钙信号参与动物细胞减数分裂或有丝分裂的特定事件的程度是持久争议的主题。我们先前已经证明,用1,2-双(2-氨基苯氧基)乙烷-N缓冲细胞内钙,N,N\',N-四乙酸(BAPTA,一种快速的钙螯合剂),但不是亚乙基双(氧亚乙基亚硝基)四乙酸(EGTA,缓慢的钙螯合剂),快速解聚非洲爪狼卵母细胞中的纺锤体微管,这表明纺锤体的组装和/或稳定性需要在极其有限的时空尺度上的钙纳米域-钙瞬变。在这项研究中,我们研究了肌醇-1,4,5-三磷酸受体(IP3R)的功能,内质网(ER)钙通道,在使用Trim21介导的IP3R耗竭的主轴组装中。耗尽IP3R的卵母细胞经历了胚泡分解,但未能发出第一极体,也未能组装适当的减数分裂纺锤体。Further,我们开发了一种无细胞纺锤体组装试验,其中从单个卵母细胞中抽吸细胞质。在这个无细胞系统中组装的纺锤体被包裹在ER膜中,两极富含IP3R,而ER组织或钙信号的破坏导致纺锤体快速分解。和完整的卵母细胞一样,无细胞卵母细胞提取物中纺锤体的形成也需要IP3R。我们得出的结论是,涉及IP3R介导的钙释放的细胞内钙信号传导是非洲爪的卵母细胞减数分裂纺锤体组装所必需的。
The extent to which calcium signaling participates in specific events of animal cell meiosis or mitosis is a subject of enduring controversy. We have previously demonstrated that buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N\',N\'-tetraacetic acid (BAPTA, a fast calcium chelator), but not ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA, a slow calcium chelator), rapidly depolymerizes spindle microtubules in Xenopus oocytes, suggesting that spindle assembly and/or stability requires calcium nanodomains-calcium transients at extremely restricted spatial-temporal scales. In this study, we have investigated the function of inositol-1,4,5-trisphosphate receptor (IP3R), an endoplasmic reticulum (ER) calcium channel, in spindle assembly using Trim21-mediated depletion of IP3R. Oocytes depleted of IP3R underwent germinal vesicle breakdown but failed to emit the first polar body and failed to assemble proper meiotic spindles. Further, we developed a cell-free spindle assembly assay in which cytoplasm was aspirated from single oocytes. Spindles assembled in this cell-free system were encased in ER membranes, with IP3R enriched at the poles, while disruption of either ER organization or calcium signaling resulted in rapid spindle disassembly. As in intact oocytes, formation of spindles in cell-free oocyte extracts also required IP3R. We conclude that intracellular calcium signaling involving IP3R-mediated calcium release is required for meiotic spindle assembly in Xenopus oocytes.