TY - JOUR
T1 - Secretory vesicle cholesterol : correlating lipid domain organization and Ca2+ triggered fusion
AU - Mahadeo, Mark
AU - Furber, Kendra L.
AU - Lam, Simon
AU - Coorssen, Jens R.
AU - Prenner, Elmar J.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
KW - cell membranes
KW - cholesterol
KW - lipids
KW - membrane fusion
UR - http://handle.uws.edu.au:8081/1959.7/uws:29759
U2 - 10.1016/j.bbamem.2015.02.005
DO - 10.1016/j.bbamem.2015.02.005
M3 - Article
SN - 0005-2736
VL - 1848
SP - 1165
EP - 1174
JO - Biochimica et Biophysica Acta
JF - Biochimica et Biophysica Acta
IS - 5
ER -