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Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA - Nature (2010)

Bottom Line: The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template.The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins.The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, and Keck Advanced Microscopy Center, University of California, San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

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Models of nucleation complex attachment and activationa) In the absence of γTuRC-specific components, as in Saccharomyces, Spc110p, or its equivalent, directly attaches γTuSC to microtubule organizing centres, promoting ring assembly. We hypothesize a conformational change in Spc98p promotes nucleation by rearranging γ-tubulin into an exact microtubule template. b) In organisms with complete γTuRCs, active complexes attach to organizing centres directly via γTuSCs, or potentially through unique sites in the γTuRC-specific components. Localization of γTuRCs at non-MTOC locations, for example within the mitotic spindle, is mediated through the γTuRC-specific proteins. In both scenarios, γTuSC interactions define the geometry of the nucleating template.
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Figure 5: Models of nucleation complex attachment and activationa) In the absence of γTuRC-specific components, as in Saccharomyces, Spc110p, or its equivalent, directly attaches γTuSC to microtubule organizing centres, promoting ring assembly. We hypothesize a conformational change in Spc98p promotes nucleation by rearranging γ-tubulin into an exact microtubule template. b) In organisms with complete γTuRCs, active complexes attach to organizing centres directly via γTuSCs, or potentially through unique sites in the γTuRC-specific components. Localization of γTuRCs at non-MTOC locations, for example within the mitotic spindle, is mediated through the γTuRC-specific proteins. In both scenarios, γTuSC interactions define the geometry of the nucleating template.

Mentions: The dramatic enhancement of γTuSC oligomer stability by Spc110p, combined with its role in γTuSC localization, likely serves to ensure that microtubule template assembly in yeast occurs only at the spindle pole body. We propose a general model for microtubule nucleation in which Spc110p or its functional equivalent directly attaches γTuSC to microtubule organizing centres, promoting template assembly. A subsequent activation step then fully activates nucleation by rearranging the γ-tubulin network (Fig. 5a). In a template with seven γTuSCs, the location of the half γTuSC overlap defines the position of the 13 protofilament microtubule seam; a single lateral contact between γ-tubulin and α-tubulin would be made at the overlap, as well. It is unclear how many γTuSCs are required to nucleate a microtubule – an incomplete ring may be sufficient to initiate growth.


Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA - Nature (2010)

Models of nucleation complex attachment and activationa) In the absence of γTuRC-specific components, as in Saccharomyces, Spc110p, or its equivalent, directly attaches γTuSC to microtubule organizing centres, promoting ring assembly. We hypothesize a conformational change in Spc98p promotes nucleation by rearranging γ-tubulin into an exact microtubule template. b) In organisms with complete γTuRCs, active complexes attach to organizing centres directly via γTuSCs, or potentially through unique sites in the γTuRC-specific components. Localization of γTuRCs at non-MTOC locations, for example within the mitotic spindle, is mediated through the γTuRC-specific proteins. In both scenarios, γTuSC interactions define the geometry of the nucleating template.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921000&req=5

Figure 5: Models of nucleation complex attachment and activationa) In the absence of γTuRC-specific components, as in Saccharomyces, Spc110p, or its equivalent, directly attaches γTuSC to microtubule organizing centres, promoting ring assembly. We hypothesize a conformational change in Spc98p promotes nucleation by rearranging γ-tubulin into an exact microtubule template. b) In organisms with complete γTuRCs, active complexes attach to organizing centres directly via γTuSCs, or potentially through unique sites in the γTuRC-specific components. Localization of γTuRCs at non-MTOC locations, for example within the mitotic spindle, is mediated through the γTuRC-specific proteins. In both scenarios, γTuSC interactions define the geometry of the nucleating template.
Mentions: The dramatic enhancement of γTuSC oligomer stability by Spc110p, combined with its role in γTuSC localization, likely serves to ensure that microtubule template assembly in yeast occurs only at the spindle pole body. We propose a general model for microtubule nucleation in which Spc110p or its functional equivalent directly attaches γTuSC to microtubule organizing centres, promoting template assembly. A subsequent activation step then fully activates nucleation by rearranging the γ-tubulin network (Fig. 5a). In a template with seven γTuSCs, the location of the half γTuSC overlap defines the position of the 13 protofilament microtubule seam; a single lateral contact between γ-tubulin and α-tubulin would be made at the overlap, as well. It is unclear how many γTuSCs are required to nucleate a microtubule – an incomplete ring may be sufficient to initiate growth.

Bottom Line: The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template.The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins.The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, and Keck Advanced Microscopy Center, University of California, San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Show MeSH
Related in: MedlinePlus