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: 10558623
• 负责人: Hsin-Yi Henry Ho
• 金额: $ 43.13万
• 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/10558623
• 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; Cytoplasm; Cytoskeleton; Cytosol; DNA Sequence Alteration; Defect; Development; Disease; Dsh protein; Elements; Exhibits; Face; Genetic; Goals; Human; Knock-out; Ligands; Limb structure; Measurement; Mediating; Morphogenesis; Mutation; Pathogenesis; Pathogenicity; Pathway interactions; 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; cell behavior; cell motility; experimental study; human disease; insight; interest; novel; pharmacologic; receptor; three dimensional cell culture; transmission process

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.

项目摘要/摘要 WNT5A-ROR信号传导是一种控制控制的发育信号通路 形态学细胞和组织行为。脊椎动物中途径的不正体导致深层组织 伸长缺陷，包括缩短和扩大体轴，四肢和面部。在人类中，突变 在该路径的关键节点中 细胞质信号传感器染色（DVL）1和DVL3，产生robinow综合征，先天性 具有高度相似组织伸长表型的疾病。值得注意的是，斗牛犬暴露了类似的物理 特征并携带DVL2中的突变，类似于DVL1和DVL3中的人类突变，该突变降低了 它响应WNT5A-ROR信号的能力。尽管其身体和临床重要性，但生化 介导Wnt5a-ror信号传导的步骤和细胞骨架机制在很大程度上保持了未表征。 因此，对疾病机制驱动robinow综合征的见解尚不清楚。总体 我们研究的目的是在生化，细胞和 生物水平。具体而言，我们在此提案中提出：1）ROR/FZD共受体复合物如何传输 Wnt5a在细胞表面的信号，ROR2中的致病突变如何改变接收器复杂函数？ 2）DVL脚手架蛋白如何中继WNT5A-ROR信号在细胞质中以及人类DVL1中的突变如何 DVL3和犬DVL2破坏DVL功能？ 3）WNT5A-ROR途径如何参与细胞骨架 改变细胞行为和生物力学，途径中的疾病突变如何扰动这些过程？ 为了解决这些问题，我们开发了新的记者测定法，以实现定量测量 活细胞中的Wnt5a-ror信号传导活性。我们还在 我们可以很容易地在近源水平上敲出感兴趣的蛋白质以挽救信号传导。 使用这种方法，我们将进行详细的ROR2和DVL结构功能分析以识别结构 这些蛋白质各自功能所需的要素和机制。这些实验将是 由蛋白质结合研究完成，以定义ROR2和DVL蛋白质相互作用网络以及它们如何 破坏有助于疾病发病机理。为了阐明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菌素的收缩力的信号传导。这些 观察结果与Rhoa-MLC-肌球蛋白的生化和亚细胞定位变化一致 监管网络。我们将进行药物和遗传扰动实验以剖析 该网络在正常和致病的WNT5A指导细胞行为中的功能。成功完成 该项目的第一个详细的机械肖像是WNT5A-ROR信号网络并亮起的 WNT5A-ROR驱动疾病的致病机制。