Genetic Indicators for Calcium Signaling Studies in Toxoplasma Gondii

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2019 Nov 24

PMID 31758454

Citations 11

Authors

Stephen A Vella

Abigail Calixto

Beejan Asady

Zhu-Hong Li

Silvia N J Moreno

Affiliations

Soon will be listed here.

Abstract

Fluctuations of the cytosolic calcium ion (Ca) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca levels and the signals that elevate Ca in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca-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 Ca 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 Ca 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.

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