Structural changes in tubulin sheets upon removal of microtubule-associated proteins

Zinc-induced tubulin sheets without microtubule-associated proteins (MAPs) were assembled from tubulin purified by phosphocellulose chromatography. Large, open sheets were obtained in five-minute incubations at pH 5.7. Electron micrographs of negatively stained sheets showed a protofilament arrangement similar to that observed for zinc-induced sheets with MAPs but with altered lattice parameters. The spacings measured from optical diffraction patterns demonstrated that the protofilaments were 2.2 A closer together in the sheets without MAPs. Each MAP-free sheet was also divided roughly in half by a discontinuity which was parallel to the protofilaments and the relationship between the two domains was deduced from computed transforms. Two-dimensional image processing was carried out by conventional Fourier techniques and by correlation analysis. The correlation analysis improved the reconstructions in this application, with the resolution limited by the inherent properties of the negative stain method to about 14 A. A prominent feature of the computed reconstructions was an alternation of light and dark protofilaments due to differential staining, as revealed by a study of folded sheets. Neighboring protofilaments are related by a 2-fold screw axis, as they are in zinc-induced sheets with MAPs, but the symmetry is masked by the differential staining. The major effect of MAP removal on the structure of the sheets is that the bilobed structure of alternate tubulin subunits is no longer observed. This observation and the closer spacing of protofilaments is consistent with the postulate that some of the MAP molecules lie in the groove between protofilaments and bind to several tubulin dimers.     

Human Microtubule-Associated-Protein Tau Regulates the Number of Protofilaments in Microtubules: A Synchrotron X-Ray Scattering Study

Microtubules (MTs), a major component of the eukaryotic cytoskeleton, are 25 nm protein nanotubes with walls comprised of assembled protofilaments built from αβ heterodimeric tubulin. In neural cells, different isoforms of the microtubule-associated-protein (MAP) tau regulate tubulin assembly and MT stability. Using synchrotron small angle x-ray scattering (SAXS), we have examined the effects of all six naturally occurring central nervous system tau isoforms on the assembly structure of taxol-stabilized MTs. Most notably, we found that tau regulates the distribution of protofilament numbers in MTs as reflected in the observed increase in the average radius 〈R^MT〉 of MTs with increasing Φ, the tau/tubulin-dimer molar ratio. Within experimental scatter, the change in 〈R^MT〉 seems to be isoform independent. Significantly, 〈R^MT〉 was observed to rapidly increase for 0 < Φ < 0.2 and saturate for Φ between 0.2-0.5. Thus, a local shape distortion of the tubulin dimer on tau binding, at coverages much less than a monolayer, is spread collectively over many dimers on the scale of protofilaments. This implies that tau regulates the shape of protofilaments and thus the spontaneous curvature C_o^MT of MTs leading to changes in the curvature C^MT (=1/R^MT). An important biological implication of these findings is a possible allosteric role for tau where the tau-induced shape changes of the MT surface may effect the MT binding activity of other MAPs present in neurons. Furthermore, the results, which provide insight into the regulation of the elastic properties of MTs by tau, may also impact biomaterials applications requiring radial size-controlled nanotubes.     

A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs)

The effect of the antimitotic drug taxol on the association of MAPs (microtubule-associated proteins) with microtubules was investigated. Extensive microtubule assembly occurred in the presence of Taxol at 37 degrees C. at 0 degrees C, and at 37 degrees C in the presence of 0.35 M NaCl, overcoming the inhibition of assembly normally observed under the latter two conditions. At 37 degrees C and at 0 degrees C, complete assembly of both tubulin and the MAPs was observed in the presence of Taxol. However, at elevated ionic strength, only tubulin assembled, forming microtubules devoid of MAPs. The MAPs could also be released from the surface of preformed microtubules by exposure to elevated ionic strength. These properties provided the basis for a rapid new procedure for isolating microtubules and MAPs of high purity from small amounts of biological material. The MAPs could be recovered by exposure of the microtubules to elevated ionic strength and subjected to further analysis. Microtubules and MAPs were prepared from bovine cerebral cortex (gray matter) and from HeLa cells. MAP 1, MAP2, and the tau MAPs, as well as species of Mr = 28,000 and 30,000 (LMW, or low molecular weight, MAPs) and a species of Mr = 70,000 were isolated from gray matter. Species identified as the 210,000 and 125,000 mol wt HeLa MAPs were isolated from HeLa cells. Microtubules were also prepared for the first time from white matter. All of the MAPs identified in gray matter preparations were identified in white matter, but the amounts of individual MAP species differed. The most striking difference in the two preparations was a fivefold lower level of MAP 2 relative to tubulin in white matter than in gray. The high molecular weigh MAP, MAP1, was present in equal ratio to tubulin in white and gray matter. These results indicate that MAP 1 and MAP2, as well as other MAP species, may have a different cellular or subcellular distribution.     

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.     

Reduction in Microtubule Dynamics In Vitro by Brain Microtubule-Associated Proteins and by a Microtubule-Associated Protein-2 Second Repeated Sequence Analogue

Abstract: Microtubule-associated protein (MAP) binding to assembled microtubules (MTs) can be reduced by the addition of polyglutamate without significant MT depolymerization or interference with MT elongation reactions. Ensuing polymer length redistribution in MAP-depleted MTs occurs on a time scale characteristic of that observed with MAP-free MTs. The redistribution phase occurs even in the absence of mechanical shearing and without appreciable effects from end-to-end annealing, as indicated by the time course of incremental changes in polymer length and MT number concentration. We also observed higher rates of MT length redistribution when the [MAP]/[tubulin] ratio was decreased. Together, these results demonstrate that MT length redistribution rates are greatly influenced by MAP content, and the data are compatible with the dynamic instability model. We also found that a peptide analogue corresponding to the second repeated sequence in the MT-binding region of MAP-2 can also markedly retard MT length redistribution kinetics, a finding that accords with the ability of this peptide to promote tubulin polymerization in the absence of MAPs and to displace MAP-2 from MTs. These results provide further evidence that MAPs can modulate MT assembly/disassembly dynamics and that peptide analogues can mimic the action of intact MAPs without the need for three contiguous repeated sequences in the MT-binding region.     

The in vitro assembly of flagellar outer doublet tubulin

Flagellar outer doublet microtubules were solubilized by use of sonication, and the tubulin was reassembled in vitro into single microtubules containing 14 and 15 protofilaments. The tubulin assembly was dependent on both the KCl and tubulin concentrations, exhibiting a critical concentration of 0.72 mg/ml at optimum solvent conditions. Flagellar tubulin was purified by cycles of temperature-dependent assembly-disassembly and molecular sieve chromatography, and characterized by two-dimensional gel electrophoresis. Although doublet microtubules were not formed in vitro, outer doublet tubulin assembled onto intact A- and B-subfibers of outer doublet microtubules and basal bodies of Chlamydomonas; the rate of assembly from the distal ends of these structures was greater than that from the proximal ends. Microtubule-associated proteins (MAPs) from mammalian brain stimulated outer doublet tubulin assembly, decorating the microtubules with fine filamentous projections.     

Assembly of tubulin from cultured cells and comparison with the neurotubulin model

Spontaneous microtubule assembly can be obtained in extracts from a variety of cultured cell lines by including glycerol in the assembly buffer. An analysis of the effects of cultured cell extracts on brain tubulin (neurotubulin) assembly has shown that the extracts contain initiation inhibitors whose effects are diminished by glycerol. By using glycerol during the assembly step, cultured cell tubulin can be purified by assembly-dissassembly procedures. The amount of glycerol necessary for significant spontaneous assembly varies from 1–6 M among the different cell lines and is dependent upon their content of inhibitor. Comparison of the assembly products obtained from NA, C₆ and CHO cells at increasing glycerol concentrations shows that glycerol enhances the purification of tubulin and a polypeptide of molecular weight 49,000 daltons in all three systems. These preparations contain a number of other polypeptides, including a group with gel electrophoretic mobilities characteristic of tau-factor, but lack the high molecular weight microtubule-associated proteins (MAPs) which are present in neurotubulin preparations. Phosphocellulose chromatography of NA tubulin removes several proteins from the tubulin and results in a loss of polymerizability. Among three proteins which are completely removed from the inactive tubulin, the most prominent is the 49K protein. This observation and the co-purification of the 49K protein with tubulin through two assembly-disassembly cycles suggest that it is a true MAP. The difference in MAP proteins between brain and tissue culture cells is parallelled by an absence of ring structures in NA tubulin preparations. NA tubulin, however, does form rings when brain MAPs are added. The early steps of NA tubulin assembly differ from those of neurotubulin; no rings are involved, and the first assembly intermediates are straight protofilament bundles. The differences between MAPs from cultured cells and brain and the absence of ring formation in NA tubulin preparations suggest that the assembly model based on neurotubulin is not completely general. A comparison of extracts from CHO cells grown with and without dibutyryl cAMP revealed no differences between the behavior of these extracts in spontaneous tubulin assembly or in mixture experiments with brain tubulin.     

Association of nucleosidediphosphate kinase with microtubules.

A nucleosidediphosphate kinase activity (EC 2.7.4.6) which phosphorylates GDP to GTP is present in bovine brain microtubule protein prepared by cycles of assembly-disassembly. This activity persists through 5 cycles of assembly-disassembly and sediments with microtubules in sucrose density gradients, but is not associated with the tubulin dimer. It is proposed that the kinase is an integral part of the microtubule and is therefore a microtubule associated protein (MAP). Several isozymes of nucleosidediphosphate kinase exist in our preparations with a pI 7.6 form predominant. It may be speculated that this enzyme affects tubulin assembly $\text{in vivo}$ by modulating the $\text{GTP}\text{GDP}$ ratio in the microtubule environment.     

Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.     

Promotion of MAP/MAP interaction by taxol

The effects of taxol on microtubule-associated proteins of high molecular weight (MAPs) were studied in vitro. After negative staining, microtubules reconstituted in the presence of taxol from preparations of partially purified tubulin and MAPs, besides being bundled, displayed prominent elongated or globular extensions without apparent regularity. These extensions, but not the tubulin polymer, were heavily decorated after immuno-gold-labeling using antibodies to MAP-1 and MAP-2. Microtubules reconsituted in the absence of taxol showed a much more regular, and apparently helical, arrangement of MAPs along their surfaces. The formation of polymeric structures was also observed when preparation of MAPs free of tubulin were incubated with taxol. In this case in addition to large network-type aggregates with little apparent substructure, more regular structures seemingly consisting of approximately 5-nm-thick filaments arrayed in parallel were observed. Taxol-induced MAP aggregation occurred rapidly and was directly proportional to the concentration of protein, as revealed by optical density measurements. It is concluded that taxol, aside from promoting the assembly of tubulin and stabilizing microtubules, promotes MAP/MAP interaction.     

The periodic association of MAP2 with brain microtubules in vitro

Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.     

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.     

Electron microscope studies of pH effects on assembly of tubulin free of associated proteins. Delineation of substructure by tannic acid staining

Bovine brain tubulin, purified by phosphocellulose chromatography (PC), was assembled in the presence of 10% dimethyl sulfoxide (DMSO), and the reaction was monitored turbidimetrically. Samples were fixed in glutaraldehyde-tannic acid after completion of polymerization, as indicated by no further change in absorbance, and then sectioned and studied electron microscopy, with special attention being given to the arrangement of protofilaments in the walls of formed elements. Samples of PC-tubulin were polymerized in buffer having various pH values from 6.0 to 7.7. At the lower pH values, only branched and flattened ribbons of protofilaments are formed. At intermediate values, the ribbons are unbranched, narrower, and more curved in cross section; complete microtubules are also seen. At the higher pH values, the predominate formed elements are complete microtubules. Most of the complete microtubules examined in this study had 14 wall protofilaments. The effect of pH on tubulin assembly was shown not to be an effect of DMSO. The dimers of associated protofilaments in ribbons and microtubules are conceptually viewed as having trapezoidal profiles in cross section, and, as additional dimers are added, the "C"-shaped ribbon closes to form a tube. The tilt angle of the lateral surfaces of the "trapezoidal" dimers will determine the number of wall protofilaments in the microtubules. At low pH, it is theorized that the trapezoidal profile of the dimer is shifted to a more rectangular configuration such that flat ribbons are formed by the lateral association of dimers. Also, variously shaped ribbon structures are formed at intermediate pH values, including "S"- and "W"-shaped structures, and elements shaped like a figure "6," all representing ribbons viewed in cross section. By visualizing the trapezoidal dimer in three-dimensions, and by arbitrarily indexing its six binding surfaces, it is possible to discuss interdimer binding in terms of preferred and possible binding interactions.     

Dependency of microtubule-associated proteins (MAPS) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules

Summary Microtubule-associated proteins (MAPS) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution.The addition of estramustine phosphate to microtubules reconstituted of MAPS prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4° C was dependent on intact bindings between the tubulin and MAPs.Abbreviations Pipes 1,4-Piperazinediethanesulfonic acid - EDTA Ethylenedinitrilo Tetraacetic Acid - MAPs Microtubule-Associated Proteins - SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis     

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4°C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60–68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

Promotion of Microtubule Assembly by Neurofilament-Associated Microtubule-Associated Proteins

Abstract: Intact neurofilaments (NF) purified from mammalian brain and spinal cord promote the assembly of microtubules in solutions of pure phosphocellulose (PC)-purified tubulin. This assembly is temperature-dependent and is inhibited by mitotic spindle inhibitors. The ability of NF to induce microtubule formation is 20% of that of purified microtubule-associated proteins (MAPs), whereas MAPs comprise less than 5% of the protein in the NF preparations. The inducing activity of NF is rapidly lost on boiling. When intact NF are incubated with PC-tubulin and then centrifuged, tubulin is sedimented together with the filaments. This association is inhibited by colchicine and podophyllotoxin and is cold-sensitive. NF purified to homogeneity under denaturing conditions and then reassembled completely lack the ability to promote the assembly of PC-tubulin or to bind tubulin on a centrifugation assay. No MAPs are present in these preparations, though these filaments have the ability to bind exogenous MAPs. While these experiments do not rule out an intrinsic microtubule-assembly-promoting activity, they suggest that this activity is due to nontriplet proteins in the preparation, most likely filament-associated MAPs.     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

Three-dimensional reconstruction of tubulin in zinc-induced sheets.

The three-dimensional structure of porcine brain tubulin in planar sheets formed in the presence of zinc has been determined to a resolution of approximately 20 Å by electron microscopy and image reconstruction on negatively stained samples. The samples were prepared with a mica floatation technique, which yields tubulin sheets with 36 reciprocal space maxima on lattice lines at 21, 28, 42 and 84 Å^?1 in Fourier transforms of digitized images. In order to obtain three-dimensional data, sheets were tilted with the goniometer stage of the electron microscope to provide images at various angles between 0 ° and ± 60 °. Transforms of 33 tilted images plus the transform of untilted sheets based on an average of nine untilted images were combined to give the third dimension of reciprocal space ($\text{z}{}^1$). These data, were expressed in terms of the phases and amplitudes along the $\text{z}{}^1$ lattice line for each of the 36 maxima observed in untilted samples, as well as five additional lattice lines which have zero-amplitudes in the non-tilted central section of the three-dimensional transform. Home of these zero-amplitudes arise from systematic absences which are due to a 2-fold screw axis relating adjacent protofilaments of tubulin in the zinc-induced sheets. Thus in the three-dimensional reconstructions of the sheets a polarity of the protofilaments is apparent, with adjacent protofilaments aligned in opposite directions to give an antiparallel pattern, in contrast to normal microtubules composed of protofilaments in parallel alignment. Two classes of morphological units, each with a mass corresponding to a molecular weight of about 55,000, are found to alternate along the protofilaments. These distinct morphological units are identified as the α and β subunits of tubulin, confirming the representation of tubulin as an αβ heterodimer. Furthermore, the extensive internal contact between subunits within a dimer can readily be distinguished from the less extensive contact between dimer units. Such differences in contacts were not apparent in the earlier two-dimensional reconstructions. In addition, areas of excluded stain joining one class of subunits to the subunits of the other class in adjacent protofilaments have been resolved for tubulin polymerized in zinc-induced sheets. Of the two classes of subunits one is distinguished by a prominent cleft. Identification of which class of subunits is α and which is β is not yet possible.     

Virions and the Coat Protein of the Potato Virus X Interact with Microtubules and Induce Tubulin Polymerization In Vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.     

Microtubule Assembly from Single Flared Protofilaments—Forget the Cozy Corner?

A paradigm shift for models of MT assembly is suggested by a recent cryo-electron microscopy study of microtubules (MTs). Previous assembly models have been based on the two-dimensional lattice of the MT wall, where incoming subunits can add with longitudinal and lateral bonds. The new study of McIntosh et al. concludes that the growing ends of MTs separate into flared single protofilaments. This means that incoming subunits must add onto the end of single protofilaments, forming only a longitudinal bond. How can growth of single-stranded protofilaments exhibit cooperative assembly with a critical concentration? An answer is suggested by FtsZ, the bacterial tubulin homolog, which assembles into single-stranded protofilaments. Cooperative assembly of FtsZ is thought to be based on conformational changes that switch the longitudinal bond from low to high affinity when the subunit is incorporated in a protofilament. This novel mechanism may also apply to tubulin assembly and could be the primary mechanism for assembly onto single flared protofilaments.