Collective directional migration drives the formation of heteroclonal cancer cell clusters.

Metastasisation occurs through the acquisition of invasive and survival capabilities that allow tumour cells to colonise distant sites. While the role of multicellular aggregates in cancer dissemination is acknowledged, the mechanisms that drive the formation of multiclonal cell aggregates are not fully elucidated. Here, we show that cancer cells of different tissue of origins can perform collective directional migration and can actively form heteroclonal aggregates in 3D, through a proliferation-independent mechanism. Coalescence of distant cell clusters is mediated by subcellular actin-rich protrusions and multicellular outgrowths that extend towards neighbouring aggregates. Coherently, perturbation of cytoskeletal dynamics impairs collective migration while myosin II activation is necessary for multicellular movements. We put forward the hypothesis that cluster attraction is mediated by secreted soluble factors. Such a hypothesis is consistent with the abrogation of aggregation by inhibition of PI3K/AKT/mTOR and MEK/ERK, the chemoattracting activity of conditioned culture media and with a wide screening of secreted proteins. Our results present a novel collective migration model and shed light on the mechanisms of formation of heteroclonal aggregates in cancer.

Iron metabolism in the social amoeba Dictyostelium discoideum: A role for ferric chelate reductases.

Iron is the most abundant transition metal in all living organisms and is essential for several cellular activities, including respiration, oxygen transport, energy production and regulation of gene expression. Iron starvation is used by professional phagocytes, from Dictyostelium to macrophages, as a form of defense mechanism against intracellular pathogens. Previously, we showed that Dictyostelium cells express the proton-driven iron transporter Nramp1 (Natural Resistance-Associated Macrophage Protein 1) and the homolog NrampB (Nramp2) in membranes of macropinosomes and phagosomes or of the contractile vacuole network, respectively. The Nramp-driven transport of iron across membranes is selective for ferrous ions. Since iron is mostly present as ferric ions in growth media and in engulfed bacteria, we have looked for proteins with ferric reductase activity. The Dictyostelium genome does not encode for classical STEAP (Six-Transmembrane Epithelial Antigen of Prostate) ferric reductases, but harbors three genes encoding putative ferric chelate reductase belonging to the Cytochrome b561 family containing a N terminus DOMON domain (DOpamine ß-MONooxygenase N-terminal domain). We have cloned the three genes, naming them fr1A, fr1B and fr1C. fr1A and fr1B are mainly expressed in the vegetative stage while fr1C is highly expressed in the post aggregative stage. All three reductases are localized in the endoplasmic reticulum, but Fr1A is also found in endolysosomal vesicles, in the Golgi and, to a much lower degree, in the plasma membrane, whereas Fr1C is homogeneously distributed in the plasma membrane and in macropinosomal and phagosomal membranes. To gain insight in the function of the three genes we generated KO mutants, but gene disruption was successful only for two of them (fr1A and fr1C), being very likely lethal for fr1B. fr1A- shows a slight delay in the aggregation stage of development, while fr1C- gives rise to large multi-tipped streams during aggregation and displays a strong delay in fruiting body formation. The two single mutants display altered cell growth under conditions of ferric ions overloading and, in the ability to reduce Fe3+, confirming a role of these putative ferric reductases in iron reduction and transport from endo-lysosomal vesicles to the cytosol.

Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching.

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities.Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein.This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48-72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals.The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis.

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.

Dictyostelium Host Response to Legionella Infection: Strategies and Assays.

The professional phagocyte Dictyostelium discoideum is a well-established model organism to study host-pathogen interactions. Dictyostelium amoebae grow as separate, independent cells; they divide by binary fission and take up bacteria and yeast via phagocytosis. In the year 2000, D. discoideum was described by two groups as a novel system for genetic analysis of host-pathogen interactions for the intracellular pathogen Legionella pneumophila. Since then additional microbial pathogens that can be studied in D. discoideum have been reported. The organism has various advantages for the dissection of the complex cross-talk between a host and a pathogen. A fully sequenced and well-curated genome is available, there are excellent molecular genetic tools on the market, and the generation of targeted multiple gene knock-outs as well as the realization of untargeted genetic screens is generally straightforward. Dictyostelium also offers easy cultivation, and the cells are suitable for cell biological studies, which in combination with in vivo expression of fluorescence-tagged proteins allows the investigation of the dynamics of bacterial uptake and infection. Furthermore, a large mutant collection is available at the Dictyostelium stock center, favoring the identification of host resistance or susceptibility genes. Here, we briefly describe strategies to identify host cell factors important during an infection, followed by protocols for cell culture and storage, uptake and infection, and confocal microscopy of infected cells.

Differential Effects of Iron, Zinc, and Copper on Dictyostelium discoideum Cell Growth and Resistance to Legionella pneumophila.

Iron, zinc, and copper play fundamental roles in eucaryotes and procaryotes, and their bioavailability regulates host-pathogen interactions. For intracellular pathogens, the source of metals is the cytoplasm of the host, which in turn manipulates intracellular metal traffic following pathogen recognition. It is established that iron is withheld from the pathogen-containing vacuole, whereas for copper and zinc the evidence is unclear. Most infection studies in mammals have concentrated on effects of metal deficiency/overloading at organismal level. Thus, zinc deficiency or supplementation correlate with high risk of respiratory tract infection or recovery from severe infection, respectively. Iron, zinc, and copper deficiency or overload affects lymphocyte proliferation/maturation, and thus the adaptive immune response. Whether they regulate innate immunity at macrophage level is open, except for iron. The early identification in a mouse mutant susceptible to mycobacterial infection of the iron transporter Nramp1 allowed dissecting Nramp1 role in phagocytes, from the social amoeba Dictyostelium to macrophages. Nramp1 regulates iron efflux from the phagosomes, thus starving pathogenic bacteria for iron. Similar studies for zinc or copper are scant, due to the large number of copper and zinc transporters. In Dictyostelium, zinc and copper transporters include 11 and 6 members, respectively. To assess the role of zinc or copper in Dictyostelium, cells were grown under conditions of metal depletion or excess and tested for resistance to Legionella pneumophila infection. Iron shortage or overload inhibited Dictyostelium cell growth within few generations. Surprisingly, zinc or copper depletion failed to affect growth. Zinc or copper overloading inhibited cell growth at, respectively, 50- or 500-fold the physiological concentration, suggesting very efficient control of their homeostasis, as confirmed by Inductively Coupled Plasma Mass Spectrometry quantification of cellular metals. Legionella infection was inhibited or enhanced in cells grown under iron shortage or overload, respectively, confirming a major role for iron in controlling resistance to pathogens. In contrast, zinc and copper depletion or excess during growth did not affect Legionella infection. Using Zinpyr-1 as fluorescent sensor, we show that zinc accumulates in endo-lysosomal vesicles, including phagosomes, and the contractile vacuole. Furthermore, we provide evidence for permeabilization of the Legionella-containing vacuole during bacterial proliferation.

Dasatinib modulates sensitivity to pemetrexed in malignant pleural mesothelioma cell lines.

BACKGROUND: Thymidylate synthase (TS), one of the key enzymes for thymidine synthesis, is a target of pemetrexed (PEM), a key agent for the systemic therapy of malignant pleural mesothelioma (MPM) and its overexpression has been correlated to PEM-resistance. In MPM, experimental data report activation of the c-SRC tyrosine kinase suggesting it as a potential target to be further investigated. RESULTS: MPM cell lines showed different sensitivity, being MSTO the most and REN the least sensitive to PEM. REN cells showed high levels of both TS and SRC: dasatinib inhibited SRC activation and suppressed TS protein expression, starting from 100 nM dose, blocking the PEM-induced up regulation of TS protein levels. Dasatinib treatment impaired cells migration, and both sequential and co-administration with PEM significantly increased apoptosis. Dasatinib pretreatment improved sensitivity to PEM, downregulated TS promoter activity and, in association with PEM, modulated the downstream PI3K-Akt-mTOR signaling. Cell lines and Methods: In three MPM cell lines (MPP89, REN and MSTO), the effects of c-SRC inhibition, in correlation with TS expression and PEM sensitivity, were evaluated. PEM and dasatinib, a SRC inhibitor, were administered as single agents, in combination or sequentially. Cell viability, apoptosis and migration, as well as TS expression and SRC activation have been assessed. CONCLUSIONS: These data indicate that dasatinib sensitizes mesothelioma cells to PEM through TS down-regulation.

New alternative splicing BCR/ABL-OOF shows an oncogenic role by lack of inhibition of BCR GTPase activity and an increased of persistence of Rac activation in chronic myeloid leukemia.

In Chronic Myeloid Leukemia 80% of patients present alternative splice variants involving BCR exons 1, 13 or 14 and ABL exon 4, with a consequent impairment in the reading frame of the ABL gene. Therefore BCR/ABL fusion proteins (BCR/ABL-OOF) are characterized by an in-frame BCR portion followed by an amino acids sequence arising from the out of frame (OOF) reading of the ABL gene. The product of this new transcript contains the characteristic BCR domains while lacking the COOH-terminal Rho GTPase GAP domain. The present work aims to characterize the protein functionality in terms of cytoskeleton (re-)modelling, adhesion and activation of canonical oncogenic signalling pathways. Here, we show that BCR/ABL-OOF has a peculiar endosomal localization which affects EGF receptor activation and turnover. Moreover, we demonstrate that BCR/ABL-OOF expression leads to aberrant cellular adhesion due to the activation of Rac GTPase, increase in cellular proliferation, migration and survival. When overexpressed in a BCR/ABL positive cell line, BCR/ABL-OOF induces hyperactivation of Rac signaling axis offering a therapeutic window for Rac-targeted therapy. Our data support a critical role of BCR/ABL-OOF in leukemogenesis and identify a subset of patients that may benefit from Rac-targeted therapies.

Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux.

The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and the transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, and electrophysiological and radiochemical assays, we show that Nramp1, similar to rat DMT1 (also known as SLC11A2), transports Fe(2+) and manganese, not Fe(3+) or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe(2+) in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.

Iron metabolism and resistance to infection by invasive bacteria in the social amoeba Dictyostelium discoideum.

Dictyostelium cells are forest soil amoebae, which feed on bacteria and proliferate as solitary cells until bacteria are consumed. Starvation triggers a change in life style, forcing cells to gather into aggregates to form multicellular organisms capable of cell differentiation and morphogenesis. As a soil amoeba and a phagocyte that grazes on bacteria as the obligate source of food, Dictyostelium could be a natural host of pathogenic bacteria. Indeed, many pathogens that occasionally infect humans are hosted for most of their time in protozoa or free-living amoebae, where evolution of their virulence traits occurs. Due to these features and its amenability to genetic manipulation, Dictyostelium has become a valuable model organism for studying strategies of both the host to resist infection and the pathogen to escape the defense mechanisms. Similarly to higher eukaryotes, iron homeostasis is crucial for Dictyostelium resistance to invasive bacteria. Iron is essential for Dictyostelium, as both iron deficiency or overload inhibit cell growth. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis. Iron transporters of the Nramp (Slc11A) family are represented with two genes, encoding Nramp1 and Nramp2. Like the mammalian ortholog, Nramp1 is recruited to phagosomes and macropinosomes, whereas Nramp2 is a membrane protein of the contractile vacuole network, which regulates osmolarity. Nramp1 and Nramp2 localization in distinct compartments suggests that both proteins synergistically regulate iron homeostasis. Rather than by absorption via membrane transporters, iron is likely gained by degradation of ingested bacteria and efflux via Nramp1 from phagosomes to the cytosol. Nramp gene disruption increases Dictyostelium sensitivity to infection, enhancing intracellular growth of Legionella or Mycobacteria. Generation of mutants in other "iron genes" will help identify genes essential for iron homeostasis and resistance to pathogens.

The Nramp (Slc11) proteins regulate development, resistance to pathogenic bacteria and iron homeostasis in Dictyostelium discoideum.

The Dictyostelium discoideum genome harbors two genes encoding members of the Nramp superfamily, which is conserved from bacteria (MntH proteins) to humans (Slc11 proteins). Nramps are proton-driven metal ion transporters with a preference for iron and manganese. Acquisition of these metal cations is vital for all cells, as they act as redox cofactors and regulate key cellular processes, such as DNA synthesis, electron transport, energy metabolism and oxidative stress. Dictyostelium Nramp1 (Slc11a1), like its mammalian ortholog, mediates resistance to infection by invasive bacteria. We have extended the analysis to the nramp2 gene, by generating single and double nramp1/nramp2 knockout mutants and cells expressing GFP fusion proteins. In contrast to Nramp1, which is recruited to phagosomes and macropinosomes, the Nramp2 protein is localized exclusively in the membrane of the contractile vacuole, a vesicular tubular network regulating cellular osmolarity. Both proteins colocalize with the V-H(+)-ATPase, which can provide the electrogenic force for vectorial transport. Like nramp1, nramp2 gene disruption affects resistance to Legionella pneumophila. Disrupting both genes additionally leads to defects in development, with strong delay in cell aggregation, formation of large streams and multi-tipped aggregates. Single and double mutants display differential sensitivity to cell growth under conditions of iron overload or depletion. The data favor the hypothesis that Nramp1 and Nramp2, under control of the V-H(+)-ATPase, synergistically regulate iron homeostasis, with the contractile vacuole possibly acting as a store for metal cations.

Dictyostelium host response to legionella infection: strategies and assays.

The professional phagocyte Dictyostelium discoideum is a simple eukaryotic microorganism, whose natural habitat is deciduous forest soil and decaying leaves, where the amoebae feed on bacteria and grow as separate, independent, single cells. In the last decade, the organism has been successfully used as a host for several human pathogens, including Legionella pneumophila, Mycobacterium avium and Mycobacterium marinum,Pseudomonas aeruginosa, Klebsiella pneumoniae, Cryptococcus neoformans, and Salmonella typhimurium. To dissect the complex cross-talk between host and pathogen Dictyostelium offers easy cultivation, a high quality genome sequence and excellent molecular genetic and biochemical tools. Dictyostelium cells are also extremely suitable for cell biological studies, which in combination with in vivo expression of fluorescence-tagged proteins allow investigating the dynamics of bacterial uptake and infection. Inactivation of genes by homologous recombination as well as gene rescue and overexpression are well established and a large mutant collection is available at the Dictyostelium stock center, favoring identification of host resistance or susceptibility genes. Here, we briefly introduce the organism, address the value of Dictyostelium as model host, describe strategies to identify host cell factors important for infection followed by protocols for cell culture and storage, uptake and infection, and confocal microscopy of infected cells.

Legionella pneumophila infection is enhanced in a RacH-null mutant of Dictyostelium.

Recently we reported that Dictyostelium cells ingest Legionella pneumophila by macropinocytosis, whereas other bacteria, such as Escherichia coli, Mycobacterium avium, Neisseria meningitidis or Salmonella typhimurium, are taken up by phagocytosis.1 In contrast to phagocytosis, macropinocytosis is partially inhibited by PI3K or PTEN inactivation, whereas both processes are sensitive to PLC inhibition. Independently from reduced uptake, L. pneumophila proliferates more efficiently in PI3K-null than in wild-type cells. PI3K inactivation also neutralizes resistance to infection conferred by constitutively expressing the endo-lysosomal iron transporter Nramp1. We have shown this to be due to altered recruitment of the V-H(+) ATPase, but not Nramp1, in the Legionella-containing vacuole (LCV) early during infection.1 As further evidence for impaired LCV acidification we examine here the effects of disrupting the small G protein RacH on Legionella infection.

Phosphoinositides differentially regulate bacterial uptake and Nramp1-induced resistance to Legionella infection in Dictyostelium.

Membrane phosphatidylinositides recruit cytosolic proteins to regulate phagocytosis, macropinocytosis and endolysosomal vesicle maturation. Here, we describe effects of inactivation of PI3K, PTEN or PLC on Escherichia coli and Legionella pneumophila uptake by the professional phagocyte Dictyostelium discoideum. We show that L. pneumophila is engulfed by macropinocytosis, a process that is partially sensitive to PI3K inactivation, unlike phagocytosis of E. coli. Both processes are blocked by PLC inhibition. Whereas E. coli is rapidly digested, Legionella proliferates intracellularly. Proliferation is blocked by constitutively expressing Nramp1, an endolysosomal iron transporter that confers resistance against invasive bacteria. Inactivation of PI3K, but not PTEN or PLC, enhances Legionella infection and suppresses the protective effect of Nramp1 overexpression. PI3K activity is restricted to early infection and is not mediated by effects on the actin cytoskeleton; rather L. pneumophila, in contrast to E. coli, subverts phosphoinositide-sensitive fusion of Legionella-containing macropinosomes with acidic vesicles, without affecting Nramp1 recruitment. A model is presented to explain how Legionella escapes fusion with acidic vesicles and Nramp1-induced resistance to pathogens.

Dictyostelium discoideum as a model host for meningococcal pathogenesis.

BACKGROUND: The aim of the present study was to evaluate the possibility of studying meningococcal virulence in a new model organism, Dictyostelium discoideum, a haploid social soil amoeba that is an established host model for several human pathogens, leading to the discovery of novel virulence mechanisms. MATERIAL/METHODS: A number of virulent and hyper-virulent N. meningitidis strains, including isogenic encapsulated, unencapsulated, and lipooligosaccharide (LOS) outer core-defective derivatives, were used to test the ability of D. discoideum to internalize and grow in the presence of bacteria. Intracellular survival of the internalized bacteria was also monitored. RESULTS: Meningococci were internalized and killed by D. discoideum cells. The presence of a capsule did not affect the internalization, but, as in human cells, it increased the resistance of the internalized bacteria. Although both encapsulated and unencapsulated meningococci supported the growth and development of D. discoideum on an agar surface, in liquid medium the encapsulated strains were toxic to the slime mould cells. Toxicity inversely correlated with meningococcal survival in the assay medium that was not favorable to bacterial replication, suggesting that it may be due to some toxic compound released after bacterial autolysis. Intriguingly, unencapsulated isogenic strains efficiently supported Dictyostelium growth in suspension, opening the possibility that the toxicity may be associated with the capsular polysaccharide. CONCLUSIONS: These results suggest that several meningococcal virulence determinants, such as the capsular polysaccharide, may be remarkably effective also in Dictyostelium cells, stimulating the use of this model host to search for novel meningococcal virulence determinants.

Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium.

BACKGROUND: Phagocytosis plays a major role in the defense of higher organisms against microbial infection and provides also the basis for antigen processing in the immune response. Cells of the model organism Dictyostelium are professional phagocytes that exploit phagocytosis of bacteria as the preferred way to ingest food, besides killing pathogens. We have investigated Dictyostelium differential gene expression during phagocytosis of non-pathogenic bacteria, using DNA microarrays, in order to identify molecular functions and novel genes involved in phagocytosis. RESULTS: The gene expression profiles of cells incubated for a brief time with bacteria were compared with cells either incubated in axenic medium or growing on bacteria. Transcriptional changes during exponential growth in axenic medium or on bacteria were also compared. We recognized 443 and 59 genes that are differentially regulated by phagocytosis or by the different growth conditions (growth on bacteria vs. axenic medium), respectively, and 102 genes regulated by both processes. Roughly one third of the genes are up-regulated compared to macropinocytosis and axenic growth. Functional annotation of differentially regulated genes with different tools revealed that phagocytosis induces profound changes in carbohydrate, amino acid and lipid metabolism, and in cytoskeletal components. Genes regulating translation and mitochondrial biogenesis are mostly up-regulated. Genes involved in sterol biosynthesis are selectively up-regulated, suggesting a shift in membrane lipid composition linked to phagocytosis. Very few changes were detected in genes required for vesicle fission/fusion, indicating that the intracellular traffic machinery is mostly in common between phagocytosis and macropinocytosis. A few putative receptors, including GPCR family 3 proteins, scaffolding and adhesion proteins, components of signal transduction and transcription factors have been identified, which could be part of a signalling complex regulating phagocytosis and adaptational downstream responses. CONCLUSION: The results highlight differences between phagocytosis and macropinocytosis, and provide the basis for targeted functional analysis of new candidate genes and for comparison studies with transcriptomes during infection with pathogenic bacteria.

Function and mechanism of action of Dictyostelium Nramp1 (Slc11a1) in bacterial infection.

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H(+)ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.

Temperature-sensitive inhibition of development in Dictyostelium due to a point mutation in the piaA gene.

The Dictyostelium mutant HSB1 is temperature-sensitive for development, undergoing aggregation and fruiting body formation at temperatures below 18 degrees C but not above. In vivo G protein-linked adenylyl cyclase activation is defective in HSB1, and the enzyme is not stimulated in vitro by GTPgammaS; stimulation is restored upon addition of wild-type cytosol. Transfection with the gene encoding the cytosolic regulator PIA rescued the mutant. We excluded the possibility that HSB1 cells fail to express PIA and show that the HSB1 piaA gene harbors a point mutation, resulting in the amino acid exchange G(917)D. Both wild-type and HSB1 cells were also transfected with the HSB1 piaA gene. The piaA(HSB1) gene product displayed a partial inhibitory effect on wild-type cell development. We hypothesize that PIA couples the heterotrimeric G protein to adenylyl cyclase via two binding sites, one of which is altered in a temperature-sensitive way by the HSB1 mutation. When overexpressed in the wild-type background, PIA(HSB1) competes with wild-type PIA via the nonmutated binding site, resulting in dominant-negative inhibition of development. Expression of GFP-fused PIA shows that PIA is homogeneously distributed in the cytoplasm of chemotactically moving cells.