The role of acetylated microtubules in cell migration

Project Summary/Abstract Cell migration is essential for many physiological processes including embryonic development, wound healing, and immune responses. Cells achieve efficient directional migration through two major steps: front-and-back polarization and re-arrangement of interactions between the cell and extracellular matrix (ECM). Cells adhere to the ECM using actin-based multiprotein complexes called focal adhesions (FAs) with which the cells exert forces to push or pull themselves to migrate, while long-term polarity is maintained by another cytoskeletal component, microtubules (MTs). Therefore, efficient cell polarity and migration are achieved by coordinated regulation of actin, MTs, and FAs. However, it remains unknown if there is a central regulator that orchestrates these seemingly distinct subcellular organizations. Our lab has recently found that cells lacking α-tubulin acetyltransferase 1 (αTAT1), the sole mediator of MT acetylation, display defects in FAs as well as in front-and- back polarity. In addition, preliminary data also showed that cells with an αTAT1 knockout (KO) migrate faster compared to control cells during random migration assays. We hypothesize that MT acetylation is a master regulator of cell migration by dynamically remodeling molecular constituents and their activity at the F-actin and MT cytoskeletons and FAs. To test this hypothesis, I will combine live-cell, time-lapse fluorescence microscopy with pharmacological and genetic perturbations to elucidate how acetylated MT affects single and collective migration (Aim 1), and to reveal molecular mechanisms regulating the front-and-back polarity (Aim 2) and FAs dynamics (Aim 3) in a MT acetylation dependent manner. In Aim 1, I will characterize the role of acetylated MTs in cell migration by performing single and collective migration assays under different levels of MT acetylation and quantifying migration characteristics such as velocity, directionality, and wound closure rate. Actin polymerization in the front defines the leading edge of a migrating cell; this reaction is regulated by spatially restricted activities of Rho family GTPases. Therefore, to investigate the role of acetylated MT in cell polarization (Aim 2), I will use time-lapse fluorescence microscopy to measure the dynamics of MTs and F-actins and quantify the activity of Rho GTPases using FRET sensors. In Aim 3, I will elucidate how MT acetylation modulates biophysical properties of FAs in migrating cells by examining the expression level and localization of FA proteins and measuring FA dynamics using optogenetics and live-cell fluorescence imaging. In addition, I will quantify the force exerted using traction force microscopy in cells with three different conditions where the extent of MT acetylation is varied (normal, none, or elevated). Together this proposal will illuminate a role of acetylated MTs as a regulator of directed cell migration by concertedly regulating front-and-back polarity and FAs dynamics.