highest curvature. Tracking individual microtubules demonstrated that the frequency of microtubule breakage events preceded by buckling was
increased twofold in TAT1-depleted cells compared
with control cells (Fig. 2D). The frequency of microtubule breakage events that were not preceded
by buckling (shown in fig. S9C and movie S10)
showed no significant difference between control
and TAT1-depleted cells (fig. S9D). These findings
suggest that tubulin acetylation protects microtubules from breakage resulting from compressive forces.
The two known types of forces responsible for
buckling and breakage of cytoplasmic microtubules
are microtubule motors pushing onto anchored microtubules (11) and actomyosin contractility transmitted through actin-microtubule linker proteins
(19). Contractility is likely to represent the major
factor responsible for microtubule compression in
nocodazole-treated cells because microtubule depolymerization leads to activation of Rho and Rho-associated kinase (ROCK), thereby increasing myosin activity and stress fiber assembly (22). To test
the hypothesis that long-lived microtubules in TAT1-
depleted cells break under actomyosin-mediated
compression, we treated cells with the ROCK inhibitor Y27632 or the myosin inhibitor blebbistatin
and then removed dynamic microtubules using nocodazole (Fig. 3A, and figs. S10 and S11). Pharmacological release of tension increased the mean
length of nocodazole-resistant microtubules 1.5-fold
in control cells (Fig. 3B). The effect of Y27632 on
TAT1-depleted cells was much more dramatic,
with the mean length of nocodazole-resistant microtubules increasing fourfold (Fig. 3B) and the
length distribution of nocodazole-resistant microtubules approaching that of control cells in the absence of Y27632 (Fig. 3B). A statistical test for the
rescue of microtubule length in TAT1-depleted cells
was highly significant with Y27632 (Fig. 3B) and
significant with blebbistatin (fig. S11). ROCK inhibition did not restore tubulin acetylation in TAT1-
depleted cells (fig. S10, B and C). Thus, inhibition
of the major Rho effectors largely restores the
nocodazole-resistant microtubules lost from TAT1-
Because ROCK inhibition may stabilize microtubules in TAT1-depleted cells through other
mechanisms than the release of compressive forces
[e.g., inhibitory phosphorylation of MAPs (23)], we
sought to release the compressive forces exerted
onto microtubules in a more direct and specific
manner. When cells are plated onto soft substrates
made of fibronectin-coated polyacrylamide (24, 25)
(Fig. 3C and fig. S12), the force-dependent maturation of focal adhesions is stunted, stress fiber
assembly is limited, and contractility is low (25).
Plating cells on polyacrylamide largely rescued
the length of nocodazole-resistant microtubules
in TAT1-depleted cells (Fig. 3D). Meanwhile, the
length of nocodazole-resistant microtubules was
not significantly changed by plating control cells
on polyacrylamide gel, and this indicates that adhesion signaling does not affect long-lived microtubules under these experimental conditions (Fig.
3D and fig. S12D). Together, the partial rescue of
microtubule length by pharmacological and physical
Fig. 1. Long-lived microtubules are lost in the absence of a-tubulin K40 acetylation. (A) a-Tubulin
K40 acetylation and detyrosination levels were measured by immunoblotting lysates of RPE cells
treated with two different small interfering RNAs (siRNAs) against TAT1 (si TAT1 #2 and si TAT1 #3) or
control siRNAs (siControl). (B) Immunofluorescence (IF) images of siRNA-treated RPE cells stained
for acetylated a-tubulin K40 (red), detyrosinated tubulin (green), and DNA (blue). Scale bar, 10 mm.
Insets are 7 by 7 mm. (C) IF images of siRNA-treated RPE cells treated with 2 mM nocodazole and
stained for a-tubulin (white), acetylated a-tubulin (red), and DNA (blue). (Bottom) The a-tubulin channel
alone. (Insets) The highly curved microtubules present in control cells and the very short microtubules in
TAT1-depleted cells. Scale bar, 10 mm (main panels). Insets are 10 by 10 mm. The number (D) and length
(E) of microtubules remaining after nocodazole treatment were measured in siRNA-treated RPE cells.
(D) N (30 min) = 153 (siCTRL), 157 (si TAT1 #3), and 155 (si TAT1 #2) cells, four independent experiments;
N (60 min) = 236 (siCTRL), 302 (si TAT1 #3) and 206 (si TAT #2) cells, three independent experiments.
Error bars indicate SD. Asterisks indicate t test significance values; ***P < 10−4. (E) The box is bound by
the 25th to 75th percentile, whiskers span 5th to 95th percentile, and the bar in the middle is the median.
N (40 min) = 3058 (siControl), 4659 (si TAT1#3) microtubules from at least 500 cells, six independent
experiments; N (60 min) = 880 (siControl), 1783 (si TAT1#3) and 1323 (si TAT1#2) microtubules from at
least 180 cells, three independent experiments. Asterisks indicate Mann-Whitney U test significance
values; ***P < 10−4. (F) IF images of RPE cells treated with nocodazole for 45 min and stained for
acetylated a-tubulin and a-tubulin. Scale bar, 10 mm. (G) The level of a-tubulin K40 acetylation and the
curvature were measured along microtubules in IF images of cells treated with nocodazole for 45 min.
The whiskers indicate 1.5 times the range. N = 1904 data points from 23 microtubules. Asterisks indicate
Mann-Whitney U test significance values; **P < 10−3, ***P < 10−4.