Calcium stores in differentiated Dictyostelium discoideum: prespore cells sequester calcium more efficiently than prestalk cells.

Dictyostelium discoideum pseudoplasmodia exhibit a gradient of the cytosolic free Ca2+-concentration ([Ca2+]i) along their anterior-posterior axis involved in cell-type specific differentiation. [Ca2+]i is high in prestalk and low in prespore cells. We determined the content and localization of calcium and other elements in cryosectioned cells of pseudoplasmodia and fruiting bodies by X-ray microanalysis. Granular stores rich in Ca, Mg and P were identified. Average Ca was higher in prespore than prestalk granules (225vs 111 mmol/kg dry weight). Total Ca stored in granules was also higher in prespore than prestalk cells. The amount of P and S in granules differed between the two cell types indicating different store composition. In spores mean granular Ca was 120 mmol/kg dry weight. Stalk cells had smaller granules with 360 mmol Ca/kg dry weight. Complementary to microanalysis, vesicular Ca2+-fluxes were studied in fractionated cell homogenates. The rate of Ca2+-uptake was higher in pellet fractions of prespore than prestalk amoebae (4.7 vs 3.4 nmol/min x mg). Ca2+-release was greater in supernatant fractions from prestalk than prespore cells (16.5vs 7.7 nmol/10(8)cells). In summary, prestalk and prespore cells possess qualitatively different, high-capacity stores containing distinct amounts of Ca and probably being involved in regulation of the anterior-posterior [Ca2+]i-gradient.

A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium.

Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.

Dissection of the cAMP induced cytosolic calcium response in Dictyostelium discoideum: the role of cAMP receptor subtypes and G protein subunits.

The cAMP signaling cascade leading to changes in [Ca2+]i in Dictyostelium discoideum was analyzed using cell lines overexpressing single cAMP receptor subtypes (cAR1-cAR3) or lacking the G(alpha2) or G(beta) subunit of the G protein. Imaging of fura2-dextran-loaded amoebae revealed cAMP-induced [Ca2+]i changes characteristic for each receptor subtype activated. Cells expressing distinct subtypes sort to defined zones during multicellular development suggesting involvement of the specific [Ca2+]i transients in patterning processes. Whereas generation of the [Ca2+]i increase was G(alpha2)-independent, only few cells devoid of G(beta) displayed a [Ca2+]i change after stimulation indicating its participation in the regulation of the calcium homeostasis.

Fatty acids induce release of Ca2+ from acidosomal stores and activate capacitative Ca2+ entry in Dictyostelium discoideum.

cAMP-induced Ca2+ fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating non-esterified fatty acids [Schaloske and Malchow (1997) Biochem. J. 327, 233-238]. In the present study the effect of fatty acids on Ca2+ homoeostasis in D. discoideum was investigated. Cytosolic free Ca2+ concentration ([Ca2+]i) was analysed by digital imaging of single fura2-dextran-loaded cells. Arachidonic acid and linoleic acid induced a transient increase in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells, with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of bis-(o-aminophenoxy)ethane-N, N,N',N'-tetra-acetic acid (BAPTA), no [Ca2+]i transient was detectable. In spite of this, we found that (1) arachidonic acid induced Ca2+ release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+ influx in intact cells and (2) Ca2+ entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase, indicating that intracellular Ca2+ release precedes Ca2+ entry. Inhibition studies and mutant analysis point to the acidosomal Ca2+ stores as a target of fatty acids. Although fatty acids can substitute fully for cAMP with respect to Ca2+ influx in wild-type cells, experiments with a mutant strain revealed that cAMP also sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+ influx was normal in a phospholipase C knockout mutant but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid which cause sufficient Ca2+ to be released from the stores to trigger extracellular Ca2+ entry.

cAMP-induced changes in the cytosolic free Ca2+ concentration in Dictyostelium discoideum are light sensitive.

The cytosolic free calcium concentration ([Ca2+]i) of the social amoeba Dictyostelium discoideum was analyzed after challenge with the chemoattractant cAMP. [Ca2+]i was measured by digital imaging in single cells loaded with the Ca2+ indicator Fura-2-dextran. Global stimulation with low concentrations of cAMP (0.1-1 microM) led to a global transient [Ca2+]i increase. This increase was abolished when cells were illuminated with high doses of light. However, after a short recovery period of several minutes, the cells again displayed the normal response. Inhibition of the [Ca2+]i elevation depended on the wavelength of illumination light. We compared the required recovery period of cells irradiated with either 340, 380, 405, 450 or 490 nm at defined intensities. Light of 405 nm had a pronounced effect; 340 nm alone or in combination with 380 nm was also effective, but to a lesser extent, whereas neither 450 nm nor 490 nm inhibited the [Ca2+]i increase, even at very high irradiance. The wavelength dependence matched the absorption spectrum of amoebae grown in darkness that contain a photopigment which seems to be responsible for phototaxis of single cells. Cells grown in darkness exhibited an increased sensitivity of the cAMP-induced [Ca2+]i transient towards light compared to light-grown cells. From these data we conclude that phototactic signaling could interfere with chemotactic signaling at the level of [Ca2+]i changes.

An increase in cytosolic Ca2+ delays cAMP oscillations in Dictyostelium cells.

We have shown that calmidazolium (R24571) causes a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i) in Dictyostelium discoideum [Schlatterer and Schaloske (1996) Biochem. J. 313, 661-667]. Here we have used R24571 to artifically increase [Ca2+]i during light-scattering oscillations and have found that, depending on the time of addition during the oscillatory cycle, R24571 suppressed cAMP synthesis and delayed the next spike for several minutes. Addition of Ca2+ to the medium, which also elevates [Ca2+]i, induced phase delays and resulted in a similar phase response curve as R24571. The magnitude of the phase delay was correlated with the point during the oscillatory cycle at which Ca2+ was added, indicating that an artificial increase in [Ca2+]i also resets the phase of the intrinsic oscillator.

On the role of calcium during chemotactic signalling and differentiation of the cellular slime mould Dictyostelium discoideum.

Transient cytosolic calcium elevations are required for chemotaxis and differentiation of Dictyostelium discoideum since Ca2+ chelating buffers introduced into the cells by scrape loading inhibited motility as well as orientation in a Ca2+ specific manner. Ca2+ changes are provided either by intrinsic cytosolic Ca2+ oscillations that can be determined as periodic Ca2+ efflux, or by receptor-mediated Ca2+ liberation from the InsP3-sensitive store and Ca2+ influx. Cytosolic Ca2+ homeostasis as well as oscillations seem to be regulated by two different Ca2+ stores, the acidosomes and the InsP3-sensitive store, both of which are dependent on Ca2+ pumps and V-type H+ ATPases. Ca2+ transients are sensed by calmodulin-binding proteins. The latter have been detected in Dictyostelium by 125I-calmodulin labeling. A calmodulin-dependent protein phosphatase, calcineurin A, was cloned, sequenced, purified and characterized biochemically. Overproduction of calcineurin A as well as antisense constructs will help to the elucidation of its function in signal transduction. Surprisingly, protein synthesis is also controlled by Ca2+/calmodulin. An integral ribosomal protein of the 60S subunit, L19, proved to be a calmodulin-binding protein and calmodulin antagonists of different classes, inhibited in vitro translation of Dictyostelium and wheat germ extracts.

Calmidazolium leads to an increase in the cytosolic Ca2+ concentration in Dictyostelium discoideum by induction of Ca2+ release from intracellular stores and influx of extracellular Ca2+.

The Ca2+ stores of Dictyostelium discoideum amoebae take part in control of homoeostasis of the cytosolic free Ca2+ concentration ([Ca2+]i) and the cyclic-AMP-induced [Ca2+]i-signalling cascade. In order to characterize regulatory mechanisms of these stores, we incubated cells with the calmodulin antagonist calmidazolium. Measurement of permeabilized and intact cells in suspension with a Ca(2+)-sensitive electrode revealed that calmidazolium induced Ca2+ release from intracellular stores, influx of Ca2+ across the plasma membrane and subsequent efflux. In single fura-2-loaded cells calmidazolium evoked rapid and global transient elevations of [Ca2+]i. Other calmodulin antagonists (trifluoperazine, chlorpromazine, fendiline and W7) also induced transient elevations of [Ca2+]i, which were, however, slower and observed in fewer cells. The calmidazolium-induced influx of extracellular Ca2+ was inhibited by preincubation with 2,5-di-(t-butyl)-1, 4-hydroquinone (BHQ) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl), both known to interact with pumps of the inositol 1,4,5-trisphosphate (IP3)-sensitive store, and by the V-type H(+)-ATPase inhibitor bafilomycin A1, which affects the acidosomal Ca2+ store. Incubation with pump inhibitors did not itself induce changes in [Ca2+]i. We conclude that the effects of calmidazolium are, at least in part, mediated by its calmodulin-antagonizing properties, that it acts by inducing Ca2+ release from filled storage compartments, and that its target of action is both the IP3-sensitive store and the acidosome; emptying of these stores leads to influx of extracellular Ca2+.

Lung surfactant protein A (SP-A) activates a phosphoinositide/calcium signaling pathway in alveolar macrophages.

Lung surfactant protein A (SP-A), the main protein component of lung surfactant which lines the alveoli, strongly enhances serum-independent phagocytosis of bacteria by rat alveolar macrophages. We tested if the effect of SP-A is due to interaction with the macrophages or to opsonization of the bacteria. In phagocytosis assays with fluorescein isothiocyanate labeled bacteria, SP-A had no opsonic effect on Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, but enhanced phagocytosis by acting only on the macrophages. We characterized this activation mechanism. With single cell measurements of fura-2 loaded cells we demonstrate that SP-A raises the intracellular free calcium ion concentration 6 to 8 seconds after addition. This calcium mobilization is dose-dependent in that increased SP-A concentrations lead to a higher percentage of responding cells. Additionally, SP-A leads to a dose-dependent and transient generation of inositol 1,4.5-trisphosphate. Release of intracellular stored calcium by SP-A is a prerequisite for its stimulatory effect on phagocytosis, since SP-A-induced enhancement of phagocytosis can be impaired by prior addition of thapsigargin, a Ca(2+)-ATPase inhibitor that leads to depletion of intracellular calcium stores. We conclude that SP-A activates a phosphoinositide/calcium signaling pathway in alveolar macrophages leading to enhanced serum-independent phagocytosis of bacteria.

The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes.

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein.

The human protein P1 belongs to a newly discovered class of mammalian nuclear proteins with high sequence homology to yeast replication proteins. We present the entire amino acid sequence of the human protein P1 as predicted from the cDNA sequence, and show that P1 shares three central regions of high sequence similarity (about 75%) and a highly hydrophilic carboxy-terminal region with the yeast Mcm3 replication protein. The human genome most probably contains one P1 gene which is activated when HeLa cells progress to S phase, as shown by a several-fold increase in P1-specific mRNA. However, the amounts of P1 protein do not detectably change during this period, but P1 protein becomes phosphorylated at the beginning of S phase. In contrast to the yeast Mcm proteins, which disappear from nuclei after initiation of DNA replication, protein P1 remains in the nucleus during and after S phase. P1 is dispersed in mitotic cells and may be excluded from binding to chromosomes.

Introduction of calcium buffers into the cytosol of Dictyostelium discoideum amoebae alters cell morphology and inhibits chemotaxis.

Differentiating Dictyostelium discoideum amoebae respond chemotactically towards the attractant cAMP. To test whether chemotaxis requires the establishment of a spatial gradient of the cytosolic calcium concentration ([Ca2+]i) we scrape-loaded calcium chelating agents with different affinities for Ca2+ into the cytosol of the cells. The buffers were 1,2-bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) and its derivatives. Parameters analyzed were general cell morphology and the capability to protrude pseudopods and to migrate towards a cAMP-filled capillary. The chelators dose- and time-dependently inhibited spreading of the amoebae on the substrate. Both oriented pseudopod formation and locomotion of the cells were reduced. This effect was overcome by extracellular Ca2+, but not Mg2+. The effects of BAPTA derivatives were compared to the inhibition by BAPTA. A dose-response curve was obtained; 5,5'-difluoro-BAPTA was the most potent analogue. We conclude that a [Ca2+]i-gradient is necessary for orientation and locomotion. Chemotaxis experiments performed in the presence of extracellular EGTA revealed that liberation of Ca2+ from intracellular stores is sufficient for pseudopod formation; yet under physiological conditions influx of extracellular Ca2+ is also used to establish the gradient.

Calcium-sequestering organelles of Dictyostelium discoideum: changes in element content during early development as measured by electron probe X-ray microanalysis.

Starving Dictyostelium discoideum amoebae aggregate within a few hours by chemotaxis towards the attractant cAMP to form a multicellular organism. The differentiating cells possess rapid and efficient calcium buffering and sequestration systems which enable them to restrict changes in the cytosolic free calcium concentration temporally and spatially during their chemotactic reaction and allow the continuous accumulation of Ca2+ during development. In order to identify and to characterize calcium storage compartments, we analyzed the element content of amoebae at three consecutive stages of differentiation. Determination of the element distribution was done using energy-dispersive X-ray microanalysis of freeze-dried cryosections of rapid-frozen cells. Amoebae were frozen in the vegetative and aggregation-competent state and after formation of aggregates. Aggregation-competent as well as aggregated cells contained mass dense granules with large amounts of calcium together with phosphorous and either potassium or magnesium: in aggregation-competent cells calcium was colocalized with potassium, whereas in aggregated cells the mass dense granules contained calcium and magnesium. Although mass dense granules were also present in undifferentiated, vegetative cells, they contained only low amounts of phosphorous and potassium together with little Ca and Mg. We conclude that during their differentiation D. discoideum cells use an intracellular storage compartment to sequester Ca and other cations constantly throughout development.

Challenge with high concentrations of cyclic AMP induces transient changes in the cytosolic free calcium concentration in Dictyostelium discoideum.

Dictyostelium discoideum cells use cyclic AMP (cAMP) for chemotactic signaling as well as for differentiation. The precise regulation of the cytosolic Ca2+ concentration ([Ca2+]i) seems to play a key role for both processes. We performed single cell measurements of [Ca2+]i in amoebae that were starved in suspension for various times and scrape-loaded with the Ca2+ indicator fura-2. Stimulation of cells with cAMP at the concentration required to induce gene expression (> or = 100 microM) elicited a global transient increase in [Ca2+]i that depended on the presence of external Ca2+. Both vegetative and aggregation-competent cells displayed a rise in [Ca2+]i, with aggregation-competent cells responding more often than vegetative cells. Basal [Ca2+]i in the presence of Ca2+ was high in vegetative cells and declined during development; the cAMP-induced rise in [Ca2+]i was higher and lasted longer in vegetative cells than in aggregative cells. The addition of 2'-deoxy-cAMP, which binds to the cAMP receptor, induced an increase in [Ca2+]i, whereas the membrane-permeant analogue 8-bromo-cAMP that has a low affinity for the receptor but activates cAMP-dependent protein kinase had no effect. This indicates that the change in [Ca2+]i is mediated by the cell surface cAMP receptor. Since HC85 mutant cells, which lack the G alpha 2 subunit of the G-protein that couples the receptor to phospholipase C, also responded to stimulation with cAMP, the Ca2+ influx does not seem to be triggered by the phosphoinositide signaling cascade.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of lung surfactant protein A with alveolar macrophages.

We analyzed the binding mechanism of human recombinant lung surfactant protein A (SP-A) to rat alveolar macrophages using anti-SP-A antiserum and protein A coated onto gold particles. Results were compared with our recent data on binding and uptake of SP-A-coated colloidal gold particles. The rationale for the current approach was to avoid any possible steric effects on SP-A binding to the cell surface. Binding of unlabeled SP-A depends on the presence of calcium ions in the medium and involves a mannose-specific mechanism. Binding is partly inhibited by the collagenase-resistant fragment of SP-A, representing mainly the globular part of SP-A. Taken together, these facts indicate binding of SP-A via the carbohydrate binding site on the globular region of SP-A. On the other hand, a partial inhibition of SP-A binding by fragments of C1q (representing the collagenous region of C1q) indicates a second binding site for SP-A by the collagen-like portion to the C1q receptor of macrophages. We conclude that two different mechanisms are probably involved in SP-A binding to alveolar macrophages. Specificity of the binding was shown with fluorescein-labeled SP-A. Binding was inhibited by an excess of unlabeled SP-A. Binding and uptake of SP-A are seen only with alveolar macrophages and not with other macrophage populations isolated from rat, such as liver macrophages (Kupffer cells), resident peritoneal macrophages, and peritoneal macrophages activated by Corynebacterium parvum. Therefore, binding sites for SP-A occur exclusively on alveolar macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Intercellular guanosine-5'-0-(3-thiotriphosphate) blocks chemotactic motility of Dictyostelium discoideum amoebae.

Starving amoebae of the cellular slime mold Dictyostelium discoideum react chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine-5'-0-(2-thiotriphosphate) (GTP gamma S) or guanosine-5'-0-(2-thiodiphosphate) (GDP beta S) was scrape-loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP-filled glass capillary revealed a reduced capacity of loaded cells to migrate towards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose-dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired. In cells loaded with the analogs together with the calcium indicator fura-2, stimulation with 10 microM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]i at low levels with 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed. The data suggest that chemotactic migratory activity involves GTP-dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulation of membrane traffic that contributes to the directed locomotion.

Intracellular calcium during chemotaxis of Dictyostelium discoideum: a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements.

During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.