arp2 arp3  (Bioss)

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet: ( A ) Schematic overview of CDC42-WASP-stimulated ARP2/3-dependent actin polymerization based on the cited literature. The process involves ARP2/3 complex, WASP (VCA) as nucleation promoting factor, filamentous actin (F-actin), and monomeric actin (G-actin). In the initial step, CDC42 is activated by GEF-catalyzed exchange of GDP to GTP. Active CDC42 (CDC42-GTP) binds to the GTP-binding domain (GBD) on WASP, thereby displacing the VCA domain. While the V-verpolin-like motif binds actin monomer (G-actin), C-central and A-acidic domains bind and activate the ARP2/3 complex. Conformational changes induced by the binding of the ARP2/3 complex promote its binding to the actin filament, which is strengthened by the additional interaction of the ARP2/3 complex with WASP (VCA)-G-actin. Further conformational changes will secure the ARP2/3 complex on the filament and allow its binding to the actin monomer and the polymerization of the newly nucleated filament. Actin polymerizes at the fast-growing/barbed end, elongating toward the plasma membrane and the ARP2/3 complex would cross-link newly polymerizing filament to the existing filament. ( B ) ERK3 co-precipitates with active RAC1 and CDC42 in complex with ARP2/3. Active RAC1/CDC42 pull-down was performed using control and ERK3 knockdown human mammary epithelial cells (HMECs). Levels of the active RAC1 and CDC42 were assessed as well as the co-immunoprecipitation levels of ERK3, ARP2, ARP3, and ARPC1A. Levels of the total protein expression were evaluated in the total cell lysates (TCL) and Ponceau S staining was used as a loading control. ( C–F ) ERK3 regulates F-actin levels in vitro and in vivo. ( C ) Western blot analyses of control (CRISPR Co) and ERK3 -depleted (CRISPR ERK3 ) HMECs are presented alongside with representative confocal images of F-actin staining. ( D, E ) In vivo analysis of F- and G-actin levels in HMECs upon ERK3 knockdown. ( D ) Representative western blot analyses of the enriched F- and G-actin fractions as well as the ERK3 knockdown validation and total actin levels in the TCL are presented. ( E ) F- and G-actin levels were quantified, and ratios were calculated from five (n = 5) independent experiments and are presented as mean ± SEM; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, unpaired t -test. Analyses of ERK3-dependent regulation of F-actin levels in cancerous MDA-MB231 cells is presented in . Cellular colocalization between endogenous ERK3 and the ARP2/3 was assessed in the absence of CDC42 and is presented in . ( F ) Effect of full-length ERK3 on ARP2/3-dependent pyrene actin polymerization was assessed using a pyrene actin polymerization assay. Polymerization induced by the VCA domain of WASP that served as a positive control (green) as well as the ARP2/3 (orange) and ERK3 protein alone (blue) are shown for reference. Actin alone (black) was used to establish a baseline of polymerization. Fluorescence at 360/415 was measured over time and is presented as mean fold change from at least three independent experiments after normalization to the first time point within the respective group. ARP2/3-dependent actin polymerization was measured in the presence of both ERK3 and WASP (VCA) domain, and the results are depicted in . Figure 5—source data 1. Full membrane scans for western blot images for and . Figure 5—source data 2. Prism and Excel file for . Figure 5—source data 3. Prism and Excel file for .

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Binding Assay, Immunoprecipitation, Expressing, Staining, In Vitro, In Vivo, Western Blot, CRISPR, Polymerization Assay, Positive Control, Fluorescence

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet: ( A ) Coomassie-stained 10% SDS-PAGE gel with 1 mg of the ARP2/3 protein complex (Cytoskeleton) presenting all the subunits. ( B ) Binding of increasing concentrations of recombinant GST-ERK3 to the ARP2/3 complex was measured by ELISA as described in the 'Materials and methods' section. ( C ) The interaction between GST-ERK3 and ARP3 was measured in vitro using GST-pull-down assay as described in the 'Materials and methods' section. ( D ) Binding affinity of the recombinant GST-ERK3 protein and ARP3 was assessed by ELISA as described in the 'Materials and methods' section and mean absorbance (Abs) ± SEM from three independent experiments is presented. ( E ) Co-immunoprecipitation (IP) of ARP2/3 protein complex and ERK3 was performed in HMECs using ARP3 antibody. Levels of precipitated ARP3 as well as co-IP of ARP2 and ERK3 were assessed. IgG control was included to determine the specificity of the interaction. Total cell lysate (TCL) was included to present expression levels of the verified interacting partners. Ponceau S staining was used as a loading control. ( F, G ) Actin phenotype of the human mammary epithelial cells (HMECs) was validated upon stable overexpression of the ARP3 non-phosphorylatable (S418A) and the phospho-mimicking (S418D) mutant, respectively. Wild type (WT) ARP3 was used as a control for the mutants and empty vector (EV) served negative control for the overexpression itself. ( F ) F-actin expression and organization in the negative (S418A) and phospho-mimicking (S418D) ARP3 mutant was visualized by green phalloidin and merged with the Hoechst staining of the nuclei. Four representative confocal images are presented. Images of EV-transfected and ARP3 WT-overexpressing HMECs are presented as controls. ( G ) Western blot validation of the overexpression efficiency and phosphorylation of ARP3 at S418. Anti-V5-tag antibody was used to detect levels of exogenous ARP3 WT, S418A, and S418D. Expression levels of the endogenous ARP3 were assessed as well as the phosphorylation at S418, total actin was validated. Ponceau S staining was used as a loading control. ( H ) Detection of the S418 phosphorylation of ARP3 in CRISPR ERK3 HMECs presented in . ( I, J ) Effect of the ARP3 mutant overexpression on F-actin levels was quantified using F/G actin in vivo assay. ( I ) Representative western blot analyses of F- and G-actin levels detected in fractions obtained from EV, ARP3 WT, S418A, S418D HMECs. ( J ) Quantification of the F/G actin ratios was performed for three (n = 3) independent experiments and is presented as mean ± SEM; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, one-way ANOVA, Tukey’s post-test. ( K–M ) Effect of ERK3 depletion on dense F-actin phenotype of the ARP3 S418D-overexpressing HMECs. HMECs stably overexpressing ARP3 S418D were transduced with lentiviral particles targeting ERK3 (shERK3) and stable knockdown was established as described in the 'Materials and methods' section. Cells were further subjected to analyses of the F-actin levels. ( K ) IF staining with Oregon Green Phalloidin 488 to visualize F-actin levels and organization. Scale bars 28 µm. ( L, M ) Effect of the ERK3 knockdown on F-actin levels was quantified in the ARP3 S418D mutant overexpressing HMECs using F/G actin in vivo assay. ( L ) Representative western blot analyses of F/G actin levels. ARP3 S418D-(V5-tagged) overexpression and ERK3 knockdown efficiency were validated in TCL. Actin and Ponceau S staining were used as loading controls. ( M ) Calculated ratios of F/G actin are presented as mean ± SEM from three (n = 3) independent experiments; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, paired t -test. Colocalization of endogenous ERK3 with endogenous and exogenous ARP3 mutant (S418D) was verified, and further effect of the ERK3 depletion on the RAC1 and CDC42 activity was assessed in ARP3 S418D-overexpressing HMECs and presented in . Figure 6—source data 1. Full membrane scans for western blot images for . Figure 6—source data 2. Prism and Excel file for . Figure 6—source data 3. Prism and Excel file for . Figure 6—source data 4. Prism and Excel file for . Figure 6—source data 5. Prism and Excel file for .

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Staining, SDS Page, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, In Vitro, Pull Down Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Over Expression, Mutagenesis, Plasmid Preparation, Negative Control, Transfection, Western Blot, CRISPR, In Vivo, Stable Transfection, Transduction, Activity Assay

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet: ERK3 directly binds and activates the ARP2/3 protein complex as well as the CDC42 and RAC1 Rho GTPases. Activation of the ARP2/3 complex and RAC1/CDC42 is required for nucleation of the new actin filaments, elongation, and branching into the lamellipodia and filopodia. ERK3 regulates actin-rich protrusions, which play a direct role in cell motility.

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Activation Assay

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet:

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Sequencing, shRNA, CRISPR, Recombinant, Mutagenesis, Plasmid Preparation, Cell Fractionation, In Vivo, Transduction, Concentration Assay, Software

Journal: Scientific Reports

Article Title: Rac1 inhibition regenerates wounds in mouse fetuses via altered actin dynamics

doi: 10.1038/s41598-024-78395-2

Figure Lengend Snippet: Fig. 1. Expression of Rac1 signaling in the wound during embryonic mouse development. (a) Immunostaining of Rac1 in embryonic day (E)13, E15, and E17 mouse fetuses 24 h after wounding. Rac1 is weakly expressed at E13, but is highly expressed in the wound epidermis after E15. Arrows: area of wound. Red: Rac1; blue: DAPI (nuclei). Scale bar = 100 μm. (b) Quantitative evaluation of Rac1 gene expression in E13, E15, and E17 mouse fetuses 24 h after wounding. (c) Immunostaining of ARP2/3 in E13, E15, and E17 mouse fetuses 24 h after wounding. Weak ARP2/3 expression is visible on E13, whereas high expression is visible in the wound epidermis after E15. Arrows: area of wound. Red: ARP2/3; blue: DAPI (nuclei). Scale bar = 100 μm. (d) Quantitative evaluation of ARP2 and ARP3 gene expression in E13, E15, and E17 mouse fetuses 24 h after wounding. *P < 0.05.

Article Snippet: After blocking with 3% nonfat milk at room temperature for 1 h, primary antibodies diluted in blocking solution, ARP2 (#4738, 1:100, Cell Signalling Technology, Danvers, MA, USA), ARP3 (ab49671, 1:100, Abcam, Cambridge UK), RhoA (#2117, 1:100, Cell Signalling Technology), and GAPDH (1:2000; Santa Cruz Biotechnology) were incubated overnight at 4 °C.

Techniques: Expressing, Immunostaining, Gene Expression