Project Summary/Abstract Microtubules (MTs) are dynamic cytoskeletal structures that mediate diverse processes from cell division to organelle transport to cell morphology and are built by self-assembly of αβ-tubulin heterodimers. Studies in vitro readily emphasize the direct relationship between the concentration of these heterodimers and MT abundance and dynamics. In cells, where MT structure and dynamics are further tuned by microtubule-associated proteins and modifying enzymes, the soluble tubulin pool is easily overlooked. Cells, however, closely monitor and buffer against perturbation to this pool through a unique phenomenon known as tubulin autoregulation whereby free tubulin governs the stability of its own transcript. Despite initial characterization over 40 years ago, the mechanistic basis of tubulin autoregulation, its molecular effectors (ex. the sensor of soluble tubulin or the RNA decay factor) and the functional implications of tubulin autoregulation in cellular homeostasis remain largely unknown. Independent studies within the last year identified the protein TTC5 as both a key mediator of tubulin autoregulation and an underlying cause of intellectual disability with cerebral atrophy, providing a valuable molecular inroad into the autoregulatory mechanism and a powerful rationale for characterizing its role within the nervous system. I hypothesize that neurons, as highly-polarized cells scaffolded structurally and functionally by MTs, are uniquely sensitive to dysregulation of the soluble tubulin pool. Here, I tackle tubulin autoregulation at multiple scales, in highly-mechanistic detail and in the disease-relevant context of the human neuron. To dissect tubulin autoregulation mechanistically (Aim 1), I will deploy TTC5 and tubulin as molecular handles to isolate additional effectors using proteomic and biochemical methods and will leverage bioinformatics, RNA reporter design and manipulation of RNA decay pathways to identify functional features within tubulin mRNAs using simple cell lines. To interrogate TTC5 and tubulin autoregulation functionally (Aim 2), I will utilize CRISPR methodologies to knock-down TTC5 in a human iPSC-derived neuron model. Using live-cell imaging, I will assess the impact of loss of TTC5 and tubulin autoregulation on neuronal morphology, MT dynamics and transport across neuronal differentiation, homeostasis and in response to and recovery from diverse stresses such as drug-induced depolymerization and axotomy. Overall, this work will address unanswered questions in fundamental microtubule biology and dissect the mechanism of tubulin autoregulation in neurophysiology and pathology. The proposed work represents an entirely new field of study for me and new direction for the lab, establishing independence while integrating the diverse expertise of my sponsors and collaborators in microtubule (Dr. Roll-Mecak), RNA (Dr. Hogg) biology and neurological disease (Dr. Ward). Through their mentorship, I will acquir...