Using co-cultures expressing fluorescence resonance energy transfer based protein biosensors to simultaneously image caspase-3 and Ca2+ signaling.
Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca2+ signaling events involved in cell death of COS-7 cells induced by 10 mM H2O2. Interestingly, this H2O2 stimulus induced synchronous caspase-3 activation and Ca2+ signaling. In parallel to caspase-3 activation, cytosolic Ca2+ concentration, [Ca2+]c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca2+]c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H2O2. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.
Chiang
Jason Jui-Hsuan
JJ
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, M5S 3G9, Ontario, Toronto, Canada.
Truong
Kevin
K
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015374:Biosensing Techniques / Q000379:methods*; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002522:Chlorocebus aethiops; D018920:Coculture Techniques / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008657:Metabolic Clearance Rate / Q000187:drug effects
D006861:Hydrogen Peroxide; D053148:Caspase 3; D020169:Caspases; D002118:Calcium