Heat-stable Microtubule-associated Protein Research Articles

Purification and characterization of a new, ubiquitously distributed class of microtubule-associated protein with molecular mass 250 kDa.

A heat-stable microtubule-associated protein (MAP) with relative molecular mass 250 000, termed 250-kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250-kDa MAP was able to bind to taxol-stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western-blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250-kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250-kDa MAP colocalized with free tubulin. Under electron microscopy, 250-kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.

XMAP230 is required for the assembly and organization of acetylated microtubules and spindles in Xenopus oocytes and eggs.

We used affinity-purified polyclonal antibodies to characterize the distribution and function of XMAP230, a heat-stable microtubule-associated protein isolated from Xenopus eggs, during oogenesis. Immunoblots revealed that XMAP230 was present throughout oogenesis and early development, but was most abundant in late stage oocytes, eggs, and early embryos. Immunofluorescence microscopy revealed that XMAP230 was associated with microtubules in oogonia, post-mitotic stage 0 oocytes, early stage I oocytes, and during stage IV-VI of oogenesis. However, staining of microtubules by anti-XMAP230 was not detectable during late stage I through stage III. In stage VI oocytes, anti-XMAP230 stained a large subset of microtubules that were also stained with monoclonal antibodies specific for acetylated (&agr ;)-tubulin. During oocyte maturation, XMAP230 was associated with the transient microtubule array that serves as the precursor of the first meiotic spindle, as well as both first and second meiotic spindles. The extensive array of cytoplasmic microtubules present throughout maturation was not detectably stained by anti-XMAP230. Microinjection of anti-XMAP230 locally disrupted the organization and acetylation of microtubules in stage VI oocytes, and reduced the re-acetylation of microtubules during recovery from cold-induced microtubule disassembly. Subsequent maturation of oocytes injected with anti-XMAP230 resulted in defects in the assembly of the transient microtubules array and first meiotic spindle. These observations suggest that XMAP230 is required for the stabilization and organization of cytoplasmic and spindle microtubules in Xenopus oocytes and eggs.

Biochemical and immunochemical identification of a microtubule-binding protein from bovine pancreas.

We have identified a 67 kDa heat-stable protein among the proteins which bind specifically to brain microtubules immobilized on a chromatographic support. Its relationship to tubulin and to the cytoskeleton using polyclonal antibodies has been studied. This 67 kDa protein is present in cytoskeleton and microtubule preparations from pancreas. This heat-stable microtubule-associated protein (MAP) copolymerized with phosphocellulose purified brain tubulin. The 67 kDa polypeptide was immunoreactive to antibodies against the 210 kDa MAP from HeLa cells; it also reacted with antibodies against an oligopeptide whose sequence corresponded to the second repeat of mouse brain tau.

Phosphorylated baculovirus p10 is a heat-stable microtubule-associated protein associated with process formation in Sf9 cells.

Insect ovarian Sf9 cells extend processes with complex morphologies when infected with a recombinant baculovirus encoding the catalytic subunit of protein kinase A. Within the shafts of the processes are abundant microtubules, which, in contrast to those in Sf9 cells expressing the microtubule-associated protein tau, are generally not organized into parallel bundles. During infection the late viral polypeptide p10 becomes phosphorylated by the protein kinase A catalytic subunit at its penultimate residue, Ser92. The expression or phosphorylation of other major host cell or viral polypeptides does not change, compared with polypeptides from a wild-type viral infection. Once phosphorylated, p10 associates with microtubules in the infected cells and may thereby play a role in process formation.

The 72-kDa microtubule-associated protein from porcine brain.

A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.

Tau, the Neuronal Heat-Stable Microtubule-Associated Protein, is also Present in the Cross-Linked Microtubule Network of the Testicular Spermatid Manchette1

The seminiferous tubule of the testis contains a rich variety of microtubule networks and of microtubule-associated proteins (MAPs). Tau is a heat-stable MAP previously believed to be limited in its expression in mammals to the nervous system. We have identified tau in rat and bovine testis, a unique non-neuronal location, using biochemical, molecular, and immunologic approaches. SDS-PAGE of ammonium sulfate-fractionated, testis heat-stable MAPs resulted in an enrichment of bands that comigrated with rat brain tau. Only the 35-45% precipitated ammonium sulfate fraction induced microtubule assembly. Immunoblotting with monoclonal anti-tau antibodies demonstrated tau immunoreactivity in these testis MAP preparations. Northern analysis of total rat testis RNA demonstrated a 1.7-kb band that hybridized with a 51-nucleotide oligomer complementary to a conserved portion of the tau transcript. This 51-mer identified a similar 1.7-kb minor band and an additional 6-kb major band in Northern analysis of total rat brain RNA. Finally, in the bull testis, immunohistochemistry localized tau to the spermatid manchette, a transient, cross-linked microtubule network of unknown function. As spermatid elongation begins, the manchette forms a sheath around the posterior aspect of the nucleus, but, by the completion of nuclear condensation, the manchette is largely disassembled. Tau most likely plays a structural role in the manchette; however, tau immunoreactivity also was observed in late stage I spermatids prior to manchette formation, suggesting that tau may serve a function in manchette assembly.

Mitogen-activated-protein-kinase-catalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function.

Mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPK-catalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubule-binding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPs and negatively regulating their microtubule-stabilizing function.

Use of a heat-stable microtubule-associated protein class-specific antibody to investigate the mechanism of microtubule binding.

Members of the heat-stable family of microtubule-associated proteins (MAPs), MAP 2, tau, and MAP 4, contain three or four tandem imperfect repeated sequences close to their carboxyl termini. These sequences lie within the microtubule-binding domains of the MAPs; they have been proposed to be responsible for microtubule binding and the ability of these MAPs to lower the critical concentration for microtubule assembly. Their spacing may reflect that of the regularly arrayed tubulin subunits on the microtubule surface. We here characterize the 32- and 34-kDa chymotryptic microtubule-binding fragments of MAP 2 identified in earlier work. We identify the primary chymotryptic cleavage site in high molecular weight MAP 2 as between Phe1525 and Lys1526, within 13 amino acids of the known MAP 2 splice junction. We have raised a monoclonal antibody to the 32- and 34-kDa fragments and find that it reacts with all members of the heat-stable MAPs class. To determine where it reacts, we sequenced immunoreactive subfragments of the 32- and 34-kDa fragments, selected several cDNA clones with the antibody, and tested for antibody reactivity against a series of synthetic MAP 2 and tau peptides. We identify the epitope sequence as HHVPGGG (His-His-Val-Pro-Gly-Gly-Gly). The antibody also recognized several other MAP 2 and tau repeats. Despite reacting with this highly conserved element, we find that the antibody does not block microtubule binding, but binds to the MAPs and co-sediments with microtubules. These results suggest that there are other regions besides the repeated elements which are essential for microtubule binding.

Molecular cloning of a ubiquitously distributed microtubule-associated protein with Mr 190,000.

A heat-stable microtubule-associated protein (MAP) with apparent molecular weight of 190,000 is a major non-neural MAP which distributes ubiquitously among bovine tissues (termed here MAP-U). Previously we reported that microtubule-binding chymotryptic fragments of MAP-U and tau contain a common assembly-promoting (AP) sequence of 22 amino acid residues (Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1989) J. Biol. Chem. 264, 5885-5890). We isolated cDNA clones for MAP-U containing the whole coding sequence. Northern blot analysis revealed that a major species of MAP-U mRNA is 5 kilobases in length and is expressed ubiquitously among bovine tissues. Nucleotide sequence analysis revealed the complete amino acid sequence of MAP-U which consists of 1,072 amino acid residues. Analysis of the deduced amino acid sequence of MAP-U indicated that this molecule is clearly divided into two domains in terms of electrostatic charge distribution: an amino-terminal acidic domain (residues 1-640) and a carboxyl-terminal basic domain (residues 641-1072). The amino-terminal domain of MAP-U shows no significant sequence homology with other known protein sequences including neural MAPs, tau, and MAP-2. The amino-terminal domain of MAP-U contains unique 18 1/2 repeats of 14-amino acid motif which have not been observed in other MAPs. The carboxyl-terminal domain of MAP-U is further divided into three regions: a Pro-rich region (residues 641-880), an AP sequence region (residues 881-1003), and a short hydrophobic tail (residues 1004-1072). The Pro-rich region is mainly composed of five species of amino acid residues, Pro, Ala, Lys, Ser, and Thr. The AP sequence region contains four tandem repeats of AP sequences, and thus, this region is considered to play a leading role in the interaction of MAP-U with microtubules.

A novel heat-stable 205 kDa microtubule-associated protein is involved in the neural development of the rat brain

A novel microtubule associated protein (MAP) was identified with a monoclonal antibody (mAb) among rat brain microtubule (MT) proteins. This MAP, which is found in a heat-stable MAP fraction, is co-polymerized with tubulin from rat brain homogenates both in cycled and in taxol-driven assembly purifications. When the heat-stable fraction of MT proteins and purified tubulin are assembled in vitro without taxol, 205 kDa MAP is identified in the MT pellet. Immunofluorescent studies using the mAb against this MAP on sections of a rat spinal cord, hippocampus, retina and cerebellar cortex, showed that 205 kDa MAP was localized in not only neural cell bodies, dendrites, and axons, but also in glial cells. This 205 kDa MAP is clearly distinct from previously reported MAPs as to molecular mass, heat stability, immunoreactivity, distribution among tissues or within a neuron, and the developmental aspect of the postnatal cerebellar cortex. As for development, the 205 kDa MAP has already been seen to appear at postnatal day 3. Throughout the postnatal development of the cerebellar cortex, 205 kDa MAP is expressed in developing Purkinje cells, with the staining reaction being particularly intense in the more superficially positioned parallel fiber profiles. This points out the possible important functions of 205 kDa MAP in the morphogenesis of parallel fiber axons.

Two Separate 18-Amino Acid Domains of Tau Promote the Polymerization of Tubulin

Tau is a heat-stable microtubule-associated protein which promotes tubulin polymerization. The assembly promoting region of tau was localized using synthetic peptides modeled after domains found in both human and mouse tau. The design of these synthetic peptides was based on the triple repeat motif found in mouse tau. The first peptide, Tau-(187-204), and the second peptide, Tau-(218-235), are capable of promoting the polymerization of tubulin into microtubules, at concentrations above 100 microM. Two other peptides tested, TauR and Tau-(250-267), were not able to promote the assembly of tubulin over a range of concentrations up to 800 microM. TauR is a random analog of Tau-(187-204). Although TauR is unable to promote polymerization, it can modify Tau-(187-204)-induced tubulin assembly.

Antibodies to the neuronal cytoskeleton are elicited by Alzheimer paired helical filament fractions

Antibodies were raised to paired helical filament (PHF) enriched fractions obtained from brains of individuals with Alzheimer disease by extraction with ionic detergent followed by sucrose gradient centrifugation. Electron microscopic examination showed that the fractions were enriched in Alzheimer PHF but contained also lipofuscin, amyloid, granular material and membranous elements. Analysis of these fractions with SDS-PAGE stained with Coomassie blue showed only a faint band at approximately 60 kDa while most of the material was excluded from the stacking gel. BALB/c mice were injected weekly with 100 or 200 μg of these fractions or corresponding fractions from age-matched control brains. The 3 mice injected with Alzheimer brain, but not the 5 mice injected with control brain fractions, produced antibodies that reacted with central and peripheral nervous system axons, Alzheimer neurofibrillary tangles in intact tissue as well as with isolated, SDS-treated paired helical filaments. In gel strips antibodies from all 3 mice injected with Alzheimer brain fractions reacted with the 200-kDa and 168-kDa but not the 68-kDa neurofilament subunits. The 3 antisera reacted also with some forms of the microtubule-associated protein τ. Adsorptions with the insoluble fraction from Alzheimer but not from control brains blocked staining of axons and NFT by all 3 antisera. Adsorption with highly purified neurofilament proteins or with a preparation containing the 200-kDa 168-kDa neurofilament subunits blocked axon and NFT immunostaining only in one antiserum. Adsorptions with microtubule protein, heat-stable microtubule-associated protein, or a preparation of τ did not completely block immunostaining by any of the 3 antisera. These results demonstrate that fractions enriched with Alzheimer paired helical filaments contain insoluble neurofilament, τ and other yet unidentified antigens.

Limited chymotryptic digestion of bovine adrenal 190,000-Mr microtubule-associated protein and preparation of a 27,000-Mr fragment which stimulates microtubule assembly.

A heat stable microtubule-associated protein of Mr 190,000 (190-kDa MAP) has been purified from bovine adrenal cortex (Murofushi, H., Kotani, S., Aizawa, H., Hisanaga, S., Hirokawa, N., and Sakai, H. (1986) J. Cell Biol. 103, 1911-1919). Limited chymotryptic digestion of 190-kDa MAP produced a fragment of Mr 27,000 (27-kDa fragment), which bound to microtubules reconstituted in the presence of taxol. This fragment was purified with the aid of cosedimentation with microtubules. The purified 27-kDa fragment showed an ability to stimulate tubulin polymerization in the absence of taxol. Electron microscopic observation of microtubules reconstituted from purified 27-kDa fragment and tubulin revealed that the microtubules were in the form of thick bundles and that lateral projections which can be seen in microtubules reconstituted from intact 190-kDa MAP and tubulin were not observed. These results indicate that 27-kDa fragment includes or is a part of microtubule-binding domain of 190-kDa MAP and that this fragment is active in stimulating microtubule assembly. Amino acid analysis revealed that the 27-kDa fragment was rich in lysine, proline, and alanine, the sum of these three being about 45% of the total amino acids and that the contents of methionine, tyrosine, phenylalanine, and histidine were very low. These data suggest that the microtubule binding domain of the 190-kDa MAP comprises an unique structure.

Purification and characterization of a 190-kD microtubule-associated protein from bovine adrenal cortex.

A heat-stable microtubule-associated protein (MAP) with molecular weight of 190,000, termed 190-kD MAP, was purified from bovine adrenal cortex. This MAP showed the same level of ability to promote tubulin polymerization as did MAP2 and tau from mammalian brains. Relatively high amounts of 190-kD MAP could bind to microtubules reconstituted in the presence of taxol. At maximum 1 mol of 190-kD MAP could bind to 2.3 mol of tubulin. 190-kD MAP was phosphorylated by a cAMP-dependent protein kinase prepared from sea urchin spermatozoa and by protein kinase(s) present in the microtubule protein fraction prepared from mammalian brains. The maximal numbers of incorporated phosphate were approximately 0.2 and approximately 0.4 mol per mole of 190-kD MAP, respectively. These values were lower than that of MAP2, which could be heavily phosphorylated by the endogenous protein kinase(s) up to 5 mol per mole of MAP2 under the same assay condition. 190-kD MAP had no effects on the low-shear viscosity of actin and did not induce an increase in turbidity of the actin solution. It was also revealed that 190-kD MAP does not cosediment with actin filaments. These data clearly show that, distinct from MAP2 and tau, this MAP does not interact with actin. Electron microscopic observation of the rotary-shadowed images of 190-kD MAP showed the molecular shape to be a long, thin, flexible rod with a contour length of approximately 100 nm. Quick-freeze, deep-etch replicas of the microtubules reconstituted from 190-kD MAP and brain tubulin revealed many cross-bridges connecting microtubules with each other.

Microtubule-associated protein tau (tau) is a major antigenic component of paired helical filaments in Alzheimer disease.

The detailed protein composition of the paired helical filaments (PHF) that accumulate in human neurons in aging and Alzheimer disease is unknown. However, the identity of certain components has been surmised by using immunocytochemical techniques. Whereas PHF share epitopes with neurofilament proteins and microtubule-associated protein (MAP) 2, we report evidence that the MAP tau (tau) appears to be their major antigenic component. Immunization of rabbits with NaDodSO4-extracted, partially purified PHF (free of normal cytoskeletal elements, including tau) consistently produces antibodies to tau but not, for example, to neurofilaments. Such PHF antibodies label all of the heterogeneous fetal and mature forms of tau from rat and human brain. Absorption of PHF antisera with heat-stable MAPs (rich in tau) results in almost complete loss of staining of neurofibrillary tangles (NFT) in human brain sections. An affinity-purified antibody to tau specifically labels NFT and the neurites of senile plaques in human brain sections as well as NaDodSO4-extracted NFT. tau-Immunoreactive NFT frequently extend into the apical dendrites of pyramidal neurons, suggesting an aberrant intracellular locus for this axonal protein. tau and PHF antibodies label tau proteins identically on electrophoretic transfer blots and stain the gel-excluded protein representing NaDodSO4-insoluble PHF in homogenates of human brain. The progressive accumulation of altered tau protein in neurons in Alzheimer disease may result in instability of microtubules, consequent loss of effective transport of molecules and organelles, and, ultimately, neuronal death.

Physicochemical characterization of the heat-stable microtubule-associated protein MAP2.

MAP2 purified without heat treatment was compared to MAP2 prepared with the usual boiling step. No significant differences were found by isoelectric focusing, peptide mapping, fluorescence spectroscopy and circular dichroism. Hydrodynamically, MAP2 is a monomer with s degree 20,w = 3.5 +/- 0.1 S and Mr = 220 000. The molecular mass was measured by sedimentation equilibrium and verified by gel chromatography in denaturing solvent and dodecyl sulfate gel electrophoresis at different acrylamide concentrations. The high frictional ratio, f/fmin = 3.7, indicated that MAP2 was clearly not globular but had either a very elongated shape or an unordered expanded structure. Circular dichroic results were consistent with a predominantly unordered structure independently of the preparation procedure. This supports the notion that MAP2, in solution, is a very flexible and non-compact protein. Examination of the circular dichroism of MAP2 as a function of temperature indicated that the thermal stability of this protein was probably due to its mostly unordered structure and the fact that the little ordered structure present was resistant to thermal denaturation.