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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.


The C-terminal region of γ-tubulin assures the formation of chromatin-containing nuclei. (A) Total lysate from U2OS cells expressing C-γtubGFP334–452 (CγTub), N-γtubGFP1–333 (Nterm), GFP-γ-tubulin (γTub) or empty vector (Cont.) were separately immunoprecipitated with anti-GFP or anti-lamin B. The expression levels of the various recombinant proteins were first analyzed by WB with an anti-GFP and reprobed with a mixture of two anti-γ-tubulin (T3320 and T6557) antibodies. The cellulose membranes containing immunoprecipitated GFP-tagged proteins were first analyzed by WB with an anti-lamin B antibody and reprobed with an anti-lamin A/C and −GFP. Arrowheads indicate the immunoprecipitated GFP-fused proteins (n = 3). Graph shows the protein concentration of lamin B found in C-γtubGFP334–452 and N-γtubGFP1–333 immunoprecipitates relative to the lamin B concentration found in GFP-γ-tubulin immunoprecipitates expressed in arbitrary units (AU; mean ± s.d; n = 3, * p < 0.05). To adjust for differences in protein loading, the protein concentration of lamin B was determined by its ratio with the immunoprecipitated GFP-tagged protein for each sample. The protein ratio in control extracts was set to 1. (B) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant Cγ-tubGFP334–452 (CγTub, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show chosen frames of the location of Cγ-tubGFP334–452 and lamin B1 during nuclear assembly in a mitotic cell. Images were collected every 30 sec. Scale bars, 10 μm. See also movie S4.
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fig0095: The C-terminal region of γ-tubulin assures the formation of chromatin-containing nuclei. (A) Total lysate from U2OS cells expressing C-γtubGFP334–452 (CγTub), N-γtubGFP1–333 (Nterm), GFP-γ-tubulin (γTub) or empty vector (Cont.) were separately immunoprecipitated with anti-GFP or anti-lamin B. The expression levels of the various recombinant proteins were first analyzed by WB with an anti-GFP and reprobed with a mixture of two anti-γ-tubulin (T3320 and T6557) antibodies. The cellulose membranes containing immunoprecipitated GFP-tagged proteins were first analyzed by WB with an anti-lamin B antibody and reprobed with an anti-lamin A/C and −GFP. Arrowheads indicate the immunoprecipitated GFP-fused proteins (n = 3). Graph shows the protein concentration of lamin B found in C-γtubGFP334–452 and N-γtubGFP1–333 immunoprecipitates relative to the lamin B concentration found in GFP-γ-tubulin immunoprecipitates expressed in arbitrary units (AU; mean ± s.d; n = 3, * p < 0.05). To adjust for differences in protein loading, the protein concentration of lamin B was determined by its ratio with the immunoprecipitated GFP-tagged protein for each sample. The protein ratio in control extracts was set to 1. (B) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant Cγ-tubGFP334–452 (CγTub, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show chosen frames of the location of Cγ-tubGFP334–452 and lamin B1 during nuclear assembly in a mitotic cell. Images were collected every 30 sec. Scale bars, 10 μm. See also movie S4.

Mentions: We first investigated the γ-tubulin domain necessary in the γ-tubulin–lamin complex by analyzing GFP immunoprecipitates from γTUBULINsh-U2OS cells stably expressing one of the following constructs: GFP-γ-tubulinresist, N-terminal (NγtubGFP1−333; γTUBULINsh-U2OS-GFP-Nγ-tubGFP1−333) and C-terminal (CγtubGFPresist334−452; γTUBULINsh-U2OS-GFP-Cγ-tubGFP334−452) region of γ-tubulin (Fig. 19A). Both CγtubGFP334−452 and NγtubGFP1−333 were associated with lamins (Fig. 19A), which imply that both regions contain necessary sequences for the formation of the γ-tubulin–lamin complex. It also suggested that the N-terminal region of γ-tubulin might be sufficient for triggering lamina formation.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
The C-terminal region of γ-tubulin assures the formation of chromatin-containing nuclei. (A) Total lysate from U2OS cells expressing C-γtubGFP334–452 (CγTub), N-γtubGFP1–333 (Nterm), GFP-γ-tubulin (γTub) or empty vector (Cont.) were separately immunoprecipitated with anti-GFP or anti-lamin B. The expression levels of the various recombinant proteins were first analyzed by WB with an anti-GFP and reprobed with a mixture of two anti-γ-tubulin (T3320 and T6557) antibodies. The cellulose membranes containing immunoprecipitated GFP-tagged proteins were first analyzed by WB with an anti-lamin B antibody and reprobed with an anti-lamin A/C and −GFP. Arrowheads indicate the immunoprecipitated GFP-fused proteins (n = 3). Graph shows the protein concentration of lamin B found in C-γtubGFP334–452 and N-γtubGFP1–333 immunoprecipitates relative to the lamin B concentration found in GFP-γ-tubulin immunoprecipitates expressed in arbitrary units (AU; mean ± s.d; n = 3, * p < 0.05). To adjust for differences in protein loading, the protein concentration of lamin B was determined by its ratio with the immunoprecipitated GFP-tagged protein for each sample. The protein ratio in control extracts was set to 1. (B) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant Cγ-tubGFP334–452 (CγTub, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show chosen frames of the location of Cγ-tubGFP334–452 and lamin B1 during nuclear assembly in a mitotic cell. Images were collected every 30 sec. Scale bars, 10 μm. See also movie S4.
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Related In: Results  -  Collection

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fig0095: The C-terminal region of γ-tubulin assures the formation of chromatin-containing nuclei. (A) Total lysate from U2OS cells expressing C-γtubGFP334–452 (CγTub), N-γtubGFP1–333 (Nterm), GFP-γ-tubulin (γTub) or empty vector (Cont.) were separately immunoprecipitated with anti-GFP or anti-lamin B. The expression levels of the various recombinant proteins were first analyzed by WB with an anti-GFP and reprobed with a mixture of two anti-γ-tubulin (T3320 and T6557) antibodies. The cellulose membranes containing immunoprecipitated GFP-tagged proteins were first analyzed by WB with an anti-lamin B antibody and reprobed with an anti-lamin A/C and −GFP. Arrowheads indicate the immunoprecipitated GFP-fused proteins (n = 3). Graph shows the protein concentration of lamin B found in C-γtubGFP334–452 and N-γtubGFP1–333 immunoprecipitates relative to the lamin B concentration found in GFP-γ-tubulin immunoprecipitates expressed in arbitrary units (AU; mean ± s.d; n = 3, * p < 0.05). To adjust for differences in protein loading, the protein concentration of lamin B was determined by its ratio with the immunoprecipitated GFP-tagged protein for each sample. The protein ratio in control extracts was set to 1. (B) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant Cγ-tubGFP334–452 (CγTub, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show chosen frames of the location of Cγ-tubGFP334–452 and lamin B1 during nuclear assembly in a mitotic cell. Images were collected every 30 sec. Scale bars, 10 μm. See also movie S4.
Mentions: We first investigated the γ-tubulin domain necessary in the γ-tubulin–lamin complex by analyzing GFP immunoprecipitates from γTUBULINsh-U2OS cells stably expressing one of the following constructs: GFP-γ-tubulinresist, N-terminal (NγtubGFP1−333; γTUBULINsh-U2OS-GFP-Nγ-tubGFP1−333) and C-terminal (CγtubGFPresist334−452; γTUBULINsh-U2OS-GFP-Cγ-tubGFP334−452) region of γ-tubulin (Fig. 19A). Both CγtubGFP334−452 and NγtubGFP1−333 were associated with lamins (Fig. 19A), which imply that both regions contain necessary sequences for the formation of the γ-tubulin–lamin complex. It also suggested that the N-terminal region of γ-tubulin might be sufficient for triggering lamina formation.

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.