Project Summary/Abstract Neurons form two distinct processes during polarization, axons and dendrites, each with a unique and tightly packed microtubule array of post-translationally modified microtubules coated in microtubule associated proteins (MAPs), which control the activity of kinesin and dynein motors responsible for long-range intracellular transport. MAPs are upregulated during polarization and are spatiotemporally regulated. MAPs play an integral role in establishing the process-specific microtubule arrays by controlling microtubule organization, microtubule dynamics, and the binding of molecular motors. In vitro studies demonstrated that MAPs at saturating concentrations form inhibitory patches or “islands” along microtubules, deterring kinesin and dynein binding, However, neurons still achieve long-range transport, suggesting regulation of the deposition of MAPs along microtubules. Two mechanisms through which MAP regulation can be achieved are: (1) competitive binding with other MAPs or (2) modifications to the underlying microtubule substrate. Post-translational modifications (PTMs) of microtubules regulate the interactions of molecular motors, severing enzymes and tip-tracking proteins with the microtubules, forming a “tubulin code”. Recent findings revealed a distinct organization of PTMs across the microtubule bundles in dendrites. I hypothesize that MAPs also form highly organized, stereotyped nanopatterns that specialize microtubules, or microtubule segments, facilitating efficient, dedicated high-volume transport of cargo and that these stereotyped MAP nanopatterns control microtubule organization, dynamics, and tubulin PTMs in neuronal processes. In Aim 1, I will investigate the interplay between MAPs and PTMs by utilizing iPSC- derived neurons to generate high-resolution maps of MAPs and PTMs during neuronal maturation using Expansion Microscopy (ExM). In Aim 2, I will knockdown MAPs, both individually and in combination, using CRISPRi to monitor changes to neuronal morphology, microtubule organization, intracellular trafficking, and the nanopatterns of MAPs and PTMs. This will shed light on the specific roles each MAP plays in establishing the intricate microtubule arrays necessary to maintain neuronal health. In Aim 3, I will dissect the interplay between MAPs and PTMs through in vitro reconstitution assays, offering mechanistic insights into the principles governing MAP nanopattern formation on microtubules. Overall, this work will explore fundamental questions regarding the formation of the neuronal cytoskeleton and intracellular transport, with broader implications for the field of cell biology. This proposal will give me the opportunity to gain a wide breadth of knowledge ranging from in vitro reconstitution of the tubulin code to genetic engineering of iPSC-derived neurons and super-resolution microscopy in the labs of my primary sponsor Dr. Roll-Mecak and co-sponsor Dr. Hari Shroff as well as through consultation w...