Recent progress in autophagic lysosome reformation.

Autophagic lysosome reformation (ALR) is the terminal step of autophagy and is essential for maintaining lysosome homeostasis during autophagy. During ALR, tubules are extruded from autolysosomes, and small vesicles named proto-lysosomes, which are composed of lysosomal membrane components, are generated from these tubules. Eventually, proto-lysosomes mature into functional lysosomes. In this review, we will summarize recent progress in understanding the regulation, mechanisms and physiological functions of ALR.

TNFAIP8L2/TIPE2 impairs autolysosome reformation via modulating the RAC1-MTORC1 axis.

Macroautophagy/autophagy is an evolutionarily conserved process that involves the selective degradation of cytoplasmic components within lysosomes in response to starvation. Autophagy is an ancient defense mechanism that has been closely integrated with the immune system and has multiple effects on innate and adaptive immunity. The pro-inflammatory and anti-inflammatory cytokines can activate and inhibit autophagy, respectively. TNFAIP8L2/TIPE2 (tumor necrosis factor, alpha-induced protein 8-like 2) is a newly identified immune negative regulator of innate and adaptive immunity that plays an important role in immune homeostasis. However, whether and how TNFAIP8L2 controls autophagy is still unknown. Murine TNFAIP8L2 can directly bind to and block the RAC1 GTPase activity to regulate innate immunity. RAC1 can also bind to MTOR and regulate MTORC1 cellular localization and activity. Here, we find that TNFAIP8L2 can compete with MTOR for binding to the GTP-bound state of RAC1 and negatively regulate MTORC1 activity. Interestingly, TNFAIP8L2 overexpression fails to induce autophagy flux by the suppression of the MTOR activity under glutamine and serum starvation. Instead, TNFAIP8L2 appears to impair autophagic lysosome reformation (ALR) during prolonged starvation. Finally, we demonstrate that TNFAIP8L2 overexpression leads to a defect in MTOR reactivation and disrupts autophagy flux, thereby leading to cell death. Furthermore, TNFAIP8L2 deficiency can exacerbate the inflammatory response and lung injury by controlling the MTOR activity in an LPS-induced mouse endotoxemia model. Our study reveals a novel role of TNFAIP8L2 in autophagy by regulating the RAC1-MTORC1 axis that supports its potential as a target for therapeutic treatment.Abbreviations: ALR: autophagic lysosome reformation; BafA1: bafilomycin A1; BMDMs: bone marrow-derived macrophages; Co-IP: Co-Immunoprecipitation; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase complex 1; RAPA: rapamycin; RPS6: ribosomal protein S6; SQSTM1/p62: sequestosome 1; Starv: Starvation; TNFAIP8L2/TIPE2: tumor necrosis factor-alpha-induced protein-8 like-2.

Single-molecule in vitro reconstitution assay for kinesin-1-driven membrane dynamics.

Intracellular membrane dynamics, especially the nano-tube formation, plays important roles in vesicle transportation and organelle biogenesis. Regarding the regulation mechanisms, it is well known that during the nano-tube formation, motor proteins act as the driven force moving along the cytoskeleton, lipid composition and its associated proteins serve as the linkers and key mediators, and the vesicle sizes play as one of the important regulators. In this review, we summarized the in vitro reconstitution assay method, which has been applied to reconstitute the nano-tube dynamics during autophagic lysosomal regeneration (ALR) and the morphology dynamics during mitochondria network formation (MNF) in a mimic and pure in vitro system. Combined with the single-molecule microscopy, the advantage of the in vitro reconstitution system is to study the key questions at a single-molecule or single-vesicle level with precisely tuned parameters and conditions, such as the motor mutation, ion concentration, lipid component, ATP/GTP concentration, and even in vitro protein knockout, which cannot easily be achieved by in vivo or intracellular studies.

Development of Research into Autophagic Lysosome Reformation.

Autophagy is a lysosome-dependent degradation process that is essential for maintaining cellular homeostasis. In recent years, more studies have focused on the late stages of autophagy. Our group discovered and studied the terminal step of autophagy, namely autophagic lysosome reformation (ALR). ALR is the process that regenerates functional lysosomes from autolysosomes, thus maintaining lysosome homeostasis. ALR involves clathrin-mediated membrane budding from autolysosomes, elongation of membrane tubules along microtubules with the pulling force provided by the motor protein KIF5B, proto-lysosome scission by dynamin 2, and finally maturation of proto-lysosomes to functional lysosomes. In this review, we will summarize progress in unveiling the molecular mechanisms underlying ALR and its potential pathophysiological roles.

Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems.

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)P2 , 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. © 2018 by John Wiley & Sons, Inc.