IDENTIFICATION OF RHOGAP INTERACTING PROTEINS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 有丝分裂是一个复杂的过程，其中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。胞质分裂的失败可导致多倍性和基因组不稳定性，这是许多癌症中发现的两个标志。在胞质分裂期间，后期纺锤体的信号传导产生活性RhoA的赤道区，其反过来指导收缩环组分的皮质积累。Rho家族GTP酶在活性GTP结合形式和非活性GDP结合形式之间循环。该循环由GEF和GAP控制，GEF促进GTP负载，GAP通过刺激小GTP酶的低内在GTP酶活性来抑制小GTP酶。在胞质分裂期间，已经提出RhoA GEF定位于纺锤体中间区提供了在细胞赤道处局部激活RhoA的手段。然而，由于RhoGTP酶与脂质部分锚定在质膜中，并且被认为在膜内自由扩散，因此先前的理论工作表明，全球RhoA GAP活性对于维持活性Rho的集中赤道区是必要的。In C.线虫RGA-3和RGA-4（RGA-3/4）是两种高度相似的RhoGAP，先前显示优先作用于RhoA，并定位于卵裂沟和微管星状体。我们发现，RGA-3/4是必需的，以限制积累的收缩环组件，包括苯胺和septins的赤道的细胞在胞质分裂的早期阶段。在卵裂沟侵入过程中，RGA-3/4也是重要的，它将苯胺和隔蛋白的区域限制在沟尖的一个紧密区域。总之，我们的研究结果支持一个模型，其中RhoGAP有助于限制RhoA的激活细胞赤道。RGA-3/4。为了了解RGA-3/4活性和/或定位如何通过有丝分裂进行调节，我们的目标是与生理蛋白质组学中心合作，在免疫沉淀后使用质谱法鉴定RGA-3/4的新结合伴侣。

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.