Rab5 is critical for SNAP23 regulated granule-granule fusion during compound exocytosis

Abstract

Compound exocytosis is considered the most massive mode of exocytosis, during which the membranes of secretory granules (SGs) fuse with each other to form a channel through which the entire contents of their granules is released. The underlying mechanisms of compound exocytosis remain largely unresolved. Here we show that the small GTPase Rab5, a known regulator of endocytosis, is pivotal for compound exocytosis in mast cells. Silencing of Rab5 shifts receptor-triggered secretion from a compound to a full exocytosis mode, in which SGs individually fuse with the plasma membrane. Moreover, we show that Rab5 is essential for FcεRI-triggered association of the SNARE protein SNAP23 with the SGs. Direct evidence is provided for SNAP23 involvement in homotypic SG fusion that occurs in the activated cells. Finally, we show that this fusion event is prevented by inhibition of the IKKβ2 kinase, however, neither a phosphorylation-deficient nor a phosphomimetic mutant of SNAP23 can mediate homotypic SG fusion in triggered cells. Taken together our findings identify Rab5 as a heretofore-unrecognized regulator of compound exocytosis that is essential for SNAP23-mediated granule-granule fusion. Our results also implicate phosphorylation cycles in controlling SNAP23 SNARE function in homotypic SG fusion.

An early endosome–derived retrograde trafficking pathway promotes secretory granule maturation

“…among a subset of SGs that require endosomal trafficking (Ailion et al, 2014;Azouz et al, 2014;Cattin-Ortolá et al, 2017;Klein et al, 2017;Sparvoli et al, 2018;Topalidou et al, 2016).…”

“…However, although AP-3 is required for targeting CD63 to mature WPBs and large dense core vesicles ( Grabner et al, 2006 ; Harrison-Lavoie et al, 2006 ), AP-3 is dispensable for glue granule maturation ( Burgess et al, 2012 ; this study). Nevertheless, the requirement for CD63- and PI4KII-dependent retrograde trafficking indicates that glue granules are among a subset of SGs that require endosomal trafficking ( Ailion et al, 2014 ; Azouz et al, 2014 ; Cattin-Ortolá et al, 2017 ; Klein et al, 2017 ; Sparvoli et al, 2018 ; Topalidou et al, 2016 ).…”

Endosomal and Phagosomal SNAREs

“…SNAP23 mediates exocytosis in many cell types (109,110,174,178,219,234,256,312,346), for instance in neurons where it mediates the post-synaptic surface delivery of the glutamate receptor at dendritic spines (296). The Qb-and Qc-SNARE-motifs of SNAP23 are connected by a linker region containing five palmitoylated cysteines (272) that anchor it at the plasma membrane, recycling and late endosomes, and the trans-Golgi network (88,94,104,109,166,223,275,309,312,346). SNAP23 is also found at phagosomes (38,51,79,215,221,271,286), and, although it might be recruited at several stages during phagosomal maturation (267), most quantitative proteomics show an overall decrease of phagosomal SNAP23 following uptake (FIGURE 5).…”

“…SNAP23 is also found at phagosomes (38,51,79,215,221,271,286), and, although it might be recruited at several stages during phagosomal maturation (267), most quantitative proteomics show an overall decrease of phagosomal SNAP23 following uptake (FIGURE 5). Although best understood for its role in plasma membrane trafficking, SNAP23 also catalyzes endosomal fusion events (166,309), such as for the biogenesis of lysosome-relate organelles (e.g., melanosomes) (339). The intracellular trafficking roles of SNAP23 are characterized for phagosomes, where SNAP23 is responsible for the recruitment of several proteins with key roles in phagosomal maturation from recycling endosomes (221) The subcellular localization of SNAP23 is regulated by phosphorylation by the serine IkB kinase-␤ (157,221), and this promotes SNARE complex formation and membrane fusion (157,221,223,323).…”

Mammalian diaphanous-related formin 1 (mDia1) coordinates mast cell migration and secretion through its actin-nucleating activity

“…Indeed, given the extensive turnover and constant remodeling of the plasma membrane that take place in a migrating cell and the fact that a fraction of SGs are fusion competent already in resting MCs, with cortical actin serving the only barrier for secretion, 59 such a mechanism would ensure retainment of the inflammatory mediators during cell migration and thereby increase the efficacy of secretagogueinduced secretion at the site of inflammation. In particular, in view of the fact that MCs can undergo compound exocytosis, [60][61][62] in which fused granules serve as hooks for the attachment of subsequent fusing SGs, it might be critical for the MC to develop an inhibitory mechanism for restricting SG fusion during migration. Indeed, we show that this constraint is relieved on encounter with a secretagogue, conditions under which the ''migratory'' actin phenotype is replaced by a ''secretory'' actin phenotype.…”