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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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γ-Tubulin forms a boundary on the cytosolic side of the nucleus and its disruption affects the integrity of the lamina. (A-D) Confocal images of isolated nuclei purified in the absence (A) or presence (B-D) of cytochalasin B and colcemid from U2OS (γTub) and stable γTUBULIN shRNA-GFP-γ-tubulinresist (GFPγTub) and γTUBULIN shRNA expressing cells. (A-C) White borders show the magnified areas displayed in the insets: the γ-string meshwork on the nuclear envelope (A-C), the γ-string boundary (A, B), chromatin-associated γ-strings (B) or γ-string bridges (C). In (A-D) γ-tubulin (γTub; green, T3320), α-tubulin (αTub; red) and lamin B (laminB; red) are shown as immuofluorescence staining and nuclei were detected with DAPI (blue). (D) shows the endogenous expression of lamin B and γ-tubulin in two nuclei containing either high or low γ-tubulin expression, as indicated. (A-D) Scale bars, 10 μm. (E) U2OS and NIH3T3 cells (20 × 106 cells) were biochemically divided into cytosolic [C], nuclear membrane [N], and chromatin [CH] fractions. Each fraction was subjected to immunoprecipitations (IP) with an anti-γ-tubulin (γTub; T6557), anti-lamin B or anti-GFP (Cont.) antibody, as indicated, and developed by WB with antibodies against lamin A/C, lamin B, γ-tubulin (T5192) and α-tubulin antibody (n = 5). (A-E) The figure shows representative images from at least five experiments. See also Fig. S1.
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fig0065: γ-Tubulin forms a boundary on the cytosolic side of the nucleus and its disruption affects the integrity of the lamina. (A-D) Confocal images of isolated nuclei purified in the absence (A) or presence (B-D) of cytochalasin B and colcemid from U2OS (γTub) and stable γTUBULIN shRNA-GFP-γ-tubulinresist (GFPγTub) and γTUBULIN shRNA expressing cells. (A-C) White borders show the magnified areas displayed in the insets: the γ-string meshwork on the nuclear envelope (A-C), the γ-string boundary (A, B), chromatin-associated γ-strings (B) or γ-string bridges (C). In (A-D) γ-tubulin (γTub; green, T3320), α-tubulin (αTub; red) and lamin B (laminB; red) are shown as immuofluorescence staining and nuclei were detected with DAPI (blue). (D) shows the endogenous expression of lamin B and γ-tubulin in two nuclei containing either high or low γ-tubulin expression, as indicated. (A-D) Scale bars, 10 μm. (E) U2OS and NIH3T3 cells (20 × 106 cells) were biochemically divided into cytosolic [C], nuclear membrane [N], and chromatin [CH] fractions. Each fraction was subjected to immunoprecipitations (IP) with an anti-γ-tubulin (γTub; T6557), anti-lamin B or anti-GFP (Cont.) antibody, as indicated, and developed by WB with antibodies against lamin A/C, lamin B, γ-tubulin (T5192) and α-tubulin antibody (n = 5). (A-E) The figure shows representative images from at least five experiments. See also Fig. S1.

Mentions: A chromatin-associated protein meshwork that facilitates lamina formation may provide a cell with a scaffold that maintains the NE. To test this, we isolated nuclei from both U2OS, γTUBULINsh-U2OS-GFP-γ-tubulinresist and γTUBULINsh-U2OS cells (Fig. 13A-C) (Mendez and Stillman, 2000) and found that the nuclei contained a nuclear boundary of γ-strings to which microtubule components were associated on the cytosolic side (Fig. 13A). Isolated nuclei from cytochalasin B and colcemid treated cells (Alvarado-Kristensson et al., 2009) had no attached microtubules (Fig. 13B), but still contained a γ-string boundary intertwined with the lamina (Fig. 13C). In addition, isolated nuclei from γTUBULINsh-U2OS cells showed that 86% of cells with low expression of γ-tubulin had a scattered lamin B meshwork (Fig. 13D), which imply that the γ-tubulin-nuclear boundary formed at the transition between cytosolic and chromatin-associated γ-strings may function as a supporting scaffold for the lamina.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
γ-Tubulin forms a boundary on the cytosolic side of the nucleus and its disruption affects the integrity of the lamina. (A-D) Confocal images of isolated nuclei purified in the absence (A) or presence (B-D) of cytochalasin B and colcemid from U2OS (γTub) and stable γTUBULIN shRNA-GFP-γ-tubulinresist (GFPγTub) and γTUBULIN shRNA expressing cells. (A-C) White borders show the magnified areas displayed in the insets: the γ-string meshwork on the nuclear envelope (A-C), the γ-string boundary (A, B), chromatin-associated γ-strings (B) or γ-string bridges (C). In (A-D) γ-tubulin (γTub; green, T3320), α-tubulin (αTub; red) and lamin B (laminB; red) are shown as immuofluorescence staining and nuclei were detected with DAPI (blue). (D) shows the endogenous expression of lamin B and γ-tubulin in two nuclei containing either high or low γ-tubulin expression, as indicated. (A-D) Scale bars, 10 μm. (E) U2OS and NIH3T3 cells (20 × 106 cells) were biochemically divided into cytosolic [C], nuclear membrane [N], and chromatin [CH] fractions. Each fraction was subjected to immunoprecipitations (IP) with an anti-γ-tubulin (γTub; T6557), anti-lamin B or anti-GFP (Cont.) antibody, as indicated, and developed by WB with antibodies against lamin A/C, lamin B, γ-tubulin (T5192) and α-tubulin antibody (n = 5). (A-E) The figure shows representative images from at least five experiments. See also Fig. S1.
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Related In: Results  -  Collection

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fig0065: γ-Tubulin forms a boundary on the cytosolic side of the nucleus and its disruption affects the integrity of the lamina. (A-D) Confocal images of isolated nuclei purified in the absence (A) or presence (B-D) of cytochalasin B and colcemid from U2OS (γTub) and stable γTUBULIN shRNA-GFP-γ-tubulinresist (GFPγTub) and γTUBULIN shRNA expressing cells. (A-C) White borders show the magnified areas displayed in the insets: the γ-string meshwork on the nuclear envelope (A-C), the γ-string boundary (A, B), chromatin-associated γ-strings (B) or γ-string bridges (C). In (A-D) γ-tubulin (γTub; green, T3320), α-tubulin (αTub; red) and lamin B (laminB; red) are shown as immuofluorescence staining and nuclei were detected with DAPI (blue). (D) shows the endogenous expression of lamin B and γ-tubulin in two nuclei containing either high or low γ-tubulin expression, as indicated. (A-D) Scale bars, 10 μm. (E) U2OS and NIH3T3 cells (20 × 106 cells) were biochemically divided into cytosolic [C], nuclear membrane [N], and chromatin [CH] fractions. Each fraction was subjected to immunoprecipitations (IP) with an anti-γ-tubulin (γTub; T6557), anti-lamin B or anti-GFP (Cont.) antibody, as indicated, and developed by WB with antibodies against lamin A/C, lamin B, γ-tubulin (T5192) and α-tubulin antibody (n = 5). (A-E) The figure shows representative images from at least five experiments. See also Fig. S1.
Mentions: A chromatin-associated protein meshwork that facilitates lamina formation may provide a cell with a scaffold that maintains the NE. To test this, we isolated nuclei from both U2OS, γTUBULINsh-U2OS-GFP-γ-tubulinresist and γTUBULINsh-U2OS cells (Fig. 13A-C) (Mendez and Stillman, 2000) and found that the nuclei contained a nuclear boundary of γ-strings to which microtubule components were associated on the cytosolic side (Fig. 13A). Isolated nuclei from cytochalasin B and colcemid treated cells (Alvarado-Kristensson et al., 2009) had no attached microtubules (Fig. 13B), but still contained a γ-string boundary intertwined with the lamina (Fig. 13C). In addition, isolated nuclei from γTUBULINsh-U2OS cells showed that 86% of cells with low expression of γ-tubulin had a scattered lamin B meshwork (Fig. 13D), which imply that the γ-tubulin-nuclear boundary formed at the transition between cytosolic and chromatin-associated γ-strings may function as a supporting scaffold for the lamina.

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.


Related in: MedlinePlus