A Ratiometric Two‐Photon Fluorescent Probe for Tracking the Lysosomal ATP Level: Direct in cellulo Observation of Lysosomal Membrane Fusion Processes
Authors and Affiliations
Vesicles exchange its contents through membrane fusion processes-kiss-and-run and full-collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We disclose a ratiometric two-photon probe offering real-time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two-photon live-cell imaging technique, lysosomal membrane fusion process in cells has been directly observed along with the concentration of its content-lysosomal ATP. Results show that the kiss-and-run process between lysosomes proceeds through repeating transient interactions with gradual content mixing, whereas the full-fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small-molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.
Keywords: Fluorescent probe, Two-photon microscopy, membrane fusion, lysosomal ATP, ratiometric imaging