Papers - OOSAKI Yuuki

Cytoplasmic lipid droplets are sites of convergence of proteasomal and autophagic degradation of apolipoprotein B

Yuki Ohsaki, Jinglei Cheng, Akikazu Fujita, Toshinobu Tokumoto, Toyoshi Fujimoto

MOLECULAR BIOLOGY OF THE CELL ( AMER SOC CELL BIOLOGY )  17 ( 6 ) 2674 - 2683  2006.06  [Refereed]  [International journal]

Authorship：   Lead author  , Corresponding author

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Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in < 10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal. inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.

DOI PubMed

Proteasomal and autophagic pathways converge on lipid droplets

Toyoshi Fujimoto, Yuki Ohsaki

Autophagy ( Taylor and Francis Inc. )  2 ( 4 ) 299 - 301  2006  [Refereed]  [Invited]

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Apolipoprotein B (apoB) is the primary protein of very low-density lipoproteins (VLDL). We found that apoB accumulated on the surface of cytoplasmic lipid droplets (LDs) of hepatocytes when the proteasomal or autophagic processes were suppressed. ApoB associated with LDs was poly-ubiquitinated and surrounded by autophagic vacuoles. Moreover, proteasomal subunits were concentrated around LDs. Our data suggest that apoB that is destined to be degraded remains adhered to LDs until it is broken down by the proteasomal and autophagic pathways. We speculate that the LD surface serves as a platform to prevent hydrophobic apoB from forming aggregates, and that LDs may play a similar role for other aggregation-prone hydrophobic proteins. ©2006 Landes Bioscience.

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Cytoplasmic lipid droplets - Rediscovery of an old structure as a unique platform

Toyoshi Fujimoto, Yuki Ohsaki

INTEGRATED MOLECULAR MEDICINE FOR NEURONAL AND NEOPLASTIC DISORDERS ( BLACKWELL PUBLISHING )  1086   104 - 115  2006  [Refereed]  [International journal]

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Cytoplasmic lipid droplets (LDs) exist in virtually any kind of cell. They have a core of lipid esters covered by the surface phospholipid monolayer. A number of proteins related to various cell functions are present in LDs and/or LD-rich subcellular fractions, suggesting that LDs are an independent organelle that is engaged in various cellular activities. Furthermore, a recent study suggested that LDs are a platform where the proteasomal and autophagic pathways converge. The molecular composition and architecture of LDs are discussed here, with special reference to the technical difficulties encountered when analyzing this unique organelle.

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Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

Y Ohsaki, T Maeda, T Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER )  124 ( 5 ) 445 - 452  2005.11  [Refereed]  [International journal]

Authorship：   Lead author

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The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

DOI PubMed

カベオラ，ラフトと血管

藤本豊士, 藤田豊, 大崎雄樹

血管医学   5   455-461  2004.10  [Invited]

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of niemann-pick C1-deficient cells

Yuko Sugimoto, Haruaki Ninomiya, Yuki Ohsaki, Katsumi Higaki, Joanna P. Davies, Yiannis A. Ioannou, Kousaku Ohno

Proceedings of the National Academy of Sciences of the United States of America   98 ( 22 ) 12391 - 12396  2001.10

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We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/ endoplasmic reticulum does not.

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