Pho81 is a novel regulator of pexophagy induced by phosphate starvation.

In the budding yeast Saccharomyces cerevisiae, macroautophagy/autophagy can be induced by various types of starvation. It is thought that potential autophagic substrates vary to meet specific nutritional demands under different starvation conditions. In a recent study, Gross et al. found that autophagy induced by phosphate starvation includes many selective aspects. For example, this work identified Pho81 as a regulator of pexophagy under conditions of phosphate starvation. Pho81 senses phosphate metabolites and directly interacts with Atg11 to promote Atg1-mediated Atg11 phosphorylation. This finding provides an example of how modulation of the Atg1/ULK kinase complex can convey specific metabolic information to regulate autophagic substrates.Abbreviation: AKC: Atg1/ULK kinase complex.

Detection of Autophagy in Plants by Fluorescence Microscopy.

Autophagy is a key process for degradation and recycling of proteins or organelles in eukaryotes. Autophagy in plants has been shown to function in stress responses, pathogen immunity, and senescence, while a basal level of autophagy plays a housekeeping role in cells. Upon activation of autophagy, vesicles termed autophagosomes are formed to deliver proteins or organelles to the vacuole for degradation. The number of autophagosomes can thus be used to indicate the level of autophagy. Here we describe two common methods used for detection of autophagosomes, staining of autophagosomes with the fluorescent dye monodansylcadaverine and expression of a fusion between GFP and the autophagosomal membrane protein ATG8.

Cantharidin downregulates PSD1 expression and inhibits autophagic flux in yeast cells.

Cantharidin is a terpenoid compound of insect origin, naturally produced by male blister beetles as an antipredatory mechanism. Cantharidin has anticancer properties, which are attributed to its ability to induce cell cycle arrest, DNA damage, MAPK signaling pathway, and apoptosis. Cantharidin has been reported to induce apoptosis in triple-negative breast cancer cells by suppressing autophagy via downregulation of Beclin 1 expression and autophagosome formation. However, it remains unclear which stage of the autophagic pathway is targeted by cantharidin. Herein, we report that yeast cells are sensitive to cantharidin, and external supplementation of ethanolamine (ETA) ameliorates the cytotoxicity. In addition, cantharidin downregulates phosphatidylserine decarboxylase 1 (PSD1) expression. We also report that cantharidin inhibits autophagic flux, and external administration of ETA could rescue this inhibition. Additionally, cotreatment with chloroquine sensitized the autophagy inhibitory effects of cantharidin. We conclude that yeast cells are sensitive to cantharidin due to inhibition of autophagic flux.

TOR and MAP kinase pathways synergistically regulate autophagy in response to nutrient depletion in fission yeast.

General autophagy is an evolutionarily conserved process in eukaryotes, by which intracellular materials are transported into and degraded inside lysosomes or vacuoles, with the main goal of recycling those materials during periods of starvation. The molecular bases of autophagy have been widely described in Saccharomyces cerevisiae, and the specific roles of Atg proteins in the process were first characterized in this model system. Important contributions have been made in Schizosaccharomyces pombe highlighting the evolutionary similarity and, at the same time, diversity of Atg components in autophagy. However, little is known regarding signals, pathways and role of autophagy in this distant yeast. Here, we undertake a global approach to investigate the signals, the pathways and the consequences of autophagy activation. We demonstrate that not only nitrogen but several nutritional deprivations including lack of carbon, sulfur, phosphorus or leucine sources, trigger autophagy, and that the TORC1, TORC2 and MAP kinase Sty1 pathways control the onset of autophagy. Furthermore, we identify an unexpected phenotype of autophagy-defective mutants, namely their inability to survive in the absence of leucine when biosynthesis of this amino acid is impaired.Abbreviations: ATG: autophagy-related; cAMP: cyclic adenosine monophosphate; cDNA: complementary deoxyribonucleic acid; GFP: green fluorescence protein; Gluc: glucose; Leu: leucine; MAP: mitogen-activated protein; MM: minimal medium; PI: propidium iodine; PKA: protein kinase A; RNA: ribonucleic acid; RT-qPCR: real time quantitative polymerase chain reaction; S. cerevisiae: Saccharomyces cerevisiae; S. pombe: Schizosaccharomyces pombe; TCA: trichloroacetic acid; TOR: target of rapamycin; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; YE5S: yeast extract 5 amino acid supplemented.

Methods to Study Autophagocytosis in Magnaporthe oryzae.

Autophagy is an evolutionarily conservative biological process in eukaryotes. Since the lysosomes were discovered by De Duve in the 1950s, autophagy has been studied for more than half a century and the mechanism of autophagy process has been discovered in many model organisms. In the rice blast fungus Magnaporthe oryzae, autophagy relative proteins are essential for appressorium formation, penetration, and invasive growth. The null mutants for the expression of autophagy gene homologs in M. oryzae lose their pathogenicity for infection of host plants. In this chapter, we provide some methods for monitoring autophagy process using physics and biochemistry assays in M. oryzae. Moreover, similar approaches can be used to monitor autophagy in other plant filamentous pathogenic fungi.

DeepPhagy: a deep learning framework for quantitatively measuring autophagy activity in Saccharomyces cerevisiae.

Seeing is believing. The direct observation of GFP-Atg8 vacuolar delivery under confocal microscopy is one of the most useful end-point measurements for monitoring yeast macroautophagy/autophagy. However, manually labelling individual cells from large-scale sets of images is time-consuming and labor-intensive, which has greatly hampered its extensive use in functional screens. Herein, we conducted a time-course analysis of nitrogen starvation-induced autophagy in wild-type and knockout mutants of 35 AuTophaGy-related (ATG) genes in Saccharomyces cerevisiae and obtained 1,944 confocal images containing > 200,000 cells. We manually labelled 8,078 autophagic and 18,493 non-autophagic cells as a benchmark dataset and developed a new deep learning tool for autophagy (DeepPhagy), which exhibited superior accuracy in recognizing autophagic cells compared to other existing methods, with an area under the curve (AUC) value of 0.9710 from 10-fold cross-validations. We further used DeepPhagy to automatically analyze all the images and quantitatively classified the autophagic phenotypes of the 35 atg knockout mutants into 3 classes. The high consistency in our computational and biochemical results indicated the reliability of DeepPhagy for measuring autophagic activity. Moreover, we used DeepPhagy to analyze 3 additional types of autophagic phenotypes, including the targeting of Atg1-GFP to the vacuole, the vacuolar delivery of GFP-Atg19, and the disintegration of autophagic bodies indicated by GFP-Atg8, all with satisfying accuracies. Taken together, our study not only enables the GFP-Atg8 fluorescence assay to become a quantitative measurement for analyzing autophagic phenotypes in S. cerevisiae but also demonstrates that deep learning-based methods could potentially be applied to different types of autophagy.Abbreviations:Ac: accuracy; ALP: alkaline phosphatase; ALR: autophagic lysosomal reformation; ATG: AuTophaGy-related; AUC: area under the curve; CNN: convolutional neural network; Cvt: cytoplasm-to-vacuole targeting; DeepPhagy: deep learning for autophagy; fc_2: second fully connected; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3 beta; HAT: histone acetyltransferase; HemI: Heat map Illustrator; JRE: Java Runtime Environment; KO: knockout; LRN: local response normalization; MCC: Mathew Correlation Coefficient; OS: operating system; PAS: phagophore assembly site; PC: principal component; PCA: principal component analysis; PPI: protein-protein interaction; Pr: precision; QPSO: Quantum-behaved Particle Swarm Optimization; ReLU: rectified linear unit; RF: random forest; ROC: receiver operating characteristic; ROI: region of interest; SD: systematic derivation; SGD: stochastic gradient descent; Sn: sensitivity; Sp: specificity; SRG: seeded region growing; t-SNE: t-distributed stochastic neighbor embedding; 2D: 2-dimensional; WT: wild-type.

Autophagy-related (ATG) 11, ATG9 and the phosphatidylinositol 3-kinase control ATG2-mediated formation of autophagosomes in Arabidopsis.

KEY MESSAGE: Using quantitative assays for autophagy, we analyzed 4 classes of atg mutants, discovered new atg2 phenotypes and ATG gene interactions, and proposed a model of autophagosome formation in plants. Plant and other eukaryotic cells use autophagy to target cytoplasmic constituents for degradation in the vacuole. Autophagy is regulated and executed by a conserved set of proteins called autophagy-related (ATG). In Arabidopsis, several groups of ATG proteins have been characterized using genetic approaches. However, the genetic interactions between ATG genes have not been established and the relationship between different ATG groups in plants remains unclear. Here we analyzed atg2, atg7, atg9, and atg11 mutants and their double mutants at the physiological, biochemical, and subcellular levels. Involvement of phosphatidylinositol 3-kinase (PI3K) in autophagy was also tested using wortmannin, a PI3K inhibitor. Our mutant analysis using autophagy markers showed that atg7 and atg2 phenotypes are more severe than those of atg11 and atg9. Unlike other mutants, atg2 cells accumulated several autophagic vesicles that could not be delivered to the vacuole. Analysis of atg double mutants, combined with wortmannin treatment, indicated that ATG11, PI3K, and ATG9 act upstream of ATG2. Our data support a model in which plant ATG1 and PI3K complexes play a role in the initiation of autophagy, whereas ATG2 is involved in a later step during the biogenesis of autophagic vesicles.

Analysis of Plant Autophagy.

Autophagy is an intercellular degradation/recycling system by which cytoplasmic components are sequestered in autophagic vesicles (autophagosomes) and delivered to the vacuole for breakdown. During the last decade, plant studies have revealed that autophagy is employed as a major route to recycle nutrients needed for plant growth and development, and to combat with a wide range of biotic and abiotic stresses. Another important outcome of these studies was the development and optimization of methods and techniques for monitoring autophagy activity in plants. In this chapter, methods frequently used in plant autophagy study, from physiological to biochemical and microscopical analyses, are discussed.

Quantitative Assay of Macroautophagy Using Pho8△60 Assay and GFP-Cleavage Assay in Yeast.

It is intrinsically difficult to directly measure a specific protein reduction that is mediated by nonselective autophagy, because the degradation proceeds at a relatively slow pace, and the residual nondegraded part becomes the background. Several methods for measuring nonselective autophagy have been reported in the past. One classical simple method is called bulk degradation assay, which measures the release of degraded amino acids after radioisotope labeling of the total cellular proteins. In 1995, we developed a quantitative Pho8△60 assay in the yeast, Saccharomyces cerevisiae, for studying autophagy, which is widely used for its advantages that are described in the following sections. Another method used in recent times is the GFP-based processing assay in yeast. We will describe these two methods in this chapter.

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.

Detection of Autophagy in Plants by Fluorescence Microscopy.

Autophagy is a key process for degradation and recycling of proteins or organelles in eukaryotes. Autophagy in plants has been shown to function in stress responses, pathogen immunity, and senescence, while a basal level of autophagy plays a housekeeping role in cells. Upon activation of autophagy, vesicles termed autophagosomes are formed to deliver proteins or organelles to the vacuole for degradation. The number of autophagosomes can thus be used to indicate the level of autophagy. Here, we describe two common methods used for detection of autophagosomes, staining of autophagosomes with the fluorescent dye monodansylcadaverine, and expression of a fusion between GFP and the autophagosomal membrane protein ATG8.