Microtubules acquire resistance from mechanical breakage through intralumenal acetylation^#

Main Text

How some cytoplasmic microtubules are stabilized and persist for several hours remains an open question (1). After stabilization, microtubules are post-translationally detyrosinated and acetylated on lysine 40 of α-tubulin (αK40). While detyrosination alters the binding site for microtubule associated proteins (MAPs), severing enzymes and motors to create specialized microtubule tracks (2), the molecular consequences of αK40 acetylation remain elusive and it is difficult to conceptualize how the modification of a residue inaccessible from outside the microtubule could alter MAP and motor binding (2, 3). We recently proposed that acetylation modifies microtubule mechanics by weakening interprotofilament interactions (4)

TAT1 is responsible for nearly all acetylation on αK40 in every organism studied (2). Although TAT1 depletion from RPE cells did not measurably affect global microtubule polymerization or organization (Fig. S1), tubulin detyrosination was significantly decreased at the bulk level (Fig. 1A and S2A–B) and reduced on microtubules of TAT1-depleted cells (Fig. 1B) and Tat1^−/− MEFs (Fig. S2C). Given that acetylation and detyrosination sites are separated by the microtubule wall, it is unlikely that the two modifications are enzymatically coupled. Instead, long-lived microtubules, including detyrosinated microtubules, may be lost when acetylation is reduced. After treating cells with nocodazole, dynamic microtubules were depolymerized and most remaining microtubules displayed the typical characteristics of long-lived microtubules with high levels of acetylation and detyrosination and long and curvy morphology (Fig. 1C and S2D). Under the same conditions, very few microtubules remained in TAT1-depleted cells and these were very short, dispersed throughout the cell and devoid of detyrosinated tubulin (Fig. 1C). The number of microtubules that remained after nocodazole treatment was significantly decreased upon TAT1 depletion in RPE cells (Fig. 1D) or in Tat1^−/− MEFs (Fig. S2E–F). The effect of TAT1 removal was even more striking when the length of nocodazole-resistant microtubules was examined (Fig. 1E). After 60 min of nocodazole treatment, most microtubules were normally longer than 4 μm, but shorter than 2 μm in TAT1-depleted cells.

Because pharmacological reduction of tubulin detyrosination did not affect the length of nocodazole-resistant microtubules or the levels of acetylation (Fig. S3), the effect of TAT1 depletion on nocodazole-resistant microtubules are unlikely to be caused by the observed reduction in detyrosination. Because overexpression of TAT1 –but not a catalytically dead mutant– significantly elevated the mass of nocodazole-resistant microtubules (Fig. S4) and because nocodazole-resistant microtubules are increased in MEFs that lack the tubulin deacetylase HDAC6 (5), it is αK40 acetylation rather than an acetyltransferase-independent activity of TAT1 (6–8) that is required for the maintenance of long-lived cytoplasmic microtubules in mammalian cells. Long-lived microtubules are not lost from TAT1-depleted cells due to an increased susceptibility to severing enzymes because acetylation does not influence the activity of spastin in vitro (9) and did not affect the activity of spastin or katanin in vivo (Fig. S5). Defective centrosomal microtubule anchoring in TAT1-depleted cells was ruled out by imaging microtubule re-growth after depolymerization (Fig. S6).

Long-lived microtubules display frequent buckling because of compressive forces generated by microtubule-based motors and actomyosin contractility (10–12). Because microtubules are very stiff polymers that rupture when subjected to flexural stresses (13), this highly bent morphology suggests the existence of protective mechanisms for long-lived microtubules. The repair of lattice defects has emerged as an intrinsic property of microtubules that are subjected to mechanical stress (14, 15) and acetylation protects microtubule from mechanical fatigue in vitro (4). Further suggesting that acetylation may confer mechanical protection to microtubules, removing TAT-1 from touch receptor neurons of nematodes results in profound microtubule lattice defects (6, 16) that can be rescued by paralyzing the animals (8). Finally, while detyrosination is evenly distributed along microtubules (Fig. S7A) (17), the pattern of acetylation is discontinuous with a preference for highly curved areas of nocodazole-resistant microtubules (Fig. 1F–G and S7B), suggesting that TAT1 may preferentially acetylate segments experiencing stress. Alternatively, it is conceivable that only bends at regions that are acetylated were preserved after fixation (see Supplementary Text). Localized acetylation is thus a prime candidate for the mechanical adaptation of microtubules to mechanical stresses.

We sought to test the hypothesis that long-lived microtubules disappear in the absence of TAT1 because of an increase rate of breakage under mechanical stress. In the past, imaging of microtubules breakage has been limited to very thin areas of the cell such as lamellipodia where single microtubules can be readily resolved (11, 18, 19). However, most of the microtubules in lamellipodia are dynamic and thus not acetylated (20). To specifically image the breakage of long-lived microtubules, we followed microtubules in real time in the presence of nocodazole using a triple GFP fusion with the microtubule-binding domain of esconsin (EMTB), a MAP that does not affect microtubule dynamics when expressed at low levels (21). After 15 min in nocodazole, most remaining microtubules were highly bent, acetylated and detyrosinated (Fig. S7A) and microtubule number was similar in control- and TAT1-depleted RPE-[EMTB-GFP³] cells (Fig. S8A–B). Over the next 30 min of live cell imaging, microtubules rapidly disappeared in TAT1-depleted cells whereas most microtubules persisted in control-depleted cells (Fig. S8A and C) and the mean microtubule length was significantly decreased in TAT1-depleted cells after 50 min in nocodazole (Fig. S8D). Consistent with prior observations of microtubule breakage in fibroblasts (18, 19), typical rupture events (Fig. 2A–C, S9A–B and Movies S5–S9) were preceded by local microtubule buckling with the breakage site coinciding with the region of highest curvature. Tracking individual microtubules demonstrated that the frequency of microtubule breakage events preceded by buckling was increased 2-fold in TAT1-depleted cells compared to control-depleted cells (Fig. 2D). The frequency of microtubule breakage events that were not preceded by buckling (shown in Fig. S9C and Movie S10) showed no significantly 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 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, S10 and S11). Pharmacological release of tension increased the mean length of nocodazole-resistant microtubules 1.5-fold in control-depleted cells (Fig. 3B). The effect of Y27632 on TAT1-depleted cells was much more dramatic with the mean length of nocodazole-resistant microtubules increasing 4-fold (Fig. 3B) and the length distribution of nocodazole-resistant microtubules approaching that of control-depleted 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. S10B and C). Thus, inhibition of the major Rho effectors largely restores the nocodazole-resistant microtubules lost from TAT1-depleted cells.

Since 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 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 thus indicating that adhesion signaling does not affect long-lived microtubules under these experimental conditions (Fig. 3D and S12D). Together, the partial rescue of microtubule length by pharmacological and physical treatments strongly suggests that acetylation protects long-lived microtubules from breakage resulting from the compressive forces generated by the actomyosin cytoskeleton and makes it unlikely that acetylation prevents nocodazole-induced depolymerization. The observation that microtubule length is not fully rescued is consistent with residual forces (e.g. microtubule motors) applying stress on microtubules after these treatments.

Considering that most tissues have the stiffness of polyacrylamide gels, the observation that nocodazole-resistant microtubules are largely immune to TAT1 depletion when cells are plated on polyacrylamide gels provides a cogent explanation for the very mild phenotypes of Tat1^−/− mice. The marked effects of TAT1 depletion when cells are plated on glass suggest that TAT1 will be required in specialized cell types where the stiffness is considerably higher than a few kPa (e.g. bone) or where microtubules are subjected to repeated mechanical stresses. Congruent with the latter hypothesis, microtubules appear damaged in touch receptor neurons of nematodes that lack TAT-1 (16, 26) and tubulin acetylation sets the optimal cell stiffness for touch sensation in mammalian mechanosensory neurons (27). Further work examining microtubule breakage in these specialized settings is needed to establish the role of tubulin acetylation in physiological contexts.

Because some MAPs can change the mechanical properties of microtubules (28, 29), acetylation could confer mechanical resistance to microtubules by altering the recruitment of specific MAPs. Alternatively, acetylation may change interactions within the lattice to directly alter microtubule mechanics (4). To determine if acetylation directly protects microtubules from physical rupture, we generated pure preparations of enzymatically acetylated and de-acetylated microtubules (4) and reconstituted microtubule breakage in vitro using a modification of our microfluidics- and micropatterning-based microtubule bending system (14) (Fig. 4A). First, we confirmed that flexural rigidity is decreased by acetylation (Fig. 4B–C) (4). By including large stationary beads in the path of the bending microtubules, the microtubule sharply kinked and frequently broke at the site of maximal curvature (Fig. 4D). While nearly a third of the deacetylated microtubules (Ac¹) ruptured under the mechanical stress, only 2% of the highly acetylated microtubules (Ac⁹⁷) broke under the same conditions (Fig. 4E). Thus, acetylation directly protects microtubules from rupture. We propose that, by weakening interprotofilament interactions (4), acetylation increases lattice plasticity and limits the spread of preexisting lattice damage under repeated mechanical stress, thus protecting microtubules from material fatigue (4) or mechanical breakage (Fig. 4E). Because acetylation is enriched in regions of high curvature (Fig. 1G), microtubule mechanics are likely to be modified locally. Furthermore, cyclic stretch of cells increases acetylation (30) and lattice openings are found in bent segments of microtubules (31). Stress-induced bending may thus produce transient openings that let TAT1 access the microtubule lumen in areas experiencing the highest stress (32) and result in an adaptive and local increase in mechanical resilience (Fig. 4F).

Supplementary Material