Clostridium difficile Toxin A Decreases Acetylation of Tubulin,Leading to Microtubule Depolymerization through Activation of Histone Deacetylase 6, and This Mediates Acute Inflammation

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2′,3′-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.     

Inhibition of HDAC6 Deacetylase Activity Increases Its Binding with Microtubules and Suppresses Microtubule Dynamic Instability in MCF-7 Cells

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ϵ-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.     

Rho-associated Coiled-coil Kinase (ROCK) Protein Controls Microtubule Dynamics in a Novel Signaling Pathway That Regulates Cell Migration

The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.     

Acetylation/deacetylation and microtubule associated proteins influence flagellar axonemal stability and sperm motility

PTMs and microtubule-associated proteins (MAPs) are known to regulate microtubule dynamicity in somatic cells. Reported literature on modulation of α-tubulin acetyl transferase (αTAT1) and histone deacetylase 6 (HDAC6) in animal models and cell lines illustrate disparity in correlating tubulin acetylation status with stability of MT. Our earlier studies showed reduced acetyl tubulin in sperm of asthenozoospermic individuals. Our studies on rat sperm showed that on inhibition of HDAC6 activity, although tubulin acetylation increased, sperm motility was reduced. Studies were therefore undertaken to investigate the influence of tubulin acetylation/deacetylation on MT dynamicity in sperm flagella using rat and human sperm. Our data on rat sperm revealed that HDAC6 specific inhibitor Tubastatin A (T) inhibited sperm motility and neutralized the depolymerizing and motility debilitating effect of Nocodazole. The effect on polymerization was further confirmed in vitro using pure MT and recHDAC6. Also polymerized axoneme was less in sperm of asthenozoosperm compared to normozoosperm. Deacetylase activity was reduced in sperm lysates and axonemes exposed to T and N+T but not in axonemes of sperm treated similarly suggesting that HDAC6 is associated with sperm axonemes or MT. Deacetylase activity was less in asthenozoosperm. Intriguingly, the expression of MDP3 physiologically known to bind to HDAC6 and inhibit its deacetylase activity remained unchanged. However, expression of acetyl α-tubulin, HDAC6 and microtubule stabilizing protein SAXO1 was less in asthenozoosperm. These observations suggest that MAPs and threshold levels of MT acetylation/deacetylation are important for MT dynamicity in sperm and may play a role in regulating sperm motility.     

HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.     

Interactions between two regulatory proteins of microtubule dynamics,HDAC6, TPPP/p25, and the hub protein,DYNLL/LC8

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6. Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network.     

A novel GRK2/HDAC6 interaction modulates cell spreading and motility

Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α-tubulin deacetylase. We identify G protein-coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α-tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α-tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2-K220R or GRK2-S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6-mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts.     

Extracellular Signal-regulated Kinase (ERK) Phosphorylates Histone Deacetylase 6 (HDAC6) at Serine 1035 to Stimulate Cell Migration

Histone deacetylase 6 (HDAC6) is well known for its ability to promote cell migration through deacetylation of its cytoplasmic substrates such as α-tubulin. However, how HDAC6 itself is regulated to control cell motility remains elusive. Previous studies have shown that one third of extracellular signal-regulated kinase (ERK) is associated with the microtubule cytoskeleton in cells. Yet, no connection between HDAC6 and ERK has been discovered. Here, for the first time, we reveal that ERK binds to and phosphorylates HDAC6 to promote cell migration via deacetylation of α-tubulin. We have identified two novel ERK-mediated phosphorylation sites: threonine 1031 and serine 1035 in HDAC6. Both sites were phosphorylated by ERK1 in vitro, whereas Ser-1035 was phosphorylated in response to the activation of EGFR-Ras-Raf-MEK-ERK signaling pathway in vivo. HDAC6-null mouse embryonic fibroblasts rescued by the nonphosphorylation mimicking mutant displayed significantly reduced cell migration compared with those rescued by the wild type. Consistently, the nonphosphorylation mimicking mutant exerted lower tubulin deacetylase activity in vivo compared with the wild type. These data indicate that ERK/HDAC6-mediated cell motility is through deacetylation of α-tubulin. Overall, our results suggest that HDAC6-mediated cell migration could be governed by EGFR-Ras-Raf-MEK-ERK signaling.     

aPKC Phosphorylation of HDAC6 Results in Increased Deacetylation Activity

The Class II histone deacetylase, HDAC6, has been shown to be involved in cell motility, aggresome formation and mitochondria transport. HDAC6 deacetylase activity regulates α-tubulin acetylation levels and thus plays a critical role in these processes. In turn, HDAC6 activity can be regulated by interaction with various proteins including multiple kinases. Kinase mediated phosphorylation of HDAC6 can lead to either increased or reduced activity. Our previous research has shown that sequestosome1/p62 (SQSTM1/p62) interacts with HDAC6 and regulates its activity. As SQSTM1/p62 is a scaffolding protein known to interact directly with the zeta isoform of Protein Kinase C (PKCζ), we sought to examine if HDAC6 could be a substrate for PKCζ phosphorylation and if so, how its activity might be regulated. Our data demonstrate that HDAC6 is not only present in a protein complex with PKCζ but can also be phosphorylated by PKCζ. We also show that specific phosphorylation of HDAC6 by PKCζ increases HDAC6 deacetylase activity resulting in reduced acetylated tubulin levels. Our findings provide novel insight into the molecular mechanism by which HDAC6, PKCζ and SQSTM1/p62 function together in protein aggregate clearance. These results also highlight a new research direction which may prove fruitful for understanding the underlying cause of several neurodegenerative diseases.     

HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.     

Loss of α-Tubulin Acetylation Is Associated with TGF-β-induced Epithelial-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated α-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated α-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-β-induced EMT. We found that TGF-β increased the activity of HDAC6, a major deacetylase of α-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-β type I receptor inhibitor SB431542 restored the level of acetylated α-tubulin and consequently blocked EMT. Our results demonstrate that acetylated α-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.     

Novel Interaction of Class IIb Histone Deacetylase 6 (HDAC6) with Class IIa HDAC9 Controls Gonadotropin Releasing Hormone (GnRH) Neuronal Cell Survival and Movement

The impact of histone deacetylases (HDACs) in the control of gonadotropin releasing hormone (GnRH) neuronal development is unknown. We identified an increase in many HDACs in GT1-7 (differentiated) compared with NLT (undifferentiated) GnRH neuronal cell lines. Increased HDAC9 mRNA and protein and specific deacetylase activity in GT1-7 cells suggested a functional role. Introduction of HDAC9 in NLT cells protected from serum withdrawal induced apoptosis and impaired basal neuronal cell movement. Conversely, silencing of endogenous HDAC9 in GT1-7 cells increased apoptosis and cell movement. Comparison of WT and mutant HDAC9 constructs demonstrated that the HDAC9 pro-survival effects required combined cytoplasmic and nuclear localization, whereas the effects on cell movement required a cytoplasmic site of action. Co-immunoprecipitation demonstrated a novel interaction of HDAC9 selectively with the Class IIb HDAC6. HDAC6 was also up-regulated at the mRNA and protein levels, and HDAC6 catalytic activity was significantly increased in GT1-7 compared with NLT cells. HDAC9 interacted with HDAC6 through its second catalytic domain. Silencing of HDAC6, HDAC9, or both, in GT1-7 cells augmented apoptosis compared with controls. HDAC6 and -9 had additive effects to promote cell survival via modulating the BAX/BCL2 pathway. Silencing of HDAC6 resulted in an activation of movement of GT1-7 cells with induction in acetylation of α-tubulin. Inhibition of HDAC6 and HDAC9 together resulted in an additive effect to increase cell movement but did not alter the acetylation of αtubulin. Together, these studies identify a novel interaction of Class IIa HDAC9 with Class IIb HDAC6 to modulate cell movement and survival in GnRH neurons.     

Histone deacetylase 6 regulates growth factor-induced actin remodeling and endocytosis

Histone deacetylase 6 (HDAC6) is a cytoplasmic deacetylase that uniquely catalyzes α-tubulin deacetylation and promotes cell motility. However, the mechanism underlying HDAC6-dependent cell migration and the role for microtubule acetylation in motility are not known. Here we show that HDAC6-induced global microtubule deacetylation was not sufficient to stimulate cell migration. Unexpectedly, in response to growth factor stimulation, HDAC6 underwent rapid translocation to actin-enriched membrane ruffles and subsequently became associated with macropinosomes, the vesicles for fluid-phase endocytosis. Supporting the importance of these associations, membrane ruffle formation, macropinocytosis, and cell migration were all impaired in HDAC6-deficient cells. Conversely, elevated HDAC6 levels promoted membrane ruffle formation with a concomitant increase in macropinocytosis and motility. In search for an HDAC6 target, we found that heat shock protein 90 (Hsp90), another prominent substrate of HDAC6, was also recruited to membrane ruffles and macropinosomes. Significantly, inhibition of Hsp90 activity suppressed membrane ruffling and cell migration, while expression of an acetylation-resistant Hsp90 mutant promoted ruffle formation. Our results uncover a surprising role for HDAC6 in actin remodeling-dependent processes and identify the actin cytoskeleton as an important target of HDAC6-regulated protein deacetylation.     

Acetylation as a major determinant to microtubule-dependent autophagy: Relevance to Alzheimer's and Parkinson disease pathology

Protein post-translational modifications (PTMs) that potentiate protein aggregation have been implicated in several neurological disorders, including Alzheimer's (AD) and Parkinson's disease (PD). In fact, Tau and alpha-synuclein (ASYN) undergo several PTMs potentiating their aggregation and neurotoxicity.Recent data posits a role for acetylation in Tau and ASYN aggregation. Herein we aimed to clarify the role of Sirtuin-2 (SIRT2) and HDAC6 tubulin deacetylases as well as p300 acetyltransferase in AD and PD neurodegeneration. We used transmitochondrial cybrids that recapitulate pathogenic alterations observed in sporadic PD and AD patient brains and ASYN and Tau cellular models.We confirmed that Tau protein and ASYN are microtubules (MTs)-associated proteins (MAPs). Moreover, our results suggest that α-tubulin acetylation induced by SIRT2 inhibition is functionally associated with the improvement of MT dynamic determined by decreased Tau phosphorylation and by increased Tau/tubulin and ASYN/tubulin binding. Our data provide a strong evidence for a functional role of tubulin and MAPs acetylation on autophagic vesicular traffic and cargo clearance. Additionally, we showed that an accumulation of ASYN oligomers imbalance mitochondrial dynamics, which further compromise autophagy. We also demonstrated that an increase in Tau acetylation is associated with Tau phosphorylation. We found that p300, HDAC6 and SIRT2 influences Tau phosphorylation and autophagic flux in AD. In addition, we demonstrated that p300 and HDAC6 modulate Tau and Tubulin acetylation.Overall, our data disclose the role of Tau and ASYN modifications through acetylation in AD and PD pathology, respectively. Moreover, this study indicates that MTs can be a promising therapeutic target in the field of neurodegenerative disorders in which intracellular transport is altered.     

Tau – an inhibitor of deacetylase HDAC6 function

Analysis of brain microtubule protein from patients with Alzheimer's disease showed decreased alpha tubulin levels along with increased acetylation of the alpha tubulin subunit, mainly in those microtubules from neurons containing neurofibrillary tau pathology. To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation. As expected, increased acetylation was also found in tubulin from wild-type mice compared with tubulin from mice lacking tau because of the tau-mediated inhibition of the deacetylase. In addition, we found that an excess of tau protein, as a HDAC6 inhibitor, prevents induction of autophagy by inhibiting proteasome function.     

P25alpha/Tubulin polymerization promoting protein expression by myelinating oligodendrocytes of the developing rat brain

P25alpha/tubulin polymerization promoting protein (TPPP) is a brain specific phosphoprotein that displays microtubule bundling activity. In the mature brain, p25alpha/TPPP distributes to oligodendrocytes and choroid plexus epithelium. We mapped the spatial and temporal distribution of p25alpha/TPPP in the developing rat brain. Having localized its expression to neuronal tissue by Western blot analyses, the distribution of p25alpha/TPPP to developing oligodendrocytes was confirmed using a specific antibody. In the pre-natal and post-natal brain, p25alpha/TPPP was localized to the perinuclear cytoplasm of myelinating oligodendrocytes from embryonic (E) day E20 as verified from cellular co-localization with 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Oligodendrocyte progenitor cells and pre-myelinating oligodendrocytes identified by the expression of NG2 proteoglycan and CD9, respectively, both failed to contain p25alpha/TPPP. In contrast, P25alpha/TPPP co-localized with beta(IV)-tubulin from post-natal (p) day P10 suggesting that p25alpha/TPPP plays an important role for tubulin-related transport in developing, myelinating oligodendrocytes.     

Interactions of pathological hallmark proteins: tubulin polymerization promoting protein/p25, beta-amyloid, and alpha-synuclein

The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ(42), α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.     

Luminal Localization of α-tubulin K40 Acetylation by Cryo-EM Analysis of Fab-Labeled Microtubules

The αβ-tubulin subunits of microtubules can undergo a variety of evolutionarily-conserved post-translational modifications (PTMs) that provide functional specialization to subsets of cellular microtubules. Acetylation of α-tubulin residue Lysine-40 (K40) has been correlated with increased microtubule stability, intracellular transport, and ciliary assembly, yet a mechanistic understanding of how acetylation influences these events is lacking. Using the anti-acetylated tubulin antibody 6-11B-1 and electron cryo-microscopy, we demonstrate that the K40 acetylation site is located inside the microtubule lumen and thus cannot directly influence events on the microtubule surface, including kinesin-1 binding. Surprisingly, the monoclonal 6-11B-1 antibody recognizes both acetylated and deacetylated microtubules. These results suggest that acetylation induces structural changes in the K40-containing loop that could have important functional consequences on microtubule stability, bending, and subunit interactions. This work has important implications for acetylation and deacetylation reaction mechanisms as well as for interpreting experiments based on 6-11B-1 labeling.