Function and regulatory mechanisms of the Wnt5a-Ror morphogenetic pathway

Wnt5a-Ror形态发生途径的功能和调控机制

• 批准号: 10336968
• 负责人: Hsin-Yi Henry Ho
• 金额: $ 42.85万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10336968
• 关键词: Actomyosin; Address; Automobile Driving; Behavior; Behavioral; Binding Proteins; Biochemical; Biological Assay; Biological Process; Biomechanics; Canis familiaris; Cell Culture System; Cell surface; Cells; Characteristics; Clinical; Complement; Complex; Congenital Abnormality; Congenital Disorders; Cytoskeleton; Cytosol; DNA Sequence Alteration; Defect; Development; Disease; Dsh protein; Elements; Exhibits; FZD2 gene; Face; Genetic; Goals; Human; Knock-out; Ligands; Limb structure; Measurement; Mediating; Morphogenesis; Mutation; Pathogenesis; Pathogenicity; Pathway interactions; Pharmacology; Phenotype; Physiological; Portraits; Process; Proteins; Regulation; Reporter; Research; Robinow syndrome; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Stress Fibers; Structure; Tissues; Transducers; Vertebrates; WNT5A gene; base; cell behavior; cell motility; experimental study; human disease; insight; interest; novel; receptor; three dimensional cell culture

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.

项目概要/摘要 Wnt5a-Ror 信号是一种进化上保守的发育信号通路，控制 形态发生细胞和组织行为。脊椎动物通路的失调导致深层组织 伸长缺陷，包括身体轴、四肢和面部的缩短和加宽。在人类中，突变 该通路的关键节​​点，包括 WNT5A 配体、ROR2 和卷曲 (FZD2) 共受体以及 细胞质信号转导器 Disheveled (DVL) 1 和 DVL3，引起 Robinow 综合征，这是一种先天性 具有高度相似的组织伸长表型的疾病。值得注意的是，斗牛犬表现出相似的身体特征 特征并携带 DVL2 突变，类似于人类 DVL1 和 DVL3 突变，从而减少 其响应 Wnt5a-Ror 信号的能力。尽管其生理和临床重要性，生化 介导 Wnt5a-Ror 信号传导的步骤和细胞骨架机制在很大程度上仍未得到表征； 因此，对导致 Robinow 综合征的疾病机制的了解尚不清楚。首要的 我们研究的目标是剖析 Wnt5a-Ror 通路在生化、细胞和 有机体水平。具体来说，我们在该提案中提出以下问题：1）Ror/FZD 共受体复合物如何传输 Wnt5a 在细胞表面发出信号，ROR2 的致病性突变如何改变受体复合物功能？ 2) Dvl支架蛋白如何在细胞质中传递Wnt5a-Ror信号，以及人DVL1中的突变如何发生 DVL3 和犬 DVL2 会破坏 DVL 功能吗？ 3) Wnt5a-Ror通路如何参与细胞骨架 改变细胞行为和生物力学，以及通路中的疾病突变如何扰乱这些过程？ 为了解决这些问题，我们开发了新颖的报告分析方法，可以定量测量 活细胞中的 Wnt5a-Ror 信号传导活性。我们还开发了高度生理化的细胞培养系统 我们可以很容易地敲除并以接近内源的水平重新表达感兴趣的蛋白质，以拯救信号传导。 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 这些蛋白质各自功能所需的元素和机制。这些实验将 辅之以蛋白质结合研究，以定义 ROR2 和 DVL 蛋白质相互作用网络以及它们如何 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 优化了用于细胞行为分析的 2D 和 3D 培养系统，并确定了 Wnt5a-Ror 的关键作用 控制细胞迁移、应力纤维稳定和基于肌动球蛋白的收缩性的信号传导。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一致 监管网络。我们将进行药理学和遗传扰动实验来剖析 该网络在正常和致病性 Wnt5a-Ror 定向细胞行为中的功能。顺利完成 该项目的研究将产生 Wnt5a-Ror 信号网络的第一个详细机制描述，并阐明 Wnt5a-Ror 驱动疾病的致病机制。