Aggregation in Dictyostelium discoideum

Cultured peritoneal macrophages have previously been shown to release a potent mitogen for mesenchymal cells. Peritoneal macrophages are derived from peripheral blood monocytes, one of the principal inflammatory cells associated with numerous tissue responses to injury. Cultured human monocytes can be activated by endotoxin or concanavalin A to secrete a potent growth factor(s) that is active on human smooth muscle cells, human fibroblasts and 3T3 cells. The optimal conditions for activation of monocyte release of this monocyte-derived growth factor(s) (MDGF) were to expose 5-day-old monocyte cultures (initially plated at 6.8 × 10⁵ cells/ml medium) to 10 μg/ml endotoxin or 6 μg/ml concanavalin A for approximately 20 hr. Monocytes can secrete MDGF into serum-free medium supplemented with 0.15% bovine serum albumin. MDGF stimulates both DNA synthesis and increase in cell number and is trypsin-sensitive, heat labile and nondialyzable. The relationship of MDGF to other monocyte products and its potential importance in wound repair and atherogenesis are discussed.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Aggregation during sexual development in Dictyostelium discoideum.

A microcinematographic analysis of the behaviour and movements of cells and cell masses in mated cultures (NC4 X VI2) of Dictyostelium discoideum indicates that a chemotactic process directs cell aggregation during macrocyst development. Zygote giant cells form before aggregation begins and act as the aggregation centres. Young multicellular macrocyst stages are sources of cyclic AMP, and amoebae from macrocyst cultures orient chemotactically to cyclic AMP. The data, coupled with other characteristics such as pulsatile streaming, suggest that the aggregation process leading to macrycyst development is the same as that occurring during fruit construction. Other aspects of sexual development are also discussed. Based upon these data, we propose a model for the sequence of events leading to macrocyst development in D. discoideum.     

Complexity of nuclear and polysomal RNAs in growing Dictyostelium discoideum cells

The proliferative activity of undifferentiated brain cells from either 5- or 7-day-old chick embryos has been investigated by labeling the cells with a 24-hr pulse label of [¹⁴C]- or [³H]-thymidine during the early stages (0 to 8 days) of culture. As soon as the neurons and the glial cells could be distinguished (after 4, 7, or 14 days of culture), the cultures were prepared and submitted to the activated autoradiographic method. In some experiments a continuous labeling was applied up to 2 weeks. During the first 48 hr of culture, and for both embryonic ages studied, nearly all neuronal precursors were able to proliferate. After 4 days in culture for the 7-day-old embryo and 7 days in culture for the 5-day-old embryo most of the neuronal cells stopped dividing. These two culture periods correspond to the stage of the embryonic life when the end of the mitotic activity of neuroblasts occurs in vivo. Thus, the proliferation and development in culture of most neuroblasts was found to parallel the in vivo evolution of these cells. Some neuroblasts, however, continued to multiply in vitro for a longer period of time. The astroblasts precursors were found to multiply actively from the 3rd day on, or immediately from time zero, for the 5- and 7-day-old chick embryos, respectively. These observations seem to indicate that the astroblast precursors are in a latent stage until they have reached Day 7. Thereafter, they proliferate actively during the first week of culture and therefore remain in an embryonic stage during this culture period. This fact corresponds also to the in vivo situation, where the glial cell precursors multiply actively around the same time period.     

The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation

The expression, misfolding, and aggregation of long repetitive amino acid tracts are a major contributing factor in a number of neurodegenerative diseases, including C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia, fragile X tremor ataxia syndrome, myotonic dystrophy type 1, spinocerebellar ataxia type 8, and the nine polyglutamine diseases. Protein aggregation is a hallmark of each of these diseases. In model organisms, including yeast, worms, flies, mice, rats, and human cells, expression of proteins with the long repetitive amino acid tracts associated with these diseases recapitulates the protein aggregation that occurs in human disease. Here we show that the model organism Dictyostelium discoideum has evolved to normally encode long polyglutamine tracts and express these proteins in a soluble form. We also show that Dictyostelium has the capacity to suppress aggregation of a polyglutamine-expanded Huntingtin construct that aggregates in other model organisms tested. Together, these data identify Dictyostelium as a novel model organism with the capacity to suppress aggregation of proteins with long polyglutamine tracts.     

Aggregation of Respiratory Complex Subunits Marks the Onset of Proteotoxicity in Proteasome Inhibited Cells

Proteostasis is maintained by optimal expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers protein aggregation and is implicated in many age-related pathologies. In this study, using quantitative proteomics and microscopy, we show that aggregation of many nuclear-encoded mitochondrial proteins is an early protein destabilization event during short-term proteasome inhibition. Among these, respiratory chain complex (RCC) subunits represent a group of functionally related proteins consistently forming aggregates under multiple proteostasis stresses with varying aggregation propensities. Sequence analysis reveals that several RCC subunits, irrespective of the cleavable mitochondrial targeting sequence, contain low-complexity regions at the N-terminus. Using different chimeric and mutant constructs, we show that these low-complexity regions partially contribute to the intrinsic instability of multiple RCC subunits. Taken together, we propose that physicochemically driven aggregation of unassembled RCC subunits destabilizes their functional assembly inside mitochondria. This eventually deregulates the biogenesis of respiratory complexes and marks the onset of mitochondrial dysfunction.     

Aggregation and inhibition of rat intestinal alkaline phosphatase by high concentrations of calcium. Reversibility of the processes

Intestinal alkaline phosphatase (IAP) is an enzyme of the brush border of the enterocyte. The activity of IAP biphasically depends on calcium. Although calcium increases IAP activity, when calcium is higher than 20 mmole/L, IAP activity decreases and the amount of an aggregated form increases. The reversibility of the effect of calcium and the aggregation process are unknown. The isoelectric point of the enzyme was higher in the presence of calcium, but was the same for the enzyme and the aggregated form. The treatment with EGTA after calcium addition did not restore the enzymatic activity but produced desaggregation of the enzyme and increase in the monomeric subunits of IAP. It is concluded that the binding of calcium does not produce important modifications on the structure of the protein, that the inhibitory effect is not reversible and that calcium could be involved in the stability of the structure of the enzyme.     

The search for morphogens in Dictyostelium

Classical embryological studies have led to the suggestion that cells in developing tissues may be directed to differentiate along a particular pathway by the concentrations of molecules called morphogens. Studies of the slime mould Dictyostelium discoideum, which has a simple tissue pattern consisting of only two cell types, have revealed several molecules which may act as morphogens. Cyclic AMP and ammonia promote the formation of spores, while adenosine and a novel class of compounds called DIFs promote the formation of stalk cells, the alternative cell fate. The constant proportions of the two differentiated cell types observed in this organism may result from a balance among the influences of these compounds.     

Aggregation and inhibition of rat intestinal alkaline phosphatase by high concentrations of calcium. Reversibility of the processes

Intestinal alkaline phosphatase (IAP) is an enzyme of the brush border of the enterocyte. The activity of IAP biphasically depends on calcium. Although calcium increases IAP activity, when calcium is higher than 20 mmole/L, IAP activity decreases and the amount of an aggregated form increases. The reversibility of the effect of calcium and the aggregation process are unknown. The isoelectric point of the enzyme was higher in the presence of calcium, but was the same for the enzyme and the aggregated form. The treatment with EGTA after calcium addition did not restore the enzymatic activity but produced desaggregation of the enzyme and increase in the monomeric subunits of IAP. It is concluded that the binding of calcium does not produce important modifications on the structure of the protein, that the inhibitory effect is not reversible and that calcium could be involved in the stability of the structure of the enzyme.     

The Dictyostelium model for mitochondrial disease

Mitochondrial diseases are a diverse family of genetic disorders caused by mutations affecting mitochondrial proteins encoded in either the nuclear or the mitochondrial genome. By impairing mitochondrial oxidative phosphorylation, they compromise cellular energy production and the downstream consequences in humans are a bewilderingly complex array of signs and symptoms that can affect any of the major organ systems in unpredictable combinations. This complexity and unpredictability has limited our understanding of the cytopathological consequences of mitochondrial dysfunction. By contrast, in Dictyostelium the mitochondrial disease phenotypes are consistent, measurable "readouts" of dysregulated intracellular signalling pathways. When the underlying genetic defects would produce coordinate, generalized deficiencies in multiple mitochondrial respiratory complexes, the disease phenotypes are mediated by chronic activation of an energy-sensing protein kinase, AMP-activated protein kinase (AMPK). This chronic AMPK hyperactivity maintains mitochondrial mass and cellular ATP concentrations at normal levels, but chronically impairs growth, cell cycle progression, multicellular development, photosensory and thermosensory signal transduction. It also causes the cells to support greater proliferation of the intracellular bacterial pathogen, Legionella pneumophila. Notably however, phagocytic and macropinocytic nutrient uptake are impervious both to AMPK signalling and to these types of mitochondrial dysfunction. Surprisingly, a Complex I-specific deficiency (midA knockout) not only causes the foregoing AMPK-mediated defects, but also produces a dramatic deficit in endocytic nutrient uptake accompanied by an additional secondary defect in growth. More restricted and specific phenotypic outcomes are produced by knocking out genes for nuclear-encoded mitochondrial proteins that are not required for respiration. The Dictyostelium model for mitochondrial disease has thus revealed consistent patterns of sublethal dysregulation of intracellular signalling pathways that are produced by different types of underlying mitochondrial dysfunction.     

The annexins of Dictyostelium

Annexins are a highly conserved ubiquitous family of Ca2+- and phospholipid-binding proteins present in nearly all eukaryotic cells. Analysis of the Dictyostelium genome revealed the presence of two annexin genes, the annexin C1 gene (nxnA) giving rise to two isoforms of 47 and 51 kDa (previously synexin), and the annexin C2 gene (nxnB) coding for a 56-kDa protein with 33% sequence identity to annexin C1. Annexin C2 is expressed at very low and constant levels throughout development. Quantification by real-time PCR indicated that it is present in about 35-fold lower amounts compared to annexin C1. We have used a GFP-tagged annexin C2 to study its cellular distribution and dynamics. In cell fractionation studies, annexin C2 cofractionates with annexin C1 and is enriched in the 100,000 g pellet. Like annexin C1, GFP-AnxC2 stains the plasma membrane. In addition it is present in the perinuclear region and overlaps to some degree with the Golgi apparatus, whereas annexin C1 is present on intracellular membranes resembling endosomal membranes and in the nucleus. Annexin C2 is not observed in the nucleus. An annexin C1 mutant (SYN-) which shows a defect during multicellular development can be rescued by full-length annexin C1, whereas overexpression of GFP-AnxC2 did not rescue the developmental defect The data support the concept that annexins, although having a highly conserved structure, participate in different functions in a cell.     

Aggregation of Dictyostelium discoideum is dependent on myristoylation and membrane localization of the G protein alpha-subunit, G alpha 2.

The heterotrimeric G protein, G2, from the eukaryotic organism Dictyostelium discoideum participates in signal transduction pathways which are essential to Dictyostelium's developmental life cycle. G2 is activated by cell surface cAMP receptors and in turn is required for the activation of a host of effectors, including adenylyl cyclase, guanylyl cyclase, and phospholipase C. Myristoylation of G protein alpha-subunits is known to affect alpha-subunit association with the beta gamma subunits and membrane localization. The putative site for N-terminal myristoylation of G alpha 2 was mutated from Gly to Ala (G2A) and expressed in the g alpha 2-null cell line, MYC2. Transformants expressing G alpha 2-G2A exhibit physiological and biochemical changes from wild-type cells. G alpha 2-G2A expressing cells fail to rescue the aggregation-minus phenotype of MYC2 cells on developmental agar plates. G alpha 2-G2A expressing cells are also not chemotactic to cAMP in a standard drop assay. G alpha 2-WT is found in both the pellet and supernatant fractions following lysis of the cells. G alpha 2-G2A however is found almost exclusively in the lysate supernatant. G alpha 2 is radiolabeled upon incubation of cells in [3H]myristate, while G alpha 2-G2A is not labeled. Examination of activation of the effectors adenylyl cyclase and guanylyl cyclase reveals that G alpha 2-G2A expressing cells partially activate adenylyl cyclase but show no cAMP-stimulation of guanylyl cyclase. The physiological deviations from wild-type can be explained by the variations in effector activation, possibly due to improper localization of the non-myristoylated G alpha 2-G2A to the cytosol.     

Identification of Darlin, a Dictyostelium Protein with Armadillo-like Repeats that Binds to Small GTPases and Is Important for the Proper Aggregation of Developing Cells

We purified from Dictyostelium lysates an 88-kDa protein that bound to a subset of small GTPases, including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88-kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis, or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly because of a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development.     

The regulation of myosin II in Dictyostelium

Dictyostelium conventional myosin (myosin II) is an abundant protein that plays a role in various cellular processes such as cytokinesis, cell protrusion and development. This review will focus on the signal transduction pathways that regulate myosin II during cell movement. Myosin II appears to have two modes of action in Dictyostelium: local stabilization of the cytoskeleton by myosin filament association to the actin meshwork (structural mode) and force generation by contraction of actin filaments (motor mode). Some processes, such as cell movement under restrictive environment, require only the structural mode of myosin. However, cytokinesis in suspension and uropod retraction depend on motor activity as well. Myosin II can self-assemble into bipolar filaments. The formation of these filaments is negatively regulated by heavy chain phosphorylation through the action of a set of novel alpha kinases and is relatively well understood. However, only recently it has become clear that the formation of bipolar filaments and their translocation to the cortex are separate events. Translocation depends on filamentous actin, and is regulated by a cGMP pathway and possibly also by the cAMP phosphodiesterase RegA and the p21-activated kinase PAKa. Myosin motor activity is regulated by phosphorylation of the regulatory light chain through myosin light chain kinase A. Unlike conventional light chain kinases, this enzyme is not regulated by calcium but is activated by cGMP-induced phosphorylation via an upstream kinase and subsequent autophosphorylation.     

Adolescence and the Reversibility of Maturity in Euplotes crassus

ABSTRACT. The clonal life history of ciliated protists is characterized by a sequence of phenotypes; sexual immaturity, maturity, and senescence. The distinctiveness of immaturity and maturity has been investigated. Standard assays of the onset of maturity of progeny clones from a cross between stocks EC1 and EC2 of Euplotes crassus demonstrated significant differences among clones and among testers within clones. They also revealed that the first positive test(s) of a progeny subclone were typically followed by at least one negative test. Special protocols were devised to investigate if maturity was reversible at the cellular level. In these experiments, the first mating pair of a progeny subclone was split before the consummation of mating. From these two cells as well as from control progeny and tester cells, subclones were established and every leftover cell was tested for maturity after each transfer. Both standard and split-pair progeny subclones had immature and slow- to-mate cells. The number of fissions before progeny exhibited sexual behavior indistinguishable from the testers was more than twice that to the first mating reaction of a subclone. At the first sign of maturity, progeny lines are a heterogeneous population of cells able and not able to mate, but remarkably, clonal descendants of those able to mate may become unable to mate. The development of maturity is progressive, quantitative and non-monotonic rather than an instantaneous switch.