Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling.
Spindle function in Xenopus oocytes involves possible nanodomain calcium signaling.
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DOI：
10.1091/mbc.e16-05-0338
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发表时间：
2016-11-01
影响因子：
3.3
通讯作者：
Liu XJ
中科院分区：
文献类型：
--
作者：
Li R;Leblanc J;He K;Liu XJ
注射二溴-BAPTA引起青蛙卵母细胞减数分裂纺锤体立即崩溃。与此相反，EGTA对纺锤体或极体发射没有影响。纺锤体完整性的破坏，但不是缓慢的钙螯合剂表明，减数分裂纺锤体功能的卵母细胞涉及纳米结构域钙信号。细胞内钙瞬变是受精过程中的一种普遍现象，是卵激活所必需的，但钙离子在第二极体发射中的确切作用仍不清楚。另一方面，在卵母细胞成熟过程中没有证明类似的钙瞬变，然而，操纵细胞内钙水平会干扰小鼠和青蛙的第一极体发射。为了确定钙信号在极体形成中的确切作用，我们使用了活细胞成像和时间精确的细胞内钙缓冲。我们发现，BAPTA为基础的钙螯合剂引起立即解聚的纺锤体微管在减数分裂I和减数分裂II。令人惊讶的是，EGTA在类似或更高的细胞内浓度对纺锤体功能或极体发射没有影响。使用两个钙探针含有置换的绿色荧光蛋白和钙传感器钙调蛋白（LCK-GCaMP 3和GCaMP 3），我们证明了富集的探针在纺锤体，但未能检测到钙增加在卵母细胞成熟过程中在纺锤体或其他地方。最后，内源性钙调素被发现在整个减数分裂的各个阶段与纺锤体微管共定位。我们的研究结果-最重要的是，不同的敏感性的纺锤体BAPTA和EGTA-表明，在青蛙卵母细胞减数分裂纺锤体功能需要高度本地化，或纳米结构域，钙信号。
Injection of dibromo-BAPTA caused immediate collapse of meiotic spindles in frog oocytes. In contrast, EGTA had no effect on the spindle or polar body emission. The disruption of spindle integrity by the fast but not slow calcium chelators suggests that meiotic spindle function in the oocytes involves nanodomain calcium signaling. Intracellular calcium transients are a universal phenomenon at fertilization and are required for egg activation, but the exact role of Ca2+ in second-polar-body emission remains unknown. On the other hand, similar calcium transients have not been demonstrated during oocyte maturation, and yet, manipulating intracellular calcium levels interferes with first-polar-body emission in mice and frogs. To determine the precise role of calcium signaling in polar body formation, we used live-cell imaging coupled with temporally precise intracellular calcium buffering. We found that BAPTA-based calcium chelators cause immediate depolymerization of spindle microtubules in meiosis I and meiosis II. Surprisingly, EGTA at similar or higher intracellular concentrations had no effect on spindle function or polar body emission. Using two calcium probes containing permutated GFP and the calcium sensor calmodulin (Lck-GCaMP3 and GCaMP3), we demonstrated enrichment of the probes at the spindle but failed to detect calcium increase during oocyte maturation at the spindle or elsewhere. Finally, endogenous calmodulin was found to colocalize with spindle microtubules throughout all stages of meiosis. Our results—most important, the different sensitivities of the spindle to BAPTA and EGTA—suggest that meiotic spindle function in frog oocytes requires highly localized, or nanodomain, calcium signaling.