Microtubules are stabilized in confluent epithelial cells but not in fibroblasts.

Abstract

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 x 10 -s cm2/s. This pool of soluble cytoplasmic tubulin was increased to >80% when cells were treated with nocodazole, or reduced to ,v20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t~) 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 tlrz 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 ~16 rain. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t~2 of ~9 rain and one with a t~2 of >25 rain. Correspondingly, the rate of incorporation of rnicroinjected tubulin into interphase micrombules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in micrombule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half-life was ,x,10 rain. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.

Regulation of microtubule dynamics and nucleation during polarization in MDCK II cells.

“…These results suggest that nucleating material is relocalized after the establishment of cell contacts and that microtubules become progressively more stable. This latter point is further demonstrated quantitatively by Pepperkok et al (1990).…”

The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation

“…Epithelial differentiation is known to be associated with an increase in microtubule stability (Pepperkok et al, 1990). In EB2-depleted ARPE-19 cells detyrosinated tubulin was particularly prominent in microtubule bundles (Fig.…”

Generation of noncentrosomal microtubule arrays

“…In these arrays, the plus ends of MTs are located basally, whereas the minus ends are apical (Bacallao et al, 1989). Like in muscle cells and neurons, these MTs tend to be more stable than those in radial interphase arrays (Bre et al, 1987;Pepperkok et al, 1990). In some epithelia, in addition to the apical-basal array, there is a separate apical array of shorter noncentrosomal MTs organized into a meshwork.…”

“…MTs are stabilized in both muscle and epithelial cells (Gundersen et al, 1989;Pepperkok et al, 1990), but whether a capture mechanism or bundling is involved is unclear. The muscle-specific RING finger proteins MURF-2 and MURF-3 both regulate muscle differentiation by modulating MT stability and so probably play a part (McElhinny et al, 2004;Spencer et al, 2000).…”