Visualizing Dynamics of Methyl‐β‐Cyclodextrin‐Induced Apoptosis Using Single‐Cell Imaging

Methyl‐β‐cyclodextrin (MβCD) disrupts lipid rafts by depleting cholesterol from the membrane and causes caspase‐3‐induced apoptosis at high concentrations. This study assessed the activity of other proapoptotic factors using fluorescence resonance energy transfer (FRET) assays, including the BH3‐interacting domain death agonist (Bid) and caspase‐9, which were observed to be cut and activated by MβCD in this article. The FRET efficiency revealed that the cleavage of Bid exceeded that of caspase‐9 during the same period. Both caspase‐9 and Bid are integral to mitochondria‐associated apoptotic pathways. Moreover, we analyzed spatiotemporal features of cell‐signaling regulators—reactive oxygen species (ROS), rat sarcoma protein (Ras), and Ca 2+ ions—using single‐cell imaging throughout apoptosis. A rapid, time‐dependent increase in ROS concentration was detected in the early stages of apoptosis. FRET imaging indicated a time‐dependent decline in Ras activity, commencing near the cell membrane due to MβCD. Fluorescence imaging revealed that MβCD prompted a fluctuating increase in intracellular Ca 2+ levels, enhancing Ca 2+ ion diffusion. While oscillation in the Ca 2+ concentration was also observed in the control group, MβCD‐treated cells showed remarkably large amplitudes and a reduced number of oscillation peaks. Our findings confirm that MβCD‐induced apoptosis involves mitochondria‐associated apoptotic signaling, with ROS, Ras, and Ca 2+ ions playing pivotal roles in the process.