Fig 1.
Verification of the nup211 shut-off (nup211-so) strain.
(A) Schematic of the nup211 loci of wild-type (nup211+) and nup211-so strains. Primer 1 is complementary to a region upstream of the nup211 open reading frame (ORF), primers 2 and 4 are complementary to the nup211 ORF, and primer 3 is complementary to the ura4+ gene. (B) PCR and (C) Southern blot analysis results confirmed the integration of ura4+ and P81nmt upstream of the nup211 locus.
Fig 2.
Characterization of the nup211-so strain.
(A) Comparison of Nup211 protein levels in wild-type and nup211-so cells grown in medium with or without thiamine by immunoblotting with anti-Nup211 antibodies. β-actin was used as a protein loading control. (B) Growth rate comparison between wild-type and nup211-so cells grown on medium with or without thiamine. Spot assays started with 5x104 cells, followed by tenfold series dilution.
Fig 3.
Down-regulation of Nup211 led to severe defects in cell morphology and cytokinesis.
(A) Bright-field images of wild-type and nup211-so cells grown in medium with or without thiamine. (B) Representative images of nup211-so cells grown in medium containing thiamine. Cells exhibit various morphological defects: elongated without septa (a), thick septa (b), asymmetric septa or bulged shape (c), multiseptated (d), branched morphology (e), and curved or swollen shape (f). Septa could also be observed in shorter cells (c). Arrowheads point to cells with specific defects. (C) Wild-type and nup211-so cells stained with DAPI (nuclei) and aniline blue (septa). For all panels, nup211-so cells were grown in medium containing thiamine for 48 hours prior to imaging. Bars: 10 μm.
Fig 4.
The N-terminal region of Nup211 is sufficient for cell viability.
(A) Growth analysis of nup211-so cells transformed with constructs expressing full-length or fragments of Nup211 grown in the presence or absence of thiamine. Spot assays started with 5x105 cells, followed by tenfold series dilution. (B) Examining Nup211 protein levels in the strains shown in (A) by immunoblotting with anti-Nup211 antibodies. β-actin was used as a protein loading control. (C) Representative bright-field images of (a) wild-type and (b) nup211-so cells transformed with empty vector. Panels (c-d) show images of nup211-so cells expressing full-length (c) or fragments of Nup211: (d) Nup2111-1033, (e) Nup2111-863, (f) Nup2111-655, (g) Nup2111-412, and (h) Nup2111034-1837. Bar: 10 μm. (D) Distribution chart showing the rescuing effects of strains with the representative images shown in (C). At least 500 cells were analyzed in each condition.
Fig 5.
Analyses of Nup211 truncation strains confirmed that the N-terminal 655 residues are sufficient for cell viability.
(A) Growth analysis of wild-type, nup2111-863 and nup2111-655 strains. Spot assays started with 1x105 cells, followed by tenfold series dilution. (B) Examining Nup211 protein levels in wild-type, nup2111-863, nup2111-655, and nup2111-655 + pARS4 (expressing Nup2111-655) strains by immunoblotting with anti-Nup211 antibodies. β-actin was used as a protein loading control. * high order (possible tetramer) of Nup2111-655. (C) Representative confocal images of wild-type, nup2111-863, and nup2111-655 + pARS4 cells immunostained with anti-Nup211 and mAb414 antibodies. mAb414 recognizes several nucleoporins of NPCs. Bars: 5μm.
Fig 6.
Transcript abundance was significantly altered upon down-regulation of nup211.
Fig 7.
Nup211 regulates the expression of several genes involved in cytokinesis.
(A) Analyzing the mRNA levels of representative genes (atf1, mbx1, pom1, knh1, pxl1, bgs1, agn1, agn2, adg1 and ace2) via RT-qPCR. (B) Analyzing the mRNA levels of representative genes in nup211-so cells exogenously expressing full-length Nup211 or Nup2111-655 via RT-qPCR. For both (A) and (B), each transcript was analyzed in triplicate for all experimental conditions. Changes in relative transcript levels were calculated using the ΔΔCq method, with act1 serving as the endogenous control. Error bars represent the 95% confidence interval. Individual points represent the independent data values from the biological replicates. (* p < 0.05, ** p <0.01, *** p <0.001).