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• W1979059478 abstract "B cells are activated in vivo after the B cell receptors (BCRs) bind 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 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 membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cμ4 domain failed to cluster and signal, and conversely, Cμ4 domains alone clustered spontaneously and activated B cells. These results support a unique 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 in vivo after the B cell receptors (BCRs) bind 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 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 membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cμ4 domain failed to cluster and signal, and conversely, Cμ4 domains alone clustered spontaneously and activated B cells. These results support a unique 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 Ig-α and Ig-β, the intracellular domains of which are phosphorylated and interact with effector signaling proteins when clustered into molecular proximity by antigen binding (Reth, 1992Reth M. Antigen receptors on B lymphocytes.Annu. Rev. Immunol. 1992; 10: 97-121Crossref PubMed Google Scholar). During infection, many pathogen-associated antigens are captured and retained on the surfaces of antigen-presenting cells (APCs) (Carrasco and Batista, 2006Carrasco Y.R. Batista F.D. B cell recognition of membrane-bound antigen: An exquisite way of sensing ligands.Curr. Opin. Immunol. 2006; 18: 286-291Crossref PubMed Scopus (78) Google Scholar), such as dendritic cells (Bergtold et al., 2005Bergtold A. Desai D.D. Gavhane A. Clynes R. Cell surface recycling of internalized antigen permits dendritic cell priming of B cells.Immunity. 2005; 23: 503-514Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, Qi et al., 2006Qi H. Egen J.G. Huang A.Y. Germain R.N. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells.Science. 2006; 312: 1672-1676Crossref PubMed Scopus (382) Google Scholar) and subcapsular macrophages (Carrasco and Batista, 2007Carrasco Y.R. Batista F.D. B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node.Immunity. 2007; 27: 160-171Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, Junt et al., 2007Junt T. Moseman E.A. Iannacone M. Massberg S. Lang P.A. Boes M. Fink K. Henrickson S.E. Shayakhmetov D.M. Di Paolo N.C. et al.Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells.Nature. 2007; 450: 110-114Crossref PubMed Scopus (574) Google Scholar, Phan et al., 2007Phan T.G. Grigorova I. Okada T. Cyster J.G. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells.Nat. Immunol. 2007; 8: 992-1000Crossref PubMed Scopus (447) Google Scholar). In addition, antigens ultimately reach B cell follicles in which they are retained on follicular dendritic cells to serve as a reservoir of antigen for germinal-center formation and affinity maturation (Tarlinton, 2006Tarlinton D. B-cell memory: Are subsets necessary?.Nat. Rev. Immunol. 2006; 6: 785-790Crossref PubMed Scopus (111) Google Scholar). B cells have been shown to interact with membrane-tethered antigens both in vitro and in vivo (Batista et al., 2001Batista F.D. Iber D. Neuberger M.S. B cells acquire antigen from target cells after synapse formation.Nature. 2001; 411: 489-494Crossref PubMed Scopus (452) Google Scholar, Phan et al., 2007Phan T.G. Grigorova I. Okada T. Cyster J.G. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells.Nat. Immunol. 2007; 8: 992-1000Crossref PubMed Scopus (447) Google Scholar, Qi et al., 2006Qi H. Egen J.G. Huang A.Y. Germain R.N. Extrafollicular activation of lymph node B cells by antigen-bearing dendritic cells.Science. 2006; 312: 1672-1676Crossref PubMed Scopus (382) Google Scholar, Schwickert et al., 2007Schwickert T.A. Lindquist R.L. Shakhar G. Livshits G. Skokos D. Kosco-Vilbois M.H. Dustin M.L. Nussenzweig M.C. In vivo imaging of germinal centres reveals a dynamic open structure.Nature. 2007; 446: 83-87Crossref PubMed Scopus (347) Google Scholar). In vitro, B cells spread in response to the initial contact with the antigen-containing membrane. This response is dependent on Lyn and Syk, the first two kinases in the B cell signaling cascade, and is accompanied by an intracellular calcium response (Weber et al., 2008Weber M. Treanor B. Depoil D. Shinohara H. Harwood N.E. Hikida M. Kurosaki T. Batista F.D. Phospholipase C-{gamma}2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen.J. Exp. Med. 2008; 205: 853-868Crossref PubMed Scopus (131) Google Scholar). The B cells then contract to form an immunological synapse (Depoil et al., 2007Depoil D. Fleire S. Treanor B.L. Weber M. Harwood N.E. Marchbank K.L. Tybulewicz V.L. Batista F.D. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.Nat. Immunol. 2007; 9: 63-72Crossref PubMed Scopus (243) Google Scholar, Fleire et al., 2006Fleire S.J. Goldman J.P. Carrasco Y.R. Weber M. Bray D. Batista F.D. B cell ligand discrimination through a spreading and contraction response.Science. 2006; 312: 738-741Crossref PubMed Scopus (305) Google Scholar). 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., 2007Depoil D. Fleire S. Treanor B.L. Weber M. Harwood N.E. Marchbank K.L. Tybulewicz V.L. Batista F.D. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.Nat. Immunol. 2007; 9: 63-72Crossref PubMed Scopus (243) Google Scholar, Fleire et al., 2006Fleire S.J. Goldman J.P. Carrasco Y.R. Weber M. Bray D. Batista F.D. B cell ligand discrimination through a spreading and contraction response.Science. 2006; 312: 738-741Crossref PubMed Scopus (305) Google Scholar, Weber et al., 2008Weber M. Treanor B. Depoil D. Shinohara H. Harwood N.E. Hikida M. Kurosaki T. Batista F.D. Phospholipase C-{gamma}2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen.J. Exp. Med. 2008; 205: 853-868Crossref PubMed Scopus (131) Google Scholar). 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., 2005Campi G. Varma R. Dustin M.L. Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling.J. Exp. Med. 2005; 202: 1031-1036Crossref PubMed Scopus (434) Google Scholar, Choudhuri et al., 2005Choudhuri K. Wiseman D. Brown M.H. Gould K. van der Merwe P.A. T-cell receptor triggering is critically dependent on the dimensions of its peptide-MHC ligand.Nature. 2005; 436: 578-582Crossref PubMed Scopus (230) Google Scholar, Depoil et al., 2007Depoil D. Fleire S. Treanor B.L. Weber M. Harwood N.E. Marchbank K.L. Tybulewicz V.L. Batista F.D. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.Nat. Immunol. 2007; 9: 63-72Crossref PubMed Scopus (243) Google Scholar, Fleire et al., 2006Fleire S.J. Goldman J.P. Carrasco Y.R. Weber M. Bray D. Batista F.D. B cell ligand discrimination through a spreading and contraction response.Science. 2006; 312: 738-741Crossref PubMed Scopus (305) Google Scholar, Varma et al., 2006Varma R. Campi G. Yokosuka T. Saito T. Dustin M.L. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.Immunity. 2006; 25: 117-127Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar), 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 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. To explore the mechanisms underlying the initiation of BCR signaling leading to immunological-synapse formation, we used total internal reflection fluorescence (TIRF) microscopy to image the activation of primary splenic B cells from IghB1-8/B1-8Igk−/− mice that express the B1-8 BCR specific for the NIP hapten (Hauser et al., 2007Hauser A.E. Junt T. Mempel T.R. Sneddon M.W. Kleinstein S.H. Henrickson S.E. von Andrian U.H. Shlomchik M.J. Haberman A.M. Definition of germinal-center B cell migration in vivo reveals predominant intrazonal circulation patterns.Immunity. 2007; 26: 655-667Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Sonoda et al., 1997Sonoda E. Pewzner-Jung Y. Schwers S. Taki S. Jung S. Eilat D. Rajewsky K. B cell development under the condition of allelic inclusion.Immunity. 1997; 6: 225-233Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). We first assessed the requirement for physical crosslinking of the BCRs 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 available online) 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). 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 (Figures 2A and 2B). 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 one another (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, NIP1-H12 clusters 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. 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; Movies S1–S4). The first contacts of the B cells with the bilayers appeared as one or more discrete points, in which 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., 2005Campi G. Varma R. Dustin M.L. Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling.J. Exp. Med. 2005; 202: 1031-1036Crossref PubMed Scopus (434) Google Scholar, Depoil et al., 2007Depoil D. Fleire S. Treanor B.L. Weber M. Harwood N.E. Marchbank K.L. Tybulewicz V.L. Batista F.D. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.Nat. Immunol. 2007; 9: 63-72Crossref PubMed Scopus (243) Google Scholar, Yokosuka et al., 2005Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (537) Google Scholar). Additional BCR microclusters formed at the leading edges of the spreading cells and in ruffling membranes at the cell's periphery (Movies S2 and S4), 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., 2007Depoil D. Fleire S. Treanor B.L. Weber M. Harwood N.E. Marchbank K.L. Tybulewicz V.L. Batista F.D. CD19 is essential for B cell activation by promoting B cell receptor-antigen microcluster formation in response to membrane-bound ligand.Nat. Immunol. 2007; 9: 63-72Crossref PubMed Scopus (243) Google Scholar). 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 response 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 (Figure 1D). Thus, the initial BCR microclusters were sufficient to trigger B cell calcium responses. On the basis of 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 50 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 colocalized 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). 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 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 ∼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. To determine whether antigen binding itself was sufficient to immobilize BCR or alternatively whether antigen binding induced a BCR change 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, during which newly formed antigen microclusters could be well resolved by simultaneous imaging of fluorescently labeled antigens to determine whether 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, whereas 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 and 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 NIP14-BSA was (Figure 3D). This observation suggests that the antigen-bound BCR stops when it encounters other antigen-bound BCRs. Second, we tracked single NIP1-H12 antigens bound to B cells at a very low concentration, at which only 5–15 BCRs were engaged in each cell (Figure 3E), making it highly unlikely that antigen-bound BCRs would encounter one another. In this case, the antigens slowed down from their rapid diffusion on the bilayer (∼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 whether 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's 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, whereas GFP-Syk in resting cells was highly mobile, Syk in the antigen-engaged cells was immobile (Movies S9–S11; Figure 3G). Thus, the immobilization of the BCR is accompanied by immobilization of Syk, suggesting that signaling is initiated primarily by immobile BCRs. Because 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 BCRs. Microclustering and immobilization of the BCRs 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 (Figure 4A). Blocking BCR signaling by pharmacological inhibition of Src kinases in primary B cells or deletion of the cytoplasmic domains of Ig-α and Ig-β in J558L cell lines had no effect on the microclustering and immobilization of BCR molecules induced by either the mono- or multivalent 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), which completely disrupts association of the mIgM with the Ig-α-Ig-β heterodimer (Shaw et al., 1990Shaw A.C. Mitchell R.N. Weaver Y.K. Campos-Torres J. Abbas A.K. Leder P. Mutations of immunoglobulin transmembrane and cytoplasmic domains: Effects on intracellular signaling and antigen presentation.Cell. 1990; 63: 381-392Abstract Full Text PDF PubMed Scopus (123) Google Scholar), 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 whether 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 which underlies the immobilization of the BCR by monovalent antigen. 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, given that 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, 1994Chitnis C.E. Miller L.H. Identification of the erythrocyte binding domains of Plasmodium vivax and Plasmodium knowlesi proteins involved in erythrocyte invasion.J. Exp. Med. 1994; 180: 497-506Crossref PubMed Scopus (327) Google Scholar, Cohen et al., 1988Cohen G.H. Wilcox W.C. Sodora D.L. Long D. Levin J.Z. Eisenberg R.J. Expression of herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells.J. Virol. 1988; 62: 1932-1940Crossref PubMed Google Scholar). 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, 2000Schamel W.W. Reth M. Monomeric and oligomeric complexes of the B cell antigen receptor.Immunity. 2000; 13: 5-14Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), 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 is 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 (Figures 5A and 5B). 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 (Figure 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 (Figure 5C; Movies S12 and 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 ∼300 × 300 nm (Figure 5D; Figure 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. We next determined whether 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 because we found that the Cμ4 domain is required for the association of the mIgM with Ig-αβ (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 Ig-α or Ig-β (Williams et al., 1994Williams G.T. Peaker C.J. Patel K.J. Neuberger M.S. The alpha/beta sheath and its cytoplasmic tyrosines are required for signaling by the B-cell antigen receptor but not for capping or for serine/threonine-kinase recruitment.Proc. Natl. Acad. Sci. USA. 1994; 91: 474-478Crossref PubMed Scopus (74) Google Scholar) (Figure 6A). Cotransfectio" @default.
• W1979059478 created "2016-06-24" @default.
• W1979059478 creator A5025853358 @default.
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• W1979059478 date "2009-01-01" @default.
• W1979059478 modified "2023-10-15" @default.
• W1979059478 title "The Constant Region of the Membrane Immunoglobulin Mediates B Cell-Receptor Clustering and Signaling in Response to Membrane Antigens" @default.
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