A key step in autophagy is the conjugation by the E3-like Atg12-Atg5-Atg16 complex of the ubiquitin-like protein Atg8 to phosphatidylethanolamine on the autophagosomal membrane, a process known as lipidation. Previous work in yeast showed that recruitment of the E3-like complex to the preautophagosomal structure is mediated by the interaction of Atg16 with the phosphatidylinositol 3-phosphate-binding protein Atg21, and by the association of Atg12 with the scaffold protein of the Atg1 kinase complex, Atg17. Here, we conducted a reverse two-hybrid screen to identify residues in Atg17 and Atg12 critical for Atg17-Atg12 binding, and used these data to generate a docking model of Atg12-Atg5-Atg16 with the Atg17 complex. In this model, a conserved alpha-helix in the N-terminal region of Atg12 binds to the convex side of crescent-shaped Atg17 and appears to form a four-helix bundle with the three helices of Atg17, similar to that described for the binding of Atg31 to Atg17. We further showed that, in agreement with previous work, Atg17-Atg12 and Atg21-Atg16 binding act cooperatively to mediate the recruitment of the E3-like complex, although our results show that alternative mechanisms are involved in this process. Finally, we found that phosphorylation of Atg12 by PKA prevents its interaction with Atg17, thus adding a new regulatory layer in the control of autophagy by the PKA signaling pathway.

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.

Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurodegenerative disease associated with severe cognitive and motor deficits. BPAN pathophysiology and phenotypic spectrum are still emerging due to the fact that mutations in the WDR45 (WD repeat domain 45) gene, a regulator of macroautophagy/autophagy, were only identified a decade ago. In the first international symposium dedicated to BPAN, which was held in Lyon, France, a panel of international speakers, including several researchers from the autophagy community, presented their work on human patients, cellular and animal models, carrying WDR45 mutations and their homologs. Autophagy researchers found an opportunity to explore the defective function of autophagy mechanisms associated with WDR45 mutations, which underlie neuronal dysfunction and early death. Importantly, BPAN is one of the few human monogenic neurological diseases targeting a regulator of autophagy, which raises the possibility that it is a relevant model to directly assess the roles of autophagy in neurodegeneration and to develop autophagy restorative therapeutic strategies for more common disorders.Abbreviations: ATG: autophagy related; BPAN: beta-propeller protein-associated neurodegeneration; ER: endoplasmic reticulum; KO: knockout; NBIA: neurodegeneration with brain iron accumulation; PtdIns3P: phosphatidylinositol-3-phosphate; ULK1: unc-51 like autophagy activating kinase 1; WDR45: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting.

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.

PROPPINs/WIPIs are β-propeller proteins that bind phosphoinositides and contribute to the recruitment of protein complexes involved in membrane remodelling processes such as autophagosome formation and endosomal trafficking. Yeast Atg21 and mammalian WIPI2 interact with Atg16/ATG16L1 to mediate recruitment of the lipidation machinery to the autophagosomal membrane. Here, we used the reverse double two-hybrid method (RD2H) to identify residues in Atg21 and Atg16 critical for protein–protein binding. Although our results are generally consistent with the crystal structure of the Atg21-Atg16 complex reported previously, they also reveal that dimerization of the Atg16 coiled-coil domain is required for Atg21 binding. Furthermore, most of the residues identified in Atg21 are conserved in WIPI2 and we showed that these residues also mediate ATG16L1 binding. Strikingly, these residues occupy the same position in the β-propeller structure as residues in PROPPINs/WIPIs Hsv2 and WIPI4 that mediate Atg2/ATG2A binding, supporting the idea that these proteins use different amino acids at the same position to interact with different autophagic proteins. Finally, our findings demonstrate the effectiveness of the RD2H system to identify critical residues for protein–protein interactions and the utility of this method to generate combinatory mutants with a complete loss of binding capacity.

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.

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.

Autophagy is a conserved self-degradation process that is activated under a wide variety of stresses and physiological conditions [...]

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.

Abstract PROPPINs are phosphoinositide-binding β-propeller proteins that mediate membrane recruitment of other proteins and are involved in different membrane remodeling processes. The main role of PROPPINs is their function in autophagy, where they act at different steps in phagophore formation. The human PROPPIN WIPI4 (WDR45) forms a complex with ATG2 involved in phagophore elongation, and mutations in this gene cause β-propeller protein-associated neurodegeneration (BPAN). The yeast functional counterpart of WIPI4 is Atg18, although its closest sequence homolog is another member of the PROPPIN family, Hsv2, whose function remains largely undefined. Here, we provide evidence that Hsv2, like WIPI4 and Atg18, interacts with Atg2. We show that Hsv2 and a pool of Atg2 colocalize on endosomes under basal conditions and at the pre-autophagosomal structure (PAS) upon autophagy induction. We further show that Hsv2 drives the recruitment of Atg2 to endosomes while Atg2 mediates Hsv2 recruitment to the PAS. HSV2 overexpression results in mis-sorting and secretion of carboxypeptidase CPY, suggesting that the endosomal function of this protein is related to the endosome-to-Golgi recycling pathway. Furthermore, we show that the Atg2 binding site is conserved in Hsv2 and WIPI4 but not in Atg18. Notably, two WIPI4 residues involved in ATG2 binding are mutated in patients with BPAN, and there is a correlation between the inhibitory effect of these mutations on ATG2 binding and the severity of the disease.

VMP1 and DedA proteins are conserved families of transmembrane proteins in eukaryotes and prokaryotes respectively. Despite numerous reports involving these proteins in multiple cellular processes, their molecular function is still unknown. They share the domain of unknown function PF09335, suggesting a possible functional relationship between these protein families. Here we show that VMP1 from different species contain two short motifs conserved in the bacterial DedA proteins and the yeast protein Tvp38. The hallmark of one of these motifs is a glycine residue previously shown to be strictly conserved in all the DedA proteins. Substitution of this residue to leucine, glutamate or arginine in Dictyostelium Vmp1 inactivates the protein, as shown by the inability of the mutants to rescue the phenotypes associated with the lack of Vmp1 including development and lipid homeostasis. This is the first experimental approach that supports an evolutionary relationship between Vmp1 and DedA proteins and highlights the importance of the conserved glycine residue in the PF09335 domain.

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.

BACKGROUND AND AIMS:The Diverticular Inflammation and Complication Assessment (DICA) endoscopic classification of diverticulosis and diverticular disease (DD) is currently available. It scores severity of the disease as DICA 1, DICA 2 and DICA 3. Our aim was to assess the agreement on this classification in an international endoscopists community setting.METHODS:A total of 96 doctors (82.9% endoscopists) independently scored a set of DD endoscopic videos. The percentages of overall agreement on DICA score and a free-marginal multirater kappa (κ) coefficient were reported as statistical measures of interrater agreement.RESULTS:Overall agreement in using DICA was 91.8% with a free-marginal kappa of 88% (95% CI 80-95). The overall agreement levels were: DICA 1, 85.2%; DICA 2, 96.5%; DICA 3, 99.5%. The free marginal κ was: DICA 1 = 0.753, DICA 2 = 0.958, DICA 3 = 0.919. The agreement about the main endoscopic items was 83.4% (k 67%) for diverticular extension, 62.6% (k 65%) for number of diverticula for each district, 86.8% (k 82%) for presence of inflammation, and 98.5 (k 98%) for presence of complications.CONCLUSIONS:The overall interrater agreement in this study ranges from good to very good. DICA score is a simple and reproducible endoscopic scoring system for diverticulosis and DD.

When we set out to organize this Special Issue, we faced the difficult task of gathering together a large variety of topics with the unique commonality of having been studied in a single model organism, Dictyostelium discoideum. This apparent setback turned into a wonderful opportunity to learn about an organism as a whole, which provides a more complete understanding of life processes, their natural meaning and their changes during evolution. From studies dedicated almost exclusively to cell motility, differentiation and patterning, the versatility of D. discoideum has allowed in recent years the expansion of our knowledge to other areas, including cell biology and many others related to human diseases. The present collection of papers can be considered as a journey throughout the mechanisms of life, where D. discoideum acts as a very special tourist guide.

Desde el punto de vista del comportamiento humano, se propone clarificar la relación que puede establecerse entre dos fenómenos distintos: violencia y paz. Se señala que términos tales como agresión, conflicto, conciliación y reconciliación, los cuales en algunas ocasiones han sido considerados como sinónimos de paz o de violencia, en realidad atienden a diferentes niveles de análisis de estos fenómenos. A partir de un entorno estructurado bajo condiciones de conflicto, se analiza la relación entre los participantes, calificada de violenta bajo el concepto de construcción social, y el tipo de conductas que pueden llevarla hasta una calificación de relación pacífica. Se espera con ello ofrecer una posible fundamentación conceptual que permita el desarrollo de programas de entrenamiento en estrategias comportamentales de resolución de conflictos para la convivencia pacífica.

The endoplasmic reticulum (ER) is a membranous network with an intricate dynamic architecture necessary for various essential cellular processes. Nearly one third of the proteins trafficking through the secretory pathway are folded and matured in the ER. Additionally, it acts as calcium storage, and it is a main source for lipid biosynthesis. The ER is highly connected with other organelles through regions of membrane apposition that allow organelle remodeling, as well as lipid and calcium traffic. Cells are under constant changes due to metabolic requirements and environmental conditions that challenge the ER network's maintenance. The unfolded protein response (UPR) is a signaling pathway that restores homeostasis of this intracellular compartment upon ER stress conditions by reducing the load of proteins, and by increasing the processes of protein folding and degradation. Significant progress on the study of the mechanisms that restore ER homeostasis was achieved using model organisms such as yeast, Arabidopsis, and mammalian cells. In this review, we address the current knowledge on ER architecture and ER stress response in Dictyostelium discoideum. This social amoeba alternates between unicellular and multicellular phases and is recognized as a valuable biomedical model organism and an alternative to yeast, particularly for the presence of traits conserved in animal cells that were lost in fungi.