Fig 1.
(A) Co-precipitation of FilGAP and RBM10. HEK293T cells were transiently transfected with HA-tagged RBM10 (HA-RBM10) and Flag-tagged FilGAP (Flag-FilGAP). Cell extracts were prepared and HA-RBM10 was immunoprecipitated using an anti-HA antibody. The washed immunoprecipitates were immunoblotted for the presence of FilGAP and RBM10. (B) HEK293T cells were transiently transfected with HA-RBM10 and Flag-FilGAP. Cell extracts were prepared and incubated without or with RNase inhibitor Rnasin (0.12 mg/ml), RNase A (0.05 mg/ml), or RNase A (0.1 mg/ml) for 20 min at 37°C. Then, HA-RBM10 was immunoprecipitated using an anti-HA antibody. The washed immunoprecipitates were immunoblotted for the presence of FilGAP and RBM10. Two separate samples are shown. (C) HEK293T cells were transiently transfected with HA-RBM10 and Flag-FilGAP constructs shown in the diagram. Cell extracts were prepared and HA-RBM10 was immunoprecipitated using an anti-HA antibody. The washed immunoprecipitates were immunoblotted for the presence of RBM10 and FilGAP. Two separate samples are shown. PH; Pleckstrin homology, GAP; Rho GAP, CC; coiled-coil domain (D) HEK293T cells were transiently transfected with Flag-FilGAP and HA-RBM10 constructs shown in the diagram. Cell extracts were prepared and HA-RBM10 was immunoprecipitated using an anti-HA antibody. The washed immunoprecipitates were immunoblotted for the presence of RBM10 and FilGAP. Two separate samples are shown. RRM1; RNA recognition motif 1, Zf; Zink finger; OCRE; octamer-repeat region, G-patch; G-patch domain (E) Endogenous FilGAP associates with endogenous RBM10. MDA-MB-231 cells were incubated with 1 mM dithiobis (succinimidyl propionate) at 25°C for 10 min, washed with PBS and suspended in lysis buffer. Cell lysates were prepared and FilGAP protein was immunoprecipitated using anti-FilGAP antibody and bound RBM10 was identified using anti-RBM10 antibody.
Fig 2.
Fyn regulates localization of RBM10 in A7 cells.
(A) A7 cells were transfected with constitutively activated Fyn (CA-Fyn) or kinase negative Fyn (KN-Fyn) and serum starved. After 24 h, cells were fixed and Fyn (green) and tyrosine-phosphorylated proteins (red) were localized by staining the cells with anti-Fyn and anti-pTyr antibodies. Merged fluorescent images are shown. Scale bar, 25 μm. Cells were also stained with Hoechst 33258 to label nuclei (blue). (B) A7 cells were transfected with HA-RBM10 together with CA-Fyn or KN-Fyn and serum starved. After 24 h, cells were fixed and HA-RBM10 (green) and tyrosine-phosphorylated proteins (red) were localized by staining the cells with anti-HA and anti-pTyr antibodies. Merged fluorescent images are shown. Cells were also stained with Hoechst 33258 to label nuclei (blue). Scale bar, 25 μm. (C) The percentages of cytosolic RBM10 were calculated and the data are expressed as the mean +/- s.e.m. (n = 3–6). *, p<0.05. (D) A7 cells were transfected with CA-Fyn and serum starved. After 24 h, cells were fixed and CA-Fyn (green) and endogenous RBM10 (red) were localized by staining the cells with anti-Fyn and anti-RBM10 antibodies. Merged fluorescent images are shown. Scale bar, 25 μm. Arrowheads indicate the nuclear localization of RBM10 in control cells. (E) The ratio of cytosolic and nuclear RBM10 fluorescence intensities are shown (n = 20 cells). The data are expressed as the mean +/- s.e.m. **, p<0.01.
Fig 3.
Fyn regulates localization of RBM10 and FilGAP in A7 cells.
(A) A7 cells were transfected with CA-Fyn in the absence or presence of Flag-FilGAP and serum starved. After 24h, cells were fixed and stained with anti-Fyn (green) or anti-Flag (green), and anti-pTyr (red) antibodies. Cells were also stained with Hoechst 33258 to label nuclei (blue). Merged fluorescent images were shown. Characteristic protrusive structures were indicated with dotted lines. Scale bar, 25 μm. (B) The surface area of protrusive structures as indicated by dotted lines in (A) was calculated (n = 15–20 cells), and the data are expressed as the mean +/- s.e.m. **, p<0.01. (C) A7 cells were transfected with HA-RBM10 and Flag-FilGAP in the absence or presence of CA-Fyn and serum starved. After 24h, cells were fixed and HA-RBM10 (red) and Flag-FilGAP (green) were localized by staining the cells with anti-HA and anti-Flag antibodies. Cells were also stained with Hoechst 33258 to label nuclei (blue). Merged fluorescent images were shown. Scale bar, 25 μm.
Fig 4.
Localization of endogenous FilGAP and RBM10 in MDA-MB-231 cells spreading on collagen.
(A) MDA-MB-231 cells were serum-starved for 18 hrs. Quiescent cells were trypsinized and cells in suspension were plated on coverslips coated with collagen and fixed at 30 min after plating. Cells were stained with control IgG, anti-FilGAP, or anti-RBM10 antibodies for endogenous FilGAP and RBM10 proteins (green). Cells were also stained with anti-pTyr antibody for visualization of focal complex (red). Merged fluorescent images are shown. Scale bar, 25 μm. (B) Immunoblot showing that RBM10 is depleted after 48hr of siRNAs treatment. Tubulin was used as a loading control. (C) MDA-MD-231 cells were transfected with control or RBM10 siRNA. After 30 hrs, the cells were serum-starved for 18 hrs and trypsinized, and cells in suspension were plated on coverslips coated with collagen and fixed at 30 min after plating. Cells were stained with anti-FilGAP antibody (green) for endogenous FilGAP, and anti-phosphotyrosine antibody (red) for tyrosine-phosphorylated proteins. Merged fluorescent images are also shown. Scale bar, 25 μm. (D) Fluorescent intensities of endogenous FilGAP were quantified by line scan analysis. (E) Fluorescent intensities of endogenous FilGAP at the cell periphery are shown, and the data are expressed as the mean +/- s.e.m. **, p<0.01. The statistical significance was accessed by Student’s t-test. (control: n = 11, RBM10KD: n = 9)
Fig 5.
RBM10 promotes FilGAP-dependent suppression of cell spreading on collagen.
(A) A7 cells were transiently transfected with Flag-FilGAP without (-) or with (+) HA-RBM10 and serum starved. Quiescent cells were trypsinized, and then plated on coverslips coated with collagen and fixed at 30 min after plating. Cells were fixed and stained with anti-FilGAP (green) and anti-pTyr (red) or anti-HA (red) antibodies. Merged fluorescent images are also shown. Scale bar, 25 μm. (B) The surface area of spreading cells 30 min after plating was calculated and the data are expressed as box plots. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the10–90th percentiles. **, p<0.01. The statistical significance was determined by Welch’s t-test. (n = 150 cells) (C) A7 cells were transiently transfected with Flag-FilGAP and in the presence of control or RBM10 siRNAs and serum starved. Quiescent cells were trypsinized, and then plated on coverslips coated with collagen and fixed at 30 min after plating. Cells were stained with anti-FilGAP (green) antibody. Scale bar, 25 μm. (D) A7 cells were transiently transfected with Flag-FilGAP and HA-RBM10 resistant to RBM10 siRNA (HA-RBM10WT-R) in the presence of RBM10 siRNA and serum starved. Quiescent cells were trypsinized, and then plated on coverslips coated with collagen and fixed at 30 min after plating. Cells were stained with anti-FilGAP (green) and anti-HA (red) antibodies. Merged fluorescent images are also shown. Scale bar, 25 μm. (E) The surface area of spreading cells 30 min after plating was calculated and the data are expressed as box plots. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the10–90th percentiles. **, p<0.01. The statistical significance was determined by Welch’s t-test. (n = 150–300 cells) (F) HEK293T cells were transiently transfected with HA-RBM10 or HA-RBM10WT-R in the presence of control or RBM10 siRNA. HA-RBM10 proteins were analyzed by immunoblot using anti-HA antibody. Tubulin was used as a loading control.
Fig 6.
RacGAP activity of FilGAP is stimulated by RBM10.
(A) Immunoblot showing that Rac1 is depleted after 48 hrs of siRNAs treatment of A7 cells. Tubulin was used as a loading control. (B) A7 cells were transiently transfected with constitutively activated Fyn with control or Rac1 siRNAs. After 48 hrs, the cells were fixed and stained with anti-pTyr antibody. Scale bar, 25 μm. (C) A7 cells were transiently transfected with CA-Fyn and Flag-FilGAP in the presence of control or RBM10 siRNA. After 48 hrs, the cells were fixed and stained with anti-FilGAP (green) and anti-pTyr (red) antibodies. Merged fluorescent images are also shown. Scale bar, 25 μm. (D) A7 cells were transiently transfected with CA-Fyn, Flag-FilGAP, and HA-RBM10WT-R in the presence of RBM10 KD #1 siRNA. After 48hrs, the cells were fixed and stained with anti-FilGAP (green) and anti-HA (red) antibodies. Merged fluorescent images are also shown. Scale bar, 25 μm.
Fig 7.
RBM10 promotes FilGAP-dependent suppression of membrane ruffles induced by EGF.
(A) Serum-starved A7 cells were fixed 30 min after the treatment of the cells with or without EGF (50 nM). Fixed cells were stained with Alexa Fluor568-Phalloidin for F-actin. Scale bar, 25 μm. (B) A7 cells were transiently transfected with Flag-FilGAP with control or RBM10 siRNA and serum starved. Cells were pre-incubated with or without 100 μM Rac inhibitor NSC23766 for 1hr and then fixed after the treatment of the cells with 50 nM EGF for 30 min. Fixed cells were stained with anti-FilGAP antibody for FilGAP (green) and Alexa Fluor 568-Phalloidin for F-actin (red). Merged fluorescent images are also shown. Scale bar, 25 μm. Arrowheads indicate membrane ruffles. (C) The percentages of membrane ruffling-positive cells were calculated (>300 cells counted for each experiment), and the data are expressed as the mean +/- s.e.m. (n = 3). **, p<0.01. The statistical significance was accessed by Student’s t-test.
Fig 8.
Proposed roles of RBM10 and FilGAP downstream of SFKs signaling to control Rac.
Arrows and T-junction represent stimulation and repression.