Origin and function of the stalk-cell vacuole in Dictyostelium

Large vacuoles are characteristic of plant and fungal cells, and their origin has long attracted interest. The cellular slime mould provides a unique opportunity to study the de novo formation of vacuoles because, in its life cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly develops a single large vacuole and cellulosic cell wall to become plant-like cells (stalk cells). Here we describe the origin and process of vacuole formation using live-imaging of Dictyostelium cells expressing GFP-tagged ammonium transporter A (AmtA-GFP), which was found to reside on the membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate from acidic vesicles and autophagosomes, which fuse to form autolysosomes. Their repeated fusion and expansion accompanied by concomitant cell wall formation enable the stalk cells to rapidly develop turgor pressure necessary to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which are rich in H⁺-ATPase as in plant vacuoles, remained separate from these vacuoles. We further argue that AmtA may play an important role in the control of stalk-cell differentiation by modulating the pH of autolysosomes.     

Unique Colony Housing the Coexisting Escherichia coli and Dictyostelium discoideum

Two well-characterized and phylogenetically diverse species, Escherichiacoli and Dictyostelium discoideum, were used as the modelorganisms. When the two species were mixed and allowed to grow onminimal agar plates at 22 ^°C, instead of the predator Dictyostelium exterminating E.coli, the two species remarkablyachieved a state of stable coexistence in about two weeks. In addition, theemerged colonies housing the coexisting species have a mucoidal naturethat is distinctive from its origin. The simplicity of the system and the shorttime span for the two species to develop the coexistence state, that isproven stable and reproducible on laboratory conditions, hence, providesa new model system for the study of symbiosis, particularly with referenceto the initial stages.     

How cellular movement determines the collective force generated by the Dictyostelium discoideum slug

How the collective motion of cells in a biological tissue originates in the behavior of a collection of individuals, each of which responds to the chemical and mechanical signals it receives from neighbors, is still poorly understood. Here we study this question for a particular system, the slug stage of the cellular slime mold Dictyostelium discoideum (Dd). We investigate how cells in the interior of a migrating slug can effectively transmit stress to the substrate and thereby contribute to the overall motive force. Theoretical analysis suggests necessary conditions on the behavior of individual cells, and computational results shed light on experimental results concerning the total force exerted by a migrating slug. The model predicts that only cells in contact with the substrate contribute to the translational motion of the slug. Since the model is not based specifically on the mechanical properties of Dd cells, the results suggest that this behavior will be found in many developing systems.     

Inhibition of aggregation by light in the cellular slime mold Dictyostelium discoideum

Aggregation of Dictyostelium amoebae is inhibited by light. White light intensities 10² W · cm^-2 cause an inhibition which reaches a saturation at 2 · 10³ W · cm^-2. The action spectrum, based on photon fluence-response curves, shows a major peak around 405 nm and extends through most of the visible spectrum with a secondary maximum at about 530 nm. The action spectrum of the inhibition of aggregation resembles the action spectrum of accumulations of amoebae in light traps and the action spectrum of photodispersal from light traps; it does not resemble the action spectrum of phototaxis in pseudoplasmodia.     

Temperature-sensitive inhibition of development in Dictyostelium due to a point mutation in the piaA gene

The Dictyostelium mutant HSB1 is temperature-sensitive for development, undergoing aggregation and fruiting body formation at temperatures below 18 degrees C but not above. In vivo G protein-linked adenylyl cyclase activation is defective in HSB1, and the enzyme is not stimulated in vitro by GTPgammaS; stimulation is restored upon addition of wild-type cytosol. Transfection with the gene encoding the cytosolic regulator PIA rescued the mutant. We excluded the possibility that HSB1 cells fail to express PIA and show that the HSB1 piaA gene harbors a point mutation, resulting in the amino acid exchange G(917)D. Both wild-type and HSB1 cells were also transfected with the HSB1 piaA gene. The piaA(HSB1) gene product displayed a partial inhibitory effect on wild-type cell development. We hypothesize that PIA couples the heterotrimeric G protein to adenylyl cyclase via two binding sites, one of which is altered in a temperature-sensitive way by the HSB1 mutation. When overexpressed in the wild-type background, PIA(HSB1) competes with wild-type PIA via the nonmutated binding site, resulting in dominant-negative inhibition of development. Expression of GFP-fused PIA shows that PIA is homogeneously distributed in the cytoplasm of chemotactically moving cells.     

An inverse PCR technique to rapidly isolate the flanking DNA of Dictyostelium insertion mutants

Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.     

The effect of altered glycosylation on the characteristics of lysosomal trehalase in Dictyostelium discoideum

The trehalase I of Dictyostelium discoideum exhibits characteristics of a typical lysosomal enzyme. The enzyme is glycosylated and carries a number of negatively charged components which cause it to be a very acidic protein. Strain M31, bears a recessive mutation mod A which alters the post-translational modification of several lysosomal enzymes including trehalase. A direct consequence of this mutation is a reduction of the negatively charged components on lysosomal enzymes. This reduction in negativity is observed in the altered chromatographic and electrophoretic behaviour of M31 trehalase.Trehalase I is synthesized during spore germination. Tunicamycin prevents the formation of recoverable trehalase from germinating spores but does not interfere with the germination process. These results indicate that the trehalase I synthesized during spore germination is not required for the successful completion of spore germination. Minor modification in the glycosylation, as seen in strain M31, does not affect the enzymatic activity. However, when glycosylation is greatly reduced by tunicamycin the enzyme is inactive.     

Purification and renaturation of Dictyostelium recombinant alkaline phosphatase by continuous elution electrophoresis

A 1583 bp fragment of Dictyostelium alp cDNA (94% of the gene) was cloned in pET32a+. The enzyme was expressed in an inactive form in the inclusion body of the expression host BL21-CodonPlus (DE3)-RIL. The recombinant ALP constituted more than 50% of the total protein in the inclusion body and 25-30% of the total protein in the expression host after 3 h induction with IPTG at 37 degrees C. A continuous elution polyacrylamide gel electrophoresis procedure was used to purify the recombinant enzyme. This technique yielded a homogeneous protein that retained enzymatic activity after dialysis without further treatment. A yield of 5mg per liter of culture broth was obtained with a specific activity of approximately 0.7 nmol/min/mg protein (0.7 mU/mg). Immunoinhibition studies using a polyclonal antibody produced against the recombinant protein showed complete inhibition of enzymatic activity when the enzyme was preincubated with the antibody at a 1:1000 dilution. The enzyme exhibited a pH optimum of approximately 9.0. The substrate specificity indicated that the Dictyostelium enzyme is a typical broad range alkaline phosphatase.     

Calcium regulates the expression of a<Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> asparaginyl tRNA synthetase gene

In a screen for calcium-regulated gene expression during growth and development ofDictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. TheddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the firstD. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of theAsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution ofGlnRS andAsnRS in eukaryotes.     

Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation during Dictyostelium development

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have previously demonstrated that Dictyostelium discoideum TRAP1 (Dd-TRAP1) synthesized at the vegetative growth phase is retained during the whole course of D. discoideum development, and that at the multicellular slug stage, it is located in prespore-specific vacuoles (PSVs) of prespore cells as well as in the cell membrane and mitochondria. Thereupon, we examined the function of Dd-TRAP1 in prepore and spore differentiation, using Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference method. As was expected, Dd-TRAP1 contained in the PSV was found to be exocytosed during sporulation to constitute the outer-most layer of the spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore prespore differentiation were significantly impaired, particularly under a heat stress condition. Although the TRAP1-RNAi cells formed apparently normal-shaped spores with a cellulosic wall, the spores were less resistant to heat and detergent treatments, as compared with those of parental MB35 cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1 may be closely involved in late development including spore differentiation, as well as in early development as realized by its induction of prestarvation response.     

Lumazine-like fluorescence in a mass of spores of the cellular slime mold,Dictyostelium discoideum

Using a fluorospectrophotometer, we examined the fluorescence of a crude preparation from the spore masses ofDictyostelium discoideum. Fluorescence emission spectra and excitation spectra suggested that the fluorescence of the crude preparation was a lumazine-like fluorescence rather than a pterin-like fluorescence. By using a microspectrophotometer, we observedin situ the fluorescence emission of a lumazine-like substance localized only in the spore mass of the fruiting body.     

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 inDictyostelium discoideum at a very early stage. In the present study, pre-and post-aggregative amoebae ofDictyostelium 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 inDictyostelium discoideum.     

Female Leadership During Migration and the Potential for Sex-Specific Benefits of Mass Spawning in the Brown Surgeonfish (Acanthurus nigrofuscus)

Spawning aggregations in coral-reef fishes have been hypothesized to confer any one of several mutually non-exclusive benefits, largely expected to serve the interests of both sexes simultaneously. Here we provide indication that in the brown surgeonfish, Acanthurus nigrofuscus, spawning aggregations may confer a sex-specific benefit. Following tagged individuals en-route to their daily spawning-aggregation site we found that while migrating groups (≤20 fish) consist of both males and females, females tend to occupy the lead position more often than expected by chance. In addition, we found evidence that female A. nigrofuscus divide their daily egg-stock among several spawning bouts within the aggregations. We propose that female leadership en-route to spawning aggregations, together with the potential benefits of multiple female mating, are consistent with a sex-specific benefit to spawning aggregations.     

Changes in spatial and temporal localization of Dictyostelium homologues of TRAP1 and GRP94 revealed by immunoelectron microscopy

TRAP1 (tumor necrosis factor receptor-associated protein 1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. In this study, we mainly examined the behavior of Dictyostelium TRAP1 homologue, Dd-TRAP1, during Dictyostelium development by immunoelectron microscopy. In vegetatively growing D. discoideum Ax-2 cells, Dd-TRAP1 locates in nucleolus and vesicles in addition to the cell cortex including cell membrane. Many of Dd-TRAP1 molecules moved to the mitochondrial matrix in response to differentiation, although Dd-TRAP1 on the cell membrane seems to be retained. Some Dd-TRAP1 was also found to be secreted to locate outside the cell membrane in Ax-2 cells starved for 6 h. At the multicellular slug stage, Dd-TRAP1 was primarily located in mitochondria and cell membrane in both prestalk and prespore cells. More importantly, in differentiating prespore cells, a significant number of Dd-TRAP1 locates in the PSV (prespore-specific vacuole) that is a sole cell type-specific organelle and essential for spore wall formation, whereas some Dd-TRAP1 in the cell cortical region of prestalk cells. These findings strongly suggest the importance of Dd-TRAP1 regulated temporally and spatially during Dictyostelium development. Incidentally, we also have certified that the glucose-regulated protein 94 (Dd-GRP94) is predominantly located in Golgi vesicles and cisternae, followed by its colocalization with Dd-TRAP1 in the PSV.     

A flow fluorimetric analysis of the cell cycle during growth and differentiation inDictyostelium discoideum

Summary We report a flow fluorimetric analysis of the DNA content of cells and nuclei from vegetative populations and various developmental stages of the cellular slime mouldDictyostelium discoideum using the dyes Hoechst 33258 and mithramycin. Nuclei from all of these populations showed an identical single DNA-content peak, indicating that most vegetative cells and most cells in all developmental stages are in one phase of the cell cycle. Our own data and findings in the literature indicate that this phase is G₂. On the other hand, we also found that various stages, subpopulations of cells at early stages and the different differentiated cell types in the slug stage differ in DNA content per cell. Any particular population typically has one major peak of DNA content, with a modal value that is characteristic for the cell type and for the developmental stage. These differences presumably reflect differences in mitochondrial DNA content per cell.     

Analytical studies on migrating movement of the pseudo-plasmodium ofDictyostelium discoideum

Summary Migrating movement of a pseudoplasmodium (slug) of the cellular slime mouldDictyostelium discoideum was analyzed using a time-lapse video tape recorder. Since slugs usually migrated with repeated interruptions of advance, migrating velocities were measured only within a period of forward movement. On the basis of some known facts and assumptions, a dynamical model for slug movement was formulated, which consists of motive force generated by slug cells against their intrinsic resistance and resistance of slime sheath at the tip. The migrating velocity of a slug depended neither on its width nor its volume, but solely on its length. Under any experimental conditions tested, a linear relationship always held between reciprocals of the two variables. The results were in good agreement with predictions of the model. Quantitative analyses of experimental results by the use of the model lead to the conclusions that a decrease in velocity at a low temperature is due to an increase in resistance of slime sheath at the tip, but that a decrease in velocity during prolonged migration is due to a decrease in motive force of constituent cells. An anterior isolate dissected from a slug migrated at a velocity greater than that of an intact slug of the same length. This was interpreted by the model to be due to the fact that the anterior cells have greater motive forces and intrinsic resistances than the posterior cells. The heterogeneous distributions of the two variables in the cell mass is discussed in reference to the mechanism of sorting out of cells.     

Measurement of the motive force of the migrating slug ofDictyostelium discoideum by a centrifuge method

Summary The motive force of the migrating slug of the cellular slime mouldDictyostelium discoideum was measured by the use of centrifugal force. Changes in shape of the slugs due to the use of centrifugal force were prevented by letting them migrate in an agar capillary. The motive force thus obtained was proportional to the slug volume, the value per unit volume being 5.8×10⁶ dyne/cm³ (58 N/cm³). This is in good agreement with the value measured by the use of hydrostatic pressure.     

Cloning,sequencing, and expression of the genomic DNA encoding the protein phosphatase cdc25 in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

A genomic DNA (Dd-cdc25) encoding the protein phosphatase cdc25 was isolated from the cellular slime mold Dictyostelium discoideum. The Dd-cdc25 DNA sequence, with a length of 2,958 bp, encodes a protein consisting of 986 amino acid (aa) residues. The sequence shares significant identities with cdc25 from human, mouse, Xenopus, Drosophila, and Shizosaccharomyces pombe, particularly at the C-terminal region including the catalytic site for phosphatase activity. The deduced Dictyostelium cdc25 protein (Dd-cdc25) has the highest molecular mass (109.9 kDa) in several cdc25 species so far reported and contains four regions consisting of unusually long asparagine repeats (22–31) in the sequence. Unexpectedly, however, Western blot analysis using a specific antibody raised against the C terminus (aa 892–986) of Dd-cdc25 demonstrated that the protein exists as a short form (56 kDa), which has the C-terminal active site of phosphatase, during the course of Dictyostelium development. The Western blot analysis also revealed marked changes in the phosphorylated state of the Dd-cdc25, coupling with cellular development.Electronic Supplementary Material Supplementary material to this paper is available in electronic form at The sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with the accession number AB039883Edited by N. Satoh     

Genome Re-duplication and Irregular Segregation Occur During the Cell Cycle of Entamoeba histolytica

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.     

Sanguinarine Decreases Cell Stiffness and Traction Force and Inhibits the Reactivity of Airway Smooth Muscle Cells in Culture

Airway hyperresponsiveness (AHR) is the cardinal character of asthma, which involves the biomechanical properties such as cell stiffness and traction force of airway smooth muscle cells (ASMCs). Therefore, these biomechanical properties comprise logical targets of therapy. β2-adrenergic agonist is currently the mainstream drug to target ASMCs in clinical practice for treating asthma. However, this drug is known for side effects such as desensitization and non-responsiveness in some patients. Therefore, it is desirable to search for new drug agents to be alternative of β2-adrenergic agonist. In this context, sanguinarine, a natural product derived from plants such as bloodroots, that has been reported to relax gut smooth muscle emerges as a potential candidate. So far, it is unknown whether sanguinarine can regulate the biomechanical properties of ASMCs and reactivity of ASMCs to irritants. Thus, we tested the hypothesis that sanguinarine reduce the contractile potentials of ASMCs in culture. To do so, the primary cultured rat ASMCs were first treated with different concentration of sanguinarine. Then, cell stiffness, traction force, fiber distribution, and calcium signaling of the ASMCs were evaluated by optical magnetic twisting cytometry, Fourier transform traction microscopy, atomic force microscopy, and Fluo-4/AM based fluorescence confocal scanning microscopy, respectively. The results indicated that sanguinarine (0.05 and 0.5 μmol/L) significantly decreased cell stiffness and traction force, inhibited reactivity of ASMCs to histamine, and disrupted the fiber structures in ASMCs in dose-dependent manner. These findings establish that sanguinarine can indeed change the biomechanical properties of ASMCs and may be used to treat AHR in asthma.