The major function of the developing nervous system is the establishment of appropriate synaptic connections.Toward this end, the growing axon produces a specialized structure, the growth cone, whose task is to discriminate between extracellular guidance cues. It is therefore necessary to understand the molecular mechanisms within the growth cone that transduce those signals into the intracellular responses that direct axon growth. One particularly important structure is the membrane skeleton that mediates the interactions between the cytoskeleton and plasma membrane responsible for altering growth cone morphology. We have hypothesized that the membrane skeleton acts as an integrative center within growth cones where a multiplicity of diverse guidance cues are transduced into a simpler range of intracellular responses. Furthermore, it is likely that key components of the growth cone membrane skeleton will be nervous-system specific proteins that can be regulated by extracellular guidance cues. One such component is the growth-associated protein GAPA3. Regulation of GAPA3 involves several calcium-dependant enzymes that mediate its phosphorylation, dephosphorylation and proteolysis. We have proposed that understanding how this regulation of GAPA3 affects membrane skeleton functions will provide important information regarding the behavior of the growth cone during axonogenesis and regeneration. We will therefore investigate three aspects of how the interaction between GAPA3 and the membrane skeleton of growth cones is regulated: First: Ca2+- dependent proteolysis by calpain. Calpain is an important regulator of membrane dynamics in many cell types, however its role in growth cones is unknown. We will use both intact isolated growth cones and 1 degree cultures to investigate how calpain proteolysis affects the interactions of GAPA3 with the membrane skeleton, and the consequences on growth cone membrane dynamics. Second: Association with actin and the substrate- associated plasma membrane: We will use affinity chromatography and cosedimentation techniques to characterize GAPA3 interactions with actin and the plasma membrane and then determine how they are regulated by Ca2+- dependent enzymes. Third: Regulation of GAP-43 phosphorylation during axonogenesis and regeneration.To determine whether GAPA3 phosphorylation can be stimulated by different classes of extracellular signals we will use trigeminal ganglion cocultures in which both axonogenesis and regeneration can be induced under different conditions.The results of these experiments will increase our understanding of the function of GAP- 43 within the membrane skeleton, and will serve our eventual goal of designing strategies to circumvent the failure of some neurons (such as mammalian CNS) to regenerate successfully.
