Figure 1.
Cellular localization of endogenous RPS9 and RPS9-EGFP fusion proteins.
A.) U2OS osteosarcoma cells were stained for endogenous RPS9 under normal cell culture conditions (upper panel), in cell cultures treated with a low concentration of Actinomycin D (5 nM) that selectively blocks RNA polymerase I activity (middle panel), and in cultures treated with proteasome inhibitor MG132 (lower panel). Objective 63x. B.) Plasmids encoding for EGFP-RPS9 or RPS9-EGFP was transfected into U2OS cells, fixed and counterstained with DAPI after 24 hours. Bar 10 µM. C.) U2OS cells were selected with G418 for 2 weeks to achieve uniform expression of EGFP or RPS9-EGFP, respectively. Objective 40x.
Figure 2.
Expression and localization of EGFP-RPS9 fusion proteins.
A.) U2OS osteosarcoma cells were transfected with EGFP-RPS9 deletion mutants as indicated in the figure and fixed 24 hours after transfection. Corresponding DAPI images are also shown. Bar 10 µM. B.) Western blotting demonstrates expression levels of the different EGFP-RPS9 fusion proteins. EGFP-RPS9 was detected using anti-GFP antibody and loading was verified with a β-actin antibody. C.) Schematic representation of the different fusion proteins tested and their localization pattern within cells. The patterns were defined as being predominantly nucleolar, nucleolar and cytoplasmic, even nuclear, or even throughout the entire cell. Shown is the percentage of cells with a particular staining pattern, representing the analysis of 200 transfected cells for each transfected plasmid.
Figure 3.
Expression and localization of RPS9-EGFP fusion proteins in living U2OS cells.
U2OS cells were transfected with EGFP or EGFP-RPS9 deletion mutants as indicated in the figure and monitored under the inverted microscope 24 hours after transfection. Corresponding phase contrast images are shown and zoom-in of selected representative cells are in addition displayed on the right side. Objective 20x.
Figure 4.
Localization of RPS9-FLAG and RPS9-FLAG deletion mutants.
A.) U2OS osteosarcoma cells were transfected with wt RPS9-FLAG and RPS9-FLAG deletion mutants as indicated in the figure and fixed 24 hours after transfection. Corresponding DAPI images are shown. Bar 10 µM. B.) Schematic representation of the different RPS9-FLAG chimeric proteins tested and their respective localization pattern. The patterns were defined as being cytoplasmic, predominantly nucleolar, nucleolar and cytoplasmic, even nuclear, throughout the entire cell, nucleoplasmic excluding nucleoli, or nucleoplasmic and cytoplasmic but excluding the nucleoli. Shown is the percentage of cells with a particular pattern based on 200 transfected cells analyzed for each construct.
Figure 5.
Altered localization of RPS9-FLAG NoLS domain mutant.
A.) Schematic representation of RPS9 with the NLS1, NLS2 and NoLS. Below is a partial multiple sequence alignment of RPS9/RPS4 depicting the central motif that confers nucleolar localization and with the conserved arginine residues mutated to alanine (R109A) and (R127A) shown. Abbreviations: Hs- Homo Sapiens, Mm – Mus Musculus, Xt- Xenopus Tropicalis, Dr – Danio Rerio, Dm – Drosophila Melanogaster, Ce- Caenorhabditis Elegans, Sc – Saccaromyces Cerevisiae, Ec- Escherichia Coli. B.) The combined mutation of R109 and R127 residues to alanine disrupted the cytoplasmic/nucleolar staining pattern seen in wt RPS9-FLAG expressing cells. A subset transfected cells displayed a more intensely stained sub-nucleolar structure as is indicated with an asterix (*) in the figure, while other transfected cells had such structures in the nucleolar proximity as indicated with the (¤) symbol. Plasmids were transfected transiently into U2OS cells and stained for FLAG expression 24 hours after transfection. Bar 10 µM.
Figure 6.
Binding and co-localization of different RPS9 domains with NPM1.
A.) In vitro translated RPS9-FLAG binds to GST-NPM1 but not GST. RPS9-FLAG was incubated with GST or GST-NPM1 on beads and bound product was visualized by autoradiography (30% of input is shown). B.) In vitro translated RPS9-FLAG deletion mutants and purified GST-NPM1 revealed that fragments 1–70, 1–140, 70–194, and 100–194 but not RPS9140–194-FLAG were able to bind GST-NPM1. The binding of radiolabeled RPS9-FLAG to GST or GST-NPM1 was detected by autoradiography (20% of input is shown). Coomassie brilliant blue staining of the gel demonstrated equivalent levels of input GST. C.) Co-localization of NPM1 and RPS9-FLAG. U2OS cells were transfected with wt RPS9-FLAG and RPS9-FLAG deletion mutants as indicated in the figure. Cells were double stained for FLAG and NPM1 expression using a rabbit polyclonal anti-FLAG antibody and an NPM1 monoclonal antibody. Bar 10 µM.
Figure 7.
Nucleolar translocation of RPS9 1–70 domain by co-expressed NPM1.
A.) Nucleolar localization of EGFP-RPS91–70 was induced by co-expressed Myc-NPM1 (wt), whereas a Myc-NPM1 mutant lacking its nuclear localization signal (Myc-NPM1ΔNLS) failed to do so. Cells were stained for expression of wt and mutant Myc-NPM1. B.) Myc-NPM1 (wt) or Myc-NPM1 mutant did not induce any alteration in the localization of EGFP-RPS9140–194. Bar 10 µM. U2OS cells were transfected with plasmids as indicated in the figure and fixed 24 hours after transfection. Cells were stained for Myc expression. Corresponding merged and DAPI images are also shown. Bar 10 µM.
Figure 8.
Altered nucleolar morphology in cells expressing RPS9-FLAG mutant.
A.) Double staining of wt RPS9-FLAG (green) or RPS9-FLAG100–194, and endogenous fibrillarin (red). Nuclei were counterstained with DAPI. B.) Zoom-in images of the nucleolus revealed the distribution of fibrillarin in wt and mutant expressing cells in relation to FLAG. Two different patterns of fibrillarin staining are shown for the RPS9 FLAG mutant expressing cells, namely foci and ring-like. The distinct patterns in mutant expressing cells are indicated with white arrows. Bar 10 µM. C.) Double staining of wt RPS9-FLAG (red), or RPS9-FLAG100–194 respectively, and endogenous NPM1 (green) in addition to the corresponding phase contrast images. D.) Silver staining of nucleolar organizer regions (AgNOR) in cells expressing wt or mutant RPS9-FLAG revealed the re-location of silver granules to the surface of a nucleolar expanding mass. Objective 40x. E.) Acidic Toluidine Blue O staining of nucleoli in wt and mutant RPS9-FLAG expressing cells demonstrated that nucleoli had coalesced into one large big nucleolus that was more faintly stained than in wt nucleoli. Objective 40x. F.) Quantification as expressed in percent of transfected cells that displayed altered nucleolar morphology according to DAPI staining and NPM1/fibrillarin localization. G.) Reduced incorporation of BrdU in RPS9-FLAG mutant expressing cells. Data were averaged from at least three independent experiments in which 200 or more cells were counted per sample. Error bars indicate the standard deviation. H.) Bradford measurement (OD595) of the relative protein content in cell lysates from cultures of U2OS cells transfected with wt or mutant RPS9-FLAG. Error bars indicate the standard deviation.