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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.


Related in: MedlinePlus

γ-Tubulin and α-tubulin are differently distributed. (A) γ-Tubulin (T5192), α-tubulin and centrin were immunofluorescence stained in demembranated sperm that were isolated in the presence of colcemid and pelleted onto a cushion containing SuNaSp and glycerol before the immunostaining (n = 5). (B) To study morphological changes, demembranated sperm were incubated with egg extracts during 0 min (stage 1, condensed sperm) or 60 min before fixation (stage 2, decondensed; stage 3, small almost-formed nucleus; and stage 4, nucleus with a γ-tubulin boundary). Localization of γ-tubulin was examined by immunofluorescence staining with a rabbit and a mouse produced anti-γ-tubulin antibody, as indicated. Graph shows the mean percentage of formed nuclei (open bar; stage 3 and 4; ± s.d., n = 3; * p < 0.05). (C) To test the integrity of formed nuclei, nuclear assembly reactions were incubated with TRITC-labeled dextran. Alternatively, nuclear pore complexes (NPC) were immunofluorescence stained (n = 3). (D-F) Demembranated sperm prepared as in Fig. 1B (D, E) or as in A (F) were incubated in the presence of egg extracts for 60 min before fixation in the absence (D, E) or presence (F) of colcemid. The localization of Xgrip109, microtubules (αTub), centrosomes (γTub and αTub) and γ-tubulin were examined by immunofluorescence staining (n = 3). (F) From left to right, first graph shows the mean percentage of formed nuclei in the presence of colcemid (black bar) relative to a non-treated control egg extracts and sperm (open bar). Second graph displays mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). In (A-C, E, F) γ-tubulin and centrin are shown as green, α-tubulin and NPC as red and nuclei as blue (DAPI). In (D) Xgrip109 and γTub are shown as green and red, respectively. Arrows and arrowheads indicate the location of centrosomes and the γ-tubulin nuclear boundary, respectively. Scale bars, 10 μm.
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fig0010: γ-Tubulin and α-tubulin are differently distributed. (A) γ-Tubulin (T5192), α-tubulin and centrin were immunofluorescence stained in demembranated sperm that were isolated in the presence of colcemid and pelleted onto a cushion containing SuNaSp and glycerol before the immunostaining (n = 5). (B) To study morphological changes, demembranated sperm were incubated with egg extracts during 0 min (stage 1, condensed sperm) or 60 min before fixation (stage 2, decondensed; stage 3, small almost-formed nucleus; and stage 4, nucleus with a γ-tubulin boundary). Localization of γ-tubulin was examined by immunofluorescence staining with a rabbit and a mouse produced anti-γ-tubulin antibody, as indicated. Graph shows the mean percentage of formed nuclei (open bar; stage 3 and 4; ± s.d., n = 3; * p < 0.05). (C) To test the integrity of formed nuclei, nuclear assembly reactions were incubated with TRITC-labeled dextran. Alternatively, nuclear pore complexes (NPC) were immunofluorescence stained (n = 3). (D-F) Demembranated sperm prepared as in Fig. 1B (D, E) or as in A (F) were incubated in the presence of egg extracts for 60 min before fixation in the absence (D, E) or presence (F) of colcemid. The localization of Xgrip109, microtubules (αTub), centrosomes (γTub and αTub) and γ-tubulin were examined by immunofluorescence staining (n = 3). (F) From left to right, first graph shows the mean percentage of formed nuclei in the presence of colcemid (black bar) relative to a non-treated control egg extracts and sperm (open bar). Second graph displays mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). In (A-C, E, F) γ-tubulin and centrin are shown as green, α-tubulin and NPC as red and nuclei as blue (DAPI). In (D) Xgrip109 and γTub are shown as green and red, respectively. Arrows and arrowheads indicate the location of centrosomes and the γ-tubulin nuclear boundary, respectively. Scale bars, 10 μm.

Mentions: To exclude an involvement of αβ-tubulin in the initial events leading to nuclear formation, we prepared the sperm in the presence of colcemid and removed αβ-tubulin debris by a glycerol cushion (Fig. 2A) (Felix et al., 1994). This treatment reduced the amount of αβ-tubulin and centrin associated to the sperm (Fig. 2A). Addition of egg extracts to the colcemid pretreated sperm triggered nuclear formation (Fig. 2B) (Lohka and Masui, 1983). Furthermore, 100% of the formed nuclei excluded TRITC-labeled 155-kDa dextran (99.7 ± 0.6; n = 3) and assembled nuclear pore complexes, confirming the integrity of the nuclei (Fig. 2C).


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
γ-Tubulin and α-tubulin are differently distributed. (A) γ-Tubulin (T5192), α-tubulin and centrin were immunofluorescence stained in demembranated sperm that were isolated in the presence of colcemid and pelleted onto a cushion containing SuNaSp and glycerol before the immunostaining (n = 5). (B) To study morphological changes, demembranated sperm were incubated with egg extracts during 0 min (stage 1, condensed sperm) or 60 min before fixation (stage 2, decondensed; stage 3, small almost-formed nucleus; and stage 4, nucleus with a γ-tubulin boundary). Localization of γ-tubulin was examined by immunofluorescence staining with a rabbit and a mouse produced anti-γ-tubulin antibody, as indicated. Graph shows the mean percentage of formed nuclei (open bar; stage 3 and 4; ± s.d., n = 3; * p < 0.05). (C) To test the integrity of formed nuclei, nuclear assembly reactions were incubated with TRITC-labeled dextran. Alternatively, nuclear pore complexes (NPC) were immunofluorescence stained (n = 3). (D-F) Demembranated sperm prepared as in Fig. 1B (D, E) or as in A (F) were incubated in the presence of egg extracts for 60 min before fixation in the absence (D, E) or presence (F) of colcemid. The localization of Xgrip109, microtubules (αTub), centrosomes (γTub and αTub) and γ-tubulin were examined by immunofluorescence staining (n = 3). (F) From left to right, first graph shows the mean percentage of formed nuclei in the presence of colcemid (black bar) relative to a non-treated control egg extracts and sperm (open bar). Second graph displays mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). In (A-C, E, F) γ-tubulin and centrin are shown as green, α-tubulin and NPC as red and nuclei as blue (DAPI). In (D) Xgrip109 and γTub are shown as green and red, respectively. Arrows and arrowheads indicate the location of centrosomes and the γ-tubulin nuclear boundary, respectively. Scale bars, 10 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig0010: γ-Tubulin and α-tubulin are differently distributed. (A) γ-Tubulin (T5192), α-tubulin and centrin were immunofluorescence stained in demembranated sperm that were isolated in the presence of colcemid and pelleted onto a cushion containing SuNaSp and glycerol before the immunostaining (n = 5). (B) To study morphological changes, demembranated sperm were incubated with egg extracts during 0 min (stage 1, condensed sperm) or 60 min before fixation (stage 2, decondensed; stage 3, small almost-formed nucleus; and stage 4, nucleus with a γ-tubulin boundary). Localization of γ-tubulin was examined by immunofluorescence staining with a rabbit and a mouse produced anti-γ-tubulin antibody, as indicated. Graph shows the mean percentage of formed nuclei (open bar; stage 3 and 4; ± s.d., n = 3; * p < 0.05). (C) To test the integrity of formed nuclei, nuclear assembly reactions were incubated with TRITC-labeled dextran. Alternatively, nuclear pore complexes (NPC) were immunofluorescence stained (n = 3). (D-F) Demembranated sperm prepared as in Fig. 1B (D, E) or as in A (F) were incubated in the presence of egg extracts for 60 min before fixation in the absence (D, E) or presence (F) of colcemid. The localization of Xgrip109, microtubules (αTub), centrosomes (γTub and αTub) and γ-tubulin were examined by immunofluorescence staining (n = 3). (F) From left to right, first graph shows the mean percentage of formed nuclei in the presence of colcemid (black bar) relative to a non-treated control egg extracts and sperm (open bar). Second graph displays mean percentage of formed nuclei with γ-tubulin localized throughout the nuclei (black bar) or marginalized to the nuclear envelope (open bar) (± s.d., n = 3). In (A-C, E, F) γ-tubulin and centrin are shown as green, α-tubulin and NPC as red and nuclei as blue (DAPI). In (D) Xgrip109 and γTub are shown as green and red, respectively. Arrows and arrowheads indicate the location of centrosomes and the γ-tubulin nuclear boundary, respectively. Scale bars, 10 μm.
Mentions: To exclude an involvement of αβ-tubulin in the initial events leading to nuclear formation, we prepared the sperm in the presence of colcemid and removed αβ-tubulin debris by a glycerol cushion (Fig. 2A) (Felix et al., 1994). This treatment reduced the amount of αβ-tubulin and centrin associated to the sperm (Fig. 2A). Addition of egg extracts to the colcemid pretreated sperm triggered nuclear formation (Fig. 2B) (Lohka and Masui, 1983). Furthermore, 100% of the formed nuclei excluded TRITC-labeled 155-kDa dextran (99.7 ± 0.6; n = 3) and assembled nuclear pore complexes, confirming the integrity of the nuclei (Fig. 2C).

View Article: PubMed Central - PubMed

ABSTRACT

The cytosolic role of &gamma;-tubulin as a microtubule organizer has been studied thoroughly, but its nuclear function is poorly understood. Here, we show that &gamma;-tubulin is located throughout the chromatin of demembranated Xenopus laevis sperm and, as the nucleus is formed, &gamma;-tubulin recruits lamin B3 and nuclear membranes. Immunodepletion of &gamma;-tubulin impairs X. laevis assembly of both the lamina and the nuclear membrane. During nuclear formation in mammalian cell lines, &gamma;-tubulin establishes a cellular protein boundary around chromatin that coordinates nuclear assembly of the daughter nuclei. Furthermore, expression of a &gamma;-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 &gamma;-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 &gamma;-tubulin around chromatin coordinates nuclear formation in eukaryotic cells.

No MeSH data available.


Related in: MedlinePlus