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

作者：Zhang, Wenbin; Wei, Pengfei; Liu, Liu; Ding, Tao; Yang, Yinyin; Jin, Peipei; Zhang, Li; Zhao, Zhibin; Wang, Meimei; Hu, Bochuan; Jin, Xin; Xu, Zeng; Zhang, Han; Song, Yang; Wang, Liansheng; Zhong, Suqin; Chen, Jing; Yang, Zhenyu; Chen, Ziying; Wu, Yu; Ye, Zhiming; Xu, Youcui^*; Zhang, Yunjiao^*; Wen, Long-Ping^*

来源：Autophagy, 2023, 19(12): 3062-3078.

DOI：10.1080/15548627.2023.2235197

• Aggregation-induced emission
• autophagy
• cell sorting
• flow cytometry
• LC3-interacting
• transfection-free

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.

• 单位
  1; 安徽医科大学; 南方医科大学; 广东省人民医院; 中山大学