One important example is the association of HIV, an enveloped RNA virus, with membrane domains [155, 156]. membrane of eukaryotes but could potentially be a ubiquitous membrane-organizing principle in several other biological systems. is the most studied of these and has been used for sensing cholesterol [60]. In a recent study, PFO was modified to probe the transbilayer distribution of cholesterol on membrane bilayers [61]. Other proteins have been isolated from different organisms that bind either selectively or non-selectively to different lipids. Lysenin, a protein isolated from the earthworm (reviewed in [147]). Intoxification of host cells by VacA is initiated by binding of the toxin to the plasma membrane, followed by toxin oligomerization, membrane insertion, and pore formation [148]. Current models suggest that one or more of these events occur in lipid rafts. Early studies demonstrating VacA associates with lipid rafts relied on biochemical approaches to isolate raft-enriched fractions and/or depleting cells of cholesterol to interfere with raft integrity and function [5, 149C151]. More recent work has now confirmed VacAs raft association by showing it preferentially associates with the raft phase in Microcystin-LR GPMVs [152]. How VacA is targeted to lipid rafts is currently not entirely clear and may involve multiple mechanisms. Some studies indicate that sphingomyelin, one of the receptors of VacA, acts to recruit VacA to rafts [5], while Microcystin-LR others have shown that initial binding of VacA is to receptors in non-lipid raft microdomains and the raft partitioning of VacA occurs subsequently as a result of clustering [151]. Interestingly, unlike other bacterial toxins such as CTx that depend at least in part on multivalent binding to their receptor to facilitate raft targeting, VacA DLL4 need not form oligomers in order to partition into rafts [152] (Figure 3B). Furthermore, the ability of the toxin to form pores is not required for it to associate with rafts [152]. Why then does VacA associate with rafts? Microcystin-LR One potential answer is that this is linked to VacAs internalization mechanism: VacA enters cells via clathrin-independent endocytic pathways, which are typically raft-dependent [153]. However, how rafts influence VacAs pore-forming activity is not yet known. For example, it is currently unclear whether the structure of pores formed by VacA differs in raft versus non-raft environments. This is an especially important question because there are multiple examples of pore-forming toxins that associate with rafts [154]. Future studies using VacA should help to provide insights into this question, as well as to better delineate raft targeting mechanisms for this interesting class of toxins. HIV selectively binds and fuses at raft/non-raft boundaries Not just bacteria, but also viruses are known to target lipid rafts. One important example is the association of HIV, an enveloped RNA virus, with membrane domains [155, 156]. Rafts are thought to play a role in multiple steps in HIV assembly and release. For example, cholesterol is important for viral fusion and infection of cells by HIV [157]. Furthermore, the host cell receptor for HIV, receptor CD4, has been identified as a raft-associated protein [158]. However, until recently, the exact mechanisms by which the virus targets rafts for entry into cells has remained enigmatic. In a series of interesting studies from both a membrane biology and virology standpoint, HIV has been shown to selectively bind and fuse to the interface between liquid ordered (Lo) and liquid disordered (Ld) domains [159C161]. Initial evidence in support of this idea came from studies showing that reconstitution of the fusion peptide (FP) of HIV gp41 into liposomes mimicking the composition of HIV viral membranes facilitates their fusion to supported bilayers consisting of mixtures of Lo and Ld domains [160]. Strikingly, liposomes containing HIV FP preferentially accumulated at the boundary between Lo and Ld domains. Further, both phase separation and cholesterol were found to be required to facilitate fusion. This behavior was specific to the HIV FP because liposomes containing the influenza FP showed no preference for the boundary [160]. HIV-1 psuedoviruses also preferentially bound to the domain boundary, demonstrating this behavior is not limited to the isolated FP [160]. An interesting question raised by these findings is why HIV virions prefer to fuse at domain.