RhoC GTPase regulates leading edge protrusion dynamics in breast carcinomas

描述（申请人提供）：Rho家族的p21小GTP酶在调节细胞运动背景下的细胞骨架重排中起重要作用。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 GTP酶是两种Rho同种型RhoA和RhoC。存在的文献证据表明，Rho GTP酶的这两种亚型可能对癌症转移产生相反的作用，但对可能导致这种过程的信号传导途径的详细分析严重缺乏。在这项工作中，我们阐明的机制，RhoC赋予高度特异性的下游信号传导的影响不同RhoA和在单独的细胞隔室在EGF刺激的运动过程中的细胞突起的前沿。我们将通过使用本文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动来解决这个问题。目标1：使用完全基因编码的方法在单个活细胞中同时实时可视化两种蛋白质活性。目标2：为下游Rho效应物ROCK-1和mDia 1开发新的生物传感器，可与特定的上游Rho亚型Aim 3一起同时可视化：研究信号协调RhoC及其下游效应物通路在前沿Aim 4的时空分离：研究RhoA及其下游效应子通路的时空信号调节机制，这些研究将产生有价值的新技术在Rho GT3亚型及其直接下游效应物激活的直接可视化中，使得能够进一步时空描绘信号传导机制。通过这些研究，我们将能够剖析乳腺癌中通过Rho亚型的差异活性控制的前缘突起的机制，并使我们能够解决RhoC在乳腺癌前缘产生局部和极化突起中所起的特定作用。 公共卫生相关性：p21小GTP酶的Rho亚家族已被假定为在空间和时间上表现出复杂的活动协调，这取决于刺激细胞运动的特定环境线索。然而，这些协调平衡的GTdR活动的高分辨率成像研究一直严重缺乏，主要是由于成像活细胞中的多种蛋白质“活动”的技术挑战。Rho-GTP酶在癌细胞侵袭和迁移的背景下与细胞骨架重组直接相关，是两种Rho同种型RhoA和RhoC。在这里，我们同时可视化这些Rho亚型的活动与激活其下游效应在一个单一的活乳腺癌细胞和地址的信号传导事件，协调前沿突起响应EGF刺激。我们的主要假设是，RhoC激活乳腺癌在响应生长因子的刺激，可以赋予亚细胞信号极化机制，促进有效的前沿通过隔离的“高度活跃”与“完全抑制”区的肌动蛋白聚合的前沿前突。

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.