Profiling the origin, dynamics, and function of …...traction stress map generated by the same cell (B). Scale bar, 5 mm. (C) Total traction force exerted by DT40 cells var-ied with

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IMMUNOLOGY

1China Ministry of Education Key Laboratory of Protein Sciences, CollaborativeInnovation Center for Diagnosis and Treatment of Infectious Diseases, Schoolof Life Sciences, Institute for Immunology, Tsinghua University, Beijing 100084, China.2Department of Mechanics and Engineering Science, College of Engineering, PekingUniversity, Beijing 100871, China. 3Department of Rheumatology and Immunology,Clinical Immunology Center, Peking University People’s Hospital, Beijing, China. 4StateKey Joint Laboratory of Environment Simulation and Pollution Control, School ofEnvironment, Tsinghua University, Beijing 100084, China. 5Chengdu Institute of Biol-ogy, Chinese Academy of Sciences, No. 9 Section 4, Renmin South Road, Chengdu610041, China. 6School of Life Science, Liaoning University, Shenyang 110036, China.7Center for Life Sciences, Department of Basic Medical Sciences, Institute for Immu-nology, Tsinghua University, Beijing 100084, China. 8Department of Rheumatologyand Clinical Immunology, Peking Union Medical College Hospital, Peking UnionMedical College and Chinese Academy of Medical Sciences, Beijing 100730, China.9Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871,China. 10Beijing Key Lab for Immunological Research on Chronic Diseases, Beijing100084, China.*These authors contributed equally to this work.†Corresponding author. Email: [email protected] (W.L.); [email protected] (C.X.)

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Profiling the origin, dynamics, and functionof traction force in B cell activationJunyi Wang1*, Feng Lin2*, Zhengpeng Wan1, Xiaolin Sun3, Yun Lu4, Jianyong Huang2, Fei Wang5,Yingyue Zeng6, Ying-Hua Chen1, Yan Shi7, Wenjie Zheng8, Zhanguo Li3,Chunyang Xiong2,9†, Wanli Liu1,10†

B lymphocytes use B cell receptors (BCRs) to recognize membrane-bound antigens to further initiate cellspreading and contraction responses during B cell activation. We combined traction force microscopy andlive-cell imaging to profile the origin, dynamics, and function of traction force generation in these responses.We showed that B cell activation required the generation of 10 to 20 nN of traction force when encounteringantigenspresentedby substrateswith stiffness values from0.5 to 1 kPa,whichmimic the rigidity of antigen-presentingcells in vivo. Perturbation experiments revealed that F-actin remodeling and myosin- and dynein-mediated con-tractility contributed to traction force generation and B cell activation. Moreover, membrane-proximal BCRsignaling molecules (including Lyn, Syk, Btk, PLC-g2, BLNK, and Vav3) and adaptor molecules (Grb2, Cbl, andDok-3) linking BCR microclusters and motor proteins were also required for the sustained generation of thesetraction forces. We found a positive correlation between the strength of the traction force and the mean fluorescenceintensity of the BCR microclusters. Furthermore, we demonstrated that isotype-switched memory B cells expressingimmunoglobulin G (IgG)–BCRs generated greater traction forces than did mature naïve B cells expressing IgM-BCRsduring B cell activation. Last, we observed that primary B cells from patients with rheumatoid arthritis generatedgreater traction forces than did B cells from healthy donors in response to antigen stimulation. Together, these datadelineate the origin, dynamics, and function of traction force during B cell activation.

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INTRODUCTIONB lymphocytesmediate antibody responses arising from the recognitionof antigens by the surface expressed B cell receptor (BCR) (1). The BCRcontains amembrane-bound immunoglobulin (mIg) and a heterodimerof Iga and Igb subunits (2, 3). The mIg is mainly responsible for therecognition of antigens, whereas the Iga and Igb heterodimer stimulatestransmembrane signaling through immunoreceptor tyrosine activationmotifs in the cytoplasmic domains (4, 5). Antigen binding–inducedactivation of BCR signaling is efficiently regulated by the presentation ofvariable forms of antigens that B cells encounter in vivo (5, 6). Theseantigen characteristics include, but are not limited to, antigen density(7, 8), antigen affinity (7, 8), antigen valency (9–14), the Brownianmobility feature of the antigen (15–17), the mechanical forces deliveredto the BCRs by the antigens (18, 19), and the stiffness feature of the sub-strates presenting the antigen (20, 21). All of these studies suggest thatthe BCR is a versatile receptor, which can efficiently sense both the

chemical and physical features of an antigen ligand and convert theminto a cytosolic signal to determine the fate of the cell.

Early biochemical studies extensively investigated how the chemicalcues from the antigen determine the strength of the signaling cascademediated by the BCR (4, 5). However, reports have revealed thatphysical cues are also important layers of external information thatare delivered to the BCRs by the antigens (22). For example, it wasreported that, when B cells encounter membrane-bound antigens,they first exhibit a substantial spreading response over the antigen-presenting surface, which is followed by amarked contraction response(8). Thus, dynamic traction forces are generated between the B cellsand the antigen-presenting surface during the B cell spreading andcontraction responses. However, the traction force in B cell activationis poorly characterized.

Here, we used a traction force microscopy system to profile theorigin, dynamics, and function of traction force generation withinthe B cell immunological synapse during B cell activation. We foundthat B cells generated a total traction force of 10 to 20 nN when theyencountered antigens presented by substrates with stiffness values of0.5 to 1 kPa, whichmimic the rigidity of antigen-presenting cells in vivo(23–25). The traction force generation in B cells after BCR-mediatedantigen recognition relied on the remodeling of polymerizedmicrofilamentactin (F-actin) and motor proteins, including dynein and myosin. Wealso observed a positive correlation between the strength of the tractionforces and themean fluorescence intensity (MFI) of theBCRmicroclusters.Moreover, the requirement of membrane-proximal BCR signalingmolecules and adaptormolecules linking BCRmicroclusters andmotorproteins to sustain traction force generation was also revealed. Further-more, we demonstrated that, during the initiation of immune activa-tion, isotype-switched memory B cells, which express IgG-BCRs,generated greater traction force than that of naïve B cells, which expressIgM-BCRs. These differences are likely due to the increased amountsof motor proteins in memory B cells and the formation of prominent

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IgG-BCR microclusters, which is mediated by the evolutionarily con-served cytoplasmic region of the heavy chain of mIgG-BCR. Further-more, our findings have clinical relevance because we found thatprimary B cells from patients with rheumatoid arthritis (RA) generatedexcessive amounts of traction forces in comparison with B cells fromhealthy controls during B cell activation. Together, these data promptedus to propose a three-step model, manifesting the anchoring rivet andtraction force transmitter functions of BCRmicroclusters, to explain themolecular mechanism for the generation of traction forces during B cellactivation. This model may provide useful information to better under-stand the function of mechanical forces in B cell activation, which maycontribute to the development of better vaccines and therapies for auto-immune diseases.

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RESULTSDynamic traction forces are generated by B cells onantigen-containing substratesThe physical nature of the traction forces that B cells apply to antigen-containing substrates was measured using traction force microscopy(Fig. 1A). In this approach, B cells were placed on polyacrylamide(PA) gels that were precoated with anti-BCR surrogate antigensaccording to our published protocols (20).We also anchored fluores-cent beads to the surface of the PA gel substrates to accurately trackand measure the lateral deformation changes of the PA gels as areflection of the traction forces that could be applied by the B cellsto the antigen-containing substrates. These fluorescent beads served


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as tracking markers in imaging experiments. Time lapse of both cellphase-contrast and fluorescent bead images was recorded (at 12-sintervals) by confocal fluorescence microscopy to capture and inves-tigate the spatiotemporal dynamics of the traction forces in the B cells.We performed the imaging starting from the earliest time points beforethe cells established an interaction with the substrates and continuedrecording up to 30 min. Substrate deformations were measured bycomputer-aided tracking of the fluorescent beads using a digital imagecorrelation (DIC) algorithm, as reported previously (26). The fluores-cence image of the beads after the B cells were detached from the PA gelwas used as a reference image. An improved Fourier transform tractioncytometry method was used to calculate the traction stress map fromthe measured substrate deformations (27, 28). We first examined theresponse of laboratory DT40 cells, a chicken B cell line. There wasnodetectable displacement of the fluorescent beads at a distance beyond~1 mm from these B cells (Fig. 1B). This indicates that bead displace-ments around the B cell reflect the deformation of the PA substrates thatwere induced by the traction forces generated by the B cells. The stiff-ness of the PA gel used in these experiments was about 1 kPa, which issimilar to the stiffness of antigen-presenting cells (23–25).

Using this methodology, we acquired a B cell traction stress mapconsisting of different time points (Fig. 2, A and B). As detailed inMaterials and Methods and in our previous studies (27, 28), the totaltraction force at each individual time pointwas calculated by integratingthe absolute cellular traction stress with the area of cell-substrate inter-actions. The distribution of the traction force was highly dynamic andexhibited large variations over time at different regions within theinterface of the cell’s contact with the antigen-containing surfaces(Fig. 2C). These data revealed the unexpected highly variable spatio-temporal dynamics of the traction forces thatwere applied to the antigen-containing substrates by the B cells. Further analyses showed that thetraction force curve in the total time course correlated with a two-exponential function (regression fitting showed an R value as high as0.9715; fig. S1A). We calculated the slope of the two-exponentialfunction over time, which represented the rate of the growth of the totaltraction force (fig. S1B). The value of the slope was very high within thefirst 5 min, demonstrating the marked increase in the traction forceswithin this period of time (Fig. 2C and fig. S1B). However, the increasein the traction forces was onlymild starting 10min, as demonstrated bythe lack of changes of the value of the slope over time from10 to 30min.Thus, in the present study, most of the traction force data generated bythe B cells were measured at 20 min after B cell contact with the PA gelsubstrates, unless otherwise stated. Here, we also quantified the tractionwork of B cells by multiplying the total traction force by the distancechanges, rendering this parameter a unit of joule. We used the tractionwork done by B cells to accurately quantify the total amount of workthat B cells exerted on the antigen-presenting surfaces during B cellactivation and found that B cells exerted an average total tractionwork on the order of 1.00 × 10−15 to 1.59 × 10−15 J during this dynamicprocess (fig. S2, A to C).

To verify that the observed traction forces were specific to antigenrecognition, traction forces were also examined for DT40 cells incontact with PA gel substrates lacking antigen [referred to as negativecontrol (NC)]. On antigen-free substrates, DT40 cells did not spread,and the total traction forces were statistically significantly less thanthose generated on antigen-containing surfaces (anti-BCR antigen–coated, referred to as Ag) (Fig. 2D).We also repeated these experimentswith primary B cells isolated from wild-type (WT) C57BL/6J mice(referred to as B6 B cells) and confirmed that primary B cells generated

Fig. 1. Schematic diagram of traction force microscopy. (A) Schematic repre-sentation of the traction force microscopy method used in this study. In this ap-proach, B cells were placed on PA gels that were precoated with anti-BCRsurrogate antigens (Ags). To accurately track and measure the lateral deformationchanges in the PA gels that are exerted by the B cells, we also anchored fluores-cent beads to the surface of the PA gel substrates. (B) Representative images ofDT40 cells under the following conditions: B cell spreading on the PA gel (phasecontrast), fluorescent beads linked to the PA gel surface (fluorescent beads), thedisplacement map (displacement) of the substrate calculated from the lateral de-formation of the PA gels by DIC, and the corresponding traction stress map (trac-tion stress) computed from the displacement map. Scale bar, 5 mm.

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much higher total traction forces on antigen-containing substrates thanon antigen-free substrates (Fig. 2, E and F). To further confirm thatthese traction forceswere induced by theBCR-antigen bonds to the sub-strate, rather than because antigen recognition by the BCR triggeredother B cell surface molecules, such as integrins, to bind to the substrateto exert traction force, we performed the following control experiments.First, we pretreated B cells with a short synthetic Arg-Gly-Asp acid(RGD peptide), which inhibits cell adhesion by binding to integrins(29), before loading the B cells onto the antigen-presenting PA sub-strates. The results from these experiments showed that blocking integ-


rin function with RGD peptide did not affect the generation of tractionforces by either DT40 cells or primary mouse B cells (Fig. 2, G and H).Second,we found that similar amounts of traction forceswere generatedby primary B cells from WT mice and B cells derived from CD11a(ITGAL) knockout (KO) mice (Fig. 2H), which are deficient in theintegrin lymphocyte function–associated antigen 1 (30, 31), the majortype of integrin found on B cells (32). Third, as NC, we placedmouseprimary B cells on substrates precoated with an irrelevant antibody(goat anti-chicken IgM) that cannot recognize mouse IgM-BCR, orconversely, we placed DT40 cells on substrates precoated with goat

Fig. 2. Traction forces generated by DT40 cells and B6primaryB cells. (A and B) Representative time-lapse, phase-contrast images of DT40 cells on antigen-coated PA sub-strates with a stiffness of 1 kPa (A) and the correspondingtraction stress map generated by the same cell (B). Scalebar, 5 mm. (C) Total traction force exerted by DT40 cells var-ied with time up to 30 min. Red lines represent the selectedexample cells (n = 15 cells), whereas the blue line displaysthe average of the total number of tested cells (n = 49 cells).(D) Total traction forces generated by DT40 cells incubated onsubstrates coated with goat anti-chicken IgM (Ag) or neutravidin(NC) for 20 min. Data are means ± SEM of the total tractionforce calculated from at least 15 cells in one experiment thatis 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 in-cubated on antigen-coated PA substrates with a stiffness of0.5 kPa (E) and analysis of the total traction forces exertedby B6 primary B cells incubated on PA substrates coatedwith antigen (Ag) or neutravidin (NC) for 20 min (F). Dataare means ± SEM of the total traction force calculated fromat least 25 cells in one experiment that is representative ofthree independent experiments. Scale bar, 10 mm. ***P <0.001 by two-tailed t test. (G) Total traction forces exertedby DT40 WT cells without (control) or with (RGD) pretreatmentwith the integrin inhibitor RGD peptide upon stimulation bysubstrates coated with goat anti-chicken IgM antibodies. Alsoprovided as NC are the total traction forces generated byDT40 WT cells upon stimulation by substrates coated with ir-relevant goat anti-mouse IgM antibodies (anti-mouse IgM).Data are means ± SEM of the total traction force calculatedfrom at least 21 cells in one experiment that is representativeof three independent experiments. ***P < 0.001 by two-tailed t test. (H) Total traction forces exerted by WT primaryB6 B cells without (control) or with (RGD) pretreatment withthe integrin inhibitor RGD peptide and primary B cells fromCD11a KO mice (CD11a-KO) upon stimulation by substratescoated with anti-mouse IgM antibodies. Also provided asNC are the total traction forces generated by WT primaryB6 B cells upon stimulation by substrates coated with ir-relevant goat anti-chicken IgM antibodies (anti-chickenIgM). Data are means ± SEM of the total traction forcecalculated from at least 34 cells in one experiment that is rep-resentative of three independent experiments. ***P < 0.001 bytwo-tailed t test.

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anti-mouse IgM as an irrelevant antibody (Fig. 2, G and H).Traction forcemicroscopy results showed that in both typesof cells, the values of traction force generated on the sub-strates presenting irrelevant antibodies were similar to thoseon antigen-free substrates (NC), both of which weremarkedlyless than the values of the traction forces that were generatedon stimulating surrogate antigens (Fig. 2, F and G). Together,these results indicate that the traction forcemicroscopymethodalgorithm is a reliable method to examine the generation oftraction forces from B cells. These results also showed that thetraction forces fromBcells exposed to antigen-containing sub-stratesweremainly inducedbyBCR-antigen recognition, althoughit is also evident that there are very low basal traction forces thatare exerted byB cells in the presence of antigen-free substrates.

Myosin and dynein motor proteins are involved inthe generation of traction forcesThe highly dynamic nature of the traction forces that wereapplied to the antigen-containing substrates by B cellsprompted us to examine themolecular machinery accountingfor their generation. Our primary targets were the motor pro-teins, including dynein and myosin IIA, which are highlyabundant in B cells and play important roles in antigen-triggered B cell activation responses (18, 33). To investigatethe effects of these twomotor proteins on the generation of trac-tion forces in B cells, we used hedgehog pathway inhibitor 4(HPI4), a specific inhibitor of dynein (34), blebbistatin (referredto as BLEB), a specific inhibitor of the adenosine tripohospha-tase activity ofmyosin IIA (35), andML7, amyosin light chainkinase inhibitor (36), to disrupt the function of these two typesof motor proteins. The inhibitors were used according to thecommon protocols for HPI4 (incubation at 37°C overnight),BLEB (incubation at 37°C for 30 min), and ML7 (incubationat room temperature for 20 min), as detailed in Materialsand Methods. The generation of traction forces by the inhibitor-treated cells was compared with that of the correspondingcontrol cells [which were pretreated with the vehicle dimethyl

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sulfoxide (DMSO)]. We also examined the MFI of the BCRs within thecontact interface between the B cells and the antigen-containing sub-strates because the BCR MFI value has been widely used as a parameterto characterize the extent of B cell activation (21, 37). The quantificationof the MFI of the BCRs demonstrated that the addition of myosin IIAinhibitors statistically significantly decreased the BCR MFI as comparedto that of the control cells (DMSO), and the dynein inhibitor had a simi-lar, but relatively,mild effect (Fig. 3A).When examining the generationofthe traction forces and tractionwork, we found that both types ofmyosininhibitor, BLEB and ML7, markedly impaired the dynamics of tractionforce generation and the tractionwork (Fig. 3B and fig. S3A, respectively)compared to those of the DMSO-treated control cells (Fig. 3B). Further-more, the total traction forces and traction work were markedlyinhibited in BLEB- orML7-treated cells (20min after B cell contact withthe antigen-presenting substrates; Fig. 3C and fig. S3B, respectively) incomparison to those of the DMSO-treated control cells. Similar to theexperiments inhibiting myosin IIA, experiments with the dynein inhibi-tor showed that dynein is involved in traction force generation (Fig. 3, Band C). These observations were also confirmed in experiments withB6 primary mouse B cells (Fig. 3D and fig. S3C). These results dem-onstrate that both the myosin and dynein motor proteins are involvedin the generation of traction forces from B cells.


Dynamic F-actin remodeling regulates traction force,whereas the MFI of BCR microclusters determines thestrength of traction forceBCR microclusters are the basic platform in the initiation of B cell ac-tivation, and their formation is highly dependent on the remodeling ofthe F-actin cytoskeleton (18, 38). Thus, we analyzed the codistributionof the traction force with both F-actin and BCR molecules within thecontact interface between the B cells and the antigen-presenting sub-strates. To this end,wedetected F-actinwith a Lifeact-mCherry–derivedlabeling strategy in the B cells that were prestained with an Alexa Fluor647–conjugated Fab anti-chicken IgM antibody. We then imaged F-actin, IgM-BCR, and traction stress by traction forcemicroscopy at 2, 5,10, 15, 20, 25, and 30min after placing the B cells on antigen-presentingand fluorescein isothiocyanate (FITC) bead–containing substrates(Fig. 4A). We found that the response of the F-actin cytoskeleton waspertinent to the behavior of BCR during B cell activation because theF-actin cytoskeleton was remodeled during the progress of B cell ac-tivation. First, F-actin was diffusely distributed in the contact interfacebetween B cell and substrate surface in the early stage of the B cell ac-tivation progress (the first 5 min), whereas F-actin was then remodeledto form an integrative and stable architecture in the peripheral area of thecontact interface between the B cell and substrate surface in the middle

Fig. 3. Myosin and dynein are involved in BCR activation and traction force genera-tion. (A) DT40 cells were pretreated with DMSO (vehicle control) or with the indicated in-hibitors before being incubated on PA surfaces coated with goat anti-chicken IgM. The MFIsof the BCRs from each indicated group of DT40 cells were quantitated as the fluorescenceintensity of the labeled BCRs averaged over the area of the B cell. Data are means ± SEM ofthe MFIs analyzed from at least 36 cells in one experiment that is representative of threeindependent experiments. **P < 0.01 and ***P < 0.001 by two-tailed t test. (B) Time-lapse anal-ysis 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 isrepresentative of three independent experiments. (C and D) Total traction force exerted byDT40 (C) or B6 primary B cells (D) that were pretreated with DMSO (vehicle) or the indicatedmotor 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 tractionforce calculated from at least 21 cells in one experiment that is representative of threeindependent experiments. ***P < 0.001 by two-tailed t test.

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to late stage of B cell activation progress (refers to the following 5 to10 min). Disrupting the polymerization of F-actin by treating the cellswith latrunculin-B (39) impaired the generation of traction forceduring B cell activation (Fig. 4B). These results demonstrate thatF-actin remodeling is required for the generation of traction force dur-ing B cell activation.

Subsequently, we acquired theMFI of both F-actin and BCR and thevalue of the traction stresswithin the same regionof interest (ROI) (Fig. 4C;see the three indicated ROIs). With these data, we profiled the spatio-temporal dynamics of the correlation of traction force with either F-actinorBCRat each timepoint by themethodsof both linear correlationand thePearson correlation coefficient (PCC) (Fig. 4, D andE). The results showedthat there was only weak correlation between traction force and F-actin

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because both the R value of linear regression and the PCC value werelow and failed to exhibit dynamic changes at each time point (Fig. 4D).In contrast, the correlation between traction force value and the MFI ofBCR microclusters was initially low in both the linear regression andPCCanalysis at the 2-min timepoint; however, both correlationsmarkedlyincreased to maximal values at 10 min and then decreased at later timepoints (Fig. 4E). Together, these results suggest that the MFI of the BCRmicroclusters positively correlates with the strength of the traction force.

The generation of traction forces is dependent on theactivation of membrane-proximal BCR signaling moleculesHaving identified motor proteins and BCR microclusters as the sourceof traction force generation, we continued to investigate the molecular

Fig. 4. The correlation between the BCR MFand the strength of the traction forces. (A) Representative time-lapse traction stress map (topand the corresponding fluorescence images oF-actin (middle) and the BCR (bottom) of DT40cells incubated for up to 30min on PA substratecoated with goat anti-chicken IgM. Scale bars5 mm. (B) Total traction force exerted by DT40cells that were pretreated with either DMSO olatrunculin-B (Lat-B) and then incubated for 20minon PA substrates coated with goat anti-chickenIgM. Data are means ± SEM of the total tractionforce calculated from at least 40 cells in one experiment that is representative of three independent experiments. ***P < 0.001 by two-tailedt test. (C) Correlation between the strength of traction stress and the MFI of F-actin or BCR at 10 minafter incubation for the cells shown in (A). LeftThree representative ROIs (a, b, and c) in the threeimages represent the same ROIs that were used tocalculate the MFIs of F-actin and the BCR and thestrength of the traction stress. Scale bars, 5 mmRight: Correlation shows the linear regressionanalysis between the MFI of both F-actin andBCR with the traction stress value. Data are fromat least 12 cells (28 ROIs per cell) in one experimenthat is representative of three independenexperiments. (D) AverageRvalueof the linear regression (left) and the Pearson correlation coefficien(PCC; right) between F-actin and traction stresof 12 tested cells varied with time up to 30 minRed lines represent the selected example cells (n =12 cells), the ochre line represents the cell shownin (A), and the blue line displays the average of thetotal tested cells. (E) Average R value of linear regression (left) and the PCC (right) between BCRand traction stress of 12 tested cells varied withtimeup to30min. Red lines represent the selectedexample cells (n = 12 cells), the ochre line represents the cell shown in (A), and the blue linedisplays the average of total tested cells.

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signaling pathways that regulate traction force generationthrough the BCR complex. It has been reported that the recog-nition ofmembrane-tethered antigen by theBCR results in therearrangement of molecules proximal to the plasma mem-brane of B cells (6, 40). Subsequently, these rearranged mole-cules, such as Lck/Yes novel tyrosine kinase (Lyn), spleentyrosine kinase (Syk), Bruton’s tyrosine kinase (Btk), phospho-lipase C–g2 (PLC-g2), B cell linker (BLNK), and the guaninenucleotide exchange factor Vav3, are required for the initiation

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and regulation of B cell spreading after stimulation with membrane-tethered antigen. Therefore, we selected a panel of DT40 cell KOs foreachof these key signalingmolecules and analyzed their ability to generatetraction force on antigen-containing substrates. The results showed thatKOofmost of the BCR signalingmolecule, including Lyn, Syk, Btk, PLC-g2, BLNK, andVav3,markedly impaired the ability of DT40 cells to gen-erate traction forces and tractionwork in comparison toWTDT40 cells(Fig. 5A and fig. S4A). To further validate the conclusion that the gen-eration of the traction forces was dependent on these signaling mole-cules but not on other off-target effects during the generation of theseKO B cells, we cloned the complementary DNAs encoding Vav3 andPLC-g2 from the mRNA of WT DT40 cells and performed a rescue ex-periment by constructing plasmids encoding either Vav3 or PLC-g2fused to a green fluorescent protein (GFP) tag and using these constructsto transfect the Vav3-KO or PLC-g2–KODT40 cells, respectively (Fig. 5,B and C). We found that exogenous expression of these molecules inthe corresponding KO DT40 cells statistically significantly restored thegeneration of traction force and traction work (Fig. 5D and fig. S4B).Thus, these data indicate that the initiation of membrane-proximalBCR signaling is required for the sustained generation of traction force.


The generation of traction force requires the loading of BCRmicroclusters onto motor proteinsNext, we investigated the function of essential bridging molecules thatload BCR microclusters onto motor proteins during the generationof traction force. BCRs are transported in a retrograde manner to thecenter of the B cell immunological synapse along cytoskeletal tracksthroughmotor proteins. A report usingmass spectrometry identifiedthree key bridging molecules that load BCR microclusters onto motorproteins (33). Whereas B cells deficient in casitas B cell lymphoma(Cbl), growth factor receptor–bound protein 2 (Grb2), or the thirdmember of the Dok (docking protein) family (Dok-3) can produceBCRmicroclusters at the peripheral region of the B cell immunologicalsynapse, these BCR microclusters are not transported to the centralregion of theB cell immunological synapse because of the lack of linkagewith motor proteins. Thus, we compared side by side both BCRmicro-cluster formation and the generation of traction forces in WT DT40cells versus DT40 cells deficient in Cbl, Grb2, or Dok-3. We found thata deficiency in each of these molecules did not affect the formation ofBCR microclusters because there was no statistically significantdifference in the MFI of BCR microclusters between WT and KO cells

Fig. 5. Membrane-proximal BCR signaling molecules and adaptormolecules linking BCR microclusters and motor proteins are re-quired for the generation of traction forces. (A) Scatter diagramsof the total traction forces exerted by WT DT40 cells and indicatedKO DT40 cells when incubated for 20 min on PA substrates coated withgoat anti-chicken IgM. Data are means ± SEM of the total traction forcecalculated from at least 28 cells in one experiment that is represent-ative 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 reconstitutedwith Vav3-GFP (B) and PLC-g2 KO DT40 cells reconstituted with PLC-g2–GFP (C) after incubation for 20 min on PA substrates coated with goatanti-chicken IgM. Scale bar, 5 mm. (D) Scatter diagrams showing the totaltraction forces generated by Vav3-KO DT40 cells, Vav3 KO DT40 cells re-constituted with Vav3-GFP, PLC-g2–KO DT40 cells, and PLC-g2 KO DT40cells reconstituted with PLC-g2–GFP after incubation as described in (C).Data are means ± SEM of the total traction force calculated from at least30 cells in one experiment that is representative of three independentexperiments. ***P < 0.001 by two-tailed t test. (E and F) Fluorescenceintensity (FI) of BCR microclusters (E) and scatter diagrams of total tractionforces (F) exerted by WT DT40 cells and the indicated KO DT40 cell linesafter incubation for 20 min on PA substrates coated with goat anti-chickenIgM. Data are means ± SEM of the fluorescence intensity calculated fromat least 2311 BCR microclusters analyzed from at least 37 cells (E) and thetotal traction force calculated from at least 45 cells (F) in one experimentthat is representative of three independent experiments, respectively. **P <0.01 and ***P < 0.001 by two-tailed t test.

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in response to antigen stimulation (Fig. 5E). However, loss of any ofthese molecules markedly impaired the ability of the cells to generatetraction force on antigen-presenting substrates (Fig. 5F). Among these,Grb2-KO B cells showed a more marked defect in force generationcompared to the Cbl-KO and Dok-3-KO cells. This result is consistentwith the reports showing that the recruitment of both Dok-3 and Cbl toBCR microclusters depends on Grb2 (33). Furthermore, Grb2 is a keysignaling molecule that mediates multiple cell functions, includingremodeling of F-actin by interacting with molecules such as Sos1 (sonof sevenless homolog 1), Rho (Ras-like guanosine 5′-triphosphate–binding protein), and Vav3 (41–43). Thus, Grb2-KO cells would beexpected to exhibit more severe effects than would Cbl-KO and Dok-3–KO B cells in the generation of traction forces in response to antigenstimulation. These results suggest that the loading of BCR microclustersontomotor proteins and the retrogrademovement of BCRmicroclustersare both essential for the generation of traction force.

IgG-BCR–expressing memory B cells generate more tractionforces than do IgM-BCR–expressing naïve B cellsB cells use different isotypes of BCRs to recognize antigens duringthe initiation of B cell activation. Mature naïve B cells use IgM-BCRs,whereas memory B cells mainly use class-switched IgG-BCRs. MemoryB cells, but notmature naïve B cells, aremainly responsible for the highlyeffective antigen recall antibody responses upon vaccine immunization(4, 44). However, there has been a lack of investigation into quantifyingtraction forces during the activation of IgG-BCR–expressing memoryB cells compared to IgM-BCR–expressing naïve B cells. Here, we ad-dressed this question by using primarymature naïve B cells expressingthe B1-8–IgM-BCR from IgH B1-8/B1-8 Igk−/− transgenic (Tg) mice(45) and B1-8 primary B cells expressingmemory IgG-BCRs that werederived from an in vitro class-switch response according to our pub-lished protocol (46). To achieve an unbiased comparison, we prelabeledthe IgM-BCR–expressing mature naïve B cells with the DyLight 649–conjugated Fab fragment from goat anti-mouse IgM (heavy chain–specific) antibodies, whereas the IgG-BCR–expressing cells were prelabeledwith the Alexa Fluor 488–conjugated Fab fragment from goat anti-mouse IgG (heavy chain–specific) antibodies. We placed a mixture ofboth types of prelabeled B cells into the traction force–measuringchambers with substrates coated with 4-hydroxy-3-nitrophenylacetylantigen, which is the specific antigen for the B1-8–BCRs. We observedthat the IgG-BCR–expressing memory B cells generated more tractionforces and tractionwork thandid the IgM-BCR–expressing naïve B cells(Fig. 6A and fig. S5A). Next, we investigated the mechanism potentiallyresponsible for the different responses in the naïve andmemory B cells.This mechanism is especially intriguing because both the IgG-BCR andthe IgM-BCR use the exact same signaling initiation component: theIga and Igb heterodimer. Thus, we focused on the BCR component thatrecognizes the antigens in these two types of BCRs, which are mIgG-BCR and mIgM-BCR (2, 3). mIgG and mIgM differ greatly in the cy-toplasmic domains of their respective heavy chains. The heavy chain ofmIgMhas only 3 amino acids in its cytoplasmic tail (KVK), whereas theheavy chains of all mIgG subtypes have 28 amino acids in their cyto-plasmic tails, which are conserved across species (7, 47, 48). Earlymousemodel studies using biochemical assays and live-cell imagingdemonstratedthat the cytoplasmic tail of the heavy chain of mIgG is both necessaryand sufficient to confer the enhanced activation of IgG-BCR–expressingB cells compared to that of IgM-BCR–expressing B cells (46, 49–55).Because the generation of traction forces is dependent on the activationof membrane-proximal BCR signaling, as we demonstrated earlier, it is


of interest to examine the contribution of the cytoplasmic tail of themIgG heavy chain in generating more traction forces in IgG-BCR–expressing B cells compared to IgM-BCR–expressing B cells. To explorethis phenomenon, we took advantage of the two types of hen egg lyso-zyme (HEL)–specific BCR Tg mice (51): (i) Tg mice with mature naïveB cells expressing HEL-specific IgM-BCRs (referred to as IgM) and (ii)Tg mice with mature naïve B cells expressing HEL-specific IgM-BCRswith the cytoplasmic tail of the heavy chain of mIgM swapped with amIgG cytoplasmic tail (referred to as IgMG). By using a similar tractionforce–measuring system with substrates presenting HEL antigens, weconfirmed that the cytoplasmic tail of the heavy chain of mIgGwas suf-ficient tomediate the enhanced generation of traction force and tractionwork during the initiation of B cell activation (Fig. 6B and fig. S5B).Together, these results suggest that IgG-BCR–expressing memory B cellsgenerate more traction forces than do IgM-BCR–expressing naïve B cellsand, furthermore, that this enhanced effect is likelymediated by the evo-lutionarily conserved cytoplasmic tail of mIgG.

Our previous studies demonstrated that the cytoplasmic tail of theIgG-BCRheavy chain promotes the formation ofmore prominent BCRmicroclusters of a higherMFI than that in the case of IgM-BCRand, as aconsequence, enhance the strength of BCR signaling (46).We speculatethat these features may facilitate the generation of traction forces, be-cause we have provided evidence here showing that the MFI of theBCRmicroclusters is positively correlated to the strength of the tractionforce. We therefore compared the MFI of the BCR microclusters inprimary B cells from these two HEL-specific Tg mice when placed onHEL-containing lipid bilayers according to our previously publishedprotocol (37, 46). We found that cells expressing an mIgM harboringthe cytoplasmic tail of mIgG formed statistically significantly largerBCR microclusters than those formed by cells expressing WTmIgM(Fig. 6, C and D). Furthermore, our previous data revealed that, afterantigen stimulation, both dynein andmyosin IIAwere required for Bcells to generate traction force. Thus, the abundances of these twomotorproteins in B cells would be another key factor determining the magni-tude of the traction force generation from B cells. According to the datasets acquired by ImmGen (www.immgen.org/databrowser/index.html)andprevious studies (56–58),memoryB cells havemoremyosin light andheavy chains and more dynein light and intermediate chains comparedto mature naïve B cells (fig. S6). These data suggest that the increasedamounts of motor proteins in memory B cells may account for theirability to generate greater traction forces than those of naïve B cells.

B cells from RA patients generate excessive amounts oftraction force compared to those of healthy controlsHaving shown that the generation of traction force was dependent onthe activation of BCR signaling molecules and that the MFI of the BCRmicrocluster positively correlated with the strength of these tractionforces, we were curious to investigate the generation of traction forcesin B cells from RA patients because numerous studies have demon-strated that B cells from RA patients exhibit enhanced BCR signalingand produce autoreactive autoantibodies [such as rheumatoid factors(RFs)] (59–61). Thus, we compared the generation of traction forcein B cells from RA patients with that in B cells from healthy individualsby placing these human primary B cells on the same antigen-containingsubstrates. To reduce intersample and interbatch variations, we chosethree age- and gender-matched pairs of healthy controls andRApatients.In each batch of the experiment, we only compared one pair of samples,a healthy control versus an RA patient. We prelabeled peripheral bloodB cells from the paired sampleswith anAlexa Fluor 647–conjugated Fab

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fragment froman anti-human IgMconstant region (Fig. 7A) andplacedthese cells on the PA gel substrates presenting anti-human Igk and anti-human Igl antibodies, which functioned as the surrogate antigens. Thecells were in contact with the antigen-coated PA gel for 20 min. TheMFIs of the BCRs (Fig. 7B) were measured, and the traction forces(Fig. 7C) and traction work (fig. S7) were calculated as described earlier.The results showed that in all three paired groups, the BCRMFI and thetraction force derived from RA patient B cells were statistically signifi-


cantly greater than those of the B cells from healthy donors. These find-ings suggest that B cells fromRA patients generate an excessive amountof traction force as compared to those from healthy donors.

DISCUSSIONMechanical forces are thought to be essential for mediating the activa-tion of antigen receptors, including the T cell receptor (TCR) and the

Fig. 6. Traction forces generated by IgM-BCR–expressing naïve B cells and isotype-switched IgG-BCR–expressing memory B cells. (A) Quantification of thetraction 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 coatedwith 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 independentexperiments. **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 naïveB 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 incubationfor 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 fluorescenceintensity 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 BCRmicroclusters 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 mm.

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BCR, according to our previous studies and those of others (18, 20, 62–64).Here, we assessed the traction force exerted by B cells, which wastransmitted to elastic substrates through the BCR microclusters at thecontact interface between the B cells and the antigen-containing sub-strates. By using traction forcemicroscopy, we reported here that B cellsgenerated an average total traction force on the order of 10 to 20 nN,which varied with time in response to antigen stimulation. In markedcontrast, only very low traction forces were detected in B cells that wereplaced on antigen-free substrates. We propose that the extremely lowextent of traction force generation may be correlated with antigen-searching events by B cells, which are facilitated by membrane-probingbehaviors, as reported in our previous study (37). Here, we also quanti-fied the energy consumption during the execution of the traction workby B cells, which is termed as traction work done by B cells. Mechanicalpotential is generally believed to be one major type of free energy that acell must exert when functioning (65). However, the mechanism ofmechanical energy production and the physiological role of mechanicalenergy consumption and transmission are not known in immune cells,


let alone B cells, mainly because of the lack of a suitable measurementmethod (66). Changes in energy define the direction for spontaneouschanges in chemistry and physics. The gradients of chemical factors,such as chemokines, can induce chemotaxis, suggesting that overcom-ing the chemical concentration gradient requires work in the form ofexpenditure of energy (67, 68). Here, we quantified the traction workof B cells by multiplying total traction force with the distance changes,rendering this parameter a unit of joule to accurately quantify the totalamount of work that B cells exerted to the antigen-presenting surfacesduring B cell activation.

Cell traction force is mainly generated by the actin-myosin cyto-skeleton and is exerted on the underlying substrate or the extracellularmatrix (ECM) (69). This distinguishes various physiological and path-ological behaviors and functions of suspension cells, such as immunecells (62–64, 66), and adherent cells, such as fibroblasts, muscle cells,cancer cells, and mesenchymal stem cells (70–72). In suspension cells,especially immune cells, traction force generation is used to recognizeantigens to further activate immune cells and kill target cells (62). Inadherent cells, traction force generation is also used to sense signalsfrom the cellularmicroenvironment, which is essential for cellmorphol-ogy maintenance, cell migration, cardiomyocyte contraction, and mes-enchymal stem cell differentiation (70–72). Traction force can stimulatedownstream signaling pathways, which further regulates cell behaviorand function (73). On the basis of this model, we propose that tractionforce is generated to initially enable the B cell to search and capture anti-gens through theBCRs. It is likely that traction forces can further enhanceB cell activation by recruiting more BCRs and membrane-proximalBCR signalingmolecules to form larger BCRmicroclusters, which, inturn, help B cells exert more traction force on the underlying substratesor the ECM, resulting inmore efficient antigen acquisition. This positivefeedbackmechanism,whichoriginates from the cell interior andproceedsto the cell exterior and then back to the cell interior again, is similar toa mechanism reported in adherent cells (73). Taking these findingstogether, we propose that the higher the traction force that a B cellcan generate in response to antigen stimulation, the stronger the B cellactivation would be.

Cell contraction is also generally mediated by the actomyosincytoskeletal network through the sliding of myosin IIA on actin fila-ments and the moving of dynein along microtubules (33, 74, 75). Themotor proteins contribute to the actomyosin cytoskeletal system bygenerating the contraction force, which is necessary for controlling cellmorphology, which, in turn, regulates cell functions. In B cell studies, itis well documented that B cells use dynein to induce retrograde BCRmicrocluster movement into the center of the B cell immunologicalsynapse, whereasmyosin IIA is used by B cells to disrupt the interactionbetween the BCR and antigens (18). Thus, we investigated the contri-bution of dynein and myosin IIA to B cell activation and traction forcegeneration.Weobserved that blockingmyosin IIA– anddynein-mediatedcontractility substantially reduced the generation of traction force andimpaired B cell activation.

BCR microclusters are first formed on the cell periphery and arelater transported toward a central aggregate through actin filamentpolymerization and contraction (8). Simultaneously, it is observed thatF-actin is also mainly distributed at the periphery of the spreading Bcells (76). We assessed whether the F-actin cytoskeleton colocalizedwith the traction force. Unexpectedly, F-actin did not correlate withthe traction force very well, but instead, we observed that the MFI ofthe BCRmicroclusters strongly correlatedwith the traction force. Theseresults lead us to speculate that the BCR microclusters might have a

Fig. 7. Traction forces generated by B cells from healthy donors and pa-tients with RA. (A) Representative phase-contrast and BCR fluorescence imagesof 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 mm. (B and C) BCR MFIs (B) and total traction forces (C) of primary B cellsfrom three pairs of healthy human controls and RA patients after incubation andspreading for 20 min on PA substrates coated with goat anti-human Igk and Igl lightchain. Data are means ± SEM of BCR MFI (B) or the total traction force (C) calculatedfrom at least 26 cells (per sample) from three pairs of donors. **P < 0.01 and ***P <0.001 by two-tailed t test.

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similar function to that of focal adhesion molecules or integrins. Bothintegrins and the focal adhesion molecules serve multiple functions,including the regulation of adhesion and migration in many adherentcells (77), the regulation of T cell activation (66, 78, 79), and thetransmission of traction force from cells to their substrates (64). Thesepossible parallels in function between BCR microclusters and integ-rins or the focal adhesion proteins may help uncover at least part ofthe mechanisms that regulate B cell activation and B cell traction forcegeneration.

We found that F-actin contractility was needed for the generationof traction force. This conclusion is supported by the observation thatdynamical F-actin remodeling from a diffusely distributed structure inthe early stage of B cell activation (before 5 min; Fig. 4A) to an integra-tive and stable architecture mainly located in the peripheral area of thecontact interface between the B cell and the substrate surface in themid-dle to late stage of B cell activation (5 to 10 min; Fig. 4A). Moreover, wefound that disrupting the polymerization of F-actin by latrunculin-Bimpaired the generation of traction force during B cell activation(Fig. 4B), although there was no strong spatiotemporal correlation be-tween F-actin and force. We speculate that motor proteins are thegenerator of the traction forces, a process that is performed on the trackof actin filaments. It was reported that the motor proteins myosin IIAand dynein interact with their cargo BCR microclusters through themajor histocompatibility complex class II invariant chain II (80) andthe E3 ubiquitin ligase Cbl and adaptors Grb2 and Dok-3 (33), respec-tively. We found that the generation of traction forces required thelinking of BCR microclusters with motor proteins and the pulling ofBCR microclusters on the tracks of the cytoskeleton toward the centralregion of the B cell immunological synapse. In this case, traction forcewas transmitted to the force-calculating beads through bonds betweenthe BCR microclusters and the antigen on the substrate. As expected,the traction force was mainly applied to the force-transmitting site thatis the circular-shaped BCR microcluster, showing as a positive correla-tion between the strength of the traction force and the MFI of the BCRmicroclusters (Fig. 4C). However, it is reported that there is lack of cor-relation betweenF-actin filaments andBCRmicroclusters and that BCRmicroclusters are mainly located at the F-actin–poor region, with F-actinfilaments usually forming a coral-like structure outside of BCR micro-clusters (81, 82). Thus, it is reasonable to observe that the linear corre-lation between F-actin and traction force is poor.

This speculation is also supported by the fact that there are nostudies supporting the high colocalization between traction force andF-actin in conventionalmechanosensing biology studies in other typesof cells (83). As for the loss of correlation between BCR MFI and trac-tion stress (decrease ofR value) in later stages of activation (after 10min;Fig. 4D), we think that it could be ascribed to the retrograding move-ment of BCR microclusters to the center of the B cell immunologicalsynapse (Fig. 4A) because there were BCR microclusters in the centralarea, whereas there was almost no traction stress. The measured displa-cements of fluorescent beads in the cell center and horizontal directionparallel to the contact interface between the cell and the substrate wereespecially experimentally small, whichwould lead to the absence of localtraction forces during the retrograde transport of the BCR to the centerof the B cell. Traction force is mainly located at the peripheral region ofthe contact between the cell and activating substrates according to var-ious studies (28, 84–86). Thus, in our model, the correlation betweenBCRmicroclusters and traction forces would decrease in the later stageof the time coursewhen the BCRmicroclusters translocate to the centralregion through the movement of motor proteins.


On the basis of these findings, we propose a three-step model, man-ifesting the anchoring rivets and traction force–transmitter function ofBCR microclusters, to explain the molecular mechanism for the gener-ation of traction forces during B cell activation (Fig. 8). These three stepsinclude the following: (i) the formation and growth of BCR microclus-ters in the peripheral area of the contact interface between the B celland the antigen-presenting substrate surface, which function as theanchoring rivets and the traction force transmitters (Fig. 8A); (ii) theremodeling of the F-actin structure and the establishment of stable in-teractions between BCRmicroclusters andmotor proteins, which is thestep of preparing the “track” and loading the cargo (BCRmicroclusters)onto the motor proteins for the generation of traction forces (Fig. 8B;magnified region indicates the loading of BCR microclusters onto mo-tor proteins); and (iii) the retrograde movement of BCR microclustersto the center of the B cell immunological synapse on the tracks of F-actin andmicrotubules by relatedmotor proteins.Our data also indicatethat the cytoskeleton- and motor protein–mediated BCR microclustermovement is of fundamental importance for the production andmain-tenance of traction forces that have been experimentally measured intraction force microscopy (Fig. 8C; red arrows in the magnified regionindicate the generation of traction forces during the retrograde move-ment of BCR microclusters to the center of B cell immunological syn-apse through the motor proteins).

Another observation was made when comparing the traction forcesdelivered by IgM- and IgG-BCRs. As we delineated, more tractionforces were generated by IgG-BCR–expressing memory B cells thanwere generated by IgM-BCR–expressing naïve B cells. Similarly, it isfound that, compared to naïve B cells, germinal center B cells applymore persistent and stronger tensile forces on the BCR, which inhibitsantigen binding by using myosin II contractility to achieve more strictaffinity discrimination when extracting antigens from immune synaps-es (87). We found that the evolutionarily conserved cytoplasmic tail ofmIgG was likely responsible for the enhanced generation of tractionforce. Mechanistically, we propose the following hypotheses to explainthe increased traction force generation in IgG-BCR–expressing memoryB cells compared to IgM-BCR–expressing mature naïve B cells. First, theIgG-BCRmicroclusters aremuch larger than the IgM-BCRmicroclustersbecause the cytoplasmic tail of mIgG promotes IgG-BCR microclusterformation, strengthens the initiation of signaling, and consequentlyreinforces the generation of traction force, all of which are supported byour published studies (21, 37). Second, memory B cells have greateramounts of motor proteins compared tomature naïve B cells, and thesemotor proteins promote traction force exertion. A report on T cells alsorevealed the key role of myosin light chain phosphorylation on thedynamics of microtubule and F-actin structures, which are essentialfor the generation of traction force (88). We compared the abundancesof myosin and dyneinmRNAs inmature naïve B cells andmemory Bcells from humans andmice.We found that the murine myosin lightchain 6 mRNA was 1.6-fold more abundant in memory B cells thanin mature naïve B cells. Furthermore, the abundance of myosin lightchain 9 mRNA was 1.4- and 5.3-fold greater in memory B cells thaninmature naïve B cells frommice andhumans, respectively. In addition,themRNAabundances of severalmurine dynein light chains, includingLC-8 (light chain–8 kDa), roadblock, and Tctex (T complex–associatedtestis–expressed 1–like), were 2.2- to 2.8-fold greater in memory B cellsthan in mature naïve B cells. Together, these data suggest that memoryB cells have increased amounts of motor protein mRNAs compared tothose of mature naïve B cells (fig. S6). Thus, the increased production ofthe traction forces mediated by these motor proteins may lower the

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threshold of IgG-BCR activation and further promote antigen acquisi-tion by IgG-BCR–expressing memory B cells during the initiation ofB cell activation. These data enable a better understanding of the rela-tively more potent activation of memory B cells.

It is well known that contractile prestress (that is, cell tractionforce) carried by the actomyosin cytoskeleton can be transmitted to theunderlying substrate and the ECM in most adherent cells through thestructure of focal adhesions (69). Focal adhesions are also regulated bymyosin, which functions as a mechanical output regulator and also amechanosensor, because myosin is linked to focal adhesions through


F-actin (75, 89). Myosin can also regulatethe formation of focal adhesions duringthe mechanotransduction process. On theother hand, myosin participates in theformation of micro-adhesion rings to in-duce the formation ofTCRmicroclusters,which are essential for initial T cell acti-vation through the outside-in signalingof integrins (90). On the basis of thesefindings and our experimental results,we propose that myosinmay also functionas a mechanical output regulator and amechanosensor in B cells. Thus, myosinmay participate in the positive feedbackloop, as described earlier. Various myosinisotypes, especially the light chains, are ac-tivated by phosphorylation to generateand sense cytoskeletal mechanics. Thus,the increased or decreased amounts ofthese myosin isotypes can lead to en-hanced or reduced activation of such apositive feedback loop to further stimu-late or inhibit the activation of differentsubsets of B cells, respectively.

In terms of the potential clinicalrelevance of our findings, we found thatB cells fromRApatients generated greatertraction force during activation than didB cells from healthy individuals, whichmay help, at least in a part, to uncoverthe pathological mechanism of the pro-duction of autoreactive antibodies in RApatients. RA is a chronic autoimmunedis-ease with complex pathological mecha-nisms involving the interplay of multiplecell types and the cross-linking ofmultiplesignaling pathways. B cells play severalcritical roles in the pathogenesis of RA be-cause B cells can both respond to and pro-duce the chemokines and cytokines thatassemble at the sites of inflammation(59, 91). Furthermore, B cells produceRFs, which are autoantibodies specificfor the constant region of self-IgG antibo-dies (59, 91). The dysfunctionalmolecularsignaling pathways involved in B cell acti-vation, the changes in expression profilesof genes important for B cell function andbehavior (59, 60), and the abnormality of

B cell spreading– and force generating–related molecules (such as focaladhesion kinase families) are all responsible for the progression of RA(92). B cells may respond to the changes in the biochemical and bio-physical properties of the cartilage because of the remodeled ECMmicroenvironment during the autoimmune reactions that occurduring RA progression (93). This scenario is similar to the aforemen-tioned positive feedback loop. Autoantigen-reactive B cells are naturallypresent in both healthy individuals and patients with autoimmune dis-ease; however, it is not completely understood why these autoreactiveB cells can remain quiescent in healthy individuals but are activated in

Fig. 8. Proposed molecular mechanism of traction force generation during B cell activation. (A) Formationand 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) ontothe motor proteins. (C) Retrograde movement of BCR microclusters to the center of the B cell immunological syn-apse along the tracks of F-actin and microtubules. Red arrows in the magnified regions indicate the generation oftraction forces during the retrograde movement of BCR microclusters to the center of the B cell immunologicalsynapse by motor proteins.

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autoimmune patients. Our data support the hypothesis that the changedbiophysical and biochemical properties of the cartilage ECM micro-environment in RA patients may potentially induce B cells to generateincreased traction forces. This increase in traction force may be greaterthan the force barrier that determineswhether B cells remain quiescent orbecome activated to commence the production of autoantibodies, suchas RFs. Themechanism of CD8+ T cell–mediated killing of target cells iscoupled to a concept known as “mechanopotentitation,”which predictsthat mechanical force modulates information flow out of the cell by po-tentiating cytotoxicity (62). Consistent with our observations here, theenhanced activation of autoreactive B cells from RA patients is definedby excessive BCR microcluster formation, downstream signaling initia-tion, and further differentiation of autoantibody-producing plasma cells.Therefore, we propose that excessive traction force generation may serveas another index of the dysregulated activation of autoreactive B cells fromRApatients. Excessive traction force generationmay play a role in the en-hanced activation of autoreactive B cells from RA patients. Together, thesefindings suggest a three-stepworkingmodel as themolecularmechanismtodefine the origin, spatiotemporal dynamics, and function of traction forcegeneration duringB cell activation. Furthermore, these studies revealed thatB cell traction force generation is markedly increased in the physiologicalconditionof class-switched IgG-BCR–expressingmemoryBcells and in thepathological condition of primary B cells from RA patients.

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MATERIALS AND METHODSCells and reagentsAll of the chickenDT40 cell lines were provided by T. Kurosaki (RIKEN,Japan). All human primary naïve B cells isolated from the peripheralblood of RA patients and healthy donors, as well as mouse primary naïveB cells, were cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum, 50 mM b-mercaptoethanol (Sigma-Aldrich), andpenicillin/streptomycin antibiotics (Invitrogen). TheWTDT40 cell lineand all of the DT40 KO cell lines used in this study were maintained at37°C in RPMI 1640 medium supplemented with 1% chicken serum.Biotin-conjugated goat anti-chicken IgM antibody was purchased fromRockland Inc. Biotin-conjugated goat anti-human Igk and Igl lightchains were purchased from Southern Biotech. DyLight 649–conjugatedFab anti-mouse IgM constant region antibody and biotin-conjugatedgoat F(ab)2 anti-mouse IgMantibodywere purchased from Jackson Im-munoResearch. The labeling of mouse anti-chicken IgM antibody(clone M1) with Alexa Fluor 647, the labeling of HEL with biotin,and the digestion of the Fab fragment of mouse anti-chicken IgM an-tibody (clone M1) were performed as previously described (46).

ImmunostainingTo evaluate the abundance and distribution of the BCR, DT40 cells, B6mouse cells, or human primary B cells were prestained with an appro-priate anti-BCR antibody conjugated with a specific fluorochrome(100 nM) on ice for 5 min before extensive washing, as outlined earlier,respectively. TheMFIs of BCRswithin the contact interface between theB cell and the antigen-tethered gel were processed and analyzed withImageJ (National Institutes of Health) or MATLAB (MathWorks)software, as described previously (20).

PA gel preparation and surface conjugation offluorescent beadsTomeasure weak traction forces more accurately, we improved the tra-ditional traction forcemicroscopymethod using surface chemicalmod-


ifications to fabricate a PA gel with fluorescent beads on surface, asdescribed previously (94, 95). Briefly, the glass bottoms of 35-mmdisheswere pretreatedwith bind-silane to ensure PAgel attachment.Here, gelswith acrylamide/bis ratios of 3:0.03 (for a stiffness of 0.5 kPa) and 3:0.1(for a stiffness of 1 kPa) were prepared to examine the traction forcegeneration of primary human and mouse B cells or DT40 cells, respec-tively. After polymerization, the PA gel surface was completely coveredby 0.2-mm-diameter fluorescent beads (rhodamine/FITC carboxylate-modified, diluted at 1:400 inwater) for 18min, and then, the beads werecovalently linked to the gel surface with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide, hydrochloride [Invitrogen; 3.8 mg/ml in 2-(N-morpholino)ethanesulfonic acid (MES; pH 5.5; Sigma-Aldrich)]and hydroxy-2,5-dioxopyrrolidine-3-sulfonic acid [Sigma-Aldrich;7.6 mg/ml in MES (pH 5.5)] solution for 2 hours and then in phosphate-buffered saline (pH 7.4) for 2 hours at room temperature, as previouslydescribed (94). The gel substrates were activated with sulfo-SANPAH(Pierce), were subsequently coated overnight at 4°C with neutravidin,and were then incubated with biotin-conjugated goat anti-chickenIgM for DT40 cells, biotin-conjugated goat F(ab)2 anti-mouse IgMfor B6 mouse primary B cells, and biotin-conjugated goat anti-humanIgk and Igl light chains for human primary B cells for 2 hours at 37°C.Before measuring the traction force, the gels were blocked with 1%casein for 30 min at room temperature.

Measurement of traction forcesTo measure the traction force generated by B cells, movies of live cellsand fluorescent beads were acquired every 12 s during B cell spreadingfor up to 30 min in phase contrast for the cells, in the 488 or 561 chan-nels for the beads, and in the 640 channel for the BCRwith an epifluor-escence microscope (Ti-E, Nikon) combined with a spinning-disk laserconfocal scanning microscope (PerkinElmer). For the traction forceexperiments, all of the data were acquired at 40× magnification. Forthe correlation experiments, the data were recorded at 60× and 100×magnifications, together with prestaining of the BCR with Alexa Fluor647–conjugated mouse Fab anti-chicken IgM and staining of F-actinwith Lifeact-mCherry. During image acquisition, the dishes were keptat 37°C by means of a live-cell station. After image acquisition, fluores-cence images of fluorescent beads were acquired as reference imagesafter the B cells were detached from the gel. A series of dynamic fluo-rescence images of the substrates were first recorded by the microscopysystemduring the cell-substrate interactions. Sample drift was correctedfor by tracking the displacement of beads located at the marginal area,which were far away from any cell, and the displacement field of the gelexerted by the B cells was measured by analyzing the positions of thefluorescent beads with DIC before and after cell detachment (26). Onthe basis of the substrate displacement field and Young’smoduli of sub-strates, which had been measured in advance, we then reconstructedcellular traction stress fields (in units of pascal) by the optimal filteringapproach founded on Fourier transform traction cytometry implemen-ted in MATLAB, as previously described (27, 28). In this context, weobtained the total traction force Ftotal (in nanonewton) by means ofthe following expression:

Ftotal ¼ ∬AjTjdA ð1Þ

where T denotes the local cellular traction stress (in pascal), and A isthe area of the cell-substrate interaction. Accordingly, we further

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defined mechanical traction work (in joule) during the cell-substrateinterplay as:

Wm ¼ ∬AðT • dÞdA ð2Þ

where d is the displacement field of the substrate. In experimentswith inhibitors, both DT40 cells and B6 mouse primary B cells werepretreated with inhibitors as follows: 20 mM RGD peptide to blockintegrin at 37°C for 2 hours, 30 mM HPI4 at 37°C overnight, 50 mMBLEB to block dynein at 37°C for 30 min, and 10 mM ML7 to blockmyosin IIA and myosin light chain kinase at room temperature for20 min. As a control, DT40 cells and B6 mouse primary B cells werepretreated with DMSO at 37°C overnight as the control for HPI4, at37°C for 30min as the control for BLEB, and at room temperature for20 min as the control for ML7. In experiments to disrupt the polymer-ization of F-actin, the DT40 cells were treated with 1 mM latrunculin-Bfor 10 min.

Data analysisFor all assays, the values shown in the figures are means ± SEM. Statis-tically significant differences were determined by one-way analysis ofvariance (ANOVA), followed by a t test for multiple comparisons be-tween groups, and two-exponential function regression, together withcurve slopes, was calculated with GraphPad Prism software.

on March

p://stke.sciencemag.org/

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/11/542/eaai9192/DC1Fig. 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-switchedIgG-BCR–expressing memory B cells.Fig. S6. Myosin and dynein mRNA abundances in IgM-BCR–expressing naïve B cells andIgG-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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Acknowledgments: We thank S. K. Pierce (National Institute of Allergy and InfectiousDiseases, NIH), K. Rajewsky (Immune Regulation and Cancer, Max Delbrück Center forMolecular Medicine), M. Shlomchik (University of Pittsburgh), C. Goodnow (Garvan Institute ofMedical Research), and T. Kurosaki and H. Shinohara (World Premier International ImmunologyFrontier Research Center, Osaka University) for providing experimental materials. Funding:This work was supported by funds from the Ministry of Science and Technology of China(2014CB542500 to W.L. and 2013CB933702 to C.X.), the National Natural Science Foundationof China (81422020 and 81621002 to W.L. and 11472013 to C.X.). Author contributions: J.W.and F.L. designed, carried out, and analyzed all experiments and wrote the manuscript.Z.W., Y.L., and Y.Z. commented on the study to assist with its improvement. J.H. calculated thetraction work. X.S., W.Z., and Z.L. provided patient blood samples. F.W. and Y.-H.C. helped withthe revisions of the manuscript. Y.S. provided the Tg mouse cells. W.L. and C.X. supervisedthe study and helped with the writing and editing of the manuscript. Competing interests: Theauthors declare that they have no competing interests. Data and materials availability: Alldata needed to evaluate the conclusions in the paper are present in the paper or theSupplementary Materials.

Submitted 31 August 2016Accepted 20 July 2018Published 7 August 201810.1126/scisignal.aai9192

Citation: J. Wang, F. Lin, Z. Wan, X. Sun, Y. Lu, J. Huang, F. Wang, Y. Zeng, Y.-H. Chen, Y. Shi,W. Zheng, Z. Li, C. Xiong, W. Liu, Profiling the origin, dynamics, and function of traction force inB cell activation. Sci. Signal. 11, eaai9192 (2018).

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

Shi, Wenjie Zheng, Zhanguo Li, Chunyang Xiong and Wanli LiuJunyi Wang, Feng Lin, Zhengpeng Wan, Xiaolin Sun, Yun Lu, Jianyong Huang, Fei Wang, Yingyue Zeng, Ying-Hua Chen, Yan

DOI: 10.1126/scisignal.aai9192 (542), eaai9192.11Sci. Signal.

may play a role in the enhanced activation of autoreactive B cells observed in these patients.cells from patients with rheumatoid arthritis exerted greater traction forces than did B cells from healthy donors, whichthe strength of these forces was correlated with increased BCR microcluster mean fluorescent intensity. In addition, B stiffness values. The authors found that memory B cells generated greater traction forces than did naïve B cells and thatdisplacing forces exerted by B cells on fluorescent beads coated onto antigen-containing gel surfaces of different

. used traction force microscopy to measure theet alspread over antigen-presenting surfaces before contracting, Wang recognition stimulates BCR-dependent intracellular signaling that is required for B cell activation. Noting that B cells first

B cells recognize antigens through membrane-bound antibodies that are part of the B cell receptor (BCR). AntigenB cells use the force

ARTICLE TOOLS http://stke.sciencemag.org/content/11/542/eaai9192

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http://stke.sciencemag.org/content/sigtrans/9/434/ra66.full

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http://stke.sciencemag.org/content/sigtrans/11/532/eaal1506.full

http://science.sciencemag.org/content/sci/358/6360/eaao2602.full

http://stm.sciencemag.org/content/scitransmed/10/428/eaal1571.full

http://immunology.sciencemag.org/content/immunology/2/17/eaan0787.full

http://immunology.sciencemag.org/content/immunology/1/1/aaf7153.full

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