CROSS-LINKED PLASMALEMMAL CHOLESTEROL IS SEQUESTERED TO CAVEOLAE - ANALYSIS WITH A NEW CYTOCHEMICAL PROBE

Theta-Toxin (perfringolysin O), a cholesterol-binding toxin, was parti ally proteolyzed and biotinylated (BC Theta) to eliminate hemolyzing a ctivity and was used as a cytochemical probe. In fixed cells, binding of BC Theta was intense in the plasma membrane, especially at the base of apical microvilli and in lateral processes. The labeling was aboli shed by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BC Theta and then with either fluores cein-avidin D or colloidal gold-streptavidin, the labeling in fine dot s was distributed on the cell-surface without local concentration as l ong as cells were kept on ice. When the temperature was raised to 37C after treatment, the probe formed discrete large patches and became se questered to caveolae. Binding of BC Theta alone without the secondary reagents did not cause redistribution even at 37C. Because the plasma membrane maintains integrity even after binding of BC Theta, the prob e can be used not only for cytochemical labeling of fixed cells but fo r pursuing the behavior of crosslinked cholesterol molecules in living cells. By use of this new probe, the present study revealed that cros s[inked cholesterol in the plasma membrane is sequestered to caveolae.