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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.


Anti-γ-tubulin antibodies against Ser385-γ-tubulin recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained U2OS and stable γTUBULINsh-U2OS cells with 385Ab (green) in interphase and mitosis. Nuclei were detected with DAPI (blue). Scale bars, 10 μm. (A) Microtubules and centrosomes were stained with an anti-α-tubulin antibody (red). Arrowheads indicate the location of centrosomes. (C, D) Endogenous γ-tubulin detection in immunoelectron microscopy using 385Ab. Images show the plasma membrane [PM], cytosol, nuclear envelope [NE] and γ-tubulin bridges [γTB] of an interphase U2OS cell. (A-D) White and black boxes show the magnified areas displayed in the insets. (E) The γ-string meshwork is composed of treads (γ-string), which are attached to the plasma membrane [PM], found in the cytosolic [C] compartment and enter into the nuclear compartment [N] through the nuclear envelope [NE]. In the nuclear compartment, γ-strings are intertwined with the lamina [lam.] meshwork and with chromatin. γ-Tubulin bridges [γTB] connect the cytosolic and the nuclear γ-tubulin pools creating a nuclear boundary of γ-tubulin [γTuB].
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fig0090: Anti-γ-tubulin antibodies against Ser385-γ-tubulin recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained U2OS and stable γTUBULINsh-U2OS cells with 385Ab (green) in interphase and mitosis. Nuclei were detected with DAPI (blue). Scale bars, 10 μm. (A) Microtubules and centrosomes were stained with an anti-α-tubulin antibody (red). Arrowheads indicate the location of centrosomes. (C, D) Endogenous γ-tubulin detection in immunoelectron microscopy using 385Ab. Images show the plasma membrane [PM], cytosol, nuclear envelope [NE] and γ-tubulin bridges [γTB] of an interphase U2OS cell. (A-D) White and black boxes show the magnified areas displayed in the insets. (E) The γ-string meshwork is composed of treads (γ-string), which are attached to the plasma membrane [PM], found in the cytosolic [C] compartment and enter into the nuclear compartment [N] through the nuclear envelope [NE]. In the nuclear compartment, γ-strings are intertwined with the lamina [lam.] meshwork and with chromatin. γ-Tubulin bridges [γTB] connect the cytosolic and the nuclear γ-tubulin pools creating a nuclear boundary of γ-tubulin [γTuB].

Mentions: Immunofluorescence analysis showed that in comparison to other anti-γ-tubulin antibodies (Fig. 17A), 385Ab only stained partially the γ-tubulin pools associated with centrosomes and microtubules (Fig. 18A, B), but recognized instead a γ-tubulin pool that was evenly distributed throughout interphase and mitotic cells (Fig. 18A, B). Moreover, the immunofluorescence staining recognized with 385Ab was decreased in U2OS cells expressing γTUBULIN shRNA (Fig. 18B). Finally, immunoelectron microscopy of U2OS cells using 385Ab showed that the antibody fully recognized γ-tubulin bridges (Fig. 18C) and cytosolic and nuclear γ-strings (Fig. 18D). The data presented here confirm that during cell division, there is a γ-tubulin boundary formed of γ-strings around chromatin (Fig. 18E).


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Anti-γ-tubulin antibodies against Ser385-γ-tubulin recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained U2OS and stable γTUBULINsh-U2OS cells with 385Ab (green) in interphase and mitosis. Nuclei were detected with DAPI (blue). Scale bars, 10 μm. (A) Microtubules and centrosomes were stained with an anti-α-tubulin antibody (red). Arrowheads indicate the location of centrosomes. (C, D) Endogenous γ-tubulin detection in immunoelectron microscopy using 385Ab. Images show the plasma membrane [PM], cytosol, nuclear envelope [NE] and γ-tubulin bridges [γTB] of an interphase U2OS cell. (A-D) White and black boxes show the magnified areas displayed in the insets. (E) The γ-string meshwork is composed of treads (γ-string), which are attached to the plasma membrane [PM], found in the cytosolic [C] compartment and enter into the nuclear compartment [N] through the nuclear envelope [NE]. In the nuclear compartment, γ-strings are intertwined with the lamina [lam.] meshwork and with chromatin. γ-Tubulin bridges [γTB] connect the cytosolic and the nuclear γ-tubulin pools creating a nuclear boundary of γ-tubulin [γTuB].
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig0090: Anti-γ-tubulin antibodies against Ser385-γ-tubulin recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained U2OS and stable γTUBULINsh-U2OS cells with 385Ab (green) in interphase and mitosis. Nuclei were detected with DAPI (blue). Scale bars, 10 μm. (A) Microtubules and centrosomes were stained with an anti-α-tubulin antibody (red). Arrowheads indicate the location of centrosomes. (C, D) Endogenous γ-tubulin detection in immunoelectron microscopy using 385Ab. Images show the plasma membrane [PM], cytosol, nuclear envelope [NE] and γ-tubulin bridges [γTB] of an interphase U2OS cell. (A-D) White and black boxes show the magnified areas displayed in the insets. (E) The γ-string meshwork is composed of treads (γ-string), which are attached to the plasma membrane [PM], found in the cytosolic [C] compartment and enter into the nuclear compartment [N] through the nuclear envelope [NE]. In the nuclear compartment, γ-strings are intertwined with the lamina [lam.] meshwork and with chromatin. γ-Tubulin bridges [γTB] connect the cytosolic and the nuclear γ-tubulin pools creating a nuclear boundary of γ-tubulin [γTuB].
Mentions: Immunofluorescence analysis showed that in comparison to other anti-γ-tubulin antibodies (Fig. 17A), 385Ab only stained partially the γ-tubulin pools associated with centrosomes and microtubules (Fig. 18A, B), but recognized instead a γ-tubulin pool that was evenly distributed throughout interphase and mitotic cells (Fig. 18A, B). Moreover, the immunofluorescence staining recognized with 385Ab was decreased in U2OS cells expressing γTUBULIN shRNA (Fig. 18B). Finally, immunoelectron microscopy of U2OS cells using 385Ab showed that the antibody fully recognized γ-tubulin bridges (Fig. 18C) and cytosolic and nuclear γ-strings (Fig. 18D). The data presented here confirm that during cell division, there is a γ-tubulin boundary formed of γ-strings around chromatin (Fig. 18E).

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.