We employed our recently developed immuno-electron microscopic method (W. Möbius, Y. Ohno-Iwashita, E. G. van Donselaar, V. M. Oorschot, Y. Shimada, T. Fujimoto, H. F. Heijnen, H. J. Geuze and J. W. Slot, J Histochem Cytochem 2002; 50: 43-55) to analyze the distribution of cholesterol in the endocytic pathway of human B lymphocytes. We could distinguish 6 categories of endocytic compartments on the basis of morphology, BSA gold uptake kinetics and organelle marker analysis. Of all cholesterol detected in the endocytic pathway, we found 20% in the recycling tubulo-vesicles and 63% present in two types of multivesicular bodies. In the multivesicular bodies, most of the cholesterol was contained in the internal membrane vesicles, the precursors of exosomes secreted by B cells. Cholesterol was almost absent from lysosomes, that contained the bulk of the lipid bis(monoacylglycero)phosphate, also termed lysobisphosphatidic acid. Thus, cholesterol displays a highly differential distribution in the various membrane domains of the endocytic pathway.

We used a proteolytically modified and biotinylated derivative of the cholesterol-binding Theta-toxin (perfringolysin O) to localize cholesterol-rich membranes in cryosections of cultured human lymphoblastoid cells (RN) by electron microscopy. We developed a fixation and immunolabeling procedure to improve the preservation of membranes and minimize the extraction and dislocalization of cholesterol on thin sections. We also labeled the surface of living cells and applied high-pressure freezing and subsequent fixation of cryosections during thawing. Cholesterol labeling was found at the plasma membrane, with strongest labeling on filopodium-like processes. Strong labeling was also associated with internal vesicles of multivesicular bodies (MVBs) and similar vesicles at the cell surface after secretion (exosomes). Tubulovesicular elements in close vicinity of endosomes and the Golgi complex were often positive as well, but the surrounding membrane of MVBs and the Golgi cisternae appeared mostly negative. Treatment of cells with methyl-beta-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the Theta-toxin derivative, when used in combination with improved fixation and high-pressure freezing, represents a useful tool for the localization of membrane cholesterol in ultrathin cryosections.

There is increasing evidence that sphingolipid-and cholesterol-rich microdomains (rafts) exist in the plasma membrane. Specific proteins assemble in these membrane domains and play a role in signal transduction and many other cellular events. Cholesterol depletion causes disassembly of the raft-associated proteins, suggesting an essential role of cholesterol in the structural maintenance and function of rafts. However, no tool has been available for the detection and monitoring of raft cholesterol in living cells. Here we show that a protease-nicked and biotinylated derivative (BC) of perfringolysin O (-toxin) binds selectively to cholesterol-rich microdomains of intact cells, the domains that fulfill the criteria of rafts. We fractionated the homogenates of nontreated and Triton X-100-treated platelets after incubation with BC on a sucrose gradient. BC was predominantly localized in the floating lowdensity fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BC binds to a subpopulation of vesicles in FLDF. Depletion of 35% cholesterol from platelets with cyclodextrin, which accompanied 76% reduction in cholesterol from FLDF, almost completely abolished BC binding to FLDF. The staining patterns of BC and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BC binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BC binding does not cause any damage to cell membranes, indicating that BC is a useful probe for the detection of membrane rafts in living cells.

f Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SMspecific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP 2 -rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP 2 and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP 2 to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.

There is much evidence to indicate that cholesterol forms lateral membrane microdomains (rafts), and to suggest their important role in cellular signaling. However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of θ‐toxin (perfringolysin O), a cholesterol‐binding cytolysin, whose chemically modified form has been recently shown to bind selectively to rafts. BIAcore and structural analyses demonstrate that the C‐terminal domain (domain 4) of the toxin is the smallest functional unit that has the same cholesterol‐binding activity as the full‐size toxin with structural stability. Cell membrane‐bound recombinant domain 4 was detected in the floating low‐density fractions and was found to be cofractionated with the raft‐associated protein Lck, indicating that recombinant domain 4 also binds selectively to cholesterol‐rich rafts. Furthermore, an enhanced green fluorescent protein‐domain 4 fusion protein stains membrane surfaces in a cholesterol‐dependent manner in living cells. Therefore, domain 4 of θ‐toxin is an essential cholesterol‐binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells.