研究者詳細 - 大﨑　雄樹

論文 - 大﨑　雄樹

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Imaging Lipid Droplets by Electron Microscopy

Toyoshi Fujimoto, Yuki Ohsaki, Michitaka Suzuki, Jinglei Cheng

LIPID DROPLETS ( ELSEVIER ACADEMIC PRESS INC ) 116 227 - 251 2013年 [査読有り] [国際誌]

The lipid droplet (LD) is different from other cellular organelles in that most of its volume is made of lipid esters and its surface is lined by a phospholipid monolayer. This uniquely lipid-dominant structure poses a problem for electron microscopy (EM) because the aldehydes commonly used as a fixative do not react with most lipids. To circumvent this difficulty and utilize the high resolving power of EM, many methods have been developed. In this chapter, we discuss methods that have been used and/or are potentially useful to study LDs. The methods include conventional EM to observe the LD core, cryoelectron microscopy to observe the LD surface, freeze-substitution, immunoelectron microscopy (pre-embedding, post-embedding, and cryosectioning methods), and freeze-fracture. Each method has strong and weak points and therefore some caution is necessary in interpreting the obtained results. In combination with methods of other disciplines, the electron microscopic techniques should contribute significantly to solving the remaining questions on LDs.

DOI PubMed

ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion

Eija Nissila, Yuki Ohsaki, Marion Weber-Boyvat, Julia Perttila, Elina Ikonen, Vesa M. Olkkonen

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS ( ELSEVIER SCIENCE BV ) 1821 ( 12 ) 1472 - 1484 2012年12月 [査読有り] [国際誌]

ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1-318) localizes at Golgi membranes. Removal of the C-terminal aa 712-764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [S-35]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in beta-lipoprotein secretion by human hepatocytes. (C) 2012 Elsevier B.V. All rights reserved.

DOI PubMed

Derlin-1 and UBXD8 are engaged in dislocation and degradation of lipidated ApoB-100 at lipid droplets

Michitaka Suzuki, Toshihiko Otsuka, Yuki Ohsaki, Jinglei Cheng, Takako Taniguchi, Hisashi Hashimoto, Hisaaki Taniguchi, Toyoshi Fujimoto

MOLECULAR BIOLOGY OF THE CELL ( AMER SOC CELL BIOLOGY ) 23 ( 5 ) 800 - 810 2012年03月 [査読有り] [国際誌]

Apolipoprotein B-100 (ApoB) is the principal component of very low density lipoprotein. Poorly lipidated nascent ApoB is extracted from the Sec61 translocon and degraded by proteasomes. ApoB lipidated in the endoplasmic reticulum (ER) lumen is also subjected to proteasomal degradation, but where and how it dislocates to the cytoplasm remain unknown. In the present study, we demonstrate that ApoB after lipidation is dislocated to the cytoplasmic surface of lipid droplets (LDs) and accumulates as ubiquitinated ApoB in Huh7 cells. Depletion of UBXD8, which is almost confined to LDs in this cell type, decreases recruitment of p97 to LDs and causes an increase of both ubiquitinated ApoB on the LD surface and lipidated ApoB in the ER lumen. In contrast, abrogation of Derlin-1 function induces an accumulation of lipidated ApoB in the ER lumen but does not increase ubiquitinated ApoB on the LD surface. UBXD8 and Derlin-1 bind with each other and with lipidated ApoB and show colocalization around LDs. These results indicate that ApoB after lipidation is dislocated from the ER lumen to the LD surface for proteasomal degradation and that Derlin-1 and UBXD8 are engaged in the predislocation and postdislocation steps, respectively.

DOI PubMed

Translation inhibitors induce formation of cholesterol ester-rich lipid droplets.

Michitaka Suzuki, Yuki Ohsaki, Tsuyako Tatematsu, Yuki Shinohara, Takashi Maeda, Jinglei Cheng, Toyoshi Fujimoto

PloS one 7 ( 8 ) e42379 2012年 [国際誌]

Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy.

DOI PubMed

Lipid droplet biogenesis: when the endoplasmic reticulum starts to fatten up

Veijo T. Salo, Yuki Ohsaki, Elina Ikonen

CURRENT OPINION IN LIPIDOLOGY ( LIPPINCOTT WILLIAMS & WILKINS ) 22 ( 6 ) 505 - 506 2011年12月 [査読有り] [国際誌]

DOI PubMed

Cytoplasmic oxysterol-binding proteins: sterol sensors or transporters?

Terhi Vihervaara, Maurice Jansen, Riikka-Liisa Uronen, Yuki Ohsaki, Elina Ikonen, Vesa M. Olkkonen

CHEMISTRY AND PHYSICS OF LIPIDS ( ELSEVIER IRELAND LTD ) 164 ( 6 ) 443 - 450 2011年09月 [査読有り] [国際誌]

Families of oxysterol-binding protein (ORP) homologues are present in eukaryotes from yeast to man. Their hallmark feature is a characteristic ligand binding domain that, for several family members, has been shown to accommodate different oxysterols and/or cholesterol. ORPs of the "long" subtype contain targeting determinants for the endoplasmic reticulum and to other organelle membranes, the most prominent of which are phosphoinositide-binding pleckstrin homology domains, while "short" ORPs comprise a ligand binding domain with little additional sequences. There is increasing evidence that both long and short ORPs can be enriched at membrane contact sites, junctions of the endoplasmic reticulum with other organelles, where they are suggested to execute regulatory or sterol transfer functions. In this review we discuss the current evidence for putative roles of ORPs as sterol sensors or transporters. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

DOI PubMed

Lipid droplets: size matters

Michitaka Suzuki, Yuki Shinohara, Yuki Ohsaki, Toyoshi Fujimoto

JOURNAL OF ELECTRON MICROSCOPY ( OXFORD UNIV PRESS ) 60 ( 1 ) S101 - S116 2011年08月 [査読有り] [国内誌]

The lipid droplet (LD), an organelle that exists ubiquitously in various organisms, from bacteria to mammals, has attracted much attention from both medical and cell biology fields. The LD in white adipocytes is often treated as the prototype LD, but is rather a special example, considering that its size, intracellular localization and molecular composition are vastly different from those of non-adipocyte LDs. These differences confer distinct properties on adipocyte and non-adipocyte LDs. In this article, we address the current understanding of LDs by discussing the differences between adipocyte and non-adipocyte LDs.

DOI PubMed

Role of ORPs in Sterol Transport from Plasma Membrane to ER and Lipid Droplets in Mammalian Cells

Maurice Jansen, Yuki Ohsaki, Laura Rita Rega, Robert Bittman, Vesa M. Olkkonen, Elina Ikonen

TRAFFIC ( WILEY-BLACKWELL PUBLISHING, INC ) 12 ( 2 ) 218 - 231 2011年02月 [査読有り] [国際誌]

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.

DOI PubMed

Lysosomal accumulation of mTOR is enhanced by rapamycin

Yuki Ohsaki, Michitaka Suzuki, Yuki Shinohara, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 134 ( 6 ) 537 - 544 2010年12月 [査読有り] [国際誌]

担当区分：筆頭著者 , 責任著者

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth that integrates signals from growth factors and nutrients. Recent studies have shown that an mTOR-containing complex, mTORC1, is targeted to lysosomes in the presence of amino acids and activated by Rheb GTPase resident in that compartment. In this study, we found that treatment with the mTOR inhibitors rapamycin and Torin1 significantly enhanced lysosomal accumulation of mTOR and Raptor. This phenomenon was not observed in the absence of amino acids but was restored upon addition of l-leucine or protein synthesis inhibitors. mTOR was not concentrated in autophagosomes that were induced by rapamycin. These results suggest that the lysosome harbors both active and inactive forms of mTOR in the presence of amino acids.

DOI PubMed

Lipoprotein lipase and hepatic triglyceride lipase reduce the infectivity of hepatitis C virus (HCV) through their catalytic activities on HCV-associated lipoproteins

Yuko Shimizu, Takayuki Hishiki, Kazuo Sugiyama, Kazuya Ogawa, Kenji Funami, Atsushi Kato, Yuki Ohsaki, Toyoshi Fujimoto, Hiroshi Takaku, Kunitada Shimotohno

VIROLOGY ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 407 ( 1 ) 152 - 159 2010年11月 [査読有り] [国際誌]

The effect of lipolysis by lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) on hepatitis C virus (HCV) infection was evaluated. First, medium from HuH7.5 cells bearing HCV genome replication was treated with LPL. LPL treatment led to reduced HCV infectivity, shifted HCV to higher densities, and lowered the amount of apolipoprotein E-associated HCV. The effect of endogenous HTGL secreted from HuH7.5 on HCV infectivity was next examined. Neutralization of HTGL by an anti-HTGL antibody resulted in suppression of LPL-induced reduction in infectivity of HCV-bearing medium, while knockdown of HTGL by siRNA led to increased HCV infectivity irrespective of LPL HCV in medium from HTGL knockdown cells was found in fractions with a lower density. These results indicate that changes in the nature of HCV-associated lipoproteins by LPL and/or HTGL affect HCV infectivity, suggesting that association of HCV with specific lipoproteins is important for HCV infectivity. (C) 2010 Elsevier Inc. All rights reserved.

DOI PubMed

Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Takayuki Hishiki, Yuko Shimizu, Reiri Tobita, Kazuo Sugiyama, Kazuya Ogawa, Kenji Funami, Yuki Ohsaki, Toyoshi Fujimoto, Hiroshi Takaku, Takaji Wakita, Thomas F. Baumert, Yusuke Miyanari, Kunitada Shimotohno

JOURNAL OF VIROLOGY ( AMER SOC MICROBIOLOGY ) 84 ( 22 ) 12048 - 12057 2010年11月 [査読有り] [国際誌]

Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.

DOI PubMed

A pitfall in using BODIPY dyes to label lipid droplets for fluorescence microscopy

Yuki Ohsaki, Yuki Shinohara, Michitaka Suzuki, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 133 ( 4 ) 477 - 480 2010年04月 [査読有り] [国際誌]

担当区分：筆頭著者

The lipid droplet (LD) has become a focus of intense research. Fluorescence labeling is indispensable for the cell biological analysis of the LD, and a lipophilic fluorescence dye, BODIPY 493/503, which emits bright green fluorescence has been used extensively for LD labeling. The dye is convenient for double fluorescence labeling, but we noticed that it emits red fluorescence under certain conditions, which could lead to erroneous interpretations. We propose a protocol to preclude such a possibility.

DOI PubMed

Quantitative electron microscopy shows uniform incorporation of triglycerides into existing lipid droplets

Jinglei Cheng, Akikazu Fujita, Yuki Ohsaki, Michitaka Suzuki, Yuki Shinohara, Toyoshi Fujimoto

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 132 ( 3 ) 281 - 291 2009年09月 [査読有り] [国際誌]

The lipid droplet (LD) is an organelle with a lipid ester core and a surface phospholipid monolayer. The mechanism of LD biogenesis is not well understood. The present study aimed to elucidate the LD growth process, for which we developed a new electron microscopic method that quantifies the proportion of existing and newly synthesized triglycerides in individual LDs. Our method takes advantage of the reactivity of unsaturated fatty acids and osmium tetroxide, which imparts LDs an electron density that reflects fatty acid composition. With this method, existing triglyceride-rich LDs in 3Y1 fibroblasts were observed to incorporate newly synthesized triglycerides at a highly uniform rate. This uniformity and its persistence even after microtubules were depolymerized suggest that triglycerides in fibroblasts are synthesized in the local vicinity of individual LDs and then incorporated. In contrast, LDs in 3T3-L1 adipocytes showed heterogeneity in the rate at which lipid esters were incorporated, indicating different mechanisms of LD growth in fibroblasts and adipocytes.

DOI PubMed

Biogenesis of cytoplasmic lipid droplets: From the lipid ester globule in the membrane to the visible structure

Yuki Ohsaki, Jinglei Cheng, Michitaka Suzuki, Yuki Shinohara, Akikazu Fujita, Toyoshi Fujimoto

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS ( ELSEVIER SCIENCE BV ) 1791 ( 6 ) 399 - 407 2009年06月 [査読有り] [国際誌]

担当区分：筆頭著者

The cytoplasmic lipid droplet (CLD) and very low-density lipoprotein are generated from the lipid ester synthesized in the endoplasmic reticulum. The lipid ester deposited between the two membrane leaflets is supposed to bulge toward the cytoplasm to make a nascent CLD, but its size must be below the resolution limit of conventional techniques and the detectable CLD should only form after acquisition of additional lipid esters. The CLD is different from vesicular organelles in that the internal content is highly hydrophobic and the shape is invariably spherical. Due to its unique characteristics, quantitative discordance between the surface and the volume may occur in the growth and/or involution processes of the CLD. The possibility that these processes may give rise to the structural and functional diversities of the CLD is discussed. (C) 2008 Elsevier B.V. All rights reserved.

DOI PubMed

脂質を観察する方法

藤田秋一, 大崎雄樹, 藤本豊士

日本組織細胞化学会編組織細胞化学2009 35-43 2009年

Lipid Droplet-Associated Proteins Protect Renal Tubular Cells from Fatty Acid-Induced Apoptosis

Yoshimichi Urahama, Yuki Ohsaki, Yutaka Fujita, Shoichi Maruyama, Yukio Yuzawa, Seiichi Matsuo, Toyoshi Fujimoto

AMERICAN JOURNAL OF PATHOLOGY ( ELSEVIER SCIENCE INC ) 173 ( 5 ) 1286 - 1294 2008年11月 [査読有り] [国際誌]

Proteinuria is a major cause of tubulointerstitial kidney damage, and free fatty acids bound to albumin are thought to play an important role in its pathogenesis. However, the mechanism whereby proteinuria causes tubulointerstitial damage to the kidney is unclear. Using primary human renal proximal tubular cells, we observed that albumin replete with fatty acids (rBSA) and defatted albumin (dBSA) complexed with linoleic acid (LA) induced significantly more apoptosis than did defatted albumin alone. Oxidative stress was partially involved in apoptotic induction by LA/dBSA but not by rBSA. Administration of fatty acid-bound BSA increased the number of lipid droplets (LDs) and the LD-associated proteins, adipocyte differentiation-related protein and TIP47. LDs are organelles that store esterified fatty acids, and the LD-associated proteins are presumed to facilitate LD formation. Knockdown of adipocyte differentiation-related protein or TIP47 by RNA interference enhanced induction of apoptosis by both rBSA and LA/dBSA. Apoptotic induction was observed similarly when either rBSA or LA/dBSA was applied to only the apical surfaces of polarized LLC-PK1 cells. The present results suggest that LDs and LD-associated proteins have protective effects against apoptosis induced by fatty acid-bound albumin by sequestering free fatty acids. Therapeutic manipulation of these LD-associated proteins could aid in the amelioration of nephritic diseases. (Anti J Pathol 2008, 173:1286-1294; DOI: 10.2353/ajpath.2008.080137)

DOI PubMed

Lipid droplets: a classic organelle with new outfits

Toyoshi Fujimoto, Yuki Ohsaki, Jinglei Cheng, Michitaka Suzuki, Yuki Shinohara

HISTOCHEMISTRY AND CELL BIOLOGY ( SPRINGER ) 130 ( 2 ) 263 - 279 2008年08月 [査読有り] [国際誌]

Lipid droplets are depots of neutral lipids that exist virtually in any kind of cell. Recent studies have revealed that the lipid droplet is not a mere lipid blob, but a major contributor not only to lipid homeostasis but also to diverse cellular functions. Because of the unique structure as well as the functional importance in relation to obesity, steatosis, and other prevailing diseases, the lipid droplet is now reborn as a brand new organelle, attracting interests from researchers of many disciplines.

DOI PubMed

*Lipid droplets are arrested in the ER membrane by tight binding of lipidated apolipoprotein B-100

Ohsaki, Y. Cheng, J. Suzuki, M. Fujita, A. Fujimoto, T

J Cell Sci 121 ( 14 ) 2415-22 - 22 2008年06月 [査読有り] [国際誌]

担当区分：筆頭著者

Apolipoprotein B-100 (ApoB) is a major component of very-low-density lipoproteins, and is deposited in a region around lipid droplets (LDs) called the ;ApoB-crescent'. The ApoB-crescent is thought to be related to ApoB degradation because it drastically increases when proteasome or autophagy is inhibited. In the present study, we found that ApoB-crescents were significantly reduced when ApoB lipidation was suppressed by either the inhibition or knockdown of the microsomal triglyceride-transfer protein. By contrast, ApoB-crescents increased under conditions that are presumed to cause lipidated ApoB abnormalities in secretory compartments. By electron microscopic analyses, we identified the ApoB-crescent as a thin cholesterol-rich ER cistern fused to an LD, and - topologically - this structure is equivalent to a lipid-ester globule between the two leaflets of the ER membrane. ApoB localized in the thin cisternal lumen, and its binding to LDs was resistant to alkaline treatment. Overexpression of ADRP or TIP47 suppressed the increase in the number of ApoB-crescents, whereas knockdown of these proteins had the opposite effect. From these results, we inferred that the ApoB-crescent is f

DOI PubMed

脂肪滴形成のメカニズム

藤本豊士, 大崎雄樹, 鈴木倫毅

膜 MEMBRANE 33 ( 1 ) 17-23 2008年01月 [招待有り]

Platelet-derived growth factor signals play critical roles in differentiation of longitudinal smooth muscle cells in mouse embryonic gut.

Kurahashi, M, Niwa, Y, Cheng, J, Ohsaki, Y, Fujita, A, Goto, H, Fujimoto, Y, Torihashi, S

Neurogastroenterol Motil. 20 ( 5 ) 521-531 2008年01月 [査読有り]

In the development of mouse gut, longitudinal smooth muscle cells (LMC) and interstitial cells of Cajal (ICC) originate from common precursor cells expressing c-Kit. Recently, some gastrointestinal stromal tumours, which develop from smooth muscle layers of the gut and have gain-of-function mutations of c-kit, have been reported to have gain-of-function mutations of platelet-derived growth factor (PDGF) receptor alpha gene. These data raise the possibility that PDGF signalling might be involved in the development of LMC. Therefore, we examined the expression pattern of the PDGF signal family of embryonic gut by immunohistochemistry and in situ hybridization, and investigated the role of PDGF signals in the development of smooth muscle layers in mouse gut using a new organ culture system. During embryonic development, the circular muscle layer expressed PDGF-A, enteric neurons expressed PDGF-B and common precursor cells of LMC and ICC expressed both PDGF receptor alpha and beta. The selective PDGF receptor inhibitor AG1295 suppressed the differentiation of LMC in gut explants. We conclude that PDGF signals play critical roles in the differentiation of LMC in mouse embryonic gut.

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