Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific.

Abstract

Abstract. Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtKI epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CliO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 + 10.8 #m/min, 32.2 • 17.7/zm/min, 11.9 • 6.5/zm/min, and 19.7 + 8.1 /zm/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtKI cells. Thus, individual microtubules in PtK~ lameUae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.

Suppression of Microtubule Dynamics by LY290181

“…At or near steady state, microtubules in vitro and in cells spend a considerable fraction of time in an attenuated or pause phase (19,20,22,33). LY290181 significantly increased the percentage of time that the microtubules spent in the attenuated state (Table I).…”

Vimentin Intermediate Filaments Template Microtubule Networks to Enhance Persistence in Cell Polarity and Directed Migration

“…Microtubules grow and shrink in cycles that yield a turnover rate of 3-5 minutes (Shelden and Wadsworth, 1993) at the population level. Conceptually, this dynamic instability offers a powerful mechanism to translate long-range directional cues on a time scale of several minutes, whereas the actin machinery turns over on a faster time scale (~30-120 seconds), which is necessary to implement an efficient protrusion -adhesioncontraction cycle.…”

Regulation of microtubule dynamic instability

“…Recent observations have demonstrated that dynamic instability is the major pathway of microtubule turnover in living cells [Cassimeris et al, 1988;Sammak and Borisy, 1988;Schulze and Kirschner, 1988;Sheldon and Wadsworth, 1993;Tanaka and Kirschner;19911. For a variety of different cells, direct observation of either fluorescently labeled tubulin subunits or endogenous microtubules has shown that during interphase elongation typically proceeds at rates of 4-7 p d m i n [Schulze and Kirschner, 1988;Sammak and Borisy, 1988;Cassimeris et al, 1988;Tanaka and Kirschner, 19911 although rates up to -20 pni/min have been observed [Sheldon and Wadsworth, 19931.…”

“…It is also possible that the variations in the measured elongation rates result from different experimental protocols, particularly image acquisition intervals [see Sheldon and Wadsworth, 19931. Rapid shortening occurs at rates of 10-20 p d m i n [Sammak and Borisy, 1988;Cassimeris et al, 1988;Sheldon and Wadsworth, 1993;Tanaka and Kirschner, 19911. Typically these dynamic microtubules transit between elongation and rapid shortening phases approximately 1-3 times per min [Cassimeris et al, 1988;Sheldon and Wadsworth, 1993;Tanaka and Kirschner, 19911. While dynamic instability originally described two states (elongation and rapid shortening), a third state may also exist where no detectable elongation or rapid shortening is observed.…”

“…Rapid shortening occurs at rates of 10-20 p d m i n [Sammak and Borisy, 1988;Cassimeris et al, 1988;Sheldon and Wadsworth, 1993;Tanaka and Kirschner, 19911. Typically these dynamic microtubules transit between elongation and rapid shortening phases approximately 1-3 times per min [Cassimeris et al, 1988;Sheldon and Wadsworth, 1993;Tanaka and Kirschner, 19911. While dynamic instability originally described two states (elongation and rapid shortening), a third state may also exist where no detectable elongation or rapid shortening is observed. This state has been referred to as a pause [Walker et al, 19881 and it is rarely observed with pure tubulin [Walker et al, 1988;Toso et al, 1993;Drechsel et al, 19921.…”