Perfringolysin O, a cholesterol-binding cytolysin, as a probe for lipid rafts.
ABSTRACT Gaining an understanding of the structural and functional roles of cholesterol in membrane lipid rafts is a critical issue in studies on cellular signaling and because of the possible involvement of lipid rafts in various diseases. We have focused on the potential of perfringolysin O (theta-toxin), a cholesterol-binding cytolysin produced by Clostridium perfringens, as a probe for studies on membrane cholesterol. We prepared a protease-nicked and biotinylated derivative of perfringolysin O (BCtheta) that binds selectively to cholesterol in cholesterol-rich microdomains of cell membranes without causing membrane lesions. Since the domains fulfill the criteria of lipid rafts, BCtheta can be used to detect cholesterol-rich lipid rafts. This is in marked contrast to filipin, another cholesterol-binding reagent, which binds indiscriminately to cell cholesterol. Using BCtheta, we are now searching for molecules that localize specifically in cholesterol-rich lipid rafts. Recently, we demonstrated that the C-terminal domain of perfringolysin O, domain 4 (D4), possesses the same binding characteristics as BCtheta. BIAcore analysis showed that D4 binds specifically to cholesterol with the same binding affinity as the full-size toxin. Cell-bound D4 is recovered predominantly from detergent-insoluble, low-density membrane fractions where raft markers, such as cholesterol, flotillin and Src family kinases, are enriched, indicating that D4 also binds selectively to lipid rafts. Furthermore, a green fluorescent protein-D4 fusion protein (GFP-D4) was revealed to be useful for real-time monitoring of cholesterol in lipid rafts in the plasma membrane. In addition, the expression of GFP-D4 in the cytoplasm might allow the investigations of intracellular trafficking of lipid rafts. The simultaneous visualization of lipid rafts in plasma membranes and inside cells might help in gaining a total understanding of the dynamic behavior of lipid rafts.
- SourceAvailable from: Vimal Selvaraj[Show abstract] [Hide abstract]
ABSTRACT: We demonstrate for the first time that a stable, micron-scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, G(M1), localizes to this same domain in live sperm. Interestingly, the G(M1) undergoes an unexplained redistribution upon cell death. We now demonstrate that G(M1) is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing G(M1) in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the "acrosome reaction." Comparison of the dynamics of CTB-bound endogenous G(M1) and exogenous BODIPY-G(M1) in live murine sperm demonstrate that the sub-acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol- and G(M1)-enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or G(M1) that can be separated biochemically.Journal of Cellular Physiology 03/2009; 218(3):522-36. DOI:10.1002/jcp.21624 · 3.87 Impact Factor
Article: Lipid sensing and lipid sensors[Show abstract] [Hide abstract]
ABSTRACT: The field of bile acids has witnessed an impulse in the last two decades. This has been the result of cloning the genes encoding enzymes of bile acid synthesis and their transporters. There is no doubt that the identification of Farnesoid X Receptor (FXR, NR1H4) as the bile acid receptor has contributed substantially to attract the interest of scientists in this area. When FXR was cloned by Forman et al. , farnesol metabolites were initially considered the physiological ligands. After identifying FXR and other nuclear receptors as bile acid sensors [2—4], it has become clear that bile acids are involved in the regulation of lipid and glucose metabolism and that these molecules are eclectic regulators of diverse cellular functions. In this review, we will summarize the current knowledge of the functions regulated by bile acids and how their physiological receptors mediate the signaling underlying numerous cellular responses.Cellular and Molecular Life Sciences CMLS 10/2007; 64(19):2477-2491. DOI:10.1007/s00018-007-7280-y · 5.86 Impact Factor
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ABSTRACT: Ostreolysin, a 15 kDa pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is lytic to membranes containing both cholesterol and sphingomyelin. Its cytotoxicity to Chinese hamster ovary cells correlates with their cholesterol contents and with the occurrence of ostreolysin in the cells detergent resistant membranes. Moreover, ostreolysin binds to supported monolayers and efficiently permeabilizes sonicated lipid vesicles, only if cholesterol is combined with either sphingomyelin or dipalmitoylphosphatidylcholine. Addition of mono- or di-unsaturated phosphatidylcholine to the cholesterol/sphingomyelin vesicles dramatically reduces the ostreolysin's activity. It appears that the protein recognizes specifically a cholesterol-rich lipid phase, probably the liquid-ordered phase.FEBS Letters 10/2004; 575(1-3):81-5. DOI:10.1016/j.febslet.2004.07.093 · 3.34 Impact Factor