{"created":"2023-06-20T13:20:57.155870+00:00","id":1019,"links":{},"metadata":{"_buckets":{"deposit":"8d529481-c756-476d-9daa-386938a317f3"},"_deposit":{"created_by":1,"id":"1019","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1019"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001019","sets":["2:430:20"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"木村, 俊介"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"キムラ, シュンスケ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2006-03-24"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"　　　Autophagy is an intracellular bulk degradation system in which cytoplasmic components, such as proteins or organelles, are directed to the lytic compartment, lysosome or vacuole, by a membrane-mediated process. In the autophagic process, part of the cytosol or organelles is first enclosed by double-or multiple-membrane structures called 'autophagosome'.Eventually, the autophagosomes fuse with lysosomes and the sequestered contents and the inner membranes are degraded by lysosomal hydrolases. Recent studies revealed that autophagy is crucial for various physiological processes.  However,the mechanism of autophagy is still left to ve solved. For example, molecular mechanisms underlying the targeting process of autophagosome to lysosome have been unclear.<br />　　　In the present study,Ishowed that a movement of autophagosome along microtubules is required for its targeting to lysosomes, and that IL3, an autophagosome binding protein,is a key molecule in assembly of autophagosome, dynein motor, and microtubules. To study the dynamics of the autophagic process, I have developed a microscopic system for real-time observation of autophagosomes. Time-lapese observation using this system indicated that autophagosomes labeled with GFL-LC3 moved in the cytosol, whereas pre-autophagosomal isolation membranes labeled with GFP-Atg5 scarcely moved. Autophagosome showed both a rapid long-range motion and random short-range motion. The average speed of autophagosome is 5.3±2.6μm/sec. Nocodazole inhibited the long rantge motion almost completely,shereas other cytoskeletal inhibitors did not inhibit this movement. Immunofluorescent microscopic study showed that endogenous LC3 arranged along the tracks of microtubules. These findings indicate that autophagosome moves along microtubules.<br />　　　Cytoplasmic dynein and kinesin are microtubule-dependent motor proteins responsible for transport of a variety of organelles and vesicles. Dynein drives the cargo to minus-end of microtubules, while kinesin drives to plus-end of microtubules. Considering that autophagosomes were often directed to the perinuclear region, the minus end of microtubules, it seemed likely that dynein are involved in the autophagosome movement. Overexpression of dynamitin,which is known to disrupt dynein-and dynactin-dependent organelle movement, inhibited autophagosome movements. In addition, endogenous LC3 colocalized with dynein-dynactin complex. Coimmunoprecipitation and GST pull-down assay showed that LC3 directly interacts with the intermediate chain of cytoplasmic dynein, which is a subunit of dynein motor comples. These results indicate that autophagosomes are linked with the dynein-dynactin comples through the interaction between LC3 and dynein intermediate chain.<br />　　　To clarify the involvement of LC3 in autophagosome movement, microinjection experiments of anti-LC3 antibodies were performed. While injection of neither anti-GFP nor control IgG had effect on autophagosome movement injection of anti-LC3 antibodies inhibited the movement. Moreover the anti-LC3 pepide antibodies raised against the N-terminal 1-15 residues, which is known as a microtubule binding region,also inhibited the movement. THese results suggest that LC3 is involved in the autophagosome movement, and interaction of micro tubule with LC3 is important for this movement.<br />　　　To assess whether microtubule-dependent movement is neccessary for the autophagic delivery of cytoplasmic components to lysosome, I established a novel transport assay using fluorescence recovery after photobleaching (FRAP). Using this FRAP assay, the transport and fusion of autophagosomes to lysosomes were measured. The transport and fusion were significantly inhibited by the injection of anti-LC3 antibody, whereas control IgG had no effects on the transport and fusion. THis FRAP assay showed that microtubule-dependent movement is necessary for targeting of peripherally-formed autophagosomes to lysosomes localizing to perinuclear region.<br />　　　From these results, I concluded that 1) autophagosome movement is dependent on icrotubule and dynein/dynactin motor complex, 2) this movement is necessary for targeting of autophagosomes to lysosomes in mammalian cells,and 3) LC3 plays an important role in assembly of the transport machinery. LC3 may link dynein motor. microtubule and autophagosome. The present study is the first report elucidating the function of LC3 in autophagy in mammalian cells.","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第956号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"18 遺伝学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2005"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"KIMURA, Shunsuke","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲956_要旨.pdf","filesize":[{"value":"260.6 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/1019/files/甲956_要旨.pdf"},"version_id":"b29c81d2-8c48-4099-a1f8-70f03164dc26"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Transition Metal-Catalyzed Asymmetric Allylic Etherifications","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Transition Metal-Catalyzed Asymmetric Allylic Etherifications"},{"subitem_title":"Transition Metal-Catalyzed Asymmetric Allylic Etherifications","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["20"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1019","relation_version_is_last":true,"title":["Transition Metal-Catalyzed Asymmetric Allylic Etherifications"],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T15:54:28.619070+00:00"}