Immunohistochemical Localization of Cyclic GMP in Aggregating Polysphondylium violaceum

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.     

Immunohistochemical localization of cyclic GMP in aggregating Polysphondylium violaceum.

Using indirect immunofluorescence technique, it has been possible to localize cyclic GMP in Polysphondylium violaceum cells. The bound cyclic nucleotide is localized throughout the cell during early stages, however, this staining increases and there is marked localization of cyclic GMP in the nuclear areas of the cells when aggregation is in full swing. Over 90% of the cells exhibited intense nuclear staining by 6 h and this decreased to less than 10% by 10 h.     

Whorl Formation in Polysphondylium violaceum: Relevance to Organization by Cyclic AMP

Whorl formation in Polysphondylium is a simple and good system for the study of pattern formation. The first step of whorl formation is characterized by separation of the rear cell mass from an advancing primary mass during culmination. Using the iontophoresis method, it has been shown that after the establishment of multicellular organization cells respond chemotactically to 3',5' -cyclic adenosine monophosphate (cAMP), but not to glorin, the chemoattaractant at the aggregating stage. P. violaceum cell masses were also found to secrete actively cAMP. Therefore, morphogenetic movement of P. violaceum cells after aggregation could be controlled mainly by the cAMP signalling system. Vital staining of cells with neutral red (NR) revealed that there are anterior-like cells stained well with NR in the posterior region of a migrating P. violaceum slug, and that the staining pattern changes markedly during whorl formation. Just before separation of the whorl mass, the anterior-like cells altered their distribution, and eventually were arranged as an equatorial band on the surface of the presumptive whorl mass, which probably would turn to organizing tip cells of the secondary masses left on the primary stalk. Thus whorl formation may be caused by separation of the strongest cAMP-source into two regions; the primary (anterior) and secondary (posterior) tips.     

Acidic protein kinases of Polysphondylium violaceum: Characterization and developmental regulation

Summary The cellular slime mould Polysphondylium violaceum contains two vegetative stage specific acidic (casein) kinases. These two enzymes have been partially purified and their properties investigated. Both utilise casein as their preferred substrate but they can be distinguished in a number of ways, including their responses to spermine, heparin and salt. In addition, they have different affinities for their substrates and different pH activity profiles. It is suggested that they may play a role in a vegetative specific function such as cell division.     

Cytosolic cAMP-dependent protein kinase of Polysphondylium violaceum: developmental regulation and properties

Summary The cellular slime mould Polysphondylium violaceum contains a cAMP-dependent protein kinase resembling the mammalian type I enzyme. The appearance of this enzyme is developmentally regulated. The level of kinase activity is very low in vegetative cell and increases more than tenfold during differentiation.The catalytic subunit of this cAMP-dependent protein kinase has a native molecular weight of 60–80 kDa, an isoelectric point of 5.7 and an apparent K_m for ATP and Kemptide of 50 and 13.4 µM respectively. It is characterised by its sensitivity to a synthetic inhibitor specific for cAMP-dependent protein kinase. The regulatory subunit has a molecular weight of 50 kDa.Abbreviations HEPES N-2-Hydroxyethylpiperazine-N-2-ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(ßaminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulphate     

Analysis of developmentally defective chemical signaling mutants of Polysphondylium violaceum.

Six aggregation-defective mutants of Polysphondylium violaceum dependent on external addition of the pheromone D factor for aggregation were isolated after nitrosoguanidine mutagenesis. With a screening technique based on synergistic development, D-factor-dependent mutants can be separated from other kinds of aggregateless mutants. Genetic complementation analyses of the newly isolated mutants showed them to be mutant at the aggA locus. Individual mutants exhibited different sensitivities to D factor(s), responding maximally over a 300-fold range of concentrations.     

Developmental regulation of production of an aggregation-stimulating factor from the cellular slime mold Polysphondylium violaceum

An excreted, dialyzable component(s) produced during development of wild-type Polysphondylium violaceum has been previously shown to stimulate aggregation of aggregateless mutants in the complementation group aggA. Production of this aggregation-stimulating factor, called D factor, is greater during development in liquid culture than during development on a surface. after partial purification of crude D factor using high performance liquid chromatography, multiple species are found that retain the ability to stimulate aggregation of the aggA mutants. The three major components (DfA, DfB, and DfC) show decreasing polarity based on purification using reverse-phase chromatography. The proportion of each component secreted varies, depending on the developmental conditions (surface versus liquid) and the time after starvation when the factors are isolated. Preliminary physical and chemical characterization of the three D factor components suggests that they are related.     

The regulation of cellular slime mold development: a factor causing development of Polysphondylium violaceum aggregation-defective mutants.

aggA mutants of Polysphondylium violaceum develop normally in synergistic mixtures with other aggregation-defective mutants. Cell to cell contact is not necessary for development. A small dialyzable factor(s) produced by wild-type and other aggregation-defective mutants triggers development of aggA mutants. This factor (D factor) is developmentally regulated, appearing early in development and then disappearing. Mutants require D factor until aggregation has just begun and then they can continue even in the absence of added factor. D factor is produced by many, but not all species of cellular slime molds and is developmentally regulated in Dictyostelium discoideum as well as P. violaceum.     

Chemotactic response of wild-type and aggregation-defective mutants of Polysphondylium violaceum

Abstract. The aggregation-specific chemoattractant for Polysphondylium violaceum is N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester, or glorin. Wild-type amoebae allowed to develop in liquid culture acquire increased ability to respond to glorin shortly after starvation, i.e., just prior to the time they become aggregation competent. Similarly, as development proceeds, the amoebae show decreased sensitivity to folic acid, but they show almost no response to cyclic AMP at any time during their development in liquid culture. The optimum concentrations for the chemotactic response are 10^-8 M for glorin and 10^-5–10^-6 M for folic acid. A class of aggregation-defective mutants, aggA , will not aggregate in the absence of an excreted pheromone, D factor. During development in liquid culture in the presence or absence of D factor, these aggA mutants show a chemotactic response similar to that of wild-type amoebae to folic acid and glorin. However, D factor does enhance the chemotactic response of aggA mutants to glorin. In the absence of D factor, mutant amoebae will form fruiting bodies if exposed to a chemotactic gradient of either folic acid or glorin. Under these conditions, the mutant amoebae circumvent the requirement for D factor in order to develop.     

Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum

The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with Kd values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 10(-5) M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10-100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTP gamma S binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.     

Syngenic Divisions of the Cellular Slime Mold Polysphondylium violaceum*

SYNOPSIS. Mating type analysis of new isolates of Polysphondylium violaceum supports the subdivision of this morphological species into two reproductively isolated breeding groups or syngens. Representatives of both syngens have been identified in soil samples taken from widely separated geographical locations. Intersyngenic cross reactions have been observed in some stocks.     

Cyclic GMP and photoreceptor function.

A single photon can be detected by a rod photoreceptor cell. The absorption of light by rhodopsin triggers a cascade of reactions that amplifies the photon signal and results in ion channel closure with hyperpolarization of the rod photoreceptor cell. Light-induced conformational changes in rhodopsin facilitate the binding of a guanosine nucleotide-binding protein, transducin, which then undergoes a GTP-GDP exchange reaction and dissociation of the transducin complex. A subunit of transducin then activates a phosphodiesterase complex that hydrolyzes cyclic GMP. In darkness, cyclic GMP binds to cation channels of the photoreceptor plasma membrane, maintaining them in an open configuration. The light-induced reduction in cyclic GMP concentration dissociates the bound cyclic GMP, resulting in channel closure and hyperpolarization. Down-regulation of the cascade involves other proteins that block the interaction of transducin with rhodopsin and another protein that may interfere with transducin recycling. Cone photoreceptors possess a light-activated cascade that follows the rod format, but it is composed of proteins that are homologous to those of rod photoreceptors. Phototransduction in invertebrate photoreceptors uses rhodopsin to activate a cascade that uses phosphoinositides and calcium ion to regulate membrane polarization.     

Cyclic GMP and lectin-induced lymphocyte activation.

Purified human peripheral lymphocytes incubated with the mitogenic plant lectins phytohemagglutinin and concanavalin A were examined for alterations in intracellular cGMP and cAMP under a variety of experimental conditions and using multiple techniques for the isolation and purification of cGMP and cAMP before assay of the cyclic nucleotides by radioimmunoassay. In contrast to work reported by others, we have been unable to demonstrate consistent increases in cGMP under any of the experimental conditions used and with any of the various purification schemes. In these same experiments exogenous cGMP added to the lymphocytes could be measured, and the immunoreactive material was destroyed by cyclic nucleotide phosphodiesterase, indicating that our inability to measure increases in cGMP was not caused by our inability to measure cGMP. Under identical experimental conditions, small but consistent and statistically significant increases in cAMP were noted. In addition, other parameters of lymphocyte activation, 45Ca uptake (an early parameter), and incorporation of 3H thymidine into DNA were unimpaired. These data call to question the concept of cGMP as the second messenger in lectin-stimulated human peripheral lymphocytes.     

Cyclic GMP signaling and regulation of SERCA activity during cardiac myocyte contraction

We tested the hypothesis that cGMP-induced reductions in cardiac myocyte function were related to activation of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and cGMP-dependent phosphorylation of phospholamban. Ventricular myocyte function was measured using a video edge detector (n = 11 rabbits). Thapsigargin (TG) or cyclopiazonic acid (CPA) were used to inhibit SERCA. 8-Bromo-cGMP was added at 10(-6), 10(-5) M followed by TG 10(-8) M or KT5823 (cGMP-protein kinase inhibitor, 10(-6) M) prior to TG or CPA. Cyclic GMP-dependent protein phosphorylation and immunoblotting with anti-phospholamban antibody were examined. TG 10(-8) M significantly increased percent shortening (from 6.6+/-0.7 to 9.1+/-1.3%). Cyclic GMP 10(-5) M significantly decreased cell shortening from 9.3+/-0.9 to 5.1+/-0.6%. This was partially reversed by KT5823 (5.1+/-0.6 to 8.2+/-1.4%) suggesting that negative functional effects of cGMP were partially through the cGMP-dependent protein kinase. Addition of TG after cGMP also reduced the negative effects of cGMP on myocyte shortening suggesting involvement of SERCA in cGMP signaling. TG after cGMP and KT5823 treatment did not alter myocyte contractility (8.2+/-1.4 to 7.2+/-1.3%). CPA had similar effects as those of TG. Protein phosphorylation and immunoblotting showed that phospholamban was a target of the cGMP protein kinase. These results indicated that the cyclic GMP-induced reductions in myocyte function were partially mediated through the action of SERCA. It further suggested that cGMP signaling affects myocyte function through phosphorylation of phospholamban which regulates SERCA activity.     

Purification, characterization, and partial structure of D factor from Polysphondylium violaceum

The A component of D factor (DfA) was overproduced during development of wild type Polyspondylium violaceum strain China after starvation in liquid medium. Crude DfA excreted by strain China was partially purified by ultrafiltration using Amicon YM10 and YM2 filters with DfA extracted from the filtrate by absorption onto a preparative grade C-18 resin. The concentrated material was further purified on a C-18 analytical column using both acetonitrile:water and methanol:water gradients. This highly purified fraction was a single component with a final specific activity of greater than 10(6) units per mg dry weight. Purified DfA is red having a broad visible absorbance at 500 nm and a ultraviolet (uv) absorbance at 290-300 nm. The red chromophore is sensitive to pH and to oxidation-reduction. 1H and 13C nmr studies with purified DfA indicate that it is a C11 compound with both polar and non-polar regions. The non-polar region has been identified as a hexanone and is the same as the side chain of DIF from Dictyostelium discoideum. Purified DfA has been used in studies with the D factor non-producing mutant, tsg-119 cyc-1 aggA586 (A586), to show that neither production of glorin nor chemotactic sensitivity to glorin are affected by D factor. However, founder cells develop in A586 mutant populations only after addition of D factor. These data suggest that DfA may be necessary for induction of aggregate formation by aggregation-competent amoebae.     

Cyclic GMP regulation of a voltage-activated K channel in dissociated Enterocytes

Summary Enterrocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from –23 to – 39 mV and 29 to 13 M, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at –80 mV and subsequently stepped to a series of depolarizing voltages (from–70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above –20 mV. This current was sensitive to holding potential and was not activated when the cells were held at –40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the currentcarrying ion. BaCl₂ (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC₅₀ for charybdotoxin was 50 nM. Cyclic. GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above–40 mV. Previous studies with intact flounder epithelium showed that K recycling and net K secretion were inhibited by Ba²⁺ and by cGMP. We suggest that the channel responsible for the whole-cell current described in this study may be important in K recycling and secretion.     

Association of a cGMP-binding activity with nuclei isolated from amoebae of Dictyostelium discoideum and Polysphondylium violaceum.

When vegetative and early slug stage amoebae of Dictyostelium discoideum or Polysphondylium violaceum were lysed by filter breakage in a nuclear isolation buffer not containing detergents, substantial levels of a cGMP-binding activity with slow-dissociation kinetics were detected. After fractionation by centrifugation, 50% or more of this binding activity was associated with isolated nuclei. In addition, with Polysphondylium cells, the fraction of stable, nuclear-associated binding activity appeared to increase during cell aggregation. These results support the idea that cGMP might function in the nucleus during early development.