Defining microtubule cytoskeleton regulatory pathways in development and disease

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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 disrupt cell function and ultimately lead to pathological conditions. The DYRK1a kinase signaling pathway is implicated in intellectual disability disorders, neurodegenerative disease, and cancer. DYRK1a has important roles in a range of cellular processes; however, the downstream molecular mechanisms of this kinase are largely unknown. Our published and preliminary data have shown that DYRK1a regulates the microtubule cytoskeleton directly by phosphorylating β-tubulin, and indirectly through phosphorylation of multiple microtubule-associated proteins (MAPs). We have found that these MAPs exhibit diverse binding behaviors on the microtubule lattice and differentially affect microtubule motors, highlighting an essential role for MAPs in gating access to the lattice. 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 three kinases, eight MAPs, and five motors both in vivo and in vitro. We aspire to construct a comprehensive network to elucidate the multiple ways in which disease-relevant kinases modulate the microtubule cytoskeleton during different cellular processes, from neuronal polarization to the establishment of specific dendritic morphologies to the maintenance of neuronal architecture. 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 the dendritic arborization neurons of the Drosophila peripheral nervous system to study neuronal morphogenesis, dendritic pruning, and polarized transport, combined with mammalian neuronal cell culture and expansion microscopy to analyze MAP localization patterns under various conditions and determine how MAPs differentially direct molecular motors to ensure proper targeting of cargoes to specific compartments. To complement these in vivo experiments, we plan to perform in vitro reconstitution experiments using TIRF microscopy with purified MAPs, microtubule motors, and kinases in order to elucidate their individual and collective effects on MAP binding, microtubule dynamics and microtubule-based transport. We endeavor to comprehensively dissect kinase-MAP networks at the protein level and at the cellular level in order to understand how these pathways contribute to a diversity of human pathologies.