On the use of heat stability as a criterion for the identification of microtubule associated proteins (MAPs)

Solubility at elevated temperature is a striking biochemical property exhibited by a restricted number of the known microtubule associated proteins. This property has been extremely useful in the identification of these proteins and in their purification as well. It is reported here that heat stability is a function of the composition of proteins present during exposure to elevated temperature. All non-tubulin proteins in bovine microtubule preparations were found to remain soluble when tubulin was removed prior to heating. Addition of purified tubulin or bovine serum albumin to the preparation restored the selective heat stability normally seen in microtubule protein preparations.     

Three-dimensional reconstruction of tubulin in zinc-induced sheets: II. Consequences of removal of microtubule associated proteins

The three-dimensional structure of zinc-induced tubulin sheets freed of microtubule associated proteins has been determined to 20 Å resolution by electron microscopy and image reconstruction. The determination was carried out with porcine brain tubulin separated from microtubule associated proteins by phosphocellulose chromatography. Negatively stained samples were tilted using the goniometer stage of the electron microscope to provide images of the tubulin sheets ranging in tilt from ?60 ° to +60 °. The micrographs were digitized and subjected to a cross-correlation analysis to compensate for smooth curvature of the lattice in the sheets. For each angle of tilt, an average unit cell was obtained from the cross-correlation analysis and subsequently a Fourier transform was computed for inclusion in the three-dimensional Fourier data set. The transforms of 47 tilted images plus the average of five untilted sheets were combined and an inverse Fourier transform was applied to give a threedimensional reconstruction of the microtubule associated protein-free tubulin sheets. Comparison of the protofilament structure in these sheets with the previously published protofilament structure of zinc-induced tubulin sheets containing microtubule associated proteins reveals a number of consequences of the removal of microtubule associated proteins. (1) The extensive internal contact along the protofilament observed in microtubule associated protein-containing tubulin sheets is maintained in microtubule associated protein-free tubulin sheets. (2) In projection, the protofilaments in microtubule associated protein-free tubulin sheets are 2.2 Å closer together than in microtubule associated protein-tubulin sheets. (3) The deviations of adjacent protofilaments from the plane of the sheets when viewed end-on are more pronounced in the absence of microtubule associated proteins. Differences are also observed at the level of individual tubulin subunits. In particular, the distinct cleft which was found in one class of subunits in tubulin sheets with microtubule associated proteins is absent in the microtubule associated protein-free tubulin sheets. The loss of this cleft and some changes in the shape of the tubulin subunits upon removal of microtubule associated proteins suggest a possible site for the interaction of tubulin with microtubule associated proteins.     

Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequencial affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behavior of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.Abbreviations MAPs microtubule-associated proteins - C-terminal carboxyl-terminal - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - DTT dithiotreitol - BSA bovine serum albumin     

Binding of microtubule proteins to DNA: specificity of the interaction.

Tubulin is detected among the DNA-binding proteins when an extract from fibroblasts is chromatographed on DNA-cellulose. Further purification of the colchicine-binding activity shows that purified tubulin from fibroblasts does not bind to DNA. Depolymerized brain microtubule proteins show a high affinity for DNA. The fraction bound is composed of tubulin and microtubule-associated proteins. Experiments with fractionated microtubule proteins indicate that tubulin-free microtubule associated proteins bind to DNA, while tubulin free of microtubule-associated proteins does not. Microtubule-associated proteins bind better to eukaryotic than to phage DNA suggesting a specificity of the interaction.     

Microtubule associated proteins of goat brain

The microtubule associated proteins of goat brain were separated from tubulin on the basis of their thermostability and then fractionated by chromatography on Sepharose 4B column. Analysis of the fractions by SDS-Polyacrylamide gel electrophoresis and assay of their tubulin-assembly-promoting activity indicate that this activity resides primarily in the tauproteins (mol. wt. 55,000–70,000) and a class of even lower molecular weight (25,000–35,000) proteins. Electrophoresis of the microtubule associated protein fractions separated from tubulin by phosphocellulose chromatography are in agreement with the results obtained from fractionation on Sepharose 4B columns.     

Vesikin, a vesicle associated ATPase from squid axoplasm and optic lobe, has characteristics in common with vertebrate brain MAP 1 and MAP 2

Vesikin, a protein that can associate with squid axoplasmic vesicles or optic lobe microtubules, has been implicated as a force-generating molecule involved in microtubule-dependent vesicle transport [Gilbert and Sloboda, 1986, 1988]. Because vesikin crossreacts with an antibody to porcine brain microtubule associated protein 2 (MAP 2), studies were conducted to compare squid vesikin and brain MAPs. When taxol stabilized microtubules containing vesikin as a microtubule associated protein were incubated in the presence of ATP, vesikin dissociated from the microtubule subunit lattice. This behavior would be expected for an ATP-dependent, force generating molecule that serves as a crossbridge between vesicles and microtubules. When chick brain microtubules were treated under the same conditions, MAP 2 remained bound to the microtubules while MAP 1 dissociated in a manner similar to vesikin. One dimensional peptide mapping procedures revealed that, although digestion of vesikin and MAP 2 generated several peptides common to both proteins, vesikin and MAP 2 are clearly not identical. Furthermore, the addition of vesikin or MAPS 1 and 2 to purified tubulin stimulated microtubule assembly in a manner dependent on the concentration of added protein. These findings demonstrate that brain MAPs share characteristics common to squid vesikin and support the suggestion that brain MAPs 1 and 2 might act as a force generating complex for vesicle transport in higher organisms.     

G protein β subunit is closely associated with microtubules

Previously, we have identified the association of G protein β subunit (Gβ) with mitotic spindles in various mammalian cells. Since microtubules are the main component of mitotic spindles, here we have isolated bovine brain microtubules and purified Gβ subunit to identify the close association of Gβ subunit with purified brain microtubules and have shown the direct incorporation of Gβ subunit into the microtubules both in vitro and in vivo. It was found that: (1) microtubular fraction isolated from bovine brain contained Gβ subunit, (2) coimmunoprecipitation demonstrated that Gβ subunit could be coprecipitated with tubulin, (3) addition of purified Gβ subunit into cytosolic extract for microtubule assembly caused direct incorporation of Gβ subunit into assembled microtubules and increased the association of microtubule-associated proteins with microtubules, and (4) incubation of exogenous Gβ subunit with detergent-permeabilized cells resulted in direct incorporation of Gβ subunit into microtubule fibers and depolymerized tubulin molecules. We conclude that G protein β subunit is closely associated with microtubules and may play an important role in the regulation of microtubule formation in addition to its regulatory role in cellular signal transduction. J. Cell. Biochem. 70:553–562, 1998. © 1998 Wiley-Liss, Inc.     

Identification and characterization of two novel proteins affecting fission yeast gamma-tubulin complex function

The gamma-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe gamma-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to gamma-tubulin-associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1(+) or gfh1(+) are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Delta and gfh1Delta cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of gamma-tubulin complex components in organizing different types of microtubule structures in S. pombe.     

Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta''-iminodipropionitrile-intoxicated axon as a model system

Axons from rats treated with the neurotoxic agent beta,beta'-iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue. To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN-treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy using colloidal gold-labeled second antibodies revealed that both anti-MAP 1A and anti-MAP 1B stained fuzzy filamentous structures between microtubules. In view of earlier work indicating that rapid transport is associated with the microtubule domain in the IDPN-treated axon, it now appears that MAP 1A and MAP 1B may play a role in this process. We believe that MAP 1A and MAP 1B are major components of the microtubule-associated fibrillar matrix in the axon.     

Molecular weight dependency of heparin inhibition of microtubule assembly in vitro

Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.     

Induction of polymerization of purified tubulin by sulfonate buffers. Marked differences between 4-morpholineethanesulfonate (Mes) and 1,4-piperazineethanesulfonate (Pipes)

Interactions of both purified tubulin and microtubule protein (tubulin plus associated proteins) with two commonly used sulfonate buffers were examined. 1,4-Piperazineethanesulfonate (Pipes) and 4-morpholineethanesulfonate (Mes) at high concentrations induce the polymerization of purified tubulin in reactions requiring only buffer, tubulin and GTP. While both reactions were temperature-dependent, cold-reversible and inhibited by GDP, colchicine or Ca2+, there were significant differences between them. Substantially lower tubulin and buffer concentrations were required for Pipes-induced polymerization; and turbidity was much more intense in the Pipes-induced than in the Mes-induced reaction at the same protein concentration. Electron microscopy demonstrated that for the most part typical smooth-walled microtubules were formed in Mes, while aberrant forms were the predominant structures formed in Pipes. When the polymerization of microtubule protein was examined as a function of buffer concentration, biphasic patterns were observed with both Pipes and Mes: polymerization occurred at both low and high, but not intermediate, buffer concentrations. The turbidity observed at high concentrations of Pipes greatly exceeded that at low concentrations. With Mes, equivalent turbidity developed at both high and low buffer concentrations. Although associated proteins copolymerized with tubulin at low buffer concentrations, they were excluded from the polymerized material at high buffer concentrations. Pipes and Mes were compared to sodium phosphate, Tris/HCl and imidazole/HCl buffers at 0.1 M in several polymerization systems using both purified tubulin and microtubule protein. The sulfonate buffers were invariably associated with more vigorous reactions than the other buffers.     

Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation

Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells. In animal cells, they are organized by the centrosome, the major microtubule-organizing center. Many centrosomal proteins act coordinately to modulate microtubule assembly and organization. Our previous work has shown that Cep70, a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells. However, the molecular details remain to be investigated. In this study, we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins. Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system. These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation.     

Analysis of microtubule polymerization inhibitors in sea urchin egg extracts: Evidence for a protease

Inhibitors of microtubule polymerization have been found in extracts of unfertilized sea urchin eggs using neural tubulin polymerization assays without glycerol. The inhibitory activity is partially destroyed by boiling or by reduction and carboxymethylation and is nondialyzable. When chromatographed on DEAE-cellulose, the inhibitory activity is eluted over a broad NaCl gradient and is in association with several peaks. This partially purified inhibitor is not destroyed by incubation with RNase A. When the partially purified inhibitor is incubated with brain microtubule protein under conditions which support microtubule polymerization, both high molecular weight-microtubule associated proteins and tubulin appear to be digested when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteolytic digestion as well as inhibition of microtubule polymerization depend upon similar concentrations of partially purified inhibitor present in the polymerization reaction. It appears as though at least part of the microtubule polymerization inhibitory activity present in unfertilized sea urchin eggs is due to this protease.     

Structural differences of microtubule associated proteins from brain probed by tryptic peptide mapping

Microtubules were purified from porcine brain by two cycles of temperature-dependent assembly and disassembly, then microtubule associated proteins, MAP-1, MAP-2, and tau, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional tryptic peptide maps of radioiodinated polypeptides were compared with each other by means of mixed sample experiments, and the following results were obtained. Subspecies of MAP-1 (355-345 and 325 kDa) showed about 33% homology in the tryptic peptide maps. Structural homology of MAP-1 and MAP-2 was very low; only 3 out of 40 peptide spots of MAP-2 were identical with those of MAP-1-C. Subspecies of tau proteins (65 and 60 kDa) were very closely related. Structural similarity between MAP-2 and tau was very low. MAP-1 from porcine brain and rat brain showed very high structural homology.     

Proteins associated with tubulin.

The distribution of proteins on SDS-urea polyacrylamide (7.5%) disc gel electrophoresis is studied from rat brain tubulin purified by three different procedures, including ammonium sulfate precipitation followed by DEAE cellulose chromotography, three cycles of polymerization-depolymerization and colchicinecontaining agarose affinity columns. Three tubulin-associated proteins other than the principal tubulin dimer are identified and characterized with respect to molecular weight, behavior on gel filtration chromatography and method of tubulin purification. One of these proteins (H₁) is released from the tubulin complex when colchicine is irreversibly bound to tubulin. These proteins may participate in processes related to microtubule assembly or function.     

Purification and characterization of native conventional kinesin, HSET, and CENP-E from mitotic hela cells

We have developed a strategy for the purification of native microtubule motor proteins from mitotic HeLa cells and describe here the purification and characterization of human conventional kinesin and two human kinesin-related proteins, HSET and CENP-E. We found that the 120-kDa HeLa cell conventional kinesin is an active motor that induces microtubule gliding at approximately 30 microm/min at room temperature. This active form of HeLa cell kinesin does not contain light chains, although light chains were detected in other fractions. HSET, a member of the C-terminal kinesin subfamily, was also purified in native form for the first time, and the protein migrates as a single band at approximately 75 kDa. The purified HSET is an active motor that induces microtubule gliding at a rate of approximately 5 microm/min, and microtubules glide for an average of 3 microm before ceasing movement. Finally, we purified native CENP-E, a kinesin-related protein that has been implicated in chromosome congression during mitosis, and we found that this form of CENP-E does not induce microtubule gliding but is able to bind to microtubules.     

A single step purification for recombinant proteins. Characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase.

A 167 base pair DNA cassette has been constructed to facilitate the detection and purification of recombinant proteins. This cassette, kfc, encodes three distinct peptide units: a phosphorylation site for the cAMP-dependent protein kinase (PKA), called kemptide, a factor Xa cleavage site, and a calmodulin-binding peptide. Expressed kfc fusion proteins can be purified from bacterial lysates in one step by affinity chromatography on calmodulin-agarose using EGTA as eluant. As a test of this system, we describe the expression, purification and characterization of the PKA binding domain of the microtubule associated protein (MAP 2).     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.     

Lipid-protein ratios by infrared spectroscopy

A method for measuring the ratio of diacyl phospholipid to protein in lipid-protein mixtures and membranes by infrared spectroscopy is described. Samples made of diacyl phospholipid and proteins mixed in known ratios were analyzed for lipid-protein ratio by the infrared (ir) method. Results had a standard deviation of less than +/- 4% over the lipid-protein molar ratio range of 9:1 to 320:1. Calculations of the ratio of total lipid to protein require that the diacyl phospholipid-to-protein ratio be divided by the mole fraction of diacyl phospholipid in the total lipid. Phospholipid-protein ratios for various sarcoplasmic reticulum membrane preparations (R1-washed, octylglucoside purified, deoxycholate treated) were determined by the ir method and compared to literature values. Also, phospholipid-protein ratios were determined for R1-washed sarcoplasmic reticulum by three chemical analyses using different protein assays and were compared with ratios obtained by the infrared method. The infrared results were closest to those of a chemical method designed specifically for membrane proteins.     

Factors implicated in determining the structure of zinc tubulin-sheets: Lateral tubulin–tubulin interaction is promoted by the presence of zinc

Addition of increasing amounts of zinc to a cold microtubule protein solution results in the disappearance of 30 S oligomer found in the absence of that cation and in the appearance of new tubulin oligomers, 90 S and 23 S. When a microtubule protein solution is warmed in the presence of zinc, tubulin-sheets are assembled. We have tested the influence of microtubule associated proteins and the zinc:tubulin ratio on the polymerization process. Depletion of microtubule associated proteins results in wider and longer tubulin-sheets than those polymerized in the presence of microtubule associated proteins. However by increasing zinc concentration wider but shorter tubulin-sheets were found. These results suggest that microtubule associated proteins and zinc could promote nucleation of tubulin-sheets, but zinc also promotes lateral tubulin-tubulin interaction. This interpretation was confirmed when microtubule protein was assembled at a low zinctubulin ratio. In such conditions composite structures of microtubules and zinc tubulin-sheets arc formed. These composite structures are consequence of a lateral attachment of a zinc tubulin-sheet on a microtubule protofilament.