Abstract：
BACKGROUND: ADD1 (adducin-1) and TPX2 (targeting protein for Xklp2) are centrosomal proteins and regulate mitotic spindle assembly. Mammalian oocytes that segregate homologous chromosomes in Meiosis I and sister chromatids in Meiosis II with a spindle lacking centrosomes are more prone to chromosome segregation errors than in mitosis. However, the regulatory mechanisms of oocyte spindle assembly and the functions of ADD1 and TPX2 in this process remain elusive.
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.
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.
摘要：
背景：ADD1（内收蛋白-1）和TPX2（Xklp2的靶向蛋白）是中心体蛋白并调节有丝分裂纺锤体的组装。与有丝分裂相比，在减数分裂I中分离同源染色体和在减数分裂II中分离姐妹染色单体的哺乳动物卵母细胞更容易出现染色体分离错误。然而,卵母细胞纺锤体组装的调控机制以及ADD1和TPX2在这一过程中的功能仍然难以捉摸。
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。
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。
