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At least 19 recordsLinked to original sources
PubMed1977

Binding of adenovirus to microtubules. II. Depletion of high-molecular-weight microtubule-associated protein content reduces specificity of in vitro binding.

A specific in vitro association between adenovirus and pruified rat brain microtubules has been previously demonstrated (R. B. Luftig and R. R. Weihing, 1975). When examined by negative-staining electron microscopy, approximately 90% of the virus associated with microtubules was edge bound, i.e., associated within +/-4 nm of the microtubule edge. Similar results are now found for the association of adenovirus with purified chick brain microtubules. When the content of the high-molecular-weight proteins (MAPs) normally present as projections on the surface of microtubules is depleted by fractionation of cold-depolymerized microtubules on agarose A-15M columns or by brief treatment of polymerized microtubules with trypsin, the percentage of edge-bound microtubule-associated viruses is reduced to a level close to that found for particles such as reovirus, coliphage f2, or polystyrene latex spheres, which randomly associate with microtubules (54 to 64% for column-fractionated microtubules; 45 to 68% for trypsin-treated microtubules). Counts of adenovirus particles specifically bound to microtubules, corrected for variations in microtubule and virus concentrations, gave values 2.5 to 3.5 times higher for unfractionated microtubules than for microtubule-associated protein-depleted microtubules. These results are consistent with the suggestion that the specific association between adenovirus and microtubules is mediated by microtubule-associated proteins.

Adenoviruses, Human↗
PubMed1977

The oral apparatus of Tetrahymena pyriformis, strain WH-6. IV. Observations on the organization of microtubules and filaments in the isolated oral apparatus and the differential effect of potassium chloride on the stability of oral apparatus microtubules.

This report is an ultrastructural analysis of the organization of the isolated oral apparatus of Tetrahymena pyriformis, strain WH-6, syngen 1. Attention has been focused on the organization of microtubules and filaments in oral apparatus membranelles. Oral apparatus membranellar basal bodies were characterized with respect to structural differentiations at the distal and proximal ends. The distal region of membranellar basal bodies contains the basal plate, accessory microtubules and filaments. The proximal end contains a dense material from which emanate accessory microtubules and filaments. There are at least two possibly three different arrangements of accessory structures at the proximal end of membranellar basal bodies. All membranellar basal bodies appear to have a dense material at the proximal end from which filaments emanate. Some of these basal bodies have accessory microtubules and filaments emanating from this dense material. A possible third arrangement is represented by basal bodies which have lateral projections, from the proximal end, of accessory microtubules and filaments which constitute cross or peripheral connectives. There are at least three examples of direct associations between oral apparatus microtubules and filaments: (1) filaments which form links between basal body triplet microtubules, (2) filaments which link the material of the basal plate to internal basal body microtubules, (3) filaments which link together microtubule bundles from membranellar connectives. KCl extraction of the isolated oral apparatus resulted in the selective solubilization of oral apparatus basal bodies, remnants of ciliary axonemes and fused basal plates. Based on their response to KCl extraction two distinct sets of morphologically similar micro tubules can be identified: (a) microtubules which constitute the internal structure of basal bodies and ciliary axonemes, (b) microtubules which constitute the fiber connectives between basal bodies.

Animals↗
PubMed1979

Differences in surface morphology of microtubules reconstituted from pure brain tubulin using two different microtubule-associated proteins: the high molecular weight MAP 2 proteins and tau proteins.

Microtubules were reconstituted from homogeneous brain tubulin and homogeneous preparations of two different microtubule associated proteins, the high molecular weight MAP 2 proteins or the tau proteins. The resulting microtubules were characterized by three electron microscopical procedures: Thin sectional analysis of embeded material, negative staining analysis using a STEM microscope and high resolution metal-shadowing analysis. By all three procedures MAP 2 microtubules have a much rougher surface morphology than tau microtubules, in agreement with the much higher molecular weight of the MAP 2 proteins. Tau microtubules, however, do not show the very smooth surface of microtubules assembled from pure tubulin in the absence of any microtubule associated proteins. In the case of MAP 2 microtubules thin sectional analysis as well as metal shadowing reveals that the globular protrusions seen in negative staining analysis appear as linear side arms which may extend by as much as 30 nm on both sides from the microtubular wall proper, giving rise to an overall structure with a diameter close to 100 nm. The possible implication of such structures for in vivo situations is briefly discussed as is the possibility that the "halo-effect" around microtubules seen in vivo may be due to a structural organization similar to that of MAP 2 tubules in vitro.

Animals↗
PubMed1979

Identification of the major 68,000-dalton protein of microtubule preparations as a 10-nm filament protein and its effects on microtubule assembly in vitro.

The major 68,000-dalton protein present in cycled microtubule preparations from bovine brain can be isolated in a rapidly sedimenting fraction consisting of filaments 10 nm in diameter. This 68,000-dalton protein remains in the filament fraction after gel filtration, phosphocellulose chromatography, or salt extraction of microtubule protein. Microtubule protein devoid of 10-nm filaments contains ring structures under depolymerizing conditions, and it polymerizes into microtubules with a characteristically low critical concentration, although all of the 68,000-dalton protein has been removed from it. When cycled microtubule protein is subjected to chromatography on phosphocellulose, the tubulin fraction (PC-tubulin) assembles into microtubules only at concentrations greater than 2 mg/mL. The other fraction, eluted from phosphocellulose at high ionic strength, contains the major 68,000-dalton protein and can be further resolved into two components by centrifugation. The supernatant, which consists mainly of high molecular weight microtubule-associated proteins, stimulates low concentrations of PC-tubulin to assemble. The pellet contains all of the 68,000-dalton protein, consists of 10-nm filaments, and does not stimulate assembly of PC-tublin. Boiling of purified filaments, however, releases several proteins, including the 68,000-dalton protein, and these released proteins stimulate the assembly of PC-tubulin. The morphology and protein composition of the filaments isolated from microtubule preparations by these techniques are very similar to those of mammalian neurofilaments. These results suggest that the major 68,000-dalton protein in cycled microtubule preparations, which may correspond to tubulin assembly protein [Lockwood, A.H. (1978) Cell 13, 613--627], is a constituent of neurofilaments.

Animals↗
PubMed1978

The role of spindle pole bodies and modified microtubule ends in the initiation of microtubule assembly in Saccharomyces cerevisiae.

The spindle poles of the budding yeast, Saccharomyces cerevisiae, have been removed from mitotic and meiotic cells by osmotic lysis of spheroplasts. The spindle pole bodies (SPBs)--diskoidal structures also termed 'spindle plaques'--have been analysed for their ability to potentiate the polymerization of microtubules in vitro. Free SPBs were completely deprived of any detectable native microtubules by incubation in the absence of added tubulin and were then challenged with chick neurotubulin, which had been rendered partially defective in self-initiation of repolymerization. Electron microscopy revealed that these SPBs served as foci for the initiation of microtubule polymerization in vitro. Because the attached microtubules elongated linearly with time but did not increase in numbers after the first stage of the reaction, it is apparent that there are a limited number of sites for initiation. The initiating potential of the SPBs was found to be inhibited by enzymic hydrolysis of protein but not of DNA. The microtubule end proximal to the site of initiation on the SPB is distinguished by a 'closed' appearance because of a terminal component which is continuous with the microtubule wall, whereas the distal end has the 'open' appearance characteristic of freely repolymerized neurotubules. SPBs which were partially purified on sucrose gradients retained their ability to initiate the assembly of microtubules with the same structural differentiation of their ends. The occurrence of closed proximal ends on native yeast microtubules suggests that closed ends may play a role in the initiation of microtubule polymerization in vivo, as well as in vitro.

Cell Differentiation↗
PubMed1979

The mechanism of microtubule associated cytoplasmic transport. Isolation and preliminary characterisation of a microtubule transport system.

The nutritive tubes of telotrophic insect ovaries are cytoplasmic channels along which ribosomes are transported over distances of several mm from trophic cells to the developing oocytes. The presence within the nutritive tubes of a massive number of orientated microtubules renders them strongly birefringent in polarised light, a property which, together with their size, rendered them amenable to isolation by microdissection. Ultrastructurally the isolated tubes were indistinguishable from undissected controls. Polyacrylamide gels revealed a consistent pattern of some 30 bands of which tubulin was the most prominent. The tubes also contained a band which comigrated with the major high molecular weight microtubule associated protein (MAP) from mouse brain but no detectable actin, myosin or dynein. Microtubules in the isolated tubes were not depolymerised by treatments (cold, calcium and colchicine) which typically disrupt cytoplasmic microtubules. Following extraction of the membrane enclosing the tubes and the cytoplasmic matrix the microtubule cytoskeleton persisted, retaining its cylindrical organisation although no bridges between the microtubules were detected in the electron microscope. The possibility that the stability and spatial deployment of the nutritive tube microtubules is conferred by specific microtubule accessory proteins is discussed.

Animals↗
PubMed1976

The fine structure of a microplate-microtubule array, microfilaments and polyhedral body associated microtubules in several species of Anabaena.

A microplate-microtubule array was observed in Anabaena sp. (B-378). This structure consists of an arched plate, about 8 nm thick, and various microtubules, 12 nm in diameter and 50 nm long, arranged in rows. The microtubules project at right angles from one side of the plate into the cytoplasm or towards the plasma membrane. Up to twelve microplate-microtubule arrays were observed in a single section of a cell. Microfilaments, about 2.8 nm in diameter and of undetermined length, were observed in four isolates of Anabaena. The microfilaments were always found in bundles, nwhich varied in size, up to 0.63 mum across and 0.91 mum long. Microtubules, 10 nm in diameter and about 150 nm in length, were observed associated with one facet of polyhedral bodies in 8 out of 20 isolates of Anabaena. The microtubules occurred in groups of up to 20 or more, and were always oriented with the long axis parallel to the facet of a polyhedral body. In cross section, the microtubules had an electron transparent lumen 5 nm wide and a wall 2.5 nm thick. These structures are compared to previously described microtubules and microfilaments.

Cyanobacteria↗
PubMed1979

Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins.

In this paper we describe a procedure for detecting proteins associated with cytoplasmic microtubules in vivo. Detergent-extracted cytoskeletons of NIL8 hamster cells are prepared under conditions which preserve the microtubules. The cytoskeletons are then extracted in the presence of calcium, which depolymerizes the microtubules and quantitatively extracted cytoskeletons are prepared from cells that have been incubated with colchicine. The cytoskeletons from these cells contain no microtubules or tubulin. Electrophoretic analysis of the calcium extracts of the colchicine-treated and untreated cells reveals several radioactively labeled polypeptides. There is, however, no apparent quantitative or qualitative difference between the two extracts other than the tubulin polypeptides. Each of the extracts is mixed with an excess of unlabeled calf brain microtubule protein and carried through cycles of temperature-dependent microtubule assembly. Distinct species from each extract co-assemble at a constant ratio, but only one polypeptide is uniquely derived from cells containing intact microtubules. The molecular weight of this polypeptide is similar to that proposed for the tau species detected in brain microtubule preparations.

Animals↗
PubMed1977

Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly.

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.

Chlamydomonas↗
PubMed1975

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

High-molecular-weight components (HMW) specifically associated with microtubule protein purified from porcine brain tissue were separated from tubulin by DEAE-Sephadex ion exchange chromatography. Analysis by viscometry, sedimentation, and electron microscopy of the unfractionated microtubule protein, separated HMW and tubulin fractions, and reconstituted mixtures showed that HMW promoted formation of ring structures at 5 degrees and tubule polymerization at 37 degrees. The HMW reassociated with tubulin and was identified in thin sections as 18.9 x 5.6 nm projections attached to the microtubules with a longitudinal periodicity of 32.5 nm. These studies: (1) indicate that the HMW fraction stimulates microtubule assembly by facilitating the formation of ring structures which are apparently intermediates in polymerization, and (2) demonstrate that the HMW associates with microtubules as a structural component projecting from the surface of the microtubule wall.

Animals↗
PubMed1977

In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol.

Microtubule protein from porcine cerebrum was fractionate into pure tubulin and microtubule-associated proteins by chromatography on phosphocellulose. In agreement with previous studies, pure tubulin does not form microtubules to a significant extent at 37 degrees in normal assembly buffers, which are characterized by a low concentration of Mg2+ ions. If, however, the Mg2+ concentration is raised to approximately 10 mM, rapid and extensive self-assembly of pure tubulin into microtubules is observed, provided the tubulin concentration is above 2.5 mg/ml. At a protein concentration of 3 mg/ml, the lag period is 1.5 min and the assembly process is virtually complete after 6 min at 37 degrees. These microtubules are like normal microtubules--sensitive to calcium ions, colchicine, and low temperature.

Animals↗
PubMed1979

Purification of cytoplasmic tubulin and microtubule organizing center proteins functioning in microtubule initiation from the alga Polytomella.

Cytoplasmic tubulin and the microtubule organizing centers (MTOCs) for the cytoskeletal microtubule system of the flagellate Polytomella have been isolated. The isolated MTOCs serve as sites for the in vitro assembly of the purified tubulin protein. The major proteins (four polypeptides of molecular weights 190,000-210,000) functioning in this assembly have been extracted from the MTOCs and purified. Kinetic studies and experiments with in vivo 35S-labeled MTOC proteins (or 35S-labeled tubulin) demonstrate that these proteins function specifically in microtubule initiation and do not contribute to microtubule elongation. The results indicate that microtubule assembly in vivo is controlled by microtubule initiating proteins associated with the organelles termed MTOCs.

Carrier Proteins↗
PubMed1979

Effect of microtubule-associated proteins on the interaction of vincristine with microtubules and tubulin.

The influence of microtubule-associated proteins on the interaction of vincristine with microtubules and tubulin was examined. Microtubules containing associated proteins were prepared in vitro from tubulin preparations which contained the proteins or from pure 6S tubulin and isolated microtubule-associated proteins. The presence of the associated proteins caused microtubules to be converted to stable spiral structures upon reacting with vincristine. When the proteins were absent, spirals were not formed, and the microtubules were completely disassembled by vincristine. At 0 degrees, 6S tubulin was converted to amorphous aggregates by vincristine, whereas if the associated proteins were present spirals were formed.

Animals↗
PubMed1977

Control of shape and pattern during the assembly of a large microtubule bundle. Evidence for a microtubule-nucleating-template.

Microtubules are packed and linked together in a well defined hexagonal arrangement in the cytopharyngeal microtubule bundles of the ciliate Nassula. Early stages in the morphogenesis of these bundles have been examined. Elements which nucleate assembly of bundle microtubules are apparently closely associated before tubule assembly commences. These nucleating elements seem to be bound together in highly ordered arrays to form microtubule-nucleating-templetes. Each array of elements is attached to the proximal end of a basal body and appears to establish the pattern of tubule packing and cross-sectional shape of a tubule bundle. A self-assembly procedure which accounts for the anisometric growth and shaping of a template and its microtubule bundle is proposed.

Animals↗
PubMed1979

Self-assembly of microtubules in extracts of cultured HeLa cells and the identification of HeLa microtubule-associated proteins.

Microtubule protein from HeLa cell extracts was purified by multiple cycles of polymerization and depolymerization in the absence of glycerol or other exogenous polymerization-stimulatory agents. Approximately 4-5% of the extract protein was tubulin, of which more than one-half was competent to participate in polymerization-depolymerization cycles. The purified HeLa microtubule protein preparations contained 95% tubulin after the second cycle of polymerization and depolymerization. Additional protein species bound specifically to and copurified quantitatively with microtubules throughout at least four cycles of polymerization and depolymerization. These microtubule-associated proteins (MAPs) were separated from tubulin by DEAE column chromatography. When added to purified brain or HeLa tubulin, these MAPs stimulated the polymerization of microtubules as assayed by electron microscopy and a quantitative sedimentation assay. The most prominent HeLa MAPs had molecular weights of approximately 210,000 and 120,000.

HeLa Cells↗
PubMed1975

Microtubules and axoplasmic transport. Inhibition of transport by podophyllotoxin: an interaction with microtubule protein.

Pharmacological evidence is presented for the involvement of microtubules in the process of fast axoplasmic transport. A quantitative measure of the inhibition of axoplasmic transport in an in vitro preparation of rat sciatic nerve is described. The alkaloids colchicine, podophyllotoxin, and vinblastine, which are known both to disrupt microtubules and to bind to the protein subunit of microtubules, are inhibitors of axoplasmic transport. Lumicolchine and picropodophyllin, unlike their respective isomers colchicine and podophyllotoxin, are poor inhibitors of axoplasmic transport. The dissociation constants for the binding of colchicine, lumicolchicine, podophyllotoxin, and picropodophyllin to purified microtubule protein from rat brain have been measured. Inhibition of axoplasmic transport by these drugs correlates favorably with their affinities of microtubule protein.

Animals↗
PubMed1976

The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187.

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.

Animals↗
PubMed1978

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins.

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.

Actins↗