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.

Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24.

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.

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.

RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA (dsRNA) to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the dsRNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically posttranscriptional gene silencing (PTGS) and chromatin-dependent gene silencing 1 . Although endogenous small RNAs play critical roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymeraseassociated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model where Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing (RITS) complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of Reprints and permissions information is available at www.nature.com/reprints. A patent application has been filed.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsCorrespondence and requests for materials should be addressed to M.B. (marc.buehler@fmi.ch). * Correspondence to: marc.buehler@fmi.ch. Author Contributions Y.S., K.M.K., V.F., and J.B. generated strains and performed experiments; Y.S. performed the sms screen; genome-wide small RNA and gene expression data was analyzed by K.M.K.; M.B.S. designed and performed the computational analysis of the mutant genome resequencing data; M.B. designed experiments and prepared the manuscript. All authors discussed the results and commented on the manuscript.

Cytoskeletal rearrangements during the cell cycle and in response to signals are regulated by small Rho-type GTPases, but it is not known how these GTPases are activated in a spatial and temporal manner. Here we show that Cdc24, the guanine-nucleotide exchange factor for the yeast GTPase Cdc42, is sequestered in the cell nucleus by Far1. Export of Cdc24 to a site of cell polarization is mediated by two mechanisms. At bud emergence, activation of the G1 cyclin-dependent kinase Cdc28-Cln triggers degradation of Far1 and, as a result, relocation of Cdc24 to the cytoplasm. Cells overexpressing a non-degradable Far1 were unable to polarize their actin cytoskeleton because they failed to relocate Cdc24 to the incipient bud site. In contrast, in response to mating pheromones, the Far1-Cdc24 complex is exported from the nucleus by Msn5. This mechanism ensures that Cdc24 is targeted to the site of receptor-associated heterotrimeric G-protein activation at the plasma membrane, thereby allowing polarization of the actin cytoskeleton along the morphogenetic gradient of pheromone. Either degradation of Far1 or its nuclear export by Msn5 was sufficient for cell growth, suggesting that the two mechanisms are redundant for cell viability. Taken together, our results indicate that Far1 functions as a nuclear anchor for Cdc24. This sequestration regulates cell polarity in response to pheromones by restricting activation of Cdc42 to the site of pheromone receptor activation.

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.

Lipid rafts on the cell surface are believed to be very important as platforms for various cellular functions. The aim of this study was to know whether defective lipid efflux may influence lipid rafts on the cell surface and their related cellular functions. We investigated macrophages with defective lipid efflux from ATP binding cassette transporter A1-deficient (Abca1-KO) mice. Lipid rafts were evaluated by the following two novel probes: a biotinylated and protease (subtilisin Carlsberg)-nicked derivative of u-toxin and a fluorescein ester of polyethylene glycol-derived cholesterol. Lipid rafts in Abca1-KO macrophages were increased, as demonstrated by both probes. Moreover, activities of nuclear factor kB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-a (TNF-a) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor kB and TNF-a were more prompt and accelerated in the Abca1-KO macrophages compared with wild-type macrophages. Modification of lipid rafts by cyclodextrin and nystatin corrected the abnormal response, suggesting an association between the increased lipid rafts and abnormal TNF-a secretion.We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-a secretion. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role as a shuttle carrying cholesterol derived from peripheral tissues to the liver (1). Cholesterol efflux from the cells is the initial step of RCT, in which free apolipoprotein A-I (apoA-I) or small HDLs take up cholesterol from the peripheral cells. We have been trying to elucidate the molecular mechanism for RCT and cholesterol efflux by analyzing the pathophysiology of patients with abnormal HDL metabolism. We have identified molecules involved in cellular cholesterol efflux and apoA-I and HDL binding proteins on macrophages (2-5).Tangier disease (TD) is a model for the impairment of cholesterol efflux from the cells (6, 7). Patients with TD suffer from genetic HDL deficiency, hepatosplenomegaly, orange tonsils, and premature atherosclerosis (8, 9). Many laboratories including ours have reported that mutations in the Abca1 gene lead to defective cholesterol efflux from the cells (10-12). As a result of the mutation(s) in the Abca1 gene, cells from TD patients exhibited a deficiency of apoA-I-mediated cholesterol efflux and a subsequent accumulation of intracellular lipids as lipid droplets, which is closely related to the development of atherosclerosis in this disorder.On the other hand, in the plasma membrane, cholesterol is distributed abundantly in some domain structures Abbreviations: Abca1-KO, ATP binding cassette transporter A1-deficient; apoA-I, apolipoprotein A-I; BCu, biotinylated and protease (subtilisin Carlsberg)-nicked derivative of u-toxin; fPEG-chol, fluorescent polyethylene glycol cholesteryl ether; L...