Journal: bioRxiv

Article Title: CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

doi: 10.1101/2023.11.26.568719

Figure Lengend Snippet: A. Representative immunofluorescence image of a HeLa cell infected with vaccinia virus at 9 hours post-infection and labelled with an antibody against cortactin and stained with phalloidin to visualize F-actin. Extra-cellular virus attached to plasma membrane are labelled with an antibody against the viral protein B5. Scale bar = 10 μm. B. Representative images showing localization of cortactin in HeLa cells infected with vaccinia at 9 hours post-infection after 1 hour treatment with CK-666 or CK-869 at the specified concentrations. Scale bar = 10 μm. The inset colour images show 2x magnifications of the yellow boxed regions with cortactin (green) and extra-cellular virus (magenta). C. The graph shows quantification of the mean number of extracellular virus co-localising with cortactin from three independent repeats with the error bar representing standard deviation. Two tailed unpaired t-test was used to analyse the statistical significance. ns = p-value > 0.05. * = p-value < 0.05. **** = p-value < 0.0001.

Article Snippet: To mimic the use of drugs in cells, Arp2/3 iso-complex was incubated with 100 µM CK-666 or CK-869 (Sigma, Bio-Techne), or equivalent amount of DMSO at room temperature for 1 hour before being added to the experimental system.

Techniques: Immunofluorescence, Infection, Virus, Staining, Membrane, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

doi: 10.1101/2023.11.26.568719

Figure Lengend Snippet: A . Representative plots showing the polymerization of pyrene labelled actin (Fluorescence Intensity) when Arp2/3 iso-complexes containing ArpC1B/C5 (left) or ArpC1B/C5L (right) are activated by GST-N-WASP-VCA in the absence (blue) or presence of 100 µM CK-666 (yellow) or CK-869 (green). B. Left shows representative TIRF images showing mother filaments (magenta) and new daughter branches (cyan) 2 minutes after Arp2/3 iso-complexes containing ArpC1B/C5 (top row) or ArpC1B/C5L (bottom row) were activated by GST-N-WASP-VCA in the absence (DMSO) or presence of 100 µM CK-666 or CK-869. Scale bar = 5 μm. The right graph shows the quantification of the mean branching rate for each condition from three independent experiments with the error bar representing standard deviation. Two tailed paired t-test has been applied to analyse the statistical significance. * = p-value < 0.05. *** = p-value < 0.001. ns = p > 0.05.

Article Snippet: To mimic the use of drugs in cells, Arp2/3 iso-complex was incubated with 100 µM CK-666 or CK-869 (Sigma, Bio-Techne), or equivalent amount of DMSO at room temperature for 1 hour before being added to the experimental system.

Techniques: Fluorescence, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

doi: 10.1101/2023.11.26.568719

Figure Lengend Snippet: A . Representative plots showing the polymerization of pyrene labelled actin (Fluorescence Intensity) when Arp2/3 iso-complexes containing ArpC1A/C5 (left) or ArpC1A/C5L (right) are activated by GST-N-WASP-VCA in the absence (blue) or presence of 100 µM CK-666 (yellow) or CK-869 (green). B. Left shows representative TIRF images showing mother filaments (magenta) and new daughter branches (cyan) 2 minutes after Arp2/3 iso-complexes containing ArpC1A/C5 (top row) or ArpC1A/C5L (bottom row) were activated by GST-N-WASP-VCA in the absence (DMSO) or presence of 100 µM CK-666 or CK-869. Scale bar = 5 μm. The right graph shows the quantification of the mean branching rate for each condition from three independent experiments with the error bar representing standard deviation. Two tailed paired t-test was used to analyse the statistical significance. * = p-value < 0.05. ** = p-value < 0.01.

Article Snippet: To mimic the use of drugs in cells, Arp2/3 iso-complex was incubated with 100 µM CK-666 or CK-869 (Sigma, Bio-Techne), or equivalent amount of DMSO at room temperature for 1 hour before being added to the experimental system.

Techniques: Fluorescence, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

doi: 10.1101/2023.11.26.568719

Figure Lengend Snippet: A. Treatment of murine bone marrow-derived macrophages (BMDM) with 100 µM CK-869 but not DMSO or CK-666 induces cell rounding. The graphs show the mean ratio of length and width as well as cell thickness from five independent experiments with error bars representing the standard deviation. Two tailed unpaired t-test was used to analyse the statistical significance. B. Representative phase images of BMDM treated with DMSO, CK-666 or CK-869 over 20 minutes. Scale bar = 20 µm. C. Quantification of the mean migration speed of BMDM treated with DMSO, CK-666 or CK-869 from five independent experiments with error bars representing the standard deviation. Two tailed unpaired t-test was used to analyse the statistical significance. D . The graph shows the quantification of the phagocytosis efficiency of zymosan by murine bone marrow-derived macrophages treated with DMSO, CK-666 or CK-869 over 120 minutes. Each point represents the mean of three independent technical repeats and error bars represent the standard deviation. Two-way ANOVA test has been applied to analyse the statistical significance. ** = p-value < 0.01. *** = p-value < 0.001. **** means p-value < 0.0001. ns = p > 0.05.

Article Snippet: To mimic the use of drugs in cells, Arp2/3 iso-complex was incubated with 100 µM CK-666 or CK-869 (Sigma, Bio-Techne), or equivalent amount of DMSO at room temperature for 1 hour before being added to the experimental system.

Techniques: Derivative Assay, Standard Deviation, Two Tailed Test, Migration

Journal: bioRxiv

Article Title: CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

doi: 10.1101/2023.11.26.568719

Figure Lengend Snippet: A. Representative Coomassie stained protein gels showing the co-sedimentation of Arp2/3 complexes containing ArpC1A (left) or ArpC1B (right) with actin filaments (P = pellet) in the absence (DMSO) or presence of 100 µM CK-666 or CK-869. The supernatant (S) contains unbound Arp2/3 complexes and non-pelleted G-actin. MK represents the molecular weight markers. B. Quantification of the mean co-sedimentation fraction of the indicated Arp2/3 complexes in the absence (DMSO) or presence of CK-666 or CK-869 from three independent experiments, with the error bars indicating the standard deviation. Two tailed paired t-test was used to analyse the statistical significance. * = p-value < 0.05. ns = p-value > 0.05. C. Immunoblots using an ArpC2 antibody demonstrate that 100 µM CK-666 or CK-869 does not impact the interaction of Arp2/3 complexes containing ArpC1A (top) or ArpC1B (bottom) with GST-N-WASP-VCA (Ponceau - red).

Article Snippet: To mimic the use of drugs in cells, Arp2/3 iso-complex was incubated with 100 µM CK-666 or CK-869 (Sigma, Bio-Techne), or equivalent amount of DMSO at room temperature for 1 hour before being added to the experimental system.

Techniques: Staining, Sedimentation, Molecular Weight, Standard Deviation, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

doi: 10.1101/2023.11.26.568719

Figure Lengend Snippet: A . Representative plots showing the polymerization of pyrene actin (Fluorescence Intensity) when Arp2/3 complexes containing ArpC1A/C5L (left) or ArpC1B/C5L (right) are activated by SPIN90-Cter in the absence (blue) or presence of 100 µM CK-666 (yellow) or CK-869 (green). B. Left shows representative TIRF images of actin filament assembly induced by Arp2/3 complexes containing ArpC1A/C5L or ArpC1B/C5L after activation by SPIN90-Cter in the absence (DMSO) or presence of 100 µM CK-666 or CK-869 at the indicated times. Scale bar = 10 μm. The right graph shows the quantification of the mean filament density at 240 s from three independent experiments with the error bar representing standard deviation. Two tailed unpaired t-test was used to analyse the statistical significance and *** = p-value < 0.001. C. The left panels show immunoblots of GST-SPIN90-Cter (Ponceau - red) pulldown assays of the indicated Arp2/3 iso-complexes (detected by ArpC2 antibody) in the absence (DMSO) or presence of 100 µM CK-666 or CK-869. The right panel shows the quantification of the mean bound Arp2/3 complex normalized to the DMSO control for three independent pulldown assays with the error bar representing standard deviation. Two tailed paired t-test was used to analyse the statistical significance. ns = p-value > 0.5. * = p-value < 0.5.

Article Snippet: To mimic the use of drugs in cells, Arp2/3 iso-complex was incubated with 100 µM CK-666 or CK-869 (Sigma, Bio-Techne), or equivalent amount of DMSO at room temperature for 1 hour before being added to the experimental system.

Techniques: Fluorescence, Activation Assay, Standard Deviation, Two Tailed Test, Western Blot

Journal: bioRxiv

Article Title: CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

doi: 10.1101/2023.11.26.568719

Figure Lengend Snippet: A Left shows representative TIRF images showing mother filaments (magenta) and new daughter branches (cyan) 2 minutes after Arp2/3 iso-complexes containing Arp3B/ArpC1B/C5L were activated by GST-N-WASP-VCA in the absence (DMSO) or presence of 100 µM CK-666 or CK-869. Scale bar = 5 μm. The right graph shows the quantification of the mean branching rate for each condition from three independent experiments with the error bar representing standard deviation. Two tailed paired t-test was used to analyse the statistical significance and ns = p-value > 0.05. B. Left shows representative TIRF images of actin filament assembly induced by Arp2/3 complexes containing Arp3B/ArpC1B/C5L after activation by SPIN90-Cter in the absence (DMSO) or presence of 100 µM CK-666 or CK-869 at the indicated times. Scale bar = 10 μm. The right graph shows the quantification of the mean filament density at 240 s from three independent experiments with the error bar representing standard deviation. Two tailed unpaired t-test was used to analyse the statistical significance. ns means p-value > 0.05. *** presents p-value < 0.001. C . The left image shows that in the Arp2/3 complex (PDB: 3ULE), CK-869 has hydrophobic interactions with Leu 112 and Thr 126 of Arp3. The right image shows that in Arp3B, Leu 112 and Thr 126 are replaced by Met 112 and Leu 126, the latter of which is predicted to have clashes with CK-869.

Article Snippet: To mimic the use of drugs in cells, Arp2/3 iso-complex was incubated with 100 µM CK-666 or CK-869 (Sigma, Bio-Techne), or equivalent amount of DMSO at room temperature for 1 hour before being added to the experimental system.

Techniques: Standard Deviation, Two Tailed Test, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Type V myosin focuses the polarisome and shapes the tip of yeast cells

doi: 10.1083/jcb.202006193

Figure Lengend Snippet: Actin is required for polarisome focusing. (A) myo2-66 cells carrying an empty plasmid or expressing MYO2 or myo2 RD from the genomic URA3 locus were spotted in 10-fold serial dilutions on SD-Ura − plates containing 70 µM methionine and incubated for 3 d at either 25°C (upper panel) or 30°C (lower panel). (B) myo2-66 cells carrying an empty plasmid or expressing MYO2 or myo2 RD from the genomic URA3 locus and coexpressing Spa2-mCherry were analyzed by fluorescence microscopy at either 27°C (left panel) or 31°C (right panel). Insets show the DIC image of the same cell in reduced magnification. (C) Intensity profiles of the Spa2-mCherry signals of the bud circumference of the cells in B at 27°C. (D) The polarisome distribution in the bud of the cells from B was classified as focused (gray), cortically spread (spread, blue), or diffuse (black; 27°C: n empty = 226, n MYO2 = 232, n myo2RD = 225; 31°C: n MYO2 = 223, n myo2RD = 224). (E) Left: Fluorescence microscopy of WT, bni1Δ , or bni1 Δ 1240–end cells expressing Spa2-GFP. Right: Intensity profiles of Spa2-GFP of the bud circumference of the cells from the left panel. (F) The polarisome distribution of the cells from E were classified as either focused (gray) or cortically spread (spread, blue; n WT = 234, n bni1Δ = 234, n bni1Δ1240–end = 227). (G) The actin cytoskeleton of WT (upper panel) or myo2 RD cells (lower panel) was stained with Alexa Fluor 488–phalloidin in the absence (left) or presence (right) of CK-666. All scale bars: 5 µm.

Article Snippet: Actin patches were removed by addition of 100 µM CK-666 (Merck) to the cell medium at 30°C, 10 min before cells were fixed.

Techniques: Plasmid Preparation, Expressing, Incubation, Fluorescence, Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: Endophilin A1 drives acute structural plasticity of dendritic spines in response to Ca 2+ /calmodulin

doi: 10.1083/jcb.202007172

Figure Lengend Snippet: Membrane unfolding and branched actin polymerization are required for initiation of sLTP. (A) Mouse hippocampal neurons transfected with pLL3.7-DsRed on DIV12 were pretreated with DMSO or tetanus toxin (TeTx) for 10 min before cLTP induction with glycine on DIV16. Neurons were fixed at different time points after glycine application and imaged by confocal microscopy. Scale bar, 5 µm. (B) Quantification of spine size in A. Data are expressed as mean ± SEM at each point (− TeTx: n = 12, n = 531 for 0 min; n = 12, n = 536 for 0.5 min; n = 15, n = 685 for 1 min; n = 13, n = 558 for 2 min; n = 12, n = 538 for 3 min; n = 15, n = 675 for 4 min; n = 14, n = 580 for 5 min + TeTx: n = 14, n = 561 for 0 min; n = 14, n = 546 for 0.5 min; n = 14, n = 549 for 1 min; n = 15, n = 642 for 2 min; n = 15, n = 687 for 3 min; n = 15, n = 698 for 4 min; n = 15, n = 588 for 5 min). P values were calculated using two-tailed unpaired t test. ***, P < 0.001. (C) Mouse hippocampal neurons were transfected with pLL3.7-DsRed and expression construct for GFP fusion of Rab11 WT or DN (S25N) mutant on DIV12 and pretreated with DMSO or MK801 before cLTP induction on DIV16. Neurons were fixed 1 min after glycine application and imaged by confocal microscopy. Scale bar, 5 µm. (D) Quantification of spine size in C. Data are expressed as mean ± SEM for each group (GFP: n = 10, n = 222 for − Gly; n = 11, n = 274 for + Gly; n = 10, n = 228 for + MK801 + Gly; Rab11-GFP: n = 11, n = 273 for − Gly; n = 10, n = 220 for + Gly; n = 11, n = 236 for + MK801 + Gly; Rab11DN-GFP: n = 10, n = 228 for − Gly; n = 11, n = 234 for + Gly; n = 11, n = 237 for + MK801 + Gly). P values were calculated using one-way ANOVA by Newman–Keuls post hoc test. ***, P < 0.001. (E) Mouse hippocampal neurons transfected with pLL3.7-DsRed and expression construct for FLAG-tagged SNAP DN mutant on DIV12 and induced cLTP on DIV16. Neurons were fixed 1 min after glycine application, immunostained with antibodies to FLAG, and imaged by confocal microscopy. Scale bar, 5 µm. (F) Quantification of spine size in E. Data are expressed as mean ± SEM for each group (vector: n = 10, n = 378 for − Gly; n = 11, n = 391 for + Gly; SNAP23 DN: n = 11, n = 374 for − Gly; n = 11, n = 386 for + Gly; SNAP25 DN: n = 10, n = 364 for − Gly; n = 11, n = 394 for + Gly; SNAP29 DN: n = 11, n = 393 for − Gly; n = 11, n = 385 for + Gly; SNAP47 DN: n = 11, n = 379 for − Gly; n = 11, n = 382 for + Gly). P values were calculated using one-way ANOVA by Newman–Keuls post hoc test. ***, P < 0.001. (G) Effect of osmotic shock on spine enlargement 1 min after glycine application. Neurons were pretreated with iso-osmotic, hypo-osmotic, or hyperosmotic solution, respectively, for 10 min, and cLTP was performed in the same solution on DIV16. Neurons were fixed 1 min after glycine application and imaged by 3D-SIM microscopy. Scale bar, 4 µm. (H) Quantification of spine size in G. Data are expressed as mean ± SEM for each group (iso: n = 14, n = 388 for − Gly; n = 12, n = 382 for + Gly; hypo: n = 14, n = 398 for − Gly; n = 15, n = 488 for + Gly; hyper: n = 14, n = 464 for − Gly; n = 13, n = 434 for + Gly). P values were calculated using two-tailed unpaired t test. ***, P < 0.001. (I) Effects of inhibitors for actin remodeling regulators on spine enlargement 1 min after glycine application. Shown are representative confocal images. Scale bar, 5 µm. CK-666, Arp2/3 inhibitor; SMIFH2, formin inhibitor; CT04, RhoA inhibitor; NSC 23766, Rac1 inhibitor; ML141, Cdc42 inhibitor. (J) Quantification of spine size (left) and changes in spine size (right) in I. Data are expressed as mean ± SEM for each group (Ctrl: n = 13, n = 523 for − Gly; n = 13, n = 537 for + Gly. LatA: n = 12, n = 516 for − Gly; n = 12, n = 519 for + Gly; CK-666: n = 13, n = 520 for − Gly; n = 13, n = 533 for + Gly; SMIFH2: n = 13, n = 552 for − Gly; n = 13, n = 561 for + Gly; CT04: n = 13, n = 546 for − Gly; n = 13, n = 522 for + Gly; NSC 23766: n = 13, n = 524 for − Gly; n = 14, n = 562 for + Gly; ML141: n = 13, n = 532 for − Gly; n = 13, n = 530 for + Gly). P values were calculated using one-way ANOVA by Newman–Keuls post hoc test. ***, P < 0.001 when compared with Ctrl.

Article Snippet: For drug treatment, HeLa cells or primary neurons cultured on coverslips were preincubated with MK801 (10 µM; Sigma-Aldrich), LatA (100 nM; Sigma-Aldrich), NSC 23766 trihydrochloride (100 µM; Abcam), CK-666 (100 µM; Sigma-Aldrich), W-7 (20 µM; TOCRIS), KN-62 (4 µM; TOCRIS), and BAPTA-AM (10 µM; Sigma-Aldrich) for 30 min. Neurons were preincubated with ML141 (15 µM; Sigma-Aldrich) or SMIFH2 (30 µM; Sigma-Aldrich; ) for 2 h or with CT04 (2 µg/ml; Cytoskeleton) for 3 h. For tetanus toxin treatment (10 nM; Sigma-Aldrich), neurons were preincubated for 10 min.

Techniques: Transfection, Confocal Microscopy, Two Tailed Test, Expressing, Construct, Mutagenesis, Plasmid Preparation, Microscopy

Journal: The Journal of Cell Biology

Article Title: Endophilin A1 drives acute structural plasticity of dendritic spines in response to Ca 2+ /calmodulin

doi: 10.1083/jcb.202007172

Figure Lengend Snippet: In response to Ca 2+ /calmodulin, endophilin A1 associates with the PM and recruits Arp2/3 via p140Cap. (A) HeLa cells were cotransfected with constructs expressing mGFP, myc-p140Cap, and FLAG-EEN1 for 24 h; preincubated with DMSO, W-7, LatA, or CK-666 for 30 min; treated with ionomycin for 20 min; and immunostained for FLAG for confocal microscopy. Shown are representative images. Magnified images are shown in white boxes. Scale bar, 10 µm. (B) Quantification of subcellular distribution of FLAG-EEN1 and mGFP in cells by linescan plots of the fluorescence intensity along the white lines indicated in the images in A. AU, arbitrary units. Ctrl: n = 34 for − ionomycin, n = 39 for + ionomycin; W-7: n = 35 for − ionomycin, n = 38 for + ionomycin; LatA: n = 30 for − ionomycin, n = 34 for + ionomycin; CK666: n = 34 for − ionomycin, n = 39 for + ionomycin. (C) Quantification of FLAG-EEN1 distribution in ionomycin-treated cells in B. n = 39 for Ctrl, n = 38 for W-7, n = 34 for LatA, n = 39 for CK666. (D) HeLa cells were cotransfected with constructs expressing mGFP, myc-p140Cap, Arp1b-mCherry and FLAG-EEN1 WT, Y343A, KKK-EEE, or DM for 24 h, treated with ionomycin for 20 min, and immunostained for FLAG for confocal microscopy. Scale bar, 10 µm. (E) Quantification of subcellular distribution of mGFP, FLAG-EEN1 and Arp1b-mCherry in cells in D. n = 32 for vector, n = 31 for WT, n = 31 for KKK-EEE, n = 32 for DM, and n = 31 for Y343A. (F) Neuro-2a cells expressing EGFP-EEN1 were imaged live by confocal microscopy before and after ionomycin application. Shown are still images of Neuro-2a cells at 0, 1, and 15 min of ionomycin treatment. (G) Quantification of EGFP-EEN1 distribution by linescan plots of fluorescence intensity along the white lines indicated in the images in F. Ctrl: 9 cells; W-7: 11 cells. Scale bar, 10 µm.

Article Snippet: For drug treatment, HeLa cells or primary neurons cultured on coverslips were preincubated with MK801 (10 µM; Sigma-Aldrich), LatA (100 nM; Sigma-Aldrich), NSC 23766 trihydrochloride (100 µM; Abcam), CK-666 (100 µM; Sigma-Aldrich), W-7 (20 µM; TOCRIS), KN-62 (4 µM; TOCRIS), and BAPTA-AM (10 µM; Sigma-Aldrich) for 30 min. Neurons were preincubated with ML141 (15 µM; Sigma-Aldrich) or SMIFH2 (30 µM; Sigma-Aldrich; ) for 2 h or with CT04 (2 µg/ml; Cytoskeleton) for 3 h. For tetanus toxin treatment (10 nM; Sigma-Aldrich), neurons were preincubated for 10 min.

Techniques: Construct, Expressing, Confocal Microscopy, Fluorescence, Plasmid Preparation