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overexpression is provided in Fig. 4.3 . Although the fragments produced by detach-
ment ( Fig. 4.3 A) and class VI
-tubulin expression ( Fig. 4.3 B) have a similar appear-
ance, the fragments produced by severing ( Fig. 4.3 C) have readily discernable
differences. Thus, with a little experience, it is possible to estimate whether a given
treatment increases detachment by looking at the pattern of microtubule fragments
that are formed.
b
4.6 MECHANISM OF DETACHMENT
The observation that microtubule detachment increases during mitosis suggests that
it is a regulated process facilitated by specific proteins. Given where detachment
takes place, it makes sense that proteins involved in the process should be located
at centrosomes and spindle poles. Severing proteins such as katanin would seem
to be obvious candidates, but as mentioned already, overexpression of these proteins
produces a microtubule fragmentation pattern that is quite distinct from the one pro-
duced by detachment. Moreover, we have found that mutations in tubulin as well as
overexpression of class V or class III
b
-tubulins that stimulate detachment confer
resistance to paclitaxel ( Bhattacharya et al., 2011; Ganguly, Yang, Pedroza, et al.,
2011; Ganguly et al., 2010 ), yet overexpression of severing proteins do not (unpub-
lished data). A protein that does appear to be involved in detachment is mitotic
centromere-associated kinesin (MCAK). This kinesin family protein is known to
cause microtubule disassembly in vitro ( Desai, Verma, Mitchison, & Walczak,
1999 ) and is not only associated with mitotic centromeres but also with centrosomes
and spindle poles ( Ems-McClung & Walczak, 2010 ). As predicted for a protein in-
volved in detachment, overexpression of MCAK produces a typical pattern of micro-
tubule fragments, increases the detachment frequency, and confers paclitaxel
resistance ( Ganguly et al., 2011b ). Mitotic cells depleted of MCAK produce “hairy”
spindles that contain longer and more abundant astral and interpolar microtubules
consistent with a decrease in detachment ( Kline-Smith & Walczak, 2002 ). In addi-
tion, depletion of MCAK is able to fully or partially correct the abnormally high de-
tachment frequencies associated with class III
-tubulin overexpression and with
tubulin mutations in paclitaxel-dependent cell lines ( Ganguly, Yang, Pedroza,
et al., 2011 ). Because some paclitaxel-dependent cell lines with abnormally high de-
tachment frequencies can only be partially rescued by MCAK depletion, it is plau-
sible that additional proteins may be involved in the process.
It should be noted that MCAK levels are highest during mitosis, the same time
that detachment frequency is highest ( Ganguly, Bhattacharya, & Cabral, 2008 ).
At mitotic centromeres, MCAK acts to sever microtubule attachments to misa-
ligned chromosomes, but its role at the spindle poles remains unsettled ( Kline-
Smith, Khodjakov, Hergert, & Walczak, 2004 ). We propose that one of its major
functions at spindle poles is to facilitate the detachment of microtubules to pro-
duce fragments needed for the construction of the mitotic spindle apparatus
( Ganguly et al., 2011b ).
b
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