Individual microtubules viewed by immunofluorescence and electron microscopy in the same PtK2 cell

PtK2 cells were grown on gold grids and treated with Triton X-100 in a microtubule stabilizing buffer. The resulting cytoskeletons were fixed with glutaraldehyde and subjected to the indirect immunofluorescence procedure using monospecific tubulin antibodies. Grids were examined first by fluorescence microscopy, and the display of fluorescent cytoplasmic microtubules was recorded. The grids were then stained with uranyl acetate and the display of fibrous structures recorded by electron microscopy. Thus the display of cytoplasmic microtubular structures in the light microscope and the electron microscope can be compared within the same cytoskeleton. The results show a direct correspondence of the fluorescent fibers in the light microscope with uninterrupted fibers of diameter approximately 550 A in the electron microscope. This is the diameter reported for a single microtubule decorated around its circumference by two layers of antibody molecules. Thus under optimal conditions immunofluorescence microscopy can visualize individual microtubules.     

Substructure of thermally unfolded chromatin subunits or nucleosomes

Mononucleosomes containing 143 ± 6 base pairs of duplex DNA and approximately two copies each of the histones H2a, H2b, H3 and H4 were examined during thermal denaturation by high resolution electron microscopy using both bright- and dark-field (tilted beam) modes. Co-operative destabilization and unfolding of the 13.2 ± 1.4 nm diameter toroids occurred only after the second of the two major melting transitions. The unfolding patterns are consistent with about 1.5–1.8 turns of supercoiled DNA in intact nucleosomes. The dominant unfolded structure of samples post-fixed with glutaraldehyde is a 17.5 ± 2.1 nm diameter open ring. Both sister DNA strands remain associated with protein. The distribution and shape of the protein patches are more irregular in unfixed, unstained samples visualized by darkfield microscopy. Image reconstruction studies on fixed and stained ring-shaped specimens indicates that there are 6–10 globular protein elements or patches, each about 3.9 ± 0.5 nm in diameter, per DNA moiety.     

Stability of microtubule protein over the pH range: 6.9--9.5.

The pH stability range of a microtubule protein preparation has been investigated between 6.9 and 9.5. Microtubule protein was exposed to various pH values in this range and then returned to pH 6.9. The appearance of microtubules as verified by electron microscopy and sedimentation analysis under polymerizing conditions was taken as an indication of a conformationally stable protein. Between pH 6.9 and pH 8.0 the loss in the ability to form microtubules was found to be reversible, at pH 8.2 it was partially reversible, above pH 8.2 it was irreversible. Tubulin and the microtubule-associated protein fraction were separately exposed to high pH. It was observed that tubulin exposed to high pH can still form microtubules in the presence of untreated microtubule-associated protein. On the other hand, microtubule-associated protein exposed to high pH could not initiate microtubule assembly with untreated tubulin. It was concluded from these observations that the loss in the ability of a microtubule protein preparation to assemble at high pH is due to a change in the microtubule-associated protein fraction and that tubulin is conformationally stable even after exposure to pH 9.5.     

浮游病毒的电镜观察

直接用戊二醛固定水样中的浮游病毒,通过超速离心使已固定的浮游病毒沉淀到覆有Formvar膜和碳支持膜的铜网上,经醋酸双氧铀染色后,利用透射电镜对湖水中的浮游病毒进行观察．结果可观察到球形、杆状和蝌蚪状等形态各异的浮游病毒颗粒及球形病毒的囊膜子粒、杆状病毒的核衣壳、具有不同尾部的蝌蚪状病毒等的精细超微结构．从而建立了一种简便、快捷和高效研究浮游病毒的电镜方法．     

Biochemical and Structural Analyses of Microtubules in the Pellicular Membrane of Leishmania tropica1

The structure of the major protein of the pellicular membrane of Leishmania tropica was investigated. This protein is composed of two polypeptides, of ca. 50,000 d molecular weight, that were found to cross-react immunologically with the α and β subunits of pig brain tubulin. The polypeptides and pig brain tubulin subunits were partially digested with S. aureus V8 protease, and the peptides obtained analysed by SDS-polyacrylamide gel electrophoresis. A comparison of the patterns showed that the β subunits of Leishmania and pig tubulin have very similar primary structures, while the α subunits have evolved divergently. These experiments demonstrate that the major polypeptides found in the pellicular membrane of L. tropica are α and β subunits of tubulin. Immuno-electron microscopy indicates that the tubulin is located in the microtubules associated with the pellicular membrane of Leishmania. Arrays of microtubules were prepared by nonionic detergent treatment of the cells and observed by electron microscopy after negative staining. Optical diffraction reveals a 5 nm spacing between protofilaments in the microtubule and a 4 nm axial periodicity corresponding to the tubulin subunits. The pitch of the shallow left-hand three-start helix is 12°. A distance of 47 nm separates each microtubule from the next. These data show that the dimensions and supramolecular organization of the tubulin subunits in the microtubules are identical in the pellicular membrane of L. tropica and in mammalian brain.     

The cryptophycin-tubulin ring structure indicates two points of curvature in the tubulin dimer

Cryptophycin-1 is the parent compound of a group of cyclic peptides with potent antineoplastic activity. Cryptophycins are thought to function by modulating the dynamic instability of spindle microtubules, and in vitro are known to bind in an equimolar ratio to the beta-tubulin subunit and to induce the formation of ring-like complexes. However, the detailed mechanisms whereby the cryptophycins interact with tubulin are not known. We have investigated the origin of the conformational changes in tubulin both biochemically and by electron microscopy and image analysis. Cryptophycin was found to protect both alpha- and beta-tubulin against proteolysis by trypsin, indicating conformational changes in specific regions of both subunits. The ring mass was determined to be approximately 0.81 MDa by sedimentation velocity combined with dynamic light scattering and by STEM, indicating a complex of eight alphabeta dimers. Statistical analysis of rings imaged by cryoelectron microscopy revealed 16-fold symmetry, corresponding to eight dimers. Computational averaging based on this symmetry yielded an image of a 24 nm diameter ring, at 2.6 nm resolution, that clearly distinguishes intradimer contacts from interdimer contacts, and allows discrimination of alpha-subunits from beta-subunits. Fitting of the tubulin dimer crystal structure into this projected density map indicates two points of curvature: a 13 degrees intradimer bend and a 32 degrees interdimer bend. We conclude that drug binding to one subunit (beta) results in two bends per dimer, affecting both subunits.     

Arabidopsis tubulin folding cofactor B interacts with alpha-tubulin in vivo

Microtubule biogenesis requires alphabeta tubulin dimers that are generated from alpha and beta tubulin following post-translational modification by several tubulin folding cofactors (TFCs). Here we report the isolation and characterization of Arabidopsis TFCB (AtTFCB). AtTFCB is expressed in all organs of Arabidopsis. The subcellular localization of AtTFCB is mainly cytosolic. AtTFCB-overexpressing cells have fewer microtubules compared with the controls. Multimode fluorescence resonance energy transfer (FRET) microscopy reveals a direct physical interaction of AtTFCB with alpha tubulin in living plant cells. We conclude that AtTFCB interacts with alpha tubulin in vivo and its overexpression reduces the number of microtubules.     

Microtubule nucleation

Microtubule nucleation is the process in which several tubulin molecules interact to form a microtubule seed. Microtubule nucleation occurs spontaneously in purified tubulin solutions, and molecular intermediates between tubulin dimers and microtubules have been identified. Microtubule nucleation is enhanced in tubulin solutions by the addition of gamma-tubulin or various gamma-tubulin complexes. In vivo, microtubule assembly is usually seeded by gamma-tubulin ring complexes. Recent studies suggest, however, that microtubule nucleation can occur in the absence of gamma-tubulin, and that gamma-tubulin may have other cell functions apart from being a major component of the gamma-tubulin ring complex.     

EFFECTS OF LIDOCAINE ON RABBIT BRAIN MICROTUBULAR PROTEIN1

Microtubule repolymerization from a crude supernatant fraction prepared from rabbit brain was followed quantitatively by viscometry and electron microscopy. Lidocaine inhibits this repolymerization in a dose-dependent fashion and the effect is completely reversible upon removal of the lidocaine by dialysis. Direct counting of microtubules by electron microscopy indicates that the local anesthetic decreases the number of tubules without significantly affecting their length. An association constant of lidocaine for tubulin is estimated at 7.5 mm . Procaine and etidocaine were also found to affect the polymerization of microtubules with results similar to those found with lidocaine.     

Structural intermediates in the assembly of taxoid-induced microtubules and GDP-tubulin double rings: time-resolved X-ray scattering.

We have studied the self-association reactions of purified GDP-liganded tubulin into double rings and taxoid-induced microtubules, employing synchrotron time-resolved x-ray solution scattering. The experimental scattering profiles have been interpreted by reference to the known scattering profiles to 3 nm resolution and to the low-resolution structures of the tubulin dimer, tubulin double rings, and microtubules, and by comparison with oligomer models and model mixtures. The time courses of the scattering bands corresponding to the different structural features were monitored during the assembly reactions under varying biochemical conditions. GDP-tubulin essentially stays as a dimer at low Mg(2+) ion activity, in either the absence or presence of taxoid. Upon addition of the divalent cations, it associates into either double-ring aggregates or taxoid-induced microtubules by different pathways. Both processes have the formation of small linear (short protofilament-like) tubulin oligomers in common. Tubulin double-ring aggregate formation, which is shown by x-ray scattering to be favored in the GDP- versus the GTP-liganded protein, can actually block microtubule assembly. The tubulin self-association leading to double rings, as determined by sedimentation velocity, is endothermic. The formation of the double-ring aggregates from oligomers, which involves additional intermolecular contacts, is exothermic, as shown by x-ray and light scattering. Microtubule assembly can be initiated from GDP-tubulin dimers or oligomers. Under fast polymerization conditions, after a short lag time, open taxoid-induced microtubular sheets have been clearly detected (monitored by the central scattering and the maximum corresponding to the J(n) Bessel function), which slowly close into microtubules (monitored by the appearance of their characteristic J(0), J(3), and J (n) - (3) Bessel function maxima). This provides direct evidence for the bidimensional assembly of taxoid-induced microtubule polymers in solution and argues against helical growth. The rate of microtubule formation was increased by the same factors known to enhance taxoid-induced microtubule stability. The results suggest that taxoids induce the accretion of the existing Mg(2+)-induced GDP-tubulin oligomers, thus forming small bidimensional polymers that are necessary to nucleate the microtubular sheets, possibly by binding to or modifying the lateral interaction sites between tubulin dimers.     

Direct incorporation of microtubule oligomers at high GTP concentrations

Chick brain microtubule protein consists primarily of a mixture of MAP2:tubulin oligomers and dimeric tubulin. The assembly of this protein is described by a single pseudofirst-order reaction at 20 microM GTP, but by the summation of two pseudofirst-order reactions at 1 mM GTP. The protein contains two GTP-binding species, corresponding to the tubulin dimers and the oligomers, and conditions which alter the dimer: oligomer equilibrium, affect the kinetics of microtubule assembly. The results indicate that the oligomers are only direct assembly intermediates at high GTP concentrations.     

The use of glutaraldehyde and tannic acid to preserve reconstituted collagen for electron microscopy

The effects of glutaraldehyde and tannic acid on the axial periodicity of collagen have measured. Both fixatives produce axial shrinkage of the collagen but whereas glutaraldehyde produces 7% shrinkage, tannic acid produces only 2% shrinkage. The technique of carbon/platinum shadowing was used to estimate the extent to which the collagen fibrils flatten down when they are dried onto grids for electron microscopy without prior embedding and sectioning. The influence of fixation was studied and it was found that minimum distortion occurred when both tannic acid and glutaraldehyde were used to preserve the protein structure.     

The use of glutaraldehyde and tannic acid to preserve reconstituted collagen for electron microscopy

Summary The effects of glutaraldehyde and tannic acid on the axial periodicity of collagen have been measured. Both fixatives produce axial shrinkage of the collagen but whereas glutaraldehyde produces 7% shrinkage, tannic acid produces only 2% shrinkage. The technique of carbon/platinum shadowing was used to estimate the extent to which the collagen fibrils flatten down when they are dried onto grids for electron microscopy without prior embedding and sectioning. The influence of fixation was studied and it was found that minimum distortion occurred when both tannic acid and glutaraldehyde were used to preserve the protein structure.     

In situ electron microscopic observation of negatively stained tissue culture cells contaminated with mycoplasmas

A simple, fast, and in situ method of detecting the inapparent infection of cultured cells with mycoplasmas is reported. Animal cells grown on Formvar-coated electron microscopic grids were directly fixed with glutaraldehyde, negatively stained with phosphotungstic acid and examined by transmission electron microscopy. Cells contaminated with mycoplasmas could be discriminated from uncontaminated cells. The micro-organisms in the negatively stained preparations corresponded with those revealed by thin sectioning, and the distribution of mycoplasmas in cultured cells coincided with those revealed by the Hoechst staining method. Most of the highly resolved mycoplasmas were polymorphic, and closely associated with host cells; often more than 500 organisms per host cell were seen.     

Changes in the hydrodynamic properties of microtubules induced by taxol

Microtubule assembly was followed and monitored by (1) the turbidity at 350 nm, (2) the weight of the pelleted microtubules, (3) linear dichroism, LD tau, of the turbidity upon flow orientation, (4) the specific viscosity, eta spec, and (5) electron microscopy. These five methods showed the same features for normal microtubule assembly, but were different in the presence of taxol, a drug which binds to tubulin. The The apparent steady state of microtubule assembly in the presence of taxol as found by turbidity or the weight of pelleted polymer did not represent a stable state, as both LD tau and eta spec continued to change for a much longer time. Microtubules assembled in the presence of taxol from microtubule proteins as well as from purified tubulin were difficult to orient, as high flow gradients were needed and the maximal LD tau value represented only 20% of the LD tau for normal microtubules. In contrast to the slow relaxation of normal microtubules, rapid relaxation to random orientation was found in the presence of taxol. Low orientability was also indicated by electron micrographs, in which pelleted microtubules were seen to be randomly oriented in the presence of taxol. Taxol induced a very high eta spec, 4-times the steady-state value in the initial phase of assembly, which slowly declined again to a steady state, an effect which was also found for assembly of purified tubulin assembled in the absence of the microtubule-associated proteins. The presence of taxol did not change the relative amount and composition of the microtubule-associated proteins in the assembled microtubules. The results therefore suggest that taxol alters the hydrodynamic properties of the microtubules due to its interaction with tubulin and that this alteration is not an effect of the microtubule-associated proteins.     

Direct observation of microtubules with the scanning tunneling microscope

To observe surface topography of microtubules, we have applied scanning tunneling microscopy (STM), which can image metal and semiconductive surfaces with atomic resolution. Isolated microtubules fixed in 0.1% glutaraldehyde in reassembly buffer containing 0.8 M glycerol were imaged in air on a graphite substrate. The presence of microtubules in solution was verified by electron microscopy. At atmospheric pressure and room temperature, STM probing of both freeze-dried and hydrated microtubules reveals structures approximately 25 nm in width, consisting of longitudinal filaments about 4 nm in width. These structures match electron microscopy images of microtubules and their component protofilaments. Microtubules imaged by STM frequently appear buckled and semiflattened. Top-view shaded scans show what appear to be individual tubulin subunits within protofilaments. We believe these results represent the first direct STM observation of protein assemblies in which components can be identified. Although the microtubule image resolution described here is no better than that presently obtainable by other techniques (e.g., electron microscopy with freeze-drying, shadowing, and/or negative staining), it is significant that suitably prepared biomolecules may be sufficiently conductive and stable for STM imaging, which is ultimately capable of atomic resolution. Further development of STM technology, computer-enhanced image processing, and elucidation of optimal STM sample preparation indicate that STM and related applications will offer unique opportunities for the study of biomolecular surfaces.     

Cytoskeletal architecture of isolated mitotic spindle with special reference to microtubule-associated proteins and cytoplasmic dynein

We have studied cytoskeletal architectures of isolated mitotic apparatus from sea urchin eggs using quick-freeze, deep-etch electron microscopy. This method revealed the existence of an extensive three- dimensional network of straight and branching crossbridges between spindle microtubules. The surface of the spindle microtubules was almost entirely covered with hexagonally packed, small, round button- like structures which were very uniform in shape and size (approximately 8 nm in diameter), and these microtubule buttons frequently provided bases for crossbridges between adjacent microtubules. These structures were removed from the surface of microtubules by high salt (0.6 M NaCl) extraction. Microtubule- associated proteins (MAPs) and microtubules isolated from mitotic spindles which were mainly composed of a large amount of 75-kD protein and some high molecular mass (250 kD, 245 kD) proteins were polymerized in vitro and examined by quick-freeze, deep-etch electron microscopy. The surfaces of microtubules were entirely covered with the same hexagonally packed round buttons, the arrangement of which is intimately related to that of tubulin dimers. Short crossbridges and some longer crossbridges were also observed. High salt treatment (0.6 M NaCl) extracted both 75-kD protein and high molecular weight proteins and removed microtubule buttons and most of crossbridges from the surface of microtubules. Considering the relatively high amount of 75- kD protein among MAPs isolated from mitotic spindles, it is concluded that these microtubule buttons probably consist of 75-kD MAP and that some of the crossbridges in vivo could belong to MAPs. Another kind of granule, larger in size (11-26 nm in diameter), was also on occasion associated with the surface of microtubules of mitotic spindles. A fine sidearm sometimes connected the larger granule to adjacent microtubules. Localization of cytoplasmic dynein ATPase in the mitotic spindle was investigated by electron microscopic immunocytochemistry with a monoclonal antibody (D57) against sea urchin sperm flagellar 21S dynein and colloidal gold-labeled second antibody. Immunogold particles were closely associated with spindle microtubules. 76% of these were within 50 nm and 55% were within 20 nm from the surface of the microtubules. These gold particles were sporadically found on both polar and kinetochore microtubules of half-spindles at both metaphase and anaphase. They localized also on the microtubules between sister chromatids in late anaphase. These data indicate that cytoplasmic dynein is attached to the microtubules in sea urchin mitotic spindles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of C-terminal tyrosine from tubulin and microtubules at steady state.

Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase.     

Arrangement of high molecular weight associated proteins on purified mammalian brain microtubules

The arrangement of the high molecular weight proteins associated with the walls of reconstituted mammalian brain microtubules has been investigated by electron microscopy of negatively stained preparations. The images are found to be consistent with an arrangement whereby the high molecular weight molecules are spaced 12 tubulin dimers apart, i.e., 960 A, along each protofilament of the microtubule, in agreement with the relative stoichiometry of tubulin and high molecular weight protein. Molecules on neighbouring protofilaments seem to be staggered so that they give rise to a helical superlattice, which can be superimposed on the underlying tubulin lattice. In micrographs of disintegrating tubules there is some indication of lateral interactions between neighbouring high molecular weight molecules. When the microtubules are depolymerized into a mixture of short spirals and rings, the high molecular weight proteins appear to remain attached to their respective protofilaments.     

Microtubule nucleation from stable tubulin oligomers

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.