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Gamma-tubulin coordinates nuclear envelope assembly around chromatin

View Article: PubMed Central - PubMed

ABSTRACT

The cytosolic role of γ-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that γ-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, γ-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of γ-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, γ-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a γ-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of γ-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by γ-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

No MeSH data available.


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Nuclear γ-tubulin is necessary for nuclear assembly. (A) Nuclear assembly was performed as in Fig. 2B. Egg extracts (egg extr.) were immunodepleted in the presence (Depl. γTub) or absence (Depl. Cont.) of an anti-γ-tubulin antibody by immunoprecipitation and protein levels of γ-tubulin and α-tubulin were analyzed by WB (n = 6). (B) Graph shows the mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) in nuclear assembly reactions that were performed under the conditions described in D (± s.d., n = 3, ** p < 0.01). (C) Proteins associated with the demembranated sperm were degraded with proteinase K (ptK; Ksperm), as indicated and remaining protein levels of γ-tubulin and histone 2B were analyzed by western blotting (WB). The localization of γ-tubulin and chromatin in sperm were assayed by immunofluorescence with an anti-γ-tubulin antibody (green; ab27074) and by DAPI staining (blue; n = 3). (D) Schematic representation describing the conditions used during nuclear assembly assays. The figure shows representative images from at least ten experiments. Scale bars, 10 μm.
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fig0015: Nuclear γ-tubulin is necessary for nuclear assembly. (A) Nuclear assembly was performed as in Fig. 2B. Egg extracts (egg extr.) were immunodepleted in the presence (Depl. γTub) or absence (Depl. Cont.) of an anti-γ-tubulin antibody by immunoprecipitation and protein levels of γ-tubulin and α-tubulin were analyzed by WB (n = 6). (B) Graph shows the mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) in nuclear assembly reactions that were performed under the conditions described in D (± s.d., n = 3, ** p < 0.01). (C) Proteins associated with the demembranated sperm were degraded with proteinase K (ptK; Ksperm), as indicated and remaining protein levels of γ-tubulin and histone 2B were analyzed by western blotting (WB). The localization of γ-tubulin and chromatin in sperm were assayed by immunofluorescence with an anti-γ-tubulin antibody (green; ab27074) and by DAPI staining (blue; n = 3). (D) Schematic representation describing the conditions used during nuclear assembly assays. The figure shows representative images from at least ten experiments. Scale bars, 10 μm.

Mentions: Immunodepletion of γ-tubulin from egg extracts reduced the amount of γ-tubulin by 54 ± 6% (Fig. 3A; n = 6) and caused a trend towards decrease nuclear formation (Fig. 3B). To further improve the degree of γ-tubulin immunodepletion in the sperm (Fig. 1B-E; stage 1), we removed chromatin-bound proteins with proteinase K (Ksperm; Fig. 3C) and found that only depletion of γ-tubulin in both sperm and egg extracts significantly impaired nuclear formation (Fig. 3A-D). Furthermore, supplementation of the egg extracts with recombinant γ-tubulin partially re-established nuclear formation, but it was not as efficient as when added to the sperm (Fig. 4A). Indeed, supplementation of Ksperm with recombinant γ-tubulin-1 or the transcription factor E2F1 mutant, E2F1Δ194−426 (Hoog et al., 2011) proved that only γ-tubulin re-established nuclear formation (Fig. 4A). These data imply that γ-tubulin is necessary for nuclear assembly.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Nuclear γ-tubulin is necessary for nuclear assembly. (A) Nuclear assembly was performed as in Fig. 2B. Egg extracts (egg extr.) were immunodepleted in the presence (Depl. γTub) or absence (Depl. Cont.) of an anti-γ-tubulin antibody by immunoprecipitation and protein levels of γ-tubulin and α-tubulin were analyzed by WB (n = 6). (B) Graph shows the mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) in nuclear assembly reactions that were performed under the conditions described in D (± s.d., n = 3, ** p < 0.01). (C) Proteins associated with the demembranated sperm were degraded with proteinase K (ptK; Ksperm), as indicated and remaining protein levels of γ-tubulin and histone 2B were analyzed by western blotting (WB). The localization of γ-tubulin and chromatin in sperm were assayed by immunofluorescence with an anti-γ-tubulin antibody (green; ab27074) and by DAPI staining (blue; n = 3). (D) Schematic representation describing the conditions used during nuclear assembly assays. The figure shows representative images from at least ten experiments. Scale bars, 10 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037270&req=5

fig0015: Nuclear γ-tubulin is necessary for nuclear assembly. (A) Nuclear assembly was performed as in Fig. 2B. Egg extracts (egg extr.) were immunodepleted in the presence (Depl. γTub) or absence (Depl. Cont.) of an anti-γ-tubulin antibody by immunoprecipitation and protein levels of γ-tubulin and α-tubulin were analyzed by WB (n = 6). (B) Graph shows the mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) in nuclear assembly reactions that were performed under the conditions described in D (± s.d., n = 3, ** p < 0.01). (C) Proteins associated with the demembranated sperm were degraded with proteinase K (ptK; Ksperm), as indicated and remaining protein levels of γ-tubulin and histone 2B were analyzed by western blotting (WB). The localization of γ-tubulin and chromatin in sperm were assayed by immunofluorescence with an anti-γ-tubulin antibody (green; ab27074) and by DAPI staining (blue; n = 3). (D) Schematic representation describing the conditions used during nuclear assembly assays. The figure shows representative images from at least ten experiments. Scale bars, 10 μm.
Mentions: Immunodepletion of γ-tubulin from egg extracts reduced the amount of γ-tubulin by 54 ± 6% (Fig. 3A; n = 6) and caused a trend towards decrease nuclear formation (Fig. 3B). To further improve the degree of γ-tubulin immunodepletion in the sperm (Fig. 1B-E; stage 1), we removed chromatin-bound proteins with proteinase K (Ksperm; Fig. 3C) and found that only depletion of γ-tubulin in both sperm and egg extracts significantly impaired nuclear formation (Fig. 3A-D). Furthermore, supplementation of the egg extracts with recombinant γ-tubulin partially re-established nuclear formation, but it was not as efficient as when added to the sperm (Fig. 4A). Indeed, supplementation of Ksperm with recombinant γ-tubulin-1 or the transcription factor E2F1 mutant, E2F1Δ194−426 (Hoog et al., 2011) proved that only γ-tubulin re-established nuclear formation (Fig. 4A). These data imply that γ-tubulin is necessary for nuclear assembly.

View Article: PubMed Central - PubMed

ABSTRACT

The cytosolic role of &gamma;-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that &gamma;-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, &gamma;-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of &gamma;-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, &gamma;-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a &gamma;-tubulin mutant that lacks the DNA-binding domain forms chromatin-empty nuclear like structures and demonstrate that a constant interplay between the chromatin-associated and the cytosolic pools of &gamma;-tubulin is required and, when the balance between pools is impaired, aberrant nuclei are formed. We therefore propose that the nuclear protein meshwork formed by &gamma;-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

No MeSH data available.


Related in: MedlinePlus