Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages
- Charité - Universitätsmedizin, Germany
- Deutsches Rheumaforschungszentrum (DRFZ), Germany
- University of Saarland, Germany
- Friedrich-Alexander-University Erlangen, Germany
- Charité-University of Medicine, Germany
- Charité - Universitätsmedizin and Deutsches Rheumaforschungszentrum, Germany
Abstract
Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca2+ ions drives a number of signaling-related processes, stress-responses or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the FRET-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial and detrimental (auto)immunity.
Data availability
Source data for flow cytometric Analysis, in vitro confocal imaging, ratiometric in vivo Imaging and fluorescence lifetime in vivo Imaging are deposited at Dryad Digital Repository 10.5061/dryad.cc2fqz63d. Analyzed absolute calcium concentration for all cells measured out of 5 experiments have also been deposited there. Source code for phasor based analysis of fluorescence lifetime data has been provided with full submission upload and will be made available to the public via github after publication.
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Intravital quantification of cytoplasmic B cell calcium reveals dynamic signaling across B cell differentiation stagesDryad Digital Repository, doi:10.5061/dryad.cc2fqz63d.
Article and author information
Author details
Asylkhan Rakhymzhan
Funding
Deutsche Forschungsgemeinschaft (TRR130 P17)
- Helena Radbruch
- Anja E Hauser
Deutsche Forschungsgemeinschaft (TRR130 C01)
- Raluca A Niesner
- Anja E Hauser
Deutsche Forschungsgemeinschaft (TRR130 P04)
- Lars Nitschke
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: The study was approved by the Berlin Landesamt für Gesundheit und Soziales under the registration # G00158/16. All surgeries and experimental procedures were conducted following the principle of minimization of suffering and 3R means were used where possible.
Copyright
© 2021, Ulbricht et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages
eLife 10:e56020.
https://doi.org/10.7554/eLife.56020
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