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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.


Nuclear-γ-tubulin promotes the formation of the nuclear envelope and the lamina meshwork. (A, B) Ksperm were incubated in the presence of γ-tubulin immunodepleted egg extracts (Depl.) during 90 min before fixation or before spun down through a sucrose cushion for analysis by WB (ass. nuclei). Representative confocal fluorescence images of morphological changes of nuclei in stage 1 or 4 showing the location of endogenous γ-tubulin (eγTub, green) or bacterially produced His6–γ-tubulin (γTub, green), His6–C-γtub334–452 (CγTub, green) or His6–N-γtub1–333 (NγTub, green) of nuclei in stage 1 or 4. Localization of lamin B3 (lamin B; red) were examined by immunofluorescence staining with an anti-lamin B3 antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. Localization of γ-tubulin, His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 were immunofluorescence stained with either T3320 (eγTub, γTub, CγTub) or T5192 (NγTub). The figure shows representative images from at least five experiments. Scale bars, 10 μm. (B) Graph shows the mean percentage of assembled nuclei in stage 3 and 4 versus a control (black bar), with an anti-γ-tubulin antibody (open and grey bars) and with each form of His6–γ-tubulin (grey bars) added back (n = 3; * p < 0.05), as indicated. The amount of protein added back (input), the γ-tubulin level present in the extracts and the association of His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 with assembled nuclei were analyzed by WB with the indicated antibodies. Numbers on WBs indicate the level of depletion of γ-tubulin in the extracts relative to control. To adjust for differences in protein loading, the protein concentration of γ-tubulin was determined by its ratio with α-tubulin for each sample. The protein ratio in control extracts was set to 1.
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fig0025: Nuclear-γ-tubulin promotes the formation of the nuclear envelope and the lamina meshwork. (A, B) Ksperm were incubated in the presence of γ-tubulin immunodepleted egg extracts (Depl.) during 90 min before fixation or before spun down through a sucrose cushion for analysis by WB (ass. nuclei). Representative confocal fluorescence images of morphological changes of nuclei in stage 1 or 4 showing the location of endogenous γ-tubulin (eγTub, green) or bacterially produced His6–γ-tubulin (γTub, green), His6–C-γtub334–452 (CγTub, green) or His6–N-γtub1–333 (NγTub, green) of nuclei in stage 1 or 4. Localization of lamin B3 (lamin B; red) were examined by immunofluorescence staining with an anti-lamin B3 antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. Localization of γ-tubulin, His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 were immunofluorescence stained with either T3320 (eγTub, γTub, CγTub) or T5192 (NγTub). The figure shows representative images from at least five experiments. Scale bars, 10 μm. (B) Graph shows the mean percentage of assembled nuclei in stage 3 and 4 versus a control (black bar), with an anti-γ-tubulin antibody (open and grey bars) and with each form of His6–γ-tubulin (grey bars) added back (n = 3; * p < 0.05), as indicated. The amount of protein added back (input), the γ-tubulin level present in the extracts and the association of His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 with assembled nuclei were analyzed by WB with the indicated antibodies. Numbers on WBs indicate the level of depletion of γ-tubulin in the extracts relative to control. To adjust for differences in protein loading, the protein concentration of γ-tubulin was determined by its ratio with α-tubulin for each sample. The protein ratio in control extracts was set to 1.

Mentions: To elucidate the role of γ-tubulin, we monitored nuclear formation in the absence of γ-tubulin and found that the chromatin was neither able to form lamina or to recruit NM (Fig. 5A). Addition of human γ-tubulin to the Ksperm enabled the formation of the NE and of the lamina meshwork again (Fig. 5A).


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Nuclear-γ-tubulin promotes the formation of the nuclear envelope and the lamina meshwork. (A, B) Ksperm were incubated in the presence of γ-tubulin immunodepleted egg extracts (Depl.) during 90 min before fixation or before spun down through a sucrose cushion for analysis by WB (ass. nuclei). Representative confocal fluorescence images of morphological changes of nuclei in stage 1 or 4 showing the location of endogenous γ-tubulin (eγTub, green) or bacterially produced His6–γ-tubulin (γTub, green), His6–C-γtub334–452 (CγTub, green) or His6–N-γtub1–333 (NγTub, green) of nuclei in stage 1 or 4. Localization of lamin B3 (lamin B; red) were examined by immunofluorescence staining with an anti-lamin B3 antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. Localization of γ-tubulin, His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 were immunofluorescence stained with either T3320 (eγTub, γTub, CγTub) or T5192 (NγTub). The figure shows representative images from at least five experiments. Scale bars, 10 μm. (B) Graph shows the mean percentage of assembled nuclei in stage 3 and 4 versus a control (black bar), with an anti-γ-tubulin antibody (open and grey bars) and with each form of His6–γ-tubulin (grey bars) added back (n = 3; * p < 0.05), as indicated. The amount of protein added back (input), the γ-tubulin level present in the extracts and the association of His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 with assembled nuclei were analyzed by WB with the indicated antibodies. Numbers on WBs indicate the level of depletion of γ-tubulin in the extracts relative to control. To adjust for differences in protein loading, the protein concentration of γ-tubulin was determined by its ratio with α-tubulin for each sample. The protein ratio in control extracts was set to 1.
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Related In: Results  -  Collection

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fig0025: Nuclear-γ-tubulin promotes the formation of the nuclear envelope and the lamina meshwork. (A, B) Ksperm were incubated in the presence of γ-tubulin immunodepleted egg extracts (Depl.) during 90 min before fixation or before spun down through a sucrose cushion for analysis by WB (ass. nuclei). Representative confocal fluorescence images of morphological changes of nuclei in stage 1 or 4 showing the location of endogenous γ-tubulin (eγTub, green) or bacterially produced His6–γ-tubulin (γTub, green), His6–C-γtub334–452 (CγTub, green) or His6–N-γtub1–333 (NγTub, green) of nuclei in stage 1 or 4. Localization of lamin B3 (lamin B; red) were examined by immunofluorescence staining with an anti-lamin B3 antibody and nuclear membranes and nuclei were detected with Nile red (red) and DAPI (blue), respectively. Localization of γ-tubulin, His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 were immunofluorescence stained with either T3320 (eγTub, γTub, CγTub) or T5192 (NγTub). The figure shows representative images from at least five experiments. Scale bars, 10 μm. (B) Graph shows the mean percentage of assembled nuclei in stage 3 and 4 versus a control (black bar), with an anti-γ-tubulin antibody (open and grey bars) and with each form of His6–γ-tubulin (grey bars) added back (n = 3; * p < 0.05), as indicated. The amount of protein added back (input), the γ-tubulin level present in the extracts and the association of His6–γ-tubulin, His6–C-γtub334–452 and His6–N-γtub1–333 with assembled nuclei were analyzed by WB with the indicated antibodies. Numbers on WBs indicate the level of depletion of γ-tubulin in the extracts relative to control. To adjust for differences in protein loading, the protein concentration of γ-tubulin was determined by its ratio with α-tubulin for each sample. The protein ratio in control extracts was set to 1.
Mentions: To elucidate the role of γ-tubulin, we monitored nuclear formation in the absence of γ-tubulin and found that the chromatin was neither able to form lamina or to recruit NM (Fig. 5A). Addition of human γ-tubulin to the Ksperm enabled the formation of the NE and of the lamina meshwork again (Fig. 5A).

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.