Abstract

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca2+, and a transgenic Ca2+ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca2+ signals reported by Salsa6f during T cell receptor activation in naïve T cells, helper Th17 T cells and regulatory T cells, and Ca2+ signals mediated in T cells by an activator of mechanosensitive Piezo1channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca2+ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca2+ transients ('sparkles') as cells migrate.

Article and author information

Author details

  1. Tobias X Dong

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5500-7099
  2. Shivashankar Othy

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6832-5547
  3. Amit Jairaman

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Jonathan Skupsky

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Angel Zavala

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ian Parker

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Joseph L Dynes

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Michael D Cahalan

    Department of Physiology and Biophysics, University of California, Irvine, Irvine, United States
    For correspondence
    mcahalan@uci.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4987-2526

Funding

National Institutes of Health (AI117555)

  • Joseph L Dynes
  • Michael D Cahalan

National Institutes of Health (NS14609)

  • Michael D Cahalan

National Institutes of Health (AI121945)

  • Michael D Cahalan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Use of blood samples from healthy human subjects has been approved by the University of California, Irvine Institutional Review Board (UCI IRB HS #1995-459). All animal procedures were approved by the UCI Institutional Animal Care and Use committee (IACUC) (protocol #1998-1366-11).

Human subjects: Human blood was prepared using support from the National Center for Research Resources and the National Center for Advancing Translational Sciences (NIH Grant UL1 TR000153).

Copyright

© 2017, Dong 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.

Metrics

  • 7,094
    views
  • 778
    downloads
  • 65
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Tobias X Dong
  2. Shivashankar Othy
  3. Amit Jairaman
  4. Jonathan Skupsky
  5. Angel Zavala
  6. Ian Parker
  7. Joseph L Dynes
  8. Michael D Cahalan
(2017)
T cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse
eLife 6:e32417.
https://doi.org/10.7554/eLife.32417

Share this article

https://doi.org/10.7554/eLife.32417

Further reading

    1. Cancer Biology
    2. Cell Biology
    Xiangning Bu, Nathanael Ashby ... Inhee Chung
    Research Article

    Cell crowding is a common microenvironmental factor influencing various disease processes, but its role in promoting cell invasiveness remains unclear. This study investigates the biomechanical changes induced by cell crowding, focusing on pro-invasive cell volume reduction in ductal carcinoma in situ (DCIS). Crowding specifically enhanced invasiveness in high-grade DCIS cells through significant volume reduction compared to hyperplasia-mimicking or normal cells. Mass spectrometry revealed that crowding selectively relocated ion channels, including TRPV4, to the plasma membrane in high-grade DCIS cells. TRPV4 inhibition triggered by crowding decreased intracellular calcium levels, reduced cell volume, and increased invasion and motility. During this process, TRPV4 membrane relocation primed the channel for later activation, compensating for calcium loss. Analyses of patient-derived breast cancer tissues confirmed that plasma membrane-associated TRPV4 is specific to high-grade DCIS and indicates the presence of a pro-invasive cell volume reduction mechanotransduction pathway. Hyperosmotic conditions and pharmacologic TRPV4 inhibition mimicked crowding-induced effects, while TRPV4 activation reversed them. Silencing TRPV4 diminished mechanotransduction in high-grade DCIS cells, reducing calcium depletion, volume reduction, and motility. This study uncovers a novel pro-invasive mechanotransduction pathway driven by cell crowding and identifies TRPV4 as a potential biomarker for predicting invasion risk in DCIS patients.

    1. Cancer Biology
    2. Cell Biology
    Rui Hua, Jean X Jiang
    Insight

    Cell crowding causes high-grade breast cancer cells to become more invasive by activating a molecular switch that causes the cells to shrink and spread.