? The active transport of b-actin mRNAs to growth cones of developing neurons may provide a basic mechanism to enrich b-actin protein and influence growth cone motility. Using high resolution fluorescence in situ hybridization and digital imaging microscopy methods, we have shown that b-actin mRNAs were localized to growth cones of developing axonal and dendritic processes in the form of granules that were associated with microtubules. The molecular mechanism of b-actin mRNA localization in these cultured neurons was shown to be dependent on a 54 nt sequence in the 3'UTR, termed the zipcode, which is bound by two mRNA binding proteins, ZBP1 and ZBP2. Live cell imaging of neurons transfected with EGFP-ZBP1 revealed fast, bi-directional movements of granules in processes. This proposal will test the hypothesis that ZBP1 and/or ZBP2 function as adapter molecules between the b-actin zipcode and distinct types of cytoskeletal motors involved in both microtubule and microfilament-dependent movements. Preliminary findings presented in this application document the association of ZBP1 with microtubules. Experiments in Specific Aim 1 will elucidate the roles of ZBP1, ZBP2 and cytoskeletal motors in the transport of b-actin mRNA in processes and growth cones of cultured neurons. High resolution fluorescence microscopy and live cell imaging technology will be developed to visualize the co-transport of b-actin mRNA and ZBPs. Molecular and biochemical methods will be used to characterize the interactions between ZBPs and cytoskeletal motors. We will determine whether disruption of the expression or function of motors, and their interaction with ZBPs, can impair b-actin mRNA localization into processes and growth cones. Experiments in Specific Aim 2 will elucidate the function for ZBP/motor dependent transport of b-actin mRNA in the local synthesis of b-actin and growth cone motility. We will determine whether disruption of ZBPs or interacting motors can inhibit the local synthesis of b-actin within growth cones and impair regulation of growth cone motility and dynamics. The elucidation of molecular mechanisms involved in mRNA transport and their perturbation will provide new insight into protein sorting mechanisms that regulate growth cone motility and process outgrowth during neuronal development. ? ? ?
