Identification of chemicals breaking the USP8 interaction with its endocytic substrate CHMP1B.

The ubiquitin-specific protease USP8 plays a major role in controlling the stability and intracellular trafficking of numerous cell surface proteins among which the EGF receptor that regulates cell growth and proliferation in many physio-pathological processes. The function of USP8 at the endocytic pathway level partly relies on binding to and deubiquitination of the Endosomal Sorting Complex Required for Transport (ESCRT) protein CHMP1B. In the aim of finding chemical inhibitors of the USP8::CHMP1B interaction, we performed a high-throughput screening campaign using an HTRF® assay to monitor the interaction directly in lysates of cells co-expressing both partners. The assay was carried out in an automated format to screen the academic Fr-PPIChem library (Bosc N et al., 2020), which includes 10,314 compounds dedicated to the targeting of protein-protein interactions (PPIs). Eleven confirmed hits inhibited the USP8::CHMP1B interaction within a range of 30% to 70% inhibition at 50 µM, while they were inactive on a set of other PPI interfaces demonstrating the feasibility of specifically disrupting this particular interface. In parallel, we adapted this HTRF® assay to compare the USP8 interacting capacity of CHMP1B variants. As anticipated from earlier studies, a deletion of the MIM (Microtubule Interacting and Trafficking domain Interacting Motif) domain or mutation of two conserved leucine residues, L192 and L195, in this domain respectively abolished or strongly impeded the USP8::CHMP1B interaction. By contrast, a CHMP1B mutant that displays a highly decreased ubiquitination level following mutation of four lysine residues in arginine interacted at a similar level as the wild-type form with USP8. Therefore, conserved leucine residues within the MIT domain rather than its ubiquitinated status triggers CHMP1B substrate recognition by USP8.

Quantitative Automated Assays in Living Cells to Screen for Inhibitors of Hemichannel Function.

In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

CAP-Gly proteins contribute to microtubule-dependent trafficking via interactions with the C-terminal aromatic residue of α-tubulin.

In mammals, the C-terminal tyrosine residue of α-tubulin is subjected to removal/re-addition cycles resulting in tyrosinated microtubules and detyrosinated Glu-microtubules. CLIP170 and its yeast ortholog (Bik1) interact weakly with Glu-microtubules. Recently, we described a Microtubule- Rho1- and Bik1-dependent mechanism involved in Snc1 routing. Here, we further show a contribution of the yeast p150Glued ortholog (Nip100) in Snc1 trafficking. Both CLIP170 and p150Glued are CAP-Gly-containing proteins that belong to the microtubule +end-tracking protein family (known as +Tips). We discuss the +Tips-dependent role of microtubules in trafficking, the role of CAP-Gly proteins as possible molecular links between microtubules and vesicles, as well as the contribution of the Rho1-GTPase to the regulation of the +Tips repertoire and the partners associated with microtubules.

Calcium influx mediates the chemoattractant-induced translocation of the arrestin-related protein AdcC in Dictyostelium.

Arrestins are key adaptor proteins that control the fate of cell-surface membrane proteins and modulate downstream signaling cascades. The Dictyostelium discoideum genome encodes six arrestin-related proteins, harboring additional modules besides the arrestin domain. Here, we studied AdcB and AdcC, two homologs that contain C2 and SAM domains. We showed that AdcC - in contrast to AdcB - responds to various stimuli (such as the chemoattractants cAMP and folate) known to induce an increase in cytosolic calcium by transiently translocating to the plasma membrane, and that calcium is a direct regulator of AdcC localization. This response requires the calcium-dependent membrane-targeting C2 domain and the double SAM domain involved in AdcC oligomerization, revealing a mode of membrane targeting and regulation unique among members of the arrestin clan. AdcB shares several biochemical properties with AdcC, including in vitro binding to anionic lipids in a calcium-dependent manner and auto-assembly as large homo-oligomers. AdcB can interact with AdcC; however, its intracellular localization is insensitive to calcium. Therefore, despite their high degree of homology and common characteristics, AdcB and AdcC are likely to fulfill distinct functions in amoebae.

Collective regulation of cell motility using an accurate density-sensing system.

The capacity of living cells to sense their population density and to migrate accordingly is essential for the regulation of many physiological processes. However, the mechanisms used to achieve such functions are poorly known. Here, based on the analysis of multiple trajectories of vegetative Dictyostelium discoideum cells, we investigate such a system extensively. We show that the cells secrete a high-molecular-weight quorum-sensing factor (QSF) in their medium. This extracellular signal induces, in turn, a reduction of the cell movements, in particular, through the downregulation of a mode of motility with high persistence time. This response appears independent of cAMP and involves a G-protein-dependent pathway. Using a mathematical analysis of the cells' response function, we evidence a negative feedback on the QSF secretion, which unveils a powerful generic mechanism for the cells to detect when they exceed a density threshold. Altogether, our results provide a comprehensive and dynamical view of this system enabling cells in a scattered population to adapt their motion to their neighbours without physical contact.

A role for the yeast CLIP170 ortholog, the plus-end-tracking protein Bik1, and the Rho1 GTPase in Snc1 trafficking.

The diversity of microtubule functions is dependent on the status of tubulin C-termini. To address the physiological role of the C-terminal aromatic residue of α-tubulin, a tub1-Glu yeast strain expressing an α-tubulin devoid of its C-terminal amino acid was used to perform a genome-wide-lethality screen. The identified synthetic lethal genes suggested links with endocytosis and related processes. In the tub1-Glu strain, the routing of the v-SNARE Snc1 was strongly impaired, with a loss of its polarized distribution in the bud, and Abp1, an actin patch or endocytic marker, developed comet-tail structures. Snc1 trafficking required dynamic microtubules but not dynein and kinesin motors. Interestingly, deletion of the microtubule plus-end-tracking protein Bik1 (a CLIP170 ortholog), which is preferentially recruited to the C-terminal residue of α-tubulin, similarly resulted in Snc1 trafficking defects. Finally, constitutively active Rho1 rescued both Bik1 localization at the microtubule plus-ends in tub1-Glu strain and a correct Snc1 trafficking in a Bik1-dependent manner. Our results provide the first evidence for a role of microtubule plus-ends in membrane cargo trafficking in yeast, through Rho1- and Bik1-dependent mechanisms, and highlight the importance of the C-terminal α-tubulin amino acid in this process.

Pycnosomes: Condensed Endosomal Structures Secreted by Dictyostelium Amoebae.

Dictyostelium discoideum has been used largely as a model organism to study the organization and function of the endocytic pathway. Here we describe dense structures present in D. discoideum endocytic compartments, which we named pycnosomes. Pycnosomes are constitutively secreted in the extracellular medium, from which they can be recovered by differential centrifugation. We identified the most abundant protein present in secreted pycnosomes, that we designated SctA. SctA defines a new family of proteins with four members in D. discoideum, and homologous proteins in other protists and eumetazoa. We developed a monoclonal antibody specific for SctA and used it to further characterize secreted and intracellular pycnosomes. Within cells, immunofluorescence as well as electron microscopy identified pycnosomes as SctA-enriched dense structures in the lumen of endocytic compartments. Pycnosomes are occasionally seen in continuity with intra-endosomal membranes, particularly in U18666A-treated cells where intraluminal budding is highly enhanced. While the exact nature, origin and cellular function of pycnosomes remain to be established, this study provides a first description of these structures as well as a characterization of reagents that can be used for further studies.

The arrestin-domain containing protein AdcA is a response element to stress.

BACKGROUND: Cell behaviour is tightly determined by sensing and integration of extracellular changes through membrane detectors such as receptors and transporters and activation of downstream signalling cascades. Arrestin proteins act as scaffolds at the plasma membrane and along the endocytic pathway, where they regulate the activity and the fate of some of these detectors. Members of the arrestin clan are widely present from unicellular to metazoa, with roles in signal transduction and metabolism. As a soil amoeba, Dictyostelium is frequently confronted with environmental changes likely to compromise survival. Here, we investigated whether the recently described arrestin-related protein AdcA is part of the cell response to stresses. RESULTS: Our data provide evidence that AdcA responds to a variety of stresses including hyperosmolarity by a transient phosphorylation. Analysis in different mutant backgrounds revealed that AdcA phosphorylation involves pathways other than the DokA and cGMP-dependent osmostress pathways, respectively known to regulate PKA and STATc, key actors in the cellular response to conditions of hyperosmolarity. Interestingly, however, both AdcA and STATc are sensitive to changes in the F-actin polymerization status, suggesting a common primary sensor/trigger and linking the stress-sensitive kinase responsive for AdcA phosphorylation to the actin cytoskeleton. We also show that STATc-dependent transcriptional activity is involved for the timely dephosphorylation of AdcA in cells under stress. CONCLUSION: Under osmotic stress, AdcA undergoes a phosphorylation-dephosphorylation cycle involving a stress-sensitive kinase and the transcription regulator STATc. This transient post-transcriptional modification may allow a regulation of AdcA function possibly to optimize the cellular stress response.

True arrestins and arrestin-fold proteins: a structure-based appraisal.

Arrestin-clan proteins are folded alike, a feature responsible for their recent grouping in a single clan. In human, it includes the well-characterized visual and ß-arrestins, the arrestin domain-containing proteins (ARRDCs), isoforms of the retromer subunit VPS26, and DSCR3, a protein involved in Down syndrome. A new arrestin-fold-predicted protein, RGP1, described here may join the clan. Unicellular organisms like the yeast Saccharomyces cerevisiae or the amoeba Dictyostelium discoideum harbor VPS26, DSCR3, and RGP1 isoforms as well as arrestin-related trafficking adaptors or ADCs, but true arrestins are missing. Functionally, members of the arrestin clan have generally a scaffolding role in various membrane protein trafficking events. Despite their similar structure, the mechanism of cargo recognition and internalization and the nature of recruited partners differ for the different members. Based on the recent literature, true arrestins (visual and ß-arrestins), ARRDCs, and yeast ARTS are the closest from a functional point of view.

Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry.

The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.

A hybrid model to study pathological mutations of the human ADP/ATP carriers.

The adenine nucleotide carrier (Ancp) plays an essential role in the metabolism of cellular energy by catalyzing the transport of ADP and ATP across the inner mitochondrial membrane. Previous reports have indicated that mutations in the HANC1 gene, encoding the muscle isoform of human Ancp (HAnc1p), are directly involved in several diseases, including autosomal dominant progressive external ophthalmoplegia and cardiomyopathies. In this work, we studied three pathogenic HANC1 mutations at the biochemical level. To do so, we expressed the DdANCA gene, encoding the unique Ancp carrier of Dictyostelium discoideum (DdAncAp), in a yeast strain lacking all endogenous ANC genes. Our results indicate that DdAncAp is a good model for the human protein. It allows the carrier to be studied in yeast, and provides information on how the HANC1 mutations impair ADP/ATP transport in humans. A94D, A126D and V291M mutations, corresponding to A90D, A123D and V289M in HAnc1p, respectively, did not affect levels of DdAncAp in yeast mitochondria. However, while the wild-type DdAncAp fully restored growth of the ANC-null yeast strain on a non-fermentable carbon source, the carriers encompassing either the A94D or the A126D mutation failed to complement the null strain. The effect of the V291M mutation was not as pronounced, but led to impairment mainly of the nucleotide translocation process per se. These findings provide new insights into the mechanisms responsible for the diseases induced by HAnc1p mutations.

FYVE-dependent endosomal targeting of an arrestin-related protein in amoeba.

BACKGROUND: Visual and ß-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. METHODOLOGY AND PRINCIPAL FINDINGS: A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. SIGNIFICANCE: This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.

The arrestin fold: variations on a theme.

Endocytosis of ligand-activated plasma membrane receptors has been shown to contribute to the regulation of their downstream signaling. beta-arrestins interact with the phosphorylated tail of activated receptors and act as scaffolds for the recruitment of adaptor proteins and clathrin, that constitute the machinery used for receptor endocytosis. Visual- and beta-arrestins have a two-lobe, immunoglobulin-like, beta-strand sandwich structure. The recent resolution of the crystal structure of VPS26, one of the retromer subunits, unexpectedly evidences an arrestin fold in this protein, which is otherwise unrelated to arrestins. From a functional point of view, VPS26 is involved in the retrograde transport of the mannose 6-P receptor from the endosomes to the trans-Golgi network. In addition to the group of genuine arrestins and Vps26, mammalian cells harbor a vast repertoire of proteins that are related to arrestins on the basis of their PFAM Nter and Cter arrestin- domains, which are named Arrestin Domain- Containing proteins (ADCs). The biological role of ADC proteins is still poorly understood. The three subfamilies have been merged into an arrestin-related protein clan.This paper provides an overall analysis of arrestin clan proteins. The structures and functions of members of the subfamilies are reviewed in mammals and model organisms such as Drosophila, Caenorhabditis, Saccharomyces and Dictyostelium.

Dictyostelium Tom1 participates to an ancestral ESCRT-0 complex.

Sorting of ubiquitinated proteins to multivesicular bodies (MVBs) in mammalian cells relies on proteins with a Vps27/Hrs/STAM (VHS) domain. Here, we show that the amoeba Dictyostelium presents only one protein with a VHS domain: DdTom1. We demonstrate that the VHS domain of DdTom1 is followed by a Golgi-localized, gamma-ear-containing, ADP-ribosylation-factor-binding and Tom1 (GAT) domain that binds ubiquitin, and by a non-conserved C-terminal domain that can recruit clathrin, EGFr pathway substrate 15 and tumor susceptibility gene 101, a component of the MVB biogenesis machinery [endosomal complexes required for transport (ESCRT) complexes]. Both VHS and GAT domains interact with phospholipids and therefore could ensure the recruitment of DdTom1 to endosomal membranes. We propose that DdTom1 participates in an ancestral ESCRT-0 complex implicated in the sorting of ubiquitinated proteins into MVBs.

Necrotic cell death: From reversible mitochondrial uncoupling to irreversible lysosomal permeabilization.

Dictyostelium atg1- mutant cells provide an experimentally and genetically favorable model to study necrotic cell death (NCD) with no interference from apoptosis or autophagy. In such cells subjected to starvation and cAMP, induction by the differentiation-inducing factor DIF or by classical uncouplers led within minutes to mitochondrial uncoupling, which causally initiated NCD. We now report that (1) in this model, NCD included a mitochondrial-lysosomal cascade of events, (2) mitochondrial uncoupling and therefore initial stages of death showed reversibility for a surprisingly long time, (3) subsequent lysosomal permeabilization could be demonstrated using Lysosensor blue, acridin orange, Texas red-dextran and cathepsin B substrate, (4) this lysosomal permeabilization was irreversible, and (5) the presence of the uncoupler was required to maintain mitochondrial lesions but also to induce lysosomal lesions, suggesting that signaling from mitochondria to lysosomes must be sustained by the continuous presence of the uncoupler. These results further characterized the NCD pathway in this priviledged model, contributed to a definition of NCD at the lysosomal level, and suggested that in mammalian NCD even late reversibility attempts by removal of the inducer may be of therapeutic interest.

Mitochondrial carrier family: repertoire and peculiarities of the cellular slime mould Dictyostelium discoideum.

Proteins of the mitochondrial carrier family (MCF) mediate the transport of a large range of compounds, including metabolites and cofactors. They are localized mainly in the inner mitochondrial membrane, except for a few members found in the membranes of peroxisomes. Similarity searches among Dictyostelium discoideum protein sequences identified a total of 31 MCF members. All these are membrane proteins that possess three characteristic repeats of a domain of approximately 100 residues. Among them, three proteins have supplementary structural domains consisting of Ca(2+)-binding motifs made up of 2 or 4 EF-hand units localized on the N-terminal end, facing the mitochondrial intermembrane space. The nature of transported substrates is proposed on the basis of sequence comparison with orthologs characterized biochemically in other organisms, of phylogenetic analysis, and of the conservation of discriminating amino acid residues belonging to the substrate binding sites. Carriers have been grouped in subclasses based on their specificity for the transport of nucleotides, amino acids or keto acids. Furthermore, we have identified an iron carrier of the mitoferrin type, an inorganic phosphate carrier, and three carriers with similarity to uncoupler proteins. This study provides a focus for mitochondrial carrier analysis in Dictyostelium discoideum.

Purification techniques of subcellular compartments for analytical and preparative purposes.

In the following protocols, broken cells are the starting material of all downstream purifications of functional organelles or intact subcellular membranes. The choice of the breakage method has direct and deep repercussions on the quality of subsequent steps. Breaking vegetative amoebae by shear stress with a steel ball cell cracker preserves the integrity of subcellular organelles and in particular that of lysosomes, the rupture of which is very deleterious to further purifications. In this chapter, we propose purification schemes for plasma membrane, nuclei, mitochondria, and endocytic compartments. Plasma membranes are purified without any cell coating by partition between aqueous polymer phases. Nuclei and mitochondria are purified by differential centrifugations in adequate buffer conditions. Endosomes are magnetically isolated after feeding the cells with colloidal iron dextran and phagosomes by flotation on a sucrose gradient after feeding amoebae with latex beads. As analytical approaches, we propose procedures to label the plasma membrane and the endo-lysosomal compartments by biotinylation and to separate early and late compartments on a Percoll gradient.

Trafficking and developmental signaling: Alix at the crossroads.

Alix is a phylogenetically conserved protein that participates in mammals in programmed cell death in association with ALG-2, a penta-EF-hand calciprotein. It contains an N-terminal Bro1 domain, a coiled-coil region and a C-terminal proline-rich domain containing several SH3- and WW-binding sites that contribute to its scaffolding properties. Recent data showed that by virtue of its Bro1 domain, Alix is functionally associated to the ESCRT complexes involved in the biogenesis of the multivesicular body and sorting of transmembrane proteins within this specific endosomal compartment. In Dictyostelium, an alx null strain shows a markedly perturbed starvation-induced morphogenetic program while ALG-2 disruptants remain unaffected. This review summarizes Dictyostelium data on Alix and ALG-2 homologues and evaluates whether known functions of Alix in other organisms can account for the developmental arrest of the alx null mutant and how Dictyostelium studies can substantiate the current understanding of the function(s) of this versatile and conserved signaling molecule.

NADPH oxidase homologs are required for normal cell differentiation and morphogenesis in Dictyostelium discoideum.

Membrane-associated NADPH oxidase complexes catalyse the production of the superoxide anion radical from oxygen and NADPH. In mammalian systems, NADPH oxidases form a family of at least seven isoforms that participate in host defence and signalling pathways. We report here the cloning and the characterisation of slime mould Dictyostelium discoideum homologs of the mammalian heme-containing subunit of flavocytochrome b (gp91(phox)) (NoxA, NoxB and NoxC), of the small subunit of flavocytochrome b (p22(phox)) and of the cytosolic factor p67(phox). Null-mutants of either noxA, noxB, noxC or p22(phox) show aberrant starvation-induced development and are unable to produce spores. The overexpression of NoxA(myc2) in noxA null strain restores spore formation. Remarkably, the gene alg-2B, coding for one of the two penta EF-hand proteins in Dictyostelium, acts as a suppressor in noxA, noxB, and p22(phox) null-mutant strains. Knockout of alg-2B allows noxA, noxB or p22(phox) null-mutants to return to normal development. However, the knockout of gene encoding NoxC, which contains two penta EF-hands, is not rescued by the invalidation of alg-2B. These data are consistent with a hypothesis connecting superoxide and calcium signalling during Dictyostelium development.