RhoC GTPase regulates leading edge protrusion dynamics in breast carcinomas

描述(由申请人提供)：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 GTP酶是两种Rho亚型RhoA和RhoC。有文献证据表明，这两种Rho GTP酶亚型可能在癌症转移中发挥相反的作用，但对可能促进这一过程的信号通路的详细分析一直非常缺乏。在这项工作中，我们阐明了在EGF刺激的运动过程中，RhoC给予不同于RhoA的高度特异的下游信号作用的机制，以及在细胞突起前沿的单独细胞间隔中的作用。我们将使用这里提出的新型生物传感器，通过直接可视化活细胞中的多种蛋白质活动来解决这个问题。目的：利用完全基因编码的方法，实时可视化单个活细胞中两种蛋白质的活性。目的：为下游Rho效应子ROCK-1和mDia1开发新的生物传感器，与特定的上游Rho异构体一起同时可视化。Aim3：研究在前沿协调RhoC及其下游效应通路的信号的时空分离。Aim4：研究在前沿协调RhoA及其下游效应通路的时空信号。这些研究将产生在直接可视化Rho GTP酶亚型及其直接下游效应激活方面有价值的新技术，使进一步描述信号机制成为可能。通过这些研究，我们将能够剖析乳腺癌前沿突起受Rho亚型不同活性控制的机制，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所起的具体作用。 与公共卫生相关：p21小GTP酶的Rho亚家族被认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，对这些GTPase活性的协调平衡的高分辨率成像研究一直非常缺乏，主要是因为在活细胞中成像多个蛋白质“活动”的技术挑战。在癌细胞侵袭和迁移的背景下，Rho-GTP酶直接与细胞骨架重组有关，是两种Rho亚型RhoA和RhoC。在这里，我们同时可视化了这些Rho异构体活动及其下游效应器在单个活的乳腺癌细胞中的激活，并解决了协调前沿突起响应EGF刺激的信号事件。我们的关键假设是，乳腺癌中RhoC在生长因子刺激下的激活可以传递一种亚细胞信号极化机制，该机制通过分离前沿内肌动蛋白聚合区的“高活性”和“完全抑制”区来促进前沿的有效向前突出。

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.