Gucek, A. | Search bioRxiv

bioRxiv2022

Local PI(4,5)P2 generation controls fusion pore expansion during exocytosis

Phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) is an important signaling phospholipid that is required for regulated exocytosis and some forms of endocytosis. The two processes share a topologically similar pore structure that connects the vesicle lumen with the outside. Widening of the fusion pore during exocytosis leads to cargo release, while its closure initiates kiss&run or cavicapture endocytosis. We show here, using live cell TIRF microscopy of insulin granule exocytosis, that transient accumulation of PI(4,5)P2 at the release site recruits components of the endocytic fission machinery, and stalls the late fusion pore expansion that is required for peptide release. The absence of clathrin differentiates this mechanism from clathrin-mediated endocytosis. The PI(4,5)P2 transients result from local phosphatidylinositol-phosphate-5-kinase-1c (PIP5K1c) activity, and knockdown of PIP5K1c, or optogenetic ablation of PI(4,5)P2 promotes fusion pore expansion. Thus, local phospholipid signaling controls fusion pore expansion peptide release through an unconventional endocytic mechanism.

cell biology↗

bioRxiv2020

Redistribution of cholesterol from vesicle to plasmalemma controls fusion pore geometry

Eukaryotic vesicles fuse with the plasmalemma to form the fusion pore, previously considered to be unstable with widening of the pore diameter. Recent studies established that the pore diameter is stable, reflecting balanced forces of widening and closure. Proteins are considered key regulators of the fusion pore, whereas the role of membrane lipids remains unclear. Super-resolution microscopy revealed that lactotroph secretory vesicles discharge cholesterol after stimulation of exocytosis; subsequently, vesicle cholesterol redistributes to the outer leaflet of the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes evokes release of vesicle hormone, indicating that cholesterol constricts the fusion pore. A new model of cholesterol-dependent fusion pore diameter regulation is proposed. High-resolution measurements of fusion pore conductance confirmed that the fusion pore widens with cholesterol depletion and constricts with cholesterol enrichment. In fibroblasts lacking the Npc1 protein, in which cholesterol accumulates in vesicles, the fusion pore is narrower than in controls, showing that cholesterol regulates fusion pore geometry. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/027169v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@73168borg.highwire.dtl.DTLVardef@1cd2afdorg.highwire.dtl.DTLVardef@c170f9org.highwire.dtl.DTLVardef@1dfff4f_HPS_FORMAT_FIGEXP M_FIG C_FIG Top: stages through which a vesicle interacts with the plasmalemma. Stage A denotes hemifusion, which proceeds to stage B, with a narrow fusion pore, which can then reversibly open (stage C), before widening fully (stage D). Bottom: redistribution of cholesterol from the vesicle to the outer leaflet of the plasmalemma controls fusion pore constriction. In BriefA membrane pore is formed when the vesicle membrane fuses with the plasmalemma. Proteins were considered key regulators of the opening and closing of this fusion pore. Here, evidence is provided to show that cholesterol, a membrane constituent, determines a radial force constricting the fusion pore, revealing that the fusion pore functions as a proteolipidic structure. HighlightsO_LIIntravesicular cholesterol redistributes to the outer leaflet of the plasmalemma. C_LIO_LICholesterol depletion widens the fusion pore, whereas cholesterol enrichment constricts the fusion pore. C_LIO_LIA model of cholesterol-dependent force preventing fusion pore widening is developed. C_LIO_LIDisease-related increase in vesicle cholesterol constricts the fusion pore. C_LI

cell biology↗

bioRxiv2018

Fusion pore regulation by Epac2/cAMP controls cargo release during insulin exocytosis

Regulated exocytosis establishes a narrow fusion pore as the initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of larger peptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in type-2 diabetes and neurodegenerative disease. Here we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation leads to pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- mice. Conversely, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.

cell biology↗