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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 https://www.biorxiv.org/content/10.1101/2023.09.06.556511v1

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

Background

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.

Summary

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)?

References:

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

doi: https://doi.org/10.1242/prelights.35977

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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.

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