In the past year we have continued to advance our systematic analysis of how the receptor Notch regulates the Abl tyrosine kinase signaling pathway to control axon growth and guidance in Drosophila. Our major advance was to publish the results of a long-term series of experiments dissecting the molecular mechanism by which Notch regulates Abl. Notch has been characterized exhaustively for the mechanism by which it directly controls nuclear gene transcription to specify cell fate. We have now show that there is a specialized population of Notch protein molecules in the fly that is tyrosine-phosphorylated, and this population of Notch associates with two cofactors of Abl tyrosine kinase prior to ligand binding. Upon ligand binding, Notch is cleaved proteolytically, both extra- and intra-cellularly, releasing the Notch intracellular domain from the plasma membrane. When that occurs, the Notch-associated Abl-pathway proteins are also released from the membrane, thereby disrupting Abl signaling complexes and thus turning off Abl signaling locally and acutely. This mechanism accounts molecularly for the phenotypic observations of our previous studies, where we showed that the key role of Notch in several different axon patterning decisions is to reduce Abl pathway activity in particular axons at precise times and places in development. We have also been extending our live-imaging analysis of axon growth in the fly wing. Analysis of these data was exceptionally challenging due to the extremely high stochastic variation in quantified distributions of actin intensity in axons in vivo. However, we have now significantly advanced our understanding of this process through the application of various analytical tools from information theory. In particular, investigation of the information content along spatial dimensions of the actin distribution in wildtype axons vs those with altered Abl signaling revealed that a key function of Abl kinase in axon patterning is to minimize the disorder of the cytoskeletal distribution in the axon at any given time, while investigation of the divergence of the actin distribution over the temporal axis revealed that wildtype Abl is also required for the orderly evolution of the actin pattern over time. Our quantitative analysis now shows that both of these properties are likely to be critical for accurate and efficient axon growth in vivo. We note that these are new approaches for quantifying cytoskeletal structure and dynamics and that they should be widely applicable across many aspects of cell biology.
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