Journal: eLife
Article Title: N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters
doi: 10.7554/eLife.87833
Figure Lengend Snippet: ( A–C ) B-cells from mice expressing the GFP fusion of non-muscle myosin IIA (GFP-NMIIA) transgene were treated with DMSO or Wisko (10 µM) 10 min before and during incubation with Fab’-PLB. The B-cell contact zones were imaged live using total internal reflection fluorescence microscopy (TIRF). Shown are representative TIRF images of DMSO- and Wisko-treated B-cells at 30 s (during spreading) and 2 min 30 s (after maximal spreading) post landing ( A Scale bars, 2 µm), the averaged GFP-NMIIA MFI (± SEM) ( B ), and the initial rates of increasing (± SEM) of GFP-NMIIA in the contact zone (the slope of the initial GFP-NMIIA MFI versus time curves of individual cells) ( C ). Data points represent individual cells from three independent experiments with ~6 cells per condition per experiment. *p<0.05, ***p<0.001, by Kolmogorov-Smirnov test ( B ) or non-parametric student’s t -test ( C ). ( D ) Primary B-cells from mice expressing both GFP-NMIIA and LifeAct-RFP transgenes were incubated with Fab’-PLB at 37 °C and imaged live by TIRF. Shown are a representative TIRF image of a cell and a kymograph generated from time-lapse TIRF images at the yellow line. The purple arrow indicates the starting point of contraction, the white arrow GFP-NMIIA recruitment proximal to the spreading membrane, and the yellow arrow an F-actin (LifeAct-RFP) focus originating at the lamellipodia and moving away from the spreading membrane. ( E, F ) Primary B-cells from flox control, Wasp germline knockout (WKO), and B-cell-specific N-wasp knockout mice (cNKO) mice were incubated with Fab’-PLB for indicated times. Cells were fixed, permeabilized, stained for non-muscle myosin II (NMII) light chain, and imaged by interference reflection microscopy (IRM) and TIRF. Shown are representative IRM and TIRF images ( E ) and percentages (± SD) of B-cells with the NMII ring-like structure in individual images ( F ), identified by visual inspection. The data were generated from three independent experiments with five images per condition per experiment. Scale bars, 2 µm. *p<0.05, **p<0.01, ***p<0.001, by non-parametric student’s t -test. ( G, H ) Wild-type (WT) splenic B-cells were treated with DMSO or Blebbistatin (Bleb, 50 µM) 20 min before and during incubation with Fab’-PLB at 37 °C. Cells were fixed at 2 and 4 min, permeabilized, stained with phalloidin, and imaged by TIRF. Shown are representative TIRF images of the B-cell contact zone ( G ) and percentages of cells (± SEM) with inner F-actin foci forming ring-like distribution ( H ), determined as described in . Data were generated from three independent experiments with ~50 cells per condition per experiment with different color dots representing individual experiments. Scale bar, 2 µm. *p<0.05, **p<0.01 by paired student’s t -test. MATLAB codes were used for detecting the B-cell contact zone and quantifying NMII FI [ , and ].
Article Snippet: Blebbistatin (Bleb, 50 μM, Cayman Chemicals) was used to inhibit the NMII motor activity ( ).
Techniques: Expressing, Incubation, Fluorescence, Microscopy, Generated, Membrane, Control, Knock-Out, Staining
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![( A–C ) B-cells from mice expressing the GFP fusion of non-muscle myosin IIA (GFP-NMIIA) transgene were treated with DMSO or Wisko (10 µM) 10 min before and during incubation with Fab’-PLB. The B-cell contact zones were imaged live using total internal reflection fluorescence microscopy (TIRF). Shown are representative TIRF images of DMSO- and Wisko-treated B-cells at 30 s (during spreading) and 2 min 30 s (after maximal spreading) post landing ( A Scale bars, 2 µm), the averaged GFP-NMIIA MFI (± SEM) ( B ), and the initial rates of increasing (± SEM) of GFP-NMIIA in the contact zone (the slope of the initial GFP-NMIIA MFI versus time curves of individual cells) ( C ). Data points represent individual cells from three independent experiments with ~6 cells per condition per experiment. *p<0.05, ***p<0.001, by Kolmogorov-Smirnov test ( B ) or non-parametric student’s t -test ( C ). ( D ) Primary B-cells from mice expressing both GFP-NMIIA and LifeAct-RFP transgenes were incubated with Fab’-PLB at 37 °C and imaged live by TIRF. Shown are a representative TIRF image of a cell and a kymograph generated from time-lapse TIRF images at the yellow line. The purple arrow indicates the starting point of contraction, the white arrow GFP-NMIIA recruitment proximal to the spreading membrane, and the yellow arrow an F-actin (LifeAct-RFP) focus originating at the lamellipodia and moving away from the spreading membrane. ( E, F ) Primary B-cells from flox control, Wasp germline knockout (WKO), and B-cell-specific N-wasp knockout mice (cNKO) mice were incubated with Fab’-PLB for indicated times. Cells were fixed, permeabilized, stained for non-muscle myosin II (NMII) light chain, and imaged by interference reflection microscopy (IRM) and TIRF. Shown are representative IRM and TIRF images ( E ) and percentages (± SD) of B-cells with the NMII ring-like structure in individual images ( F ), identified by visual inspection. The data were generated from three independent experiments with five images per condition per experiment. Scale bars, 2 µm. *p<0.05, **p<0.01, ***p<0.001, by non-parametric student’s t -test. ( G, H ) Wild-type (WT) splenic B-cells were treated with DMSO or <t>Blebbistatin</t> (Bleb, 50 µM) 20 min before and during incubation with Fab’-PLB at 37 °C. Cells were fixed at 2 and 4 min, permeabilized, stained with phalloidin, and imaged by TIRF. Shown are representative TIRF images of the B-cell contact zone ( G ) and percentages of cells (± SEM) with inner F-actin foci forming ring-like distribution ( H ), determined as described in . Data were generated from three independent experiments with ~50 cells per condition per experiment with different color dots representing individual experiments. Scale bar, 2 µm. *p<0.05, **p<0.01 by paired student’s t -test. MATLAB codes were used for detecting the B-cell contact zone and quantifying NMII FI [ , and ].](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5734/pmc10715734/pmc10715734__elife-87833-fig5.jpg)
