Pierre Cosson

Anthrax toxin triggers endocytosis of its receptor via a lipid raft–mediated clathrin-dependent process

The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

Late endosome motility depends on lipids via the small GTPase Rab7

We report that lipids contribute to regulate the bidirectional motility of late endocytic compartments. Late endocytic vesicles loaded with cholesterol lose their dynamic properties, and become essentially immobile, including in cells from Niemann–Pick C patients. These vesicles then retain cytoplasmic dynein activity, but seem to be unable to acquire kinesin activity, eventually leading to paralysis. Our data suggest that this defect depends on the small GTPase Rab7, since the motility of vesicles loaded with cholesterol can be restored by the Rab7 inhibitory mutant N125I. Conversely, wild‐type Rab7 overexpression mimics the effects of cholesterol on motility in control cells. Consistently, cholesterol accumulation increases the amounts of membrane‐associated Rab7, and inhibits Rab7 membrane extraction by the guanine nucleotide dissociation inhibitor. Our observations thus indicate that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 in turn controls the net movement of late endocytic elements. We conclude that motor functions can be regulated by the membrane lipid composition via the Rab7 cycle.

Separation and Characterization of Late Endosomal Membrane Domains

Very little is known about the biophysical properties and the lipid or protein composition of membrane domains presumably present in endocytic and biosynthetic organelles. Here we analyzed the membrane composition of late endosomes by suborganellar fractionation in the absence of detergent. We found that the internal membranes of this multivesicular organelle can be separated from the limiting membrane and that each membrane population exhibited a defined composition. Our data also indicated that internal membranes may consist of at least two populations, containing primarily phosphatidylcholine or lysobisphosphatidic acid as major phospholipid, arguing for the existence of significant microheterogeneity within late endosomal membranes. We also found that lysobisphosphatidic acid exhibited unique pH-dependent fusogenic properties, and we speculated that this lipid is an ideal candidate to regulate the dynamic properties of this internal membrane mosaic.

Pseudomonas aeruginosa Virulence Analyzed in a Dictyostelium discoideum Host System

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.

DIRECT INTERACTION BETWEEN THE ENVELOPE AND MATRIX PROTEINS OF HIV-1

The incorporation of the envelope (env) glycoprotein of the human immunodeficiency virus type 1 (HIV‐1) into budding virions has been proposed to be mediated by an interaction between its cytoplasmic domain and the matrix protein of HIV‐1. However, this interaction was never directly demonstrated and its role in the biogenesis of HIV‐1 virions is still debated. Here, a direct interaction is reported between the matrix protein of HIV‐1 and the cytoplasmic domain of the env protein of HIV‐1. No interaction was seen with the env cytoplasmic domain of other retroviruses. The region of the HIV‐1 env involved in the interaction was delineated by mutagenesis and is comprised of the C‐terminal 67 amino acid residues of env. These results, as well as the analysis of mutants of the matrix protein, suggest that the interaction between the HIV‐1 env and matrix proteins accounts for the specific incorporation of the env glycoprotein into HIV‐1 virions.

Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains

The intracellular fate of T cell antigen receptor (TCR) subunits (alpha beta gamma delta epsilon zeta 2) is determined by their assembly in the endoplasmic reticulum (ER). To study the structural bases for this tight correlation between assembly and intracellular fate, we sought to define the nature of determinants for both ER degradation and subunit assembly within the TCR-alpha chain. We found that a 9 amino acid transmembrane sequence of the TCR-alpha chain, containing 2 critical charged residues, was sufficient to cause ER degradation when placed in the context of the Tac antigen, used here as a reporter protein. CD3-delta assembled with chimeric proteins containing this short transmembrane sequence, and this assembly resulted in abrogation of targeting for ER degradation. Thus, the colocalization of determinants for ER degradation and sites of subunit interactions explains how the fate of some newly synthesized TCR chains can be decided on the basis of their assembly status.

Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum.

The selective breakdown of newly synthesized proteins retained within the endoplasmic reticulum (ER) is probably mediated by the specific recognition of structural features of protein substrates by components of a degradative system. Within the alpha chain of the multisubunit T‐cell antigen receptor (TCR) complex, a transmembrane sequence containing two basic amino acid residues has been shown to act as a determinant for retention and rapid degradation in the ER. We now demonstrate that single basic or acidic amino acid residues can cause targeting for retention and degradation in the ER when placed within the transmembrane domain of an integral membrane protein normally destined for the cell surface. The effect of such potentially charged residues is dependent on their relative position within the transmembrane sequence and on the nature of the amino acid side chains. The phenotypic changes induced by potentially charged transmembrane residues occur without apparent alterations of the global folding or transmembrane topology of the mutant proteins. These observations test the hypothesis that potentially charged residues within transmembrane domains can provide the basis for a motif for ER degradation and explain the selective breakdown of some proteins retained within the ER.

Eat, kill or die: when amoeba meets bacteria.

The core function of the innate immune response, phagocytosis, did not evolve first in metazoans but rather in primitive unicellular eukaryotes. Thus, though amoebae separated from the tree leading to metazoan shortly after the divergence of plants, they share many specific functions with mammalian phagocytic cells. Dictyostelium discoideum is by far the most studied amoeba, and it is proving useful to analyze phagocytosis and intracellular killing of bacteria. Since the basic mechanisms involved appear extremely conserved, Dictyostelium provides novel insights into the function of many new gene products. Bacterial pathogenicity was certainly largely developed to resist predatory amoebae in the environment, and this accounts for the fact that a large number of bacterial virulence traits can be studied using Dictyostelium as a host. This provides a particularly powerful system to analyze the complex interactions between pathogenic bacteria and host cells, where both the Dictyostelium host and the bacteria can be manipulated genetically with relative ease.

From the Cover: STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum

Store-operated calcium entry relies on the formation of a specialized compartment derived from the endoplasmic reticulum (ER) and closely apposed to the plasma membrane. In this study, detailed ultrastructural analysis revealed the existence of three distinct structures derived from conventional ER: precortical ER, cortical ER, and thin cortical ER. Precortical subdomains of the ER enriched in STIM1 can form without contacting the plasma membrane. Upon ER calcium depletion, these subdomains are translocated to the plasma membrane to form cortical ER, which is still connected to the conventional ER. Thin cortical ER, depleted of BiP and deprived of attached ribosomes, may represent a specialized region dedicated to calcium regulation and not engaged in protein translocation and folding. These observations form the basis for future structure-function analysis of cortical ER.

Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval.

Two new thermosensitive yeast mutants defective in retrieval of dilysine‐tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2–1 and ret3–1 were defective for ER retrieval, only ret2–1 exhibited a defect in forward ER‐to‐Golgi transport at the non‐permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro. The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively. Both proteins show significant homology to clathrin adaptor subunits. These results emphasize the role of coatomer in retrieval of dilysine‐tagged proteins back to the ER, and the similarity between clathrin and coatomer coats.