Canonical and non-canonical autophagy: variations on a common theme of self-eating?

The autophagosome is the central organelle in macroautophagy, a vacuolar lysosomal catabolic pathway that degrades cytoplasmic material to fuel starving cells and eliminates intracellular pathogens. Macroautophagy has important physiological roles during development, ageing and the immune response, and its cytoprotective function is compromised in various diseases. A set of autophagy-related (ATG) proteins is hierarchically recruited to the phagophore, the initial membrane template in the construction of the autophagosome. However, recent findings suggest that macroautophagy can also occur in the absence of some of these key autophagy proteins, through the unconventional biogenesis of canonical autophagosomes. Such alternatives to the evolutionarily conserved scheme might provide additional therapeutic opportunities.

A mouse model for SPG48 reveals a block of autophagic flux upon disruption of adaptor protein complex five.

Hereditary spastic paraplegia is a spastic gait disorder that arises from degeneration of corticospinal axons. The subtype SPG48 is associated with mutations in the zeta subunit of the adaptor protein complex five (AP5). AP5 function and the pathophysiology of SPG48 are only poorly understood. Here, we report an AP5 zeta knockout mouse, which shows an age-dependent degeneration of corticospinal axons. Our analysis of knockout fibroblasts supports a trafficking defect from late endosomes to the transGolgi network and reveals a structural defect of the Golgi. We further show that both autophagic flux and the recycling of lysosomes from autolysosomes were impaired in knockout cells. In vivo, we observe an increase of autophagosomes and autolysosomes and, at later stages, the accumulation of intracellular waste in neurons. Taken together, we propose that loss of AP5 function blocks autophagy and thus leads to the aberrant accumulation of autophagic cargo, which finally results in axon degeneration.

WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome.

Autophagy is a pivotal cytoprotective process that secures cellular homeostasis, fulfills essential roles in development, immunity and defence against pathogens, and determines the lifespan of eukaryotic organisms. However, autophagy also crucially contributes to the development of age-related human pathologies, including cancer and neurodegeneration. Macroautophagy (hereafter referred to as autophagy) clears the cytoplasm by stochastic or specific cargo recognition and destruction, and is initiated and executed by autophagy related (ATG) proteins functioning in dynamical hierarchies to form autophagosomes. Autophagosomes sequester cytoplasmic cargo material, including proteins, lipids and organelles, and acquire acidic hydrolases from the lysosomal compartment for cargo degradation. Prerequisite and essential for autophagosome formation is the production of phosphatidylinositol 3-phosphate (PtdIns3P) by phosphatidylinositol 3-kinase class III (PI3KC3, also known as PIK3C3) in complex with beclin 1, p150 (also known as PIK3R4; Vps15 in yeast) and ATG14L. Members of the human WD-repeat protein interacting with phosphoinositides (WIPI) family play an important role in recognizing and decoding the PtdIns3P signal at the nascent autophagosome, and hence function as autophagy-specific PtdIns3P-binding effectors, similar to their ancestral yeast Atg18 homolog. The PtdIns3P effector function of human WIPI proteins appears to be compromised in cancer and neurodegeneration, and WIPI genes and proteins might present novel targets for rational therapies. Here, we summarize the current knowledge on the roles of the four human WIPI proteins, WIPI1-4, in autophagy. This article is part of a Focus on Autophagosome biogenesis. For further reading, please see related articles: 'ERES: sites for autophagosome biogenesis and maturation?' by Jana Sanchez-Wandelmer et al. (J. Cell Sci. 128, 185-192) and 'Membrane dynamics in autophagosome biogenesis' by Sven R. Carlsson and Anne Simonsen (J. Cell Sci. 128, 193-205).

Fluorescence-based imaging of autophagy progression by human WIPI protein detection.

Central to the process of macroautophagy (hereafter autophagy) is the formation of autophagosomes, double-membrane vesicles that sequester cytoplasmic cargo, including proteins, lipids and organelles, for lysosomal degradation and macromolecule recycling. Tight regulation of both autophagic activity and capacity is crucial to secure cellular homeostasis and aberrant autophagy is tightly linked to the development of many human diseases. Hence it is of great importance to accurately measure autophagy progression in health and disease. Members of the human WIPI ß-propeller proteins associate with autophagosomal membranes due to specific phosphatidylinositol 3-phosphate (PtdIns3P) binding at the onset of autophagy. The specific autophagosomal localization of both WIPI1 and WIPI2 (refered to as WIPI puncta) has been employed to assess autophagy using fluorescence microscopy methods, such as confocal and live-cell video microscopy and was extended for automated high-throughput image acquisition and analyses procedures. We here provide an overview on the employment of human WIPI members for the assessment of autophagy in higher eukaryotic cells, suitable for systems biology approaches such as mathematical modelling.

Atg18 function in autophagy is regulated by specific sites within its ß-propeller.

Autophagy is a conserved degradative transport pathway. It is characterized by the formation of double-membrane autophagosomes at the phagophore assembly site (PAS). Atg18 is essential for autophagy but also for vacuole homeostasis and probably endosomal functions. This protein is basically a ß-propeller, formed by seven WD40 repeats, that contains a conserved FRRG motif that binds to phosphoinositides and promotes Atg18 recruitment to the PAS, endosomes and vacuoles. However, it is unknown how Atg18 association with these organelles is regulated, as the phosphoinositides bound by this protein are present on the surface of all of them. We have investigated Atg18 recruitment to the PAS and found that Atg18 binds to Atg2 through a specific stretch of amino acids in the ß-propeller on the opposite surface to the FRRG motif. As in the absence of the FRRG sequence, the inability of Atg18 to interact with Atg2 impairs its association with the PAS, causing an autophagy block. Our data provide a model whereby the Atg18 ß-propeller provides organelle specificity by binding to two determinants on the target membrane.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

WIPI ß-propellers at the crossroads of autophagosome and lipid droplet dynamics.

Macroautophagy (autophagy hereafter) is an evolutionarily highly conserved catabolic process activated by eukaryotes in order to counteract cellular starvation. Autophagy leads to bulk degradation of cytoplasmic content in the lysosomal compartment, thereby clearing the cytoplasm and generating nutrients and energy. Upon autophagy initiation, cytoplasmic material becomes sequestered in newly formed double-membrane vesicles termed 'autophagosomes' that subsequently acquire acidic hydrolases for content destruction. The de novo biogenesis of autophagosomes often occurs at the endoplasmic reticulum (ER) and, in many cases, in close proximity to lipid droplets (LDs), intracellular neutral lipid storage reservoirs. LDs are targets of autophagic destruction, but have recently also been shown to contribute to autophagosome formation. In fact, some autophagy-related (Atg) proteins, such as microtubule-associated protein light chain 3 (LC3), Atg2 and Atg14L, functionally contribute to both LD and autophagosome biogenesis. In the present paper, we discuss Atg proteins, including members of the human WD-repeat protein interacting with phosphoinositides (WIPI) family that co-localize prominently with LC3, Atg2 and Atg14L to conceivably integrate LD and autophagosome dynamics.

Ferritinophagy: Assessing the Selective Degradation of Iron by Autophagy in Human Fibroblasts.

Mutations in the autophagy gene WDR45/WIPI4 are the cause of beta-propeller-associated neurodegeneration (BPAN), a subtype of human diseases known as neurodegeneration with brain iron accumulation (NBIA) due to the presence of iron deposits in the brains of patients. Intracellular iron levels are tightly regulated by a number of cellular mechanisms, including the critical mechanism of ferritinophagy. This paper describes how ferritinophagy can be assessed in primary, skin-derived human fibroblasts. In this protocol, we use iron-modulating conditions for inducing or inhibiting ferritinophagy at the cellular level, such as the administration of bafilomycin A1 to inhibit lysosome function and ferric ammonium citrate (FAC) or deferasiox (DFX) treatments to overload or deplete iron, respectively. Such treated fibroblasts are then subjected to high-throughput imaging and CellProfiler-based quantitative localization analysis of endogenous ferritin and autophagosomal/lysosomal markers, here LAMP2. Based on the level of autophagosomal/lysosomal ferritin, conclusions can be drawn regarding the level of ferritinophagy. This protocol can be used to assess ferritinophagy in BPAN patient-derived primary fibroblasts or other types of mammalian cells.

Human WIPI ß-propeller function in autophagy and neurodegeneration.

The four human WIPI ß-propellers, WIPI1 through WIPI4, belong to the ancient PROPPIN family and fulfill scaffold functions in the control of autophagy. In this context, WIPI ß-propellers function as PI3P effectors during autophagosome formation and loss of WIPI function negatively impacts autophagy and contributes to neurodegeneration. Of particular interest are mutations in WDR45, the human gene that encodes WIPI4. Sporadic WDR45 mutations are the cause of a rare human neurodegenerative disease called BPAN, hallmarked by high brain iron accumulation. Here, we discuss the current understanding of the functions of human WIPI ß-propellers and address unanswered questions with a particular focus on the role of WIPI4 in autophagy and BPAN.

Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy.

The majority of studies on autophagy, a cytoplasmic homeostasis pathway of broad biological and medical significance, have been hitherto focused on the phosphatidylinositol 3-kinases as the regulators of autophagy. Here, we addressed the reverse process driven by phosphoinositide phosphatases and uncovered a key negative regulatory role in autophagy of a phosphatidylinositol 3-phosphate (PI3P) phosphatase Jumpy (MTMR14). Jumpy associated with autophagic isolation membranes and early autophagosomes, defined by the key factor Atg16 necessary for proper localization and development of autophagic organelles. Jumpy orchestrated orderly succession of Atg factors by controlling recruitment to autophagic membranes of the sole mammalian Atg factor that interacts with PI3P, WIPI-1 (Atg18), and by affecting the distribution of Atg9 and LC3, the two Atg factors controlling organization and growth of autophagic membranes. A catalytically inactive Jumpy mutant, R336Q, found in congenital disease centronuclear myopathy, lost the ability to negatively regulate autophagy. This work reports for the first time that initiation of autophagy is controlled not only by the forward reaction of generating PI3P through a lipid kinase but that its levels are controlled by a specific PI3P phosphatase, which when defective can lead to human disease.

WIPI ß-propellers in autophagy-related diseases and longevity.

Autophagy is a catabolic pathway in which the cell sequesters cytoplasmic material, including long-lived proteins, lipids and organelles, in specialized double-membrane vesicles, called autophagosomes. Subsequently, autophagosomes communicate with the lysosomal compartment and acquire acidic hydrolases for final cargo degradation. This process of partial self-eating secures the survival of eukaryotic cells during starvation periods and is critically regulated by mTORC1 (mammalian target of rapamycin complex 1). Under nutrient-poor conditions, inhibited mTORC1 permits localized PtdIns(3)P production at particular membranes that contribute to autophagosome formation. Members of the human WIPI (WD-repeat protein interacting with phosphoinositides) family fulfil an essential role as PtdIns(3)P effectors at the initiation step of autophagosome formation. In the present article, we discuss the role of human WIPIs in autophagy, and the identification of evolutionarily conserved amino acids of WIPI-1 that confer PtdIns(3)P binding downstream of mTORC1 inhibition. We also discuss the PtdIns(3)P effector function of WIPIs in the context of longevity and autophagy-related human diseases, such as cancer and neurodegeneration.

Autophagy profiling in single cells with open source CellProfiler-based image analysis.

Single cell-based analysis of macroautophagy/autophagy is largely achieved through the use of fluorescence microscopy to detect autophagy-related proteins that associate with autophagic membranes and therefore can be quantified as fluorescent puncta. In this context, an automated analysis of the number and size of recognized puncta is preferable to a manual count, because more reliable results can be generated in a short time. Here we present a method for open source CellProfiler software-based analysis for quantitative autophagy assessments using GFP-tagged WIPI1 (WD repeat domain, phosphoinositide interacting 1) images acquired with Airyscan or confocal laser-scanning microscopy. The CellProfiler protocol is provided as a ready-to-use software pipeline, and the creation of this pipeline is detailed in both text and video formats. In addition, we provide CellProfiler pipelines for endogenous SQSTM1/p62 (sequestosome 1) or intracellular lipid droplet (LD) analysis, suitable to assess forms of selective autophagy. All protocols and software pipelines can be quickly and easily adapted for the use of alternative autophagy markers or cell types, and can also be used for high-throughput purposes.Abbreviations: AF Alexa Fluor ATG autophagy related BafA1 bafilomycin A1 BSA bovine serum albumin DAPI 4,6-diamidino-2-phenylindole DMEM Dulbecco's modified Eagle's medium DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid EBSS Earle's balanced salt solution FBS fetal bovine serum GFP green fluorescent protein LD lipid droplet LSM laser scanning microscope MAP1LC3B microtubule associated protein 1 light chain 3 beta MTOR mechanistic target of rapamycin kinase PBS phosphate-buffered saline PIK3C3/VPS34 phosphatidylinositol 3-kinase catalytic subunit type 3 SQSTM1 sequestosome 1 TIFF tagged image file format U2OS U-2 OS cell line WIPI WD repeat domain, phosphoinositide interacting.

The ABL-MYC axis controls WIPI1-enhanced autophagy in lifespan extension.

Human WIPI ß-propellers function as PI3P effectors in autophagy, with WIPI4 and WIPI3 being able to link autophagy control by AMPK and TORC1 to the formation of autophagosomes. WIPI1, instead, assists WIPI2 in efficiently recruiting the ATG16L1 complex at the nascent autophagosome, which in turn promotes lipidation of LC3/GABARAP and autophagosome maturation. However, the specific role of WIPI1 and its regulation are unknown. Here, we discovered the ABL-ERK-MYC signalling axis controlling WIPI1. As a result of this signalling, MYC binds to the WIPI1 promoter and represses WIPI1 gene expression. When ABL-ERK-MYC signalling is counteracted, increased WIPI1 gene expression enhances the formation of autophagic membranes capable of migrating through tunnelling nanotubes to neighbouring cells with low autophagic activity. ABL-regulated WIPI1 function is relevant to lifespan control, as ABL deficiency in C. elegans increased gene expression of the WIPI1 orthologue ATG-18 and prolonged lifespan in a manner dependent on ATG-18. We propose that WIPI1 acts as an enhancer of autophagy that is physiologically relevant for regulating the level of autophagic activity over the lifespan.

The ménage à trois of autophagy, lipid droplets and liver disease.

Autophagic pathways cross with lipid homeostasis and thus provide energy and essential building blocks that are indispensable for liver functions. Energy deficiencies are compensated by breaking down lipid droplets (LDs), intracellular organelles that store neutral lipids, in part by a selective type of autophagy, referred to as lipophagy. The process of lipophagy does not appear to be properly regulated in fatty liver diseases (FLDs), an important risk factor for the development of hepatocellular carcinomas (HCC). Here we provide an overview on our current knowledge of the biogenesis and functions of LDs, and the mechanisms underlying their lysosomal turnover by autophagic processes. This review also focuses on nonalcoholic steatohepatitis (NASH), a specific type of FLD characterized by steatosis, chronic inflammation and cell death. Particular attention is paid to the role of macroautophagy and macrolipophagy in relation to the parenchymal and non-parenchymal cells of the liver in NASH, as this disease has been associated with inappropriate lipophagy in various cell types of the liver.Abbreviations: ACAT: acetyl-CoA acetyltransferase; ACAC/ACC: acetyl-CoA carboxylase; AKT: AKT serine/threonine kinase; ATG: autophagy related; AUP1: AUP1 lipid droplet regulating VLDL assembly factor; BECN1/Vps30/Atg6: beclin 1; BSCL2/seipin: BSCL2 lipid droplet biogenesis associated, seipin; CMA: chaperone-mediated autophagy; CREB1/CREB: cAMP responsive element binding protein 1; CXCR3: C-X-C motif chemokine receptor 3; DAGs: diacylglycerols; DAMPs: danger/damage-associated molecular patterns; DEN: diethylnitrosamine; DGAT: diacylglycerol O-acyltransferase; DNL: de novo lipogenesis; EHBP1/NACSIN (EH domain binding protein 1); EHD2/PAST2: EH domain containing 2; CoA: coenzyme A; CCL/chemokines: chemokine ligands; CCl4: carbon tetrachloride; ER: endoplasmic reticulum; ESCRT: endosomal sorting complexes required for transport; FA: fatty acid; FFAs: free fatty acids; FFC: high saturated fats, fructose and cholesterol; FGF21: fibroblast growth factor 21; FITM/FIT: fat storage inducing transmembrane protein; FLD: fatty liver diseases; FOXO: forkhead box O; GABARAP: GABA type A receptor-associated protein; GPAT: glycerol-3-phosphate acyltransferase; HCC: hepatocellular carcinoma; HDAC6: histone deacetylase 6; HECT: homologous to E6-AP C-terminus; HFCD: high fat, choline deficient; HFD: high-fat diet; HSCs: hepatic stellate cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; ITCH/AIP4: itchy E3 ubiquitin protein ligase; KCs: Kupffer cells; LAMP2A: lysosomal associated membrane protein 2A; LDs: lipid droplets; LDL: low density lipoprotein; LEP/OB: leptin; LEPR/OBR: leptin receptor; LIPA/LAL: lipase A, lysosomal acid type; LIPE/HSL: lipase E, hormone sensitive type; LIR: LC3-interacting region; LPS: lipopolysaccharide; LSECs: liver sinusoidal endothelial cells; MAGs: monoacylglycerols; MAPK: mitogen-activated protein kinase; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCD: methionine-choline deficient; MGLL/MGL: monoglyceride lipase; MLXIPL/ChREBP: MLX interacting protein like; MTORC1: mechanistic target of rapamycin kinase complex 1; NAFLD: nonalcoholic fatty liver disease; NAS: NAFLD activity score; NASH: nonalcoholic steatohepatitis; NPC: NPC intracellular cholesterol transporter; NR1H3/LXRα: nuclear receptor subfamily 1 group H member 3; NR1H4/FXR: nuclear receptor subfamily 1 group H member 4; PDGF: platelet derived growth factor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLIN: perilipin; PNPLA: patatin like phospholipase domain containing; PNPLA2/ATGL: patatin like phospholipase domain containing 2; PNPLA3/adiponutrin: patatin like phospholipase domain containing 3; PPAR: peroxisome proliferator activated receptor; PPARA/PPARα: peroxisome proliferator activated receptor alpha; PPARD/PPARδ: peroxisome proliferator activated receptor delta; PPARG/PPARγ: peroxisome proliferator activated receptor gamma; PPARGC1A/PGC1α: PPARG coactivator 1 alpha; PRKAA/AMPK: protein kinase AMP-activated catalytic subunit; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; PTEN: phosphatase and tensin homolog; ROS: reactive oxygen species; SE: sterol esters; SIRT1: sirtuin 1; SPART/SPG20: spartin; SQSTM1/p62: sequestosome 1; SREBF1/SREBP1c: sterol regulatory element binding transcription factor 1; TAGs: triacylglycerols; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TGFB1/TGFß: transforming growth factor beta 1; Ub: ubiquitin; UBE2G2/UBC7: ubiquitin conjugating enzyme E2 G2; ULK1/Atg1: unc-51 like autophagy activating kinase 1; USF1: upstream transcription factor 1; VLDL: very-low density lipoprotein; VPS: vacuolar protein sorting; WIPI: WD-repeat domain, phosphoinositide interacting; WDR: WD repeat domain.

IKCa channels control breast cancer metabolism including AMPK-driven autophagy.

Ca2+-activated K+ channels of intermediate conductance (IK) are frequently overexpressed in breast cancer (BC) cells, while IK channel depletion reduces BC cell proliferation and tumorigenesis. This raises the question, of whether and mechanistically how IK activity interferes with the metabolic activity and energy consumption rates, which are fundamental for rapidly growing cells. Using BC cells obtained from MMTV-PyMT tumor-bearing mice, we show that both, glycolysis and mitochondrial ATP-production are reduced in cells derived from IK-deficient breast tumors. Loss of IK altered the sub-/cellular K+- and Ca2+- homeostasis and mitochondrial membrane potential, ultimately resulting in reduced ATP-production and metabolic activity. Consequently, we find that BC cells lacking IK upregulate AMP-activated protein kinase activity to induce autophagy compensating the glycolytic and mitochondrial energy shortage. Our results emphasize that IK by modulating cellular Ca2+- and K+-dynamics contributes to the remodeling of metabolic pathways in cancer. Thus, targeting IK channel might disturb the metabolic activity of BC cells and reduce malignancy.

The mechanism of macroautophagy: The movie.

This animated movie presents the mechanism of macroautophagy, hereafter autophagy, by showing the molecular features of the formation of autophagosomes, the hallmark organelle of this intracellular catabolic pathway. It is based on our current knowledge and it also illustrates how autophagosomes can recognize and eliminate selected cargoes.

Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy.

Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca(2+)-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5-50 µM), sorafenib (1-40 µM), staurosporine (20-500 nM), or thapsigargin (20-500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca(2+) chelation by BAPTA-AM (10 and 30 µM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) α/ß and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10-30 µg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1-10 µM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca(2+) mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α(1)/α(2)-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down-regulation of AMPK by siRNA resulted in an increased basal level of both WIPI-1 puncta and LC3 lipidation, and nutrient-starvation induced autophagy was sensitive to STO-609/KN-93. Our data provide evidence that pharmacological compound-modulated and starvation-induced autophagy involves Ca(2+)-dependent signaling, including CaMKI independent of AMPKα(1)/α(2). Our data also suggest that AMPKα(1)/α(2) might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.

Freeze-fracture replica immunolabelling reveals human WIPI-1 and WIPI-2 as membrane proteins of autophagosomes.

Autophagy defines the lifespan of eukaryotic organisms by ensuring cellular survival through regulated bulk clearance of proteins, organelles and membranes. Pathophysiological consequences of improper autophagy give rise to a variety of age-related human diseases such as cancer and neurodegeneration. Rational therapeutic implementation of autophagy modulation remains problematic, as fundamental molecular details such as the generation of autophagosomes, unique double-membrane vesicles formed to permit the process of autophagy, are insufficiently understood. Here, freeze-fracture replica immunolabelling reveals WD-repeat protein interacting with phosphoinositides 1 and 2 (WIPI-1 and WIPI-2) as membrane components of autophagosomes and the plasma membrane (PM). In addition, WIPI-1 is also present in membranes of the endoplasmic reticulum (ER) and WIPI-2 was further detected in membranes close to the Golgi cisternae. Our results identify WIPI-1 and WIPI-2 as novel protein components of autophagosomes, and of membrane sites from which autophagosomes might originate (ER, PM, Golgi area). Hence therapeutic modulation of autophagy could involve approaches that functionally target human WIPI proteins.

Starvation-induced hyperacetylation of tubulin is required for the stimulation of autophagy by nutrient deprivation.

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.

The Bcl-2 homology domain 3 mimetic gossypol induces both Beclin 1-dependent and Beclin 1-independent cytoprotective autophagy in cancer cells.

Gossypol, a natural Bcl-2 homology domain 3 mimetic compound isolated from cottonseeds, is currently being evaluated in clinical trials. Here, we provide evidence that gossypol induces autophagy followed by apoptotic cell death in both the MCF-7 human breast adenocarcinoma and HeLa cell lines. We first show that knockdown of the Bcl-2 homology domain 3-only protein Beclin 1 reduces gossypol-induced autophagy in MCF-7 cells, but not in HeLa cells. Gossypol inhibits the interaction between Beclin 1 and Bcl-2 (B-cell leukemia/lymphoma 2), antagonizes the inhibition of autophagy by Bcl-2, and hence stimulates autophagy. We then show that knockdown of Vps34 reduces gossypol-induced autophagy in both cell lines, and consistent with this, the phosphatidylinositol 3-phosphate-binding protein WIPI-1 is recruited to autophagosomal membranes. Further, Atg5 knockdown also reduces gossypol-mediated autophagy. We conclude that gossypol induces autophagy in both a canonical and a noncanonical manner. Notably, we found that gossypol-mediated apoptotic cell death was potentiated by treatment with the autophagy inhibitor wortmannin or with small interfering RNA against essential autophagy genes (Vps34, Beclin 1, and Atg5). Our findings support the notion that gossypol-induced autophagy is cytoprotective and not part of the cell death process induced by this compound.