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Identifying Critical Components of Native Ca 2+ ‐triggered Membrane Fusion
Author(s) -
Furber Kendra L.,
Churchward Matthew A.,
Rogasevskaia Tatiana P.,
Coorssen Jens R.
Publication year - 2009
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2008.03993.x
Subject(s) - lipid bilayer fusion , biophysics , membrane curvature , vesicle , chemistry , fusion , biochemistry , sphingomyelin , exocytosis , membrane , lipid bilayer , biology , linguistics , philosophy
Ca 2+ ‐triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage‐specific preparation of Ca 2+ ‐sensitive release‐ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk‐pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high‐negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca 2+ sensitivity and kinetics). Conversely, cholesterol‐ and sphingomyelin‐enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca 2+ sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol‐reactive reagent iodoacetamide (IA), potentiation of the Ca 2+ sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol‐enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca 2+ ‐triggered membrane fusion.