Structural principles of B cell antigen receptor assembly.

The B cell antigen receptor (BCR) is composed of a membrane-bound class M, D, G, E or A immunoglobulin for antigen recognition1-3 and a disulfide-linked Igα (also known as CD79A) and Igß (also known as CD79B) heterodimer (Igα/ß) that functions as the signalling entity through intracellular immunoreceptor tyrosine-based activation motifs (ITAMs)4,5. The organizing principle of the BCR remains unknown. Here we report cryo-electron microscopy structures of mouse full-length IgM BCR and its Fab-deleted form. At the ectodomain (ECD), the Igα/ß heterodimer mainly uses Igα to associate with Cµ3 and Cµ4 domains of one heavy chain (µHC) while leaving the other heavy chain (µHC') unbound. The transmembrane domain (TMD) helices of µHC and µHC' interact with those of the Igα/ß heterodimer to form a tight four-helix bundle. The asymmetry at the TMD prevents the recruitment of two Igα/ß heterodimers. Notably, the connecting peptide between the ECD and TMD of µHC intervenes in between those of Igα and Igß to guide TMD assembly through charge complementarity. Weaker but distinct density for the Igß ITAM nestles next to the TMD, suggesting potential autoinhibition of ITAM phosphorylation. Interfacial analyses suggest that all BCR classes utilize a general organizational architecture. Our studies provide a structural platform for understanding B cell signalling and designing rational therapies against BCR-mediated diseases.

Network pharmacology-based strategy to investigate the mechanisms of artemisinin in treating primary Sjögren's syndrome.

OBJECTIVE: The study aimed to explore the mechanism of artemisinin in treating primary Sjögren's syndrome (pSS) based on network pharmacology and experimental validation. METHODS: Relevant targets of the artemisinin and pSS-related targets were integrated by public databases online. An artemisinin-pSS network was constructed by Cytoscape. The genes of artemisinin regulating pSS were imported into STRING database to construct a protein-protein interaction (PPI) network in order to predict the key targets. The enrichment analyses were performed to predict the crucial mechanism and pathway of artemisinin against pSS. The active component of artemisinin underwent molecular docking with the key proteins. Artemisinin was administered intragastrically to SS-like NOD/Ltj mice to validate the efficacy and critical mechanisms. RESULTS: Network Pharmacology analysis revealed that artemisinin corresponded to 412 targets, and pSS related to 1495 genes. There were 40 intersection genes between artemisinin and pSS. KEGG indicated that therapeutic effects of artemisinin on pSS involves IL-17 signaling pathway, HIF-1 signaling pathway, apoptosis signaling pathway, Th17 cell differentiation, PI3K-Akt signaling pathway, and MAPK signaling pathway. Molecular docking results further showed that the artemisinin molecule had higher binding energy by combining with the key nodes in IL-17 signaling pathway. In vivo experiments suggested artemisinin can restored salivary gland secretory function and improve the level of glandular damage of NOD/Ltj mice. It contributed to the increase of regulatory T cells (Tregs) and the downregulated secretion of IL-17 in NOD/Ltj model. CONCLUSION: The treatment of pSS with artemisinin is closely related to modulating the balance of Tregs and Th17 cells via T cell differentiation.

Disease Duration Affects the Clinical Phenotype of Primary Sjögren Syndrome: A Medical Records Review Study of 952 Cases.

OBJECTIVES: To investigate the impact of disease duration on clinical phenotypes in Chinese patients with primary Sjögren syndrome (pSS) and examine the correlation between clinical phenotypes and onset age, age at diagnosis, and disease duration. METHODS: Data from 952 patients diagnosed with pSS in China between January 2013 and March 2022 were analyzed based on medical records. Patients were categorized into 3 groups based on disease duration: short (<5 years), moderate (≥5 and <10 years), and long (≥10 years) group. Clinical characteristics were compared among the 3 groups, and pSS patients with a long disease duration were compared with the other patients after matching age at diagnosis and age at onset. RESULTS: Among the patients, 20.4% had a disease duration over 10 years. After matching for age at onset and age at diagnosis, pSS patients with a long disease duration exhibited a significantly higher prevalence of dry mouth ( p <0.001), dry eyes ( p <0.001), fatigue ( p <0.001), arthralgia ( p <0.001), and dental caries ( p <0.001) and higher rates of anti-Sjögren syndrome A ( p < 0.05), anti-Ro52 ( p < 0.05), and anti-SSB ( p < 0.05) positivity than their control groups, with prevalence increasing with disease duration ( ptrend < 0.001). However, no differences were noted in the prevalence of interstitial lung disease and leukopenia between different disease duration groups after matching for age at onset, although differences were shown when matching for age at diagnosis. CONCLUSION: Longer disease duration in pSS patients correlates with increased prevalence of sicca symptoms, fatigue, and arthralgia and higher positivity of autoantibodies associated with pSS. However, the prevalence of interstitial lung disease and leukopenia did not correlate with disease duration after matching for age at onset.

Disaster process-based spatiotemporal characteristics of apricot frost in the warm temperate zone (WTZ), China.

Frost stress is a major environmental factor that limits apricot growth in the warm temperate zone (WTZ) of China, and is always triggered by extreme low temperature weather processes. In this study, the characteristics of the apricot frost processes f(D, Tcum), which were identified from historical disaster representation, were analyzed and apricot frost evaluation indicators were developed, thus facilitating the process-based assessment and spatiotemporal analysis of apricot frost processes. Periods of low temperature that persist for 1~2, 3, and ≥4 days (i.e., duration days, D) provide the initial identification indicator for light, moderate, and severe apricot frost. The threshold ranges for Tcum are 0~3.9, 9.2~12.0, and >16.2 for D values of 1~2, 3, and ≥4, respectively. The northwest of the WTZ is dominated by apricot frost, with approximately 80% of apricot frost being light, followed by moderate and severe. Regional apricot frost exhibited a significant decreasing trend over the last four decades. A total of 29.65% of stations, which were mainly located in the northwest and middle parts of the study region, detected an increasing trend in apricot frost. The results provide technical support for targeted apricot frost level detection, and the process-based spatiotemporal characteristics of apricot frost can provide basic information for the prevention and mitigation of apricot frost.

Sex Difference in Primary Sjögren Syndrome: A Medical Records Review Study.

OBJECTIVES: The aim of this study was to study clinical and biological differences between men and women with primary Sjögren syndrome (pSS) in China and perform a literature review to confirm if the clinical phenotypes are affected by sex in patients with pSS. METHODS: Data from 961 patients with pSS treated at a tertiary hospital in China between January 2013 and March 2022 were analyzed based on medical records. Clinical characteristics, including disease manifestations and serological parameters of the disease, were compared between men and women with pSS using the Mann-Whitney U test and χ 2 test. RESULTS: This study included 140 (14.6%) men and 821 (85.4%) women with pSS. Women with pSS demonstrated a higher prevalence of dry mouth, dry eyes, arthralgia, and dental caries ( p < 0.05); higher erythrocyte sedimentation rate and immunoglobulin M levels ( p < 0.05); higher prevalence of leukopenia, neutropenia, anemia, low complement 3, and low complement 4 ( p < 0.05); and higher titers of antinuclear antibody, anti-Sjögren syndrome A, anti-Ro52, and rheumatoid factor positivity ( p < 0.05) than men, whereas men with pSS had a higher prevalence of parotid enlargement and interstitial lung disease ( p < 0.05). CONCLUSIONS: Women with pSS are associated with more dryness, cytopenia, hypocomplementemia, and autoantibody positivity. Although men with pSS probably have lighter sicca symptoms and lower immunoactivity and serologic responses, regular monitoring of interstitial lung disease in men is vital.

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells.

Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igß (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igß can be expressed on the B-cell surface, where it is found in close proximity to CD19 and signals in an ITAM-dependent manner. These data suggest that Igß and CD19 are part of an alternative B-cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.

A new branched proximity hybridization assay for the quantification of nanoscale protein-protein proximity.

Membrane proteins are organized in nanoscale compartments. Their reorganization plays a crucial role in receptor activation and cell signaling. To monitor the organization and reorganization of membrane proteins, we developed a new branched proximity hybridization assay (bPHA) allowing better quantification of the nanoscale protein-protein proximity. In this assay, oligo-coupled binding probes, such as aptamer, nanobody, and antibodies, are used to translate the proximity of target proteins to the proximity of oligos. The closely positioned oligos then serve as a template for a maximum of 400-fold branched DNA (bDNA) signal amplification. The amplified bPHA signal is recorded by flow cytometer, thus enabling proximity studies with high throughput, multiplexing, and single-cell resolution. To demonstrate the potential of the bPHA method, we measured the reorganization of the immunoglobulin M (IgM)- and immunoglobulin D (IgD)-class B cell antigen receptor (BCR) on the plasma membrane and the recruitment of spleen tyrosine kinase (Syk) to the BCR upon B lymphocyte activation.

Factors associated with secondary immune thrombocytopenia in patients with primary Sjögren's syndrome: a retrospective study of 639 cases.

OBJECTIVES: To investigate the clinical characteristics and relevant factors of secondary immune thrombocytopenia (ITP) in patients with primary Sjögren's syndrome (pSS). METHODS: Patients with pSS being treated between 2013 and 2020 in China-Japan Friendship Hospital were retrospectively analysed. Clinical characteristics were compared between pSS patients with and without secondary ITP. Logistic regression analysis was performed to identify factors associated with secondary ITP in patients with pSS. RESULTS: 639 patients with pSS were included in this study, among which 566 (88.6%) were women. The prevalence of secondary ITP in patients with pSS were 12.4%. Among pSS patients with secondary ITP, 55.7% had mucocutaneous bleeding and 8.9% experienced visceral bleeding. Lymphopenia (OR=3.154, 95% CI 1.185-8.395, p=0.021), anaemia (OR=2.416, 95% CI 1.250-4.668, p=0.009), low C4 (OR=2.904, 95% CI 1.563-5.394, p=0.001), and positive anti-RNP (OR=2.777, 95% CI 1.070-7.202, p=0.036) were significantly related to secondary ITP, while interstitial lung disease (ILD, OR=0.429, 95% CI 0.203-0.907, p=0.027), ANA ≥1:320 (OR=0.469, 95% CI 0.221-0.996, p=0.049) and positive anti-SSB (OR=0.288, 95% CI 0.126-0.685, p=0.003) were negatively associated with secondary ITP in patients with pSS. CONCLUSIONS: Over 10% of patients with pSS had secondary ITP, among whom visceral bleeding was comparatively rare. Lymphopenia and anaemia were positively related to secondary ITP, while ILD was negatively associated with secondary ITP. Low C4 and positive anti-RNP seem to be two potential risk factors for secondary ITP in patients with pSS, while ANA ≥1:320 and positive anti-SSB may be two potential protective factors.

Distinct clinical phenotypes of primary Sjögren's syndrome differ by onset age: a retrospective study of 742 cases and review of the literature.

OBJECTIVES: To study the clinical characteristics of primary Sjögren's syndrome (pSS) with different onset age, and perform a review of the literature to confirm if the clinical phenotypes are affected by onset age in patients with pSS. METHODS: Data of 742 patients with pSS were retrospectively analysed. Patients were divided into three groups according to onset age: young-onset pSS (YopSS, <35 years), adult-onset pSS (AopSS, ≥35 and ≤65 years), and elderly-onset pSS (EopSS, >65 years). Clinical characteristics were compared among three groups and further multiple comparisons were conducted by Bonferroni adjustment. The Chi-squared test for linear-by-linear association was used to explore variation tendency. RESULTS: This study included 105 (14.2%), 533 (71.8%), and 104 (14.0%) cases of YopSS, AopSS, and EopSS, respectively. YopSS demonstrated lower prevalence of dry mouth, abnormal Schirmer I tests, and interstitial lung disease (ILD), but higher proportions of low C3 and C4 levels, and ANA, anti-SSA, anti-SSB, and rheumatoid factor (RF) positivity than AopSS and EopSS. The proportions of dry mouth (p=0.004), abnormal Schirmer I tests (p=0.002), and ILD (p<0.001) tended to increase with the increase of onset age, while the prevalence of leukopenia (p=0.011), low C3 (p=0.001), low C4 (p=0.001), and ANA (p<0.001), anti-SSA (p<0.001), anti-SSB (p<0.001) and RF (p<0.001) positivity tended to decrease with an increase in onset age. CONCLUSIONS: YopSS demonstrated less dryness and ILD, but more immunologic disorders. ILD prevalence were directly proportional to onset age of pSS; however, leukopenia, hypocomplementaemia, and autoantibody positivity showed opposite trends.

The validity and reliability of quality of life questionnaires in patients with ankylosing spondylitis and non-radiographic axial spondyloarthritis: a systematic review and meta-analysis.

BACKGROUND: Patients who suffered from ankylosing spondylitis (AS) or non-radiographic axial spondyloarthritis (nr-axSpA) often have poor quality of life (QoL) and there has been a substantial increase in research on acceptable questionnaires for assessment of QoL. This systematic review aims at examining the validity and reliability of QoL questionnaires in patients with AS/nr-axSpA. METHODS: Randomized controlled trials (RCTs), cohort trials, and cross-sectional trails were retrieved by searching seven databases. Primary outcomes included test-retest reliability and construct validity. Secondary outcomes included internal consistency, structural validity, responsiveness and so on. Data extraction and analyses were conducted according to the Cochrane standards. The Agency for Healthcare Research and Quality (AHRQ) checklists was used to assess the risk of bias for each included study. We used the Consensus-based Standards for the Selection of Health Status Measurement Instruments (COSMIN) to assess the methodological quality and measurement property of included instruments. The quality of evidence on pre-specified outcomes were assessed by the Grades of Recommendations, Development and Evaluation (GRADE) approach. RESULTS: 22 publications containing 10 self-rating instruments were included in this study. Most studies were cross-sectional in design and a total of 3,085 participants were enrolled. 19 studies had moderate to high test-retest reliability. Cronbach's alpha (α) Coefficients were generally high (0.79-0.97) for overall scales. The ankylosing spondylitis quality of life (ASQOL) and evaluation of ankylosing spondylitis quality of life (EASi-QoL) questionnaires showed the strongest measurement properties in high-quality studies. The correlation coefficient for test-retest reliability of the ASQOL questionnaire was 0.85 (95% CI 0.80 to 0.89). The pooled Cronbach's α coefficients of the ASQOL questionnaire and the EASi-QoL questionnaire were high. Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and Bath Ankylosing Spondylitis Functional Index (BASFI) were considered as two validity criteria. For the ASQOL and EASi-QoL questionnaire, pooled convergent validity associations with BASDAI and BASFI were low to strong (0.24-0.81). CONCLUSIONS: This study indicated acceptable reliability and stability of included QoL questionnaires. The ASQOL and the EASi-QoL questionnaires are validated and reliable disease-specific questionnaires for the assessment of QoL in patients with AS/nr-axSpA.

Research on primary Sjögren's syndrome in 2004-2021: a Web of Science-based cross-sectional bibliometric analysis.

The extent, range, and nature of available research in the field of primary Sjögren's syndrome (pSS) have not been understood fully. This study aimed to map the literature available on pSS, and identify global hotspots and trends in the research. Papers on pSS published between 2004 and 2021 were searched from Web of Science Core Collection. The quantity and citations of publications, and the research hotspots and trends in the field of pSS were analyzed and presented visually by Microsoft Excel and Citespace software. A total of 3606 papers mainly from 526 institutions in 83 countries/regions were included for analysis. The number of publications presented an overall upward trend in the field of pSS from 2004 to 2021. The USA ranked first in the number of publications (n = 661), followed by China (n = 491), Italy (n = 405), France (n = 351), and Japan (n = 292). Moreover, seven of the top ten countries by the number of publications on pSS were from Europe. The University of Groningen (n = 661), Xavier Mariette (n = 95), and Clinical and Experimental Rheumatology (n = 184) were the most prolific affiliation, author, and journal, respectively. Vitali C (n = 2009) and Arthritis and Rheumatism (n = 3918) held the record for the most cited papers by an author and journal, respectively. At present, the hot keywords in the field of pSS include disease activity, ultrasonography, management, consensus, and data-driven. Lymphoid organization, clinical phenotypes outcome, salivary gland ultrasonography, and Toll-like receptor are the emerging research trends in pSS. Research on pSS is flourishing. Current research of pSS mainly focuses on disease activity, ultrasonography, and management. While, the emerging research trends in pSS are lymphoid organization, clinical phenotypes outcome, salivary gland ultrasonography, and Toll-like receptor.

A symmetric geometry of transmembrane domains inside the B cell antigen receptor complex.

B lymphocytes have the ability to sense thousands of structurally different antigens and produce cognate antibodies against these molecules. For this they carry on their surface multiple copies of the B cell antigen receptor (BCR) comprising the membrane-bound Ig (mIg) molecule and the Igα/Igß heterodimer functioning as antigen binding and signal transducing components, respectively. The mIg is a symmetric complex of 2 identical membrane-bound heavy chains (mHC) and 2 identical light chains. How the symmetric mIg molecule is asymmetrically associated with only one Igα/Igß heterodimer has been a puzzle. Here we describe that Igα and Igß both carry on one side of their α-helical transmembrane domain a conserved amino acid motif. By a mutational analysis in combination with a BCR rebuilding approach, we show that this motif is required for the retention of unassembled Igα or Igß molecules inside the endoplasmic reticulum and the binding of the Igα/Igß heterodimer to the mIg molecule. We suggest that the BCR forms within the lipid bilayer of the membrane a symmetric Igα-mHC:mHC-Igß complex that is stabilized by an aromatic proline-tyrosine interaction. Outside the membrane this symmetry is broken by the disulfide-bridged dimerization of the extracellular Ig domains of Igα and Igß. However, symmetry of the receptor can be regained by a dimerization of 2 BCR complexes as suggested by the dissociation activation model.

Sensitive photoelectrochemical detection of colitoxin DNA based on NCDs@CuO/ZnO heterostructured nanocomposites with efficient separation capacity of photo-induced carriers.

A metal-organic framework (MOF) of Cu-TPA (terephthalic acid) microsphere was prepared, followed by calcinating the MOF precursor of Cu-TPA/ZIF-8 mixture to obtain the CuO/ZnO. N-doped carbon dots (NCDs) were employed to combine the CuO/ZnO composite to form a tripartite heterostructured architecture of NCDs@CuO/ZnO, which led to a fierce enlargement of the photocurrent response. This  was ascribed to the thinner-shell structure of the CuO microsphere and the fact that hollow ZnO particles could sharply promote the incidence intensity of visible light. The more porous defectiveness exposed on CuO/ZnO surface was in favor of rapidly infiltrating electrolyte ions. The p-n type CuO/ZnO composite with more contact interface could abridge the transfer distance of photo-induced electron (e-1)/hole (h+) pairs and repress their recombination availably. NCDs not only could boost electron transfer rate on the electrode interface but also successfully sensitized the CuO/ZnO composite, which resulted in high conversion efficiency of photon-to-electron. The probe DNA (S1) was firmly assembled on the modified ITO electrode surface (S1/NCDs@CuO/ZnO) through an amidation reaction. Under optimal conditions, the prepared DNA biosensor displayed a wide linear range of 1.0 × 10-6 ~ 7.5 × 10-1 nM and a low limit of detection (LOD) of 1.81 × 10-7 nM for colitoxin DNA (S2) measure, which exhibited a better photoelectrochemistry (PEC) analysis performance than that obtained by differential pulse voltammetry techniques. The relative standard deviation (RSD) of the sensing platform for target DNA detection of 5.0 × 10-2 nM was 6.3%. This proposed DNA biosensor also showed good selectivity, stability, and reproducibility, demonstrating that the well-designed and synthesized photoactive materials of NCDs@CuO/ZnO are promising candidates for PEC analysis.

Molecular requirements of the B-cell antigen receptor for sensing monovalent antigens.

How the B-cell antigen receptor (BCR) is activated upon interaction with its cognate antigen or with anti-BCR antibodies is not fully understood. We have recently shown that B-cell activation is accompanied by the opening of the pre-organized BCR oligomers, an observation that strengthens the role of receptor reorganization in signalling. We have now analysed the BCR oligomer opening and signalling upon treatment with different monovalent stimuli. Our results indicate that monovalent antigens are able to disturb and open the BCR oligomer, but that this requires the presence and activity of the Src family kinase (SFK) Lyn. We have also shown that monovalent Fab fragments of anti-BCR antibodies can open the BCR oligomers as long as they directly interact with the antigen-binding site. We found that monovalent antigen binding opens both the IgM-BCR and IgD-BCR, but calcium signalling is only seen in cells expressing IgM-BCR; this provides a molecular basis for IgM- and IgD-BCR functional segregation.

Dynamic changes in circulating levels of metabolites in the portal-drained viscera of finishing pigs receiving acute administration of l-arginine.

In this study, we examined the effects of acute intravenous administration of l-arginine on circulating levels of metabolites in the portal-drained viscera (PDV) of 12 barrows surgically fitted with chronic catheters in the portal vein. At day 14 post-surgery, the pigs were fasted for 12 hr and then randomly allocated to one of three groups to receive administration of normal saline, l-alanine [103 mg/kg body weight (BW), isonitrogenous control] or l-arginine-HCl (61 mg/kg BW), via the portal vein. Blood samples were obtained from the carotid artery before and at 30-min intervals for 5 hr after the administration of saline or amino acid in order to determine metabolic profiles. The results showed that, compared with the saline treatment, arginine infusion increased plasma concentrations of insulin-like growth factor-I, arginine and cystine in the portal vein plasma, whereas plasma concentrations of threonine, serine, leucine and methionine were reduced. These findings indicate that increasing arginine concentrations in the portal vein alters the metabolic profile in swine, an established animal model for studying human nutrition and metabolism.

Synthetic biology of B cell activation: understanding signal amplification at the B cell antigen receptor using a rebuilding approach.

Upon activation of the B cell antigen receptor (BCR), the spleen tyrosine kinase (Syk) and the Src family kinase Lyn phosphorylate tyrosines of the immunoreceptor tyrosine-based activation motif (ITAM) of Igα and Igß which further serve as binding sites for the SH2 domains of these kinases. Using a synthetic biology approach, we dissect the roles of different ITAM residues of Igα in Syk activation. We found that a leucine to glycine mutation at the Y+3 position after the first ITAM tyrosine prevents Syk binding and activation. However, a pre-activated Syk can still phosphorylate this tyrosine in trans. Our data show that the formation of a Syk/ITAM initiation complex and trans-ITAM phosphorylation is crucial for BCR signal amplification. In contrast, the interaction of Lyn with the first ITAM tyrosine is not altered by the leucine to glycine mutation. In addition, our study suggests that an ITAM-bound Syk phosphorylates the non-ITAM tyrosine Y204 of Igα only in cis. Collectively, our reconstitution experiments suggest a model whereby first trans-phosphorylation amplifies the BCR signal and subsequently cis-phosphorylation couples the receptor to downstream signaling elements.

A photoelectrochemical aptasensor for thrombin based on the use of carbon quantum dot-sensitized TiO2 and visible-light photoelectrochemical activity.

A photoelectrochemical (PEC) aptasensor for the highly sensitive and specific detection of thrombin is described. This aptasensor is based on an indium tin oxide (ITO) support that is covered with carbon quantum dot (CQD)-sensitized TiO2 and acts as a photoactive matrix. The ITO/TiO2/CQD electrode was prepared by impregnation assembly. It displays an enhanced and steady photocurrent response under irradiation by visible light. A carboxyl-functionalized thrombin-binding aptamer was covalently immobilized on the modified ITO to obtain a PEC aptasensor whose photocurrent decreases with increasing concentration of thrombin. Under 420 nm irradiation at a bias voltage of 0 V, the aptasensor has a linear response in the 1.0 to 250 pM thrombin concentration range, with a 0.83 pM detection limit. Conceivably, this approach can be extended to numerous other PEC aptasensors for the detection of targets for which appropriate aptamers are available. Graphical abstract Schematic of a PEC aptasensor for thrombin. It is based on the use of CQD as the sensitizer, TiO2/CQDs as the photoactive matrix, and the thrombin aptamer as the recognition element.

Receptor Dissociation and B-Cell Activation.

The B-cell antigen receptor (BCR) is one of the most abundant receptors on the surface of B cells with roughly 100,000-200,000 copies per cell. Signaling through the BCR is crucial for the activation and differentiation of B cells. Unlike other receptors, the BCR can be activated by a large set of structurally different ligands, but the molecular mechanism of BCR activation is still a matter of controversy. Although dominant for a long time, the cross-link model (CLM) of BCR activation is not supported by recent studies of the nanoscale organization of the BCR on the surface of resting B cells. In contrast to the prediction of CLM, the numerous BCR complexes on these cells are not randomly distributed monomers but rather form oligomers which reside within membrane confinements. This finding is more in line with the dissociation activation model (DAM), wherein B-cell activation is accompanied by an opening of the auto-inhibited BCR oligomers instead of a cross-linking of the BCR monomers. In this review, we discuss in detail the new findings and their implications for BCR signaling.

The nanoscale organization of the B lymphocyte membrane.

The fluid mosaic model of Singer and Nicolson correctly predicted that the plasma membrane (PM) forms a lipid bi-layer containing many integral trans-membrane proteins. This model also suggested that most of these proteins were randomly dispersed and freely diffusing moieties. Initially, this view of a dynamic and rather unorganized membrane was supported by early observations of the cell surfaces using the light microscope. However, recent studies on the PM below the diffraction limit of visible light (~250nm) revealed that, at nanoscale dimensions, membranes are highly organized and compartmentalized structures. Lymphocytes are particularly useful to study this nanoscale membrane organization because they grow as single cells and are not permanently engaged in cell:cell contacts within a tissue that can influence membrane organization. In this review, we describe the methods that can be used to better study the protein:protein interaction and nanoscale organization of lymphocyte membrane proteins, with a focus on the B cell antigen receptor (BCR). Furthermore, we discuss the factors that may generate and maintain these membrane structures.

Oligomeric organization of the B-cell antigen receptor on resting cells.

B lymphocytes are activated by many different antigens to produce specific antibodies protecting higher organisms from infection. To detect its cognate antigen, each B cell contains up to 120,000 B-cell antigen receptor (BCR) complexes on its cell surface. How these abundant receptors stay silent on resting B cells and how they can be activated by a molecularly diverse set of ligands is poorly understood. Here we show, with the use of a quantitative bifluorescence complementation assay (BiFC), that the BCR has an intrinsic ability to form oligomers on the surface of living cells. A BCR mutant that fails to form oligomers is more active and cannot be expressed stably on the B-cell surface, whereas BiFC-stabilized BCR oligomers are less active and more strongly expressed on the surface. We propose that oligomers are the autoinhibited form of the BCR and that it is the shift from closed BCR oligomers to clustered monomers that drives B-cell activation in a way that is independent of the structural input from the antigen.