LMBD1 蛋白質經由調控微管組裝參與細胞有絲分裂

LMBD1 protein participates in cell mitosis by regulating microtubule assembly

孫樵隱(Chiao-Yin Sun)

指導教授 ： 張明富

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2021年)

https://doi.org/10.6342/NTU202102028

摘要

先前研究已證明LMBD1可調節細胞表面胰島素受體的內吞作用並介導鈷胺素 (cobalamin) 從溶酶體向細胞質的輸出，但對其在有絲分裂中的功能知之甚少。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。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。在這項研究中，相互作用組 (interactome) 分析數據顯示 LMBD1 參與細胞骨架調節。免疫沉澱實驗亦證明了 LMBD1 與微管蛋白的關聯。此外，LMBD1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。免疫螢光染色還顯示 LMBD1 與微管在間期和有絲分裂細胞中有共位的現象。微管組裝的體外實驗發現 LMBD1 在體外特異性地加速微管組裝動力學並拮抗長春鹼 (vinblastine) 的微管破壞作用。此外，LMBRD1 敲低(knockdown) 會損害有絲分裂紡錘體的形成，抑制微管蛋白聚合，並減少與有絲分裂相關的微管蛋白乙醯化。而 LMBD1 蛋白質的異位表達可以逆轉乙醯化減少。這些結果顯示 LMBD1 蛋白質穩定微管中間體。此外，來自 Lmbrd1 雜合基因敲除 (Lmbrd1+/-) 小鼠的胚胎成纖維細胞在微管形成、有絲分裂和細胞生長方面表現出異常。總之，LMBD1 在調節微管組裝方面起著關鍵作用，這對細胞有絲分裂過程至關重要。

關鍵字

LMBRD1 gene ； LMBD1 蛋白質 ； 微管組裝 ； 有絲分裂

並列摘要

LMBD1 was previously demonstrated to regulate the endocytosis of insulin receptor on cell surface and to mediate the export of cobalamin from the lysosomes to cytosol, but little is known about its function in mitosis. In this study, interactome analysis data indicate that LMBD1 is involved in cytoskeleton regulation. Both immunoprecipitation and GST pulldown assays demonstrated association of LMBD1 with tubulin. Immunofluorescence staining also showed the colocalization of LMBD1 with microtubule in both interphase and mitotic cells. LMBD1 specifically accelerates microtubule assembly dynamics in vitro and antagonizes the microtubule-disruptive effect of vinblastine. In addition, LMBRD1-knockdown impairs mitotic spindle formation, inhibits tubulin polymerization, and diminishes the mitosis-associated tubulin acetylation. The reduced acetylation can be reversed by ectopic expression of LMBD1 protein. These results suggest that LMBD1 protein stabilizes microtubule intermediates. Furthermore, embryonic fibroblasts derived from Lmbrd1 heterozygous knockout mice showed abnormality in microtubule formation, mitosis, and cell growth. Taken together, LMBD1 plays a pivotal role in regulating microtubule assembly that is essential for the process of cell mitosis.

並列關鍵字

LMBRD1 gene ； LMBD1 protein ； microtubule formation ； mitosis

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