MARCKS Phosphorylation in Growth Cone Signaling

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DESCRIPTION (provided by applicant): Repellent action requires a partial, asymmetric collapse of the growth cone to effectively "steer" the developing neurite to its target. Determination of the molecular events that underlie the collapse response will provide a greater understanding of neurite pathfinding, a process of fundamental importance in the development of the functional nervous system. The main goal of the proposed research is to elucidate the signaling events of semaphorin-mediated growth cone collapse. This research will 1) analyze the role of protein kinase C (PKC) signaling in the disassembly of adhesion sites required for semaphorin-induced growth cone collapse and 2) characterize the distribution and dynamics of the predominant PKC substrate in growth cones, the myristoylated, alanine-rich C kinase substrate (MARCKS). Confocal immunofluorescence microscopy and reflection interference microscopy (RIM) will be used to determine the spatial relationships between known adhesion site proteins, MARCKS, and adhesive areas within the growth cone. Biochemical isolation of growth cone adhesion sites will be used to analyze the effect of MARCKS phosphorylation on adhesion complex integrity. MARCKS dynamics within the growth cone will be characterized by observing repellent-induced changes in the distribution of a MARCKS-green fluorescent protein (GFP) fusion protein. Functional consequences of aberrant PKC signaling will be determined by observing the turning response of growth cones from DRG neurons expressing a dominant-negative, phosphorylation-deficient MARCKS mutant.