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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Immunity. Author manuscript; available in PMC 2010 January 1.
Published in final edited form as:
PMCID: PMC2656684

The constant region of the membrane immunoglobulin mediates B-cell receptor clustering and signaling in response to membrane antigens


B cells are activated in vivo following the B-cell receptors (BCRs) binding to antigens captured on the surfaces of antigen presenting cells. Antigen binding results in BCR microclustering and signaling, however, the molecular nature of the signaling-active BCR clusters is not well understood. Using new single molecule imaging techniques we provide evidence that within microclusters, the binding of monovalent membrane antigens results in the assembly of immobile signaling-active BCR oligomers. The oligomerization depends on interactions between the membrane-proximal Cμ4 domains of the mIg that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cμ4 domain failed to cluster and signal and conversely, Cμ4 domain alone clustered spontaneously and activated B cells. These results support a novel mechanism for the initiation of BCR signaling in which antigen binding induces a conformational change in the Fc portion of the BCR revealing an interface that promotes BCR clustering.


B cells are activated by pathogens via their specific B-cell receptors (BCRs) binding directly to pathogen-associated antigens. Antigen binding initiates BCR signaling and a program of B-cell activation that ultimately results in B-cell differentiation and antibody production. The BCR is composed of an antigen-specific membrane immunoglobulin (mIg) paired with a heterodimer of Igα/Igβ, the intracellular domains of which are phosphorylated and interact with effector signaling proteins when clustered into molecular proximity by antigen binding (Reth, 1992).

During infection, many pathogen-associated antigens are captured and retained on the surfaces of antigen-presenting cells (APCs) (Carrasco and Batista, 2006), such as dendritic cells (Bergtold et al., 2005; Qi et al., 2006) and subcapsular macrophages (Carrasco and Batista, 2007; Junt et al., 2007; Phan et al., 2007). In addition, antigens ultimately reach B-cell follicles where they are retained on follicular dendritic cells to serve as a reservoir of antigen for germinal center formation and affinity maturation (Tarlinton, 2006). B cells have been shown to interact with membrane-tethered antigens both in vitro and in vivo (Batista et al., 2001; Phan et al., 2007; Qi et al., 2006; Schwickert et al., 2007). In vitro, the initial contact with the antigen-containing membrane triggers B cells to spread, a response that is dependent on Lyn and Syk, the first two kinases in the B cell signaling cascade and accompanied by an intracellular calcium response (Weber et al., 2008). The B cells then contract to form an immunological synapse (Depoil et al., 2007; Fleire et al., 2006).

Although the cascade of events that follow the initiation of signaling by the BCR’s intracellular domains are becoming better characterized, at present we know little about the molecular mechanism by which antigen binding to the extracellular domains triggers these events. We neither understand the repercussions of antigen binding on the BCR, nor do we understand what structural features of the BCR are important for the initiation of signaling. In the first moments of B-cell contact with membrane antigens, the BCR redistributes to microclusters that accumulate proximal signaling molecules (Depoil et al., 2007; Fleire et al., 2006; Weber et al., 2008). Microclustering of the BCR by membrane antigens could simply be the repercussion of a physical crosslinking of the BCR by the binding of multivalent antigens as has been observed in response to multivalent antigens bound by B cells from solution. However, there is some evidence indicating that the membrane topology and the actin cytoskeleton contribute to the formation of immunoreceptor microclusters in response to membrane associated antigens (Campi et al., 2005; Choudhuri et al., 2005; Depoil et al., 2007; Fleire et al., 2006; Varma et al., 2006) suggesting that the molecular mechanisms underlying BCR microclustering in response to membrane associated antigens may not be the same as those mediating soluble-antigen induced clustering.

Here we use single molecule imaging to describe the events that follow the binding of membrane antigens to the BCR and that are required for the initiation of signaling. We provide evidence for a novel mechanism for antigen-induced BCR oligomerization that occurs inside synaptic microclusters and requires the Cμ4 domain and the N-terminal part of the transmembrane helix of the BCR’s mIgM. These results suggest an unexpected involvement of BCR’s extracellular and transmembrane domains in the initiation of signaling.


B-cell activation by membrane antigens

To explore the mechanisms underlying the initiation of BCR signaling leading to immune synapse formation, we used total internal reflection fluorescence (TIRF) microscopy to image the activation of primary splenic B cells from B1-8 +/+ Igκ −/− mice that express the B1-8 BCR specific for the NIP hapten (Sonoda et al., 1997). We first assessed the requirement for physical crosslinking of the BCR by multivalent antigens when antigen was presented on a fluid lipid bilayer. To do so, we attached either a multivalent antigen (4-Hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP)14-BSA, or a monovalent NIP1-H12 peptide, together with intercellular adhesion molecule 1 (ICAM-1) to planar lipid bilayers via engineered histidine tags binding to nickel-containing lipids. The B cells responded to both the multivalent and the monovalent antigens attached to the bilayers both in terms of inducing calcium fluxes (Figure 1A) and upregulation of CD69 (Figure 1B) as well as CD86 (Figure S1A) expression. This was in contrast to stimulation of the B cells with soluble forms of the antigens, in which case only the NIP14-BSA induced elevation of intracellular calcium, whereas the NIP1-H12 was unable to stimulate the B cells, even at concentrations of NIP1-H12 that insured equivalent BCR occupancy of the two antigens (Figure S1B).

Figure 1
B-cell activation and BCR clustering in response to multi- and monovalent membrane antigens

Time-lapse TIRF imaging showed that the B cells spread on the bilayers containing either the monovalent or the multivalent antigens with similar dynamics and redistribution of the BCR (Figure 1C, Movie S1-4). The first contacts of the B cells with the bilayers appeared as one or more discrete points, where BCR were concentrated into small areas that persisted as the cell spread. These areas of antigen-concentrated BCRs have been referred to as ‘microclusters’ (Campi et al., 2005; Depoil et al., 2007; Yokosuka et al., 2005). Additional BCR microclusters formed at the leading edges of the spreading cells and in ruffling membranes at the cell’s periphery (Movie S2, 4), and then moved to the center of the synapse, fusing with one another on their way and accumulating in the center of the synapse as described (Depoil et al., 2007).

The BCR microclusters that formed in response to either monovalent or multivalent antigen appeared to be signaling active structures. First, simultaneous imaging of B-cell intracellular calcium levels and the antigen in the bilayers showed that the calcium response was initiated at the same time as the initial microclustering of the BCR-bound antigens both of which preceded B-cell spreading (Fig. 1D). Thus, the initial BCR microclusters were sufficient to trigger B-cell calcium responses. Based on quantification of the fluorescence of the antigen microclusters at the time of elevation of intracellular calcium (Figure 1D), we estimated that the binding of as few as fifty NIP1-H12 peptides was sufficient to initiate the calcium response. Similar analyses for B cells encountering NIP14-BSA-containing membranes showed that only five NIP14-BSA molecules were sufficient to induce a calcium flux. Thus, both forms of the antigen initiate signaling highly efficiently. Second, we examined the recruitment of Syk to the BCR microclusters by visualizing GFP-Syk transfected into primary B-cell blasts. In B cells spreading on bilayers without antigen, no specific co-redistribution of Syk and the BCR was observed (data not shown). However, Syk co-localized with the BCR microclusters induced by either NIP1-H12 or NIP14-BSA (Figure S1C). In addition, the microclusters of the BCR were sites of tyrosine phosphorylation, as detected by intracellular staining (Figure S1D).

Several lines of evidence verified that the individual NIP1-H12 peptides were monomeric when attached to the bilayers. We analyzed the peptides conjugated with a single fluorescent label at a single molecule level, and showed that the peptides appeared as individual mobile spots of similar intensities that bleached in single steps (Figure 2A, B). Moreover, FRET between donor and acceptor labeled NIP1-H12 peptides on the bilayer was low at concentrations used to activate B cells indicating little interactions of the membrane associated peptides with each other (Figure 2C). FRET rose linearly only with increasing concentration of the antigen, as is consistent with random bumping of the peptides without specific interactions. Lastly, clusters of NIP1-H12 on the bilayer that formed during the contact with B cells were rapidly dispersed by the addition of soluble NIP hapten that competed out binding of the BCR and released the B cells from the bilayer (Figure 2D), indicating that even when highly concentrated in clusters, the NIP1-H12 peptide does not self aggregate. Thus, BCR microclustering and B-cell activation by membrane antigens is not driven by the ability of the antigens to crosslink the BCR, as is the case with soluble antigens.

Figure 2
Monomeric nature of the NIP1-H12 peptide antigen

Single molecule observations of BCR microclustering

To provide insights into the mechanism by which the BCRs formed microclusters, we analyzed the dynamics of individual BCRs by single molecule TIRF microscopy. We labeled BCRs with fluorescent monovalent Fab fragments of anti-Ig antibodies under conditions that allowed tracking of individual BCR spots. The fluorescence intensity and bleaching characteristics of the spots indicated that they correspond to single BCR molecules. Tracking of the single BCR molecules in the absence of antigen showed that their short-range mean square displacements (MSDs) were linearly dependent on time, indicating that their short-range movement was consistent with simple diffusion (Figure S2). We calculated short-range diffusion coefficients of thousands of individual BCR molecules and analyzed their distribution. In the absence of antigen on bilayers, most BCRs were mobile (Figure 3A, Movie S5) with a median diffusion coefficient of about 0.1 μm2/s. Using a cutoff of diffusion slower then 0.01 μm2/s, we estimated from the cumulative probability plots of the diffusion coefficients that approximately 18% of BCR molecules were immobile. In contrast, after cells contacted bilayers containing NIP1-H12 (Movie S6) or NIP14BSA (Movie S7) the fraction of immobile BCRs increased to more than 50% for both monovalent and multivalent antigens (Figure 3A), indicating that the immobilization of the BCR was independent of antigen valency.

Figure 3
Microclusters arrest diffusion of BCR and Syk

To determine if antigen binding itself was sufficient to immobilize BCR or alternatively if antigen binding induced a change in the BCR that allowed it to cluster with other antigen-bound BCRs we carried out two analyses. First, we tracked single BCR molecules in B cells during the initial phase of cell spreading, when newly-formed antigen microclusters could be well resolved by simultaneous imaging of fluorescently labeled antigens to determine if BCR were more likely to be immobilized when they entered existing BCR clusters. The tracking revealed that most immobile BCRs were localized inside the antigen microclusters, while the BCR outside of the antigen microclusters were still mobile (Figure 3B). In some trajectories we observed that when the mobile BCRs entered the existing BCR clusters, they abruptly decreased their mobility (Figure 3C, Figure S3, Movie S8). An analysis of all trajectories confirmed that the probability of a BCR stopping was highest within antigen clusters as compared to the membrane outside of these clusters (Figure 3D). The monovalent NIP1-H12 was as potent in inducing stopping of the BCR as was NIP14-BSA (Figure 3D). This observation suggests that the antigen-bound BCR stops when it encounters other antigen bound BCRs.

Secondly, we tracked single NIP1-H12 antigens bound to B cells at a very low concentration, when only 5–15 BCRs were engaged in each cell (Figure 3E) making it highly unlikely that antigen bound BCRs would encounter each other. In this case, the antigens slowed down from their rapid diffusion on the bilayer (approximately 1 μm2/s) to the mobility of the BCRs (0.1 μm2/s) indicating that most of the antigens were bound to BCRs (Figure 3E). However, only ~ 12% of the antigens were immobilized. Immobilization of a larger fraction of the antigens, ~ 30%, required simultaneous, monovalent engagement of a larger number of BCRs achieved by increasing the antigen concentration (Figure 3E). Collectively, these results indicate that immobilization of the BCR, although dependent on antigen binding, represents a discrete step, distinct from antigen binding that requires the engagement of several BCRs in close proximity.

To determine if the immobilization of the BCR resulted in immobilization of proximal signaling molecules at the plasma membrane, we tracked single molecules of GFP-Syk in transfected primary B-cell blasts. In resting B cells, the appearance of GFP-Syk molecules in the TIRF field was rare (Movie S9), but the numbers of GFP-Syk molecules dramatically increased in cells stimulated with either NIP1-H12 (Movie S10) or NIP14-BSA (Movie S11). Calculation of the time that individual GFP-Syk molecules spent at the plasma membrane showed that Syk lifetime at the plasma membrane was significantly increased in cells stimulated with either NIP14-BSA or NIP1-H12 (Figure 3F). The membrane lifetime of GFP-Syk in antigen-stimulated cells approached the lifetime of control transmembrane GFP molecules disappearing only by photobleaching. Importantly, while GFP-Syk in resting cells was highly mobile, Syk in the antigen-engaged cells was immobile (Movie S9-11, Figure 3G). Thus, the immobilization of the BCR is accompanied by immobilization of Syk, suggesting that signaling is initiated primarily by immobile BCRs.

The Cμ4 and the transmembrane domain of the mIg regulate immobilization of BCRs in microclusters

As BCRs were efficiently immobilized and activated in microclusters without physical crosslinking by multivalent antigens, we next searched for the molecular basis of such clustering of the BCR. Microclustering and immobilization of the BCR was readily observed after BCR expression in J588L cells, which lack many of the B-cell surface proteins such as LFA-1, CD45, CD19 and CD22 (Fig 4A). Blocking BCR signaling by pharmacological inhibition of Src-kinases in primary B cells or deletion of the cytoplasmic domains of Igα and Igβ in J558L cell lines had no effect on the microclustering and immobilization of BCR molecules induced by either the mono- or multi-valent antigens, despite inhibition of cell spreading and impairment of the active movement of the BCR clusters to the center of the synapse (data not shown). Furthermore, mutation of a tyrosine-serine motif to valine (YS/VV) in the transmembrane domain of mIgM (Figure 4B), that completely disrupts association of the mIgM with the Igα/Igβ heterodimer (Shaw et al., 1990), had no effect on the immobilization and clustering of single BCR molecules in response to either NIP1-H12 or NIP14-BSA. Thus, the signal transduction components of the BCR do not play a role in BCR immobilization in microclusters. We next investigated if the ectodomains of the mIgM are important for the immobilization of the BCR. We found that deletion of two extracellular, membrane proximal domains of mIgM YS/VV, together with the peptide connecting to the transmembrane domain, (YS/VV Cμ3-4Δ) severely impaired the immobilization of single BCR molecules induced by NIP1-H12, but not the immobilization after crosslinking by the multivalent antigen, NIP14-BSA (Figure 4C). This observation indicates that the physical crosslinking of the BCR by multivalent antigen immobilizes BCR by a mechanism distinct from that underlying the immobilization of the BCR by monovalent antigen.

Figure 4
Cμ4 domain and the transmembrane region of the mIgM are required for BCR clustering in response to monovalent membrane antigens

The effect of the Cμ3-Cμ4 deletion could not be explained simply by a difference in the size of the extracellular portion of the BCR, as a similar behavior was observed when the Cμ3-Cμ4 region in YS/VV was replaced with a stalk derived from glycoprotein D of herpes simplex virus (HSVgD) (Figure S4). The stalk of HSVgD is a highly hydrophilic, proline-rich domain that has been used in chimeric recombinant membrane proteins to increase the distance of the protein from the plasma membrane (Chitnis and Miller, 1994; Cohen et al., 1988). Therefore, we searched for a specific region within the Fc portion of the mIgM responsible for the immobilization of the BCR. The deletion of the Cμ3 domain alone had no effect on BCR immobilization by either monovalent or multivalent antigen (Figure 4D). Similarly, mutations in the connecting peptide were without an effect on the immobilization (data not shown). In contrast, deletion of the Cμ4 domain impaired specifically immobilization induced by the monovalent antigen, although to a slightly lesser extent than full deletion of the Cμ3-Cμ4 region (Figure 4E). In addition, mutation of a motif WTxxST to VVxxVV in the N-terminal part of the transmembrane helix (termed here the TM mutation), predicted to be on the opposite side of the transmembrane helix from the YS/VV mutation (Schamel and Reth, 2000), also had a partial effect on the immobilization of the BCR by the monovalent antigen (Figure 4F). The combination of a deletion of the Cμ4 domain with the TM mutation completely blocked immobilization of the BCR by the monovalent antigen without affecting immobilization by the multivalent antigen (Figure 4G). Thus, the Cμ4 domain together with the N-terminal part of the transmembrane helix are critical for the intrinsic immobilization of the BCR by antigens in microclusters.

The failure of both the YS/VV Cμ3-4Δ and the YS/VV Cμ4ΔTM to become immobilized in response to NIP1-H12 was not due to the failure to bind the monovalent antigen. Quantitative measurements of the amount of the BCR and antigen in the contact area between the B cells and the bilayers showed that the YS/VV Cμ3-4Δ and the YS/VV Cμ4ΔTM constructs accumulated in the area of contact similarly to the YS/VV construct and that all constructs bound similar amounts of antigen (Figure 5A, B). Microscopically, in response to either the monovalent or the multivalent antigens, the YS/VV, YS/VV Cμ3-4Δ and YS/VV Cμ4ΔTM constructs appeared to form microclusters in the first minutes. The microclusters then grew over time, passively fused and eventually covered most of the contact area (Fig S5A). However, dual color imaging of single YS/VV Cμ3-4Δ or YS/VV Cμ4ΔTM molecules within the clusters induced by NIP1-H12 binding showed that molecules of the mutated constructs were not immobilized in the microclusters, but rather were only confined in their movement inside the microclusters (Fig 5C, Movie S12, S13). Analysis of the MSD plots of the YS/VV and YS/VV Cμ4ΔTM molecules binding to NIP1-H12 showed a plateau at 0.09 μm2 confirming confined diffusion of the constructs in clusters of approximately 300×300 nm (Fig 5D, S5B). Taken together these results indicate that the antigen-mediated accumulation and retention of the YS/VV Cμ3-4Δ and YS/VV Cμ4ΔTM molecules in clusters occurred by confinement rather then by immobilization, suggesting the existence of mechanisms that confine BCR movement in the clusters independently of mIg structure, most likely dependent on membrane topology. In contrast, the structural features of the BCR’s IgM Cμ4 and transmembrane domains appear to play a specific role in the assembly of the BCRs into immobile oligomeric complexes.

Figure 5
Accumulation in synapses, antigen binding, and type of movement of BCR constructs in synapses

The Cμ4 domain and the transmembrane region of mIg are important for the initiation of BCR signaling

We next determined if the Cμ4 and the transmembrane domains of the mIgM that were necessary for the oligomerization and immobilization of the BCR after monovalent antigen binding were necessary for the initiation of BCR signaling. The ability of constructs lacking the Cμ4 domain to initiate signaling could not be evaluated directly as we found that the Cμ4 domain is required for the association of the mIgM with Igα/β (data not shown). Therefore, to evaluate the function the Cμ4 domain in the initiation of signaling, we produced fusion constructs of the YS/VV mIgM, YS/VV Cμ3 -4Δ and the YS/VV Cμ4ΔTM that contained the intracellular domains of either Igα or Igβ (Williams et al., 1994) (Figure 6A). Co-transfection of the Igα– and Igβ-fusion constructs into either J558L cells or primary B-cell blasts resulted in similar expression levels of NIP-specific mIgM regardless of the presence or absence of the Cμ4 domain (Fig S6A, B). We first analyzed intracellular tyrosine phosphorylation as an indicator of B-cell activation. Co-transfection of mIgM-YS/VV-Igα and mIgM-YS/VV-Igβ (mIgM-Igα/β) in J558L cells resulted in expression of a functional receptor that induced phosphorylation in response to either NIP1-H12 or NIP14-BSA (Figure 6B). However, cells expressing either mIgM Cμ3 -4Δ-Igα/β or mIgM Cμ4ΔTM -Igα/β were significantly impaired in tyrosine phosphorylation induced by NIP1-H12. Binding of the multivalent antigen, NIP14-BSA, induced similar protein phosphorylation in all cells indicating the signaling defect is specific for the response to monovalent antigen. Similar impairment of tyrosine phosphorylation was observed in retrovirally transfected primary wild-type B-cell blasts expressing mIgM Cμ3– 4Δ-Igα/β or mIgM Cμ4Δ-Igα/β as compared to mIgM- Igα/β (Figure S6C). We also measured upregulation of CD69 in these transfected B-cell blasts and found that as compared to cells expressing the mIgM-Igα/β both mIgM Cμ3 -4Δ-Igα/β and mIgM Cμ4ΔTM -Igα/β were poor mediators of CD69 upregulation in response to NIP1-H12. In contrast, binding of NIP14-BSA induced comparable CD69 upregulation in all cells (Fig 6C). Collectively these results demonstrate that monovalent antigen binding could neither immobilize, nor efficiently induce signaling and B-cell activation in receptors lacking Cμ4, indicating that the Fc region mediates assembly of the BCR into signaling active oligomers.

Figure 6
Fc region of mIgM is important for the initiation of BCR signaling in response to membrane antigens

The Cμ4 domain of mIg spontaneously clusters and activates B cells

Taken together these results suggest that antigen binding in some way changes the mIg so that it oligomerizes with other antigen-bound mIgs through their Cμ4 and transmembrane domains, leading to the initiation of signaling. We hypothesized that a potential clustering interface in the Fc region may be hidden or sterically blocked by the configuration of the unbound receptor. To test this possibility, we created a series of YS/VV-containing constructs in which the Fab and an increasing number of the constant domains of mIgM were deleted. These constructs contained the Cμ2-Cμ4 domains, Cμ3-Cμ4 or just the Cμ4 domain (Figure 7A). We found that the YS/VV Cμ2-Cμ4 and YS/VV Cμ3-Cμ4 constructs were homogenously distributed on the cell surface similarly to the full-length mIgM YS/VV or wild-type IgM BCR. In contrast, the YS/VV Cμ4 domain appeared clustered in structures similar to those induced by antigen binding to the full-length mIgM (Fig 7A). Single molecule tracking revealed that the YS/VV Cμ2-Cμ4 and YS/VV Cμ3-Cμ4 constructs were highly mobile on the cell surface, whereas approximately 35 % of the YS/VV Cμ4 molecules were immobilized (Figure 7B). The immobilization was a dynamic process as we observed the YS/VV Cμ4 molecules both stopping and starting movement. The clustering was specific for the YS/VV Cμ4 domain, as single domain constructs of either YS/VV Cμ2 or YS/VV Cμ3 were diffusely distributed and mobile on the cell surface (Figure S7A). Introduction of the TM mutation into YS/VV Cμ4 slightly increased the mobility of the construct (data not shown), similarly to the effect of the TM mutation on the NIP1H12-induced immobilization of a full length YS/VV IgM. The spontaneous oligomerization appeared to be a general feature of the membrane proximal domains of mIgs as we also observed spontaneous immobilization of a construct containing Cγ3, the membrane proximal domain of mIgG that is homologous to the Cμ4 domain, but not wild-type mIgG or a construct containing Cγ2-Cγ3 (Fig. S7B).

Figure 7
Spontaneous clustering and B-cell activation by the Cμ4 domain

To determine if the spontaneous clustering of the Cμ4 domain resulted in B cell activation, we determined whether the N-terminally truncated mIgM constructs associate with Igα/β when expressed with a wild-type transmembrane domain and if so if the BCR-like complex was immobilized and activated B cells. We found that the Cμ2-Cμ4, Cμ3-Cμ4 and Cμ4 constructs with wild-type transmembrane domains were transported to the cell surface only in cells expressing Igα/β (Figure S8A) as is the case for wild-type mIgM (Shaw et al., 1990; Tolar et al., 2005) indicating that all the constructs associated with Igα/β. Flow cytometry as well as TIRF microscopy showed that the three constructs were not expressed at the same levels, with the Cμ2-Cμ4 showing the highest and the Cμ4 the lowest expression (Fig S8A, B). Single molecule measurements of the constructs associated with Igα/β showed that while the Cμ2-Cμ4 and Cμ3-Cμ4 constructs were mobile on the cell surface, a substantial fraction of the Cμ4 was immobilized (Figure S8C). Moreover, TIRF microscopy revealed that the Cμ4 domain was co-localized in clusters with Igα, whereas the Cμ2-Cμ4 and Cμ3-Cμ4 constructs with Igα were distributed homogenously over the cell surface (Figure 7C). We next investigated whether the spontaneous clustering of the Cμ4/Igα/β complex induced BCR signaling and B-cell activation. We found that co-expression of the GFP-Syk with the Cμ4/Igα/β in J558L cells lead to recruitment of GFP-Syk to the Cμ4 clusters (Fig 7D inset, Fig S8D) and a modest, but significant increase of GFP-Syk lifetime at the plasma membrane as compared to co-expression of GFP-Syk with Cμ2-Cμ4/Igα/β and Cμ3-Cμ4/Igα/β (Figure 7D) suggesting that Cμ4/Igα/β is constitutively bound to Syk. In addition, analysis of surface CD69 expression in primary B-cell blasts showed that transfection of Cμ4 induced significant upregulation of CD69 in approximately 20% of the transfected cells (Figure 7E). In contrast, B cells transfected with either Cμ2-Cμ4 or Cμ3-Cμ4 showed no increase in the frequency of CD69 positive cells over those of untransfected cells. Thus, despite the relatively lower expression of Cμ4/Igα/β it appeared signaling active as compared to Cμ3-Cμ4/Igα/β or Cμ2-Cμ4/Igα/β. Taken together these results show that the Cμ4 spontaneously clustered when assembled into a BCR-like complex with Igα/β and induced chronic BCR signaling and B-cell activation that was weaker, but qualitatively similar to that induced by antigens.


A number of recent studies showed that the activation of antigen receptors in B and T cells occurs in receptor microclusters that form after the lymphocytes contact antigen presenting surfaces prior to the formation of mature immune synapses (Campi et al., 2005; Depoil et al., 2007; Fleire et al., 2006; Mossman et al., 2005; Varma et al., 2006; Weber et al., 2008; Yokosuka et al., 2005). The exclusive recruitment of signaling proteins into these microclusters suggests that microclusters are sites of the initiation of immunoreceptor signaling. However, the molecular nature of the microclusters is not well understood. Using new single molecule imaging techniques, we provide evidence that there are two levels of organization of BCRs in the immune synapse, first the accumulation of the BCRs and antigen in microscopic microclusters in which the BCRs are confined in their movement but not immobile and second, within the microclusters the submicroscopic molecular-level oligomerization of the antigen bound BCRs mediated by interactions through BCR’s mIg. We show that after binding of monovalent membrane antigens, the immobile oligomers form in a process dependent on the Cμ4 domain and on the N-terminal part of the transmembrane region of the mIgM of the BCR. BCRs with deletion of the Cμ4 domain together with mutation of the transmembrane region of the mIgM were not immobilized by antigen and did not signal. Tracking of single BCR molecules during the initial phase of B cell contact with the antigen containing bilayer when newly formed fluorescent antigen microclusters could be well resolved at the microscopic level, showed that immobile oligomers formed within these microclusters. In contrast to the requirement for the Cμ4 domain in the formation of the immobile oligomers, the accumulation of the BCR and antigen in such microclusters was not affected by deletion of the Cμ4 domain. Collectively, these results indicate that homotypic BCR oligomerization through the Cμ4 and transmembrane domain occurs within synaptic microclusters and is required for initiation of signaling.

Although we have not specifically focused on the basis of the formation of microscopic-size BCR microclusters, we hypothesize that membrane curvature at the contact points between the B cell and the antigen presenting surface leads to a local accumulation of antigen-bound BCRs, promoting oligomerization of antigen-engaged BCRs through the Cμ4 and transmembrane domains. Both our findings and previous reports (Campi et al., 2005; Depoil et al., 2007; Fleire et al., 2006; Varma et al., 2006) indicate that microcluster formation is aided by the topology of the synaptic membranes of the two interacting cells. Here, we observed microclustering of the BCR exclusively in filopodia that first contact the antigen presenting membrane and in membrane ruffles in the periphery of the spreading cell. These membrane structures and their dynamics during B-cell spreading are dependent on the actin cytoskeleton; the involvement of actin is consistent with experiments in T cells, where disruption of actin reduces the number and size of the microclusters and inhibits intracellular signaling (Campi et al., 2005; Varma et al., 2006). Similarly, disruption of actin leads to an impairment of membrane ruffling, resulting in reduced numbers of BCR clusters generated actively at the periphery of the cell (Depoil et al., 2007; Fleire et al., 2006). Thus, membrane topology supported by the actin cytoskeleton is important for BCR accumulation in microclusters. It has also been proposed that in T cells, segregation of the TCR in the synapse can result from differences in the size of the TCR-MHC-peptide complexes as compared to the larger LFA-1 and CD45 molecules (Choudhuri et al., 2005; Davis and van der Merwe, 2006). However, as reported earlier (Fleire et al., 2006), we observed BCR microclustering and oligomerization in the absence of LFA-1 and even in the absence of the large phosphatase, CD45, that are not expressed in J558L cells. In addition, we show that microclustering and oligomerization of mutated forms of the mIgM does not correlate with the size of the constructs. Thus, the role of the size of B-cell surface proteins in BCR microclustering remains unclear. Instead, our results support a role for specific homotypic assembly of the BCR into ordered structures.

For the most part, our understanding of the dynamics of the formation of immunoreceptor microclusters has been limited by the fact that their small size makes them difficult to interrogate by conventional imaging techniques. Here we used single molecule tracking of individual BCRs to overcome such limits. Single molecule imaging determines the position of individual molecules with a precision that exceeds the typical resolution of optical systems. Therefore, BCRs can be tracked even within the ~ 0.5 μm structures such as the microclusters. Our findings that the immobilization of the BCR in synaptic microclusters depends on the Cμ4 domain and the transmembrane region of the mIgM indicate that the immobilization is specific and is not related to molecular crowding or impingement of the BCR between the B cell and the lipid bilayer. What accounts for the immobilization of the BCR oligomer? Although theoretical calculations of diffusion predict a weak dependence of mobility on protein mass (Saffman and Delbrück, 1975), recent experiments in artificial membranes show that the diffusion of membrane proteins is inversely proportional to their cross-sectional radii, leading to a strong reduction of mobility of oligomerized proteins (Gambin et al., 2006; Lee and Petersen, 2003). Possibly, the hydrodynamic continuum model used in the theoretical predictions may not be valid for protein complexes diffusing in a heterogeneous mixture of lipids of finite sizes (Gambin et al., 2006). Alternatively, oligomerization of the BCR may lead to immobilization by oligomerization-induced trapping in corrals of other membrane proteins (Kusumi et al., 2005). In either case, single molecule tracking proves to be a new and insightful way of detecting dynamic oligomerization of membrane receptors. In addition, newly emerging high-resolution techniques based on single molecule imaging (Betzig et al., 2006; Rust et al., 2006) might provide even the size and the structure of such BCR oligomers in the near future.

Our results indicate that the membrane proximal domain of the mIg mediates oligomerization together with the WTxxST motif in the transmembrane region. Interestingly, the transmembrane helix of the mIg has been a subject of several previous studies. It has been shown that, in contrast to the YS/VV mutation that abrogates association with Igα/β and renders the BCR inactive, mutation of only the ST motif had no effect on BCR signaling or internalization (Shaw et al., 1990). However, a more extensive mutation covering the WTxxST motif and extending along the corresponding side of the transmembrane helix in IgD was found to partially inhibit the formation of large BCR complexes in limiting concentrations of detergent (Schamel and Reth, 2000), although the mutation still had little effect on BCR signaling (Pracht et al., 2002). Our data indicate that mutation of the WTxxST motif inhibits the oligomerization of the BCR induced by monovalent antigens. Notably, the mutation was mostly compensated by the presence of the membrane proximal domain of the mIg, explaining the absence of a strong phenotype of the mutation in the above studies. In addition, the effects of the mutation of WTxxST motif were specific for activation by monovalent membrane antigens and therefore were unlikely detectable in the experiments where B cells had been stimulated by soluble multivalent antigens. Together with our results, these studies suggest that the transmembrane region of the mIg plays a role in both association with Igα/β and antigen-induced oligomerization. Exactly how the transmembrane region contributes to BCR oligomerization remains unknown, but it is possible that the transmembrane helices either oligomerize by direct interactions, or indirectly by regulating interactions of the BCR with membrane lipids (Sohn et al., 2006; Sohn et al., 2008; Tolar et al., 2008).

How can the binding of a monovalent membrane antigen oligomerize the BCR? Available crystal structures of antigens bound to the Fab regions of antibodies suggested that antigen binding does not propagate conformational changes to the rest of the Ig molecule and led to the view that the mIg serves as an inert antigen-binding unit in the BCR (Metzger, 1974, 1992). The involvement of the membrane proximal region of mIgM in oligmerization of the BCR described here suggests a more active role for the mIg in the initiation of BCR signaling, at least for membrane antigens. Thus, in analogy with secreted antibody molecules in which both the Fab and the Fc region have discrete biological functions, the results presented here indicate that the Fab domains of the BCR mIg bind antigen, and the Fc domains function to oligmerize the BCR and initiate signaling. Although at present we do not know how the BCR’s binding of monovalent antigens on membranes initiates oligomerization through the Fc domains, we speculate that forces and constrains induced by binding of the BCR to membrane-tethered antigens promote changes in conformation or orientation in the BCR’s extracellular domains that expose a clustering interface on the Cμ4 domain. This hypothesis is supported by our data showing that deletion of the Vμ-Cμ3 domains frees the Cμ4 domain to cluster spontaneously. The role of the Cμ4 domain could be also more complex, involving for example a change in conformation or orientation of the associated Igα/Igβ, important for oligomerization and/or initiation of signaling as proposed for the T-cell receptor (TCR) (Kuhns and Davis, 2007; Kuhns et al., 2006). Thus, the structure of the membrane-proximal extracellular domains of the BCR will be of great future interest. Given the dependence of the putative conformational change on the engagement of membrane antigens and our results showing that the full Fc region of mIg does not cluster spontaneously, it is not surprising that the crystal structures of the immunoglobulin bound to soluble antigens have not yet provided evidence for such oligomerization of the BCR. Further structural studies of the Fc region will be necessary to resolve this issue. Notably, the spatial configuration of the Cμ4 domains of the IgM is shared with other Ig heavy chains suggesting that similar oligomerization may occur with other Ig classes. Indeed, we observed spontaneous clustering of the corresponding domain of mIgG, Cγ3. However, differences in this region may also underlie possible variations in the clustering and function of the different classes of the BCR, as observed in the case of IgD (Depoil et al., 2007).

Spontaneous clustering and activation of B cells by the expression of the Cμ4 domain is reminiscent of the activation of B cells by N-terminally truncated forms of the mIgμ during the heavy chain disease (Corcos, 2007). The heavy chain disease is a neoplastic proliferation of B cells containing mutations in the Igμ gene leading to accumulation of a truncated Igμ protein that cannot pair with a light chain or bind antigen. Although the mechanism, by which the truncated Igμ contributes to the proliferation of the malignant B cells is not well understood, several groups reported that expression of N-terminally truncated Igμ in mice can drive B-cell development without pairing of the truncated Igμ with a surrogate light chain (Corcos et al., 1995; Corcos et al., 1991; Shaffer and Schlissel, 1997). Presumably, the truncated Igμ spontaneously activates BCR signaling pathways. In some cases, the truncated Igμ has been found clustered on the cell surface (Corcos et al., 1995). However, the truncations of the Igμ found in the heavy chain disease are limited to the V1-Cμ1 region that we would not predict to activate the BCR according to our basic model. Nonetheless, it is possible that these mutations perturb the Ig in a way that results in clustering. Future studies will be necessary to understand the mechanism by which the N-terminally truncated mIgμ contributes to the disease.

Our data indicate that despite the importance of membrane topology, simple local accumulation of the BCRs in microclusters is not enough for efficient activation of BCR signaling. Rather, we found that immobilization of the BCR in oligomers, dependent on the constant region of the mIg, was required for efficient signaling. Antigen-induced oligomerization through membrane proximal regions may be a general feature of immunoreceptors (Reich et al., 1997). In this regard, immunoreceptors may be similar to a variety of other receptors, including c-Kit (Yuzawa et al., 2007), Toll-like receptor 3 (Liu et al., 2008) and IL-6Rα/gp130 (Skiniotis et al., 2005), where the crystal structures showed interactions between membrane-proximal extracellular domains in receptor dimers that are proposed to be important for reorientation of the cytoplasmic domains and the initiation of signaling. In the case of B cells, the intrinsic oligomerization of the BCR following monovalent interactions with membrane antigens may allow B cells to respond to antigens in which the B-cell epitopes are not in an optimal configuration for crosslinking of the BCR directly into a signaling-active oligomer (Reth et al., 2000). Thus, the formation of the immunological synapse and the intrinsic microclustering of the BCR may help explain how B cells are able to respond efficiently to the vast range of antigen structures the pathogen world likely presents.


Preparation of planar lipid bilayers containing ICAM-1 and NIP-based antigens

Planar lipid bilayers were prepared by fusing small unilamellar lipid vesicles with clean glass coverslip surface as described (Brian and McConnell, 1984), using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-Dioleoyl-sn-Glycero-3-{[N(5-Amino-1-Carboxypentyl)iminodiAcetic Acid]Succinyl} (Nickel Salt) (DOGS-NiNTA, both from Avanti) at 10:1 ratio. Small unilamellar vesicles were obtained by sonication and clarified by ultracentrifugation and filtering. Glass coverslips were cleaned in Nanostrip (Cyantek), washed and dried. Lipid bilayers were prepared from 0.1 mM lipid solution on the coverslips attached to the bottom of Labtek imaging chambers (Nalgene Nunc). Imaging was performed in HBSS supplemented with 1% fetal calf serum. NIP14-BSA was prepared as described (Tolar et al., 2005) and conjugated to a cystein-containing peptide terminated with twelve-histidine tag (ASTGTASACTSGASSTGSH12) using Succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Pierce) according to manufacturer’s protocols. NIP1-H12 and NIP1-H12- Hylight647 were from Anaspec (NIP-ASTGKTASACTSGASSTGSH12 and NIP-ASTGKTASAC[HyLight647]TSGASSTGSH12). NIP1-H12- DyLight547 was created by conjugation of NIP1-H12 with DyLight547 maleimide (Pierce) and was HPLC purified and verified by mass spectroscopy. Conjugation of NIP14-BSA to succinimidyl AlexaFluor647 and ICAM-1-H12 to AlexaFluor488 (both Molecular Probes) was performed according to manufacturer’s protocols. Attachment of recombinant ICAM-1 (a gift of M. Davis and J. Huppa, Stanford University, Stanford, CA) was through a twelve-histidine tag at the protein’s C-terminus. To quantify the amount of antigen and ICAM-1 bound to the bilayers we titrated the concentration of fluorescently labeled proteins low enough to resolve single molecules; protein concentration on the bilayer at the experimental conditions was then calculated as:


where C is the concentration (number of molecules per area), N is the number of counted protein molecules at single molecule resolution per area A, IS is the average intensity of the single molecule image illuminated with intensity LS and IC is the average intensity of the sample illuminated at intensity LC. Laser illumination intensity was calibrated using a wand laser meter. Unless stated otherwise NIP1-H12 was used approximately at 150 molecules per μm2, NIP14-BSA at 50/μm2 and ICAM-1 at 50/μm2.

Cells and transfections

B cells were isolated from spleens of Balb/c or B1-8+/+ Igκ−/− mice (B1-8 mice with inactivated Igκ locus, a gift of M. Shlomchik, Yale University, New Haven, CT) by negative selection. For transfections, B cells were stimulated for 1 day with CpG and LPS (Calbiochem) and infected by a 90 min centrifugation with retrovirus-containing supernatants in the presence of polybrene (Sigma). Recombinant retroviruses were produced by transient transfection of PLAT-E packaging cells (Morita et al., 2000). B cells were then cultured for 2 days either in the presence of CpG and LPS, or, for analyses of CD69 expression without CpG and LPS. Responses of the B-cell blasts to soluble and membrane antigens were weaker, but qualitatively similar to those of freshly isolated primary B cells. J558L cells were used and transfected as described (Tolar et al., 2005).

Constructs and IgM mutations

GFP-Syk was created as described (Tolar et al., 2005) using a monomeric version of GFP and cloned into pMSCV vector (BD Clontech). The YS/VV mutation (Shaw et al., 1990) of the transmembrane region of B1-8μ-CFP in pcDNA3 (Tolar et al., 2005) and the deletions of the Cμ3- Cμ4 domains (amino acids 368–582), Cμ3 domain only (amino acids 366–460), Cμ4 domain only (amino acids 465–568), Vμ-Cμ1 (amino acids 20–246), Vμ-Cμ2 (amino acids 20–366), Vμ-Cμ3 (amino acids 20–460), and the mutation of the transmembrane domain (WT/VVxxST/VV), as well as the deletion of Vγ1 -Cγ1 1 (amino acids 20–248) and Vγ1 -Cγ12 (amino acids 20–355) from B1-8γ1 -CFP (Tolar et al., 2005) were performed using Quickchange (Stratagene). Constructs containing only Cμ2 or Cμ3 were generated by deleting Cμ3-Cμ4 or Cμ4 from Cμ2-Cμ4 and Cμ3-Cμ4, respectively. To swap the Cμ3- Cμ4 domains with HSVgD stalk (Chitnis and Miller, 1994), the Cμ3- Cμ4 region was replaced with an EcoRV site to which amino acids of the HSVgD (Chitnis and Miller, 1994) (a gift of L. Miller, NIH, Rockville, MD) were ligated. Transient expression in J558L cells that contain an endogenous Igλ1 light chain was as described (Tolar et al., 2005). For functional assays, the mutated B1-8μ heavy chain was fused with the entire intracellular domains of either Igα or Igβ tagged with monomeric CFP, and the resulting constructs were cloned into both pcDNA6 for expression in J558L cells, and into a pMIGR vector modified to contain the Igλ1 light chain after an internal ribosome entry sequence for retroviral transfection into Balb/c B-cell blasts. Constructs containing Igα and Igβ were co-transfected in 1:1 ratio. To express the Cμ2-Cμ4, Cμ3-Cμ4, and Cμ4 in primary B cell blasts, the constructs tagged intracellularly with monomeric YFP were cloned into pMIGR- Igλ1.

TIRF microscopy

TIRF imaging was performed on an Olympus IX-81 microscope equipped with a TIRF port, 100x objective (Carl Zeiss), and CascadeII 512 electron-multiplying camera (Roper Scientific). Image acquisition was controlled using Metamorph (Molecular Devices). Simultaneous dual color imaging was performed using Dual-View image splitter (Optical Insights). All imaging was performed at 37 °C. Cell surface IgM was labeled with IgM-specific Cy2-conjugated rat monoclonal Fab (clone II/41, Rockland), which binds to the Cμ2 domain. Plasma membrane was stained with DiIC16 (Molecular Probes). Before analysis, images were split, aligned, background subtracted and corrected for spectral bleedthroughs using Image Pro Plus (Media Cybernetics).

FRET between NIP1-H12-DyLight547 and NIP1-H12-HyLight647 was measured by acceptor photobleaching. Intracellular tyrosine phosphorylation in synapses was measured by fixing B cells with 2% paraformaldehyde, permeabilizing and staining with anti-phosphotyrosine antibody (4G10-FITC, Millipore). Phosphorylation was expressed as a ratio of the fluorescence of the phosphotyrosine staining and the fluorescence of BCR’s CFP tag. The amounts of bound antigen were calculated as average antigen fluorescence in the synapses minus average antigen fluorescence in the surrounding bilayer.

Measurements of intracellular calcium

For measurements of intracellular calcium, B cells were loaded with Fluor-4AM and FuraRed-AM in the presence of probenecid (all Molecular Probes). Stimulation of the B cells was performed with NIP14-BSA-AlexaFluor647 or NIP1-H12-HyLight647 in HBSS containing 1% serum using a flow cytometer (FACS Calibur, Becton Dickinson). Measurements of intracellular calcium in response to antigens on the bilayers were performed using epifluorescence illumination combined with 647 nm laser TIRF illumination through a prism to allow rapid alternation of excitation of the fluorescently labeled antigen on the bilayers and the calcium indicators inside the cells. Calcium levels were expressed as ratios of Fluo4 to FuraRed fluorescence.

Single molecule tracking and analysis

To track single BCR molecules, primary B cells or transfected J558L cells were labeled with 10 ng/ml of Cμ2 domain-specific Cy3-cojugated monoclonal Fab for 10 min, which allowed resolution of individual fluorescence spots without the need for photobleaching. To label constructs of the isolated Fc region, some of which did not bind the monoclonal antibody, Cy3-labeled polyclonal anti-IgM Fab fragments were used (Jackson). Both antibodies produced identical results in tracking of wild type BCR. Cells were incubated with bilayers for 3–10 min and imaged using TIRF illumination with 514 nm laser (~ 5mW per 100 μm2) and a 590/60 emission filter. Residual laser light was blocked using a notch filter (Semrock). Continuous image streams capturing single BCR molecules were acquired with 35 ms exposures for about 10 s. We found that this time resolution was sufficient to reliably track the BCRs. Data from 5–20 cells were acquired for each condition in each experiment. In the indicated experiments, NIP14-BSA-AlexaFluor647 or NIP1-H12-HyLight647 were imaged simultaneously with Cy3 by adding 647 nm laser illumination and using the Dual-View. In this case, cells were imaged within 1 min of the contact with the bilayer. To minimize fluorescent background in the Cy3 channel when using the labeled antigens, only ~ 1% of the antigen on the bilayer was fluorescently labeled and was supplemented with unlabeled antigens to obtain 15 and 5 μm−2 of NIP1-H12 and NIP14-BSA, respectively. Dual color imaging of single molecules of mIgM constructs in J558L cells was performed with simultaneous visualization of the BCR microclusters by means of their CFP tags. Single molecule tracking and analysis was performed using Matlab (Mathworks) code based on available tracking algorithms (Douglass and Vale, 2005) ( The resulting trajectories were visually inspected and occasional errors in tracking were manually corrected. The positions of the diffraction-limited spots in the trajectories were refined using 2D Gaussian fit. Mean square displacements were calculated from positional coordinates as described (Douglass and Vale, 2005). Short-range diffusion coefficients were calculated from linear fits to mean square displacement data of individual molecules for time intervals 35–140 ms and plotted in cumulative distribution graphs.

To determine the relationship of the BCR trajectories to antigen clusters, the antigen images were averaged through the time, enhanced using a Gaussian high pass filter, and tresholded. Each point in the trajectories was then assigned as being inside or outside of the tresholded image of the antigen clusters. Instant diffusion coefficients were calculated from square displacements during a single time frame. To identify stopping of the BCR, instant diffusion data were scanned for a change from a moving spot (at least 210 ms with instant diffusion in at least 40% of frames > 0.15 μm2/s) to an immobile spot (at least 210 ms with instant diffusion in all frames < 0.05 μm2/s). Results were expressed as probability that a moving particle inside or outside of clusters will stop in the next frame.

Analysis of single molecules of NIP1-H12-HyLight647 on the bilayers at 0.05/μm2 was performed with 10 ms time resolution and fluorescence intensities were measured as averages in 5×5 pixel areas centered on the spots. Lifetime of GFP-Syk molecules at the plasma membrane was calculated as lengths of single molecule tracks.

Supplementary Material


Figure S1. (A) Upregulation of CD86 activation marker in B cells stimulated with bilayers containing the indicated antigens. (B) Intracellular calcium levels in B cells exposed to the indicated concentrations of soluble antigens. The apparent affinity for NIP14BSA and NIP1H12 binding was 0.1 nM and 50 nM, respectively. (C) Co-localization of GFP-Syk in B1-8 B-cell blasts with BCR during spreading on bilayers containing the indicated antigens. (D) Tyrosine phosphorylation in BCR clusters of B1-8 B-cell blasts as determined by intracellular staining with anti-phosphotyrosine antibodies. Scale bars in 5 μm.

Figure S2. Mean square displacements of single BCR molecules in synapses formed with the indicated antigens. The data represent means and SEM (n of molecules in parentheses) from three experiments. Lines represent linear fit to the data based on simple diffusion. Diffusion coefficients (D) derived from the fits are shown.

Figure S3. Examples of single BCR molecules diffusing into antigen clusters and stopping. The trajectories of single BCR molecules (green) are superimposed on the thresholded images of the antigen (red). Plots show instant diffusion coefficient over time. Top bars indicate corresponding localization of the BCR outside (black) or inside (red) of the antigen cluster.

Figure S4. Impaired immobilization of mIgM with Cμ3-Cμ4 swapped with a stalk derived from HSVgD. Data show single molecule diffusion of 724–1286 molecules from three experiments. * indicates p<0.0001 in Kolmogorov-Smirnov tests.

Figure S5. Microclustering and the type of movement of BCR constructs in immune synapses. (A) TIRF images of CFP-tagged BCR constructs in synapses with the indicated antigens. Cells with the YS/VV, and YS/VV Cμ3-4Δ and YS/VV Cμ4ΔTM constructs do not actively spread or transport clusters to the center of the synapse. (B) Confined diffusion of the YS/VV Cμ3-4Δ construct in synapses with NIP1-H12 antigen. Plots show MSDs over time steps up to 0.7 s in BCR trajectories of the mIgM YS/VV or YS/VV Cμ3-4Δ constructs with or without binding to the NIP1-H12 antigen. Data were acquired at 1–3 min of cell spreading when clusters were well resolved. Linearity of the plots indicates random walk, plateau indicates confined diffusion. Data represent means and SEM from 1369–1784 trajectories from three experiments.

Figure S6. The Fc region of the mIgM is important for BCR activation by monovalent membrane antigen in primary B cells. (A) Surface expression levels of IgM-Igα/β fusion constructs in J558L cells as measured by staining with anti-IgM antibodies and flow cytometry. Data represent mean fluorescence of IgM-positive cells. Mean and SEM of four experiments are shown. (B) Expression of IgM-Igα/β fusion constructs in primary B-cell blasts as measured by staining with NIP14BSA-AlexaFluor647. The data represent mean fluorescence of NIP14BSA-binding cells. Means and SEM of seven experiments are shown. (C) Tyrosine phosphorylation in synapses of primary wild type B-cell blasts expressing the indicated constructs. Data represent median and 95% confidence intervals from 24–67 cells from at least two experiments.*, p < 0.05 in Mann-Whitney tests.

Figure S7. Spontaneous immobilization is specific for the Cμ4 domain and is similar with Cγ3 domain. (A) Single molecule diffusion of constructs containing the individual domains of the Fc region of the mIgμ. (B) Schematic representation and single molecule diffusion of constructs of the Fc region of Igγ. In (A) and (B), data are from 627–2503 molecules from at least three experiments. * indicates p<0.0001 in Kolmogorov-Smirnov tests.

Figure S8. Coexpression of Cμ4 domain with Igα and spontaneous immobilization on the cell surface. (A) Cell surface expression of Fc region constructs transfected into J558L alone, or together with Igα-YFP. Surface IgM was detected by staining with anti-IgM antibodies and flow cytometry. Note that J558L cells express endogenous Igβ. Although the anti-IgM antibodies bind less well to the construct containing Cμ4 only, TIRF measurements of the membrane fluorescence of the constructs tagged with CFP confirmed that Cμ4 is expressed at ~30% lower levels then Cμ2-Cμ4 and Cμ3-Cμ4. (B) Expression levels of the indicated constructs in primary B-cell blasts as determined by fluorescence of their YFP tags and flow cytometry. The data show mean and SEM of fluorescence of YFP-positive cells from three experiments. (C) Spontaneous immobilization of the Cμ4 domain associated with Igα/β. Data show diffusion of 655–1604 molecules from four experiments. * indicates p<0.0001 in Kolmogorov-Smirnov tests. (D) Clustering of Cμ4/Igα/β is accompanied by recruitment of Syk. TIRF images show the distribution of the indicated constructs together with GFP-Syk.


Movie S1. Distribution of the BCR (green) as compared to plasma membrane staining with DiIC16 (red) during B-cell spreading on bilayers containing NIP14-BSA.


Movie S2. Formation of a BCR cluster (green) in a small membrane contact of ruffling membranes (red) at the periphery of the immune synapse with bilayers containing NIP14-BSA.


Movie S3. Distribution of the BCR (green) as compared to plasma membrane staining with DiIC16 (red) during B-cell spreading on bilayers containing NIP1-H12.


Movie S4. Formation of a BCR cluster (green) in a small membrane contact of ruffling membranes (red) at the periphery of the immune synapse with bilayers containing NIP1-H12.


Movie S5. Single BCR molecules labeled with anti-IgM-Cy3 Fab on bilayers containing ICAM-1 only.


Movie S6. Single BCR molecules labeled with anti-IgM-Cy3 Fab on bilayers containing NIP1-H12.


Movie S7. Single BCR molecules labeled with anti-IgM-Cy3 Fab on bilayers containing NIP14-BSA.


Movie S8. Single BCR molecule (green) stopping in a cluster of NIP1-H12 (red).


Movie S9. Single GFP-Syk molecules in a primary B-cell blast on a bilayer containing ICAM-1 only.


Movie S10. Single GFP-Syk molecules in a primary B-cell blast on a bilayer containing NIP1-H12.


Movie S11. Single GFP-Syk molecules in a primary B-cell blast on a bilayer containing NIP14-BSA.


Movie S12. Confinement of mIgM Cμ4ΔTM molecules (green) in a cluster (red) formed in a synapse with NIP1-H12.


Movie S13. Confinement of mIgM Cμ3-Cμ4Δ molecules (green) in a cluster (red) formed in a synapse with NIP1-H12.


We thank M. Davis and J. Huppa for advice on bilayer preparation, T. Meckel for advice on single molecule data analysis, Chiung-Yu Huang for advice on statistical analysis and J. Brzostowski for help with imaging and comments on the manuscript. This work has been supported by the Intramural Research program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.


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