Serological Similarity of Flagellar and Mitotic Microtubules

Fulton, Chandler; Kane, Robert E.; Stephens, R. E.

doi:10.1083/jcb.50.3.762

Cited by 75 publications

(15 citation statements)

References 44 publications

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“…Studies using colchicine have shown that microtubules from many different sources bind to this drug with similar affinities, implying the conservation of at least one common site (4,(7)(8)(9). Immunological studies have given ambiguous results; some of these studies suggest the presence of a microtubular antigen in a wide variety of organisms (10,11) and another study suggests that microtubular antigens may vary widely from one organism to another (12).…”

mentioning

confidence: 96%

Isolation and Partial Characterization of α- and β-Tubulin from Outer Doublets of Sea-Urchin Sperm and Microtubules of Chick-Embryo Brain

Ludueña

Woodward

1973

Proc. Natl. Acad. Sci. U.S.A.

145

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Two kinds of tubulin (a and ,3) have been described in microtubules from many different systems. In this study a discontinuous acrylamide-gel system containing sodium dodecyl sulfate was used to separate milligram quantities of a-and fl-tubulin from microtubules of chick-embryo brain and from outer doublets of sea-urchin sperm. The isolated tubulins were characterized by peptide mapping and automated sequencing of the first 25 NH2-terminal amino acids. Our results show that a-and #-tubulin are related but distinctly different proteins and that each one has been highly conserved in the course of evolution.The structural subunit of microtubules is a 100,000-120,000 molecular weight dimer of two 55,000-60,000 molecular weight monomers (1). Recent evidence suggests that there may be at least two kinds of tubulin monomers in microtubules, although it is not clear how they are arranged within the microtubular structure. When microtubules from various sources are solubilized and subjected to electrophoresis on acrylamide gels containing urea or Na dodecyl sulfate, two bands are obtained which contain proteins differing in amino-acid composition and in cyanogen bromide and tryptic peptide maps (2-6).Despite the morphological similarity of microtubules from different sources, it is not known how closely tubulin monomers from different organisms and different types of microtubules resemble each other. Studies using colchicine have shown that microtubules from many different sources bind to this drug with similar affinities, implying the conservation of at least one common site (4,(7)(8)(9). Immunological studies have given ambiguous results; some of these studies suggest the presence of a microtubular antigen in a wide variety of organisms (10, 11) and another study suggests that microtubular antigens may vary widely from one organism to another (12).A major obstacle to analyzing the differences between the two forms of tubulin and between tubulins from different sources has hitherto been the difficulty in separating and isolating them in milligram quantities. We report here a method for isolating preparative amounts of each tubulin species on a discontinuous Na dodecyl sulfate gel system, using as sources microtubules from embryonic chick brain and outer doublets of sea-urchin sperm tails. The amino-acid sequences of the two tubulins differ considerably, but relatedness is apparent from their primary structure. Comparison of corresponding tubulin species from each organism indicated that the primary structure of each type of tubulin is strongly conserved in evolution.MATERIAL AND METHODS Preparation of Microtubules. Outer doublets were isolated from sperm flagella of several hundred male sea urchins of the species Strongylocentrotus purpuratus by the method of Stephens (13).. Brain microtubule protein was the generous gift of Dr. Leslie Wilson. It was prepared from 16-to 18-day-old chick-embryo brains by the method of Bryan and Wilson (4).Protein Was Determined by the method of Lowry et al. (14) as modified by Ba...

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confidence: 96%

Isolation and Partial Characterization of α- and β-Tubulin from Outer Doublets of Sea-Urchin Sperm and Microtubules of Chick-Embryo Brain

Ludueña

Woodward

1973

Proc. Natl. Acad. Sci. U.S.A.

145

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“…The suggestion that these differences in stability indicate different tubulin compositions (Behnke and Forer, 1967) has been supported by biochemical (Stephens, 1970) and serological studies (Fulton et al, 1971) and recently Olmsted et al (1971) have clearly demonstrated the presence of distinctive tubulins in cytoplasmic (neurotubules) and flagellar microtubules. It is possible that the differences in stability of flagellar and cytoplasmic microtubules in Ochromonas, and the differential sensitivity of the beak and rhizoplast sets of cytoplasmic tubules, might also reflect unique tubulin compositions.…”

Section: The Independent Roles Of Beak and Rhizoplast Microtubulesmentioning

confidence: 87%

MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS

Brown

Bouck

1973

The Journal of Cell Biology

109

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A B S T R A C TThe proposal made in the preceding paper that the species-specific shape of Ochromonas is mediated by cytoplasmic microtubules which are related to two nucleating sites has been experimentally verified. Exposure of cells to colchicine or hydrostatic pressure causes microtubule disassembly and a correlative loss of cell shape in a posterior to anterior direction. Upon removal of colchicine or release of pressure, cell shape regenerates and microtubules reappear, first in association with the kineto-beak site concomitant with regeneration of the anterior asymmetry, and later at the rhizoplast site concomitant with formation of the posterior tail. It is concluded that two separate sets of cytoplasmic tubules function in formation and maintenance of specific portions of the total cell shape. On the basis of the following observations, we further suggest that the beak and rhizoplast sites could exert control over the position and timing of the appearance, the orientation, and the pattern of microtubule distribution in Ochromonas. (a) the two sites are accurately positioned in the cell relative to other cell organelles; (b) in regenerating cells microtubules reform first at these sites and appear to elongate to the cell posterior; (c) microtubules initially reappear in the orientation characteristic of the fully differentiated cell; (d) the two sets of tubules are polymerized at different times, in the same sequence, during reassembly or resynthesis of the microtubular system. Experiments using cycloheximide, after a treatment with colchicine, have demonstrated that Ochromonas cannot reassume its normal shape without new protein synthesis. This suggests that microtubule protein once exposed to colchicine cannot be reassembled into microtubules. Pressure-treated cells, on the other hand, reassemble tubules and regenerate the normal shape in the presence or absence of cycloheximide. The use of these two agents in analyzing nucleating site function and the independent processes of synthesis and assembly of microtubules is discussed.The preceding paper (Bouck and Brown, 1973) examines the fine structure of vegetative and dividing cells of Ochromonas and describes, (a) a close correlation between the distribution of cytoplasmic microtubules and the asymmetric cell shape, and (b) the association of these microtubules with two distinct sites in the cell anterior. These observations implied a cytoskeletal role for microtubules and further suggested that control of microtubule distribution, and hence control of cell shape, may be effected by restricting microtubule assembly to specific nucleating sites (terminology of Tilney and Goddard, 1970).The present study was undertaken to determine if the observed microtubule distribution is obligatory to development and/or maintenance of cell shape, as well as to clarify whether the presumptive nucleating sites do in fact function as sites of initial microtubule assembly. It was found that microtubule depolymerizing agents (colchicine 860

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“…Antibodies produced against different classes of tubulin have been found to show high degree of crossreactivity with tubulins from other sources (Dales et al 1973;Fulton et al 1971;Weber et al 1975). Morgan et al (1978) examined the antigenic similarities between tubulin of brain from lamb, mouse and chick embryo, using rabbit antisera prepared against each of these tubulins.…”

Section: Discussionmentioning

confidence: 99%

Extraction and immunochemical assays of a tubulin-like factor in cotton seedlings

Rikin

Atsmon

Gitler

1982

Planta

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Antibodies to tubulin were prepared in rabbits by immunization with reduced-carboxymethylated calf-brain tubulin. In immunodiffusion tests the antibodies showed full cross reactivity with the immunogen as well as with native calf-brain tubulin. The same antibodies showed cross reactivity with a factor in extract of cotton (Gossypium hirsutum L.) cotyledons but there was no full immunological identity between calf-brain tubulin and this factor. A solid-phase radioimmunoassay for quantitative estimation of this plant tubulin-like factor was developed. It measured the binding of antibodies to immobilized calf-native tubulin. Competition between the unknown soluble tubulin-like factor, and immobilized tubulin was assayed at serum dilution of 1:50. Extraction conditions which preserved the antigenic properties of the tubulin-like factor from cotton cotyledons were defined. The radioimmunoassay measured quantities of the tubulin-like factor in the range of 0.1-10 μg-equivalents of calf-brain tubulin. Immediately after homogenization of the tissue only 25% of the total amount of tubulin-like activity was present in soluble form, while most of it remained in the insoluble fraction. Apparent maximal solubilization was achieved spontaneously 10 h after homogenization or by treatment with guanidine hydrochloride. These results indicate that in this material, tubulin is not released immediately by homogenization but remains assembled in microtubules and-or in a bound or sequestered form.

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Serological Similarity of Flagellar and Mitotic Microtubules

Abstract: An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers .

Cited by 75 publications

References 44 publications

Isolation and Partial Characterization of α- and β-Tubulin from Outer Doublets of Sea-Urchin Sperm and Microtubules of Chick-Embryo Brain

Isolation and Partial Characterization of α- and β-Tubulin from Outer Doublets of Sea-Urchin Sperm and Microtubules of Chick-Embryo Brain

MICROTUBULE BIOGENESIS AND CELL SHAPE IN OCHROMONAS

Extraction and immunochemical assays of a tubulin-like factor in cotton seedlings

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