A sensitive and quantitative autolysosome probe for detecting autophagic activity in live and prestained fixed cells

Abstract

Autophagy is a complex, multi-step and biologically important pathway mediated by autophagosomes and autolysosomes. Accurately dissecting and detecting different stages of autophagy is important to elucidate its molecular mechanism and thereby facilitate the discovery of pharmaceutical molecules. We herein reported a small-molecule synthetic probe, Zn-G 4, which is only fluorescent upon starvation- or chemical agent-induced autophagy within the autolysosome or possible the late endosome/lysosome networks. The probe can be detected by one-photon microscopy, which gives a high signal-to-noise ratio readout of autophagic activity. The pH gradient-independent fluorescence can be detected both in live and prestained fixed cells. Moreover, the fluorescent recording can be used to quantify autophagic activity at a single point without transfection or false positive signals due to protein aggregation. Furthermore, autophagy-induced fluorescence in autolysosomes can also be detected by two-photon microscopy, suggesting potential applications in deep tissue and in vivo. In conclusion, we have developed a sensitive and specific autolysosomal probe that can be used for monitoring autophagy during later stages along with quantitative assays together with widely used early markers or microtubule-associated protein 1 light chain 3 (LC3)-based probes.

Fibroblast Growth Factor 21 Protects Against High Glucose Induced Cellular Damage and Dysfunction of Endothelial Nitric-Oxide Synthase in Endothelial Cells

“…After migration for 12 and 24 hours, nonmigrating cells on the upper side of the membrane were removed with a cotton swab [30]. Cells that had migrated and were attached to the lower side were fixed with 10% formalin and stained with DAPI for 2 min and counted in five different random fields using a fluorescent microscopy [31]. …”

ZnII Complexes for Bioimaging and Correlated Applications

“…This effect restrains the pathways of metal complexes exposure to cells and thus influence the intracellular distribution. ZnSalens might be applied as fewer cytotoxicity agents with high fluorescence intensity in particular organelles presenting more hydrophobic environments than in cytosol or cell culture media . As ZnSalen tends to aggregate in aqueous media, it is likely to examine whether ZnSalen also aggregates in biological environments and how the aggregation influences its biological behaviour including cellular uptake and intracellular localization.…”

“…ZnSalens might be applied as fewer cytotoxicity agentsw ith high fluorescencei ntensityi n particularo rganelles presenting more hydrophobic environmentst han in cytosol or cell culture media. [83][84] As ZnSalen tends to aggregate in aqueous media, it is likely to examine whether ZnSalen also aggregates in biological environments and how the aggregation influences its biological behaviour including cellular uptake and intracellularl ocalization. The discernmento btained from the coordination chemistry of ZnSalens are very useful tools for further developing efficient fluorescent ZnSalen complexes to act as bioimaging probes.…”

Introducing Metallosalens into Biological Studies: The Renaissance of Traditional Coordination Complexes

“…Autophagy is an important biological process involving digestion of worn‐out organelles by the cell's own lysosomes. d ‐Glucose‐modified Zn(salen) can be used as a specific and quantitative autolysosome tracker for monitoring later stages of autophagy by one‐ or two‐photon fluorescence microscopy . This probe only exhibits “turn‐on” fluorescence upon starvation‐ or chemical‐agent‐induced autophagy, with localization in the autolysosome or possibly in late endosome/lysosome networks.…”