TY - JOUR
T1 - Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems
AU - Chen, Yang
AU - Su, Qian Peter
AU - Sun, Yujie
AU - Yu, Li
N1 - Publisher Copyright:
© 2018 John Wiley & Sons, Inc.
PY - 2018/3
Y1 - 2018/3
N2 - Autophagy is a lysosome-based degradation pathway. Autophagic lysosome reformation (ALR) is a lysosomal membrane recycling process that marks the terminal step of autophagy. During ALR, LAMP1-positive tubules, named reformation tubules, are extruded from autolysosomes, and nascent lysosomes are generated from these tubules. By combining proteomic analysis of purified autolysosomes and RNA interference screening of identified candidates, we systematically elucidated the ALR pathway at the molecular level. Based on the key components clathrin, PtdIns(4,5)P 2 , and the motor protein KIF5B, among others, we reconstituted this process in vitro. This unit describes a detailed method for visualizing ALR in cells during the autophagy process. This unit also present a protocol for reconstituting the ALR tubular protrusion and elongation process in vitro and three methods for preparing materials for in vitro reconstitution: (1) autolysosome purification from cultured cells, (2) liposome preparation, and (3) KIF5B purification and quality testing.
AB - Autophagy is a lysosome-based degradation pathway. Autophagic lysosome reformation (ALR) is a lysosomal membrane recycling process that marks the terminal step of autophagy. During ALR, LAMP1-positive tubules, named reformation tubules, are extruded from autolysosomes, and nascent lysosomes are generated from these tubules. By combining proteomic analysis of purified autolysosomes and RNA interference screening of identified candidates, we systematically elucidated the ALR pathway at the molecular level. Based on the key components clathrin, PtdIns(4,5)P 2 , and the motor protein KIF5B, among others, we reconstituted this process in vitro. This unit describes a detailed method for visualizing ALR in cells during the autophagy process. This unit also present a protocol for reconstituting the ALR tubular protrusion and elongation process in vitro and three methods for preparing materials for in vitro reconstitution: (1) autolysosome purification from cultured cells, (2) liposome preparation, and (3) KIF5B purification and quality testing.
KW - autolysosome purification
KW - autophagic lysosome reformation (ALR)
KW - autophagy
KW - density-gradient centrifugation
KW - in vitro reconstitution
KW - liposome preparation
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U2 - 10.1002/cpcb.44
DO - 10.1002/cpcb.44
M3 - Article
C2 - 30040179
AN - SCOPUS:85050817569
SN - 1934-2500
VL - 78
SP - 11.24.1-11.24.15
JO - Current Protocols in Cell Biology
JF - Current Protocols in Cell Biology
IS - 1
ER -