Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate

Abstract

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, t(1/2), 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 t(1/2) (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.

Figure 1. Fold-purification of plasma membranes and caveolae/raft domains

Left-hand panel: plasma membranes were isolated and the purity was compared with that of cell homogenates by analysis of Western blots with anti-Na⁺,K⁺-ATPase as described in the Materials and methods section. Right-hand panel: caveolae/raft domains were isolated from the plasma membranes without the use of detergents as described in the Materials and methods section. The purity of caveolae/rafts was compared with that of plasma membranes by analysis of Western blots with anti-caveolin-1 or measurement of DHE/mg of protein as described in the Materials and methods section.

Figure 2. Sterol exchange between membranes

(A) Effects of SCP-2 on sterol transfer from plasma membranes. DHE exchange between plasma membrane donors and plasma membrane acceptors was measured by monitoring polarization as described in the Materials and methods section. Open circles show the spontaneous sterol transfer from donor plasma membranes after the addition of a 10-fold excess of acceptor plasma membranes. Closed circles show the effects of 1.5 μM SCP-2 on the sterol transfer from donor plasma membranes to a 10-fold excess of acceptor plasma membranes. (B) Effects of SCP-2 on sterol transfer from caveolae/raft domains. DHE exchange was measured as in (A), except that caveolar/raft domain donor and caveolar/raft domain acceptor membranes were used as described in the Materials and methods section. Open circles show the spontaneous sterol transfer from donor caveolae after addition of a 10-fold excess of acceptor caveolae/raft domains. Closed circles show the effect of 1.5 μM SCP-2 on the sterol transfer from donor caveolae/rafts to a 10-fold excess of acceptor caveolae/rafts.