Brain Tubulin Research Articles

Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: Implications for chemotherapy-induced peripheral neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is a serious, painful and dose-limiting side effect of cancer drugs that target microtubules. The mechanisms underlying the neuronal damage are unknown, but may include disruption of fast axonal transport, an essential microtubule-based process that moves cellular components over long distances between neuronal cell bodies and nerve terminals. This idea is supported by the “dying back” pattern of degeneration observed in CIPN, and by the selective vulnerability of sensory neurons bearing the longest axonal projections. In this study, we test the hypothesis that microtubule-targeting drugs disrupt fast axonal transport using vesicle motility assays in isolated squid axoplasm and a cell-free microtubule gliding assay with defined components. We compare four clinically-used drugs, eribulin, vincristine, paclitaxel and ixabepilone. Of these, eribulin is associated with a relatively low incidence of severe neuropathy, while vincristine has a relatively high incidence. In vesicle motility assays, we found that all four drugs inhibited anterograde (conventional kinesin-dependent) fast axonal transport, with the potency being vincristine=ixabepilone>paclitaxel=eribulin. Interestingly, eribulin and paclitaxel did not inhibit retrograde (cytoplasmic dynein-dependent) fast axonal transport, in contrast to vincristine and ixabepilone. Similarly, vincristine and ixabepilone both exerted significant inhibitory effects in an in vitro microtubule gliding assay consisting of recombinant kinesin (kinesin-1) and microtubules composed of purified bovine brain tubulin, whereas paclitaxel and eribulin had negligible effects. Our results suggest that (i) inhibition of microtubule-based fast axonal transport may be a significant contributor to neurotoxicity induced by microtubule-targeting drugs, and (ii) that individual microtubule-targeting drugs affect fast axonal transport through different mechanisms.

Displacement-Weighted Velocity Analysis of Gliding Assays Reveals that Chlamydomonas Axonemal Dynein Preferentially Moves Conspecific Microtubules

In vitro gliding assays, in which microtubules are observed to glide over surfaces coated with motor proteins, are important tools for studying the biophysics of motility. Gliding assays with axonemal dyneins have the unusual feature that the microtubules exhibit large variations in gliding speed despite measures taken to eliminate unsteadiness. Because axonemal dynein gliding assays are usually done using heterologous proteins, i.e., dynein and tubulin from different organisms, we asked whether the source of tubulin could underlie the unsteadiness. By comparing gliding assays with microtubules polymerized from Chlamydomonas axonemal tubulin with those from porcine brain tubulin, we found that the unsteadiness is present despite matching the source of tubulin to the source of dynein. We developed a novel, to our knowledge, displacement-weighted velocity analysis to quantify both the velocity and the unsteadiness of gliding assays systematically and without introducing bias toward low motility. We found that the quantified unsteadiness is independent of tubulin source. In addition, we found that the short Chlamydomonas microtubules translocate significantly faster than their porcine counterparts. By modeling the effect of length on velocity, we propose that the observed effect may be due to a higher rate of binding of Chlamydomonas axonemal dynein to Chlamydomonas microtubules than to porcine microtubules.

Abstract 5682: Design and preclinical evaluation of single agents with combination chemotherapy potential.

Abstract Antiangiogenic agents target tumor mechanisms involved in blood vessel formation vital for tumor growth and metastases. However, these agents are mainly cytostatic and usually not tumoricidal. Several combinations of antiangiogenic and cytotoxic agents have shown significant promise in the clinic, and several clinical trials are in progress. Single agents with both antiangiogenic activities and cytotoxicity would afford agents that would circumvent pharmacokinetic problems of multiple agents, avoid drug-drug interactions, could be used at lower doses to alleviate toxicity, be devoid of overlapping toxicities, and could delay or prevent tumor cell resistance. We designed, synthesized and evaluated a novel compound AG119 that inhibits both vascular endothelial growth factor receptor-2 (VEGFR-2) affording antiangiogenic effects and tubulin for cytotoxic effects in a single agent. Structural modifications of AG119 afforded AG321 that inhibited platelet derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) in addition to VEGFR-2 and tubulin. The novel compounds AG119 and AG321 were potent inhibitors (activity comparable to that of combretastatin A-4) of bovine brain tubulin assembly and of [3H]colchicine binding to tubulin, indicating that these compounds bind at the colchicine site on tubulin. Both compounds have potency comparable to those of sunitinib and semaxinib in tumor cell lines overexpressing VEGFR-2. AG321 has potency comparable to that of erlotinib in tumor cell lines overexpressing EGFR and potency comparable to those of erlotinib, DMBI and PD153035 in cell lines overexpressing PDGFR-β. Both AG119 and AG321 also inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. AG321 is a unique compound with four separate mechanisms of action in a single agent including inhibition of tubulin. This is unprecedented and to our knowledge, is the first of its kind. In mice, AG119 reduced tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 model and U251 glioma model] and in a 4T1 triple negative breast orthotopic allograft model. In these in vivo models, the activity of AG119 was superior to those of temozolomide (U251), docetaxel and sunitinib (MDA-MB-435 and 4T1) without overt toxicity to the animals. Citation Format: Aleem Gangjee, Roheeth K. Pavana, Michael A. Ihnat, Jessica E. Thorpe, Bryan C. Disch, Anja Bastian, Lora C. Bailey-Downs, Ernest Hamel, Rouli Bai. Design and preclinical evaluation of single agents with combination chemotherapy potential. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5682. doi:10.1158/1538-7445.AM2013-5682

Abstract 4496: Differential inhibition by microtubule stabilizing agents of 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulins from different eukaryotic sources.

Abstract Distinct α- and β-tubulin isotypes are present in different quantities in diverse eukaryotic cells. The tritium-labeled photoaffinity Taxol analog, 2-(m-azidobenzoyl)Taxol, was used to photolabel tubulins prepared from HeLa cells, bovine brain and chicken erythrocytes. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of tubulin by >90%, demonstrating the specificity of this photolabeling. Tubulin isotype content varies from one source to another, particularly for chicken erythrocytes that contain only one α-tubulin and one β-tubulin (α1 and βVI). 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulin was carried out in the presence of six different microtubule stabilizing agents, including Taxol, epothilone B, discodermolide, ixabepilone, laulimalide and peloruside A. The inhibitory effects elicited by Taxol, epothilone B, discodermolide and ixabepilone, at a 5-fold molar excess, on photolabeling are different for tubulins prepared from different sources. Taxol had a minimal inhibitory effect on HeLa and bovine brain tubulin, but a strong inhibitory effect on chicken erythrocyte tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. This is most likely due to the presence of distinct tubulin isotypes in different sources. However, laulimalide and peloruside A that are known to bind to a different site than Taxol in β-tubulin, stimulated photolabeling of tubulins from all three sources. Stathmin (S) plays an important role in the regulation of microtubule dynamics through sequestering tubulin (T) into a T2S complex. When stathmin was incubated, in the presence of 1 mM CaCl2, with chicken erythrocyte tubulin at a tubulin:stathmin ratio of 2:1, followed by incubation with tritium-labeled 2-(m-azidobenzoyl)Taxol in a buffer containing EGTA, photolabeling of β-tubulin was markedly reduced, suggesting that stathmin inhibited binding of Taxol to tubulin. Radiolabeled photoaffinity drug analogs are useful tools for determining drug binding affinities for the different tubulin isotypes. We are currently analyzing binding affinities of 2-(m-azidobenzoyl)Taxol to different bovine brain tubulin isotypes resolved by isoelectrofocusing. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Differential inhibition by microtubule stabilizing agents of 2-(m-azidobenzoyl)Taxol photoaffinity labeling of tubulins from different eukaryotic sources. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4496. doi:10.1158/1538-7445.AM2013-4496

In vitro study on the alterations of brain tubulin structure and assembly affected by magnetite nanoparticles

In recent decades, considerable efforts have been made to understand the mechanism of memory, cognition, and relevant neurodegenerative diseases in the human brain. Several studies have shown the importance of microtubule proteins in the memory mechanism and memory dysfunction. Microtubules possess dynamicity, which is essential for functions of neuronal networks. Microtubule-associated proteins, i.e., tau, play vital roles in microtubule stability. On the other hand, the ferromagnetic mineral magnetite (Fe(3)O(4)) has been detected in the normal human brain, and elevated levels of magnetite are also observed in the brains of Alzheimer's disease patients. Therefore, we propose that a relationship between microtubule organization in axons and brain magnetite nanoparticles is possible. In this study we found alterations of microtubule polymerization in the presence of increasing concentrations of magnetite through transmission electron microscopy images and a turbidimetry method. Structural changes of microtubule and tau protein, as an essential microtubule-associated protein for tubulin assembly, were detected via circular dichroism spectroscopy, intrinsic fluorescence, and 8-anilino-1-naphthalenesulfonic acid fluorometry. We predicted three possible binding sites on tau protein and one possible binding site on tubulin dimer for magnetite nanoparticles. Magnetite also causes the morphology of PC12 cells to change abnormally and cell viability to decrease. Finally, we suggest that magnetite changes microtubule dynamics and polymerization through two paths: (1) changing the secondary and tertiary structure of tubulin and (2) binding to either tubulin dimer or tau protein and preventing tau-tubulin interaction.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF SULFUR COMPOUNDS SHOWING STRUCTURAL ANALOGY WITH COMBRETASTATIN A-4.

We extended our previous exploration of sulfur bridges as bioisosteric replacements for atoms forming the bridge between the aromatic rings of combretastatin A-4. Employing coupling reactions between 5-iodo-1,2,3-trimethoxybenzene and substituted thiols, followed by oxidation to sulfones with m-CPBA, different locations for attaching the sulfur atom to ring A through the synthesis of nine compounds were examined. Antitubulin activity was performed with electrophoretically homogenous bovine brain tubulin, and activity occurred with the 1,2,3-trimethoxy-4-[(4-methoxyphenyl)thio]benzene (12), while the other compounds were inactive. The compounds were also tested for leishmanicidal activity using promastigote forms of Leishmania braziliensis (MHOM/BR175/M2904), and the greatest activity was observed with 1,2,3-trimethoxy-4-(phenylthio)benzene (10) and 1,2,3-trimethoxy-4-[(4-methoxyphenyl) sulfinyl]benzene (15).

Tubulin isoforms identified in the brain by MALDI in-source decay

Identification of biomarkers is a major issue for enhancement of chemotherapies. The molecular characterization of tissues necessitates the identification of thousands of biomolecules each participating in physiopathological processes. MALDI in-source decay (ISD) fragmentation has already been proven to be effective for protein characterization. However, the difficulty to identify proteins from complex mixtures such as tissue sections can limit the applications of this technique. In this study, we evidenced that tubulin has an unusual fragmentation pathway in the MALDI source. This striking property allowed the detecting of several mouse brain tubulin isotypes simultaneously by simply using laser fragmentation. Tubulin isoforms are consistent markers of a bad prognosis of solid tumors and could be the target of targeted chemotherapies. Such a direct molecular printout of tubulin in tissues is a milestone that should be useful either at preclinical or clinical stage.

Streptococcus and rheumatic fever

To give an overview of the current hypotheses of the pathogenesis of rheumatic fever and group A streptococcal autoimmune sequelae of the heart valve and brain. Human monoclonal antibodies (mAbs) derived from rheumatic heart disease have provided evidence for crossreactive autoantibodies that target the dominant group A streptococcal epitope of the group A carbohydrate, N-acetyl-beta-D-glucosamine (GlcNAc), and heart valve endothelium, laminin and laminar basement membrane. T cells in peripheral blood and in rheumatic heart valves revealed the presence of T cells crossreactive with streptococcal M protein and cardiac myosin. For initiation of disease, evidence suggests a two-hit hypothesis for antibody attack on the valve endothelium with subsequent extravasation of T cells through activated endothelium into the valve to form granulomatous lesions and Aschoff bodies. Autoantibodies against the group A streptococcal carbohydrate epitope GlcNAc and cardiac myosin and its peptides appear during progression of rheumatic heart disease. However, autoantibodies against collagen that are not crossreactive may form because of the release of collagen from damaged valve or to responses to collagen bound in vitro by certain serotypes of streptococci. In Sydenham chorea, human mAbs derived from disease target the group A carbohydrate epitope GlcNAc and gangliosides and dopamine receptors found on the surface of neuronal cells in the brain. Human mAbs and autoantibodies in Sydenham chorea were found to signal neuronal cells and activate calcium calmodulin-dependent protein kinase II (CaMKII) in neuronal cells and recognize the intracellular protein biomarker tubulin. To summarize, pathogenic mechanisms of crossreactive autoantibodies which target the valve in rheumatic heart disease and the neuronal cell in Sydenham chorea share a common streptococcal epitope GlcNAc and target intracellular biomarkers of disease including cardiac myosin in the myocardium and tubulin, a protein abundant in the brain. However, intracellular antigens are not believed to be the basis for disease. The theme of molecular mimicry in streptococcal autoimmune sequelae is the recognition of targeted intracellular biomarker antigens such as cardiac myosin and brain tubulin, while targeting extracellular membrane antigens such as laminin on the valve surface endothelium or lysoganglioside and dopamine receptors in the brain. Antibody binding to these cell surface antigens may lead to valve damage in rheumatic heart disease or neuropsychiatric behaviors and involuntary movements in Sydenham chorea.

Abstract 2789: Interaction of (+)-discodermolide-Taxol hybrids with microtubules

Abstract Taxol and discodermolide are both microtubule stabilizing agents that bind to the β-tubulin subunit of microtubules. Based on our results that the hydrophobic binding pocket of β-tubulin is occupied by the Taxol side chain but not by (+)-discodermolide, and that the two drugs act synergistically in cancer cell lines, a small library of (+)-discodermolide-Taxol hybrids was synthesized (J. Med. Chem. 54:6319, 2011). Cytotoxicity assays demonstrated a 2-9-fold increase in antiproliferative activity by the hybrids compared to discodermolide in the human cancer cell lines, A549 and MCF-7. Hybrids containing a tether of 3 carbons that connect discodermolide with the Taxol side chain, exhibited the best activity. Tubulin assembly assays were performed with the two most potent hybrid molecules. Like discodermolide, these two hybrids increased tubulin assembly rapidly without a lag period. Tubulin polymerization studies using purified bovine brain tubulin demonstrated that they also had the greatest effect on the formation of polymerized microtubules. These results also were observed in intact A549 cells and in 100,000 x g supernatants prepared from these cells. We have previously shown that [3H]2-(m-azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217-231 of β-tubulin. A 5-fold molar excess of unlabeled compound inhibited the photolabeling of purified bovine brain tubulin by 94%, demonstrating the specificity of this photolabeling. Microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, discodermolide and ixabepilone, each at a 5-fold molar excess, inhibited the photolabeling by 24%, 92%, 100% and 41%, respectively, indicating that discodermolide is the most potent inhibitor of the photolabeling. In contrast, two other MSAs, laulimalide and peloruside that are known to bind to a different site in β-tubulin, exhibited stimulatory effects on the photolabeling. Both drugs, at a 5-fold molar excess, increased the labeling by 30-40%. [3H]2-(m-azidobenzoyl)Taxol (0.5 - 20 µM) was used to study the kinetics of the inhibitory effects of the hybrid molecules on photoaffinity labeling of tubulin. Discodermolide-Taxol hybrids inhibited the photolabeling of bovine brain tubulin in a dose dependent manner. The concentrations that inhibited by 50% were lowest for the two most potent hybrid molecules. Therefore, the tubulin polymerization activity and the binding affinity of the hybrids to β-tubulin correlated with their antiproliferative activity. Other biological properties of the discodermolide-Taxol hybrids including senescence and antitumor activity are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2789. doi:1538-7445.AM2012-2789

Abstract 3897: Design and preclinical evaluation of single agents with antitubulin and antiangiogenic activity as antitumor agents

Abstract Agents that interfere with microtubules are important antitumor agents. Tumor angiogenic mechanisms that are vital for tumor growth and metastasis are targeted by antiangiogenic agents. Antiangiogenic agents are usually not tumoricidal but are mainly cytostatic. Combination chemotherapy with antiangiogenic and cytotoxic agents has shown significant promise, and several studies with such combinations are in progress in the clinic. Single agents with both antiangiogenic activities and cytotoxicity could afford single agents that would circumvent the pharmacokinetic problems of multiple agents, avoid drug-drug interactions, be used at lower doses to alleviate toxicity, be devoid of overlapping toxicities, and delay or prevent tumor cell resistance. We have designed, synthesized and evaluated two novel compounds, AG119 and AG425, that inhibit both vascular endothelial growth factor receptor-2 (VEGFR-2) for antiangiogenic effects and tubulin for cytotoxic effects in single agents. Both compounds have comparable potency as sunitinib and semaxinib against cell lines overexpressing VEGFR-2 and were equipotent with semaxinib on VEGFR-2 mediated endothelial cell proliferation, indicating a functional effect of these agents on VEGFR-2 inhibition (IC50: semaxanib 68.2 ± 4.2 µM, AG119: 60.2 ± 2.4 µM, AG425: 52.3 ± 3.8 µM). The novel compounds are effective and potent inhibitors (comparable to combretastatin A-4) of bovine brain tubulin assembly (IC50: combretastatin A-4 1.2 ± 0.01 µM, AG119: 3.5 µM, AG425: 1.9 ± 0.1 µM). The compounds were potent inhibitors of [3H]colchicine binding to tubulin, indicating that these compounds, particularly AG425, bind at the colchicine site. These agents cause microtubule depolymerization, arrest cells in late G2/M phase and trigger apoptotic cell death. Moreover, AG119 and AG425 are potent anticancer compounds in aggressive basal like breast cancer (BLBC) and triple negative breast cancer (TNBC) cell lines. The novel compounds showed sensitivity in taxol resistant cells, indicating that they are unlikely to be Pgp or MRP substrates [unlike taxol], and these compounds possess relative selectivity of kill on tumor versus normal cell types. In vivo these agents reduce tumor size and vascularity in two flank xenograft models [the BLBC MDA-MB-435 model and U251 glioma model] in immunodeficient mice, superior to docetaxel and sunitinib, without overt toxicity to the animals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3897. doi:1538-7445.AM2012-3897

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis.

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of β-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z= 1287.77), a β-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.

Microtubules as a Critical Target for Arsenic Toxicity in Lung Cells in Vitro and in Vivo

To understand mechanisms for arsenic toxicity in the lung, we examined effects of sodium m-arsenite (As3+) on microtubule (MT) assembly in vitro (0–40 µM), in cultured rat lung fibroblasts (RFL6, 0–20 µM for 24 h) and in the rat animal model (intratracheal instillation of 2.02 mg As/kg body weight, once a week for 5 weeks). As3+ induced a dose-dependent disassembly of cellular MTs and enhancement of the free tubulin pool, initiating an autoregulation of tubulin synthesis manifest as inhibition of steady-state mRNA levels of βI-tubulin in dosed lung cells and tissues. Spindle MT injuries by As3+ were concomitant with chromosomal disorientations. As3+ reduced the binding to tubulin of [3H]N-ethylmaleimide (NEM), an -SH group reagent, resulting in inhibition of MT polymerization in vitro with bovine brain tubulins which was abolished by addition of dithiothreitol (DTT) suggesting As3+ action upon tubulin through -SH groups. In response to As3+, cells elevated cellular thiols such as metallothionein. Taxol, a tubulin polymerization agent, antagonized both As3+ and NEM induced MT depolymerization. MT–associated proteins (MAPs) essential for the MT stability were markedly suppressed in As3+-treated cells. Thus, tubulin sulfhydryls and MAPs are major molecular targets for As3+ damage to the lung triggering MT disassembly cascades.

Microtubule Dynamics of S. Pombe Tubulin

We have developed a fission yeast system for expression and purification of single isoform tubulin. Replacing the non-essential S. pombe α2 tubulin gene with a second copy of the α1 encoding gene creates a strain expressing single α1 and β tubulin isoforms. We obtain yields of about 10 mg of highly purified (>99%) single isoform tubulin from an 80 l culture. Mass spectroscopy of the purified tubulin detects no post-translational modifications. Dynamic microtubules were nucleated from axonemes, recorded by video enhanced DIC microscopy and microtubule lengths measured. Microtubule growth rates increase linearly with tubulin concentration at both the fast and slow growing microtubule ends. Rate constants were determined using a simple bimolecular model. α1β tubulin has fast end kon 5.5 uM−1 s−1 and koff 6.7 s−1, and slow end kon 3.1 uM−1 s−1 and koff 2.8 s−1. Mixed isoform α1α2β has fast end kon 7.6 uM s−1 and koff 15.1 s−1 and slow end kon 2.6 uM−1 s−1 and koff 5.0 s−1. The Kd for both microtubule ends was ∼2uM for α1α2β and ∼1.1 uM for α1β suggesting similar binding affinities for tubulin heterodimers at fast and slow ends, but different kinetics. Following catastrophe the rate of rapid shrinkage was independent of the free tubulin concentration and about 2x faster at the fast compared to the slow ends: 218 ± 16 s−1 compared to 124 ± 23 s−1 for α1α2β and 263 ± 26 s−1 compared to 111 ± 29 s−1 for α1β. We conclude that S. pombe microtubule dynamics are qualitatively similar to those of brain tubulin microtubules but the kinetic rates are different, consistent with S. pombe tubulin assembling at lower concentrations than brain tubulin. We are now comparing S. pombe microtubule growth with that of brain tubulin at higher resolution.

Alp7 Potentiates Microtubule Tip Tracking by the Processive Plus End Polymerase Alp14

TOG-family polymerases track microtubule plus ends in vivo and modulate microtubule dynamics. Several different model mechanisms for the molecular mechanism of action of these proteins have been proposed (Kerssemakers et al. (2006) Nature 442:709; Al-Bassam et al (2006) JCB 172:1009; Brouhard et al. (2008) Cell 132:79). To test candidate models in more detail, we have expressed and purified Alp14 and Alp7 using a baculovirus system and reconstituted their activities in vitro using dynamically unstable microtubules built from purified single isoform (a1b) S.pombe tubulin. Alp14 deletion mutants show an in vivo defect in microtubule assembly (Sato et al. (2004) MCB 15:1609), consistent with an effect on microtubule dynamics. We find that in vitro, Alp14 accelerates the shrinkage of GMPCPP microtubules 2x and the growth of GTP-microtubules up to 10x. Changes in growth rate within a growth phase correspond to changes in the amount of Alp14 at the tip. The acceleration of growth by Alp14 causes a decrease in catastrophe frequency consistent with extension of the GTP-cap. Remarkably, although Alp14 binds to mammalian brain tubulin, it does not accelerate the growth of mammalian brain microtubules. Instead, Alp14 is competitively inhibited by mammalian brain tubulin. Tip tracking by Alp14 is tightly linked to the catalysis of microtubule growth: Alp14 loses its tip tracking ability upon the addition of 10% mammalian brain tubulin. The addition of the TACC-protein Alp7 restores the tip-tracking ability of Alp14, but not its ability to enhance the microtubule growth rate. This result is consistent with reports that Alp7 is a localization factor of Alp14 in vivo (Sato et al. (2004) MCB 15:1609). On dynamic S.pombe microtubules, Alp7 enhances the processivity of Alp14, causing sustained fast growth and correspondingly reduced catastrophe.

In Vitro Gliding Assays Indicate that Chlamydomonas Dynein Moves Microtubules Polymerized from Chlamydomonas Axonemal Tubulin Faster than those Polymerized from Porcine Brain Tubulin

The axonemal dyneins drive the motility of cilia and flagella. Dynein activity must be coordinated spatially and temporally within the axoneme to generate the regular, repeating waveforms characteristic of cilial motion. Recent studies have suggested that the mechanochemical properties of dynein, in particular the dependance of velocity and dissociation constants on force, may be sufficient to achieve coordination. Because the processes involving these properties require interactions between the dyneins and the microtubules, it is likely that they are dependent on the specific dynein-tubulin interactions. We investigated this possibility using microtubule gliding assays with outer arm dynein from Chlamydomonas. We found that that the translocation speed of microtubules polymerized from Chlamydomonas tubulin is 1.6 times faster than the speed of porcine brain microtubules. Additionally, we found that the source of the tubulin also influenced how translocation speed depended on microtubule length. Both of these results suggest that dynein from Chlamydomonas prefers Chlamydomonas axonemal tubulin despite greater than 85% similarity in primary structure. Finally, we found that the instantaneous translocation speed of microtubules in the gliding assay is unsteady. Our analysis suggests that the source of this unsteadiness may arise from the same mechanochemical properties of dynein that have been predicted to be sufficient for coordination. Together these results suggest that the interactions between dynein and tubulin are important factors in axonemal dynein coordination.

Cellular studies reveal mechanistic differences between taccalonolide A and paclitaxel

Taccalonolide A is a microtubule stabilizer that has cellular effects almost identical to paclitaxel. However, biochemical studies show that, unlike paclitaxel, taccalonolide A does not enhance purified tubulin polymerization or bind tubulin/microtubules. Mechanistic studies aimed at understanding the nature of the differences between taccalonolide A and paclitaxel were conducted. Our results show that taccalonolide A causes bundling of interphase microtubules at concentrations that cause antiproliferative effects. In contrast, the concentration of paclitaxel that initiates microtubule bundling is 31-fold higher than its IC50. Taccalonolide A’s effects are further differentiated from paclitaxel in that it is unable to enhance the polymerization of tubulin in cellular extracts. This finding extends previous biochemical results with purified brain tubulin to demonstrate that taccalonolide A requires more than tubulin and a full complement of cytosolic proteins to cause microtubule stabilization. Reversibility studies were conducted and show that the cellular effects of taccalonolide A persist after drug washout. In contrast, other microtubule stabilizers, including paclitaxel and laulimalide, demonstrate a much higher degree of cellular reversibility in both short-term proliferation and long-term clonogenic assays. The propensity of taccalonolide A to alter interphase microtubules at antiproliferative concentrations as well as its high degree of cellular persistence may explain why taccalonolide A is more potent in vivo than would be expected from cellular studies. The close linkage between the microtubule bundling and antiproliferative effects of taccalonolide A is of interest given the recent hypothesis that the effects of microtubule targeting agents on interphase microtubules might play a prominent role in their clinical anticancer efficacy.

Abstract 664: Polymerization of human βIII-tubulin is distinct from βI-tubulin in a cell-free system

Abstract Tubulins are a family of proteins consisting of 7 α- and 6 β-tubulin isotypes. Many studies have demonstrated that tumors overexpressing βIII-tubulin have a poor response to chemotherapy. Suppression of βIII-tubulin sensitizes non-small cell lung cancer (NSCLC) cells to microtubule-interacting agents, such as Taxol. βIII-tubulin has also been shown to mediate the incidence and progression of lung cancer. Tubulin isotype content of βI-, βIII-, βIV- and βV-tubulin in the human lung carcinoma cell line A549 is 50%, 8%, 36.5%, and 5.5%, respectively. In our study, the ability of six microtubule stabilizing agents, Taxol, epothilone B (EpoB), discodermolide (Disco), Ixabepilone, laulimalide and peloruside A, to polymerize tubulin in a 100,000 × g supernatant prepared from A549 cells was examined. The level of different tubulin isotypes in the drug-polymerized tubulin was determined by Western blot analysis with tubulin isotype specific antibodies. In the absence of drug, the level of polymerized βI-, βIII-, βIV- and βV-tubulin was 55%, 9%, 25%, and 48%, respectively. Ten µM Taxol caused a smalll increase (<15%) in βI- and βV- tubulin polymerization, but it induced a 367% and 228% increase in βIII- and βIV-tubulin polymerization. Among the six drugs tested, Disco and EpoB exhibited the greatest ability to polymerize all isotypes. The differential βI- and βIII-tubulin polymerization effects also were observed when intact A549 cells were lysed in the presence of drugs, followed by centrifugation. In the absence of drug, the level of polymerized βI- and βIII-tubulin was 9% and 0.2%, respectively. In the presence of 10 µM Taxol, the level of polymerized βI- and βIII-tubulin was increased to 28% and 7% which corresponded to a 3- and 35-fold increase in βI- and βIII-tubulin polymerization, respectively. However, when purified bovine brain tubulin in the absence of all endogenous proteins was used, there was no differential effect of drugs on the polymerization of βI+II- and βIII-tubulin. The βI-, βII-, βIII-, and βIV-tubulin content in bovine brain is 3%, 58%, 25% and 14%, respectively. Disco and EpoB also have the strongest effects on the polymerization of all bovine brain tubulin isotypes. Our results indicate that polymerization of βIII-tubulin is distinct from that of βI-tubulin in a cell-free system and that endogenous cellular components may influence the polymerization of tubulin isotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2011-664

Hallmarks of Molecular Action of Microtubule Stabilizing Agents: EFFECTS OF EPOTHILONE B, IXABEPILONE, PELORUSIDE A, AND LAULIMALIDE ON MICROTUBULE CONFORMATION

Microtubule stabilizing agents (MSAs) comprise a class of drugs that bind to microtubule (MT) polymers and stabilize them against disassembly. Several of these agents are currently in clinical use as anticancer drugs, whereas others are in various stages of development. Nonetheless, there is insufficient knowledge about the molecular modes of their action. Recent studies from our laboratory utilizing hydrogen-deuterium exchange in combination with mass spectrometry (MS) provide new information on the conformational effects of Taxol and discodermolide on microtubules isolated from chicken erythrocytes (CET). We report here a comprehensive analysis of the effects of epothilone B, ixabepilone (IXEMPRA(TM)), laulimalide, and peloruside A on CET conformation. The results of our comparative hydrogen-deuterium exchange MS studies indicate that all MSAs have significant conformational effects on the C-terminal H12 helix of α-tubulin, which is a likely molecular mechanism for the previously observed modulations of MT interactions with microtubule-associated and motor proteins. More importantly, the major mode of MT stabilization by MSAs is the tightening of the longitudinal interactions between two adjacent αβ-tubulin heterodimers at the interdimer interface. In contrast to previous observations reported with bovine brain tubulin, the lateral interactions between the adjacent protofilaments in CET are particularly strongly stabilized by peloruside A and laulimalide, drugs that bind outside the taxane site. This not only highlights the significance of tubulin isotype composition in modulating drug effects on MT conformation and stability but also provides a potential explanation for the synergy observed when combinations of taxane and alternative site binding drugs are used.