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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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Nup mislocalization in sth1-F793S cells requires ongoing transcription. The RPB4 deletion allele was integrated into the sth1-F793S strains expressing GFP-tagged Nic96 (SWY4243), Nup133 (SWY4245), or Nup60 (SWY4247). These strains and the corresponding parental sth1-F793S RPB4 strains (SWY4244, SWY4246, and SWY4248, respectively) were shifted to 34°C for 5 h. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. Bar, 5 μm.
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Figure 6: Nup mislocalization in sth1-F793S cells requires ongoing transcription. The RPB4 deletion allele was integrated into the sth1-F793S strains expressing GFP-tagged Nic96 (SWY4243), Nup133 (SWY4245), or Nup60 (SWY4247). These strains and the corresponding parental sth1-F793S RPB4 strains (SWY4244, SWY4246, and SWY4248, respectively) were shifted to 34°C for 5 h. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. Bar, 5 μm.

Mentions: Because the RSC complex is functionally linked to gene expression (Angus-Hill et al., 2001; Damelin et al., 2002; Ng et al., 2002; Kasten et al., 2004; Soutourina et al., 2006; Badis et al., 2008; Parnell et al., 2008; Hartley and Madhani, 2009; Mas et al., 2009), we speculated that some of the defects in the sth1-F793S mutant might be linked to altered expression of RSC-controlled genes that encode proteins involved in NE and/or NPC biogenesis. To globally assess the role of transcription in the sth1-F793S Nup mislocalization phenotype, we used a RNA polymerase II temperature-sensitive mutant. The RBP4 gene encodes a nonessential RNA polymerase II subunit (Woychik and Young, 1989); however, the rbp4Δ is temperature sensitive for growth above 32°C and after 45 min at 37°C, 96% of RNA polymerase II transcription is lost (Woychik and Young, 1989; Miyao et al., 2001). The sth1-F793S rbp4Δ double mutant was evaluated for NPC localization by monitoring GFP-tagged Nic96, Nup60, or Nup133 (Figure 6). After shifting to growth at 34°C for 5 h, the respective GFP-tagged Nups remained localized at the nuclear rim, and mislocalization was not detected. GFP-tagged Nups also remained rim localized in the rpb4Δ single mutant (data not shown). This observation was further confirmed using thiolutin, an inhibitor of global RNA synthesis. Treatment with thiolutin blocked GFP-tagged Nic96 mislocalization in TetO7-STH1 cells grown in the presence of doxycycline (Supplemental Figure S4) and GFP-tagged Nic96, Nup60, Nup133 mislocalization in the sth1-F793S mutant (data not shown). Together, both ongoing transcription and translation were required for the NPC/NE defects.


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)

Nup mislocalization in sth1-F793S cells requires ongoing transcription. The RPB4 deletion allele was integrated into the sth1-F793S strains expressing GFP-tagged Nic96 (SWY4243), Nup133 (SWY4245), or Nup60 (SWY4247). These strains and the corresponding parental sth1-F793S RPB4 strains (SWY4244, SWY4246, and SWY4248, respectively) were shifted to 34°C for 5 h. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2836959&req=5

Figure 6: Nup mislocalization in sth1-F793S cells requires ongoing transcription. The RPB4 deletion allele was integrated into the sth1-F793S strains expressing GFP-tagged Nic96 (SWY4243), Nup133 (SWY4245), or Nup60 (SWY4247). These strains and the corresponding parental sth1-F793S RPB4 strains (SWY4244, SWY4246, and SWY4248, respectively) were shifted to 34°C for 5 h. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. Bar, 5 μm.
Mentions: Because the RSC complex is functionally linked to gene expression (Angus-Hill et al., 2001; Damelin et al., 2002; Ng et al., 2002; Kasten et al., 2004; Soutourina et al., 2006; Badis et al., 2008; Parnell et al., 2008; Hartley and Madhani, 2009; Mas et al., 2009), we speculated that some of the defects in the sth1-F793S mutant might be linked to altered expression of RSC-controlled genes that encode proteins involved in NE and/or NPC biogenesis. To globally assess the role of transcription in the sth1-F793S Nup mislocalization phenotype, we used a RNA polymerase II temperature-sensitive mutant. The RBP4 gene encodes a nonessential RNA polymerase II subunit (Woychik and Young, 1989); however, the rbp4Δ is temperature sensitive for growth above 32°C and after 45 min at 37°C, 96% of RNA polymerase II transcription is lost (Woychik and Young, 1989; Miyao et al., 2001). The sth1-F793S rbp4Δ double mutant was evaluated for NPC localization by monitoring GFP-tagged Nic96, Nup60, or Nup133 (Figure 6). After shifting to growth at 34°C for 5 h, the respective GFP-tagged Nups remained localized at the nuclear rim, and mislocalization was not detected. GFP-tagged Nups also remained rim localized in the rpb4Δ single mutant (data not shown). This observation was further confirmed using thiolutin, an inhibitor of global RNA synthesis. Treatment with thiolutin blocked GFP-tagged Nic96 mislocalization in TetO7-STH1 cells grown in the presence of doxycycline (Supplemental Figure S4) and GFP-tagged Nic96, Nup60, Nup133 mislocalization in the sth1-F793S mutant (data not shown). Together, both ongoing transcription and translation were required for the NPC/NE defects.

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