Role of the class I Myosin, MyoK, and the actin binding protein 1, Abp1, in phagocytosis in dictyostelium discoideum

Le but de ce travail était de mieux comprendre le rôle des myosines de classe I (M1) dans l'endocytose. L'endocytose englobe l'ingestion et la digestion par la cellule de matériel exogène. Les M1s sont des moteurs moléculaires qui génèrent une tension ou un mouvement. Elles sont formées d'un domaine moteur conservé liant l'actine et d'un domaine de queue de longueur variable qui contient au moins un domaine de liaison aux membranes et, éventuellement, un à deux domaines spécifiques d'interaction protéine-protéine. L'actine, le complexe Arp2/3 de nucléation de l'actine et les M1s sont présentes lors de fermeture d'une vésicule dans tout processus endocytaire. Néanmoins, leur coordination et leur organisation spatiale sont peu connues. Le cycle de polymérisation/dépolymérisation de l'actine ainsi que l'activité contractile des myosines sont également essentiels au processus d'ingestion de particules ou phagocytose. Nous avons étudié le rôle de la M1, MyoK, dans la phagocytose et la dynamique de l'actine dans le modèle de cellule phagocytaire aisément manipulable génétiquement, l'amibe Dictyostelium discoideum

Phagosome Proteomes Unite! A Virtual Model of Maturation as a Tool to Study Pathogen-Induced Changes

Proteomics is the science that studies the complete set of proteins expressed in a given biological system at a given time. One of its key aims is to measure dynamic changes in protein quantities and posttranslational modifications as a function of physiological, pathological or experimental perturbations. Together with ultrastructural studies, comparative analysis of the protein composition of the organelles defines their identity and their relationships within the cell. Therefore, the exact identity of the phagosome is crucial to understanding its biology. The phagosome is a cytoplasmic organelle composed of a particle of extracellular origin enclosed by a membrane. It is built de novo upon particle uptake and its composition changes over time due to maturation. As early as the first published purification, the question of the phagosome identity was addressed and its lipid composition and enzymatic activities were compared with those of the plasma membrane [1, 2]. The proteomic characterization of phagosomes led to the stepwise identification of over 600 proteins in Drosophila and 800 in the mouse, among a total estimated number of about 1000 proteins [3, 4]. From these efforts have emerged the concept of a virtual model of the phagosome [5]. Groups of proteins can be recognized as functional entities, allowing phylogenetic reconstruction of their acquisition during evolution. The model has been used as a reference for comparisons with physiological [5] or pathogen-induced alterations [6] as well as a predictive tool for genes affecting uptake of bacteria [3]. The predictive power and biological validation of this proteomic-derived model depends both on the mastering of biological variation and on the robustness of the methodology used. On the one hand, the origin of the phagocytic cell, the type of particle and the time of maturation are the main sources of biological variations. Phagocytic cells can be phylogenetically diverse, from protozoan to higher vertebrates. In addition, the particle can be synthetic or natural. Its physical and biological properties impact on the receptors triggered, rate of uptake and subsequent j107 maturation. After engulfment, the process of maturation, which is naturally dedicated to microbe killing and degradation, will progressively change the environment around the particle, making the phagosome a transient and very dynamic organelle. On the other hand, because phagosome isolation from the cell is an obvious prerequisite of proteomic studies, organelle isolation as well as protein separation and identification techniques are known to introduce biases. In view of such complexity, we will discuss how the knowledge of the protein content of the phagosome has evolved and what are the advantages and drawbacks of a virtual phagosome as a comparative tool to study microbe-containing phagosomes or vacuoles (MCVs)

Monitoring time-dependent maturation changes in purified phagosomes from Dictyostelium discoideum

The amoeba Dictyostelium discoideum is an established model to study phagocytosis. The sequence of events leading to the internalization and degradation of a particle is conserved in D. discoideum compared to metazoan cells. As its small haploid genome has been sequenced, it is now amenable to genome-wide analysis including organelle proteomics. Therefore, we adapted to Dictyostelium the classical protocol to purify phagosomes formed by ingestion of latex beads particles. The pulse-chase protocol detailed here gives easy access to pure, intact, and synchronized phagosomes from representative stages of the entire process of phagosome maturation. Recently, this protocol was used to generate individual temporal profiles of proteins and lipids during phagosome maturation generating a proteomic fingerprint of six maturation stages (1). In addition, immunolabeling of phagosomes on a coverslip was developed to visualize and quantitate antigen distribution at the level of individual phagosomes