Imaging bioluminescent indicators shows Ca2+ and ATP permeability thresholds in live cells attacked by complement

Area of Science:

• Cellular biology
• Immunology
• Biochemistry

Background:

• The complement system plays a crucial role in innate immunity.
• Understanding complement-mediated cell damage is vital for treating inflammatory diseases.
• Previous studies suggested propidium iodide uptake as a measure of cell death.

Purpose of the Study:

• To investigate the temporal sequence of cellular responses to complement attack.
• To establish permeability thresholds to different molecules during complement-mediated cell damage.
• To re-evaluate the reliability of propidium iodide as an indicator of cell death.

Main Methods:

• Utilized HeLa cells expressing a luciferase-aequorin fusion protein to monitor ATP levels and cytosolic free calcium (Ca2+).
• Employed nuclear propidium fluorescence to measure permeability to small molecules.
• Assessed cell lysis through imaging luciferase activity and monitoring ATP reduction.
• Investigated the dose-dependent effects of complement C9 on cellular responses.

Main Results:

• A rise in cytosolic free Ca2+ was observed at least 60 seconds before propidium uptake and ATP reduction following C9 attack.
• These Ca2+ changes and subsequent events were dependent on the concentration of C9.
• Cells remained impermeable to the luciferase-aequorin protein, even when permeable to propidium, indicating intact membrane integrity.
• No loss of intracellular protein was detected at sub-lytic C9 concentrations.

Conclusions:

• Propidium uptake is not a reliable indicator of cell death during complement attack, as cells can be permeable to propidium while maintaining membrane integrity.
• The influx of Ca2+ is an early event preceding membrane permeabilization and lysis.
• Cellular heterogeneity must be considered when interpreting cellular responses to membrane pore-forming attacks by the complement system.