Isabelle Fanget

Rab27a and Rab27b control different steps of the exosome secretion pathway

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

Myosin Va (MyoVa) is a prime candidate for controlling actin-based organelle motion in neurons and neuroendocrine cells. Its function in secretory granule (SG) trafficking was investigated in enterochromaffin cells by wide-field and total internal reflection fluorescence microscopy. The distribution of endogenous MyoVa partially overlapped with SGs and microtubules. Impairing MyoVa function by means of a truncated construct (MyoVa tail) or RNA interference prevented the formation of SG-rich regions at the cell periphery and reduced SG density in the subplasmalemmal region. Individual SG trajectories were tracked to analyze SG mobility. A wide distribution of their diffusion coefficient, Dxy, was observed. Almost immobile SGs (Dxy < 5 × 10−4 μm2 · s−1) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a Dxy below this threshold value for at least 2 s before fusion; (2) a negative autocorrelation of the vertical motion was found in subtrajectories with a Dxy below the threshold. Using this criterion of docking, we found that the main effect of MyoVa inhibition was to reduce the number of docked granules, leading to reduced secretory responses. Surprisingly, this reduction was not attributable to a decreased transport of SGs toward release sites. In contrast, MyoVa silencing reduced the occurrence of long-lasting, but not short-lasting, docking periods. We thus propose that, despite its known motor activity, MyoVa directly mediates stable attachment of SGs at the plasma membrane.

Coupling Amperometry and Total Internal Reflection Fluorescence Microscopy at ITO Surfaces for Monitoring Exocytosis of Single Vesicles

Water-soluble hormones and neurotransmitters are packaged in secretory vesicles and secreted into the extracellular medium by exocytosis, a process involving the fusion of the vesicle membrane with the cell membrane. Transport of the secretory vesicles to the cell s periphery, the maturation stages they undergo there to acquire fusion competence, and the factors controlling the fusion process itself (including the dynamics of the fusion pore) are important biological questions that are not fully understood. To elucidate secretory mechanisms at the single-vesicle level, currently only a few analytical methods exist, which can be grouped into electrical or optical recordings. The great advantage of electrical recordings (patch–clamp membrane capacitance and electrochemical amperometry) is their excellent time resolution (ca. tens of microseconds), which allows studies of the dynamics of the fusion pore itself. However, a major disadvantage is the fact that signals appear only after fusion has commenced; that is, the dynamics of the secretory vesicle itself or any labeled regulatory protein prior to the fusion event cannot be detected. In contrast, optical recordings allow secretory vesicles or regulatory proteins to be visualized and tracked prior to their fusion, yet generally they lack the time resolution required to follow the dynamics of the fusion pore (typical time resolution is ca. 100 ms). In addition, depending on the technique, secretion may be probed from different areas of a cell (top or bottom), which makes comparison of the results obtained by different approaches difficult. Because of their complementary nature, it would be a great advance if electrical and optical measurements could be made simultaneously from the same side of a cell at the singlevesicle level. This will enable a comprehensive and precise analysis of the whole exocytotic event, from predocking through fusion steps up to the dynamics of vesicular release. Herein, we report a device based on transparent indium tin oxide (ITO) electrodes, which allows simultaneous total internal reflection fluorescence microscopy (TIRFM) and amperometric measurements (Figure 1). As a proof of concept, the ability of our device in the coupled optical and electrochemical detections of exocytotic events is demonstrated using enterochromaffin BON cells. Amperometry is based on detection at a microelectrode surface positioned near the emitting cell of electroactive vesicular contents that are released into the extracellular medium. With very high temporal resolution and sensitivity, the flux of the vesicular content (released through an initial fusion pore that is only a few nanometers wide) corresponding to an exocytotic event appears as a current spike, which features (frequency, time length, area, magnitude) the dynamics of release from single vesicles. Generally, amperometry involves placing a large collecting electrode near the investigated cell. The whole cell active surface area is covered so the spatial localization of a particular exocytotic event cannot be achieved. Nevertheless, a few studies involving smaller microelectrodes or microelectrode arrays allowed amperometric signals from different releasing sites to be identified, but with a random positioning for the small microelectrode and a spatial resolution necessarily limited by the array dimensions, respectively. Coupling of amperometric and optical recordings would allow precise localization of exocytosis events in space and time. The most widely used optical approach to study exocytosis, TIRFM, is based on the total internal reflection of a laser beam at the glass/water interface, which creates an evanescent field in the aqueous medium whose characteristic decay length (ca. 100 nm) provides a high signal-to-noise ratio and an axial resolution of about 10 nm. When a vesicle fuses with the plasma membrane, its labeled contents are released toward the glass/water interface where the excitation [*] A. Meunier, Dr. R. Fulcrand, Dr. S. Arbault, Dr. M. Guille, Dr. F. Lema tre, Prof. C. Amatore D partement de Chimie, Ecole Normale Sup rieure UMR 8640 (CNRS-ENS-UPMC Univ Paris 06) 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-3863 E-mail: Christian.Amatore@ens.fr

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

On exocytosis, membrane fusion starts with the formation of a narrow fusion pore that must expand to allow the release of secretory compounds. The GTPase Cdc42 promotes fusion pore dilation in neuroendocrine cells by controlling membrane tension.

A 20-nm Step toward the Cell Membrane Preceding Exocytosis May Correspond to Docking of Tethered Granules

In endocrine cells, plasma membrane (PM)-bound secretory granules must undergo a number of maturation stages (i.e., priming) to become fusion-competent. Despite identification of several molecules involved in binding granules to the PM and priming them, the exact nature of events occurring at the PM still largely remains a mystery. In stimulated BON cells, we used evanescent wave microscopy to study trajectories of granules shortly before their exocytoses, which provided a physical description of vesicle-PM interactions at an unprecedented level of detail, and directly lead to an original mechanistic model. In these cells, tethered (T), nonfusogenic, vesicles are prevented from converting to fusogenic, docked (D) ones in resting conditions. Upon elevation of calcium, T-vesicles perform a 21-nm step toward the PM to become D, and fuse approximately 3 s thereafter. Our ability to directly visualize different modes of PM-attachment paves the way for clarifying the exact role of various molecules implicated in attachment and priming of granules in future studies.

Myrip Couples the Capture of Secretory Granules by the Actin-Rich Cell Cortex and Their Attachment to the Plasma Membrane

Exocytosis of secretory granules (SGs) requires their delivery to the actin-rich cell cortex followed by their attachment to the plasma membrane (PM). How these reactions are executed and coordinated is still unclear. Myrip, which is also known as Slac-2c, binds to the SG-associated GTPase Rab27 and is thought to promote the delivery of SGs to the PM by recruiting the molecular motor myosin Va. Myrip also interacts with actin and the exocyst complex, suggesting that it may exert multiple roles in the secretory process. By combining total internal reflection fluorescence microscopy, single-particle tracking, a photoconversion-based assay, and mathematical modeling, we show that, in human enterochromaffin cells, Myrip (1) inhibits a class of SG motion characterized by fast and directed movement, suggesting that it facilitates the dissociation of SGs from microtubules; (2) enhances their motion toward the PM and the probability of SG attachment to the PM; and (3) increases the characteristic time of immobilization at the PM, indicating that it is a component of the molecular machinery that tether SGs to the PM. Remarkably, while the first two effects of Myrip depend on its ability to recruit myosin Va on SGs, the third is myosin Va independent but relies on the C-terminal domain of Myrip. We conclude that Myrip couples the retention of SGs in the cell cortex, their transport to the PM, and their attachment to the PM, and thus promotes secretion. These three steps of the secretory process are thus intimately coordinated.