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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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Analysis of centrosome tubulins. Centrosome preparations were  analyzed by two-dimensional gel electrophoresis (A)  and quantitative immunoblot  (B–D). Three tubulin types  were found in centrosomes,  α, β, and γ (A). γ-Tubulin  has a major isoform with a pI  of 5.52 and is the major form  of tubulin found in centrosome fractions. SDS-gel  analysis of glutamate-purified Spisula oocyte tubulin  stained with Coomassie blue  (B, lane 1) provided an α- and  β-tubulin standard (B, lane  2). Stoichiometrically equivalent amounts of α- and β-tubulin (B, lane 1) were probed simultaneously with antibodies  specific to either α-tubulin  (DM1A) or β-tubulin (DM1B)  (B, lane 2). In these samples,  the α-tubulin–specific signal  is more intense than the  β-tubulin–specific signal, indicating that DM1A is a  more sensitive probe than  DM1B (B, lane 2). In contrast, probing centrosome fractions resulted in a higher β-tubulin–specific (DM1B) signal than the α-tubulin–specific (DM1A) signal  (B, lane 3). The tubulin content of centrosomes was determined using quantitative immunoblot analysis. (D) 5 μg of centrosome protein (lane 1) and series dilution of purified Spisula tubulin standards (lane 2, 1 mg/ml; lane 3, 0.3 mg/ml; lane 4, 0.1 mg/ml; lane 5, 0.03 mg/ ml; lane 6, 0.01 mg/ml) were separated in 10% polyacrylamide gels and processed for immunoblot analysis using DM1A and DM1B (see  Materials and Methods for details). The IOD of tubulin stain for centrosome samples and each standard lane in D was determined by  densitometry and plotted (C). The IOD of only the top band of the two stained by DM1B in D, lane 1, was used in this analysis, the  lower band being an artifact in this particular blot. The linear range (C, solid lines) for DM1A (C, filled squares) and DM1B (C, open  circles) ranges from 0.01 to 0.1 mg. The IOD obtained for the tubulin signals in the centrosome sample for both DM1A and DM1B lay  within the linear range of detection (C, arrows). First order regression analysis was used to calculate the tubulin content of centrosomes  (results presented in text and Table II). The r2 value for the regression for DM1A was 0.962, and for DM1B was 0.893.
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Figure 5: Analysis of centrosome tubulins. Centrosome preparations were analyzed by two-dimensional gel electrophoresis (A) and quantitative immunoblot (B–D). Three tubulin types were found in centrosomes, α, β, and γ (A). γ-Tubulin has a major isoform with a pI of 5.52 and is the major form of tubulin found in centrosome fractions. SDS-gel analysis of glutamate-purified Spisula oocyte tubulin stained with Coomassie blue (B, lane 1) provided an α- and β-tubulin standard (B, lane 2). Stoichiometrically equivalent amounts of α- and β-tubulin (B, lane 1) were probed simultaneously with antibodies specific to either α-tubulin (DM1A) or β-tubulin (DM1B) (B, lane 2). In these samples, the α-tubulin–specific signal is more intense than the β-tubulin–specific signal, indicating that DM1A is a more sensitive probe than DM1B (B, lane 2). In contrast, probing centrosome fractions resulted in a higher β-tubulin–specific (DM1B) signal than the α-tubulin–specific (DM1A) signal (B, lane 3). The tubulin content of centrosomes was determined using quantitative immunoblot analysis. (D) 5 μg of centrosome protein (lane 1) and series dilution of purified Spisula tubulin standards (lane 2, 1 mg/ml; lane 3, 0.3 mg/ml; lane 4, 0.1 mg/ml; lane 5, 0.03 mg/ ml; lane 6, 0.01 mg/ml) were separated in 10% polyacrylamide gels and processed for immunoblot analysis using DM1A and DM1B (see Materials and Methods for details). The IOD of tubulin stain for centrosome samples and each standard lane in D was determined by densitometry and plotted (C). The IOD of only the top band of the two stained by DM1B in D, lane 1, was used in this analysis, the lower band being an artifact in this particular blot. The linear range (C, solid lines) for DM1A (C, filled squares) and DM1B (C, open circles) ranges from 0.01 to 0.1 mg. The IOD obtained for the tubulin signals in the centrosome sample for both DM1A and DM1B lay within the linear range of detection (C, arrows). First order regression analysis was used to calculate the tubulin content of centrosomes (results presented in text and Table II). The r2 value for the regression for DM1A was 0.962, and for DM1B was 0.893.

Mentions: Two-dimensional gel analysis of centrosome proteins visualized with silver stain revealed that the major form of tubulin to copurify with centrosomes was γ-tubulin (Fig. 5 A). The staining intensity of γ-tubulin was higher than that observed for either α- or β-tubulin (Fig. 5 A). Densitometric analysis of silver-stained gels revealed that centrosomes contain ∼10-fold more γ-tubulin than β-tubulin, consistent with the tubulin ratios reported for γ-tubulin ring complexes isolated from Xenopus oocytes (Zheng et al., 1995). Surprisingly, the results of two-dimensional gel analysis suggested that centrosomes contain more β- than α-tubulin (Fig. 5 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)

Analysis of centrosome tubulins. Centrosome preparations were  analyzed by two-dimensional gel electrophoresis (A)  and quantitative immunoblot  (B–D). Three tubulin types  were found in centrosomes,  α, β, and γ (A). γ-Tubulin  has a major isoform with a pI  of 5.52 and is the major form  of tubulin found in centrosome fractions. SDS-gel  analysis of glutamate-purified Spisula oocyte tubulin  stained with Coomassie blue  (B, lane 1) provided an α- and  β-tubulin standard (B, lane  2). Stoichiometrically equivalent amounts of α- and β-tubulin (B, lane 1) were probed simultaneously with antibodies  specific to either α-tubulin  (DM1A) or β-tubulin (DM1B)  (B, lane 2). In these samples,  the α-tubulin–specific signal  is more intense than the  β-tubulin–specific signal, indicating that DM1A is a  more sensitive probe than  DM1B (B, lane 2). In contrast, probing centrosome fractions resulted in a higher β-tubulin–specific (DM1B) signal than the α-tubulin–specific (DM1A) signal  (B, lane 3). The tubulin content of centrosomes was determined using quantitative immunoblot analysis. (D) 5 μg of centrosome protein (lane 1) and series dilution of purified Spisula tubulin standards (lane 2, 1 mg/ml; lane 3, 0.3 mg/ml; lane 4, 0.1 mg/ml; lane 5, 0.03 mg/ ml; lane 6, 0.01 mg/ml) were separated in 10% polyacrylamide gels and processed for immunoblot analysis using DM1A and DM1B (see  Materials and Methods for details). The IOD of tubulin stain for centrosome samples and each standard lane in D was determined by  densitometry and plotted (C). The IOD of only the top band of the two stained by DM1B in D, lane 1, was used in this analysis, the  lower band being an artifact in this particular blot. The linear range (C, solid lines) for DM1A (C, filled squares) and DM1B (C, open  circles) ranges from 0.01 to 0.1 mg. The IOD obtained for the tubulin signals in the centrosome sample for both DM1A and DM1B lay  within the linear range of detection (C, arrows). First order regression analysis was used to calculate the tubulin content of centrosomes  (results presented in text and Table II). The r2 value for the regression for DM1A was 0.962, and for DM1B was 0.893.
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Figure 5: Analysis of centrosome tubulins. Centrosome preparations were analyzed by two-dimensional gel electrophoresis (A) and quantitative immunoblot (B–D). Three tubulin types were found in centrosomes, α, β, and γ (A). γ-Tubulin has a major isoform with a pI of 5.52 and is the major form of tubulin found in centrosome fractions. SDS-gel analysis of glutamate-purified Spisula oocyte tubulin stained with Coomassie blue (B, lane 1) provided an α- and β-tubulin standard (B, lane 2). Stoichiometrically equivalent amounts of α- and β-tubulin (B, lane 1) were probed simultaneously with antibodies specific to either α-tubulin (DM1A) or β-tubulin (DM1B) (B, lane 2). In these samples, the α-tubulin–specific signal is more intense than the β-tubulin–specific signal, indicating that DM1A is a more sensitive probe than DM1B (B, lane 2). In contrast, probing centrosome fractions resulted in a higher β-tubulin–specific (DM1B) signal than the α-tubulin–specific (DM1A) signal (B, lane 3). The tubulin content of centrosomes was determined using quantitative immunoblot analysis. (D) 5 μg of centrosome protein (lane 1) and series dilution of purified Spisula tubulin standards (lane 2, 1 mg/ml; lane 3, 0.3 mg/ml; lane 4, 0.1 mg/ml; lane 5, 0.03 mg/ ml; lane 6, 0.01 mg/ml) were separated in 10% polyacrylamide gels and processed for immunoblot analysis using DM1A and DM1B (see Materials and Methods for details). The IOD of tubulin stain for centrosome samples and each standard lane in D was determined by densitometry and plotted (C). The IOD of only the top band of the two stained by DM1B in D, lane 1, was used in this analysis, the lower band being an artifact in this particular blot. The linear range (C, solid lines) for DM1A (C, filled squares) and DM1B (C, open circles) ranges from 0.01 to 0.1 mg. The IOD obtained for the tubulin signals in the centrosome sample for both DM1A and DM1B lay within the linear range of detection (C, arrows). First order regression analysis was used to calculate the tubulin content of centrosomes (results presented in text and Table II). The r2 value for the regression for DM1A was 0.962, and for DM1B was 0.893.
Mentions: Two-dimensional gel analysis of centrosome proteins visualized with silver stain revealed that the major form of tubulin to copurify with centrosomes was γ-tubulin (Fig. 5 A). The staining intensity of γ-tubulin was higher than that observed for either α- or β-tubulin (Fig. 5 A). Densitometric analysis of silver-stained gels revealed that centrosomes contain ∼10-fold more γ-tubulin than β-tubulin, consistent with the tubulin ratios reported for γ-tubulin ring complexes isolated from Xenopus oocytes (Zheng et al., 1995). Surprisingly, the results of two-dimensional gel analysis suggested that centrosomes contain more β- than α-tubulin (Fig. 5 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
Related in: MedlinePlus