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

生物学中的一个长期存在的问题是，组织和器官是如何在 发展。Wnt5a-Ror信号通路是胚胎组织形态发生的主要调节因子。 解除对该途径的管制已被发现会导致广泛的人类病理状况， 包括先天性疾病罗比诺综合征和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不同， Wnt5a-Ror通路通过细胞骨架调节来控制关键的形态发生细胞行为， 如细胞迁移、极化和黏附。然而，Wnt5a背后的分子机制- 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)什么是 调节Wnt5a-Ror信号处理和细胞内传播的生化相互作用？(2)如何 WNT5A-ROR信号使细胞骨架参与控制形态发生细胞的行为？(3)这些是如何实现的 Wnt5A/Ror驱动的过程最终控制体内组织的形态发生？为此，我们已经整合了 小鼠遗传学和比较蛋白质组学构建Wnt5a-Ror途径的第一个扩展清单 组件。这项工作不仅为Wnt5a-RoR信号的分子机制提供了至关重要的见解 转导，但也发现Kif26b(运动蛋白微管运动家族的成员)是一个关键的 该途径的细胞骨架效应因子。通过功能得失研究，我们证明了Kif26b 介导Wnt5a-Ror通路控制细胞迁移的能力，Kif26b的这一功能是 从线虫到人类的遗传。从机制上讲，我们已经确定了Wnt5a-Ror的关键发现 信号通过泛素参与调控细胞内Kif26b的稳态浓度。 蛋白酶体系统。利用这种新的Wnt5a-Ror-Kif26b信号范式，我们成功地开发了一种 记者化验首次允许定量测量活体中Wnt5a-Ror信号活性 细胞。在这一应用中，我们建议使用蛋白质生物化学、显微镜和遗传学的组合来 阐明ROR受体激活与Kif26b降解有关的分子机制 Kif26b调节细胞骨架动力学和细胞迁移的机制以及Kif26b在体内的作用 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- 使用基于CRISPR/Cas9的大规模遗传筛查来识别其他成分的RoR信号报告 在这条小路上。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，以及(3)提出新的生物标志物和治疗方法 Wnt5a-Ror驱动的疾病的靶点。

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.