Molecular Mechanisms Orchestrating the Dynamics of Secretory Vesicle Pools.

Abstract

The secretion of chemical messengers via Ca2+-dependent exocytosis of vesicles is fundamental to a wide-range of physiological events. Rab GTPases and SNARE proteins govern the temporal and spatial precision of transmitter release. Yet, little is known about their role in specifying the size and filling kinetics of functionally defined vesicle pools, which impact the strength and efficiency of exocytosis. We first sought to delineate the distinct vs. overlapping roles of highly homologous Rab GTPase proteins, Rab3 and Rab27, which display high sequence homology, share protein-effectors, and may functionally compensate. To define their actions, we overexpressed Rab3GAP and/or EPI64A GTPase-activating protein in wild-type or Rab27-null cells to transit the Rab3 family or Rab27A to a GDP-bound inactive state. We found Rab27A is essential for generation of the functionally defined immediately releasable pool, Rab3 is essential for a kinetically rapid filling of the RRP, and both cooperate in populating the readily releasable granule pool (RRP). We conclude that while Rab3 and Rab27A cooperate to generate release-ready vesicles in β-cells, they also direct unique kinetic and functional properties of the exocytotic pathway. We also investigated how the SNARE Tomosyn1 (Tomo1) regulates the partitioning of synaptic vesicle (SV) pools in hippocampal neurons. Tomo1 inhibits SV priming at the plasma membrane. Yet, its localization to SVs and cytosol uniquely positions it to coordinate SV pool partitioning. We that find that Tomo1 controls SV transition between the Resting Pool and Total Recycling Pool (TRP), and modulates the RRP size. Tomo1’s regulation of SV distribution between pools is sensitive to neural activity and requires Cdk5. We provide novel evidence for an interaction between Tomo1 and Rab3A-GTP, and through this with Synapsin1 proteins, known regulators of SV recruitment. In addition, Tomo1 regulatory control over the TRP occurred independent of its C-terminal SNARE domain. Hence, Tomo1 actions on neurotransmission extend beyond its known inhibition of SV priming into the RRP and may involve other effector proteins. Altogether, our results advance the understanding of how Rab and Tomosyn proteins coordinate steps of the vesicle cycle that lead to functional heterogeneity among vesicles and thus may determine modes of transmitter release.