以線蟲為模型探討AIR-2及GSP-2在雄性減數分裂中染色體分離的調控

Abstract

在細胞的複製過程中，準確且正確的將遺傳物質分離非常重要。若染色體出現異常或數量錯誤，可能導致個體細胞癌化，或在發育前期即死亡；而在生殖細胞中，這種異常則可能導致不孕。許多研究顯示在細胞分裂中，細胞內磷酸化會隨著不同時期的演進而有不同程度的改變。其 中的Aurora B蛋白激酶（Aurora B kinase）藉由磷酸化調控染色體的轉向及分離；同時蛋白去磷酸酶（Protein Phosphatase）拮抗Aurora B蛋白激酶以維持染色體上磷酸化的平衡。 以線蟲為模式生物，在雄性減數分裂中，生殖細胞會進行兩次染色體分離。第一次減數分裂時，同源染色體分離；第二次減數分裂時，姊妹染色分體（sister chromatids）被分配到不同的子細胞。目前仍不清楚在減數分裂中兩次連續的分裂是否與細胞分裂調控機制相同，且兩次分裂為同一套調控機制。我們利用化學及遺傳學的方法，發現在第一次減數分裂AIR-2剔除或是受到抑制時，染色體會發生不規則的排列轉向，且無法進行分離，並且在第二次減數分裂時也觀察到相同現象。暗示著在兩次減數分裂中AIR-2有相同的調控標的。 然而在雄性減數分裂中，我們發現兩次減數分裂中可能是由不同的蛋白去磷酸酶所平衡。在gsp-2 （Protein Phosphatase 1）基因突變的精原母細胞中，染色體在減數分裂的兩次分裂中皆無法在染色體分離前整齊的排列在赤道版上（equatorial template）。利用Protein phosphatase抑制劑okadaic acid 後，染色體也出現與在gsp-2 基因突變的細胞中相似無法在分離前整齊排列的狀況。在第二次減數分裂中，OA抑制劑造成細胞染色體除了排列混亂之外更嚴重的缺陷：例如chromosome bridge及lagging chromosome的現象。由此推斷GSP-2在第一次分裂當中是主要的PP1，而在第二次分裂中則可能還有其他PP1參與蛋白激酶與去磷酸酶之間的調控平衡。

Accurate and precise segregation of chromosome is crucial for ensuring cell integrity during cell division. If happened during mitosis, defective segregation lead to aneuploidy, which subsequently leads to cell death or even cancer. Likewise, in meiosis, mis-segregation results in infertility. During mitotic cell division, Aurora B kinase plays crucial roles in modulating the orientation and separation of chromosomes. PP1 phosphatases counteract Aurora B kinase to maintain proper phosphorylation levels during the dynamic chromosome movements. In male meiotic cells, two rounds of chromosome separation events take place to separate homologous and then sister chromosomes sequentially. Whether the same Aurora B kinase/phosphatase-dependent phosphorylation is repeated between the two chromosome segregation events during male meiosis remains unclear. Using chemical genetic approaches, we found inhibition of Aurora B kinase AIR-2 in meiosis I caused segregation defects, such as mis-biorientation and nondisjunction, which are comparable to inhibition of Aurora B kinase in meiosis II. These results imply that Aurora B kinase targets the same molecules in both divisions. In contrast to Aurora B kinase, we found male MI and MII divisions might employ different phosphatases for regulation. Deletion of PP1 GSP-2 caused significant mal-alignment of chromosomes at metaphase onset in MI and the subsequent MII. Primary spermatocytes treated with Pan-PP1 phosphatases inhibitor okadaic acid exhibited chromosome orientation defects comparable to gsp-2 mutants, indicating GSP-2 is the major phosphatase during meiosis I. However, okadaic acid treatment induced a different set of chromosome segregation defects in male meiosis II: chromosomes not only failed to align bi-orientally prior to separation but also showed either chromosome bridge or nondisjunction. These results support that GSP-2 is not the only PP1 involved in the regulation of MII division. Our study reveals Aurora B kinase is regulating MI and MII in a kinase/phosphatase-dependent phosphorylation with different PP1s.