title: Secretory vesicle cholesterol: Correlating lipid domain organization and Ca2+ triggered fusion.
abstract: Membrane organization has received substantial research interest since the degree of ordering in membrane regions is relevant in many biological processes. Here we relate the impact of varying cholesterol concentrations on native secretory vesicle fusion and the lateral domain organization of membrane extracts from these vesicles. Membranes of isolated cortical secretory vesicles were either depleted of cholesterol, had cholesterol loaded to excess of native levels, or were depleted of cholesterol but subsequently reloaded to restore native cholesterol levels. Lipid analyses confirmed cholesterol was the only species significantly altered by these treatments. Treated vesicles were characterized for their ability to undergo fusion. Cholesterol depletion resulted in a decrease of Ca2+ sensitivity and the extent of fusion, while cholesterol loading had no effect on fusion parameters. Membrane extracts were characterized in terms of lipid packing by surface pressure-area isotherms whereas the lateral membrane organization was analyzed by Brewster angle microscopy. While no differences in the isotherms were observed, imaging revealed drastic differences in domain size, shape and frequency between the various conditions. Cholesterol depletion induced larger but fewer domains, suggesting that domain coalescence into larger structures may disrupt the native temporal-spatial organization of the fusion machinery and thus inhibit vesicle docking, priming, and fusion. In contrast, adding excess cholesterol, or rescuing with exogenous cholesterol after sterol depletion, resulted in more but smaller domains. Therefore, cholesterol is an important membrane organizer in the process of Ca2+ triggered vesicular fusion, which can be related to specific physical effects on native membrane substructure.
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors: Mark
authors: M
authors: Mahadeo
authors: Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada.
authors: Kendra L
authors: KL
authors: Furber
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors: Simon
authors: S
authors: Lam
authors: Molecular Physiology Department, School of Medicine, University of Western Sydney, Campbelltown, NSW, Australia; Molecular Medicine Research Group, School of Medicine, University of Western Sydney, Campbelltown, NSW, Australia. Electronic address: J.Coorssen@uws.edu.au.
authors: Jens R
authors: JR
authors: Coorssen
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada. Electronic address: eprenner@ucalgary.ca.
authors: Elmar J
authors: EJ
authors: Prenner
mesh_terms: D000818:Animals; D020013:Calcium Signaling; D002784:Cholesterol / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008561:Membrane Fusion; D008853:Microscopy; D015394:Molecular Structure; D011312:Pressure; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D013314:Stress, Mechanical; D047270:Strongylocentrotus purpuratus
chemical_list: D002784:Cholesterol
keywords: Brewster angle microscopy; Cholesterol; Membrane domain; Membrane fusion