Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins.

Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin-like proteins (mATG8s). We used phage display to isolate peptides that possess bona fide LIR (LC3-interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C-specific sensor, we were able to monitor recruitment of endogenous LC3C to Salmonella during xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs.

Semisynthesis of autophagy protein LC3 conjugates.

Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens. Autophagosome formation is the key process in autophagy. Lipidated Atg8/LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. To understand the function of Atg8/LC3-PE in autophagosome formation and host-pathogen interaction requires preparation and structural manipulation of lipidated Atg8/LC3 proteins. Herein, we report the semisynthesis of LC3 proteins and mutants with modifications of different PE fragments or lipids using native chemical ligation and aminolysis approaches.

Selective Autophagy: ATG8 Family Proteins, LIR Motifs and Cargo Receptors.

Selective autophagy relies on soluble or membrane-bound cargo receptors that recognize cargo and bring about autophagosome formation at the cargo. The cargo-bound receptors interact with lipidated ATG8 family proteins anchored in the membrane at the concave side of the forming autophagosome. The interaction is mediated by 15- to 20-amino-acid-long sequence motifs called LC3-interacting region (LIR) motifs that bind to the LIR docking site (LDS) of ATG8 proteins. In this review, we focus on LIR-ATG8 interactions and the soluble mammalian selective autophagy receptors. We discuss the roles of ATG8 family proteins as membrane scaffolds in autophagy and the LIR-LDS interaction and how specificity for binding to GABARAP or LC3 subfamily proteins is achieved. We also discuss atypical LIR-LDS interactions and a novel LIR-independent interaction. Recently, it has become clear that several of the soluble cargo receptors are able to recruit components of the core autophagy apparatus to aid in assembling autophagosome formation at the site of cargo sequestration. A model on phagophore recruitment and expansion on a selective autophagy receptor-coated cargo incorporating the latest findings is presented.

Imaging Noncanonical Autophagy and LC3-Associated Phagocytosis in Cultured Cells.

Monitoring of ATG8 proteins by western blotting and immunofluorescence microscopy are the most common methods to monitor the autophagy pathway. However, it has recently been shown that ATG8 proteins can be lipidated to non-autophagosome, single-membrane compartments through a noncanonical autophagy pathway. This is commonly found to occur during macro-endocytic processes such as phagocytosis, where it has been termed LC3-associated phagocytosis, and upon lysosomotropic drug treatment. Therefore, care is required when interpreting data based on ATG8 in order to conclude whether a signal relates to the canonical or noncanonical pathway. Here we provide methods to monitor noncanonical autophagy through fluorescence microscopy.

Biochemical Methods to Monitor Autophagic Responses in Plants.

The study of autophagy in plants is rapidly increasing, due to its pivotal and fundamental roles in responding to stressful stimuli, recycling nutrients during senescence, and maintaining growth under normal conditions. Assays for detecting autophagy in plants have generally been based on microscopic observations, providing qualitative information on autophagy activity. Here, we discuss biochemical assays for detecting autophagy, which have the potential for providing more quantitative information, with a focus on immunoblotting with antibodies against ATG8, NBR1, or epitope tags fused to ATG proteins.

Methods to Assess Autophagy and Chronological Aging in Yeast.

Autophagy is a catabolic process that is crucial for cellular homeostasis and adaptive response to changing environments. Importantly, autophagy has been shown to be induced in many longevity-associated scenarios and to be required to maintain lifespan extension. Notably, autophagy is a highly conserved cellular process among eukaryotes, and the yeast Saccharomyces cerevisiae has become a universal model system for unraveling the molecular machinery underlying autophagic mechanisms. Here, we discuss different protocols to monitor survival and autophagy of yeast cells upon chronological aging. These include the use of propidium iodide to assess the loss of cell membrane integrity, as well as clonogenic assays to directly determine survival rates. Additionally, we describe methods to quantify autophagic flux, including the alkaline phosphatase activity or the GFP liberation assays, which measure the delivery of autophagosomal cargo to the vacuole. In sum, we have recapped established protocols used to evaluate a link between lifespan extension and autophagy in yeast.