Mechanism of ECM regulation of actin nucleation during morphogenesis.

项目总结/摘要 癌细胞的生长和扩散需要改变细胞极性， 肌动蛋白细胞骨架的组织。虽然我们的研究已经确定了复合物， 控制肌动蛋白细胞骨架，我们现在需要确定这些肌动蛋白成核 复合体在发育过程中被极化。我们正在识别细胞外信号， 调节这些复合物组织能力的信号通路组分 细胞骨架极性和启动迁移。在模式生物中使用敲除突变体， C.我们已经证明，GTCED-10/Rac 1的缺失， 肌动蛋白成核Arp 2/3复合物，或其激活剂WAVE/SCAR复合物，导致 相同的表型：胚胎细胞迁移失败，形态发生和改变 上皮极性我们将Rac 1编码的肌动蛋白成核盒称为- WAVE/SCAR-Arp 2/3作为格克斯（GTX/成核增强剂/肌动蛋白成核 eXtreme）复合物。哺乳动物中格克斯分子的同源物在 癌的例如，WAVE 2在恶性人类肺癌中错误表达， 转移性结肠直肠癌（Semba et al. 2006; Iwaya et al. 2007）。然而，这些 肌动蛋白成核蛋白在转移过程中被错误调节还不清楚。我们有 确定了一些细胞外基质（ECM）轴突导向分子，除了 它们在轴突中的作用，影响胚胎迁移，并调节格克斯的水平 proteins. 目的/假设：我们假设ECM信号通过以下方式调节细胞骨架极性： 募集、稳定和/或激活特定区域中的格克斯肌动蛋白成核复合物 信元我们建议确定的指导线索，并分析如何这些上游 信号激活格克斯组件以启动迁移。 具体目的：（1）确定细胞外基质受体是否调节肌动蛋白 成核(2)To确定哪些组织正在发送和接收信号， 胚胎中的细胞迁移。(3)使用靶向遗传学来识别新的调节因子， 格克斯复合体。研究设计：在目的1中，我们确定ECM受体在 极化的F-肌动蛋白富集，并测试ECM受体是否需要格克斯复合物来影响 肌动蛋白在形态发生中的富集。在目标2中，我们确定组织的结构 通过测试哪些组织需要格克斯和ECM组分的活性来进行信号传导， 形态发生运动在目标3中，我们确定了胚胎细胞的新调节因子， 通过分子克隆新的格克斯突变体进行迁移。临床相关性： 癌细胞迁移与恶性进展和转移密切相关。 我们在C. elegans，WAVE3， 在神经母细胞瘤中下调，WAVE 3的突变与 神经母细胞瘤和肌动蛋白细胞骨架的变化。我们的研究可能会发现 用于癌症检测的靶点，并提供对转移的机制性洞察。

Project Summary/Abstract The growth and spread of cancerous cells requires alterations in cell polarity, and in the organization of the actin cytoskeleton. While our studies have identified complexes that control the actin cytoskeleton, we now need to determine how these actin nucleation complexes are polarized during development. We are identifying extracellular cues and components of signaling pathways that regulate the ability of these complexes to organize cytoskeletal polarity and initiate migrations. Using knockout mutants in a model organism, C. elegans, we have shown that loss of the GTPase CED-10/Rac1, any component of the actin nucleating Arp2/3 complex, or of its activator, the WAVE/SCAR complex, results in the same phenotype: failure in embryonic cell migrations, morphogenesis and altered epithelial polarity. We refer to the actin nucleation cassette encoded by Rac1- WAVE/SCAR-Arp2/3 as the GEX (GTPase/Enhancer of nucleation/actin nucleation eXecution) complex. Mammalian homologs of GEX molecules are misregulated in cancers. For example, WAVE2 is misexpressed in malignant human lung cancers and metastatic colorectal cancers (Semba et al. 2006; Iwaya et al. 2007). However, how these actin nucleation proteins are misregulated during metastasis is not understood. We have determined that some extracellular matrix (ECM) axonal guidance molecules, in addition to their role in axons, affect embryonic migrations, and they regulate the levels of GEX proteins. Objective/Hypothesis: We hypothesize that ECM signals regulate cytoskeletal polarity by recruiting, stabilizing, and/or activating GEX actin nucleation complexes in specific regions of cells. We propose to identify the guidance cues and to analyze how these upstream signals activate the GEX components to initiate migrations. Specific Aims: (1) To determine whether extracellular matrix receptors regulate actin nucleation. (2)To determine which tissues are sending and receiving the signals that lead to cell migrations in the embryo. (3) To use targeted genetics to identify new regulators of the GEX complex. Study design: In Aim 1 we determine the role of ECM receptors in polarized F-actin enrichment, and test if ECM receptors require the GEX complex to affect actin enrichment during morphogenesis. In Aim 2 we determine the architecture of tissue signaling by testing which tissues require the activity of GEX and ECM components during morphogenetic movements. In Aim 3 we identify new regulators of embryonic cell migrations by molecularly cloning new gex mutants. Clinical relevance: The ability of cancer cells to migrate is strongly correlated with malignant progression and metastasis. The human homolog of one of the genes we have identified in C. elegans, WAVE3, is down regulated in neuroblastomas and a mutation in WAVE3 has been connected to neuroblastoma and to changes in the actin cytoskeleton. Our studies may identify genetic targets for cancer detection and provide mechanistic insight into metastasis.