Quantitative Imaging of the Exocytosis Machinery Assembly

Abstract

The assembly of the secretory machinery is a poorly understood prerequisite for regulated exocytosis. Current models propose that the arriving vesicle docks at the plasma membrane by binding to either raft-like clusters (nanodomains) of SNARE proteins or to structural proteins such as RIM1, in both cases implying at least partial assembly of the secretory machinery prior to docking. In contrast, we recently showed that docking coincides with and requires recruitment of syntaxin and munc18 into nanodomains at the docking site, suggesting assembly after docking. Here we extend on this work and present live cell imaging-based quantification for many exocytosis proteins (including syntaxin, SNAP25, Munc18, Munc13, Rab3+27, Rabphilin, Granuphilin, RIM1, CaV1.2, EPAC, NSF, alfaSNAP; all tagged with EGFP) at the insulin granule release site during docking, priming and exocytosis. We find that the Rab3 interacting protein RIM1 was the only protein enriched at docking sites prior to vesicle tethering and docking. Further recruitment of RIM1 to the docking site occurred during granule maturation into the releasable pool (priming), suggesting roles in both docking and priming. None of the other proteins were present before granule arrival, but these were instead recruited during docking or even later during priming. Granules that successfully docked carried Rab3 and Rabphilin, whereas those that only temporarily tethered did not. In contrast, Rab27 and its effector Granuphilin were present on both types of granules. We conclude that sites enriched in RIM1 at the plasma membrane may facilitate docking by weakly tethering the incoming granule through interaction with rab3/rabphilin. Successful docking requires acute clustering of syntaxin/munc18, and we propose that this cluster then nucleates assembly of the exocytosis machinery.