Endocytic transit inDictyostelium discoideum

Summary The cells ofDictyostelium discoideum are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells andD. discoideum, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway.     

A potentially exhaustive screening strategy reveals two novel divergent myosins inDictyostelium

In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging. Therefore,Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the actomyosin cytoskeleton in a variety of cellular tasks. Because of the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism. Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family inD. discoideum. Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library. Preliminary inspection ofmyoK andmyoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins.     

Accumulation of unsulphated precursors inDictyostelium discoideum during selenate inhibition of growth

Incubation ofDictyostelium discoideum cells with selenate is known to inhibit vegetative growth. In this paper we show that in the presence of selenate macromolecules accumulate which can be converted to sulphated products once the selenate is removed. The presence of cycloheximide, an inhibitor of protein synthesis, during the subsequent incubation does not prevent this conversion but tunicamycin, an inhibitor of glycosylation does. It is concluded that, in the presence of selenate, precursors accumulate as unglycosylated proteins, suggesting that feedback inhibition of glycosylation may be operated.     

Environmental factors inducing sexual development inDictyostelium discoideum

In the heterothallic strains NC4 and HM1 ofDictyostelium discoideum, sexual development is initiated by the formation of diploid zygotic giant cells produced through the fusion of these two opposite mating-type haploid cells. For sexual cell fusion, amoeboid cells must first acquire fusion competence, which requires culture under certain environmental conditions, such as darkness, excessive water, and sufficient bacteria as food. However, in the subsequent stages of cell fusion and development of the giant cells into mature macrocysts, cells do not require the above conditions. Cell fusion and development into macrocysts were able to occur even in light with minimum water and in the absence of bacteria. For cell fusion calcium ions were required.     

Periodic stimuli are more successful than randomly spaced ones for inducing development inDictyostelium discoideum

Aggregation in the cellular slime moldDictyostelium discoideum is due to chemotaxis. The chemoattractant, cyclic AMP, is synthesised and released periodically by the cells. Externally applied periodic pulses of cyclic AMP can also induce differentiation in this organism. The present work examines the role of periodicityper se in cyclic AMP-mediated stimulation of cell differentiation. For this purpose we use Agip53, aDictyostelium mutant which does not develop beyond the vegetative state but can be made to aggregate and differentiate by reiterated applications of cyclic AMP. Importantly, Agip53 cells do not make or release any cyclic AMP themselves even in response to an increase in extracellular cyclic AMP. A comparison of the relative efficiencies of periodic and aperiodic stimulation shows that whereas the two patterns of stimulation are equally effective in inducing the formation of EDTA-stable cell contacts, periodic stimuli are significantly superior for inducing terminal differentiation. This suggests that there must be molecular pathways which can only function when stimulation occurs at regular intervals.     

Localization of a DNA topoisomerase II to mitochondria inDictyostelium discoideum: Deletion mutant analysis and mitochondrial targeting signal presequence

DNA topoisomerase II ofDictyostelium discoideum (TopA), the gene (topA) encoding which we cloned, was shown to have an additional N-terminal region which contains a putative mitochondrial targeting signal presequence. We constructed overexpression mutants which expressed the wild-type or the N-terminally deleted enzyme, and examined its localization by immunofluorescence microscopy and proteinase K digestion experiment. These experiments revealed that the enzyme is located in the mitochondria by virtue of the additional N-terminal region. Furthermore, in the cell extract depleted the enzyme by immunoprecipitation, nuclear DNA topoisomerase II activity was not decreased. These results confirmed that TopA is located in the mitochondria, even through its amino acid sequence is highly similar to those of nuclear type topoisomerase II of other organisms. Thus, this report is the first to establish the location of the mitochondrial targeting signal presequence in DNA topoisomerase II and in proteins ofD. discoideum directly by analyzing deletion mutants. Tsukuba Advanced Research Alliance (TARA researcher for the Sakabe project)     

Development of prespore vacuoles inDictyostelium cells differentiating in a liquid shake culture

Summary When shaken in a glucose-albumin-cyclic AMP medium, dissociated aggregative cells form small clumps in which prespore cells differentiate fairly synchronously (Okamoto 1981). Formation of prespore vacuoles (PSVs) in differentiating prespore cells was examined in these culture conditions, by electronmicroscopy and immunocytochemistry.After 6 hours of culture, a typical Golgi apparatus composed of vesicles and stacked flat cisternae develops near the nucleus. FITC-conjugated antispore serum stains a crescent-shaped region in the cells which seems to correspond to the Golgi area. After 9 hours, flat sacs which contain electron dense lining membrane similar to that of PSVs appear alongside Golgi cisternae. Later, partially and fully round PSVs are observed in this region, suggesting that flat sacs round up to become mature PSVs. After 12 hours, as mature PSVs increase in number, they become dispersed throughout the cytoplasm and a typical Golgi apparatus with cisternae disappears. When cultured in a medium devoid of cyclic AMP, cells develop neither Golgi cisternae nor PSVs. These results strongly suggest that PSVs form from Golgi cisternae.     

A novel PCR-mediated method for one-tube generation of a gene disruption construct

A novel PCR-based method is reported for generating a gene disruption construct which requires no purification of PCR fragments and enables the whole procedure to be completed in one tube very rapidly. The procedure starts with PCR amplification of both the 5 and 3 regions of a particular gene in one tube. Then, exonuclease I is added to the tube to remove the residual primers. After heat inactivation of the enzyme, a marker cassette DNA fragment is added and fusion PCR is performed to build up a gene disruption construct. The gene disruption construct is subsequently amplified with the outermost primers in the amount necessary for transformation. In order to distinguish the gene disruption construct from the remaining intact gene allele, the outermost primers are designed to have GC-rich tag sequences that anneal at a higher temperature, ensuring the specific amplification of the gene disruption construct.     

Guanine nucleotides modulate the function of chemotactic cyclic AMP receptors inDictyostelium discoideum

Summary Guanosine di- and triphosphates specifically decrease the affinity of chemotactic cAMP receptors in isolatedDictyostelium discoideum membranes. The K_0.5 was increased from 50 nM to 150 nM. Receptors were shown to be heterogeneous in dissociation kinetics. In the absence of guanine nucleotides three dissociation processes could be resolved, having first order rate constants of 8.7 x 10⁻⁴, 1.3 X 10⁻², and higher than 0.1 s⁻¹. Guanine nucleotides decreased the affinity for cAMP by transforming the slowest dissociating receptor form (K_D is 8 nM) to forms dissociating more rapidly. Our data indicate that a guanine nucleotide binding protein (G-protein) is involved in the transduction of the cAMP signal inD. discoideum.     

Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

Dictyostelium discoideum chemotaxis: Threshold for directed motion

The chemotactic response of Dictyostelium discoideum cells to stationary, linear gradients of cyclic adenosine 3′,5′-monophosphate (cAMP) was studied using microfluidic devices. In shallow gradients of less than 10⁻³ nM/μm, the cells showed no directional response and exhibited a constant basal motility. In steeper gradients, cells moved up the gradient on average. The chemotactic speed and the motility increased with increasing steepness up to a plateau at around 10⁻¹ nM/μm. In very steep gradients, above 10 nM/μm, the cells lost directionality and the motility returned to the sub-threshold level. In the regime of optimal response the difference in receptor occupancy at the front and back of the cell is estimated to be only about 100 molecules.     

Co-loss of profilin I, II and cofilin with actin from maturing phagosomes inDictyostelium discoideum

Summary Although it is known that actin polymerizes rapidly at the plasma membrane during the ingestion phase of phagocytosis, not yet fully understood are the mechanisms by which actin is recruited to form a phagoeytic cup and subsequently is dissociated from the phagosome. The aim of this study was to identify actin-binding proteins that mediated actin filament dynamics during phagosome formation and processing. We report that profilins I and II, which promote filament assembly, and cofilin, which stimulates filament disassembly, were constituents of phagosomes isolated fromDictyostelium discoideum fed latex beads, and associated with actin. Biochemical analyses detected one isoform only of cofilin, which bound actin in unstimulated cells as well as in cells engaged in phagocytosis, subjected to various stress treatments, and through development. At membranes of young phagosomes, profilins I and II colocalized with monomeric actin labeled with fluorescent DNase I, and cofilin colocalized with filamentous actin labeled with rhodamine phalloidin. Both immunocytochemical and quantitative immunoblotting data indicated that the kinetic loss of profilins I, II, and cofilin of maturing phagosomes closely followed the falling levels of actin associated with the vesicles. As evidence of vesicle processing,D. discoideum crystal protein (an esterase) was recruited rapidly to phagosomes and its levels increased while those of actin, profilins I, II, and cofilin jointly decreased. The localization data and concurrent losses of profilins and cofilin with actin from phagosomes are consistent with the roles of these actin-binding proteins in filament dynamics and indicated that they were involved in regulating the assembly and disassembly of the actin coat of phagosomes.Abbreviations DNase deoxyribonuclease - FITC fluorescein isothiocyanate - NEpHGE nonequilibrium pH gradient gel electrophoresis - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Use of the transposable element Ac/Ds in conjunction with Spm/dSpm for gene tagging allows extensive genome coverage with a limited number of starter lines: functional analysis of a four-element system in Arabidopsis thaliana

We have developed a novel four-element based gene tagging system in Arabidopsis to minimize the number of starter lines required to generate genome-wide insertions for saturation mutagenesis. In this system, the non-autonomous cassette, Ds(dSpm), comprises of both Ds and dSpm elements cloned one within the other along with appropriate selection markers to allow efficient monitoring of excision and re-integration of the transposons. Trans-activation of the outer borders (Ds) and selection against the negative selection marker (iaaH) linked to the cassette ensures unlinked spread of the Ds(dSpm) cassette from the initial site of integration of the T-DNA. This creates several launch pads within the genome from where the internal element (dSpm) can be subsequently mobilized to generate secondary insertions. In this study, starting from a single T-DNA integration we could spread the Ds(dSpm) cassette to 11 different locations over all the five chromosomes of Arabidopsis. The frequency of unlinked Ds transpositions in the F2 generation varied between 0.05 and 3.35%. Three of these lines were then deployed to trans-activate the internal dSpm element which led to the selection of 29 dSpm insertions. The study conclusively shows the feasibility of deploying Ds and the dSpm elements in a single construct for insertional mutagenesis.     

The prestalk/prespore differentiation and polarized cell movement inDictyostelium discoideum slugs. A possible involvement of the intracellular Ca2+-concentration

Summary Intracellular free calcium ion concentrations ([Ca²⁺]_i) in the anterior prestalk and posterior prespore cells of theDictyostelium discoideum slug were determined, using the highly selective Ca²⁺ indicators, quin-2/AM and fura-2/AM. Temporal changes in [Ca²⁺]_i in response to chemotactic stimulation with cAMP were also monitored at the single-cell level and compared between the two types of cells. The results obtained showed that resting [Ca²⁺]_i in the prestalk cells is considerably higher than that in the prespore cells. Moreover, transient increase in [Ca²⁺]_i upon stimulation with a low concentration of cAMP (20 nM) was noticed only in the prestalk cells, but not in the prespore cells. These facts are discussed in relation to the polarized movement and cellular differentiation in the migrating slug.Abbreviations cAMP 3,5-cyclic adenosine monophosphate - DIF differentiation-inducing factors - IP₃ inositol 1,4,5-triphosphate     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid     

An improved method for Beauveria bassiana transformation using phosphinothricin acetlytransferase and green fluorescent protein fusion gene as a selectable and visible marker

An improved transformation method for the biocontrol agent, Beauveria bassiana, was developed. For convenience of transformation selection and detection, the coding regions of the genes for phosphinothricin acetyltransferase and green fluorescent protein were fused and an expression vector, pBFT, carrying this fusion was constructed. Under optimum conditions, over 60 transformants microg(-1) plasmid DNA were obtained. B. bassiana conidia frozen 1 month at -80 degrees C were fully competent for transformation. The method was significantly less laborious and more rapid than current methods for B. bassiana. The bar::egfp provides a selectable and visible marker which may expedite future genetic engineering of this fungus.     

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.     

RFLP tagging of a new semidwarfing gene in rice

A new rice semidwarfing gene which is not allelic tosd1, temporarily designated assdg, might be of use as a new source of semidwarfism in rice breeding programs. We report here the identification of a DNA marker closely linked to this gene. The DNA marker was identified by testing 120 mapped rice RFLP makers as hybridization probes for Southern analysis of a pair of nearly isogenic lines with or withoutsdg. Linkage association of the marker with the gene was verified using a F₂ population segregating for semidwarfism. RFLP analysis showed thatsdg is closely linked to a single-copy DNA clone RZ182 on chromosome 5, with a distance of 4.3 centiMorgans between them. This marker may facilitate early selection for the semidwarfing gene in rice breeding programs     

The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.