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.

生物学中一个长期存在的问题是组织和器官如何在生命过程中获得其定型形状。 发展。 Wnt5a-Ror 信号通路是胚胎组织形态发生的主要调节因子，并且 已发现该通路的失调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 型短指畸形，以及癌症转移。 与大多数通过基因转录发挥作用的发育信号通路相比， Wnt5a-Ror 通路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。然而，Wnt5a-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 Wnt5a-Ror 信号处理和细胞内传播的生化相互作用？ (2) 如何 Wnt5a-Ror 信号传导参与细胞骨架来控制形态发生细胞行为？ (3) 这些是如何实现的 Wnt5a/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第一个 Wnt5a-Ror 通路扩展库 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，但也确定 Kif26b（驱动蛋白微管运动家族的成员）是关键的 该途径的细胞骨架效应子。通过功能获得和丧失的研究，我们证明 Kif26b 介导 Wnt5a-Ror 通路控制细胞迁移的能力，Kif26b 的这种功能是 从秀丽隐杆线虫到人类都是保守的。从机制上讲，我们已经确定了 Wnt5a-Ror 的关键发现 信号传导通过涉及泛素的机制控制 Kif26b 的细胞稳态浓度 蛋白酶体系统。利用这种新颖的 Wnt5a-Ror-Kif26b 信号范式，我们成功开发了一种 报告基因检测首次允许定量测量活体中的 Wnt5a-Ror 信号活性 细胞。在此应用中，我们建议结合使用蛋白质生物化学、显微镜和遗传学来 阐明 Ror 受体激活与 Kif26b 降解之间的分子机制，细胞生物学 Kif26b 调节细胞骨架动力学和细胞迁移的机制，以及 Kif26b 的体内作用 Wnt5a-Ror-Kif26b 信号盒在胚胎组织形态发生中的作用。此外，我们将配对我们的 Wnt5a- Ror 信号报告基因，采用基于 CRISPR/Cas9 的大规模遗传筛选来识别其他成分 的路径。该项目的成功完成将（1）提供第一个详细的分子肖像 Wnt5a-Ror 信号网络，(2) 揭示 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，并且（3）提出新的生物标志物和治疗方法 Wnt5a-Ror 驱动疾病的目标。