Gotta Monica, Dong Yan, Peterson Yuri K, Lanier Stephen M, Ahringer Julie
Wellcome/Cancer Research United Kingdom Institute, Tennis Court Road, University of Cambridge, United Kingdom.
Curr Biol. 2003 Jun 17;13(12):1029-37. doi: 10.1016/s0960-9822(03)00371-3.
Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric Galpha subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown.
Here we show that the Galpha subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). Finally, we show that the GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3.
Our results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins.
在不对称细胞分裂过程中，纺锤体定位对于确保子细胞的正确大小和决定因素的分离至关重要。在秀丽隐杆线虫胚胎中，第一个纺锤体不对称定位，这种不对称由两个异源三聚体Gα亚基GOA-1和GPA-16冗余控制。Gα亚基在PAR极性蛋白的下游起作用，PAR极性蛋白控制作用于纺锤体两极的相对拉力。这些异源三聚体G蛋白如何被调节以及它们如何控制纺锤体位置仍然未知。
我们在此表明，Gα亚基受一种不依赖受体的机制调节。对gpr-1和gpr-2（哺乳动物AGS3和果蝇PINS（不依赖受体的G蛋白调节剂）的同源物）进行RNA干扰缺失，导致的表型与同时缺失GPA-16和GOA-1的胚胎相同；第一次分裂是对称的，但极性不受影响。对gpr-1和gpr-2进行RNA干扰后纺锤体不对称性的丧失似乎是作用于两极的拉力减弱的结果。GPR蛋白定位于单细胞胚胎的皮层周围，并在后侧富集。因此，不对称的G蛋白调节可以解释纺锤体的向后位移。在没有PAR极性蛋白PAR-2或PAR-3的情况下，后侧富集被消除。此外，LIN-5（一种纺锤体定位也需要的卷曲螺旋蛋白）与GPR蛋白结合并参与其皮层结合。最后，我们表明GPR-1和GPR-2的GPR结构域表现为GOA-1的GDP解离抑制剂，因此其活性与哺乳动物AGS3相似。
我们的结果表明，GPR-1和/或GPR-2通过响应PAR蛋白的信号不对称调节异源三聚体G蛋白的活性，从而控制施加在纺锤体两极上的力的不对称性。
