Laurence Aubry

Cell-death alternative model organisms: why and which?

Classical model organisms have helped greatly in our understanding of cell death but, at the same time, might have constrained it. The use of other, non-classical model organisms from all biological kingdoms could reveal undetected molecular pathways and better-defined morphological types of cell death. Here we discuss what is known and what might be learned from these alternative model systems.

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. β-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 β-arrestins have a two-lobe, immunoglobulin-like, β-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.

A necrotic cell death model in a protist.

While necrotic cell death is attracting considerable interest, its molecular bases are still poorly understood. Investigations in simple biological models, taken for instance outside the animal kingdom, may benefit from less interference from other cell death mechanisms and from better experimental accessibility, while providing phylogenetic information. Can necrotic cell death occur outside the animal kingdom? In the protist Dictyostelium, developmental stimuli induced in an autophagy mutant a stereotyped sequence of events characteristic of necrotic cell death. This sequence included swift mitochondrial uncoupling with mitochondrial 2′,7′-dichlorofluorescein diacetate fluorescence, ATP depletion and increased oxygen consumption. This was followed by perinuclear clustering of dilated mitochondria. Rapid plasma membrane rupture then occurred, which was evidenced by time-lapse videos and quantified by FACS. Of additional interest, developmental stimuli and classical mitochondrial uncouplers triggered a similar sequence of events, and exogenous glucose delayed plasma membrane rupture in a nonglycolytic manner. The occurrence of necrotic cell death in the protist Dictyostelium (1) provides a very favorable model for further study of this type of cell death, and (2) strongly suggests that the mechanism underlying necrotic cell death was present in an ancestor common to the Amoebozoa protists and to animals and has been conserved in evolution.

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.

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.

FYVE-Dependent Endosomal Targeting of an Arrestin-Related Protein in Amoeba

Background Visual and β-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.

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.

Modelling of fluid-phase endocytosis kinetics in the amoebae of the cellular slime mouldDictyostelium discoideum. A multicompartmental approach

Fluid-phase endocytosis (pinocytosis) kinetics were studied inDictyostelium discoideum amoebae from the axenic strain Ax-2 that exhibits high rates of fluid-phase endocytosis when cultured in liquid nutrient media. Fluorescein-labelled dextran (FITC-dextran) was used as a marker in continuous uptake- and in pulse-chase exocytosis experiments. In the latter case, efflux of the marker was monitored on cells loaded for short periods of time and resuspended in marker-free medium. A multicompartmental model was developed which describes satisfactorily fluid-phase endocytosis kinetics. In particular, it accounts correctly for the extended latency period before exocytosis in pulse-chase experiments and it suggests the existence of some sorts of maturation stages in the pathway.

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.

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.