Phg2, a kinase involved in adhesion and focal site modeling in Dictyostelium

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.     

Synergistic control of cellular adhesion by transmembrane 9 proteins

The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Herein, the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in D. discoideum. The phenotypes of PHG1a, PHG1b, and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth, and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Together, these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface.     

SadA,a novel adhesion receptor in Dictyostelium

Little is known about cell-substrate adhesion and how motile and adhesive forces work together in moving cells. The ability to rapidly screen a large number of insertional mutants prompted us to perform a genetic screen in Dictyostelium to isolate adhesion-deficient mutants. The resulting substrate adhesion-deficient (sad) mutants grew in plastic dishes without attaching to the substrate. The cells were often larger than their wild-type parents and displayed a rough surface with many apparent blebs. One of these mutants, sadA-, completely lacked substrate adhesion in growth medium. The sadA- mutant also showed slightly impaired cytokinesis, an aberrant F-actin organization, and a phagocytosis defect. Deletion of the sadA gene by homologous recombination recreated the original mutant phenotype. Expression of sadA-GFP in sadA-null cells restored the wild-type phenotype. In sadA-GFP-rescued mutant cells, sadA-GFP localized to the cell surface, appropriate for an adhesion molecule. SadA contains nine putative transmembrane domains and three conserved EGF-like repeats in a predicted extracellular domain. The EGF repeats are similar to corresponding regions in proteins known to be involved in adhesion, such as tenascins and integrins. Our data combined suggest that sadA is the first substrate adhesion receptor to be identified in Dictyostelium.     

Dictyostelium discoideum mutants with conditional defects in phagocytosis

We have isolated and characterized Dictyostelium discoideum mutants with conditional defects in phagocytosis. Under suspension conditions, the mutants exhibited dramatic reductions in the uptake of bacteria and polystyrene latex beads. The initial binding of these ligands was unaffected, however, indicating that the defect was not in a plasma membrane receptor: Because of the phagocytosis defect, the mutants were unable to grow when cultured in suspensions of heat-killed bacteria. The mutants exhibited normal capacities for fluid phase endocytosis and grew as rapidly as parental (AX4) cells in axenic medium. Both the defects in phagocytosis and growth on bacteria were corrected when the mutant Dictyostelium cells were cultured on solid substrates. Reversion and genetic complementation analysis suggested that the mutant phenotypes were caused by single gene defects. While the precise site of action of the mutations was not established, the mutations are likely to affect an early signaling event because the binding of bacteria to mutant cells in suspension was unable to trigger the localized polymerization of actin filaments required for ingestion; other aspects of actin function appeared normal. This class of conditional phagocytosis mutant should prove to be useful for the expression cloning of the affected gene(s).     

Specific host genes required for the killing of Klebsiella bacteria by phagocytes

The amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes (PHG1 and KIL1) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae. Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.     

Mediation of cell-substratum adhesion by RasG in Dictyostelium

Previous studies on the functions of the RasG gene in the cellular slime mold, Dictyostelium discoideum, have revealed that it is required for normal motility and cytokinesis. To further understand how the RasG gene regulates various cellular processes, we transformed an activated form of RasG, that is, RasG (G12T), a mutation from glycine to threonine at amino acid position 12 into wild type KAX-3 cells. This produced moderate but constitutive RasG(G12T) protein expression, which causes cells to become significantly more adherent to the substratum than are wild type cells. The RasG(G12T) transformants also grow slowly on bacterial plates, and engulf fewer bacteria on filter surfaces, indicating a defect in phagocytosis when cells are adhered. The expression of the activated RasG also dramatically reduces the number of filopodia on the cell surface. Tyrosine phosphorylation on a 43 kDa protein (most likely actin) of the RasG (G12T) transformants is highly elevated. Taken together, our observations suggest that RasG is crucial for Dictyostelium cell-substratum adhesion during growth and that RasG may play a role in adhesion-mediated phagocytosis. Our results also suggest that RasG is important in filopodial formation and that RasG is involved in the signal pathway that is regulated by tyrosine phosphorylation.     

An adhesion molecule in free-living Dictyostelium amoebae with integrin beta features

The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin beta chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin beta chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.     

Cell-cell signaling and adhesion in phagocytosis and early development of Dictyostelium

Cell-cell signaling and adhesion regulate transition from the unicellular to the multicellular stage of development in the cellular slime mold Dictyostelium. Essential gene networks involved in these processes have been identified and their interplay dissected. Heterotrimeric G protein-linked signal transduction plays a key role in regulating expression of genes mediating chemotaxis or cell adhesion, as well as coordinating actin-based cell motility during phagocytosis and chemotaxis. Two classes of cell adhesion molecules, one cadherin-like and the second belonging to the IgG superfamily, contribute to the strength of adhesion in Dictyostelium aggregates. The developmental role of genes involved in motility and adhesion, and their degree of redundancy, have been re-assessed by using novel developmental assay conditions which are closer to development in nature.     

Post-translational glycosylation of the contact site A protein of Dictyostelium discoideum is important for stability but not for its function in cell adhesion

The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-surface-exposed csA glycoprotein against proteolytic cleavage. HG220 cells were still capable of forming EDTA-stable contacts to a reduced extent, consistent with the low amounts of the 68-kd glycoprotein present on their surfaces. Thus type 1 rather than type 2 carbohydrate appears to be directly involved in intercellular adhesion that is mediated by the csA glycoprotein. Tunicamycin-treated wild-type and mutant cells produce a 53-kd protein that lacks both type 1 and 2 carbohydrates. While this protein is stable and not transported to the cell surface in the wild type, it is cleaved in the mutants and fragments of it are released into the extracellular medium. These results suggest that the primary defect in the two mutants studied is relief from a restriction in protein transport to the cell surface, and that the defect in type 2 glycosylation is secondary.     

The isoform B of the Dictyostelium long-chain fatty-acyl-coenzyme A synthetase is initially inserted into the ER and subsequently provides peroxisomes with an activity important for efficient phagocytosis

Long-chain fatty-acyl-coenzyme A synthetases activate fatty acids for anabolic or catabolic metabolism. They often localize to more than one organelle within eukaryotic cells. Dictyostelium contains two of these proteins, FcsA and FcsB with the latter being targeted to the membrane of the endoplasmic reticulum by virtue of an N-terminal signal sequence and from there appears to move on to peroxisomes. Deletion of this signal favors the peripheral association of the protein with the mitochondrial surface instead. A strain lacking the activity of the FcsB enzyme was constructed by homologous recombination. It has a severe deficiency in the phagocytic uptake of particles, which can be partially alleviated by a peroxisomally targeted, soluble FcsA enzyme. It is, however, not rescued by expressing FcsA in the cytoplasm or targeting it to the ER, indicating that peroxisomal β-oxidation is important for phagocytosis. In a fcsA(-)/B(-) double mutant phagocytosis efficiency is similar to fcsB(-) cells. However, unlike the single mutants, the fcsA(-)/B(-) strain is delayed in morphogenesis, but forms viable spores, albeit within a small fruiting body. This developmental defect is also seen in other mutants affecting peroxisomal enzymes involved in β-oxidation and the glyoxylate cycle.     

The role of unconventional myosins in Dictyostelium endocytosis

Dictyostelium discoideum is a simple eukaryote amenable to detailed molecular studies of the endocytic processes phagocytosis and macropinocytosis. Both the actin cytoskeleton and associated myosin motors are well-described and a range of mutants are now available that enable characterization of the role of the cytoskeleton in a range of cellular functions. Molecular genetic studies have uncovered roles for two different classes of Dictyostelium unconventional myosins in endocytosis. The class I myosins contribute to both macropinocytosis and phagocytosis by playing a general role in controlling actin-dependent manipulations of the actin-rich cortex. A class VII myosin has been shown to be important for phagocytosis. This brief review summarizes what is known about the role of these different myosins in both fluid and particle uptake in this system.     

Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis.

Calreticulin and calnexin are Ca2+-binding proteins with chaperone activity in the endoplasmic reticulum. These proteins have been eliminated by gene replacement in Dictyostelium, the only microorganism known to harbor both proteins; family members in Dictyostelium are located at the base of phylogenetic trees. A dramatic decline in the rate of phagocytosis was observed in double mutants lacking calreticulin and calnexin, whereas only mild changes occurred in single mutants. Dictyostelium cells are professional phagocytes, capable of internalizing particles by a sequence of activities: adhesion of the particle to the cell surface, actin-dependent outgrowth of a phagocytic cup, and separation of the phagosome from the plasma membrane. In the double-null mutants, particles still adhered to the cell surface, but the outgrowth of phagocytic cups was compromised. Green fluorescent protein-tagged calreticulin and calnexin, expressed in wild-type cells, revealed a direct link of the endoplasmic reticulum to the phagocytic cup enclosing a particle, such that the Ca2+ storage capacity of calreticulin and calnexin might directly modulate activities of the actin system during particle uptake.     

Dictyostelium LvsB mutants model the lysosomal defects associated with Chediak-Higashi syndrome

Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans ("lysosomal trafficking regulator") or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideum contains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal alpha-mannosidase were normal, although lvsB mutants inefficiently retained alpha-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis.     

α-Mannosidase-1 mutants of Dictyostelium dkoideum: Early aggregation-essential genes regulate enzyme precursor synthesis, modification, and processing

The lysosomal enzyme alpha-mannosidase-1 is one of the earliest developmentally controlled gene products in Dictyostelium discoideum. Although this enzyme is synthesized throughout the first 20 h of development, it is not required for complete morphogenesis, since structural gene (manA) mutants lacking activity develop normally. We isolated six strains deficient in alpha-mannosidase-1 activity which, unlike structural gene mutants, fail to aggregate. Fruiting revertants of these strains accumulate wild-type levels of alpha-mannosidase-1 activity, suggesting that both the enzymatic and morphological defects are caused by single mutations in nonstructural genes essential for early development. Direct genetic evidence for mutations outside of the structural gene was obtained by complementation analysis. We used alpha-mannosidase-1-specific monoclonal antibodies to analyze the biochemical defects in these mad (alpha-mannosidase-1-deficient) mutants. All mad mutants show a significantly reduced relative rate of enzyme precursor biosynthesis. The mad-404 mutation results in a complete lack of precursor biosynthesis, as well as a lack of functional alpha-mannosidase-1 mRNA. In some cases, however, the enzymatic defect results from improper post-translational modification which affects precursor processing. We conclude that a small number of aggregation-essential genes are involved in regulating the synthesis, modification, and processing of alpha-mannosidase-1 during development.     

The coronin family of actin-associated proteins.

Coronin was first isolated from Dictyostelium, but similar proteins have been identified in many species and individual cell types. The coronin-like protein in yeast promotes actin polymerization and also interacts with microtubules. Dictyostelium mutants lacking coronin are impaired in cytokinesis and all actin-mediated processes. Analysis of coronin-GFP (green-fluorescent protein) fusions and knockout mutants shows that coronin participates in the remodelling of the cortical actin cytoskeleton that is responsible for phagocytosis and macropinocytosis. Likewise, in mammalian neutrophils, a coronin-like protein is also associated with the phagocytic apparatus. The diversity of function in this family of actin-associated proteins is just beginning to be explored.     

The identification of pats1, a novel gene locus required for cytokinesis in Dictyostelium discoideum

Here, we describe the identification and characterization of the cytokinesis-deficient mutant cell line 17HG5, which was generated in a restriction enzyme-mediated integration mutagenesis screen designed to isolate genes required for cytokinesis in Dictyostelium discoideum. Phenotypic characterization of the 17HG5 cell line revealed no apparent defects in the global functionality of the actomyosin cytoskeleton except for the observed cytokinesis defect when grown in suspension culture. Plasmid rescue was used to identify the disrupted gene locus (pats1; protein associated with the transduction of signal 1). that caused the cytokinesis defect. Disruption of the pats1 locus was recreated through homologous recombination in several independent cell lines, each recapitulating the cytokinesis-defective phenotype and thereby confirming that this gene locus is important for proper cytokinesis. Sequence data obtained by analysis of the genomic region flanking the inserted restriction enzyme-mediated integration plasmid revealed an 8892-bp genomic open reading frame encoding a 2964-amino-acid protein. The putative pats1 protein contains 3 regulatory domains (RI-phosphatase, RII-GTP-binding, R-III protein kinase), 13 leucine-rich repeats, and 8 WD-40 repeats. These regulatory domains coupled with the protein-protein interacting domains suggest that pats1 is involved in signal transduction during cytokinesis in Dictyostelium.     

A novel member of the rho family of small GTP-binding proteins is specifically required for cytokinesis

Several members of the rho/rac family of small GTP-binding proteins are known to regulate the distribution of the actin cytoskeleton in various subcellular processes. We describe here a novel rac protein, racE, which is specifically required for cytokinesis, an actomyosin-mediated process. The racE gene was isolated in a molecular genetic screen devised to isolate genes required for cytokinesis in Dictyostelium. Phenotypic characterization of racE mutants revealed that racE is not essential for any other cell motility event, including phagocytosis, chemotaxis, capping, or development. Our data provide the first genetic evidence for the essential requirement of a rho-like protein, specifically in cytokinesis, and suggest a role for these proteins in coordinating cytokinesis with the mitotic events of the cell cycle.     

Redundancy in the microfilament system: abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins.

We generated by gene disruption Dictyostelium cells that lacked both the F-actin cross-linking proteins, alpha-actinin and gelation factor. Several major cell functions, such as growth, chemotaxis, phagocytosis, and pinocytosis, were apparently unaltered. However, in all double mutants, development was greatly impaired. After formation of aggregates, cells were very rarely able to form fruiting bodies. This ability was rescued when mutant and wild-type strains were mixed in a ratio of 70 to 30. The developmental program in the mutant was not arrested, since the expression pattern of early and late genes remained unchanged. Development of the mutant was rendered normal when a functional alpha-actinin gene was introduced and expressed, showing the morphogenetic defect to be due to the absence of the two F-actin cross-linking proteins. These findings suggest the existence of a functional network allowing mutual complementation of certain actin-binding proteins.     

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.     

Dictyostelium macroautophagy mutants vary in the severity of their developmental defects

Macroautophagy is the major mechanism that eukaryotes use to recycle cellular components during stressful conditions. We have shown previously that the Atg12-Atg5 conjugation system, required for autophagosome formation in yeast, is necessary for Dictyostelium development. A second conjugation reaction, Aut7/Atg8 lipidation with phosphatidylethanolamine, as well as a protein kinase complex and a phosphatidylinositol 3-kinase complex are also required for macroautophagy in yeast. In this study, we characterize mutations in the putative Dictyostelium discoideum orthologues of budding yeast genes that are involved in one of each of these functions, ATG1, ATG6, and ATG8. All three genes are required for macroautophagy in Dictyostelium. Mutant amoebae display reduced survival during nitrogen starvation and reduced protein degradation during development. Mutations in the three genes produce aberrant development with defects of varying severity. As with other Dictyostelium macroautophagy mutants, development of atg1-1, atg6(-), and atg8(-) is more aberrant in plaques on bacterial lawns than on nitrocellulose filters. The most severe defect is observed in the atg1-1 mutant, which does not aggregate on bacterial lawns and arrests as loose mounds on nitrocellulose filters. The atg6(-) and atg8(-) mutants display almost normal development on nitrocellulose filters, producing multi-tipped aggregates that mature into small fruiting bodies. The distribution of a green fluorescent protein fusion of the autophagosome marker, Atg8, is aberrant in both atg1-1 and atg6(-) mutants.