Metabolic regulation of cytoskeleton functions by HDAC6-catalyzed α-tubulin lactylation

Abstract

Posttranslational modifications (PTMs) of tubulin, termed the "tubulin code", play important roles in regulating microtubule functions within subcellular compartments for specialized cellular activities. While numerous tubulin PTMs have been identified, a comprehensive understanding of the complete repertoire is still underway. In this study, we report that α-tubulin lactylation is catalyzed by HDAC6 by using lactate to increase microtubule dynamics in neurons. We identify lactylation on lysine 40 of α-tubulin in the soluble tubulin dimers. Notably, lactylated α-tubulin enhances microtubule dynamics and facilitates neurite outgrowth and branching in cultured hippocampal neurons. Moreover, we discover an unexpected function of HDAC6, acting as the primary lactyltransferase to catalyze α-tubulin lactylation. HDAC6-catalyzed lactylation is a reversible process, dependent on lactate concentrations. Intracellular lactate concentration triggers HDAC6 to lactylate α-tubulin, a process dependent on its deacetylase activity. Additionally, the lactyltransferase activity may be conserved in HDAC family proteins. Our study reveals the primary role of HDAC6 in regulating α-tubulin lactylation, establishing a link between cell metabolism and cytoskeleton functions.

Fig. 1. Identification of α-tubulin lactylation on K40.

a Schematic diagram of purification and proteomic analysis of lactylated peptides from cultured neurons. b Top 10 significantly enriched molecular function of lactylated proteins. Two-sided Fisher’s Exact Test, p < 0.05. c Mass spectrometry analysis showing lactylation at K40 on α-tubulin from cultured cortical neurons. Expected molecular weights of lactylated peptides from N terminus and C terminus are shown as peaks in red and blue, respectively. The CycIm ion at m/z 156.103 is shown as a peak in green. d Shown are amino acid sequences of tubulins encoded by different genes, including TUBA1A, TUBA1B, TUBA1C, TUBA3, TUBA4A, and TUBA8. The variant amino acids are indicated in red. e Lactylation of α-tubulin encoded by different genes. HEK293T cells were transfected with HA-tagged α-tubulins encoded by indicated tubulin genes. HA-α-tubulin was purified by immunoprecipitation with anti-HA antibody and α-tubulin lactylation was revealed by anti-Lac-K antibody. n = 3 experiments. f Abolished α-tubulin lactylation in K40A mutant. HEK293T cells were transfected with HA-tagged α-tubulin or α-tubulin K40A mutant. g Quantification analysis of data in (f). n = 6 experiments. Two-sided paired student’s t-test, p < 0.0001. Data are shown as mean ± SEM. ***p < 0.001. Source data are provided as a Source Data file.

Fig. 2. Identification of HDAC6 as a primary lactyltransferase for α-tubulin lactylation.

a, b HDAC6 overexpression leads to increased α-tubulin lactylation. HEK293T cells were transfected with Flag-tagged HDAC family proteins, together with HA-α-tubulin. HA-α-tubulin was immunoprecipitated with anti-HA antibody and α-tubulin lactylation was revealed by anti-Lac-K antibody. n = 3 experiments. Two-sided paired student’s t-test, HDAC6 vs control, p = 0.0170. c, d α-Tubulin lactylation is largely reduced in HDAC6-deficient cortex. n = 4 experiments. Two-sided paired student’s t-test, HDAC6^-/- vs HDAC6^+/+, p = 0.0038. e, f HDAC6-induced α-tubulin lactylation occurs on K40 residue. n = 3 experiments. Two-sided paired student’s t-test, K40A vs WT, p = 0.0001; WT + HDAC6 vs WT, p = 0.0473; K40A + HDAC6 vs WT + HDAC6, p = 0.0369. g, h HDAC6-induced α-tubulin lactylation is independent of α-tubulin acetylation. n = 3 experiments. Two-sided paired student’s t-test, WT + HDAC6 vs WT, p = 0.0171; MEC-17^-/- + HDAC6 vs MEC-17^-/-, p = 0.0326. i, j HDAC6 plays the primary role in regulating α-tubulin lactylation. n = 4 experiments. Two-sided paired student’s t-test, MEC-17^-/- vs WT, p = 0.0146; HDAC6^-/- vs WT, p = 0.0014; dKO vs WT, p = 0.0001; HDAC6^-/- vs MEC-17^-/-, p = 0.0055. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 3. HDAC6 catalyzes α-tubulin lactylation through a reversible reaction dependent on its deacetylase activity.

a, b In vitro assays using purified HDAC6 and tubulins in the presence of various concentrations of lactate. 4 μM Flag-HDAC6 and 2 μM tubulin dimers were incubated in the lactylation buffer with the indicated concentrations of lactate at 37 °C for 1 h, and α-tubulin lactylation was revealed by immunoblot. n = 3 experiments. One-way ANOVA, HDAC6 vs control, p = 0.0022; HDAC6 + 0.3 mM Lactate vs control, p = 0.0013; HDAC6 + 10 mM Lactate vs control, p = 0.0286; HDAC6 + 30 mM Lactate vs control, p = 0.0083. c, d HDAC6 catalyzes α-tubulin lactylation in a dose-dependent manner in the in vitro assay. The tubulin dimers at 2 μM and lactate at 10 mM were incubated with the indicated concentrations of Flag-HDAC6 at 37 °C for 1 h. n = 3 experiments. One-way ANOVA, Lactate + 0.3 μM HDAC6 vs control, p = 0.0061; Lactate + 1 μM HDAC6 vs control, p < 0.0001; Lactate + 3 μM HDAC6 vs control, p < 0.0001. e The K_m and K_cat of recombinant Flag-HDAC6 toward lactate were determined in the in vitro assay. f, g The catalytic preference of HDAC6 for tubulin dimers over microtubules (MTs) in the in vitro assay. 4 μM Flag-HDAC6 was incubated with 10 mM lactate, and 2 μM tubulin dimers or microtubules at 37 °C for the indicated time points. n = 3 experiments. Two-way ANOVA, Dimers vs MTs, p = 0.0057. h, i HDAC inhibitor TSA disrupts HDAC6 catalytic activity for α-tubulin lactylation in the in vitro assay. The in vitro assay was performed in the presence of 10 μM TSA. n = 3 experiments. Two-sided paired student’s t-test, HDAC6 + Lactate vs control, p = 0.0012; HDAC6 + Lactate + TSA vs HDAC6 + Lactate, p = 0.0005. j, k HDAC inhibitor TSA and HDAC6 inhibitor TST attenuates HDAC6-induced α-tubulin lactylation in HEK293T cells. Cells were treated with 2 μM TSA or 2 μM TST for 20 h. n = 3 experiments. Two-sided paired student’s t-test, HDAC6 vs control, p = 0.0386; HDAC6 + TSA vs HDAC6, p = 0.0718; HDAC6 + TST vs HDAC6, p = 0.0160. l, m HDAC6-catalyzed α-tubulin lactylation requires its deacetylase activity. n = 4 experiments. Two-sided paired student’s t-test, for Lac-tub, WT vs Ctrl, p = 0.0279; H216A vs Ctrl, p = 0.0299. For Ac-tub, WT vs Ctrl, p = 0.0045; H216A vs Ctrl, p = 0.0008. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 4. Distribution of lactylated α-tubulin in tubulin dimers.

a, b Distribution of lactylated α-tubulin in the soluble tubulin dimers (S), but not the polymerized microtubules (P). n = 4 experiments. Two-sided paired student’s t-test, for Lac-tub, p = 0.0037. For Ac-tub, p = 0.0193. c, d HEK293T cells were treated with Nocodazole (Noco) or Taxol for 12 h. Immunoblotting revealed α-tubulin lactylation levels in HEK293T cells after treatment. The concentration of Nocodazole or Taxol was 1 μM. n = 9 experiments. Two-sided paired student’s t-test, for Lac-tub, Noco vs Ctrl, p = 0.0272; Taxol vs Ctrl, p = 0.0078. For Ac-tub, Noco vs Ctrl, p = 0.0006; Taxol vs Ctrl, p = 0.0018. e–h Representative images of cortical neurons at DIV1, stained using anti-lactylated-α-tubulin (green), and anti-acetylated-α-tubulin (red) in (e), and Phalloidin (red) to label F-actin in (g). The top left region indicates higher-magnification images. The arrow head indicates the growth cone. The fluorescence-intensity profile of lactylated-α-tubulin (blue), and acetylated-α-tubulin (red) in (f), or lactylated-α-tubulin (blue), and Phalloidin (red) in (h) were obtained along the while line. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 5. α-Tubulin lactylation promotes microtubule polymerization and dynamics.

a Representative images of tubulin nucleation assay using 15 μM native or in vitro lactylated α-tubulin. Free tubulin comprising unlabeled and HiLyte-488-tubulin at a 9:1 ratio were incubated with 1 mM GTP and 5% glycerol at 37 °C for 30 min. b Quantitative analysis of data in (a). Ctrl T, n = 9; Lac T, n = 6. Two-sided unpaired student’s t-test, p = 0.0210. c Representative Kymographs of dynamic microtubules from α-tubulin or lactylated-α-tubulin by time-lapse imaging with TIRF microscopy. Quantitative analysis of microtubule growth rate in (d), shrinkage rate in (e), catastrophe frequency in (f), and maximum length in (g). n = 6 experiments. Two-sided paired student’s t-test, for growth rate, p = 0.0026; for shrinkage rate, p = 0.0042; for maximum length, p = 0.0295. h Representative images and kymographs of EB3-tdTomato in cultured hippocampus neurons at DIV3. The neurons were transfected with EB3-tdTomato together with α-tubulin or α-tubulin K40A mutant, were then cultured with or without 30 mM lactate. i Quantitative analysis of number of EB3 comets and anterograde velocity. EB3 comets: WT, n = 20; WT + Lac, n = 21; K40A, n = 31; and K40A + Lac, n = 17 neurons from 3 experiments. Anterograde velocity: WT, n = 112; WT + Lac, n = 301; K40A, n = 194; and K40A + Lac, n = 236 EB3 comets from 3 experiments. One-way ANOVA, for EB3 comets, WT + Lac vs WT, p < 0.0001; K40A + Lac vs WT + Lac, p < 0.0001. For anterograde velocity, WT + Lac vs WT, p < 0.0001; K40A + Lac vs WT + Lac, p < 0.0001. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 6. α-Tubulin lactylation promotes axon outgrowth and branching.

a Representative images of hippocampal neurons at DIV3. The hippocampal neurons were transfected with α-tubulin or α-tubulin K40A mutant. The neurons were treated with 30 mM lactate for 72 h, and stained with anti-SMI-312 (green) and anti-Tuj-1 (red) antibodies. b Quantitative analysis of the longest axon length, number of axonal branching points, number of neurite tips, and percentage of polarized neurons in cultured neurons in (a). For WT, WT + Lac, K40A, and K40A + Lac, n = 66, 74, 54, and 62 neurons, respectively, from 3 experiments. One-way ANOVA, for axonal branching points, WT + Lac vs WT, p = 0.0265; K40A + Lac vs WT + Lac, p = 0.0024. For neurite tips, WT + Lac vs WT, p < 0.0001; K40A + Lac vs WT + Lac, p < 0.0001. For polarized neurons, WT + Lac vs WT, p = 0.0039; K40A + Lac vs WT + Lac, p = 0.0035. c Representative images of axon regeneration. The cortical neurons were infected with lentiviruses encoding α-tubulin or α-tubulin K40A mutant. The axons were severed after being treated with or without 30 mM lactate for 4 h at DIV7 and axon regeneration was assessed 1 day later. d Quantitative analysis of data in (c). For WT, WT + Lac, K40A, and K40A + Lac, n = 222, 221, 175, and 159 axons, respectively, from 3 experiments. One-way ANOVA, WT + Lac vs WT, p < 0.0001; K40A + Lac vs WT + Lac, p < 0.0001. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 7. A proposed model for the regulation of α-tubulin lactylation.

The glycolysis is crucial for energy generation in cells and results in the production of lactate. HDAC6 functions as a lactyltransferase to lactylate the α-tubulin K40 residue by using lactate, facilitating microtubule dynamics. HDAC3 catalyzes lactate to enhance lactylation of microtubule-associated proteins that may also affect cytoskeleton functions.