Coiled-coil-mediated dimerization of Atg16 is required for binding to the PROPPIN Atg21.

PROPPINs/WIPIs are ß-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 ß-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.

Anomalous ionic transport in tunable angstrom-size water films on silica.

Liquid and ionic transport through nanometric structures is central to many phenomena, ranging from cellular exchanges to water resource management or green energy conversion. While pushing down toward molecular scales progressively unveils novel transport behaviors, reaching ultimate confinement in controlled systems remains challenging and has often involved 2D Van der Waals materials. Here, we propose an alternative route which circumvents demanding nanofabrication steps, partially releases material constraints, and offers continuously tunable molecular confinement. This soft-matter-inspired approach is based on the spontaneous formation of a molecularly thin liquid film onto fully wettable substrates in contact with the vapor phase of the liquid. Using silicon dioxide substrates, water films ranging from angstrom to nanometric thicknesses are formed in this manner, and ionic transport within the film can then be measured. Performing conductance measurements as a function of confinement in these ultimate regimes reveals a one-molecule thick layer of fully hindered transport nearby the silica, above which continuum, bulk-like approaches account for experimental results. Overall, this work paves the way for future investigations of molecular scale nanofluidics and provides insights into ionic transport nearby high surface energy materials such as natural rocks and clays, building concretes, or nanoscale silica membranes used for separation and filtering.

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

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.

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.

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.

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

WIPIs are a conserved family of proteins with a characteristic 7-bladed ß-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.

A conserved ATG2 binding site in WIPI4 and yeast Hsv2 is disrupted by mutations causing ß-propeller protein-associated neurodegeneration.

PROPPINs are phosphoinositide-binding ß-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 ß-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.

A novel reverse two-hybrid method for the identification of missense mutations that disrupt protein-protein binding.

The reverse two-hybrid system is a powerful method to select mutations that disrupt the interaction between two proteins and therefore to identify the residues involved in this interaction. However, the usefulness of this technique has been limited by its relative complexity when compared to the classical two-hybrid system, since an additional selection step is required to eliminate the high background of uninformative truncation mutants. We have developed a new method that combines the classical and reverse two-hybrid systems to select loss-of-binding missense mutations in a single step. The strategy used to select against truncation mutants is based on the two-hybrid interaction between a C-terminal fusion peptide and the Tsg101 protein. We have applied this method to identify mutations in human glucokinase (GK) that disrupt glucokinase regulatory protein (GKRP) binding. Our results indicate that this method is very efficient and eliminates all the truncation mutants and false positives. The mutated residues identified in GK are involved in the GKRP binding interface or in stabilizing the super-open conformation of GK that binds GKRP. This technique offers an improvement over existing methods in terms of speed, efficiency and simplicity and can be used to study any detectable protein interaction in the two-hybrid system.

Evidence for an evolutionary relationship between Vmp1 and bacterial DedA proteins.

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.

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

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.

Adsorption, Desorption, and Crystallization of Aqueous Solutions in Nanopores.

Probing nanoconfined solutions in tortuous, mesoporous media is challenging because of pore size, complex pore connectivity, and the coexistence of multiple components and phases. Here, we use optical reflectance to experimentally investigate the wetting and drying of a mesoporous medium with ∼3-nm-diameter pores containing aqueous solutions of sodium chloride and lithium chloride. We show that the vapor activities (i.e., relative humidities) that correspond to optical features in the isotherms for solutions can be used to deduce the thermodynamic state of a nanoscopic solution that undergoes evaporation and crystallization upon drying and condensation and deliquescence when increasing the relative humidity. We emphasize specific equilibrium states of the system: the onset of draining during desorption and the end of filling during adsorption as well as percolation-induced scattering and crystallization. We find that theoretical arguments involving classical thermodynamics (a modified Kelvin-Laplace equation and classical nucleation theory) explain quantitatively the evolution of the optical features and thereby the state of the solution as a function of imposed vapor activity and solute concentration.

How Solutes Modify the Thermodynamics and Dynamics of Filling and Emptying in Extreme Ink-Bottle Pores.

We investigate the filling and emptying of extreme ink-bottle porous media-micrometer-scale pores connected by nanometer-scale pores-when changing the pressure of the external vapor, in a case where the pore liquid contains solutes. These phenomena are relevant in diverse contexts, such as the weathering of building materials and artwork, aerosol formation in the atmosphere, and the hydration of soils and plants. Using model systems made of vein-shaped microcavities interconnected by a mesoporous matrix, we show experimentally that the presence of a nonvolatile solute shifts the condensation and evaporation transitions and in a way that is consistent with a modified Kelvin-Laplace equation that takes into account the osmotic pressure of the solution. Emptying occurs far below saturation, when the Kelvin stress, mediated by the large curvature of the liquid-vapor interfaces in the nanopores, is negative enough to induce spontaneous bubble nucleation in the microveins. Filling, on the other hand, occurs close to equilibrium (i.e., at saturation, psat for pure water and ps < psat for a solution), driven by the weak capillary pressure of the liquid-vapor interface in the microveins. Interestingly, solutes allow the system to reach situations where the vapor is supersaturated with respect to the solution ( ps < p < psat). We show that in that latter situation, a condensation layer covers the outer surface of the porous system, preventing the generation of Kelvin stresses but inducing osmotic stresses and flows that are vapor pressure-dependent. The timescales and dynamics reflect these different driving forces: emptying proceeds through discrete, stochastic nucleation events with very fast, unsteady bubble growth associated with a poroelastic relaxation process, while filling occurs collectively in all veins of the sample through a slower steady-state process driven by a combination of osmosis and capillarity. The dynamics can however be rendered symmetrical between filling and emptying if bubbles pre-exist during emptying, a case that we explore using cycling of the vapor pressure around equilibrium.

Fibroblast growth factor 2 lethally sensitizes cancer cells to stress-targeted therapeutic inhibitors

In malignant transformation, cellular stress-response pathways are dynami-cally mobilized to counterbalance oncogenic activity, keeping cancer cellsviable. Therapeutic disruption of this vulnerable homeostasis might changethe outcome of many human cancers, particularly those for which no effec-tive therapy is available. Here, we report the use of fibroblast growth factor2 (FGF2) to demonstrate that further mitogenic activation disrupts cellularhomeostasis and strongly sensitizes cancer cells to stress-targeted therapeu-tic inhibitors. We show that FGF2 enhanced replication and proteotoxicstresses in a K-Ras-driven murine cancer cell model, and combinations ofFGF2 and proteasome or DNA damage response-checkpoint inhibitorstriggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressedthe malignant phenotype and prevented these synergic toxicities in thesemurine cells. Moreover, in a panel of human Ewing’s sarcoma family tumorcells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sus-tained MAPK-ERK1/2 overactivation induced by FGF2 appears to under-lie these synthetic lethalities, as late pharmacological inhibition of thispathway restored cell homeostasis and prevented these described synergies.Our results highlight how mitotic signaling pathways which are frequentlyoverridden in malignant transformation might be exploited to disrupt therobustness of cancer cells, ultimately sensitizing them to stress-targeted ther-apies. This approach provides a new therapeutic rationale for human can-cers, with important implications for tumors still lacking effectivetreatment, and for those that frequently relapse after treatment with avail-able therapies.

Fibroblast growth factor 2 lethally sensitizes cancer cells to stress-targeted therapeutic inhibitors

In malignant transformation, cellular stress-response pathways are dynami-cally mobilized to counterbalance oncogenic activity, keeping cancer cellsviable. Therapeutic disruption of this vulnerable homeostasis might changethe outcome of many human cancers, particularly those for which no effec-tive therapy is available. Here, we report the use of fibroblast growth factor2 (FGF2) to demonstrate that further mitogenic activation disrupts cellularhomeostasis and strongly sensitizes cancer cells to stress-targeted therapeu-tic inhibitors. We show that FGF2 enhanced replication and proteotoxicstresses in a K-Ras-driven murine cancer cell model, and combinations ofFGF2 and proteasome or DNA damage response-checkpoint inhibitorstriggered cell death. CRISPR/Cas9-mediated K-Ras depletion suppressedthe malignant phenotype and prevented these synergic toxicities in thesemurine cells. Moreover, in a panel of human Ewing’s sarcoma family tumorcells, sublethal concentrations of bortezomib (proteasome inhibitor) or VE-821 (ATR inhibitor) induced cell death when combined with FGF2. Sus-tained MAPK-ERK1/2 overactivation induced by FGF2 appears to under-lie these synthetic lethalities, as late pharmacological inhibition of thispathway restored cell homeostasis and prevented these described synergies.Our results highlight how mitotic signaling pathways which are frequentlyoverridden in malignant transformation might be exploited to disrupt therobustness of cancer cells, ultimately sensitizing them to stress-targeted ther-apies. This approach provides a new therapeutic rationale for human can-cers, with important implications for tumors still lacking effectivetreatment, and for those that frequently relapse after treatment with avail-able therapies.

Unfolding the Endoplasmic Reticulum of a Social Amoeba: Dictyostelium discoideum as a New Model for the Study of Endoplasmic Reticulum Stress.

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.

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)-domains associated with diverse ER-organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1-enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1-deficient mammalian cells and other model systems.

IreA Controls Endoplasmic Reticulum Stress-Induced Autophagy and Survival through Homeostasis Recovery.

The unfolded protein response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress. The ER-resident kinase/RNase Ire1 is the only UPR sensor conserved during evolution. Autophagy, a lysosomal degradative pathway, also contributes to the recovery of cell homeostasis after ER stress, but the interplay between these two pathways is still poorly understood. We describe the Dictyostelium discoideum ER stress response and characterize its single bona fide Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression reprogramming that increases the protein folding capacity of the ER and alleviates ER protein load. Further, IreA is required for cell survival after TN-induced ER stress and is responsible for nearly 40% of the transcriptional changes induced by TN. The response of Dictyostelium cells to ER stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway. These two pathways are independently activated in response to ER stress but, interestingly, autophagy requires IreA at a later stage for proper autophagosome formation. We propose that unresolved ER stress in cells lacking IreA causes structural alterations of the ER, leading to a late-stage blockade of autophagy clearance. This unexpected functional link may critically affect eukaryotic cell survival under ER stress.

Functional characterization of MODY2 mutations in the nuclear export signal of glucokinase.

Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence. Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning. In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.

Imbibition Triggered by Capillary Condensation in Nanopores.

We study the spatiotemporal dynamics of water uptake by capillary condensation from unsaturated vapor in mesoporous silicon layers (pore radius rp ≃ 2 nm), taking advantage of the local changes in optical reflectance as a function of water saturation. Our experiments elucidate two qualitatively different regimes as a function of the imposed external vapor pressure: at low vapor pressures, equilibration occurs via a diffusion-like process; at high vapor pressures, an imbibition-like wetting front results in fast equilibration toward a fully saturated sample. We show that the imbibition dynamics can be described by a modified Lucas-Washburn equation that takes into account the liquid stresses implied by Kelvin equation.

Autophagy in Dictyostelium: Mechanisms, regulation and disease in a simple biomedical model.

Autophagy is a fast-moving field with an enormous impact on human health and disease. Understanding the complexity of the mechanism and regulation of this process often benefits from the use of simple experimental models such as the social amoeba Dictyostelium discoideum. Since the publication of the first review describing the potential of D. discoideum in autophagy, significant advances have been made that demonstrate both the experimental advantages and interest in using this model. Since our previous review, research in D. discoideum has shed light on the mechanisms that regulate autophagosome formation and contributed significantly to the study of autophagy-related pathologies. Here, we review these advances, as well as the current techniques to monitor autophagy in D. discoideum. The comprehensive bioinformatics search of autophagic proteins that was a substantial part of the previous review has not been revisited here except for those aspects that challenged previous predictions such as the composition of the Atg1 complex. In recent years our understanding of, and ability to investigate, autophagy in D. discoideum has evolved significantly and will surely enable and accelerate future research using this model.

Capillarity-driven flows at the continuum limit.

We experimentally investigate the dynamics of capillary-driven flows at the nanoscale, using an original platform that combines nanoscale pores (⋍3 nm in diameter) and microfluidic features. In particular, we show that drying involves a fine coupling between thermodynamics and fluid mechanics that can be used to generate precisely controlled nanoflows driven by extreme stresses - up to 100 MPa of tension. We exploit these tunable flows to provide quantitative tests of continuum theories (e.g. Kelvin-Laplace equation and Poiseuille flow) across an unprecedented range and we isolate the breakdown of continuum as a negative slip length of molecular dimension. Our results show a coherent picture across multiple experiments including drying-induced permeation flows, imbibition and poroelastic transients.