Limits...
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.


Demembranated sperm contains γ-tubulin. (A) Sequence alignment of the variable region of human γ-tubulin 1 and γ-tubulin 2 and Xenopus laevis γ-tubulin, showing residues 334–451 of the C terminal region. Bold letters indicate differences. (B, C) The localization of γ-tubulin, histone 3, γ-tubulin ring complex (Xgrip109) and centrosome (centrin and αTub) were immunofluorescence stained in demembranated sperm (n = 5) with an anti-γ-tubulin antibody that was produced either in rabbit (γ-TubR; T3320) or mouse (γ-TubM; ab27074). (Da) Total lysates of egg extract and demembranated sperm were analyzed by western blotting with the indicated antibodies (n = 3; T3320). An actin and α-tubulin loading controls are shown. (Db) The indicated amount of egg extract (egg) and demembranated sperm (sperm) was analyzed by western blotting with the indicated antibodies (n = 6; T3320). An α-tubulin loading control is shown. (E) The anti-γ-tubulin antibody produced in rabbit or mouse (T3320 and ab27074) were preincubatd for 2 h with Ni2+ affinity resin (control) or with Ni2+ affinity resin associated with His6-tagged γ-tubulin (absorb. His6-γ-Tub) before immunofluorescence staining the sperm. In (B, C, E) γ-tubulin is shown as green and histone 3 as red and nuclei as blue (DAPI). In (C) Xgrip109 and γTub are shown as green and red, respectively. Scale bars, 10 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037270&req=5

fig0005: Demembranated sperm contains γ-tubulin. (A) Sequence alignment of the variable region of human γ-tubulin 1 and γ-tubulin 2 and Xenopus laevis γ-tubulin, showing residues 334–451 of the C terminal region. Bold letters indicate differences. (B, C) The localization of γ-tubulin, histone 3, γ-tubulin ring complex (Xgrip109) and centrosome (centrin and αTub) were immunofluorescence stained in demembranated sperm (n = 5) with an anti-γ-tubulin antibody that was produced either in rabbit (γ-TubR; T3320) or mouse (γ-TubM; ab27074). (Da) Total lysates of egg extract and demembranated sperm were analyzed by western blotting with the indicated antibodies (n = 3; T3320). An actin and α-tubulin loading controls are shown. (Db) The indicated amount of egg extract (egg) and demembranated sperm (sperm) was analyzed by western blotting with the indicated antibodies (n = 6; T3320). An α-tubulin loading control is shown. (E) The anti-γ-tubulin antibody produced in rabbit or mouse (T3320 and ab27074) were preincubatd for 2 h with Ni2+ affinity resin (control) or with Ni2+ affinity resin associated with His6-tagged γ-tubulin (absorb. His6-γ-Tub) before immunofluorescence staining the sperm. In (B, C, E) γ-tubulin is shown as green and histone 3 as red and nuclei as blue (DAPI). In (C) Xgrip109 and γTub are shown as green and red, respectively. Scale bars, 10 μm.

Mentions: In cell lines, TUBULIN-shRNA expression only partially reduces the γ-tubulin protein levels (Eklund et al., 2014; Hoog et al., 2011), making depletion experiments difficult to interpret. γ-Tubulin is highly conserved among species; at the protein level human and X. laevis γ-tubulin-1 are 98% homologous (Fig. 1A). For this reason, X. laevis egg extracts have extensively been used to study the role of γ-tubulin in microtubule nucleation (Felix et al., 1994).


Gamma-tubulin coordinates nuclear envelope assembly around chromatin
Demembranated sperm contains γ-tubulin. (A) Sequence alignment of the variable region of human γ-tubulin 1 and γ-tubulin 2 and Xenopus laevis γ-tubulin, showing residues 334–451 of the C terminal region. Bold letters indicate differences. (B, C) The localization of γ-tubulin, histone 3, γ-tubulin ring complex (Xgrip109) and centrosome (centrin and αTub) were immunofluorescence stained in demembranated sperm (n = 5) with an anti-γ-tubulin antibody that was produced either in rabbit (γ-TubR; T3320) or mouse (γ-TubM; ab27074). (Da) Total lysates of egg extract and demembranated sperm were analyzed by western blotting with the indicated antibodies (n = 3; T3320). An actin and α-tubulin loading controls are shown. (Db) The indicated amount of egg extract (egg) and demembranated sperm (sperm) was analyzed by western blotting with the indicated antibodies (n = 6; T3320). An α-tubulin loading control is shown. (E) The anti-γ-tubulin antibody produced in rabbit or mouse (T3320 and ab27074) were preincubatd for 2 h with Ni2+ affinity resin (control) or with Ni2+ affinity resin associated with His6-tagged γ-tubulin (absorb. His6-γ-Tub) before immunofluorescence staining the sperm. In (B, C, E) γ-tubulin is shown as green and histone 3 as red and nuclei as blue (DAPI). In (C) Xgrip109 and γTub are shown as green and red, respectively. Scale bars, 10 μm.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

fig0005: Demembranated sperm contains γ-tubulin. (A) Sequence alignment of the variable region of human γ-tubulin 1 and γ-tubulin 2 and Xenopus laevis γ-tubulin, showing residues 334–451 of the C terminal region. Bold letters indicate differences. (B, C) The localization of γ-tubulin, histone 3, γ-tubulin ring complex (Xgrip109) and centrosome (centrin and αTub) were immunofluorescence stained in demembranated sperm (n = 5) with an anti-γ-tubulin antibody that was produced either in rabbit (γ-TubR; T3320) or mouse (γ-TubM; ab27074). (Da) Total lysates of egg extract and demembranated sperm were analyzed by western blotting with the indicated antibodies (n = 3; T3320). An actin and α-tubulin loading controls are shown. (Db) The indicated amount of egg extract (egg) and demembranated sperm (sperm) was analyzed by western blotting with the indicated antibodies (n = 6; T3320). An α-tubulin loading control is shown. (E) The anti-γ-tubulin antibody produced in rabbit or mouse (T3320 and ab27074) were preincubatd for 2 h with Ni2+ affinity resin (control) or with Ni2+ affinity resin associated with His6-tagged γ-tubulin (absorb. His6-γ-Tub) before immunofluorescence staining the sperm. In (B, C, E) γ-tubulin is shown as green and histone 3 as red and nuclei as blue (DAPI). In (C) Xgrip109 and γTub are shown as green and red, respectively. Scale bars, 10 μm.
Mentions: In cell lines, TUBULIN-shRNA expression only partially reduces the γ-tubulin protein levels (Eklund et al., 2014; Hoog et al., 2011), making depletion experiments difficult to interpret. γ-Tubulin is highly conserved among species; at the protein level human and X. laevis γ-tubulin-1 are 98% homologous (Fig. 1A). For this reason, X. laevis egg extracts have extensively been used to study the role of γ-tubulin in microtubule nucleation (Felix et al., 1994).

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.