Researcher Interviews

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Assistant Prof. Joseph Michael Hyser & Dr. Alexandra Leigh Chang-Graham

Virology and Microbiology
Baylor College of Medicine
Houston, Texas, USA

Please tell us about your research.

Rotavirus is an enteric virus that causes severe gastroenteritis in children, resulting in life-threatening diarrhea and vomiting. Rotavirus primarily infects intestinal epithelial cells in the small intestine, particularly the mature enterocytes at the tip of intestinal villi. While there are multiple vaccines that haven been approved, they are less effective in developing countries, especially in those where rotavirus disease burden is greatest. Currently, there are no specific antiviral or antidiarrheal drugs available to treat rotavirus infection. The standard treatment is oral rehydration solutions to prevent potentially fatal dehydration, but does not reduce the duration or severity of disease. This underscores a need to develop more targeted treatments to reduce fluid loss.

A hallmark of rotavirus infection is a global dysregulation of intracellular calcium homeostasis. This increase in cytosolic calcium is necessary for rotavirus replication and for rotavirus pathogenesis, including activation of secretory pathways leading to diarrhea. The diarrheal disease caused by rotavirus infection is multifactorial and includes increased fluid secretion, decreased absorption, and a breakdown in the intestinal barrier. However, the molecular mechanisms underlying these disease manifestations are not well understood, which represents a significant barrier to developing more effective treatments for rotavirus diarrheal disease. Thus, deciphering the mechanisms of how rotavirus dysregulates intracellular calcium during infection is key to understanding rotavirus disease.

MA104 (monkey kidney) cells stably expressing the fluorescent calcium biosensor GCaMP6s are infected with rotavirus (expressing a mRuby3 reporter). The rotavirus-infected cell generates a signal that propagates from the infected cell to the surrounding uninfected cells.

How does microscope-based imaging support your research?

Calcium (Ca2+) is an essential intracellular second messenger in cells and governs many essential biological processes including endo/exocytosis, contraction, cell motility, metabolism, transcription, apoptosis, and proliferation. To understand how rotavirus dysregulates calcium to cause disease, we need to measure calcium changes in infected cells and neighboring, uninfected cells during infection. Genetically-encoded calcium indicators have been developed using green or red fluorescent protein-based biosensors in cells and thus report calcium concentration changes as changes in fluorescence intensity. Microscope-based imaging best meets these requirements for live cell measurements with spatiotemporal relationships preserved. The Nikon Ti-E inverted microscope has enabled fast, gentle epifluorescence imaging of our rotavirus-infected cells at single-cell resolution. The Okolab stage-top incubator system, which is mounted onto the Ti-E microscope stage, has been essential for measuring the changes in calcium in these cells throughout infection, about 18hrs. Furthermore, the NIS-Elements software has allowed us to measure multiple wells and experimental conditions within a single imaging run, increasing our research productivity.

Please tell us about technical challenges presented by calcium imaging.

Calcium signaling is a dynamic process in living cells, in which the location, duration, and frequency have important consequences for cell biology. These aspects make studying calcium signaling particularly challenging. First, the imaging must be gentle to avoid causing phototoxicity in the cells, which could confound interpretation of the calcium signals. This gentle imaging must be robust enough to capture the changes reported by the calcium sensor. Second, the imaging must be fast enough to capture the changes in intracellular calcium as they can rapidly flux, on the order of seconds to milliseconds depending on the particular signal. Third, the imaging must have sufficient resolution to determine where the calcium signal is, on the single-cell or even subcellular levels. In our experimental system, all of these challenges must be met over ~18 hrs in a system that also keeps the cells alive, and the microscope stays in focus over multiple positions to measure many different conditions in the same experiment.

How does the Nikon Ti-E microscope with NIS-Elements software address these challenges?

The Nikon Ti-E inverted microscope has been ideal for our calcium imaging experiments during rotavirus infection. Epifluorescence microscopy provides excellent resolution for cell monolayers as well as gentle excitation for fluorescence imaging. The NIS-Elements software allows multipoint acquisitions so that we can choose specific areas of wells to image repeatedly throughout the experiment, allowing comparison of multiple experimental conditions. These aspects have been key for measuring changes in fluorescence intensity, and thus calcium, in cells throughout rotavirus infection. The NIS-Elements software has also enabled easy image analysis of relative fluorescence increases of the cells through selections of regions of interest and export of data to spreadsheet for further analysis.

3D Jejunum-derived human intestinal organoids stably expressing the fluorescent calcium biosensor GCaMP6s are mock-infected (left) or rotavirus-infected (right). While there is basal signaling in the uninfected organoid, rotavirus infection drastically increases the frequency and area of multicellular calcium signaling events in the infected organoids.

You recently published a research article in the journal Science using this type of system. Can you please briefly tell us about this publication?

A leading model of rotavirus-induced diarrhea is that virus-infected cells activate paracrine signaling pathways to dysregulate neighboring, uninfected cells. Using this strategy, the virus amplifies its disease effects beyond the directly infected cell and results in excessive fluid secretion and diarrhea. However, this type of infected cell to neighboring cell signaling has not been directly observed in an infection. In our recent Science paper, we found that rotavirus triggered a paracrine signal from infected to uninfected cells and then characterized this paracrine signaling pathway (also see this perspective about the paper). Using the long-term calcium imaging techniques we had developed using the Nikon microscope and software, we demonstrated that individual rotavirus-infected cells produced a cytosolic calcium-raising signal that propagated from the infected cell to the surrounding uninfected cells—a pattern of signaling called intercellular calcium waves (ICWs). Surprisingly, the rotavirus-induced ICWs were not caused by any previously identified paracrine signaling pathway upregulated during rotavirus infection. Rather, the rotavirus-infected cells repeatedly secreted the extracellular purine ADP that in turn activated purinergic receptors on neighboring cells, specifically the P2Y1 receptor. The activation of the P2Y1 receptor caused the increase in cytosolic calcium in the uninfected cells, which we observed using a fluorescent biosensor in cells via epifluorescence microscopy. Rotavirus-infected cells initiated hundreds of these ICWs during the course of infection, yielding stunning time-lapse fluorescence microscopy videos.

Blocking this paracrine purinergic signal also blocked the other pathways associated with rotavirus pathophysiology. Pharmacological blockers of the P2Y1 receptor also reduced rotavirus diarrhea in a mouse model, which demonstrated the potential clinical significance of this work. Together, these studies confirmed the long-held premise that rotavirus-infected cells elicit a paracrine signal involved in diarrhea and vomiting responses. Since paracrine purinergic signaling was involved in rotavirus pathophysiology, the P2Y1 receptor is a potential therapeutic target for host-targeted antiviral or anti-diarrheal drugs. Furthermore, this was the first known example that viruses can exploit purinergic signaling and ICWs to amplify pathophysiological signaling important for diarrhea. These observations and findings were made possible through live cell calcium imaging of rotavirus-infected cells.

What are your future research plans?

Our studies of rotavirus-induced paracrine signaling that manifests as intercellular calcium waves (ICWs) raises many new research directions. First, the mechanism by which rotavirus induces the intercellular calcium waves is unknown. Identifying the viral proteins that dysregulate host cell processes will provide important further insights into rotavirus pathophysiology. Second, the mechanism by which ADP leaves the infected cell is also unknown. Using rotavirus to study purine release mechanisms will advance understanding of basic epithelial biology and cell to cell communication. Finally, it is possible that other enteric or epithelial viruses also exploit intercellular calcium waves for their pathophysiology and have yet to be observed.