AIE-enabled transfection-free identification and isolation of viable cell subpopulations differing in the level of autophagy

Elevated macroautophagy/autophagy, typically characterized by increased autophagosome accumulation, occurs in a wide variety of physiological and pathophysiological processes, but the current methodology for studying autophagy aberration in native non-transfected cells is rather limited. Here we show that LKT, an engineered molecular probe composed of a cell-penetrating peptide, an LC3-interacting motif and the aggregation-inducedemission (AIE) luminogen tetraphenylethylene, achieved robust identification and isolation of viable autophagy-varying cell subpopulations without the need of foreign reporter gene expression. Non-fluorescent in water, LKT fluorescence is activated upon interaction with liposomes in an AIE-dependent fashion, and the presence of LC3 on the liposome membrane dramatically boosted LKT fluorescence enhancement. In LKT-treated GFP-LC3 HeLa cells, induction of autophagy with rapamycin or trehalose, and blockade of autophagy with chloroquine, both produced bright GFP-LC3-colocalizing LKT puncta, leading to an increase in LKT fluorescence that facilitated efficient separation of cells based on the level of autophagosome accumulation. Using fluorescence-activated cell sorting, we were able to isolate cell subpopulations varying in the level of basal autophagy from a variety of cultured cell lines and primary cells. In a proof-of-concept study, we isolated autophagy-high and autophagy-low subpopulations from differentiated THP-1 cells and revealed that the autophagy-high THP-1 cells, compared to their autophagy-low counterparts, exhibited a higher level of NLRP3 protein expression and a stronger NLRP3 inflammasome activation following nigericin challenge. Our work demonstrated the unique power of the AIE technology and LKT, filling a void, should prove valuable for autophagy research.