Molecular Events Within the Neuronal Growth Cone

在神经系统发育以及损伤后的再生过程中，许多神经元的轴突必须在复杂的地形中长出很长的距离，才能与合适的靶点进行突触连接。轴突的生长受其活动末端--生长锥体的活动控制。因此，阐明生长锥内导致轴突延长的分子机制以及外部线索影响该机制的分子机制是神经系统发育和再生研究的重要目标。这个项目的工作旨在帮助理解微管是如何调节生长锥细胞骨架的主要组成部分，以影响轴突生长的速度和方向的。尽管微管的重要性正在变得越来越明显，但对微管的这种调节在分子上却知之甚少。实验将集中在生长锥体中微管行为的变化，生长锥体是分化轴突快速、流线型生长的基础。最近的工作指出，分子马达，细胞质动力蛋白，在调节这种行为的各个方面发挥着重要作用。特别是，这个项目中的实验将检验这样一个假设，即动力蛋白在微管末端和亚浆肌动蛋白皮质的界面上发挥作用，以捕获微管，促进它们的捆绑和轴突的协调流线型。实验将确定动力蛋白失活如何影响轴突的延长。然后将确定生长锥体中微管行为的哪些特定方面对动力蛋白失活敏感，以了解轴突延长的调节是如何实现的。此外，还将研究动力蛋白在生长锥体转向环境线索过程中的潜在作用。高分辨率荧光显微镜将是这一项目的主要实验技术。这将包括观察在XFP-蛋白质构建体转染后活的生长锥体内的微管。通过显微注射或RNAi引入的功能阻断抗体将被用于灭活生长锥体内的特定蛋白质。这个项目解决了神经元发育中的基本问题，涉及轴突的生长和引导，即神经元的长期投射过程。这项工作也与促进脊髓损伤和中风后神经再生的策略设计有关，因为刺激长距离轴突生长是重要的。这些都是对整个社会的潜在好处。此外，该项目将在两个方面整合研究和教育。理科专业的本科生将在暑假期间参与这项研究，可能也会在学年参与。此外，中学理科教师可能会在暑期参加一项旨在丰富他们教学内容的项目。视频显微镜的广泛使用促进了本科生和教师在研究中的有效结合，这是一种既有吸引力又相对容易学习的技术。这项工作可以探索将视频显微镜作为一种工具纳入中学生物课程的可能性。最后，预计夏季就业的本科生中将有一名是少数族裔学生，这是加强未被充分代表的群体参与研究科学的计划的一部分。

During development of the nervous system, as well as regeneration after injury, the axons of many neurons must grow long distances through complicated terrain to make synaptic connection with appropriate targets. Growth of an axon is controlled by activities of its motile ending, the growth cone. Thus, elucidating the molecular machinery within the growth cone that causes axonal elongation and the molecular mechanisms whereby external cues affect that machinery are very important goals of the study of development and regeneration of the nervous system. Work in this project is designed to help understand how microtubules, major components of the cytoskeleton of the growth cone, are regulated to affect the rate and direction of axonal growth. There is little molecular understanding of this regulation of microtubules, though its importance is becoming evident. Experiments will focus on changes in microtubule behavior in the growth cone that underlie rapid, streamlined growth of differentiated axons. Recent work points to the molecular motor, cytoplasmic dynein, as playing an important role in regulating aspects of this behavior. In particular, experiments in this project will test the hypothesis that dynein functions at the interface of the ends of microtubules and the subplasmalemmal actin cortex to capture microtubules and facilitate their bundling and the coordinated streamlining of the axon. Experiments will determine how the elongation of axons is affected by inactivation of dynein. Which specific aspects of microtubule behavior in the growth cone are sensitive to inactivation of dynein will then be determined so as to understand how the regulation of axonal elongation is achieved. The potential involvement of dynein in the turning of growth cones towards environmental cues will also be examined. High resolution fluorescence microscopy will be a major experimental technique for this project. This will include observations of microtubules within living growth cones after transfection of XFP-protein constructs. Function-blocking antibodies introduced by microinjection or RNAi will be used to inactivate specific proteins within growth cones. This project addresses basic issues in neuronal development involving the growth and guidance of the axon, the long projecting process of the neuron. The work is also relevant to the design of strategies to foster nerve regeneration after spinal cord injury and stroke, when the stimulation of long distance axonal growth is important. These are potential benefits to society at large. In addition, this project will integrate research and education in two ways. Undergraduate science majors will participate in the research during the summer and, probably, the academic year as well. Also, secondary school science teachers may participate in the research during the summer in a program designed to enrich their teaching. The productive incorporation of the undergraduates and teachers in the research is facilitated by the pervasive use of video microscopy, a technique both engaging to do and relatively easy to learn. The work could explore the possibility of incorporating video microscopy as a tool into the secondary school biology curriculum. Lastly, it is expected that one of the undergraduates employed during the summer will be a minority student, as part of a program to enhance the participation of underrepresented groups in research science.