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Tubulin acetylation protects long-lived microtubules against mechanical ageing

Nature Cell Biology volume 19, pages 391–398 (2017)Cite this article

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Abstract

Long-lived microtubules endow the eukaryotic cell with long-range transport abilities. While long-lived microtubules are acetylated on Lys40 of α-tubulin (αK40), acetylation takes place after stabilization1 and does not protect against depolymerization2. Instead, αK40 acetylation has been proposed to mechanically stabilize microtubules3. Yet how modification of αK40, a residue exposed to the microtubule lumen and inaccessible to microtubule-associated proteins and motors1,4, could affect microtubule mechanics remains an open question. Here we develop FRET-based assays that report on the lateral interactions between protofilaments and find that αK40 acetylation directly weakens inter-protofilament interactions. Congruently, αK40 acetylation affects two processes largely governed by inter-protofilament interactions, reducing the nucleation frequency and accelerating the shrinkage rate. Most relevant to the biological function of acetylation, microfluidics manipulations demonstrate that αK40 acetylation enhances flexibility and confers resilience against repeated mechanical stresses. Thus, unlike deacetylated microtubules that accumulate damage when subjected to repeated stresses, long-lived microtubules are protected from mechanical ageing through their acquisition of αK40 acetylation. In contrast to other tubulin post-translational modifications that act through microtubule-associated proteins, motors and severing enzymes, intraluminal acetylation directly tunes the compliance and resilience of microtubules.

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Figure 1: αK40 acetylation impairs microtubule nucleation and accelerates depolymerization.
Figure 2: Tubulin acetylation affects tubulin self-assembly.
Figure 3: αK40 acetylation weakens inter-protofilament interactions.
Figure 4: The protofilament interaction assay produces parallel sheets.
Figure 5: Acetylation at αK40 protects microtubules against stress-induced material fatigue.

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Acknowledgements

This work was supported by HFSP Grant RGY0088/2012 to M.V.N. and M.T., ERC grant 310472 to M.T. and a Stanford School of Medicine Dean’s Postdoctoral Fellowship to D.P. We thank B. K. Kobilka for the use of his fluorimeter, J. Al-Bassam for advice and assistance on imaging of microtubule dynamics, E. Nogales for comments on the manuscript and E. Verdin (UCSF/Gladstone, USA) for the SIRT2 expression construct. The Jeol JEM1400 TEM was funded by NIH grant 1S10RR02678001 to the Stanford Microscopy Facility.

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Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, California 94305, USA

    Didier Portran, Zhenjie Xu & Maxence V. Nachury

  2. Laboratoire de Physiologie Cellulaire et Végétale, Institut de Recherche en Technologie et Science pour le Vivant, UMR5168, CEA/INRA/CNRS/UGA, 38054 Grenoble, France

    Laura Schaedel & Manuel Théry

  3. Unité de Thérapie Cellulaire, Hôpital Saint Louis, Institut Universitaire d’Hématologie, UMRS1160, INSERM/AP-HP/Université Paris Diderot, 75010 Paris, France

    Manuel Théry

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Contributions

Z.X. was involved in the initial conceptualization of the project. M.V.N. and M.T. designed the study. D.P. developed and conducted the enzymatic modifications of tubulin, self-assembly assays and electron microscopy. L.S. performed the measurement and analysis of microtubule persistence length and material fatigue. M.V.N. and D.P. wrote the manuscript with input from all authors.

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Correspondence to Maxence V. Nachury.

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Integrated supplementary information

Supplementary Figure 1 The effect of tubulin acetylation on microtubule assembly is reversible.

a, Diagram of the experimental procedure to produce enzyme-free acetylated or deacetylated tubulin. b, Coomassie-stained SDS-PAGE gel showing the acetylated and deacetylated tubulin, free of modifying enzymes (representative of 3 independent purifications). 10 μg were loaded in each lane. c, Uncropped scans of the gel and immunoblot shown in Fig. 1a. Red boxes denote the cropped regions shown in Fig. 1a. d, Diagram of the experimental procedure to re-acetylate or deacetylate previously deacetylated or acetylated tubulin. e, Samples were resolved on SDS-PAGE and Coomassie-stained (top) or immunoblotted for K40 acetylated α-tubulin (bottom) (1 experiment performed). Axonemal preparations from Tetrahymena cilia provide a 100% acetylation calibrator. f, Polymer formation was monitored by following the turbidity, or absorbance at 350 nm, of solutions containing 40 μM tubulin incubated at 37 °C (1 experiment performed). The levels of acetylation and not the order of acetylation/deacetylation affect polymer assembly.

Supplementary Figure 2 Tubulin acetylation affects microtubules nucleation and depolymerization speed.

a, The experiment imaged in Fig. 1c was analyzed and microtubule nucleation was plotted against time. Data were linearly fitted. Error bars: SEM, (from n = 3 independent experiments). b, Table showing the mean rates of nucleation (±SEM), and the dynamic instability parameters (mean ± SD) measured using the TIRF assay for n = 117 Ac96 microtubules and n = 156 Ac0 microtubules (pooled from 3 independent experiments). P-values are from two-tailed unpaired Student’s t-tests. c, The pre-nucleation self-assembly assays were imaged by fluorescence microscopy to visualize the presence or absence of rhodamine labeled microtubules. Scale bar, 5 μm. Representative images from 3 independent experiments. Microtubule nucleation is observed when 5 μM tubulin is mixed with either GMPCPP or GTP and taxol. For this reason, the self-assembly assays in Fig. 2c, e were conducted in the presence of 0.5 μM tubulin. d, Tubulin self-assembly assayed by inter-dimer FRET. Assay was conducted as in Fig. 2b–e. 5 μM tubulin was mixed with 1 mM GDP. Data points are mean ± SEM. n = 4 independent experiments for Ac96 tubulin and n = 3 independent experiments for Ac0 tubulin. e, GTPase assay. Dot plot showing the molar amount of inorganic phosphate released from the hydrolysis of GTP by Ac0 or Ac96 tubulin during a 2.5 h reaction performed at 4 °C, 37 °C or at 37 °C in the presence of GMPCPP seeds. The rates of GTP hydrolysis largely mirror the rates of bulk polymerization reported in Fig. 1b and d. Notably, once the polymerization rates of Ac96 and Ac0 tubulin are normalized by addition of nucleation seeds, GTP hydrolysis is not significantly affected by the degree of tubulin acetylation. The bar indicates the mean, n = 4 independent experiments for each condition. P-values are from two-tailed unpaired Student’s t-tests. f, Diagram of the protofilament assembly experiments. Protofilaments were assembled at 4 °C in presence of GTP, GTPγS, GMPCPP or GTP + Taxol.

Supplementary Figure 3 Neither acetylation nor dialysis affect the shape or length of protofilaments.

Representative EM images of protofilaments assembled from 5 μM of free tubulin (representative images from 2 independent experiments for each conditions) with 1 mM GTP (a), 1 mM GTPγS (b), 1 mM GTP + 5 μM taxol (c), or 0.5 mM GMPCPP (d). The median values of the protofilament length and radius are shown below the corresponding EM images for Ac0 protofilament (in blue) and Ac96 protofilaments (in red). e,f, Tukey box plots of the length (e) and radius (f) of the Ac0 (blue boxes) and Ac96 (red boxes) protofilaments assembled in the presence of GTPγS or GMPCPP. n = 627 Ac0 protofilaments and n = 717 Ac96 protofilaments were measured after assembly in the presence of GTPγS and n = 532 Ac0 protofilaments and n = 554 Ac96 protofilaments were measured after assembly in the presence of GMPCPP (pooled from 2 independent experiments). A Mann-Whitney test was used to compare Ac0 and Ac96 protofilaments populations in each condition. No significant differences were observed between Ac0 and Ac96 protofilaments in length (P = 0.97 for GTPγS and P = 0.49 for GMPCPP) or radius (P = 0.59 for GTPγS and P = 0.20 for GMPCPP). All box plots are Tukey boxplots, the box represents the 25th–75th percentile, whiskers indicate 1.5 times the range, bar in the middle is the median. g, Dot plot of the amount of tubulin pelleted at 424,000gave for 1 h at 4 °C with protofilaments or free tubulin (5 μM of tubulin initial concentration). Protofilaments were assembled in the presence of taxol and GTP at 4 °C and dialyzed to replace GTP with GDP. The pelleting assay was conducted in the presence of taxol and GDP (n = 3 independent experiments). h, Protofilament dialysis is diagramed (left) and representative EM images from Ac96 and Ac0 protofilaments (representative images from 2 independent experiments) are shown (right). i, Box plots showing the length and radii of the Ac96 and Ac0 protofilaments before (n = 479 and 475 respectively) and after dialysis (n = 457 and 489 respectively) (pooled from 2 independent experiments). All box plots are Tukey boxplots, the box represents the 25th–75th percentile, whiskers indicate 1.5 times the range, bar in the middle is the median. A Mann-Whitney test comparing Ac0 and Ac96 protofilament lengths or radii failed to find a significant difference in length (P = 0.08 before dialysis and P = 0.52 after dialysis) or radius (P = 0.16 before dialysis and P = 0.19 after dialysis).

Supplementary Figure 4 Controls for the FRET assay and gel images of the pelleting assays.

a, Coomassie-stained SDS-PAGE gels of the pelleting assays (representative of 3 independent experiments for each condition) plotted in Supplementary Fig. 3g. b, Dot plot representing the amount of tubulin (protofilament preparations, 0.5 μM of tubulin initial concentration) pelleted at 86,000gave for 30 min at 32 °C. c, Coomassie-stained SDS-PAGE gels of the pelleting assays plotted in Fig. 3f and Supplementary Fig. 4b (representative of 3 independent experiments for each condition).

Supplementary Figure 5 Acetylation at αK40 protects microtubules against stress-induced material fatigue.

a,b, EM micrographs of the protofilaments interaction assays. Protofilaments were incubated at 32 °C for 30 min with 0.5 μM taxol and 1 mM GDP, and imaged by negative-stain EM. Scale bar, 100 nm, (representative images from 2 independent experiments). c–f, Measurements of Ac96 (c and e) and Ac0 (d and f) microtubule persistence length evolution over successive bending cycles (n = 11 Ac96 microtubules and n = 17 Ac0 microtubules). Delay between cycles was 10 s. Spearman correlation tests were performed on the persistence length values over successive cycles to test for tendency. Green curves are for microtubules whose persistence length was not significantly affected over the bending cycles. Magenta curves are for microtubules that had softened during the cyclic stress. In e and f, Microtubule persistence lengths were normalized to their initial value. All data were used to generate the graphs in Fig. 5b, d.

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Time lapse of the material fatigue experiment.

An Ac0 microtubule is shown in the top panel and an Ac96 microtubule in the bottom panel. Microtubules were elongated from GMPCPP seeds grafted onto micropatterns, bent using a perpendicular flow for 10 s and then allowed to relax for 10 s. The microtubules are kept dynamic during the experiment by maintaining tubulin concentration at 14 μM in the flowing solution. Microtubule persistence lengths measured during the first bending cycle. The left top and bottom panel are the experimental data, the middle top and bottom panel represent the snake generated by FilamentJ (ImageJ) of the bending microtubules, the right top and bottom panel are pseudocolor images of a single representative microtubule at the end of each bending cycle. Time is represented in (min:sec), Scale bar, 5 μm. (AVI 1295 kb)

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Portran, D., Schaedel, L., Xu, Z. et al. Tubulin acetylation protects long-lived microtubules against mechanical ageing. Nat Cell Biol 19, 391–398 (2017). https://doi.org/10.1038/ncb3481

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