A study of wound repair in Dictyostelium cells by using novel laserporation.

We examined the mechanism of cell membrane repair in Dictyostelium cells by using a novel laser-based cell poration method. The dynamics of wound pores opening and closing were characterized by live imaging of fluorescent cell membrane proteins, influx of fluorescent dye, and Ca2+ imaging. The wound closed within 2-4 sec, depending on the wound size. Cells could tolerate a wound size of less than 2.0 µm. In the absence of Ca2+ in the external medium, the wound pore did not close and cells ruptured. The release of Ca2+ from intracellular stores also contributed to the elevation of cytoplasmic Ca2+ but not to wound repair. Annexin C1 immediately accumulated at the wound site depending on the external Ca2+ concentration, and annexin C1 knockout cells had a defect in wound repair, but it was not essential. Dictyostelium cells were able to respond to multiple repeated wounds with the same time courses, in contrast to previous reports showing that the first wound accelerates the second wound repair in fibroblasts.

A novel low-power laser-mediated transfer of foreign molecules into cells.

Efficiently introducing molecules such as chemical drugs, proteins, or nucleic acids into cells is a central technique in cell and molecular biology, gene therapy and regenerative medicine. The cell membrane is a critical barrier for this purpose. While many approaches exist, some of which are applicable to single cells that researchers specify under microscopy, no reliable and efficient technique has been invented. In this study, cells were cultured on a coverslip that had been coated with carbon by vapor deposition, and a laser beam was focused on a small local spot beneath a single cell under microscopy. The absorbed energy of the laser beam by the carbon made a pore only in the cell membrane that was attached to the carbon coat, which resulted in an efficient introduction. An inexpensive and lower-power laser could be used for this method, and the introduction efficiency was 100% without any loss of cell viability. This new technique will provide a powerful tool not only to research but also to many applied fields.

Systematic evaluation of buffer influences on the development of Dictyostelium discoideum.

Development and cell differentiation are key features of the social amoeba Dictyostelium discoideum. Already at early developmental stages, the gene expression profile changes in the amoebae to make the cells aggregation competent. In the laboratory, development starts when the cells are washed free of nutrients. For this purpose, various non-nutrient buffers are used in different laboratories. However, to date, it is not clear if different buffers have different influences on the development of D. discoideum. Therefore, we investigated systematically the influence of six widely used buffers on the development of D. discoideum. Investigation was done at the phenotypical, biochemical, and molecular level. The results show that some of the investigated buffers show clear differences in the phenotypical outcome of the developmental cycle, at a biochemical level as measured in the response to cAMP, and/or at a molecular level as measured in the expression of early developmental marker genes. According to our results buffer compositions should be considered carefully for all developmental experiments with D. discoideum, especially when gene expression will be investigated.

Response of Dictyostelium discoideum to UV-C and involvement of poly (ADP-ribose) polymerase.

OBJECTIVES: Radiation and chemical mutagens are direct DNA-damaging agents and ultraviolet (UV) radiation is frequently used in biological studies. Consequent to ozone depletion, UV-C could become a great challenge to living organisms on earth, in the near future. The present study has focused on the role of poly (ADP-ribose) polymerase (PARP) during UV-C-induced growth and developmental changes in Dictyostelium discoideum, a phylogenetically important unicellular eukaryote. MATERIALS AND METHODS: Dictyostelium discoideum cells were exposed to different doses of UV-C and PARP activity, and effects of its inhibition were studied. Expression of developmentally regulated genes yakA, car1, aca, csA, regA, ctnA, ctnB, gp24, hspD and dsn were analysed using semiquantitative RT-PCR. RESULTS: We report that the D. discoideum cells displayed PARP activation within 2 min of UV-C irradiation and there was increase in NO levels in a dose-dependent manner. UV-C-irradiated cells had impaired growth, delayed or blocked development and delayed germination compared to control cells. In our previous studies we have shown that inhibition of PARP recovered oxidative stress-induced changes in D. discoideum; however, intriguingly PARP inhibition did not correct all defects as effectively in UV-C-irradiated cells. This possibly was due to interplay with increased NO signalling. CONCLUSIONS: Our results signify that UV-C and oxidative stress affected growth and development in D. discoideum by different mechanisms; these studies could provide major clues to complex mechanisms of growth and development in higher organisms.

Effects of an extremely low-frequency electromagnetic field on stress factors: a study in Dictyostelium discoideum cells.

The development of technologies that generate environmental electromagnetic fields (EMFs) has led public opinion and the scientific community to debate upon the existence of possible effects caused by man-made EMFs on the human population and, more generally, on terrestrial ecosystems. Protozoa are known to be excellent bioassay systems in bioelectromagnetic studies because of their features that combine the reliability of in vivo results with the practicality of in vitro ones. For this reason, we examined the possible stressful effects of a 50-Hz, 300-µT extremely low-frequency electromagnetic field (ELF-EMF) on the protozoan Dictyostelium discoideum, which was used as it is included in the eight bioassay alternatives to vertebrate models for the study of human disease by the U.S. National Institutes of Health. Our results show how a 24-h exposure of D. discoideum cells to ELF-EMF can affect the net fission rate, the activity and presence of the pseudocholinesterase as well as the presence of the heat shock protein-70, while no change in the catalase and glutathione peroxidase activities was observed. However, this effect seems to be transient and all the altered parameters returned to their respective control value after a 24-h stay under dummy exposure conditions.

Dictyostelium slug phototaxis.

Dictyostelium slugs are able to respond to environmental stimuli in an extremely sensitive and efficient way. This enables a slug to migrate to more favourable locations for formation of fruiting bodies and dispersal of spores. Phototaxis is a readily assayed phenotype and reflects the interactions of environmental stimuli with morphogenetic signalling systems controlling the movement of the slug. The methods for assaying phototaxis are described here. Qualitative phototaxis tests are described and can be used for rapid screening of potential mutants or effects of pharmacological agents. These tests are simple to conduct yet care must be taken in order to avoid the effects of high cell density which can be misleading when interpreting results. Quantitative phototaxis tests can be performed with known cell densities of amoebae which ensures that any effects seen are caused by the mutation or pharmacological agent and not simply due to differences in cell densities.

A fast Ca2+-induced Ca2+-release mechanism in Dictyostelium discoideum.

In vertebrate cells calcium-induced calcium release (CICR) is thought to be responsible for rapid cytosolic Ca(2+) elevations despite the occurrence of strong Ca(2+) buffering within the cytosol. In Dictyostelium, a CICR mechanism has not been reported. While analyzing Ca(2+) regulation in a vesicular fraction of Dictyostelium rich in Ca(2+)-flux activity, containing contractile vacuoles (CV) as the main component of acidic Ca(2+) stores and ER, we detected a rapid Ca(2+) change upon addition of Ca(2+) (CIC). CIC was three times larger in active stores accumulating Ca(2+) than before Ca(2+) uptake and in inactivated stores. Ca(2+) release was demonstrated with the calmodulin antagonist W7 that inhibits the V-type H(+)ATPase activity and Ca(2+) uptake of acidic Ca(2+) stores. W7 caused a rapid and large increase of extravesicular Ca(2+) ([Ca(2+)](e)), much faster and larger than thapsigargin (Tg), a Ca(2+)-uptake inhibitor of the ER. W7 treatment blocked CIC indicating that a large part of CIC is due to Ca(2+) release. The height of CIC depended on the filling state of the Ca(2+) stores. CIC was virtually unchanged in the iplA(-) strain that lacks a putative IP(3) or ryanodine receptor thought to be located at the endoplasmic reticulum. By contrast, CIC was reduced in two mutants, HGR8 and lvsA(-), that are impaired in acidic Ca(2+)-store function. Purified Ca(2+) stores enriched in CV still displayed CIC, indicating that CV are a source of Ca(2+)-induced Ca(2+) release. CIC-defective mutants were altered in their oscillatory properties. The irregularity of the HGR8 oscillation suggests that the principal oscillator is affected in this mutant.

Filamin repeat segments required for photosensory signalling in Dictyostelium discoideum.

BACKGROUND: Filamin is an actin binding protein which is ubiquitous in eukaryotes and its basic structure is well conserved - an N-terminal actin binding domain followed by a series of repeated segments which vary in number in different organisms. D. discoideum is a well established model organism for the study of signalling pathways and the actin cytoskeleton and as such makes an excellent organism in which to study filamin. Ddfilamin plays a putative role as a scaffolding protein in a photosensory signalling pathway and this role is thought to be mediated by the unusual repeat segments in the rod domain. RESULTS: To study the role of filamin in phototaxis, a filamin null mutant, HG1264, was transformed with constructs each of which expressed wild type filamin or a mutant filamin with a deletion of one of the repeat segments. Transformants expressing the full length filamin to wild type levels completely rescued the phototaxis defect in HG1264, however if filamin was expressed at lower than wild type levels the phototaxis defect was not restored. The transformants lacking any one of the repeat segments 2-6 retained defective phototaxis and thermotaxis phenotypes, whereas transformants expressing filaminDelta1 exhibited a range of partial complementation of the phototaxis phenotype which was related to expression levels. Immunofluorescence microscopy showed that filamin lacking any of the repeat segments still localised to the same actin rich areas as wild type filamin. Ddfilamin interacts with RasD and IP experiments demonstrated that this interaction did not rely upon any single repeat segment or the actin binding domain. CONCLUSION: This paper demonstrates that wild type levels of filamin expression are essential for the formation of functional photosensory signalling complexes and that each of the repeat segments 2-6 are essential for filamins role in phototaxis. By contrast, repeat segment 1 is not essential provided the mutated filamin lacking repeat segment 1 is expressed at a high enough level. The defects in photo/thermosensory signal transduction caused by the absence of the repeats are due neither to mislocalisation of filamin nor to the loss of RasD recruitment to the previously described photosensory signalling complex.

Slug phototaxis, thermotaxis, and spontaneous turning behavior.

Dictyostelium slug phototaxis and thermotaxis are readily assayed phenotypes that reflect with great sensitivity and specificity the interactions of environmental stimuli with morphogenetic signaling systems controlling the collective movement of slug cells. Methods are described for conducting and recording phototaxis, thermotaxis, and spontaneous turning experiments, although it is pointed out that spontaneous turning rates are not easily measured in most strains of molecular biological interest. Both phototaxis and thermotaxis can be assayed qualitatively for rapid screening of prospective mutants and quantitatively for detailed phenotypic analysis. Both types of assay are simple to conduct, but require care to avoid the potentially misleading effects of other factors such as cell density and extraneous thermal and chemical gradients that might influence slug behavior. The quantitative analysis and statistical testing of conclusions are carried out using directional statistics, because traditional statistical methods for linear data are inappropriate and potentially misleading when applied to directional data. The appropriate statistical methods are given for measuring maximum likelihood estimates and confidence intervals for the average direction (mu) and the accuracy of orientation (kappa) for unidirectional orientation, as well as the two preferred directions (+/-alpha) and accuracy of orientation in bidirectional phototaxis. In addition, tests for uniformity (kappa = 0), for equality of kappa in the two-sample and multisample cases, and for bidirectional phototaxis vs unidirectional phototaxis are described. These methods are readily implemented in the R environment for statistical computing and the functions required to do so are described and provided.

Effects of a 50 Hz magnetic field on Dictyostelium discoideum (Protista).

Some studies have demonstrated that a few biological systems are affected by weak, extremely low frequency (ELF) electromagnetic fields (EMFs), lower than 10 mT. However, to date there is scanty evidence of this effect on Protists in the literature. Due to their peculiarity as single-cell eukaryotic organisms, Protists respond directly to environmental stimuli, thus appearing as very suitable experimental systems. Recently, we showed the presence of propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum. This enzyme activity was assumed to be involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation. In this work, we have exposed single-cell amoebae of D. discoideum to an ELF-EMF of about 200 microT, 50 Hz, for 3 h or 24 h at 21 degrees C. A delay in the early phase of the differentiation was observed in 3 h exposed cells, and a significant decrease in the fission rate appeared in 24 h exposed cells. The PrChE activity was significantly lower in 3 h exposed cells than in the controls, whereas 24 h exposed cells exhibited an increase in this enzyme activity. However, such effects appeared to be transient, as the fission rate and PrChE activity values returned to the respective control values after a 24 h stay under standard conditions.

Dictyostelium discoideum transformation by oscillating electric field electroporation.

Dictyostelium discoideum has been used as a genetically tractable model organism to study many biological phenomena. High-efficiency transformation is a prerequisite for successful genetic screens such as mutant complementation, identification of suppressor genes, or insertional mutagenesis. Although exponential decay electroporation is the standard transformation technique for D. discoideum, its efficiency is relatively low and its reproducibility is weak. Here we optimized the oscillating electroporation technique for D. discoideum transformation and compared it to the exponential decay electroporation. A 20-fold increase in the efficiency was resproducibly achieved. This alternative electroporation technique should facilitate future genetic approaches in D. discoideum.

Analysis of the gene encoding copper/zinc superoxide dismutase homolog in Dictyostelium discoideum.

SodD, a Cu/Zn superoxide dismutase in Dictyostelium discoideum, shows 48% identity to the cytosolic Cu/Zn superoxide dismutase (SOD) of Saccharomyces cerevisiae (SOD1). The sodD gene is expressed in D. discoideum cells at late-developmental stages. However, gene expression was not detected in the sporeless mutant, indicating that sodD is a spore cell-specific gene. The D. discoideum mutant, in which sodD was disrupted, grew and formed a multicellular structure normally, therefore the gene is not essential for growth and development. The mutant spores were sensitive to UV-light compared to the wild-type spores, indicating that SodD protects spores from cellular damage caused by UV-light.

Differentiation of Dictyostelium discoideum vegetative cells into spores during Earth orbit in space.

We reported previously that emerged amoebae of Dictyostelium (D.) discoideum grew, aggregated and differentiated to fruiting bodies with normal morphology in space. Here, we investigated the effects of space radiation and/or microgravity on the number, viability, kinetics of germination, growth rate and mutation frequency of spores formed in space in a radiation-sensitive strain, gamma s13, and the parental strain, NC4. In gamma s13, there were hardly spores in the fruiting bodies formed in space. In NC4, we found a decrease in the number of spores, a delay in germination of the spores and delayed start of cell growth of the spores formed in space when compared to the ground control. However, the mutation frequency of the NC4 spores formed in space was similar to that of the ground control. We conclude that the depression of spore formation might be induced by microgravity and/or space radiation through the depression of some stage(s) of DNA repair during cell differentiation in the slime mold.

Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light.

Cells of Dictyostelium discoideum are highly resistant to DNA damaging agents such as UV-light, gamma-radiation and chemicals. The genes encoding nucleotide excision repair (NER) and base excision repair (BER) enzymes are rapidly upregulated in response to UV-irradiation and DNA-damaging chemicals, suggesting that this is at least partially responsible for the resistance of this organism to these agents. Although Dictyostelium is also unusually resistant to high concentrations of H(2)O(2), little is known about the response of this organism to oxidative stress. To determine if transcriptional upregulation is a common mechanism for responding to DNA-damaging agents, we have studied the Dictyostelium catalase and Cu/Zn superoxide dismutase antioxidant enzymes. We show that there are two catalase genes and that each is differentially regulated both temporally and spatially during multicellular development. The catA gene is expressed throughout growth and development and its corresponding enzyme is maintained at a steady level. In contrast, the catB gene encodes a larger protein and is only expressed during the final stages of morphogenesis. Cell type fractionation showed that the CatB enzyme is exclusively localized to the prespore cells and the CatA enzyme is found exclusively in the prestalk cells. Each enzyme has a different subcellular localization. The unique developmental timing and cell type distribution suggest that the role for catB in cell differentiation is to protect the dormant spores from oxidative damage. We found that exposure to H(2)O(2) does not result in the induction of the catalase, superoxide dismutase, NER or BER mRNAs. A mutant with greatly reduced levels of catA mRNA and enzyme has greatly increased sensitivity to H(2)O(2) but normal sensitivity to UV. These results indicate that the natural resistance to oxidative stress is not due to an ability to rapidly raise the level of antioxidant or DNA repair enzymes and that the response to UV-light is independent from the response to reactive oxygen compounds.

Light affects cAMP signaling and cell movement activity in Dictyostelium discoideum.

The multicellular, slug stage of the slime mould Dictyostelium discoideum lacks specific sensory cells and organs but can nevertheless respond in a very sensitive manner to external stimuli such as temperature and light. Within the migrating slug, the behavior of up to 100,000 individual amoebae is coordinated by cAMP mediated cell-cell signaling and chemotaxis. We report here the striking result that light directly modulates the cAMP cell-cell signaling system. Light-induced secretion of cAMP from the slug tips decreased the period length of optical density waves and speeded up cell movement. A local effect of light on cAMP release within the slug tip could modulate cell movement within the slug and thus control its phototactic turning and orientation toward a light source.

The use of nonlinear dielectric spectroscopy to monitor the bioelectromagnetic effects of a weak pulsed magnetic field in real time.

Nonlinear dielectric spectroscopy (NLDS) was used to detect interaction of a pulsed magnetic field (PMF) with membrane protein dynamics in aggregating Dictyostelium discoideum amoebae. In the experiments reported here, a strong nonlinear dielectric response of Dictyostelium discoideum cells is shown, and a distinctive nonlinear dielectric response of cells previously exposed to PMF is shown. The method of NLDS is shown to be capable of monitoring and charting the dynamic frequency response of the cell to an electromagnetic field.

Phototactic migration of Dictyostelium cells is linked to a new type of gelsolin-related protein.

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100-insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

A mutation in repB, the dictyostelium homolog of the human xeroderma pigmentosum B gene, has increased sensitivity to UV-light but normal morphogenesis.

Nucleotide excision repair (NER) is an important cellular defense mechanism which protects the integrity of the genome by removing DNA damage caused by UV-light or chemical agents. In humans, defects in the NER pathway result in the disease xeroderma pigmentosum (XP) which is characterized by increased UV-sensitivity, with increased propensity for skin cancer, and an array of developmental abnormalities. Some XP patients exhibit, in addition, symptoms of Cockayne's syndrome (CS) and trichothiodystrophy (TTD), which are characterized by increased UV-sensitivity, without increased cancer incidence, and an array of developmental abnormalities. Some NER genes, including the DNA helicases XPB and XPD, have been shown to function in transcription as well as repair, by virtue of being an integral part of the transcription initiation factor TFIIH. This dual function may account for the above-mentioned wide pleiotropy of phenotypes associated with defects in NER genes, and may explain why some XP patients exhibit developmental abnormalities in addition to XP symptoms. To date, only five XPB patients with three different mutations in the XPB gene have been reported. One of these mutations is a C to A transversion at the splice site at the beginning of the last exon, which resulted in a frameshift throughout the last exon. This patient shows combined clinical symptoms of XP and CS. The recent cloning of the repB gene, the Dictyostelium discoideum homolog of XPB, allowed us to generate a similar C-terminal mutation in the Dictyostelium, in order to test whether the defect in this NER gene has an effect on growth or development. To this end, we have constructed a C-terminal deletion repB mutant in Dictyostelium. To avoid the possibility that a null mutant would be lethal, we used direct homologous recombination to create a 46 amino acid C-terminal deletion mutant. Indeed, we were unable to obtain mutants with a longer 95 amino acid deletion. The repB delta C46 mutants showed an increased sensitivity to UV-light, but a normal pattern of UV-induced expression of repair genes, and no immediately obvious defect in either growth rate or development. The results suggest that the associated developmental defects in the human XPB patients may be due to mutations in another gene.

Rapid changes of nucleotide excision repair gene expression following UV-irradiation and cisplatin treatment of Dictyostelium discoideum.

Organisms use different mechanisms to detect and repair different types of DNA damage, and different species vary in their sensitivity to DNA damaging agents. The cellular slime mold Dictyostelium discoideum has long been recognized for its unusual resistance to UV and ionizing radiation. We have recently cloned three nucleotide excision repair (NER) genes from Dictyostelium , the rep B, D and E genes (the homologs of the human xeroderma pigmentosum group B, D and E genes, respectively). Each of these genes has a unique pattern of expression during the multicellular development of this organism. We have now examined the response of these genes to DNA damage. The rep B and D DNA helicase genes are rapidly and transiently induced in a dose dependent manner following exposure to both UV-light and the widely used chemotherapeutic agent cisplatin. Interestingly, the rep E mRNA level is repressed by UV but not by cisplatin, implying unique signal transduction pathways for recognizing and repairing different types of damage. Cells from all stages of growth and development display the same pattern of NER gene expression following exposure to UV-light. These results suggest that the response to UV is independent of DNA replication, and that all the factors necessary for rapid transcription of these NER genes are either stable throughout development, or are continuously synthesized. It is significant that the up-regulation of the rep B and D genes in response to UV and chemical damage has not been observed to occur in cells from other species. We suggest that this rapid expression of NER genes is at least in part responsible for the unusual resistance of Dictyostelium to DNA damage.

Mutation frequency of Dictyostelium discoideum spores exposed to the space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiosensitive mutant and the parental wild-type strain, were used to investigate the effects of cosmic radiation on viability and mutation frequency at the spore stage for about 9 days in Space Shuttle of NASA. We measured little effect of space environment on viability and cell growth in the both strains as compared to ground controls. The mutation frequency of the flown spores were similar to that of ground control. These results suggest that there could be no effect of cosmic radiation, containing high linear energy transfer radiation at about 0.9 mSv/day as detected by real-time radiation monitoring device on the induction of mutation at the spore stage.