The Sensing of Membrane Microdomains Based on Pore-Forming Toxins

Abstract

Membrane rafts are transient and unstable membrane microdomains that are enriched in sphingolipids, cholesterol, and specific proteins. They are involved in intracellular trafficking, signal transduction, pathogen entry, and attachment of various ligands. Increasing experimental evidence on the crucial biological roles of membrane rafts under normal and pathological conditions require new techniques for their structural and functional characterization. In particular, fluorescence-labeled cytolytic proteins that interact specifically with molecules enriched in rafts are of increasing interest. Cholera toxin subunit B interacts specifically with raft-residing ganglioside G(M1), and it has long been the lipid probe of choice for membrane rafts. Recently, four new pore-forming toxins have been proposed as selective raft markers: (i) equinatoxin II, a cytolysin from the sea anemone Actinia equina, which specifically recognizes free and membrane-embedded sphingomyelin; (ii) a truncated non-toxic mutant of a cytolytic protein, lysenin, from the earthworm Eisenia foetida, which specifically recognizes sphingomyelin-enriched membrane domains; (iii) a non-toxic derivative of the cholesterol-dependent cytolysin perfringolysin O, from the bacterium Clostridium perfringens, which selectively binds to membrane domains enriched in cholesterol; and (iv) ostreolysin, from the mushroom Pleurotus ostreatus, which does not bind to a single raft-enriched lipid component, but requires a specific combination of two of the most important raft-residing lipids: sphingomyelin and cholesterol. Nontoxic, raft-binding derivatives of cytolytic proteins have already been successfully used to explore both the structure and function of membrane rafts, and of raft-associated molecules. Here, we review these four new derivatives of pore-forming toxins as new putative markers of these membrane microdomains.

Dynamic label-free imaging of lipid nanodomains

“…S3). iSCAT does not suffer from the uncertainties in studying lipid nanodomains by fluorescence as it is an inherently label-free technique and avoids the potential problems of fluorescent probes perturbing the phase behavior one would seek to study (14,15). Even when taking advantage of FRET, current methods are restricted by the affinity of lipid probes for specific lipid phases (13) and cannot image lipid nanodomains.…”

“…This approach is successful for micrometer-sized domains but inevitably fails on the tens to few hundreds of nanometers scale due to limitations in phase specificity, the limited residence time of a label within a specific nanoscopic domain, and the achievable optical resolution (13). The fluorescent probe is itself an additional component that can perturb phase behavior, either directly or through photooxidation (14,15). As a result, lipid nanodomain dynamics have not been observed directly even in artificial systems, although recent ensemblebased techniques report lipid heterogeneity on the appropriate length scales (13).…”

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

“…Alexa Fluor), have proved useful in confocal or super-resolution microscopy analyses [22, 23, 26, 114] (see Table 1). For further general information on lysenin, please see [110, 111, 115]. Regarding equinatoxin II, produced from the sea anemone Actinia equine , the full-length toxin has been fused to fluorescent proteins in order to analyze SM distribution in cell membranes.…”

Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane

“…However, only a few lipid-specifi c fl uorescent toxins are validated so far ( 45 ); and admittedly, their size is much greater than the targeted lipid (e.g., projected diameter of lysenin* is ‫ف‬ 15 times larger than endogenous SM). This size discrepancy predicts steric hindrance but does not preclude specifi city, as perhaps best exemplifi ed by EGFferritin conjugates for which grafting a ‫ف‬ 450 kDa ferritin moiety allowed us to faithfully follow the fate of the small EGF molecule [ ‫ف‬ 6 kDa; ( 46 )].…”