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: 9749980
• 负责人: Hsin-Yi Henry Ho
• 金额: $ 39.25万
• 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/9749980
• 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.

生物学上的一个长期问题涉及组织和器官如何在期间获得其定型形状 发展。 Wnt5a-ror信号通路是胚胎组织形态发生的主要调节剂，并且 已经发现对途径的放松管制会导致广泛的人类病理状况， 包括先天性疾病robinow综合征和B型型B型，以及癌症转移。 与最有特征性的发育信号通路相反，该信号通路通过基因转录起作用， WNT5A-ROR途径通过细胞骨架调节发挥作用，以控制关键的形态发生细胞行为， 例如细胞迁移，极化和粘附。但是，Wnt5a-的分子机制 ROR功能仍然神秘。我们的研究计划旨在填补该领域的三个主要空白：（1） 介导细胞内Wnt5a-ror信号处理和传播的生化相互作用？ （2）如何 WNT5A-ROR信号传导使细胞骨架接合以控制形态发生细胞行为？ （3）如何 WNT5A/ROR驱动过程最终控制体内组织形态发生？为此，我们已经整合了 小鼠遗传学和比较蛋白质组学，以构建Wnt5a-Ror途径的第一个扩展清单 成分。这项工作不仅为WNT5A-ROR信号的分子机制提供了重要的见解 转导，但也将KIF26B（驱动蛋白微管运动家族的成员）确定为关键 途径的细胞骨架效应子。通过获得功能丧失研究，我们证明了KIF26B 介导Wnt5a-Ror途径控制细胞迁移的能力，并且KIF26B的这一功能为 从秀丽隐杆线虫到人类保守。从机械上讲，我们已经建立了Wnt5a-ror的关键发现 信号传导通过涉及泛素的机制来控制KIF26B的细胞稳态浓度 蛋白酶体系统。使用这种新颖的Wnt5a-ror-kif26b信号传导范式，我们成功地开发了一个 记者测定，首次允许定量测量现场的Wnt5a-ror信号传导活性 细胞。在此应用中，我们建议将蛋白质生物化学，显微镜和遗传学的组合结合到 阐明将ROR受体激活与KIF26B降解的分子机制，细胞生物学 KIF26b细胞骨架动力学和细胞迁移的调节的机制，以及体内的作用 WNT5A-ROR-KIF26B信号盒在胚胎组织形态发生中。而且，我们将配对我们的Wnt5a- 带有大规模CRISPR/CAS9的基于遗传筛选的ROR信号记者，以识别其他成分 路径。项目的成功完成（1）将提供第一张详细的分子肖像 WNT5A-ROR信号网络（2）揭示了WNT5A-ROR信号传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，（3）提出了新型生物标志物和治疗性 WNT5A-ROR驱动疾病的目标。