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Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.

Vogel JM, Stearns T, Rieder CL, Palazzo RE - J. Cell Biol. (1997)

Bottom Line: To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes.A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes.Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

View Article: PubMed Central - PubMed

Affiliation: The Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045, USA.

ABSTRACT
Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

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In vitro aster formation using centrosome-containing  sucrose fractions. Aliquots of centrosome fractions were reconstituted with tubulin media to assemble asters. (A) Aster-forming  activity was quantified using polarized light microscopy. (B) Asters  were composed of microtubules (green) stained by β-tubulin antibody TU27, which project from discrete central foci (red) stained  by γ-tubulin antibody EAD24. (C) EM analysis revealed that  centrosome fractions contained bona fide centrosomes composed  of centrioles (arrows) surrounded by a mass of pericentriolar material ∼2 μm in diameter. Bar, (A and B) 10 μm; (C) 2.0 μm.
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Figure 2: In vitro aster formation using centrosome-containing sucrose fractions. Aliquots of centrosome fractions were reconstituted with tubulin media to assemble asters. (A) Aster-forming activity was quantified using polarized light microscopy. (B) Asters were composed of microtubules (green) stained by β-tubulin antibody TU27, which project from discrete central foci (red) stained by γ-tubulin antibody EAD24. (C) EM analysis revealed that centrosome fractions contained bona fide centrosomes composed of centrioles (arrows) surrounded by a mass of pericentriolar material ∼2 μm in diameter. Bar, (A and B) 10 μm; (C) 2.0 μm.

Mentions: Centrosomes were isolated from Spisula oocyte lysates by a discontinuous two-step sucrose gradient centrifugation system, consisting of 66 and 52.5% sucrose steps, similar to that used for isolation of mammalian centrosomes (Mitchison and Kirschner, 1984). The centrosome content of gradient fractions was determined using a functional reconstitution assay based on the ability of centrosomes to organize Mts into astral arrays (Mitchison and Kirschner, 1984; Palazzo et al., 1988, 1992). Thus, aliquots of gradient fractions were diluted into tubulin-containing media, and aster formation was visualized by polarized light microscopy (see Fig. 2 A).


Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.

Vogel JM, Stearns T, Rieder CL, Palazzo RE - J. Cell Biol. (1997)

In vitro aster formation using centrosome-containing  sucrose fractions. Aliquots of centrosome fractions were reconstituted with tubulin media to assemble asters. (A) Aster-forming  activity was quantified using polarized light microscopy. (B) Asters  were composed of microtubules (green) stained by β-tubulin antibody TU27, which project from discrete central foci (red) stained  by γ-tubulin antibody EAD24. (C) EM analysis revealed that  centrosome fractions contained bona fide centrosomes composed  of centrioles (arrows) surrounded by a mass of pericentriolar material ∼2 μm in diameter. Bar, (A and B) 10 μm; (C) 2.0 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139867&req=5

Figure 2: In vitro aster formation using centrosome-containing sucrose fractions. Aliquots of centrosome fractions were reconstituted with tubulin media to assemble asters. (A) Aster-forming activity was quantified using polarized light microscopy. (B) Asters were composed of microtubules (green) stained by β-tubulin antibody TU27, which project from discrete central foci (red) stained by γ-tubulin antibody EAD24. (C) EM analysis revealed that centrosome fractions contained bona fide centrosomes composed of centrioles (arrows) surrounded by a mass of pericentriolar material ∼2 μm in diameter. Bar, (A and B) 10 μm; (C) 2.0 μm.
Mentions: Centrosomes were isolated from Spisula oocyte lysates by a discontinuous two-step sucrose gradient centrifugation system, consisting of 66 and 52.5% sucrose steps, similar to that used for isolation of mammalian centrosomes (Mitchison and Kirschner, 1984). The centrosome content of gradient fractions was determined using a functional reconstitution assay based on the ability of centrosomes to organize Mts into astral arrays (Mitchison and Kirschner, 1984; Palazzo et al., 1988, 1992). Thus, aliquots of gradient fractions were diluted into tubulin-containing media, and aster formation was visualized by polarized light microscopy (see Fig. 2 A).

Bottom Line: To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes.A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes.Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

View Article: PubMed Central - PubMed

Affiliation: The Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045, USA.

ABSTRACT
Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

Show MeSH