Microtubules acquire resistance from mechanical breakage through intralumenal acetylation

Abstract

Eukaryotic cells rely on long-lived microtubules for intracellular transport and as compression-bearing elements. We considered that long-lived microtubules are acetylated inside their lumen and that microtubule acetylation may modify microtubule mechanics. Here, we found that tubulin acetylation is required for the mechanical stabilization of long-lived microtubules in cells. Depletion of the tubulin acetyltransferase TAT1 led to a significant increase in the frequency of microtubule breakage. Nocodazole-resistant microtubules lost upon removal of acetylation were largely restored by either pharmacological or physical removal of compressive forces. In in vitro reconstitution experiments, acetylation was sufficient to protect microtubules from mechanical breakage. Thus, acetylation increases mechanical resilience to ensure the persistence of long-lived microtubules.

Fig. 1. Long-lived microtubules are lost in the absence of α-tubulin K40 acetylation

(A) α-tubulin K40 acetylation and detyrosination levels were measured by immunoblotting lysates of RPE cells treated with two different siRNAs against TAT1 (siTAT1#2 and siTAT1#3) or control siRNAs (siControl). (B) Immunofluorescence (IF) images of siRNA-treated RPE cells stained for acetylated α-tubulin K40 (red), detyrosinated tubulin (green) and DNA (blue). Scale bar: 10 μm. (C) IF images of siRNA-treated RPE cells treated with 2 μM nocodazole and stained for α-tubulin (white), acetylated α-tubulin (red) and DNA (blue). Bottom panels show the α-tubulin channel alone. Insets show the highly curved microtubules present in control-depleted cells and the very short microtubules in TAT1-depleted cells. Scale bar, 10 μm (main panels). Insets are 10 × 10 μm. 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 (siTAT1#3) and 155 (siTAT1#2) cells, 4 independent experiments; N (60 min) = 236 (siCTRL), 302 (siTAT1#3) and 206 (siTAT#2) cells, 3 independent experiments. Error bars indicate SD. Asterisks indicate t test significance values; ***P < 10⁻⁴. (E) The box is bound by the 25^th–75^th percentile, whiskers span 5^th to 95^th percentile and the bar in the middle is the median. N (40 min) = 3,058 (siControl), 4,659 (siTAT1#3) microtubules from at least 500 cells, 6 independent experiments; N (60 min) = 880 (siControl), 1,783 (siTAT1#3) and 1323 (siTAT1#2) microtubules from at least 180 cells, 3 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 α-tubulin K40 and α-tubulin. Scale bar, 10 μm. (G) The level of α-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 = 1,904 data points from 23 microtubules. Asterisks indicate Mann-Whitney U test significance values; **P < 10⁻³, ***P < 10⁻⁴.

Fig. 2. TAT1 depletion sensitizes nocodazole-resistant microtubules to mechanical breakage

(A–C) Microtubules were imaged in real-time in siRNA-treated RPE-[EMTB-GFP³] cells after at least 15 min in the presence of 2 μM nocodazole. Projection images were generated to capture microtubules across the entire cell thickness and to avoid missing microtubule segments because they leave the focal plane. The yellow lines highlight microtubule behavior and the red box indicates the first frame where rupture is clearly detected. Scale bar, 1 μm. The time series are extracted from Movies S5–S7. (D) Microtubule breakage events preceded by buckling were counted in control- and TAT1-depleted RPE-[EMTB-GFP³] cells during the 15 to 80 min period of nocodazole treatment. N = 46 (siControl) and 52 (siTAT1) cells, 6 independent experiments. The bar marks the mean. Asterisks indicate t test significance values; ***P < 10⁻⁴.

Fig. 3. Release of cell tension restores the length of nocodazole-resistant microtubules in TAT1-depleted cells

(A) Control- and TAT1-depleted cells were treated with Y27632 or vehicle for 1 h, then nocodazole was added for 40 min and cells were fixed and stained for α-tubulin. Insets show the detailed morphology of nocodazole-resistant microtubules. Scale bar: 10 μm. Insets are 10 × 10 μm. Cells before nocodazole treatment are shown in Fig. S10B. While Nocodazole-resistant microtubules are few and short in the absence of TAT1, the addition of Y27632 leads to the presence of numerous long nocodazole-resistant microtubules in TAT1-depleted cells. (B) Measurement of individual microtubule length. The box plots follow the same conventions as Fig. 1E. N = 128 cells (2,371 microtubules) siControl/DMSO, 369 cells (2,879 microtubules) siTAT1/DMSO, 152 cells (2,462 microtubules) siControl/Y27632 and 359 cells (2,446 microtubules) siTAT1/Y27632, 3 independent experiments. Asterisks indicate multiple regression test significance values. ***P < 10⁻⁴. (C) Control- and TAT1-depleted cells plated on glass coverslips (elastic modulus 50 GPa) or polyacrymide-gel (PA) coated coverslips (elastic modulus 7 kPa) were treated with nocodazole for 40 min, fixed with PFA and stained for α-tubulin. Insets show the detailed morphology of nocodazole-resistant microtubules. Scale bar: 10 μm (main panels). Insets are 10 × 10 μm. Cells before nocodazole treatment are shown in Fig. S12. Nocodazole-resistant microtubules in TAT1-depleted cells are nearly absent when cells are plated onto glass but largely intact when cells are plated onto soft substrates. (D) Measurement of individual microtubule length. The box plots follow the same conventions as Fig. 1E. N = 1,489 microtubules (siControl/glass), 2,201 (siTAT1/glass), 1,739 (siControl/PA) and 2,138 (siTAT1/PA), 3 independent experiments. Asterisks indicate multiple regression test significance values. ***P < 10⁻⁴. n.s. indicates Mann-Whitney U test significance value P > 0.01.

Fig. 4. Acetylation protects microtubules from mechanical breakage

(A) The microfluidic device used to reconstitute microtubule bending and breaking comprised two inlets and two outlets to control fluid flow along two orthogonal axes. By flowing them along the long axis, microtubule seeds (red) were grafted normally to the micropatterned lines, thus forcing microtubules to elongate parallel to the long axis. For the breakage assay, large beads (pink) that nonspecifically adhere to the surface were included to serve as fixed obstacles. A controlled fluid flow was applied along the short axis to subject microtubules to a normal bending force (right). The solution applied during the bending step contained free tubulin to keep microtubules dynamic and small beads (red) were added to the flowed solution to measure the flow in situ. (B) Time series showing the progressive bending of a microtubule (green) upon application of fluid flow. Scale bar: 5 μm. The pseudocolored image shows the overlay of successive time points. (C) Quantitation of the persistence length of microtubules made from enzymatically acetylated and deacetylated tubulin. The box plot follows the conventions of Fig. 1G. The levels of αK40 acetylation were 97.2 % (Ac⁹⁷) or 0.8 % (Ac¹). N = 29 (Ac¹) and 25 (Ac⁹⁷) microtubules, 3 independent experiments. Asterisks indicate Mann-Whitney U test significance values. ***P < 10⁻⁴. (D) Time series showing the breaking of a microtubule (green) upon application of fluid flow. Large beads nonspecifically adhering to the surface (arrowhead) were used as fixed obstacles to enhance microtubule bending upon flow thus resulting in microtubule rupture at the site of maximal bending. Scale bar: 10 μm. (E) Time taken for microtubules to break after application of flow. The shortest experimental application of flow was 9.55 s and all microtubules not broken at 9.55 s are displayed as dots. N = 46 (Ac¹) and 42 (Ac⁹⁷) microtubules, 2 independent experiments. The frequency of breakage is 28% for Ac¹ microtubules and 2% for Ac⁹⁷ microtubules. A Mann-Whitney U test was conducted on the entire data set and asterisks indicate significance values. **P < 0.005. (F) Model for regulation of microtubules mechanics by TAT1-mediated acetylation. We propose a two-step adaptive model for the mechanical stabilization of microtubules where bending results in sidewall breathing and lets TAT1 enter the lumen. Subsequent acetylation locally modifies the mechanical properties of the microtubule to protect it against flexural breakage.