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because other mechanisms can also cause microtubule fragmentation. We have
looked for spontaneous fractures due to mechanical stress, but these occur much
more rarely and are seldom seen even in microtubules that form hairpin loops. More-
over, the frequency of these events is not cell cycle-dependent nor is it altered by
drugs or other perturbations that affect detachment (
Bhattacharya et al., 2011;
Ganguly et al., 2010; Yang et al., 2010
). We have also looked for spontaneous nu-
cleation away from the centrosome and have failed to find any evidence for this
mechanism of fragment formation. On the other hand, we have found large numbers
of microtubule fragments in cells that overexpress class VI
-tubulin, an isotype
whose expression is normally limited to platelets in mammals (
Schwer et al.,
2001; Yang, Ganguly, Yin, & Cabral, 2011
). Unlike the fragments produced by
detachment, the formation of fragments due to class VI
b
-tubulin expression appears
to be limited to mitotic cells. Because of their extraordinary stability due to incor-
poration of this unusual isotype, the fragments are carried forward into subsequent
phases of the cell cycle. We do not see increased frequencies of detachment in these
cells indicating that they arise by a distinct mechanism (
Yang et al., 2011
). Similarly,
we have seen that overexpression of katanin and other microtubule severing proteins
can also produce microtubule fragments, but these fragments tend to be much more
uniform in size and are frequently arranged in a linear array as would be expected for
multiple severing of a linear filament. A side-by-side comparison of microtubule
fragments produced by detachment, class VI
b
-tubulin overexpression, and katanin
b
FIGURE 4.3
Mechanisms that generate microtubule fragments. Paclitaxel-dependent CHO
b
-tubulin
mutant Tax 18 grown without the drug (A), CHO cells transfected with class VI
-tubulin (B),
and CHO cells transfected with the catalytic subunit of katanin (C) are shown. The cells were
stained with an antibody to
b
-tubulin. Arrows indicate the transfected cells. Note that the
pattern of fragments are similar in Tax 18 (A) and class VI b-tubulin-transfected cells (B) even
though the mechanisms by which they are generated differ (
Ganguly et al., 2010; Yang et al.,
2011
). On the other hand, the pattern of fragments generated by katanin overexpression is
distinct (C). In contrast to (A) and (B), the fragments in panel C are shorter, often follow a
linear path due to severing along the length of the microtubule, and long microtubules
remaining attached to the centrosome are seldom seen. In addition, the centrosome itself is
much less distinct. Scale bar
a
10
m
m.
ΒΌ
