The interaction of triethyl lead with tubulin and microtubules

The impact of triethyl lead chloride was studied on: (i) the in vitro assembly and disassembly of microtubules from porcine brain by turbidometry and electron microscopy, (ii) the microtubule system of living mammalian cells using immunofluorescence microscopy, (iii) cell motility and chemotaxis employing the methods of phagokinetic track formation and the Boyden chamber assay, respectively, and (iv) thiol groups of the protein tubulin by their titration in the presence and absence of the organic lead compound. Triethyl lead chloride inhibited microtubule assembly and depolymerized preformed microtubules in vitro and in living cells. Random motility of cells was not markedly inhibited by triethyl lead chloride, whereas chemotaxis (directed cellular movement) was strongly inhibited. Triethyl lead chloride was found to interact with 2 thiol groups of the tubulin dimer. The interaction of triethyl lead chloride with the tubulin/microtubule system in vivo likely causes aneuploidy and is at least partly responsible for the cytotoxicity of the drug.     

Phosphorylation controls the interaction of the connexin43 C-terminal domain with tubulin and microtubules

Connexins are structurally related transmembrane proteins that assemble to form gap junction channels involved in the mediation of intercellular communication. It has been shown that the intracellular tail of connexin43 (Cx43) interacts with tubulin and microtubules with putative impacts on its own intracellular trafficking, its activity in channel communication, and its interference with specific growth factor signal transduction cascades. We demonstrate here that the microtubule binding of Cx43 is mainly driven by a short region of 26 amino acid residues located within the intracellular tail of Cx43. The nuclear magnetic resonance structural analysis of a peptide (K26D) corresponding to this region shows that this peptide is unstructured when free in solution and adopts a helix conformation upon binding with tubulin. In addition, the resulting K26D-tubulin molecular complex defines a new structural organization that could be shared by other microtubule partners. Interestingly, the K26D-tubulin interaction is prevented by the phosphorylation of K26D at a src kinase specific site. Altogether, the results elucidate the mechanism of the interaction of Cx43 with the microtubule cytoskeleton and propose a pathway for understanding the microtubule-dependent regulation of Cx43 gap junctional communications and the involvement of Cx43 in TGF-β signal transduction.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Mechanical properties of brain tubulin and microtubules

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.     

Secondary structure analysis of tubulin and microtubules with Raman spectroscopy

Raman spectroscopy is used to study the secondary structure of tubulin in the assembled and the dissociated states from the analysis of the amide-I band. Essentially two states are recognized: the GTP- and the GDP-bound state, differing in alpha-helix and antiparallel beta-sheet content. Microtubules give a spectrum which is very similar to the GDP-bound state. MAPs and temperature have minor effects, while increasing the pH up to 8 causes a reduction in alpha-helix content and a increase in antiparallel beta-sheet. The binding of demecolcine also induces structural changes which are similar to the GDP-bound state.     

Interactions of enolase isoforms with tubulin and microtubules during myogenesis

Enolase is a glycolytic enzyme, expressed as cell-type specific isoforms in higher vertebrates. Herein we demonstrated for the first time that enolase isoforms interact with microtubules during muscle satellite cell differentiation. While in undifferentiated myoblasts the ubiquitous alphaalpha enolase isoform, expressed at high level, exhibited extensive co-localization with microtubules, the muscle-specific betabeta isoform, expressed at low level, did not. During differentiation, the level of beta subunit increased significantly; the alpha and beta enolase immunoreactivities were detected both in cytosol and along the microtubules. We identified tubulin from muscle extract as an interacting protein for immobilized betabeta enolase. ELISA and surface plasmon resonance measurements demonstrated the direct binding of enolase isoforms to tubulin with an apparent KD below the micromolar range, and indicated that the presence of 0.8 mM 2-phosphoglycerate abolished the interaction. Our data showed that, at various stages of myogenic differentiation, microtubules were decorated by different enolase isoforms, which was controlled by the abundance of both partners. We suggest that the binding of enolase to microtubules could contribute to the regulation of the dynamism of the cytoskeletal filaments known to occur during the transition from myoblast to myotubes.     

Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp

The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.     

The interaction of cystamine with bovine brain tubulin

Microtubule assembly in vitro is sensitive to a variety of non-physiological sulfhydryl-oxidizing agents, but the physiological significance of this phenomenon is unknown, since no physiological sulfhydryl-oxidizing agent has been shown to affect microtubule assembly in vitro. We have accordingly investigated the interaction of tubulin with cystamine. We have found that millimolar concentrations of cystamine inhibit microtubule assembly and induce an abnormal form of tubulin polymerization. Cystamine-induced polymerization does not occur at cold temperature. Formation of the polymer requires reaction of cystamine with two sulfhydryls which become available at 37 degrees C. In addition, cystamine reacts with about three sulfhydryls at 0 degrees C without inducing polymerization. This latter set of sulfhydryls appear to include one or both of the previously defined beta s sulfhydryls whose reaction with N, N'-ethylene-bis(iodoacetamide) is markedly inhibited by GTP, maytansine and vinblastine [Roach, M. C. & Luduena, R. F. (1984) J. Biol. Chem. 259, 12063-12071]. Cystamine's specific manner of interacting with tubulin suggests that it may mimic an endogenous sulfhydryl-directed regulator of microtubule assembly.     

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.     

The association of carbohydrate with tubulin and in vitro assembled microtubules from bovine brain

Summary Phosphocellulose-purified tubulin (PC tubulin) was analyzed for neutral and amino sugar content, which was found to be 8.3±0.11 and 0.8±0.02 mol/mol dimer, respectively. A histochemical-electron-microscopic investigation was undertaken to attempt to localize carbohydrate associated with polymerized microtubules (MT). Outer diameters of MT assembled in vitro from bovine brain MT protein (tubulin and microtubule associated proteins) were found to increase upon treatment with ruthenium red, Alcian blue, and lanthanum hydroxide, which have been reported to possess specificity for complex carbohydrates. Concanavalin A-reactive sites were detected on the surface and in the lumen of MT assembled from MT protein and from PC tubulin.This work was supported by grants from Statens Naturvetenskapliga. Forsknigsråd (No. B0294-020), Stiftelsen Lars Hiertas Minne, Adlerbertska Forskningsfonden, Hierta-Retzius' Stipendiefond, Anna Ahrenbergs Fond, and Wilhelm och Martina Lundgrens Vetenskapsfond. Appreciation is extended to Inger Holmqvist for her excellent technical assistance     

The interaction of myelin basic protein with tubulin and the inhibition of tubulin carboxypeptidase activity

Tubulin carboxypeptidase was found to be inhibited by myelin basic protein in a concentration dependent manner. The inhibition was produced by the interaction between myelin basic protein with the substrate. As a consequence of this interaction, turbid insoluble aggregates were formed at either 5 degrees or 37 degrees C. The turbidity increased by increasing the myelin basic protein concentration and it reached a plateau at a molar ratio of myelin basic protein to tubulin dimer of about 6. At plateau, the molar ration in the insoluble aggregates was about 6. When tubulin was in excess, the formation of the insoluble aggregates was diminished. However, if the excess of tubulin was added after the formation of the aggregates, the turbidity was not significantly affected. Turbidity was diminished by increasing the ionic strength.     

HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo

Microtubules are cylindrical cytoskeletal structures found in almost all eukaryotic cell types which are involved in a great variety of cellular processes. Reversible acetylation on the epsilon-amino group of alpha-tubulin Lys40 marks stabilized microtubule structures and may contribute to regulating microtubule dynamics. Yet, the enzymes catalysing this acetylation/deacetylation have remained unidentified until recently. Here we report that beta-tubulin interacts with histone deacetylase-6 (HDAC-6) in a yeast two-hybrid assay and in vitro. We find that HDAC-6 is a micro tubule-associated protein capable of deacetylating alpha-tubulin in vivo and in vitro. HDAC-6's microtubule binding and deacetylation functions both depend on the hdac domains. Overexpression of HDAC-6 in mammalian cells leads to tubulin hypoacetylation. In contrast, inhibition of HDAC-6 function by two independent mechanisms--pharmacological (HDAC inhibitors) or genetic (targeted inactivation of HDAC-6 in embryonic stem cells)--leads to hyperacetylation of tubulin and microtubules. Taken together, our data provide evidence that HDAC-6 might act as a dual deacetylase for tubulin and histones, and suggest the possibility that acetylated non-histone proteins might represent novel targets for pharmacological therapy by HDAC inhibitors.     

The interaction of actin filaments with microtubules and microtubule-associated proteins

Purified actin and microtubule proteins polymerized together form a gel, while mixtures of actin with tubulin polymers lacking microtubule-associated proteins (MAPs) have low viscosities close to the sum of the viscosities of the constituents. Mixtures of actin and MAPs also have high viscosities. Our interpretation of these observations was that there is interaction of actin filaments and microtubules which is mediated by MAPs (Griffith, L. M., and Pollard, T. D. (1978) J. Cell Biol. 78, 958-965). We report here further evidence for this interaction. 1) Actin filaments and microtubules can form gels at physiological ionic strength providing the anion is glutamate rather than chloride. Both glutamate and chloride inhibit actin-MAPs interaction, but this is compensated for in glutamate where the microtubules are longer than in chloride. 2) The low shear viscosity of mixtures of isolated MAPs and actin filaments is enhanced by acidic pH and inhibited by high ionic strength. 3) MAPs can be fractionated to yield four different fractions with actin cross-linking activity: a subset of high molecular weight MAPs, purified "MAP-2" and two different fractions of tau polypeptides. 4) We have reconstituted a gel from actin, purified tubulin, and whole MAPs, but have not yet been successful with actin, purified tubulin, and any single purified MAP.     

The interaction of phomopsin A with bovine brain tubulin

Phomopsin A is an anti-mitotic compound from the fungus Phomopsis leptostroniformis which is a potent inhibitor of microtubule assembly in vitro; like maytansine, it is known to compete with vinblastine for binding to tubulin (E. Lacey, J. A. Edgar, and C. C. J. Culvenor (1987) Biochem. Pharmacol. 36, 2133-2138). A major difference between the effects of maytansine and vinblastine is that vinblastine is a potent inhibitor of tubulin decay, whereas maytansine has little or no effect on decay. Since phomopsin A is structurally distinct from either maytansine or vinblastine, tubulin decay may be measured by either the time-dependent loss of the ability to bind to [3H]colchicine or the time-dependent increase in the binding of bis(8-anilinonaphthalene 1-sulfonate) (BisANS) to tubulin. By either method, phomopsin A was found to be a much stronger inhibitor of tubulin decay than is vinblastine or any other drug yet tested, and in fact, when decay is measured by the increase of BisANS binding, phomopsin A appears to stop the process entirely. This may prove to be useful in the determination of the higher-order structure of the tubulin molecule.     

Parkin-co-regulated gene (PACRG) product interacts with tubulin and microtubules

Parkin-co-regulated gene (PACRG) is a gene that shares a bidirectional promoter with Parkinson's disease-related Parkin/Park2 gene. Recently, the PACRG gene product was implicated in the function of flagella. However, its exact function remains unknown. Here, I assessed the interaction between PACRG and tubulin. Co-sedimentation experiments revealed that PACRG directly binds to microtubules and alpha/beta-tubulin heterodimers with high affinity. Microscopic studies showed that PACRG bundles microtubules and forms branched aggregates with unpolymerized tubulin dimers. The amino acid sequence of the microtubule-binding region of PACRG is highly conserved among various organisms, suggesting that tubulin binding is a basic property of PACRG.     

The association of carbohydrate with tubulin and in vitro assembled microtubules from bovine brain.

Phosphocellulose-purified tubulin (PC tubulin) was analyzed for neutral and amino sugar content, which was found to be 8.3 +/- 0.11 and 0.8 +/- 0.02 mol/mol dimer, respectively. A histochemical-electron-microscopic investigation was undertaken to attempt to localize carbohydrate associated with polymerized microtubules (MT). Outer diameters of MT assembled in vitro from bovine brain MT protein (tubulin and microtubule associated proteins) were found to increase upon treatment with ruthenium red, Alcian blue, and lanthanum hydroxide, which have been reported to possess specificity for complex carbohydrates. Concanavalin A-reactive sites were detected on the surface and in the lumen of MT assembled from MT protein and from PC tubulin.     

Axonal tubulin and microtubules: morphologic evidence for stable regions on axonal microtubules

Biochemical studies indicate that axonal tubulin is composed of at least two distinct pools that differ in cold solubility and biochemical composition [Brady et al: J. Cell Biol. 99:1716-1724]. To determine the morphologic correlate of cold-insoluble tubulin, segments of rat optic nerves were exposed to a series of in vitro experimental conditions that affect microtubules (MTs), including cold, podophyllotoxin (PT), triflupromazine (TFP), and taxol, and then examined by electron microscopy. Longitudinal sections of control axons showed MTs oriented parallel to the long axis of the axons. Axons exposed to cold, PT, and TFP showed short segments of MTs in association with cytoskeletal disarray. Morphometric studies were used to distinguish between a simple malorientation of MTs (undulation or zigzags in their course) and the loss of labile segments of MTs, leaving the stable portions behind. The lengths of MT segments were measured in longitudinal sections, and the numbers of MTs were determined in the cross sections. All MT segment-length histograms showed a unimodal distribution. Cold and PT produced a simple shift of the control histogram to the shorter length MTs. In cross sections the numbers of MTs in cold- and PT-exposed axons were significantly decreased, indicating that the presence of short segments of MTs in the longitudinal plane of sections was due to a loss of portions of MTs. Taxol, an agent that promotes MT assembly, reversed the cold effect partially and resulted in increases in both MT segment length and number. These studies indicate that stable MT segments are portions of longer MTs containing both stable and labile regions. Furthermore, these findings are consistent with the hypothesis that cold-insoluble tubulin functions as a transportable MT-organizing complex in the axon.     

Tyrosination of microtubules and non-assembled tubulin in brain slices

Brain slices were used to examine comparatively the incorporation of [14C]tyrosine into the C terminus of alpha-tubulin of the microtubule and non-assembled tubulin pools. We found that the incorporation of [14C]tyrosine from 5 min up to 60 min of incubation was higher in microtubules than in non-assembled tubulin. The possibility that this result was due to the activity of tubulin carboxypeptidase or tubulin:tyrosine ligase during the in vitro isolation of tubulin was discarded. We also found that tubulin:tyrosine ligase was mainly associated with microtubules when brain slices were homogenized under microtubule-preserving conditions. Conversely the enzyme behaved as a soluble entity when homogenization was performed under conditions that do not preserve microtubules. In addition, soluble tubulin:tyrosine ligase did not become sedimentable when in vitro conditions were changed to induce the formation of microtubules. The results presented in this work indicate the possibility that, in vivo, microtubules and not tubulin dimers are the major substrate for tubulin:tyrosine ligase. This is in contrast with previous findings from in vitro experiments, which showed a preference of the ligase for non-assembled tubulin.