Figure 1.
RacGAP activity of ARHGAP22 suppresses lamellae formation.
(A) EGF-induced lamellae formation. Serum-starved A7 cells were fixed 30 min after the treatment of the cells without (control) or with 50 nM EGF (+EGF) and stained with phalloidin for F-actin (red). The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm. (B) A7 cells were either not transfected (control) or transfected with ARHGAP22 constructs (WT, ΔGAP, or R211A) for 5 h and serum-starved. The cells were fixed after treatment without (control) or with 50 nM EGF (+EGF) for 30 min. Representative images of cells stained with anti-HA antibody for HA-ARHGAP22 (green) and phalloidin (red) are shown. Merged fluorescent images are shown. The cells were also stained with hoechst 33258 (blue). Scale bar, 20 µm. (C) The percentages of lamellipod-positive cells (n = 100) were calculated, and the data are expressed as the mean ± s.e.m. (N = 3). **, p<0.01. Statistical significance was determined by Student's t-test.
Figure 2.
ARHGAP22 does not interact with FLNa.
(A) A7 cells were transfected with FLAG-ARHGAP22 or FLAG-FilGAP. After 24 h, cells were fixed and ARHGAP22 and FilGAP (green) or FLNa (red) was localized by staining the cells with anti-FLAG and anti-FLNa antibodies. Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm. (B) HEK cells were transfected with HA-ARHGAP22 or HA-FilGAP. HA-ARHGAP22 or HA-FilGAP was immunoprecipitated from cell extracts using anti-HA agarose, and bound proteins were identified by immunoblot using anti-HA and anti-FLNa antibodies. (C) HEK cells were transfected with HA-ARHGAP22 or HA-FilGAP. Cell extracts were prepared and then incubated with GST-FLNa-Repeat 23–24 or GST alone, and precipitated with glutathione-Sepharose beads. Bound proteins were analyzed by immunoblot using anti-HA antibody. Asterisks indicate non-specific bands. (D) HEK cells were transfected with HA-FilGAP (or HA-ARHGAP22) in the presence or absence of FLAG-FilGAP (or FLAG-ARHGAP22). Then, HA-FilGAP (or HA-ARHGAP22) was immunoprecipitated from cell extracts using anti-HA agarose, and bound proteins were identified by immunoblot using anti-HA and anti-FLAG antibodies. Asterisk indicates a non-specific band. (E) HEK cells were transfected with HA-ARHGAP22 or HA-FilGAP. The cells were lysed after treatment with indicated concentrations of DSP, boiled with 1% SDS in the absence or presence of 2-mercaptoethanol (2ME) and analyzed by immunoblot using anti-ARHGAP22 or anti-FilGAP antibody.
Figure 3.
Subcellular distribution of ARHGAP22.
(A) Schematic diagram of HA-ARHGAP22 constructs. (B) Ectopic expression of HA-ARHGAP22 constructs. HEK cells were transfected with HA-ARHGAP22 constructs. HA-ARHGAP22 proteins were analyzed by immunoblot using anti-HA antibody. Tubulin was used as a loading control. (C) A7 cells were transfected with HA-ARHGAP22 constructs. After 24 h, the cells were fixed and stained with anti-HA (green) and anti-FLNa (red) antibodies. Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm.
Figure 4.
Colocalization of ARHGAP22 with endocytic markers.
(A) A7 cells were transfected with HA-ARHGAP22. After 24 h, the cells were fixed and stained with anti-HA antibody for HA-ARHGAP22 (green) and antibodies for Rab11, Rab5, EEA1, LAMP-1, GM130, or TGN46 (red). Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm. Insets show magnification images of the boxed regions. (B) C2C12 cells were transfected with HA-ARHGAP22. After 24 h, the cells were fixed and stained with anti-HA antibody for HA-ARHGAP22 (green) and antibodies for Rab11 or Rab5 (green). Merged fluorescent images are shown. The cells were also stained with hoechst 33258 (blue). Scale bar, 20 µm. Insets show magnification images of the boxed regions.
Figure 5.
Coiled-coil domain of ARHGAP22 is responsible for targeting to Rab11-positive vesicle structures.
(A) A7 cells were transfected with HA-tagged coiled-coil (CC) domain of ARHGAP22 (HA-ARHGAP22CC). After 24 h, the cells were fixed and stained with anti-HA antibody for HA-ARHGAP22CC (green) and anti-Rab11 antibody (red). Merged fluorescent image is shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm. Inset shows magnification image of the boxed region. (B) A7 cells were transfected with HA- ARHGAP22ΔPH. After 24 h, the cells were fixed and stained with anti-HA for HA- ARHGAP22ΔPH (green) and antibodies for GM130 or Rab11 (red). Merge fluorescent images are shown. The cells were also stained with hoechst 33258 (blue). Scale bar, 20 µm. Insets show magnification images of the boxed regions.
Figure 6.
Localization of endogenous ARHGAP22 in C2C12 cells.
(A) C2C12 cells were fixed and stained with anti-ARHGAP22 (green) or anti-Rab11 (red) antibodies, which was non-treated (control) or preabsorbed with lysates from non-transfected (−) or HA-ARHGAP22-transfected (+) HEK cells. Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm. Inset shows a magnification image of the boxed region. (B) C2C12 cells were treated with control or ARHGAP22 siRNAs for 48 h and serum-starved. The cells were fixed and stained with anti-ARHGAP22 (green) and anti-Rab11 (red) antibodies. Merged fluorescent images are shown. The cells were also stained with hoechst 33258 (blue). Scale bar, 20 µm. (C) C2C12 cells were fixed and stained with anti-ARHGAP22 antibody (green) and antibodies for TGN46 or EEA1 (red). Merged fluorescent images are shown. The cells were also stained with hoechst 33258 (blue). Scale bar, 20 µm. (D) Pearson's Colocalization Coefficient (PCC) was calculated by ImageJ (NIH). The data are expressed as the mean ± s.e.m. (N = 3). Ten cells were analyzed for each experiment. **, p<0.01. Statistical significance was determined by Student's t-test.
Figure 7.
ARHGAP22 suppresses cell spreading.
(A) A7 cells were transfected with HA-ARHGAP22 constructs (WT, ΔGAP, R211A, or ΔCC,) and serum-starved for 20 h. Quiescent cells were trypsinized and then plated on collagen-coated coverslips and fixed 20 min after plating. The cells were stained with anti-HA antibody for HA-ARHGAP22 (green) and phalloidin for F-actin (red). Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Arrowheads indicate the transfected cells. Scale bar, 20 µm. (B) The surface area of spreading cells (n = 100) 20 min after plating were calculated and shown as box and whisker plots. **, p<0.01. Statistical significance was determined by Welch's t-test.
Figure 8.
Depletion of ARHGAP22 stimulates cell spreading on fibronectin.
(A) Immunoblot showing that ARHGAP22 is depleted after 48 h of siRNA treatment of C2C12 cells. ARHGAP22 and tubulin were detected by immunoblot using anti-ARHGAP22 and anti-tubulin antibodies, respectively. (B) C2C12 cells were treated with control or ARHGAP22 siRNAs for 48 h and serum-starved. The cells were trypsinized and then plated on fibronectin-coated coverslips and fixed at 10, 20, 30, and 40 min after plating. The cells were stained with phalloidin for F-actin. Scale bar, 20 µm. (C) The percentage of spread cells (n = 200) were calculated and plotted as the mean ± s.e.m. (N = 3). (D) The surface area of spreading cells (n = 100) 10 min after plating was calculated and shown as box and whisker plots. **, p<0.01. Statistical significance was determined by Welch's t-test. (E) HEK cells were treated with control or ARHGAP22 siRNA for 24 h followed by transfection with HA-tagged ARHGAP22 constructs. The cells were cultured for another 24 h. ARHGAP22 and tubulin were analyzed by immunoblot using anti-HA and anti-tubulin antibodies, respectively. (F) C2C12 cells were treated with control or ARHGAP22 siRNA KD#1 for 24 h followed by a transfection with rescue constructs (KDr). The cells were cultured for another 24 h and serum-starved. The cells were fixed and stained with anti-HA antibody for HA-KDr (green) and phalloidin (red). Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm.
Figure 9.
ARHGAP22 co-localizes with constitutively activated Rac at the plasma membrane.
A7 cells were transfected with HA-ARHGAP22 constructs (WT, R211A, or ΔGAP) and constitutively activated Rac mutant (EGFP-Rac1 Q61L). After 24 h, the cells were fixed and stained with anti-HA for HA-ARHGAP22 (red). The GFP signal for Rac Q61L (green) was observed in the fixed cells. Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm. Insets show magnification images of the boxed regions.
Figure 10.
Forced expression of ARHGAP22 does not affect endocytic trafficking of transferrin.
A7 cells were either not transfected (control) or transfected with HA-ARHGAP22. After 24 h, the cells were incubated in serum-free growth medium for 1 h. The cells were then incubated with 20 µg/ml Alexa Fluor 568-transferrin at 37°C for 30 min and fixed immediately after incubation. Cells were stained with antibodies for Rab11 or HA-ARHGAP22 (green). Internalized transferrin signals (red) were detected in the fixed cells. Merged fluorescent images are shown. The cells were also stained with hoechst 33258 for nuclei (blue). Scale bar, 20 µm. (B) Quantification of the internalized transferrin signal. The transferrin intensity was calculated as fluorescence intensity of Alexa Fluor 568-transferrin per cell divided by the surface area of this cell. The fluorescence intensity and cell area were measured by ImageJ (NIH), and the data are expressed as the mean ± s.e.m. (n = 50 cells). Statistical significance was determined by Student's t-test.