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Journal of Lipid Research, Vol. 44, 1033-1041, May 2003
Copyright © 2003 by Lipid Research, Inc.

Methods

Biotinylated -toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

Shigeki Sugii^*, Patrick C. Reid^*, Nobutaka Ohgami^*, Yukiko Shimada^**, Robert A. Maue, Haruaki Ninomiya, Yoshiko Ohno-Iwashita^** and Ta-Yuan Chang^1,*

^* Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755
Departments of Physiology and Biochemistry, Dartmouth Medical School, Hanover, NH 03755
Department of Neurobiology, Tottori University Faculty of Medicine, Yonago 683-8503, Japan
^** Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan

¹ To whom correspondence should be addressed. e-mail: ta.yuan.chang{at}dartmouth.edu

BC is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BC can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BC signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BC. Under this condition, BC mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BC staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to ß-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BC staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin.

Thus, BC is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

Abbreviations: CD, ß-cyclodextrin; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; GFP, green fluorescent protein; hpCD, 2-hydroxypropyl-ß-cyclodextrin; LDLR, low density lipoprotein receptor; NPC1, Niemann-Pick type C1; PFA, paraformaldehyde; PM, plasma membrane

Supplementary key words BC • perfringolysin O toxin • cholesterol metabolism • cholesterol stain • intracellular cholesterol trafficking • cholesterol mutants • lipid rafts

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