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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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IVEM-tomography of centrosomes. (A) IVEM micrograph, (B) 2.6-nm-thick slice, and (C–F) rendered projections through  the volume of a tomographic reconstruction from a 0.25-μm section through a Spisula centrosome isolated from a 4-min extract. C and D  and E and F show highly magnified sections from this tomogram in untilted (C and E) and tilted (D and F) views. Numerous ringshaped structures (e.g., arrowheads), completely contained within the section, are found throughout the centrosome, including regions  near the centriole (C and D) and near its periphery (E and F). A 2.6-nm-thick slice from a second tomogram, obtained from a 0.25-μmthick section cut from one end of the centriole in an isolated centrosome, is pictured in G and at higher magnification in H. Note that a  conspicuous electron-opaque “vacuole” is associated with the centriolar end. Bars: (A, B, and G) 0.50 μm; (C–F, and H) 250 nm.
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Figure 3: IVEM-tomography of centrosomes. (A) IVEM micrograph, (B) 2.6-nm-thick slice, and (C–F) rendered projections through the volume of a tomographic reconstruction from a 0.25-μm section through a Spisula centrosome isolated from a 4-min extract. C and D and E and F show highly magnified sections from this tomogram in untilted (C and E) and tilted (D and F) views. Numerous ringshaped structures (e.g., arrowheads), completely contained within the section, are found throughout the centrosome, including regions near the centriole (C and D) and near its periphery (E and F). A 2.6-nm-thick slice from a second tomogram, obtained from a 0.25-μmthick section cut from one end of the centriole in an isolated centrosome, is pictured in G and at higher magnification in H. Note that a conspicuous electron-opaque “vacuole” is associated with the centriolar end. Bars: (A, B, and G) 0.50 μm; (C–F, and H) 250 nm.

Mentions: IVEM analyses of serial semi-thick sections confirmed our original finding (Palazzo et al., 1992) that centrosomes isolated from Spisula oocytes shortly after activation contain a single centriole embedded in the middle of an extensive sphere of PCM (e.g., Fig. 3, A and G). When viewed by tomography, this PCM was seen to be studded throughout its volume with numerous, conspicuous ring-shaped structures (Fig. 3, C–F, arrowheads). The depth (z-axis dimension) of each ring was less than its 25–28-nm diameter, and most were found to be completely contained within the 150–200-nm-thick (after irradiation) volume of each tomogram. These rings were similar, if not identical, to those recently detected by IVEM tomography in the PCM of isolated Drosophila centrosomes (Moritz et al., 1995a). IVEM also revealed that the great majority of centrosomes isolated from Spisula oocytes shortly after activation contained an unusual vesicular structure at the base of the centriole (Fig. 3, G and H). It is possible that this structure is involved in assembling the second centriole, which we have previously shown to occur near this time point.


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)

IVEM-tomography of centrosomes. (A) IVEM micrograph, (B) 2.6-nm-thick slice, and (C–F) rendered projections through  the volume of a tomographic reconstruction from a 0.25-μm section through a Spisula centrosome isolated from a 4-min extract. C and D  and E and F show highly magnified sections from this tomogram in untilted (C and E) and tilted (D and F) views. Numerous ringshaped structures (e.g., arrowheads), completely contained within the section, are found throughout the centrosome, including regions  near the centriole (C and D) and near its periphery (E and F). A 2.6-nm-thick slice from a second tomogram, obtained from a 0.25-μmthick section cut from one end of the centriole in an isolated centrosome, is pictured in G and at higher magnification in H. Note that a  conspicuous electron-opaque “vacuole” is associated with the centriolar end. Bars: (A, B, and G) 0.50 μm; (C–F, and H) 250 nm.
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Related In: Results  -  Collection

Show All Figures
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Figure 3: IVEM-tomography of centrosomes. (A) IVEM micrograph, (B) 2.6-nm-thick slice, and (C–F) rendered projections through the volume of a tomographic reconstruction from a 0.25-μm section through a Spisula centrosome isolated from a 4-min extract. C and D and E and F show highly magnified sections from this tomogram in untilted (C and E) and tilted (D and F) views. Numerous ringshaped structures (e.g., arrowheads), completely contained within the section, are found throughout the centrosome, including regions near the centriole (C and D) and near its periphery (E and F). A 2.6-nm-thick slice from a second tomogram, obtained from a 0.25-μmthick section cut from one end of the centriole in an isolated centrosome, is pictured in G and at higher magnification in H. Note that a conspicuous electron-opaque “vacuole” is associated with the centriolar end. Bars: (A, B, and G) 0.50 μm; (C–F, and H) 250 nm.
Mentions: IVEM analyses of serial semi-thick sections confirmed our original finding (Palazzo et al., 1992) that centrosomes isolated from Spisula oocytes shortly after activation contain a single centriole embedded in the middle of an extensive sphere of PCM (e.g., Fig. 3, A and G). When viewed by tomography, this PCM was seen to be studded throughout its volume with numerous, conspicuous ring-shaped structures (Fig. 3, C–F, arrowheads). The depth (z-axis dimension) of each ring was less than its 25–28-nm diameter, and most were found to be completely contained within the 150–200-nm-thick (after irradiation) volume of each tomogram. These rings were similar, if not identical, to those recently detected by IVEM tomography in the PCM of isolated Drosophila centrosomes (Moritz et al., 1995a). IVEM also revealed that the great majority of centrosomes isolated from Spisula oocytes shortly after activation contained an unusual vesicular structure at the base of the centriole (Fig. 3, G and H). It is possible that this structure is involved in assembling the second centriole, which we have previously shown to occur near this time point.

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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