Nerve cells are the quintessential asymmetric cell and differentiate two morphologically and functionally distinct types of processes: axons and dendrites. The development of the nervous system proceeds by the extension of axons over long distances to reach their appropriate target cells. The growth cone is a specialized structure at the termini of neuronal processes that is essential for directed process outgrowth of both axons and dendrites. The ability of the growth cone to reach its correct targets may depend on mRNA transport and local protein synthesis to modulate cytoskeletal composition in response to external signals. In recent years, mechanisms for fast and slow transport systems been elucidated for membrane bound vesicles and cytoskeletal complexes, yet contemporary models for protein sorting into axons and dendrites have not considered mRNA transport as an additional mechanism. Using high resolution in situ hybridization and digital imaging microscopy methods, we have shown that beta actin mRNAs were localized to growth cones of developing dendritic and axonal processes. Beta actin mRNAs were detected in the form of spatially distinct granules that were associated with microtubules. The localization of beta actin mRNAs to neuronal growth cones of cultured neurons is proposed to be dependent on cis-acting sequences within the 3'UTR. Preliminary data suggests that short zip code sequences within the 3'UTR are involved in beta actin mRNA localization within neurons. The hypothesis is that these cis-acting sequences are necessary and sufficient to localize beta actin mRNA and that perturbation of these elements disrupts protein localization within the growth cone and has deleterious effects on process outgrowth. The spatial component of translation will be studied by expression of epitope tagged beta actin constructs. Trans-acting factors are proposed to bind the cis-acting elements and to be involved in beta actin mRNA localization and its association with cytoskeletal filaments. Using ultrastructural and fluorescence in situ hybridization methods, the localization and cytoskeletal association of beta actin mRNA complexes will be determined. The dynamic aspects of RNA transport in processes will be studied by microinjection of fluorescent mRNA sequences in living neurons. Proteins which bind the RNA localization sequences will be isolated, cloned and sequenced. The elucidation of a mechanism to regulate mRNA localization to growth cones will provide insight into the regulation of growth cone structure and mechanisms that may underlie process outgrowth.
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