Yukiko Shimada

Recycling Compartments and the Internal Vesicles of Multivesicular Bodies Harbor Most of the Cholesterol Found in the Endocytic Pathway

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.

Selective binding of perfringolysin O derivative to cholesterol-rich membrane microdomains (rafts)

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 (BCθ) of perfringolysin O (θ-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 BCθ on a sucrose gradient. BCθ was predominantly localized in the floating low-density fractions (FLDF) where cholesterol, sphingomyelin, and Src family kinases are enriched. Immunoelectron microscopy demonstrated that BCθ 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 BCθ binding to FLDF. The staining patterns of BCθ and filipin in human epidermoid carcinoma A431 cells with and without cholesterol depletion suggest that BCθ binds to specific membrane domains on the cell surface, whereas filipin binding is indiscriminate to cell cholesterol. Furthermore, BCθ binding does not cause any damage to cell membranes, indicating that BCθ is a useful probe for the detection of membrane rafts in living cells.

Immunoelectron Microscopic Localization of Cholesterol Using Biotinylated and Non-cytolytic Perfringolysin O:

We used a proteolytically modified and biotinylated derivative of the cholesterol-binding θ-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-β-cyclodextrin completely abolished the labeling for cholesterol. Our results show that the θ-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.

Increased lipid rafts and accelerated lipopolysaccharide-induced tumor necrosis factor-α secretion in Abca1-deficient macrophages

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 𝛉-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 κB, mRNA and intracellular distribution, and secretion of tumor necrosis factor-α (TNF-α) were examined after stimulation by lipopolysaccharides (LPSs). LPS-induced responses of the activation of nuclear factor κB and TNF-α 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-α secretion. We report here that Abca1-KO macrophages with defective lipid efflux exhibited increased lipid rafts on the cell surface and accelerated TNF-α secretion.

Accelerated Aβ aggregation in the presence of GM1‐ganglioside‐accumulated synaptosomes of aged apoE4‐knock‐in mouse brain

Aging and apolipoprotein E4 (apoE4) expression are strong risk factors for the development of Alzheimers disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid β‐protein (Aβ) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that Aβ aggregation is accelerated through the generation of GM1 ganglioside (GM1)‐bound Aβ which acts as a seed for Aβ fibril formation. Here we report that GM1 level in detergent‐resistant membrane microdomains (DRMs) of synaptosomes increased with age and that this increase was significantly pronounced in the apoE4‐ than the apoE3‐knock‐in mouse brain. Furthermore, we show that Aβ aggregation is markedly accelerated in the presence of the synaptosomes of the aged apoE4‐knock‐in mouse brain. These observations suggest that aging and apoE4 expression cooperatively accelerate Aβ aggregation in the brain through an increase in the level of GM1 in neuronal membranes.

Ultrastructural localization of flotillin-1 to cholesterol-rich membrane microdomains, rafts, in rat brain tissue.

There is much interest in research on cholesterol-rich membrane microdomains, rafts, in the field of neurobiology. However, no one has shown the ultrastructure of rafts in tissues. We examined the ultrastructure of rafts in rat brain tissue by pre-embedding immunoelectron microscopy using flotillin-1 antibody, which is a biochemical marker of lipid rafts, and BCtheta, which is nicked and biotinylated theta-toxin, and binds to membrane cholesterol of rafts. Flotillin-1- and BCtheta-labeled areas were patchy and prominent on the plasma membranes of small processes and synapses in the neuropil. The size of flotillin-1 labeling was 40-200 nm. In addition, the membrane of lysosome and Golgi apparatus were frequently labeled for flotillin-1 with a patchy pattern. Flotillin-1 and BCtheta were mostly colocalized in double immunolabeling on a part of the plasma membranes of small processes and secondary lysosome membranes. We first indicate that flotillin-1 localizes to BCtheta-positive cholesterol-rich membrane microdomains in vivo, and that flotillin-1 and BCtheta could be ultrastructural raft markers in neural tissue.

Reduction of Glycosphingolipid Levels in Lipid Rafts Affects the Expression State and Function of Glycosylphosphatidylinositol-anchored Proteins but Does Not Impair Signal Transduction via the T Cell Receptor

Lipid rafts are highly enriched in cholesterol and sphingolipids. In contrast to many reports that verify the importance of cholesterol among raft lipid components, studies that address the role of sphingolipids in raft organization and function are scarce. Here, we investigate the role of glycosphingolipids (GSLs) in raft structure and raft-mediated signal transduction in T lymphocytes by the usage of a specific GSL synthesis inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-PDMP). Surface GM1 expression and the expression of GSLs in rafts were profoundly reduced by d-PDMP treatment, whereas the expression of other lipid and protein constituents, such as cholesterol, sphingomyelin, Lck, and linker for activation of T cells, was not affected. T cell receptor-mediated signal transduction induced by antigen stimulation or by antibody cross-linking was normal in d-PDMP-treated T cells. In contrast, the signal through glycosylphosphatidylinositol (GPI)-anchored proteins was clearly augmented by d-PDMP treatment. Moreover, GPI-anchored proteins became more susceptible to phosphatidylinositol-specific phospholipase C cleavage in d-PDMP-treated cells, demonstrating that GSL depletion from rafts primarily influences the expression state and function of GPI-anchored proteins. Finally, by comparing the effect of d-PDMP with that of methyl-β-cyclodextrin, we identified that compared with cholesterol depletion, GSL depletion has the opposite effect on the phosphatidylinositol-specific phospholipase C sensitivity and signaling ability of GPI-anchored proteins. These results indicate a specific role of GSLs in T cell membrane rafts that is dispensable for T cell receptor signaling but is important for the signal via GPI-anchored proteins.

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

Perfringolysin O (θ-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the β-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in θ-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native θ-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 m guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in β-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of θ-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.

Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

BCθ is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (θ-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCθ can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCθ signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCθ. Under this condition, BCθ mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCθ staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to β-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCθ staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCθ is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

Plasma membrane microdomains in aging and disease.

The plasma membrane of eukaryotic cells participates in signal transduction and many other cellular events to maintain the physiological state of cells. In recent decades, much attention has been paid to membrane microdomains, called lipid rafts or membrane rafts, as signaling platforms in the plasma membrane. Lipid rafts are lateral lipid clusters enriched in cholesterol and sphingolipids in which particular molecules are concentrated and participate in membrane‐mediated signaling events. Recent studies have shown a close relationship between lipid rafts and the age‐associated decline and dysregulation of cellular signaling pathways, such as T‐cell receptor signaling and cellular senescence‐related signaling. Lipid rafts have also been implicated in senile diseases and in lifestyle‐related diseases whose incidences increase with age. Geriatr Gerontol Int 2010; 10 (Suppl. 1): S41–S52.