酵母菌產生孢子過程中Hsp26相關紡錘體檢控點的調控機制

Abstract

我們實驗室在研究Hsp26蛋白於出芽酵母菌孢子形成過程中所扮演的角色時，發現有一個和Hsp26有關的紡錘體檢控點 (Hsp26-dependent spindle checkpoint)，監控第二次減數分裂到孢子形成過程中紡錘體的形態是否適合形成孢子，藉此確保孢子的存活。先前發現Tem1和Ady3這兩個和Hsp26有交互作用的蛋白也參與此調控機制，Tem1蛋白是有絲分裂中mitotic exit network (MEN) pathway的重要成員，扮演類似感應器的角色，而Ady3蛋白則位於原生孢子膜 (prospore membrane) 前端，協助孢子形成。我們推測Tem1蛋白在Hsp26-dependent spindle checkpoint中可能也扮演著類似感應器的角色去偵測第二次減數分裂中紡錘體的形態是否適合形成孢子，並透過Hsp26蛋白來影響Ady3和Ssp1兩個前緣蛋白質 (leading edge protein) 的位置，以調控孢子形成。 為了瞭解其機制，本篇論文利用benomyl干擾紡錘絲的形成，發現不論在野生型酵母菌還是在hsp26突變細胞中，原生孢子膜的形成都不會受到影響，由此結果推論檢控點並不會調控原生孢子膜的生成，而是直接調控Ady3和Ssp1蛋白的定位以控制孢子可否形成。另外觀察到無論是Ady3、Ssp1或是Hsp26蛋白本身的表現並不會受到 benomyl處理影響，顯示其他蛋白質的共同參與此檢控點機制。 之前發現當細胞進行減數分裂時Tem1蛋白會被磷酸化，本實驗也發現此磷酸化並不受benomyl處理影響。綜合所有結果，本篇研究排除一些常見調控方式的可能性，幫助確定未來對此檢控點機制的研究方向。

Previous studies in our laboratory confirmed that there is a novel Hsp26-dependent spindle checkpoint during meiosis. The checkpoint monitors meiotic II spindle formation and regulates spore formation. In addition, two Hsp26-interacting proteins, Tem1 and Ady3, are involved in the checkpoint. Tem1 is an important component in the mitotic exit network (MEN) pathway, it plays a role of sensor for proper spindle positioning in a dividing cell. Ady3 is a component of the leading edge complex (LEP), associates with the growing prospore membrane (PSM) to assist proper spore formation. It was proposed that Tem1 might act as a sensor in the Hsp26-dependent spindle checkpoint to regulate the localization of Ady3 and Ssp1 into LEP. In order to clarify the mechanism of the checkpoint, I used benomyl to interfere meiotic II spindle and found that PSM formation was not affected in the wild type, neither in the hsp26 mutant, indicating that PSM formation was not a target of the Hsp26-dependent spindle checkpoint. In addition, there was no detectable difference in the expression of Ady3, Ssp1 or Hsp26 after benomyl treatment, suggesting that there is other protein(s) involved in the checkpoint. It has been reported that Tem1 was phosphorylated during meiosis; however, phosphorylation of Tem1 was not affected in responding to spindle defects. In this study, I excluded some possibilities for certain regulatory mechanisms and provided useful clues for studying the mechanism of the Hsp26-dependent spindle checkpoint in the future.