Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Tuesday Speaker Abstracts

DYNAMICS OF MEMBRANE FUSION AND ENDOCYTOSIS IN SECRETORY CELLS

Ling-Gang Wu 1 ; 1 NINDS, NIH, Bethesda, MD, USA

Membrane fusion and budding mediate fundamental processes like intracellular trafficking, exo-, and endocytosis, and viral infection. Their dynamic membrane transformations have not been observed in live cells, but speculated from electron microscopy of fixed or frozen tissues. Here I will describe recent experiments that visualized in real-time exo- and endocytosis membrane transformations in neuroendocrine secretory cells, the adrenal chromaffin cells. Exocytosis involves hemi-fusion, hemi-to-full fusion, pore expansion, constriction and/or closure while fusing vesicular W-profiles may shrink, enlarge, or receive another vesicle fusion. Endocytic vesicle formation involves flat- to L-, W-, and O-shape transformation. I will describe the molecular mechanisms that control these exo- and endocytosis membrane dynamics. MEMBRANE LIPIDS COUPLE SYNAPTOTAGMIN TO SNARE-MEDIATED GRANULE FUSION IN INSULIN SECRETING CELLS Chase Amos 1 ; Volker Kiessling 1 ; Arun Anantharam 2 ; Lukas K. Tamm 1 ; 1 University of Virginia, Molecular Physiology and Biological Physics, Charlottesville, VA, USA 2 University of Toledo, Neuroscience, Toledo, OH, USA Insulin secretion depends on the Ca 2+ -regulated fusion of granules with the plasma membrane. A recent model of Ca 2+ -triggered exocytosis in secretory cells proposes that lipids in the plasma membrane couple the calcium sensor Syt1 to membrane fusion machinery (Kiessling et al., Nature Struct Mol Biol 25:911 [2018]). Specifically, Ca 2+ mediated binding of Syt1’s C2 domains to the cell membrane shifts the membrane-anchored SNARE syntaxin-1a to a more fusogenic conformation, straightening its juxtamembrane linker. To test this model in live cells and extend it to insulin secretion, we enriched INS1 cells with a panel of lipids with different properties. Fluorescence lifetime measurements demonstrate that cells with more disordered membranes show an increase in fusion efficiency, and vice versa. Experiments with granules purified from INS1 cells and recombinant SNARE proteins reconstituted in supported membranes confirmed that lipid acyl chain composition determines SNARE conformation and correlates with fusion. Addition of Syt1’s C2AB domains significantly decreased lipid order in target membranes. Strikingly, Syt’s action on both fusion and lipid order could be bypassed by artificially increasing unsaturated phosphatidylserines in the target membrane. Thus, plasma membrane lipids actively participate in coupling Ca 2+ /synaptotagmin-sensing to the SNARE fusion machinery in living cells.

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