Biology Reference
In-Depth Information
that arise from the methodologies that are used in different laboratories. Nonetheless,
the effects of various treatments on microtubule detachment can be accurately
assessed provided that appropriate controls are used, and the procedure is standard-
ized within a given laboratory.
4.5
MICROTUBULE FRAGMENTS AS A SURROGATE
FOR DETACHMENT
Direct measurements of microtubule detachment can be tricky and time consuming.
However, we have seen that cells with high frequencies of detachment invariably
exhibit microtubule fragments in the cytoplasm. For example, paclitaxel-dependent
CHO cells with mutations in tubulin not only have been found to have high frequencies
of microtubule detachment when they are grown without the drug, but they also have
fewer than normal microtubules as well as abundant microtubule fragments (
Ganguly
et al., 2010
). Similar results have been seen in cells that overexpress class III or class V
b
-tubulin (
Bhattacharya et al., 2011; Ganguly, Yang, Pedroza, et al., 2011
) and in cells
treated with drugs that are known to inhibit microtubule assembly and block cell di-
vision (
Yang et al., 2010
). Treatment of paclitaxel-dependent cells with concentrations
of paclitaxel (or any other drug known to stabilize microtubules) that restores cell di-
vision has also been found to suppress microtubule detachment and eliminate the pres-
ence of cytoplasmic microtubule fragments. The explanation for these observations is
that microtubules that detach from the centrosome expose their minus ends which are
seldom, if ever, seen to elongate, but are frequently seen to shorten (
Keating et al.,
1997; Yang et al., 2010
). Because unattached microtubules are able to shorten from
both the plus and minus ends, fewer intact microtubules are seen in the cell, but many
more fragments are present. These fragments vary in length depending on the initial
size of the detachedmicrotubule and how long it has been unattached. In addition, frag-
ments are often seen to translocate toward the outer edges of the cell. This translocation
may be powered by treadmilling (addition of subunits at the plus end balanced by loss
of subunits at the minus end), but the involvement of microtubule motor molecules
cannot be ruled out. These processes result in a relatively uniform distribution of
fragments with varying size scattered throughout the cytoplasm. Because of the link
between microtubule detachment and fragment formation, it is often possible to
gauge the effects of a treatment on microtubule detachment by simply quantifying
the number of cells with greater than a threshold number of fragments in the cytoplasm
(
Yang et al., 2010
).
The ability to detect microtubule fragments resulting from detachment can some-
times be problematic because treatments that alter detachment produce varying mi-
totic defects in different cell types. Some cell lines undergo apoptosis when they are
blocked in mitosis and are lost from the cell culture, whereas others slip through the
mitotic block and reenter the cell cycle to produce larger and flatter cells that make it
easier to detect any fragments that are present. In addition, the use of microtubule
fragments as a surrogate for detachment has to be exercised with some caution
