One of the key problems in developmental neuroscience is understanding how different types of neurons respond to external signals to establish synapses with their appropriate targets and to generate their specific axonal and dendritic architecture. The cytoskeletal network of actin filaments and microtubules acts as a scaffolding to allow navigating growth cones to alter their trajectories in response to attractive and repulsive guidance cues, to define the morphology of mature neurons, and to remodel in response to injury. For all of these processes to proceed normally, neurons must coordinate the dynamics of actin filaments and microtubules in a highly regulated manner. The goal of this proposal is to understand how neuronal cytoskeletal dynamics are regulated by studying the Drosophila spectraplakin Short stop (Shot). Flies lacking Shot exhibit axon outgrowth defects and CNS malformation. Spectraplakins are conserved among animal lineages and their mutation causes sensory neuron degeneration and developmental brain malformations in mice and neurodegenerative disease in humans. At the cellular level, spectraplakins act as cross-linkers to physically bridge actin and microtubules. Despite these essential functions in the nervous system, we know very little about how spectraplakins are regulated at the molecular level. In this proposal, we will use Shot as a paradigm to ask how spectraplakins are regulated using cutting-edge single molecule visualization techniques. 1) We will establish an assay for visualizing Shot actin-microtubule cross-linking at single molecule resolution. 2) We will test the hypothesis that Shot is regulated by an intramolecular inhibition mechanism. Given the high degree of conservation among members of this family of proteins, we anticipate that these regulatory mechanisms will be conserved with vertebrate spectraplakins, as well.
The proposed research is relevant to public health as it will advance our understanding of neuronal development and regeneration at the molecular level. Axon outgrowth defects underlie many diseases such as birth defects, neurodegenerative diseases, and paralysis. A deeper understanding of this process will allow us to design therapeutic approaches that curb these pathological cell behaviors.