Mechanism of ECM regulation of actin nucleation during morphogenesis.

描述(申请人提供)：癌细胞的生长和扩散需要改变细胞的极性和肌动蛋白细胞骨架的组织。虽然我们的研究已经确定了控制肌动蛋白细胞骨架的复合体，但我们现在需要确定这些肌动蛋白核复合体在发育过程中是如何极化的。我们正在确定细胞外信号和信号通路的组成部分，这些信号通路调节这些复合体组织细胞骨架极性和启动迁移的能力。利用模式生物秀丽线虫的基因敲除突变体，我们已经证明了GTPase CED-10/rac1(肌动蛋白核化Arp2/3复合体的任何组成部分)或其激活剂WAVE/SCAR复合体的缺失会导致相同的表型：胚胎细胞迁移失败、形态发生和上皮极性改变。我们将由rac1-Wave/scar-Arp2/3编码的肌动蛋白核心盒称为Gex(GTP酶/肌动蛋白核化执行增强因子)复合体。哺乳动物中Gex分子的同源物在癌症中受到错误调控。例如，WAVE2在恶性肺癌和转移性结直肠癌中错误表达(Semba等人。2006年；岩屋等人。2007)。然而，这些肌动蛋白核蛋白是如何在转移过程中被错误调控的还不清楚。我们已经确定，一些细胞外基质(ECM)轴突引导分子，除了在轴突中的作用外，还影响胚胎的迁移，并调节Gex蛋白的水平。目的/假设：我们假设ECM信号通过重新招募、稳定和/或激活细胞特定区域的Gex肌动蛋白核复合体来调节细胞骨架的极性。我们建议识别引导线索，并分析这些上游信号如何激活Gex组件来启动迁徙。具体目的：(1)确定细胞外基质受体是否调节肌动蛋白的核化。(2)确定哪些组织正在发送和接收导致胚胎细胞迁移的信号。(3)利用靶向遗传学识别Gex复合体的新调控因子。研究设计：在目标1中，我们确定ECM受体在极化F-肌动蛋白浓缩中的作用，并测试ECM受体是否需要Gex复合体在形态发生过程中影响肌动蛋白浓缩。在目标2中，我们通过测试哪些组织在形态发生运动中需要Gex和ECM成分的活性来确定组织信号的架构。在目标3中，我们通过分子克隆新的Gex突变体来鉴定新的胚胎细胞迁移调节因子。临床意义：癌细胞的迁移能力与恶性进展和转移密切相关。我们在线虫中发现的基因之一的人类同源基因WAVE3在神经母细胞瘤中下调，WAVE3的一个突变与神经母细胞瘤和肌动蛋白细胞骨架的变化有关。我们的研究可能确定癌症检测的基因靶点，并提供对转移的机械性洞察。公共卫生相关性：这些关于细胞迁移调控的研究解决了发展和癌症研究中的一个主要问题：如何启动和控制细胞迁移以实现健康生长。在人类发育过程中，我们需要确定确保健康成长的分子。为了在癌症扩散之前进行治疗，我们需要了解转移过程是如何调控的。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: These studies on the regulation of cell migrations address a major question in development and in cancer research: how cell migrations are initiated and controlled for healthy growth. During human development we need to identify the molecules that ensure healthy growth. To treat cancers before they spread, we need to understand how the process of metastasis is regulated.