Cell cycle-dependent changes in microtubule dynamics in living cells expressing green fluorescent protein-alpha tubulin

LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-alpha tubulin construct and a cell line permanently expressing GFP-alpha tubulin was established (LLCPK-1 alpha). The mitotic index and doubling time for L LCPK-1 alpha were not significantly different from parental cells. Quantita tive immunoblotting showed that 17% of the tubulin in LLCPK-1 alpha cells w as GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that i n parental cells. The parameters of microtubule dynamic instability were co mpared for interphase LLCPK-1 alpha and parental cells injected with rhodam ine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1 alpha cells are a useful tool for analysis of m icrotubule dynamics throughout the cell cycle. Comparison of astral microtu bule behavior in mitosis with microtubule behavior in interphase demonstrat ed that the frequency of catastrophe increased twofold and that the frequen cy of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated stat e, or pause, was also dramatically reduced, from 73.5% in interphase to 11. 4% in mitosis. The rates of microtubule elongation and rapid shortening wer e not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubul e 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.