Structured RhoGEF recruitment drives myosin II organization on large exocytotic vesicles

Kumari Kamalesh, Dagan Segal, Ori Avinoam, Eyal D. Schejter, Ben-Zion Shilo

Preprint posted on 6 September 2023

A coordinated recruitment of actin and myosin at large vesicles allows for the "exocytosis by vesicle crumpling" in Drosophila Larval Salivary Glands

Selected by Ines Jmel-Boyer

Categories: cell biology


Drosophila undergo a metamorphosis, that happen during the pupal stage. Larvae climb to a solid surface and start secreting “glue” for the forming pupae to stick. Drosophila Larval Salivary Glands (LSG) use large vesicles to transport the “glue” proteins into the lumen allowing the “glue” to be secreted. The secretion, taking place in a two-hour window, involves large vesicle migration and vesicle fusion with the apical membrane allowing the ejection of the “glue” proteins into the lumen, a process taking three to four minutes only.

Adapted from Figure 1.

The author of this preprint previously found that the vesicles fused in a new type of exocytosis called “exocytosis by vesicle crumpling” (Kamalesh et al 2021). During this process, the “glue” is ejected from the vesicle by its anisotropic contraction (thus the “crumpled” vesicle). This type of exocytosis does not result in membrane expansion.

Members of the Rho family of GTPases are known to be very important in the control of cytoskeletal architecture and thus play a crucial role in eukaryotic cells. Rho proteins cycle between the Rho-GTP and GDP state and this cycling is controlled by RhoGEF (activators) and RhoGAP (inhibitors). Tight spatiotemporal control of Rho pathway activation is necessary and is tissue (and organism) specific.

The preprint authors already established that Rho1 is the trigger for the actomyosin reorganisation in the LSG vesicles. Rho1 acts on two different branches of the pathway: Rho1 activates the formin Dia resulting in actin polymerisation, but Rho1 also activates Rock (Rho-kinase) resulting in the activation and recruitment of myosin II (Rousso et al 2013). Nonetheless, the mechanisms driving the initiation and regulation of the actomyosin changes during the vesicle “crumpling” phase remain unknown.


Kamalesh et al. first observed that F-actin and Sqh (the Drosophila regulatory light chain of myosin II) are present in different patterns on the vesicles. Using a Rho-GTP reporter line (Munjal et al 2015), they showed that RhoGEF2 is essential to get the correct Rho-GTP levels on the vesicles. Knocking down RhoGEF2 did not perturb actin polymerisation. Surprisingly, perturbing actin polymerisation with latrunculin A resulted in the loss of RhoGEF2 recruitment. Furthermore, RhoGEF2 knockdown did perturb myosin II recruitment to the vesicles.

The preprint authors could further show that RhoGEF2, RhoGTP, and Sqh colocalised on the vesicles and that the punctuated pattern they displayed was necessary for the contraction of the vesicle (using a phosphomimetic Sqh vesicle that stalled and did not release its content into the lumen). They further pursued this by showing a causal relationship between myosin II recruitment and the sites of vesicle membrane folding (during the “crumpling”). Finally, they were able to explain initial Rho1 activation in the vesicle with a FRAP experiment showing that the recovery of a fluorescent membrane reporter happened quickly and before actin polymerisation.

Model proposed:

At the time of fusion, Rho1 diffuses from the apical membrane to the vesicle and activates the formin Dia allowing the polymerisation of actin during a first wave. F-actin then triggers the recruitment of RhoGEF2 on the vesicle in a punctate pattern. This starts a second wave of Rho1 activation in a punctate pattern resulting in the punctuated activation of myosin II via Rock phosphorylation. Myosin II accumulates at different points leading to uneven contraction and to the formation of “crumpled” vesicles, constricting and thereby ejecting their “glue” into the lumen.

Proposed model (Figure 5)

Questions & Comments:

  • I really enjoyed reading the preprint as it describes a mechanism requiring precise spatiotemporal control of actomyosin contractility. I found the preprint easy to follow and the figures convincing.
  • What happens to the crumpled vesicle once the “glue” has been ejected? Is having remnants of empty “crumpled” vesicles not an issue for the new incoming vesicles?
  • Are more RhoGEFs involved in this process?
  • In the sqhEEX line, the vesicles are stalled – do you know why? Do the vesicles still try to contract albeit in an isotropic manner?
  • Does the fusion happen as usual with LatA treatment? Or does it induce changes in the vesicle and/or the apical membrane?
  • Would it be possible to measure the contraction of the vesicle (the diameter over time for example)?


Kamalesh K, Scher N, Biton T, Schejter ED, Shilo BZ, Avinoam O. 2021. Exocytosis by vesicle crumpling maintains apical membrane homeostasis during exocrine secretion. Dev Cell 56: 1603-16.e6

Munjal A, Philippe JM, Munro E, Lecuit T. 2015. A self-organized biomechanical network drives shape changes during tissue morphogenesis. Nature 524: 351-5

Rousso T, Shewan AM, Mostov KE, Schejter ED, Shilo BZ. 2013. Apical targeting of the formin Diaphanous in Drosophila tubular epithelia. Elife 2: e00666


Posted on: 15 November 2023


Read preprint (No Ratings Yet)

Author's response

The author team shared

  • What happens to the crumpled vesicle once the “glue” has been ejected? Is having remnants of empty “crumpled” vesicles not an issue for the new incoming vesicles?

The crumpled vesicle is a large piece of membrane that needs to be resolved as it becomes an issue for new coming vesicles (although not simple because to spatial hinderance). We have explored this aspect in Kamalesh et. al 2021, where we used a combination of live imaging, genetic manipulations and CLEM to show that the clathrin mode of endocytosis specifically recycles this crumpled vesicular membrane after content extrusion. This phase on endocytic recycling is a long latent phase of ~ 20mins or more, occurring after content release that ensures the clearance of the crumpled vesicles. We have demonstrated that crumpling-mediated exocytosis take place in mouse exocrine pancreas implicating this to be a conserved mechanism. Mammalian exocrine glands undergo repeated cycles of secretion unlike a single cycle in Drosophila larval salivary glands, and such endocytic recycling of the crumpled vesicle membrane is probably even more crucial in those contexts.

  • Are more RhoGEFs involved in this process?

Yes, very likely, other RhoGEFs are involved in this process. We believe there will be other RhoGEFs for regulating F-actin assembly and patterning on fused vesicles apart from RhoGEF2 described here.  We have been preforming a genetic screen towards this direction.

  • In the sqhEEX line, the vesicles are stalled – do you know why? Do the vesicles still try to contract albeit in an isotropic manner?

SqhEEX -GFP is a phosphomimetic constitutively active allele for Sqh, that does not need to rely on the underlying signals for recruitment and patterning on the vesicles. However, it is less active than wild type Sqh allele. Also, in our experiments there is a wild type Sqh allele in the background. We use this Sqh-EEX line to show that, when the underlying signals are not read, the Sqh pattern is more uniform and not as punctate like the WT Sqh. The stalling could either be due to the loss in the punctate pattern or the lower activity of SqhEEX, the exact cause will be difficult to ascertain due to the nature of the experiment. Also, the WT Sqh in the background will be patterned and can also hetro-dimerise with the SqhEEX to form some functional complexes (but will not be visible as WT allele is not tagged) that might be responsible for the vesicular contraction observed.

  • Does the fusion happen as usual with LatA treatment? Or does it induce changes in the vesicle and/or the apical membrane?

The concentration of LatA we use (1 µM) is such that it specifically prevents actin polymerization on vesicles post-fusion but does not prevent cortical actin assembly on apical surface. In these experimental conditions and for the duration for which we perform the live imaging (within 30 min of dissecting out the glands), we have verified that it does not affect vesicle fusion to the surface or induce any other gross structural changes to the secretory gland.

  • Would it be possible to measure the contraction of the vesicle (the diameter over time for example)?

Yes, we have measured the contraction of the vesicle over time, in various ways. In Rousso et al. 2015, Fig 1 e, f and in Kamalesh et. al 2021, Fig 1. H, one can find the changing diameter or volume of vesicles over time that depicts the rate of vesicular contraction.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023


List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.


List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.


List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23


List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.


List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.


List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage