Axon extension during development and following injury is a prerequisite for the formation of functional neuronal circuitry. Growth promoting and inhibitory influences appear to be important for directing axon extension through the complex tissue environment. These influences exert their effects by regulating the motility of the growth cone, the structure at the tips of extending axons. Considerable progress has been made in identifying and characterizing inhibitory and facilatory molecules, and in some cases, second messengers for transducing their effects have been suggested. In contrast, little progress has been made in identifying the molecular effectors of motility that are the ultimate targets of these extracellular influences. The overall aim of this project is to identify and characterize these effectors and the molecules that regulate them, and determine how they influence growth cone navigation and axon extension. Since tension produced by growth cones appears to have an important role in regulating the rate and direction of axon extension, myosins I and II. Although other mechanoenzymes are present in growth cones, the abundance, localization, and mechanochemical properties of myosin I and II make them leading candidates for effectors of tension production. The first emphasis will be on the precise isoform specific location of these myosins, determined using quantitative immunofluorescence and immunoelectron microscopy. The second emphasis of this proposal is to investigate the role of myosin I and II in axon extension, and to examine the correspondence between effects on axon extension and tension production by growth cones. To this end, myosin I and II function will be disrupted with antibodies and antisense oligonucleotide that inhibit their mechanoenzymatic activity or synthesis, respectively. Inhibiting the function of myosin II in non- neuronal systems had significant effects on cell motility and morphology, but it was not possible to determine whether these effects correlated with a decrease in tension production. Therefore, this work will assess the influence of these perturbations on: 1. gross aspects of axon extension, such as axon extension rate, 2. subtle features of growth cone motility that may indicate the site at which myosins I and II act, such as protrusion and retraction of growth cone periphery, and 3. tension production by growth cones. By characterizing not only the initial steps in the regulation of growth cone locomotion, but also the final (effector) steps, the potential sites for successful intervention during abnormal development or in regenerative failure can be identified.
Showing the most recent 10 out of 21 publications
