Cortical localization of the Gα protein GPA-16 requires RIC-8 function during C-elegans asymmetric cell division
Cortical localization of the Gα protein GPA-16 requires RIC-8 function during C-elegans asymmetric cell division
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DOI：
10.1242/dev.02039
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发表时间：
2005-10-01
期刊：
影响因子：
4.6
通讯作者：
Gönczy, P
中科院分区：
文献类型：
--
作者：
Afshar, K;Willard, FS;Gönczy, P
在不对称分裂过程中，对主轴定位机制的理解仍然不完全。在单细胞期线虫胚胎的不均等分裂过程中，Got蛋白果阿-1和GPA-16以部分冗余的方式起作用，沿沿着星形微管产生拉力。以前的工作主要集中在果阿-1上，而GPA-16参与这一过程的机制还没有得到很好的理解。在这里，我们报告说，GPA-16主要存在于一个细胞阶段胚胎的皮质。使用免疫共沉淀和表面等离子体共振结合测定，我们发现GPA-16与RIC-8和GPR-1/2，两种蛋白质已知是所需的拉力产生。使用纺锤体切断作为拉力的测定，我们证明GP蛋白GPB-1的失活使得GPA-16和果阿-1完全多余。这表明两种G α蛋白可以激活相同的途径，并且通常需要它们的双重存在来对抗G β γ。使用核苷酸交换试验，我们确定，而GPR-1/2作为一个鸟嘌呤核苷酸解离抑制剂（GDI）的GPA 16，因为它对果阿-1，RIC-8不表现出鸟嘌呤核苷酸交换因子（GEF）对GPA-16的活性，相反，它对果阿-1的影响。此外，我们还确定RIC-8是GPA-16皮质定位所必需的，而果阿-1则不需要。我们的分析表明，这种要求对GPA-16是不同的已知功能的RIC-8之间的相互作用，使得到的蛋白质和GPR-1/2，从而提供了新的见解不对称主轴定位的机制。
Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C elegans embryos, the Got proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using coimmunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the GP protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two G alpha proteins can activate the same pathway and that their dual presence is normally needed to counter G beta gamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Got proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.