连接蛋白lrap35a调节Wnt信号通路和细胞运动的作用机理

It was shown that leucine repeat adaptor protein 35a (lrap35a) regulates cell protrusion and migration through binding to and activation of MRCKα and MRCK? (Myotonic dystrophy Kinase-related Cdc42-binding Kinase αand ?). However, whether and how it plays a role during early development is actually not known. We have recently made the interesting finding that lrap35a physically interacts with Dishevelled (Dsh or DVL), a key component of both canonical and non-canonical Wnt signaling pathways. In addition, it is also able to modulate the activity of Dsh in canonical Wnt signaling. Talen-mediated disruption of lrap35a gene in zebrafish indicates that mutation of lrap35a affects epiboly movement, suggesting that it may be required for cell movements during gastrulation in zebrafish embryo. Based on these observations, we plan first to determine how lrap35a regulates the activation of canonical and non-canonical Wnt pathways, we will also analyze how it is involved in the regulation of different cell behaviors during gastrulation, such as epiboly, cell adhesion and migration, and convergence and extension. The main objective of our study is to understand how the interaction between lrap35a and its partners, such as Dsh and MRCK, as well as other novel proteins that we plan to identify in this project, regulates the ability and polarity of movement cells in the whole embryo. Furthermore, our results should help to provide insight into the regulatory network operating in morphogenesis. We also expect to uncover novel regulators of Wnt signaling pathway, and novel mechanism that controls cell movements. Since the protein partners of lrap35a, such as Dsh and MRCK, are implicated in tumorigenesis and cancer cell invasion, our results may be also potentially important for a better understanding of human diseases and should have a broad impact on developmental biology and medicine.

已有的体外研究显示连接蛋白lrap35a通过结合并激活MRCK参与调节细胞的运动，但它在胚胎发育过程中的功能还没有报道。我们的工作首次发现lrap35a能与Wnt通路重要成员Dishevelled相结合并能抑制其激活经典Wnt信号的活性。在斑马鱼中靶向敲除lrap35a显示突变体胚胎的下包运动延迟，说明它在早期发育中有重要作用。本项目将依据这些工作基础在斑马鱼中深入研究lrap35a在胚胎发育过程中调节经典和非经典Wnt信号的机制，确定它在原肠作用过程中调节各种细胞行为的作用。我们还将通过解决lrap35a与其结合蛋白的相互作用如何调节胚胎细胞的运动能力和极向这个关键问题，来达到进一步阐明形态发生运动调控网络和分子机制的目标。这项研究将有助于鉴别出在发育过程中调节不同Wnt信号通路和细胞定向运动的新成员以及新机理，其成果能为了解人类疾病的病因和采取干预手段等方面提供有价值的线索。

连接蛋白lrap35a（现更名为Lurap1)对细胞的方向性运动有重要作用，但其调节机制及其在早期发育过程中的功能还没有报道。我们在斑马鱼胚胎发育过程中研究了Lurap1调节原肠胚形成过程中各种细胞行为的作用。对Lurap1母源-合子突变体的分析显示它们表现出细胞定向运动和胚胎前后轴延伸的缺陷，但胚胎背腹轴的图式化没有受到影响，说明Lurap1特异性地调节早期胚胎发育过程中的细胞运动。生化分析发现Lurap1通过其羧基端与Dishevelled的PDZ结构域相作用，影响Dishevelled的细胞膜定位以及对非经典Wnt信号的激活。高表达和基因敲除分析显示Lurap1负调节非经典Wnt信号途径，在细胞中抑制JNK的激活。通过延时活体成像，我们发现Lurap1在进行不对称运动的细胞中（脊索中胚层和神经板外胚层）呈现一定程度的不对称分布。它与Dishevelled的功能性作用调节细胞突触（例如板足和丝足）以及微管组织中心的极性形成，因此调节细胞的极性和定向运动。这一工作鉴别出一个新的Dishevelled作用蛋白，并且证明Lurap1参与调节非经典Wnt信号途径（Nature Communications 2017）。.对这个工作的延伸使得我们详细阐明了Dishevelled（Dvl）旁系同源基因调节集中延伸运动和体轴形成的差异性和协同性功能。我们首次在斑马鱼中得到了5个dvl基因的母源-合子突变体，进一步的研究发现dvl2基因对细胞的定向运动和胚胎体轴的延伸有主要的作用，而dvl3a在这些过程中与dvl2协同作用，显示出重要的剂量依赖性效应。这一工作还突出了dvl2和dvl3a基因的母源产物对合子发育过程的重要性。此外，dvl2基因的突变引起剂量依赖的颅面发育缺陷，显示出Dvl介导的各种Wnt信号途径在软骨形态发生过程中的调节性功能（Plos Genetics 2018）。