Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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B-Lymphocytes / metabolism
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Cell Line
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Cell Membrane / metabolism
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Female
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Humans
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Immunoglobulin D
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Immunoglobulin M
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Intracellular Signaling Peptides and Proteins / metabolism
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Lymphocyte Activation / immunology
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Male
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Membrane Microdomains / metabolism*
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Membrane Proteins / metabolism
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Membrane Proteins / physiology*
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Mice
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Mice, Inbred C57BL
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Protein Interaction Mapping / methods*
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Receptors, Antigen, B-Cell / genetics
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Signal Transduction / immunology
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Syk Kinase
Substances
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Immunoglobulin D
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Immunoglobulin M
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Intracellular Signaling Peptides and Proteins
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Membrane Proteins
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Receptors, Antigen, B-Cell
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Syk Kinase
Grants and funding
Funding for this work was provided by Deutsche Forschungsgemeinschaft TRR130-P02 (B-Zellen: Immunität und Autoimmunität,
http://www.trr130.forschung.uni-erlangen.de/index.php/en/home.html, MR), Max Planck Society (
https://www.ie-freiburg.mpg.de/de, MR and JY), Excellence Initiative of the German Federal and State Governments EXC294 (
https://www.bioss.uni-freiburg.de, MR and JY), and Spemann Graduate School for Biology and Medicine (SGBM,
https://www.sgbm.uni-freiburg.de, MS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.