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Journal of Histochemistry and Cytochemistry, Vol. 45, 1197-1206, Copyright © 1997 by The Histochemical Society, Inc.

ARTICLE

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

Toyoshi Fujimotoa, Masami Hayashib, Machiko Iwamotob, and Yoshiko Ohno-Iwashitab
a Department of Anatomy and Cell Biology, Gunma University School of Medicine, Maebashi, Japan
b Department of Enzyme Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

Correspondence to: Toyoshi Fujimoto, Dept. of Anatomy and Cell Biology, Gunma Univ. School of Medicine, Maebashi 371, Japan.

{Theta}-Toxin (perfringolysin O), a cholesterol-binding toxin, was partially proteolyzed and biotinylated (BC{Theta}) to eliminate hemolyzing activity 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 abolished by pretreatment with filipin, digitonin, or tomatin. When living cultured cells were treated with BC{Theta} 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 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 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. (J Histochem Cytochem 45:1197-1205, 1997)

Key Words: cholesterol, {Theta}-toxin, caveolae, plasma membrane, histochemistry, electron microscopy, freeze-fracture


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