title: Pre-clustering of the B cell antigen receptor demonstrated by mathematically extended electron microscopy.
abstract: The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation.
authors.affiliation: Faculty of Biology, Department of Molecular Immunology, Albert Ludwigs University Freiburg , Freiburg , Germany ; Spemann Graduate School of Biology and Medicine (SGBM), Albert Ludwigs University Freiburg , Freiburg , Germany ; Centre for Biological Signalling Studies BIOSS, Albert Ludwigs University Freiburg , Freiburg , Germany.
authors.forename: Gina J
authors.initials: GJ
authors.lastname: Fiala
authors.affiliation: Institute of Physics, Albert Ludwigs University Freiburg , Freiburg , Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kaschek
authors.affiliation: Faculty of Biology, Department of Molecular Immunology, Albert Ludwigs University Freiburg , Freiburg , Germany ; Medical Faculty, Centre for Chronic Immunodeficiency CCI, University Clinics Freiburg, Albert Ludwigs University Freiburg , Freiburg , Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Blumenthal
authors.affiliation: Faculty of Biology, Department of Molecular Immunology, Albert Ludwigs University Freiburg , Freiburg , Germany ; Centre for Biological Signalling Studies BIOSS, Albert Ludwigs University Freiburg , Freiburg , Germany ; Max Planck-Institute of Immunobiology and Epigenetics , Freiburg , Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Reth
authors.affiliation: Centre for Biological Signalling Studies BIOSS, Albert Ludwigs University Freiburg , Freiburg , Germany ; Institute of Physics, Albert Ludwigs University Freiburg , Freiburg , Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Timmer
authors.affiliation: Faculty of Biology, Department of Molecular Immunology, Albert Ludwigs University Freiburg , Freiburg , Germany ; Centre for Biological Signalling Studies BIOSS, Albert Ludwigs University Freiburg , Freiburg , Germany ; Medical Faculty, Centre for Chronic Immunodeficiency CCI, University Clinics Freiburg, Albert Ludwigs University Freiburg , Freiburg , Germany.
authors.forename: Wolfgang W A
authors.initials: WW
authors.lastname: Schamel
keywords: BCR; Monte Carlo simulation; electron microscopy; immuno-gold-labeling; maximum-likelihood method; oligomerization