Abstract
The hypothesis of lipid domains in cellular plasma membranes is well established. However, direct visualization of the domains has been difficult. Here we report direct visualization of plasma membrane cholesterol microdomains modulated by agents that affect cholesterol trafficking to and from the plasma membrane. The cholesterol microdomains were visualized with a monoclonal antibody that specifically detects ordered cholesterol arrays. These unique cholesterol microdomains were induced on macrophages and fibroblasts when they were enriched with cholesterol in the presence of an ACAT inhibitor, to block esterification of excess cellular cholesterol. Induction of the plasma membrane cholesterol microdomains could be blocked by agents that inhibit trafficking of cholesterol to the plasma membrane and by cholesterol acceptors that remove cholesterol from the plasma membrane. In addition, plasma membrane cholesterol microdomains did not develop in mutant Niemann-Pick type C fibroblasts, consistent with the defect in cholesterol trafficking reported for these cells. The induction of plasma membrane cholesterol microdomains on inhibition of ACAT helps explain how ACAT inhibition promotes cholesterol efflux from cells in the presence of cholesterol acceptors such as HDL. The anti-cholesterol monoclonal antibody also detected extracellular cholesterol-containing particles that accumulated most prominently during cholesterol enrichment of less differentiated human monocyte-macrophages. For the first time, cholesterol microdomains have been visualized that function in cholesterol trafficking to and from the plasma membrane. —Kruth, H. S., I. Ifrim, J. Chang, L. Addadi, D. Perl-Treves, and W-Y. Zhang. Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking.
Highlights
The hypothesis of lipid domains in cellular plasma membranes is well established
Human HDL, LDL, apolipoprotein A-I (apoA-I), lipoprotein-deficient serum, and fetal bovine serum were obtained from PerImmune (Rockville, MD); cholesterol oxidase was obtained from Boehringer Mannheim (Indianapolis, IN); bovine pancreas tr ypsin (3,000 U/mg) was from Life Technologies (Rockville, MD); cholesterol (C8667), progesterone, ketoconazole, chloroquine, 2-hydroxypropyl--cyclodextrin, and fatty acid-free BSA for rinsing cells before fixation were obtained from Sigma
When macrophages were incubated with acetylated LDL (AcLDL) or AcLDL plus an ACAT inhibitor (S58-035) did macrophage plasma membranes label with monoclonal antibody (MAb) 58B1
Summary
The hypothesis of lipid domains in cellular plasma membranes is well established. direct visualization of the domains has been difficult. We report direct visualization of plasma membrane cholesterol microdomains modulated by agents that affect cholesterol trafficking to and from the plasma membrane. The cholesterol microdomains were visualized with a monoclonal antibody that detects ordered cholesterol arrays. These unique cholesterol microdomains were induced on macrophages and fibroblasts when they were enriched with cholesterol in the presence of an ACAT inhibitor, to block esterification of excess cellular cholesterol. Plasma membrane cholesterol microdomains did not develop in mutant Niemann-Pick type C fibroblasts, consistent with the defect in cholesterol trafficking reported for these cells. Cholesterol microdomains have been visualized that function in cholesterol trafficking to and from the plasma membrane.—Kruth, H. Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking.
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