Title: Defining microtubule cytoskeleton regulatory pathways in development and disease P.I. Kassandra Ori-McKenney Project Summary: Cellular architecture is governed by the organization of cytoskeletal networks and determines the functional output of a cell. It is therefore essential to understand the regulatory mechanisms of cytoskeleton organization as a cell develops, changes, or maintains its internal structure, because altering these processes can hinder cell function and ultimately lead to pathological conditions. In particular, disrupting the activities of microtubule- associated proteins (MAPs) is correlated with a range of neurodevelopmental disorders, neurodegenerative diseases, and cancers. In published and preliminary data, we have found that a subset of MAPs exhibit diverse binding behaviors on the microtubule lattice and differentially affect microtubule motors and tubulin posttranslational modifications, highlighting an essential role for MAPs in gating access to the lattice. Furthermore, we have observed that cells utilize specific MAPs to remodel their microtubule landscapes in order to efficiently adapt to changes in their environment. We are only beginning to understand how MAPs in their unaltered states control tubulin posttranslational modifications and microtubule-based transport under different cellular contexts, but MAPs themselves are heavily modified by a handful of kinases whose regulatory effects remain unclear. We have identified DYRK1a kinase as a key regulator of the microtubule cytoskeleton due to its role in phosphorylating multiple MAPs. DYRK1a is involved in a range of cellular processes and is implicated in many neuropathologies and cancers; however, the downstream molecular mechanisms of this kinase are largely unexplored. The goal of this project is to dissect the multiple layers of regulation of microtubule-based processes by studying the biochemical and genetic relationships between kinases, MAPs, tubulin posttranslational modifications, and motors both in vivo and in vitro. We aspire to construct a comprehensive network to elucidate the multiple ways in which disease-relevant kinases and MAPs modulate the microtubule cytoskeleton during different cellular processes. To accomplish these goals, we will use an interdisciplinary approach combining in vivo and ex vivo imaging techniques with in vitro biochemical assays. We will utilize epithelial cell lines to study how cells tailor their microtubule cytoskeletons for specific functions, the dendritic arborization neurons of the Drosophila peripheral nervous system to study neuronal morphogenesis, dendritic pruning, and polarized transport, and mammalian neuronal cell culture to analyze patterns of MAPs, tubulin posttranslational modifications and cargo transport under various conditions. To complement these in vivo experiments, we plan to perform in vitro reconstitution experiments using TIRF microscopy with purified MAPs, microtubule motors, tubulin modifyin...