Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Thursday Speaker Abstracts

BIRTH OF A NANODOMAIN: VESICLE DOCKING IS INITIATED BY RAB3 POSITIVE VESICLES IDENTIFYING RIM SITES TO TETHER AT THE PLASMA MEMBRANE Nikhil R. Gandasi ; Sebastian Barg 1 ; 1 Uppsala University, Biomedical Centrum, Institute of Medical Cell Biology, Uppsala, Uppsala län, Sweden Insulin is released by regulated exocytosis, which requires secretory vesicles to be docked at the plasma membrane. The number of release ready vesicles at the plasma membrane is therefore rate limiting for hormone secretion. Stable docking is preceded by a loosely tethered state, and we showed recently that this transition occurs within seconds after arrival of the vesicle by recruitment of syntaxin and munc18 to the docking site. The molecular nature of the tethered state is not known and it remains elusive whether the vesicles tether to a preexisiting receptor complex in the plasma membrane or attach to random sites. To answer this we quantified GTP- binding Rab proteins and their effectors at the docking site by imaging GFP-tagged proteins using TIRF microscopy. Clusters of the Rab3 interacting protein RIM and Rabphilin existed at docking sites prior to vesicle tethering and docking. A further increase in RIM fluorescence was seen at vesicles during their maturation into the releasable pool, confirming a role of RIM in priming. Vesicles 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 vesicles. These results suggest that Rab3 and Rabphilin act on the incoming granules as signal to initiate the docking process. Since RIM is thus far the only protein found to be enriched at the docking site it may act as a docking receptor for the incoming vesicle.

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