The objective of the proposal is to define the mechanisms by which the number and length of filopodia are regulated in pathfinding neuronal growth cones. Filopodia are important organelles of the growth cone, and function in growth cone steering and movement. Since the number and length of filopodia determine the sensory radius that a growth cone can survey, these parameters will affect the cell s ability to obtain information about its environment and consequently to orientate correctly in vivo. The proposed research has 3 main aims: (1) To test the hypothesis that the length and number of growth cone filopodia are regulated by physiological changes in intracellular calcium concentrations, and to identify the location of calcium s action; (2) To test the hypothesis that intracelullar calcium acts via calmodulin to affect growth cone filopodia; and (3) To determine whether filopodial elongation triggered by tyrosine kinase inhibition is elicited by calcium or by a mechanism independent of calcium. The proposed experiments will take advantage of a well-characterized in vitro model system, identified neurons B5 and B19 of the snail Helisoma. These neurons have large growth cones and filopodia that can be reproducibly manipulated in both length and number. A combination of video microscopy and quantitative calcium imaging techniques will be used to quantify filopodial behavior and measure calcium concentration in both growth cones and filopodia.
