Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for ...degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A
1
(BafA
1
), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in in vitro experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA
1
. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not bona fide surrogates for other types of late stage lysosomal inhibitors for in vivo experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signalling motivated us to ...explore the possible functional interactions between this signalling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signalling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. Whereas abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signalling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie some common ciliopathies.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The vast number of implications of autophagy in multiple areas of life sciences and medicine has attracted the interest of numerous scientists that aim to unveil the role of this process in specific ...physiological and pathological contexts. Cell cultures are one of the most frequently used experimental setup for the investigation of autophagy. As a result, it is essential to assess this highly regulated molecular pathway with efficient and reliable methods. Each method has its own advantages and disadvantages. Here, we present a review summarizing the most established assays used to monitor autophagy induction and progression in cell cultures, in order to guide researchers in the selection of the most optimal solution for their experimental setup and design.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Relatively quiescent tissues like salivary glands (SGs) respond to stimuli such as injury to expand, replace and regenerate. Resident stem/progenitor cells are key in this process because, upon ...activation, they possess the ability to self-renew. Macroautophagy/autophagy contributes to and regulates differentiation in adult tissues, but an important question is whether this pathway promotes stem cell self-renewal in tissues. We took advantage of a 3D organoid system that allows assessing the self-renewal of mouse SGs stem cells (SGSCs). We found that autophagy in dormant SGSCs has slower flux than self-renewing SGSCs. Importantly, autophagy enhancement upon SGSCs activation is a self-renewal feature in 3D organoid cultures and SGs regenerating in vivo. Accordingly, autophagy ablation in SGSCs inhibits self-renewal whereas pharmacological stimulation promotes self-renewal of mouse and human SGSCs. Thus, autophagy is a key pathway for self-renewal activation in low proliferative adult tissues, and its pharmacological manipulation has the potential to promote tissue regeneration.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic ...pifithrin-), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-XL with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G1 phase, less so in the S phase and spares the G2/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
The maintenance of cellular homeostasis in response to extracellular stresses by autophagy is vital for the health of various tissues. Extracellular stimuli may include nutrient starvation, ...endoplasmic reticulum stress, hypoxia, cytotoxic agents, or mechanical stress. The primary cilium (PC) is a microtubule-based sensory organelle that regulates the integration of various extracellular stimuli. The interconnection between macroautophagy/autophagy and the PC is beginning to be illuminated. In this punctum, we discuss our recent study of PC-dependent autophagy in response to fluid flow in kidney epithelial cells. Urinary flow in kidney tubules creates a shear stress that regulates epithelial cell volume. PC-mediated autophagy is necessary for the regulation of cell size. The signal from the PC is transduced by the activation of STK11/LKB1 and by MTOR inhibition. Our results clarify the physiological role of PC-dependent autophagy in the kidney and suggest that autophagy manipulation may provide a route to the treatment of ciliopathies.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Autophagy is an adaptation mechanism that is vital for cellular homeostasis in response to various stress conditions. Previous reports indicate that there is a functional interaction between the ...primary cilium (PC) and autophagy. The PC, a microtubule-based structure present at the surface of numerous cell types, is a mechanical sensor. Here we show that autophagy induced by fluid flow regulates kidney epithelial cell volume in vitro and in vivo. PC ablation blocked autophagy induction and cell-volume regulation. In addition, inhibition of autophagy in ciliated cells impaired the flow-dependent regulation of cell volume. PC-dependent autophagy can be triggered either by mTOR inhibition or a mechanism dependent on the polycystin 2 channel. Only the LKB1-AMPK-mTOR signalling pathway was required for the flow-dependent regulation of cell volume by autophagy. These findings suggest that therapies regulating autophagy should be considered in developing treatments for PC-related diseases.
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IJS, NUK, SBMB, UL, UM, UPUK
Fluidic shear stress applied to epithelial cells inside the kidney tubules affects cell size in an autophagy-related manner. Here, we describe the technical equipment that we routinely use to apply ...shear stress on cells, as well as immunoblotting, immunofluorescence, and three-dimensional cell volume reconstruction techniques used in analysis of the influence of this stress on cells and cellular components. By pointing out details of experimental techniques and potential pitfalls, this review will serve as a guide for those interested in study of how shear stress influences cells.
Les cils primaires et motiles sont des structures microtubulaires présentent à la surface de nombreux types cellulaires. Les structures ciliées contrôlent de nombreuses fonctions allant de la ...motilité cellulaire à l’intégration par la cellule de stimuli chimiques et mécaniques. Au cours de cette thèse, nous avons étudié le dialogue entre le cil primaire et l’autophagie, un processus d’autodigestion qui permet à la cellule de s’adapter à des situations de stress. L’hypothèse de ce dialogue reposait sur l’analyse de la littérature montrant que de nombreux médiateurs (calcium, carence en sérum, arrêt du cycle cellulaire) stimulent à la fois l’activité ciliaire et l’autophagie. Dans un premier temps de notre étude nous avons montré que l’inhibition de la ciliogenèse altère l’induction de l’autophagie en réponse à la carence en sérum dans des fibroblastes d’embryon de souris, des cellules épithéliales rénales et des lignées de neurone. Nous avons aussi montré que la carence en sérum induisait une redistribution de nombreuses protéines Atg (Autophagy-related), protéines impliquées dans la biogenèse de l’autophagosome, au niveau du cil primaire (soit au niveau du corps basal soit au niveau de l’axonème). Particulièrement la protéine Atg16L1 est co-transportée vésiculairement au corps basal avec la protéine ciliaire IFT20. L’inhibition génétique ou pharmacologique de la voie de signalisation Hedgehog inhibe à la fois le transport de la protéine Atg16L1 au corps basal et l’induction de l’autophagie en absence de sérum. Nous avons aussi montré que l’invalidation de gènes ATG est associée à une ciliogenèse accrue. Dans ces conditions nous avons conclu sur des bases morphologiques et biochimiques que ces cils primaires sont fonctionnels. La protéine IFT20 s ‘accumule dans les cellules déficientes en autophagie et est dégradée par autophagie dans les cellules sauvage en présence de sérum. Ces résultats montre que l’autophagie basale (autophagie observée en présence de sérum) est un mécanisme qui contribue au contrôle de la croissance du cil primaire. Dans une deuxième partie du travail nous avons étudié l’importance de l’autophagie dans la réponse cellulaire à stress mécanique. Le contrôle de la taille et du volume des cellules épithéliales rénales est un élément important pour maintenir la polarité planaire des cellules tubulaires. Cette propriété est dépendante du cil primaire. Au cours de l’application d’un flux de liquide (1 dyn/cm2) concomitamment à la réduction du volume et de la taille cellulaire nous avons observé une stimulation de l’autophagie. Cette réponse autophagique dépend du cil primaire. L’invalidation de l’autophagie dans des cellules épithéliales ciliées abolit le contrôle du volume et de la taille cellulaire dans les cellules épithéliales rénales. L’ensemble de ces résultats montre le dialogue qui existe en l’autophagie et le cil primaire et l’importance de ce dialogue dans l’intégration par la cellule du stress mécanique.
Motile and primary cilia are microtubule-based structures located at the cell surface of many cell types. Cilia govern cellular functions ranging from motility to integration of mechanical and chemical signaling from the environment. In this work we investigate the potential cross-talk between the primary cilium and macroautophagy. Macroautophagy or self-eating is a lysosomal degradative pathway that allows cells to adapt to various stress situations. The rational for the study was based on the survey of the literature showing that many stress situations that trigger primary cilium signaling also stimulates autophagy (serum starvation, calcium mobilization, cell cycle arrest). In the first part of the study we showed that inhibition of ciliogenesis severely impairs serum-induced autophagy in mouse embryo fibroblasts, kidney epithelial cells and neurons. We also showed that in response to serum deprivation many Autophagy-related proteins (Atg proteins) involved in autophagosome formation are co-localized with cilium subdomains (axoneme and basal body). Notably the protein Atg16L1 is co-transported to the basal body with the ciliary protein IFT20. The localization of Atg16L1 to the basal body as well as serum-induced autophagy were severely impaired by inhibiting the Hedgehog signaling pathway either genetic or pharmacological approaches. We also showed that invalidation of ATG genes induced an increase in primary cilium length in basal condition. Cilia were functional in ATG-deficient cells because of the presence of a ciliary pocket and the activation of the Hedgehog signaling pathway. Finally we identified IFT20 as a substrate for autophagy. Thus autophagy is required to regulate the level of IFT20 and consequently that of the length of the primary cilium. In the second part of the work we investigate the role of the cross-talk between autophagy and the primary cilium in regulating the size of kidney epithelial cells. Previous studies have shown that the primary cilium plays a central role in regulating cell size and cell volume. This regulation is important to keep the physiological functions of tubular renal cells by maintaining the planar polarity in kidney tubule. By applying a liquid flow of 1 dyn/cm2 to MDCK or mouse kidney epithelial cells to mimic physiological conditions, we show that the flow induces autophagy and reduction of the cell volume. In absence of cilium we observed that autophagy is not induced and that the cell size/volume is not responsive to the mechanical stress. Finally we showed that ablation of autophagy led also to an impairment of flow-dependent regulation of cell size/volume in ciliated kidney epithelial cells. In conclusion primary cilium-dependent autophagy plays a major role in controlling the epithelial kidney cell size/volume during mechanical stress induced by fluid flow.
Macroautophagy (hereafter referred to as autophagy), a central mechanism mediating the lysosomal degradation of cytoplasmic components, can be stimulated by a wide panel of adverse stimuli, including ...a panoply of anticancer agents. The central autophagic organelle is the autophagosome, a double membrane-bound vacuole that sequesters the cytoplasmic material destined to disposal. The ultimate destiny of the autophagosome is to fuse with a lysosome, resulting in the degradation of the autophagic cargo. In this setting, it is important to discriminate whether a particular stimulus actually promotes autophagy or it simply blocks the fusion of autophagosomes with lysosomes. To this aim, the methods that assess autophagy should assess not only the number of autophagosomes but also the so-called autophagic flux, that is, the clearance of the autophagy cargo from the lysosomal compartment. Here, we present a compendium of methods to assess the autophagic flux in cultured malignant cells. This approach should allow for the study of the intimate link between autophagy and oncometabolism in several experimental paradigms.
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