The long-term objectives of this project are to understand at the subcellular and molecular levels how events in the growth cone (the specialized ending of a growing neuronal process) lead to neuritic growth and mediate interactions with environmental cues important in directing that growth. The current proposal focuses on protrusive structures of the growth cone, especially filopodia, which are so important in transducing cues into changes in the rate or direction of growth. The proposed experiments will also begin to apply an understanding of events in the growth cone to the analysis of selective synapse formation. Based on previous work in this project, it is hypothesized that protein- tyrosine phosphorylation is involved in mediating or facilitating interactions of filopodia with environmental cues. This hypothesis will be examined by using high resolution video microscopy and immunocytochemistry to determine whether there are changes in tyrosine phosphorylation in filopodia of growth cones interacting with important cell-bound cues in culture. Two model culture systems will be used which approximate important aspects of in vivo development, one involving a pathway decision by embryonic mouse retinal ganglion cell axons when they interact with cells of the optic chiasm and the other in which growth cones of identified Aplysia neurons make decisions that contribute to the selective formation of chemical synapses. In addition, immunocytochemical and biochemical techniques will be used to identify proteins associated with the phosphotyrosine in filopodia. The other major set of experiments seeks to identify molecular changes underlying the activation of the actin-based motility of the growth cone, which is responsible for all protrusive activities, such as the formation and movement of filopodia. These experiments are based on a strategy of eliciting motile activity synchronously in a large number of cells (specifically, by readding nerve growth factor to primed PC12 cells from which it has been withheld for several hours) and then determining whether certain proteins of interest become associated with, or phosphorylated in, networks that contain actin filaments. Gel electrophoresis, immunoprecipitation and Western blotting will be the main techniques employed here. The role in growth cone motile activities of any implicated protein will be assessed by depleting the protein through the use of antisense oligonucleotides and measuring the consequences by high resolution video microscopy. By focusing on basic mechanisms whereby neuronal connectivity is established during development and after nerve injury, this work is of potential relevance to the understanding of, and the development of therapies for, certain developmental disorders of the central nervous system and traumas such as spinal cord injury.
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