Molecular Events Within the Neuronal Growth Cone

在神经系统的发育以及损伤后的再生过程中，许多神经元的轴突必须长距离穿过复杂的地形，以与适当的靶点进行突触连接。 轴突的生长是由其运动末梢生长锥的活动控制的。 因此，阐明生长锥内引起轴突伸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非常重要的目标。 这个项目的工作旨在帮助理解微管，生长锥细胞骨架的主要组成部分，是如何调节影响轴突生长的速度和方向的。 尽管微管的重要性越来越明显，但对微管的这种调节的分子理解很少。 实验将集中在微管行为的变化，在生长锥的基础上快速，流线型的分化轴突的生长。 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，在这个项目中的实验将测试的假设，动力蛋白的功能在微管和质膜下肌动蛋白皮层的末端的接口，以捕获微管，促进他们的捆绑和协调流线型的轴突。 实验将确定动力蛋白失活如何影响轴突的伸长。 然后将确定生长锥中微管行为的哪些特定方面对动力蛋白的失活敏感，以便了解如何实现轴突伸长的调节。 动力蛋白的潜在参与转向对环境线索的生长锥也将被检查。 高分辨率荧光显微镜将是该项目的主要实验技术。 这将包括在转染XFP-蛋白构建体后观察活生长锥内的微管。 通过显微注射或RNAi引入的功能阻断抗体将用于抑制生长锥内的特异性蛋白质。 这个项目涉及神经元发育的基本问题，包括轴突的生长和指导，神经元的长投射过程。 这项工作也与脊髓损伤和中风后促进神经再生的策略设计有关，此时长距离轴突生长的刺激很重要。 这些都是对整个社会的潜在好处。 此外，该项目将以两种方式整合研究和教育。 本科科学专业的学生将在夏季参加研究，可能也会在学年参加。 此外，中学科学教师可以在夏季参加一个旨在丰富他们教学的项目。 在研究中的本科生和教师的生产纳入促进了视频显微镜的广泛使用，一种技术既从事做和相对容易学习。 这项工作可以探讨将视频显微镜作为工具纳入中学生物课程的可能性。 最后，预计在夏季雇用的本科生之一将是少数民族学生，作为加强代表性不足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.