RhoC GTPase regulates leading edge protrusion dynamics in breast carcinomas

RhoC GTPase 调节乳腺癌前缘突起动力学

• 批准号: 7861954
• 负责人: Louis Hodgson
• 金额: $ 31.54万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7861954
• 关键词: Accounting; Actins; Address; Binding; Biological Markers; Biosensor; Breast Carcinoma; Cells; Cessation of life; Clinical; Complex; Cues; Cytoskeletal Modeling; Cytoskeleton; DNA Sequence Rearrangement; Diagnosis; Diagnostic Procedure; Disease; EGF gene; Equilibrium; Event; Exhibits; Family; Functional disorder; GTP Binding; Growth Factor; Guanosine Triphosphate Phosphohydrolases; Image; Imagery; In Vitro; Life; Link; Literature; Malignant Epithelial Cell; Malignant Neoplasms; Membrane; Molecular; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; Pathway interactions; Play; Process; Protein Isoforms; Proteins; Resolution; Role; Signal Pathway; Signal Transduction; Staging; Time; Up-Regulation; Woman; Work; cancer cell; cancer diagnosis; cell motility; in vivo; malignant breast neoplasm; migration; new technology; novel; polymerization; public health relevance; response; rho; rho GTP-Binding Proteins; segregation; spatiotemporal; time use; tumor progression

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.

描述（由申请人提供）：P21小GTPases的Rho家族在调节细胞运动性的背景下在调节细胞骨架重排方面起着重要作用。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 GTPases是两种Rho同工型RhoA和RHOC。文献证据表明，Rho GTPases的这两种同工型可能会对癌症转移产生相反的影响，但对可能导致这种过程的信号传导途径的详细分析一直缺乏。在这项工作中，我们阐明了RHOC在EGF刺激的运动中赋予与RhoA高度特异性的下游信号传导效应不同，而在细胞突起的前缘的单独细胞隔室中。我们将使用此处提出的新型生物传感器直接在活细胞中同时可视化多种蛋白质活性来解决这个问题。 AIM1：使用完全遗传编码的方法在单个活细胞中同时和实时地观察两种蛋白质活性。 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研究将产生在直接可视化Rho GTPase同工型及其直接下游效应子激活中有价值的新技术，从而实现了信号传导机制的进一步时空描述。通过这些研究，我们将能够剖析通过Rho同工型在乳腺癌中的差异活性控制的前缘突起的机制，并使我们能够解决RHOC在乳腺癌的领先边缘产生局部和极化突起方面的特定作用。 公共卫生相关性：假定P21小型GTPases的Rho-Subframily根据刺激细胞运动的特定环境提示，表现出其在时空活动的复杂协调。然而，主要缺乏对GTPase活动的这些协调平衡的高分辨率成像研究，这主要是由于成像活细胞中多种蛋白“活性”的技术挑战。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同工型RhoA和RhoC。在这里，我们同时可视化这些RHO同工型活动，以及它们在单个活乳腺癌细胞中其下游效应子的激活，并解决信号事件，以响应EGF刺激，以协调前缘突起。我们的关键假设是，乳腺癌中的RHOC激活对生长因子刺激响应生长因子刺激可能会赋予细胞信号极化机制，该机制通过隔离“高度活性”和“完全抑制性”的肌动蛋白聚合区域内促进前缘的有效向前突出。