IDENTIFICATION OF RHOGAP INTERACTING PROTEINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 有丝分裂是一个复杂的过程，复制的基因组被分离成两个子细胞。胞质分裂是有丝分裂的最后一步，是两个子细胞的物理分离。胞质分裂失败会导致多倍体和基因组不稳定，这是许多癌症的两个特征。在细胞质分裂过程中，后期纺锤体发出信号，产生一个赤道区的活性RhoA，进而引导收缩环成分的皮质积累。Rho家族GTP酶在活跃的GTP结合形式和非活跃的GDP结合形式之间循环。这个循环由促进GTP负荷的GEF和GAP控制，GAP通过刺激小的GTP酶的低内在GTP酶活性来使其失活。在细胞质分裂过程中，有人提出将RhoA全环定位于纺锤体中区提供了一种在细胞赤道局部激活RhoA的方法。然而，由于RhoGTP酶以脂质部分锚定在质膜上，并被认为在膜内自由扩散，以前的理论工作表明，全球RhoA间隙活性对于维持赤道区的活性Rho是必要的。在线虫RGA-3和RGA-4(RGA-3/4)中，两个高度相似的RhoGAP先前被证明优先作用于RhoA，并定位于卵裂沟和微管。我们发现，在细胞质分裂的早期阶段，需要RGA-3/4来限制收缩环成分的积累，包括香草素和细胞赤道的隔膜。在解理沟道中，RGA-3/4的引入也很重要，可以将香兰素和间隔蛋白的区域限制在沟尖端的紧密区。综上所述，我们的发现支持一个模型，在该模型中，RhoGAP有助于将RhoA的激活限制在细胞赤道。为了了解RGA-3/4的活性和/或定位是如何通过有丝分裂进行调节的，我们的目标是与生理蛋白质组学中心合作，通过免疫沉淀后的质谱学来鉴定新的RGA-3/4的结合伙伴。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mitosis is an elaborate process where the duplicated genome is segregated into two daughter cells. Cytokinesis, the final step of mitosis, is the physical separation of the two daughter cells. Failure in cytokinesis can result in polyploidy and genome instability, two hallmarks found in many cancers. During cytokinesis, signaling by the anaphase spindle generates an equatorial zone of active RhoA, which in turn directs the cortical accumulation of contractile ring components. Rho family GTPases cycle between an active GTP-bound and inactive GDP-bound form. This cycle is controlled by GEFs, which promote GTP loading, and GAPs, which inactivate small GTPases by stimulating their low intrinsic GTPase activity. During cytokinesis, it has been proposed that localization of the RhoA GEF to the spindle midzone provides a means to locally activate RhoA at the cell equator. However, as RhoGTPases are anchored in the plasma membrane with a lipid moiety and are thought to freely diffuse within the membrane, previous theoretical work has suggested that a global RhoA GAP activity would be necessary to maintain a focused equatorial zone of active Rho. In C. elegans RGA-3 and RGA-4 (RGA-3/4), two highly similar RhoGAPs, were previously shown to act preferentially on RhoA and localizes to the cleavage furrow and the microtubule asters. We find that RGA-3/4 are required to restrict the accumulation of contractile ring components including anillin and the septins to the equator of the cell during early stages of cytokinesis. During cleavage furrow ingression RGA-3/4 are also important to limit the zone of anillin and the septins to a tight region at the furrow tip. Taken together our findings support a model in which a RhoGAP helps to restrict the activation of RhoA to the cell equator. RGA-3/4. To understand how RGA-3/4 activity and/or localization are regulated through mitosis we aim to identify new binding partners of RGA-3/4 using mass spectrometry after immunoprecipitation in collaboration with The Center for Physiological Proteomics.