熱休克蛋白70對於有絲分裂紡錘體組裝過程的調控

The Regulation of Mitotic Spindle Assembly by HSP70

方玠鼎(Chieh-Ting Fang)

指導教授 ： 易玲輝

國立臺灣大學/生命科學院/生命科學系/博士(2020年)

https://doi.org/10.6342/NTU202002175

摘要

有絲分裂紡錘體的組裝過程為有絲分裂時期確保染色體正確分離的關鍵，因而此過程對於子細胞的存活、基因組完整性的保留、個體生物的發育、以及腫瘤生成的預防是非常必要的。紡錘體組裝過程的精確調控需要仰賴許多胞器，例如中心體 (centrosome)、微管 (microtubule)、著絲粒 (kinetochore) 之間彼此協調的作用，然而調控此協調作用的機制仍未完全釐清。由於這些胞器內皆含有大量的蛋白質，因此有可能亦需要非常細緻的蛋白質品質調控機制來維持其在紡錘體組裝時的正常功能。可誘導性熱休克蛋白70 (inducible HSP70) 為一極其重要的伴護蛋白及蛋白質恆定調控者，並且在細胞內進行各方面的蛋白質品質調控。在我們先前的報導中，我們曾發現HSP70對於紡錘體組裝、有絲分裂進行以及子細胞的存活皆扮演重要角色。因此在後續的研究中，我們著重在進一步描述HSP70對於紡錘體組裝調控的作用機制。透過次繞射極限顯微影像 (subdiffraction resolution microscopy)，我們證明HSP70對於中心體裡兩個重要的蛋白質pericentrin及CEP215彼此之間的互動是必要的，並因此確保分裂期中心體的精確三維組裝、完整成熟化以及正常的功能。透過單分子影像技術、藥物所促之蛋白質交聯及譜線輪廓分析，我們證明HSP70對於維持關鍵分裂期動力蛋白Eg5的正確低聚物型態及正確的紡錘體內分布是必要的，並因此使得仰賴於Eg5的微管動態調控及紡錘體完整性得以確保。另外，運用變異型HSP70所進行的基因敲落並回復的實驗，我們揭示了HSP70的「腺苷三磷酸非依賴性的被動式受質結合 (ATP-independent passive substrate binding)」能力能夠確保Eg5在紡錘體中的正確分布、支持pericentrin在分裂期中心體的堆積，並可能藉此來協助紡錘體組裝。最後，我們也發現HSP70的去活化能夠強化抗有絲分裂藥物所造成的分裂期缺陷及細胞毒性。根據我們的發現總結，HSP70運用腺苷三磷酸非依賴性的被動式受質結合能力來調控pericentrin、CEP215及Eg5的功能，而此調控應是透過協助各蛋白質之間的互動及組裝來確保其形成之蛋白質複合體的正常功能。此調控機制則進一步確保分裂期中心體的正常功能、微管動態的精確控制以及正確的紡錘體組裝。我們的研究揭示了有絲分裂期間的一種由HSP70所斡旋的蛋白質恆定的調控途徑，而此途徑可能支撐著細胞對於有絲分裂異常或抗有絲分裂藥物所帶來之逆境的抗性。

關鍵字

有絲分裂紡錘體 ； HSP70蛋白質 ； 分裂期中心體 ； 紡錘體極點 ； 中心體周圍物質 ； 微管 ； 動力蛋白Eg5

並列摘要

The process of mitotic spindle assembly is central to faithful chromosome segregation during mitotic cell division, and is thus essential to survival of daughter cells, preservation of genomic integrity, development of organisms and prevention of tumorigenesis. The precisely regulated assembly of this structure depends on coordinated actions between multiple organelles such as centrosome, microtubule or kinetochore by the mechanism which is not fully understood. Since these organelles all harbor enormous amount of proteins, it is conceivable that they require a delicate protein quality control for their proper functions during spindle assembly. The inducible HSP70 is a master proteostasis regulating molecular chaperone that mediates all aspects of protein quality control and has been reported to be critical for mitotic spindle assembly, mitosis progression and mitotic cell survival in our previous study. In our following studies, we focused on delineating the mechanism regarding how HSP70 regulates the mitotic spindle assembly. By subdiffraction resolution microscopy, we demonstrated that HSP70 is required for adequate interactions between the critical centrosomal proteins pericentrin and CEP215 to ensure proper 3D assembly, complete maturation, and accurate functions of the mitotic centrosome. By single molecule imaging, chemical-mediated protein crosslinking, and line profile analysis of the key mitotic kinesin Eg5, we showed that HSP70 is required for correct oligomeric assembly and distribution of Eg5 within the spindle, thus ensuring Eg5-dependent control of microtubule dynamics and spindle integrity. In addition, by knockdown-rescue experiments using HSP70 mutants, we revealed that the ATP-independent passive substrate binding activity of HSP70 ensures accurate Eg5 distribution within the spindle, supports pericentrin accumulation at the mitotic centrosome, and may thus assist spindle assembly. Finally, we found that HSP70 inactivation enhanced the mitotic defects and the cytotoxicity induced by the antimitotic drugs. Collectively, our findings led us to conclude that HSP70 may rely on its passive binding activity to regulate the functions of pericentrin, CEP215 and Eg5, possibly by assisting their interaction and assembly into functional protein complexes; these regulations thus ensure properly functioning mitotic centrosome, precisely regulated microtubule dynamics, and thus faithful mitotic spindle assembly. Our studies revealed one of the potential HSP70-mediated proteostasis regulation pathways during mitosis, which may underlie cellular resistance to mitotic error- or antimitotic drug-induced stressful conditions.

並列關鍵字

Mitotic spindle ； HSP70 ； Mitotic centrosome ； spindle pole ； pericentriolar material ； microtubule ； Eg5

參考文獻

1. Prosser SL, Pelletier L (2017) Mitotic spindle assembly in animal cells: a fine balancing act. Nature reviews Molecular cell biology 18 (3):187-201.

2. Levine MS, Holland AJ (2018) The impact of mitotic errors on cell proliferation and tumorigenesis. Genes development 32 (9-10):620-638.

3. Funk LC, Zasadil LM, Weaver BA (2016) Living in CIN: Mitotic Infidelity and Its Consequences for Tumor Promotion and Suppression. Developmental cell 39 (6):638-652.

4. Bakhoum SF, Silkworth WT, Nardi IK, Nicholson JM, Compton DA, Cimini D (2014) The mitotic origin of chromosomal instability. Current biology : CB 24 (4):R148-149.

5. Negrini S, Gorgoulis VG, Halazonetis TD (2010) Genomic instability--an evolving hallmark of cancer. Nature reviews Molecular cell biology 11 (3):220-228.