蛋白激酶LIMK1活性在小鼠卵母细胞染色体分离过程中的作用和分子机制

Aneuploid embryos are mainly caused by oocyte chromosome segregation errors, leading to miscarriage and birth defects. The spindle functions to drive chromosome division, while spindle assembly checkpoints (SAC) on kinetochore control the onset of anaphase and timely separation of chromosome. In our previous work, the protein kinase Lim kinase 1(LIMK1) was found specially localized on spindle while phosphorylated LIMK1 is present in the microtubule organizing centers (MTOCs) and chromosome kinetochores, a physiological interaction was also observed between phosphorylated LIMK1 and gamma-tubulin in mouse oocytes during meiosis, implying LIMK1 activity is involved in spindle formation and SAC function. In this project, we will continue to explore the role of LIMK1 activity in the formation and maintenance of MTOC and the establishment of SAC function on kinetochore; establish limk1 oocytes conditional knockout mouse model, studying the role of LIMK1 in oocytes in vivo. The project findings will help to throw light on the molecular mechanism ensuring accurate chromosome segregation in oocyte, with high significance for understanding the formation of human aneuploid embryos.

非整倍体胚胎常由卵母细胞染色体分离错误所致，引起流产或出生缺陷。纺锤体牵引染色体分离，而动粒上纺锤体组装检验点(Spindle Assembly Checkpoint, SAC)则调控细胞分裂后期启动和染色体适时分离。我们在前期工作中发现蛋白激酶LIMK1(Lim kinase 1)分布于小鼠卵母细胞减数分裂纺锤体上，磷酸化LIMK1同时存在于微管组织中心(microtubule organizing centers，MTOCs)和动粒上，并与gamma-tubulin 之间存在生理性相互作用，提示LIMK1活性参与纺锤体形成并与SAC功能相关。本项目继续探索LIMK1在MTOC形成和维持以及SAC功能建立过程中的作用；构建卵母细胞limk1条件性敲除小鼠模型，在体性地研究LIMK1的作用。研究结果将有助于揭示卵母细胞内确保染色体精确分离的分子机制，对于认识非整倍体胚胎形成具有重要意义。

卵母细胞减数分裂过程中染色体分离错误会导致产生非整倍体胚胎，是临床不孕不育、自发性流产和出生缺陷的最主要原因。纺锤体牵引染色体运动和分离，其形成依赖于特异的微管组织中心（microtubule organizing center, MTOC），而纺锤体组装检验点（spindle assembly checkpoint, SAC）则阻止后期启动直至纺锤体组装完成并与染色体有效联结，在后期进程中染色体臂间黏结素REC8发生磷酸化并被降解，同源染色体所受约束解除而相互分离。迄今， MTOC的分子构成、SAC功能建立机制和磷酸化REC8的激酶仍旧不甚清楚。在本研究项目中，我们发现LIMK1（Lim kinase 1)参与调控小鼠卵母细胞纺锤体的形成，活化的LIMK1（pLIMK1Thr508）是MTOC相关蛋白，LIMK1活性缺失会阻止MTOC的迁移和双极性定位，从而破坏微管组装成纺锤体的过程。LIMK1与调控纺锤体双极性的核心分子EG5存在时空共定位特性并对微管聚合状态的改变呈现出相似的反应，两者活性抑制后纺锤体表型一致，提示LIMK1可能通过参与EG5的功能而调控纺锤体的组装。pLIMK1Thr508持续存在于染色体动粒上，并动态地聚集在染色体臂间，提示其与SAC功能和黏结素解离相关。LIMK1相关蛋白Plk1（polo-like kinase 1）、PAK1（p21-activated kinase 1）、Stau2（Staufen 2）和RPB1（polymerase (RNA) II (DNA directed) polypeptide A）也参与卵母细胞染色体分离过程，阻止Plk1会破坏黏结素蛋白REC8的降解和染色体分离；活化的PAK1（pPAK1Thr423）亚细胞定位与pLIMK1Thr508相似，抑制PAK1会阻止染色体的凝集和纺锤体形成，并可破坏SAC蛋白MAD1（mitotic arrest deficient 1）在着丝粒部位的募集，导致染色体提前分离；Stau2调控纺锤体微管稳定性的维持、微管与动粒之间联结的建立以及胞质分裂的有序完成；RPB1羧基端的磷酸化在卵母细胞减数分裂中仍旧存在，发挥转录以外的功能并与调控染色体分离的分子事件相关。这些研究结果有助于揭示卵母细胞内确保染色体精确分离的分子机制，对于认识非整倍体胚胎形成具有一定的意义。