Ciccotosto, G. D.; Jana, M.; Galluzzi, L.; Mitchell, L.; Gupta, P.; Furness, S. G. B.; Lazarou, M.; Hare, D. L.; Wookey, P. J.

Autophagy is a highly-conserved mechanism that ensures the lysosomal degradation of disposable or potential toxic cytosolic entities in support of cell survival and adaptation to stress. Autophagy is deregulated in various pathological conditions including cardiovascular, neurological, neoplastic- autoimmune and degenerative disorders. However, clinically relevant pharmacological modulators of autophagy remain elusive, calling for the development of novel screening approaches that are amenable to high throughput/content applications. Here, we describe a simple method to detect autophagy in cultured mammalian cells based on the pH-sensitive fluorescent conjugate CalRexin:pHrodo Red and high throughput/content imaging. CalRexin:pHrodo Red is promptly taken up by endosome- amphisome-autolysosome (low pH ~4.5) pathway, which is connected to canonical autophagy, culminating with a bright red fluorescence. Importantly, this system allows for the discrimination between autophagic responses coupled with the endosome-amphisome-autolysosome pathways and the endosome-lysosome pathway as elicited by receptor-driven endocytosis and phagocytosis. In human cervical carcinoma sHeLa cells, the accumulation of CalRexin:pHrodo Red as elicited by canonical autophagy activators like the mTOR inhibitor rapamycin or serum starvation was suppressed by conventional inhibitors 3-methyladenine or chloroquine. Similarly, RB1CC1-/- (FIP200) as well as ATG5-/- sHeLa cells (which bear genetic defects in two different steps of autophagy) were unable to accumulate CalRexin:pHrodo Red upon exposure to autophagy activators. Thus, CalRexin:pHrodo Red provides a novel approach for imaging autophagy on high throughout/content platforms to screen large libraries for the identification of novel autophagy-targeting agents.