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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.


Lamin B1 assembles at the γ-tubulin boundary. The DIC/fluorescence images show time-lapse series from a stable γTUBULINsh-U2OS cell co-expressing GFP-γ-tubulinresist (γTub; green) that was transiently expressing mCherry-lamin B1 (lamB1; red). The image series show chosen frames of the lamina (laminB1) formation at the γ-string boundary (cytosolic and chromatin-associated γ-strings) formed by GFP-γ-tubulinresist during nuclear assembly in a mitotic cell. The mitotic chromosomes/daughter nuclei are magnified below the merged images. Arrowheads and arrows show the γ-string-boundary around the mitotic chromosomes and the mitotic spindle, respectively. The graph shows the time-dependent changes in fluorescence intensity across the white box at the γ-string-boundary of γtubGFP (green) and mCherry-lamin B1 (mChlmB1; red) expressed in arbitrary units (AU; mean ± s.d.; n = 3). Right, structured illumination microscope (SIM) images show immunofluorescence staining of endogenous γ-tubulin and lamin B with an anti-γ-tubulin (green; T6557) and anti-lamin B1 (red) antibody of newly formed U2OS daughter cells. Nuclei were detected with DAPI (blue). The yellow box shows colocalization pixel-map (CM) of the red and green channels. White areas in CM denote colocalized pixels between channels. Scale bars, 10 μm. See also movie S1.
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fig0070: Lamin B1 assembles at the γ-tubulin boundary. The DIC/fluorescence images show time-lapse series from a stable γTUBULINsh-U2OS cell co-expressing GFP-γ-tubulinresist (γTub; green) that was transiently expressing mCherry-lamin B1 (lamB1; red). The image series show chosen frames of the lamina (laminB1) formation at the γ-string boundary (cytosolic and chromatin-associated γ-strings) formed by GFP-γ-tubulinresist during nuclear assembly in a mitotic cell. The mitotic chromosomes/daughter nuclei are magnified below the merged images. Arrowheads and arrows show the γ-string-boundary around the mitotic chromosomes and the mitotic spindle, respectively. The graph shows the time-dependent changes in fluorescence intensity across the white box at the γ-string-boundary of γtubGFP (green) and mCherry-lamin B1 (mChlmB1; red) expressed in arbitrary units (AU; mean ± s.d.; n = 3). Right, structured illumination microscope (SIM) images show immunofluorescence staining of endogenous γ-tubulin and lamin B with an anti-γ-tubulin (green; T6557) and anti-lamin B1 (red) antibody of newly formed U2OS daughter cells. Nuclei were detected with DAPI (blue). The yellow box shows colocalization pixel-map (CM) of the red and green channels. White areas in CM denote colocalized pixels between channels. Scale bars, 10 μm. See also movie S1.

Mentions: Based on the finding of the γ-string boundary, we hypothesized that it may provide a cell with a tool to structure synchronized cytosolic and nuclear events during cell division. To visualize the interconnection between γ-strings and lamina formation during nuclear assembly, we analyzed time-lapse images of dividing γTUBULINsh-U2OSGFP-γ-tubulinresist (with C-terminal tagged γ-tubulin; Fig. 14; movie S1), U2OS (Fig. 15A; movie S2) and γTUBULINsh-U2OS cells (Fig. 15B; movie S3) that transiently co-expressed mCherrylamin B1. In metaphase, γ-tubulin was evenly distributed throughout the cell (Fig. 14, 0 min) and a γ-tubulin-boundary composed of cytosolic and chromatin-associated γ-strings around the mitotic chromosomes (Fig. 14, 0–3 min) was observed. This γ-tubulin-boundary remained in the newly formed daughter nuclei (Fig. 14, 4–12 min). In contrast, in metaphase lamin B remained non-chromatin bound (Fig. 14, 0–3 min). During early telophase the lamina became visible and grew over time intertwined with the γ-tubulin-boundary of γ-strings (Fig. 14, 4–12 min). Structured illumination microscopy confirmed the location of the γ-tubulin-boundary in the newly assembled daughter nuclei (Fig. 14), which resembled the γ-tubulin nuclear boundary formed in stage 4 in a newly assembled X. laevis nucleus (Fig. 4B). Similar mCherry-lamin B1 localization was observed in U2OS cells (Fig. 15A) and in γTUBULINsh-U2OS cells, despite the lower expression of endogenous γ-tubulin and mCherry-lamin B1 in the latter cell line (Fig. 15B). These data further support that in living cells γ-strings may function as a scaffold meshwork that assists the formation of the lamina around mitotic chromosomes.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Lamin B1 assembles at the γ-tubulin boundary. The DIC/fluorescence images show time-lapse series from a stable γTUBULINsh-U2OS cell co-expressing GFP-γ-tubulinresist (γTub; green) that was transiently expressing mCherry-lamin B1 (lamB1; red). The image series show chosen frames of the lamina (laminB1) formation at the γ-string boundary (cytosolic and chromatin-associated γ-strings) formed by GFP-γ-tubulinresist during nuclear assembly in a mitotic cell. The mitotic chromosomes/daughter nuclei are magnified below the merged images. Arrowheads and arrows show the γ-string-boundary around the mitotic chromosomes and the mitotic spindle, respectively. The graph shows the time-dependent changes in fluorescence intensity across the white box at the γ-string-boundary of γtubGFP (green) and mCherry-lamin B1 (mChlmB1; red) expressed in arbitrary units (AU; mean ± s.d.; n = 3). Right, structured illumination microscope (SIM) images show immunofluorescence staining of endogenous γ-tubulin and lamin B with an anti-γ-tubulin (green; T6557) and anti-lamin B1 (red) antibody of newly formed U2OS daughter cells. Nuclei were detected with DAPI (blue). The yellow box shows colocalization pixel-map (CM) of the red and green channels. White areas in CM denote colocalized pixels between channels. Scale bars, 10 μm. See also movie S1.
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fig0070: Lamin B1 assembles at the γ-tubulin boundary. The DIC/fluorescence images show time-lapse series from a stable γTUBULINsh-U2OS cell co-expressing GFP-γ-tubulinresist (γTub; green) that was transiently expressing mCherry-lamin B1 (lamB1; red). The image series show chosen frames of the lamina (laminB1) formation at the γ-string boundary (cytosolic and chromatin-associated γ-strings) formed by GFP-γ-tubulinresist during nuclear assembly in a mitotic cell. The mitotic chromosomes/daughter nuclei are magnified below the merged images. Arrowheads and arrows show the γ-string-boundary around the mitotic chromosomes and the mitotic spindle, respectively. The graph shows the time-dependent changes in fluorescence intensity across the white box at the γ-string-boundary of γtubGFP (green) and mCherry-lamin B1 (mChlmB1; red) expressed in arbitrary units (AU; mean ± s.d.; n = 3). Right, structured illumination microscope (SIM) images show immunofluorescence staining of endogenous γ-tubulin and lamin B with an anti-γ-tubulin (green; T6557) and anti-lamin B1 (red) antibody of newly formed U2OS daughter cells. Nuclei were detected with DAPI (blue). The yellow box shows colocalization pixel-map (CM) of the red and green channels. White areas in CM denote colocalized pixels between channels. Scale bars, 10 μm. See also movie S1.
Mentions: Based on the finding of the γ-string boundary, we hypothesized that it may provide a cell with a tool to structure synchronized cytosolic and nuclear events during cell division. To visualize the interconnection between γ-strings and lamina formation during nuclear assembly, we analyzed time-lapse images of dividing γTUBULINsh-U2OSGFP-γ-tubulinresist (with C-terminal tagged γ-tubulin; Fig. 14; movie S1), U2OS (Fig. 15A; movie S2) and γTUBULINsh-U2OS cells (Fig. 15B; movie S3) that transiently co-expressed mCherrylamin B1. In metaphase, γ-tubulin was evenly distributed throughout the cell (Fig. 14, 0 min) and a γ-tubulin-boundary composed of cytosolic and chromatin-associated γ-strings around the mitotic chromosomes (Fig. 14, 0–3 min) was observed. This γ-tubulin-boundary remained in the newly formed daughter nuclei (Fig. 14, 4–12 min). In contrast, in metaphase lamin B remained non-chromatin bound (Fig. 14, 0–3 min). During early telophase the lamina became visible and grew over time intertwined with the γ-tubulin-boundary of γ-strings (Fig. 14, 4–12 min). Structured illumination microscopy confirmed the location of the γ-tubulin-boundary in the newly assembled daughter nuclei (Fig. 14), which resembled the γ-tubulin nuclear boundary formed in stage 4 in a newly assembled X. laevis nucleus (Fig. 4B). Similar mCherry-lamin B1 localization was observed in U2OS cells (Fig. 15A) and in γTUBULINsh-U2OS cells, despite the lower expression of endogenous γ-tubulin and mCherry-lamin B1 in the latter cell line (Fig. 15B). These data further support that in living cells γ-strings may function as a scaffold meshwork that assists the formation of the lamina around 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.