Dictyostelium

 The cellular slime mold Dictyostelium discoideum has been studied in the laboratory extensively as a model eukaryotic organism. This soil amoeba has provided extraordinary opportunities for molecular biologists and biochemists to examine a wide variety of fundamental cellular functions. Developmental biologists have characterized at the molecular level Dictyostelium's unique developmental pathway. Single free-living cells can respond to a number of chemical signals, aggregate, and then differentiate into a complex multicellular organism.

A powerful library of techniques has been developed for manipulating this organism in the laboratory. Large numbers of cells can be grown, and proteins can be purified for biochemical characterization and, in some cases, structural characterization. The recent utilization of peptide proteinase inhibitors have made this increasingly easy. Genetic techniques have been developed for making mutations, and sophisticated methods have been applied for analyzing them. Genetic selection can be carried out with a variety of metabolic and drug resistance markers. Plasmids that either integrate into the chromosome or replicate extrachromosomally have been constructed from fragments of naturally occurring ones, and a variety of promoters are available for obtaining heterologous gene expression. Cell biological techniques are also well developed, yielding information on expression and localization in fixed and live cells.

A simplified schematic of the Dictyostelium discoideum developmental pathway is shown in Figure 1. The free-living amoebae feed on bacteria, and they can be found outside the laboratory in forest soils and detritus. In the laboratory, they can be grown in liquid media in shaking flasks or in petri plates, either in liquid media or on agar surfaces. Strains of Dictyostelium have been developed that can grow in synthetic and fully characterized media. On surfaces, the amoebae are motile and can use a variety of chemotaxis signals to find food and to optimize growth conditions (1).

Figure 1. A schematic diagram of the Dictyostelium developmental cycle.

Under certain conditions, such as the onset of starvation, a population of amoebae can suspend their cell cycle and begin a highly regulated set of gene inductions, which initiates the developmental pathway. In the laboratory, this can be done synchronously. Early in this process, a series of new enzymes are synthesized that are responsible for the synthesis, detection, and degradation of cyclic AMP (cAMP). These enzymes are used to regulate the chemotactic response of the cells in such a way that the cells aggregate. A founder cell initiates the aggregation process by secreting pulses of cAMP. Surrounding cells, if they are competent to respond, do so in two ways. First, they migrate toward the cAMP source. Second, they secrete a cAMP pulse themselves, which serves to amplify the signal and to increase the area over which it can travel. An extracellular phosphodiesterase degrades the cAMP between the waves.

As the cells migrate, they form cell–cell contacts that serve to establish polarity signals, which are used as the aggregate forms a mound and the cells differentiate into different types and sort into different locations. The tip of the mound becomes an organizing center which, based on environmental factors, decides whether to proceed in development to a fruiting body or to search for more favorable conditions by becoming a slug. If the organism becomes a slug, the mound tips over on the surface, and the outside cells secrete an extracellular matrix, commonly known as slime, which is left behind on the surface after the organism has passed. The tip of the slug controls its migration direction chemotactically, as well as by detecting gradients of temperature and light. Migration rates can exceed 1 cm/h.

When the mound or slug is ready to proceed to form a fruiting body, the cells have differentiated, through a pattern of differential gene expression, into either of two distinct cell types: pre-stalk or pre-spore. As the pre-stalk cells migrate to the bottom of the forming structure, they differentiate terminally into a plant-like cell with a rigid cell wall. A clorinated alkyl phenone, DIF-1, is released to induce stalk cell differentiation. Pre-spore cells differentiate into small environmentally resistant spores, which are carried up by the forming stalk. The spore sac is quite sticky, which facilitates its use of insect legs as transport in the wild. When conditions are optimal, the spores can germinate and begin dividing once again as amoebae.

The haploid genome of Dictyostelium is about 54 × 10⁶ base pairs in six chromosomes, and a detailed map has been assembled (2). The DNA has an unusually high content of A and T nucleotides. Complete characterization of the genome is underway. A wide variety of genetic tools have be developed for manipulation of Dictyostelium. Mutations can be created, and the resulting phenotypes can be analyzed in many ways. Naturally occurring extrachromosomal plasmids have been isolated and reconstructed into a family of vectors useful in stable or transient expression of heterologous genes. Multiple selectable markers are available, allowing the construction of strains in which complementation can be used to analyze protein–protein interactions. Homologous recombination has been shown to occur, allowing gene knockouts and gene replacements to be made.

Dictyostelium has been particularly useful in the characterization of the fundamental aspects of transmembrane signaling and cell–cell communication in the regulation of motility (3). The organism has the ability to detect and respond to a whole series of environmental factors, such as light, temperature, and moisture. It also can detect a wide variety of chemical signals, from sugars to hormones. Dictyostelium uses a variety of mechanisms of intracellular signaling mechanisms, including proton gradients and electrochemical gradients, as well as Ca2+ waves. Analysis of a variety of protein kinases and phosphatases and their target have illuminated a number of regulatory pathways. The dynamic cytoskeleton of Dictyostelium has been extensively studied, particularly actin, actin-binding proteins, and the myosins, as well as tubulin, tubulin-binding proteins, and microtubule-based motor proteins. Major rearrangements of the cytoskeleton are required during cytokinesis and cell motility. Analysis of genetic knockouts of individual genes and combinations of those for cytoskeletal proteins have been particularly useful in attempting to determine their cellular roles. Targeted gene disruptions have also been used to characterize the pathways of endocytosis and exocytosis. Dictyostelium feeds on bacteria using phagocytosis that is highly regulated and specific (4).

 The highly regulated developmental pathway of Dictyostelium has been extremely valuable in terms of understanding gene induction and regulation. The appearance and disappearance of specific messenger RNAs have been mapped to specific time points during development, and many of the gene products have been characterized and their function determined. The appearance of different cell types in the aggregate, along with their sorting to yield the final fruiting body, has given insights into positional information and the cell cycle in morphogenesis.

Study of the cellular slime mold Dictyostelium discoideum, as a model eukaryotic organism, has produced many insights into basic cellular function.

References  

1. S. J. McRobbie (1986) Chemotaxis and cell motility in the cellular slime molds. Crit. Rev. Microbiol. 13, 335–375. 

2. W. F. Loomis, D. Welker, J. Huges, D. Meghakian, and A. Kuspa (1995) Genetics 141, 147–157.

3. P. Devroetes (1989) Dictyostelium discoideum: a model system for cell–cell interactions in development. Science 245, 1054–1058. 

4. G. Vogel (1983) Dictyostelium discoideum as a model system to study recognition mechanisms in phagocytosis. Methods Enzymol. 98, 431–430.