TY - JOUR
AU - Wang, Qiong
AB - Abstract Axon growth is tightly controlled to establish functional neural circuits during brain development. Despite the belief that cytoskeletal dynamics is critical for cell morphology, how microtubule acetylation regulates axon development in the mammalian central nervous system remains unclear. Here, we report that loss of α-tubulin acetylation by ablation of MEC-17 in mice predisposes neurons to axon overbranching and overgrowth. Introduction of MEC-17F183A lacking α-tubulin acetyltransferase activity into MEC-17-deficient neurons failed to rescue axon defects. Moreover, loss of α-tubulin acetylation led to increases in microtubule debundling, microtubule invasion into filopodia and growth cones, and microtubule plus-end dynamics along the axon. Taxol application dampened microtubule hyperdynamics and suppressed axon overbranching and overgrowth in MEC-17-deficient neurons. Thus, our study reveals that α-tubulin acetylation acts as a brake for axon overbranching and overgrowth by dampening microtubule dynamics, providing insight into the role of microtubule post-translational modifications in regulating neural development. axon branching, MEC-17, microtubule dynamics, α-tubulin acetylation Introduction In the central nervous system, neurons wire together by sending axons to their targets to establish functional neural circuits (Gallo 2011; Kalil and Dent 2014). The axon from a single neuron can widely connect to divergent brain regions. For example, a cortical neuron sends axon collateral branches to diverse regions of the ipsilateral and contralateral cortex. Recently, more attention has been paid to the molecular mechanisms underlying axon branching in establishing neural circuitry, ranging from extracellular cues to intracellular signaling pathways that regulate cytoskeletal dynamics (Poulain and Sobel 2010; Gallo 2011; Lewis et al. 2013; Kalil and Dent 2014). However, the role of post-translational cytoskeletal modifications such as microtubule acetylation in axon development in the mammalian central nervous system remains elusive. Microtubules, composed of α- and β-tubulin heterodimers, dynamically switch between growing and shrinking phases, which is required for the establishment of mature neuronal morphology (Conde and Caceres 2009). Post-translational modifications of tubulin, such as acetylation, detyrosination, glycylation, and glutamylation, regulate the organization and dynamics of microtubules (Wloga et al. 2010; Song et al. 2013). Despite being a major post-translational modification of tubulin, the function of α-tubulin acetylation in regulating microtubule dynamics is still controversial. Loss of α-tubulin acetylation increases the resistance of microtubules to chemical-induced depolymerization in MEC-17-deficient mouse fibroblasts (Kalebic et al. 2013b), but loss of α-tubulin acetylation induces microtubule instability in the axons of nematode neurons (Neumann and Hilliard 2014). Given that previously identified deacetylases, HDAC6 and SIRT2, deacetylate molecular substrates other than α-tubulin, the recent identification of the specific α-tubulin acetyltransferase MEC-17/αTAT-1 (α-tubulin acetyltransferase 1) (Akella et al. 2010; Shida et al. 2010) provides a good tool to study the microtubule-related function of α-tubulin acetylation. Previous studies report that genetic deletion of Atat1 (encoding MEC-17) causes a subtle disorganization of the dentate gyrus in the mouse brain (Kim et al. 2013). Here, we reveal that loss of α-tubulin acetylation in MEC-17 knockout mice leads to axon overbranching and overgrowth both in vivo and in vitro, and this effect can be rescued by the re-expression of MEC-17 or of acetylation-mimicking α-tubulinK40Q but not of MEC-17F183A, which lacks α-tubulin acetyltransferase activity, in cultured MEC-17-deficient neurons. Importantly, loss of α-tubulin acetylation in the neuron increases microtubule plus-end dynamics and microtubule debundling along the axon shaft, and allows microtubules to easily invade filopodia and growth cones. Chemical manipulation to suppress microtubule plus-end dynamics rescues axon overbranching and overgrowth in MEC-17-deficient neurons. This study provides evidence that α-tubulin acetylation restricts axon overbranching by dampening microtubule plus-end dynamics in mammalian neuronal development. Materials and Methods Animals All experiments were approved by the Committee for the Use of Laboratory Animals and Common Facility, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The C57BL6 mice of either sex at postnatal day 0 (P0) were provided by the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. Plasmids The expression constructs for MEC-17 (NM_001142745.1), MEC-171–310 (NM_028476.4), MEC-171–421 (NM_001142744.1), and cortactin (NM_007803.5) were cloned from cDNA of mouse brain tissue into the vector pEGFP-N3. The rat MEC-17-GFP and Myc-α-tubulin, GFP-α-tubulin and GFP-α-tubulinK40Q plasmids have been described previously (Li et al. 2012a). The α-tubulin construct was prepared using the PCR-amplified rat Tuba1a (NM_022298.1), and the protein sequence of Tuba1a is identical between rat (NP_071634.1) and mouse (NP_035783.1). The end-binding protein 3 (EB3) (NM_001007656.1) plasmid was cloned from the cDNA of rat dorsal root ganglion into the vector pRFP-N3. The plasmids containing MEC-17-GFP with point mutations were prepared using the KOD-Plus mutagenesis kit (Toyobo). The plasmids containing MEC-17-GFP and MEC-171–421-GFP with various truncations were constructed using PCR-amplified fragments inserted into the C-terminus of pEGFP-N3. Cell Culture, Transfection, and Drug Treatment HEK293 cells (American Type Culture Collection) were maintained in MEM (GIBCO) supplemented with 10% fetal bovine serum (Biochrom) and antibiotics. The PtK1 cells (female Potorous tridactylus kidney cells) were maintained in MEM supplemented with 10% fetal bovine serum and 0.11 g/L sodium pyruvate (Sigma). The HEK293 and PtK1 cells were transiently transfected with plasmids using Lipofectamine 2000 (Invitrogen) and prepared for various assays after 48 h. Hippocampal or cortical tissues of P0 mice were dissected in Hank’s balanced salt solution (HBSS) and digested with 10 U/mL papain (Worthington Bio Corp) and 0.1 mg/mL DNase I (Sigma) for 18 min at 37 °C, washed in HBSS, and dissociated by repeated passaging through a 1 mL pipette. Directly after isolation, plasmids were added to the suspension of neurons in Nucleofector buffer (0.1 mL), and the mixture was electroporated in an Amaxa Nucleofector II (Lonza) using program O-005. Subsequently, neurons were plated with MEM containing 5% fetal bovine serum on 35-mm glass bottom dishes coated with poly-d-lysine (0.01 mg/mL, Sigma). After 4 h, the medium was replaced with Neurobasal medium (GIBCO) containing 2% B27 supplement and 2 mM l-Glutamax (GIBCO), and then neurons were fixed for staining at days 2–3 or used for time-lapse imaging at day 2. For the drug treatment of neurons, nocodazole (165 nM or 5 μM, Sigma) was added to the neurons for 5 or 30 min before fixation; taxol (0.1 nM, R&D Systems) was applied to the neurons 36 h after plating, followed by incubation for a further 12 h for the detection of microtubule debundling or for a further 36 h for the examination of neuronal morphology. Immunohistochemistry and Immunocytochemistry P0 and P14 mice of either sex were transcardially perfused with 4% paraformaldehyde. The brains were dissected and post-fixed in the same fixative and then sequentially dehydrated in 20% and 30% sucrose at 4 °C overnight. The 50 μm-thick sections were cut using a cryostat and mounted on gelatin-coated slides. Brain sections were incubated with primary antibodies against GFP (1:1000; Molecular Probes), Cux1 (1:500; Santa Cruz), Tbr1 (1:1000; Abcam), cleaved caspase-3 (1:500; Cell Signaling Technology), and Ki-67 (1:500; Invitrogen) overnight at 4 °C, followed by incubation with the corresponding Alexa-Fluor-conjugated secondary antibodies (1:500; Molecular Probe) for 45 min at 37 °C or together with DAPI (1:2000; Sigma). The cultured neurons were fixed with 4% paraformaldehyde in microtubule stabilizing buffer (0.1 M PIPES, 10 mM EDTA and 10 mM MgSO4, pH 6.9) for 15 min at room temperature and incubated with primary antibodies against GFP (1:1000; Molecular Probe), K40-acetylated α-tubulin (clone 6-11B, 1:5000; Sigma) and α-tubulin (1:1000; Abcam; 1:1000; Sigma) overnight at 4 °C, followed by incubation with Alexa-Fluor-conjugated secondary antibodies for 45 min at 37 °C. Actin was labeled with Alexa Fluor 546 phalloidin (1:300; Invitrogen). For quantitative analysis of axon branches, 20–30 neurons from each experiment and 60–100 neurons from 3 to 4 independent experiments were collected for each group. A process longer than 20 μm was defined as a branch. The longest axon and the primary, secondary and tertiary axon branches were defined according to previous papers (Krylova et al. 2002; Ko et al. 2005). The longest axon shaft with abundant branches was regarded as the longest axon. The numbers of total axon branches and primary axon branches per 100 μm were counted, and the lengths of total axon, axon branches and the longest axon were measured in hippocampal neurons. For quantitative analysis of growth cones or nascent branches, 20–30 cases from each experiment and 70–100 cases from 3 to 4 independent experiments were collected for each group. The sections and cells were mounted and scanned using a Leica TCS SP5 or SP8 confocal microscope (Leica). The microtubules in growth cones were imaged with a Leica TCS SP8 STED 3X super-resolution microscope (Leica). Neuronal morphology was analyzed using Neurolucida V9.0 (MBF Bioscience), Neurolucida Explorer V9.0 (MBF Bioscience), and ImageJ 1.46j (NIH). In Utero Electroporation The uteruses of mice at gestation day 15.5 were exposed, and 0.75 μg of GFP plasmid and 0.1 μL of Fast Green (2 mg/mL; Sigma) were injected into the lateral ventricle of each embryo. Next, electric pulses were generated by an ElectroSquireportator T830 (BTX) and applied to the cerebral wall for 5 repetitions of 50 V for 50 ms with an interval of 1 s. The uterine horns were then replaced in the abdominal cavity, and the abdominal wall and skin were sutured. At P14, the mouse brain was processed for immunohistochemistry. For quantitative analysis, 3 sections from each of 4 mice were collected for each group. The intensity of immunofluorescence was analyzed using ImageJ 1.46j. Total fluorescence intensity of contralateral branching was normalized to the total area of labeled cell bodies to avoid the effects of variations in electroporation efficiency. The fluorescence intensity along the radial axis of cortical wall in the ipsilateral and contralateral cortex was measured according to a previous study (Courchet et al. 2013). Regions with similar intensity of cortical layers 2–3, representing the distribution of labeled cell bodies along the radial axis, were selected for comparing the fluorescence intensity of ipsilateral cortex between MEC-17+/+ and MEC-17−/− mice. Time-Lapse Imaging Hippocampal neurons were cultured for 48 h on glass bottom dishes and then placed on a temperature-controlled workstation (37 °C, 5% CO2) with an inverted microscope. To observe new branches, lost branches and transient branches along the axon, hippocampal neurons were imaged at 5-min intervals for 3 h using a Nikon A1 microscope and a ×60 oil lens. An axon was defined as a process that was at least double the length of the next longest minor process (Dent et al. 2004). A process longer than 10 μm throughout the 3 h was defined as a branch. The numbers of new branches, lost branches and transient branches per axon were counted, and the axon growth rate was calculated from the increase in total axon length according to a previous study (Courchet et al. 2013). For quantitative analysis, 20–30 neurons from each experiment and 80–100 neurons from 4 independent experiments were collected for each group. To detect the movement of EB3 tagged with monomeric red fluorescence protein (EB3-RFP), hippocampal neurons were cultured for 48 or 36 h followed by 24-h taxol treatment. Axons were imaged using the PerkinElmer UltraView Vox system (PerkinElmer Inc.) with a ×60 oil lens. Regions of the axon segments were selected randomly, and 60 frames were collected for 2 min at 2-s intervals. The kymograph was generated using ImageJ 1.44 software (NIH). The analysis of mean EB3 velocity and other parameters in a particular kymograph was performed using Image-Pro Plus 5.1 software (Media Cybernetics). For quantitative analysis of EB3-RFP, approximately 10 neurons from each experiment and approximately 30 neurons from 3 independent experiments were collected for each group. Coimmunoprecipitation The HEK293 cells were lysed in ice-cold RIPA buffer [150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1% Triton X-100, 10% glycerol, 0.1 mM PMSF, 1 mg/mL pepstatin A, and 1 mg/mL leupeptin]. Then, the samples were incubated with specific Myc antibody-conjugated beads (Sigma) for 5 h at 4 °C. The immunoprecipitates and 5–10% total lysates were analyzed by immunoblotting. Immunoblotting The samples were separated by SDS-PAGE, transferred, probed with specific antibodies, and visualized with enhanced chemiluminescence (Bio-Rad). The primary antibodies included mouse antibodies against GFP (1:2000; Roche), Myc (1:1000; Developmental Studies Hybridoma Bank), α-tubulin (1:20 000; Sigma), and K40-acetylated α-tubulin (1:20 000; Sigma) and rabbit antibodies against MEC-17 (1:5000; homemade), kinesin-1 (1:500; Millipore), and dynein intermediate chain (1:500; Chemicon). The immunoreactive bands were quantified from 3 to 5 independent experiments using Image-Pro Plus 5.1 software. The acetylation level of α-tubulin by MEC-17 mutants and the level of α-tubulin binding with MEC-17 mutants were normalized to that of MEC-17-GFP or MEC-171-421-GFP. And the acetylation level of cortactin by MEC-17 mutants was normalized to that of MEC-17-Myc. Behavioral tests Male adult mice at 8–12 weeks old were subjected to behavioral tests. For the open field test, exploratory locomotor activity within 30 min was recorded and analyzed in an open field (45 × 45 cm) using EthoVision software (Noldus). The distance and duration of locomotor activity in the central and peripheral areas were recorded. Evaluation of anxiety-like behavior included the light-dark box test and the elevated maze test (File et al. 2004). Anxiety-like activity was assessed by the number of entries into the light compartment or open arms and the time spent within the open arms. The light-dark box consists of a light side (27 × 27 × 30 cm) and a fully dark side (18 × 27 × 30 cm) separated by a partition with a small opening (12 × 5 cm). The light compartment was brightly illuminated (550–850 lux). At the beginning of the test, the mouse was placed in the dark compartment and allowed to freely explore both compartments for 10 min. Entries into the light compartments were recorded. The elevated maze comprised 2 open arms (50 × 10 × 0.5 cm) across from each other and perpendicular to 2 closed arms (50 × 10 × 40 cm) with a center platform (10 × 10 cm). The maze was 50 cm above the floor and dimly illuminated. The animals were placed individually on the central platform facing an open arm and allowed to explore the apparatus for 6 min. Statistical Analysis Data were acquired from at least 3 independent experiments and are presented as the mean ± SEM. Statistical significance was analyzed using Student’s paired or unpaired t tests for 2-group comparisons using Prism 5 software (GraphPad). Comparisons of branching distribution curves, the distance distribution curves in the open field test and the learning curves in the rotarod test and Morris water maze test between 2 groups were performed using two-way ANOVA. Differences were considered significant at the level of P < 0.05. Results Loss of MEC-17 Causes Excessive Axon Branching in the Cerebral Cortex To investigate the role of α-tubulin acetylation, we obtained MEC-17tm1(KOMP)Vlcg heterozygous mice from the knockout mouse project (KOMP) repository at the University of California at Davis. The major coding component of Atat1, comprising exons 4–13, was deleted by homologous recombination to generate a knockout allele (Supplementary Fig. 1A). Conventional PCR and quantitative real-time PCR were applied to identify the MEC-17 knockout mice (MEC-17−/−; Supplementary Fig. 1B, C). Furthermore, immunoblotting using our homemade antibody against MEC-17 showed that MEC-17 was absent, and the level of α-tubulin acetylation was dramatically diminished in the brains of MEC-17−/− mice at P0 and in cultured cortical neurons (Supplementary Fig. 1D–F), indicating that MEC-17 is the major acetyltransferase of α-tubulin at lysine (K) 40, which is consistent with previous reports (Kalebic et al. 2013b; Kim et al. 2013). Thus, MEC-17 knockout mice were successfully constructed. Subsequently, wild-type mice (MEC-17+/+) and MEC-17−/− mice were electroporated with eGFP reporter plasmids at embryonic day 15.5 (E15.5) in utero to label the neural progenitors of callosal projection neurons in cortical layers 2–3. We then analyzed the axon growth of unilaterally electroporated neurons in the somatosensory cortex. As shown previously (Courchet et al. 2013), callosal axons cross the corpus callosum, reach the contralateral primary (S1) and secondary (S2) somatosensory cortex, and branch extensively for synaptic connection at P14 (Fig. 1A, C, D). After ablation of MEC-17, callosal axons displayed excessive ipsilateral and contralateral branching (Fig. 1A–D). Quantitative analysis of axon branching showed that loss of MEC-17 in cortical neurons particularly increased the collateral branching ipsilaterally in layer 5 and the terminal arborization of axons contralaterally both in layers 2–3 and in layer 5 of the S1 and S2 cortex (Fig. 1E–G). In somatosensory cortex with MEC-17 deficiency, the neuronal migration at P0 was not significantly affected, as indicated by the layers 2–3 marker Cux1 and the layers 5–6 marker Tbr1 (Supplementary Fig. 2A, B). Loss of MEC-17 at P0 did not alter neuronal survival and proliferation, which were assessed using the apoptosis marker cleaved caspase-3 and the cell division marker Ki-67, respectively (Supplementary Fig. 2C, D). Therefore, these data reveal the function of MEC-17 in the control of axon branching in cortical neurons of the somatosensory cortex. Figure 1. View largeDownload slide Loss of MEC-17 increased axon branching in the cerebral cortex. E15.5 pups were in utero electroporated with GFP plasmid and processed for DAPI (blue), GFP (green), and Cux1 (red) staining at P14. Representative images of coronal brain sections at low magnification showing the distribution of axonal projections in the cerebral cortex of MEC-17+/+ and MEC-17−/− mice at P14. (A) S1 and S2 (asterisk) represent 2 areas innervated by contralateral axons. Scale bar: 1 mm. (B–D) Representative images of the ipsilateral (B) and contralateral (C and D) somatosensory cortex at higher magnification. Scale bar: 200 μm. (E–G) Quantitative analysis of branching distribution along the radial axis of the cortical wall showing significantly increased axon branching in ipsilateral layer 5 (E) and contralateral layers 2–3 and 5 (G) of somatosensory cortex. The total contralateral branching shown in C was quantified and plotted as normalized values versus MEC-17+/+ mice. All error bars represent the SEM. *P < 0.05 and ***P < 0.001, n = 4. See also Supplementary Fig 1 and 2. Figure 1. View largeDownload slide Loss of MEC-17 increased axon branching in the cerebral cortex. E15.5 pups were in utero electroporated with GFP plasmid and processed for DAPI (blue), GFP (green), and Cux1 (red) staining at P14. Representative images of coronal brain sections at low magnification showing the distribution of axonal projections in the cerebral cortex of MEC-17+/+ and MEC-17−/− mice at P14. (A) S1 and S2 (asterisk) represent 2 areas innervated by contralateral axons. Scale bar: 1 mm. (B–D) Representative images of the ipsilateral (B) and contralateral (C and D) somatosensory cortex at higher magnification. Scale bar: 200 μm. (E–G) Quantitative analysis of branching distribution along the radial axis of the cortical wall showing significantly increased axon branching in ipsilateral layer 5 (E) and contralateral layers 2–3 and 5 (G) of somatosensory cortex. The total contralateral branching shown in C was quantified and plotted as normalized values versus MEC-17+/+ mice. All error bars represent the SEM. *P < 0.05 and ***P < 0.001, n = 4. See also Supplementary Fig 1 and 2. Loss of MEC-17 Leads to Axon Overbranching and Overgrowth in Hippocampal Neurons In parallel, we evaluated the roles of MEC-17 and α-tubulin acetylation in vitro to quantify axonal morphology at single-cell resolution. Cultured hippocampal neurons exhibit typical axonal and dendritic structures and are commonly used to analyze single-neuron morphology. Hippocampal neurons from P0 mice were cultured for 3 days in vitro and imaged after the neurons displayed a typical axon and several dendrites. Immunostaining showed that, in the cultured neurons, loss of MEC-17 specifically abolished α-tubulin acetylation but maintained its tyrosination (Fig. 2A). Consistent with the in vivo results, neurons deficient in MEC-17 developed abnormally abundant and long axon branches (Fig. 2A). Quantitative data showed that loss of MEC-17 increased the numbers of primary, secondary and tertiary axon branches and the lengths of primary and secondary axon branches (Fig. 2B). The number of primary axon branches per 100 μm in the longest axon was also increased in MEC-17-deficient neurons (Fig. 2B), suggesting a higher density of axon branches. However, the length of the longest axon was not significantly changed (Fig. 2B). Total axon length was markedly increased in neurons deficient in MEC-17 (Fig. 2B), mainly due to axon overbranching. A previous study also reported increased neuronal branch length without a change in the length of the longest axon (Dent et al. 2004). This phenomenon may be explained by competition between the longest axon and the rest of the axon branches for growth. Thus, depletion of MEC-17 affects axon branching and axon growth. Figure 2. View largeDownload slide Loss of MEC-17 induced increases in axon branching and growth in hippocampal neurons. (A) Representative images of hippocampal neurons cultured from P0 MEC-17+/+ and MEC-17−/− mice for 3 days. The neurons were stained for α-tubulin (green), Ac-tubulin (red), and Tyr-tubulin (blue). Scale bar: 50 μm. (B) A schematic drawing showing the longest axon (black) and the axon branch order containing primary (red), secondary (green), and tertiary (blue) branches in hippocampal neurons stained for α-tubulin. Quantitative analysis of total axon length, number of axon branches, primary branches per 100 μm, axon branch length, and longest axon length showed that loss of MEC-17 induced increases in axon branching and growth in hippocampal neurons. (C) Time-lapse imaging of axon branching and growth over 3 h in hippocampal neurons from P0 MEC-17+/+ and MEC-17−/− mice after 48 h in culture. A schematic drawing showing the events of a new branch (red), axon growth (green), and axon retraction (blue). Scale bar: 25 μm. (D) Quantitative analysis of axon dynamics including the numbers of new, lost and transient branches and the axon growth rate showed that loss of MEC-17 increased new branching and axon growth of hippocampal neurons. All error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 70–100 neurons from 3 to 4 experiments. Figure 2. View largeDownload slide Loss of MEC-17 induced increases in axon branching and growth in hippocampal neurons. (A) Representative images of hippocampal neurons cultured from P0 MEC-17+/+ and MEC-17−/− mice for 3 days. The neurons were stained for α-tubulin (green), Ac-tubulin (red), and Tyr-tubulin (blue). Scale bar: 50 μm. (B) A schematic drawing showing the longest axon (black) and the axon branch order containing primary (red), secondary (green), and tertiary (blue) branches in hippocampal neurons stained for α-tubulin. Quantitative analysis of total axon length, number of axon branches, primary branches per 100 μm, axon branch length, and longest axon length showed that loss of MEC-17 induced increases in axon branching and growth in hippocampal neurons. (C) Time-lapse imaging of axon branching and growth over 3 h in hippocampal neurons from P0 MEC-17+/+ and MEC-17−/− mice after 48 h in culture. A schematic drawing showing the events of a new branch (red), axon growth (green), and axon retraction (blue). Scale bar: 25 μm. (D) Quantitative analysis of axon dynamics including the numbers of new, lost and transient branches and the axon growth rate showed that loss of MEC-17 increased new branching and axon growth of hippocampal neurons. All error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001, n = 70–100 neurons from 3 to 4 experiments. To study whether loss of MEC-17 increases branch budding or decreases branch pruning, we performed time-lapse imaging to monitor the dynamic behavior of axon branching in cultured hippocampal neurons. During 3-h live cell observations, the number of new branches budded from the axon and the axon growth rate were increased in MEC-17-deficient neurons, while the number of pruned branches, including lost and transient branches, was not changed (Fig. 2C, D), suggesting that the increase in new branches is responsible for the excessive axon branching of neurons deficient in MEC-17. Taken together, MEC-17 is critical for proper axon branching and growth during neuronal development. MEC-17 Performs Two Roles: α-Tubulin Acetylation and Binding To explore whether loss of α-tubulin acetylation is responsible for the axon overbranching and overgrowth induced by MEC-17 deficiency, we carefully dissected the molecular basis of MEC-17. Of 5 alternative splicing isoforms, both the Atat1-004 isoform encoding MEC-17 with 333 aa and the Atat1-005 isoform encoding MEC-17 with 310 aa (MEC-171–310) were abundantly expressed in neonatal mouse brains (Supplementary Figs 1E and 3). As Atat1-005 has not been used in previous studies, we chose the frequently used Atat1-004 (Shida et al. 2010; Aguilar et al. 2014; Davenport et al. 2014; Mackeh et al. 2014; Xu et al. 2017) for further experiments in order to compare results with other studies, and we also evaluated the effect of Atat1-005 in the later part of this study. Previous studies on the molecular structure of MEC-17 (Friedmann et al. 2012; Li et al. 2012b; Taschner et al. 2012; Szyk et al. 2014) report that the N-terminus (1–193 aa) is a conservative acetyltransferase domain belonging to the GCN5 family and that several putative residues, such as Q58, K102, E111, D157, and F183, are critical for the acetyltransferase activity. Therefore, we detected the level of α-tubulin acetylation in PtK1 cells and HEK293 cells expressing various MEC-17 mutants (Fig. 3A). PtK1 cells displayed a very low level of endogenous α-tubulin acetylation (Fig. 3B) but were not easy to transfect with exogenous genes. However, HEK293 cells had a moderate level of endogenous α-tubulin acetylation and exhibited higher transfection efficiency (Fig. 3D). Immunoblotting showed that, in PtK1 cells, full-length MEC-17 (1–333 aa) significantly enhanced α-tubulin acetylation (Fig. 3B). Consistent with previous reports, the N-terminus but not the C-terminus (194–333 aa) of MEC-17 was required for α-tubulin acetylation in cells (Fig. 3B). However, the level of α-tubulin acetylation catalyzed by MEC-171–193 only reached 43% of that produced by full-length MEC-17 (Fig. 3B). Importantly, the molecular basis of the acetyltransferase activity of MEC-17 was confirmed in MEC-17-deficient cortical neurons (Fig. 3C). Furthermore, interruption within the 1–193 aa severely influenced the acetyltransferase activity (Fig. 3D) in HEK293 cells, indicating that the integrity of the N-terminus is indispensable for the acetyltransferase activity of MEC-17. Notably, MEC-17F183A displayed an almost complete loss of the acetyltransferase activity, while MEC-17Q58A, MEC-17K102E, MEC-17E111A, and MEC-17D157N retained partial enzymatic activity (Fig. 3B, C, and Supplementary Fig. 4A). Therefore, MEC-17F183A was the best choice to serve as an acetylation-deficient mutant for further study. Figure 3. View largeDownload slide MEC-17 demonstrated α-tubulin acetylation and binding activities. (A) A schematic sketch showing a functional diagram of MEC-17 and various plasmids for MEC-17 mutants. (B–D) Immunoblot (IB) of protein extracts from PtK1 cells (B), MEC-17−/− cortical neurons (C), and HEK293 cells (D) transfected with various MEC-17 mutant plasmids. The level of α-tubulin acetylation (Ac-tubulin) was compared. α-Tubulin and GFP served as controls for protein loading and transfection, respectively. Data were quantified and plotted as normalized values versus MEC-17-GFP. All error bars represent the SEM. *P < 0.05 and **P < 0.01 versus GFP control, n = 3 for PtK1 cells, n = 6 for cortical neurons and n = 4 for HEK293 cells. (E–G) Immunoblot showing that the C-terminus of MEC-17, especially amino acids 291–302, was essential for α-tubulin binding. Co-IP was performed with protein extracts from HEK293 cells co-transfected Myc-α-tubulin and the indicated plasmids, using Myc antibody-conjugated beads. Data were quantified and plotted as normalized values versus MEC-17-GFP. All error bars represent the SEM. *P < 0.05 and **P < 0.01 versus GFP control, n = 5 for E and F, n = 4 for G. See also Supplementary Figs 3 and 4. Figure 3. View largeDownload slide MEC-17 demonstrated α-tubulin acetylation and binding activities. (A) A schematic sketch showing a functional diagram of MEC-17 and various plasmids for MEC-17 mutants. (B–D) Immunoblot (IB) of protein extracts from PtK1 cells (B), MEC-17−/− cortical neurons (C), and HEK293 cells (D) transfected with various MEC-17 mutant plasmids. The level of α-tubulin acetylation (Ac-tubulin) was compared. α-Tubulin and GFP served as controls for protein loading and transfection, respectively. Data were quantified and plotted as normalized values versus MEC-17-GFP. All error bars represent the SEM. *P < 0.05 and **P < 0.01 versus GFP control, n = 3 for PtK1 cells, n = 6 for cortical neurons and n = 4 for HEK293 cells. (E–G) Immunoblot showing that the C-terminus of MEC-17, especially amino acids 291–302, was essential for α-tubulin binding. Co-IP was performed with protein extracts from HEK293 cells co-transfected Myc-α-tubulin and the indicated plasmids, using Myc antibody-conjugated beads. Data were quantified and plotted as normalized values versus MEC-17-GFP. All error bars represent the SEM. *P < 0.05 and **P < 0.01 versus GFP control, n = 5 for E and F, n = 4 for G. See also Supplementary Figs 3 and 4. Next, we dissected the interaction domain between MEC-17 and α-tubulin in HEK293 cells using a coimmunoprecipitation (Co-IP) assay. Immunoblotting showed that MEC-17194–333 but not MEC-171–193 interacted with α-tubulin (Fig. 3E–G). The binding affinity of the site-specific mutant MEC-17F183A to α-tubulin was not changed (Fig. 3E). Thus, the catalytic activity of MEC-17 and its ability to bind to α-tubulin are structurally separated. However, tight binding of MEC-17 to α-tubulin favors its catalytic effect because the level of α-tubulin acetylation by full-length MEC-17 is much higher than that by MEC-171–193 (Fig. 3B, C). By introducing a series of MEC-17 mutants, we identified the sequence 291–302 aa in MEC-17 as the α-tubulin binding domain (Fig. 3A, F, G). A previous study reported that in the Atat1-001 isoform encoding MEC-17 with 421 aa (MEC-171–421), the sequence 307–387 aa is responsible for its interaction with α-tubulin (Montagnac et al. 2013). Co-IP showed that the interaction between MEC-171–421 and α-tubulin was significantly reduced by deletion of either amino acids 291–302 or amino acids 307–387, but the binding capacity of MEC-171–421 to α-tubulin was further impaired when both sequences were absent (Supplementary Fig. 4B). Thus, different MEC-17 isoforms display different α-tubulin binding capacity and contain conservative and unique domains for substrate binding. Taken together, clear dissection of the MEC-17 sequences for α-tubulin acetylation and binding enables us to investigate the role of α-tubulin acetylation in the axon branching and growth of neurons in the mammalian central nervous system. α-Tubulin Acetylation Restricts Axon Overbranching and Overgrowth in Neurons of the Central Nervous System To explore whether the defect caused by MEC-17 deficiency is induced by loss of α-tubulin acetylation, we introduced MEC-17, MEC-171–310, MEC-17F183A, and MEC-17D157N into MEC-17-deficient neurons to observe axonal development. Importantly, MEC-17 restored axon overbranching and overgrowth to normal levels in MEC-17-deficient hippocampal neurons, while MEC-17F183A did not rescue the defect (Fig. 4A, B). Interestingly, compared with MEC-17, MEC-171–310 displayed an equal ability to rescue α-tubulin acetylation and neuronal morphology (Supplementary Fig. 5A–C), indicating an important role for MEC-171–310 in MEC-17-mediated function because among the 5 alternative splicing isoforms in neonatal mouse brain, this is the most abundant. Although the level of α-tubulin acetylation by MEC-17D157N was partially retained in PtK1 cells (12% of that produced by MEC-17; Supplementary Fig. 4A), a low level of α-tubulin acetylation by MEC-17D157N in neurons (7.5% of that produced by MEC-17) did not rescue the morphological defect caused by loss of MEC-17 (Supplementary Fig. 5D–F), similar to MEC-17F183A. Moreover, MEC-171–193 displayed partial rescue ability compared with full-length MEC-17 (Fig. 4A, B), consistent with its weaker acetyltransferase activity than full-length MEC-17 (Fig. 3B, C). MEC-17194–333 was not able to rescue axon overbranching and overgrowth in MEC-17-deficient neurons (Fig. 4A, B), indicating that the ability of MEC-17 to bind with α-tubulin alone does not influence axon growth but enhances α-tubulin acetylation by the N-terminus of MEC-17 to regulate axonal development. These data suggest that the axon overbranching and overgrowth in MEC-17-deficient neurons are caused by loss of MEC-17 acetylation activity. Figure 4. View largeDownload slide MEC-17-mediated acetylation of α-tubulin was required for its regulation of axon branching and growth in hippocampal neurons. (A) Representative images of MEC-17-deficient hippocampal neurons expressing MEC-17-GFP and various MEC-17 mutant plasmids. Neurons from P0 MEC-17−/− mice were electroporated and cultured for 3 days. Scale bar: 50 μm. (B, C) Quantitative analysis of total axon length, number of axon branches, primary branches per 100 μm, axon branch length and longest axon length showed that the increases in axon branching and growth were rescued by the re-expression of MEC-17-GFP (B) and GFP-α-tubulinK40Q (C) but not of MEC-17F183A-GFP (B), MEC-17194–333-GFP (B) and GFP-α-tubulin (C) in MEC-17−/− neurons, and MEC-171–193-GFP (B) partially rescued the defects caused by MEC-17 deficiency. All error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus GFP control or GFP-α-tubulin and #P < 0.05, ##P < 0.01, ###P < 0.001 versus the indicated conditions, n = 60–100 neurons from 3 to 4 experiments. See also Supplementary Figs 4 and 5. Figure 4. View largeDownload slide MEC-17-mediated acetylation of α-tubulin was required for its regulation of axon branching and growth in hippocampal neurons. (A) Representative images of MEC-17-deficient hippocampal neurons expressing MEC-17-GFP and various MEC-17 mutant plasmids. Neurons from P0 MEC-17−/− mice were electroporated and cultured for 3 days. Scale bar: 50 μm. (B, C) Quantitative analysis of total axon length, number of axon branches, primary branches per 100 μm, axon branch length and longest axon length showed that the increases in axon branching and growth were rescued by the re-expression of MEC-17-GFP (B) and GFP-α-tubulinK40Q (C) but not of MEC-17F183A-GFP (B), MEC-17194–333-GFP (B) and GFP-α-tubulin (C) in MEC-17−/− neurons, and MEC-171–193-GFP (B) partially rescued the defects caused by MEC-17 deficiency. All error bars represent the SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus GFP control or GFP-α-tubulin and #P < 0.05, ##P < 0.01, ###P < 0.001 versus the indicated conditions, n = 60–100 neurons from 3 to 4 experiments. See also Supplementary Figs 4 and 5. To further investigate the relationship between the absence of α-tubulin acetylation and the abnormal axon morphology associated with loss of MEC-17, we expressed α-tubulinK40Q, an acetylation-mimicking mutant (Shida et al. 2010; Li et al. 2012a), in MEC-17-deficient neurons. Immunostaining showed that the expression of α-tubulinK40Q dramatically rescued axon overbranching and overgrowth of MEC-17-deficient hippocampal neurons, but the expression of wild-type α-tubulin did not have this effect (Fig. 4C). Taken together, these data indicate that the effect of MEC-17 is specifically due to the regulation of α-tubulin acetylation. In addition to α-tubulin, MEC-17 has been reported to acetylate the actin regulatory protein cortactin and MEC-17 itself (Castro-Castro et al. 2012; Kalebic et al. 2013a). In HEK293 cells co-expressing cortactin-GFP and MEC-17-Myc or MEC-17 mutants, the level of cortactin acetylation was detected through immunoprecipitation assays (Supplementary Fig. 4C). Immunoblotting confirmed the acetylation of cortactin by MEC-17 and showed that the α-tubulin acetylation-deficient mutants (MEC-17F183A and MEC-17D157N) also lost the ability to acetylate cortactin. Most importantly, MEC-171–193 did not present obvious acetyltransferase activity towards cortactin. However, MEC-171–193 exhibited a significant ability to acetylate α-tubulin, and the expression of MEC-171–193 partially rescued the axon overbranching and overgrowth of MEC-17-deficient hippocampal neurons. Thus, the axon overbranching and overgrowth of MEC-17-deficient hippocampal neurons are not primarily induced by loss of cortactin acetylation. Additionally, the number of filopodia represented by F-actin-marked nascent protrusions per 100 μm was not changed by loss of MEC-17 (Fig. 5A, C), precluding significant changes to the actin cytoskeleton. Figure 5. View largeDownload slide Loss of α-tubulin acetylation induced microtubule debundling and invasion as well as microtubule hyperdynamics in the axons of hippocampal neurons. (A) Representative images of microtubule debundling (white brace) and entry into filopodia (arrowheads) along the axon shaft. The neurons were stained for F-actin (red) and α-tubulin (green). Scale bar: 5 μm. (B–D) Quantitative analysis showed that loss of MEC-17 induced microtubule debundling, including increases in the total number of microtubule debundling sites in the axon and the number of microtubule debundling sites per 100 μm along the longest axon (B), and enhanced microtubule protrusion, including increased percentages of filopodia invaded by microtubules (MTs) in the axon and an unchanged number of filopodia per 100 μm along the longest axon in hippocampal neurons (C). Impaired microtubule organization in the axons of MEC-17−/− hippocampal neurons could be rescued by re-expression of MEC-17-GFP but not of MEC-17F183A-GFP (D). n = 60–100 neurons from 3 to 4 experiments. (E) Representative images of the growth cones of MEC-17+/+ and MEC-17−/− hippocampal neurons. The neurons were stained for F-actin (red) and α-tubulin (green). Scale bar: 5 μm. (F) Quantitative analysis showed enlarged growth cone area and increased numbers of growth cones and free MTs within growth cones in MEC-17−/− neurons. n = 80–100. (G) Representative kymographs of microtubule plus ends in the axons of hippocampal neurons expressing EB3-RFP after 48 h in culture (G, upper). The schematic sketch shows microtubule dynamics including growth lifetime, pause and catastrophe (G, lower). Scale bar: 5 μm. (H) Quantitative analysis showed that loss of MEC-17 changed the microtubule dynamics, including increases in growth velocity, number of comets per 10 μm and catastrophe frequency, and decreased the growth lifetime of microtubule plus ends in the axons of hippocampal neurons. These effects were rescued by re-expression of MEC-17-GFP but not of MEC-17F183A-GFP. n = 28–34. (I) Representative images of the microtubules that remained in the nascent branches of hippocampal neurons treated with 165 nM nocodazole for 5 min. Neurons were stained for α-tubulin (green) and F-actin (indicated by a white line). Scale bar: 2 μm. (J) Quantitative analysis showed that loss of MEC-17 reduced the proportion of microtubules remaining in nascent branches and growth cones after nocodazole treatment. n = 90. All error bars represent the SEM. **P < 0.01, ***P < 0.001 versus MEC-17+/+ , and ###P < 0.001 versus the indicated conditions. See also Supplementary Fig. 6. Figure 5. View largeDownload slide Loss of α-tubulin acetylation induced microtubule debundling and invasion as well as microtubule hyperdynamics in the axons of hippocampal neurons. (A) Representative images of microtubule debundling (white brace) and entry into filopodia (arrowheads) along the axon shaft. The neurons were stained for F-actin (red) and α-tubulin (green). Scale bar: 5 μm. (B–D) Quantitative analysis showed that loss of MEC-17 induced microtubule debundling, including increases in the total number of microtubule debundling sites in the axon and the number of microtubule debundling sites per 100 μm along the longest axon (B), and enhanced microtubule protrusion, including increased percentages of filopodia invaded by microtubules (MTs) in the axon and an unchanged number of filopodia per 100 μm along the longest axon in hippocampal neurons (C). Impaired microtubule organization in the axons of MEC-17−/− hippocampal neurons could be rescued by re-expression of MEC-17-GFP but not of MEC-17F183A-GFP (D). n = 60–100 neurons from 3 to 4 experiments. (E) Representative images of the growth cones of MEC-17+/+ and MEC-17−/− hippocampal neurons. The neurons were stained for F-actin (red) and α-tubulin (green). Scale bar: 5 μm. (F) Quantitative analysis showed enlarged growth cone area and increased numbers of growth cones and free MTs within growth cones in MEC-17−/− neurons. n = 80–100. (G) Representative kymographs of microtubule plus ends in the axons of hippocampal neurons expressing EB3-RFP after 48 h in culture (G, upper). The schematic sketch shows microtubule dynamics including growth lifetime, pause and catastrophe (G, lower). Scale bar: 5 μm. (H) Quantitative analysis showed that loss of MEC-17 changed the microtubule dynamics, including increases in growth velocity, number of comets per 10 μm and catastrophe frequency, and decreased the growth lifetime of microtubule plus ends in the axons of hippocampal neurons. These effects were rescued by re-expression of MEC-17-GFP but not of MEC-17F183A-GFP. n = 28–34. (I) Representative images of the microtubules that remained in the nascent branches of hippocampal neurons treated with 165 nM nocodazole for 5 min. Neurons were stained for α-tubulin (green) and F-actin (indicated by a white line). Scale bar: 2 μm. (J) Quantitative analysis showed that loss of MEC-17 reduced the proportion of microtubules remaining in nascent branches and growth cones after nocodazole treatment. n = 90. All error bars represent the SEM. **P < 0.01, ***P < 0.001 versus MEC-17+/+ , and ###P < 0.001 versus the indicated conditions. See also Supplementary Fig. 6. Deficiency in α-Tubulin Acetylation Leads to Hyperdynamic Microtubules The initiation and growth of axon branches require the regulation of cytoskeletal dynamics. An initial step in branch formation involves the focal accumulation of actin filaments at a filopodial protrusion, followed by microtubule invasion to stabilize the nascent branch (Gallo 2011). Notably, debundling of microtubules frequently occurs at the point of axonal collateral branching (Ketschek et al. 2015). To assess the change in microtubule organization in MEC-17-deficient neurons, we labeled the cultured hippocampal neurons for both α-tubulin and actin (Fig. 5A). Immunostaining showed that the total number of microtubule debundling sites in the axon and the number of microtubule debundling sites per 100 μm along the longest axon were significantly increased in MEC-17-deficient neurons (Fig. 5A, B). Although the number of filopodia represented by nascent protrusions along the longest axon was not changed, the percentage of filopodia invaded by microtubules was significantly increased in MEC-17-deficient neurons (Fig. 5A, C). Furthermore, the increases in microtubule debundling and microtubule invasion into filopodia in MEC-17-deficient neurons were rescued by re-expression of MEC-17 but not of MEC-17F183A (Fig. 5D). Additionally, electropration caused attenuated microtubule debundling and invasion in MEC-17-deficient neurons, consistent with decreased number of axon branches after electroporation (Figs 2B and 4B). At the same time, immunostaining combined with super-resolution imaging showed that loss of MEC-17 increased the number and area of growth cones, as well as the number of free microtubule ends per growth cone in the axons of hippocampal neurons (Fig. 5E, F). Previous studies support the correlation of similar changes in growth cones with the acceleration of axon growth (Chen et al. 2011; Wu et al. 2012). Thus, loss of MEC-17-mediated α-tubulin acetylation impairs microtubule organization in the axons of hippocampal neurons. The above microtubule organization defect caused by loss of MEC-17 implies that microtubules without α-tubulin acetylation are more dynamic. To detect microtubule dynamics along the axon shaft, we expressed EB3-RFP to clearly label the plus end of growing microtubules in hippocampal neurons cultured for 48 h. Time-lapse imaging of EB3-RFP along the axon shaft showed that microtubule growth was increased with indications of increases in the velocity and number of EB3 comets, and microtubule dynamics was elevated with the observation of the shortened lifetime of microtubule growth and the increased frequency of microtubule catastrophe in MEC-17-deficient neurons (Fig. 5G, H). The detection of EB3-RFP in the axon shaft provides evidence of hyperdynamic microtubules in neurons with loss of MEC-17. Moreover, the microtubule hyperdynamics in MEC-17-deficient neurons was rescued by the introduction of MEC-17 but not of MEC-17F183A (Fig. 5H). Thus, loss of MEC-17-mediated α-tubulin acetylation causes the microtubule hyperdynamics in the axons of hippocampal neurons. Microtubule dynamics and stability were further evaluated by observing the microtubules remaining in nascent branches, growth cones, and axons after nocodazole treatment. Previous work showed that nanomolar concentrations of nocodazole suppressed microtubule dynamics without affecting microtubule disassembly while micromolar concentrations rapidly depolymerize microtubules (Rochlin et al. 1996; Vasquez et al. 1997; Witte et al. 2008). Immunostaining showed that the area occupied by microtubules after a low dose (165 nM) of nocodazole treatment for 5 min was significantly decreased in the nascent branch and growth cone of MEC-17-deficient neurons (Fig. 5I, J). This result may imply that a low dose of nocodazole treatment suppresses microtubule hyperdynamics caused by loss of MEC-17 to result in a decrease of microtubule targeting. However, the mean staining intensity per axon and the distance of microtubule retraction from the growth cone after a high dose (5 μM) of nocodazole treatment for 5 or 30 min did not differ between MEC-17-deficient and wild-type neurons, suggesting an insignificant change in microtubule stability (Supplementary Fig. 6A–D). Taken together, MEC-17-mediated α-tubulin acetylation acts to maintain moderate microtubule bundling and dynamics in neuronal axons, especially at branch points and growth cones. As microtubules serve as the structural basis for axonal transport, we also investigated whether microtubule-based axonal transport would be affected by loss of MEC-17 (Supplementary Fig. 6E–H). The binding affinity of molecular motors to microtubules was examined by microtubule co-sedimentation assays. Immunoblotting showed that the microtubule binding ability of kinesin-1 and dynein intermediate chain was not significantly altered in MEC-17-deficient neurons compared with wild-type ones (Supplementary Fig. 6E, F). Moreover, time-lapse imaging of DsRed-mito-labeled mitochondria showed that the percentage of mobile mitochondria and the velocity of mitochondrial movement in the anterograde and retrograde directions were not altered in the axons of MEC-17-deficient neurons (Supplementary Fig. 6G, H). These data rule out significant changes in microtubule-based axonal transport in MEC-17-deficient neurons. Suppression of Microtubule Hyperdynamics Rescues Axonal Development in MEC-17-Deficient Neurons Low concentrations of taxol have been widely used to suppress microtubule dynamics without altering the microtubule polymer mass in living cells (De Brabander et al. 1982; Yvon et al. 1999; Orr et al. 2003). Therefore, we cultured hippocampal neurons and performed taxol treatment at a low concentration that did not affect the microtubule organization and dynamics of neurons from wild-type mice (Fig. 6A, B). The microtubule organization defects in MEC-17-deficient neurons, including increases in the total number of microtubule debundling sites in the axon, the number of microtubule debundling sites per 100 μm along the longest axon and the percentage of filopodia invaded by microtubules along the longest axon, were rescued by the application of 0.1 nM taxol (Fig. 6A), suggesting consolidation of microtubules by taxol. Moreover, we monitored the microtubule dynamics in neurons cultured for 36 h and treated with taxol for a further 24 h. Notably, time-lapse imaging of EB3-RFP showed that the changes in microtubule growth and dynamic instability along the axon shaft induced by loss of MEC-17 were rescued after 0.1 nM taxol treatment (Fig. 6B). Additionally, a longer culture time (60 h) caused attenuated microtubule dynamics, observed as decreases in the velocity of EB3 comets, number of EB3 comets per 10 μm of axon and catastrophe frequency in hippocampal neurons (Figs 5G and 6B). Thus, taxol application suppresses the hyperdynamics of acetylation-deficient microtubules. Figure 6. View largeDownload slide Suppression of microtubule hyperdynamics rescued the defects in microtubule organization and dynamics and in axon branching and growth in MEC-17-deficient hippocampal neurons. (A and B) Quantitative analysis showed that impaired microtubule organization (A) and changed microtubule dynamics (B) in the axons of MEC-17−/− hippocampal neurons could be rescued by treatment with 0.1 nM taxol, which did not affect MEC-17+/+ neurons. n = 60–80 for A, and 30–35 for B. (C) Representative images of hippocampal neurons from P0 MEC-17+/+ and MEC-17−/− mice after culture for 36 h, treatment with 0.1 nM taxol for 36 h and then staining for α-tubulin (green). Scale bar: 50 μm. (D) Quantitative analysis of total axon length, numbers of axon branches, and primary branches per 100 μm, axon branch length and longest axon length showed that the increases in axon branching and growth induced by loss of MEC-17 were rescued in hippocampal neurons after treatment with 0.1 nM taxol, which did not affect MEC-17+/+ neurons. n = 75–95. All error bars represent the SEM. **P < 0.01, ***P < 0.001 versus MEC-17+/+ , and #P < 0.05, ##P < 0.01, ###P < 0.001 versus the indicated conditions. Figure 6. View largeDownload slide Suppression of microtubule hyperdynamics rescued the defects in microtubule organization and dynamics and in axon branching and growth in MEC-17-deficient hippocampal neurons. (A and B) Quantitative analysis showed that impaired microtubule organization (A) and changed microtubule dynamics (B) in the axons of MEC-17−/− hippocampal neurons could be rescued by treatment with 0.1 nM taxol, which did not affect MEC-17+/+ neurons. n = 60–80 for A, and 30–35 for B. (C) Representative images of hippocampal neurons from P0 MEC-17+/+ and MEC-17−/− mice after culture for 36 h, treatment with 0.1 nM taxol for 36 h and then staining for α-tubulin (green). Scale bar: 50 μm. (D) Quantitative analysis of total axon length, numbers of axon branches, and primary branches per 100 μm, axon branch length and longest axon length showed that the increases in axon branching and growth induced by loss of MEC-17 were rescued in hippocampal neurons after treatment with 0.1 nM taxol, which did not affect MEC-17+/+ neurons. n = 75–95. All error bars represent the SEM. **P < 0.01, ***P < 0.001 versus MEC-17+/+ , and #P < 0.05, ##P < 0.01, ###P < 0.001 versus the indicated conditions. Next, we analyzed axon branching and growth after taxol treatment of cultured hippocampal neurons. Notably, the suppression of microtubule hyperdynamics by treatment with 0.1 nM taxol rescued the increases in the total axon length, the number of axon branches, the number of primary branches per 100 μm and the length of axon branches in MEC-17-deficient neurons (Fig. 6C, D). However, axon branching and growth were not affected by 0.1 nM taxol in cultured hippocampal neurons from wild-type mice (Fig. 6C, D). All of these data support a model in which axon overbranching and overgrowth are caused by microtubule plus-end hyperdynamics induced by loss of α-tubulin acetylation. Loss of MEC-17 and α-Tubulin Acetylation Induces Anxiety-Like Behavior in Mice Given that the collateral and terminal axon branching determines the wiring of a neural circuit in the brain, we wondered whether loss of MEC-17-mediated α-tubulin acetylation impairs the behavior of animals. Interestingly, in the open field test, MEC-17-deficient mice favored the peripheral area more than their wild-type littermates (Fig. 7A). The mutant mice also spent less time than wild-type littermates in the anxiogenic environment during the light-dark box test and the elevated maze test (Fig. 7B, C). Nevertheless, spatial learning and memory were not altered in MEC-17-deficient mice (Supplementary Fig. 7). A previous study also detected mild anxiety in MEC-17 knockout mice (Kalebic et al. 2013b). Our data combined with the previous study suggest that the developmental defects caused by loss of MEC-17 and α-tubulin acetylation produce this disorder of brain function in mice. Figure 7. View largeDownload slide MEC-17 deficiency induced anxiety-like behavior in mice. (A–C) Quantitative analysis showing anxiety-like behavior in MEC-17−/− mice. In the open field test, loss of MEC-17 decreased the duration in the central area and increased the time in the periphery (n = 8 mice for MEC-17+/+ and n = 11 mice for MEC-17−/−) (A). During 10 min of the light-dark box test, loss of MEC-17 led to a slight but not significant decrease in entries into the light box (n = 7 mice for MEC-17+/+ and n = 10 mice for MEC-17−/−) (B). In the elevated maze test, loss of MEC-17 reduced the entries into open arms and the time spent in open arms (n = 8 mice for MEC-17+/+ and n = 11 mice for MEC-17−/−) (C). All error bars represent the SEM. *P < 0.05, ***P < 0.001 versus MEC-17−/− mice. (D) A proposed model of α-tubulin acetylation in regulating microtubule dynamics in nascent branches and growth cones during axon development. In the presence of MEC-17, α-tubulin is acetylated, and the microtubules remain moderately dynamic and consolidated, allowing the neuron to maintain proper axon branching and growth during neuronal development. In the absence of MEC-17, acetylation of α-tubulin is lost, resulting in microtubule hyperdynamics and debundling. Then, the microtubules easily invade the filopodia and growth cones, thereby leading to axon overbranching and overgrowth in neurons. See also Supplementary Fig. 7. Figure 7. View largeDownload slide MEC-17 deficiency induced anxiety-like behavior in mice. (A–C) Quantitative analysis showing anxiety-like behavior in MEC-17−/− mice. In the open field test, loss of MEC-17 decreased the duration in the central area and increased the time in the periphery (n = 8 mice for MEC-17+/+ and n = 11 mice for MEC-17−/−) (A). During 10 min of the light-dark box test, loss of MEC-17 led to a slight but not significant decrease in entries into the light box (n = 7 mice for MEC-17+/+ and n = 10 mice for MEC-17−/−) (B). In the elevated maze test, loss of MEC-17 reduced the entries into open arms and the time spent in open arms (n = 8 mice for MEC-17+/+ and n = 11 mice for MEC-17−/−) (C). All error bars represent the SEM. *P < 0.05, ***P < 0.001 versus MEC-17−/− mice. (D) A proposed model of α-tubulin acetylation in regulating microtubule dynamics in nascent branches and growth cones during axon development. In the presence of MEC-17, α-tubulin is acetylated, and the microtubules remain moderately dynamic and consolidated, allowing the neuron to maintain proper axon branching and growth during neuronal development. In the absence of MEC-17, acetylation of α-tubulin is lost, resulting in microtubule hyperdynamics and debundling. Then, the microtubules easily invade the filopodia and growth cones, thereby leading to axon overbranching and overgrowth in neurons. See also Supplementary Fig. 7. Discussion The spatiotemporally controlled dynamics of the microtubule cytoskeleton, regulated by tubulin post-translational modifications, is critical for neural development. Here, we reveal that MEC-17-mediated α-tubulin acetylation restrains axon branching and growth by dampening microtubule plus-end dynamics in the mouse central nervous system (Fig. 7D). In the presence of MEC-17, α-tubulin acetylation keeps microtubules moderately dynamic and consolidated, and axon branching and growth are restricted during neuronal development. Loss of MEC-17 causes a deficiency in α-tubulin acetylation, which leads to microtubule hyperdynamics and debundling, thereby resulting in axon overbranching and overgrowth in neurons. These findings expand our current knowledge of the physiological function of α-tubulin acetylation in the mammalian nervous system. α-Tubulin Acetylation at K40 is Required for Microtubule Dynamics Acetylation of α-tubulin has been linked to stable microtubules, but whether α-tubulin acetylation can regulate microtubules remains controversial. Recently, it has been reported that MEC-17 overexpression destabilizes microtubules in NIH3T3 cells (Kalebic et al. 2013a) and that loss of MEC-17 makes microtubules more resistant to treatment with 10 μM nocodazole in mouse embryonic fibroblasts (Kalebic et al. 2013b), indicating a role for MEC-17 in destabilizing microtubules. However, another study reported that loss of MEC-17 leads to microtubule instability based on time-lapse imaging of EBP-2 in the axons of Caenorhabditis elegans (Neumann and Hilliard 2014). In the present study, loss of MEC-17 caused microtubule debundling and invasion into filopodia and growth cones of hippocampal neurons. Most importantly, in the axons of MEC-17-deficient neuron, the microtubules are hyperdynamic, showing increases in growth velocity, the number of growth comets and catastrophe frequency as well as a decreased growth lifetime. Furthermore, the elevated microtubule dynamics could be rescued by full-length MEC-17 but not by MEC-17F183A, which almost completely lost the acetyltransferase activity. Therefore, α-tubulin acetylation at K40 is necessary for microtubule dynamics in neurons of the mouse central nervous system. Notably, previous studies used MEC-17D157N as a loss-of-function mutant to reach the conclusion that MEC-17 regulated microtubule dynamics independently of its acetyltransferase activity (Kalebic et al. 2013a). Although MEC-17D157N partially retained the ability to acetylate α-tubulin, the low level of its acetyltransferase activity in MEC-17-deficient neurons (7.5% of that produced by MEC-17) was unable to rescue the morphological defects induced by loss of MEC-17. The effects of MEC-17-mediated α-tubulin acetylation on microtubule dynamics in various cells and systems need to be evaluated using multiple approaches, such as MEC-17F183A and α-tubulinK40Q. In the most recent studies, α-tubulin acetylation has been reported to enhance microtubule flexibility and mechanical resilience to ensure the persistence of long-lived microtubules in cells (Portran et al. 2017; Xu et al. 2017); therefore, further evaluation needs to be investigated in neurons. α-Tubulin Acetylation Constrains Axon Branching and Growth by Regulating Microtubule Plus-End Dynamics Recent studies have shown that MEC-17 knockout in Caenorhabditis elegans leads to morphological abnormalities, including extended and new processes and infrequent ectopic branches in touch-response neurons, and the extended and new processes could be rescued by the expression of MEC-17D157N (Topalidou et al. 2012). In the present study, loss of MEC-17 significantly induced axon overbranching both in the somatosensory cortex and in cultured hippocampal neurons, and this effect was rescued by the re-expression of full-length MEC-17 but not of MEC-17F183A in MEC-17-deficient neurons. Interestingly, only the N-terminus of MEC-17, MEC-171–193, exhibited acetyltransferase activity but retained partial enzymatic activity compared with full-length MEC-17, indicating that the C-terminus, which is responsible for the interaction between MEC-17 and α-tubulin, also contributes to the acetylation effect of this enzyme. Consistent with this, MEC-171–193 was able to partially rescue axon overbranching and overgrowth in MEC-17-deficient neurons. Furthermore, the expression of the acetylation-mimicking mutant α-tubulinK40Q dramatically rescued the axon overbranching and overgrowth of MEC-17-deficient neurons. Thus, MEC-17-mediated α-tubulin acetylation is essential to constrain axon branching and growth in the neurons of the central nervous system. Coordinated dynamics of the microtubule network is critical for the generation and maintenance of neuronal morphology. Previous studies have reported that microtubule polymerization is required for axon branching (Letourneau et al. 1987; Baas and Ahmad 1993) and that specific suppression of microtubule dynamics by taxol inhibits the formation of axon branching (Gallo and Letourneau 1999; Dent and Kalil 2001), suggesting a critical role of proper microtubule dynamics in axon branching. We found that an increase in the microtubule plus-end dynamics accompanied axon overbranching and overgrowth in MEC-17-deficient neurons. Restoration of the microtubule organization and dynamics by the re-expression of MEC-17 correlated with the rescue of the axon overbranching and overgrowth caused by loss of MEC-17. A mutant deficient in acetyltransferase activity, MEC-17F183A, neither recovered the microtubule organization and hyperdynamics nor rescued the axonal defect in MEC-17-deficient neurons. Importantly, treatment with a low concentration of taxol that did not affect wild-type hippocampal neurons rescued the defects in microtubule organization and dynamics, as well as axon branching and growth in MEC-17-deficient neurons. These data further support a model in which MEC-17-mediated α-tubulin acetylation maintains moderate microtubule plus-end dynamics to keep the normal axon morphology. Functional Implications of α-Tubulin Acetylation During the Development of the Central Nervous System Post-translational modifications of tubulin may regulate the interaction between microtubule and microtubule-associated proteins or motor proteins, thereby controlling neuronal migration, synapse formation and neural network formation (Fukushima et al. 2009). Recent advances regarding α-tubulin acetylation reveal that deficiency in the α-tubulin acetyltransferase MEC-17 causes a delay in neuronal migration in the cortex and a neuronal disorganization in the dentate gyrus (Li et al. 2012a; Kim et al. 2013). Maternal application of the α-tubulin deacetylase inhibitor tubacin rescues the neuronal migratory defect induced by silencing Srpx2 (Salmi et al. 2013). However, in the present study, neuronal migration in the somatosensory cortex was not delayed at P0 in MEC-17-deficient mice, possibly due to a compensatory effect in the embryonic stage. Intriguingly, a role of α-tubulin acetylation was further uncovered in regulating axon branching and growth. Loss of MEC-17 caused excessive axonal branching in cortical neurons in the somatosensory cortex. Given the pronounced correlation between axon overbranching and neuronal miswiring, behavioral abnormalities were expected in MEC-17-deficient mice. Indeed, the knockout mice displayed an anxiety-like behavior despite showing intact spatial learning and memory. Previous studies have suggested that alterations in neuronal morphology and synaptic structure in the amygdala, hippocampus and prefrontal cortex are involved in anxiety (Leuner and Shors 2013). Further experiments to genetically delete MEC-17 in specific brain regions will extend our current understanding of how α-tubulin acetylation in neurons regulates anxiety-like behavior. Overall, our previous and current studies demonstrate that α-tubulin acetylation is required for normal neuronal morphology during brain development. Loss of α-tubulin acetylation disrupts the brain structure and alters the behavior of animals. The role of α-tubulin acetylation in synapse formation, synaptic transmission and neuronal plasticity remains to be explored. Supplementary Material Supplementary data are available at Cerebral Cortex online. Funding National Natural Science Foundation of China (31330046), National Basic Research Program of China (2014CB942802), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19000000). Authors' Contribution D.W. did experiments of axon growth and MT structure in vitro and in vivo, and immunoblotting of neurons. N.N.G. did the majority of molecular, biochemical and behavioral experiments, and time-lapse detection of MT dynamics in live cells. L.L. helped to initiate this project. J.X.Z., J.S.H., Q.J.H., S.G.W., Q.W., H.Q.X. helped molecular, biochemical, and behavioral experiments. L.D. helped detection of MT dynamics. L.B., N.N.G., D.W. wrote the article. X.Z. helped the project design and Q.F.W. helped the article revision. L.B. supervised the whole project. Notes We thank the knockout mouse project (KOMP) repository at the University of California at Davis to breed MEC-17tm1(KOMP)Vlcg heterozygous mice, Dr. Jianping Ding for providing GST-MEC-171–193 plasmid, Dr. Jiansheng Kang for providing DsRed-mito plasmid and Dr. Yuanyuan Su for helping to image with Leica TCS SP8 STED 3X super-resolution microscopy. Conflict of Interest: None declared. References Aguilar A , Becker L , Tedeschi T , Heller S , Iomini C , Nachury MV . 2014 . α-tubulin K40 acetylation is required for contact inhibition of proliferation and cell-substrate adhesion . Mol Biol Cell . 25 : 1854 – 1866 . Google Scholar CrossRef Search ADS PubMed Akella JS , Wloga D , Kim J , Starostina NG , Lyons-Abbott S , Morrissette NS , Dougan ST , Kipreos ET , Gaertig J . 2010 . MEC-17 is an α-tubulin acetyltransferase . Nature . 467 : 218 – 222 . Google Scholar CrossRef Search ADS PubMed Baas PW , Ahmad FJ . 1993 . The transport properties of axonal microtubules establish their polarity orientation . J Cell Biol . 120 : 1427 – 1437 . Google Scholar CrossRef Search ADS PubMed Castro-Castro A , Janke C , Montagnac G , Paul-Gilloteaux P , Chavrier P . 2012 . ATAT1/MEC-17 acetyltransferase and HDAC6 deacetylase control a balance of acetylation of alpha-tubulin and cortactin and regulate MT1-MMP trafficking and breast tumor cell invasion . Eur J Cell Biol . 91 : 950 – 960 . Google Scholar CrossRef Search ADS PubMed Chen Y , Tian X , Kim WY , Snider WD . 2011 . Adenomatous polyposis coli regulates axon arborization and cytoskeleton organization via its N-terminus . PLoS One . 6 : e24335 . Google Scholar CrossRef Search ADS PubMed Conde C , Caceres A . 2009 . Microtubule assembly, organization and dynamics in axons and dendrites . Nat Rev Neurosci . 10 : 319 – 332 . Google Scholar CrossRef Search ADS PubMed Courchet J , Lewis TL Jr , Lee S , Courchet V , Liou DY , Aizawa S , Polleux F . 2013 . Terminal axon branching is regulated by the LKB1-NUAK1 kinase pathway via presynaptic mitochondrial capture . Cell . 153 : 1510 – 1525 . Google Scholar CrossRef Search ADS PubMed Davenport AM , Collins LN , Chiu H , Minor PJ , Sternberg PW , Hoelz A . 2014 . Structural and functional characterization of the α-tubulin acetyltransferase MEC-17 . J Mol Biol . 426 : 2605 – 2616 . Google Scholar CrossRef Search ADS PubMed De Brabander M , Geuens G , Nuydens R , Willebrords R , De Mey J . 1982 . Microtubule stability and assembly in living cells: the influence of metabolic inhibitors, taxol and pH . Cold Spring Harb Symp Quant Biol . 46 ( Pt 1 ): 227 – 240 . Google Scholar CrossRef Search ADS PubMed Dent EW , Barnes AM , Tang F , Kalil K . 2004 . Netrin-1 and semaphorin 3A promote or inhibit cortical axon branching, respectively, by reorganization of the cytoskeleton . J Neurosci . 24 : 3002 – 3012 . Google Scholar CrossRef Search ADS PubMed Dent EW , Kalil K . 2001 . Axon branching requires interactions between dynamic microtubules and actin filaments . J Neurosci . 21 : 9757 – 9769 . Google Scholar CrossRef Search ADS PubMed File SE , Lippa AS , Beer B , Lippa MT . 2004 . Animal tests of anxiety. In: Current protocols in neuroscience . New York : John Wiley & Sons . p. 8.3.1 – 8.3.22 . Friedmann DR , Aguilar A , Fan J , Nachury MV , Marmorstein R . 2012 . Structure of the α-tubulin acetyltransferase, αTAT1, and implications for tubulin-specific acetylation . Proc Natl Acad Sci USA . 109 : 19655 – 19660 . Google Scholar CrossRef Search ADS PubMed Fukushima N , Furuta D , Hidaka Y , Moriyama R , Tsujiuchi T . 2009 . Post-translational modifications of tubulin in the nervous system . J Neurochem . 109 : 683 – 693 . Google Scholar CrossRef Search ADS PubMed Gallo G . 2011 . The cytoskeletal and signaling mechanisms of axon collateral branching . Dev Neurobiol . 71 : 201 – 220 . Google Scholar CrossRef Search ADS PubMed Gallo G , Letourneau PC . 1999 . Different contributions of microtubule dynamics and transport to the growth of axons and collateral sprouts . J Neurosci . 19 : 3860 – 3873 . Google Scholar CrossRef Search ADS PubMed Kalebic N , Martinez C , Perlas E , Hublitz P , Bilbao-Cortes D , Fiedorczuk K , Andolfo A , Heppenstall PA . 2013 a. Tubulin acetyltransferase αTAT1 destabilizes microtubules independently of its acetylation activity . Mol Biol Cell . 33 : 1114 – 1123 . Google Scholar CrossRef Search ADS Kalebic N , Sorrentino S , Perlas E , Bolasco G , Martinez C , Heppenstall PA . 2013 b. αTAT1 is the major α-tubulin acetyltransferase in mice . Nat Commun . 4 : 1962 . Google Scholar CrossRef Search ADS PubMed Kalil K , Dent EW . 2014 . Branch management: mechanisms of axon branching in the developing vertebrate CNS . Nat Rev Neurosci . 15 : 7 – 18 . Google Scholar CrossRef Search ADS PubMed Ketschek A , Jones S , Spillane M , Korobova F , Svitkina T , Gallo G . 2015 . Nerve growth factor promotes reorganization of the axonal microtubule array at sites of axon collateral branching . Dev Neurobiol . 75 : 1441 – 1461 . Google Scholar CrossRef Search ADS PubMed Kim GW , Li L , Gorbani M , You L , Yang XJ . 2013 . Mice lacking α-tubulin acetyltransferase 1 are viable but display α-tubulin acetylation deficiency and dentate gyrus distortion . J Biol Chem . 288 : 20334 – 20350 . Google Scholar CrossRef Search ADS PubMed Ko M , Zou K , Minagawa H , Yu W , Gong JS , Yanagisawa K , Michikawa M . 2005 . Cholesterol-mediated neurite outgrowth is differently regulated between cortical and hippocampal neurons . J Biol Chem . 280 : 42759 – 42765 . Google Scholar CrossRef Search ADS PubMed Krylova O , Herreros J , Cleverley KE , Ehler E , Henriquez JP , Hughes SM , Salinas PC . 2002 . WNT-3, expressed by motoneurons, regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons . Neuron . 35 : 1043 – 1056 . Google Scholar CrossRef Search ADS PubMed Letourneau PC , Shattuck TA , Ressler AH . 1987 . “Pull” and “push” in neurite elongation: observations on the effects of different concentrations of cytochalasin B and taxol . Cell Motil Cytoskeleton . 8 : 193 – 209 . Google Scholar CrossRef Search ADS PubMed Leuner B , Shors TJ . 2013 . Stress, anxiety, and dendritic spines: what are the connections? Neuroscience . 251 : 108 – 119 . Google Scholar CrossRef Search ADS PubMed Lewis TL Jr. , Courchet J , Polleux F . 2013 . Cell biology in neuroscience: cellular and molecular mechanisms underlying axon formation, growth, and branching . J Cell Biol . 202 : 837 – 848 . Google Scholar CrossRef Search ADS PubMed Li L , Wei D , Wang Q , Pan J , Liu R , Zhang X , Bao L . 2012 a. MEC-17 deficiency leads to reduced α-tubulin acetylation and impaired migration of cortical neurons . J Neurosci . 32 : 12673 – 12683 . Google Scholar CrossRef Search ADS PubMed Li W , Zhong C , Li L , Sun B , Wang W , Xu S , Zhang T , Wang C , Bao L , Ding J . 2012 b. Molecular basis of the acetyltransferase activity of MEC-17 towards α-tubulin . Cell Res . 22 : 1707 – 1711 . Google Scholar CrossRef Search ADS PubMed Mackeh R , Lorin S , Ratier A , Mejdoubi-Charef N , Baillet A , Bruneel A , Hamai A , Codogno P , Pous C , Perdiz D . 2014 . Reactive oxygen species, AMP-activated protein kinase, and the transcription cofactor p300 regulate alpha-tubulin acetyltransferase-1 (αTAT-1/MEC-17)-dependent microtubule hyperacetylation during cell stress . J Biol Chem . 289 : 11816 – 11828 . Google Scholar CrossRef Search ADS PubMed Montagnac G , Meas-Yedid V , Irondelle M , Castro-Castro A , Franco M , Shida T , Nachury MV , Benmerah A , Olivo-Marin JC , Chavrier P . 2013 . αTAT1 catalyses microtubule acetylation at clathrin-coated pits . Nature . 502 : 567 – 570 . Google Scholar CrossRef Search ADS PubMed Neumann B , Hilliard MA . 2014 . Loss of MEC-17 leads to microtubule instability and axonal degeneration . Cell Rep . 6 : 93 – 103 . Google Scholar CrossRef Search ADS PubMed Orr GA , Verdier-Pinard P , McDaid H , Horwitz SB . 2003 . Mechanisms of Taxol resistance related to microtubules . Oncogene . 22 : 7280 – 7295 . Google Scholar CrossRef Search ADS PubMed Portran D , Schaedel L , Xu Z , Thery M , Nachury MV . 2017 . Tubulin acetylation protects long-lived microtubules against mechanical ageing . Nat Cell Biol . 19 : 391 – 398 . Google Scholar CrossRef Search ADS PubMed Poulain FE , Sobel A . 2010 . The microtubule network and neuronal morphogenesis: dynamic and coordinated orchestration through multiple players . Mol Cell Neurosci . 43 : 15 – 32 . Google Scholar CrossRef Search ADS PubMed Rochlin MW , Wickline KM , Bridgman PC . 1996 . Microtubule stability decreases axon elongation but not axoplasm production . J Neurosci . 16 : 3236 – 3246 . Google Scholar CrossRef Search ADS PubMed Salmi M , Bruneau N , Cillario J , Lozovaya N , Massacrier A , Buhler E , Cloarec R , Tsintsadze T , Watrin F , Tsintsadze V , et al. . 2013 . Tubacin prevents neuronal migration defects and epileptic activity caused by rat Srpx2 silencing in utero . Brain . 136 : 2457 – 2473 . Google Scholar CrossRef Search ADS PubMed Shida T , Cueva JG , Xu Z , Goodman MB , Nachury MV . 2010 . The major α-tubulin K40 acetyltransferase αTAT1 promotes rapid ciliogenesis and efficient mechanosensation . Proc Natl Acad Sci USA . 107 : 21517 – 21522 . Google Scholar CrossRef Search ADS PubMed Song Y , Kirkpatrick LL , Schilling AB , Helseth DL , Chabot N , Keillor JW , Johnson GV , Brady ST . 2013 . Transglutaminase and polyamination of tubulin: posttranslational modification for stabilizing axonal microtubules . Neuron . 78 : 109 – 123 . Google Scholar CrossRef Search ADS PubMed Szyk A , Deaconescu AM , Spector J , Goodman B , Valenstein ML , Ziolkowska NE , Kormendi V , Grigorieff N , Roll-Mecak A . 2014 . Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase . Cell . 157 : 1405 – 1415 . Google Scholar CrossRef Search ADS PubMed Taschner M , Vetter M , Lorentzen E . 2012 . Atomic resolution structure of human α-tubulin acetyltransferase bound to acetyl-CoA . Proc Natl Acad Sci USA . 109 : 19649 – 19654 . Google Scholar CrossRef Search ADS PubMed Topalidou I , Keller C , Kalebic N , Nguyen KC , Somhegyi H , Politi KA , Heppenstall P , Hall DH , Chalfie M . 2012 . Genetically separable functions of the MEC-17 tubulin acetyltransferase affect microtubule organization . Curr Biol . 22 : 1057 – 1065 . Google Scholar CrossRef Search ADS PubMed Vasquez RJ , Howell B , Yvon AM , Wadsworth P , Cassimeris L . 1997 . Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro . Mol Biol Cell . 8 : 973 – 985 . Google Scholar CrossRef Search ADS PubMed Witte H , Neukirchen D , Bradke F . 2008 . Microtubule stabilization specifies initial neuronal polarization . J Cell Biol . 180 : 619 – 632 . Google Scholar CrossRef Search ADS PubMed Wloga D , Dave D , Meagley J , Rogowski K , Jerka-Dziadosz M , Gaertig J . 2010 . Hyperglutamylation of tubulin can either stabilize or destabilize microtubules in the same cell . Eukaryot Cell . 9 : 184 – 193 . Google Scholar CrossRef Search ADS PubMed Wu CH , Fallini C , Ticozzi N , Keagle PJ , Sapp PC , Piotrowska K , Lowe P , Koppers M , McKenna-Yasek D , Baron DM , et al. . 2012 . Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis . Nature . 488 : 499 – 503 . Google Scholar CrossRef Search ADS PubMed Xu Z , Schaedel L , Portran D , Aguilar A , Gaillard J , Marinkovich MP , Thery M , Nachury MV . 2017 . Microtubules acquire resistance from mechanical breakage through intralumenal acetylation . Science . 356 : 328 – 332 . Google Scholar CrossRef Search ADS PubMed Yvon AM , Wadsworth P , Jordan MA . 1999 . Taxol suppresses dynamics of individual microtubules in living human tumor cells . Mol Biol Cell . 10 : 947 – 959 . Google Scholar CrossRef Search ADS PubMed © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)
TI - α-Tubulin Acetylation Restricts Axon Overbranching by Dampening Microtubule Plus-End Dynamics in Neurons
JO - Cerebral Cortex
DO - 10.1093/cercor/bhx225
DA - 2018-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/tubulin-acetylation-restricts-axon-overbranching-by-dampening-lUTHyKXpiM
SP - 3332
EP - 3346
VL - 28
IS - 9
DP - DeepDyve
ER -