Negative chemotaxis in Dictyostelium and Polysphondylium

A method has been described to measure negative chemotaxis in the cellular slime molds directly and to purify the repellents. Conclusive evidence is given that negative chemotaxis exists in the cellular slime molds and that it occurs generally in Dictyostelium and Polysphondylium. Amoebae respond shortly after their exposure to repellents, which are secreted by vegetative and not by preaggregative cells. The amoebae are sensitive to repellents in both development stages and contain enzyme(s) to inactivate them. Cross reactions of different species indicate that there is more than one repellent, although it cannot be excluded that the variability in response depends on the balancing effect of attractants and repellents.     

Dictyostelium aggregate-less mutant plasma membranes

This investigation concerns a freeze-fracture study of the plasma membranes of aggregate-less mutants 67 and 20-2 derived from the wild-type slime mold Dictyostelium discoideum V12/M2. These mutants cannot respond chemotactically to cAMP and consequently are incapable of normal fruiting body formation. Freeze-fracture studies of Agg 67 and 20-2 amoebae revealed that the average diameters of the plasma membrane particles were almost identical to the wild-type strain V12/M2 vegetative amoebae. Although exposure to cAMP effected a 1.4 × increase in average particle size in the V12/M2 amoebae membranes, those in the mutant cell types did not increase in average diameters. The state of the plasma membrane and its regulatory role in the cell cycle and cell differentiation is discussed.     

Chromatin-associated heat shock proteins of Dictyostelium

A heat shock response has been observed in a wide variety of eukaryotic organisms and may be universal. In Drosophila four heat shock proteins (hsp 22, 23, 26, and 27) have been found in nuclei (A. Arrigo, S. Fakan, and A. Tissieres, 1980, Develop. Biol. 78, 86–103). Eight heat shock-induced proteins of Dictyostelium discoideum were found to be preferentially localized in nuclei. They ranged in size from 26,000 to 32,000 daltons and could be recognized among the chromatin-associated proteins. Partial degradation of the chromatin released the low-molecular-weight heat shock proteins to the same extent as the histones. The heat shock response has been shown to result in protection of cells to the lethal effects of high temperature in a variety of organisms including Dictyostelium. We found that this response is extremely rapid in Dictyostelium being maximal by 30 min. The low-molecular-weight heat shock proteins enter the nuclei rapidly and so could play a role there in thermal protection. A mutant strain was isolated which is impaired in the protection afforded by a heat shock. This strain synthesizes most proteins normally but specifically fails to synthesize the low-molecular-weight heat shock proteins under conditions which result in their induction in wild-type cells.     

Signal transduction pathways controlling multicellular development in Dictyostelium

The importance of signal transduction pathways in regulating developmental processes in a number of organisms has become evident in recent years. This is exceptionally clear for Dictyostelium, which uses soluble factors to regulate morphogenesis and cellular differentiation. It is now known that many of these processes are controlled by signal transduction pathways mediated by cyclic AMP through cell surface receptors coupled to G proteins, and that others are mediated by the morphogen DIF.     

The regulation of glycoprotein synthesis by cAMP in Dictyostelium

Dictyostelium discoideum (Dd) 1-H vegetative amoebae exposed to cAMP differentiate into mature stalk cells within 48 h [6]. It was of interest to monitor the patterns of glycoprotein synthesis in the amoebae during the first 5 h of exposure to cAMP and phosphate buffer (PB) controls. Following the exposure period the amoebae were labeled with -[6-³H]fucose. It was determined by both silver grain counts of autoradiographs and scintillation spectroscopy that within minutes cAMP effects an inhibition of [³H]fucose incorporation. However, by 5 h of exposure both experimentals and controls lose a major amount of their labeling capacity based upon the initial PB control value. Vegetative amoebae exposed to cAMP mimics the sparse labeling found in prestalk cells. Prestalk cells synthesize cellulose as a result of cAMP-induced gluconeogenesis and consequently glycoprotein synthesis is reduced. Cellular interactions promoted by cAMP appears to initiate prestalk cell differentiation during the pre-aggregation phase of development. This event is accompanied by a loss in the ability of the aggregating cells to synthesize glycoprotein.     

Cyclic AMP induces a transient alkalinization in Dictyostelium

In a wide range of cell types, stimulus-response coupling is accompanied by a rise in cytoplasmic pH (pH_i). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pH_i. About 1.5 min after stimulation, pH_i starts increasing from pH_i7.45 to pH_i7.60, as is revealed independently by two different pH null-point methods. The rise in pH_i is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pH_i is accomplished by the DES-sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.     

The Dictyostelium repertoire of seven transmembrane domain receptors

The availability of fully sequenced genomes allows the in silico analysis of whole gene families in a given genome. A particularly large and interesting gene family is the G-protein-coupled receptor family. These receptors detect a variety of extracellular signals and transduce them, generally via heterotrimeric G-proteins, to effector proteins inside the cell and thus elicit a physiological response. G-protein-coupled receptors are found in all eukaryotes and constitute in vertebrates 3–5% of all genes. They are also very important drug targets and approximately 25 of the top 100 selling drugs are directed against these receptors. The Dictyostelium discoideum genome contains a surprisingly high number of 55 such receptors, approximately 0.5% of the encoded genes. Besides the four well-studied cAMP receptors the genome encodes eight additional cAMP receptor-like proteins and one of these is distinguished by a novel domain structure, one secretin-like receptor, 17 GABA_B-like and 25 Frizzled-like receptors. The existence of the latter three types of receptors in D. discoideum was surprising because they had not been observed outside the animal kingdom before. Their presence suggests unprecedentedly complex and so far unknown signaling activities in this lower eukaryote.     

Unit membrane structural changes following cell association in Dictyostelium

1. 1. A study of the plasma membranes of Dictyostelium discoideum during development was conducted by freeze-fracture and thin-section electron microscopy.

2. 2. The plasma membranes exhibited paniculate structures ranging from 41–188 Å in diameter. The particles increase significantly in average size from the vegetative stage until mature spore formation at which time a decrease occurs.

3. 3. The increase in particle size during development has been attributed to the fusion of mobile particles within the matrix of the membrane.

4. 4. The possibility that cell differentiation is dependent upon an adhesive-stimulatory mechanism provided by the plasma membranes upon aggregation is discussed.

     

Variables Controlling the Expression Level of Exogenous Genes in Dictyostelium

Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.     

gbpC和gbpD基因在盘基网柄菌细胞趋化性和趋电性运动中的差异研究

目的 趋化性和趋电性是细胞定向迁移的主要方式，并在生物有机体的生理和病理过程中发挥重要作用，但二者存在差异。本文对盘基网柄菌gbpC和gbpD基因在细胞趋电性和趋化性中的作用进行对比研究，以寻找两种迁移方式之间的新差异。方法 将gbpC基因突变株gefT-和gbpD基因突变株gefU-分别置于场强为12 V/cm的直流电场中，分析细胞在电场中的运动方向及运动速度，探讨细胞的趋电性变化；利用电穿孔技术将标记F-actin的Lifeact-GFP质粒转化进入细胞，用荧光显微镜观察活细胞运动时F-actin的分布；用蛋白质印迹技术定量分析细胞的肌球蛋白调节轻链（RLC）在受直流电场刺激后的磷酸化变化情况。结果 gefT-突变株细胞极化消失，但保持与野生型类似的趋电性；gefU-突变株细胞发生超极化，但趋电性显著降低。在直流电场中，突变株细胞和野生型细胞的F-actin主要分布在伪足部位。在电场作用下，细胞株的肌球蛋白RLC磷酸化变化情况存在差异，即野生型细胞以时间依赖的方式发生磷酸化，gefT-突变株细胞先急剧下降，然后再上升，gefU-突变株细胞却以时间依赖方式脱磷酸化。结论 本研究表明gbpC和gbpD基因在盘基网柄菌趋化性和趋电性中的作用不同，暗示了电信号与化学信号确实通过不同的机理指导细胞的定向迁移。     

A simple method of generating axenic derivatives of Dictyostelium strains

We describe a generally applicable method of adapting Dictyostelium from growth on a bacterial food source to axenic growth. Cells are initially selected by growth on a plastic substratum but subsequently acquire the ability to grow in suspension culture. These strains can be transformed efficiently by DNA-mediated gene transfer.     

Cell motility and SCAR localisation in axenically growing Dictyostelium cells

Dictyostelium is a popular experimental organism, in particular for studies of actin dynamics, cell motility and chemotaxis. We find that the motility of axenic cells is unexpectedly different from other strains during growth. In particular, vegetative AX3 cells do not show detectable localisation of SCAR and its regulatory complex to actin-rich protrusions such as filopodia and pseudopodia. Similarly, a range of different mutations, in particular knockouts of members of the SCAR complex and Ras proteins, cause different phenotypes during vegetative growth in different parental strains. Development reverses this unusual behaviour; aggregation-competent AX3 cells localise SCAR in the same way as cells of other strains and species. Studies on cell motility using vegetative cells should therefore be interpreted with caution.     

Characterisation of serum-induced intracellular Ca2+ oscillations in primary bone marrow stromal cells

Intracellular Ca2+ signalling is pivotal to cell function and [Ca2+]i oscillations permit precise and prolonged modulation of an array of Ca2+-sensitive processes without the need for extended, global elevations in [Ca2+]i. We have studied [Ca2+]i signalling in primary rat marrow stromal cells exposed to foetal calf serum (FCS) constituents at concentrations up to those required to promote growth and differentiation in culture. Spontaneous [Ca2+]i signalling was not observed, but exposure to 1% FCS induced regular, sustained Ca2+ oscillations in 41 +/- 3% of cells. Incidence of FCS-induced oscillations was dose-dependent, saturating at 0.5%. These oscillations were arrested by disruption of Ca2+ stores with 100 nM-1 microM thapsigargin or discharge of mitochondrial membrane potential and were sensitive to blockade of IP3-receptors by 50 microM 2-amino-ethoxydiphenyl borate (2-APB) and inhibition of phospholipase C with 5 microM U73122. The oscillations decreased in frequency and amplitude following inhibition of Ca2+ influx with EGTA or La3+ but were poorly sensitive to nifedipine (1-10 microM) and Bay K 8644 (300 nM). The factor(s) responsible for inducing [Ca2+]i oscillations are heat stable, insensitive to disulphide bond reduction with 20 mM dithioerythritol and retained by a 30 kDa molecular weight filter. Serum is routinely present in culture medium at 10%-15% [v/v] and marrow stromal cells maintained under culture conditions exhibited sustained oscillations. This is the first demonstration of agonist-induced complex Ca2+ signals in marrow stromal cells. We conclude that Ca2+ oscillations occur constantly in these cells in culture and are potentially important regulators of cell proliferation and differentiation.     

Transformation with Vectors Harboring the NEO Selection Marker Induces Germination-Specific Adenylylcyclase Activity in Dictyostelium Cells

Dictyostelium cells express an aggregative adenylylcyclase (ACA), responsible for oscillatory cAMP signaling, and a spore germination-specific adenylylcyclase (ACG). Overexpression of PKA regulatory (R) subunits blocks oscillatory cAMP signaling but increases basal cAMP levels, while neither ACA nor ACG mRNA could be detected. To test whether a novel type of adenylylcyclase (AC) was responsible for cAMP synthesis, dominant-negative PKA-R subunits (PKA-R_M) and control R-subunits (PKA-R_C) were overexpressed in ACA null routants. Both transformations as well as transformation with an unrelated vector, carrying the same NEO^R selection marker, induced an AC activity in growing cells with the biochemical characteristics of ACG. Similar vectors with a different URA selection marker did not increase AC activity. We conclude that the amino-glycoside phosphotransferase encoded by the very commonly used NEO^R selection marker induces ectopic ACG activity in Dictyostelium cells.