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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 partially recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained GFP-γ-tubulin (green; γTubGFP) with an anti-γ-tubulin antibody (red; γTubAb; T3320) in interphase (A) and mitotic (B) U2OS cells stably expressing both γTUBULIN shRNA (shRNA) and GFP-γ-tubulinresist (γTubGFP). Four different antibodies were tested (ab27074, T5192, T3320 and T6557) and the one that best recognized chromatin-associated γ-tubulin (T3320) is shown. (A) The specificity of the polyclonal anti-γ-tubulin antibody (T3320) used was tested in U2OS cells stably expressing both γTUBULIN shRNA and GFP-γ-tubulinresist (γTUBULINsh-U2OS-GFP-γ-tubulinresist cells). The dashed line follows one of three γTUBULINsh-U2OS-GFP-γ-tubulinresist cells with low expression of both endogenous γ-tubulin and GFP-γ-tubulinresist. Arrows and arrowheads indicate a cell with high and low expression of γ-tubulin and the location of centrosomes, respectively. (C) Structured illumination images of a fixed U2OS immunostained with an anti-γ-tubulin (T6557). (A-C) White borders show the magnified areas displayed in the insets. Scale bars, 10 μm. (B, C) Nuclei were detected with DAPI (blue). (D) Total lysate from U2OS and U2OS cells stably expressing γTUBULIN shRNA (shγTUB) or NGFP-γ-tubulin were first analyzed by WB with a polyclonal anti-γ-tubulin antibody (385Ab) and reprobed with a polyclonal anti-γ-tubulin antibody (131Ab), a mixture of two anti-γ-tubulin (T5192 and T3320) or of two anti-GFP antibodies (sc-53882 and sc-8334) and an anti-α-tubulin antibody. Arrowheads indicate the 60-kDa and the 90-kDa bands recognized by 385Ab (n = 3). Numbers on WBs indicate the level of depletion or enrichment of the amount or proteins recognized by the indicated antibody relative to control. To adjust for differences in protein loading, the protein concentration of the indicated proteins was determined by its ratio with endogenous γ-tubulin for each sample. The protein ratio in control extracts was set to 1.
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fig0085: Anti-γ-tubulin antibodies partially recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained GFP-γ-tubulin (green; γTubGFP) with an anti-γ-tubulin antibody (red; γTubAb; T3320) in interphase (A) and mitotic (B) U2OS cells stably expressing both γTUBULIN shRNA (shRNA) and GFP-γ-tubulinresist (γTubGFP). Four different antibodies were tested (ab27074, T5192, T3320 and T6557) and the one that best recognized chromatin-associated γ-tubulin (T3320) is shown. (A) The specificity of the polyclonal anti-γ-tubulin antibody (T3320) used was tested in U2OS cells stably expressing both γTUBULIN shRNA and GFP-γ-tubulinresist (γTUBULINsh-U2OS-GFP-γ-tubulinresist cells). The dashed line follows one of three γTUBULINsh-U2OS-GFP-γ-tubulinresist cells with low expression of both endogenous γ-tubulin and GFP-γ-tubulinresist. Arrows and arrowheads indicate a cell with high and low expression of γ-tubulin and the location of centrosomes, respectively. (C) Structured illumination images of a fixed U2OS immunostained with an anti-γ-tubulin (T6557). (A-C) White borders show the magnified areas displayed in the insets. Scale bars, 10 μm. (B, C) Nuclei were detected with DAPI (blue). (D) Total lysate from U2OS and U2OS cells stably expressing γTUBULIN shRNA (shγTUB) or NGFP-γ-tubulin were first analyzed by WB with a polyclonal anti-γ-tubulin antibody (385Ab) and reprobed with a polyclonal anti-γ-tubulin antibody (131Ab), a mixture of two anti-γ-tubulin (T5192 and T3320) or of two anti-GFP antibodies (sc-53882 and sc-8334) and an anti-α-tubulin antibody. Arrowheads indicate the 60-kDa and the 90-kDa bands recognized by 385Ab (n = 3). Numbers on WBs indicate the level of depletion or enrichment of the amount or proteins recognized by the indicated antibody relative to control. To adjust for differences in protein loading, the protein concentration of the indicated proteins was determined by its ratio with endogenous γ-tubulin for each sample. The protein ratio in control extracts was set to 1.

Mentions: One plausible reason for the underestimation of the amount of chromatin-associated γ-tubulin is that available antibodies may not fully recognize this pool, as previously reported (Eklund et al., 2014). Indeed, interphase (Fig. 17A) and mitotic (Fig. 17B) γ-tubulin immunostained γTUBULINsh-U2OS-GFP-γ-tubulinresist cells showed that the antibody recognized only part of the chromatin-associated pool (Fig. 17A, B). Nonetheless, structured illumination microscopy detected the chromatin-associated γ-tubulin pool in mitotic cells (Fig. 17C), confirming the association of γ-tubulin with mitotic chromosomes.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Anti-γ-tubulin antibodies partially recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained GFP-γ-tubulin (green; γTubGFP) with an anti-γ-tubulin antibody (red; γTubAb; T3320) in interphase (A) and mitotic (B) U2OS cells stably expressing both γTUBULIN shRNA (shRNA) and GFP-γ-tubulinresist (γTubGFP). Four different antibodies were tested (ab27074, T5192, T3320 and T6557) and the one that best recognized chromatin-associated γ-tubulin (T3320) is shown. (A) The specificity of the polyclonal anti-γ-tubulin antibody (T3320) used was tested in U2OS cells stably expressing both γTUBULIN shRNA and GFP-γ-tubulinresist (γTUBULINsh-U2OS-GFP-γ-tubulinresist cells). The dashed line follows one of three γTUBULINsh-U2OS-GFP-γ-tubulinresist cells with low expression of both endogenous γ-tubulin and GFP-γ-tubulinresist. Arrows and arrowheads indicate a cell with high and low expression of γ-tubulin and the location of centrosomes, respectively. (C) Structured illumination images of a fixed U2OS immunostained with an anti-γ-tubulin (T6557). (A-C) White borders show the magnified areas displayed in the insets. Scale bars, 10 μm. (B, C) Nuclei were detected with DAPI (blue). (D) Total lysate from U2OS and U2OS cells stably expressing γTUBULIN shRNA (shγTUB) or NGFP-γ-tubulin were first analyzed by WB with a polyclonal anti-γ-tubulin antibody (385Ab) and reprobed with a polyclonal anti-γ-tubulin antibody (131Ab), a mixture of two anti-γ-tubulin (T5192 and T3320) or of two anti-GFP antibodies (sc-53882 and sc-8334) and an anti-α-tubulin antibody. Arrowheads indicate the 60-kDa and the 90-kDa bands recognized by 385Ab (n = 3). Numbers on WBs indicate the level of depletion or enrichment of the amount or proteins recognized by the indicated antibody relative to control. To adjust for differences in protein loading, the protein concentration of the indicated proteins was determined by its ratio with endogenous γ-tubulin for each sample. The protein ratio in control extracts was set to 1.
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fig0085: Anti-γ-tubulin antibodies partially recognize chromatin-associated γ-strings. (A, B) The fluorescence images show representative images of immunostained GFP-γ-tubulin (green; γTubGFP) with an anti-γ-tubulin antibody (red; γTubAb; T3320) in interphase (A) and mitotic (B) U2OS cells stably expressing both γTUBULIN shRNA (shRNA) and GFP-γ-tubulinresist (γTubGFP). Four different antibodies were tested (ab27074, T5192, T3320 and T6557) and the one that best recognized chromatin-associated γ-tubulin (T3320) is shown. (A) The specificity of the polyclonal anti-γ-tubulin antibody (T3320) used was tested in U2OS cells stably expressing both γTUBULIN shRNA and GFP-γ-tubulinresist (γTUBULINsh-U2OS-GFP-γ-tubulinresist cells). The dashed line follows one of three γTUBULINsh-U2OS-GFP-γ-tubulinresist cells with low expression of both endogenous γ-tubulin and GFP-γ-tubulinresist. Arrows and arrowheads indicate a cell with high and low expression of γ-tubulin and the location of centrosomes, respectively. (C) Structured illumination images of a fixed U2OS immunostained with an anti-γ-tubulin (T6557). (A-C) White borders show the magnified areas displayed in the insets. Scale bars, 10 μm. (B, C) Nuclei were detected with DAPI (blue). (D) Total lysate from U2OS and U2OS cells stably expressing γTUBULIN shRNA (shγTUB) or NGFP-γ-tubulin were first analyzed by WB with a polyclonal anti-γ-tubulin antibody (385Ab) and reprobed with a polyclonal anti-γ-tubulin antibody (131Ab), a mixture of two anti-γ-tubulin (T5192 and T3320) or of two anti-GFP antibodies (sc-53882 and sc-8334) and an anti-α-tubulin antibody. Arrowheads indicate the 60-kDa and the 90-kDa bands recognized by 385Ab (n = 3). Numbers on WBs indicate the level of depletion or enrichment of the amount or proteins recognized by the indicated antibody relative to control. To adjust for differences in protein loading, the protein concentration of the indicated proteins was determined by its ratio with endogenous γ-tubulin for each sample. The protein ratio in control extracts was set to 1.
Mentions: One plausible reason for the underestimation of the amount of chromatin-associated γ-tubulin is that available antibodies may not fully recognize this pool, as previously reported (Eklund et al., 2014). Indeed, interphase (Fig. 17A) and mitotic (Fig. 17B) γ-tubulin immunostained γTUBULINsh-U2OS-GFP-γ-tubulinresist cells showed that the antibody recognized only part of the chromatin-associated pool (Fig. 17A, B). Nonetheless, structured illumination microscopy detected the chromatin-associated γ-tubulin pool in mitotic cells (Fig. 17C), confirming the association of γ-tubulin with mitotic chromosomes.

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.