Journal: The Journal of Cell Biology

Article Title: FMNL2 regulates dynamics of fascin in filopodia

doi: 10.1083/jcb.201906111

Figure Lengend Snippet: Disturbing the organization of F-actin alters filopodia and fascin movement. (A) Filopodia length in micrometers (left) and filopodia lifetime in seconds (right) of fascin knockdown HeLa cells expressing GFP-fascin. n = 22 cells, three independent experiments. (B) Filopodia length (normalized to DMSO control) and coverage (relative filopodia number/perimeter) was evaluated after long-time (left, n = 22, 28, or 25 cells) or short-time (right, n = 51, 28, or 22 cells) treatment with SMIFH2 and CK666. One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant. (C) Percentage of fast-moving fascin was extracted from filopodia photoconversion data from HeLa cells treated with DMSO, SMIFH2, or CK666. Shown are data from three independent experiments ( n = 16, 18, or 11 filopodia respectively), one-way ANOVA. (D) Morphology of GFP-fascin–expressing HeLa cells embedded in soft 3D ECM after inhibition of formins (SMIFH2) and Arp2/3 complex (CK666). Images were captured using LLSM. Scale bar = 10 µm. (E) Inhibition of formins decreases pulling forces on ECM. Velocity magnitude of cell-induced bead displacement in 3D collagen matrix without ( n = 220 bead velocity vectors) and with inhibitor treatment. Analysis was performed as described in (SMIFH2, n = 140; CK666, n = 120; Jasplakinolide, n = 137; beads only, n = 60). **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. (F) Evaluation of fascin-S39A speed in comparison to WT fascin by FRAP. Filopodia were bleached in RFP-fascin WT (black) and RFP-fascin-S39A (green) expressing fascin knockdown HeLa cells, and FRAP intensities were determined over time. Representative of three independent experiments. n = 17 filopodia, two-way ANOVA with Bonferroni posttest; **, P ≤ 0.01. (G) Photoconversion of mEOS2-fascin in HeLa cells on 2D was performed to compare fascin recovery in lamellipodia and filopodia. n = 3 ROI measurements per cell area from 18 cells from three experiments. (H) Retention of fascin in lamellipodia is dependent on F-actin density. Small lamellipodial regions of DMSO control (black) and CK666-treated (magenta) GFP-fascin–expressing HeLa cells were photobleached, and FRAP was analyzed over time; n = 10 from one of three experiments. (I–K) Characterization of the CS-fascin. Filopodia length (I) and number (filopodia/perimeter; J) were measured for GFP-fascin–expressing cells and CS-fascin expressing cells. CS-fascin (tagged with both GFP and mScarlet) or GFP-fascin and mScarlet-fascin (K) were coexpressed in fascin knockdown HeLa cells; cells were fixed, and lifetime was measured by FLIM-FRET. Shown are data from two independent experiments ( n = 15, 16, or 12 cells). One-way ANOVA with Bonferroni posttest; ***, P ≤ 0.001; ****, P ≤ 0.0001. (L) CS-fascin–expressing cells were treated with the Arp2/3 inhibitor CK666 and imaged on a confocal microscope to measure ratiometric FRET. Bar graph shows FRET/donor (in %) for control ( n = 40 cells) and CK666 treatment ( n = 15 cells). One-way ANOVA with uncorrected Fisher posttest. (M) FRET signal of HeLa cells expressing CS-fascin from time binned datasets. Binning of time-lapse images shows increased FRET signal of CS-fascin both in filopodia and at the base of filopodia after combining 50 time-lapse frames. High FRET is shown in yellow/green, and low FRET is shown in dark blue/black. Imaging was performed by SIM; representative example is shown; scale bar = 5 µm. (N) Representative image of the GFP-fascin donor-only control for 3D FRET experiment shows the lack of filopodia and no FRET signal. Scale bar = 2 µm.

Article Snippet: The following compounds were used: CK666 and Jasplakinolide (Santa Cruz), SMIFH2 (Tocris), Cytochalasin D (Sigma-Aldrich), BIM (Sigma-Aldrich), phorbol 12,13-dibutyrate (Sigma-Aldrich), tetradecanoyl phorbol acetate (Sigma-Aldrich), and Kinesore (kindly provided by Mark Dodding, University of Bristol, UK).

Techniques: Knockdown, Expressing, Control, Inhibition, Comparison, Microscopy, Imaging

Journal: The Journal of Cell Biology

Article Title: FMNL2 regulates dynamics of fascin in filopodia

doi: 10.1083/jcb.201906111

Figure Lengend Snippet: Local F-actin organization regulates fascin movement into and out of filopodia. (A) Filopodia of mEOS2-fascin–expressing HeLa cells were photoconverted, and exit of photoconverted fascin was monitored over time using a confocal microscope at a 1-s frame rate. Upper panel, control cells (DMSO treated cells); middle panel, formin inhibition (SMIFH2-treated cells); bottom panel, Arp 2/3 complex inhibition (CK666-treated cells). Shown are pseudocolored ratios of photoconverted/unconverted intensities before (first column, −1 s) and after (shown are every 5 s) conversion. Scale bar = 5 µm. (B) Fascin movement out of filopodia. Photoconversion intensities plotted versus time for control and SMIFH2- and CK666-treated cells. Early and late phases were compared using two-way ANOVA with uncorrected Fisher’s posttest; shown are data ( n = 15, 24, or 17 cells, one ROI per cell, and two to four filopodia per ROI) from three independent experiments; *, P ≤ 0.05; **, P ≤ 0.01; ns, not significant. (C) Fascin t 1/2 values and mobility versus immobility (in %) extracted from monoexponential single-curve photoconversion fitting ( n = 15, 24, or 17 cells, one ROI per cell, and two to four filopodia per ROI). One-way ANOVA; *, P ≤ 0.05; **, P ≤ 0.01. (D) Fascin movement into filopodia. Filopodia of GFP-fascin–expressing HeLa were bleached (FRAP), and recovery intensities were plotted over time for control and SMIFH2- and CK666-treated cells. Early and late phases were compared using two-way ANOVA with uncorrected Fisher’s posttest; shown are data ( n = 18, 21, or 17 cells) from three independent experiments; *, P ≤ 0.05; **, P ≤ 0.01. (E) Fascin t 1/2 values and mobile versus immobile fractions extracted from monoexponential single-curve FRAP fitting ( n = 18, 21, or 17 cells). One-way ANOVA; *, P ≤ 0.05; ****, P ≤ 0.0001. (F) Small regions within single filopodia were bleached in control and SMIFH2- and CK666-treated cells and displayed as pseudocolored kymographs (space vs. time). Arrowheads point to the base or the tip of the filopodium. Scale bar, 1 μm. (G) Analysis of kymographs shown in F. Relative base to tip (left) and tip to base (right) speeds are shown. Data are from three independent experiments ( n = 18, 21, or 17 single filopodia from ≥15 individual cells per condition), one-way ANOVA; *, P ≤ 0.05; ***, P ≤ 0.001. (H) Interaction of GFP-fascin and RFP-actin measured in live cells by multiconfocal FLIM-FRET imaging. Examples of control cells (top panel) and SMIFH2 treated cells (bottom panel) are shown. Intensity images on the left and pseudocolored FLIM image on the right. Warm colors indicate FRET (low lifetime) and cool colors indicate no FRET (high lifetime). Scale bar = 2 µm. (I) Mean FRET efficiency (left, n = 16) and lifetime Lowess residuals (right, n = 28) of fascin–actin interaction of single filopodia measured by live FLIM in cells with and without SMIFH2 treatment. Student’s t test; *, P ≤ 0.05.

Article Snippet: The following compounds were used: CK666 and Jasplakinolide (Santa Cruz), SMIFH2 (Tocris), Cytochalasin D (Sigma-Aldrich), BIM (Sigma-Aldrich), phorbol 12,13-dibutyrate (Sigma-Aldrich), tetradecanoyl phorbol acetate (Sigma-Aldrich), and Kinesore (kindly provided by Mark Dodding, University of Bristol, UK).

Techniques: Expressing, Microscopy, Control, Inhibition, Imaging

Journal: Developmental cell

Article Title: Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1 P29S Melanoma Cells

doi: 10.1016/j.devcel.2019.04.007

Figure Lengend Snippet: (A) Proliferation assay applied to A375 cells expressing Rac1 P29S(+P29S), Rac1WT(+WT), or empty vector (+EV) treated with DMSO (0.003%), the Arp2/3 complex Inhibitor CK666 (200 μM), the control peptide CK689 (200 μM), and the pan-formin inhibitor SMIFH2 (25 μM). Treatment of cells with CK666 or SMIFH2 alone suppresses proliferation to the same degree and similarly across all cell lines regardless of Rac1 status.

Article Snippet: Drugs were acquired from the following sources: Dabrafenib, Trametinib, SB203580, and YAP-TEAD Inhibitor 1 (Peptide 17) from Selleckchem, SMIFH2 from Tocris, CK666, CK689, Blebbistatin, and Y27632 ROCK Inhibitor from Sigma, and SCH772984 from ChemieTek.

Techniques: Proliferation Assay, Expressing, Plasmid Preparation, Control

Journal: Developmental cell

Article Title: Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1 P29S Melanoma Cells

doi: 10.1016/j.devcel.2019.04.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Drugs were acquired from the following sources: Dabrafenib, Trametinib, SB203580, and YAP-TEAD Inhibitor 1 (Peptide 17) from Selleckchem, SMIFH2 from Tocris, CK666, CK689, Blebbistatin, and Y27632 ROCK Inhibitor from Sigma, and SCH772984 from ChemieTek.

Techniques: Recombinant, Mutagenesis, Blocking Assay, Plasmid Preparation, Immunodetection, Staining, Imaging, Flow Cytometry, CRISPR, Sequencing, Knock-Out, Software, Immunofluorescence

Journal: Microorganisms

Article Title: Herpes Simplex Virus Type 1 Infection Induces the Formation of Tunneling Nanotubes

doi: 10.3390/microorganisms11081916

Figure Lengend Snippet: Fluorescence microscopy images showed the effect of CK666 on the formation of TNTs. ( A , B ) HCECs and Vero cells were stained with phalloidin green to visualize the nanotubes. Three images (per experiment) were taken per condition, with −100 cells examined, and the number of TNTs was counted. ( C ) Graph shows a significant reduction in the number of TNTs in the presence of CK666 (50 μM). Data are presented as mean ± SD. Student’s t -test was performed to compare between the groups, and the following significance values were considered: * p < 0.05, *** p < 0.001. ( D ) Cell viability of human corneal epithelial cells and Vero cells in the presence of CK666. Cells were treated with different concentrations of inhibitors, 0.1–100 μM, for 24 h, and the cell viability was measured by MTT assays. Only 100 μM was found to be toxic in two types of cells. Data shown are of three replicates.

Article Snippet: The 10 4 HCECs and Vero cells in 96-wells plate were treated with various concentrations of CK666 (AdooQ Bioscience LLC., Irvine, CA, USA) (0, 0.1, 1, 5, 10, 20, 50, 100 μM) and 0.1% dimethyl sulfoxide (DMSO, as a drugs vehicle) (Sigma, Cream Ridge, NJ, USA) for 24 h. After incubation with CK666 and DMSO, cells were incubated with 5 mg/mL concentration of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Beyotime Biotechnology Co., Ltd., Haimen, China) reagent in media for 4 h. Then, cells were incubated with Formazan solvent for 4 h and absorbance recorded using Multiskan Spectrum at 570 nm (Multiskan GO, Thermo Scientific, Waltham, MA, USA).

Techniques: Fluorescence, Microscopy, Staining

Journal: Microorganisms

Article Title: Herpes Simplex Virus Type 1 Infection Induces the Formation of Tunneling Nanotubes

doi: 10.3390/microorganisms11081916

Figure Lengend Snippet: CK666 affects the transmission of HSV-1. Confluent monolayers of cell plates in 24-well culture were infected with HSV-1 for 1 h, then treated with/without 50 μM CK666 for 24 h in HCECs ( A ) and Vero cells ( B ) and processed for the plaque assay. ( C ) Graph shows a reduction in the number of plaques in HCECs and Vero cells treated with CK666 compared to control cells. ( D ) HCECs and Veros were infected with wild-type HSV-1 (MOI of 1), and the effect of the addition of 50 μM CK666 on the HSV-1 DNA level in HCECs and Vero cells was quantified with qPCR. Data represent mean ± SD of triplicate assays. Student’s t -test was used to compare between the groups. ** p < 0.01, *** p < 0.001.

Article Snippet: The 10 4 HCECs and Vero cells in 96-wells plate were treated with various concentrations of CK666 (AdooQ Bioscience LLC., Irvine, CA, USA) (0, 0.1, 1, 5, 10, 20, 50, 100 μM) and 0.1% dimethyl sulfoxide (DMSO, as a drugs vehicle) (Sigma, Cream Ridge, NJ, USA) for 24 h. After incubation with CK666 and DMSO, cells were incubated with 5 mg/mL concentration of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Beyotime Biotechnology Co., Ltd., Haimen, China) reagent in media for 4 h. Then, cells were incubated with Formazan solvent for 4 h and absorbance recorded using Multiskan Spectrum at 570 nm (Multiskan GO, Thermo Scientific, Waltham, MA, USA).

Techniques: Transmission Assay, Infection, Plaque Assay, Control

Journal: EMBO Reports

Article Title: Nesprin‐2 accumulates at the front of the nucleus during confined cell migration

doi: 10.15252/embr.201949910

Figure Lengend Snippet: Reagents and Tools table

Article Snippet: CK666 (VWR) was dissolved to a stock concentration of 250 mM in dry DMSO and used at a working concentration of 0.25 mM.

Techniques: Sequencing, Stable Transfection, Expressing, Construct, Mutagenesis, Recombinant, Plasmid Preparation, Imaging, Software

Journal: eLife

Article Title: Keratins and plakin family cytolinker proteins control the length of epithelial microridge protrusions

doi: 10.7554/eLife.58149

Figure Lengend Snippet: ( A ) Krt17-GFP[BAC]- and Lifeact-mRuby-expressing cells in 48hpf fish, at time 0 and 30 min after treatment with DMSO or the Arp2/3 inhibitor CK666. Arrows show that the F-actin microridge pattern was disrupted after 30 min of CK666 treatment. Arrowheads show that the keratin microridge pattern was retained after 30 min of CK666 or DMSO treatment. ( B ) GFP-PH-PLC (membrane) and Lifeact-mRuby in 48hpf periderm cells at time 0 and 30 min after treatment with DMSO or CK666. GFP-PH-PLC was expressed by injecting Krt5:Gal4 into UAS:GFP-PH-PLC fish; Krt5:Lifeact-mRuby was expressed by transient transgenesis. Arrows show that the membrane and actin microridge patterns were disrupted after 30 min of CK666 treatment. Arrowheads show that the membrane and actin microridge patterns were retained in DMSO controls. ( C ) Projection and orthogonal views of GFP-PH-PLC and Krt17-mRuby[BAC] in 48hpf periderm cells at time 0 and 30 min after treatment with DMSO or CK666. GFP-PH-PLC was expressed by injecting Krt5:Gal4 into UAS:GFP-PH-PLC fish; Krt17-mRuby[BAC] was expressed by transient transgenesis. Arrowheads show that membrane protrusion and Krt17 microridge patterns were retained after 30 min of DMSO or CK666 treatment. Orthogonal views (XZ) show that Krt17 preserved the protrusive membrane structure after 30 min of CK666 treatment. ( D ) Dot plot of average microridge length per cell at 48hpf after 30 min treatment with DMSO or CK666. Line indicates average. ***p<0.005, the Wilcoxon rank-sum test. n = 11–12 cells from three fish. ( E ) Dot plot of microridge and keratin overlap coefficients, comparing the colocalization of each protein at 0 min with its localization after 30 min DMSO or CK666 treatment. Line indicates average. **p<0.01, ***p<0.005, the Wilcoxon rank-sum test. n = 11–12 cells from three fish. Black-and-white images were inverted so that high-intensity fluorescence appears black and low-intensity fluorescence is white. Scale bars: 10 µm ( A–C ) and 1 µm (orthogonal images in C).

Article Snippet: CK666 (Fisher Scientific) was dissolved in DMSO.

Techniques: Expressing, Membrane, Fluorescence

Journal: eLife

Article Title: Keratins and plakin family cytolinker proteins control the length of epithelial microridge protrusions

doi: 10.7554/eLife.58149

Figure Lengend Snippet:

Article Snippet: CK666 (Fisher Scientific) was dissolved in DMSO.

Techniques: Mutagenesis, Recombinant, Sequencing, Homologous Recombination, Plasmid Preparation, Software, Microscopy