Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

21-POS Board 21 FUSION PORE REGULATION BY CAMP AND EPAC2 IN PANCREATIC BETA CELLS Alenka Gucek 1 ; Nikhil R Gandasi 1 ; Muhmmad O Hmeadi 1 ; Marit Bakke 2 ; Stein O Doskeland 2 ; Anders Tengholm 1 ; Sebastian Barg 1 ; 1 Uppsala University, Medical Cell Biology, Uppsala, Uppsala län, Sweden 2 University of Bergen, Biomedicine, Bergen, Hordaland, Norway The central feature of regulated exocytosis is formation of a fusion pore, an aqueous channel connecting vesicular lumen with the extracellular space. After exocytosis has been triggered, this complex proteo-lipidic structure acts as a molecular sieve that regulates cargo release based on size. There is evidence that dysregulated fusion pore expansion is involved in common diseases, including type-2 diabetes and Parkinson’s disease. Here we have used high-resolution TIRF imaging in pancreatic beta-cells to study molecular mechanisms that regulate fusion pore behavior. A pH-sensitive granule marker was used to measure fusion pore behavior in real time. The time course of GFP-labeled proteins at the release site was quantified in parallel, and the data correlated with pore behavior. We report that elevated cytosolic cAMP favours stable fusion pores that allow rapid release of nucleotides, but restrict exit of peptides. The effect is mediated by the cAMP-dependent Rap-GEF Epac2, because cAMP-dependent fusion pore regulation is absent when Epac2 is inactivated pharmacologically or in Epac2-knockout mice. We show further that overexpression of Epac2 and several proteins involved in endocytosis favor cAMP- dependent pore restriction, and are recruited specifically to restricted fusion pores during exocytosis. We conclude that Epac2 controls cAMP-dependent fusion pore restriction and thereby affects differentially the release of hormones and transmitter molecules during exocytosis.

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