Level Of Acetylated Α-tubulin Research Articles

Asymmetric Synthesis of Saturated and Unsaturated Hydroxy Fatty Acids (HFAs) and Study of Their Antiproliferative Activity.

Hydroxy fatty acids (HFAs) constitute a class of lipids, distinguished by the presence of a hydroxyl on a long aliphatic chain. This study aims to expand our insights into HFA bioactivities, while also introducing new methods for asymmetrically synthesizing unsaturated and saturated HFAs. Simultaneously, a procedure previously established by us was adapted to generate new HFA regioisomers. An organocatalytic step was employed for the synthesis of chiral terminal epoxides, which either by alkynylation or by Grignard reagents resulted in unsaturated or saturated chiral secondary alcohols and, ultimately, HFAs. 7-(S)-Hydroxyoleic acid (7SHOA), 7-(S)-hydroxypalmitoleic acid (7SHPOA) and 7-(R)- and (S)-hydroxymargaric acids (7HMAs) were synthesized for the first time and, together with regioisomers of (R)- and (S)-hydroxypalmitic acids (HPAs) and hydroxystearic acids (HSAs), whose biological activity has not been tested so far, were studied for their antiproliferative activities. The unsaturation of the long chain, as well as an odd-numbered (C17) fatty acid chain, led to reduced activity, while the new 6-(S)-HPA regioisomer was identified as exhibiting potent antiproliferative activity in A549 cells. 6SHPA induced acetylation of histone 3 in A549 cells, without affecting acetylated α-tubulin levels, suggesting the selective inhibition of histone deacetylase (HDAC) class I enzymes, and was found to inhibit signal transducer and activator of transcription 3 (STAT3) expression.

Reduced acetylated α-tubulin in SPAST hereditary spastic paraplegia patient PBMCs.

HSP-SPAST is the most common form of hereditary spastic paraplegia (HSP), a neurodegenerative disease causing lower limb spasticity. Previous studies using HSP-SPAST patient-derived induced pluripotent stem cell cortical neurons have shown that patient neurons have reduced levels of acetylated α-tubulin, a form of stabilized microtubules, leading to a chain of downstream effects eventuating in increased vulnerability to axonal degeneration. Noscapine treatment rescued these downstream effects by restoring the levels of acetylated α-tubulin in patient neurons. Here we show that HSP-SPAST patient non-neuronal cells, peripheral blood mononuclear cells (PBMCs), also have the disease-associated effect of reduced levels of acetylated α-tubulin. Evaluation of multiple PBMC subtypes showed that patient T cell lymphocytes had reduced levels of acetylated α-tubulin. T cells make up to 80% of all PBMCs and likely contributed to the effect of reduced acetylated α-tubulin levels seen in overall PBMCs. We further showed that mouse administered orally with increasing concentrations of noscapine exhibited a dose-dependent increase of noscapine levels and acetylated α-tubulin in the brain. A similar effect of noscapine treatment is anticipated in HSP-SPAST patients. To measure acetylated α-tubulin levels, we used a homogeneous time resolved fluorescence technology-based assay. This assay was sensitive to noscapine-induced changes in acetylated α-tubulin levels in multiple sample types. The assay is high throughput and uses nano-molar protein concentrations, making it an ideal assay for evaluation of noscapine-induced changes in acetylated α-tubulin levels. This study shows that HSP-SPAST patient PBMCs exhibit disease-associated effects. This finding can help expedite the drug discovery and testing process.

The Role of Runx2 in Microtubule Acetylation in Bone Metastatic Breast Cancer Cells.

Simple SummaryBreast cancer is the most commonly diagnosed cancer type, making up a quarter of all cases among women. While modern-day research has shown tremendous progress in the development of treatment options, complications related to bone metastasis remain an obstacle that demands further attention. Cancer cells that migrate into the bone microenvironment show significant alterations in their metabolism and gene expression profiles. In this study, we set out to better understand the changes induced by Runx2 in bone-derived breast cancer cells. We identified key changes in the stability of the microtubule cytoskeleton of bone-derived cells expressing Runx2. These changes have implications on a variety of processes related to metabolism, cellular stress response, and response to common chemotherapies. These studies help to shed light on the changes that take place as a tumor begins to metastasize and to better predict which therapies will benefit patients with bone metastasis.Bone metastasis of breast cancer results in severe bone loss, fractures, and death. Crosstalk between breast cancer cells and bone resident cells promotes osteoclast activity and the release of growth factors from the bone matrix resulting in aggressive tumor growth and bone loss. We and others have shown that Runt-related transcription factor-2 (Runx2) promotes metastatic tumor growth-associated bone loss. Breast cancer cells also induce autophagy to survive metabolic stress at the metastatic site. Recently, we reported a Runx2-dependent increase in autophagy. In this study, to examine the underlying mechanisms of metastasis and tumor resistance to stress, we used a bone metastatic isogenic variant of breast cancer MDA-MB-231 cells isolated from a xenograft tumor mouse model of metastasis. Our results with immunofluorescence and biochemical approaches revealed that Runx2 promotes microtubule (MT) stability to facilitate autophagy. Stable MTs are critical for autophagosome trafficking and display increased acetylation at Lysine 40 of α-tubulin. Runx2 silencing decreases acetylated α-tubulin levels. The expression levels of HDAC6 and αTAT1, which serve to regulate the acetylation of α-tubulin, were not altered with Runx2 silencing. We found that HDAC6 interaction with α-tubulin is inhibited by Runt-related factor-2 (Runx2). We show that the expression of wild-type Runx2 can restore the acetylated polymer of MTs in Runx2 knockdown cells, while the C-terminal deletion mutant fails to rescue the polymer of MTs. Importantly, cellular stress, such as glucose starvation also increases the acetylation of α-tubulin. We found that the loss of Runx2 increases the sensitivity of breast cancer cells to MT-targeting agents. Overall, our results indicate a novel regulatory mechanism of microtubule acetylation and suggest that Runx2 and acetylated microtubules may serve as therapeutic targets for bone metastatic tumors.

HDAC6 Inhibition Corrects Electrophysiological and Axonal Transport Deficits in a Human Stem Cell-Based Model of Charcot-Marie-Tooth Disease (Type 2D).

Charcot-Marie-Tooth disease type 2D (CMT2D), is a hereditary peripheral neuropathy caused by mutations in the gene encoding glycyl-tRNA synthetase (GARS1). Here, human induced pluripotent stem cell (hiPSC)-based models of CMT2D bearing mutations in GARS1 and their use for the identification of predictive biomarkers amenable to therapeutic efficacy screening is described. Cultures containing spinal cord motor neurons generated from this line exhibit network activity marked by significant deficiencies in spontaneous action potential firing and burst fire behavior. This result matches clinical data collected from a patient bearing a GARS1P724H mutation and is coupled with significant decreases in acetylated α-tubulin levels and mitochondrial movement within axons. Treatment with histone deacetylase 6 inhibitors, tubastatin A and CKD504, improves mitochondrial movement and α-tubulin acetylation in these cells. Furthermore, CKD504 treatment enhances population-level electrophysiological activity, highlighting its potential as an effective treatment for CMT2D.

Pathogenic tauR406W‐induced neurodegeneration and memory deficits rescued by HDAC6‐null mutation in Drosophila melanogaster

Genetic screening was performed by Drosophila melanogaster to observe how different variants of HDAC6 regulate tauR406W mediated neurodegeneration. The versatile Gal4-UAS gene expression system was used to express variants of HDAC6 and tauR406W with the help of pan-eye specific GMR-Gal4, and pan-neuronal elav-Gal4 in the flies. The extent of neurodegeneration was quantified by automated analysis of degenerated tissues, the number of aggregates, immunofluorescence, and also with the help of various behavioral and memory assays. Live imaging was performed on the wing with GCamP6f to study the role of HDAC6 on axonal transport. On expressing the HDAC6 mutants (H237A, H664A) the levels of total tau were increased followed by a decrease in acetylated α-tubulin levels. We have also noticed a reduction in mRNA levels of Atg1, Atg8, and Atg12. On analyzing the various regions of the eye, there was a significant surface degeneration as well as an increase in the number of aggregates. The wings of flies expressing a GCamp6f and eb1-eGFP displayed a defective axonal transport as well as damaged neuronal morphology. Upon expressing HDAC6[KO], there was amelioration in memory deficits, neurodegeneration and the flies regained a better retinal surface morphology. HDAC6 can interact with tau mutations outside the microtubule-binding region (MTBR) and can cause memory deficits, behavioral anomalies. Inhibition of HDAC6 via gene therapy can be an effective therapy to treat Alzheimer's disease and other associated dementias.

Transthyretin Promotes Axon Growth via Regulation of Microtubule Dynamics and Tubulin Acetylation.

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics. We found that TTR KO mice have an intrinsic increase in dynamic MTs and reduced levels of acetylated α-tubulin in peripheral axons. In addition, they failed to modulate MT dynamics in response to sciatic nerve injury, leading to decreased regenerative capacity. Importantly, restoring acetylated α-tubulin levels of TTR KO dorsal root ganglia (DRG) neurons using an HDAC6 inhibitor is sufficient to completely revert defective MT dynamics and neurite outgrowth. In summary, our results reveal a new role for TTR in the modulation of MT dynamics by regulating α-tubulin acetylation via modulation of the acetylase ATAT1, and suggest that this activity underlies TTR neuritogenic function.

Synthesis and biological evaluation of selective histone deacetylase 6 inhibitors as multifunctional agents against Alzheimer's disease

Histone deacetylase 6 (HDAC6) is a potential target for Alzheimer's disease (AD). In this study, a series of novel phenothiazine-, memantine-, and 1,2,3,4-tetrahydro-γ-carboline-based HDAC6 inhibitors with a variety of linker moieties were designed and synthesized. As a hydrochloride salt, the phenothiazine-based hydroxamic acid W5 with a pyridyl-containing linker motif was identified as a high potent and selective HDAC6 inhibitor. It inhibited HDAC6 with an IC50 of 2.54 nM and was more than 290- to 3300-fold selective over other HDAC isoforms. In SH-SY5Y cells, W5 dose-dependently increased the acetylated α-tubulin levels and reduced the hyperphosphorylated tau proteins at Ser396. As an effective metal chelator, W5 inhibited Cu2+-induced Aβ1–42 aggregation and disaggregated Cu2+-Aβ1−42 oligomers, and showed protective effects on the SH-SY5Y cells against Aβ1–42- as well as Cu2+-Aβ1–42 induced cell damages, serving as a potential ligand to target AD metal dyshomeostasis. Moreover, W5 promoted the differentiated neuronal neurite outgrowth, increased the mRNA expression of the recognized neurogenesis markers, GAP43, N-myc, and MAP-2. Therefore, W5 might be a good lead for the development of novel HDAC6 inhibitors targeting multi-facets of AD.

Protein Quality Control Activation and Microtubule Remodeling in Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder. It is mainly caused by mutations in genes encoding sarcomere proteins. Mutant forms of these highly abundant proteins likely stress the protein quality control (PQC) system of cardiomyocytes. The PQC system, together with a functional microtubule network, maintains proteostasis. We compared left ventricular (LV) tissue of nine donors (controls) with 38 sarcomere mutation-positive (HCMSMP) and 14 sarcomere mutation-negative (HCMSMN) patients to define HCM and mutation-specific changes in PQC. Mutations in HCMSMP result in poison polypeptides or reduced protein levels (haploinsufficiency, HI). The main findings were (1) several key PQC players were more abundant in HCM compared to controls, (2) after correction for sex and age, stabilizing heat shock protein (HSP)B1, and refolding, HSPD1 and HSPA2 were increased in HCMSMP compared to controls, (3) α-tubulin and acetylated α-tubulin levels were higher in HCM compared to controls, especially in HCMHI, (4) myosin-binding protein-C (cMyBP-C) levels were inversely correlated with α-tubulin, and (5) α-tubulin levels correlated with acetylated α-tubulin and HSPs. Overall, carrying a mutation affects PQC and α-tubulin acetylation. The haploinsufficiency of cMyBP-C may trigger HSPs and α-tubulin acetylation. Our study indicates that proliferation of the microtubular network may represent a novel pathomechanism in cMyBP-C haploinsufficiency-mediated HCM.

Brain Penetrable Histone Deacetylase 6 Inhibitor SW-100 Ameliorates Memory and Learning Impairments in a Mouse Model of Fragile X Syndrome.

Disease-modifying therapies are needed for Fragile X Syndrome (FXS), as at present there are no effective treatments or cures. Herein, we report on a tetrahydroquinoline-based selective histone deacetylase 6 (HDAC6) inhibitor SW-100, its pharmacological and ADMET properties, and its ability to improve upon memory performance in a mouse model of FXS, Fmr1-/- mice. This small molecule demonstrates good brain penetrance, low-nanomolar potency for the inhibition of HDAC6 (IC50 = 2.3 nM), with at least a thousand-fold selectivity over all other class I, II, and IV HDAC isoforms. Moreover, through its inhibition of the α-tubulin deacetylase domain of HDAC6 (CD2), in cells SW-100 upregulates α-tubulin acetylation with no effect on histone acetylation and selectively restores the impaired acetylated α-tubulin levels in the hippocampus of Fmr1-/- mice. Lastly, SW-100 ameliorates several memory and learning impairments in Fmr1-/- mice, thus modeling the intellectual deficiencies associated with FXS, and hence providing a strong rationale for pursuing HDAC6-based therapies for the treatment of this rare disease.

HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease.

Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

RCCD1 depletion attenuates TGF-β-induced EMT and cell migration by stabilizing cytoskeletal microtubules in NSCLC cells

Lung cancer is one of the most lethal cancers due to its highly metastatic spreading. The motility of lung cancer cells is regulated by paracrine factors, such as TGF-β, in the tumor microenvironment through the induction of epithelial-to-mesenchymal transition (EMT). The stability of microtubules is reported to be associated with the EMT process and the migration of cancer cells. Here, we observed that RCC1 domain-containing protein 1 (RCCD1) is highly expressed in non-small cell lung cancer (NSCLC) patients with poor prognosis, and RCCD1 is much higher expressed in tumor tissues compared with adjacent normal tissues. Depletion of RCCD1 using siRNAs significantly inhibits the migration of lung cancer cells. Subsequent studies reveal that the loss of RCCD1 results in upregulation of acetylated α-tubulin levels and stabilizes cytoskeletal microtubules. Mechanistically, we observed that RCCD1 modulates the stability of microtubules through interacting with JMJD5. Furthermore, RCCD1 depletion significantly attenuates the TGF-β-induced EMT process, as assessed by altered expression of epithelial and mesenchymal markers (Occludin, Vimentin and Snail), and inhibits TGF-β-induced cell migration. Collectively, these findings support RCCD1 as a novel regulator of TGF-β-induced EMT in NSCLC.

The tubulin deacetylase sirtuin-2 regulates neuronal differentiation through the ERK/CREB signaling pathway

Modification of microtubule (MT) dynamics is important for diverse aspects of cellular function including differentiation, cargo trafficking, migration, and adhesion. MTs also play a crucial role in the progression of neuronal development. The MT deacetylase Sirtuin 2 (Sirt2) and histone deacetylase 6 (HDAC6) regulate MT dynamics by deacetylating alpha-tubulin (α-tubulin). In this study, we investigated the role of MT deacetylation in the progression of neuronal differentiation. For this, we examined acetylated α-tubulin levels during the differentiation of stem cells into neurons. Acetylated α-tubulin levels were significantly altered during differentiation, and these changes were abolished following treatment with 10 μM AGK2 (Sirt2 inhibitor) or 3 μM tubastatin A (HDAC6 inhibitor). However, neural-specific protein expression (Nestin, NF-M, and MAP-2) was reduced in AGK2-treated hBM-MSCs (AGK-MSCs), but not in tubastatin A-treated hBM-MSCs (Tubastatin A-MSCs). Inhibition of Sirt2 led to a decrease in ERK phosphorylation (p-ERK) level, but HDAC6 inhibition had no such effect. Similar results were obtained for CREB phosphorylation (p-CREB). The results suggest that Sirt2 plays a crucial role in neuronal differentiation via the ERK-CREB signaling pathway.

Rescue of CAMDI deletion-induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor.

The DISC1-interacting protein CAMDI has been suggested to promote radial migration through centrosome regulation. However, its physiological relevance is unclear. Here, we report the generation and characterization of CAMDI-deficient mice. CAMDI-deficient mice exhibit delayed radial migration with aberrant neural circuit formation and psychiatric behaviors including hyperactivity, repetitive behavior, and social abnormality typically observed in autism spectrum disorder patients. Analyses of direct targets of CAMDI identify HDAC6 whose α-tubulin deacetylase activity is inhibited by CAMDI at the centrosome. CAMDI deficiency increases HDAC6 activity, leading to unstable centrosomes with reduced γ-tubulin and acetylated α-tubulin levels. Most importantly, psychiatric behaviors as well as delayed migration are significantly rescued by treatment with Tubastatin A, a specific inhibitor of HDAC6. Our findings indicate that HDAC6 hyperactivation by CAMDI deletion causes psychiatric behaviors, at least in part, through delayed radial migration due to impaired centrosomes.

α-Tubulin acetylation elevated in metastatic and basal-like breast cancer cells promotes microtentacle formation, adhesion, and invasive migration.

Metastatic cases of breast cancer pose the primary challenge in clinical management of this disease, demanding the identification of effective therapeutic strategies that remain wanting. In this study, we report that elevated levels of α-tubulin acetylation are a sufficient cause of metastatic potential in breast cancer. In suspended cell culture conditions, metastatic breast cancer cells exhibited high α-tubulin acetylation levels that extended along microtentacle (McTN) protrusions. Mutation of the acetylation site on α-tubulin and enzymatic modulation of this posttranslational modification exerted a significant impact on McTN frequency and the reattachment of suspended tumor cells. Reducing α-tubulin acetylation significantly inhibited migration but did not affect proliferation. In an analysis of more than 140 matched primary and metastatic tumors from patients, we found that acetylation was maintained and in many cases increased in lymph node metastases compared with primary tumors. Proteomic analysis of an independent cohort of more than 390 patient specimens further documented the relationship between increased α-tubulin acetylation and the aggressive behaviors of basal-like breast cancers, with a trend toward increased risk of disease progression and death in patients with high-intensity α-tubulin acetylation in primary tumors. Taken together, our results identify a tight correlation between acetylated α-tubulin levels and aggressive metastatic behavior in breast cancer, with potential implications for the definition of a simple prognostic biomarker in patients with breast cancer.

A patient-derived stem cell model of hereditary spastic paraplegia with SPAST mutations

SUMMARYHereditary spastic paraplegia (HSP) leads to progressive gait disturbances with lower limb muscle weakness and spasticity. Mutations in SPAST are a major cause of adult-onset, autosomal-dominant HSP. Spastin, the protein encoded by SPAST, is a microtubule-severing protein that is enriched in the distal axon of corticospinal motor neurons, which degenerate in HSP patients. Animal and cell models have identified functions of spastin and mutated spastin but these models lack the gene dosage, mutation variability and genetic background that characterize patients with the disease. In this study, this genetic variability is encompassed by comparing neural progenitor cells derived from biopsies of the olfactory mucosa from healthy controls with similar cells from HSP patients with SPAST mutations, in order to identify cell functions altered in HSP. Patient-derived cells were similar to control-derived cells in proliferation and multiple metabolic functions but had major dysregulation of gene expression, with 57% of all mRNA transcripts affected, including many associated with microtubule dynamics. Compared to control cells, patient-derived cells had 50% spastin, 50% acetylated α-tubulin and 150% stathmin, a microtubule-destabilizing enzyme. Patient-derived cells were smaller than control cells. They had altered intracellular distributions of peroxisomes and mitochondria and they had slower moving peroxisomes. These results suggest that patient-derived cells might compensate for reduced spastin, but their increased stathmin expression reduced stabilized microtubules and altered organelle trafficking. Sub-nanomolar concentrations of the microtubule-binding drugs, paclitaxel and vinblastine, increased acetylated α-tubulin levels in patient cells to control levels, indicating the utility of this cell model for screening other candidate compounds for drug therapies.

The interrelationship between selective tau phosphorylation and microtubule association

The purpose of this study was to examine the modulation of tau phosphorylation mediated by protein kinase A, a kinase with low intrinsic activity, and by the constitutively active glycogen synthase kinase, as well as to examine the subsequent effects on tau-microtubule association in differentiated human SH-SY5Y neuroblastoma cells. Activation of protein kinase A with forskolin and rolipram significantly increased tau phosphorylation at Ser262/356 only in the presence of okadaic acid, indicating that phosphates at these sites are normally turned over rapidly. In contrast, glycogen synthase kinase appears to maintain tau phosphorylation at Thr181 and Ser396/404 since inhibition of glycogen synthase kinase with lithium reduced phosphorylation at these sites. Lithium treatment also significantly decreased tau and tyrosinated α-tubulin levels. Perturbation of microtubules with nocodazole or taxol induced tau dephosphorylation at Tau-1 sites, Thr181 and Ser396/404, indicating that both constitutive kinase activity and microtubule state modulate tau phosphorylation at these sites. Nocodazole- or taxol-induced tau dephosphorylation was blocked by the protein phosphatase 2A/1 inhibitor okadaic acid, but not by the protein phosphatase 2B inhibitor cyclosporin A. In addition, osmotic stress, such as treatment with 20 mM NaCl, selectively increased tau phosphorylation at the Tau-1 epitope. To investigate the effect of phosphorylation on tau association with microtubules and microtubule stability in situ, a Triton X-100 extraction assay was utilized to separate the detergent-soluble cytosolic components from the detergent-insoluble cytoskeletal components. In control cells or cells treated with lithium very little tau was detected in the cytosolic fraction. Activation of protein kinase A in the presence of okadaic acid elevated tau levels in the detergent-soluble fraction, which contained all the tau phosphorylated at Ser262/356, and also decreased microtubule stability, as indicated by decreased acetylated α-tubulin levels. In conclusion, the phosphorylation state of tau in differentiated SH-SY5Y cells is regulated by glycogen synthase kinase, microtubule dynamics and osmotic stress at overlapping sites which apparently have little influence on tau-microtubule association. In contrast, phosphorylation of tau at Ser262/356 within the microtubule-binding, which was mediated in part by protein kinase A, prevented the association of tau with microtubules in situ.

Nickel (Ni2+) Enhancement of α-Tubulin Acetylation in Cultured 3T3 Cells

Stable microtubules (MTs) are well known to contain acetylated α-tubulin. Ni2+-induced MT bundling may be accompanied with such tubulin post-translational modification. To explore this possibility as a mechanism of Ni2+-induced cytoskeletal injury, we have examined acetylated α-tubulin levels in cultured 3T3 cells by both immunoprecipitation assays and fluorescent staining of MTs using a monoclonal antibody (clone 6-11B-1) specific for acetylated α-tubulin. Cell extracts prepared from [35S]methionine labeled cultures in the presence or absence of Ni2+were immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. A predominant protein band (molecular mass 55 kDa), representing acetylated α-tubulin, appeared in Ni2+-treated cells in a dose and time-dependent manner whereas the corresponding labeled protein band was only barely detectable in control cells. Consistent with the immunoblot findings, MTs in control 3T3 cells in the absence of Ni2+were not labeled by the 6-11B-1 antibody except for some short, discontinuous segments localized in the cell center or the perinuclear MT organizing center area. In contrast, treatment of cells with NiCl2(2 mMfor 20 hr) resulted in, as expected, the formation of MT bundles that were intensely stained by the 6-11B-1 monoclonal antibody specific for acetylated α-tubulin. Furthermore, MTs containing acetylated α-tubulin in Ni2+-treated cells were resistant to disassembly induced by nocodazole, and at least partially resistant to cold temperature (0°C), which also depolymerizes MTs. Since acetylated α-tubulin serves as a marker for the presence of stable MTs, the marked enhancement of α-tubulin acetylation in Ni2+-treated cells indicates that stabilization of MTs may be an important mechanism by which Ni2+induces cell injury since stabilized MTs in turn should favor MT bundling, an unusual form of cytoskeletal perturbation in response to Ni2+exposure.