Quantification of microtubule nucleation, growth and dynamics in wound-edge cells

Abstract

Mammalian cells develop a polarized morphology and migrate directionally into a wound in a monolayer culture. To understand how microtubules contribute to these processes, we used GFP-tubulin to measure dynamic instability and GFP-EB1, a protein that marks microtubule plus-ends, to measure microtubule growth events at the centrosome and cell periphery. Growth events at the centrosome, or nucleation, do not show directional bias, but are equivalent toward and away from the wound. Cells with two centrosomes nucleated approximately twice as many microtubules/minute as cells with one centrosome. The average number of growing microtubules per microm2 at the cell periphery is similar for leading and trailing edges and for cells containing one or two centrosomes. In contrast to microtubule growth, measurement of the parameters of microtubule dynamic instability demonstrate that microtubules in the trailing edge are more dynamic than those in the leading edge. Inhibition of Rho with C3 transferase had no detectable effect on microtubule dynamics in the leading edge, but stimulated microtubule turnover in the trailing edge. Our data demonstrate that in wound-edge cells, microtubule nucleation is non-polarized, in contrast to microtubule dynamic instability, which is highly polarized, and that factors in addition to Rho contribute to microtubule stabilization.