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Yukiko Shimada

Publications and source records attributed to Yukiko Shimada.

24 records · Page 2Linked to original sources
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↗