Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells.
Calcium signaling is a ubiquitous and versatile process involved in nearly every cellular process, and exploitation of host calcium signals is a common strategy used by viruses to facilitate replication and cause disease. Small molecule fluorescent calcium dyes have been used by many to examine changes in host cell calcium signaling and calcium channel activation during virus infections, but disadvantages of these dyes, including poor loading and poor long-term retention, complicate analysis of calcium imaging in virus-infected cells due to changes in cell physiology and membrane integrity. The recent expansion of genetically-encoded calcium indicators (GECIs), including blue and red-shifted color variants and variants with calcium affinities appropriate for calcium storage organelles like the endoplasmic reticulum (ER), make the use of GECIs an attractive alternative for calcium imaging in the context of virus infections. Here we describe the development and testing of cell lines stably expressing both green cytoplasmic (GCaMP5G and GCaMP6s) and red ER-targeted (RCEPIAer) GECIs. Using three viruses (rotavirus, poliovirus and respiratory syncytial virus) previously shown to disrupt host calcium homeostasis, we show the GECI cell lines can be used to detect simultaneous cytoplasmic and ER calcium signals. Further, we demonstrate the GECI expression has sufficient stability to enable long-term confocal imaging of both cytoplasmic and ER calcium during the course of virus infections.
Department of Molecular Virology and Microbiology and Alkek Center for Metagenomic and Microbiome Research, Baylor College of Medicine, Houston, TX 77030, United States.
Jacob L
JL
Perry
Department of Molecular Virology and Microbiology and Alkek Center for Metagenomic and Microbiome Research, Baylor College of Medicine, Houston, TX 77030, United States.
Nina K
NK
Ramachandran
Shared Equipment Authority, Rice University, Houston, TX 77030, United States.
Budi
B
Utama
Department of Molecular Virology and Microbiology and Alkek Center for Metagenomic and Microbiome Research, Baylor College of Medicine, Houston, TX 77030, United States. Electronic address: joseph.hyser@bcm.edu.
Joseph M
JM
Hyser
D000818:Animals; D002118:Calcium / Q000032:analysis*; D020013:Calcium Signaling; D002460:Cell Line; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D017955:Poliovirus / Q000378:metabolism; D012136:Respiratory Syncytial Viruses / Q000378:metabolism; D012401:Rotavirus / Q000378:metabolism
D007202:Indicators and Reagents; D002118:Calcium
Endoplasmic reticulum; Enterovirus; GCaMP5G; GCaMP6s; RCEPIAer; Respiratory syncytial virus; Rotavirus