Journal: Frontiers in Cell and Developmental Biology

Article Title: Feedback-Driven Mechanisms Between Phosphorylated Caveolin-1 and Contractile Actin Assemblies Instruct Persistent Cell Migration

doi: 10.3389/fcell.2021.665919

Figure Lengend Snippet: Actin protrusive network is upregulated by AMPK-Rac1-PAK1-Cofilin signaling cascade in cells lacking CAV-1. (A) Immunofluorescence microscopy analysis demonstrating that more pronounced endogenous ARPC2 colocalizes with F-actin (visualized by Alexa 568 phalloidin) on the lamellipodia protrusions in CAV-1 deficient cells. Magnified regions of cell edges on the right show the distribution of ARPC2 in WT and CAV-1 KO cells. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). Quantification of width of lamellipodial protrusions are shown on the right. n = 16 regions from 16 cells for each group. (B) Representative images of membrane ruffling by time-lapse microscope. 1-pixel-wide areas were cut out to generate a 300 frame 2 s interval kymograph. Yellow dashed lines indicate the track of cell movement. An enlarged region is displayed on the right, vertical dashed lines show the membrane protrusion distance, while horizontal dashed lines mark the duration of protrusion. Bars, 10 μm (in cell images) and 2 μm (in the magnified box). Quantification of protrusion rate are shown on the right. n = 16 regions from 16 cells for each group. (C) P-AMPK (Thr172) and total AMPK were detected from the lysates of each group by western blotting. Please note that CAV-1(Y14F)-mEGFP can’t be detected by using phospho-CAV-1(Tyr14) antibody. Asterisk denotes the non-specific band. Quantification of P-AMPK (Thr172) levels (compared to total AMPK) from each group was shown on the right panel. n = 3. (D) Pull-down assays were performed for WT, CAV-1 KO, and CAV-1 KO; CAV-1-mEGFP re-expressed cells. Proteins bound to GST-PAK binding domain were analyzed by western blots and further quantified (compared to total Rac1) based on the band’s intensity. n = 3. (E) Western blot analysis and quantification (compared to total PAK1 and Cofilin) of the levels of phosphorylated PAK1 (Thr423) and Cofilin (Ser3) in WT and CAV-1 KO cell lysates. n = 3. (F) Immunostaining and quantification of endogenous P-Cofilin (Ser3) and F-actin distribution in WT and CAV-1 KO cells. A 16 μm length line was used to generate a line profile to illustrate the co-localization of P-Cofilin (Ser3) and F-actin. The lamellipodia region was enlarged on the right, and 2 μm width region was chosen to analyze the mean intensity of P-Cofilin (Ser3) on the leading edge. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (G) Immunostaining and quantification of endogenous P-cofilin (Ser3) distribution upon compound C treatment in WT and CAV-1 KO cells. Magnified regions represent the lamellipodia region. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (H) Western blot analysis and quantification of the phosphorylated levels of AMPK (Thr172), PAK1 (Thr423), Cofilin (Ser3) and activity of Rac1 and RhoA upon compound C treatment. n = 3. In panel (C–E,H) , the obtained intensity value from wild-type cells was set to 1. All the data are presented as mean ± SD. In (C,G,H) , *** P < 0.001; * P < 0.05; N.A., not significant (one-way ANOVA). In (D–F) , *** P < 0.001; ** P < 0.01 (unpaired t -test). All the data are from three independent experiments.

Article Snippet: The following antibodies were used in this study: CAV-1 (D46G3) rabbit antibody (1:1,000 dilution for WB, 1:200 for IF; #3267, Cell Signaling, Beverly, MO, United States); Phospho-CAV-1 (Tyr14) rabbit antibody (dilution 1:1,000 for WB; #3251, Cell signaling); AMPK rabbit antibody (dilution 1:500 for WB; #SAB4502329, Sigma, St. Louis, MO, United States); P-AMPK (Thr172) rabbit antibody (dilution 1:500 for WB, 1:100 for IF; #2531S, Cell Signaling); Cofilin (E-8) mouse antibody (dilution 1:1,000 for WB; #sc-376476, Santa Cruz, Dallas, TX, United States); Phospho-Cofilin (Ser3) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #3313, Cell signaling); p190RhoGAP rabbit antibody (dilution 1:2,000 for WB; #26789, Proteintech, Rosemont, IL, United States); FAK rabbit antibody (dilution 1:1,000 for WB; #3285, Cell Signaling); Phospho-FAK (Tyr397) rabbit antibody (dilution 1:1,000 for WB; #3283, Cell Signaling); PAK1 rabbit antibody (dilution 1:1,000; #2602, Cell Signaling); Phospho-PAK1 (Thr423)/PAK2 (Thr402) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #2601, Cell Signaling); Tpm4.2 (LC24) mouse antibody (dilution 1:500 for WB and IF; a kind gift from Peter W. Gunning, UNSW Australia); Phospho-myosin light chain 2 (Thr18/Ser19) rabbit antibody (dilution 1:500 for WB, 1:200 for IF; #3674, Cell Signaling); Myosin light chain mouse antibody (dilution 1:1,000 for WB; #M4401, Sigma); Vinculin mouse antibody (dilution 1:100 for IF; #V9131, Sigma); ARPC2 rabbit antibody (dilution 1:1,000 for WB and IF; #15058, Proteintech, Rosemont, IL, United States); Myosin-18B rabbit antibody (dilution 1:500 for WB; #HPA000953, Sigma); Rab8 rabbit antibody (dilution 1:100 for IF; #R5530, Sigma); and GAPDH mouse polyclonal antibody (dilution 1:1,000 for WB; #G8795, Sigma).

Techniques: Immunofluorescence, Microscopy, Membrane, Western Blot, Binding Assay, Immunostaining, Activity Assay

Journal: Oncotarget

Article Title: Identification of the PAK4 interactome reveals PAK4 phosphorylation of N-WASP and promotion of Arp2/3-dependent actin polymerization

doi: 10.18632/oncotarget.20352

Figure Lengend Snippet: (A) Several subunits of the Arp2/3 and CCT complexes were identified in the PAK4 interactome. White nodes: proteins passed QMS cut-off; Grey nodes: proteins appeared in MS but did not pass the QMS cut-off; Darker grey node: not in the MS list. (B) After GFP-Trap IP of H1299 cell lysates transiently expressing EGFP (control) or EGFP-PAK4, samples were subjected to immunoblot analysis for the indicated proteins. The upper panel is blotted with anti-CCTε, the middle panel with anti-ARPC2, while anti-GFP was used to control the IP efficiency in the lower panel. Input lanes are direct immunoblot of the used cell lysates. (C) After anti-CCTε IP of H1299 cell lysates transiently expressing EGFP-PAK4, blots were probed with an anti-GFP antibody in the upper panel. Anti-CCTε was used to control the IP efficiency in the lower panel. Input lane shows direct immunoblotting of the used lysate.

Article Snippet: Antibodies for ARPC2 (HPA008352) were obtained from Atlas Antibodies.

Techniques: Expressing, Control, Western Blot

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet: ( A ) dSTORM reconstruction of F-actin stained with phalloidin-AlexaFluor647 in synapses of primary mouse B cells interacting for 20 min with PMS loaded with anti-Igκ F(ab’) 2 . The cells were treated with the indicated inhibitors from 5 min after initial spreading. Scale bar, 2 μm. ( B ) Quantification of foci characteristics obtained from TIRF timelapses of live cells treated with inhibitors as in ( A ). Plots show foci numbers, mean lifetimes, and mean displacements per cell. ( C ) Immunoblot of lysates of Cas9 Ramos cells transfected with the indicated gRNAs, developed with anti-ARPC2 and anti-GAPDH antibodies. ( D, F ) TIRF images of F-actin stained with phalloidin-AlexaFluor647 in synapses of a Cas9-expressing Ramos B cells transduced with the indicated gRNAs. Cells were imaged after interacting for 20 min with PMS loaded with anti-IgM F(ab’) 2 . Scale bars, 5 μm. ( E, G ) Quantification of Ramos actin foci numbers per cell area. Data in B, E, G show values from individual cells pooled from two ( B, G ) or three ( E ) experiments. Bars indicate means and SDs. P, significance in one-way ANOVA compared to DMSO or control, *, p<0.0001.

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Staining, Western Blot, Transfection, Expressing, Transduction, Control

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet: ( A ) Ramos cells expressing ARPC2-mRuby (magenta) were imaged by TIRF microscopy on PMSs loaded with anti-IgM F(ab’) 2 . F-actin was stained with phalloidin-AlexaFluor647 (green). Scale bar, 5 μm. Panels on the right show magnified area in the white box. Arrows show ARPC2 clusters colocalized with actin foci. Scale bar 1 μm. ( B ) Example of dynamics of ARPC2-mRuby in a single actin focus visualized with Lifeact-GFP. Time zero corresponds to initial focus formation. Scalebar 1 μm. ( C ) Example of a dynamic filament growth from ARPC2-positive actin foci in Ramos cells co-expressing ARPC2-mRuby and Lifeact-GFP. Bottom panel shows results of actin and fiber segmentation. Scalebar 1 μm. ( D ) Ramos cells expressing DIAPH1-mRuby (magenta) were imaged as in ( A ). Scale bar, 5 μm. Panels on the right show magnified area in white box. Arrows show clusters of DIAPH1 colocalized with actin foci. Scale bars 1 μm. ( E ) Example of dynamics of DIAPH1-mRuby in a single actin focus visualized with Lifeact-GFP. Time zero corresponds to initial focus formation. Scalebar 1 μm. ( F ) Example of a fiber outgrow from a DIAPH1 cluster in . Scalebar 1 μm. ( G ) Quantification of relative enrichment or depletion of ARPC2-mRuby and DIAPH1-mRuby fluorescence in actin foci and filaments. Data are mean ± SEM from n = 4 experiments each containing 12–213 cells. P, significance from one-way ANOVA. ( H, I ) Fraction of actin foci colocalized with ARPC2 ( G ) or DIAPH1 ( H ) spots and vice versa. Total bar heights show rates of colocalization, white bars indicate colocalization obtained after randomization of locations of the corresponding structures. Values are mean ± SEM of n = 4 of the same experiments as in F. P, significance in two-way repeated measures ANOVA comparing the measured with the randomized data.

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Expressing, Microscopy, Staining, Fluorescence

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet: TIRF images of a Ramos cell expressing ARPC2-mRuby and Lifeact-GFP, and labeled with the plasma membrane dye DiD on PMS loaded with anti-IgM F(ab’) 2 . Magnified inset shows an ARPC2-negative actin fiber associated with an ARPC2-positive actin focus in the absence of DiD membrane signal (arrow), in contrast to DiD-labeled filopodia at the cell periphery (arrowheads). Scalebar 5 μm (1 μm in inset). Contrast was enhanced in inset images.

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Expressing, Labeling, Clinical Proteomics, Membrane

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet:

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: Concentration Assay, Staining

Journal: eLife

Article Title: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments

doi: 10.7554/eLife.48093

Figure Lengend Snippet:

Article Snippet: ARPC2 antibody , Polyclonal , GeneTex , 1:1000 , IB.

Techniques: