Function and regulatory mechanisms of the Wnt5a-Ror morphogenetic pathway

项目摘要/摘要 Wnt5A-Ror信号是一条进化上保守的发育信号通路，控制着 形态发生细胞和组织行为。脊椎动物中该途径的错误调节导致深层组织 伸长缺陷，包括体轴、四肢和面部的缩短和加宽。在人类中，突变 在该途径的关键节点，包括Wnt5A配体、ROR2和Frizzled2(FZD2)共受体，以及 胞浆信号转导不整齐(DVL)1和DVL3，导致罗比诺综合征，先天性 具有高度相似的组织伸长表型的疾病。值得注意的是，斗牛犬表现出类似的身体状况 DVL2的特征和携带的突变，类似于人类DVL1和DVL3的突变，该突变减少 它对Wnt5a-Ror信号的响应能力。尽管它在生理和临床上很重要，但生化 介导Wnt5a-Ror信号转导的步骤和细胞骨架机制在很大程度上仍未确定； 因此，对推动罗比诺综合症的疾病机制(S)的见解尚不清楚。最重要的是 我们的研究目的是从生化、细胞和生物化学角度剖析Wnt5a-Ror通路的功能和调控。 生物体水平。具体地说，我们在这个提案中问：1)ROR/FZD共受体复合体是如何传递 Wnt5A信号位于细胞表面，ROR2的致病突变如何改变受体复合体的功能？ 2)DVL支架蛋白如何在胞浆中传递Wnt5a-Ror信号，以及人类DVL1中的突变是如何 3)Wnt5a-Ror通路是如何与细胞骨架结合的？ 来改变细胞行为和生物力学，以及该途径中的疾病突变如何扰乱这些过程？ 为了解决这些问题，我们开发了新的报告分析，使定量测量 WNT5A-ROR信号在活细胞中的活性。我们还开发了一种高度生理的细胞培养系统 我们可以很容易地敲除并在接近内源性的水平上重新表达感兴趣的蛋白质，以拯救信号。 使用这种方法，我们将进行详细的ROR2和DVL结构-功能分析，以确定结构 这些蛋白质各自的功能所需的要素和机制。这些实验将是 辅以蛋白质结合研究，以定义ROR2和DVL蛋白质相互作用网络及其如何 破坏有助于疾病的发病机制。为了阐明该途径的细胞生物学功能，我们有 优化2D和3D培养系统以进行细胞行为分析并确定Wnt5a-Ror的关键作用 控制细胞迁移、应激纤维稳定和肌动球蛋白收缩的信号转导。这些 观察结果与RhoA-MLC-肌动球蛋白的生化和亚细胞定位变化相吻合 监管网络。我们将进行药理和基因扰动实验来解剖 该网络在正常和致病的Wnt5a-Ror导向的细胞行为中的作用。圆满完成 将产生WNT5a-Ror信令网络的第一个详细的机械肖像，并说明 Wnt5a-Ror致病机制的研究。

Project Summary/Abstract Wnt5a-Ror signaling is an evolutionarily conserved developmental signaling pathway that controls morphogenetic cell and tissue behavior. Misregulation of the pathway in vertebrates results in profound tissue elongation defects, including shortening and widening of the body axis, limbs, and face. In humans, mutations in key nodes of the pathway, including the WNT5A ligand, the ROR2 and Frizzled (FZD2) co-receptors, and the cytoplasmic signal transducers Dishevelled (DVL) 1 and DVL3, give rise to Robinow syndrome, a congenital disorder with highly similar tissue elongation phenotypes. Notably, bulldogs exhibit similar physical characteristics and carry a mutation in DVL2, analogous to the human mutations in DVL1 and DVL3, that reduces its capacity to respond to Wnt5a-Ror signals. Despite its physiological and clinical importance, the biochemical steps and cytoskeletal mechanisms that mediate Wnt5a-Ror signaling remain largely uncharacterized; consequently, insights into the disease mechanism(s) driving Robinow syndrome are unknown. The overarching goal of our research is to dissect Wnt5a-Ror pathway function and regulation at the biochemical, cellular and organismal levels. Specifically, we ask in this proposal: 1) How does the Ror/FZD co-receptor complex transmit Wnt5a signals at the cell surface, and how do pathogenic mutations in ROR2 alter receptor complex function? 2) How do Dvl scaffolding proteins relay Wnt5a-Ror signals in the cytosol, and how do mutations in human DVL1 and DVL3 and canine DVL2 disrupt DVL function? 3) How does the Wnt5a-Ror pathway engage the cytoskeleton to alter cell behavior and biomechanics, and how do disease mutations in the pathway perturb these processes? To address these questions, we have developed novel reporter assays that enable quantitative measurement of Wnt5a-Ror signaling activity in live cells. We have also developed a highly physiological cell culture system in which we can readily knock out and re-express proteins of interest at near-endogenous levels to rescue signaling. Using this approach, we will conduct detailed ROR2 and DVL structure-function analyses to identify the structural elements and mechanisms required for these proteins’ respective functions. These experiments will be complemented by protein binding studies to define ROR2 and DVL protein interaction networks and how their disruption contributes to disease pathogenesis. To elucidate the cell biological function of the pathway, we have optimized 2D and 3D culture systems for cell behavioral analyses and identified a critical role for Wnt5a-Ror signaling in controlling cell migration, stress fiber stabilization and actomyosin-based contractility. These observations coincide with biochemical and subcellular localization changes in the RhoA-MLC-actomyosin regulatory network. We will conduct pharmacological and genetic perturbation experiments to dissect the function of this network in normal and pathogenic Wnt5a-Ror-directed cell behaviors. The successful completion of this project will yield the first detailed mechanistic portrait of the Wnt5a-Ror signaling network and illuminate the pathogenic mechanisms of Wnt5a-Ror-driven diseases.