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.
