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Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.

Oegema K, Wiese C, Martin OC, Milligan RA, Iwamatsu A, Mitchison TJ, Zheng Y - J. Cell Biol. (1999)

Bottom Line: Mitchison. 1995.The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity.Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. Karen.Omega@EMBL-Heidelburg.DE

ABSTRACT
gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

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Dgrip91 and Dgrip84 associate and colocalize with  γ-tubulin. (A) Clarified Drosophila embryo extract was immunoblotted for Dgrip91, Dgrip84, and γ-tubulin after separation on  10% SDS-PAGE gels (left). Each antibody recognizes a band of  the expected molecular weight. Both Dgrip91 and Dgrip84 comigrate with γ-tubulin on sucrose gradients of embryo extract  (right). (B) Immunofluorescence of early Drosophila embryos  with antibodies against Dgrip91, Dgrip84, and γ-tubulin. Both  Dgrip84 (top) and Dgrip91 (bottom) localize to centrosomes and  to the mitotic spindle in a fashion that is indistinguishable from  the localization of γ-tubulin. Examples of embryos in interphase  and mitosis are shown for each antibody. Bar, 15 μm.
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Figure 7: Dgrip91 and Dgrip84 associate and colocalize with γ-tubulin. (A) Clarified Drosophila embryo extract was immunoblotted for Dgrip91, Dgrip84, and γ-tubulin after separation on 10% SDS-PAGE gels (left). Each antibody recognizes a band of the expected molecular weight. Both Dgrip91 and Dgrip84 comigrate with γ-tubulin on sucrose gradients of embryo extract (right). (B) Immunofluorescence of early Drosophila embryos with antibodies against Dgrip91, Dgrip84, and γ-tubulin. Both Dgrip84 (top) and Dgrip91 (bottom) localize to centrosomes and to the mitotic spindle in a fashion that is indistinguishable from the localization of γ-tubulin. Examples of embryos in interphase and mitosis are shown for each antibody. Bar, 15 μm.

Mentions: If γ-tubulin in Drosophila embryos primarily exists associated with Dgrip84 and Dgrip91 in either γTuSC or γTuRC, we would expect these three proteins to cofractionate on sucrose gradients of embryo extract and to colocalize in embryos. To test this hypothesis, we raised and affinity-purified rabbit polyclonal antibodies that recognize Dgrip84 and Dgrip91. Each antibody recognizes a band of the expected molecular weight on Western blots of embryo extract (Fig. 7 A, left). As expected, both Dgrip84 and Dgrip91 comigrate with γ-tubulin in γTuSC and γTuRC when embryo extract is fractionated on sucrose gradients (Fig. 7 A, right). In addition, the localizations of Dgrip84 and Dgrip91 in Drosophila embryos are indistinguishable from that of γ-tubulin. Each antibody recognizes the centrosome throughout the cell cycle and shows some spindle staining during mitosis with enrichment at the spindle poles (Fig. 7 B), regardless of its cognate antigen. We propose that Drosophila γ-tubulin is stably associated with Dgrip91/84. Interestingly, we found no evidence for a non–γ-tubulin associated pool of either Dgrip84 or Dgrip91.


Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.

Oegema K, Wiese C, Martin OC, Milligan RA, Iwamatsu A, Mitchison TJ, Zheng Y - J. Cell Biol. (1999)

Dgrip91 and Dgrip84 associate and colocalize with  γ-tubulin. (A) Clarified Drosophila embryo extract was immunoblotted for Dgrip91, Dgrip84, and γ-tubulin after separation on  10% SDS-PAGE gels (left). Each antibody recognizes a band of  the expected molecular weight. Both Dgrip91 and Dgrip84 comigrate with γ-tubulin on sucrose gradients of embryo extract  (right). (B) Immunofluorescence of early Drosophila embryos  with antibodies against Dgrip91, Dgrip84, and γ-tubulin. Both  Dgrip84 (top) and Dgrip91 (bottom) localize to centrosomes and  to the mitotic spindle in a fashion that is indistinguishable from  the localization of γ-tubulin. Examples of embryos in interphase  and mitosis are shown for each antibody. Bar, 15 μm.
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Related In: Results  -  Collection

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Figure 7: Dgrip91 and Dgrip84 associate and colocalize with γ-tubulin. (A) Clarified Drosophila embryo extract was immunoblotted for Dgrip91, Dgrip84, and γ-tubulin after separation on 10% SDS-PAGE gels (left). Each antibody recognizes a band of the expected molecular weight. Both Dgrip91 and Dgrip84 comigrate with γ-tubulin on sucrose gradients of embryo extract (right). (B) Immunofluorescence of early Drosophila embryos with antibodies against Dgrip91, Dgrip84, and γ-tubulin. Both Dgrip84 (top) and Dgrip91 (bottom) localize to centrosomes and to the mitotic spindle in a fashion that is indistinguishable from the localization of γ-tubulin. Examples of embryos in interphase and mitosis are shown for each antibody. Bar, 15 μm.
Mentions: If γ-tubulin in Drosophila embryos primarily exists associated with Dgrip84 and Dgrip91 in either γTuSC or γTuRC, we would expect these three proteins to cofractionate on sucrose gradients of embryo extract and to colocalize in embryos. To test this hypothesis, we raised and affinity-purified rabbit polyclonal antibodies that recognize Dgrip84 and Dgrip91. Each antibody recognizes a band of the expected molecular weight on Western blots of embryo extract (Fig. 7 A, left). As expected, both Dgrip84 and Dgrip91 comigrate with γ-tubulin in γTuSC and γTuRC when embryo extract is fractionated on sucrose gradients (Fig. 7 A, right). In addition, the localizations of Dgrip84 and Dgrip91 in Drosophila embryos are indistinguishable from that of γ-tubulin. Each antibody recognizes the centrosome throughout the cell cycle and shows some spindle staining during mitosis with enrichment at the spindle poles (Fig. 7 B), regardless of its cognate antigen. We propose that Drosophila γ-tubulin is stably associated with Dgrip91/84. Interestingly, we found no evidence for a non–γ-tubulin associated pool of either Dgrip84 or Dgrip91.

Bottom Line: Mitchison. 1995.The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity.Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. Karen.Omega@EMBL-Heidelburg.DE

ABSTRACT
gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

Show MeSH
Related in: MedlinePlus