Biophysical Society Thematic Meeting | Ascona, Switzerland

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

Tuesday Speaker Abstracts

Plasma Membrane PI(4,5)P2 Is Critical for Secretory Granule Exocytosis Muhmmad Omar Hmeadi , Sebastian Barg.Nikhil Gandasi. Uppsala University, Uppsala, Sweden.

Phosphoinositides, which include PI(4)P (phosphatidylinositol- 4-phosphate), PI(4,5)P2 (phosphatidylinositol- 4,5-bisphosphate) and PI(3,4,5)P3 (phosphatidylinositol-3,4,5- trisphosphate), are involved in multiple signalling cascades but their role in the regulation of exocytosis is not fully understood. Recent studies indicate that phosphoinositides, in particular PI(4,5)P2 and PI(3,4,5)P3, are enriched near docked secretory granules in cell free systems. Here we have studied phosphoinositide dynamics near secretory granules in live pancreatic β-cells using TIRF microscopy. Phosphoinositides were detected with EGFP-labelled PH domains of phospholipase C (PLC) δ1 (high affinity PI(4,5)P2 sensor), PLC δ4 (low affinity PI(4,5)P2 sensor), Grp1 (PI(3,4,5)P3), and EGFP-labelled P4M domains (PI(4)P). Secretory granules were labelled with NPY-mCherry or NPY-td-Orange2. In live rat insulin secreting (INS1) cells we found all tested phosphoinositide markers to be evenly distributed across the cell membrane, without discernible clustering or accumulation at granule docking sites. However, when the cells were permeabilized with alpha-toxin, the phosphoinositide markers formed a punctate pattern with partial co-localization with docked secretory granules. In live cells, exposure to insulin promoted synthesis of PI(3,4,5)P3, but did not influence secretory granule distribution or exocytosis. Chemically-induced recruitment of a 5’-phosphatase to the plasma membrane decreased PI(4,5)P2 levels, and resulted in an 85% decrease in secretory granule exocytosis. Our data indicate that plasma membrane PI(4,5)P2 does not influence secretory granule recruitment or distribution. However, since PI(4,5)P2 is the predominant phosphoinositide, we hypothesize that it plays a role in the priming of secretory granules prior to exocytosis.

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