Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 4. Rab35 and centaurin-b2 colocalize with Arf6. (A) Subcellular localization of endogenous Rab35 and Arf6 after NGF stimulation for 6 hours. NGF-stimulated PC12 cells were fixed, and then stained with anti-Rab35 antibody (green; 1:100 dilution), anti-Arf6 antibody (red; 1:100 dilution), and DAPI (nuclei; blue). Note that Rab35 and Arf6 are colocalized in the perinuclear area (arrowheads). Scale bar: 10 mm. The arrowheads indicate colocalization between Rab35 and Arf6. (B) Subcellular localization of endogenous centaurin-b2 (Centb2) and Arf6 after NGF stimulation. After NGF stimulation for 6 hours PC12 cells were fixed, and then stained with anti- centaurin-b2 antibody (green; 1:100 dilution), anti-Arf6 antibody (red; 1:100 dilution) and DAPI (nuclei; blue). Note that centaurin-b2 and Arf6 are colocalized in the perinuclear area (arrowheads). Scale bar: 10 mm. The lower panels in A and B are magnified views of the boxed areas in the upper right panels. The arrowheads indicate colocalization between centaurin-b2 and Arf6. (C) Intensity scatterplot of Rab35 signals (green) and Arf6 signals (red) in PC12 cells after NGF stimulation for 6 hours. The Pearson’s correlation coefficient (PCC) (mean and s.d.) for the relation between them is shown at the bottom (n530 from three independent experiments). (D) Intensity scatterplot of centaurin-b2 signals (green) and Arf6 signals (red) in PC12 cells after NGF stimulation for 6 hours. The PCC (mean and s.d.) for the relation between them is shown at the bottom (n530 from three independent experiments).

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Staining

Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 5. Arf6-GAP activity of centaurin-b2 is required for NGF-induced neurite outgrowth of PC12 cells after Rab35-dependent recruitment. (A) Colocalization analysis of wild-type centaurin-b2 (Centb2-WT), centaurin-b2(DANKR) (Centb2-DANKR), and centaurin-b2(RQ) (Centb2- RQ) with endogenous Arf6 in PC12 cells after NGF stimulation for 36 hours. PC12 cells transiently expressing either Myc-Centb2(WT), Myc- Centb2(DANKR), or Myc-Centb2(RQ) (left panels) were fixed, and then stained with anti-Myc antibody (left panels; 1:500 dilution) and anti-Arf6 antibody (middle panels; 1:100 dilution). Merged images are shown in the right panels. Fluorescence intensity along white dashed lines (right panels) is shown on the right. Note that the Rab35-binding-deficient Centb2(DANKR) mutant had impaired ability to localize at the Arf6-positive pericentrosomal compartment. Scale bar: 5 mm. (B) Typical images of EGFP–Centb2(WT)SR- expressing and EGFP–Centb2(DANKR)SR-expressing PC12 cells after NGF stimulation for 36 hours under centaurin-b2-depleted conditions. Scale bar: 30 mm. (C) Effect of EGFP–Centb2(WT)SR and EGFP–Centb2(DANKR)SR

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Activity Assay, Expressing, Staining, Fluorescence, Binding Assay, Mutagenesis

Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 7. A model of the crosstalk between Rab35 and Arf6 through centaurin-b2 during neurite outgrowth. Rab35 is recruited to the pericentrosomal Arf6-positive endosomes in response to NGF stimulation and recruits its effector, centaurin-b2, to the same compartment in PC12 cells (Figs 2–4). Centaurin-b2 then inactivates Arf6 at the pericentrosomal endosomes through its Arf6-GAP activity during neurite outgrowth (Fig. 5). Inactivation of Arf6 at the pericentrosomal endosomes is required for the Rab35-mediated neurite outgrowth (Figs 1,6).

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Activity Assay

Journal: Journal of cell science

Article Title: Rab35 regulates Arf6 activity through centaurin-β2 (ACAP2) during neurite outgrowth.

doi: 10.1242/jcs.098657

Figure Lengend Snippet: Fig. 6. Arf6 functions downstream of Rab35 in neurite outgrowth of PC12 cells. (A) Typical images of EGFP–Rab35(QL) + control siRNA (siControl)-expressing and EGFP–Rab35(QL) + Arf6 siRNA (siArf6)- expressing PC12 cells after NGF stimulation for 36 hours. Scale bar: 30 mm. (B) Typical images of EGFP–Rab35(QL) + mStr-expressing and EGFP– Rab35–(QL) + Arf6(QL)–mStr-expressing PC12 cells after NGF stimulation for 36 hours. Scale bar: 30 mm. (C) Effect of Arf6 knockdown on active Rab35-promoted neurite outgrowth of PC12 cells. Bars represent the total neurite length values (mean and s.e.) of EGFP + siControl-expressing (control; black bar), EGFP + siArf6-expressing (left white bar), EGFP– Rab35(QL) + siControl-expressing (central white bar) and EGFP–Rab35(QL) + siArf6-expressing (right white bar) cells (n.100). **P,0.01, in comparison with the control cells (Student’s unpaired t-test). Note that Rab35(QL)-dependent promotion of neurite outgrowth was dramatically inhibited by knockdown of endogenous Arf6. (D) Effect of expression of Arf6(QL) on active Rab35-promoted neurite outgrowth of PC12 cells. Bars represent the total neurite length values (mean and s.e.) of EGFP + mStr- expressing (control; black bar), Arf6(QL)–mStr + EGFP-expressing (left white bar), mStr + EGFP–Rab35(QL)-expressing (central white bar) and Arf6(QL)–mStr + EGFP–Rab35(QL)-expressing (right white bar) cells (n.100). **P,0.01, in comparison with the control cells (Student’s unpaired t-test). Note that the Rab35(QL)-dependent promotion of neurite outgrowth was completely inhibited by co-expression with Arf6(QL).

Article Snippet: Anti-actin goat polyclonal antibody, anti-Arf6 mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GM130 mouse monoclonal antibody, anti-EEA-1 mouse monoclonal antibody (BD Biosciences, San Jose, CA), anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA), anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan), anti-c-tubulin mouse monoclonal antibody, and anti-Myc rabbit polyclonal antibody (Sigma, St Louis, MO) were obtained commercially.

Techniques: Control, Expressing, Knockdown, Comparison

Journal: The EMBO Journal

Article Title: A Brucella effector modulates the Arf6‐Rab8a GTPase cascade to promote intravacuolar replication

doi: 10.15252/embj.2021107664

Figure Lengend Snippet:

Article Snippet: For Western blotting, primary antibodies used were rabbit monoclonal anti‐Arf6 (1:1,000; Cell Signaling), anti‐Rab8a (1:1,000; Cell Signaling), anti‐Rab6 (1:250; Cell Signaling), anti‐Stx6 (1:1,000; Cell Signaling), rabbit polyclonal anti‐Calnexin (1:20,000; Stressgen), anti‐Lamin A/C (1:5,000; Cell Signaling), anti‐β‐actin (1:20,000; Cell Signaling) antibodies; rabbit monoclonal anti‐HA, (1:10,000; Cell Signaling), anti‐myc (1:10,000; Cell Signaling), anti‐Hsp27 (1:10,000; clone G31, Cell Signaling) antibodies.

Techniques: Derivative Assay, Recombinant, Transduction, Sequencing, Software, cDNA Library Assay, Transformation Assay, Plasmid Preparation, Isolation, Activation Assay

Journal: PLoS ONE

Article Title: The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

doi: 10.1371/journal.pone.0018458

Figure Lengend Snippet: A. Transient over-expression of ARF6(Q67L) in GFP-GAP273(3.2) promotes cell spreading (left panels). Actin foci are also absent from the cell. Transient over-expression of ARF6(T27N) in GFP-GAP273(3.2) cells (right panels) promotes or stabilizes the formation of actin foci. Images of GFP-GAP273 fluorescence of the same cells are shown in the bottom panels. The GFP-GAP273 fluorescence co-localizes with ARF6 staining in the cell transfected with ARF6(T27N). B. Transient over-expression of Rac1(G12V) (top left) promotes cell spreading and induces membrane ruffling in GFP-GAP273(3.2) cells but suppresses the localization of peripheral concentrations of GFP-GAP273 at the actin foci (compare the cell transfected with Rac1(G12V) with the surrounding non-transfected cells, bottom left). Transient over-expression of Rac1(T17N) (top right) in GFP-GAP273(3.2) cells inhibits cell spreading and prolongs the appearance of membrane projections containing GFP-GAP273. The GFP image (bottom right) shows that surrounding non-transfected cells are more spread and contain little, if any, peripheral concentrations of GFP-GAP273. For cells transfected with Rac1(G12V) or ARF6(Q67L), the cells had been re-plated on glass cover slips for about 20 hours before fixation and immunofluorescence labeling. For cells transfected with Rac1(T17N) or ARF6(T27N), the cells had been re-plated for 40 hours. Scale bar = 25 µm. C. Quantification of the effect of Rac1(T17N) and ARF6(T27N) on the formation of actin foci. The percentage of cells transiently over-expressing Rac1(T17N) or ARF6(T27N) in GFP-GAP273(3.2) cells was measured. In each of the three independent experiments, one hundred cells were counted. Error bar = S.D. D. Quantification of the effect of Rac1(G12V) and ARF6(Q67L) on cell spreading. The area covered by GFP-GAP273(3.2) cells transiently over-expressed with Rac1(G12V) or ARF6(Q67L) was measured. The mean area covered were obtained from 41, 60 and 123 cells, for ARF6(Q67L), Rac1(G12V) or non-transfected GFP-GAP273(3.2), respectively. Error bar = S.D.

Article Snippet: Goat anti-rabbit IgG (for ARF6) and goat anti-mouse IgG (for Rac1) secondary antibodies (Invitrogen) conjugated with Alexa fluorophores were used to generate fluorescence signals.

Techniques: Over Expression, Fluorescence, Staining, Transfection, Immunofluorescence, Labeling, Expressing

Journal: PLoS ONE

Article Title: The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

doi: 10.1371/journal.pone.0018458

Figure Lengend Snippet: A. Activation of Rac1 is inhibited in CHO cells stably expressing GFP-GAP273, but not in wild type CHO cells. The result shown is a representative of three independent experiments. B. GFP-GAP273 did not inhibit the activation of RhoA. C. Serum caused little change in binding of GTP by ARF6 in either cell type and GFP-GAP273 did not affect ARF6 activation. The percent of total GTP-binding protein that was bound to GTP in each sample is listed below the image of the immunoblots.

Article Snippet: Goat anti-rabbit IgG (for ARF6) and goat anti-mouse IgG (for Rac1) secondary antibodies (Invitrogen) conjugated with Alexa fluorophores were used to generate fluorescence signals.

Techniques: Activation Assay, Stable Transfection, Expressing, Binding Assay, Western Blot

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: ARF1 and ARF6 knock down impairs HGF-stimulated recycling of GRASP and Dock180. (A-C) MDCK cells were transfected with control siRNA or siRNA against either ARF6 (B) or ARF1 (C) by Neon transfection and plated on fibronectin-coated coverslips as described in the Materials and Methods. The following day, cells were transfected with GRASP and Dock180 by Lipofectamine 3000 and allowed to express for 10–12 hours. Cell were switched to serum-free media overnight and treated with HGF (10 ng/mL) the next morning. Cells were fixed at the indicated time points and imaged by deconvolution microscopy. Scale bars: 10 um. (D, E) Slidebook 6.0 imaging software was used to calculate levels of Dock180 (D) and GRASP (E) at the cell periphery in 62–78 cells as described in the Materials and Methods. Data are means ± standard error of the peripheral sum intensity normalized to the whole cell sum intensity. Asterisk indicates statistically significant compared to zero hour of set. * = p < 5 × 10−5, T test.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Transfection, Microscopy, Imaging, Software

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: Inhibition of ARF-associated recycling pathways impairs HGF-stimulated Rac activation. (A, B) Expression of Rab8 T22N or Rab11 S25N impairs HGF-stimulated Rac activation. MDCK cells were transfected with mTurquoise 2-Rab8 T22N, Rab11 S25N, or empty vector using Lipofectamine 3000. Cells were allowed to express for 10 hours and switched to serum-free media overnight. Cells were incubated in the presence or absence of HGF (20ng/mL) and lysed after 6 hours. GST-PBD pull downs were performed to isolate Rac-GTP. Starting lysate and pull down samples were run on a Western and blotted for Rac and GFP (Rabs). Representative gels are shown. Experiments were quantified by the LI-COR imaging system; Rab8 T22N (A, n = 8) and Rab11 S25N (B, n=7). Data shown are means ± standard error. * = p < 0.05, paired T test. (C, D) ARF1 and ARF6 knockdowns impair HGF-mediated Rac activation. MDCK cells were transfected with control siRNA or siRNA against ARF1 or ARF6 by Neon transfection. After 30 hours of expression, cells were switched to serum-free media overnight. The next day, cells were incubated in the absence or presence of 20 ng/mL HGF. After 6 hours, cells were lysed and Rac-GTP was isolated by GST-PBD pull down as described above. ARF6 (C) n = 7, ARF1 (D) n = 9. Data shown are means ± standard error. * = p < 0.05, paired T test.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Inhibition, Activation Assay, Expressing, Transfection, Plasmid Preparation, Incubation, Western Blot, Imaging, Isolation

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: Inhibition of ARF trafficking pathways traps GRASP and Dock180 together. (A-C) Expression of Rab8 T22N and Rab11 S25N traps GRASP and Dock180 in resting cells. MDCK cells were plated on fibronectin-coated coverslips and transfected with YPET-GRASP, mCherry-Dock180, and either mTurquoise 2-Rab8 T22N, Rab11 S25N or empty vector by Lipofectamine 3000. Cells were allowed to express for 10–12 hours, serum-starved overnight, and treated with HGF (10 ng/mL) the following morning. Cell were fixed and imaged by deconvolution microscopy as described in the Materials and Methods. (D) Extended inhibition of cytohesin GEF activity promotes GRASP and Dock180 binding. Following transfection and 10–12 hours of expression as described previously, cells were switched to serum-free media and treated with SecinH3 (15 uM). The next morning, cells were treated with HGF (10 ng/mL) and fixed at the designated time points. Scale bar: 10 um. (E-G) Knockdown of ARF1 and ARF6 promotes GRASP and Dock180 interaction. MDCK cells were Neon transfected with control siRNA or siRNA against either ARF1 or ARF6 and plated on fibronectin-coated coverslips. The next day, cells were transfected with YPET-GRASP and mCherry-Dock180 by Lipofectamine 3000, allowed to express for 10–12 hours, and switched to serum-free media overnight. Cells were treated with HGF (10 ng/mL) the next morning and fixed at the indicated time points. Scale bar: 10 um. (H) Quantification of corrected FRET signal by Slidebook 6.0 in 67–70 cells expressing either Rab8 T22N, Rab11 S22N or treated with SecinH3. Data are means ± standard error, * = p < 5 × 10−4, ** p < 5 × 10−7, *** p < 5 × 10−20, T test. Corrected FRET intensity values in scale bar are in arbitrary units. (I) Whole cell FRET sum intensity was calculated using Slidebook 6.0 in 62–78 cells. Data are means ± standard error. Asterisk indicates statistically significant compared to zero hour of set. * p < 5 × 10−9, T test. Corrected FRET intensity values in scale bar are in arbitrary units.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Microscopy, Activity Assay, Binding Assay

Journal: Small GTPases

Article Title: ARF1 and ARF6 regulate recycling of GRASP/Tamalin and the Rac1-GEF Dock180 during HGF-induced Rac1 activation

doi: 10.1080/21541248.2016.1219186

Figure Lengend Snippet: Trafficking of GRASP and Dock180 during ARF6 to Rac cross talk. GRASP and Dock180 localize to recycling endosomes in resting cells. HGF stimulation promotes association of GRASP and Dock180 and trafficking to the plasma membrane. Movement to the plasma membrane positions Dock180 to activate the membrane-localized Rac1. Inhibition of ARF6-associated trafficking pathways blocks HGF-stimulated movement of GRASP and Dock180 to the periphery and Rac1 activation. Inhibition of trafficking also traps GRASP and Dock180 together in the cell.

Article Snippet: Rabbit anti-ARF6 was purchased from Sigma.

Techniques: Inhibition, Activation Assay