RESULTS: We found that the expression levels and localization of ADD1, S726 phosphorylated ADD1 (p-ADD1), and TPX2 proteins exhibited spindle assembly-dependent dynamic changes during mouse oocyte meiosis. Taxol treatment, which stabilizes the microtubule polymer and protects it from disassembly, made the signals of ADD1, p-ADD1, and TPX2 present in the microtubule organizing centers of small asters and spindles. Knockdown of approximately 60% of ADD1 protein levels destabilized interpolar microtubules in the meiotic spindle, resulting in aberrant chromosome alignment, reduced first polar body extrusion, and increased aneuploidy in metaphase II oocytes, but did not affect K-fiber homeostasis and the expression and localization of TPX2. Strikingly, TPX2 deficiency caused increased protein content of ADD1, but decreased expression and detachment of p-ADD1 from the spindle, thereby arresting mouse oocytes at the metaphase I stage with collapsed spindles.
CONCLUSIONS: Phosphorylation of ADD1 at S726 by TPX2 mediates acentriolar spindle assembly and precise chromosome segregation in mouse oocytes.
结果:我们发现ADD1,S726磷酸化ADD1(p-ADD1)的表达水平和定位,TPX2蛋白在小鼠卵母细胞减数分裂过程中表现出纺锤体组装依赖性动态变化。紫杉醇治疗,稳定微管聚合物并保护其免受拆卸,使ADD1,p-ADD1和TPX2的信号存在于小紫苑和纺锤体的微管组织中心。大约60%的ADD1蛋白水平的敲除使减数分裂纺锤体中的极间微管不稳定,导致异常的染色体排列,减少第一极体挤出,中期II卵母细胞的非整倍性增加,但不影响K纤维稳态和TPX2的表达和定位。引人注目的是,TPX2缺乏导致ADD1蛋白含量增加,但p-ADD1表达减少,与纺锤体脱离,从而在中期I阶段用塌陷的纺锤体逮捕小鼠卵母细胞。
结论:在小鼠卵母细胞中,TPX2在S726对ADD1的磷酸化介导了Acentriolar纺锤体的组装和精确的染色体分离。