In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Organelles compartmentalize eukaryotic cells, enhancing their ability to respond to environmental and developmental changes. One way in which organelles communicate and integrate their activities is by forming close contacts, often called 'membrane contact sites' (MCSs). Interest in MCSs has grown dramatically in the past decade as it is has become clear that they are ubiquitous and have a much broader range of critical roles in cells than was initially thought. Indeed, functions for MCSs in intracellular signalling (particularly calcium signalling, reactive oxygen species signalling and lipid signalling), autophagy, lipid metabolism, membrane dynamics, cellular stress responses and organelle trafficking and biogenesis have now been reported.

The functional universe of membrane contact sites.

Regulation and Function of Mitochondria-Lysosome Membrane Contact Sites in Cellular Homeostasis.

Mitochondria are key determinants of cellular health. However, the functional role of mitochondria varies from cell to cell depending on the relative demands for energy distribution, metabolite biosynthesis, and/or signaling. In order to support the specific needs of different cell types, mitochondrial functional capacity can be optimized in part by modulating mitochondrial structure across several different spatial scales. Here we discuss the functional implications of altering mitochondrial structure with an emphasis on the physiological trade-offs associated with different mitochondrial configurations. Within a mitochondrion, increasing the amount of cristae in the inner membrane improves capacity for energy conversion and free radical-mediated signaling but may come at the expense of matrix space where enzymes critical for metabolite biosynthesis and signaling reside. Electrically isolating individual cristae could provide a protective mechanism to limit the spread of dysfunction within a mitochondrion but may also slow the response time to an increase in cellular energy demand. For individual mitochondria, those with relatively greater surface areas can facilitate interactions with the cytosol or other organelles but may be more costly to remove through mitophagy due to the need for larger phagophore membranes. At the network scale, a large, stable mitochondrial reticulum can provide a structural pathway for energy distribution and communication across long distances yet also enable rapid spreading of localized dysfunction. Highly dynamic mitochondrial networks allow for frequent content mixing and communication but require constant cellular remodeling to accommodate the movement of mitochondria. The formation of contact sites between mitochondria and several other organelles provides a mechanism for specialized communication and direct content transfer between organelles. However, increasing the number of contact sites between mitochondria and any given organelle reduces the mitochondrial surface area available for contact sites with other organelles as well as for metabolite exchange with cytosol. Though the precise mechanisms guiding the coordinated multi-scale mitochondrial configurations observed in different cell types have yet to be elucidated, it is clear that mitochondrial structure is tailored at every level to optimize mitochondrial function to meet specific cellular demands.

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The Functional Impact of Mitochondrial Structure Across Subcellular Scales.

Cellular organelles are not static but show dynamism-a property that is likely relevant for their function. In addition, they interact with other organelles in a highly dynamic manner. In this review, we analyze the proteins involved in the interaction between mitochondria and other cellular organelles, especially the endoplasmic reticulum, lipid droplets, and lysosomes. Recent results indicate that, on one hand, metabolic alterations perturb the interaction between mitochondria and other organelles, and, on the other hand, that deficiency in proteins involved in the tethering between mitochondria and the ER or in specific functions of the interaction leads to metabolic alterations in a variety of tissues. The interaction between organelles is an emerging field that will permit to identify key proteins, to delineate novel modulation pathways, and to elucidate their implications in human disease.

https://www.embopress.org/doi/pdf/10.15252/embr.201947928

Metabolic implications of organelle-mitochondria communication.

Members of the VPS13 family are associated with various human diseases. In particular, the loss of function of VPS13A leads to chorea-acanthocytosis (ChAc), a rare neurodegenerative disease without available curative treatments. Autophagy has been considered a promising therapeutic target because the absence of VPS13A causes a defective autophagy flux. However, the mechanistic details of this deficiency are unknown. Here, we identified Rab7A as an interactor of one of the VPS13 family members in Dictyostelium discoideum and showed that this interaction is conserved between the human homologs VPS13A and RAB7A in HeLa cells. As RAB7A is a key player in endosome trafficking, we addressed the possible function of VPS13A in endosome dynamics and lysosome degradation. Our results suggest that the decrease in autophagy observed in the absence of VPS13A may be the result of a more general defect in endocytic trafficking and lysosomal degradation. Unexpectedly, we found that VPS13A is closely localized to mitochondria, suggesting that the role of VPS13A in the endolysosomal pathway might be related to inter-organelle communication. We show that VPS13A localizes at the interface between mitochondria-endosomes and mitochondria-endoplasmic reticulum and that the presence of membrane contact sites is altered in the absence of VPS13A. Based on these findings, we propose that therapeutic strategies aimed at modulating the endolysosomal pathway could be beneficial in the treatment of ChAc.This article has an associated First Person interview with the first author of the paper.

VPS13A is closely associated with mitochondria and is required for efficient lysosomal degradation

WIPIs are a conserved family of proteins with a characteristic 7-bladed β-propeller structure. They play a prominent role in autophagy, but also in other membrane trafficking processes. Mutations in human WIPI4 cause several neurodegenerative diseases. One of them is BPAN, a rare disease characterized by developmental delay, motor disorders, and seizures. Autophagy dysfunction is thought to play an important role in this disease but the precise pathological consequences of the mutations are not well established. The use of simple models such as the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum provides valuable information on the molecular and cellular function of these proteins, but also sheds light on possible pathways that may be relevant in the search for potential therapies. Here, we review the function of WIPIs as well as disease-causing mutations with a special focus on the information provided by these simple models.

/pdf/the-wipi-gene-family-and-neurodegenerative-diseases-insights-4ev88flvk9.pdf

The WIPI Gene Family and Neurodegenerative Diseases: Insights From Yeast and Dictyostelium Models.

PROPPINs are conserved PtdIns3P-binding proteins required for autophagosome biogenesis that fold into a characteristic group of seven-bladed beta-propellers. Mutations in WDR45/WIPI4, a human member of this family, lead to BPAN, a rare form of neurodegeneration. We have generated mutants for the two PROPPIN proteins present in the model system Dictyostelium discoideum (Atg18 and Wdr45l) and characterized their function. Lack of Wdr45l greatly impairs autophagy, while Atg18 only causes subtle defects in the maturation of autolysosomes. The strong phenotype of the Wdr45l mutant is strikingly similar to that observed in Dictyostelium cells lacking Vmp1, an ER protein required for omegasome formation. Common phenotypes include impaired growth in axenic medium, lack of aggregation, and local enrichment of PtdIns3P as determined by the use of lipid reporters. In addition, Vmp1 and Wdr45l mutants show a chronically active response to ER stress. For both mutants, this altered PtdIns3P localization can be prevented by the additional mutation of the upstream regulator Atg1, which also leads to recovery of axenic growth and reduction of ER stress. We propose that, in addition to an autophagy defect, local autophagy-associated PtdIns3P accumulation might contribute to the pathogenesis of BPAN by disrupting ER homeostasis. The introduction of BPAN-associated mutations in Dictyostelium Wdr45l reveals the impact of pathogenic residues on the function and localization of the protein.

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2021.1953262

A Dictyostelium model for BPAN disease reveals a functional relationship between the WDR45/WIPI4 homolog Wdr45l and Vmp1 in the regulation of autophagy-associated PtdIns3P and ER stress.

VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea‐acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo‐lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild‐type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long‐term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo‐lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/jcmm.17768

Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9‐based cellular model of VPS13A deficiency

Binding of sorting nexin SNX5 to the VAB domain of VPS13A mediates its recruitment to membrane contact sites between SNX5-containing endosomal membranes, mitochondria, and ER. Human VPS13 proteins are implicated in severe neurological diseases. These proteins play an important role in lipid transport at membrane contact sites between different organelles. Identification of adaptors that regulate the subcellular localization of these proteins at specific membrane contact sites is essential to understand their function and role in disease. We have identified the sorting nexin SNX5 as an interactor of VPS13A that mediates its association with endosomal subdomains. As for the yeast sorting nexin and Vps13 endosomal adaptor Ypt35, this association involves the VPS13 adaptor-binding (VAB) domain in VPS13A and a PxP motif in SNX5. Notably, this interaction is impaired by mutation of a conserved asparagine residue in the VAB domain, which is also required for Vps13-adaptor binding in yeast and is pathogenic in VPS13D. VPS13A fragments containing the VAB domain co-localize with SNX5, whereas the more C-terminal part of VPS13A directs its localization to the mitochondria. Overall, our results suggest that a fraction of VPS13A localizes to junctions between the endoplasmic reticulum, mitochondria, and SNX5-containing endosomes.

/pdf/the-association-of-lipid-transfer-protein-vps13a-with-6lbxg9g4.pdf

Alba Tornero-Écija

Papers

VPS13A is closely associated with mitochondria and is required for efficient lysosomal degradation

The WIPI Gene Family and Neurodegenerative Diseases: Insights From Yeast and Dictyostelium Models.

A Dictyostelium model for BPAN disease reveals a functional relationship between the WDR45/WIPI4 homolog Wdr45l and Vmp1 in the regulation of autophagy-associated PtdIns3P and ER stress.

Effect of rapamycin on lysosomal accumulation in a CRISPR/Cas9‐based cellular model of VPS13A deficiency

The association of lipid transfer protein VPS13A with endosomes is mediated by sorting nexin SNX5