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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 endogenous protein levels of γ-tubulin affect the association of GFP-γ-tubulin to chromatin. (A, B) The DIC/fluorescence images show time-lapse series from U2OS cells that stably expressed either C-terminal tagged GFP-γ-tubulin (γTub; A) or both γTUBULIN shRNA and N-terminal tagged GFP-γ-tubulinresist (γTubresistShγTUB; B). The image series show chosen frames of the variation over time in the amount of chromatin-associated GFP-γ-tubulin. Images were collected every 30 sec. Scale bars, 10 μm. (C) The graph shows the time dependent changes in fluorescence intensity across the chromatin of GFP-γ-tubulin expressed in arbitrary units (AU; mean ± s.d.; n = 3).
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fig0080: The endogenous protein levels of γ-tubulin affect the association of GFP-γ-tubulin to chromatin. (A, B) The DIC/fluorescence images show time-lapse series from U2OS cells that stably expressed either C-terminal tagged GFP-γ-tubulin (γTub; A) or both γTUBULIN shRNA and N-terminal tagged GFP-γ-tubulinresist (γTubresistShγTUB; B). The image series show chosen frames of the variation over time in the amount of chromatin-associated GFP-γ-tubulin. Images were collected every 30 sec. Scale bars, 10 μm. (C) The graph shows the time dependent changes in fluorescence intensity across the chromatin of GFP-γ-tubulin expressed in arbitrary units (AU; mean ± s.d.; n = 3).

Mentions: As the amount of GFP-γ-tubulin associated with mitotic chromosomes was higher than anticipated in γTUBULINsh-U2OS-GFP-γ-tubulinresist cells (Fig. 14), we tested the effect of the position of the GFP-tag and the stable expression of γTUBULIN shRNA on the cellular location of GFP-γ-tubulin in γtubGFP-U2OS (U2OS cells that stably expressed human GFP-γ-tubulin; Fig. 16A) and in γTUBULINsh-U2OS-NGFP-γ-tubulinresist cells (γTUBULINsh-U2OS cells that stably expressed human GFP-N terminal-tagged sh-resistant γ-tubulin; Fig. 16B) and monitored the amount of chromatin-associated GFP-γ-tubulin pool by time-lapse microscopy. We noticed that the association of GFP-γ-tubulin with chromatin was independent of the position of the GFP-tag. Also, the lower the endogenous protein levels of γ-tubulin, the higher levels of chromatin-associated GFP-γ-tubulin were found (Fig. 14 and Fig. 16C). These findings suggested that endogenous γ-tubulin interfered with GFP-γ-tubulin binding to chromatin and that the amount of chromatin-associated γ-tubulin might be higher than expected.


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
The endogenous protein levels of γ-tubulin affect the association of GFP-γ-tubulin to chromatin. (A, B) The DIC/fluorescence images show time-lapse series from U2OS cells that stably expressed either C-terminal tagged GFP-γ-tubulin (γTub; A) or both γTUBULIN shRNA and N-terminal tagged GFP-γ-tubulinresist (γTubresistShγTUB; B). The image series show chosen frames of the variation over time in the amount of chromatin-associated GFP-γ-tubulin. Images were collected every 30 sec. Scale bars, 10 μm. (C) The graph shows the time dependent changes in fluorescence intensity across the chromatin of GFP-γ-tubulin expressed in arbitrary units (AU; mean ± s.d.; n = 3).
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Related In: Results  -  Collection

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fig0080: The endogenous protein levels of γ-tubulin affect the association of GFP-γ-tubulin to chromatin. (A, B) The DIC/fluorescence images show time-lapse series from U2OS cells that stably expressed either C-terminal tagged GFP-γ-tubulin (γTub; A) or both γTUBULIN shRNA and N-terminal tagged GFP-γ-tubulinresist (γTubresistShγTUB; B). The image series show chosen frames of the variation over time in the amount of chromatin-associated GFP-γ-tubulin. Images were collected every 30 sec. Scale bars, 10 μm. (C) The graph shows the time dependent changes in fluorescence intensity across the chromatin of GFP-γ-tubulin expressed in arbitrary units (AU; mean ± s.d.; n = 3).
Mentions: As the amount of GFP-γ-tubulin associated with mitotic chromosomes was higher than anticipated in γTUBULINsh-U2OS-GFP-γ-tubulinresist cells (Fig. 14), we tested the effect of the position of the GFP-tag and the stable expression of γTUBULIN shRNA on the cellular location of GFP-γ-tubulin in γtubGFP-U2OS (U2OS cells that stably expressed human GFP-γ-tubulin; Fig. 16A) and in γTUBULINsh-U2OS-NGFP-γ-tubulinresist cells (γTUBULINsh-U2OS cells that stably expressed human GFP-N terminal-tagged sh-resistant γ-tubulin; Fig. 16B) and monitored the amount of chromatin-associated GFP-γ-tubulin pool by time-lapse microscopy. We noticed that the association of GFP-γ-tubulin with chromatin was independent of the position of the GFP-tag. Also, the lower the endogenous protein levels of γ-tubulin, the higher levels of chromatin-associated GFP-γ-tubulin were found (Fig. 14 and Fig. 16C). These findings suggested that endogenous γ-tubulin interfered with GFP-γ-tubulin binding to chromatin and that the amount of chromatin-associated γ-tubulin might be higher than expected.

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.