Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

Perry, Jacob L.; Ramachandran, Nina K.; Utama, Budi; Hyser, Joseph M.

doi:10.1016/j.ymeth.2015.09.004

Cited by 32 publications

(34 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…RGECO1.2 (Addgene plasmid #45494), R-CEPIA1er (Addgene plasmid #58216), and GCaMP6s were cloned into pLVX-IRES-Hygro. Lentivirus vectors for the GECI constructs were packaged in HEK293T cells as previously described 59 or produced commercially (Cyagen Biosciences, Inc.). MA104-GCaMP5G, MA104-GCaMP5G/RCEPIAer, MA104-RGECO1/GCEPIAer, and the MA104-GCaMP6s-shRNA lines and jHIE-CaMP6s enteroids (from patient J3) were generated as previously described 26, 59 .…”

Section: Methodsmentioning

confidence: 99%

Rotavirus Induces Intercellular Calcium Waves through ADP Signaling

Chang-Graham

Perry

Engevik

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

26Rotavirus causes severe diarrheal disease in children worldwide. A hallmark of rotavirus 27 infection is an increase in cytosolic calcium in infected small intestine epithelial cells. However, 28 the underlying mechanism(s) of rotavirus-cell signaling remains incompletely characterized. Here 29 we show that rotavirus-infected cells produce paracrine signals that manifest as intercellular 30 calcium waves (ICWs); which are observed in both cell lines and human intestinal enteroids 31 (HIEs). Rotavirus ICWs are caused by the release of extracellular adenosine diphosphate (ADP) 32 that activates P2Y1 purinergic receptors on neighboring cells and are blocked by P2Y1 33 antagonists or CRISPR/Cas9 knockout of P2Y1. This paracrine purinergic signal is critical for 34 rotavirus replication and diarrhea. Blocking the ICW signal reduces rotavirus replication; inhibits 35 rotavirus-induced serotonin release and fluid secretion; and reduces diarrhea severity in neonatal 36 mice. This is the first evidence that viruses exploit intercellular calcium waves to amplify diarrheal 37 signaling; a finding which have broad implications for gastrointestinal physiology. signaling molecules during RV infection, including enterotoxin NSP4, prostaglandins (PGE2), and 57 nitric oxide (NO) 7,15-17 . In this model, enterotoxin NSP4 can bind to neighboring, uninfected 58 enterocytes to activate Ca 2+ -activated chloride channels and cause secretory diarrhea 18,19 , and 59 PGE2 and NO may further activate fluid secretion processes 20,21 . Dysregulation of neighboring 60 enteroendocrine cells triggers the Ca 2+ -dependent release of serotonin, which stimulates the 61 enteric nervous system both to activate vomiting centers in the central nervous system and to 62 activate secretory reflex pathways in the gastrointestinal (GI) tract 5,22 . Thus, this model of RV-63 induced diarrhea addresses how limited infection at the middle-to-upper villi may cause 64 widespread dysregulation of host physiology and life-threatening disease. 65Herein we demonstrate that RV-infected cells signal to uninfected cells via an extracellular 66 purinergic signaling pathway. This newly identified pathway is a dominant driver of observed RV 67 disease processes, including replication, upregulation of PGE2-and NO-producing enzymes, 68 serotonin secretion, fluid secretion, and diarrhea in a neonatal mouse model. Our findings provide 69 new insights into the mechanism(s) of viral diarrhea and gastrointestinal physiology. 70 Results 71 Low multiplicity infection reveals intercellular calcium waves 72Previous studies have shown that RV significantly increases cytosolic Ca 2+ during infection 73 and disrupts host Ca 2+ -dependent processes to cause disease [23][24][25] . We used African Green 74 monkey kidney MA104 cells stably expressing the genetically-encoded calcium indicator (GECI) 75GCaMP5G or GCaMP6s to observe changes in cytosolic Ca 2+ during RV infection using live-cell 76 time-lapse epifluorescence imaging. We did not observe differences in response using either...

show abstract

Section: Methodsmentioning

confidence: 99%

Rotavirus Induces Intercellular Calcium Waves through ADP Signaling

Chang-Graham

Perry

Engevik

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…RGECO1.2 (Addgene plasmid #45494), R-CEPIA1er (Addgene plasmid #58216), and GCaMP6s were cloned into pLVX-IRES-Hygro. Lentivirus vectors for the GECI constructs were packaged in HEK293T cells as previously described 23 or produced commercially (Cyagen Biosciences, Inc.). Production of the MA104-GCaMP5G and MA104-GCaMP5G/RCEPIAer cell lines were previously described and similar methods were used to generate MA104-RGECO1/GCEPIAer and the MA104-GCaMP6s-shRNA lines 23 .…”

Section: Methodsmentioning

confidence: 99%

“…Lentivirus vectors for the GECI constructs were packaged in HEK293T cells as previously described 23 or produced commercially (Cyagen Biosciences, Inc.). Production of the MA104-GCaMP5G and MA104-GCaMP5G/RCEPIAer cell lines were previously described and similar methods were used to generate MA104-RGECO1/GCEPIAer and the MA104-GCaMP6s-shRNA lines 23 . Human intestinal enteroids expressing GCaMP6s (G6S-HIEs) were created using lentivirus transduction as described previously and grown in high Wnt3a CMGF+ with 1 µg/mL puromycin for selection 32 .…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…For many years, technical limitations of Ca 2+ indicator dyes ( e.g ., photobleaching, uneven loading, and dye leakage) made single-cell measurements of Ca 2+ signaling throughout the entire RV infection not feasible. However, we recently developed the use of genetically-encoded calcium indicators (GECIs) for the study of Ca 2+ signaling in virus-infected cells 23 . GECIs are dynamic fluorescent protein-based Ca 2+ sensors, and variants have been developed for simultaneous Ca 2+ measurements in multiple cellular compartments ( e.g ., cytoplasm and ER) 24 .…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Chang‐Graham

Perry

Strtak

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

28 Like many viruses, rotavirus (RV) dysregulates calcium homeostasis by elevating 29 cytosolic calcium ([Ca 2+ ]cyt) and decreasing endoplasmic reticulum (ER) stores. While 30 an overall, monophasic increase in [Ca 2+ ]cyt during RV infection has been shown, the 31 nature of the RV-induced aberrant calcium signals and how they manifest over time at 32 the single-cell level have not been characterized. Thus, we generated cell lines and 33 human intestinal enteroids (HIEs) stably expressing cytosolic and/or ER-targeted 34 genetically-encoded calcium indicators to characterize calcium signaling throughout RV 35 infection by time-lapse imaging. We found that RV induces highly dynamic [Ca 2+ ]cyt 36 signaling that manifest as hundreds of discrete [Ca 2+ ]cyt spikes, which increase during 37 peak infection. Knockdown of nonstructural protein 4 (NSP4) attenuates the [Ca 2+ ]cyt 38 spikes, consistent with its role in dysregulating calcium homeostasis. RV-induced 39 [Ca 2+ ]cyt spikes were primarily from ER calcium release and were attenuated by 40 inhibiting the store-operated calcium entry (SOCE) channel Orai1. RV-infected HIEs 41 also exhibited prominent [Ca 2+ ]cyt spikes that were attenuated by inhibiting SOCE, 42 underlining the relevance of these [Ca 2+ ]cyt spikes to gastrointestinal physiology and 43 role of SOCE in RV pathophysiology. Thus, our discovery that RV increases [Ca 2+ ]cyt 44 by dynamic Ca 2+ signaling, establishes a new, paradigm-shifting understanding of the 45 spatial and temporal complexity of virus-induced Ca 2+ signaling. 46 47 48 Eukaryotic signal transduction pathways employ a variety of signaling molecules 49 to regulate cellular processes. Calcium (Ca 2+ ) is one of the most ubiquitous secondary 50 messengers in the cell, which tightly regulates Ca 2+ movement through the coordinated 51 function of Ca 2+ channels, transporters, and pumps. Since Ca 2+ signaling modulates a 52 wide array of cellular processes, it is not surprising that many different viruses exploit 53 Ca 2+ signaling to facilitate their replication, and the resulting dysregulation of Ca 2+ 54 signaling causes pathogenesis. Rotavirus (RV), a member of the Reoviridae family, is 55 one of the first viruses shown to elevate cellular Ca 2+ levels and has become a widely-56 used model system to characterize mechanisms by which viruses dysregulate host Ca 2+ 57 homeostasis 1 . RV is a clinically important enteric virus that causes severe diarrhea and 58 vomiting in children, resulting in over approximately 258 million diarrhea episodes and 59 198,000 deaths in 2016 2 . Hyperactivation of cyclic nucleotide (e.g., cAMP/cGMP) and 60 Ca 2+ signaling pathways is a common strategy among enteric pathogens 3 . Thus, 61 understanding how RV exploits Ca 2+ signaling is key to understanding and combating 62 RV-induced diarrhea. 63 RV was first reported to elevate cytosolic [Ca 2+ ] by Michelangeli et al. (1991), 64 which stimulated subsequent research into how RV alters cellular Ca 2+ levels 4 . RV 65 causes a 2-fold steady-state increase in ...

show abstract

Sensing Intra‐ and Extra‐Cellular Ca²⁺ in the Islet of Langerhans

Huang

Xie

et al. 2021

Adv Funct Materials

View full text Add to dashboard Cite

Calcium ions (Ca2+) take part in intra‐ and inter‐cellular signaling to mediate cellular functions. Sensing this ubiquitous messenger is instrumental in disentangling the specific functions of cellular sub‐compartments and/or intercellular communications. In this review, the authors first describe intra‐ and inter‐cellular Ca2+ signaling in relation to insulin secretion from the pancreatic islets, and then outline the development of diverse sensors, for example, chemically synthesized indicators, genetically encoded proteins, and ion‐selective microelectrodes, for intra‐ and extra‐cellular sensing of Ca2+. Particular emphasis is placed on emerging approaches in this field, such as low‐affinity Ca2+ indicators and unique Ca2+‐responsive composite materials. The authors conclude by remarking on the challenges and opportunities for further developments in this field, which may facilitate a more comprehensive understanding of Ca2+ signaling within and outside the islets, and its relevance in health and disease.

show abstract

Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

Cited by 32 publications

References 38 publications

Rotavirus Induces Intercellular Calcium Waves through ADP Signaling

Rotavirus Induces Intercellular Calcium Waves through ADP Signaling

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Sensing Intra‐ and Extra‐Cellular Ca²⁺ in the Islet of Langerhans

Contact Info

Product

Resources

About

Use of genetically-encoded calcium indicators for live cell calcium imaging and localization in virus-infected cells

Cited by 32 publications

References 38 publications

Rotavirus Induces Intercellular Calcium Waves through ADP Signaling

Rotavirus Induces Intercellular Calcium Waves through ADP Signaling

Rotavirus Calcium Dysregulation Manifests as Dynamic Calcium Signaling in the Cytoplasm and Endoplasmic Reticulum

Sensing Intra‐ and Extra‐Cellular Ca2+ in the Islet of Langerhans

Contact Info

Product

Resources

About

Sensing Intra‐ and Extra‐Cellular Ca²⁺ in the Islet of Langerhans