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.

项目总结/摘要 Wnt 5a-Ror信号传导是一种进化上保守的发育信号传导途径， 形态发生细胞和组织行为。在脊椎动物中，该途径的失调导致深层组织 伸长缺陷，包括体轴、四肢和面部的缩短和加宽。在人类中， 在途径的关键节点，包括WNT 5A配体，ROR 2和Frizzled（FZD 2）共受体，以及 细胞质信号转导紊乱（DVL）1和DVL 3，引起Robinow综合征，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，斗牛犬表现出类似的身体 在DVL 2中携带突变，类似于DVL 1和DVL 3中的人类突变， 其响应Wnt 5a-Ror信号的能力。尽管其在生理和临床上的重要性， 介导Wnt 5a-Ror信号传导的步骤和细胞骨架机制仍然在很大程度上未被表征; 因此，对驱动Robinow综合征的疾病机制的了解是未知的。总体 我们的研究目标是从生物化学、细胞学和免疫学角度剖析Wnt 5a-Ror通路的功能和调控， 有机体水平。具体来说，我们在这个建议中提出：1）Ror/FZD共受体复合物如何传递 Wnt 5a在细胞表面发出信号，ROR 2中的致病性突变如何改变受体复合物的功能？ 2)Dvl支架蛋白如何在胞质溶胶中传递Wnt 5a-Ror信号，以及人DVL 1中的突变如何 DVL 3和犬DVL 2会破坏DVL功能？3)Wnt 5a-Ror通路如何参与细胞骨架 改变细胞行为和生物力学，以及疾病突变如何干扰这些过程？ 为了解决这些问题，我们开发了新的报告基因测定， 活细胞中的Wnt 5a-Ror信号传导活性。我们还开发了一种高度生理化的细胞培养系统， 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传导。 使用这种方法，我们将进行详细的ROR 2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件和机制。这些实验将 通过蛋白质结合研究来补充，以定义ROR 2和DVL蛋白质相互作用网络，以及它们如何相互作用。 破坏有助于疾病的发病机理。为了阐明该通路的细胞生物学功能，我们 优化了用于细胞行为分析的二维和三维培养系统，并确定了Wnt 5a-Ror的关键作用。 在控制细胞迁移、应力纤维稳定和基于肌动球蛋白的收缩性中的信号传导。这些 观察结果与RhoA-MLC-肌动球蛋白中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， 该网络在正常和致病性Wnt 5a-Ror指导的细胞行为中的功能。圆满完成 该项目将产生Wnt 5a-Ror信令网络的第一个详细的机械图，并阐明 Wnt 5a-Ror驱动的疾病的致病机制。