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Members of the RSC chromatin-remodeling complex are required for maintaining proper nuclear envelope structure and pore complex localization.

Titus LC, Dawson TR, Rexer DJ, Ryan KJ, Wente SR - Mol. Biol. Cell (2010)

Bottom Line: NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes.Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation.We speculate that NE structure is functionally linked to proper chromatin architecture.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-8240, USA.

ABSTRACT
The assembly, distribution, and functional integrity of nuclear pore complexes (NPCs) in the nuclear envelope (NE) are key determinants in the nuclear periphery architecture. However, the mechanisms controlling proper NPC and NE structure are not fully defined. We used two different genetic screening approaches to identify Saccharomyces cerevisiae mutants with defects in NPC localization. The first approach examined green fluorescent protein (GFP)-Nic96 in 531 strains from the yeast Tet-promoters Hughes Collection with individual essential genes expressed from a doxycycline-regulated promoter (TetO(7)-orf). Under repressive conditions, depletion of the protein encoded by 44 TetO(7)-orf strains resulted in mislocalized GFP-Nic96. These included STH1, RSC4, RSC8, RSC9, RSC58, ARP7, and ARP9, each encoding components of the RSC chromatin remodeling complex. Second, a temperature-sensitive sth1-F793S (npa18-1) mutant was identified in an independent genetic screen for NPC assembly (npa) mutants. NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes. Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation. Strikingly, increasing membrane fluidity with benzyl alcohol treatment prevented the sth1-F793S NE structural defects and NPC mislocalization. We speculate that NE structure is functionally linked to proper chromatin architecture.

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The sth1-F793S allele is distinct from other sth1 alleles. (A) NPC mislocalization defect is specific to the sth1-F793S allele. Indirect immunofluorescence microscopy for Nup116 localization was conducted on logarithmically growing parental (WT) and designated sth1 mutant cells cultured at 30°C or 37°C for 4 h. Bar, 5 μm. (B) The growth phenotypes of the sth1-F793S allele are distinct from those for the sth1-3 allele. Serial diluted sth1-F793S and sth1-3 mutant cells and the corresponding WT strains, W303 (SWY518), and S288C (YOL183) respectively, were spotted onto YP agar plates with different carbon sources: TBZ (60 μg/ml) or HU (50 mM). The plates were incubated at semipermissive growth temperatures (30°C for sth1-F793S; 35°C for sth1-3) and monitored for growth after 2 d. EtOH, ethanol. (C) The sth1-F793S allele is an effective  at 34°C. The wild-type (SWY518) and sth1-F793S (SWY4143) strains were grown for 5 h at 23 or 34°C in the presence or absence of 0.4% BA. Total cell lysates were separated by SDS-PAGE and immunoblotted with a rabbit anti-Sth1 polyclonal antibody.
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Figure 3: The sth1-F793S allele is distinct from other sth1 alleles. (A) NPC mislocalization defect is specific to the sth1-F793S allele. Indirect immunofluorescence microscopy for Nup116 localization was conducted on logarithmically growing parental (WT) and designated sth1 mutant cells cultured at 30°C or 37°C for 4 h. Bar, 5 μm. (B) The growth phenotypes of the sth1-F793S allele are distinct from those for the sth1-3 allele. Serial diluted sth1-F793S and sth1-3 mutant cells and the corresponding WT strains, W303 (SWY518), and S288C (YOL183) respectively, were spotted onto YP agar plates with different carbon sources: TBZ (60 μg/ml) or HU (50 mM). The plates were incubated at semipermissive growth temperatures (30°C for sth1-F793S; 35°C for sth1-3) and monitored for growth after 2 d. EtOH, ethanol. (C) The sth1-F793S allele is an effective at 34°C. The wild-type (SWY518) and sth1-F793S (SWY4143) strains were grown for 5 h at 23 or 34°C in the presence or absence of 0.4% BA. Total cell lysates were separated by SDS-PAGE and immunoblotted with a rabbit anti-Sth1 polyclonal antibody.

Mentions: Previous studies of STH1 have reported four temperature-sensitive sth1 alleles (sth1-1, sth1-2, sth1-3, and sth1-L1346A) (Du et al., 1998; Huang et al., 2004). The sth1-1, sth1-2, and sth1-3 alleles each have mutations in the sequence region corresponding to the ATPase domain, although distinct from the sth1-F793S allele. To determine whether these other sth1 alleles perturb Nup localization, we conducted indirect immunofluorescence microscopy for Nup116 localization. After 4 h at 37°C, Nup116 remained predominantly at the nuclear rim in each of these strains (Figure 3A), whereas Nup116 mislocalized under similar conditions in the strain expressing sth1-F793S (Figure 2B). Similar results were obtained after 9 h at 37°C, with only slight mislocalization of Nup116 detectable in cells expressing sth1-3 (data not shown). Therefore, the sth1-F793S allele had a specific effect on Nup localization.


Members of the RSC chromatin-remodeling complex are required for maintaining proper nuclear envelope structure and pore complex localization.

Titus LC, Dawson TR, Rexer DJ, Ryan KJ, Wente SR - Mol. Biol. Cell (2010)

The sth1-F793S allele is distinct from other sth1 alleles. (A) NPC mislocalization defect is specific to the sth1-F793S allele. Indirect immunofluorescence microscopy for Nup116 localization was conducted on logarithmically growing parental (WT) and designated sth1 mutant cells cultured at 30°C or 37°C for 4 h. Bar, 5 μm. (B) The growth phenotypes of the sth1-F793S allele are distinct from those for the sth1-3 allele. Serial diluted sth1-F793S and sth1-3 mutant cells and the corresponding WT strains, W303 (SWY518), and S288C (YOL183) respectively, were spotted onto YP agar plates with different carbon sources: TBZ (60 μg/ml) or HU (50 mM). The plates were incubated at semipermissive growth temperatures (30°C for sth1-F793S; 35°C for sth1-3) and monitored for growth after 2 d. EtOH, ethanol. (C) The sth1-F793S allele is an effective  at 34°C. The wild-type (SWY518) and sth1-F793S (SWY4143) strains were grown for 5 h at 23 or 34°C in the presence or absence of 0.4% BA. Total cell lysates were separated by SDS-PAGE and immunoblotted with a rabbit anti-Sth1 polyclonal antibody.
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Related In: Results  -  Collection

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Figure 3: The sth1-F793S allele is distinct from other sth1 alleles. (A) NPC mislocalization defect is specific to the sth1-F793S allele. Indirect immunofluorescence microscopy for Nup116 localization was conducted on logarithmically growing parental (WT) and designated sth1 mutant cells cultured at 30°C or 37°C for 4 h. Bar, 5 μm. (B) The growth phenotypes of the sth1-F793S allele are distinct from those for the sth1-3 allele. Serial diluted sth1-F793S and sth1-3 mutant cells and the corresponding WT strains, W303 (SWY518), and S288C (YOL183) respectively, were spotted onto YP agar plates with different carbon sources: TBZ (60 μg/ml) or HU (50 mM). The plates were incubated at semipermissive growth temperatures (30°C for sth1-F793S; 35°C for sth1-3) and monitored for growth after 2 d. EtOH, ethanol. (C) The sth1-F793S allele is an effective at 34°C. The wild-type (SWY518) and sth1-F793S (SWY4143) strains were grown for 5 h at 23 or 34°C in the presence or absence of 0.4% BA. Total cell lysates were separated by SDS-PAGE and immunoblotted with a rabbit anti-Sth1 polyclonal antibody.
Mentions: Previous studies of STH1 have reported four temperature-sensitive sth1 alleles (sth1-1, sth1-2, sth1-3, and sth1-L1346A) (Du et al., 1998; Huang et al., 2004). The sth1-1, sth1-2, and sth1-3 alleles each have mutations in the sequence region corresponding to the ATPase domain, although distinct from the sth1-F793S allele. To determine whether these other sth1 alleles perturb Nup localization, we conducted indirect immunofluorescence microscopy for Nup116 localization. After 4 h at 37°C, Nup116 remained predominantly at the nuclear rim in each of these strains (Figure 3A), whereas Nup116 mislocalized under similar conditions in the strain expressing sth1-F793S (Figure 2B). Similar results were obtained after 9 h at 37°C, with only slight mislocalization of Nup116 detectable in cells expressing sth1-3 (data not shown). Therefore, the sth1-F793S allele had a specific effect on Nup localization.

Bottom Line: NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes.Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation.We speculate that NE structure is functionally linked to proper chromatin architecture.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-8240, USA.

ABSTRACT
The assembly, distribution, and functional integrity of nuclear pore complexes (NPCs) in the nuclear envelope (NE) are key determinants in the nuclear periphery architecture. However, the mechanisms controlling proper NPC and NE structure are not fully defined. We used two different genetic screening approaches to identify Saccharomyces cerevisiae mutants with defects in NPC localization. The first approach examined green fluorescent protein (GFP)-Nic96 in 531 strains from the yeast Tet-promoters Hughes Collection with individual essential genes expressed from a doxycycline-regulated promoter (TetO(7)-orf). Under repressive conditions, depletion of the protein encoded by 44 TetO(7)-orf strains resulted in mislocalized GFP-Nic96. These included STH1, RSC4, RSC8, RSC9, RSC58, ARP7, and ARP9, each encoding components of the RSC chromatin remodeling complex. Second, a temperature-sensitive sth1-F793S (npa18-1) mutant was identified in an independent genetic screen for NPC assembly (npa) mutants. NPC mislocalization in the RSC mutants required new protein synthesis and ongoing transcription, confirming that lack of global transcription did not underlie the phenotypes. Electron microscopy studies showed significantly altered NEs and nuclear morphology, with coincident cytoplasmic membrane sheet accumulation. Strikingly, increasing membrane fluidity with benzyl alcohol treatment prevented the sth1-F793S NE structural defects and NPC mislocalization. We speculate that NE structure is functionally linked to proper chromatin architecture.

Show MeSH
Related in: MedlinePlus