Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation.

Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80% homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium.

Classification and expression analyses of homeobox genes from Dictyostelium discoideum.

Homeobox genes are compared between genomes in an attempt to understand the evolution of animal development. The ability of the protist, Dictyostelium discoideum, to shift between uni- and multicellularity makes this group ideal for studying the genetic changes that may have occurred during this transition. We present here the first genome-wide classification and comparative genomic analysis of the 14 homeobox genes present in D. discoideum. Based on the structural alignment of the homeodomains, they can be broadly divided into TALE and non-TALE classes. When individual homeobox genes were compared with members of known class or family, we could further classify them into 3 groups, namely, TALE, OTHER and NOVEL classes, but no HOX family was found. The 5 members of TALE class could be further divided into PBX, PKNOX, IRX and CUP families; 4 homeobox genes classified as NOVEL did not show any similarity to any known homeobox genes; while the remaining 5 were classified as OTHERS as they did show certain degree of similarity to few known homeobox genes. No unique RNA expression pattern during development of D. discoideum emerged for members of an individual group. Putative promoter analysis revealed binding sites for few homeobox transcription factors among many probable factors.

Caspase-like proteins: Acanthamoeba castellanii metacaspase and Dictyostelium discoideum paracaspase, what are their functions.

Caspases are cysteine proteases that are important regulators of programmed cell death in animals. Two novel relatives to members of the caspase families metacaspases and paracaspase have been discovered. Metacaspase type-1 was identified in Acanthamoeba castellanii, an opportunistic protozoan parasite that causes severe diseases in humans. Paracaspase was found in the non-pathogenic protozoan Dictyostelium discoideum. Since their discovery in Acanthamoeba and Dictyostelium, metacaspases and paracaspases have remained poorly characterized. At present we do not have sufficient data about the molecular function of these caspase-like proteins or their role, if any, in programmed cell death. How these caspase proteins function at the molecular level is an important area of study that will provide insight into their potential for treatment therapies against Acanthamoeba infection and other similar parasitic protozoan. Additionally, finding the molecular functions of these caspase-like proteins will provide information concerning their role in more complex organisms.The aim of this article was to review recent discoveries about metacaspases and paracaspases as regulators of apoptotic and non-apoptotic processes.

Analysis of rapamycin induced autophagy in Dictyostelium discoideum.

Natural autophagy and autophagic cell death is being studied in the model system, D. discoideum, which has well known genetic and experimental advantages over the other known systems. There is no apoptotic machinery present in this organism which could interfere with the non-apoptotic cell death. The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway which when inhibited by the drug rapamycin induces autophagy. Rapamycin was originally discovered as an anti-fungal agent but its use was abandoned when it was discovered to have potent immunosuppressive and anti-proliferative properties. It is a known drug used today for various cancer treatments and also for increasing longevity in many model organisms. It has a wide usage but its effects on other pathways or molecules are not known. This model system was used to study the action of rapamycin on autophagy induction. Using the GFP-Atg8, an autophagosome marker, it was shown that rapamycin treatment can induce autophagy by an accumulation of reactive oxygen species and intracellular free calcium. Rapamycin suppresses proliferation by induction of cell cycle arrest in the G1 phase. Taken together, the results suggest that the core machinery for autophagy is conserved in D. discoideum and it can serve as a good model system to delineate the action of rapamycin induced autophagy.

Peptide: N- glycanase is expressed in prestalk cells and plays a role in the differentiation of prespore cells during development of Dictyostelium discoideum.

Peptide: N- glycanase (PNGase) enzyme is found throughout eukaryotes and plays an important role in the misfolded glycoprotein degradation pathway. This communication reports the expression patterns of the pngase transcript (as studied by the analysis of β- galactosidase reporter driven by the putative pngase promoter) and protein (as studied by the analysis of β- galactosidase reporter expressed under the putative pngase promoter as a fusion with the pngase ORF) during development and further elucidated the developmental defects of the cells lacking PNGase (png-). The results show that the DdPNGase is an essential protein expressed throughout development and β- galactosidase activity was present in the anterior part of the slug. In structures derived from a null mutant for pngase, the prestalk A and AO patterning was expanded and covered a large section of the prespore region of the slugs. When developed as chimeras with wild type, the png- cells preferentially populate the prestalk/stalk region. When the mutants were mixed in higher ratios, they also tend to form the prespore/spore cells. The results emphasize that the DdPNGase has an essential role during development and the mutants have defects in a system that changes the physiological dynamics in the prespore cells. DdPNGase play a role in development both during aggregation and in the differentiation of prespore cells.

Analysis of Rheb in the cellular slime mold Dictyostelium discoideum: Cellular localization, spatial expression and overexpression.

Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52% identity and 100% similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the β-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.

A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase.

Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.

Spatial patterning of the distribution of Ca2+ in Dictyostelium discoideum as assayed in fine glass capillaries.

We have shown previously that the Ca2+-specific fluorescent dyes chlortetracycline (CTC) and indo-1/AM can be used to distinguish between prestalk and prespore cells in Dictyostelium discoideum at a very early stage. In the present study, pre- and post-aggregative amoebae of Dictyostelium discoideum were labelled with CTC or indo-1 and their fluorescence monitored after being drawn into a fine glass capillary. The cells rapidly form two zones of Ca2+-CTC or Ca2+-indo-1 fluorescence. Anterior (air side) cells display a high level of fluorescence; the level drops in the middle portion of the capillary and rises again to a lesser extent in the posteriormost cells (oil side). When bounded by air on both sides, the cells display high fluorescence at both ends. When oil is present at both ends of the capillary, there is little fluorescence except for small regions at the ends. These outcomes are evident within a couple of minutes of the start of the experiment and the fluorescence pattern intensifies over the course of time. By using the indicator neutral red, as well as with CTC and indo-1, we show that a band displaying strong fluorescence moves away from the anterior end before stabilizing at the anterior-posterior boundary. We discuss our findings in relation to the role of Ca2+ in cell-type differentiation in Dictyostelium discoideum.

Why does slug length correlate with speed during migration in Dictyostelium discoideum.

Taking advantage of the fact that static electricity in plastic Petri dishes will produce very long, thin migrating slugs of Dictyostelium discoideum, it was shown that these slugs moved particularly rapidly. This is consistent with the demonstration of Inouye and Takeuchi that speed varies with length for slugs migrating on agar. Based on these observations it is suggested that slug speed is controlled by both the resistance at the tip and some factor that correlates With slug size, such as the concentration of endogenously produced ammonia.

Social insects and social amoebae.

The evolution of social groupings in insects, especially wasps, is compared to that of social amoebae (cellular slime moulds). They both show a gamut of colony sizes, from solitary forms to complex colonies with a division of labour. The various ideas as to how there might have been an evolution of complexity within insect societies, such as the role of genetic relatedness, the role of mutualism, the origin of sterility, the manipulation and exploitation of some individuals by others within a colony, are discussed, and then applied to social amoebae. The result is both interesting and instructive: despite some differences, there are many striking parallels, which suggests that there are some common denominators in the formation and evolution of a social existence among organisms.

The determination of spatial pattern in Dictyostelium discoideum.

Free-living amoebae of the cellular slime mould Dictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case of Dictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) 'coin tossing', whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) 'chemical kinetics', whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other; and (iii) 'positional information', in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arises in situ.

Patterning in the cellular slime moulds.

The aim of the present article is to derive and illustrate in a simple form some of the important concepts in developmental biology. The development of the cellular slime mould Dictyostelium discoideum is an ideal model system for this purpose. I will outline the development of this organism at its multicellular stages and review some relevant studies focusing on the control of cell differentiation and pattern formation while deriving some key concepts in the current thinking about the control of development.