, with diagrams. In addition, provide a comprehensive step by step description of each experimental protocol, providing the reader with the rationale for each step in the protocol with explanatory diagrams. The authors should also more clearly explain the rationale and logic that was utilized to make the conclusions that they did from the depicted observations. Only then can a broader audience determine if the authors' conclusions are justified.

Significance

In summary, I will reiterate that the reported experiments need to be much better explained to make the study understandable to a broader audience and for that audience to determine whether the conclusions are justified.

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Referee #1

Evidence, reproducibility and clarity

The manuscript, using different live and fixed cell trafficking assays, demonstrates that incorporation of poly-unsaturated, but not saturated, free fatty acids in the membrane phospholipids reduce agonist induced internalization of the D2 dopamine receptor but not the adrenergic beta2 receptors or the transferrin receptor. Pulsed pH (ppH) live microscopy further demonstrated that the reduced internalization by incorporation of free fatty acid was accompanied by a blunted recruitment of Beta-arrestin for the D2R.

I believe said claims put forward in the manuscript are overall well supported by the data and as such I do not believe that further experiments are necessarily needed to uphold these key claims. Also, the methodology is satisfactorily reported, and statistics are robust, although two-way Anova like used in Fig 1 seems appropriate for Fig 2 and 3

That said, I suggest that the fixed cell internalization experiments (Fig 2 and 3), which relate the effect on the D2R to B2AR and transferrin are revised. This is important since this is relevant to judge whether the effect is a general or a selective molecular mechanism since this is the one of the three assay which this comparison relies on. Alternatively, I suggest omitting this data and include the B2AR in the Live DERET assay and both B2AR and TfR in the ppH assay. Specifically, my concerns with the fixed cell internalization are:

• The analysis is based on counting the number of endosomes, which is not necessarily equivalent to the number of receptors internalized
• The analysis relies on fully effective stripping of the surface pool of receptors - i.e clustered surface receptors not stripped by the protocol will be assessed as internalized. It is often very difficult to obtain full efficiency of the Flag-tag stripping and this is somewhat expression dependent.
• The protocol for the constitutive and agonist induced internalization is different and yet shown on the same absolute graph. Although I take it the microscope gain setting are unaltered between the constitutive and agonist induced internalization I don't believe the quantification can be directly related. This is confusing at the very least. More critically however, the membrane signal from the non-stripped condition of constitutive internalization will likely fully shield internalized receptors in the Rab4 membrane proximal recycling pathway leading to under-estimation of the in the constitutive endocytosis. I believe this methodological limitation underlies the massive relative difference in the constitutive endocytosis between panel 2A,B and 2C,D. For comparison, by a quantitative dual color FACS endocytosis assay, we have previously demonstrated the ligand endocytosis a ~4 fold increased over constitutive (in concert with Fig 2A,B here) (Schmidt et al 20XX). Importantly, high relative variability by this methodology could well shield an actual effect of incorporation of FFAs on the constitutive endocytosis.

'Optional' Also, it would be informative to see the ppH Beta-arrestin experiments with the B2AR to assess, whether the putative discrepancy between D2R and B2AR is upstream or downstream of the blunted Beta-arrestin recruitment. To the same point, it would be very informative to assess how the incorporation of the free fatty acids affect receptor signalling, which would also help relate the effect of incorporation of the FFA's in the phospholipids to previous experiment using short term incubation with FFA's

References overall seem appropriate although Schmidt et al would be relevant for reference of the constitutive vs agonist induced endocytosis of D2R and B2AR. Overall, the figures are well composed and convey the messages fairly well. Specific point that would strengthen the rigor include:

• Chosing actual representative pictures of the qunatiative data in Fig 2 and 3 (e.g. har to see 25 endocytic events in Fig 2A constitutive endo, EtOH)
• Showing actual p values for the statistical comparisions

Moreover, for ease of reading the figures (without consulting the legend repeatedly) it would be very helpful to headline individual panel with what the experiments assesses. Figure 1a and 1b for example can't be distinguished at all before reading the figure legend. Also, y-axis could be more informative on what I measured rather than just giving the unit.

Finally, the figure presentation and description of S1 is very hard to follow. I cannot really make out what is assessed in the different panels.

Significance

The strength of the manuscript is the use and validation of incorporation of FFA's in the plasma membrane, which more closely mimics the physiological situation than brief application of FFAs as often done. Is addition, the blunted recruitment of beta-arrestin as assessed by the ppH protocol is quite intriguing mechanistically. The limitation are the relative narrow focus on the D2 receptor (and not multiple GPCRs) that does not really speak to as or assess the physiological, pathophysiological or therapeutic role of the observations (except from referring the relation between FFAs and disease). Also, despite the putative role of Beta-arrestin recruitment in the process, the actual causation in the process is not clear. This shortcoming is underscored by the putative effect on the constitutive internalization described above.

My specific expertise for assessing the paper is within general trafficking processes (including the trafficking methodology applied), trafficking of GPCRs and function of the dopamine system including the role of D2 receptors.

Essentially saying that the == operator only compares the references stored in the variables, which are basically memory locations and they are always different unless both the given variables are storing the same reference. Making it pretty unreliable when it comes to strings. BUT, you can use it without a hitch when working with primitive types.

The Equals method however is actually going to the end of that reference and then comparing the strings.

leeswijzer

Wouhou it' s working!

test

Law give ethics strength and backing

Objective moral value trumps subjective moral value. It must be taken into consideration what will benefit the majority, while somewhat adhering to societal values to an extent that is ethical

lived experiences rather than theory alone

Health ethics helps guide decision making in the context of healthcare and promotion.

What is the relationship between health ethics and law? e.g. a law prohibiting public health authorities from imposing “unreasonable” restrictions on individual liberty.

What is the difference between ethical, social and personal values in health?

What health ethics adds to the analysis is the incorporation of value-oriented questions, such as the equity of the vaccine distribution system and its impact on vulnerable groups.

Nicht dass jüdische Studierende sich aus Angst vor dem Mob nicht mehr an die Unis trauen.

deneme

In den USA führen wachsende Datencenter, die u.a. durch die Industriepolitik steigende Produktion in Fabriken und immer mehr Elektrofahrzeuge zu einem steilen Anstieg des Bedarfs nach Elektrizität. Der zusätzliche Verbrauch wird in fünf Jahren etwa dem jetzigen Kaliforniens entsprechen. Die bestehenden Klimaziele werden dadurch gefährdet. Viele neue Gaskraftwerke werden bereits projektiert, unter anderem, weil die Regulierungen fossile Energien begünstigen. https://www.nytimes.com/interactive/2024/03/13/climate/electric-power-climate-change.html

https://eprint.iacr.org/2024/375.pdf

https://eprint.iacr.org/2024/599.pdf

https://eprint.iacr.org/2023/1559.pdf

https://eprint.iacr.org/2024/312.pdf

I think there is still an impact between social media and personal life. When people are swiped by a lot of screens, the information that is easy to see is misleading. Many times, people's views are often the views that people want to see, even if they are not correct.

The article discusses "Munchausen by internet" (MBI) which is a mental disorder formerly known as Munchausen syndrome, where people pretend to have an illness or actually make themselves sick for sympathy and attention. The author discusses the disorder through the case of Marissa Marchand, who pretended to have terminal cancer in an online support group, however she was actually healthy and so was promptly arrested for fraud. Due to the internet's anonymity it is easy to deceive, which can impact genuine cancer survivors and their support communities. Though there hasn't been many investigations over the motivations of those who are caught faking cancers, the author explains how cancer-support groups can supply high levels of attention and sympathy that are rarely found elsewhere. It is believed that MBI is attributed to a need for attention and control.

Die Stadt Paris geht juristisch dagegen vor, dass die französische Regierung eine Ölbohrkonzession für ein Gebiet bei Nonville in der Nähe der Hauptstadt erweitert hat. Befürchtet wird eine Verschmutzung des Trinkwassers. In dem Interview mit dem für Wasser Verantwortlichen der Stadt Paris werden auch andere Konflikte um die Ressource Wasser in Frankreich angesprochen. https://www.liberation.fr/environnement/pollution/cest-une-catastrophe-ecologique-en-puissance-une-menace-sur-leau-potable-20240505_TCX42POIMJHAJJWNIF6GXTWC5A/

What are some different considerations when collecting and preparing data?

Are there any particularly helpful figures or tables that assist in summarising key points?

Figure 1 summarises key points of 3 step process.

What are the three steps of the AIHW's Assessment Framework?

Collect, Identify, and Assess

I have experienced this firsthand for many months, until my laptop got away and I failed to stay productive due to my own limiting beliefs and stupidity... Now it's hard to get back into the lifestyle of the great again.

About anti-goals

"The worst day working on your own goals is still better than the best day working on someone else's." -- Dan Koe

100X goals force one to filter action... Impossible goals = Mental Clarity of the HIGHEST degree.

100X come from vision which in turn comes from future identity (future-self)

Gamify one's life to get progress if necessary. Integrate into systems.

Such is the risk of limiting beliefs.

"He who looks for external validation is not properly grounded in life." -- Marcus Aurelius

In other words, do not care about what others think... Heed their advice, take it into account, but ultimately you must make the decision yourself.

I agree with this approach. If a person who wants to commit suicide continues to receive other information about suicide on the Internet, it is likely to increase the possibility of suicide. However, when the information he receives becomes more POSITIVE information, he is likely that he will not choose to commit suicide.

A layman's guide.

Philhellenism as a return to of Greek culture (see "Hellenism" in the word".

Feedback Ole Neijenhuis (studentassistent vak): "De notatie in Exercise 5.6 is R_N, moet dit niet R_n zijn?"

Feedback vanuit Ole Neijenhuis (Studentassistent vak): "1. In Exercise 5.6 staat de longwave radiation als R_B beschreven, moet dit niet 'R_l,out - R_l,in' zijn?"

Feedback Ole Neijenhuis (Studentassistent vak): "Eq. 5.13 geeft T+272 in het eerste deel van de formule voor R_l, maar dit moet 273 zijn volgens mij."

Emphasis on the requirement of consent. The focus is on sovereignty of the state.

Else-if syntax

I definitely believe that a digital detox, or simply a social media detox, is necessary. However, I think it's much harder than we realize. I find it challenging to detox because I usually use social media to catch up with my friends and even to read the news by following reputable sources. I fear being left behind the rest of the world in trends and news. It's not something that comes easily to us. As many of the authors below have described, we're so comfortable and dependent on technology and social media that we can't seem to live without it.

.

Torre di Pisa page note

My point exactly.

Good to know. No. Really important to know.

Better to always use curly braces. As shown in the next highlight.

If-Else Condition Syntax

If Condition Syntax

There should be a != here instead of a >.

Since a negative odd number would fetch the remainder -1 and thus using the > relational operator would bring about faulty results.