Mechanisms of mechanical transduction in the guidance of neuronal axons

描述（由申请人提供）：成人神经系统可以被认为是一个神经元细胞体的网络，通过称为轴突的薄过程相互连接。在发育过程中，这些轴突延伸被环境中有图案的化学线索引导到它们的目标。不能重建这些轴突连接是许多疾病持续残疾的主要原因，包括：脊髓损伤、创伤性脑损伤和中风。轴突的尖端，被称为生长锥，利用细胞表面受体来识别这些化学信号。生长锥也是一种机械结构，它可以拉动周围的环境向前移动。理论上，线索可能间接影响生长锥的动力机制与周围环境的耦合。然而，我最近已经证明，这些线索中至少有一种-神经网络-1 -直接用于牵引。换句话说，netrin-1吸引生长锥的能力反映了其支持生长锥机械拉力的能力。我目前的工作是研究生长锥内一种叫做局灶黏附激酶（FAK）的蛋白质，它将这个线索与细胞骨架联系起来。我发现，这种蛋白质感受到的机械张力激活了它的催化活性，导致生化级联反应，加强了与线索的联系。在这个应用中，我建议使用光学激光镊子、磁镊子、超分辨率成像技术和纳米柱阵列来解决我以前工作中出现的其他四个基本重要的研究方向：(1)确定FAK是如何附着在细胞骨架上的，以及它是否在细胞中被物理拉伸。(2)测试化学成分、环境刚度以及不同神经元种群之间的变化对生长锥拉力的影响。(3)检查是否有其他有吸引力的轴突引导线索用于牵引。(4)探讨线索及其受体感受到的机械张力是否通过诱导构象变化而改变其功能。Michael Sheetz的实验室为学习和利用解决这些问题所需的技术提供了一个独特的环境。另外两位共同导师将提供额外的专业知识：约翰·亨特是蛋白质纯化和结构方面的世界领导者，而詹姆斯·霍恩是制造尖端纳米制造设备的专家。从这个项目中获得的见解将进一步加深我们对轴突引导的基本理解，因此有助于在神经系统损伤后开发更好的再生策略。

DESCRIPTION (provided by applicant): The adult nervous system can be thought of as a network of neuron cell bodies connected to each other by thin processes called axons. During development, these axonal extensions are guided to their target by patterned chemical cues in their environment. Failure to reestablish these axonal connections is a major cause of the persistent disabilities of a number of disease conditions, including: spinal cord injury, traumatic brain injury and stroke. The leading tip of an axon, known as a growth cone, uses cell surface receptors to recognize these chemical cues. The growth cone is also a mechanical structure that pulls on its surroundings to move forward. In theory, cues could have an indirect role in influencing the coupling of the growth cone's locomotive machinery to its surroundings. However, I have recently shown that at least one of these cues - netrin-1 - is directly used for traction. In other words, the ability of netrin-1 to attract the growth cone reflects its ability support the growth cone's mechanical pulling. My current work examines a protein called focal adhesion kinase (FAK) within the growth cone that links this cue to the cytoskeleton. I have discovered that the mechanical tension felt on this protein activates its catalytic activity resulting in biochemical cascades that reinforces the link to the cue. In this application, I propose to use optical laser tweezers, magnetic tweezers, super resolution imaging techniques and nano-fabricated pillar arrays to address four other fundamentally important lines of investigation that have emerged from my previous work: (1) Determine how FAK is attached to cytoskeleton and whether it is physical stretched in cells. (2) Test the effect of chemical composition and rigidity of the environment, as well as, changes between different neuronal populations on the pulling strength of the growth cone. (3) Examine whether other attractive axon guidance cues used for traction. (4) Explore whether mechanical tension felt on the cue and its receptor alters their function by inducing conformational changes. Michael Sheetz' lab offers a unique environment to learn and utilize the techniques necessary to address these questions. Two additional co-mentors will provide complimentary expertise: John Hunt is world leader on protein purification and structure, while James Hone is an expert on generating cutting-edge nanofabricated devices. Insights gained from this project will further our basic understanding of axon guidance and therefore contribute to the development of better regenerative strategies following injury of the nervous system.