Abstract. The two centrioles that are localized close to each other and to the nucleus in single MadinDarby Canine kidney cells (MDCK) move apart by distances as large as 13/xm after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.

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.

Abstract. The micmtubnle-nucleating activity of centrosomes was analyzed in fibroblastic

Abstract. MDCK cells form a polarized epithelium when they reach confluence in tissue culture. We have previously shown that concomitantly with the establishment of intercellular junctions, centrioles separate and microtubules lose their radial organization (Bacallao, R., C. Antony, C. Dotti, E. Karsenti, E. H. K. Stelzer, and K. Simons. 1989. J. Cell Biol. 109:2817-2832. Buendia, B., M. H. Brt, G. Griffiths, and E. Karsenti. 1990Karsenti. . 110:1123Karsenti. -1136. In this work, we have examined the pattern of microtubule nucleation before and after the establishment of intercellular contacts. We analyzed the elongation rate and stability of microtubules in single and confluent cells. This was achieved by microinjection of Paramecium axonemal tubulin and detection of the newly incorporated subunits by an antibody directed specifically against the Paramecium axonemal tubulin. The determination of newly nucleated microtubule localization has been made possible by the use of advanced doubleimmunofluorescence confocal microscopy. We have shown that in single cells, newly nucleated microtubules originate from several sites concentrated in a region localized close to the nucleus and not from a single spot that could correspond to a pair of centrioles. In confluent cells, newly nucleated microtubules were still more dispersed. The microtubule elongation rate of individual microtubules was not different in single and confluent cells (4/xm/min). However, in confluent cells, the population of long lived microtubules was strongly increased. In single or subconfluent cells most microtubules showed a tla of < 30 min, whereas in confluent monolayers, a large population of microtubules had a t~a of >2 h. These results, together with previous observations cited above, indicate that during the establishment of polarity in MDCK cells, microtubule reorganization involves both a relocalization of microtubule-nucleating activity and increased microtubule stabilization.

In this paper, we report on the effect of brain microtubule-associated proteins (MAPs) on the dynamic instability of microtubules as well as on the nucleation activity of purified centrosomes. Under our experimental conditions, tau and MAP2 have similar effects on microtubule nucleation and dynamic instability. Tau increases the apparent elongation rate of microtubules in proportion to its molar ratio to tubulin, and we present evidence indicating that this is due to a reduction of microtubule instability rather than to an increase of the on rate of tubulin subunits at the end of growing microtubules. Increasing the molar ratio of tau over tubulin leads also to an increase in the average number of microtubules nucleated per centrosome. This number remains constant with time. This suggests that the number of centrosome-nucleated microtubules at steady state can be determined by factors that are not necessarily irreversibly bound to centrosomes but, rather, affect the dynamic properties of microtubules.