Arp2/3-generated branched F-actin is known to drive lamellipodial expansion for B-cell spreading (Bolger-Munro et al. 2019). However, whether branched F-actin is involved in the subsequent contraction phase is unknown. To address this, we perturbed the polymerization of branched F-actin using the Arp2/3 inhibitor CK-666 (50 µM) while using its inactive derivative CK-689 as a control. Pre-warmed splenic B-cells from WT C57BL/6 mice were incubated with monobiotinylated Fab’ fragment of anti-mouse IgM+G attached to planar lipid bilayers (Fab’-PLB) by biotin-streptavidin interaction and imaged live at 37°C using interference reflection microscopy (IRM). The contact area of B-cells treated with CK-689 rapidly increased upon contacting Fab’-PLB and reached a maximum at ≥0.5 min after the initial contact (Figure 1A, Figure 1-figure supplement 1, and Figure 1-Video 1A). Following maximal spreading, most B-cells reduced the area of their contact zone, indicating contraction (Figure 1A, Figure 1-figure supplement 1, and Figure 1-Video 1A). We classified a B-cell as contracting if its contact zone area reduced by ≥5% for at least 10 sec after reaching a maximum value. Based on the average timing for B-cell maximal spreading, we treated B-cells with CK-666 at the beginning of the incubation (Time 0), before B-cell spreading initiation, or at 2 min, when all B-cells had already spread. The effectiveness of CK-666 was detected by reduced Arp2/3 staining in the contact zone (Figure 1-figure supplement 2). As expected, CK-666 treatment at 0 but not 2 min reduced the kinetics of B-cell spreading (Figure 1-figure supplement 3). Importantly, CK-666 treatment at 0 and 2 min both significantly reduced the percentage of B-cells undergoing contraction (Figure 1A-C and Figure 1-Video 1A). The inhibitory effect of CK-666 on B-cell contraction, particularly CK-666 treatment after B-cell spreading, suggests that Arp2/3-mediated branched actin polymerization is required for B-cell contraction.

WASP and N-WASP are actin nucleation-promoting factors upstream of Arp2/3 that are expressed in B-cells (Padrick and Rosen 2010). To determine if either or both were responsible for activating Arp2/3 for B-cell contraction, we utilized the N-WASP inhibitor wiskostatin (Wisko), and splenic B-cells from B-cell-specific N-WASP knockout mice (cNKO) and germline WASP knockout (WKO) mice (Westerberg et al. 2012; Liu et al. 2013a). Wisko has been shown to inhibit N-WASP activation while enhancing WASP activation in B-cells (Figure 1-figure supplement 4) (Liu et al. 2013a). We found that both Wisko (10 µM) (Figure 1D and E and Figure 1-Video 1B) and cNKO (Figure 1F and G and Figure 1-Video 1C), but not WKO (Figure 1H and I and Figure 1-Video 1D), significantly reduced the percentage of B-cells undergoing contraction, compared to the vehicle, flox, or WT controls. These results suggest that N-WASP- but not WASP-activated Arp2/3 mediates branched actin polymerization for B-cell contraction.

To understand how Arp2/3 drives B-cells to transition from spreading to contraction, we identified F-actin structures associated with contracting B-cells that were sensitive to CK-666 treatment. We visualized F-actin by phalloidin staining and compared F-actin organization in the contact zone of B-cells at the spreading (2 min) and contraction (4 min) phases using TIRF. While B-cells in both spreading and contraction phases exhibited phalloidin staining outlining the contact zone (Fig. 2A, green arrows), only B-cells in the contracting phase showed interior phalloidin patches brighter than the phalloidin staining at the periphery of the contact zone (Figure 2A, purple arrows). These F-actin patches were organized into a ring-like structure and resided ∼1 μm behind the spreading front, surrounding an F-actin-poor center (Figure 2A, purple arrows). Here, we refer these F-actin patches as inner F-actin foci. We identified inner F-actin foci based on whether their peak fluorescence intensity (FI) was ≥2 fold of the mean fluorescence intensity (MFI) of phalloidin in the no-foci area, had diameters of ≥250 nm, and were located 1 µm away from the edge of the contact zone. We found that such inner F-actin foci were detected in >60% of B-cells in the contracting phase (4 min) but only in <20% of B-cells in the spreading phase (2 min) (Figure 2B). CK-666 treatment at 0 min, which inhibited B-cell contraction, significantly reduced the percentage of B-cells showing inner F-actin foci at 4 min but not at 2 min (Figure 2B). CK-666 treatment did not affect the phalloidin staining outlining the contact zone (Figure 2A). Similarly, the percentage of cNKO B-cells showing inner F-actin foci was drastically reduced at 4 min but not at 2 min (Figure 2C). These results suggest that the formation of these inner F-actin foci is associated with B-cell contraction.

We further quantified the number of inner F-actin foci in individual B-cells from mice expressing the LifeAct-GFP transgene (Riedl et al. 2010) (Figure 2D, E, and G), which allowed us to monitor F-actin reorganization using live-cell imaging (Figure 2-Video 1), or using phalloidin staining in flox control and cNKO B-cells (Figure 2F). Consistent with the results with phalloidin staining, CK-666 treatment at time 0 significantly reduced the number of inner F-actin foci in the contact zone (Figure 2D and Figure 1-Video 1A and B). When we followed the same B-cells before and after CK-666 treatment at 2 min, many of the inner F-actin foci formed before the treatment (1 min 50 sec) disappeared after the treatment (2 min 20 sec), significantly reducing the number of inner F-actin foci (Figure 2E). Similar to the CK-666 treatment, cNKO cells significantly reduced the number of inner F-actin foci in the contact zone, compared to flox controls (Figure 2F). In contrast, WKO, which does not affect B-cell contraction, did not significantly change the number of inner F-actin foci, compared to WT control B-cells (Figure 2G and Figure 2-Video 1C and D). Thus, N-WASP- but not WASP-activated Arp2/3 drives the formation and the maintenance of contraction-associated inner F-actin foci.

We next examined the formation of contraction-associated inner F-actin foci utilizing live-cell TIRF imaging of B-cells from mice expressing LifeAct-GFP. We generated kymographs along eight lines from the center of each contact zone using time-lapse images of LifeAct-GFP (Figure 3A and B and Figure 3-figure supplement 1). Analysis of these kymographs showed that most F-actin foci were first detected closely behind lamellipodial F-actin networks. Following maximal spreading, these F-actin foci moved centripetally while increasing in intensity, becoming inner F-actin foci, in cells transitioning from spreading to contraction (Figure 3B, top panel, and Figure 3-figure supplement 1). However, such centripetally moving F-actin foci were not detected in B-cells that did not undergo contraction (Figure 3B, bottom panel). We quantified the percentage of the eight kymographs from each cell that exhibited lamellipodia-derived inner F-actin foci and found, on average, that six out of eight kymographs from contracting cells showed lamellipodia-derived inner F-actin foci, compared to only one or two kymographs from non-contracting cells (Figure 3C). To examine the temporal relationship between the generation of lamellipodia-derived inner F-actin foci and contraction, we plotted the percentage of kymographs with lamellipodia-derived inner F-actin foci over time with the spreading to contracting transition time set as 0 (Figure 3D). We found that the percentage of kymographs showing lamellipodia-derived inner F-actin foci peaked at almost the same time when the spreading transitioned to contraction (Figure 3D), suggesting a close temporal relationship between the two events. Furthermore, the N-WASP inhibitor Wisko, but not WKO, significantly inhibited the formation of lamellipodia-derived inner F-actin foci (Figure 3E). Thus, inner F-actin foci originate from branched actin driven lamellipodia and form simultaneously with the transition of B-cell spreading to contraction.

We examined the mechanism by which N-WASP and Arp2/3 generate inner F-actin foci by measuring their relative lifetime and mobility using kymographs generated from TIRF time-lapse images of B-cells expressing LifeAct-GFP. Inner F-actin foci were identified as described above, and their tracks were manually determined (Figure 4A, black dashed lines). The time window in which an inner F-actin patch could be detected in a kymograph was measured as the relative lifetime, as actin foci could move away from the kymograph line or the TIRF evanescent field (Figure 4A, right panels). The distance each F-actin focus moved during its lifetime was used to calculate its speed (Figure 4A, right panels). Compared to CK-689-treated B-cells, CK-666 treatment at time 0 significantly reduced the relative lifetime and the centripetal speed of inner F-actin foci (Figure 4B-D). After B-cells were treated with CK-666 at 2 min (the average time for B-cells to reach the maximal spreading), the relative lifetime and the centripetal speed of inner F-actin foci were significantly lower than before the treatment in the same cell (Figure 4E-G, line-linked dots). Similarly, the N-WASP inhibitor Wisko significantly reduced the relative lifetime and the centripetal speed of inner F-actin foci (Figure 4H-J); however, WKO had no significant effect (Figure 4K-M). These results show that N-WASP and Arp2/3 mediated branched actin polymerization prolongs the lifetime of lamellipodia-derived F-actin foci and drives them to move inward in the B-cell contact zone.

As non-muscle myosin II (NMII) is required for B-cell contraction (Seeley-Fallen et al. 2022), we examined the relationship between the formation of inner F-actin foci and the recruitment and reorganization of NMII in the contact zone using TIRF imaging of B-cells from mice expressing a GFP-NMIIA transgene. Upon interacting with Fab’-PLB, the GFP-NMIIA MFI in the contact zone of untreated WT B-cells increased rapidly in the first minute and slowly afterward (Figure 5A and B and Figure 5-Video 1). Wiskostatin treatment significantly reduced the GFP-NMIIA MFI in the contact zone (Figure 5B) and its initial rate of increase (as determined by the slope of the GFP-NMIIA MFI increase at 0∼30 sec time window) (Figure 5C). Kymographs generated from time-lapse TIRF images of B-cells from mice expressing GFP-NMIIA and LifeAct-RFP showed that recruited NMIIA accumulated between lamellipodia and inner F-actin foci when the foci moved centripetally away from lamellipodia (Figure 5D, white arrow, and Figure 5-Video 1). Recruited NMIIA reorganized with inner F-actin foci to form a ring-like structure in the contact zone (Figure 5A, D, and E, and Figure 5-Video 1). The percentage of B-cells with NMIIA ring-like structures, visualized by immunostaining, increased over time as more B-cells underwent contraction (Figure 5E and F). Compared to flox controls, the percentage of cNKO B-cells with NMIIA ring-like structures was significantly decreased (Figure 5E and F). Wiskostatin treatment also reduced NMIIA recruitment and ring-like structure formation (Figure 5-Video 1). Surprisingly, the percentage of WKO B-cells with NMIIA ring-like structure was higher than that of flox control B-cells (Figure 5E and F). Thus, N-WASP and Arp2/3 mediated branched actin polymerization promotes the recruitment and the reorganization of NMII ring-like structures by generating inner F-actin foci in the contact zone.

To understand how B-cell contraction promotes BCR signaling attenuation, we examined the impact of B-cell contraction on the properties of BCR clusters. We first measured the MFI of AF546-Fab’ attached to PLB gathered by B-cells into the contact zone as an indication of the overall BCR molecular density. The clustering of AF546-Fab’ on PLB by B-cell binding reflects surface BCR clustering, as B-cell binding to transferrin (Tf)-tethered PLB does not cause surface BCRs to cluster and be phosphorylated (Figure 6-figure supplement 1) (Liu et al. 2011; Liu et al. 2013a). The MFI of AF546-Fab’ in the B-cell contact zone increased over time.

Treatment with CK-666, Wisko, or cNKO all reduced the AF546-Fab’ MFI, particularly during the time window of B-cell contraction in controls (Figure 6A-G, purple rectangles, and Figure 6-Video 1). Notably, the rates of increase in AF546-Fab’ MFI, calculated from the slopes of AF546-Fab’ MFI versus time plots in individual cells, were significantly higher during B-cell contraction than before B-cell contraction in control cells and conditions (Figure 6D-K). Significantly, inhibiting B-cell contraction by CK-666 treatment at 0 (Figure 6A, D, and H, and Figure 6-Video 1A and D) or 2 min (Figure 6A, E, and I), Wisko (Figure 6B, F, and J, and Figure 6-Video 1B and E), and cNKO (Figure 6C, G, and K, and Figure 6-Video 1C and F) abolished the increases in AF546-Fab’ accumulation rates. We further examined the peak FI of AF546-Fab’ in individual microclusters as a measure of the BCR molecular density in individual clusters. AF546-Fab’ clusters were identified based on their diameters ≥250 nm, peak FI ≥1.1 fold outside the B-cell contact zone, and trackable for ≥20 sec (Figure 6L and Figure 6-figure supplement 2). AF546-Fab’ microclusters could not be identified during the early stage of B-cell spreading. Time-lapse imaging by TIRF enabled us to measure the rate of increase in AF546-Fab’ peak FI in individual clusters (Figure 6M and Figure 6-figure supplement 2). Consistent with our observation of AF546-Fab’ MFI increase in the contact zone, the peak FI of individual clusters increased at a faster rate during contraction than after contraction (when the contact area no longer decreased) (Figure 6N). Furthermore, CK-666 (Figure 6O), Wisko (Figure 6P), and cNKO (Figure 6Q) all significantly reduced the rate of increase in AF546-Fab’ peak FI in individual clusters. These results show that B-cell contraction significantly increases the molecular density of BCRs in BCR clusters.

To examine how BCR molecular density influenced BCR signaling capability, we immunostained B-cells interacting with AF546-Fab’-PLB for 1, 3, 5, and 7 min, for phosphorylated CD79a (pCD79a, Y182) and performed IRM and TIRF imaging (Figure 7A and H). We analyzed equal numbers of AF546-Fab’ clusters selected randomly, in the contact zone of B-cells interacting with Fab’-PLB for 1, 3, 5, and 7 min, using a gradient threshold of their AF546-Fab’ MFI, 1.1 to 4.1 fold of the background, and with diameter ≥250 nm (Figure 6-figure supplement 2). We determined the MFI of pCD79a and AF546-Fab’ in individual clusters and plotted the MFI ratio of pCD79a relative to AF546-Fab’ (reflecting the relative level of BCR phosphorylation) versus the AF546-Fab’ peak FI (reflecting BCR molecular density) in individual clusters (Figure 7B and C). The dot plots show that when the AF546-Fab’ peak FI of individual clusters was relatively low, the MFI ratios of pCD79a to AF546-Fab’ increased with the AF546-Fab’ peak FI in individual clusters of flox control B-cells (Figure 7B). When the AF546-Fab’ peak FI reached a certain level, the MFI ratios of pCD79a to AF546-Fab’ decreased as the AF546-Fab’ peak FI in individual clusters further increased (Figure 7A and B). Inhibition of B-cell contraction by cNKO reduced the AF546-Fab’ peak FI in individual clusters, maintaining it within a relatively low range, where the MFI ratios of pCD79a to AF546-Fab’ increased with the AF546-Fab’ peak FI (Figure 7C and D). Additionally, the average pCD79a to Fab’ MFI ratios were much higher in cNKO B-cells than flox control B-cells, when comparing AF546-Fab’ clusters with the same range of peak FI (Figure 7B-D). During the contraction stage (5 and 7 min), flox control B-cells exhibited increased AF546-Fab’ MFI (Figure 7E) but decreased pCD79a MFI (Figure 7F) and the MFI ratios of pCD79a relative to AF54-Fab’ (Figure 7G) in individual clusters, compared to the spreading stage (1 and 3 min). In contrast, non-contracting cNKO B-cells only slightly increased AF546-Fab’ MFI (Figure 7E) but significantly increased pCD79a MFI (Figure 7F) and the pCD79a to AF546-Fab’ MFI ratios (Figure 7G) in individual clusters at the contraction stage compared to the spreading stage. Consequently, individual clusters in non-contracting cNKO B-cells had significantly lower AF546-Fab’ MFI but significantly higher pCD79a MFI and pCD79a to AF546-Fab’ FIRs than contracting flox control B-cells at 5 and 7 min but not at 1 and 3 min (Figure 7E-G). Similarly, inhibiting B-cell contraction by treatment with the Arp2/3 inhibitor CK-666 at 2 min post-stimulation reduced AF546-Fab’ MFI (Figure 7I) but increased pCD79a MFI (Figure 7J) and the MFI ratios of pCD79a relative to AF546-Fab’ (Figure 7K) in individual clusters. These data suggest that increases in BCR molecular density in BCR clusters during B-cell contraction inhibit BCR phosphorylation.

Increased BCR molecular density may promote signaling attenuation by inducing the disassociation and/or dephosphorylation of stimulatory kinases from and at BCR clusters. To test this hypothesis, we analyzed the relative amounts of Syk, a major stimulatory kinase in the BCR signaling pathway, and its phosphorylated form pSyk (Y519/520) in individual BCR clusters in relation to the molecular density of BCRs. Splenic B-cells were incubated with Fab’-PLB for 3 and 7 min (when most cells were at the spreading and contraction phase, respectively), fixed, permeabilized, stained for Syk or pSyk, and imaged using TIRF. We measured MFI ratios of Syk relative to AF546-Fab’ in individual BCR clusters to reflect the relative amount of Syk associated with individual BCR clusters and analyzed their relationship with AF546-Fab’ peak FI (reflecting the molecular density within BCR clusters). AF546-Fab’ clusters were detected and analyzed as described above. In flox control B-cells, the highest fractions of BCR clusters had an AF546-Fab’ peak FI at the150∼200 range (Figure 8A, brown line and symbols). The average MFI ratios of Syk to Fab’ in individual clusters increased at a low peak FI range (<140) and did not significantly decrease until Fab’ peak FI reached a relatively high range (>280) (Figure 8A, black line and symbol). B-cells from cNKO mice exhibited a reduction in the Fab’ peak FI of clusters (Figure 8B and C, brown line and symbol) but an increase in the average Syk to Fab’ MFI ratios in clusters in a wide range of Fab’ peak FI, when compared to flox control B-cells (Figure 8B and C, black lines and symbols). However, the average Syk to Fab’ MFI ratios of cNKO B-cells decreased in clusters with Fab’ peak FI ≥220 and reduced to levels similar to those in flox control B-cells in clusters with Fab’ peak FI ≥240 (Figure 8B and C, black lines and symbols). These data suggest that increases in the molecular density of BCR clusters, reflected by Fab’ peak FI, induce disassociation of Syk from BCR clusters in both flox control and cNKO B-cells. Our results also show that BCR clusters in cNKO B-cells have significantly higher levels of Syk association than in flox control B-cells, even though they have similar Fab’ peak intensities.

We next analyzed the relationship of the pSyk level in individual clusters with BCR molecular density using the method described above. We found that the MFI ratios of pSyk relative to AF546-Fab’ gradually decreased with increases in Fab’ peak FI in both flox control and cNKO B-cells (Figure 8D and E, black lines and symbols), even though we did not observe an increase in pSyk to Fab’ MFI ratio at the low Fab’ peak FI range. Similar to the Syk to Fab’ MFI ratio, the average pSyk to Fab’ MFI ratios of individual clusters in cNKO B-cells were much higher than those in flox control B-cells, except for those at the high Fab’ peak FI range (Figure 8D-F). To confirm this result, we analyzed equal numbers of pSyk clusters in the same cells, based on the criteria of ≥1.3 fold increase in the peak FI compared to the background outside the contact zone with a diameter of ≥250 nm. Similar to clusters identified by AF546-Fab’, the average MFI ratios of pSyk to Fab’ in these pSyk clusters decreased with increase in their Fab’ peak FI in both flox control and cNKO B-cells (Figure 8G-I, black lines and symbols). Again, the average pSyk to Fab’ MFI ratios of pSyk clusters were much higher in cNKO than flox control B-cells, but were reduced to similar levels in clusters with relatively high Fab’ peak FI (Figure 8I, black line and symbols). Similar reductions of total Syk and pSyk with increasing molecular density of BCR clusters suggest that the disassociation of Syk from BCR clusters, caused by cell contraction-induced increases in molecular density, contributes to BCR signaling attenuation.

The inhibitory phosphatase SHIP-1 is essential for B-cell signaling attenuation (Brauweiler et al. 2000; Liu et al. 2011), suggesting that increases in the molecular density of BCR clusters by B-cell contraction may promote SHIP-1 recruitment. We used the methods described above to address this hypothesis, staining cells for total SHIP-1 and phosphorylated SHIP-1 (pSHIP-1 Y1020). We found that the average SHIP-1 to Fab’ MFI ratios in both flox control and cNKO B-cells decreased with Fab’ peak FI at similar rates (Figure 9A-C, black lines and symbols), even though inhibition of contraction by cNKO reduced the Fab’ peak FI of BCR clusters (Figure 9A-C, brown lines and symbols). Notably, the reduction in the SHIP-1 to Fab’ MFI ratios with increasing Fab’ peak FI occurred at the lowest detectable Fab’ peak PI (Figure 9A-C, brown lines and symbols), when the Syk to Fab’ MFI ratios increased and were sustained (Figure 8A-C). Furthermore, this reciprocal relationship between the SHIP-1 to Fab’ MFI ratio and Fab’ peak FI continued over the entire Fab’ peak FI range. It also remained the same in both flox control and cNKO B-cells (Figure 9A-C). These results suggest that SHIP-1 disassociates from BCR clusters as their molecular density increases, and that the SHIP-1 disassociation is more sensitive to the molecular density of BCR clusters than Syk disassociation.

Similar to the relationship of total SHIP-1 with BCR molecular density, the average MFI ratios of pSHIP-1 relative to AF546-Fab’ in individual Fab’ clusters decreased with increases in Fab’ peak FI at similar rates in flox control and cNKO B-cells (Figure 9D-F, black lines and symbols), even though inhibition of contraction by cNKO reduced Fab’ peak FI of BCR clusters (Figure 9D-F, brown lines and symbols). The average pSHIP-1 to Fab’ MFI ratios in individual pSHIP-1 clusters, detected and analyzed in the same way as pSyk clusters, showed the same decrease with increases in their Fab’ peak FI in both flox control and cNKO B-cells (Figure 9G-I, black lines and symbols). Notably, the average pSHIP-1 to Fab’ MFI ratios in individual Fab’ or pSHIP-1 clusters in flox control and cNKO B-cells were at similar levels at the same Fab’ peak FI ranges (Figure 9F and I, black lines and symbols). These results indicate that contraction-induced molecular density increases within individual BCR clusters do not induce preferential recruitment of SHIP-1; instead, it promotes the disassociation of SHIP-1 from BCR clusters.

Wild-type (WT) C57BL/6 mice and WASP knockout (WKO) mice on the C57BL/6 background were purchased from Jackson Laboratories. C56BL/6 mice expressing LifeAct-GFP transgene (Riedl et al. 2010) were kindly provided by Dr. Roberto Weigert at National Cancer Institute, USA. C56BL/6 mice expressing the LifeAct-RFP transgene were kindly provided by Dr. Klaus Ley at La Jolla Institute for Allergy & Immunology. C57BL/6 mice expressing the GFP-non-muscle myosin IIA (NMIIA) transgene were kindly provided by Dr. Robert Adelstein at the National Heart, Lung, and Blood Institute, USA. WKO mice were bred with LifeAct-GFP mice to obtain LifeAct-GFP-expressing WKO mice. GFP-NMIIA mice were crossed with LifeAct-RFP mice to generate mice expressing both transgenes. N-WASP^flox/flox on a 129Sv background (Cotta-de-Almeida et al. 2007) were kindly provided by Dr. Lisa Westerberg at Karolinska Institute, Sweden. B-cell-specific N-WASP knockout mice (cNKO, CD19^Cre/+N-WASP^flox/flox) and floxed littermate controls (N-WASP^flox/flox) were obtained by breeding N-WASP^flox/flox mice with CD19^Cre/+ mice on a C57BL/6 background.

Primary B-cells were isolated from the spleens of 6 to 18 week-old male or female mice, using a previously published protocol (Sharma et al. 2009). Briefly, mononuclear cells were isolated by Ficoll density-gradient centrifugation (Millipore Sigma), and T-cells were eliminated by complement-mediated cytolysis with anti-mouse CD90.2 mAb (BD Biosciences) and guinea pig complement (Innovative Research Inc). Monocytes and dendritic cells were eliminated by panning at 37°C and 5% CO₂. Isolated B-cells were kept on ice in DMEM (Lonza) supplemented with 0.6% BSA (Thermo Fisher Scientific). All work involving mice was approved by the Institutional Animal Care and Usage Committee of the University of Maryland.

Planar lipid bilayers (PLB) were prepared using a previously described method (Dustin et al. 2007; Liu et al. 2012a). Briefly, liposomes were generated from a mixture of 5 mM (total concentration) 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-cap-biotin (Avanti Polar Lipids) at a 100:1 molar ratio by sonication. Glass coverslips, cleaned overnight with Piranha solution (KMG chemicals), were attached to 8-well chambers (Lab-Tek) and incubated with liposomes (0.05 mM) in PBS for 20 min at room temperature and washed with PBS. The chambers were then incubated with 1 µg/ml streptavidin (Jackson ImmunoResearch Laboratories) for 10 min, washed with PBS, and then incubated with 10 µg/ml mono-biotinylated Fab’ fragment of goat anti-mouse Ig(G+M) (pseudo-antigen) (Fab’-PLB) for 10 min, followed by PBS wash. For transferrin-coated PLB (Tf-PLB), 16 16 µg/ml biotinylated holoTF was used. To visualize Fab’ clustering, a mixture of 0.5 µg/ml Alexa Fluor (AF) 546-labeled and 9.5 µg/ml unlabeled mono-biotinylated Fab’ fragment of goat anti-mouse Ig(G+M) was used. The lateral mobility of AF546-Fab’ on the PLB was tested using fluorescence recovery after photobleaching (FRAP) to ensure ≥85% AF546 fluorescence recovery within 1 min after photobleaching using a Zeiss LSM 710 equipped with a 60X oil-immersion objective.

Mono-biotinylated Fab’ fragments were generated as previously described (Liu et al. 2011). Briefly, the disulfide bond linking the two Fab fragments of F(ab’)₂ goat anti-mouse Ig(G+M) was reduced using 2-mercaptoethylamine HCL (Thermo Fisher Scientific) and biotinylated by using maleimide-PEG₂-biotin (Thermo Fisher Scientific). Mono-biotinylated Fab’ fragments of goat anti-mouse Ig(G+M) were labeled with AF546 using an Alexa Fluor 546 antibody labeling kit (Thermo Fisher Scientific). The molar ratio of AF546 to Fab’ in the AF546-labeled Fab’ was ∼2 determined by a Nanodrop spectrophotometer (Nanodrop Technologies).

To visualize molecules proximal to interacting sites between B-cells and Fab’- or Tf-PLB, we utilized total internal reflection fluorescence microscopy (TIRF) and interference reflection microscopy (IRM). Images were acquired using a Nikon TIRF system on an inverted microscope (Nikon TE2000-PFS, Nikon Instruments Inc.) equipped with a 60X, NA 1.49 Apochromat TIRF objective (Nikon), a Coolsnap HQ2 CCD camera (Roper Scientific), and two solid-state lasers of wavelength 491 and 561 nm. IRM, AF488, and AF546 images were acquired sequentially.

The plasma membrane area of B-cells contacting PLB was determined using IRM images and custom MATLAB codes. Whether a B-cell contracted or not was determined using the area versus time plots, wherein if a B-cell’s contact zone area reduced by ≥5% for at least 10 sec after reaching a maximum value, it was classified as contracting. To image intracellular molecules, B-cells were incubated with Fab’-PLBs for varying lengths of time in PBS at 37°C, fixed with 4% paraformaldehyde, permeabilized with 0.05% saponin, and stained for various molecules. For live-cell imaging, B-cells were pre-warmed to 37°C and imaged as soon as B-cells were dropped into coverslip chambers coated with Fab’-PLB containing PBS in a humidity chamber at 37°C, at 2 sec per frame, up to 7 min. All images from multiple independent experiments were analyzed using NIH ImageJ and custom MatLab codes. Acquired fluorescence intensity (FI) data were normalized to the one with the lowest FI.

CK-666 (50 µM, Millipore Sigma) was used to perturb Arp2/3 activity (Nolen et al. 2009), and its non-functional derivative CK-689 (50 µM, Millipore Sigma) as a control. CK-666 or CK-689 was added at either 0 min, the start of incubation with Fab’-PLB, or at 2 min when most B-cells reached maximum spreading. Notably, B-cells take approximately 1 to 1.5 min to land on PLB. The time when B-cells were added to Fab’-PLB is referred to as 0 min, and the time when B-cells landed on PLB as the start of spreading. The effectiveness of CK-666 was determined by its inhibitory effects on the recruitment of Arp2/3, stained by an anti-Arp2 antibody (Abcam), to the B-cell contact zone using TIRF (Figure 1-figure supplement 2). Wiskostatin (Wisko, 10 µM, Millipore Sigma) was used to perturb N-WASP activity in B-cells (Peterson et al. 2004), with DMSO used as a vehicle control. Splenic B-cells were pretreated with Wisko for 10 min at 37°C before and during incubation with Fab’-PLB. The effectiveness of Wisko was determined by its inhibitory effects on the level of phosphorylated N-WASP in the B-cell contact zone using immunostaining and TIRF (Figure 1-figure supplement 4A and C). Possible effects of Wisko on WASP activation were evaluated by measuring the mean fluorescence intensity (MFI) of phosphorylated WASP in the contact zone of B-cells treated with or without Wisko for 10 min (Figure 1-figure supplement 4B and D).

F-actin was visualized by phalloidin staining in fixed cells and by LifeAct-GFP or LifeAct-RFP expressed by primary B-cells from transgenic mice in live cells. Inner F-actin foci, visualized by both phalloidin staining and LifeAct-GFP, were identified using NIH ImageJ based on the following three criteria: 1) ≥250 nm in diameter, 2) peak fluorescence intensity (FI) ≥2 fold higher than the FI of a nearby area containing no foci, and 3) ≥1 µm away from the outer edge of the B-cell contact zone. The horizontal or vertical length of a focus was measured, and the lesser of the two values was used as its diameter. To analyze the spatiotemporal relationship between inner F-actin foci and the lamellipodial F-actin network, we generated 8 radially and equally spaced kymographs from each cell using time-lapse images and MATLAB. Each kymograph was either classified as contracting or not contracting based on the movement of the leading edge of the B-cell contact zone. The percentage of 8 kymographs from each cell exhibiting inner F-actin foci that could be traced back to lamellipodial F-actin when the B-cell switched from spreading to contraction was determined.

The relative speeds and lifetimes of F-actin foci were determined using three randomly positioned kymographs from each B-cell and F-actin focuses emerging during a 60-sec window right after B-cell maximal spreading. Only those remaining visible in individual kymographs for at least 4 sec were analyzed. The time duration of each F-actin foci detected in a kymograph was used to determine the relative lifetime. The distances individual F-actin foci moved during their lifetimes were used to determine the relative speed. The term ‘relative’ has been used because the disappearance of an F-actin focus could be due to its movement away from the region used for the kymograph or vertically from the TIRF evanescent field of excitation.

To determine the rate of myosin recruitment, GFP-NMIIA MFI in the B-cell contact zone was plotted over time, and the slope for the initial segment of the MFI versus the time plot was determined using linear regression. The percentage of B-cells with the NMIIA ring was determined by visual inspection.

BCR clusters were identified by clustering of AF546-Fab’ on PLBs using custom code by MATLAB. We utilized the median FI of the Fab’-PLB within the same area outside but near the B-cell contact zone as the background. The median, but not mean FI value was used for background FI calculations to minimize fluctuations due to debris. To ensure that individual clusters with varying FI were detected as distinct objects, we used 16 graded thresholds from 1.1 to 4.1 fold of the background (0.2 fold apart) to acquire 16 sets of binary masks for each frame of time-lapse images from each cell. When clusters were detected by multiple thresholds at the same location, only the one identified by the highest threshold was retained. Objects that were smaller than 250 nm in diameter or could not be tracked for at least 20 sec were eliminated. This allowed us to detect the position of BCR-Fab’ clusters and track them over time until they merged with other clusters. The horizontal or vertical length of a focus was measured, and the lesser of the two values was used as its diameter. We chose the peak FI as a metric for the extent of Fab’ clustering by the BCR, because it does not rely on the area occupied by each cluster. The rate of peak FI increase was determined by linear regression of peak FI versus time curves for each cluster for a given window of time.

Splenic B-cells were incubated with AF546-Fab’-PLB at 37°C and fixed at 1, 3, 5, 7, and 9 min. After fixation, cells were stained for phosphorylated CD79a (pCD79a, Y182) (Cell Signaling Technology), Syk (pSyk, Y519/520) (Cell Signaling Technology), SHIP-1 (pSHIP-1, Y1020) (Cell Signaling Technology), or total Syk or SHIP-1 proteins (Thermo Fisher Scientific). We identified BCR-Fab’ clusters as described. We determined the peak FI of AF546-Fab’ and the MFI of AF546-Fab’, pCD79a, pSyk, pSHIP-1, Syk, or SHIP-1 within each cluster. The ratio of pCD79a MFI to AF546-Fab’ MFI in the same cluster was used to estimate the relative phosphorylation level of BCRs in that cluster. This MFI ratio was plotted against AF546-Fab’ peak FI to analyze the relationship between BCR phosphorylation and BCR density of individual clusters. We calculated the MFI ratio of Syk to Fab’ or SHIP-1 to Fab’ in individual clusters to estimate the relative recruitment level of Syk or SHIP-1 to BCR clusters. We plotted these MFI ratios against Fab’ peak FI to determine the relationship between Syk and SHIP-1 recruitment levels and BCR density in individual clusters. We calculated the MFI ratio of pSyk to Fab’ or pSHIP-1 to Fab’ to estimate the amount of pSyk or pSHIP-1 relative to BCR in individual clusters. We plotted these MFI ratios against Fab’ peak FI to determine the relationship between Syk or SHIP-1 phosphorylation and BCR density in individual clusters. We identified pSyk or pSHIP-1 puncta using the criteria: ≥1.3 fold of the background staining outside the B-cell contact zone and ≥250 nm in diameter. We calculated the MFI ratio of pSyk to Fab’ or pSHIP-1 to Fab’ in individual puncta to estimate the amount of pSyk or pSHIP-1 relative to BCR in individual puncta. We plotted these MFI ratios against Fab’ peak FI to determine the relationship between Syk or SHIP-1 phosphorylation and BCR density in individual puncta.

Statistical analysis was performed using the Mann-Whitney U non-parametric test for unpaired groups having different sample sizes or the Student’s t-test for paired groups with the same sample size. To compare curves, Kolmogorov-Smirnov test was used. Statistical analyses were performed in Microsoft Excel, GraphPad Prism, and MATLAB. All data are presented as mean ± SEM (standard error of the mean). When testing multiple hypotheses, p--values acquired using t-tests were corrected using the Benjamini-Hochberg/Yekutieli method for false discovery rate control.

All MATLAB scripts used for this study are available as supplemental materials.