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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.


The N-terminal region of γ-tubulin leads to the formation of chromatin-empty nuclei. (A) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant N-γtubGFP1–333 (NTerm, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show the location of N-γtubGFP1–333 and lamin B1 in a mitotic cell that during nuclear assembly transiently formed two nuclear-like structures, which lack chromatin (arrowhead and arrow). Chromatin-lacking nuclear-like structures (white borders) are shown on the right images. Graph shows the percentage of filmed cells that formed chromatin lacking nuclear like structures. Images were collected every 30 sec. See also movie S5. (B) Fixed U2OS cells that are stably expressing γTUBULIN shRNA and N-γtubGFP1–333 were immunofluorescence stained with an anti-lamin B antibody (laminB; red) and nuclei were detected with DAPI (blue). (A, B) Scale bars, 10 μm.
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fig0100: The N-terminal region of γ-tubulin leads to the formation of chromatin-empty nuclei. (A) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant N-γtubGFP1–333 (NTerm, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show the location of N-γtubGFP1–333 and lamin B1 in a mitotic cell that during nuclear assembly transiently formed two nuclear-like structures, which lack chromatin (arrowhead and arrow). Chromatin-lacking nuclear-like structures (white borders) are shown on the right images. Graph shows the percentage of filmed cells that formed chromatin lacking nuclear like structures. Images were collected every 30 sec. See also movie S5. (B) Fixed U2OS cells that are stably expressing γTUBULIN shRNA and N-γtubGFP1–333 were immunofluorescence stained with an anti-lamin B antibody (laminB; red) and nuclei were detected with DAPI (blue). (A, B) Scale bars, 10 μm.

Mentions: To understand the impact of the interactions of the N-terminal region of γ-tubulin with lamin B1 on lamina formation in U2OS cells, we monitored mitotic γTUBULINsh-U2OS-GFP-Cγ-tubGFP334−452 and γTUBULINsh-U2OS-GFP-Nγ-tubGFP1−333 (Eklund et al., 2014; Hoog et al., 2011) cells that transiently co-expressed mCherry-lamin B1 by time-lapse microscopy. Mitotic U2OS cells expressing Cγ-tubGFP334−452 divided similarly to γTUBULINsh-U2OS-GFP-γ-tubulinresist cells (Fig. 14 and Fig. 19B; movie S4; n = 16). By contrast, in 39% (13 cells) of the studied mitotic γTUBULINsh-U2OS-GFP-Nγ-tubGFP1−333 cells (n = 33), daughter cells formed an additional lamina in the absence of chromatin (movie S5; Fig. 20A, 6–59 min). Moreover, we found chromatin empty nuclear like structures in 15% of interphase cells stably expressing N-γtubGFP (4 ± 1%, n = 3; Fig. 20B). Together, our results demonstrate that both the N- and C-terminal regions of γ-tubulin assist in the formation of the lamina, but only the DNA-binding C terminus has the ability to assure the formation of a lamina around chromatin.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
The N-terminal region of γ-tubulin leads to the formation of chromatin-empty nuclei. (A) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant N-γtubGFP1–333 (NTerm, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show the location of N-γtubGFP1–333 and lamin B1 in a mitotic cell that during nuclear assembly transiently formed two nuclear-like structures, which lack chromatin (arrowhead and arrow). Chromatin-lacking nuclear-like structures (white borders) are shown on the right images. Graph shows the percentage of filmed cells that formed chromatin lacking nuclear like structures. Images were collected every 30 sec. See also movie S5. (B) Fixed U2OS cells that are stably expressing γTUBULIN shRNA and N-γtubGFP1–333 were immunofluorescence stained with an anti-lamin B antibody (laminB; red) and nuclei were detected with DAPI (blue). (A, B) Scale bars, 10 μm.
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fig0100: The N-terminal region of γ-tubulin leads to the formation of chromatin-empty nuclei. (A) DIC/fluorescence images of time-lapse from a U2OS cell that is stably expressing both γTUBULIN shRNA and sh-resistant N-γtubGFP1–333 (NTerm, green), and transiently expressing mCherry-lamin B1 (lamB1; red) with Hoechst 33258 stained chromatin (blue), as indicated. The image series show the location of N-γtubGFP1–333 and lamin B1 in a mitotic cell that during nuclear assembly transiently formed two nuclear-like structures, which lack chromatin (arrowhead and arrow). Chromatin-lacking nuclear-like structures (white borders) are shown on the right images. Graph shows the percentage of filmed cells that formed chromatin lacking nuclear like structures. Images were collected every 30 sec. See also movie S5. (B) Fixed U2OS cells that are stably expressing γTUBULIN shRNA and N-γtubGFP1–333 were immunofluorescence stained with an anti-lamin B antibody (laminB; red) and nuclei were detected with DAPI (blue). (A, B) Scale bars, 10 μm.
Mentions: To understand the impact of the interactions of the N-terminal region of γ-tubulin with lamin B1 on lamina formation in U2OS cells, we monitored mitotic γTUBULINsh-U2OS-GFP-Cγ-tubGFP334−452 and γTUBULINsh-U2OS-GFP-Nγ-tubGFP1−333 (Eklund et al., 2014; Hoog et al., 2011) cells that transiently co-expressed mCherry-lamin B1 by time-lapse microscopy. Mitotic U2OS cells expressing Cγ-tubGFP334−452 divided similarly to γTUBULINsh-U2OS-GFP-γ-tubulinresist cells (Fig. 14 and Fig. 19B; movie S4; n = 16). By contrast, in 39% (13 cells) of the studied mitotic γTUBULINsh-U2OS-GFP-Nγ-tubGFP1−333 cells (n = 33), daughter cells formed an additional lamina in the absence of chromatin (movie S5; Fig. 20A, 6–59 min). Moreover, we found chromatin empty nuclear like structures in 15% of interphase cells stably expressing N-γtubGFP (4 ± 1%, n = 3; Fig. 20B). Together, our results demonstrate that both the N- and C-terminal regions of γ-tubulin assist in the formation of the lamina, but only the DNA-binding C terminus has the ability to assure the formation of a lamina 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.