Ratiometric and Discriminative Visualization of Autophagy and Apoptosis with a Single Fluorescent Probe Based on the Aggregation/Monomer Principle

Abstract

Autophagy and apoptosis play a central role in maintaining homeostasis in mammals. Therefore, discriminative visualization of the two cellular processes is an important and challenging task. However, fluorescent probes enabling ratiometric visualization of both autophagy and apoptosis with different sets of fluorescence signals have not been developed yet. In this work, we constructed a versatile single fluorescent probe (NKLR) based on the aggregation/monomer principle for the ratiometric and discriminative visualization of autophagy and apoptosis. NKLR can simultaneously perform two-color imaging of RNA (deep red channel) and lysosomes (yellow channel) in aggregation and monomer states, respectively. During autophagy, NKLR migrated from cytoplasmic RNA and nuclear RNA to lysosomes, showing enhanced yellow emission and sharply decreased deep red fluorescence. Moreover, this migration process was reversible upon the recovery of autophagy. Comparatively, during apoptosis, NKLR immigrated from lysosomes to RNA, and the yellow emission decreased and even disappeared, while the fluorescence of the deep red channel slightly increased. Overall, autophagy and apoptosis could be discriminatively visualized via the fluorescence intensity ratios of the two channels. Meanwhile, the cells in three different states (healthy, autophagic, apoptotic) could be distinguished by three point-to-point fluorescence images via the localization and emission color of NKLR. Therefore, the probe NKLR can serve as a desirable molecular tool to reveal the in-depth relation between autophagy and apoptosis and facilitate the study on the two cellular processes.

Dual-Labeled Single Fluorescent Probes for the Simultaneous Two-Color Visualization of Dual Organelles and for Monitoring Cell Autophagy

“…Up to now, small-molecule fluorescent probes have been widely used in monitoring the interplay between organelles during autophagy due to the advantages of the simple staining process, remarkable sensitive response, flexible molecular design strategies, accurate spatiotemporal information, and nondestructive detection. − Recently, there have been several reports on autophagy fluorescent probes, − which mainly need to be combined with lysosomes and other autophagy-related organelle probes with different emissions. − However, this strategy tends to have undesirable effects such as uneven probe distribution, additional risks of cytotoxicity, different metabolism profiles, and tedious staining and washing procedures. In contrast, dual-labeled single fluorescent probes − that separately label lysosomes and other autophagy-related organelles in the dual-color model at the same time can avoid the above problems of composite probes, but only a few such dual-labeled probes have been reported so far. − Theoretically, for realizing the targeting of multiple organelles, different organelle-targeting groups can be simply introduced into a single probe molecule. However, this plausible approach inevitably leads to targeting competition between complex intracellular organelles.…”