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RemodelingCluster Formation by Controlling ActinSHIP Activation Regulates B Cell Receptor A Balance of Bruton's Tyrosine Kinase and

SongGrooman, Silvia Bolland, Arpita Upadhyaya and Wenxia Chaohong Liu, Heather Miller, King Lam Hui, Brian

http://www.jimmunol.org/content/187/1/230doi: 10.4049/jimmunol.11001572011;

2011; 187:230-239; Prepublished online 27 MayJ Immunol 

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7.DC1http://www.jimmunol.org/content/suppl/2011/05/27/jimmunol.110015

Referenceshttp://www.jimmunol.org/content/187/1/230.full#ref-list-1

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The Journal of Immunology

A Balance of Bruton’s Tyrosine Kinase and SHIP ActivationRegulates B Cell Receptor Cluster Formation by ControllingActin Remodeling

Chaohong Liu,* Heather Miller,* King Lam Hui,† Brian Grooman,† Silvia Bolland,‡

Arpita Upadhyaya,† and Wenxia Song*

The activation of the BCR, which initiates B cell activation, is triggered by Ag-induced self-aggregation and clustering of receptors

at the cell surface. Although Ag-induced actin reorganization is known to be involved in BCR clustering in response to membrane-

associated Ag, the underlying mechanism that links actin reorganization to BCR activation remains unknown. In this study, we

show that both the stimulatory Bruton’s tyrosine kinase (Btk) and the inhibitory SHIP-1 are required for efficient BCR self-

aggregation. In Btk-deficient B cells, the magnitude of BCR aggregation into clusters and B cell spreading in response to an Ag-

tethered lipid bilayer is drastically reduced, compared with BCR aggregation observed in wild-type B cells. In SHIP-12/2 B cells,

although surface BCRs aggregate into microclusters, the centripetal movement and growth of BCR clusters are inhibited, and

B cell spreading is increased. The persistent BCR microclusters in SHIP-12/2 B cells exhibit higher levels of signaling than merged

BCR clusters. In contrast to the inhibition of actin remodeling in Btk-deficient B cells, actin polymerization, F-actin accumulation,

and Wiskott–Aldrich symptom protein phosphorylation are enhanced in SHIP-12/2 B cells in a Btk-dependent manner. Thus,

a balance between positive and negative signaling regulates the spatiotemporal organization of the BCR at the cell surface by

controlling actin remodeling, which potentially regulates the signal transduction of the BCR. This study suggests a novel feedback

loop between BCR signaling and the actin cytoskeleton. The Journal of Immunology, 2011, 187: 230–239.

The BCR induces signaling cascades and Ag processingand presentation in response to Ag binding. These BCR-induced cellular activities combine with signals from the

microenvironment to determine the fate of B cells. Biochemicaland genetic studies in the past two decades (1–3) have shown thatupon cross-linking by Ag, surface BCRs aggregate and associatewith lipid rafts (4), where they are phosphorylated by Src kinases,such as Lyn. The binding of tyrosine kinase Syk to phosphorylatedITAMs in the cytoplasmic tails of the BCR activates Syk, which inturn activates downstream signaling components including phos-pholipase Cg2 (PLCg2), Ras, PI3Ks, and Bruton’s tyrosine kinase(Btk). Ag binding to the BCR also activates negative signalingcomponents, in particular, SHIP-1 (5–7). SHIP-1 converts phos-phatidylinositol-3,4,5-triphosphate into phosphatidylinositol-3,4-biphosphate, eliminating the docking sites of PLCg2, Btk, and

Akt at the plasma membrane and turning down BCR signaling(7, 8).Recent studies using advanced cell imaging technologies have

begun to reveal the molecular details of the initiation events in BCR

activation (9–11). Ag binding induces conformational changes of

the BCR, which potentially expose the Cm4 domain of membrane

IgM for BCR self-aggregation (12) and ITAMs for signaling

molecules to bind (13). Self-aggregation reduces the lateral mo-

bility of the BCR and induces the formation of BCR microclusters

(12). Newly formed BCR microclusters reside in lipid rafts (14)

and recruit signaling molecules, including Lyn, Syk (13), PLCg2,

Vav (15), and the costimulatory receptor CD19 (16). BCR

microclusters grow in size by trapping more BCRs and merging

into each other. This leads to the formation of a polarized central

cluster, similar to the immunological synapse formed between

T cells and APCs (17). Therefore, the control of BCR mobility and

self-aggregation is essential for signal initiation and transduction.The surface mobility and aggregation of the BCR has been

shown to require Ag-induced actin reorganization. The actin cy-toskeleton is known to control cell morphology (18, 19) and lateraldiffusion of transmembrane proteins (19). Recent studies haveshown that membrane-associated Ags induce B cell spreading,which is followed by cell contraction. These morphologicalchanges of B cells enhance the formation of BCR clusters. Dis-rupting the actin cytoskeleton inhibits this enhanced BCR clusterformation (20). However, in the absence of Ag, actin disruptionincreases the lateral diffusion rate of surface BCRs and inducesspontaneous signaling in B cells (21). These findings suggest thatAg-induced actin remodeling can regulate BCR self-aggregationby controlling B cell morphology and BCR lateral mobility at thecell surface.Ag-induced actin reorganization, BCR microcluster formation

and B cell spreading all are signaling-dependent processes.

*Department of Cell Biology and Molecular Genetics, University of Maryland, Col-lege Park, MD 20742; †Department of Physics, University of Maryland, CollegePark, MD 20742; and ‡Laboratory of Immunogenetics, National Institute of Allergyand Infectious Diseases, National Institutes of Health, Rockville, MD 20852

Received for publication January 18, 2011. Accepted for publication April 29, 2011.

This work was supported by Grant AI059617 (to W.S.) and Intramural ResearchProgram (to S.B.) of the National Institute of Allergy and Infectious Diseases, Na-tional Institutes of Health.

Address correspondence and reprint requests to Dr. Wenxia Song, 1133A Microbi-ology Building, Department of Cell Biology and Molecular Genetics, University ofMaryland, College Park, MD 20742. E-mail address: wenxsong@umd.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: AF, Alexa Fluor; Btk, Bruton’s tyrosine kinase; FI,fluorescence intensity; IRM, interference reflection microscopy; LOWESS, locallyweighted scatterplot smoothing; MFI, mean fluorescence intensity; NS-Ag, nonspe-cific Ag; pAkt, phosphorylated Akt; pBtk, phosphorylated Btk; PLCg2, phospholi-pase Cg2; pWASP, phosphorylated Wiskott–Aldrich symptom protein; pY,phosphotyrosine; Tf, transferrin; TFI, total fluorescence intensity; TIRFm, total in-ternal reflection microscopy; wt, wild-type.

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Multiple BCR signaling molecules, including CD19, PLCg2, Vav,and Rac2, promote BCR cluster formation and B cell spreading(15, 16, 22). In contrast, coengagement of the BCR and FcgRIIB,which activates SHIP-1, inhibits the formation of BCR clustersand BCR signaling (23, 24). We have previously shown that Btkcan relay BCR signaling to the actin cytoskeleton by activating anactin nucleation promoting factor, Wiskott–Aldrich symptomprotein (WASP), via Vav and phosphatidylinositol (25). Thesefindings point to an intimate cooperativity between BCR signalingand the actin cytoskeleton. However, the underlying mechanismfor the signaling–actin interplay during BCR activation remainsunknown.In this study, we used live cell imaging, total internal reflec-

tion fluorescence microscopy (TIRFm), interference reflectionmicroscopy (IRM), and genetically altered mice to examine themolecular mechanism by which the actin cytoskeleton cooperateswith early BCR signaling at the cell surface during BCR activation.Our results show that the positive and negative downstream sig-naling molecules of the BCR, Btk and SHIP-1, have distinct rolesin self-aggregation and cluster formation of surface BCRs, B cellmorphology, and actin reorganization. The activation of Btk pro-motes B cell spreading, BCR microcluster formation, and actinpolymerization and accumulation. In contrast, SHIP-1 promotesthe merger of BCRmicroclusters and B cell contraction but inhibitsactin polymerization and accumulation. Our results suggest a bal-ance of Btk and SHIP-1 activation controls the nature of actinreorganization, which in turn regulates the spatiotemporal orga-nization of surface BCRs.

Materials and MethodsMice and cells

Wild-type (wt) (CBA/CaJ), xid (CBA/CaHNBtkxid/J), and CD19Cre/+

(B6.129P2(C)-Cd19tm1(cre)Cgn/J) mice were purchased from The JacksonLaboratory (Bar Harbor, ME). B cell-specific SHIP-1–knockout miceCD19Cre/+SHIP-1Flox/Flox were generated by crossing CD19Cre/+ withSHIP-1Flox/Flox mice (26). WASP2/2 mice on a 129 SvEv background wereprovided by Dr. S. Snapper (Harvard Medical School, Boston, MA) (27),and 129 SvEv wt mice were from The Jackson Laboratory. Splenic B cellswere isolated as described previously (25). All animal work was reviewedand proved by the Institutional Animal Care and Usage Committee ofUniversity of Maryland.

Preparation of monobiotinylated Fab9 Ab

Monobiotinylated Fab9 fragment of anti-mouse IgM+G Ab (mB-Fab9–anti-Ig) was generated from the F(ab9)2 fragment (Jackson ImmunoResearchLaboratories, West Grove, PA) using a published protocol (28). Thedisulfide bond that links the two Fab9 was reduced using 20 mM 2-mer-captoethylamine, and the reduced cysteine was biotinylated by maleimide-activated biotin (Thermo Scientific, Odessa, TX). Fab9 was further purifiedusing Amicon Ultracentrifugal filters (Millipore, Temecula, CA). One bi-otin per Fab9 was confirmed by a biotin quantification kit (Thermo Sci-entific). Fab9 was labeled with Alexa Fluor (AF) 546 (Invitrogen, Carlsbad,CA).

Preparation of Ag-tethered planar lipid bilayers

The planar lipid bilayer was prepared as described previously (14, 29).Liposomes were made by sonicating 1,2-dioleoyl-sn-glycero-3-phospho-choline and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-cap-biotin(Avanti Polar Lipids, Alabaster, AL) in a 100:1 molar ratio in PBS at alipid concentration of 5 mM. Aggregated liposomes were removed byultracentrifugation and filtration. Coverslip chambers (Nalge Nunc In-ternational, Rochester, NY) were coated with the planar lipid bilayer byincubating with the liposomes (0.05 mM) for 10 min. After extensivewashes, the coated coverslip chamber was incubated with 1 mg/ml strep-tavidin (Jackson ImmunoResearch Laboratories), followed by 2 mg/mlAF546-mB-Fab9–anti-Ig mixed with 8 mg/ml mB-Fab9–anti-Ig Ab. Asa nonspecific Ag (NS-Ag), the coated coverslip was incubated with 1 mg/ml streptavidin (Invitrogen), followed by 10 mg/ml AF546-labeled bio-tinylated Fab-anti–rabbit IgG Ab. For a nonantigenic control, surface

BCRs were labeled by incubating with AF546-Fab–anti-Ig (2 mg/ml) onice for 30 min. The labeled B cells were then incubated with biotinylatedholo-transferrin (Tf; 16 mg/ml, which gave an equal molar concentration of10 mg/ml mB-Fab9–anti-Ig; Sigma-Aldrich, St. Louis, MO) tethered tolipid bilayers by streptavidin.

Total internal reflection fluorescence microscopy

Images were acquired using a Nikon laser TIRF system on an invertedmicroscope (Nikon TE2000-PFS), equipped with a 603, NA 1.49 Apo-chromat TIRF objective (Nikon Instruments, Melville, NY), a CoolsnapHQ2 charge-coupled device camera (Roper Scientific, Sarasota, FL), andtwo solid-state lasers of wavelengths 491 and 561 nm. For live cell im-aging, time lapse images were acquired at the rate of one frame every 3 s.Image acquisition started upon the addition of B cells onto an Ag-tetheredlipid bilayer and continued for 5–10 min at 37˚C. Interference refectionimages (IRM) and AF488 and AF546 images were acquired sequentially.

To image intracellular molecules, B cells were incubated with an Ag-tethered lipid bilayer at 37˚C for varying lengths of time. Cells werethen fixed with 4% paraformaldehyde, permeabilized with 0.05% saponin,and stained for phosphotyrosine (pY) (Millipore), phosphorylated Btk(pBtk, Y551; BD Biosciences, San Jose, CA), Akt (S473; Cell SignalingTechnology, Danvers, MA), and WASP (S483/S484; Bethyl Laboratory,Montgomery, TX). F-actin was stained using AF488-phalloidin. In the caseof Btk inhibition, splenic B cells were pretreated with LFM A-13 [2-cyano-N-(2,5-dibromophenyl)-3-hydroxy-2-butenamide, 50 or 400 mg/ml;EMD Bioscience, Gibbstown, NJ] for 1 h at 37˚C before incubation withlipid bilayer-tethered Ag. The inhibitor was also included in the incubationmedia. The B cell contact area was determined using IRM images andMATLAB software (MathWorks, Natick, MA). The total fluorescenceintensity (TFI) and mean fluorescence intensity (MFI) of each staining inthe B cell contact zone and relative fluorescence and IRM intensity alonga line across cells were determined using Andor iQ software (AndorTechnology, Belfast, U.K.). Background fluorescence generated by Agtethered to lipid bilayers in the absence of B cells or secondary Ab controlswas subtracted. For each set of data,.20 individual cells from two or threeindependent experiments were analyzed.

Analysis of the mobility and fluorescence intensity of BCRclusters

To analyze the mobility of BCR microclusters, kymographs of time lapseimages by TIRFm were generated using Andor iQ software (AndorTechnology). The moving velocity of BCR microclusters were calculatedusing the slope of moving streaks of individual clusters in kymographs. Thelength of time that each emerging microcluster required to merge with thecentral cluster was calculated as the life span.

The TFI of BCR and pY staining in individual BCR clusters was de-termined using TIRFm images of B cells that were incubated with an Ag-tethered lipid bilayer for 3 and 7 min, using Andor iQ software. The datawere plotted as the TFI ratio of pY to the BCR in individual BCR clustersversus the TFI of the BCR in individual clusters. Smooth curves of the plotwere generated using a nonparametric regression method, locally weightedscatterplot smoothing (LOWESS) (30, 31), by Stata software (StataCorp,College Station, TX). The bwidth or the smoothing factor used for theLOWESS analysis is 0.8.

Analysis of actin nucleation sites

Actin nucleation sites were detected as described previously (32). B cellswere incubated with AF546-mB-Fab9–anti-Ig tethered to lipid bilayers inthe presence of AF488-G-actin (Invitrogen) and 0.025% saponin at 37˚C.Time lapse images were acquired for 5 min using TIRFm. The TFI andMFI of incorporated AF488-G-actin in the B cell contact zone and relativefluorescence and IRM intensity along a line across cells were determinedusing Andor iQ software (Andor Technology).

Statistical analysis

Statistical significance was assessed using the Mann–Whitney U test byPrism software (GraphPad Software, San Diego, CA). The p values weredetermined in comparison with wt or control B cells.

ResultsBtk and SHIP-1 regulate B cell spreading and BCRaggregation

Ag engagement of the BCR induces the activation of both Btk (2,33) and SHIP-1 (5–7), the key positive and negative signaling

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molecules downstream of the BCR. To determine how BCR sig-naling regulates the organization of surface BCRs, we used xidmice, which express inactive Btk that has a point mutation in itspleckstrin homology domain (34), and B cell-specific SHIP-1–knockout mice (26). We examined the effects of Btk or SHIP-1deficiency on BCR aggregation at the cell surface and B cellspreading in response to membrane-associated Ag in the absenceof adhesion molecules. The model Ag consisted of a fluorescentlylabeled, monobiotinylated Fab9 fragment of anti-mouse IgG+MAb (mB-Fab9–anti-Ig) tethered to planar lipid bilayers by strep-tavidin. TIRFm was used to evaluate the aggregation of surfaceBCRs and IRM to identify and determine the areas of B cellscontacting an Ag-tethered lipid bilayer (B cell contact zone).Upon incubation with an Ag-tethered lipid bilayer, the contact

zone of wt and CD19Cre/+SHIP-1+/+ control B cells rapidly ex-panded, peaked at ∼6 and ∼3 min, respectively, and then slightlydecreased (Fig. 1A–D, Supplemental Videos 1, 2). Concurrently,surface BCRs formed microclusters, appearing as puncta, in thefirst few minutes, which then merged into each other, forminga central cluster in the B cell contact zone (Fig. 1A, 1B, Supple-mental Videos 1, 2). The TFI of Ag in the contact zone of wt andcontrol B cells increased over time and reached a plateau at ∼7 or5 min, respectively (Fig. 1A, 1B, 1E, 1F, Supplemental Videos 1,2). Fab-anti–rabbit Ab tethered to lipid bilayers was used as anNS-Ag control, and Tf tethered to lipid bilayers, which binds to Tfreceptor on the B cell surface, was used as a nonantigenic control.For the NS-Ag control, wt B cells established limited contact withlipid bilayers, but neither spread further nor recruited the Fab tothe B cell contact zone (Fig. 1A, 1C, 1E). For the nonantigeniccontrol, the B cell contact area was larger than that observed withnonspecific Fab, but smaller than that observed with specific Ag(Fig. 1A–D). Similarly, BCR staining in the contact zone on a Tf-tethered lipid bilayer was much lower than that on an Ag-tetheredlipid bilayer (Fig. 1A, 1B, 1E, 1F). Because only BCR-specific Aginduces cluster formation, the Ag accumulation shown in thisstudy reflects the aggregation of surface BCRs. Therefore, BCRcluster formation and B cell spreading are events induced byspecific Ag.When incubated with an Ag-tethered lipid bilayer, the xid splenic

B cells only established loose attachment to the lipid bilayerand failed to spread (Fig. 1A, 1C, Supplemental Video 1). Com-pared with that of wt B cells, the amount of the BCR accumulatedin the contact zone of xid B cells was reduced drastically (Fig. 1A,1E). The behavior of xid B cells was similar to that of wt wheninteracting with a Tf-tethered lipid bilayer (Fig. 1A, 1C, 1E). Thisindicates that Btk deficiency inhibits Ag-induced BCR clusterformation and B cell spreading but not Tf-induced B cellspreading. In contrast to xid B cells, SHIP-12/2 B cells spreadmore extensively than CD19Cre/+SHIP-1+/+ control B cells (Fig.1B, 1D, Supplemental Video 2). Despite the increase in the contactarea, the amount of the BCR in the contact zone of SHIP-12/2

B cells was substantially reduced (Fig. 1B, 1F, SupplementalVideo 2), showing that BCR aggregation efficiency is dis-associated from the B cell spreading process. Moreover, the BCRat the contact zone of SHIP-12/2 B cells appeared as punctateclusters and failed to form a central cluster as in control B cells(Fig. 1B, Supplemental Video 2).To determine whether B cell developmental defects caused

by Btk mutation affect BCR microcluster formation and B cellspreading, we sorted mature and immature B cells from the spleensof wt and xid mice based on the CD93 expression level. Whenincubated with an Ag-tethered lipid bilayer, mature (CD932) andimmature (CD93+) B cells spread and accumulated BCRs in thecontact zone in a similar kinetics and to a similar magnitude.

Furthermore, Btk deficiency inhibited BCR microcluster forma-tion and B cell spreading in both mature and immature B cells(Supplemental Fig. 1). B cell-specific SHIP-1 knockout did notcause any major alterations in immature transitional and maturefollicular B cell subsets in the spleen (W.-H. Leung, T. Tarasenko,Z. Biesova, H. Kole, and S. Bolland, manuscript in preparation).This indicates that the effect of Btk and SHIP-1 deficiency onBCR cluster formation and B cell spreading are not due to alter-ations of B cell subsets in the spleens of xid and SHIP-12/2 mice.

FIGURE 1. Both Btk and SHIP-1 regulate B cell spreading and BCR

cluster formation and accumulation in response to membrane-associated

Ag. Splenic B cells from wt CBA, xid, CD19Cre/+SHIP-1+/+ (control), and

CD19Cre/+SHIP-1Flox/Flox (SHIP knockout [ko]) mice were incubated with

AF546-mB-Fab9–anti-Ig (Ag) tethered to lipid bilayers at 37˚C. As

a nonantigen control, splenic B cells were labeled with AF546-Fab–anti-Ig

for the BCR before incubation with biotinylated Tf tethered to lipid

bilayers. As an NS-Ag control, splenic B cells from wt CBA or xid mice

were incubated with biotinylated AF546-Fab–anti-rabbit IgG tethered to

lipid bilayers. Time lapse images were acquired using TIRFm and IRM.

The B cell contact area and the TFI of Ag in the contact zone were

quantified. Shown are representative images of cells at 7 min (A, B) and the

average values (6 SD) of the contact area (C, D) and the TFI (E, F) from

∼20 cells of three independent experiments. Scale bars, 2.5 mm.

232 Btk AND SHIP ACTIVATION REGULATES BCR CLUSTER FORMATION

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Taken together, these results indicate that both positive andnegative signaling mediated by Btk and SHIP-1 are involved inregulating Ag-induced B cell spreading and BCR aggregation andaccumulation in the contact zone. Btk induces B cell spreading andBCR microcluster formation, and SHIP-1 is involved in inhibitingB cell spreading and promoting the formation of the central cluster.

The centripetal movement of BCR microclusters is inhibited inSHIP-12/2 B cells

To understand why SHIP-12/2 B cells fail to form BCR centralclusters, we analyzed the lateral movement of BCR microclus-ters using kymographs. Kymographs generated from time lapseimages by TIRFm (Supplemental Videos 1, 2) provide graphicalrepresentations of spatial positions of BCR microclusters in theB cell contact zone. Using kymographs, we determined the mo-bility of individual BCR microclusters and the time span that anemerging BCR microcluster takes to merge with the central cluster(life span). The results show that in CD19Cre/+SHIP-1+/+ controlB cells, BCR microclusters moved at an average mobility of ∼10nm/s or ∼0.6 mm/min toward the center of the B cell contact zone(Fig. 2A, 2B). It took ∼1 min for emerging BCR microclusters tomerge with central clusters (Fig. 2C). In SHIP-12/2 B cells, themobility of BCR microclusters was reduced to 1.2 nm/s or 0.072mm/min, and therefore, they took six times longer to merge intothe central cluster (Fig. 2). These data demonstrate a critical rolefor SHIP-1 in the centripetal movement of BCR microclusters andthe merger of BCR microclusters into the central cluster.

The growth of BCR microclusters is inhibited, but the signalingcapability of BCR microclusters is enhanced in SHIP-12/2

B cells

BCR self-aggregation is essential for receptor activation. Althoughthe contribution of SHIP-1 to BCR signaling via its phosphataseactivity is well-known, the effects of SHIP-1 deficiency on surfaceBCR aggregation suggest a role for SHIP-1 beyond its enzymaticactivity. To investigate this hypothesis, we examined the levels and

distribution of pY, pBtk, and pAkt in relation to BCR microclus-ters in the B cell contact zone. In CD19+/+SHIP-1Flox/Flox controlB cells, the level of pY staining in the contact zone increased overtime and peaked at ∼3 min (Fig. 3A). As the pY level rose, the pYstaining largely colocalized with BCR microclusters (Fig. 3D,3F). After 3 min, the pY level in the B cell contact zone decreased(Fig. 3A). Concurrent with this decrease, BCR microclustersmerged into a central cluster, and the pY staining redistributedaway from BCR clusters to the outer edge of the B cell contactzone (Fig. 3D, 3F). Similarly, the levels of pBtk and pAkt in thecontact zone of control B cells peaked ∼3 min and decreasedafterward (Fig. 3B, 3C). This suggests a negative correlation be-tween signaling activity and the merger of BCR microclusters atthe cell surface. In SHIP-12/2 B cells, the pY level in the contactzone increased at a rate similar to that of control B cells, but thepeak level was sustained ∼2 min longer than that in the controlB cells (Fig. 3A). The higher level of pY in the contact zone wasnot simply due to an increase in cell spreading, because the MFIof pY staining in the contact zone of SHIP-12/2 B cells was alsohigher than that in control B cells (data not shown). In addition,the levels of pBtk and pAkt in the contact zone of SHIP-12/2

B cells were markedly higher than those in control B cells (Fig.3B, 3C). Moreover, the pBtk level in the contact zone of SHIP-12/2

B cells continuously increased until ∼5 min (Fig. 3B), and thehigh level of pAkt persisted rather than returning to the basal levellike in the control B cells (Fig. 3C). The surface distribution of pYwas also altered in SHIP-12/2 B cells, appearing as puncta andcolocalizing with BCR clusters at all the time points tested (Fig.3E, 3G).To further investigate the relationship between the growth of

BCR clusters and their signaling activities, we determined therelative size of BCR clusters based on the TFI of BCR labeling inindividual clusters and the relative signaling levels of BCR clustersbased on the fluorescence intensity ratio of the pY to the BCR inindividual clusters. We used a nonparametric regression method,LOWESS, to analyze the trend of the data. In CD19+/+SHIP-1Flox/Flox control B cells, we found a two-phase correlation be-tween the sizes of BCR clusters and their tyrosine phosphorylationactivity. First, when the sizes of BCR clusters were relativelysmall, the pY-to-BCR ratio increased as emerging microclustersgrew (Fig. 3H). However, after the sizes of the BCR clustersreached a certain level, the pY-to-BCR ratio decreased as BCRclusters further expanded, likely via the merger of BCR micro-clusters (Fig. 3H). In contrast, BCR clusters formed in the contactzone of SHIP-12/2 B cells were limited to smaller sizes incomparison with those in control B cells (Fig. 3I). The fluores-cence intensity ratio of pY to BCR in individual microclusters ofSHIP-12/2 B cells was much higher than that of control B cells.These results suggest that SHIP-1 promotes the growth of BCRclusters, which contributes to signaling downregulation.

Btk and SHIP-1 play opposing roles in actin reorganization

Both BCR aggregation and B cell spreading are shown to depend onthe actin cytoskeleton. The effects of Btk and SHIP-1 deficiency onBCR cluster formation and B cell spreading imply a role for thesesignaling molecules in regulating actin dynamics. We have pre-viously shown that Btk deficiency inhibits actin polymerization andWASP activation in response to soluble Ag (25). In this study, weexamined the effect of SHIP-1 gene knockout on the levels anddistribution of F-actin and actin polymerization in the contactzone. Although the level of F-actin in the contact zone of bothCD19+/+SHIP-1Flox/Flox control and SHIP-12/2 B cells increasedin response to an Ag-tethered lipid bilayer, the increase was sig-nificantly greater in SHIP-12/2 B cells (Fig. 4A–C). In the contact

FIGURE 2. The centripetal movement of BCR microclusters is inhibited

in SHIP-12/2 B cells. Splenic B cells from CD19Cre/+SHIP-1+/+ (control)

and CD19Cre/+SHIP-1Flox/Flox (SHIP knockout [ko]) mice were incubated

with AF546-mB-Fab9–anti-Ig tethered to lipid bilayers at 37˚C. Time lapse

images were acquired using TIRFm. Kymographs of individual clusters

were generated using time lapse images. Shown are two representative

kymographs depicting movement of BCR microclusters (A). Arrowheads

point to individual moving microclusters. The moving velocity of BCR

microclusters was calculated using the slope of the moving streak in

kymographs. The time span that each emerging microcluster required to

merge with a central cluster was calculated as the life span. Shown are the

average velocity (6 SD) (B) and the average life span (6 SD) (C) cal-

culated from 30 BCR microclusters of three independent experiments.

Scale bar, 2.5 mm. *p , 0.01.

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zone of control B cells, F-actin was largely colocalized with BCRsupon B cell interaction with an Ag-tethered lipid bilayer (Fig. 4A).When BCR microclusters merged into each other, F-actin movedaway from BCR clusters to BCR poor regions and the outer edgeof the contact zone (Fig. 4A, 4D). In SHIP-12/2 B cells, F-actindid redistribute away from BCR clusters, but it formed a wide ringin the middle of the contact zone and did not accumulate at theouter edge of the contact zone (Fig. 4B, 4E).F-actin accumulation suggests an increase in actin polymeri-

zation. To compare actin polymerization activity of Btk and SHIP-1–deficient B cells with wt and CD19+/+SHIP1Flox/Flox control

B cells, we used G-actin incorporation assay. In this assay, theincorporation of fluorescently labeled G-actin to the polymerizingend of F-actin indicates the location and level of actin polymeri-zation (32). We found that in xid B cells, the MFI of incorporatedAF488-G-actin in the contact zone was significantly decreased,compared with that in wt B cells. In contrast, G-actin in-corporation was significantly increased in the contact zone ofSHIP-12/2 B cells, compared with that in control B cells (Fig.4F). Despite the enhanced actin polymerization in SHIP-12/2

B cells, actin polymerization sites were distributed in a patternsimilar to what was seen in control B cells (Fig. 4G, 4H).

FIGURE 3. The signaling capability of BCR microclusters is increased, but the growth of BCR microclusters is inhibited in SHIP-12/2 B cells. Splenic B

cells from CD19+/+SHIP-1Flox/Flox (control) and CD19Cre/+SHIP-1Flox/Flox (SHIP knockout [ko]) mice were incubated with AF546-mB-Fab9–anti-Ig tethered

to lipid bilayers at 37˚C for the indicated times. Cells were fixed, permeabilized, and stained for pY, pBtk, and pAkt using specific Abs and AF488-

conjugated secondary Abs. Cells were analyzed using TIRFm. The TFI (A–C) of pY, pBtk, and pAkt in the B cell contact zone was quantified. The average

TFI (6 SD) were determined from 34 to 87 cells of two independent experiments. Shown are representative images (D, E) and the relative intensity of IRM,

BCRs, and pYacross the cells (blue lines) (F, G). Green dashed lines indicate the major peaks of pY, and red arrows point to BCR peaks in histograms. The

TFI of the BCR and the fluorescence intensity ratio of pY to the BCR in individual BCR clusters were determined (H, I). Each open symbol represents

a BCR cluster, and solid symbols represent the LOWESS curve that was generated by Stata software. The data were generated from 40 cells of each strain

of mice and two independent experiments. Scale bars, 2.5 mm. *p , 0.01.

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Actin polymerization is activated by actin nucleation promotingfactors. Our previous study shows that Btk promotes actin poly-merization by activating a hematopoietic-specific actin nucleationpromoting factor, WASP (25). In this study, we determined theeffect of SHIP-1 deficiency on WASP activation using an Abspecific for the phosphorylated WASP (pWASP, S483/S484).Similar to F-actin, the level of pWASP in the contact zone ofCD19+/+SHIP-1Flox/Flox control B cells increased over time in re-sponse to an Ag-tethered lipid bilayer and reached a peak at ∼5min (Fig. 5A, 5F). In SHIP-12/2 B cells, the pWASP level in thecontact zone was significantly increased, especially at 1–3 min(Fig. 5B, top panels, and 5F). The pWASP staining in the contactzone of both control and SHIP-12/2 B cells appeared as punctaand colocalized with BCR microclusters early during the stimu-lation (Fig. 5A, 5C; data not shown). As BCR microclustersmerged into each other, pWASP in control B cells redistributedaway from BCR clusters to the outer edge of the contact zone andto BCR poor regions (Fig. 5A, 5D), but pWASP in SHIP-12/2

B cells failed to do so (Fig. 5B, top panels, 5E).

These results collectively indicate that Btk and SHIP-1 positivelyand negatively regulate actin reorganization, respectively. Btkinduces whereas SHIP-1 inhibits WASP activation, actin poly-merization, and F-actin accumulation in the B cell contact zone.

SHIP-1 regulates WASP activation, B cell spreading, and BCRcluster formation in a Btk-dependent manner

Btk is one of the downstream targets of SHIP-1. SHIP-1dephosphorylates phosphatidylinositol-3,4,5-triphosphate at itsfive position, removing the docking site of Btk at the plasmamembrane. Therefore, SHIP-1 deficiency increases Btk activation(Fig. 3B) (8). To examine the functional interrelationship betweenSHIP-1 and Btk in initiation steps of BCR activation, we reducedBtk activity in SHIP-12/2 B cells using a Btk-specific inhibitor,LFM A-13. Our previous study shows that this inhibitor causesreductions in BCR and WASP activation similar to Btk deficiency(25). An intermediate and a high concentration of LFM A-13 wereused to manipulate the level of the kinase activity of Btk. At an

FIGURE 4. Btk and SHIP-1 have opposing roles in Ag-induced actin reorganization. A–E, Splenic B cells from CD19Cre/+SHIP-1+/+ (control or cont) and

CD19Cre/+SHIP-1Flox/Flox (SHIP-1 ko) mice were incubated with AF546-mB-Fab9–anti-Ig tethered to lipid bilayers at 37˚C for indicated times. Cells were

fixed, permeabilized, and stained for F-actin using AF488-phalloidin. Cells were analyzed by TIRFm. Shown are representative images (A, B), the MFI of

F-actin in the contact zone (C), and the relative intensity of IRM, F-actin, and the BCR across the cells (blue lines) (D, E). Green dashed lines indicate the

major peaks of F-actin, and red arrows point to BCR peaks in histograms. The average values (6 SD) of the MFI were generated from 20 to 90 cells of three

independent experiments. F–H, Splenic B cells were incubated with AF546-mB-Fab9–anti-Ig tethered to lipid bilayers at 37˚C for 5 min in the presence of

AF488-G-actin and 0.025% saponin. Cells were fixed and analyzed using TIRFm. Shown are representative images (G, H) and the average MFI (6 SD) of

incorporated AF488-G-actin in the contact zone (F), generated from 22 to 24 cells of two or three independent experiments. Scale bars, 2.5 mm. *p, 0.01.

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intermediate concentration, the Btk inhibitor restored the mag-nitudes of B cell spreading (Fig. 5G), BCR accumulation (Fig.5H), and WASP phosphorylation (Fig. 5B, 5F) in the contact zoneof SHIP-12/2 B cells to levels similar to those in CD19+/+SHIP-1Flox/Flox control B cells. At a high concentration, the Btk inhibitorreduced B cell spreading (Fig. 5G), BCR accumulation (Fig. 5H),and WASP phosphorylation in the contact zone (Fig. 5B, 5F) tolevels close to those in Btk-deficient B cells. These data indicatethat SHIP-1 can regulate actin reorganization, B cell spreading,and BCR aggregation and accumulation via inhibiting Btk.

B cell spreading, BCR cluster formation, and tyrosinephosphorylation are reduced in WASP2/2 B cells

Our finding that WASP activation is regulated by both Btk andSHIP-1 suggests that WASP contributes to BCR activation. Todetermine the role of WASP in the early event of BCR activation,we compared B cell spreading, BCR cluster formation, and tyrosinephosphorylation in WASP2/2 B cells with those in wt B cells. InWASP2/2 B cells, the magnitude of B cell spreading, the extent ofBCR clustering, and the level of tyrosine phosphorylation in theB cell contact zone were significantly reduced, compared with wtB cells (Fig. 6). However, the inhibitory effects of WASP de-ficiency were less remarkable than those of Btk deficiency and Btkinhibition (Figs. 1, 5). These results suggest that WASP is im-portant, but not essential, for B cell spreading and BCR aggre-gation in response to membrane-associated Ag.

DiscussionIn this study, we have examined the underlying mechanism forthe functional interplay between signaling and the actin cytoskele-ton during BCR activation. Our results show that the stimulatorykinase Btk promotes B cell spreading, BCR microcluster forma-tion, and actin polymerization and recruitment in response tomembrane-associated Ag. In contrast, the inhibitory phosphataseSHIP-1 inhibits B cell spreading and actin polymerization andrecruitment but promotes the centripetal movement and merger ofBCR microclusters. These results suggest that a balance betweenpositive and negative signaling regulates the dynamics and mag-nitude of BCR self-aggregation at the B cell surface via controllingactin reorganization.The unique finding of this study is that positive and negative

signals have distinct roles in regulating BCR aggregation. Previousstudies show that the activation of proximal signaling molecules,including PLCg2, Vav, Rac2, and costimulatory molecule CD19(15, 16, 22), can have significant impact on BCR aggregation onthe cell surface. Consistent with these findings, we show in thisstudy that Btk, a downstream molecule of CD19 and an upstreammolecule of Vav and Rac, is required to induce the formation ofBCR microclusters in response to membrane-associated Ag.However, the inhibitory phosphatase SHIP-1 is not essential forthe formation of BCR microclusters, but it is involved in thecentripetal movement and merger of BCR microclusters after theirformation. Our results suggest that the relative activation levelsand timing of stimulatory kinases, such as Btk, and inhibitory

FIGURE 5. SHIP-1 regulates WASP activation,

B cell spreading, and BCR cluster formation and

accumulation in a Btk-dependent manner. Splenic

B cells from CD19+/+SHIP-1Flox/Flox (control) and

CD19Cre/+SHIP-1Flox/Flox (SHIP knockout [ko])

mice were pretreated with or without LFM A-13

(A-13) for 1 h and incubated with AF546-mB-

Fab9–anti-Ig tethered to lipid bilayers at 37˚C for

indicated times in the presence or absence of A-13.

Cells were fixed, permeabilized, and stained for

phosphorylated WASP (pWASP) using a specific

Ab and an AF488-conjugated secondary Ab. Cells

were analyzed by TIRFm. Shown are representative

images (A, B) and the relative intensity of IRM,

pWASP, and the BCR across the cells (blue lines)

(C–E). Green dashed lines indicate the major peaks

of pWASP, and red arrows point to BCR peaks in

histograms. The MFI of pWASP (F), the B cell

contact area (G), and the TFI of the BCR (H) in the

contact zone were quantified. Shown are average

values (6 SD) of 20–90 cells from three in-

dependent experiments. Scale bars, 2.5 mm. *p ,0.01 in comparison with controls.

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phosphatases, such as SHIP-1, can control the formation andgrowth of BCR microclusters. Therefore, signaling triggered byAg-induced aggregation of BCRs in turn regulates the kinetics andmagnitude of BCR self-aggregation, providing feedbacks to signalinitiation.BCR activation is triggered by self-aggregation of surface

receptors into microclusters. The formation and growth of BCRmicroclusters has been positively linked to the signaling capabilityof the receptor (35). Contrary to this positive link, proximal sig-naling molecules have been shown to be primarily recruited toTCR microclusters in the periphery but not the center of the im-munological synapse (36). In line with the previous study, wedetected tyrosine phosphorylation primarily at BCR microclustersand the outer edge of the BCR central cluster. This supports thenotion that signal initiation occurs at receptor microclusters ratherthan the central cluster. By analyzing the ratio between the amountof BCRs and the level of tyrosine phosphorylation in individualclusters, our results suggest a two-phase relationship between thesize and signaling capability of BCR clusters. In the first phase,the tyrosine phosphorylation level increases as BCR microclustersemerge and undergo initial growth, which can be considered as thesignal activation phase. In the second phase, the tyrosine phos-phorylation level decreases as the size of the BCR clusters furtherexpands, which can be considered as the signal transitional ordownregulation phase. We found that SHIP-1 deficiency inhibitedthe growth but not the formation of BCR microclusters, whichseems to limit the size of BCR microclusters to the signal acti-vation phase and to prevent BCR microclusters from moving tothe signal downregulation phase. Therefore, Btk plays a dominantrole in the signal activation phase and SHIP-1 in the signaldownregulation phase of BCR clusters. This suggests that in ad-dition to their enzymatic activity, Btk and SHIP-1 could upregu-late and downregulate BCR activation by promoting the formationof BCR microclusters or the growth and merger of BCR micro-clusters into the central cluster. How the merger of BCR micro-clusters contributes to signaling downregulation remains to beelucidated.The effects of Btk and SHIP-1 deficiency on the organization of

the actin cytoskeleton suggest that these signaling molecules canregulate BCR aggregation by controlling actin dynamics. In ad-dition to determine cell morphology, the actin cytoskeleton alsocreates a barrier for the lateral movement of transmembrane pro-teins whose cytoplasmic tails are extended into the cortical actinnetwork (19). Perturbing this barrier has been shown to increasethe lateral mobility of surface BCRs and to induce signalingwithout Ag (21). We found in this study that Btk and SHIP-1 had

opposite roles in actin reorganization. Btk and SHIP-1 deficiencydecrease and increase actin polymerization activity and F-actinaccumulation in the B cell contact zone, respectively. The de-creased actin polymerization in Btk-deficient B cells is associatedwith a marked reduction in B cell spreading and surface BCRcluster formation. This suggests that Btk-induced actin polymer-ization is required for BCR aggregation and B cell spreading. Theinhibition of BCR aggregation in Btk-deficient B cells is likelycaused by the reduction in B cell spreading, because B cellspreading has been shown to enhance the formation of BCRmicroclusters by increasing the number of surface BCRs engagingAg (20). In SHIP-12/2 B cells, enhanced actin polymerizationand F-actin accumulation are concurrent with increased B cellspreading and reduced centripetal movement and merger of BCRmicroclusters. This suggests that in response to antigenic stimu-lation, SHIP-1–suppressed actin polymerization is important forpreventing B cells from further spreading and for driving thecentripetal movement of BCR microclusters. The timing and levelof SHIP-1 activation may negatively control the magnitude ofB cell spreading and the mobility of BCR aggregates by alteringthe dynamics and organization of the actin cytoskeleton. There-fore, a balance of positive and negative signals can regulate thenature of actin reorganization, which in turn controls the mor-phology of B cells and the mobility and growth of BCR aggre-gates.How the proximal signaling molecules regulate actin dynamics

is not fully understood. In this study, we show that WASP, an actinnucleation promoting factor, is a common downstream target ofBtk and SHIP-1 during antigenic activation of the BCR. WASPhas been shown to be involved in the formation of immunologicalsynapses in both B and T cells (37–39). We have previously shownthat Btk can induce the activation of WASP via increasing thephosphorylation of Vav and the level of phosphatidylinositol-4,5-biphosphate (25). This study shows that SHIP-1 inhibits WASPphosphorylation, counteracting against Btk. The opposing effectof Btk and SHIP-1 on WASP activation is consistent with theiropposite impact on actin polymerization. Moreover, phosphory-lated WASP and F-actin were found to exhibit similar distributionpatterns in the B cell contact zone. These results suggest thatWASP controls de novo actin polymerization induced by BCRactivation. Our finding that the SHIP-1–mediated inhibition ofWASP depends on its ability to inhibit Btk indicates that SHIP-1prevents WASP from activating by turning off the kinase thatinduces WASP activation. Therefore, BCR signaling can regulateactin reorganization by controlling the activity of actin regulatorslike WASP.

FIGURE 6. BCR cluster formation, B cell spreading,

and tyrosine phosphorylation are reduced in WASP2/2

B cells. Splenic B cells from wt and WASP2/2 mice

were incubated with AF546-mB-Fab9–anti-Ig tethered

to lipid bilayers at 37˚C for indicated times. Cells were

fixed, permeabilized, and stained for pYusing a specific

mAb and an AF488-conjugated secondary Ab. Cells

were analyzed using TIRFm. Shown are representative

images (A, B) and the average values (6 SD) of the B

cell contact area (C), the TFI of the BCR (D), and the

MFI of the pY (E) in the contact zone. The data were

generated using 20–90 cells from three independent

experiments. Scale bars, 2.5 mm. *p , 0.01.

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In support of a role forWASP in BCR activation, this study foundthat B cell spreading, BCR cluster formation, and tyrosine phos-phorylation in the contact zone induced by membrane-associatedAg were inhibited by WASP deficiency. However, the inhibitionwas only partial, suggesting that there are additional actin regu-lators that have overlapping functions with WASP in the process ofsignal initiation. WASP belongs to a family of proteins (40). Theother members of WASP family proteins, such as N-WASP andWAVE, may compensate for the function of WASP in WASP2/2

B cells. Such compensational roles could explain the partial in-hibition observed in this study as well as the mild defects in B cellactivation and B cell responses in WASP2/2 mice reported pre-viously (27, 37). It is also noted that the inhibitory effects ofWASP deficiency on B cell spreading and BCR clustering are lessdramatic than those of Btk deficiency. This suggests that Btk canregulate these processes through multiple downstream moleculesin addition to WASP. Identification of additional actin regulatorsdownstream of Btk and SHIP-1 that are involved in actinremodeling during BCR activation is a subject of our future in-vestigation.In summary, this study demonstrates a close cooperation be-

tween signaling and the actin cytoskeleton during BCR activation.Positive and negative signals mediated through Btk and SHIP-1regulate B cell membrane dynamics and spatiotemporal organi-zation of surface BCRs via controlling actin reorganization. Themagnitude of BCR aggregation and the mobility of BCR aggre-gates regulate the signaling capability of the receptor. This inter-play between actin reorganization with signaling forms a mecha-nistic basis for feedback regulation of BCR signaling. Such afeedback is potentially a general regulatory mechanism of recep-tor signaling. Further studies are required to identify additionalmolecular linkers and to define the molecular nature of the inter-action between the actin cytoskeleton and BCR signaling pathway.

AcknowledgmentsWe thank Drs. Hae Won Sohn and Wanli Liu at the National Institute of

Allergy and Infectious Diseases, National Institutes of Health, for technical

assistance on generating lipid bilayers.We also thank Kenneth Class and the

Maryland Pathogen Research Institute Flow Cytometry Facility for tech-

nical assistance on cell sorting.

DisclosuresThe authors have no financial conflicts of interest.

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