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Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor
Published on: April 1, 2011
Monitoring Calcium Fluxes and Lysosome Exocytosis During Pyroptosis
Wendy P Loomis1, Tessa Bergsbaken2
1Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.
Abstract:
Inflammasome-mediated activation of inflammatory caspases (caspase-1, caspase-4, caspase-5, caspase-11) initiates a cascade of cellular events that lead to proinflammatory cell death, or pyroptosis. Proteolytic cleavage of gasdermin D results in the formation of transmembrane pores that allow the release of mature cytokines IL-1β and IL-18. Gasdermin pores also allow calcium influx through the plasma membrane, triggering the fusion of lysosomal compartments with the cell surface and release of their contents into the extracellular milieu in a process termed lysosome exocytosis. This chapter outlines methods for measuring calcium flux, lysosome exocytosis, and membrane disruption after inflammatory caspase activation.
Insights
Inflammasome activation triggers pyroptosis, a proinflammatory cell death. Methods are presented to measure gasdermin D pore formation, calcium influx, and lysosome exocytosis following inflammatory caspase activation.
Area of Science:
- Cellular biology
- Immunology
- Molecular mechanisms of cell death
Background:
- Inflammasomes activate inflammatory caspases, initiating pyroptosis, a programmed inflammatory cell death.
- Gasdermin D cleavage by caspases forms pores, releasing IL-1β and IL-18 and causing membrane disruption.
- These pores also facilitate calcium influx, leading to lysosome exocytosis.
Purpose of the Study:
- To outline methods for assessing cellular events downstream of inflammatory caspase activation.
- To provide techniques for measuring calcium flux, lysosome exocytosis, and membrane integrity.
Main Methods:
- Measurement of calcium influx using fluorescent indicators.
- Assessment of lysosome exocytosis via marker release or imaging.
- Quantification of membrane disruption and pore formation.
Main Results:
- Established protocols for quantifying key pyroptosis events.
- Demonstrated links between caspase activation, gasdermin D pore formation, and downstream cellular responses.
- Provided a framework for studying inflammasome-mediated cell death.
Conclusions:
- The presented methods enable detailed analysis of pyroptosis.
- Understanding these mechanisms is crucial for studying inflammatory diseases.
- This work facilitates research into inflammasome signaling pathways.
