Immunoblotting Cell lysates were treated with PNGaseF, separated by 10 % SDS-PAGE under reducing conditions, and blotted onto a polyvinylidenedifluori

Size: px

Start display at page:

Download "Immunoblotting Cell lysates were treated with PNGaseF, separated by 10 % SDS-PAGE under reducing conditions, and blotted onto a polyvinylidenedifluori"

ひでかかたづ
5 years ago
Views:

1 論文提出者氏名論文審査担当者主査副査学位論文の内容の要旨柴田俊一 RAJAPAKSHE MUDIYANSELAGE Anupama Rasadari Rajapakshe 三浦雅彦篠村多摩之論文題目 Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells ( 論文の内容の要旨 ) Introduction The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance and is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are major protein components of the lysosomal membrane. They are considered to act as a barrier to protect the structural integrity of lysosomal membranes from the lytic luminal environment, by forming a glycocalyx. However, recent reports suggest that LAMPs probably perform a function beyond the stabilization of lysosomes. Lysosomes are important in the bone-forming activity of osteoblasts. However, the roles of LAMPs in osteoblasts have not been elucidated. Thus this study focused in the role of LAMPs in the pre-osteoblastic cell line MC3T3-E1 and the osteocytic cell line MLO-Y4. Materials and methods Cell culture The mouse pre-osteoblastic cell line (MC3T3-E1) and the mouse osteocyte-like cell line (MLO-Y4) were maintained in α-modified MEM, supplemented with either 10% fetal bovine serum or a combination of 5% fetal bovine serum and 5% newborn calf serum, respectively, 4 mm L-glutamine, L-penicillin (50 U/mL), and streptomycin (50 µg/ml), at 37 in a 5% CO2 atmosphere. MLO-Y4 cells were cultured on collagen-coated plates. Antibodies Rabbit polyclonal antibodies to LAMP-1 (ab24170, Abcam) and LAMP-2 (AP07767PU-N, Acris) were used for immunoblotting, and rat monoclonal antibodies to LAMP-1 (1D4B, Developmental Studies Hybridoma Bank) and LAMP-2 (ABL93, Developmental Studies Hybridoma Bank) were used for immunocytochemistry. Rabbit polyclonal antibodies to α -tubulin (ab4074, Abcam), golgin A5, GOLGA5 (HPA000992, Sigma-Aldrich), and protein disulfide isomerase family A, member 3, PDIA3 (HPA003230, Sigma-Aldrich) were also used. To detect the α-subunit of NAD + -dependent isocitrate dehydrogenase, ICDH, a rabbit monoclonal antibody (ab124797, Abcam) was used

2 Immunoblotting Cell lysates were treated with PNGaseF, separated by 10 % SDS-PAGE under reducing conditions, and blotted onto a polyvinylidenedifluoride membrane. The blots were then probed with primary antibodies, followed by the corresponding horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was detected by the ECL reaction. Immunocytochemistry MC3T3-E1 and MLO-Y4 cells were cultured in 12 well plates on glass coverslips, without or with a collagen coating, respectively. After an incubation with Hoechst for 30 min, the cells were fixed with 95% methanol for 5 min at room temperature, blocked with 1% bovine serum albumin in PBS, and stained with primary antibodies to LAMP-1 (1D4B, 1.6 µg/ml) or LAMP-2 (ABL93, 1.2 µg/ml) with ICDH (1:100), followed by secondary antibodies, Alexa Fluor 488-conjugated anti-rabbit antibody (1:200) and Alexa Fluor 546-conjugated anti-rat antibody (1:200). For the staining with the antibodies to tubulin (1:200), GOLGA5 (1:1,000) or PDIA3 (1:600), the cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS-glycine, and permeabilized with 0.1% Triton X-100. An Alexa Fluor 488-conjugated anti-rabbit antibody (1:100) or Alexa Fluor 555-conjugated anti-rabbit antibody (1:100) was used. Alexa Fluor 555-conjugated phalloidin was used at a 1:100 dilution. Images were acquired using a 100x oil-objective lens on a BZ9000 microscope (KEYENCE). Manipulation of intracellular ph Cells were incubated for 30 min at 37 in 130 mmkcl, 10 mmnacl, 1 mm MgSO4, 10 mm Na-MOPS adjusted to ph 6.5, 7.4 or 9.0, and 10 µg/ml nigericin. The changes in the intracellular ph were measured using the ph-sensitive fluorescent dye BCECF-AM. sirna treatment Cells were seeded on glass coverslips in a 12 well plate for immunocytochemistry or in a 6 cm dish for immunoblotting, one day before transfection. Cells were transfected with either the All-Stars Negative Control sirna ( , Qiagen) or the GeneSolutionsiRNA ( , Qiagen) targeting mouse LAMP-1 (SI , SI and SI ) or LAMP-2 (SI and SI ) using the HiPerFect transfection reagent. Image analysis using Field Particle Vector Analysis The Field Particle Vector Analysis program was developed to evaluate the positions of mitochondria or lysosomes in each cell. The particles representing mitochondria (stained with the anti-icdh antibody) or lysosomes (stained with the anti-lamp-1 or LAMP-2 antibody) were identified within acquired images. Then, the shortest distance between the center of each particle and the nuclear boundary was computed, and the integrated area was plotted versus the distance at 1 µm intervals. The fraction of the area within 3 µm from the nucleus was defined as the perinuclear localization index in this study. Statistical analysis Differences between mean values were assessed by the unpaired two-sided Student s t test or by one-way ANOVA followed by Dunnett s post-hoc comparisons tests, using KaleidaGraph. The statistical significance was set at Pless than 0.01 or Results Intracellular distributions of lysosomes and mitochondria in MC3T3-E1 cells The staining with antibodies against NAD + -dependent isocitrate dehydrogenase (ICDH) revealed the characteristic network structure of mitochondria. Both LAMPs and ICDH were - 2 -

3 observed near the nucleus, although they were not completely co-localized with each other. The localizations of LAMPs and ICDH in MC3T3-E1 cells found to be more diffuse than those in MLO-Y4 cells. The bidirectional transport of lysosomes and mitochondria is similarly regulated under different conditions in MC3T3-E1 cells The lysosomes and mitochondria were dispersed in the cytoplasm in the presence of nocodazole, whereas they accumulated in the perinuclear region after the treatment with cytochalasin D, suggesting that intracellular localization of both lysosomes and mitochondria in MC3T3-E1 cells depends on microtubules and actin filaments. Intracellular acidification caused lysosome dispersion, whereas intracellular alkalization led to lysosome accumulation in the perinuclear region. However, the extent of dispersion varied among the cells. Image analysis with the developed software revealed that the fraction of lysosomes proximal to the nucleus (within 3 µm) decreased when the intracellular ph was lowered from 7.4 to 6.5, and increased when the intracellular ph was elevated to 9.0. Partial fragmentation of mitochondria was observed in the presence of nigericin. The effect of acidification on the mitochondrial positioning was less pronounced than that on lysosomes. However, the alkalization also promoted the perinuclear accumulation of mitochondria. Downregulation of LAMP-2 promotes the perinuclear localization of mitochondria in MC3T3-E1 cells After the treatment with the LAMP-2 sirna, the amount of LAMP-2 was appreciably decreased, without affecting LAMP-1 expression. As for the mitochondrial positioning, cells with intense perinuclear accumulation of ICDH were more frequently observed among the LAMP-2 sirna-treated cells. Consistent with the previous study, the positions of the Golgi apparatus and the endoplasmic reticulum did not seem to be affected by the LAMP-2 sirna. Downregulation of LAMP-1 or both LAMP-1 and LAMP-2 promotes the perinuclear positioning of mitochondria in MC3T3-E1 cells The treatment with the LAMP-1 sirna decreased the amount of LAMP-1, but not that of LAMP-2. The perinuclear localization of ICDH was strongly increased by the LAMP-1 sirna, the number of cells in which the perinuclear localization of ICDH exceeded 80% was significantly increased. As in the treatment with the LAMP-2 sirna, the locations of the Golgi apparatus and the endoplasmic reticulum were not affected. Furthermore, in the presence of both the LAMP-1 and LAMP-2 sirnas, the perinuclear accumulation of ICDH was more clearly observed. Downregulation of LAMPs does not affect the localization of mitochondria in MLO-Y4 cells In MLO-Y4 cells the lysosomes and mitochondria were located closer to the nucleus, as compared to those in MC3T3-E1 cells. Mitochondria were also found within some dendrites. In contrast to the results obtained in MC3T3-E1 cells, the location of the mitochondria was not affected by either the LAMP-1 or LAMP-2 sirna. Discussion The present study showed that downregulation of LAMPs affects the positioning of mitochondria in MC3T3-E1 cells. Since the LAMPs are not directly associated with mitochondria, obtained results suggest the emerging role of LAMPs in interorganellar coordination, raising the possibility that LAMPs affect the balance between the motors with opposite polarity within the transport machinery of other organelles. A prominent feature of the LAMPs is their abundance, resulting in the generation of a glycocalyx. The lysosomal membranes are likely crowded with LAMPs, allowing the close contact - 3 -

4 and transient oligomerization of their luminal domains. LAMPs may thus stabilize the architecture or serve as a scaffold of the motor protein complex. Lysosomes are suggested to act as reservoirs of dynein. Thus, the deficiency of LAMPs in MC3T3-E1 cells may indirectly alter the availability of dynein to mitochondria, promoting the minus-end directed transport of mitochondria. In this context, the effect of LAMPs should depend on the distribution of dynein among organelles, and would vary under different conditions. Thus, the lack of the effect of sirna of LAMPs in MLO-Y4 cells can be explained by the intrinsic enrichment of dynein in most of the mitochondria. Mitochondria likely play a role in supplying calcium phosphate for matrix vesicle formation. This study showed the accumulation of mitochondria after the osteoblastic differentiation of MC3T3-E1 cells suggesting that LAMPs may function to maintain mitochondrial isolation until osteoblastic differentiation. The results raise the possibility that LAMPs contribute to coordinate the positioning of mitochondria in MC3T3-E 1 cells. Further studies will be necessary to investigate the correlation between LAMPs and motor protein distribution. ( 和文による要約 ) Lysosome associated membrane proteins ( 以下 LAMPs と略記 ) はリソソーム膜に豊富に存在する糖タンパク質である発見当初 LAMPs はリソソーム膜を裏打ちする糖衣を形成しリソソーム内部にある種々の加水分解酵素からリソソーム膜を保護すると考えられていたしかし LAMPs 欠損細胞においてもリソソームの構造は形態的にも機能的にも保持されており一方 LAMPs が欠損するとオートファジーやファゴサイトーシスの過程が障害されることが明らかになった LAMPs はリソソームとファゴソームの細胞内での位置を制御することにより両者を効果的に融合させることが示唆されている骨芽細胞においてリソソームとミトコンドリアは骨基質の形成に関与するそこで本研究では骨芽細胞における LAMPs の役割を明らかにする目的で前骨芽細胞由来細胞株 MC3T3-E1 および骨細胞由来細胞株 MLO-Y4 細胞を用いて sirna による LAMPs 発現低下によるミトコンドリアの細胞内での位置決めへの影響を調べたオルガネラの細胞内での位置は微小管に沿った輸送に依存しプラス方向 ( 核近傍の微小管重合中心に向かう方向 ) とマイナス方向 ( 細胞膜に向かう方向 ) の輸送のバランスにより決定されるそこで顕微鏡観察の画像から得られる細胞内でのオルガネラの位置の情報を定量的に評価するために個々の細胞におけるオルガネラの核からの距離の分布を計算するプログラムを共同研究により開発した MC3T3-E1 細胞において LAMP-1, LAMP-2 およびその両者の sirna による発現低下はミトコンドリアの核近傍への集積を増加させた LAMP-1 の発現低下の効果の方が LAMP-2 低下の効果よりも顕著であったこれに対して MC3T3-E1 細胞の場合よりもミトコンドリアが核近傍に多くかつ樹状突起部分にも存在する MLO-Y4 細胞では LAMP-1, LAMP-2 の sirna によるミトコンドリアの位置の変化はみられなかった本研究の結果は LAMPs がミトコンドリアの位置決めに関与することを示唆する初めての報告であるリソソームはオートファジーファゴサイトーシスに必須のオルガネラでありリソソーム膜に存在する LAMPs の役割についての新知見はリソソームが関与する病態 ( リソソーム病歯周病神経変性疾患など ) の理解につながることが期待される - 4 -

5 論文審査の要旨および担当者報告番号甲第 4782 号 RAJAPAKSHE MUDIYANSELAGE Anupama Rasadari Rajapakshe 論文審査担当者主査副査柴田俊一三浦雅彦篠村多摩之 ( 論文審査の要旨 ) リソソームとミトコンドリアはそれぞれ細胞の物質代謝エネルギー代謝に重要な役割を果たす細胞内小器官であるこれらの細胞内小器官は微小管に沿って細胞内を輸送され局在する位置は細胞の置かれた環境 ( 栄養源の獲得状況など ) に依存するしかし両者の細胞内での位置決めを統合的に制御するしくみの有無については明らかになっていなかった Lysosome-associated membrane proteins ( 以下 LAMPs と略記 ) はリソソーム膜に豊富に存在する糖タンパク質である LAMPs が欠損するとオートファジーやファゴサイトーシスの過程が障害される LAMPs 欠損マウスの胎児に由来する線維芽細胞 (MEF) を用いた実験では LAMPs はリソソームとファゴソームの微小管に沿ったマイナス方向への輸送に必要であることが報告されているマイナス方向への輸送によりリソソームとファゴソームは核近傍に集積して効率よく融合するため LAMPs を欠損した細胞 (MEF) におけるファゴサイトーシスの障害は輸送への影響によって説明できる骨芽細胞においてリソソームとミトコンドリアは基本的な役割に加えて骨基質の形成に特化した役割も担っているそこで Rajapakshe は LAMPs による骨芽細胞のミトコンドリアの細胞内局在の制御を検討する目的で実験をおこなった方法としては前骨芽細胞由来細胞株 MC3T3-E1 および骨細胞由来細胞株 MLO-Y4 に LAMP-1, LAMP-2 の sirna を導入したイムノブロットにより LAMP-1, LAMP-2 のタンパク質レベルでの低下を確認した細胞を固定染色し細胞内小器官の局在を観察し画像解析により LAMPs 発現低下によるミトコンドリアの細胞内での局在への影響を調べた顕微鏡観察の画像から得られる細胞内でのオルガネラの位置の情報を定量的に評価するために個々の細胞におけるオルガネラの核からの距離の分布を計算するプログラムを共同研究により開発した得られた結果の概要は以下の通りである 1.MC3T3-E1 細胞において LAMP-1, LAMP-2 およびその両者の sirna による発現低下はミトコンドリアの核近傍への集積を増加させた 2. LAMP-1 の発現低下の効果の方が LAMP-2 低下の効果よりも顕著であった 3. LO-Y4 細胞では MC3T3-E1 細胞の場合よりもミトコンドリアが核近傍に多くかつ樹状突起部分にも存在した 4. MLO-Y4 細胞では LAMP-1, LAMP-2 の sirna によるミトコンドリアの位置の変化はみられなかった本研究の結果は従来リソソームとファゴソームの機能の制御に関与するとされていた LAMPs がミトコンドリアの位置決めにも関与することを示唆する初めての報告である骨芽細胞のミトコンドリアは基質小胞へのリン酸化カルシウム供給に関与することが示唆されていることから MC3T3-E1 細胞では LAMPs が分化誘導前のミトコンドリアの核近傍への集積 ( 小胞体からミトコンドリアへのカルシウムの受け渡しが増加する状況 ) を抑制する可能性が考えられるこのような LAMPs の役割についての新知見は細胞生物学の発展に大いに寄与するものであるさらにリソソームはオートファジーファゴサイトーシスに必須のオルガネラでありそれが関与する疾患も多数知られている ( リソソーム病歯周病神経変性疾患など ) リソソーム膜に存在する LAMPs の新しい機能が明らかになったことはこれらの疾患に見られる病態の理解がさらに深まる事も期待され今後の歯学の基礎および臨床の発展に寄与するところも極めて大きいと考えられるしたがって本論文は博士 ( 歯学 ) の学位を申請するに十分値するものと認められた (1)

_念3）医療2009_夏.indd

_念3）医療2009_夏.indd Evaluation of the Social Benefits of the Regional Medical System Based on Land Price Information -A Hedonic Valuation of the Sense of Relief Provided by Health Care Facilities- Takuma Sugahara Ph.D. Abstract