Vol. 89, Issue 2, 731-741, August 2000
In situ localization of cholesterol in skeletal muscle by use
of a monoclonal antibody
Mark S. F.
Clarke1,4,
Charles R.
Vanderburg2,
Marcas M.
Bamman3,
Robert W.
Caldwell4, and
Daniel L.
Feeback5
1 Division of Space Life Sciences, Universities Space
Research Association, and 5 Life Sciences Research Laboratories,
National Aeronautics and Space Administration/Johnson Space Center,
Houston, Texas 77058; 2 Research Space Management Group,
Massachusetts General Hospital, Boston, Massachusetts 02114;
3 Division of Exercise Physiology, University of Alabama at
Birmingham, Birmingham, Alabama 35294; and 4 Department of
Pharmacology and Toxicology, Medical College of Georgia, Augusta,
Georgia 30912
A common perception is that cholesterol,
the major structural lipid found in mammalian membranes, is localized
nearly exclusively to the plasma membrane of living cells and that it
is found in much smaller quantities in internal membranes. This
perception is based almost exclusively on cell fractionation studies,
in which density gradient centrifugation is used for purification of
discrete subcellular membrane fractions. Here we describe a monoclonal
antibody, MAb 2C5-6, previously reported to detect purified cholesterol
in synthetic membranes (Swartz GM Jr, Gentry MK, Amende LM,
Blanchette-Mackie EJ, and Alving CR. Proc Natl Acad Sci USA
85: 1902-1906, 1988), that is capable of detecting cholesterol in situ
in the membranes of skeletal muscle sections. Localization of
cholesterol, the dihydropyridine receptor of the T tubule, and the
Ca2+-ATPase of the sarcoplasmic reticulum (SERCA2) by means
of double and triple immunostaining protocols clearly demonstrates that cholesterol is primarily localized to the sarcoplasmic reticulum membranes of skeletal muscle rather than the sarcolemmal or T tubule
membranes. The availability of this reagent and its ability to
spatially localize cholesterol in situ may provide a greater understanding of the relationship between membrane cholesterol content
and transmembrane signaling in skeletal muscle.
immunolocalization; sarcoplasmic reticulum
