The hypothesis of lipid domains in cellular plasma membranes is well establ
ished. However, direct visualization of the domains has been difficult. Her
e we report direct visualization of plasma membrane cholesterol microdomain
s modulated by agents that affect cholesterol trafficking to and from the p
lasma membrane. The cholesterol microdomains were visualized with a monoclo
nal antibody that specifically detects ordered cholesterol arrays. These un
ique cholesterol microdomains were induced on macrophages and fibroblasts w
hen they were enriched with cholesterol in the presence of an ACAT inhibito
r, to block esterification of excess cellular cholesterol. Induction of the
plasma membrane cholesterol microdomains could be blocked by agents that i
nhibit trafficking of cholesterol to the plasma membrane and by cholesterol
acceptors that remove cholesterol from the plasma membrane. In addition, p
lasma membrane cholesterol microdomains did not develop in mutant Niemann-P
ick type C fibroblasts, consistent with the defect in cholesterol trafficki
ng reported for these cells. The induction of plasma membrane cholesterol m
icrodomains on inhibition of ACAT helps explain how AC-AT inhibition promot
es cholesterol efflux from cells in the presence of cholesterol acceptors s
uch as HDL. The anti-cholesterol monoclonal antibody also detected extracel
lular cholesterol-containing particles that accumulated most prominently du
ring cholesterol enrichment of less differentiated human monocyte-macrophag
es. For the first time, cholesterol microdomains have been visualized that
function in cholesterol trafficking to and from the plasma membrane.