Yapping at the autophagy door? The answer is flowing in the kidney proximal tubule.

Shear stress induced by urinary flow stimulates macroautophagy (hereafter referred to as autophagy) in kidney proximal tubule epithelial cells. Autophagy and selective degradation of lipid droplets by lipophagy contribute to tubule homeostasis by the production of ATP and control of epithelial cell size. Autophagy/lipophagy is controlled by a signaling cascade emanating from the primary cilium, localized at the apical side of epithelial cells. Downstream of the primary cilium, AMPK controls mitochondrial biogenesis on the one hand and autophagy/lipophagy on the other hand, which together increase fatty acid production that fuels oxidative phosphorylation to increase energy production. Recently, we reported that the co-transcriptional factors YAP1 and WWTR1/TAZ act downstream of AMPK to control autophagy. In fact, YAP1 and the transcription factor TEAD control the expression of RUBCN/rubicon. Under shear stress, YAP1 is excluded from the nucleus in a SIRT1-dependent manner to favor autophagic flux by downregulating the expression of RUBCN. When simulating in vitro a pathological urinary flow in murine proximal tubule kidney epithelial cells, we observe the nuclear retention of YAP1 and, consequently, high expression of RUBCN and inhibition of autophagic flux. Importantly, these findings were confirmed in biopsies of patients suffering from diabetic nephropathy, a major cause of chronic kidney disease.

To squeeze or not: Regulation of cell size by mechanical forces in development and human diseases.

Physical constraints, such as compression, shear stress, stretching and tension play major roles during development and tissue homeostasis. Mechanics directly impact physiology, and their alteration is also recognized as having an active role in driving human diseases. Recently, growing evidence has accumulated on how mechanical forces are translated into a wide panel of biological responses, including metabolism and changes in cell morphology. The aim of this review is to summarize and discuss our knowledge on the impact of mechanical forces on cell size regulation. Other biological consequences of mechanical forces will not be covered by this review. Moreover, wherever possible, we also discuss mechanosensors and molecular and cellular signaling pathways upstream of cell size regulation. We finally highlight the relevance of mechanical forces acting on cell size in physiology and human diseases.

A historical perspective of macroautophagy regulation by biochemical and biomechanical stimuli.

Macroautophagy is a lysosomal degradative pathway for intracellular macromolecules, protein aggregates, and organelles. The formation of the autophagosome, a double membrane-bound structure that sequesters cargoes before their delivery to the lysosome, is regulated by several stimuli in multicellular organisms. Pioneering studies in rat liver showed the importance of amino acids, insulin, and glucagon in controlling macroautophagy. Thereafter, many studies have deciphered the signaling pathways downstream of these biochemical stimuli to control autophagosome formation. Two signaling hubs have emerged: the kinase mTOR, in a complex at the surface of lysosomes which is sensitive to nutrients and hormones; and AMPK, which is sensitive to the cellular energetic status. Besides nutritional, hormonal, and energetic fluctuations, many organs have to respond to mechanical forces (compression, stretching, and shear stress). Recent studies have shown the importance of mechanotransduction in controlling macroautophagy. This regulation engages cell surface sensors, such as the primary cilium, in order to translate mechanical stimuli into biological responses.

PI3KCIIα-Dependent Autophagy Program Protects From Endothelial Dysfunction and Atherosclerosis in Response to Low Shear Stress in Mice.

BACKGROUND: The ability to respond to mechanical forces is a basic requirement for maintaining endothelial cell (ECs) homeostasis, which is continuously subjected to low shear stress (LSS) and high shear stress (HSS). In arteries, LSS and HSS have a differential impact on EC autophagy processes. However, it is still unclear whether LSS and HSS differently tune unique autophagic machinery or trigger specific autophagic responses in ECs. METHODS: Using fluid flow system to generate forces on EC and multiscale imaging analyses on ApoE-/- mice whole arteries, we studied the cellular and molecular mechanism involved in autophagic response to LSS or HSS on the endothelium. RESULTS: We found that LSS and HSS trigger autophagy activation by mobilizing specific autophagic signaling modules. Indeed, LSS-induced autophagy in endothelium was independent of the class III PI3K (phosphoinositide 3-kinase) VPS34 (vacuolar sorting protein 34) but controlled by the α isoform of class II PI3K (phosphoinositide 3-kinase class II α [PI3KCIIα]). Accordingly, reduced PI3KCIIα expression in ApoE-/- mice (ApoE-/-PI3KCIIα+/-) led to EC dysfunctions associated with increased plaque deposition in the LSS regions. Mechanistically, we revealed that PI3KCIIα inhibits mTORC1 (mammalian target of rapamycin complex 1) activation and that rapamycin treatment in ApoE-/-PI3KCIIα+/- mice specifically rescue autophagy in arterial LSS regions. Finally, we demonstrated that absence of PI3KCIIα led to decreased endothelial primary cilium biogenesis in response to LSS and that ablation of primary cilium mimics PI3KCIIα-decreased expression in EC dysfunction, suggesting that this organelle could be the mechanosensor linking PI3KCIIα and EC homeostasis. CONCLUSIONS: Our data reveal that mechanical forces variability within the arterial system determines EC autophagic response and supports a central role of PI3KCIIα/mTORC1 axis to prevent EC dysfunction in LSS regions.

The AMPK-Sirtuin 1-YAP axis is regulated by fluid flow intensity and controls autophagy flux in kidney epithelial cells.

Shear stress generated by urinary fluid flow is an important regulator of renal function. Its dysregulation is observed in various chronic and acute kidney diseases. Previously, we demonstrated that primary cilium-dependent autophagy allows kidney epithelial cells to adapt their metabolism in response to fluid flow. Here, we show that nuclear YAP/TAZ negatively regulates autophagy flux in kidney epithelial cells subjected to fluid flow. This crosstalk is supported by a primary cilium-dependent activation of AMPK and SIRT1, independently of the Hippo pathway. We confirm the relevance of the YAP/TAZ-autophagy molecular dialog in vivo using a zebrafish model of kidney development and a unilateral ureteral obstruction mouse model. In addition, an in vitro assay simulating pathological accelerated flow observed at early stages of chronic kidney disease (CKD) activates YAP, leading to a primary cilium-dependent inhibition of autophagic flux. We confirm this YAP/autophagy relationship in renal biopsies from patients suffering from diabetic kidney disease (DKD), the leading cause of CKD. Our findings demonstrate the importance of YAP/TAZ and autophagy in the translation of fluid flow into cellular and physiological responses. Dysregulation of this pathway is associated with the early onset of CKD.

Fluid shear stress triggers cholesterol biosynthesis and uptake in inner medullary collecting duct cells, independently of nephrocystin-1 and nephrocystin-4.

Renal epithelial cells are subjected to fluid shear stress of urine flow. Several cellular structures act as mechanosensors-the primary cilium, microvilli and cell adhesion complexes-that directly relay signals to the cytoskeleton to regulate various processes including cell differentiation and renal cell functions. Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy leading to end-stage kidney failure before adulthood. NPHP1 and NPHP4 are the major genes which code for proteins that form a complex at the transition zone of the primary cilium, a crucial region required for the maintenance of the ciliary composition integrity. These two proteins also interact with signaling components and proteins associated with the actin cytoskeleton at cell junctions. Due to their specific subcellular localization, we wondered whether NPHP1 and NPHP4 could ensure mechanosensory functions. Using a microfluidic set up, we showed that murine inner medullary collecting ductal cells invalidated for Nphp1 or Nphp4 are more responsive to immediate shear exposure with a fast calcium influx, and upon a prolonged shear condition, an inability to properly regulate cilium length and actin cytoskeleton remodeling. Following a transcriptomic study highlighting shear stress-induced gene expression changes, we showed that prolonged shear triggers both cholesterol biosynthesis pathway and uptake, processes that do not seem to involve neither NPHP1 nor NPHP4. To conclude, our study allowed us to determine a moderate role of NPHP1 and NPHP4 in flow sensation, and to highlight a new signaling pathway induced by shear stress, the cholesterol biosynthesis and uptake pathways, which would allow cells to cope with mechanical stress by strengthening their plasma membrane through the supply of cholesterol.

Autophagy and the primary cilium in cell metabolism: What's upstream?

The maintenance of cellular homeostasis in response to extracellular stimuli, i.e., nutrient and hormone signaling, hypoxia, or mechanical forces by autophagy, is vital for the health of various tissues. The primary cilium (PC) is a microtubule-based sensory organelle that regulates the integration of several extracellular stimuli. Over the past decade, an interconnection between autophagy and PC has begun to be revealed. Indeed, the PC regulates autophagy and in turn, a selective form of autophagy called ciliophagy contributes to the regulation of ciliogenesis. Moreover, the PC regulates both mitochondrial biogenesis and lipophagy to produce free fatty acids. These two pathways converge to activate oxidative phosphorylation and produce ATP, which is mandatory for cell metabolism and membrane transport. The autophagy-dependent production of energy is fully efficient when the PC senses shear stress induced by fluid flow. In this review, we discuss the cross-talk between autophagy, the PC and physical forces in the regulation of cell biology and physiology.

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.

Primary cilium-dependent autophagy in the response to shear stress.

Mechanical forces, such as compression, shear stress and stretching, play major roles during development, tissue homeostasis and immune processes. These forces are translated into a wide panel of biological responses, ranging from changes in cell morphology, membrane transport, metabolism, energy production and gene expression. Recent studies demonstrate the role of autophagy in the integration of these physical constraints. Here we focus on the role of autophagy in the integration of shear stress induced by blood and urine flows in the circulatory system and the kidney, respectively. Many studies highlight the involvement of the primary cilium, a microtubule-based antenna present at the surface of many cell types, in the integration of extracellular stimuli. The cross-talk between the molecular machinery of autophagy and that of the primary cilium in the context of shear stress is revealed to be an important dialog in cell biology.

Links between autophagy and tissue mechanics.

Physical constraints, such as compression, shear stress, stretching and tension, play major roles during development, tissue homeostasis, immune responses and pathologies. Cells and organelles also face mechanical forces during migration and extravasation, and investigations into how mechanical forces are translated into a wide panel of biological responses, including changes in cell morphology, membrane transport, metabolism, energy production and gene expression, is a flourishing field. Recent studies demonstrate the role of macroautophagy in the integration of physical constraints. The aim of this Review is to summarize and discuss our knowledge of the role of macroautophagy in controlling a large panel of cell responses, from morphological and metabolic changes, to inflammation and senescence, for the integration of mechanical forces. Moreover, wherever possible, we also discuss the cell surface molecules and structures that sense mechanical forces upstream of macroautophagy.

Monitoring lipophagy in kidney epithelial cells in response to shear stress.

Mechanical stress has been shown to induce the degradation of lipid droplets in kidney epithelial cells. Here, we illustrate the technical equipment and devices that are currently used in our laboratory to apply shear stress on cells. We provide a detailed protocol to monitor lipophagy in response to shear stress. The aim of this review is to guide and help people understand the challenges in studying acidic lipolysis in cells subjected to fluid flow.

When the autophagy protein ATG16L1 met the ciliary protein IFT20.

The primary cilium (PC), a plasma membrane microtubule-based structure, is a sensor of extracellular chemical and mechanical stress stimuli. Upon ciliogenesis, the autophagy protein ATG16L1 and the ciliary protein IFT20 are co-transported to the PC. We demonstrated in a recent study that IFT20 and ATG16L1 interact in a multiprotein complex. This interaction is mediated by the ATG16L1 WD40 domain and an ATG16L1-binding motif newly identified in IFT20. ATG16L1-deficient cells are decorated by giant ciliary structures hallmarked by defects in PC-associated signaling. These structures uncommonly accumulate phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2) while phosphatidylinositol-4-phosphate (PtdIns4P), a lipid normally concentrated in the PC, is excluded. We show that INPP5E, a phosphoinositide-associated phosphatase responsible for PtdIns4P generation, is a partner of ATG16L1 in this context. Perturbation of the ATG16L1-IFT20 complex alters INPP5E trafficking and proper function at the ciliary membrane. Altogether, these results reveal a novel autophagy-independent function of ATG16L1 that contributes to proper PC dynamics and function.

The autophagy protein ATG16L1 cooperates with IFT20 and INPP5E to regulate the turnover of phosphoinositides at the primary cilium.

The primary cilium (PC) regulates signalization linked to external stress sensing. Previous works established a functional interplay between the PC and the autophagic machinery. When ciliogenesis is promoted by serum deprivation, the autophagy protein ATG16L1 and the ciliary protein IFT20 are co-transported to the PC. Here, we demonstrate that IFT20 and ATG16L1 are part of the same complex requiring the WD40 domain of ATG16L1 and a Y-E-F-I motif in IFT20. We show that ATG16L1-deficient cells exhibit aberrant ciliary structures, which accumulate PI4,5P2, whereas PI4P, a lipid normally concentrated in the PC, is absent. Finally, we demonstrate that INPP5E, a phosphoinositide-associated phosphatase responsible for PI4P generation, interacts with ATG16L1 and that a perturbation of the ATG16L1/IFT20 complex alters its trafficking to the PC. Altogether, our results reveal a function of ATG16L1 in ciliary lipid and protein trafficking, thus directly contributing to proper PC dynamics and functions.

ATG4D is the main ATG8 delipidating enzyme in mammalian cells and protects against cerebellar neurodegeneration.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

The primary cilium and lipophagy translate mechanical forces to direct metabolic adaptation of kidney epithelial cells.

Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through ß-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.

Fluid flow-induced shear stress controls the metabolism of proximal tubule kidney epithelial cells through primary cilium-dependent lipophagy and mitochondria biogenesis.

The kidney, similar to many other organs, has to face shear stress induced by biological fluids. How epithelial kidney cells respond to shear stress is poorly understood. Recently we showed in vitro and in vivo that proximal tubule epithelial cells use lipophagy to fuel mitochondria with fatty acids. Lipophagy is stimulated by a primary cilium-dependent signaling that converges at AMP kinase. AMP kinase is a central signaling hub to trigger lipophagy and also to stimulate mitochondrial biogenesis. These two pathways contribute to generate ATP needed to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis. These findings demonstrate the role of the primary cilium and selective macroautophagy/autophagy to integrate shear stress and to sustain the execution of a specific cellular program.

Primary cilium-dependent autophagy drafts PIK3C2A to generate PtdIns3P in response to shear stress.

Primary cilium-dependent macroautophagy/autophagy is induced by the urinary flow in epithelial cells of the kidney proximal tubule. A major physiological outcome of this cascade is the control of cell size. Some components of the ATG machinery are recruited at the primary cilium to generate autophagic structures. Shear stress induced by the liquid flow promotes PtdIns3P synthesis at the primary cilium, and this lipid is required both for ciliogenesis and initiation of autophagy. We showed that PtdIns3P is generated by PIK3C2A, but not by PIK3C3/VPS34, during flow-associated primary cilium-dependent autophagy, in a ULK1-independent manner. Along the same line BECN1 (beclin 1), a partner of PIK3C3 in starvation-induced autophagy, is not recruited at the primary cilium under shear stress. Thus, kidney epithelial cells mobilize different PtdIns 3-kinases, i.e., PIK3C2A or PIK3C3, to produce PtdIns3P in order to initiate autophagy depending on the stimuli (shear stress or starvation). ABBREVIATIONS: PtdIns3P: phosphatidylinositol-3-phosphate; PIK3C2A: class two alpha phosphatidylinositol 3-kinase; PIK3C3/VPS34: class three phosphatidylinositol 3-kinase; ATG: autophagy associated genes.