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 a RNA interference screen, we identified five RabGTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to act 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 play different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (SYTL4) and Slac2b (EXPH5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and open ways to manipulate exosome secretion in vivo.3

Engagement of the B cell receptor (BCR) by surface-tethered antigens (Ag) leads to formation of a synapse that promotes Ag uptake for presentation onto major histocompatibility complex class II (MHCII) molecules. We have highlighted the membrane trafficking events and associated molecular mechanisms involved in Ag extraction and processing at the B cell synapse. MHCII-containing lysosomes are recruited to the synapse where they locally undergo exocytosis, allowing synapse acidification and the extracellular release of hydrolases that promote the extraction of the immobilized Ag. Lysosome recruitment and secretion results from the polarization of the microtubule-organizing center (MTOC), which relies on the cell division cycle (Cdc42)-downstream effector, atypical protein kinase C (aPKCζ). aPKCζ is phosphorylated upon BCR engagement, associates to lysosomal vesicles, and is required for their polarized secretion at the B cell synapse. Regulation of B lymphocyte polarity therefore emerges as a central mechanism that couples Ag extraction to Ag processing and presentation.

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

Analysis of trajectories of dynamical biological objects, such as breeding ants or cell organelles, is essential to reveal the interactions they develop with their environments. Many previous works used a global characterization based on parameters calculated for entire trajectories. In cases where transient behavior was detected, this usually concerned only a particular type, such as confinement or directed motion. However, these approaches are not appropriate in situations in which the tracked objects may display many different types of transient motion. We have developed a method to exhaustively analyze different kinds of transient behavior that the tracked objects may exhibit. The method discriminates stalled periods, constrained and directed motions from random dynamics by evaluating the diffusion coefficient, the mean-square displacement curvature, and the trajectory asymmetry along individual trajectories. To detect transient motions of various durations, these parameters are calculated along trajectories using a rolling analysis window whose width is variable. The method was applied to the study of secretory vesicle dynamics in the subplasmalemmal region of human carcinoid BON cells. Analysis of transitions between transient motion periods, combined with plausible assumptions about the origin of each motion type, leads to a model of dynamical subplasmalemmal organization.

Ϫ4 m 2 ⅐ s Ϫ1 ) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a D xy 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 D xy 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.

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.

More transparency in bioanalysis: A microdevice based on transparent indium tin oxide (ITO) electrodes allows simultaneous total internal reflection fluorescence microscopy and amperometric measurements. Use of the device in the coupled optical and electrochemical detection of single exocytotic events is demonstrated with enterochromaffin BON cells (see picture).

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.