Advantages

Dictyostelium discoideum offers unique and attractive features in addition to its powerful molecular genetics

Time-lapse images of living cells. One of the incredible strengths of this model system is the capacity to track the dynamic behaviors of individual cells. For example, the normal cytokinesis displayed by wild-type cells and the aborted cytokinesis displayed by mutant cells can be filmed in suspension. There is a considerable collection of mutants displaying similar defects in cytokinesis in suspension. The optical clarity displayed by the cells facilitates digitized three-dimensional imaging. Studies of the chemotactic movements of wild-type cells have revealed features of normal motility such as the tendency of pseudopods to be initially extended above the substratum and the limited contact that a moving cell maintains with the substrate. Digitized imaging has been used to compare the movements of a wild-type and  mutant cells. In the example presented here, it is clear that the mutant moves poorly because it cannot suppress pseudopod formation in the rear end of the cell.

The capacity for tracking living cells and phenotypic complementation of null mutants with GFP fusion proteins is providing extremely useful tools for cell biology. One of the significant conclusions of these studies has been the demonstration that cytokinesis, motility, and phagocytosis share features and molecular components. Phenotypic rescue provides assurance that the GFP fusion protein is functional and the behavior of the protein can be followed in living cells under a variety of conditions. Some of the most interesting observations made in these experiments have been the rapid assembly of cytoskeletal proteins in the tips of newly extended pseudopods. For example, coronin, actin, talin, and a variety of other cytoskeletal proteins concentrate in the cortex of nascent pseudopods. Many of these proteins have also been tracked during cytokinesis and phagocytosis and found to translocate to the rims of phagocytic cups and to the distal edges of dividing cells.

Signal transduction proteins have also been tracked to discover how cells sense spatial gradients. While actin and actin binding proteins accumulate in the cortex of new pseudopods at the cell's leading edge, surface receptors and G-protein subunits remain uniformly distributed around the cell perimeter. Thus, the key decisions for directional sensing must occur at intermediary steps. One of these steps appears to be a rapid and transient appearance of binding sites for PH domains on the inner face of the membrane elicited by increases in receptor occupancy. In gradients of chemoattractant these sites are persistently present on the side of the cell facing the higher concentration. The local formation of these sites is independent of the actin cytoskeleton and may be an early event in directional sensing.

Elegant studies of dynamics of groups of cells are being produced in D. discoideum. Although the multicellular stages are unusual, studies have provided interesting information about the interactions of large groups of cells. The spontaneous aggregation of hundreds of thousands of cells occurs in a highly coordinated manner. The chemotactic movements of the cells are organized by periodic waves of cAMP that propagate through the cell monolayer. The waves are the result of a regulated production and secretion of extracellular cAMP and a spontaneous biological oscillator that initiates the waves at centers of territories. The periodic stimuli are critical for proper timing of developmental gene expression and they control the morphogenetic movements  in three-dimensional structures of the multicellular stages. The differentiated cells provide a fantastic system for studies of chemotactic cell sorting. Mixed cells will form chimeric organisms and individual fluorescently labeled cells can be tracked. Prestalk cells sort to the anterior region of the structure while prespore cells sort to the posterior and various mutants sort to specific locations.

Accessible phenotypes and biochemistry. The simplicity of the life cycle facilitates mutant selection. The growth and developmental stages are completely independent, and switching between the two states is achieved by removing nutrients. Many mutations can be screened by clonally plating cells on bacterial lawns. As the amoebae grow, they ingest the bacteria and form a plaque. The cells within the plaque starve and enter the developmental program. Aberrant phenotypes can be scored by visual inspection of the plaques. Since the early stages of development are readily reversible, mutants can be selected and then propagated by returning them to nutrients. Strains are preserved in liquid nitrogen and can be recovered by scraping some frozen cells directly onto a lawn of bacteria or into axenic medium. Spores remain viable on silica gel at -20° C for 5-10 years and for longer when lyophilized. The Nomenclature Guidelines describe how to name strains and alleles.

Developmental and cell-type gene expression and differentiation have been extensively characterized. The timing and conditions that control the expression of numerous genes are known. Specific regimens of application of extracellular cAMP and differentiation inducing factor (DIF) regulate gene expression in predictable ways. Gene expression has been characterized in a large collection of gene deletion strains under a variety of conditions. This rich repertoire of conditions and extracellular stimuli that control gene induction can now be rationally applied to DNA arrays to gain a comprehensive correlation of gene expression patterns to cellular phenotype.

The amoebae are easy to grow, lyse, and process for a multitude of biochemical assays or subcellular fractionations, as decribed in the General Dictyostelium Techniques page. The amoebae grow on bacterial lawns or in liquid cultures of defined media with doubling times of 4 and 12 hours, respectively. Over 10¹¹ clonal D. discoideum amoebae can be grown and harvested in a few days without sophisticated equipment. A small-scale industrial facility could increase this number to 5 x 10¹² identical cells (5 kilograms) per week. The cells can be harvested from growth or any of the developmental stages. In the early stages of development, the genetically identical cells differentiate synchronously and the population remains homogeneous. This allows for biochemical analyses to be performed using a variety of physiologically relevant conditions. The high levels of exogenous protein expression obtained in transformed cells makes this system suitable for protein purification.