Department of Molecular Biology, University of Wyoming, 1000 E. University Ave., Laramie, WY 82071, USA; Marine Biological Laboratory, Cell Division and Organization Group, 7 MBL Street, Woods Hole, MA 02543, USA.
Department of Molecular Biology, University of Wyoming, 1000 E. University Ave., Laramie, WY 82071, USA.
Curr Biol. 2019 Apr 22;29(8):1273-1285.e5. doi: 10.1016/j.cub.2019.02.061. Epub 2019 Mar 28.
Normal mitotic spindle assembly is a prerequisite for faithful chromosome segregation and unperturbed cell-cycle progression. Precise functioning of the spindle machinery relies on conserved architectural features, such as focused poles, chromosome alignment at the metaphase plate, and proper spindle length. These morphological requirements can be achieved only within a compositionally distinct cytoplasm that results from cell-cycle-dependent regulation of specific protein levels and specific post-translational modifications. Here, we used cell-free extracts derived from Xenopus laevis eggs to recapitulate different phases of the cell cycle in vitro and to determine which components are required to render interphase cytoplasm spindle-assembly competent in the absence of protein translation. We found that addition of a nondegradable form of the master cell-cycle regulator cyclin B1 can indeed induce some biochemical and phenomenological characteristics of mitosis, but cyclin B1 alone is insufficient and actually deleterious at high levels for normal spindle assembly. In contrast, addition of a phosphomimetic form of the Greatwall-kinase effector Arpp19 with a specific concentration of nondegradable cyclin B1 rescued spindle bipolarity but resulted in larger-than-normal bipolar spindles with a misalignment of chromosomes. Both were corrected by the addition of exogenous Xkid (Xenopus homolog of human Kid/KIF22), indicating a role for this chromokinesin in regulating spindle length. These observations suggest that, of the many components degraded at mitotic exit and then replenished during the subsequent interphase, only a few are required to induce a cell-cycle transition that produces a spindle-assembly-competent cytoplasm.
正常的有丝分裂纺锤体组装是忠实染色体分离和细胞周期正常进行的前提。纺锤体机制的精确功能依赖于保守的结构特征，如聚焦的两极、中期板上的染色体对齐和适当的纺锤体长度。这些形态要求只能在组成上不同的细胞质中实现，这种细胞质是由细胞周期依赖性的特定蛋白质水平和特定翻译后修饰的调节产生的。在这里，我们使用从小耳蝾螈卵中提取的无细胞提取物在体外重现细胞周期的不同阶段，并确定哪些成分是在没有蛋白质翻译的情况下使间期细胞质具有纺锤体组装能力所必需的。我们发现，添加不可降解形式的主细胞周期调节剂 cyclin B1 确实可以诱导有丝分裂的一些生化和表型特征，但 cyclin B1 本身是不足够的，而且在高水平时实际上对正常纺锤体组装是有害的。相比之下，添加具有特定浓度不可降解 cyclin B1 的 Greatwall-kinase 效应物 Arpp19 的磷酸模拟形式可以挽救纺锤体的两极性，但会导致染色体不对齐的大于正常的两极纺锤体。这两种情况都可以通过添加外源 Xkid（人类 Kid/KIF22 的 Xenopus 同源物）来纠正，这表明这种染色质动力蛋白在调节纺锤体长度方面起着作用。这些观察结果表明，在有丝分裂退出时降解的许多成分中，只有少数成分是必需的，以诱导产生具有纺锤体组装能力的细胞质的细胞周期转变。
