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Figures
- Fig. 1.
SVIL is a cytokinesis-associated protein. (A) HeLa cells cultured on 24-well plates were transfected with siRNAs, and 72 hours later, the ratio of multinucleated cells was evaluated. In the first screen, two different siRNAs were used for 88 genes. In the second screen, four different siRNAs were used for the indicated genes. The graph shows the ratio of multinucleated cells after treatment with each siRNA. Two independent experiments were performed for the first and second screen. (B) The cells were transfected with siRNAs, and 72 hours later, the cells were lysed and immunoblotted with anti-SVIL antibody. (C) The cells were transfected with GFP-SVIL, GFP-SVIL-Res#1, or GFP-SVIL-Res#2 together with the indicated siRNAs, and 72 hours later, the cells were lysed and immunoblotted using the indicated antibodies. GFP-SVIL-Res#1 has silent mutations to confer resistance to SVIL siRNA #1 and GFP-SVIL-Res#2 to SVIL siRNA#2. (D) The cells were treated as in C, and the ratio of multinucleated cells was evaluated. Three independent experiments were performed, and 200 cells were evaluated for each experiment (for this and all subsequent figures the data are means ± s.d.; **P<0.01, n.s; not significant). (E) HeLa cells were fixed with methanol/acetone and immunostained for SVIL, α-tubulin and the nucleus (Hoechst). Ana1, Ana2 and Ana3 are different stages in anaphase.
- Fig. 2.
PLK1 phosphorylates Ser238 of SVIL. (A) The cells were synchronized by double thymidine block and lysed at the indicated time points. The cell lysates were separated by SDS-PAGE with Phos-tag. The graph shows the relative thickness of each band of SVIL. (B) Ser238 of SVIL is in the consensus sequence of PLK1 phosphorylation. (C) HA-tagged full-length and deletion mutants of SVIL were expressed in HEK293T cells with or without GFP–PLK1, and the mobility shift was examined by immunoblotting. (D) HA-tagged aa1–345 SVIL was expressed in HEK293T cells together with wild-type or kinase-dead (K82M) PLK1 and immunoprecipitated with anti-HA antibody. The immunoprecipitates were treated with or without λ-phosphatase, and the mobility shift was examined by immunoblotting. (E) HA-tagged aa1–345 of wild-type or S238A-SVIL was expressed in HEK293T cells with wild-type or kinase-dead PLK1, and the mobility shift was examined by immunoblotting. (F) GST-fused residues 228–256 of wild-type or S238A-SVIL was incubated with PLK1 for 30 minutes at 30°C in the presence of [γ-32P]ATP and separated by SDS-PAGE. The upper panel shows the phosphorylation of recombinant proteins, and the lower panel shows Coomassie Blue (CBB) staining of the recombinant proteins. The arrow in the upper panel indicates the phosphorylated GST-SVIL (aa228–256). The arrow in the lower panel indicates GST–SVIL (aa228–256). The asterisks indicate degraded forms of GST–SVIL (aa228–256), and the arrowhead indicates GST. (G) HA-tagged full-length SVIL was transiently expressed in HEK293T cells and immunoprecipitated with anti-HA antibody and subjected to an in vitro kinase assay. The arrow indicates phosphorylated SVIL.
- Fig. 3.
PLK1 is required for the localization of SVIL to the central spindle. (A) Nocodazole-arrested HeLa cells were released in the presence of 25 µM of MG132 for 1 hour. Cells were then incubated with DMSO or PLK1 inhibitor for 1 hour. The cells were fixed with methanol/acetone and immunostained for the indicated proteins. The graph shows the ratio of cells with SVIL at the central spindle (n = 30, **P<0.01). (B) HeLa cells transfected with GFP-tagged wild-type or ST4AA-PRC1 were nocodazole-arrested and released. The cells were fixed with methanol/acetone and immunostained for the indicated proteins. In ST4AA-PRC, Ser577, Thr578, Ser601 and Thr602 were replaced with alanines. The graph shows the ratio of cells with SVIL at the central spindle (n = 30, **P<0.01).
- Fig. 4.
SVIL associates with PRC1. (A) GFP-tagged full-length and deletion mutants of SVIL were expressed in HEK293T cells together with HA–PRC1 and immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to immunoblotting with the indicated antibodies. (B) GFP-tagged full-length and deletion mutants of PRC1 were expressed in HEK293T cells together with HA–SVIL and immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to immunoblotting with anti-HA and anti-GFP antibodies. (C) In vitro translated HA–SVIL (aa676–1009) was affinity precipitated with GST or GST-fused residues 1–200 of PRC1 bound to glutathione–agarose beads. The precipitates were subjected to immunoblotting with anti-HA antibody. The lower panel shows Coomassie Blue staining of recombinant proteins. (D) Double thymidine-blocked HeLa cells were released and lysed at the indicated time points. The cell lysates were immunoprecipitated with anti-SVIL antibody, and the immunoprecipitates were blotted with the indicated antibodies.
- Fig. 5.
Phosphorylation of Ser238 regulates the interaction of SVIL and PRC1. (A) HA–SVIL was expressed in HEK293T cells with GFP-tagged wild-type or kinase-dead PLK1, and phosphorylated or non-phosphorylated HA–SVIL was affinity precipitated by anti-HA antibody. The precipitates were mixed with lysates of HeLa cells that transiently expressed GFP–PRC1, and proteins bound to HA–SVIL were affinity precipitated and subjected to immunoblot analysis. Band intensities were measured using ImageJ software, and the relative intensities are indicated. (B) HA-tagged wild-type, S238A- and S238D-SVIL were transiently expressed in HEK293T cells with GFP–PRC1. The cells were lysed and immunoprecipitated with anti-HA antibody. The immunoprecipitates were blotted with anti-HA and anti-GFP antibody. The relative band intensities are indicated. (C) GFP-tagged wild-type, ST4AA-, ST4DD-PRC1 were transiently expressed in HEK293T cells with HA–SVIL. The cells were lysed and immunoprecipitated with anti-HA antibody. The immunoprecipitates were blotted with anti-HA and anti-GFP antibodies. (D) HeLa cells that constitutively expressed GFP-tagged wild-type, S238A- and S238D-SVIL were fixed with methanol/acetone and immunostained for GFP and PRC1. (E) The fluorescence intensities across the anaphase cells, as indicated by the dashed white lines in D, were measured using ImageJ software (n = 10).
- Fig. 6.
SVIL is required to confine the furrow in the restricted zone. (A) MCF10A cells that constitutively expressed both GFP–RLC and DsRed–H2B were transfected with siRNAs. Twenty-four hours later, cell division was monitored by time-lapse microcopy. (B) The ratio of the furrow length (furrow) to the distance between the polar cortexes (total) was measured. The ratio of furrow/total of each cell was plotted, and the average ratios are indicated in the graph (**P<0.01). (C) MCF10A cells that constitutively expressed siRNA-resistant GFP-tagged wild-type, S238A- and S238D-SVIL were established. The cells were transfected with SVIL siRNA to deplete endogenous protein, and cell division was monitored by time-lapse microscopy. (D) The ratio of furrow/total was evaluated for each cell and plotted on the graph. The average ratios are indicated (**P<0.01, n.s; not significant). (E) Schematic representation of ΔAct-SVIL and ΔAyo-SVIL. (F) MCF10A cells that constitutively expressed siRNA-resistant GFP-tagged wild-type, ΔAct- and ΔMyo-SVIL were established, and endogenous SVIL was depleted by siRNA transfection. The ratio of furrow/total was evaluated in each cell and plotted on the graph. The average ratios are indicated by horizontal bars (**P<0.01, n.s; not significant).
- Fig. 7.
SVIL regulates the activation of myosin II at the cleavage furrow. (A) HeLa cells transfected with control or SVIL siRNA were nocodazole-arrested and released. The cells were lysed at the indicated time points, and the phosphorylation of RLC was examined by immunoblot. (B) HeLa cells, transfected with siRNAs, were fixed with 4% paraformaldehyde and immunostained. The graph shows the ratio of cells with phosphorylated RLC at the equatorial cortex (n = 30). (C) HeLa cells that constitutively expressed siRNA-resistant GFP-tagged wild-type, ΔAct- and ΔMyo-SVIL were generated and transfected with SVIL siRNA. The cells were fixed with methanol/acetone or 4% paraformaldehyde and immunostained. Note that the methanol/acetone-fixed cells and paraformaldehyde-fixed cells are different. The graph shows the ratio of cells with phosphorylated RLC at the equatorial cortex (n = 30). (D) HeLa cells that constitutively expressed siRNA-resistant GFP-tagged wild-type, S238A- and S238D-SVIL were established and transfected with SVIL siRNA. The cells were fixed with 4% paraformaldehyde and immunostained. Note that the central spindle localization of SVIL is not visible after 4% paraformaldehyde fixation. The graph shows the ratio of cells with phosphorylated RLC at the equatorial cortex (n = 30). (E) Schematic representation of PLK1-mediated myosin II activation at the cleavage furrow.
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