From cell-cell adhesion and cellular oscillations to spectacular views inside the cell--50 years of research with Dictyostelium.
Explore the source record for details and available documents.
Actins↗
Biomedical subjects
Salvatore Bozzaro
Publications and source records attributed to Salvatore Bozzaro.
Cloning of Dictyostelium eIF6 (p27BBP) and mapping its nucle(ol)ar localization subdomains.
Eukaryotic translation initiation factor 6 (eIF6), also termed p27BBP, is an evolutionary conserved regulator of ribosomal function. The protein is involved in maturation and/or export from the nucleus of the 60S ribosomal subunit. Regulated binding to and release from the 60S subunit also regulates formation of 80S ribosomes, and thus translation. The protein is also found in hemidesmosomes of epithelial cells expressing beta4 integrin and is assumed to regulate cross-talk between beta4 integrin, intermediate filaments and ribosomes. In the present study we show that the Dictyostelium eIF6 (also called p27BBP) gene is expressed during growth, down-regulated during the first hours of starvation, and up-regulated again at the end of aggregation. Phagocytosis, and to a lesser extent pinocytic uptake of axenic medium, stimulate gene expression in starving cells. The eIF6 gene is present in single copy and its ablation is lethal. We utilized the green fluorescent protein (GFT) as fusion protein marker to investigate sequences responsible for eIF6 subcellular localization. The protein is found both in cytoplasm and nucleus, and is enriched in nucleoli. Deletion sequence analysis shows that nucle(ol)ar localization sequences are located within the N- and C-terminal subdomains of the protein.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Assaying cell-cell adhesion.
A major feature in Dictyostelium development is the transition from the unicellular to the multicellular stage, a process brought about by chemotaxis and cell-cell adhesion. Growth-phase cells are weakly cohesive, whereas aggregation-competent cells adhere strongly to each other. In addition, aggregating cells display an ethylenediamine tetraacetic acid (EDTA)-resistant form of adhesion, which is developmentally regulated. Measuring cell-cell adhesion can thus be a simple and convenient method by which to assess the developmental progression of cells, to characterize mutants, and to discriminate between development and functional defects in cell adhesion molecules or membrane-cytoskeletal interactions. A quantitative cell adhesion assay is obviously crucial for identifying novel cell adhesion factors.
Animals↗
A novel Dictyostelium RasGEF required for chemotaxis and development.
BACKGROUND: Ras proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM. RESULTS: RasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein. CONCLUSION: The data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.
Actins↗
Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton.
Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.
Actins↗
Guenther Gerisch and Dictyostelium, the microbial model for ameboid motility and multicellular morphogenesis.
Beginning in 1960 and continuing to this day, Guenther Gerisch's work on the social ameba Dictyostelium discoideum has helped to make it the model organism of choice for studies of cellular activities that depend upon the actomyosin cytoskeleton. Gerisch has brought insight and quantitative rigor to cell biology by developing novel assays and by applying advanced genetic, biochemical and microscopic techniques to topics as varied as cell-cell adhesion, chemotaxis, motility, endocytosis and cytokinesis.
Amoeba↗
Calnexin, calreticulin and cytoskeleton-associated proteins modulate uptake and growth of Legionella pneumophila in Dictyostelium discoideum.
The haploid amoeba Dictyostelium discoideum is a versatile host system for studying cellular aspects of Legionella pathogenicity. Previous studies have shown that the internalization of L. pneumophila leads to an endoplasmic reticulum (ER)-derived organelle that supports intracellular replication of the bacteria. In this study a roadmap of host-cell factors involved in this process was developed. Phagocytosis assays with specific cellular inhibitors and the effects of well defined host-cell mutants revealed that cytoplasmic calcium levels, cytoskeleton-associated proteins and the calcium-binding proteins of the ER, calreticulin and calnexin, specifically influence the uptake and intracellular growth of L. pneumophila. Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup of L. pneumophila-infected host cells. In contrast to the control experiment with Escherichia coli-containing phagosomes, both proteins decorated the replicative vacuole of L. pneumophila during the entire growth phase of the bacteria. The cumulative effects of cytosolic calcium levels, the spatial distribution of calnexin and calreticulin, and the defective invasion and replication of L. pneumophila in calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole of L. pneumophila.
Animals↗
Single-gene greenbeard effects in the social amoeba Dictyostelium discoideum.
Selection can favor reproductive altruism if an altruism allele aids copies of itself by helping relatives. The alternative "greenbeard" mechanism, in which an allele directly recognizes and aids copies of itself in others, is generally thought to be too complex for a single gene to carry out. The csA gene in Dictyostelium discoideum acts as a single-gene greenbeard. When wild-type cells are mixed with csA-knockout cells, the wild type is more altruistic, but is also able preferentially to direct the benefits to other wild-type cells. Both properties derive directly from homophilic cell adhesion of the protein encoded by csA.
Alleles↗
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.
Animals↗