Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Tuesday Speaker Abstracts

RECONSTITUTION OF REGULATED EXOCYTOSIS OF DIFFERENT SECRETORY VESICLE TYPES Alex J.B. Kreutzberger 1,2 ; Volker Kiessling 1,2 ; J David Castle 1,3 ; Reinhard Jahn 4 ; Lukas K Tamm 1,2 ; 1 University of Virginia, Center for Membrane and Cell Physiology, Charlottesville, Virginia, USA 2 University of Virginia, Department of Molecular Physiology and Biological Physics, Charlottesville, Virginia, USA 3 University of Virginia, Cell Biology, Charlottesville, Virginia, USA 4 Max Planck Institute for Biophysical Chemistry, Neurobiology, Göttingen, Niedersachsen, Germany Dense core vesicles have previously been purified from an immortalized rat chromaffin cell line (PC12 cells) and incorporated into a fusion assay with planar supported bilayers reconstituted with the complete required target membrane fusion machinery (Kreutzberger et al. Sci Adv. 2017). This assay has been extended to be used with synaptic vesicles purified from rat brains and insulin vesicles purified from an immortalized rat beta cell line (INS-1 cells). Docking and fusion of all three secretory vesicles can be observed using different content labeling strategies. All three secretory vesicles dock in an arrested state when the planar supported bilayers contain syntaxin-1a and SNAP-25 incubated with Munc18 and complexin-1. Perfusion of calcium stimulates these vesicles to fuse. Three major differences were observed for the different secretory vesicle types. First, a differential requirement for a recombinant fragment of Munc13 was observed, with it being necessary for a robust calcium response of synaptic vesicles but not required for dense core vesicles. Second, a large disparity in fusion rates after calcium arrival was observed with synaptic vesicles being the fastest, dense core vesicles intermediate, and insulin vesicles being the slowest. Finally, there was a different affinity for calcium for each secretory vesicle type due to the different synaptotagmin isoforms present on the vesicles. Calcium responses of fusion depended strongly on the concentration of PI(4,5)P 2 present in the planar supported bilayer.

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