Language

English

Publication Date

7-1-2026

Journal

Cell Calcium

DOI

10.1016/j.ceca.2026.103160

PMID

42330797

PMCID

PMC13496235

PubMedCentral® Posted Date

8-22-2026

PubMedCentral® Full Text Version

Author MSS

Abstract

Many intracellular pathogens manipulate host cell calcium to facilitate their survival and replication. Live-cell microscopy using fluorescent calcium indicators has become an indispensable tool for characterizing the mechanisms underlying both homeostatic and pathogen-induced cellular calcium dynamics, but such imaging must be coupled with robust quantitative analysis. Further, calcium imaging is most powerful when paired with reductive studies targeting calcium-modulating proteins. The lack of specific inhibitors or agonists to directly target most pathogen-induced calcium signals precludes many of the approaches that have allowed for robust characterization of major eukaryotic cell calcium signaling mechanisms, such as ER Ca2+ release by inositol triphosphate receptors. Given this, we sought to develop quantitative imaging pipelines tailored for the characterization of pathogen-induced calcium signals. Using rotavirus as a prototypical calcium-modulating pathogen, we developed and optimized a suite of computational tools for automated quantitation of both intra- and inter-cellular calcium signals detected via live-cell imaging of infected epithelial monolayers expressing genetically encoded calcium indicators. Using recombinant strains of rotavirus that express fluorescent markers, we developed a system that allows for automated detection of rotavirus-infected cells and normalization of signals to infectivity. All tools were built in ImageJ, making them freely available and adaptable across operating systems and microscope setups. These tools required minimal active time from the user and allowed for the extraction of signal parameters previously unquantifiable, increasing the speed and breadth of characterization.

Keywords

Calcium Signaling, Rotavirus, Microscopy, Fluorescence, Animals, Calcium, Humans, High-Throughput Screening Assays, virus, pathogen, calcium signaling, analysis, quantitation, microscopy

Published Open-Access

yes

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