Porcine Brain Tubulin Research Articles

Immunofluorescence microscopy of organized microtubule arrays in structurally stabilized meristematic plant cells.

Cells were prepared for indirect immunofluorescence microscopy after paraformaldehyde fixation of multicellular root apices and brief incubation in cell wall-digesting enzymes. This allowed subsequent separation of the tissue into individual cells or short files of cells which were put onto coverslips coated with polylysine. Unlike spherical protoplasts made from living tissues, these preparations retain the same polyhedral shape as the cells from which they are derived. Cellular contents, including organized arrays of microtubules, are likewise structurally stabilized. Antibodies to porcine brain tubulin react with all types of microtubule array known to occur in plant meristematic cells, namely, interphase cortical microtubules, pre-prophase bands, the mitotic spindle, and phragmoplast microtubules. The retention of antigenicity in permeabilized, isolated, stabilized cells from typical, wall-enclosed plant cells has much potential for plant immunocytochemistry, and in particular should facilitate work on the role of microtubules in the morphogenesis of organized plant tissues.

Evidence for rapid structural and functional changes of the melanophore microtubule-organizing center upon pigment movements.

Melanophores of the angelfish, pterophyllum scalare, have previously been shown to display approximately 2,400 microtubules in cells wih pigment dispersed; these microtubules radiate from a presumptive organizing center, the central apparatus (CA), and their number is reduced to approximately 1,000 in the state with aggregated pigment (M. Schliwa and U. Euteneuer, 1978, J. Supramol. Struct. 8:177-190). In an attempt to elucidate the factors controlling this rapid reorganization of the microtubule apparatus, structure and function of the CA have been investigated under different physiological conditions. As a function of the state of pigment distribution, melanophores differ markedly with respect to CA organization. A complex of dense amorphous aggregates and associated fuzzy material, several micrometers in diameter, surrounds the centrioles in cells with pigment dispersed, and numerous microtubules emanate from this complex in a radial fashion. In the aggregated state, on the other hand, few microtubules are observed in the pericentiolar region, and the amount of fibrous material is greatly reduced. These changes in CA morphology as a function of the state of pigment distribution are associated with a marked difference in its capacity to initiatiate the assembly of microtubules from exogenous pure porcine brain tubulin in lysed cell preparations. After complete removal of preexisting microtubules, cells lysed in the dispersed state into a solution of 1-2 mg/ml pure tubulin have numerous microtubules associated with the CA in radial fashion, while cells lysed in the aggregated state nucleate the assembly of only a few microtubules. We conclude that it is the activity of the CA that basically regulates the expression of microtubules. This regulation is achieved through a variation in the capacity to initiate microtubule assembly. Increase or decrease in the amount of dense material, as readily observed in the cell system studied here, seems to be a morphologic expression of such a physiologic function.

Cytoskeletal proteins of the cell surface in Tetrahymena: I. Identification and localization of major proteins

Isolated pellicles (cell ‘ghosts’) have been prepared from Tetrahymena thermophila strain B by two different methods. Using differential solubilization in combination with polyacrylamide gel electrophoresis and electron microscopy, we have tentatively identified the major proteins found in the surface-associated cytoskeleton. The ‘epiplasm’, a continuous layer of fibrous material found just beneath the surface membranes, appears to contain two major proteins. The smaller of the two (mol. wt 122 000 D) is believed to be present throughout the layer, whereas the larger protein (mol. wt 145 000 D) appears to be localized in the regions where ciliary basal bodies connect to the epiplasmic layer and to surface membranes. Evidence is presented which suggests that actin may also be present in this structure. Tubulin has been isolated from the cytosol of Tetrahymena and compared with cytoskeletal tubulin and porcine brain tubulin. A major protein of mol. wt 250 000 D which is found in Tetrahymena pellicles appears to be the major component of kinetodesmal fibers (striated elements which attach to the ciliary basal bodies).

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 ( z ∗ ). These data, were expressed in terms of the phases and amplitudes along the z ∗ 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.

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence.

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.

Identification of tubulin from the yeast Saccharomyces cerevisiae.

A tubulin-like protein was identified in the lower eukaryote Saccharomyces cerevisiae. The following criteria were used: (i) copolymerization of the 35S-labeled yeast protein with porcine brain tubulin; (ii) immunoprecipitation of the 35S-labeled yeast protein with antiflagellar tubulin antibody; (iii) the presence of the yeast protein as a constituent of isolated yeast nuclei; and (iv) splitting of the yeast protein in a gel electrophoretic system containing sodium dodecyl sulfate that resolved the alpha- and beta-tubulin chains from other sources. This protein did not appear to have significant affinity for the plant alkaloid, Colcemid.

Identification of a gene for β-tubulin in aspergillus nidulans

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.

Quantitative initiation of microtubule assembly by chromosomes from Chinese hamster ovary cells

The microtubule nucleating capacity of chromosomes was tested in vitro in lysates of Chinese hamster ovary cells. Colcemid-blocked mitotic cells were lysed with the detergent Triton X-100, incubated with exogenous porcine brain tubulin, attached to electron microscope grids and observed as whole-mounts. Under suitable conditions, greater than 98% of the chromosomes gave rise to microtubules at their kinetochore regions, thus unequivocally demonstrating that chromosomes are competent to initiate specifically microtubule formation. The average number of microtubules that polymerized onto a chromosome was 8 ± 5, and greater than 36% of the chromosomes had between 10 and 19 microtubules per kinetochore region. We conclude that under the lysis conditions employed, virtually all the chromosomes retain their kinetochores, and that the kinetochores retain a substantial fraction of their microtubule nucleating capacity.

Indirect Immunofluorescence Microscopy of Microtubular Structures in Male Germ Cells of Wildtype and l(3)pl (lethal-polyploid) Drosophila hydei

Tubulin-containing structures of the male germ cells of Drosophila hydei crossreact in indirect immunofluorescence microscopy with antibody directed against homogeneous porcine brain tubulin. There is no detectable difference in reactivity between germ cells of wildtype flies and the mutant l(3)pl (lethal-polyploid) which is characterized by microtubular abnormalities. However, the technique of indirect immunofluorescence microscopy allows the direct visualization of several abnormalities in the arrangement of the microtubular system of the mutant, particularly in the axonemal complex.

Characterization and in vitro polymerization of Tetrahymena tubulin.

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.

Microtubule system of isolated fish melanophores as revealed by immunofluorescence microscopy.

The microtubule system of melanophores of the angelfish, Pterophyllum scalare, has been studied using antibodies prepared against purified porcine brain tubulin in indirect immunofluorescence microscopy. Melanophores were freed from the surrounding tissue components of isolated scales by mild enzymatic digestion and then allowed to settle on a glass cover slip. In both the dispersed and the aggregated states large numbers of fluorescent fibers are seen. The number and the astral arrangement of these fibers, which run from the central region to the periphery of the cell, are striking. The system of fluorescent fibers is replaced by diffuse fluorescence of moderate intensity after cold treatment, but is restored after rewarming the cells. Differences in the immunofluorescence profiles between cells with dispersed and aggregated pigment are discussed in relation to electron microscopic data available for this system.

Interactions of Tetrahymena dynein with microtubule protein. Tubulin-induced stimulation of dynein ATPase activity

The ATPase (EC 3.6.1.3) activity of 30 S dynein from Tetrahymena cilia was remarkably stimulated by porcine brain tubulin at pH 10. The activity increased with increasing concentration of tubulin until the molar ratio of tubulin dimer to 30 S dynein reached approx. 10. The optimum of the ATPase activity of 30 S dynein in the presence of tubulin was 1−2 mM for MgCl 2 and 2 mM for CaCl 2. Increasing ionic strength gradually inhibited the stimulation effects of tubulin. Activation energies of 30 S dynein in the presence and absence of tubulin were almost the same. At the temperatures beyond 25 °C stimulation effects of tubulin disappeared. ATP was a specific substrate even in the presence of tubulin. In kinetic investigations parallel reciprocal plots were observed in a constant ratio of divalent cations to ATP of 2, indicating that tubulin was less tightly bound to 30 S dynein in the presence of ATP than in the absence. The similar results were obtained at pH 8.2. 14 S dynein and the 12 S fragment which have poor ability to recombine with outer fibers were also activated with brain tubulin.

Structural studies on porcine brain tubulin in extended sheets

Structural studies have been conducted on porcine brain tubulin, assembled into microtubule-related structures, by electron microscopy in conjunction with optical diffraction and image reconstruction techniques. By minimizing background noise and sample damage, we have improved the resolution on negatively stained samples, extending the data from the previous limit of a 42 Å layer line to an additional layer line at 21 Å. The new reflections confirm the basic surface lattice proposed from the earlier studies and extend the structural features that can be assigned to individual tubulin molecules. Data are obtained for microtubules in standard buffers for both the helical form and flat sheets of up to 13 protofilaments. When zinc is added to the preparations, sheets with more than 13 protofilaments are formed and the extended lattices provide more reflections on both the 42 Å and 21 Å layer lines, as well as the equator. The lattice in the presence of zinc differs considerably from the normal lattice, with adjacent protofilaments staggered by 21 Å, compared to the staggering of adjacent filaments of about 10 Å in the absence of zinc. There is also a distinct pairing of adjacent protofilaments in the zinc-induced sheets. Initial studies with the Unwin-Henderson method on unstained zinc-tubulin sheets suggest that the adjacent protofilaments may be related by a dyad axis, either perpendicular or parallel to the protofilament axes.

Fluorescent Guanosine‐Nucleotide. Analogs Suitable for Photoaffinity‐Labeling Experiments

The synthesis and properties of strongly fluorescent derivative of inosine and its nucleotides are described. Reaction of 2-chloro-inosinic acid with sodium azide leads to a product bearing the tetrazole ring between position 2 and 3. Methylation at N1 was effected with dimethyl sulfate. The corresponding nucleosides and their 5'-triphosphates were also prepared. Only the non-methylated series is at equilibrium with small concentrations of their azido forms, and can be photolyzed by wavelengths above 300 nm. Both series are strongly fluorescent, their emission and excitation fluorescence spectra, as well as their quantum yields were measured. The nucleoside 5'-triphosphates are able to initiate and sustain polymerization of porcine brain tubulin.

Tubulin-like protein from [formula omitted

[ 35S] labeled extracts of the fungus Aspergillus nidulans were copolymerized with purified porcine brain tubulin. The [ 35S] A. nidulans protein which copurified with porcine microtubules was found to be similar to [ 3H] chick tubulin when the two were coelectrophoresed on several polyacrylamide gel electrophoresis systems. These results strongly suggest the presence in A. nidulans of a tubulin-like protein.

Quantitative electron microscopy of microtubule assembly in vitro.

The kinetics of microtubule assembly from purified porcine brain tubulin were examined by quantitative electron microscopy. Assembly at 37°C was initiated by the addition of GTP to 1 mm to the protein solution at ∼1 mg/ml in ammonium acetate buffer, pH 6·4. A solution of partially polymerized protein was mixed with a suspension of tomato bushy stunt virus at known particle concentration, and spray-deposited, in negative stain, upon specimen films. The resulting drop patterns were micrographed and the percent protein seen in distinguishable polymeric forms was calculated from the numbers, lengths, and known masses and mass/unit lengths of these entities. Clumping of protein by the uranyl acetate stain was avoided by use of a dual nebulizer which mixed protein and stain only upon spray droplet formation. The micrographs displayed ring-like forms, microtubules, and aggregates of parallel protofilaments—arrayed as flat, curved, and helical ribbons. Protein found as rings rapidly decreased from an initial 30% to a negligible amount at 2 minutes; the amount as microtubules increased asymptotically to an equilibrium value of 50% by 8 minutes; the quantity of ribbons was largest at 3 minutes (∼20%), but vanishingly small by 8 minutes. The results show that the fully formed microtubules consisted of protein which was initially both in the ring form and in the tubulin-dimer (6 S) form. They also quantitatively demonstrate that the twisted ribbons are assembly intermediates, those with a full protofilament complement presumably converting to microtubules by cylindrical folding.

A reexamination of the reaction between colchicine and tubulin.

The rate of reversible dissociation of the colchicine-tubulin complex has been determined for purified porcine brain tubulin and for sea urchin tubulin from 100,000 times g supernatant. The rate constant, which is essentially the same for both species, is extremely slow; it corresponds to a half-life of approximately 36 hours for the reaction. This rate was the same whether or not sucrose was present, and was not influenced by low concentrations of vinblastine or by millimolar concentrations of colchicine. The association rate constant was determined for porcine brain tubulin with and without CaCl2. The sample without CaCl2 had been maximally polymerized prior to the addition of colchicine. A 4.7-fold difference was observed between the association rate constants under these two conditions. Equilibrium constants calculated from the measured rate constants are 5 to 20 times greater than directly measured values; this suggests that the interaction of microtubules and colchicine is more complex than was previously thought.