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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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Benzyl alcohol treatment prevents GFP-Nup mislocalization in sth1-F793S cells. Logarithmically growing cultures of the sth1-F793S GFP-nic96 nup170-GFP (SWY3202) strain (A) and the sth1-F793S (SWY4143) strains with GFP-tagged Nic96, Nup60, Nup133, or Pom34 (B) were grown for 5 h at 23°C (left column) and then shifted to 34°C in the absence (middle column) or presence (right column) of 0.4% BA. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. For A, the corresponding DIC images are shown. Bars, 5 μm.
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Figure 7: Benzyl alcohol treatment prevents GFP-Nup mislocalization in sth1-F793S cells. Logarithmically growing cultures of the sth1-F793S GFP-nic96 nup170-GFP (SWY3202) strain (A) and the sth1-F793S (SWY4143) strains with GFP-tagged Nic96, Nup60, Nup133, or Pom34 (B) were grown for 5 h at 23°C (left column) and then shifted to 34°C in the absence (middle column) or presence (right column) of 0.4% BA. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. For A, the corresponding DIC images are shown. Bars, 5 μm.

Mentions: We used an independent assay to investigate whether NE membrane composition or fluidity was connected to the sth1-F793S mechanism of perturbation. Benzyl alcohol (BA) is an established membrane fluidizer (Colley and Metcalfe, 1972; Gordon et al., 1980) that has recently been used in S. cerevisiae to examine the role of Apq12 in NPC assembly (Scarcelli et al., 2007) and in Aspergillus nidulans to analyze functional roles for the An-Nup84-120 complex at the NE (Liu et al., 2009). To test this with the sth1-F793S mutant, 0.4% BA was added to the cells coincident with the shift to the nonpermissive growth temperature. Nuclear rim localization of GFP-tagged Nic96, Nup170, Nup60, Nup133, and Pom34 were independently evaluated in respective strains by direct fluorescence microscopy (Figure 7). Strikingly, no Nup mislocalization was observed in the BA treated sth1-F793S cells. GFP-Nic96 was also not mislocalized when TetO7-STH1 cells were treated with BA during growth in the presence of doxycycline (Supplemental Figure S4). Moreover, TEM examination of the BA-treated, temperature-shifted sth1-F793S cells revealed that the ultrastructural NE defects were also absent (Figure 8). Immunoblotting was conducted and showed that the sth1-F793S protein was still unstable in the BA-treated cells (Figure 3C). Thus, the RSC role in mediating proper NE morphology and NPC localization was compensated for by alteration in NE dynamics.


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)

Benzyl alcohol treatment prevents GFP-Nup mislocalization in sth1-F793S cells. Logarithmically growing cultures of the sth1-F793S GFP-nic96 nup170-GFP (SWY3202) strain (A) and the sth1-F793S (SWY4143) strains with GFP-tagged Nic96, Nup60, Nup133, or Pom34 (B) were grown for 5 h at 23°C (left column) and then shifted to 34°C in the absence (middle column) or presence (right column) of 0.4% BA. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. For A, the corresponding DIC images are shown. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Benzyl alcohol treatment prevents GFP-Nup mislocalization in sth1-F793S cells. Logarithmically growing cultures of the sth1-F793S GFP-nic96 nup170-GFP (SWY3202) strain (A) and the sth1-F793S (SWY4143) strains with GFP-tagged Nic96, Nup60, Nup133, or Pom34 (B) were grown for 5 h at 23°C (left column) and then shifted to 34°C in the absence (middle column) or presence (right column) of 0.4% BA. Representative live-cell, direct fluorescence images of GFP-Nup localization are shown. For A, the corresponding DIC images are shown. Bars, 5 μm.
Mentions: We used an independent assay to investigate whether NE membrane composition or fluidity was connected to the sth1-F793S mechanism of perturbation. Benzyl alcohol (BA) is an established membrane fluidizer (Colley and Metcalfe, 1972; Gordon et al., 1980) that has recently been used in S. cerevisiae to examine the role of Apq12 in NPC assembly (Scarcelli et al., 2007) and in Aspergillus nidulans to analyze functional roles for the An-Nup84-120 complex at the NE (Liu et al., 2009). To test this with the sth1-F793S mutant, 0.4% BA was added to the cells coincident with the shift to the nonpermissive growth temperature. Nuclear rim localization of GFP-tagged Nic96, Nup170, Nup60, Nup133, and Pom34 were independently evaluated in respective strains by direct fluorescence microscopy (Figure 7). Strikingly, no Nup mislocalization was observed in the BA treated sth1-F793S cells. GFP-Nic96 was also not mislocalized when TetO7-STH1 cells were treated with BA during growth in the presence of doxycycline (Supplemental Figure S4). Moreover, TEM examination of the BA-treated, temperature-shifted sth1-F793S cells revealed that the ultrastructural NE defects were also absent (Figure 8). Immunoblotting was conducted and showed that the sth1-F793S protein was still unstable in the BA-treated cells (Figure 3C). Thus, the RSC role in mediating proper NE morphology and NPC localization was compensated for by alteration in NE dynamics.

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