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 μm2 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.

The Role of Microtubules and Their Dynamics in Cell Migration

“…Drug-treated cells were still able to extend lamellipodia and elongate in the direction of migration, but they spent a long time in the elongated state unable to retract their tails. Consistent with this observation, dynamic microtubules in wild-type cells were largely restricted to the tail as previously noted (30), but trailing microtubules were far less dynamic in migration-impaired mutant cells. It thus appears that suppression of microtubule dynamics "freezes" the tail into a configuration that is not easily remodeled.…”

Amer2 Protein Interacts with EB1 Protein and Adenomatous Polyposis Coli (APC) and Controls Microtubule Stability and Cell Migration

“…In line with this, microtubules are more stable at the leading edge than at the trailing edge of migrating cells (30). Our knockdown experiments showed that Amer2, APC, and EB1 are similarly required for cell migration, probably due to their effects on microtubule stability.…”

Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site

“…To detect growing MT plus ends, EB1-GFP was expressed in PtK2 cells. Fluorescent 'comets' with bright fronts and dimmer 'tails' were observed at the elongating MTs in PtK2 cells, as described in other cells (Mimori-Kiyosue et al, 2000;Ma et al, 2004;Salaycik et al, 2005). There were also faint MT images in addition to the comets.…”