Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Tuesday Speaker Abstracts

Assembly of the Secretory Machinery During Insulin Granule Docking Nikhil R. Gandasi , Sebastian Barg.

Institute of Medical Cell Biology, Box 571, Husargatan 3, BMC, Uppsala, Upplands, Sweden. The assembly of the secretory machinery at the plasma membrane is a poorly understood prerequisite for regulated exocytosis. For example, it is not known whether the required proteins are preassembled at the release site or instead recruited and assembled after vesicle docking. Current models propose that docking of the vesicle occurs through binding to either raft-like clusters 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. However, direct evidence for this is lacking. Using high resolution live cell microscopy we showed that the transition from a loosely tethered to the stably docked state occurs within seconds after vesicle arrival by recruitment of syntaxin and munc18. Here we extend on this work and present quantification of several exocytosis proteins at insulin granule release site during docking, priming and exocytosis. We find that the Rab3 interacting protein RIM1 and Rabphilin were enriched at docking sites prior to vesicle tethering and docking. A slow increase in RIM1 fluorescence was seen during granule maturation into the releasable pool (priming), suggesting roles for RIM1 in both docking and priming. None of the other proteins were present before granule arrival, but these were instead recruited during docking or 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.

27