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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.


Endogenous γ-tubulin bridges connect both the nuclear and the cytosolic γ-tubulin pools across the nuclear envelope. (A, B) Localization of endogenous γ-tubulin with an anti-γ-tubulin (T3320; green), lamina with an anti-lamin B (laminB; red) antibody and nuclei with DAPI (blue) were examined in U2OS cells in interphase. Confocal images are the mid planes of the γ-tubulin boundary at the nuclear membrane (A) or of the nuclear compartment (B) of a U2OS cell. White and yellow boxes show the magnified areas and colocalization pixel-map (CM) of the red and green channels of the magnified areas displayed in the inset, respectively. White areas in CM denote colocalized pixels between channels. The figure shows representative images from at least five experiments. Scale bars, 10 μm.
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fig0035: Endogenous γ-tubulin bridges connect both the nuclear and the cytosolic γ-tubulin pools across the nuclear envelope. (A, B) Localization of endogenous γ-tubulin with an anti-γ-tubulin (T3320; green), lamina with an anti-lamin B (laminB; red) antibody and nuclei with DAPI (blue) were examined in U2OS cells in interphase. Confocal images are the mid planes of the γ-tubulin boundary at the nuclear membrane (A) or of the nuclear compartment (B) of a U2OS cell. White and yellow boxes show the magnified areas and colocalization pixel-map (CM) of the red and green channels of the magnified areas displayed in the inset, respectively. White areas in CM denote colocalized pixels between channels. The figure shows representative images from at least five experiments. Scale bars, 10 μm.

Mentions: In an asynchronous cell population, approximately 26% of the total amount of endogenous γ-tubulin is associated with chromatin in U2OS and NIH3T3 cells (Eklund et al., 2014; Hoog et al., 2011). To understand the interconnection between the cytosolic and the nuclear γ-tubulin pools, we performed confocal microscopy and superresolution microscopy of the top plane (Fig. 7A) and mid plane (Fig. 7B and Fig. 8) of fixed U2OS cells and of living U2OS cells that stably co-expressed γTUBULIN shRNA (γTUBULINsh-U2OS) and human GFP-tagged sh-resistant γ-tubulin (γTUBULINsh-U2OS-GFP-γ-tubulinresist; Fig. 9A, B) and transiently expressed mCherry-tagged lamin B (mCherry-lamin B1; Fig. 9B). The γTUBULIN shRNA reduced the expression of endogenous γ-tubulin by approximately 50% (53 ± 6%, n = 3; Fig. 9A) and we compensated for this reduction by stably co-expressing GFP-γ-tubulinresist (Fig. 9A, B).


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Endogenous γ-tubulin bridges connect both the nuclear and the cytosolic γ-tubulin pools across the nuclear envelope. (A, B) Localization of endogenous γ-tubulin with an anti-γ-tubulin (T3320; green), lamina with an anti-lamin B (laminB; red) antibody and nuclei with DAPI (blue) were examined in U2OS cells in interphase. Confocal images are the mid planes of the γ-tubulin boundary at the nuclear membrane (A) or of the nuclear compartment (B) of a U2OS cell. White and yellow boxes show the magnified areas and colocalization pixel-map (CM) of the red and green channels of the magnified areas displayed in the inset, respectively. White areas in CM denote colocalized pixels between channels. The figure shows representative images from at least five experiments. Scale bars, 10 μm.
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Related In: Results  -  Collection

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fig0035: Endogenous γ-tubulin bridges connect both the nuclear and the cytosolic γ-tubulin pools across the nuclear envelope. (A, B) Localization of endogenous γ-tubulin with an anti-γ-tubulin (T3320; green), lamina with an anti-lamin B (laminB; red) antibody and nuclei with DAPI (blue) were examined in U2OS cells in interphase. Confocal images are the mid planes of the γ-tubulin boundary at the nuclear membrane (A) or of the nuclear compartment (B) of a U2OS cell. White and yellow boxes show the magnified areas and colocalization pixel-map (CM) of the red and green channels of the magnified areas displayed in the inset, respectively. White areas in CM denote colocalized pixels between channels. The figure shows representative images from at least five experiments. Scale bars, 10 μm.
Mentions: In an asynchronous cell population, approximately 26% of the total amount of endogenous γ-tubulin is associated with chromatin in U2OS and NIH3T3 cells (Eklund et al., 2014; Hoog et al., 2011). To understand the interconnection between the cytosolic and the nuclear γ-tubulin pools, we performed confocal microscopy and superresolution microscopy of the top plane (Fig. 7A) and mid plane (Fig. 7B and Fig. 8) of fixed U2OS cells and of living U2OS cells that stably co-expressed γTUBULIN shRNA (γTUBULINsh-U2OS) and human GFP-tagged sh-resistant γ-tubulin (γTUBULINsh-U2OS-GFP-γ-tubulinresist; Fig. 9A, B) and transiently expressed mCherry-tagged lamin B (mCherry-lamin B1; Fig. 9B). The γTUBULIN shRNA reduced the expression of endogenous γ-tubulin by approximately 50% (53 ± 6%, n = 3; Fig. 9A) and we compensated for this reduction by stably co-expressing GFP-γ-tubulinresist (Fig. 9A, B).

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.