Crosslinked Plasmalemmal Cholesterol Is Sequestered to Caveolae: Analysis with a New Cytochemical Probe

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Abstract
Θ-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolyzed and biotinylated (BCΘ) to eliminate hemolyzing activity and was used as a cytochemical probe. In fixed cells, binding of BCΘ was intense in the plasma membrane, especially at the base of apical microvilli and in lateral processes. The labeling was abolished by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BCΘ and then with either fluorescein–avidin D or colloidal gold–streptavidin, the labeling in fine dots was distributed on the cell surface without local concentration as long as cells were kept on ice. When the temperature was raised to 37C after treatment, the probe formed discrete large patches and became sequestered to caveolae. Binding of BCΘ alone without the secondary reagents did not cause redistribution even at 37C. Because the plasma membrane maintains integrity even after binding of BCΘ, the probe can be used not only for cytochemical labeling of fixed cells but for pursuing the behavior of crosslinked cholesterol molecules in living cells. By use of this new probe, the present study revealed that crosslinked cholesterol in the plasma membrane is sequestered to caveolae.