Research ArticleImmunology

Profiling the origin, dynamics, and function of traction force in B cell activation

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Science Signaling  07 Aug 2018:
Vol. 11, Issue 542, eaai9192
DOI: 10.1126/scisignal.aai9192
  • Fig. 1 Schematic diagram of traction force microscopy.

    (A) Schematic representation of the traction force microscopy method used in this study. In this approach, B cells were placed on PA gels that were precoated with anti-BCR surrogate antigens (Ags). To accurately track and measure the lateral deformation changes in the PA gels that are exerted by the B cells, we also anchored fluorescent beads to the surface of the PA gel substrates. (B) Representative images of DT40 cells under the following conditions: B cell spreading on the PA gel (phase contrast), fluorescent beads linked to the PA gel surface (fluorescent beads), the displacement map (displacement) of the substrate calculated from the lateral deformation of the PA gels by DIC, and the corresponding traction stress map (traction stress) computed from the displacement map. Scale bar, 5 μm.

  • Fig. 2 Traction forces generated by DT40 cells and B6 primary B cells.

    (A and B) Representative time-lapse, phase-contrast images of DT40 cells on antigen-coated PA substrates with a stiffness of 1 kPa (A) and the corresponding traction stress map generated by the same cell (B). Scale bar, 5 μm. (C) Total traction force exerted by DT40 cells varied with time up to 30 min. Red lines represent the selected example cells (n = 15 cells), whereas the blue line displays the average of the total number of tested cells (n = 49 cells). (D) Total traction forces generated by DT40 cells incubated on substrates coated with goat anti-chicken IgM (Ag) or neutravidin (NC) for 20 min. Data are means ± SEM of the total traction force calculated from at least 15 cells in one experiment that is representative of three independent experiments. ***P < 0.001 by two-tailed t test. (E and F) Representative phase-contrast and fluorescence images of B6 primary B cells incubated on antigen-coated PA substrates with a stiffness of 0.5 kPa (E) and analysis of the total traction forces exerted by B6 primary B cells incubated on PA substrates coated with antigen (Ag) or neutravidin (NC) for 20 min (F). Data are means ± SEM of the total traction force calculated from at least 25 cells in one experiment that is representative of three independent experiments. Scale bar, 10 μm. ***P < 0.001 by two-tailed t test. (G) Total traction forces exerted by DT40 WT cells without (control) or with (RGD) pretreatment with the integrin inhibitor RGD peptide upon stimulation by substrates coated with goat anti-chicken IgM antibodies. Also provided as NC are the total traction forces generated by DT40 WT cells upon stimulation by substrates coated with irrelevant goat anti-mouse IgM antibodies (anti-mouse IgM). Data are means ± SEM of the total traction force calculated from at least 21 cells in one experiment that is representative of three independent experiments. ***P < 0.001 by two-tailed t test. (H) Total traction forces exerted by WT primary B6 B cells without (control) or with (RGD) pretreatment with the integrin inhibitor RGD peptide and primary B cells from CD11a KO mice (CD11a-KO) upon stimulation by substrates coated with anti-mouse IgM antibodies. Also provided as NC are the total traction forces generated by WT primary B6 B cells upon stimulation by substrates coated with irrelevant goat anti-chicken IgM antibodies (anti-chicken IgM). Data are means ± SEM of the total traction force calculated from at least 34 cells in one experiment that is representative of three independent experiments. ***P < 0.001 by two-tailed t test.

  • Fig. 3 Myosin and dynein are involved in BCR activation and traction force generation.

    (A) DT40 cells were pretreated with DMSO (vehicle control) or with the indicated inhibitors before being incubated on PA surfaces coated with goat anti-chicken IgM. The MFIs of the BCRs from each indicated group of DT40 cells were quantitated as the fluorescence intensity of the labeled BCRs averaged over the area of the B cell. Data are means ± SEM of the MFIs analyzed from at least 36 cells in one experiment that is representative of three independent experiments. **P < 0.01 and ***P < 0.001 by two-tailed t test. (B) Time-lapse analysis of the average total traction force generated by DT40 cells treated with DMSO (black), HPI4 (red), BLEB (ochre), and ML7 (blue). Data are from at least 14 cells in one experiment that is representative of three independent experiments. (C and D) Total traction force exerted by DT40 (C) or B6 primary B cells (D) that were pretreated with DMSO (vehicle) or the indicated motor protein inhibitors and then incubated for 20 min on PA substrates coated with goat anti-chicken IgM or goat anti-mouse IgM, respectively. Data are means ± SEM of the total traction force calculated from at least 21 cells in one experiment that is representative of three independent experiments. ***P < 0.001 by two-tailed t test.

  • Fig. 4 The correlation between the BCR MFI and the strength of the traction forces.

    (A) Representative time-lapse traction stress map (top) and the corresponding fluorescence images of F-actin (middle) and the BCR (bottom) of DT40 cells incubated for up to 30 min on PA substrates coated with goat anti-chicken IgM. Scale bars, 5 μm. (B) Total traction force exerted by DT40 cells that were pretreated with either DMSO or latrunculin-B (Lat-B) and then incubated for 20 min on PA substrates coated with goat anti-chicken IgM. Data are means ± SEM of the total traction force calculated from at least 40 cells in one experiment that is representative of three independent experiments. ***P < 0.001 by two-tailed t test. (C) Correlation between the strength of traction stress and the MFI of F-actin or BCR at 10 min after incubation for the cells shown in (A). Left: Three representative ROIs (a, b, and c) in the three images represent the same ROIs that were used to calculate the MFIs of F-actin and the BCR and the strength of the traction stress. Scale bars, 5 μm. Right: Correlation shows the linear regression analysis between the MFI of both F-actin and BCR with the traction stress value. Data are from at least 12 cells (28 ROIs per cell) in one experiment that is representative of three independent experiments. (D) Average R value of the linear regression (left) and the Pearson correlation coefficient (PCC; right) between F-actin and traction stress of 12 tested cells varied with time up to 30 min. Red lines represent the selected example cells (n = 12 cells), the ochre line represents the cell shown in (A), and the blue line displays the average of the total tested cells. (E) Average R value of linear regression (left) and the PCC (right) between BCR and traction stress of 12 tested cells varied with time up to 30 min. Red lines represent the selected example cells (n = 12 cells), the ochre line represents the cell shown in (A), and the blue line displays the average of total tested cells.

  • Fig. 5 Membrane-proximal BCR signaling molecules and adaptor molecules linking BCR microclusters and motor proteins are required for the generation of traction forces.

    (A) Scatter diagrams of the total traction forces exerted by WT DT40 cells and indicated KO DT40 cells when incubated for 20 min on PA substrates coated with goat anti-chicken IgM. Data are means ± SEM of the total traction force calculated from at least 28 cells in one experiment that is representative of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed t test. (B and C) Representative DT40 cell phase-contrast and fluorescence images of Vav3 KO DT40 cells reconstituted with Vav3-GFP (B) and PLC-γ2 KO DT40 cells reconstituted with PLC-γ2–GFP (C) after incubation for 20 min on PA substrates coated with goat anti-chicken IgM. Scale bar, 5 μm. (D) Scatter diagrams showing the total traction forces generated by Vav3-KO DT40 cells, Vav3 KO DT40 cells reconstituted with Vav3-GFP, PLC-γ2–KO DT40 cells, and PLC-γ2 KO DT40 cells reconstituted with PLC-γ2–GFP after incubation as described in (C). Data are means ± SEM of the total traction force calculated from at least 30 cells in one experiment that is representative of three independent experiments. ***P < 0.001 by two-tailed t test. (E and F) Fluorescence intensity (FI) of BCR microclusters (E) and scatter diagrams of total traction forces (F) exerted by WT DT40 cells and the indicated KO DT40 cell lines after incubation for 20 min on PA substrates coated with goat anti-chicken IgM. Data are means ± SEM of the fluorescence intensity calculated from at least 2311 BCR microclusters analyzed from at least 37 cells (E) and the total traction force calculated from at least 45 cells (F) in one experiment that is representative of three independent experiments, respectively. **P < 0.01 and ***P < 0.001 by two-tailed t test.

  • Fig. 6 Traction forces generated by IgM-BCR–expressing naïve B cells and isotype-switched IgG-BCR–expressing memory B cells.

    (A) Quantification of the traction forces generated by IgM-BCR–expressing and isotype-switched, IgG-BCR–expressing B1-8 primary B cells after incubation for 20 min on PA substrates coated with NP8-BSA. Data are means ± SEM of the total traction force calculated from at least 25 cells in one experiment that is representative of three independent experiments. **P < 0.01 by two-tailed t test. (B and C) Quantification of the traction force generated (B) and the FI of the BCR microclusters (C) from mature naïve B cells expressing HEL-specific IgM-BCRs (IgM) and from mature naïve B cells expressing HEL-specific IgM-BCRs with the mIgG cytoplasmic tail (IgMG) after incubation for 20 min on PA substrates coated with HEL. Data are means ± SEM of the total traction force calculated from at least 48 cells (B) and means ± SEM of the fluorescence intensity calculated from at least 3042 BCR microclusters analyzed from at least 50 cells (C) in one experiment that is representative of three independent experiments. **P < 0.01 by two-tailed t test. (D) Representative original (top rows), pseudocolored (middle rows), and 2.5-dimensional Gaussian images (bottom rows) of typical BCR microclusters from mature naïve B cells expressing HEL-specific IgM-BCRs (top) and naïve B cells expressing HEL-specific IgM-BCRs with the mIgG cytoplasmic tail (bottom) tested in (C). Scale bar, 1.5 μm.

  • Fig. 7 Traction forces generated by B cells from healthy donors and patients with RA.

    (A) Representative phase-contrast and BCR fluorescence images of B cells from a healthy control and an RA patient. Insets show contrast-enhanced, magnified views of the respective primary B cells in the dashed boxes. Scale bar, 10 μm. (B and C) BCR MFIs (B) and total traction forces (C) of primary B cells from three pairs of healthy human controls and RA patients after incubation and spreading for 20 min on PA substrates coated with goat anti-human Igκ and Igλ light chain. Data are means ± SEM of BCR MFI (B) or the total traction force (C) calculated from at least 26 cells (per sample) from three pairs of donors. **P < 0.01 and ***P < 0.001 by two-tailed t test.

  • Fig. 8 Proposed molecular mechanism of traction force generation during B cell activation.

    (A) Formation and growth of BCR microclusters in the peripheral area of the contact interface between the B cell and the antigen-presenting substrate surface. (B) Remodeling of F-actin structures and the loading of cargo (BCR microclusters) onto the motor proteins. (C) Retrograde movement of BCR microclusters to the center of the B cell immunological synapse along the tracks of F-actin and microtubules. Red arrows in the magnified regions indicate the generation of traction forces during the retrograde movement of BCR microclusters to the center of the B cell immunological synapse by motor proteins.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/542/eaai9192/DC1

    Fig. S1. Total traction force regressed to a two-exponential function of time.

    Fig. S2. Traction work done by DT40 cells and B6 primary B cells when exerting traction forces.

    Fig. S3. Myosin and dynein are involved in the production of traction work in B cells.

    Fig. S4. Membrane-proximal BCR signaling molecules are required for sustained traction work.

    Fig. S5. Traction work done by IgM-BCR–expressing naïve B cells and isotype-switched IgG-BCR–expressing memory B cells.

    Fig. S6. Myosin and dynein mRNA abundances in IgM-BCR–expressing naïve B cells and IgG-BCR–expressing memory B cells from mice and humans.

    Fig. S7. Traction work exerted by B cells from healthy controls and RA patients.

  • This PDF file includes:

    • Fig. S1. Total traction force regressed to a two-exponential function of time.
    • Fig. S2. Traction work done by DT40 cells and B6 primary B cells when exerting traction forces.
    • Fig. S3. Myosin and dynein are involved in the production of traction work in B cells.
    • Fig. S4. Membrane-proximal BCR signaling molecules are required for sustained traction work.
    • Fig. S5. Traction work done by IgM-BCR–expressing naïve B cells and isotype-switched IgG-BCR–expressing memory B cells.
    • Fig. S6. Myosin and dynein mRNA abundances in IgM-BCR–expressing naïve B cells and IgG-BCR–expressing memory B cells from mice and humans.
    • Fig. S7. Traction work exerted by B cells from healthy controls and RA patients.

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