Detecting Rafts in Cells Using Sterol Modification

One common method to identify likely raft-dependent func­tions is to define biological processes inhibited upon (partial) extraction of cholesterol from cells using cyclodextrins (Zidovetzki and Levitan, 2007). Restoration of function by replenishment of cellular cholesterol using cholesterol-cyclodextrin complexes is then used to demonstrate that it is extraction of cholesterol that altered function.

These studies have the limitation that sterol extraction, which changes overall mass balance in the membrane, changes more than just raft levels. It is also claimed that the loss of DRM upon sterol extraction demonstrates that rafts have been destroyed by cyclodextrin treatment, but this is not correct. Ordered do­mains with low levels of sterol or no sterol can sometimes dissolve in Triton X-100 (London and Brown, 2000).

A more reliable, albeit more complex, approach involves sterol substitution. The abilities of various sterols and steroids to stabilize or destabilize raft formation have been defined (Megha et al., 2006; Wang et al., 2004; Wenz and Barrantes, 2003; Xu and London, 2000; Xu et al., 2001). In sterol sub­stitution partial extraction of cellular sterols is followed by replenishment with a diverse series of exogenous sterols with different raft-forming abilities.

Overall lipid mass balance can be (roughly) maintained in these experiments. When there is a strong correlation between a biological process and the raft­forming ability of sterols with diverse chemical structures, then it becomes likely that the biological process is raft-dependent.

To be more precise, if every raft-stabilizing sterol supports a function while every raft-destablizing sterol does not, and there are no other properties that distinguish function­supporting from the function-inhibiting sterols, then it can be inferred that a sterol having raft-stabilizing properties is both necessary (i.e., function is not supported by raft-destabilizing sterols) and sufficient (i.e., function is supported by all raft- stabilizing sterols) for the function.

This pattern establishes raft-dependence as the favored working hypothesis for the process under study. Perhaps just as important, by identifying biological processes that do not show this pattern, sterol substitution is an efficient approach to define processes un­likely to be raft-dependent (Singh et al., 2009).

A similar strategy is to introduce other molecules that should stabilize or destabilize rafts into membranes, such as polyunsaturated fatty acids or ceramides (Gidwani et al., 2003; Zech et al., 2009). In all such studies additional issues to consider include rapid metabolic transformations of the added molecules and signaling events that such molecules may trigger.

Several sterol substitution studies have been carried out. Sterol substitution studies in the bacterium B. burgdorferi show raft properties were necessary and sufficient for domain for­mation. These studies were especially informative because ordered lipid domains can readily be detected in this organism by electron microscopy and FRET (LaRocca et al., 2010, 2013). In addition, sterols having raft-stabilizing properties were necessary and sufficient to maintain membrane integrity in B. burgdorferi (LaRocca etal., 2013).

In Tcells, 7-ketocholesterol has been substituted for cholesterol. The raft-forming abilities of 7-ketocholesterol may only be slightly weaker than those of cholesterol (Wang et al., 2004), but the difference appears to be sufficient to disrupt raft formation and T cell signaling (Rentero et al., 2008). In the case of HIV, it has been shown that sub­stitution of raft-destabilizing sterols into virions inhibits infectivity while substitution of raft-stabilizing sterols does not (Campbell et al., 2004).