There are three major directions to our analysis of axon growth and guidance: - revealing the mechanism by which the receptor Notch specifies a guidance decision, - dissecting the interactions among the components of the Abl signaling network that executes the guidance decision, and - analyzing how growth cone morphology and organization respond to a signal from the Abl network to produce directed motility. In the past year, most of our effort has been directed toward the second and third of these directions. The core components of the Abl signaling network have been known for over 20 years, and it has been known that this network is key to signaling by most of the ubiquitous, phylogenetically conserved families of axon guidance receptors. Nonetheless, the functional relationships among the Abl network proteins and their detailed mechanism of action have remained obscure. In the past year, we have made two major advances on this problem. First, in work published earlier this year, we found that the Abl pathway regulates the structure and dynamics of the secretory machinery in neurons, and developed evidence that this contributes to Abl-dependent axon guidance. Specifically, we found that gain or loss of function of the adaptor protein Disabled, the tyrosine kinase Abl or the actin regulator Enabled control the fragmentation state and subcellular localization of the Golgi compartment in neurons. We further showed that this occurs via Enabled-regulated modulation of the actin cytoskeleton. The significance of this discovery for axon patterning is demonstrated by the observation that mutations affecting Golgi function mimic the axon patterning defects of Abl pathway mutations, and are functionally downstream of Abl in this process, as assayed by genetic criteria. Quite unexpectedly, this forces us to consider the role of alterations in secretion as a mechanism underlying the neural connectivity defects of Abl mutants. Second, in work currently being prepared for submission, we have developed FRET reporters of the activity of the two key outputs of the Abl network, Abl kinase and Rac GTPase, and used them to show that Rac is linearly downstream of Abl activity; that the GEF Trio links Abl to Rac, and that the Trio/Rac branch of the Abl network acts in parallel to the Enabled branch. These discoveries also suggest that Abl coordinates the dynamics of the two major forms of actin structures in the neuron, Ena-dependent linear actin bundles and Rac-dependent branched actin networks. Additional experiments are in progress to confirm this. If validated, this would help explain why the Abl network is such a potent regulator of neuronal wiring. Concerning the linkage of Abl activity to growth cone morphology and motility, we have extended our live-imaging analysis of single, identified Drosophila growth cones extending in their native tissue. In collaboration with investigators in NIH/CIT, we have developed software and novel quantitative metrics for tracing single growth cones in three-dimensional images of developing tissue, and for extracting parameters that define growth cone size, speed, complexity and molecular organization. Current efforts focus on investigating the relationships among these parameters and linking them to the effects of specific components of the Abl network. Upon completion, this will tell us for the first time precisely how Abl controls axon growth, turning and branching at the level of cytoskeletal dynamics in vivo.
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