Collaborative Research: Evolution of ligand-dependent Robo receptor activation mechanisms for axon guidance

我们神经系统的信息处理细胞之间的连接是由这些细胞的长延伸形成的，称为轴突。轴突的生长和导向正确的靶细胞是胚胎发育过程中神经回路组装的关键步骤。一组重要的蛋白质位于生长的轴突上并控制它们的指导，称为Robos。这项合作研究将阐明激活Robo家族蛋白进行轴突转向的分子机制，还将确定Robo在进化过程中如何改变，以使神经系统的布线变得越来越复杂。通过深入了解轴突寻路的分子机制，这项工作可以提高我们对神经系统布线错误导致的疾病的理解，并有可能为神经回路修复的治疗方法提供信息。该项目将让本科生和研究生参与尖端研究，重点是妇女和代表性不足的少数民族的职业发展。它还包括通过在研究实验室接待学生并帮助他们获得发育神经生物学研究的早期经验，与罗德岛州普罗维登斯和伊利诺伊州芝加哥的公立高中进行外展。Slit-Robo配体-受体对是一种进化上古老的信号传导模块，通过介导排斥来控制两侧对称神经系统中线的轴突交叉。然而，我们对Robo和Slit同源物以及其他相关蛋白质的信号传导机制的理解仍然不完整。进一步复杂化的图片是一个分歧的Robo家族成员，哺乳动物Robo 3，它不能结合裂缝，但对脊髓中线的轴突穿越是必不可少的。先前的工作发现了一种分泌的Robo 3配体NELL 2，它通过排斥作用引导连合轴突。初步结果表明，活性NELL 2-Robo 3复合物的形成在机制上不同于Slit-Robo相互作用，并且在哺乳动物中，Robo 3和典型的Robos，Robo 1和Robo 2，从脊椎动物树根部的双配体结合祖先进化而来。拟议研究的驱动假设是，哺乳动物Robo家族成员的功能特化对电路布线至关重要。该项目整合了两名研究人员在结构/生物物理和功能/遗传方法方面的专业知识，以研究Robo信号传导和进化。古老的序列重建，生物化学方法和轴突指导测定将用于绘制Robo配体结合和信号传导能力的进化历史。此外，将结合结构方法、基于结构的诱变、生物物理方法和轴突引导测定来确定Robo配体结合位点趋异、细胞外结构域构象和分子拥挤对哺乳动物和非哺乳动物脊椎动物中配体-受体复合物形成和信号传导的贡献。该项目将为各级学生提供培训，并包括在当地高中开展以研究为重点的推广活动。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The connections between the information-processing cells of our nervous system are formed by long extensions of these cells called axons. The growth and guidance of axons toward their correct target cells is a critical step in neural circuit assembly during embryonic development. One important group of proteins that reside on growing axons and control their guidance is called Robos. This collaborative study will elucidate the molecular mechanisms underlying the activation of Robo family proteins for axon steering, and it will also determine how Robos have changed over the course of evolution to allow the wiring of nervous systems with increasing complexity. By providing insights into the molecular mechanisms of axon pathfinding, this work can improve our understanding of disorders resulting from nervous system miswiring, and it has the potential to inform therapeutic approaches for neural circuit repair. The project will involve undergraduate and graduate students in cutting-edge research, with an emphasis on the career development of women and underrepresented minorities. It also includes outreach to Providence, RI, and Chicago, IL, public high schools by hosting students in research laboratories and helping them gain early experience in developmental neurobiology research.The Slit-Robo ligand-receptor pair is an evolutionarily ancient signaling module that controls axon crossing of the nervous system midline in bilaterians by mediating repulsion. Yet, our understanding of the signaling mechanisms of Robo and Slit homologs and other associated proteins remains incomplete. Further complicating the picture is a divergent Robo family member, mammalian Robo3, which cannot bind Slits but is essential for axon crossing of the spinal cord midline. Previous work discovered a secreted Robo3 ligand, NELL2, which guides commissural axons via repulsion. Preliminary results indicate that the formation of an active NELL2-Robo3 complex is mechanistically distinct from Slit-Robo interactions and that, in mammals, Robo3 and the canonical Robos, Robo1, and Robo2, evolved from a dual ligand-binding ancestor at the root of the vertebrate tree. The driving hypothesis for the proposed studies is that the functional specialization of mammalian Robo family members is crucial for circuit wiring. This project integrates the expertise of two investigators in structural/biophysical and functional/genetic approaches to investigate Robo signaling and evolution. Ancient sequence reconstruction, biochemical methods, and axon guidance assays will be used to map the evolutionary history of Robo ligand binding and signaling capabilities. Further, structural approaches, structure-based mutagenesis, biophysical methods, and axon guidance assays will be combined to determine the contributions of Robo ligand binding site divergence, extracellular domain conformation, and molecular crowding to ligand-receptor complex formation and signaling in mammals and non-mammalian vertebrates. The project will provide training to students at all levels and include outreach activities, with a research focus, at local high schools.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.