Axon extension during development and following injury is a prerequisite for the formation of neuronal circuitry. Growth promoting and inhibitory influences are 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 facilitatory molecules, and in some cases, second messengers for transducing their effects have been studied. In contrast, less progress has been made in identifying the molecular effectors of motility that are the ultimate targets of the extracellular influences. The overall aim of this project is to characterize an important effector and determine how it influences growth cone navigation and axon extension. The focus of the current proposal is on myosin IIB, a mechanoenzyme likely to participate in several aspects of growth cone motility and neurite tension production. Although other mechanoenzymes are present in growth cones, the abundance, localization, and mechanical-chemical properties of myosin IIB make it a leading candidate for an effector in growth cone turning, production of traction force, regulation of protrusion and tension production. To investigate the role of myosin IIB in growth cone motility and axon extension, neurons derived from mice in which myosin IIB has been partially or completely eliminated by gene targeting will be used in cell culture experiments. Growth cones from myosin IIB knockout animals will be analyzed for deficits in protrusion and retraction, cortical tension, retrograde flow, traction force, neurite tension and the ability to turn in response to attractive or repulsive environmental cues. To directly test the role that myosin IIB bipolar filaments contribute to these processes, the applicants will determine if any deficits in these processes can be eliminated by expressing either green fluorescent protein B myosin IIB (which can form bipolar filaments) or green fluorescent protein B heavy meromyosin IIB (which cannot form bipolar filaments) fusion proteins in the cells from the knockout animals. Results from these studies will contribute to our ability to understand how a growing axon responds to its environment as it navigates to its target.
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