The long term objective of this proposal is to understand the molecular mechanisms of growth cone motility in developing neurons. Axons are guided by extracellular cues that direct filopodial motility by locally affecting cytoskeletal dynamics. Understanding the molecular basis of this is required to understand how neurocircuitry is formed during embryonic development and to establish what factors can cause neurological birth defects. Moreover, these molecular mechanisms are likely to be used in nerve regeneration and an understanding of them will aid in designing treatment after nerve injury. Considerable work in neural development, cell biology, and signal transduction have identified candidate proteins that may be involved in filopodial motility and guidance but establishing their in vivo function in neuronal growth cones has been difficult. The long term objective will be addressed by applying microscale chromophore assisted laser inactivation (micro-CALI), a method developed in this laboratory, to inactivate specific intracellular proteins with an unprecedented level of spatial and temporal resolution. CALI has been rigorously tested and used to determine the in vivo roles of membrane proteins in neural development. It is timely to use this technique to address the molecular mechanisms of growth cone motility. Specificically, the in vivo roles of talin, vinculin, pp60c-src, and the myosins in filopodial motility will be determined by focally inactivating them in growth cones and observing the resulting behavior by video- enhanced microscopy and quantitative morphometry. Experiments are also proposed to ask if these proteins play a role in substrate-mediated guidance by using micro- CALI as growth cones reach borders on patterned substrates. These studies will be done using chick dorsal root ganglion neurons in culture, a well characterized and manipulatable system for which there exist antibodies against many proteins that are potentially involved in growth cone motility. The proposed experiments are focused on these proteins because our preliminary experiments coupled with in vitro biochemical data suggest a model for how they function and interact in the extension and regulation of filopodia. Micro-CALI will be applied to test this model by inactivating these proteins in combination to give specific phenotypes that will support or refute the proposed interactions.
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