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摘要
細胞週期是細胞生長並且分裂成子細胞的動態過程，細胞骨架中的微管在細胞分裂中扮演了重要的角色。微管是由α-微管以及β-微管形成二聚體，進而聚合形成長條狀空心圓柱體。在細胞週期的過程中，微管的聚合及組裝依靠著中心體 (亦被稱作微管組合中心) ，伴隨著中心體大小的改變以及相關蛋白質的調控，微管於中心體聚合的能力在G1期最低，而在有絲分裂期達到最高。lmbrd1基因主要會表達出兩種功能性異體蛋白質－LMBD1與NESI蛋白質。本實驗室先前的研究證明NESI蛋白質與D型肝炎病毒大型delta抗原上的proline-rich細胞核輸出訊號結合並協助其輸出至細胞質，實驗室之前研究LMBD1可調控胰島素受體的內吞作用，研究也指出LMBD1蛋白質可位於溶酶體膜上並且幫助維生素B12輸出至細胞質。由於兩者在細胞內分佈位置不同，顯示LMBD1與NESI蛋白質仍可能有著尚未被發現的功能。本實驗室以LMBD1的抗體進行免疫沉澱將可能與LMBD1蛋白質交互作用的蛋白質純化出來，再透過質譜儀進行分析，發現此蛋白質的複合體中有一些細胞核蛋白質，而微管蛋白質亦包含於其中。此外，LMBD1與NESI蛋白質在細胞分裂期的表現量豐富，並且呈現類紡錘絲結構的分佈；暗示lmbrd1基因可能在細胞分裂具有功能性。本研究發現LMBD1與NESI蛋白質在不同細胞株的細胞分裂期皆會於細胞核與α-微管共位，而且是藉由此二異體蛋白質共同擁有的羧基端來與α-微管形成蛋白質複合體。利用VSV-G pseudotyped lentivirus表現lmbrd1 shRNA抑制lmbrd1 mRNA表現量時，偵測到數個微管組合中心以及細胞核出現在單一細胞的現象。同時也發現lmbrd1 mRNA表現被抑制時，會促進細胞微管在早期的聚合能力。當以流式細胞技術分析細胞在各細胞週期階段的比例時，發現lmbrd1 mRNA表現被抑制的細胞能夠較快地離開G2/M期。綜合以上結果，推測lmbrd1基因可能透過影響微管紡錘絲的形成，有效調控細胞分裂的速度，以完成正確的細胞分裂週期。
關鍵字
有絲分裂並列摘要
The cell cycle is the series of events for cells to grow and divide to daughter cells. The microtubule network is a part of the cytoskeleton that plays an essential role in functional cell division. Microtubules are long, hollow cylinders composed of polymerized α- and β-tubulin dimers. Microtubule nucleation and organization depend on the centrosome that is also known as the microtubule-organizing center (MTOC). During the cell cycle, the microtubule-nucleating activity at centrosomes is dynamic being the weakest in the G1 phase and the strongest during mitosis. This change is concurrent with alterations in centrosomal volume and other associated proteins. lmbrd1 gene encodes two major functional proteins, LMBD1 and NESI. Previous studies demonstrated that the NESI protein is involved in the nuclear export of the large hepatitis delta antigen bearing a proline-rich nuclear export signal, whereas the LMBD1 protein functions as a regulator of insulin receptor endocytosis and is a putative lysosomal exporter of vitamin B12. With different subcellular distributions, LMBD1 and NESI might possess functions that are not yet identified. By performing immunoprecipitation followed by LC-MS/MS analysis, our group has previously identified several nuclear proteins as well as tubulin that coimmunoprecipitated with LMBD1/NESI protein. In addition, abundant LMBD1 and NESI expression were found in the mitotic phase of cell cycle. Furthermore, distribution of LMBD1/NESI protein became filamentous profile during cell mitosis, indicating their possible functions in cell mitosis. In this study, LMBD1 and NESI were found to colocalized with α-tubulin mainly in the nucleus of various cell types during mitosis. In addition, LMBD1 and NESI proteins formed complexes with α-tubulin through their identical C-terminal domains. Knockdown of lmbrd1 by a recombinant VSV-G pseudotyped lentivirus expressing lmbrd1 shRNA caused multi-MTOC and multi-nuclei in a single cell. Furthermore, lmbrd1 knockdown facilitated microtubule regrowth in the early stage. Results from flow cytometry analysis indicated that lmbrd1 knockdown accelerated the progression of G2/M phase. Collectively, lmbrd1 gene might function as a regulator in cell division, which tightly regulates the formation of microtubule network in order to fulfill the accuracy of cell cycle.
並列關鍵字
lmbrd1參考文獻
延伸閱讀
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- 顏建平（2011）。抑制細胞凋亡蛋白Mcl-1與Bcl-xL在Wnt1+細胞譜系之功能探討〔碩士論文，臺北醫學大學〕。華藝線上圖書館。https://doi.org/10.6831/TMU.2011.00050
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- 林雨萱（2016）。LMBD1蛋白質參與神經突脊形成之作用機制〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU201603165
- 賴怡廷（2019）。The mechanism of LMBD1 protein involved in dopamine transport〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU201903117