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Yukiko Shimada

Publications and source records attributed to Yukiko Shimada.

17 recordsLinked to original sources
PubMed2005

Distinct localization of lipid rafts and externalized phosphatidylserine at the surface of apoptotic cells.

Externalization of phosphatidylserine (PS) takes place in apoptotic cells as well as in viable cells under certain circumstances. Recent studies showed that externalized PS is localized at the lipid raft in viable activated immune cells. We found that lipid rafts and PS existed in a mutually exclusive manner in apoptotic cells. The number of PS-exposing apoptotic cells decreased when lipid rafts were disrupted. BCtheta;, which binds selectively to cholesterol in a cholesterol-rich region, did not effectively recognize lipid rafts of apoptotic cells. Lipid rafts rich in GM1 were successfully prepared from apoptotic cells, but the lipid raft protein LAT was not enriched in the preparation. Furthermore, the amount of PS and phosphatidylethanolamine but not of cholesterol in lipid rafts appeared to change after induction of apoptosis. These results suggest that lipid rafts are structurally modified during apoptosis and, despite being localized differently from PS, are involved in the externalization of PS.

Apoptosis↗
PubMed2004

Accelerated Abeta 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 Alzheimer's disease (AD); however, their pathological roles remain to be clarified. In the process of AD development, the conversion of the nontoxic amyloid beta-protein (Abeta) monomer to its toxic aggregates is a fundamental process. We previously hypothesized that Abeta aggregation is accelerated through the generation of GM1 ganglioside (GM1)-bound Abeta which acts as a seed for Abeta 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 Abeta 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 Abeta aggregation in the brain through an increase in the level of GM1 in neuronal membranes.

Aging↗
PubMed2004

Reduced sensitivity of Niemann-Pick C1-deficient cells to theta-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles.

Theta-toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(-) that lacked Niemann-Pick C1 showed reduced sensitivity to theta-toxin, compared with wild-type (wt) cells. BCtheta is a derivative of theta-toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BCtheta, both on the cell surface and in the extracellular space of these cells. BCtheta binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4 degrees C and recovery of bound BCtheta in floating low-density fractions on sucrose density gradient fractionation. BCtheta-labeled vesicles were abolished when NPC1(-) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, theta-toxin sensitivity of NPC1(-) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to theta-toxin.

Animals↗
PubMed2004

Site-specific regulation of the GEF Cdc24p by the scaffold protein Far1p during yeast mating.

Receptor-mediated cell polarization via heterotrimeric G-proteins induces cytoskeletal rearrangements in a variety of organisms. In yeast, Far1p is required for orienting cell growth towards the mating partner by linking activated Gbetagamma to the guanine-nucleotide exchange factor (GEF) Cdc24p, which activates the Rho-type GTPase Cdc42p. Here we investigated the role of Far1p in the regulation of Cdc24p in vivo. Using time-lapse microscopy of mating cells and artificial membrane targeting of Far1p, we show that Far1p is necessary and sufficient to recruit Cdc24p to the plasma membrane. Wild-type Far1p contains a PH-like domain, which is required for its membrane localization in vivo. Interestingly, expression of membrane-targeted Far1p causes toxicity, most likely by activating Cdc42p uniformly at the cell cortex. The ability of full-length Far1p to function as an activator of Cdc24p in vivo requires its interaction with Cdc24p and Gbetagamma. Our results imply that Gbetagamma not only targets Far1p to the correct site but may also trigger a conformational change in Far1p that is required for its ability to activate Cdc24p in vivo.

Amino Acid Sequence↗
PubMed2004

The nucleotide exchange factor Cdc24p may be regulated by auto-inhibition.

Site-specific activation of the Rho-type GTPase Cdc42p by its guanine-nucleotide exchange factor (GEF) Cdc24p is critical for the establishment of cell polarity. Here we show that binding of Cdc24p to the small GTPase Rsr1p/Bud1p is required for its recruitment to the incipient bud site. Rsr1p/Bud1p binds to the CH-domain of Cdc24p, which is essential for its function in vivo. We have identified a cdc24-mutant allele, which is specifically defective for bud-site selection. Our results suggest that Cdc24p is auto-inhibited by an intramolecular interaction with its carboxy-terminal PB1-domain. Rsr1p/Bud1p appears to activate the GEF activity of Cdc24p in vivo, possibly by triggering a conformational change that dissociates the PB1-domain from its intramolecular binding site. Genetic experiments suggest that Bem1p functions as a positive regulator of Cdc24p by binding to the PB1-domain of Cdc24p, thereby preventing its re-binding to the intramolecular inhibitory site. Taken together, our results support a two-step molecular mechanism for the site-specific activation of Cdc24p, which involves Rsr1p/Bud1p and the adaptor protein Bem1p.

Adaptor Proteins, Signal Transducing↗
PubMed2004

A novel cholesterol stain reveals early neuronal cholesterol accumulation in the Niemann-Pick type C1 mouse brain.

Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by progressive accumulation of cholesterol, gangliosides, and other lipids in the central nervous system and visceral organs. In the NPC1 mouse model, neurodegeneration and neuronal cell loss occur before postnatal day 21. Whether neuronal cholesterol accumulation occurs in vivo before the first signs of neuronal cell loss has not been demonstrated. In this report, we used the NPC1 mouse model and employed a novel cholesterol binding reagent, BC theta, that enabled us to visualize cellular cholesterol accumulation at a level previously unattainable. The results demonstrate the superiority of BC theta staining over conventional filipin staining in confocal microscopy and highlight several new findings. We show that at postnatal day 9, although only mild signs of neurodegeneration are detectable, significant neuronal cholesterol accumulation has already occurred throughout the NPC1 brain. In addition, although NPC1 Purkinje neurons exhibit a normal morphology at day 9, significant cholesterol accumulation within their extensive dendritic trees has occurred. We also show that in the thalamus and cortex of NPC1 mice, activated glial cells first appear at postnatal day 9 and heavily populate by day 22, suggesting that in NPC1 mice, neuronal cholesterol accumulation precedes neuronal injury and neuronal cell loss.

Animals↗
PubMed2004

Axon-dominant localization of cell-surface cholesterol in cultured hippocampal neurons and its disappearance in Niemann-Pick type C model cells.

There is growing evidence showing the important role of cholesterol in maintaining neuronal function. In particular, much attention has been paid to the role of the cholesterol-rich microdomains called lipid rafts. However, the cholesterol distribution on neurons is not clear. Here, we investigated localization of cholesterol in cultured rat hippocampal neurons, using filipin and a novel cholesterol-binding reagent BCtheta. In our culture system, BCtheta detects only cell-surface cholesterol, whereas filipin stains both intracellular and cell-surface cholesterol. BCtheta staining appeared visible in a maturation-dependent manner and showed axon-dominant distribution of cell-surface cholesterol in fully matured neurons. A part of this cholesterol on axons was resistant to detergents at 4 degrees C, and thus might be involved in lipid rafts. Interestingly, Niemann-Pick type C model neurons induced by class 2 amphiphiles lost the cell-surface but not the intracellular cholesterol staining. Niemann-Pick type C disease is caused by the disruption of intracellular cholesterol transport and is known to induce neurodegeneration in brains accompanied by formation of neurofibrillary tangles. Our observations suggest the important role of cell-surface cholesterol in maintaining a functional axonal membrane and indicate that the observed defect in axonal surface cholesterol might lead to neurodegeneration.

Animals↗
PubMed2003

Gamma-secretase activity is present in rafts but is not cholesterol-dependent.

Cholesterol has been claimed to be involved in the generation and/or accumulation of amyloid beta protein (Abeta). However, the underlying molecular mechanisms have not been fully elucidated yet. Here, we have investigated the effect of membrane cholesterol content on gamma-secretase activity using Chinese hamster ovary cells stably expressing beta-amyloid precursor protein (APP) and either wild-type or N141I mutant-type presenilin 2. Cholesterol was acutely depleted from the isolated membrane by methyl-beta-cyclodextrin, and Abeta production was assessed in a cell-free assay system. Reduced cholesterol did not significantly alter the amounts of Abeta produced by either total cell membranes or cholesterol-rich low-density membrane domains. Even its extremely low levels in the latter domains did not affect Abeta production. This indicates that the membrane cholesterol content does not directly modulate the activity of gamma-secretase. To ascertain that gamma-secretase resides in cholesterol-rich membrane domains, low-density membrane domains were further fractionated with BCtheta (biotinylated theta-toxin nicked with subtilisin Carlsberg protease), which has recently been shown to bind selectively to rafts of intact cells. The membrane domains purified with BCtheta did indeed produce Abeta. These observations indicate that the gamma-cleavage required for generating Abeta occurs in rafts, but its activity is virtually cholesterol-independent.

Amyloid Precursor Protein Secretases↗
PubMed2003

Cellular aging-dependent decrease in cholesterol in membrane microdomains of human diploid fibroblasts.

Recently it has been shown that cholesterol plays indispensable roles in the function of cholesterol-rich microdomains (rafts), such as in ligand-mediated signal transduction. Using a perfringolysin O derivative (BCtheta) that binds selectively to cholesterol in rafts without causing membrane damage (Proc. Natl. Acad. Sci. USA 98 (2001) 4926), we have investigated the effect of in vitro replicative aging of human diploid fibroblasts, TIG-1, on the distribution of plasma membrane cholesterol. The amount of BCtheta-labeled membrane cholesterol decreased during replicative aging of TIG-1 cells, whereas total cholesterol increased somewhat. The relationship was confirmed by double staining with BCtheta and senescence-associated-beta-galactosidase, a biomarker of senescent cells. Cell fractionation experiments revealed decreases in both cholesterol in rafts and a raft marker, flotillin, during replicative aging. In addition, hydroxyurea-induced prematurely senescent cells also showed a lower level of BCtheta-labeled cholesterol than untreated cells, despite maintaining the total amount of cholesterol. When TIG-1 cells were cultured in cholesterol-deficient medium, BCtheta labeling was first diminished and then premature senescence was induced. Taken together with the reduced signaling capacity of aged cells, these results suggest that plasma membrane cholesterol in raft microdomains is more sensitive to senescence than total cholesterol and is a primary target in aging.

Apoptosis↗
PubMed2003

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-beta-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.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

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

BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-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, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta 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 BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.

Animals↗
PubMed2002

Lipid binding activity of a neuron-specific protein NAP-22 studied in vivo and in vitro.

There exists a microdomain called "raft" in the cell membrane. The enrichment of cholesterol and sphingolipids in its outer leaflet is well recognized. In contrast, little is known of the lipid composition of the inner leaflet of raft, where many acylated signal-transducing molecules, such as trimeric G proteins and protein tyrosine kinases, associate. NAP-22 is a neuronal protein localized on the inner leaflet of raft domain. This protein was found to bind cholesterol in the liposome. In this study, we further analyze the lipid binding activity of NAP-22 using eukaryotic and bacterial expression systems. In addition to cholesterol, NAP-22 showed a phosphatidylethanolamine (PE)- and polyphosphoinositide-dependent membrane binding in the liposome assay. The N-terminal myristoylation was essential for the liposome binding. The C-terminal deletion up to D61 showed little effect on the binding. The lipid binding region was hence judged to be in the N-terminal 60-amino-acid sequence. NAP-22 was then expressed in COS7 cells, and the intracellular localization was studied. Biochemical analysis showed the localization of NAP-22 in a Triton-insoluble low-density fraction. Cell staining analysis showed colocalization patterns of NAP-22 with PE and cholesterol in the membrane.

Amino Acid Sequence↗
PubMed2002

The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains.

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 theta-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 theta-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.

Amino Acid Sequence↗
PubMed2002

Immunoelectron microscopic localization of cholesterol using biotinylated and non-cytolytic perfringolysin O.

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.

Bacterial Toxins↗