Mechanism of a Microtubule Polymerase Complex Critical for Axon Outgrowth

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There is a fundamental gap in our knowledge to explain how microtubules polymerize in neurons. The microtubule cytoskeleton is critical for neuronal structure, dynamics, and trafficking. How factors spatially regulate the microtubule cytoskeleton to promote axon outgrowth is poorly understood. Microtubule polymerases are key regulators that localize to microtubule plus ends and promote microtubule polymerization. In neurons, microtubule polymerases associate with the factors Sentin and TACC. Knockdown of the microtubule polymerase or its associated factors compromises microtubule dynamics, yielding aberrant responses to guidance cues and defects in axon outgrowth. How the microtubule polymerase binds Sentin and TACC and how this complex collectively regulates the microtubule cytoskeleton is poorly understood. We have mapped Sentin and TACC binding to a conserved C-terminal domain of the microtubule polymerase Msps and determined that this conserved domain has microtubule-binding activity. Drawn from our preliminary data, we hypothesize that the components of the Sentin-Msps-TACC microtubule polymerase complex work synergistically to bind the microtubule lattice and drive microtubule polymerization. The rationale for the proposed research is to determine how the components of a microtubule polymerase complex potentiate microtubule polymerization, a polymer required for neuronal structure, polarity, signaling and dynamics. Two specific aims examine the structure and mechanism of the microtubule polymerase complex. The first aim is to determine the molecular architecture of the microtubule polymerase complex using X-ray crystallography, elucidate binding stoichiometry and affinity, and design and test mutations that prevent complex formation. The second aim is to map residues in the microtubule polymerase complex involved in microtubule binding and determine how the complex affects microtubule dynamics using an in vitro reconstituted microtubule dynamics assay. These two independent aims work to develop a high-resolution model for Sentin-Msps-TACC microtubule polymerase activity. The approach is innovative because it marshals a diverse set of biophysical, biochemical, and cellular assays to provide a multi-resolution model of the microtubule polymerase mechanism. The proposed research is significant because it tests a core molecular process required for neuronal shape, dynamics, and signaling. The investigation's long-term objective is to determine how the microtubule polymerase complex regulates cytoskeletal dynamics in neuronal growth cones in response to guidance cues. The proposed research will impact public health by establishing a mechanistic framework from which cytoskeletal-based defects in axon outgrowth can be investigated, providing molecular insight into mutant microtubule polymerase, Sentin, and TACC phenotypes, including spontaneous axon retraction and defects in the response to axon guidance cues.