Thierry Soldati

Rab9 functions in transport between late endosomes and the trans Golgi network.

Rab proteins represent a large family of ras‐like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion. We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9. The majority of rab9 appears to be located on the surface of late endosomes. Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate. In vitro‐prenylated rab9 protein, but not C‐terminally truncated rab9, stimulated the transport of mannose 6‐phosphate receptors from late endosomes to the trans Golgi network in a cell‐free system that reconstitutes this transport step. Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP. These results strongly suggest that rab9 functions in the transport of mannose 6‐phosphate receptors between late endosomes and the trans Golgi network. Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.

Powering membrane traffic in endocytosis and recycling

Early in evolution, the diversification of membrane-bound compartments that characterize eukaryotic cells was accompanied by the elaboration of molecular machineries that mediate intercompartmental communication and deliver materials to specific destinations. Molecular motors that move on tracks of actin filaments or microtubules mediate the movement of organelles and transport between compartments. The subjects of this review are the motors that power the transport steps along the endocytic and recycling pathways, their modes of attachment to cargo and their regulation.

Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells

In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion.

Ca2+-Triggered Peptide Secretion in Single Cells Imaged with Green Fluorescent Protein and Evanescent-Wave Microscopy

Green fluorescent protein fused to human chromogranin B or neuropeptide Y was expressed in PC12 cells and caused bright, punctate fluorescence. The fluorescent points colocalized with the endogenous secretory granule marker dopamine beta-hydroxylase. Stimulation of live PC12 cells with elevated [K+], or of permeabilized PC12 cells with Ca2+, led to Ca2+-dependent loss of fluorescence from neurites. Ca2+ stimulated secretion of both fusion proteins equally well. In living cells, single fluorescent granules were imaged by evanescent-wave fluorescence microscopy. Granules were seen to migrate; to stop, as if trapped by plasmalemmal docking sites; and then to disappear abruptly, as if through exocytosis. Evidently, GFP fused to secreted peptides is a fluorescent marker for dense-core secretory granules and may be used for time-resolved microscopy of single granules.

Toxoplasma gondii myosin A and its light chain: a fast, single‐headed, plus‐end‐directed motor

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle–laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 μm/s towards the plus end of actin filaments. TgMyoA is the first fast, single‐headed myosin and fulfils all the requirements for power parasite gliding.

Infection by Tubercular Mycobacteria Is Spread by Nonlytic Ejection from Their Amoeba Hosts

To generate efficient vaccines and cures for Mycobacterium tuberculosis, we need a far better understanding of its modes of infection, persistence, and spreading. Host cell entry and the establishment of a replication niche are well understood, but little is known about how tubercular mycobacteria exit host cells and disseminate the infection. Using the social amoeba Dictyostelium as a genetically tractable host for pathogenic mycobacteria, we discovered that M. tuberculosis and M. marinum, but not M. avium, are ejected from the cell through an actin-based structure, the ejectosome. This conserved nonlytic spreading mechanism requires a cytoskeleton regulator from the host and an intact mycobacterial ESX-1 secretion system. This insight offers new directions for research into the spreading of tubercular mycobacteria infections in mammalian cells.

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.

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

WASH coats mature lysosomes and is required for exocytosis of indigestible material.

Reactive oxygen species and mitochondria: A nexus of cellular homeostasis.

Reactive oxygen species (ROS) are integral components of multiple cellular pathways even though excessive or inappropriately localized ROS damage cells. ROS function as anti-microbial effector molecules and as signaling molecules that regulate such processes as NF-kB transcriptional activity, the production of DNA-based neutrophil extracellular traps (NETs), and autophagy. The main sources of cellular ROS are mitochondria and NADPH oxidases (NOXs). In contrast to NOX-generated ROS, ROS produced in the mitochondria (mtROS) were initially considered to be unwanted by-products of oxidative metabolism. Increasing evidence indicates that mtROS have been incorporated into signaling pathways including those regulating immune responses and autophagy. As metabolic hubs, mitochondria facilitate crosstalk between the metabolic state of the cell with these pathways. Mitochondria and ROS are thus a nexus of multiple pathways that determine the response of cells to disruptions in cellular homeostasis such as infection, sterile damage, and metabolic imbalance. In this review, we discuss the roles of mitochondria in the generation of ROS-derived anti-microbial effectors, the interplay of mitochondria and ROS with autophagy and the formation of DNA extracellular traps, and activation of the NLRP3 inflammasome by ROS and mitochondria.

Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake

Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Gα4 and Gβ, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.