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Vol. 12, Issue 4, 971-980, April 2001
Tubulin
Department of Biology and Program in Molecular and Cellular
Biology, University of Massachusetts, Amherst, Massachusetts 01003
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.
Online version of this article contains video
material and is available at www.molbiocell.org.
*
Corresponding author. E-mail address: patw{at}bio.umass.edu.
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