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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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The γ-tubulin meshwork supports formation of lamin B3 protofilaments. (A) Endogenous γ-tubulin detection in immunoelectron microscopy using an anti-γ-tubulin antibody or no primary antibody (control) on paraformaldehyde fixed (PFA) U2OS cells. Images show the plasma membrane [PM], cytosol [C], nuclear envelope [NE], inner [IN] and outer [ON] nuclear membranes and nucleus [N] and γ-tubulin bridges [γTB] of a U2OS cell. A black box shows the magnified area displayed in the inset. Black arrowheads show either immunolabeled γ-strings (RγTub) or immunolabeled microtubules (RαTub). Arrows show the indicated structure (n = 3). (B) Purified γ-tubulin and lamin B3 (lamB3) were negatively stained and imaged by electron microscopy in the absence (γ-Tub) or presence of 1 mM GTP (γ-TubGTP). The magnified area (black border) shows γ-strings and arrows and arrowheads show lamin B3 fibers and γ-strings, respectively.
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fig0060: The γ-tubulin meshwork supports formation of lamin B3 protofilaments. (A) Endogenous γ-tubulin detection in immunoelectron microscopy using an anti-γ-tubulin antibody or no primary antibody (control) on paraformaldehyde fixed (PFA) U2OS cells. Images show the plasma membrane [PM], cytosol [C], nuclear envelope [NE], inner [IN] and outer [ON] nuclear membranes and nucleus [N] and γ-tubulin bridges [γTB] of a U2OS cell. A black box shows the magnified area displayed in the inset. Black arrowheads show either immunolabeled γ-strings (RγTub) or immunolabeled microtubules (RαTub). Arrows show the indicated structure (n = 3). (B) Purified γ-tubulin and lamin B3 (lamB3) were negatively stained and imaged by electron microscopy in the absence (γ-Tub) or presence of 1 mM GTP (γ-TubGTP). The magnified area (black border) shows γ-strings and arrows and arrowheads show lamin B3 fibers and γ-strings, respectively.

Mentions: To characterize the structure of the γ-tubulin meshwork, we performed immunoelectron microscopy of U2OS cells that were prepared with two different methods. In the first method, we high-pressure frozen U2OS cells (Fig. 11). In the second method, we tested various fixation procedures and found that short fixation (5 min) of cells with 4% paraformaldehyde preserved the γ-tubulin meshwork. With both methods, we detected strings in both cytoplasm and nucleus that went across the NE (Fig. 11A and Fig. 12A). Immunoelectron microscopy confirmed that the antibody recognized strings with a 4 to 6 nm in diameter, which from now on will be referred as γ-strings (Fig. 11A and Fig. 12A; n = 19). γ-Strings were attached to the plasma membrane (Fig. 11A and Fig. 12A), occurred in both the outer, the inner nuclear membrane (Fig. 11A and Fig. 12A) and in the nuclear compartment (Fig. 11A and Fig. 12A) and connected both the nuclear and the cytosolic γ-tubulin pools across the NE (Fig. 11A and Fig. 12A). By contrast, control immunostaining with an anti-α-tubulin antibody recognized cytosolic arrays of microtubules (Fig. 11B). Together these data demonstrate the existence of a NE-associated network, the γ-string meshwork.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
The γ-tubulin meshwork supports formation of lamin B3 protofilaments. (A) Endogenous γ-tubulin detection in immunoelectron microscopy using an anti-γ-tubulin antibody or no primary antibody (control) on paraformaldehyde fixed (PFA) U2OS cells. Images show the plasma membrane [PM], cytosol [C], nuclear envelope [NE], inner [IN] and outer [ON] nuclear membranes and nucleus [N] and γ-tubulin bridges [γTB] of a U2OS cell. A black box shows the magnified area displayed in the inset. Black arrowheads show either immunolabeled γ-strings (RγTub) or immunolabeled microtubules (RαTub). Arrows show the indicated structure (n = 3). (B) Purified γ-tubulin and lamin B3 (lamB3) were negatively stained and imaged by electron microscopy in the absence (γ-Tub) or presence of 1 mM GTP (γ-TubGTP). The magnified area (black border) shows γ-strings and arrows and arrowheads show lamin B3 fibers and γ-strings, respectively.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037270&req=5

fig0060: The γ-tubulin meshwork supports formation of lamin B3 protofilaments. (A) Endogenous γ-tubulin detection in immunoelectron microscopy using an anti-γ-tubulin antibody or no primary antibody (control) on paraformaldehyde fixed (PFA) U2OS cells. Images show the plasma membrane [PM], cytosol [C], nuclear envelope [NE], inner [IN] and outer [ON] nuclear membranes and nucleus [N] and γ-tubulin bridges [γTB] of a U2OS cell. A black box shows the magnified area displayed in the inset. Black arrowheads show either immunolabeled γ-strings (RγTub) or immunolabeled microtubules (RαTub). Arrows show the indicated structure (n = 3). (B) Purified γ-tubulin and lamin B3 (lamB3) were negatively stained and imaged by electron microscopy in the absence (γ-Tub) or presence of 1 mM GTP (γ-TubGTP). The magnified area (black border) shows γ-strings and arrows and arrowheads show lamin B3 fibers and γ-strings, respectively.
Mentions: To characterize the structure of the γ-tubulin meshwork, we performed immunoelectron microscopy of U2OS cells that were prepared with two different methods. In the first method, we high-pressure frozen U2OS cells (Fig. 11). In the second method, we tested various fixation procedures and found that short fixation (5 min) of cells with 4% paraformaldehyde preserved the γ-tubulin meshwork. With both methods, we detected strings in both cytoplasm and nucleus that went across the NE (Fig. 11A and Fig. 12A). Immunoelectron microscopy confirmed that the antibody recognized strings with a 4 to 6 nm in diameter, which from now on will be referred as γ-strings (Fig. 11A and Fig. 12A; n = 19). γ-Strings were attached to the plasma membrane (Fig. 11A and Fig. 12A), occurred in both the outer, the inner nuclear membrane (Fig. 11A and Fig. 12A) and in the nuclear compartment (Fig. 11A and Fig. 12A) and connected both the nuclear and the cytosolic γ-tubulin pools across the NE (Fig. 11A and Fig. 12A). By contrast, control immunostaining with an anti-α-tubulin antibody recognized cytosolic arrays of microtubules (Fig. 11B). Together these data demonstrate the existence of a NE-associated network, the γ-string meshwork.

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