Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems

摘要

Autophagy is a lysosome-based degradation pathway. Autophagic lysosomereformation (ALR) is a lysosomal membrane recycling process that marks theterminal step of autophagy. During ALR, LAMP1-positive tubules, named ref-ormation tubules, are extruded from autolysosomes, and nascent lysosomesare generated from these tubules. By combining proteomic analysis of puri-fied autolysosomes and RNA interference screening of identified candidates,we systematically elucidated the ALR pathway at the molecular level. Basedon the key components clathrin, PtdIns(4,5)P2, and the motor protein KIF5B,among others, we reconstituted this process in vitro. This unit describes adetailed method for visualizing ALR in cells during the autophagy process.This unit also presenta protocol for reconstituting the ALR tubular protru-sion and elongation process in vitro and three methods for preparing materialsfor in vitro reconstitution: (1) autolysosome purification from cultured cells,(2) liposome preparation, and (3) KIF5B purification and quality testing.