Developmental Biology, Department of Biology, Faculty of Sciences, Utrecht University, Utrecht, Netherlands.
Cell Biology, Department of Biology, Faculty of Sciences, Utrecht University, Utrecht, Netherlands.
Elife. 2018 Aug 15;7:e38198. doi: 10.7554/eLife.38198.
The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2, and LIN-5 proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2 as a regulatable membrane anchor, and LIN-5 as a potent activator of dynein-dependent spindle-positioning forces.
有丝分裂纺锤体的位置决定了细胞分裂的平面，从而决定了子细胞的位置、大小和内容。纺锤体的定位是由星状微管上的动力蛋白介导的拉力驱动的，这需要一个进化保守的 Gα∙GDP、GPR-1/2 和 LIN-5 蛋白复合物。为了研究该复合物成分的单个功能，我们开发了一种在 胚胎中进行光控定位内源性蛋白的遗传策略。通过用光诱导的膜锚定点替代 Gα 和 GPR-1/2，我们证明了 Gα∙GDP、Gα∙GTP 和 GPR-1/2 对于产生拉力并不必需。在没有 Gα 和 GPR-1/2 的情况下，LIN-5 的皮质募集而不是动力蛋白本身诱导了高拉力。LIN-5 的光控定位推翻了正常的细胞周期和极性调节，并为纺锤体和细胞分裂平面提供了实验控制。我们的结果将 Gα∙GDP-GPR-1/2 定义为可调节的膜锚定点，将 LIN-5 定义为动力蛋白依赖性纺锤体定位力的有效激活剂。
