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Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii

  • Stephen A. Vella
  • Abigail Calixto
  • Beejan Asady
  • Zhu-Hong Li
  • Silvia N. J. MorenoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2071)

Abstract

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.

The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing “tried and true” methods of our lab who pioneered the first use of GECI’s in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

Key words

Toxoplasma Calcium signaling Genetic indicators GCaMPs GECOs Egress Time lapse Fura2 measurements 

Notes

Acknowledgments

This work was partially funded by NIH grants AI096836, AI128356, and AI110027 to S.N.J.M.

A.C. was supported by an NIH diversity supplement to AI128356. S.V. was partially supported by a fellowship of the Office of the Vice-President for Research, UGA. We would also like to thank Dr. Muthugapatti Kandasamy from the Biomedical Microscopy Core and Julie Nelson from the Cytometry Shared Resource Laboratory of the University of Georgia.

Eric Dykes helped performed the westerns of the cells expressing GCAMP-mScarlet and Christina Moore made the videos used for Fig. 4.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Stephen A. Vella
    • 1
  • Abigail Calixto
    • 1
  • Beejan Asady
    • 1
  • Zhu-Hong Li
    • 1
  • Silvia N. J. Moreno
    • 1
    • 2
    Email author
  1. 1.Center for Tropical and Emerging Global DiseasesUniversity of GeorgiaAthensUSA
  2. 2.Department of Cellular BiologyUniversity of GeorgiaAthensUSA

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