Biology Reference
In-Depth Information
spindles as well (
O'Connell & Khodjakov, 2007
). More recently, it was reported that
a protein complex called “augmin” is able to nucleate new microtubules away from
the centrosome and may thus contribute to the high density of these polymers in the
area between the spindle poles and chromosomes (
Goshima, Mayer, Zhang,
Stuurman, & Vale, 2008
). Additional microtubule fragments for spindle assembly
may be generated through detachment. It has recently been shown that a sevenfold
increase in the rate of microtubule detachment from spindle poles occurs as early
as prophase (
Yang, Ganguly, & Cabral, 2010
) and a similar and perhaps larger
increase occurs during anaphase but the mechanisms appear to differ (
Rusan &
Wadsworth, 2005
).
Although the detachment of microtubules from interphase centrosomes occurs
at a very low rate (
Yang et al., 2010
), the increased frequency in mitotic cells sug-
gests that it is a cell cycle-regulated event that may be of vital importance for spindle
structure and cell division. In support of this hypothesis, paclitaxel-dependent CHO
cells were shown to have a highly elevated frequency of detachment. When grown
without the drug, these cells have fragmented microtubules, much reduced polymer
levels, defective mitotic spindle assembly, and an inability to divide into daughter
cells (
Cabral, 1983; Cabral, Wible, Brenner, & Brinkley, 1983; Ganguly, Yang, &
Cabral, 2010; Schibler & Cabral, 1986
). All of these problems are prevented by
the presence of paclitaxel or other microtubule stabilizing drugs that also correct
the abnormally high rate of microtubule detachment. The results with paclitaxel-
dependent mutants suggested that microtubule destabilizing drugs such as col-
chicine, vinblastine, and nocodazole might act by increasing the frequency of
microtubule detachment, and this prediction was confirmed in a number of cell lines
(
Yang et al., 2010
). The observation that drugs modulate the frequency of microtu-
bule detachment at the same concentrations that interfere with cell division suggests
that microtubule detachment is an important process that is necessary for spindle
assembly and function. The additional observation that tubulin mutations and over-
expression of
-tubulin isotypes that interfere with cell division also alter the micro-
tubule detachment frequency and that detachment is corrected at drug concentrations
that allow the cells to proliferate, further supports the link between microtubule de-
tachment and spindle function (
Bhattacharya, Yang, & Cabral, 2011; Ganguly,
Yang, Pedroza, Bhattacharya, & Cabral, 2011; Ganguly et al., 2010
). Given the
growing awareness of the importance of detachment for understanding cell division
and antimitotic drug action, we will present what we have learned in the process of
studying these events as a guide for further investigations into this understudied and
poorly understood phenomenon.
b
4.1
CHOICE OF CELL LINE
The main challenge for calculating microtubule detachment is the high density of
microtubules surrounding the centrosome that make it difficult to resolve single mi-
crotubules. A related factor is the high fluorescence intensity in that area that requires
