利用線蟲為模型探討紡錘體檢查點在調控精子生成時的角色

Investigating the Spindle Assembly Checkpoint Regulation in C. elegans Spermatogenesis

陳尚暘(Shang-Yang Chen)

指導教授 ： 吳瑞菁

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201701440

摘要

細胞分裂對於多細胞生物的成長、再生及生育都極為重要。在有絲分裂的系統中，從細胞分裂中期過渡到分裂後期的過程是由紡錘體檢查點SAC(spoindle assembly checkpoint)所嚴格的控管。SAC經由抑制一種E3泛素連接酶APC/C，來防止姊妹染色體之間的黏連蛋白被降解以及染色體分離。但是在雄性減數分裂系統中，細胞只進行一次染色體複製接著連續兩次染色體分離，而目前對於這兩次染色體分離的調控機制是否由SAC所調控尚不清楚。
為了研究雄性減數分裂的過程是否有SAC的參與，我們利用一個演算法來自動且快速分析我們所拍攝的縮時攝影影像中，位於染色體周圍的蛋白質表現量。我們發現位於著絲點外層，同時也是SAC訊號的蛋白BUB-1在第一次染色體分離時會脫離染色體，接著在第二次分裂中期開始之前會回到染色體上，表示著絲點的結構，同時也是SAC訊號的模板會在兩次減數分裂之間有重新再構築的動作。相反的，APC/C的作用目標securin雖然會在第一次染色體分離時被分解，但在第二次染色體分裂的過程中並不會重新聚集回染色體附近，推論第二次染色體分離的調控不需要APC/C的參與。為了更做進一步驗證，我們對精母細胞施予蛋白酶體抑制劑，發現初級精母細胞會被抑制在第一次分裂中期，然而二級精母細胞能夠完成第二次的染色體及細胞分離，推論第二次染色體分離的調控和第一次分裂不同，不需要蛋白酶體的參與。
作為總結，我們認為對有絲分裂的系統而言相當重要的APC/C以及蛋白酶體系統，不存在於第二次減數分裂的調控機制中。

關鍵字

紡錘體檢查點 ； 雄性減數分裂 ； 縮時攝影 ； 泛素連接酶 ； 蛋白酶體

並列摘要

Cell division is important in all multicellular organisms for vital processes including growth, regeneration and reproduction. During cell cycle, the transition from metaphase to anaphase strictly controlled by Spindle Assembly Checkpoint (SAC). Until all chromosomes are properly attached by spindle microtubules, SAC prevents chromosome separation by inhibiting APC/C, an E3 ubiquitin ligase essential for releasing sister chromatid cohesion. In male meiosis, however, two consecutive chromosome separation events occur after one round of chromosome duplication. Thus far, it is unclear if both male meiotic divisions subjected to the control of canonical SAC signaling. To investigate if SAC signals function during male meiotic divisions, we developed an algorithm that allows automatic quantification of the levels of chromosome-associated proteins through the divisions in time-lapse recordings. We found that outer kinetochore SAC signaling protein BUB-1 released from chromosomes during first chromosome segregation event and recruited back to chromosome before second chromosome segregation takes place, indicating proper kinetochore structure and platform for SAC disassembled and re-assembled between two divisions. Contrarily, securin, the direct target of APC/C activity, though is degraded when first chromosome separation is initiated, fails to be recruited to chromosome during second division. These results indicate that the second male meiotic division does not required APC/C and the proteasome-dependent protein degradation. To test this, we examined the progression of division in primary and secondary spermatocytes treated with proteasome inhibitor MG132. As expected, primary spermatocytes treated with MG132 were stalled at metaphase I. Interestingly, secondary spermatocytes treated with MG132 were able to complete chromosome segregation and division. These results suggest the second male meiotic division might regulated differently compared to the first meiosis. Taken together, we hypothesize that the APC/C-proteasome system, which is crucial for canonical SAC signaling, does not participate in second chromosome segregation.

並列關鍵字

SAC ； male meiosis ； time-lapse recording ； APC/C ； proteasome

參考文獻

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