Measurement of cytoplasmic pH in Dictyostelium discoideum by using a new method for introducing macromolecules into living cells

We have developed a novel method for introducing exogenous macromolecules from solution into the cytoplasm of living amoebae of the cellular slime mold Dictyostelium discoideum and have used it to measure the cytoplasmic pH of these cells. Amoebae (strain NC-4) were loaded with fluorescein-labelled dextran by sonication in a solution containing 17 mM phosphate buffer, 1 mM CaCl2, and 10 mg/ml of fluorescein-labelled dextran, pH 6.1. The recovery of living cells was approximately 40% after sonication and washing. A significant fraction (10%) of the recovered cells were loaded and contained 10(5) to 10(7) molecules of fluorescein-labelled dextran per cell as assessed by flow cytometry. The cells loaded by sonication appeared both viable and healthy, since they exhibited normal morphology and locomotion, could differentiate to form mature fruiting bodies, could chemotax in a gradient of extracellular cAMP, and could endocytose latex microspheres. The pH of single cells was estimated by using flow cytometry to measure the fluorescence ratio (fluorescein/rhodamine) in cells loaded with a mixture of the two fluorochrome-labelled dextrans. The fluorescence ratios were calibrated in situ with the flow cytometer after treatment of the cells with either weak acid or weak base to clamp the internal pH at known values. The intracellular pH measured in cells loaded with dextran in a simple salt solution was 5.9. The intracellular pH measured in cells loaded with dextran in the same solution supplemented with amino acids and glucose was 6.7. The novel sonication loading technique described may have general utility for loading diverse types of macromolecules into suspensions of living cells.     

A cytoplasmic cAMP-binding protein in Dictyostelium discoideum

A cytoplasmic cAMP-binding protein from Dictyostelium discoideum was purified about 1200-fold. The binding protein is relatively specific for cAMP, but also binds some other adenine derivatives; it has a molecular weight of approximately 185,000 and an apparent K_D of 1 μM cAMP. The highest level of cytoplasmic cAMP-binding activity is found in amoebae which have been starved for 0–2 hr. Amoebal extracts contain inhibitors of cAMP binding which are removed by chromatography through Sephacryl S200.     

Intracellular pH control in Dictyostelium discoideum: a 31P-NMR analysis

Phosphorus metabolites and intracellular pH have been examined in the slime mold Dictyostelium discoideum by non-destructive 31P-NMR measurements. In a spectrum from a suspension of aerobic amoebae, the major peaks are inorganic phosphate, nucleotide di- and triphosphates. In the corresponding perchloric acid extract, resonances originating from purine and pyrimidine nucleotides are resolved. Adenine nucleotides are the most abundant components, but the other nucleotides are present in significant amounts. In a spectrum from intact spores in a dormant state, only inorganic phosphate and polyphosphates are detected and nucleotides are no longer present in large amounts. Of particular importance is the ability to observe separately in aerobic amoebae the resonance of inorganic phosphate localized in two different cell compartments: the cytosol and the mitochondria. The cytosolic pH and mitochondrial pH have been measured as 6.7 and 7.7, respectively, on the basis of intracellular inorganic phosphate chemical shifts. They are essentially unaffected over a large range of external pH and they are not modified transiently or permanently during the initiation of the developmental program of the organism. A weak acid, such as propionate, which modifies the progression of differentiation by favoring prestalk cells, perturbs intracellular pH gradients by selectively decreasing mitochondrial pH without any effect on cytosolic pH.     

Cytoplasmic pH and glycolysis in the Dictyostelium discoideum cell cycle

Lactate production measurements during the cell cycle of synchronized populations of Dictyostelium discoideum cells reveal cyclic variations in glycolysis which correspond with pH_i oscillations which were discovered by us previously [(1985) Cell, in press]. Aerobic lactate production varies about 6-fold during the cell cycle and the lactate maxima correlate with (~ 0.25 pH unit) cyclic increases in pH. However, artificially altering pH_i using weak acids or bases does not influence the rate of lactate production in asynchronous cell populations. This result suggests that the cyclic variations in pH_i and those in glycolytic rate are not causally related events.     

Towards a molecular understanding of human diseases using Dictyostelium discoideum

The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. This article focuses on several recent examples of Dictyostelium-based biomedical research, including the analysis of immune-cell disease and chemotaxis, centrosomal abnormalities and lissencephaly, bacterial intracellular pathogenesis, and mechanisms of neuroprotective and anti-cancer drug action. The combination of cellular, genetic and molecular biology techniques that are available in Dictyostelium often makes the analysis of these problems more amenable to study in this system than in mammalian cell culture. Findings that have been made in these areas using Dictyostelium have driven research in mammalian systems and have established Dictyostelium as a powerful model for human-disease analysis.     

Intracellular pH in Dictyostelium discoideum: a 31P nuclear magnetic resonance study

We have used phosphorus-31 nuclear magnetic resonance to determine intracellular pH in the cellular slime mold Dictyostelium discoideum. We devised an air-lift circulator to maintain the dense cell suspensions in a well-oxygenated and well-stirred state while causing minimal perturbation to the sample flowing through the detector coils. Cells continued to develop normally in this set-up. Spectra acquired under these conditions typically show two peaks in the inorganic phosphate region corresponding to pH values of 7.16 +/- 0.03 and 6.48 +/- 0.02. These peaks are believed to represent the mitochondrial and cytosolic compartments respectively, based on a comparison of these values with published data and the collapse of the two compartments upon addition of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. Dictyostelium cells show a remarkable degree of intracellular pH homeostasis. Both mitochondrial and cytosolic pH remained unchanged as extracellular pH was varied from 4.3 to 8.1. There was also no apparent change in the pH of either compartment after up to 13.5 hours' development in suspension.     

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.     

Relationships between extracellular pH, intracellular pH, and gene expression in Dictyostelium discoideum

A variety of studies have shown that differentiation of Dictyostelium discoideum amoebae in the presence of cAMP is strongly influenced by extracellular pH and various other treatments thought to act by modifying intracellular pH. Thus conditions expected to lower intracellular pH markedly enhance stalk cell formation, while treatments with the opposite effect favor spores. To directly test the idea that intracellular pH is a cell-type-specific messenger in Dictyostelium, we have measured intracellular pH in cells exposed to either low extracellular pH plus weak acid or high extracellular pH plus weak base using 31P nuclear magnetic resonance (NMR). Our results show that there is no significant difference in intracellular pH (cytosolic or mitochondrial) between pH conditions which strongly promote either stalk cell or spore formation, respectively. We have also examined the effects of external pH on the expression of various cell-type-specific markers, particularly mRNAs. Some mRNAs, such as those of the prestalk II (PL1 and 2H6) and prespore II (D19, 2H3) categories, are strongly regulated by external pH in a manner consistent with their cell-type specificity during normal development. Other markers such as mRNAs D14 (prestalk I), D18 (prespore I), 10C3 (common), or the enzyme UDP-galactose polysaccharide transferase are regulated only weakly or not at all by external pH. In sum, our results show that modulation of phenotype by extracellular pH in cell monolayers incubated with cAMP does not precisely mimic the regulation of stalk and spore pathways during normal development and that this phenotypic regulation by extracellular pH does not involve changes in intracellular pH.     

Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum.

An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP-derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O-(thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway.     

Actin on disease--studying the pathobiology of cell motility using Dictyostelium discoideum

The actin cytoskeleton in eukaryotic cells provides cell structure and organisation, and allows cells to generate forces against membranes. As such it is a central component of a variety of cellular structures involved in cell motility, cytokinesis and vesicle trafficking. In multicellular organisms these processes contribute towards embryonic development and effective functioning of cells of all types, most obviously rapidly moving cells like lymphocytes. Actin also defines and maintains the architecture of complex structures such as neuronal synapses and stereocillia, and is required for basic housekeeping tasks within the cell. It is therefore not surprising that misregulation of the actin cytoskeleton can cause a variety of disease pathologies, including compromised immunity, neurodegeneration, and cancer spread. Dictyostelium discoideum has long been used as a tool for dissecting the mechanisms by which eukaryotic cells migrate and chemotax, and recently it has gained precedence as a model organism for studying the roles of conserved pathways in disease processes. Dictyostelium's unusual lifestyle, positioned between unicellular and multicellular organisms, combined with ease of handling and strong conservation of actin regulatory machinery with higher animals, make it ideally suited for studying actin-related diseases. Here we address how research in Dictyostelium has contributed to our understanding of immune deficiencies and neurological defects in humans, and briefly discuss its future prospects for furthering our understanding of neurodegenerative disorders.     

31P-nuclear magnetic resonance analysis of the intracellular pH in the slime mold Dictyostelium discoideum

Amoebae of the slime mold D. discoideum were studied by phosphorus nuclear magnetic resonance. Under aerobic conditions, major intracellular phosphate compounds included phosphomonoesters, inorganic phosphate (Pi), ADP and ATP. Nucleotides were essentially as magnesium complexes. Two intracellular Pi resonances were clearly resolved and the corresponding pHs determined by the chemical shifts characteristics were 7.7 and 6.7. These intracellular pHs were strictly constant over an extracellular pH range between 5.0 and 7.5. The two cellular compartments defined by the Pi resonances were assigned to mitochondria (pH 7.7) and cytosol (pH 6.7) on the basis of their response to anaerobiosis or to carbonylcyanide-m-chloro phenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, which equilibrate the two intracellular pHs.     

Dictyostelium discoideum as a non-mammalian biomedical model

Dictyostelium discoideum is one of eight non-mammalian model organisms recognized by the National Institute of Health for the study of human pathology. The use of this slime mould is possible owing to similarities in cell structure, behaviour and intracellular signalling with mammalian cells. Its haploid set of chromosomes completely sequenced amenable to genetic manipulation, its unique and short life cycle with unicellular and multicellular stages, and phenotypic richness encoding many human orthologues, make Dictyostelium a representative and simple model organism to unveil cellular processes in human disease. Dictyostelium studies within the biomedical field have provided fundamental knowledge in the areas of bacterial infection, immune cell chemotaxis, autophagy/phagocytosis and mitochondrial and neurological disorders. Consequently, Dictyostelium has been used to the development of related pharmacological treatments. Herein, we review the utilization of Dictyostelium as a model organism in biomedicine.     

Temperature-sensitive mutants of Dictyostelium discoideum

Three classes of temperature-sensitive mutants of the cellular slime mold Dictyostelium discoideum have been isolated. One class contains strains able to grow at 22 C but not at 27 C. Cells of these strains can develop into sorocarps at both temperatures. Another class contains strains which can grow at both temperatures but can only develop at the lower temperature. The third class contains strains unable to grow or develop at 27 C. Those strains whose development is temperature-sensitive appear to carry mutations which affect the cells only during the period of aggregation before the construction of a multicellular sorocarp. When pairs of growth-temperature-sensitive (GTS) strains develop in mixed aggregates, temperature-resistant (TR) cells are formed at a frequency of about 10(-4). These TR cells transmit the phenotype in a relatively stable hereditary fashion. However, temperature-sensitive segregants can be isolated from TR strains even after six clonal passages. Mixed incubation of pairs of morphologically aberrant GTS strains was found to give rise to TR progeny which develop normally. These progeny clones independently segregate morphologically aberrant strains and temperature-sensitive strains. The results indicate that several temperature-sensitive and morphological mutations are recessive and nonidentical.     

Characterization of a novel calmodulin from Dictyostelium discoideum

We have purified calmodulin from the eukaryotic microorganism Dictyostelium discoideum (Clarke, M., Bazari, W. L., and Kayman, S. C. (1980) J. Bacteriol. 141, 397-400) and have compared it to calmodulin purified from bovine brain. The two proteins behaved almost identically during fractionation on ion exchange and gel filtration columns and on isoelectric focusing gels. Dictyostelium calmodulin had one-third the specific activity of brain calmodulin in the Ca2+-dependent activation of brain cyclic nucleotide phosphodiesterase; this activation was inhibited for both proteins by 25 microM trifluoperazine. Dictyostelium calmodulin also activated erythrocyte (Ca2+ + Mg2+)-ATPase and interacted with the inhibitory subunit of skeletal muscle troponin. Competition radioimmune assays showed that Dictyostelium calmodulin could compete with brain calmodulin for antibodies to brain calmodulin. These similarities indicate a close relationship between Dictyostelium and brain calmodulin and suggest that the functional capabilities of the protein have been conserved even among evolutionarily distant species. However, substantial differences in primary structure were detected by amino acid analyses and peptide mapping. Most interesting is the lack of trimethyllysine in Dictyostelium calmodulin. This unusual amino acid, which is commonly found in calmodulins, is therefore not essential for interaction between calmodulin and the calmodulin-regulated proteins tested here.     

F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge.     

Modeling the model organism Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is a fascinating organism, not only for biologists, but also for physicists. Since the Belousov-Zhabotinskii reaction pattern, a well-known non-linear phenomenon in chemistry, was observed during aggregation of Dictyostelium amoebae, Dictyostelium has been one of the major subjects of non-linear dynamics studies. Macroscopic theory, such as continuous cell density approximation, has been a common approach to studying pattern formation since the pioneering work of Turing. Recently, promising microscopic approaches, such as the cellular dynamics method, have emerged. They have shown that Dictyostelium is useful as a model system in biology, The synchronization mechanism of oscillatory production of cyclic adenosine 3',5'-monophosphate in Dictyostelium is discussed in detail to show how it is a universal feature that can explain synchronization in other organisms.     

Phagosomal Proteins of Dictyostelium discoideum

ABSTRACT. In recognizing food particles, Dictyostelium cell-surface molecules initiate cytoskeletal rearrangements that result in phagosome formation. After feeding D. discoideum cells latex beads, early phagosomes were isolated on sucrose step gradietns. Protein analyses of these vesicles showed that they contained glycoproteins and surface-labeled species corresponding to integral plasma membrane proteins. Cytoskeletal proteins also were associated with phagosomes, including myosin II, actin and a 30 kDa-actin bundling protein. As seen by the acridine orange fluorescence of vesicles containing bacteria, phagosomes were acidified rapidly by a vacuolar H⁺-ATPase that was detected by immunoblotting. Except for the loss of cytoskeletal proteins, few other changes over time were noted in the protein profiles of phagosomes, suggesting that phagosome maturation was incomplete. The indigestibility of the beads possibly inhibited further endocytic processing, which has been observed by others. Since nascent phagosomes contained molecules of both the cytoskeleton and plasma membrane, they will be useful in studies aimed at identifying specific protein associations occurring between membrane proteins and the cytoskeleton during phagocytosis.