2017-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/685571摘要：在有絲分裂中，紡綞體檢查點抑制姐妹染色體分離直到所有染色體都正確地與紡綞絲連結。當檢查點滿足時會誘發一系列蛋白質降解使分離開始。不同於有絲分裂，雄性減數分裂中包含了連續兩次的染色體分離。至今，對於雄性減數分裂是否也受紡綞體檢查點的調控仍不清楚。我們計劃在線蟲中針對紡綞體檢查點在雄性減數分裂的角色做全面性探討，而思考三種可能性：檢查點活性在第一次分裂後重新建立、檢查點活性在第一次分裂部份減低以支持第二次分裂的調控、檢查點活性在第一次分裂時完全解除。初步實驗結果支持第三種可能，暗示第二次分裂並不需要檢查點調控。為進一步區分上述三種模式，我們將由三個面向研究。首先，我們將檢視紡綞體檢查點中各別分子在雄性減數分裂中的動態分布。其次，我們將測試紡綞體檢查點下游的蛋白質降解功能是否對兩次分裂都重要。最後，我們將測試是否干擾染色體與紡綞絲的結合會導致兩次分裂的停滯。我們已發展兩項重要的實驗技術。第一，我們發展了可自動測量活體攝影中螢光訊號的程式碼。第二，我們發展了可獨立測試兩次分裂的小分子抑制劑方法。結合本實驗室已完善建立的多色活體攝影和遺傳學技術，我們將得以深入探索雄性減數分裂的分子機制。<br> Abstract: Faithful chromosome segregation plays crucial roles in development and viability and requiresstrict regulation to prevent erroneous partitioning of the genetic material. It is known in mitotic cellsspindle assembly checkpoint (SAC) prevents initiation of segregation until all chromosomes arecorrected connected to the spindle microtubules. When SAC is satisfied, a series ofproteosome-dependent protein degradation events occur to release chromosome cohesion. In malemeiosis, two consecutive chromosome segregation events take place after one round of DNAduplication. Thus far it remains controversial whether SAC also regulates male meiotic divisions. Inthis proposed study, we plan to investigate the checkpoint control during this specialized celldivision. We postulate three models to account for checkpoint activities in male meiotic divisions:re-establishment of the SAC activity after first division, stepwise reduction of SAC activity betweentwo divisions, and no checkpoint activity for the second division. Interestingly, our preliminaryresults support that the second division does not required SAC regulation. To further distinguish thethree models, we will investigate the roles of SAC pathway in male meiotic divisions using thenematode Caenorhabditis elegans as a model. First, we will investigate the dynamics of SACcomponents during the two division events. Second, we will determine if SAC-dependent proteindegradation plays crucial roles in both divisions. Finally, we will investigate if the dividing primaryand secondary spermatocytes would be arrested when perturbation of chromosome-spindleinteraction is applied. We have developed two novel methodologies to ensure the success executionof the proposed studies. First, an algorithm was developed to allow unbiased systematicquantification of the fluorescence intensity of target proteins in time-lapse recordings. Second, aprotocol for chemical inhibitor administration was developed to allow investigation of the first andthe second division separately. In combination of the fully established live-imaging and geneticapproaches, this proposed study will shed lights on the molecular mechanistic aspects of the highlyspecialized male meiotic division. We expect the results from this pioneering study would generategreat impact of the cell division field.