Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages

  1. Carolin Ulbricht
  2. Ruth Leben
  3. Asylkhan Rakhymzhan
  4. Frank Kirchhoff
  5. Lars Nitschke
  6. Helena Radbruch
  7. Raluca A Niesner
  8. Anja E Hauser  Is a corresponding author
  1. Charité - Universitätsmedizin, Germany
  2. Deutsches Rheumaforschungszentrum (DRFZ), Germany
  3. University of Saarland, Germany
  4. Friedrich-Alexander-University Erlangen, Germany
  5. Charité-University of Medicine, Germany
  6. 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.

The following data sets were generated

Article and author information

Author details

  1. Carolin Ulbricht

    Rheumatology and Clinical Immunology, Charité - Universitätsmedizin, Berlin, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2983-6242
  2. Ruth Leben

    Deutsches Rheumaforschungszentrum (DRFZ), Berlin, Germany
    Competing interests
    No competing interests declared.
  3. Asylkhan Rakhymzhan

    Deutsches Rheumaforschungszentrum (DRFZ), Berlin, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3152-1557
  4. Frank Kirchhoff

    Molecular Physiology, Center for Integrative Physiology and Molecular Medicine, University of Saarland, Homburg, Germany
    Competing interests
    Frank Kirchhoff, Reviewing editor, eLife.
  5. Lars Nitschke

    Friedrich-Alexander-University Erlangen, Erlangen, Germany
    Competing interests
    No competing interests declared.
  6. Helena Radbruch

    Charité-University of Medicine, Berlin, Germany
    Competing interests
    No competing interests declared.
  7. Raluca A Niesner

    Deutsches Rheumaforschungszentrum (DRFZ), Berlin, Germany
    Competing interests
    No competing interests declared.
  8. Anja E Hauser

    Rheumatology and Clinical Immunology, Charité - Universitätsmedizin and Deutsches Rheumaforschungszentrum, Berlin, Germany
    For correspondence
    hauser@drfz.de
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7725-9526

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.

Reviewing Editor

  1. Michael L Dustin, University of Oxford, United Kingdom

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.

Version history

  1. Received: February 14, 2020
  2. Accepted: March 19, 2021
  3. Accepted Manuscript published: March 22, 2021 (version 1)
  4. Version of Record published: April 21, 2021 (version 2)

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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  1. Carolin Ulbricht
  2. Ruth Leben
  3. Asylkhan Rakhymzhan
  4. Frank Kirchhoff
  5. Lars Nitschke
  6. Helena Radbruch
  7. Raluca A Niesner
  8. Anja E Hauser
(2021)
Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages
eLife 10:e56020.
https://doi.org/10.7554/eLife.56020

Share this article

https://doi.org/10.7554/eLife.56020

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