RhoC GTPase regulates leading edge protrusion dynamics in breast carcinomas

描述（由申请人提供）：p21小gtpase的Rho家族在细胞运动背景下调节细胞骨架重排中发挥重要作用。在癌细胞侵袭和迁移的背景下，与细胞骨架重组直接相关的Rho gtpase是两个Rho亚型RhoA和RhoC。文献证据表明，Rho gtpase的这两种亚型可能对癌症转移具有相反的作用，但对可能有助于这一过程的信号通路的详细分析严重缺乏。在这项工作中，我们阐明了在egf刺激的运动过程中，RhoC在细胞突起前沿的独立细胞室中传递与RhoA不同的高度特异性下游信号效应的机制。我们将通过使用在这里提出的新型生物传感器，直接可视化活细胞中同时存在的多种蛋白质活性来解决这个问题。目的1：利用完全基因编码的方法实时观察单个活细胞中两种蛋白质的活性。Aim2：开发下游Rho效应物ROCK-1和mDia1的新型生物传感器，可与特定的上游Rho异构体aim同时可视化；Aim3：研究信号协调RhoC及其下游效应物通路在前沿Aim4的时空隔离；研究协调RhoA及其下游效应通路的时空信号，这些研究将产生有价值的新技术，用于直接可视化Rho GTPase亚型及其直接下游效应激活，从而进一步描述信号机制的时空。通过这些研究，我们将能够剖析Rho异构体在乳腺癌中通过差异活动控制前沿突起的机制，并使我们能够解决RhoC在乳腺癌前沿局部和极化突起产生中的具体作用。

DESCRIPTION (provided by applicant): The Rho family of p21 small GTPases plays important roles in regulating cytoskeleton rearrangement in the context of cell motility. The Rho GTPases directly linked to cytoskeletal reorganization in the context of cancer cell invasion and migration is the two Rho isoforms RhoA and RhoC. Literature evidence exists that these two isoforms of Rho GTPases may impart opposing effects on cancer metastasis, yet detailed analysis of signaling pathways that could contribute to such process has been acutely lacking. In this work, we elucidate the mechanism by which RhoC imparts highly specific downstream signaling effects different than RhoA and in separate cellular compartments at the leading edge of cell protrusions during EGF-stimulated motility. We will address this problem by directly visualizing multiple protein activities simultaneously in living cells, using novel biosensors that are proposed here. Aim1: Visualize two protein activities simultaneously in single living cell and in real-time using fully genetically encoded approach. Aim2: Develop new biosensors for downstream Rho effectors ROCK-1 and mDia1, amenable to simultaneous visualization together with the specific upstream Rho isoform Aim3: Investigate the spatiotemporal segregation of signaling coordinating RhoC and its downstream effector pathways at the leading edge Aim4: Investigate the spatiotemporal signaling coordinating RhoA and its downstream effector pathways at the leading edge These studies will produce new technologies valuable in direct visualization of Rho GTPase isoforms and their immediate downstream effector activations, enabling further spatiotemporal delineation of signaling mechanisms. Through these studies, we will be able to dissect the mechanism of the leading edge protrusions controlled through differential activities of Rho isoforms in breast carcinomas and enable us to address the specific role RhoC plays in producing the localized and polarized protrusions at the leading edge of breast carcinomas. PUBLIC HEALTH RELEVANCE: Rho-subfamily of p21 small GTPases has been postulated to exhibit a complex coordination of their activities in space and time, depending on particular environmental cues stimulating cell motility. However, high-resolution imaging studies of these coordinated balance of GTPase activities have been acutely lacking, due primarily to the technical challenges of imaging multiple protein "activities" in living cells. Rho-GTPases directly linked to cytoskeletal reorganization in the context of cancer cell invasion and migration, are the two Rho isoforms RhoA and RhoC. Here, we visualize simultaneously these Rho isoform activities together with activations of their downstream effectors in a single living breast carcinoma cell and address signaling events that coordinate the leading edge protrusions in response to EGF stimulation. Our key hypothesis is that RhoC activation in breast carcinomas in response to growth factor stimulation could impart a subcellular signal polarization mechanism that promotes effective forward protrusion of the leading edge through segregation of "highly active" versus "complete inhibitive" zones of actin polymerization within the leading edge.