Mechanism of ECM regulation of actin nucleation during morphogenesis.

项目摘要/摘要 癌细胞的生长和扩散需要改变细胞的极性，以及在 肌动蛋白细胞骨架的组织。虽然我们的研究发现了一些复合体 控制肌动蛋白的细胞骨架，我们现在需要确定这些肌动蛋白是如何成核的 复合体在发育过程中被极化。我们正在识别细胞外的线索和 调节这些复合体组织能力的信号通路的组成部分 细胞骨架的极性和启动迁移。在模型生物体中使用基因敲除突变体， 秀丽线虫，我们已经证明了GTPase CED-10/rac1的丢失，任何成分 肌动蛋白核化Arp2/3复合体，或其激活剂，波/疤痕复合体，导致 相同的表型：胚胎细胞迁移、形态发生和改变失败 上皮极性。我们指的是由rac1编码的肌动蛋白成核盒- WAVE/SCAR-Arp2/3作为Gex(GTP酶/肌动蛋白成核增强子 执行)复杂。哺乳动物中Gex分子的同源物在 癌症。例如，WAVE2在恶性肺癌和肺癌中错误表达 转移性结直肠癌(Semba等人2006年；岩屋等人。2007)。然而，这些是如何 肌动蛋白核蛋白在转移过程中是否受到错误调控尚不清楚。我们有 确定了一些细胞外基质(ECM)轴突引导分子，除了 它们在轴突中的作用，影响胚胎的迁移，并调节Gex的水平 蛋白质。 目的/假设：我们假设ECM信号通过以下方式调节细胞骨架的极性 在特定区域招募、稳定和/或激活Gex肌动蛋白核复合体 细胞的数量。我们建议确定指导线索，并分析这些上游线索是如何 信号激活Gex组件以启动迁徙。 具体目的：(1)确定细胞外基质受体是否调节肌动蛋白 成核作用。(2)确定哪些组织正在发送和接收引导信号 胚胎中的细胞迁移。(3)利用靶向遗传学寻找新的调控因子 性爱情结。研究设计：在目标1中，我们确定细胞外基质受体在 极化F-肌动蛋白的丰富，并测试ECM受体是否需要Gex复合体才能影响 肌动蛋白在形态发生过程中的丰富。在目标2中，我们确定了组织的结构 通过测试哪些组织需要Gex和ECM组件的活性来传递信号 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Gex突变体进行的迁移。临床相关性：有能力 癌细胞的迁移与恶性进展和转移密切相关。 我们在线虫中发现的一个基因WAVE3的人类同源物是 神经母细胞瘤中表达下调，WAVE3突变与 神经母细胞瘤和肌动蛋白细胞骨架的变化。我们的研究可能会确定基因 癌症检测的靶点，并提供对转移的机械性洞察。

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.