Fig 1.
Chromosomal passenger protein-like staining of RUVBL1/2 during cell division.
(A) Methanol-fixed U2OS cells were stained with anti-RUVBL1 antibody (green) and DAPI (blue) in interphase and various stages of mitosis. At least 50 events were examined (N≥50). (B) Localization of GFP-mRuvBL1 at different stages of mitosis in HeLa cells. (N≥50). (C) Co-staining of RUVBL1 (red) and GFP-Anillin (green) in dividing cells. DNA is counterstained with DAPI (blue). Scale bar, 5 μm; (N≥50). (D) Co-staining of RUVBL1 (green) and β-tubulin (red) in dividing cells. DNA is counterstained with DAPI (blue). (N≥50) (E) Co-staining of RUVBL1 (red) and RUVBL2 (green) showing their different localization during late telophase. DNA is counterstained with DAPI (blue). (N≥50). (F) Whole cell extracts (1 mg) from asynchronous or double-thymidine/nocodazole-synchronized GFP-mRuvBL1 expressing HeLa cells were used for immunoprecipitation with a GFP-trap antibody. Precipitated material was analyzed with antibodies to GFP and RUVBL2. In: input (2.5%); IP: immunoprecipitated fraction; Ub: unbound fraction.
Fig 2.
RUVBL1 depletion affects the length of mitosis and results in lagging chromosomes.
(A) HeLa cells were transfected with the indicated siRNA oligos and analyzed by RT-PCR and immunoblot 48 h post-transfection. (B) Confocal live imaging after siRNA-mediated knock-down was performed. The figure shows stills of control or RUVBL1 siRNA-treated cells. Lagging chromosomes are indicated with arrowheads. DNA is shown in cyan and α-tubulin in red. (C) Early mitotic progression was analyzed by measuring the time from prophase (nuclear envelope breakdown: NEB) to anaphase onset (left panel). Mitotic exit was estimated by measuring the period of time from anaphase onset until accomplished cytokinesis (right panel). The bottom and top of the boxes represent the first and third quartiles, respectively. Horizontal lines inside the boxes represent the median of the data points (n = 100). Whiskers span the 10th and 90th percentiles, with individual dots showing data points that lie outside of these percentiles. Statistical significance was determined by Mann–Whitney test and p-values are indicated above. (D) Occurrence of aberrant mitotic phenotypes was quantified by analyzing 100 cell divisions in each cell line.
Fig 3.
In vitro phosphorylation of RUVBL1 by PLK1.
(A) Protein sequences were obtained from http://www.ncbi.nlm.nih.gov and scanned for PLK1 consensus motifs [37]. Corresponding sequences of human RUVBL2 are shown by comparison below the alignments. Crucial amino acids are shown in gray. (B) The indicated amounts of His-tagged RUVBL1 or RUVBL2 were incubated with PLK1 in the presence of [γ-32P]ATP and protein phosphorylation was monitored by autoradiography. (C) Bands corresponding to phosphorylated RUVBL1 were excised and in-gel digested with trypsin. Eluted peptides were separated in two dimensions by their hydrophobicity and charge. Potential sequences were assigned to the obtained phosphopeptide patterns after autoradiography. Trypsin digestion sites are indicated with K•X. (D) Purified His-tagged RUVBL1 mutants were incubated with PLK1 in the presence of [γ-32P]ATP and protein phosphorylation was monitored by autoradiography.
Fig 4.
RUVBL1 and PLK1 interact during mitosis.
(A) Co-staining of RUVBL1 (green) and PLK1 (red) in dividing HeLa cells. DNA is counterstained with DAPI (blue). At least 50 events were examined (N≥50). (B) Extracts of asynchronous (As) or nocodazole-arrested (M) FLAG-PLK1-transfected 293T cells were immunoprecipitated with anti-FLAG beads and bound material was analyzed for the presence of RUVBL1. Input (5%, both for As and M); IP: immunoprecipitated fraction. (C) Reciprocal immunoprecipitation of nocodazole-arrested 293T cells transfected with HA-tagged RUVBL1. In: input (5%); IP: immunoprecipitated fraction. (D) Extracts of double-thymidine/nocodazole-synchronized mouse GFP-RuvBL1 expressing HeLa cells were immunoprecipitated and proteins detected with the indicated antibodies. In: input (2.5%); IP: immunoprecipitated fraction; Ub: unbound fraction.
Fig 5.
ATPase activity of mammalian RUVBL1 is indispensable for cell proliferation.
(A) FLAG-tagged RUVBL1 was transiently expressed in 293T cells and protein extracts were prepared 48 h after transfection. Anti-FLAG beads were used to isolate the tagged RUVBL1. Silver staining PAGE shows the purity of the isolated material. (B) ATPase activity was measured by incubating purified FLAG-tagged RUVBL1 with [γ-32P]ATP for the times indicated, either in the presence or absence of ssDNA. (C) U2OS T-REx cells carrying stably-integrated, inducible shRNA-resistant murine RuvBL1 variants, were treated with doxycycline for 96 h (lane 2 and 5). In addition, the cell lines were stably-transfected with an inducible shRNA construct that enables downregulation of endogenous RUVBL1 (lane 3 and 6) after doxcycycline addition. Parental cells are shown for comparison (lanes 1 and 4). (D) Anti-FLAG immunoprecipitates confirm the interaction of exogenous RuvBL1 with endogenous RUVBL2. (E) Paraformaldehyde-fixed cells were stained with anti-FLAG antibody (green) and DAPI (blue) after 96 h of doxycycline induction. Scale bar, 5 μm. (F) Clonogenic survival assay after doxycycline-induced expression of the wild type or D302N RuvBL1 variants. The experiments were performed in triplicates and a representative image is shown (quantification of the assay is shown in S5 Fig). (G) Cells were induced with doxycycline or left untreated for 96 hours. To arrest the cells in mitosis, nocodazole was added for additional 16 h. DNA content was measured by flow cytometric analysis of propidium iodide-stained cells. (H) Paraformaldehyde-fixed cells were stained with anti-Cyclin A antibody (red) and DAPI (blue) after 96 h of doxycycline induction and 16 h of nocodazole treatment. Scale bar, 5 μm.