Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin

Abstract

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-␣ tubulin construct and a cell line permanently expressing GFP-␣ tubulin was established (LLCPK-1␣). The mitotic index and doubling time for LLCPK-1␣ were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1␣ cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1␣ and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1␣ cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.

EB1–Microtubule Interactions inXenopusEgg Extracts: Role of EB1 in Microtubule Stabilization and Mechanisms of Targeting to Microtubules

“…Such pauses have been observed by others in vivo and in vitro (Walker et al, 1988;Tirnauer et al, 1999;Davis et al, 1993;Grego et al, 2001;Rusan et al, 2001), but their origins are obscure. On pausing microtubule ends visualized by EB1 fluorescence, the high signal-to-background ratio of EB1 binding allowed visualization of a novel structure not previously reported: curved filamentous extensions with the intensity expected for EB1 bound to a few tubulin protofilaments ( Figure 6).…”

Antagonistic forces generated by myosin II and cytoplasmic dynein regulate microtubule turnover, movement, and organization in interphase cells

“…Thus, inhibition of cytoplasmic dynein-induced MTs to move further than in control cells, but increased the percentage of MTs that moved by only 5%, indicating that movement is limited, perhaps by interaction with or interference by other (Table 1). To determine the mechanism responsible for the increased half-time of turnover, we measured the dynamic instability behavior of the plus-ends of individual MTs in control, ML7-treated, and 70.1-injected cells (15,19). As shown in Table 2, the dynamic instability parameters are similar for all three groups.…”

“…The behavior of individual MTs was measured and analyzed as described (15). To measure turnover of the MT array, fluorescence intensity values, within a rectangle of sufficient dimensions to ensure that the photoactivated region was within the box for the entire sequence, were measured and corrected for background fluorescence (determined by measuring fluorescence in a rectangle of the same dimensions placed in a region of the cell that was not photoactivated).…”

Infection with Replication-deficient Adenovirus Induces Changes in the Dynamic Instability of Host Cell Microtubules

“…The MetaView software was also used to track the length changes of individual MTs at the periphery of cells expressing GFP-tubulin (Rusan et al, 2001). The selection of individual MTs for tracking was based on our ability to clearly define a plus-end tip for at least a 12-s interval.…”