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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.


γ-Tubulin forms a cellular meshwork. (A) Total lysate from U2OS, U2OS cells stably expressing γTUBULIN shRNA (shγTUB) and U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γTubresist, n = 3) were analyzed by WB for the expression of endogenous γ-tubulin (arrowhead) and GFP-γ-tubulinresist (arrow) with a mixture of an anti-γ-tubulin (T6557) and an anti-GFP (sc-8334) antibody. An α-tubulin loading control is shown. (B) Differential interference contrast (DIC)/fluorescence images of a Z-stack of U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γ-TubGFP; green) and transiently expressing mCherry-tagged lamin B (red). Images were collected at 0.34 μm intervals. Z-stack images taken in the intervals 0.34–1.36 μm and 3.06–3.74 μm show the lower and the higher γ-string meshwork formed on the nuclear envelope, respectively. Z-stack images in the interval 1.7–2.72 μm show the γ-string meshwork in the nuclear compartment. Arrows and arrowheads show the centrosomes and γ-tubulin bridges, respectively. The magnified area (white border) shows a γ-tubulin bridge (right panels). The figure shows representative images from at least eight experiments. (C, D) Localization of endogenous centrin with an anti-centrin (green) and lamina with an anti-lamin B (laminB; red) antibody were examined in U2OS cells in interphase. Confocal images are the mid planes of the nuclear membrane (C) or of the nuclear compartment (D) of U2OS cells. (B-D) Scale bars, 10 μm.
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fig0045: γ-Tubulin forms a cellular meshwork. (A) Total lysate from U2OS, U2OS cells stably expressing γTUBULIN shRNA (shγTUB) and U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γTubresist, n = 3) were analyzed by WB for the expression of endogenous γ-tubulin (arrowhead) and GFP-γ-tubulinresist (arrow) with a mixture of an anti-γ-tubulin (T6557) and an anti-GFP (sc-8334) antibody. An α-tubulin loading control is shown. (B) Differential interference contrast (DIC)/fluorescence images of a Z-stack of U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γ-TubGFP; green) and transiently expressing mCherry-tagged lamin B (red). Images were collected at 0.34 μm intervals. Z-stack images taken in the intervals 0.34–1.36 μm and 3.06–3.74 μm show the lower and the higher γ-string meshwork formed on the nuclear envelope, respectively. Z-stack images in the interval 1.7–2.72 μm show the γ-string meshwork in the nuclear compartment. Arrows and arrowheads show the centrosomes and γ-tubulin bridges, respectively. The magnified area (white border) shows a γ-tubulin bridge (right panels). The figure shows representative images from at least eight experiments. (C, D) Localization of endogenous centrin with an anti-centrin (green) and lamina with an anti-lamin B (laminB; red) antibody were examined in U2OS cells in interphase. Confocal images are the mid planes of the nuclear membrane (C) or of the nuclear compartment (D) of U2OS cells. (B-D) 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
γ-Tubulin forms a cellular meshwork. (A) Total lysate from U2OS, U2OS cells stably expressing γTUBULIN shRNA (shγTUB) and U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γTubresist, n = 3) were analyzed by WB for the expression of endogenous γ-tubulin (arrowhead) and GFP-γ-tubulinresist (arrow) with a mixture of an anti-γ-tubulin (T6557) and an anti-GFP (sc-8334) antibody. An α-tubulin loading control is shown. (B) Differential interference contrast (DIC)/fluorescence images of a Z-stack of U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γ-TubGFP; green) and transiently expressing mCherry-tagged lamin B (red). Images were collected at 0.34 μm intervals. Z-stack images taken in the intervals 0.34–1.36 μm and 3.06–3.74 μm show the lower and the higher γ-string meshwork formed on the nuclear envelope, respectively. Z-stack images in the interval 1.7–2.72 μm show the γ-string meshwork in the nuclear compartment. Arrows and arrowheads show the centrosomes and γ-tubulin bridges, respectively. The magnified area (white border) shows a γ-tubulin bridge (right panels). The figure shows representative images from at least eight experiments. (C, D) Localization of endogenous centrin with an anti-centrin (green) and lamina with an anti-lamin B (laminB; red) antibody were examined in U2OS cells in interphase. Confocal images are the mid planes of the nuclear membrane (C) or of the nuclear compartment (D) of U2OS cells. (B-D) Scale bars, 10 μm.
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fig0045: γ-Tubulin forms a cellular meshwork. (A) Total lysate from U2OS, U2OS cells stably expressing γTUBULIN shRNA (shγTUB) and U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γTubresist, n = 3) were analyzed by WB for the expression of endogenous γ-tubulin (arrowhead) and GFP-γ-tubulinresist (arrow) with a mixture of an anti-γ-tubulin (T6557) and an anti-GFP (sc-8334) antibody. An α-tubulin loading control is shown. (B) Differential interference contrast (DIC)/fluorescence images of a Z-stack of U2OS cells stably expressing γTUBULIN shRNA and GFP-γ-tubulinresist (γ-TubGFP; green) and transiently expressing mCherry-tagged lamin B (red). Images were collected at 0.34 μm intervals. Z-stack images taken in the intervals 0.34–1.36 μm and 3.06–3.74 μm show the lower and the higher γ-string meshwork formed on the nuclear envelope, respectively. Z-stack images in the interval 1.7–2.72 μm show the γ-string meshwork in the nuclear compartment. Arrows and arrowheads show the centrosomes and γ-tubulin bridges, respectively. The magnified area (white border) shows a γ-tubulin bridge (right panels). The figure shows representative images from at least eight experiments. (C, D) Localization of endogenous centrin with an anti-centrin (green) and lamina with an anti-lamin B (laminB; red) antibody were examined in U2OS cells in interphase. Confocal images are the mid planes of the nuclear membrane (C) or of the nuclear compartment (D) of U2OS cells. (B-D) 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.