Biology Reference
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region near the spindle poles. We have been able to partially overcome problems
associated with the rounded morphology of mitotic CHO cells by examining those
cells during prophase, a stage at which they remain well attached. Despite being early
in mitosis, detachment events are already elevated during prophase compared to in-
terphase (
Yang et al., 2010
). We have not been able to reliably measure detachment
at later stages of mitosis, and so we cannot say with any confidence that detachment
remains elevated following prophase. However, others have reported a high detach-
ment frequency during anaphase in LLCPK1 cells, and they proposed that detach-
ment is required for microtubule rearrangements that may be occurring during
that stage of mitosis (
Rusan &Wadsworth, 2005
). It is possible that detachment plays
a similar role during prophase to hasten the conversion of the cytoplasmic microtu-
bules into mitotic spindle fibers. Alternatively, detachment may be taking place
throughout mitosis to provide microtubule fragments for the construction and dy-
namics of the mitotic spindle apparatus (
Ganguly et al., 2010; Yang et al., 2007
).
Given the problems associated with the measurement of microtubule detachment
from spindle poles, we have used interphase cells as a substitute to follow changes
that are likely to also take place during mitosis. The rationale for this approach comes
from the observation that perturbations such as drug treatment, mutant tubulin
expression, overexpression of class V
-tubulin, and overexpression of MCAK that
interfere with spindle function and cell division alter the detachment frequency not
only during mitosis but also during interphase (
Bhattacharya et al., 2011; Ganguly,
Yang, & Cabral, 2011b; Ganguly, Yang, Pedroza, et al., 2011; Ganguly et al., 2010;
Yang et al., 2010
).
b
4.3
LABELING MICROTUBULES
4.3.1
Rhodamine tubulin microinjection
To detect microtubule detachment and other forms of microtubule behavior such as
dynamic instability, the cellular microtubules must be selectively labeled. This can
be achieved by a variety of methods. One of the earliest approaches used microin-
jection of fluorescently labeled tubulin. While this methodology is effective in label-
ing the microtubules, it suffers from a requirement for specialized microinjection
equipment, mechanical injury to the cell, and a change in the cellular composition
of tubulin. This latter problem can be especially significant because tubulin is com-
monly purified from brain tissue which has a different tubulin isotype composition
from most cultured cell lines (
Leandro-Garcia et al., 2010; Luduena, 1998
). Class III
b
-tubulin found in brain is the most troublesome isotype in this regard because it has
been shown to reduce microtubule assembly, alter drug sensitivity, and counteract
drug effects on dynamic instability (
Ganguly, Yang, & Cabral, 2011a; Hari,
Yang, Zeng, Canizales, & Cabral, 2003; Kamath, Wilson, Cabral, & Jordan,
2005
). Moreover, we have found that expression of this isotype increases the fre-
quency of detachment events (
Ganguly, Yang, Pedroza, et al., 2011
).
