Autophagy induction during stem cell activation plays a key role in salivary gland self-renewal.

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.

Monitoring of Autophagy and Cell Volume Regulation in Kidney Epithelial Cells in Response to Fluid Shear Stress.

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.

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion.

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 A1 (BafA1), 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 BafA1. 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.

Assays to Monitor Autophagy Progression in Cell Cultures.

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.

Primary cilium and autophagy: The avengers of cell-size regulation.

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.

Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow.

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.

Autophagy and autophagic flux in tumor cells.

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.

Functional interaction between autophagy and ciliogenesis.

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.

p53 represses autophagy in a cell cycle-dependent fashion.

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-alpha), 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-X(L) 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 G(1) phase, less so in the S phase and spares the G(2)/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.