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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.


Recombinant γ-tubulin restores nuclear assembly. (A) Nuclear assembly was performed as in Fig. 3. Bacterially produced His6–γ-tubulin (HisγTub) or His6–E2F1Δ194−426 (HisE2FΔ194−426) was added back (addback) to Ksperm or to immunodepleted egg extract (egg extr.) and the effects of His6–γ-tubulin on nuclear assembly were examined. The protein levels of γ-tubulin in extracts (egg extr.-Ksperm) or sperm were analyzed by WB as indicated. An α-tubulin (αTub) loading control is shown (n = 3-6). Graphs show mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) from nuclear assembly reactions that consisted of immunodepleted egg extracts and sperm or Ksperm, in which an anti-γ-tubulin antibody (open bar; T3320) or an anti-γ-tubulin antibody that after immunodepletion, either His6–γ-tubulin or His6–E2F1Δ194−426 was added to Ksperm or to depleted egg extract (grey bar; ± s.d., n = 3-6 for each graph; * p < 0.05, ** p < 0.01). (B, C) Confocal images of the morphological changes of nuclei in stage 1, 3 and 4 from a nuclear assembly of egg extracts and sperm treated as in Fig. 3 but incubated for 90 min before fixation to increase the number of nuclei in stage 4. Arrowheads show γ-tubulin boundaries around sperm and nuclei. Arrows show γ-tubulin–lamin B3 enriched areas. In (A-C) γ-tubulin (γTub; green;) and lamin B3 (laminB; red) are shown as immunofluorescence staining with an anti-γ-tubulin and an anti-lamin B3 antibody. Nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. The figure shows representative images from at least ten experiments. Scale bars, 10 μm.
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fig0020: Recombinant γ-tubulin restores nuclear assembly. (A) Nuclear assembly was performed as in Fig. 3. Bacterially produced His6–γ-tubulin (HisγTub) or His6–E2F1Δ194−426 (HisE2FΔ194−426) was added back (addback) to Ksperm or to immunodepleted egg extract (egg extr.) and the effects of His6–γ-tubulin on nuclear assembly were examined. The protein levels of γ-tubulin in extracts (egg extr.-Ksperm) or sperm were analyzed by WB as indicated. An α-tubulin (αTub) loading control is shown (n = 3-6). Graphs show mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) from nuclear assembly reactions that consisted of immunodepleted egg extracts and sperm or Ksperm, in which an anti-γ-tubulin antibody (open bar; T3320) or an anti-γ-tubulin antibody that after immunodepletion, either His6–γ-tubulin or His6–E2F1Δ194−426 was added to Ksperm or to depleted egg extract (grey bar; ± s.d., n = 3-6 for each graph; * p < 0.05, ** p < 0.01). (B, C) Confocal images of the morphological changes of nuclei in stage 1, 3 and 4 from a nuclear assembly of egg extracts and sperm treated as in Fig. 3 but incubated for 90 min before fixation to increase the number of nuclei in stage 4. Arrowheads show γ-tubulin boundaries around sperm and nuclei. Arrows show γ-tubulin–lamin B3 enriched areas. In (A-C) γ-tubulin (γTub; green;) and lamin B3 (laminB; red) are shown as immunofluorescence staining with an anti-γ-tubulin and an anti-lamin B3 antibody. Nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. 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
Recombinant γ-tubulin restores nuclear assembly. (A) Nuclear assembly was performed as in Fig. 3. Bacterially produced His6–γ-tubulin (HisγTub) or His6–E2F1Δ194−426 (HisE2FΔ194−426) was added back (addback) to Ksperm or to immunodepleted egg extract (egg extr.) and the effects of His6–γ-tubulin on nuclear assembly were examined. The protein levels of γ-tubulin in extracts (egg extr.-Ksperm) or sperm were analyzed by WB as indicated. An α-tubulin (αTub) loading control is shown (n = 3-6). Graphs show mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) from nuclear assembly reactions that consisted of immunodepleted egg extracts and sperm or Ksperm, in which an anti-γ-tubulin antibody (open bar; T3320) or an anti-γ-tubulin antibody that after immunodepletion, either His6–γ-tubulin or His6–E2F1Δ194−426 was added to Ksperm or to depleted egg extract (grey bar; ± s.d., n = 3-6 for each graph; * p < 0.05, ** p < 0.01). (B, C) Confocal images of the morphological changes of nuclei in stage 1, 3 and 4 from a nuclear assembly of egg extracts and sperm treated as in Fig. 3 but incubated for 90 min before fixation to increase the number of nuclei in stage 4. Arrowheads show γ-tubulin boundaries around sperm and nuclei. Arrows show γ-tubulin–lamin B3 enriched areas. In (A-C) γ-tubulin (γTub; green;) and lamin B3 (laminB; red) are shown as immunofluorescence staining with an anti-γ-tubulin and an anti-lamin B3 antibody. Nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. The figure shows representative images from at least ten experiments. Scale bars, 10 μm.
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fig0020: Recombinant γ-tubulin restores nuclear assembly. (A) Nuclear assembly was performed as in Fig. 3. Bacterially produced His6–γ-tubulin (HisγTub) or His6–E2F1Δ194−426 (HisE2FΔ194−426) was added back (addback) to Ksperm or to immunodepleted egg extract (egg extr.) and the effects of His6–γ-tubulin on nuclear assembly were examined. The protein levels of γ-tubulin in extracts (egg extr.-Ksperm) or sperm were analyzed by WB as indicated. An α-tubulin (αTub) loading control is shown (n = 3-6). Graphs show mean percentage of formed nuclei in stage 3 and 4 relative to a control (black bar) from nuclear assembly reactions that consisted of immunodepleted egg extracts and sperm or Ksperm, in which an anti-γ-tubulin antibody (open bar; T3320) or an anti-γ-tubulin antibody that after immunodepletion, either His6–γ-tubulin or His6–E2F1Δ194−426 was added to Ksperm or to depleted egg extract (grey bar; ± s.d., n = 3-6 for each graph; * p < 0.05, ** p < 0.01). (B, C) Confocal images of the morphological changes of nuclei in stage 1, 3 and 4 from a nuclear assembly of egg extracts and sperm treated as in Fig. 3 but incubated for 90 min before fixation to increase the number of nuclei in stage 4. Arrowheads show γ-tubulin boundaries around sperm and nuclei. Arrows show γ-tubulin–lamin B3 enriched areas. In (A-C) γ-tubulin (γTub; green;) and lamin B3 (laminB; red) are shown as immunofluorescence staining with an anti-γ-tubulin and an anti-lamin B3 antibody. Nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. 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.