2017-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/702180摘要：細胞分裂是指母細胞分裂成兩個子細胞的過程。在真核細胞有兩種分裂方式，有絲分裂及減數分裂。在細胞骨架中的微管對於細胞分裂具有相當的重要性。微管是由-微管蛋白及-微管蛋白之二聚體所組成的長條的中空管狀結構。微管的聚合及組裝是依靠著中心體(或稱為微管組合中心)來達成。在細胞週期中，微管在中心體的聚合活性是動態的，伴隨著中心體大小及其他結合蛋白質的調節而有所變化。在細胞週期的G1 期微管聚合是最弱的，而在有絲分裂期間達到最高。lmbrd1 基因主要表現兩個具功能性的蛋白質─LMBD1 及NESI 蛋白質；兩者皆含有一個推測的二部核位訊息及一個-輔肌動蛋白型態的肌動蛋白結合位。先前的研究顯示位於細胞核的NESI 蛋白質參與大型D 型肝炎抗原的核輸出，而位於細胞表面之LMBD1 蛋白質除了可以做為接合者調控clathrin 所媒介之胰島素受體的內吞作用，也具有幫助維生素B12 由溶酶體輸出至細胞質的功能。最近，經由蛋白質共沉澱和液態色層配合質譜分析，我們找到並鑑定出LMBD1 之結合蛋白質，包括-tubulin complex proteins 2 (GCP2)、nuclear mitotic apparatus protein (NuMA) 和nucleophosmin (NPM)。更進一步研究發現，LMBD1 蛋白質的分佈在有絲分裂期間會變成絲狀型態。而當lmbrd1 基因表現量下降時，在單一細胞中會出現多微管組合中心及多核的現象。未來研究的興趣是想了解LMBD1 蛋白質如何直接參與有絲分裂的過程及其中的機制為何。為了回答以上的問題，本研究的主要目標說明如下：一、 利用專一性抗體之阻斷實驗研究LMBD1 蛋白質在細胞週期中心體複製及胞質分裂之功能二、 鑑定與-或-微管蛋白結合之LMBD1 蛋白質的重要胺基酸殘基三、 利用體外及細胞培養系統分析LMBD1 蛋白質參與微管聚合之功能角色四、 探討與LMBD1 蛋白質結合之GCP2、NuMA 及NPM，在有絲分裂期間對紡錘體和中心體形成及穩定是否具重要性五、 鑑定與絲狀肌動蛋白結合之LMBD1 蛋白質的重要胺基酸殘基六、 分析LMBD1 蛋白質與絲狀肌動蛋白結合在胞質分裂之功能角色七、 研究LMBD1 蛋白質在細胞有絲分裂期間如何由細胞表面和內質網逆行運輸至細胞核的機制<br> Abstract: Cell division is the process by which a parental cell divides into two daughter cells. In eukaryotic cells,there are two types of cell division: mitosis and meiosis. The microtubule network is a part of thecytoskeleton that plays an essential role in cell division. Microtubules are long, hollow cylinders consistingof polymerized heterodimers of - and -tubuln. The nucleation and organization of microtubule depend onthe centrosome that is also known as the microtubule-organizing center (MTOC). The microtubule-nucleatingactivity at centrosomes is dynamic during the cell cycle, being the weakest in the G1 phase and the strongestduring mitosis. This change is concurrent with alterations in centrosomal volume and other associatedproteins. lmbrd1 gene encodes two major functional proteins, LMBD1 and NESI. Both proteins contain aputative bipartite nuclear localization signal and a -actinin-type actin binding site. Previous studiesdemonstrated that the nucleus-localized NESI protein is involved in the nuclear export of the large hepatitisdelta antigen, whereas the LMBD1 protein functions as an adaptor for clathrin-mediated endocytosis ofinsulin receptor on the cell surface and is a putative lysosomal exporter of vitamin B12. Recently, weidentified several LMBD1-associated proteins by coimmunoprecipitation and LC-tandem MS/MS analysis.These include -tubulin complex proteins 2 (GCP2), nuclear mitotic apparatus protein (NuMA) andnucleophosmin (NPM). In addition, the distribution of LMBD1 protein became filamentous profiles duringmitosis. Furthermore, lmbrd1 knockdown caused multiple MTOC and nuclei in a single cell. It would beinteresting to study how LMBD1 protein directly involves in the processes of cell mitosis and themechanisms behind. To address these questions, specific aims of this study are described as follows.1. To study the functions of LMBD1 protein involved in the regulation of centrosome duplication andcytokinesis by antibody blocking experiments2. To identify critical amino acids of LMBD1 protein required for the interaction with - or-tubulin3. To examine the functional roles of LMBD1 protein involved in microtubule polymerization4. To investigate whether the binding of LMBD1 to GCP2, NuMA and NPM is required for spindle andcentrosome formation and stabilization during cell mitosis5. To identify critical amino acids of LMBD1 protein required for the interaction with F-actin6. To analyze the functional roles of the interaction between LMBD1 and F-actin in cytokinesis7. To elucidate how LMBD1 protein undergoes retrograde transport from the cell surface and ER to thenucleus during cell mitosis