Molecular analysis of the chaperone Hsc70-Trio protein complex in the neuronal growth cone

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During development, the pathfinding of neurons is governed by guidance cues, such as the secreted protein netrin-1. Binding of netrin-1 to the receptor Deleted in Colorectal Cancer (DCC) expressed at the axon's distal tip, the neuronal growth cone, initiates molecular signals that modulate and change the cell cytoskeleton dynamics, leading to axon outgrowth and guidance. We identified the guanine nucleotide exchange factor (GEF) Trio as a key component of netrin-1/DCC signaling in growth cone guidance. Trio is a large (330kDa) cytoplasmic protein containing three catalytic domains, including a serine/threonine kinase domain and two GEFD1 and GEFD2 acting as activators of Rac1 and RhoA, respectively. Trio contains other motifs, including a Sec14 lipid-binding domain, eight spectrin repeats, two Src Homology 3 domains, and an immunoglobulin-like domain. The presence of these various domains suggests that Trio functions as an integrator of multiple upstream inputs and an activator of multiple downstream pathways. However, understanding how Trio integrates these signals is limited by the lack of knowledge in the structure of the protein. To characterize the mechanisms governing Trio localization and activation downstream of netrin-1/DCC, we employed a proteomic approach and identified the molecular chaperone Hsc70 as a novel Trio regulator in the developing cortex. In addition, we identified four Trio phosphorylated serine residues for which the exact function still remains to be elucidated. ***Overall, our recent work highlighted an important role for Trio in neurodevelopment. However, the molecular mechanisms by which Trio modulates the response of axons to guidance cues still remain elusive. Therefore, the goal of this research program is to investigate the regulation, localization, and molecular structure of Trio protein complexes at the neuronal growth cone, in three specific aims:***Aim 1: To characterize the role of serine phosphorylation in Trio function and regulation ***Aim 2: To identify the global Trio interactome in neurite outgrowth using a BioID approach***Aim 3. To elucidate the molecular structure of the Hsc70-Trio protein complex at growth cones using high-resolution molecular cryo-electron microscopy (cryo-EM) combined with live cell imaging using fluorescence microscopy (correlative light electron microscopy or CLEM).***These studies will provide the structural basis for understanding the molecular functions of Trio protein complexes assembled at the leading edge of neuronal growth cones. The interdisciplinary nature of this proposal will give the students the opportunity of learning a wide range of molecular and cellular techniques to analyze protein complexes in neurodevelopment. The current proposal fits within the long-term goals of my program, which are to increase the knowledge of fundamental cytoskeletal-dependent mechanisms during development.