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 a-tubulin
acetyltransferase 1 (aTAT1), 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 aTAT1 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.