Microtubules are stabilized in confluent epithelial cells but not in fibroblasts

Abstract

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 × 10-8 cm2/s. This pool of soluble cytoplasmic tubulin was increased to >80% when cells were treated with nocodazole, or reduced to ∼20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to >22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to ∼6 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of ∼9 min and one with a t1/2 of >25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half-life was ∼10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.