Journal: Cell

Article Title: Transmembrane pickets connect cyto-and pericellular-skeletons forming barriers to receptor engagement

doi: 10.1016/j.cell.2017.12.023

Figure Lengend Snippet: A) Control or LatA-treated BMDMs were exposed to 5 μm beads (asterisks = centre of bead) with or without application of centrifugal force (100–500 xg for 1 min). Glycocalyx exclusion was visualized with WGA (green) and is plotted on the right as a function of centrifugal force. Means ± SE from >100 contacts per condition, from 3 experiments. B) BMDMs challenged with 1.5 μm IgG-opsonized beads in the absence or presence of applied g force (300 xg). The number of beads bound per cell was determined, shown as means ± SE of 5 determinations. C) BMDMs were challenged for 20 min with equal amounts of dsRed-expressing ΔinvA S. typhimurium opsonized against H-(flagella) or O-(LPS) antigens. Anti-H greatly reduces the ability of the bacteria to swim (Supplementary Video 3). Number of bacteria bound per cell is shown as means ± SE of 5 determinations. D) RAW cells transiently transfected with HAS3-GFP stained for HA with a fluorescent HA-binding complex (red) and incubated with non-opsonized RBCs to visualize the exclusion area by DIC (left). Similarly transfected cells challenged with 8 μm opsonized beads in the absence or presence of applied centrifugal force. Number of beads bound per cell is shown as means ± SE of 3 determinations. E–F) Wildtype and CD44−/−BMDMs challenged with beads or ΔinvA S. typhimurium (F) as in B–C. G–H) Control or CN03-treated BMDMs (G) or RAW cells expressing a control vector, wildtype Slingshot (SSH-1L) or phosphatase-dead Slingshot (SSH-1L CS) (H) were challenged as in B. Particle binding presented as means ± SE of >50 cells from 3 experiments.

Article Snippet: Latrunculin A (Sigma) was used at 1 μM for 510 min. For experiments with suspended cells, the Rho Inhibitor I CT04 and Activator II CN03 (Cytoskeleton) were used at 2.0 μg/mL and incubated for 2 h. For adherent cell experiments, BMDMs were first lifted and incubated with 1.0 μg/mL of Rho Activator II CN03 for 10 min before plating in medium also containing 1.0 μg/mL of CN03 for 3–4 h. Antibodies for immunostaining were used as follows: mouse anti-human FcγRIIA/IV.3 (StemCell Technologies) at 5 μg/mL, Cy3-or Alexa 647-conjugated donkey anti-mouse or anti-human secondary antibodies (Jackson ImmunoResearch) were used at 1 μg/mL.

Techniques: Expressing, Transfection, Staining, Binding Assay, Incubation, Plasmid Preparation

Journal: Cell

Article Title: Transmembrane pickets connect cyto-and pericellular-skeletons forming barriers to receptor engagement

doi: 10.1016/j.cell.2017.12.023

Figure Lengend Snippet: A) BMDMs treated with or without 10 μM of ezrin inhibitor NSC668394 were extracted in actin-stabilizing buffer and centrifuged. Pellet (P) and supernatant (S) were analyzed by immunoblotting. Representative of 3 experiments. B) Schematic of the construct used in C,D and F. A transmembrane actin-binding protein was engineered by fusing the actin-binding domain of ezrin to the transmembrane domain of Fc receptor, tagged with hemagglutinin (HA) used to attach biotinylated anti-HA Fab for single-particle tracking. An otherwise identical construct bearing a R579A mutation was used as an inactive control. C) Fraction of time spent tethered by the unmodified (wt) and mutant (R579A) constructs, determined for >25 cells from 3 experiments. D) Diffusion coefficient estimates for the recordings in I. E) BMDMs treated with or without Rho activator (CN03) were fixed and stained for F-actin. Right panel image was acquired using ½ the exposure time used for the left; exposure time for inset in right panel was same as for the left panel. F) Cells expressing the actin-binding chimera were treated with 1.0 μg/mL CN03 for 2–3 h or 10 μM CK666 for 20 min. Fraction of time spent undergoing restricted or free motion determined for >35 cells from 3 experiments. Tethered and confined modes were not separated. G–H) BMDMs were treated with CN03 or CK666 as above before labeling single particles with Cy3-labeled Fab against CD44 or FcγRs. Particles tracked for 10 s at 10 Hz and median diffusion coefficients for >25 cells from 3 experiments are plotted for CD44 (G) and FcγRs (H). I) Schematic illustrating the regulation of actin filament stability by cofilin. Slingshot (SSH-1L) localizes to actin filaments and dephosphorylates cofilin to enable its binding and severing of the filament. J) CD44 mobility assessed in RAW cells transfected with wt or phosphatase-dead (CS) SSH-1L. Single CD44 molecules visualized using Qdots tracked for 30 s at 33 Hz and the fraction of time spent in the tethered state calculated for >25 cells from 3 experiments.

Article Snippet: Latrunculin A (Sigma) was used at 1 μM for 510 min. For experiments with suspended cells, the Rho Inhibitor I CT04 and Activator II CN03 (Cytoskeleton) were used at 2.0 μg/mL and incubated for 2 h. For adherent cell experiments, BMDMs were first lifted and incubated with 1.0 μg/mL of Rho Activator II CN03 for 10 min before plating in medium also containing 1.0 μg/mL of CN03 for 3–4 h. Antibodies for immunostaining were used as follows: mouse anti-human FcγRIIA/IV.3 (StemCell Technologies) at 5 μg/mL, Cy3-or Alexa 647-conjugated donkey anti-mouse or anti-human secondary antibodies (Jackson ImmunoResearch) were used at 1 μg/mL.

Techniques: Western Blot, Construct, Binding Assay, Single-particle Tracking, Mutagenesis, Diffusion-based Assay, Staining, Expressing, Labeling, Transfection

Journal: Cell

Article Title: Transmembrane pickets connect cyto-and pericellular-skeletons forming barriers to receptor engagement

doi: 10.1016/j.cell.2017.12.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Latrunculin A (Sigma) was used at 1 μM for 510 min. For experiments with suspended cells, the Rho Inhibitor I CT04 and Activator II CN03 (Cytoskeleton) were used at 2.0 μg/mL and incubated for 2 h. For adherent cell experiments, BMDMs were first lifted and incubated with 1.0 μg/mL of Rho Activator II CN03 for 10 min before plating in medium also containing 1.0 μg/mL of CN03 for 3–4 h. Antibodies for immunostaining were used as follows: mouse anti-human FcγRIIA/IV.3 (StemCell Technologies) at 5 μg/mL, Cy3-or Alexa 647-conjugated donkey anti-mouse or anti-human secondary antibodies (Jackson ImmunoResearch) were used at 1 μg/mL.

Techniques: Recombinant, Binding Assay, Isolation, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell Reports

Article Title: PAK4 Kinase Activity Plays a Crucial Role in the Podosome Ring of Myeloid Cells

doi: 10.1016/j.celrep.2019.11.016

Figure Lengend Snippet:

Article Snippet: Fibronectin HiLyte 488 , Cytoskeleton, Inc. , Cat. # FNR02-A.

Techniques: Plasmid Preparation, Recombinant, PCR Cloning, Mutagenesis, Clone Assay, shRNA, Software, Cell Tracking Assay