Deciphering Wnt-Ror signaling in cytoskeletal regulation and tissue shape control

解读细胞骨架调节和组织形状控制中的 Wnt-Ror 信号传导

• 批准号: 9323512
• 负责人: Hsin-Yi Henry Ho
• 金额: $ 38.48万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9323512
• 关键词: Adhesions; Biochemical; Biological; Biological Assay; Biology; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Congenital Abnormality; Congenital Disorders; Cytoskeleton; Development; Disease; Equipment and supply inventories; Family; Genetic; Genetic Screening; Genetic Transcription; Human; Kinesin; Knowledge; Link; Measurement; Mediating; Microscopy; Microtubules; Molecular; Morphogenesis; Motor; Mus; Neoplasm Metastasis; Organ; Pathologic; Pathway interactions; Play; Portraits; Process; Protein Biochemistry; Proteomics; Receptor Activation; Regulation; Reporter; Research; Robinow syndrome; Role; Shapes; Signal Pathway; Signal Transduction; Stereotyping; System; Time; Tissues; Ubiquitin; Work; base; cell behavior; cell motility; combat; comparative; embryo tissue; human disease; in vivo; insight; loss of function; member; multicatalytic endopeptidase complex; new therapeutic target; novel; novel marker; novel strategies; programs; signal processing

A long-standing question in biology concerns how tissues and organs acquire their stereotyped shape during development. The Wnt5a-Ror signaling pathway is a master regulator of embryonic tissue morphogenesis, and deregulation of the pathway has been found to cause a broad range of human pathological conditions, including the congenital disorders Robinow syndrome and Brachydactyly Type B, as well as cancer metastasis. In contrast to most well characterized developmental signaling pathways that function via gene transcription, the Wnt5a-Ror pathway functions through cytoskeletal regulation to control key morphogenetic cell behaviors, such as cell migration, polarization and adhesion. However, the molecular mechanisms that underlie Wnt5a- Ror function remain enigmatic. Our research program aims to fill three major gaps in the field: (1) What are the biochemical interactions that mediate Wnt5a-Ror signal processing and propagation within cells? (2) How does Wnt5a-Ror signaling engage the cytoskeleton to control morphogenetic cell behaviors? (3) How do these Wnt5a/Ror-driven processes ultimately control tissue morphogenesis in vivo? To this end, we have integrated mouse genetics and comparative proteomics to construct the first extended inventory of Wnt5a-Ror pathway components. This work not only provided crucial insights into the molecular mechanism of Wnt5a-Ror signal transduction, but also identified Kif26b (a member of the kinesin microtubule motor family) as a critical cytoskeletal effector of the pathway. Through gain- and loss-of-function studies, we demonstrated that Kif26b mediates the ability of the Wnt5a-Ror pathway to control cell migration, and that this function of Kif26b is conserved from C. elegans to humans. Mechanistically, we have established the key finding that Wnt5a-Ror signaling controls the cellular steady-state concentration of Kif26b via a mechanism involving the ubiquitin- proteasome system. Using this novel Wnt5a-Ror-Kif26b signaling paradigm, we have successfully developed a reporter assay that for the first time, allows for quantitative measurement of Wnt5a-Ror signaling activity in live cells. In this application, we propose to use a combination of protein biochemistry, microscopy and genetics to elucidate the molecular mechanism linking Ror receptor activation to Kif26b degradation, the cell biological mechanism underlying Kif26b regulation of cytoskeletal dynamics and cell migration, and the in vivo role of the Wnt5a-Ror-Kif26b signaling cassette in embryonic tissue morphogenesis. Moreover, we will pair our Wnt5a- Ror signaling reporter with large-scale CRISPR/Cas9-based genetic screens to identify additional constituents of the pathway. The successful completion of the project will (1) provide the first detailed molecular portrait of the Wnt5a-Ror signaling network, (2) reveal the cell biological mechanisms by which Wnt5a-Ror signaling regulates cytoskeletal dynamics and tissue morphogenesis, and (3) suggest novel biomarkers and therapeutic targets for Wnt5a-Ror driven diseases.

生物学中一个长期存在的问题是，组织和器官在发育过程中是如何获得它们的定型形状的。 发展Wnt 5a-Ror信号通路是胚胎组织形态发生的主要调节因子， 已经发现该途径的失调引起广泛的人类病理状况， 包括先天性疾病Robinow综合征和B型短指（趾）畸形以及癌症转移。 与大多数通过基因转录发挥功能的发育信号传导途径不同， Wnt 5a-Ror途径通过细胞骨架调节来控制关键的形态发生细胞行为， 例如细胞迁移、极化和粘附。然而，Wnt 5a的分子机制- Ror函数仍然是个谜。我们的研究计划旨在填补该领域的三个主要空白：（1） 介导Wnt 5a-Ror信号处理和细胞内传播的生物化学相互作用？(2)如何 Wnt 5a-Ror信号参与细胞骨架控制形态发生细胞行为？(3)这些怎么 Wnt 5a/Ror驱动的过程最终控制体内组织形态发生？为此，我们整合了 小鼠遗传学和比较蛋白质组学构建Wnt 5a-Ror通路的第一个扩展清单 件.这项工作不仅为Wnt 5a-Ror信号的分子机制提供了重要的见解， 转导，但也确定Kif 26 b（驱动蛋白微管运动家族的成员）作为关键的 细胞骨架效应子的途径。通过功能获得和丧失研究，我们证明Kif 26 b 介导Wnt 5a-Ror途径控制细胞迁移的能力，并且Kif 26 b的这种功能是 从C. elegans to humans人类.从机制上讲，我们已经确定了Wnt 5a-Ror 信号转导通过涉及泛素的机制控制Kif 26 b的细胞稳态浓度， 蛋白酶体系统使用这种新的Wnt 5a-Ror-Kif 26 b信号转导模式，我们成功地开发了一种新的Wnt 5a-Ror-Kif 26 b信号转导系统。 报告基因测定，首次允许定量测量活体中Wnt 5a-Ror信号传导活性， 细胞在这个应用中，我们建议使用蛋白质生物化学，显微镜和遗传学的组合， 阐明Ror受体活化与Kif 26 b降解的分子机制，细胞生物学特性， Kif 26 b调节细胞骨架动力学和细胞迁移的潜在机制，以及Kif 26 b在体内的作用。 胚胎组织形态发生中的Wnt 5a-Ror-Kif 26 b信号传导盒。此外，我们将配对我们的Wnt 5a- Ror信号转导报告基因与基于CRISPR/Cas9的大规模遗传筛选相结合，以识别其他成分 的路径。该项目的成功完成将（1）提供第一个详细的分子画像， Wnt 5a-Ror信号网络，（2）揭示Wnt 5a-Ror信号转导的细胞生物学机制 调节细胞骨架动力学和组织形态发生，和（3）提出新的生物标志物和治疗 针对Wnt 5a-Ror驱动的疾病的靶点。