分析線蟲的TLK-1蛋白在細胞分裂中對DNA濃縮與分離和在細胞質分裂中的功能

Abstract

在細胞進行有絲分裂過程中，染色體的濃縮與分離和細胞質分裂若是發生異常，就會破壞基因體的完整性並造成生物體的死亡或是癌化。線蟲裡的condensin和AIR-2蛋白，對於細胞分裂能夠正常執行是必需的；TLK-1已被證實是AIR-2的受質及活化物，在AIR-2與另一個活化物ICP-1結合的情況下，能參與它們的作用共同確保染色質分裂能正常進行。然而TLK-1是否有參與染色質濃縮或是細胞質分裂仍是未知問題。在論文裡，我們使用顯微鏡追蹤分析tlk-1突變株發現，TLK-1不只作用在染色體分離過程，也作用在染色體濃縮和細胞質分裂。從結果顯示，TLK-1以不同於ICP-1的作用方式參與染色體濃縮和細胞質分裂，既不影響AIR-2和condensin與染色體結合的能力，整個kinetochore的結構和功能也是正常的，只有染色體的排列有問題。更甚者，TLK-1在細胞分裂過程中的位置分佈也與AIR-2和ICP-1不同，在核膜破裂後TLK-1散布到整個細胞中，而不是結合到染色體上，這也與之前TLK-1的研究結果不一樣；由此可知，TLK-1雖是AIR-2的活化蛋白，但它是以不同於ICP-1的方式和AIR-2共同作用。而TLK-1影響細胞質分裂的分式，則是透過調控AIR-2在細胞分裂末期的分佈，使之能坐落到midzone microtubule以促進細胞質分裂完成。除此之外，TLK-1還可能透過磷酸化HCP-3的模式，使染色體在進行濃縮時能形成正確的結構。最後透過yeast two-hybrid screen，我們發現了一個新的可與TLK-1共同作用的蛋白，DLC-1。TLK-1可能與DLC-1共同影響microtubule和kinetochore的交互作用。

Defects in chromosome condensation, segregation or cytokinesis during mitosis disrupt genome integrity and cause organismal death or tumorigenesis. The conserved kinase protein AIR-2/Aurora B is required for normal execution of all these important mitotic events in C. elegans. TLK-1 has been recently shown to be a substrate and activator of AIR-2 in the presence of another AIR-2 activator ICP-1/INCENP, and to cooperate with AIR-2 to ensure proper mitotic chromosome segregation. However, whether TLK-1 may contribute to chromosome condensation or cytokinesis is unclear. A time-lapse microscopy analysis showed that tlk-1 mutants are defective in chromosome condensation and cytokinesis, in addition to chromosome segregation, during mitosis. Our data indicate that TLK-1 contributes to chromosome condensation and segregation, at least in part, in a manner that is distinct from the ICP-1-mediated mechanism and does not involve loading AIR-2 and condensin complexes to mitotic chromosomes. The overall structure of kinetochore is also normal accompanies with the chromosome alignment defect during metaphase in tlk-1 mutant. Moreover, the localization pattern of TLK-1 is different from the previous data and AIR-2/ICP-1 complex, which TLK-1 diffused to cytoplasm after nuclear envelope broken down. This reveals the differences in dynamic regulation and association of TLK-1 and ICP-1 towards AIR-2 in vivo. Finally, TLK-1 functions in cytokinesis by localizing AIR-2 to the midzone microtubules. Otherwise, TLK-1 may have function in chromosome architecture during chromosome condensation via phosphorylating HCP-3. And via yeast two-hybrid screen, we found a new TLK-1 interacting protein, DLC-1, which may cooperate to regulate the microtubule and kinetochore interaction during mitosis.