ARAP1 fine-tunes F-actin polymerization level in lymphocytes through RhoA inhibition

Figure 1. ARAP1 expression in lymphocytes. (A) Isoforms of ARAP1. (B) mRNA expression of ARAP1 isoforms (iso1/4, isoforms 1 and 4; iso2, isoform 2; iso3/5, isoforms 3 and 5) in lymphocytes, measured using quantitative RT-PCR. (C) The detection of 33 bp insert in PH5 domain of ARAP1 isoforms 4 and 5 using RT-PCR (D) ARAP1 protein expression in lymphocytes. Lysates from T cell, B cells, T-blasts and Ba/F3 cells were subjected to western blot analysis to detect ARAP protein expression.Arrowheads indicate long isoforms (~180kDa, isoforms 3 and 5) and intermediate isoforms (~130kDa, isoforms 1 and 4).

Figure 2. ARAP1 localization in lymphocytes after chemokine stimulation. (A) ARAP1 protein expression in Ba/F3.hLFA1 cells following ARAP1 knockout (KO). (B) CXCR4 expression of wild-type (WT) cells (red) and Arap1 knockout (KO) cells (light blue). Unstained control WT cells (dotted red) and Arap1 KO cells (dotted light blue) were also shown. (C) Integrin αL expression in WT cells (red) and Arap1 KO cells (light blue), measured using flow cytometry and TS2/4 antibody staining. Unstained control WT cells (dotted red) and Arap1 KO cells (dotted light blue) were also shown. (D) Representative Z-stack images of endogenous ARAP1 in cells unstimulated and stimulated with CXCL12 (10 nM, 1 and 10 min). ARAP1, green; F-actin: red. Arrow heads indicate ARAP1 accumulation in ruffle membranes. Scale Bar, 5 μm. (E) Representative Z-projection images of Venus-ARAP1 wild type in the enter body of cells unstimulated and stimulated with CXCL12 (10 nM, 1 and 10 min). ARAP1, green; F-actin, red. Scale Bar, 10 μm. Arrowheads indicate colocalization of Venus-ARAP1 and F-actin rich ruffles.

Figure 3. ARAP1 deficiency increased F-actin capping and polymerization in lymphocytes. (A) Quantification of F-actin level in WT (n = 3) and Arap1 KO (n = 3) cells following stimulation with CXCL12 (10 nM). F-actin level was visualized by staining with fluorescently labeled phalloidin and measured using flow cytometry. Median fluorescent intensity (MFI) was normalized to the average MFI of unstimulated WT cells. (B) The upper representative image showed F-actin and CD44 staining in unpolarized cells and chemokine-induced polarized cells (10 nM, 10 min), acquired using imaging cytometer. The lower panel shows average percentages of the F-actin capped cells among WT (n = 3) and Arap1 KO (n = 3) cells before and after CXCL12 stimulation (1, 10, 50 nM, 10 min). (C) Representative Z-projection images of WT and Arap1 KO cells stained with fluorescently labeled phalloidin. Scale Bar, 10 μm. Asterisks indicate statistical significance for (A, B) calculated using Student’s t-test; *p < 0.05; **p < 0.01.

Figure 4. ARAP1 deficiency increased RhoA-GTP in lymphocytes. (A) Detection of active RhoA in WT and Arap1 KO cells using GST-Rhotekin-RBD pull-down assay. Both active (GTP-bound, the upper image) and total RhoA (the lower image) were detected using anti-RhoA antibody. The numbers under images represent relative active RhoA expression calculated by dividing the intensity of RhoA-GTP by that of total RhoA and normalizing the calculated ratios with that of unstimulated WT cells. (B) RhoA activation levels in WT (n = 4) and Arap1 KO cells (n = 4), measured using flowcytometry with anti-RhoA-GTP. RhoA activation levels were calculated as the intensity of RhoA-GTP staining divided by intensity of total RhoA staining. Calculated ratios were normalized to the ratio of unstimulated WT cells. Asterisks indicate statistical significance for (A, B) calculated using Student’s t-test; **p < 0.01.

Figure 5. Ectopic ARAP1 expression inhibits RhoA activation via RhoGAP and the RA domain. (A) Schematic diagram of ARAP1 mutants with RhoGAP domain (ΔRhoGAP) or RA domain (ΔRA) deletion. (B) Expression of Venus alone, Venus-ARAP1 wild-type (WT), Venus-ARAP1ΔRhoGAP, and Venus-ARAP1ΔRA in Ba/F3 transfectants, detected using western blotting. The arrowhead shows endogenous ARAP1. (C) Detection of active RhoA in cells expressing Venus alone or Venus-Arap1 mutants using GST-Rhotekin-RBD pull-down assay. Both active (GTP-bound, the upper image) and total RhoA (the lower image) were detected using anti-RhoA antibody. The numbers under images represent relative active RhoA expression calculated by dividing the intensity of RhoA-GTP by that of total RhoA and normalizing the calculated ratios to that of unstimulated Venus-transfected cells. (D) RhoA activation levels in Ba/F3 transfectants expressing Venus alone (n = 3), Venus-ARAP1WT (n = 3), Venus-ARAP1ΔRhoGAP (n = 3), and Venus-ARAP1ΔRA (n = 3) mutants with and without CXCL12 stimulation (10 nM, 5 min) measured using flowcytometry with anti-RhoA-GTP and anti-total RhoA staining. RhoA activation levels were calculated as the intensity of RhoA-GTP…

Figure 6. ARAP1-RA domain bound to Rap1. (A) Co-immunoprecipitation assay of Venus-ARAP1 and SNAP-Rap1 using anti-GFP antibody. The left panel shows precipitated Venus alone and Venus-Arap1; the middle panel shows co-immunoprecipitated SNAP alone and SNAP-Rap1; the right panel shows the inputs of SNAP and SNAP-Rap1 from whole lysates. (B) The distribution of Venus-ARAP1 and Turquoise2-Rap1 in unstimulated and CXCL12-stimulated (CXCL12, 10 nM, 1 min) cells. Confocal images of Venus-ARAP1 and Turquoise2-Rap1 and the merged images are shown. Arrowheads represent the location where ARAP1 and Rap1 colocalize. Scale bar, 10 µm (C) Schematic diagram of GST, GST-RA, and GST-RA-PH5-C-terminal domains. The image shows Coomassie staining of these proteins enriched using Glutathione Sepharose beads. (D) RA domain of ARAP1 bound to Rap1. Lysates from 293T cells transfected with SNAP-Rap1 mutants were subjected to pull-down assay using GST, GST-RA, and GST-RA-PH5-C-terminal domain. Representative images of pull-down samples (upper image) and their inputs (lower image) are shown.

Figure 7. ARAP1 inhibited lymphocyte migration. (A) Chemotactic migration efficiency of WT and Arap1KO T cells. Cells (1 × 105) were loaded onto upper chamber and allowed to migrate to lower chamber for 3 h The lower chambers contained CXCL12 at concentrations of 0 (n = 5), 10 nM (n = 5). (B) Chemotactic migration efficiency of WT and Arap1KO T cells on integrin-ligand ICAM-1. The upper chambers were coated with 100 μg/ml of anti-human IgG capture antibody, followed by loading of 0 or 0.5 μg/ml of human ICAM1. The lower chambers contained CXCL12 at concentrations of 0 (n = 3), 10 nM (n = 3). (C) Migration efficiency of Ba/F3.hLFA1 cells expressing ARAP1 mutants towards CXCL12 using transwell migration assay. The lower chambers contained CXCL12 at concentrations of 0 (n = 1), 3 nM (n = 3). (D) Migration of Ba/F3.hLFA1 cells expressing ARAP1 mutants on immobilized ICAM-1 (0.5 μg/ml) and CXCL12 (50 nM). Upper images display individual cell tracks. Scale bar, 50 µm. The lower left panel showed track velocity (μm/min); the lower right panel confinement ratio calculated as the displacement divided by total path length. Statistical analysis was performed using Student’s t-test for (A, B) and…