Repeat motifs of tau bind to the insides of microtubules in the absence of taxol

The tau family of microtubule-associated proteins has a microtubule-binding domain which includes three or four conserved sequence repeats. Pelleting assays show that when tubulin and tau are co- assembled into microtubules, the presence of taxol reduces the amount of tau incorporated. In the absence of taxol, strong binding sites for tau are filled by one repeat motif per tubulin dimer; additional tau molecules bind more weakly. We have labelled a repeat motif with nanogold and used three-dimensional electron cryomicroscopy to compare images of microtubules assembled with labelled or unlabelled tau. With kinesin motor domains bound to the microtubule outer surface to distinguish between alpha- and beta-tubulin, we show that the gold label lies on the inner surface close to the taxol binding site on beta-tubulin. Loops within the repeat motifs of tau have sequence similarity to an extended loop which occupies a site in alpha-tubulin equivalent to the taxol-binding pocket in beta-tubulin. We propose that loops in bound tau stabilize microtubules in a similar way to taxol, although with lower affinity so that assembly is reversible.     

Differential Association of Tau With Subsets of Microtubules Containing Posttranslationally-Modified Tubulin Variants in Neuroblastoma Cells

Neuronal cells display different subsets of dynamic microtubules. In axons and extending neurites, this intrinsic dynamics is modulated by the microtubule-associated protein tau. Moreover, posttranslational modifications of tubulin, namely acetylation, tyrosination or glutamylation are directly involved in determining the stability of neuronal microtubules. Studies were carried out to analyze the interaction patterns of tau with subsets of microtubules in N2A neuroblastoma cells, which can differentiate in the presence of dibutyryl cAMP. Double labeling studies showed a differential pattern of tau association with microtubules containing acetylated and tyrosinated tubulin. Furthermore, studies using depolymerizing drugs revealed a selectivity in the association of tau with microtubular polymers and microfilaments, within the organization of the neuronal cytoskeleton. In order to study the association of specific tau isoforms with microtubules containing modified tubulin variants, immunoprecipitation studies were carried out. The coimmunoprecipitation data indicated a selective binding of specific tau isoforms to either modified tubulin variant. To assess the hypothesis on the roles of tau isoforms in the stabilization of microtubules containing modified tubulins, the association of those variants with tau isoforms was analyzed in overlay experiments. A preferential binding of acetylated tubulin from undifferentiated N2A cell extracts, to at least one slow-migrating tau isoform was revealed. However, acetylated tubulin from N2A cells containing long neurites displayed a preferential association with two isoforms of tau. On the other hand, tyrosinated tubulin from N2A extracts bound to the entire set of neuronal tau isoforms. These studies, along with the tau association with microtubules with different stability, indicate that tau segregates into subsets of microtubules in the axonal processes. The studies also suggest that these interactions may respond to a functional versatility of these polymers in differentiating neurons.     

Tau protein binds to microtubules through a flexible array of distributed weak sites

Tau protein plays a role in the extension and maintenance of neuronal processes through a direct association with microtubules. To characterize the nature of this association, we have synthesized a collection of tau protein fragments and studied their binding properties. The relatively weak affinity of tau protein for microtubules (approximately 10(-7) M) is concentrated in a large region containing three or four 18 amino acid repeated binding elements. These are separated by apparently flexible but less conserved linker sequences of 13-14 amino acids that do not bind. Within the repeats, the binding energy for microtubules is delocalized and derives from a series of weak interactions contributed by small groups of amino acids. These unusual characteristics suggest tau protein can assume multiple conformations and can pivot and perhaps migrate on the surface of the microtubule. The flexible structure of the tau protein binding interaction may allow it to be easily displaced from the microtubule lattice and may have important consequences for its function.     

Discodermolide interferes with the binding of tau protein to microtubules

We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.     

Interaction of microtubule-associated proteins with microtubules: yeast lysyl- and valyl-tRNA synthetases and tau 218-235 synthetic peptide as model systems.

The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS, KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a KD in the 10(-6) M range, and tau 218-235 binds with a KD in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha beta S tubulin and 1/alpha S beta S tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.     

Specific macromolecular interactions between tau and the microtubule system

The microtubule-associated protein Tau, a major component of brain microtubules, shares common repeated C-terminal sequences with the high molecular-weight protein MAP-2. It has been shown that tau peptides V¹⁸⁷-G²⁰⁴ and V²¹⁸-G²³⁵ representing two main repeats, induced brain tubulin assembly in a concentration-dependent fashion. The specific roles of these repeats in the interaction of tau with microtubules, and its antigenic nature were investigated using synthetic tau peptides and site-directed monoclonal antibodies. Tau peptides appeared to compete with MAP-2 incorporation into assembled microtubules. The interactions of the tau fragments with -tubulin peptides bearing the tau binding domain on tubulin were analyzed by fluorescence spectroscopy. The specificity of the binding was further demonstrated by the reactivity of tau and the tau peptides with a monoclonal anti-idiotypic antibody produced after immunization with the -11(422–434) tubulin peptide, as assessed by enzyme-linked immunoassay. Western blots confirmed the interaction of tau with the monoclonal antibody. In addition, immunoassays revealed a competition between the MAP-reacting monoclonal antibody and the tubulin peptide -11(422–434) for their interaction with the tau molecule.     

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.     

Molecular cloning of XTP, a tau-like microtubule-associated protein from Xenopus laevis tadpoles

The microtubules of the mammalian nervous system are stabilised by several microtubule-associated proteins (MAPs), including the tau and MAP-2 protein families. The most prominent feature of mammalian tau and MAP-2 proteins is a common and highly homologous microtubule-binding region consisting of three or four imperfect tandem repeats. In this paper we report the cloning and characterisation of a Xenopus laevis tau-like protein (XTP) from tadpole tails. This protein encompasses two isoforms of 673 or 644 amino acids with four tandem repeats that are highly homologous to mammalian tau repeats. Both isoforms share a common amino terminal half, whereas the carboxyl terminus downstream of the repeat region is unique for each isoform. Northern blot analysis revealed that both isoforms are preferentially expressed in the tail of X. laevis tadpoles, whereas a shorter version of XTP is expressed in the head. Recombinant proteins of both XTP isoforms were able to bind microtubules. The longest isoform, however, was more effective at promoting tubulin polymerisation, indicating that sequences downstream of the repeat region affect the microtubule assembling capacity. These results demonstrate that tau-like proteins are found in non-mammalian vertebrate species, where they may support the stability of microtubules.     

The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus-2 is microtubule binding.

The signal for retention in the endoplasmic reticulum of the E3/19K protein of adenovirus type 2 is located within the carboxyl-terminal cytoplasmic extension. A synthetic peptide corresponding to this sequence showed affinity for beta-tubulin, could promote tubulin polymerization in vitro, and bound to taxol-polymerized microtubules. When compared with the microtubule binding sequences from two microtubule-associated proteins (MAPs; MAP2 and tau), we found similarities suggesting that the cytoplasmic tail might bind to tubulin/microtubules in a MAPs-like fashion. A synthetic peptide corresponding to the cytoplasmic tail of an E3/19K deletion mutant not retained in the endoplasmic reticulum was also tested. It had the same net charge but did not promote tubulin polymerization in vitro nor did it show measurable affinity for tubulin or microtubules. This indicates that binding to microtubules is important for retention of the E3/19K protein in the endoplasmic reticulum.     

First tau repeat domain binding to growing and taxol-stabilized microtubules,and serine 262 residue phosphorylation

Tau phosphorylation plays a crucial role in microtubule stabilization and in Alzheimer's disease. To characterize the molecular mechanisms of tau binding on microtubules, we synthesized the peptide R1 (QTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQI), reproducing the first tau microtubule binding motif. We thermodynamically characterized the molecular mechanism of tubulin assembly with R1 in vitro, and measured, for the first time, the binding parameters of R1 on both growing and taxol-stabilized microtubules. In addition, we obtained similar binding parameters with R1 phosphorylated on Ser262. These data suggest that the consequences of Ser262 phosphorylation on tau binding to microtubules and on tubulin assembly are due to large intramolecular rearrangements of the tau protein.     

Tau induces ring and microtubule formation from alphabeta-tubulin dimers under nonassembly conditions

Tau is a neuronal microtubule-associated protein that plays a central role in many cellular processes, both physiological and pathological, such as axons stabilization and Alzheimer's disease. Despite extensive studies, very little is known about the detailed molecular basis of tau binding to microtubules. We used the four-repeat recombinant htau40 and tubulin dimers to show for the first time that tau is able to induce both microtubule and ring formation from 6S alphabeta tubulin in phosphate buffer without added magnesium (nonassembly conditions). The amount of microtubules or rings formed was protein concentration-, temperature-, and nucleotide-dependent. By means of biophysical approaches, we showed that tau binds to tubulin without global-folding change, detectable by circular dichroism. We also demonstrated that the tau-tubulin interaction follows a ligand-mediated elongation process, with two tau-binding site per tubulin dimer. Moreover, using a tubulin recombinant alpha-tubulin C-terminal fragment (404-451) and a beta-tubulin C-terminal fragment (394-445), we demonstrated the involvement of both of these tubulin regions in tau binding. From this model system, we gain new insight into the mechanisms by which tau binds to tubulin and induces microtubule formation.     

Tau protein function in living cells

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.     

Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter- repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site- directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult- specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.     

Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly.

This paper describes the physical and chemical properties of purified tau, a protein which is associated with brain microtubules and which induces assembly of microtubules from tubulin. Purified tau is composed of four polypeptides which migrate at positions equivalent to molecular weights between 55,000 and 62,000 during electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. These polypeptides are shown to be closely related by peptide mapping and by amnio acid analysis. A comparison by various techniques of the high molecular weight microtubule-associated proteins with the tau polypeptides indicates no apparent relationship. Tau is found by analytical ultracentrifugation and by sedimentation equilibrium to have a sedimentation coefficient of 2.6 S and a native molecular weight of 57,000. Tau, therefore, must be highly asymmetric (an axial ratio of 20:1 using a prolate ellipsoid model), and yet possess little α-helical structure as indicated by circular dichroism. Isoelectric focusing shows tau to be a neutral or slightly basic protein. Tau is also seen to be phosphorylated by a protein kinase which copurifies with microtubules.In the assembly process, tau apparently regulates the formation of longitudinal oligomers from tubulin dimers, and hence promotes ring formation under depolymerizing conditions and microtubule formation under polymerizing conditions. The known asymmetry of the tau molecule suggests that tau induces assembly by binding to several tubulin molecules per tau molecule, thereby effectively increasing the local concentration of tubulin and inducing the formation of longitudinal filaments. The role of tau is discussed in light of reports of polymerization induced by particular non-physiological conditions and by various polycations. The formation of normal microtubules over a wide range of tubulin and tau concentrations under mild buffer conditions suggests that tau and tubulin define a complete in vitro assembly system under conditions which approach physiological.     

Surface-decoration of microtubules by human tau

Tau is a neuronal, microtubule-associated protein that stabilizes microtubules and promotes neurite outgrowth. Tau is largely unfolded in solution and presumably forms mostly random coil. Because of its hydrophilic nature and flexible structure, tau complexed to microtubules is largely invisible by standard electron microscopy methods. We applied a combination of high-resolution metal-shadowing and cryo-electron microscopy to study the interactions between tau and microtubules. We used recombinant tau variants with different domain compositions, (1) full length tau, (2) the repeat domain that mediates microtubule binding (K19), and (3) two GFP-tau fusion proteins that contain a globular marker (GFP) attached to full-length tau at either end. All of these constructs bind exclusively to the outside of microtubules. Most of the tau-related mass appears randomly distributed, creating a "halo" of low-density mass spread across the microtubule surface. Only a small fraction of tau creates a periodic signal at an 8 nm interval, centered on alpha-tubulin subunits. Our data suggest that tau retains most of its disordered structure even when bound to the microtubule surface. Hence, it binds along, as well as across protofilaments. Nevertheless, even minute concentrations of tau have a strong stabilizing effect and effectively scavenge unpolymerized tubulin.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

Tau – an inhibitor of deacetylase HDAC6 function

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

Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau.

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.     

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.