86 results on '"Pavel Tolar"'
Search Results
2. Editorial: BCR Signaling and B Cell Activation
- Author
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Wanli Liu, Pavel Tolar, Wenxia Song, and Tae Jin Kim
- Subjects
B cell ,B cell receptor (BCR) ,B cell activation ,antibody ,antigen ,imaging ,Immunologic diseases. Allergy ,RC581-607 - Published
- 2020
- Full Text
- View/download PDF
3. B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments
- Author
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Sophie I Roper, Laabiah Wasim, Dessislava Malinova, Michael Way, Susan Cox, and Pavel Tolar
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B cell ,immune synapse ,antigen ,endocytosis ,actin cytoskeleton ,B cell receptor ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.
- Published
- 2019
- Full Text
- View/download PDF
4. Myosin II Synergizes with F-Actin to Promote DNGR-1-Dependent Cross-Presentation of Dead Cell-Associated Antigens
- Author
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Oliver Schulz, Pavel Hanč, Jan P. Böttcher, Robbert Hoogeboom, Sandra S. Diebold, Pavel Tolar, and Caetano Reis e Sousa
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Biology (General) ,QH301-705.5 - Abstract
Summary: Conventional type 1 DCs (cDC1s) excel at cross-presentation of dead cell-associated antigens partly because they express DNGR-1, a receptor that recognizes exposed actin filaments on dead cells. In vitro polymerized F-actin can be used as a synthetic ligand for DNGR-1. However, cellular F-actin is decorated with actin-binding proteins, which could affect DNGR-1 recognition. Here, we demonstrate that myosin II, an F-actin-associated motor protein, greatly potentiates the binding of DNGR-1 to F-actin. Latex beads coated with F-actin and myosin II are taken up by DNGR-1+ cDC1s, and antigen associated with those beads is efficiently cross-presented to CD8+ T cells. Myosin II-deficient necrotic cells are impaired in their ability to stimulate DNGR-1 or to serve as substrates for cDC1 cross-presentation to CD8+ T cells. These results provide insights into the nature of the DNGR-1 ligand and have implications for understanding immune responses to cell-associated antigens and for vaccine design. : Schulz et al. show that dead cells lacking myosin II have a diminished capacity to serve as antigen donors for dendritic cells that express the DNGR-1 receptor. DNGR-1 binds to F-actin exposed on cell corpses, and myosin II organizes actin filaments into bundles that cross-link the receptor more efficiently. Keywords: DNGR-1, CLEC9A, C-type lectin, dendritic cells, actin, actin-binding proteins, myosin II
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- 2018
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5. Myosin IIa Promotes Antibody Responses by Regulating B Cell Activation, Acquisition of Antigen, and Proliferation
- Author
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Robbert Hoogeboom, Elizabeth M. Natkanski, Carla R. Nowosad, Dessislava Malinova, Rajesh P. Menon, Antonio Casal, and Pavel Tolar
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Biology (General) ,QH301-705.5 - Abstract
Summary: B cell responses are regulated by antigen acquisition, processing, and presentation to helper T cells. These functions are thought to depend on contractile activity of non-muscle myosin IIa. Here, we show that B cell-specific deletion of the myosin IIa heavy chain reduced the numbers of bone marrow B cell precursors and splenic marginal zone, peritoneal B1b, and germinal center B cells. In addition, myosin IIa-deficient follicular B cells acquired an activated phenotype and were less efficient in chemokinesis and extraction of membrane-presented antigens. Moreover, myosin IIa was indispensable for cytokinesis. Consequently, mice with myosin IIa-deficient B cells harbored reduced serum immunoglobulin levels and did not mount robust antibody responses when immunized. Altogether, these data indicate that myosin IIa is a negative regulator of B cell activation but a positive regulator of antigen acquisition from antigen-presenting cells and that myosin IIa is essential for B cell development, proliferation, and antibody responses. : B cell antigen acquisition, processing, and presentation may depend on contractile activity of the actomyosin cytoskeleton. Here, Hoogeboom et al. show that non-muscle myosin IIa positively regulates B cell antigen acquisition from antigen-presenting cells in vivo. In addition, myosin IIa negatively regulates B cell activation and is required for B cell cytokinesis. Keywords: B cell response, B cell development, B cell signaling, antigen internalization, antigen presentation, cytoskeleton, non-muscle myosin
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- 2018
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- View/download PDF
6. Activation of the B cell receptor leads to increased membrane proximity of the Igα cytoplasmic domain.
- Author
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Wing-Yiu Lee and Pavel Tolar
- Subjects
Medicine ,Science - Abstract
Binding of antigen to the B cell receptor (BCR) induces conformational changes in BCR's cytoplasmic domains that are concomitant with phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs). Recently, reversible folding of the CD3ε and ξ chain ITAMs into the plasma membrane has been suggested to regulate T cell receptor signaling. Here we show that the Igα and Igβ cytoplasmic domains of the BCR do not associate with plasma membrane in resting B cells. However, antigen binding and ITAM phosphorylation specifically increased membrane proximity of Igα, but not Igβ. Thus, BCR activation is accompanied by asymmetric conformational changes, possibly promoting the binding of Igα and Igβ to differently localized signaling complexes.
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- 2013
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7. Structural basis for Fc receptor recognition of immunoglobulin M
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Qu Chen, Rajesh P. Menon, Laura Masino, Pavel Tolar, and Peter B. Rosenthal
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Structural Biology ,Molecular Biology - Published
- 2023
8. Structural basis for Fc receptor recognition of immunoglobulin M
- Author
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Qu Chen, Rajesh Menon, Pavel Tolar, and Peter B. Rosenthal
- Abstract
FcμR is the IgM-specific Fc receptor involved in the survival and activation of B cells. Using cryo-EM, we reveal eight binding sites for the human FcμR Ig domain on the IgM pentamer, one of which overlaps with the receptor binding site for the transcytosis receptor pIgR, but a different mode of binding explains Ig isotype specificity. The complex explains engagement with polymeric serum IgM and the monomeric IgM B cell receptor.
- Published
- 2022
9. Long-term retention of antigens in germinal centres is controlled by the spatial organisation of the follicular dendritic cell network
- Author
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Ana Martinez-Riano, Shenshen Wang, Stefan Boeing, Sophie Minoughan, Antonio Casal, Katelyn M Spillane, Burkhard Ludewig, and Pavel Tolar
- Abstract
Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We show the existence of two light zone (LZ) FDC populations, which differ in the duration of antigen retention. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as a long-term antigen reservoir, where different antigens arriving from subsequent immunizations co-localize. Mechanistically, central FDCs constitutively express subtly higher CR2 membrane densities than peripheral FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.
- Published
- 2022
10. Unveiling the B cell receptor structure
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Pavel Tolar and Susan K. Pierce
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Multidisciplinary ,Immunoglobulin M ,Protein Conformation ,Cryoelectron Microscopy ,Humans ,CD79 Antigens - Abstract
Molecular structures provide a road map for understanding and controlling B cell receptor activation
- Published
- 2022
11. Digital holography-based 3D particle localisation for single molecule tweezer techniques
- Author
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James L. Flewellen, Sophie Minoughan, Isabel Llorente Garcia, and Pavel Tolar
- Abstract
We present a three-dimensional imaging technique for fast tracking of microscopic objects in a fluid environment. Our technique couples digital holographic microscopy with three-dimensional localisation via parabolic masking. Compared with existing approaches, our method reconstructs 3D volumes from single-plane images, which greatly simplifies image acquisition, reduces the demand on microscope hardware, and facilitates tracking higher densities of microscopic particles while maintaining similar levels of precision. We demonstrate utility of this method in magnetic tweezer experiments, opening their use to multiplexed single-molecule force spectroscopy assays. We propose that our technique will also be useful in other applications that involve the tracking of microscopic objects in three dimensions.SIGNIFICANCETracking objects in 3D is a common task in biology, but typically requires the acquisition of image stacks, which is limited by speed, the depth of field of microscope objectives and by the presence of other objects that obscure the illumination. Here we develop HoloMiP (Holographic Microscopy with Parabolic masking), which uses digital holography to reconstruct the three-dimensional images from a single plane allowing tracking of light-scattering objects in 3D. HoloMiP outperforms existing methods in precision, speed, simplicity and tolerance to crowding. We show that it is particularly suitable for fast, multiplexed magnetic tweezer experiments, opening new avenues to high-throughput force spectroscopy.
- Published
- 2022
12. Mechanistic Insights into Deregulated TGF‐β Family Signalling in Human Disease
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Caroline S. Hill, Ilaria Gori, Svend Kjaer, Roger George, Anassuya Ramachandran, Merima Mehic, Pavel Tolar, Marko Hyvonen, and Chris Jones
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Genetics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2022
13. Cryomicroscopy reveals the structural basis for a flexible hinge motion in the immunoglobulin M pentamer
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Qu Chen, Rajesh Menon, Lesley J. Calder, Pavel Tolar, and Peter B. Rosenthal
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Model organisms ,Chemical Biology & High Throughput ,Multidisciplinary ,FOS: Clinical medicine ,Immunology ,General Physics and Astronomy ,Infectious Disease ,General Chemistry ,Cell Biology ,General Biochemistry, Genetics and Molecular Biology ,Imaging ,Immunoglobulin Fc Fragments ,Immunoglobulin A ,Immunoglobulin Fab Fragments ,Immunoglobulin M ,Immunoglobulin G ,Humans ,Structural Biology & Biophysics - Abstract
Immunoglobulin M (IgM) is the most ancient of the five isotypes of immunoglobulin (Ig) molecules and serves as the first line of defence against pathogens. Here, we use cryo-EM to image the structure of the human full-length IgM pentamer, revealing antigen binding domains flexibly attached to the asymmetric and rigid core formed by the Cμ4 and Cμ3 constant regions and the J-chain. A hinge is located at the Cμ3/Cμ2 domain interface, allowing Fabs and Cμ2 to pivot as a unit both in-plane and out-of-plane. This motion is different from that observed in IgG and IgA, where the two Fab arms are able to swing independently. A biased orientation of one pair of Fab arms results from asymmetry in the constant domain (Cμ3) at the IgM subunit interacting most extensively with the J-chain. This may influence the multi-valent binding to surface-associated antigens and complement pathway activation. By comparison, the structure of the Fc fragment in the IgM monomer is similar to that of the pentamer, but is more dynamic in the Cμ4 domain.
- Published
- 2021
14. Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses
- Author
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Pavel Tolar, Laabiah Wasim, Rebecca Newman, Ana Martínez-Riaño, Niklas Engels, and Dessislava Malinova
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Endosome ,endophilin A2 ,media_common.quotation_subject ,B‐cell responses ,Immunology ,Antigen presentation ,Infectious Disease ,Endosomes ,Endocytosis ,EMBO20 ,antigen uptake ,Biochemistry ,Article ,Imaging ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,EMBO19 ,Genetics ,medicine ,endocytosis ,Animals ,Membrane & Intracellular Transport ,Internalization ,Endophilin-A2 ,Molecular Biology ,B cell ,030304 developmental biology ,media_common ,B-Lymphocytes ,0303 health sciences ,Chemistry ,FOS: Clinical medicine ,Germinal center ,Cell Biology ,Articles ,Germinal Center ,Clathrin ,Cell biology ,medicine.anatomical_structure ,germinal center ,030217 neurology & neurosurgery ,Structural Biology & Biophysics - Abstract
Antigen‐specific B‐cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B‐cell endosomal trafficking pathways and their specific roles in B‐cell responses have not been systematically investigated. Here, we report high‐throughput identification of genes regulating B‐cell receptor (BCR)‐mediated antigen internalization using genome‐wide functional screens. We show that antigen internalization depends both on constitutive, clathrin‐mediated endocytosis and on antigen‐induced, clathrin‐independent endocytosis mediated by endophilin A2. Although endophilin A2‐mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B‐cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2‐deficient mice show defects in GC B‐cell responses and production of high‐affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin‐independent intracellular trafficking in GC B‐cell clonal expansion and antibody responses., A genome‐wide screen reveals a new clathrin‐independent mechanism of antigen uptake in B lymphocytes. The pathway is regulated by endophilin A2, a protein essential for germinal centre expansion and antibody responses.
- Published
- 2021
15. Pathogenic ACVR1R206H activation by Activin A‐induced receptor clustering and autophosphorylation
- Author
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Beata K. Blaszczyk, Marko Hyvönen, Eileen M. Shore, Anassuya Ramachandran, Pavel Tolar, Diana Carvalho, Chris Jones, Caroline S. Hill, Dessislava Malinova, Laabiah Wasim, Merima Mehić, Ilaria Gori, Ramachandran, Anassuya [0000-0002-0123-6539], Mehić, Merima [0000-0001-8249-5526], Malinova, Dessislava [0000-0003-0784-5703], Gori, Ilaria [0000-0003-1913-8520], Shore, Eileen M [0000-0003-2609-6971], Jones, Chris [0000-0001-8118-2296], Hyvönen, Marko [0000-0001-8683-4070], Tolar, Pavel [0000-0003-4693-7299], Hill, Caroline S [0000-0002-8632-0480], and Apollo - University of Cambridge Repository
- Subjects
animal structures ,Biology ,ACVR1 ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,03 medical and health sciences ,Mice ,0302 clinical medicine ,medicine ,Animals ,Cluster Analysis ,Humans ,FOP ,Phosphorylation ,Receptor ,Molecular Biology ,ACVR1B ,ACVR1R206H ,030304 developmental biology ,Cancer ,0303 health sciences ,General Immunology and Microbiology ,General Neuroscience ,Autophosphorylation ,Articles ,Activin A ,medicine.disease ,Cell biology ,Activins ,HEK293 Cells ,Myositis Ossificans ,Fibrodysplasia ossificans progressiva ,embryonic structures ,Mutation ,DIPG ,NIH 3T3 Cells ,Receptor clustering ,Activin Receptors, Type I ,030217 neurology & neurosurgery ,receptor clustering ,ACVR2A ,Signal Transduction - Abstract
Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF‐β family type I receptor. ACVR1R206H is a frequent mutation in both diseases. Pathogenic signaling via the SMAD1/5 pathway is mediated by Activin A, but how the mutation triggers aberrant signaling is not known. We show that ACVR1 is essential for Activin A‐mediated SMAD1/5 phosphorylation and is activated by two distinct mechanisms. Wild‐type ACVR1 is activated by the Activin type I receptors, ACVR1B/C. In contrast, ACVR1R206H activation does not require upstream kinases, but is predominantly activated via Activin A‐dependent receptor clustering, which induces its auto‐activation. We use optogenetics and live‐imaging approaches to demonstrate Activin A‐induced receptor clustering and show it requires the type II receptors ACVR2A/B. Our data provide molecular mechanistic insight into the pathogenesis of FOP and DIPG by linking the causal activating genetic mutation to disrupted signaling., Activation of mutated TGF‐β family type I receptors independent of upstream type II receptor kinase activity promotes SMAD signaling in human glioma cells.
- Published
- 2021
16. Genome-wide screens identify calcium signaling as a key regulator of IgE+ plasma cell differentiation and survival
- Author
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Rebecca Newman and Pavel Tolar
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biology ,Cell growth ,Plasma cell differentiation ,biology.protein ,Antibody ,Immunoglobulin E ,Receptor ,PI3K/AKT/mTOR pathway ,Calcium in biology ,Calcium signaling ,Cell biology - Abstract
SummaryIgE antibodies protect against toxins and parasites, however, they also mediate allergic reactions. In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. Although the intrinsic differences of IgE+ B cells have been linked to signaling by the IgE-B cell receptor (BCR), the molecular pathways controlling their behavior remain poorly understood. Here we employ whole-genome CRISPR screening to identify genes regulating IgE+ B cell proliferation, survival and differentiation into PCs. We show that IgE+ B cells are selectively suppressed by the IgE-BCR signaling to intracellular calcium, which inhibits PC differentiation and limits their lifespan after differentiation. Consequently, manipulation of calcium signaling in vivo enhances IgE+ PC responses. Insights from this pathway shed new light on the self-limiting character of IgE responses and open new avenues to eliminate IgE+ PCs in allergy.
- Published
- 2021
17. Chronic calcium signaling in IgE
- Author
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Rebecca, Newman and Pavel, Tolar
- Subjects
Bcl-2-Like Protein 11 ,Cell Survival ,Calcineurin ,Plasma Cells ,B-Lymphocyte Subsets ,Receptors, Antigen, B-Cell ,Apoptosis ,Cell Differentiation ,Immunoglobulin E ,Mice, Inbred C57BL ,Mice ,Phosphatidylinositol 3-Kinases ,Immunoglobulin G ,Hypersensitivity ,Animals ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Calcium Signaling ,Immunologic Memory ,Cells, Cultured - Abstract
In contrast to other antibody isotypes, B cells switched to IgE respond transiently and do not give rise to long-lived plasma cells (PCs) or memory B cells. To better understand IgE-BCR-mediated control of IgE responses, we developed whole-genome CRISPR screening that enabled comparison of IgE
- Published
- 2021
18. Endophilin A2 regulates B cell protein trafficking and humoral responses
- Author
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Pavel Tolar, Dessislava Malinova, Niklas Engels, and Laabiah Wasim
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0303 health sciences ,Chemistry ,media_common.quotation_subject ,B-cell receptor ,Germinal center ,Endocytosis ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Antigen ,medicine ,Internalization ,Receptor ,030217 neurology & neurosurgery ,Intracellular ,B cell ,030304 developmental biology ,media_common - Abstract
Antigen-specific B cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B cell endosomal trafficking pathways and their specific roles in B cell responses have not been systematically investigated. Here we report high-throughput identification of genes regulating B cell receptor (BCR)-mediated antigen internalization using genome-wide functional screens. We show that antigen internalization depends both on clathrin-coated pits and on clathrin-independent endocytosis mediated by endophilin A2. Although endophilin A2 is dispensable for presentation of the endocytosed antigen, it is required for metabolic support of germinal center (GC) B cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2 deficient mice show selective defects in GC B cell responses and production of high-affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin-independent intracellular trafficking in GC B cell clonal expansion and antibody responses.HIGHLIGHTSGenome-wide CRISPR screens comprehensively identify genes regulating antigen uptake in B cellsB cell receptor-mediated antigen internalization is mediated by both epsin1- dependent clathrin-coated pits and a novel fast endophilin A2-mediated endocytosis.Endophilin A2 is required for peripheral B cell development, antigen-specific germinal center responses and high-affinity IgG production.Endophilin A2 is broadly essential for B cell intracellular trafficking pathways providing metabolic support of germinal center B cell proliferation, in part through regulation of iron uptake.
- Published
- 2020
19. BCR Signaling and B Cell Activation
- Author
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Wanli Liu, Tae Jin Kim, Wenxia Song, and Pavel Tolar
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lcsh:Immunologic diseases. Allergy ,Co-receptor ,co-receptor ,Immunology ,B cell activation ,Receptors, Antigen, B-Cell ,Lymphocyte Activation ,03 medical and health sciences ,Mice ,0302 clinical medicine ,antigen ,Antigen ,antibody ,medicine ,Immunology and Allergy ,Animals ,Humans ,Cytoskeleton ,B cell ,030304 developmental biology ,B-cell activation ,0303 health sciences ,B-Lymphocytes ,biology ,Chemistry ,imaging ,cytoskeleton ,Bcr signaling ,B cell receptor (BCR) ,Cell biology ,medicine.anatomical_structure ,Editorial ,030220 oncology & carcinogenesis ,biology.protein ,Antibody ,lcsh:RC581-607 ,Signal Transduction - Published
- 2020
20. B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments
- Author
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Dessislava Malinova, Pavel Tolar, Susan Cox, Sophie I. Roper, Laabiah Wasim, and Michael Way
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0301 basic medicine ,actin cytoskeleton ,Immunological Synapses ,Mouse ,Cytoskeleton organization ,Imaging ,Immunology and Inflammation ,0302 clinical medicine ,Biology (General) ,Cytoskeleton ,B-Lymphocytes ,B cell ,biology ,Chemistry ,General Neuroscience ,immune synapse ,General Medicine ,Cell biology ,medicine.anatomical_structure ,Formins ,Medicine ,Research Article ,Human ,Model organisms ,QH301-705.5 ,Science ,Neuroscience(all) ,B-cell receptor ,Immunology ,Antigen-Presenting Cells ,Receptors, Antigen, B-Cell ,Mice, Transgenic ,Infectious Disease ,macromolecular substances ,Biochemistry & Proteomics ,Actin-Related Protein 2-3 Complex ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Signalling & Oncogenes ,antigen ,Antigen ,Cell Line, Tumor ,Immunology and Microbiology(all) ,medicine ,Animals ,Humans ,endocytosis ,Antigens ,Actin ,B cell receptor ,General Immunology and Microbiology ,Biochemistry, Genetics and Molecular Biology(all) ,FOS: Clinical medicine ,Cell Biology ,Actin cytoskeleton ,Actins ,Mice, Inbred C57BL ,HEK293 Cells ,030104 developmental biology ,Antibody Formation ,biology.protein ,030217 neurology & neurosurgery ,Structural Biology & Biophysics - Abstract
Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in synapses of primary mouse B cells and human B cell lines forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.
- Published
- 2020
- Full Text
- View/download PDF
21. Monocytes work harder under pressure
- Author
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Andreas Wack and Pavel Tolar
- Subjects
Work (electrical) ,Chemistry ,Macrophages ,Immunology ,Immunity ,Immunology and Allergy ,Humans ,Data science ,Lung ,Ion Channels ,Monocytes - Published
- 2019
22. Author response: B cells extract antigens at Arp2/3-generated actin foci interspersed with linear filaments
- Author
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Dessislava Malinova, Susan Cox, Pavel Tolar, Laabiah Wasim, Sophie I. Roper, and Michael Way
- Subjects
Antigen ,Chemistry ,Actin ,Cell biology - Published
- 2019
23. B cells extract antigens using Arp2/3-generated actin foci interspersed with linear filaments
- Author
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Michael Way, Susan Cox, Laabiah Wasim, Sophie I. Roper, Dessislava Malinova, and Pavel Tolar
- Subjects
biology ,Cytoskeleton organization ,Chemistry ,media_common.quotation_subject ,macromolecular substances ,Cell biology ,medicine.anatomical_structure ,Immune system ,Antigen ,Formins ,medicine ,biology.protein ,Cytoskeleton ,Internalization ,B cell ,Actin ,media_common - Abstract
Antibody production depends on B cell internalization and presentation of antigens to helper T cells. To acquire antigens displayed by antigen-presenting cells, B cells form immune synapses and extract antigens by the mechanical activity of the acto-myosin cytoskeleton. While cytoskeleton organization driving the initial formation of the B cell synapse has been studied, how the cytoskeleton supports antigen extraction remains poorly understood. Here we show that after initial cell spreading, F-actin in B cell synapses forms a highly dynamic pattern composed of actin foci interspersed with linear filaments and myosin IIa. The foci are generated by Arp2/3-mediated branched-actin polymerization and stochastically associate with antigen clusters to mediate internalization. However, antigen extraction also requires the activity of formins, which reside near the foci and produce the interspersed filaments. Thus, a cooperation of branched-actin foci supported by linear filaments underlies B cell mechanics during antigen extraction.
- Published
- 2019
- Full Text
- View/download PDF
24. Decision letter: Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T cells
- Author
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Francisco Sánchez Madrid, Pavel Tolar, and Morgan Huse
- Subjects
CD40 ,biology ,Chemistry ,Antigen receptor ,biology.protein ,Composition (combinatorics) ,Cell biology ,Helper t-cells - Published
- 2019
25. Decision letter: Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation
- Author
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Pavel Tolar and Bebhinn Treanor
- Subjects
biology ,Chemistry ,breakpoint cluster region ,biology.protein ,Arp2/3 complex ,Bcr signaling ,B-cell activation ,Immunological synapse ,Cell biology - Published
- 2019
26. MHC class II cell-autonomously regulates self-renewal and differentiation of normal and malignant B cells
- Author
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Jan Attig, Urszula Eksmond, Julia Merkenschlager, Pavel Tolar, Carla R. Nowosad, George Kassiotis, George R. Young, and Luca Danelli
- Subjects
CD4-Positive T-Lymphocytes ,Male ,Hematopoiesis and Stem Cells ,LYMPHOPOIESIS ,Cellular differentiation ,Cell ,LIPOPOLYSACCHARIDE ,Lymphocyte Activation ,Biochemistry ,Imaging ,ACTIVATION ,Mice ,MOLECULES ,Bone Marrow ,1102 Cardiorespiratory Medicine and Haematology ,TRANSGENIC MICE ,B-Lymphocytes ,Hematopoietic stem cell differentiation ,Cell Differentiation ,Hematology ,DISTINGUISHES ,Cell biology ,DNA-Binding Proteins ,medicine.anatomical_structure ,Disease Progression ,Female ,Stem cell ,Life Sciences & Biomedicine ,Genetics & Genomics ,EXPRESSION ,Model organisms ,Antigen presentation ,Immunology ,Bone Marrow Cells ,Infectious Disease ,Biology ,Cell Line, Tumor ,Leukemia, B-Cell ,ANTIGEN PRESENTATION ,medicine ,Animals ,Computational & Systems Biology ,Homeodomain Proteins ,MHC class II ,Science & Technology ,PRO-B ,FOS: Clinical medicine ,Histocompatibility Antigens Class II ,1103 Clinical Sciences ,Cell Biology ,Mice, Inbred C57BL ,Cell culture ,T-CELLS ,biology.protein ,1114 Paediatrics and Reproductive Medicine ,Bone marrow ,Structural Biology & Biophysics - Abstract
Best known for presenting antigenic peptides to CD4+ T cells, major histocompatibility complex class II (MHC II) also transmits or may modify intracellular signals. Here, we show that MHC II cell-autonomously regulates the balance between self-renewal and differentiation in B-cell precursors, as well as in malignant B cells. Initiation of MHC II expression early during bone marrow B-cell development limited the occupancy of cycling compartments by promoting differentiation, thus regulating the numerical output of B cells. MHC II deficiency preserved stem cell characteristics in developing pro-B cells in vivo, and ectopic MHC II expression accelerated hematopoietic stem cell differentiation in vitro. Moreover, MHC II expression restrained growth of murine B-cell leukemia cell lines in vitro and in vivo, independently of CD4+ T-cell surveillance. Our results highlight an important cell-intrinsic contribution of MHC II expression to establishing the differentiated B-cell phenotype.
- Published
- 2019
- Full Text
- View/download PDF
27. Decision letter: Conformational change within the extracellular domain of B cell receptor in B cell activation upon antigen binding
- Author
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Pavel Tolar and Balbino Alarcón
- Subjects
Conformational change ,Chemistry ,B-cell receptor ,Domain (ring theory) ,Extracellular ,Biophysics ,Antigen binding ,B-cell activation - Published
- 2018
28. Intrinsic properties of human germinal center B cells set antigen affinity thresholds
- Author
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Haewon Sohn, Katelyn M. Spillane, Pavel Tolar, Joseph Brzostowski, Avva Saniee, Susan K. Pierce, Kihyuck Kwak, Prasida Holla, Nicolas Quizon, Hengyi Xie, Jinghua Lu, Javier Manzella-Lapeira, Chenguang Xu, Commission of the European Communities, and European Research Council
- Subjects
0301 basic medicine ,DYNAMICS ,media_common.quotation_subject ,Naive B cell ,Immunology ,Antibody Affinity ,PROTEIN ,Article ,Affinity maturation ,Antigen-Antibody Reactions ,ACTIVATION ,03 medical and health sciences ,0302 clinical medicine ,Antigen ,REVEALS ,Synapse formation ,Humans ,Internalization ,Actin ,media_common ,Antigen Presentation ,B-Lymphocytes ,Science & Technology ,Chemistry ,breakpoint cluster region ,Germinal center ,General Medicine ,Germinal Center ,Cell biology ,030104 developmental biology ,MEMORY B ,DIFFERENTIATION ,CENTER SELECTION ,Life Sciences & Biomedicine ,030217 neurology & neurosurgery ,RESPONSES - Abstract
Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (Tfh) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to an- tigen affinity– and Tfh cell–dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.
- Published
- 2018
29. DNA-Based Probes for Measuring Mechanical Forces in Cell-Cell Contacts: Application to B Cell Antigen Extraction from Immune Synapses
- Author
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Katelyn M, Spillane and Pavel, Tolar
- Subjects
B-Lymphocytes ,Immunological Synapses ,Animals ,Humans ,Receptors, Antigen, B-Cell ,Cell Communication ,Stress, Mechanical ,Antigens - Abstract
The production of antibodies requires the expansion and selection of high-affinity B cell clones. This process is initiated by antigen uptake through the B cell receptor (BCR), which recognizes and binds antigen displayed on the surface of an antigen-presenting cell (APC). To acquire the antigen, B cells use myosin contractility to physically pull BCR-antigen clusters from the APC membrane. These mechanical forces influence association and dissociation rates of BCR-antigen bonds, resulting in affinity-dependent acquisition of antigen by B cells. Mechanical regulation of B cell antigen acquisition from APCs remains poorly understood, although the recent development of DNA-based force sensors has enabled the measurement of mechanical forces generated in B cell-APC contacts. In this chapter, we describe a protocol to design, synthesize, and purify DNA-based force sensors to measure B cell antigen extraction forces using fluorescence microscopy.
- Published
- 2018
30. Great stretches for your antibody workout
- Author
-
Pavel Tolar, Commission of the European Communities, and European Research Council
- Subjects
Technology ,Science & Technology ,biology ,Chemistry ,Materials Science ,Biomedical Engineering ,Materials Science, Multidisciplinary ,Bioengineering ,Condensed Matter Physics ,Atomic and Molecular Physics, and Optics ,Immune tolerance ,MD Multidisciplinary ,Immunology ,biology.protein ,Science & Technology - Other Topics ,General Materials Science ,Nanoscience & Nanotechnology ,Electrical and Electronic Engineering ,Antibody ,AFFINITY - Published
- 2019
31. Biomechanics of HIV and immune cell interactions investigated using magnetic tweezer force spectroscopy
- Author
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Pavel Tolar, James L. Flewellen, Commission of the European Communities, and European Research Council
- Subjects
Science & Technology ,02 Physical Sciences ,Chemistry ,Cell ,Human immunodeficiency virus (HIV) ,Biomechanics ,Force spectroscopy ,Biophysics ,06 Biological Sciences ,medicine.disease_cause ,Immune system ,medicine.anatomical_structure ,medicine ,03 Chemical Sciences ,Life Sciences & Biomedicine - Published
- 2017
32. Cytoskeletal control of B cell responses to antigens
- Author
-
Pavel Tolar and European Research Council
- Subjects
0301 basic medicine ,History ,Cell signaling ,Immunology ,B-cell receptor ,Arp2/3 complex ,Receptors, Antigen, B-Cell ,macromolecular substances ,Lymphocyte Activation ,Education ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Antigens ,Cytoskeleton ,Actin ,B cell ,B-Lymphocytes ,biology ,breakpoint cluster region ,Actin cytoskeleton ,Computer Science Applications ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,1107 Immunology ,biology.protein ,030215 immunology - Abstract
The actin cytoskeleton is essential for cell mechanics and has increasingly been implicated in the regulation of cell signalling. In B cells, the actin cytoskeleton is extensively coupled to B cell receptor (BCR) signalling pathways, and defects of the actin cytoskeleton can either promote or suppress B cell activation. Recent insights from studies using single-cell imaging and biophysical techniques suggest that actin orchestrates BCR signalling at the plasma membrane through effects on protein diffusion and that it regulates antigen discrimination through the biomechanics of immune synapses. These mechanical functions also have a role in the adaptation of B cell subsets to specialized tasks during antibody responses.
- Published
- 2017
33. Plasma Membrane Sheets for Studies of B Cell Antigen Internalization from Immune Synapses
- Author
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Pavel Tolar and Carla R. Nowosad
- Subjects
0301 basic medicine ,Trogocytosis ,Chemistry ,Antigen processing ,B-cell receptor ,Endocytosis ,Immunological Synapses ,Cell biology ,Cell membrane ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,Antigen ,medicine ,B cell - Abstract
Surrogate planar and membrane systems have been employed to study the architecture of immune synapses; however, they often do not recapitulate trans-synaptic extraction and endocytosis of ligands by the immune cells. Transendocytosis (or trogocytosis) of antigen from immune synapses is particularly critical for antigen processing and presentation by B cells. Here we describe a protocol for preparation of plasma membrane sheets (PMSs), which are flexible and fluid membrane substrates that support robust B cell antigen extraction. We show how to attach B cell antigens to the PMSs and how to investigate antigen extraction and endocytosis by fluorescent microscopy and computational image analysis. These techniques should be broadly applicable to studies of transendocytosis in a variety of cellular systems.
- Published
- 2017
- Full Text
- View/download PDF
34. Germinal center B cells recognize antigen through a specialized immune synapse architecture
- Author
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Carla R. Nowosad, Pavel Tolar, Katelyn M. Spillane, and European Research Council
- Subjects
0301 basic medicine ,Immunological Synapses ,Antigen presentation ,Naive B cell ,Immunology ,Antibody Affinity ,B-Lymphocyte Subsets ,Receptors, Antigen, B-Cell ,Mice, Transgenic ,Lymphocyte Activation ,Article ,Mice ,03 medical and health sciences ,Protein Kinase C beta ,medicine ,Animals ,Humans ,Immunology and Allergy ,Antigen-presenting cell ,B cell ,Antigen Presentation ,B-Lymphocytes ,CD40 ,biology ,NF-kappa B ,Germinal center ,T-Lymphocytes, Helper-Inducer ,Germinal Center ,Cell biology ,Mice, Inbred C57BL ,B-1 cell ,HEK293 Cells ,030104 developmental biology ,medicine.anatomical_structure ,Polyclonal B cell response ,1107 Immunology ,biology.protein ,Immunologic Memory ,Signal Transduction - Abstract
B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we show that in contrast to naive and memory B cells, which gathered antigen towards the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β (PKC-β)–NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T-cell dependent selection of high-affinity B cells in GCs.
- Published
- 2016
35. Inside the microcluster: antigen receptor signalling viewed with molecular imaging tools
- Author
-
Pavel Tolar
- Subjects
Signalling ,Antigen ,Live cell imaging ,Antigen receptor ,Signalling molecules ,Immunology ,Immunology and Allergy ,Molecular imaging ,Biology ,Molecular resolution ,Immunological synapse ,Cell biology - Abstract
Over the last decade, live cell imaging has revealed the surprisingly complex orchestration of antigen receptor signalling at the immunological synapse. The imaging studies showed that one of the earliest steps in antigen receptor activation is the formation of submicroscopic clusters, which regulate the early signalling events. However, the molecular mechanisms operating inside these microclusters have remained beyond the resolution of optical microscopy. Recent development of imaging techniques that approach molecular resolution in intact cells offers a first view of the molecular processes inside these structures. Here I review the contributions of molecular imaging of the immunological synapse to our understanding of antigen receptor clustering, binding to antigens, and recruitment of signalling molecules. Finally, I provide an outlook on the future prospects of this rapidly advancing technology.
- Published
- 2011
36. Structural and Functional Studies of Igαβ and Its Assembly with the B Cell Antigen Receptor
- Author
-
Zhongcheng Zou, Pavel Tolar, Susan K. Pierce, Sergei Radaev, Anh Nguyen, Peter D. Sun, Nicole Stutzman, Peter D. Krueger, and Khanh Q. Nguyen
- Subjects
Models, Molecular ,Protein Folding ,PROTEINS ,Protein Conformation ,DNA Mutational Analysis ,Molecular Sequence Data ,Receptors, Antigen, B-Cell ,Sequence alignment ,Plasma protein binding ,Biology ,Article ,Mice ,Protein structure ,Species Specificity ,Structural Biology ,medicine ,Animals ,Humans ,Amino Acid Sequence ,MOLIMMUNO ,Molecular Biology ,Peptide sequence ,B cell ,DNA Primers ,Crystallography ,Base Sequence ,breakpoint cluster region ,Surface Plasmon Resonance ,Cell biology ,medicine.anatomical_structure ,Biochemistry ,biology.protein ,Protein folding ,Antibody ,Dimerization ,Sequence Alignment ,CD79 Antigens - Abstract
The B cell antigen receptor (BCR) plays an essential role in all phases of B cell development. Here we show that the extracellular domains of murine and human Igbeta form an I-set immunoglobulin-like structure with an interchain disulfide between cysteines on their G strands. Structural and sequence analysis suggests that Igalpha displays a similar fold as Igbeta. An Igalphabeta heterodimer model was generated based on the unique disulfide-bonded Igbeta dimer. Solution binding studies showed that the extracellular domains of Igalphabeta preferentially recognize the constant region of BCR with mu chain specificity, suggesting a role for Igalphabeta to enhance BCRmu chain signaling. Cluster mutations on Igalpha, Igbeta, and a membrane-bound form of immunoglobulin (mIgM) based on the structural model identified distinct areas of potential contacts involving charged residues on both subunits of the coreceptor and the Cmu4 domain of mIgM. These studies provide the first structural model for understanding BCR function.
- Published
- 2010
37. Human germinal center B cells are intrinsically able to discriminate antigen affinity and with T cell help express plasma cell transcription factors
- Author
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Avva Saniee, Kihyuck Kwak, Nicolas Quizon, Haewon Sohn, Javier Manzella-Lapeira, Prasida Holla, Jinghua Lu, HengYi Xie, Chenguang Xu, Katelyn Spillane, Pavel Tolar, and Susan K. Pierce
- Subjects
Immunology ,Immunology and Allergy - Abstract
The selection of GC B cells that express high affinity B cell receptors (BCRs) is driven by the ability of B cells to both signal through the BCR and to extract antigen and present it to follicular helper T cells (T FH cells). Using anti-kappa antibodies of low (KD=3.9×10 −7 ) versus high (KD=2.4×10 −9 ) affinities attached to membranes as surrogate antigens, we show that LZ GC B cells are able to discriminate between these and responded only to the high affinity antigen. In contrast, naïve B cells responded to both high and low affinity antigens. LZ GC B cells engaged membrane associated surrogate antigens through highly dynamic F-actin and ezrin-rich pod-like structures that concentrated BCRs at their contact points. Using DNA-based mechanical force nano-sensors we observed robust pulling forces localized to the contact sites of the pods with the membrane. In contrast, naïve B cells formed flat stable contacts with the membranes and showed only defuse weak pulling forces. GC B cells optimally express transcription factors that drive plasma cell differentiation, namely IRF-4 and PRDM-1, in response to antigen in combination with T FH help while naïve B cells increased IRF-4 and PRDM-1 transcription in response to T FH alone and less so to antigen alone. Thus, LZ GC B cells appear to be intrinsically capable of antigen affinity discrimination and with the acquisition of T FH help BCR-activated LZ GC B cells differentiate into plasma cells.
- Published
- 2018
38. Antigen affinity discrimination is an intrinsic function of the B cell receptor
- Author
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Hae Won Sohn, Wanli Liu, Pavel Tolar, Tobias Meckel, and Susan K. Pierce
- Subjects
Immunology ,T-cell receptor ,B-cell receptor ,Naive B cell ,Biology ,Molecular biology ,Affinity maturation ,B-1 cell ,medicine.anatomical_structure ,Antigen ,Polyclonal B cell response ,hemic and lymphatic diseases ,medicine ,Immunology and Allergy ,B cell - Abstract
Antibody affinity maturation, a hallmark of adaptive immune responses, results from the selection of B cells expressing somatically hypermutated B cell receptors (BCRs) with increased affinity for antigens. Despite the central role of affinity maturation in antibody responses, the molecular mechanisms by which the increased affinity of a B cell for antigen is translated into a selective advantage for that B cell in immune responses is incompletely understood. We use high resolution live-cell imaging to provide evidence that the earliest BCR-intrinsic events that follow within seconds of BCR–antigen binding are highly sensitive to the affinity of the BCR for antigen. High affinity BCRs readily form oligomers and the resulting microclusters grow rapidly, resulting in enhanced recruitment of Syk kinase and calcium fluxes. Thus, B cells are able to read the affinity of antigen by BCR-intrinsic mechanisms during the earliest phases of BCR clustering, leading to the initiation of B cell responses.
- Published
- 2010
39. Change we can believe in—of the conformational type
- Author
-
Susan K. Pierce and Pavel Tolar
- Subjects
T-cell receptor ,B-cell receptor ,Receptors, Antigen, T-Cell ,breakpoint cluster region ,Receptors, Antigen, B-Cell ,Review Article ,Biology ,Acquired immune system ,Models, Biological ,Biochemistry ,Immunological synapse ,Cell biology ,Receptors, Antigen ,Antigen ,Genetics ,Animals ,Humans ,Signal transduction ,Receptor ,Molecular Biology ,Signal Transduction - Abstract
The Cantoblanco Workshop on the Initiation of Antigen Receptor Signaling took place between 20 and 22 October 2008, in Cantoblanco, Madrid, Spain, and was organized by B. Alarcon, M. Davis & A. Weiss. ![][1] See Glossary for abbreviations used in this article In the back of our minds, we all know how antigen receptors trigger the activation of T‐ and B‐lymphocytes; it seems relatively well worked out. The binding of an antigen to the T‐cell receptor (TCR) and the B‐cell receptor (BCR) results in the phosphorylation of the cytoplasmic domains of these receptors, and off the cells go. Yet how exactly does that happen? For a group of researchers gathered in Madrid, Spain, in the autumn of 2008, that was the question. How does antigen binding to the ectodomains of the TCR and the BCR change the receptors so as to initiate intracellular signalling? Since the discovery of the TCR and the BCR, immunologists have learned a tremendous amount about the signalling cascades that are triggered by antigen binding and ultimately lead to T‐cell and B‐cell activation. However, the mechanisms that translate antigen binding into intracellular signalling remain among the real mysteries of adaptive immunity. How can that be if the mechanisms underlying the activation of a range of receptors—such as growth factor receptors—have been well worked out? In contrast to these conventional receptors, several unique characteristics of antigen receptors suggest that their inner workings are more complicated, including the enormous variability of their antigen‐binding domains and the need to learn to discriminate self from non‐self. How do the numerous unique interactions of antigens with the TCRs and BCRs expressed by each lymphocyte converge on a common change in the intracellular domains of the receptors that is sensitive to foreign antigens, but not self? To foster a discussion of antigen‐receptor activation, … [1]: /embed/graphic-1.gif
- Published
- 2009
40. The Constant Region of the Membrane Immunoglobulin Mediates B Cell-Receptor Clustering and Signaling in Response to Membrane Antigens
- Author
-
Pavel Tolar, Joseph Hanna, Peter D. Krueger, and Susan K. Pierce
- Subjects
Conformational change ,B-cell receptor ,Immunology ,Receptors, Antigen, B-Cell ,Biology ,Lymphocyte Activation ,Article ,Cell membrane ,Mice ,Antigen ,hemic and lymphatic diseases ,medicine ,Animals ,Syk Kinase ,Immunology and Allergy ,Mice, Knockout ,B-Lymphocytes ,Mice, Inbred BALB C ,Cell Membrane ,Intracellular Signaling Peptides and Proteins ,breakpoint cluster region ,Nitrohydroxyiodophenylacetate ,Protein-Tyrosine Kinases ,Intercellular Adhesion Molecule-1 ,Cell biology ,MOLIMMUN ,medicine.anatomical_structure ,Infectious Diseases ,biology.protein ,Immunoglobulin Constant Region ,Signal transduction ,Antibody ,Immunoglobulin Constant Regions ,Signal Transduction - 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 Cmicro4 domains of the membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cmicro4 domain failed to cluster and signal, and conversely, Cmicro4 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.
- Published
- 2009
- Full Text
- View/download PDF
41. Membrane heterogeneities in the formation of B cell receptor–Lyn kinase microclusters and the immune synapse
- Author
-
Hae Won Sohn, Pavel Tolar, and Susan K. Pierce
- Subjects
Protein Conformation ,Immunology ,B-cell receptor ,Receptors, Antigen, B-Cell ,Biology ,Article ,Cell Line ,Immunological synapse ,Cell membrane ,Mice ,Membrane Microdomains ,LYN ,hemic and lymphatic diseases ,Fluorescence Resonance Energy Transfer ,medicine ,Animals ,Immunology and Allergy ,Src family kinase ,Lipid raft ,Research Articles ,Antigen Presentation ,B-Lymphocytes ,Cell Membrane ,Receptor Aggregation ,breakpoint cluster region ,Cell Biology ,Cell biology ,Antigens, Differentiation, B-Lymphocyte ,src-Family Kinases ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Signal transduction ,Signal Transduction - Abstract
Antigen binding to the B cell receptors (BCRs) induces BCR clustering, phosphorylation of BCRs by the Src family kinase Lyn, initiation of signaling, and formation of an immune synapse. We investigated B cells as they first encountered antigen on a membrane using live cell high resolution total internal reflection fluorescence microscopy in conjunction with fluorescence resonance energy transfer. Newly formed BCR microclusters perturb the local membrane microenvironment, leading to association with a lipid raft probe. This early event is BCR intrinsic and independent of BCR signaling. Association of BCR microclusters with membrane-tethered Lyn depends on Lyn activity and persists as microclusters accumulate and form an immune synapse. Membrane perturbation and BCR–Lyn association correlate both temporally and spatially with the transition of microclustered BCRs from a “closed” to an “open” active signaling conformation. Visualization and analysis of the earliest events in BCR signaling highlight the importance of the membrane microenvironment for formation of BCR–Lyn complexes and the B cell immune synapse.
- Published
- 2008
42. Viewing the antigen-induced initiation of B-cell activation in living cells
- Author
-
Hae Won Sohn, Pavel Tolar, and Susan K. Pierce
- Subjects
Antigen Presentation ,B-Lymphocytes ,biology ,Cell Membrane ,Immunology ,breakpoint cluster region ,Receptors, Antigen, B-Cell ,Lymphocyte Activation ,Major histocompatibility complex ,Cell biology ,medicine.anatomical_structure ,Antigen ,Live cell imaging ,hemic and lymphatic diseases ,Image Processing, Computer-Assisted ,biology.protein ,medicine ,Animals ,Humans ,Immunology and Allergy ,Receptor clustering ,Antigens ,Signal transduction ,Receptor ,B cell - Abstract
The binding of antigen to the B‐cell receptor (BCR) induces BCR clustering and signaling cascades that lead to the activation of a variety of genes associated with B‐cell activation. Over the last several years, our understanding of the molecular details of the BCR signaling pathways have been considerably advanced; what remains only poorly understood are the molecular events that initiate BCR clustering and how clustering leads to activation. Here, we review our progress using live cell imaging technologies to view the earliest events that follow the B cell's binding of antigen. We provide a model for BCR clustering and B‐cell activation that involves an intrinsic tendency of the BCR to cluster and does not require direct crosslinking of the BCR by multivalent antigens. We suggest that local membrane topology and lipid composition play key roles in BCR clustering and initiation of signaling. We believe that our model for B‐cell activation, in which receptor interactions with monovalent antigens on membrane surfaces lead to receptor clustering, may be highly relevant to the mechanisms by which other immune receptors cluster including the T‐cell receptor in response to monovalent peptide–major histocompatibility complex complexes.
- Published
- 2008
43. Molecular Mechanisms of B Cell Antigen Gathering and Endocytosis
- Author
-
Robbert, Hoogeboom and Pavel, Tolar
- Subjects
B-Lymphocytes ,Protein Transport ,Animals ,Humans ,Receptors, Antigen, B-Cell ,Endosomes ,Endocytosis ,Signal Transduction - Abstract
Generation of high-affinity, protective antibodies requires B cell receptor (BCR) signaling, as well as antigen internalization and presentation to helper T cells. B cell antigen internalization is initiated by antigen capture, either from solution or from immune synapses formed on the surface of antigen-presenting cells, and proceeds via clathrin-dependent endocytosis and intracellular routing to late endosomes. Although the components of this pathway are still being discovered, it has become clear that antigen internalization is actively regulated by BCR signaling at multiple steps and, vice versa, that localization of the BCR along the endocytic pathway modulates signaling. Accordingly, defects in BCR internalization or trafficking contribute to enhanced B cell activation in models of autoimmune diseases and in B cell lymphomas. In this review, we discuss how BCR signaling complexes regulate each of the steps of this endocytic process and why defects along this pathway manifest as hyperactive B cell responses in vivo.
- Published
- 2015
44. Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation
- Author
-
Bruce S. Hostager, Sebastian A. Wagner, Chunaram Choudhary, Rajat Gupta, Pavel Tolar, Gail A. Bishop, Petra Beli, Trine A Kristiansen, Dessislava Malinova, Shankha Satpathy, and Chiara Francavilla
- Subjects
Proteomics ,Ubiquitin-Protein Ligases ,Receptors, Antigen, B-Cell ,Biology ,General Biochemistry, Genetics and Molecular Biology ,BCL10 ,chemistry.chemical_compound ,hemic and lymphatic diseases ,Humans ,ubiquitylation ,Adaptor Proteins, Signal Transducing ,B-Lymphocytes ,General Immunology and Microbiology ,Effector ,phosphorylation ,RAB7A ,Applied Mathematics ,HEK 293 cells ,breakpoint cluster region ,NF-kappa B ,Ubiquitination ,Membrane Proteins ,Tyrosine phosphorylation ,Articles ,B-Cell CLL-Lymphoma 10 Protein ,BCR ,Cell biology ,HEK293 Cells ,Computational Theory and Mathematics ,chemistry ,Membrane protein ,Phosphorylation ,Signal transduction ,General Agricultural and Biological Sciences ,Information Systems ,HeLa Cells ,Signal Transduction - Abstract
B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association with effector proteins and with endo-lysosomal compartments. In addition, we show that BCL10 is modified by LUBAC-mediated linear ubiquitylation, and demonstrate an important function of LUBAC in BCR-induced NF-κB signaling. Our results offer a global and integrated view of BCR signaling, and the provided datasets can serve as a valuable resource for further understanding BCR signaling networks.
- Published
- 2015
45. Molecular Mechanisms of B Cell Antigen Gathering and Endocytosis
- Author
-
Robbert Hoogeboom and Pavel Tolar
- Subjects
Chemistry ,Endosome ,media_common.quotation_subject ,Endocytic cycle ,B-cell receptor ,Endocytosis ,Immunological synapse ,Cell biology ,medicine.anatomical_structure ,hemic and lymphatic diseases ,Immunology ,medicine ,Internalization ,B cell ,Late endosome ,media_common - Abstract
Generation of high-affinity, protective antibodies requires B cell receptor (BCR) signaling, as well as antigen internalization and presentation to helper T cells. B cell antigen internalization is initiated by antigen capture, either from solution or from immune synapses formed on the surface of antigen-presenting cells, and proceeds via clathrin-dependent endocytosis and intracellular routing to late endosomes. Although the components of this pathway are still being discovered, it has become clear that antigen internalization is actively regulated by BCR signaling at multiple steps and, vice versa, that localization of the BCR along the endocytic pathway modulates signaling. Accordingly, defects in BCR internalization or trafficking contribute to enhanced B cell activation in models of autoimmune diseases and in B cell lymphomas. In this review, we discuss how BCR signaling complexes regulate each of the steps of this endocytic process and why defects along this pathway manifest as hyperactive B cell responses in vivo.
- Published
- 2015
46. Characterizing Mechanical Forces during B Cell Responses
- Author
-
Pavel Tolar and Katelyn M. Spillane
- Subjects
biology ,T cell ,Antigen presentation ,B-cell receptor ,Biophysics ,Acquired immune system ,Cell biology ,medicine.anatomical_structure ,Antigen ,biology.protein ,medicine ,Antibody ,Antigen-presenting cell ,B cell - Abstract
B cells are components of the adaptive immune response that produce high-affinity antibodies that neutralize and provide immunity against infections. Their responses are initiated when the B cell receptor (BCR) binds foreign antigen, triggering intracellular signaling that leads to B cell activation. Current evidence suggests that to elicit a full immune response involving development into high-affinity memory and plasma cells, B cells need to acquire the antigen from immune synapses with antigen presenting cells, and then internalize, process and present it to receive T cell help. Recent studies from our group suggest that B cells use contractile force to physically extract antigen from the presenting membrane prior to endocytosis. In addition, it was shown that pulling on the BCR-antigen bond results in discrimination between high- and low-affinity interactions. This suggests that B cells mechanically test the strength of BCR-antigen bonds to actively regulate affinity discrimination, a process that is important for the efficient development of high-affinity antibodies. To characterize force generation in the B cell synapse, we combine live-cell imaging with molecular force sensors. For the first time, we resolve the spatiotemporal dynamics and magnitude of mechanical forces in the B cell synapse, giving us insight into how B cells use mechanical forces to actively regulate their responses to antigen binding.
- Published
- 2015
- Full Text
- View/download PDF
47. The initiation of antigen-induced B cell antigen receptor signaling viewed in living cells by fluorescence resonance energy transfer
- Author
-
Hae Won Sohn, Susan K. Pierce, and Pavel Tolar
- Subjects
Conformational change ,Immunology ,B-cell receptor ,Receptors, Antigen, B-Cell ,Syk ,Biology ,Mice ,Antigen ,hemic and lymphatic diseases ,Fluorescence Resonance Energy Transfer ,Animals ,Syk Kinase ,Immunology and Allergy ,Src family kinase ,Antigens ,Phosphorylation ,B-Lymphocytes ,Enzyme Precursors ,T-cell receptor ,Intracellular Signaling Peptides and Proteins ,breakpoint cluster region ,Protein-Tyrosine Kinases ,Protein Structure, Tertiary ,Cell biology ,src-Family Kinases ,Signal transduction ,Signal Transduction - Abstract
Binding of antigen to the B cell antigen receptor (BCR) triggers signaling that ultimately leads to B cell activation. Using quantitative fluorescence resonance energy transfer imaging, we provide evidence here that the BCR is a monomer on the surface of resting cells. Binding of multivalent antigen clustered the BCR, resulting in the simultaneous phosphorylation of and a conformational change in the BCR cytoplasmic domains from a closed to an open form. Notably, the open conformation required immunoreceptor tyrosine-activation motif and continuous Src family kinase activity but not binding of the kinase Syk. Thus, the initiation of BCR signaling is a very dynamic process accompanied by reversible conformational changes induced by Src family kinase activity.
- Published
- 2005
48. Signaling assemblies formed in mast cells activated via Fcε receptor I dimers
- Author
-
Pavel Tolar, Jan Korb, Jitka Štokrová, Lubica Dráberová, Petr Dráber, Ivana Halova, Pavel Lebduška, and Helena Tolarová
- Subjects
Cell signaling ,Immunology ,Basophil Degranulation Test ,Biology ,chemistry.chemical_compound ,medicine ,Animals ,Humans ,Syk Kinase ,Immunology and Allergy ,Mast Cells ,Phosphorylation ,Protein kinase A ,Receptor ,Adaptor Proteins, Signal Transducing ,Enzyme Precursors ,Receptors, IgE ,Cell Membrane ,Intracellular Signaling Peptides and Proteins ,Degranulation ,Membrane Proteins ,Tyrosine phosphorylation ,Immunogold labelling ,Protein-Tyrosine Kinases ,Phosphoproteins ,Mast cell ,Actins ,Rats ,Cell biology ,Microscopy, Electron ,medicine.anatomical_structure ,chemistry ,biology.protein ,Tyrosine ,GRB2 ,Carrier Proteins ,Dimerization ,Signal Transduction - Abstract
Although aggregation of the Fcepsilon receptor I (FcepsilonRI) is necessary for Ag-mediated mast cell triggering, the relationship between the extent of the FcepsilonRI aggregation and subsequent biochemical and topographical events is incompletely understood. In this study, we analyzed the activation events induced by FcepsilonRI dimers, elicited by binding of anti-FcepsilonRI mAb to rat basophilic leukemia cells. We found that, in contrast to extensively aggregated FcepsilonRI, receptor dimers (1) induced a less extensive association of FcepsilonRI with detergent-resistant membranes, (2) delayed the tyrosine phosphorylation and membrane recruitment of several signaling molecules, (3) triggered a slower but more sustained increase in concentration of free cytoplasmic calcium, (4) induced degranulation which was not inhibited at higher concentrations of the cross-linking mAb, and (5) failed to produce clusters of FcepsilonRI, Syk kinase and Grb2 adapter in osmiophilic membranes, as detected by immunogold electron microscopy on membrane sheets. Despite striking differences in the topography of FcepsilonRI dimers and multimers, biochemical differences were less pronounced. The combined data suggest that FcepsilonRI-activated mast cells propagate signals from small signaling domains formed around dimerized/oligomerized FcepsilonRI; formation of large FcepsilonRI aggregates in osmiophilic membranes seems to promote both strong receptor triggering and rapid termination of the signaling responses.
- Published
- 2004
49. Positive and negative regulation of Fcε receptor I-mediated signaling events by Lyn kinase C-terminal tyrosine phosphorylation
- Author
-
Pavel Tolar, Helena Tolarová, Lubica Dráberová, and Petr Dráber
- Subjects
Immunology ,Basophil Degranulation Test ,environment and public health ,Receptor tyrosine kinase ,chemistry.chemical_compound ,LYN ,hemic and lymphatic diseases ,medicine ,Animals ,Immunology and Allergy ,Mast Cells ,Phosphorylation ,Cells, Cultured ,Tyrosine-protein kinase CSK ,biology ,Receptors, IgE ,hemic and immune systems ,Tyrosine phosphorylation ,Mast cell ,Cell biology ,enzymes and coenzymes (carbohydrates) ,src-Family Kinases ,medicine.anatomical_structure ,chemistry ,ROR1 ,biology.protein ,Cancer research ,Tyrosine ,biological phenomena, cell phenomena, and immunity ,Tyrosine kinase ,Signal Transduction ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Although it is known that the Src family tyrosine kinase Lyn initiates Fc epsilon receptor I (Fc epsilon RI) signaling by phosphorylation of the receptor subunits, regulation of Lyn kinase activity and its consequences for receptor signaling are incompletely understood. Using a phospho-Lyn-specific antiserum, we show an increased phosphorylation of the Lyn C-terminal regulatory tyrosine and decreased Lyn kinase activity during Fc epsilon RI-mediated mast cell activation. Mutant Lyn, defective in the C-terminal tyrosine, constitutively phosphorylated several substrates in resting cells, but did not cause Fc epsilon RI internalization or spontaneous degranulation. Fc epsilon RI-induced signaling in the presence of constitutively active Lyn exhibited enhanced phosphorylation of the receptor subunits, Syk, LAT, Gab2, phospholipase C (PLC)gamma 1 and PLC gamma 2, and production of phosphatidylinositol 3,4,5-trisphosphate. Although enzymatic activities of PLC gamma 1 and PLC gamma 2 were also up-regulated, amounts of inositol 1,4,5-trisphosphate, mobilization of intracellular calcium and degranulation were suppressed. Additionally, constitutively active Lyn was strikingly less efficient than wild-type Lyn in restoring the receptor-mediated calcium responses in bone marrow mast cells derived from Lyn(-/-) mice. These findings pinpoint the tight regulation of Lyn kinase activity as a critical event in mast cell degranulation.
- Published
- 2004
50. Mechanisms of B Cell Antigen Extraction Revealed by DNA-Based Molecular Sensors
- Author
-
Katelyn M. Spillane and Pavel Tolar
- Subjects
medicine.anatomical_structure ,Antigen ,Polyclonal B cell response ,Antigen processing ,B-cell receptor ,T-cell receptor ,Antigen presentation ,Biophysics ,medicine ,Biology ,Antigen-presenting cell ,B cell ,Cell biology - Abstract
Here we show the development and use of novel DNA-based molecular sensors to investigate B cell antigen extraction from antigen presenting cells. B cells play a critical role in immune responses by producing highly specific antibodies against the foreign pathogens that they encounter. Their responses are initiated when the B cell receptor (BCR) binds antigen on the surface of an antigen-presenting cell in a cell-cell contact known as an immune synapse. This event triggers the B cell to internalize, process and present the antigen to helper T cells, which are required for B cell clonal selection. It is known that the extent of T cell help depends upon the affinity of the BCR for antigen, suggesting that affinity discrimination during antigen acquisition is essential for high-affinity antibody responses. The mechanisms of antigen recognition, discrimination and uptake remain a topic of debate, with extraction through mechanical forces and degradation through lysosome exocytosis proposed as efficient ways for B cells to acquire antigen. One limiting factor in studies to date has been the artificial substrates used to mimic antigen-presenting cell membranes. To overcome this limitation, we have developed DNA-based molecular sensors that distinguish between degradation and force-mediated antigen extraction from both artificial substrates and live antigen-presenting cells. Our results show that B cells modulate the mechanism of antigen uptake in response to the character of the antigen-presenting substrate, with extraction by mechanical force the dominant mechanism under physiological conditions. Using sensors that report on the dynamics and magnitude of forces in B cell synapses, we characterize the mechanical extraction of antigen and show that different B cell subsets have unique mechanical properties that allow them to actively regulate their responses to antigen binding.
- Published
- 2016
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