Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost nothing is known regarding the structural properties, cholesterol dynamics or intracellular factors affecting caveolar cholesterol dynamics. A non-detergent method was employed to isolate caveolae/raft domains from purified plasma membranes of murine fibroblasts. A series of fluorescent lipid probe molecules or a fluorescent cholesterol analogue, dehydroergosterol, were then incorporated into the caveolae/raft domains to show that: (i) fluorescence polarization of the multiple probe molecules {diphenylhexatriene analogues, DiI18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), parinaric acids and NBD-stearic acid {12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid} indicated that acyl chains in caveolae/raft domains were significantly less ‘fluid’ (i.e. more rigid) and the transbilayer ‘fluidity gradient’ was 4.4-fold greater than in plasma membranes; (ii) although sterol was more ordered in caveolae/raft domains than plasma membranes, spontaneous sterol transfer from caveolae/raft domains was faster (initial rate, 32%; half-time, t1/2, 57%) than from the plasma membrane; (iii) although kinetic analysis showed similar proportions of exchangeable and non-exchangeable sterol pools in caveolae/raft domains and plasma membranes, addition of SCP-2 (sterol carrier protein-2) 1.3-fold more selectively increased sterol transfer from caveolae/raft domains by decreasing the t1/2 (50%) and increasing the initial rate (5-fold); (iv) SCP-2 was also 2-fold more selective in decreasing the amount of non-exchangeable sterol in caveolae/raft domains compared with plasma membranes, such that nearly 80% of caveolar/raft sterol became exchangeable. In summary, although caveolae/raft lipids were less fluid than those of plasma membranes, sterol domains in caveolae/rafts were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes. Thus caveolae/raft domains isolated without the use of detergents display unique structure, cholesterol domain kinetics and responsiveness to SCP-2 as compared with the parent plasma membrane.
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Research Article|
August 24 2004
Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate
Adalberto M. GALLEGOS;
Adalberto M. GALLEGOS
*Department of Pathobiology, Texas A&M University, TVMC, College Station, TX 77843-4467, U.S.A.
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Avery L. McINTOSH;
Avery L. McINTOSH
†Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, U.S.A.
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Barbara P. ATSHAVES;
Barbara P. ATSHAVES
†Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, U.S.A.
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Friedhelm SCHROEDER
Friedhelm SCHROEDER
1
†Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, U.S.A.
1To whom correspondence should be addressed (email Fschroeder@cvm.tamu.edu).
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Publisher: Portland Press Ltd
Received:
October 10 2003
Revision Received:
May 14 2004
Accepted:
May 19 2004
Accepted Manuscript online:
May 19 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 382 (2): 451–461.
Article history
Received:
October 10 2003
Revision Received:
May 14 2004
Accepted:
May 19 2004
Accepted Manuscript online:
May 19 2004
Citation
Adalberto M. GALLEGOS, Avery L. McINTOSH, Barbara P. ATSHAVES, Friedhelm SCHROEDER; Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate. Biochem J 1 September 2004; 382 (2): 451–461. doi: https://doi.org/10.1042/BJ20031562
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