Aggregation-related and post-aggregation-related cohesive systems in Dictyostelium discoideum involve stage-specific, Ca-dependent, D1- or polymeric associations of membrane-bound moieties

The cells of D. discoideum acquire developmentally regulated cohesive properties during aggregation and fruiting body construction. On the bases of genetic, serological, and physiological evidence, it has been suggested that two distinct cohesive systems operate: an aggregation-related (AR) system that facilitates the formation of multicellular aggregates and post-aggregation-related (PAR) system that maintains the integrity of the aggregate thereafter. We had previously demonstrated that ghosts and membrane fragments retain the cohesive properties of the cell from which they were derived. Here, we describe a two-phase assay involving the Ca²⁺-dependent binding of ¹²⁵I-labeled cell ghosts in suspension to their unlabeled counterparts immobilized on plastic surfaces. Using this assay we show that the ghosts of newly aggregation-competent (8 h) cells and of cells from the ‘Mexican hat (18 h) stage’ of fruit construction can bind, each to its immobilized counterpart, but not heterologously. Furthermore, neither binds to the immobilized ghosts of vegetative cells. This provides direct, functional evidence demonstrating the existence of the two stage-specific cohesive systems. It also suggests that both cohesive acts involve at least dimeric associations of molecules or molecular complexes located within or on juxtaposed membranes. Using immobilized 8 and 18 h ghosts, the specific binding activities of ghosts prepared from cells harvested at stages throughout the morphogenetic sequence were assayed in order to describe the developmental kinetics of the two cohesive systems. The binding data suggest that the AR system appears soon after the start of the morphogenetic sequence, peaks during early aggregation and is progressively diminished thereafter. The PAR system makes its appearance after aggregation and accumulates thereafter. Both systems are present in migrating slugs.     

Stage-specific cohesion of cell ghosts and plasma-membrane fragments from Dictyostelium discoideum

During fruiting-body construction by Dictyostelium discoideum, the formation and subsequent maintenance of the multicellular assembly involve two stage-specific cohesive systems that are acquired sequentially and are distinguishable on serological and genetic grounds. We demonstrated that both systems, termed aggregation related (AR) and postaggregation related (PAR), can function in vitro. Ghosts prepared from cells of the wild-type and of a cohesion-defective mutant that were harvested during growth and at aggregation and postaggregative stages of fruiting-body construction exhibited the same cohesive properties as the cells from which they were derived. Membrane fragments prepared from the ghosts by mechanical disruption retained these cohesive properties.     

Coh A,a mutation affecting the post aggregative related (par) cohesive system of Dictyostelium discoideum

Three stage-specific cohesive systems operate in D. discoideum: VEG, elaborated by vegetative cells: AR, by aggregation competent cells; and PAR, by post aggregation stage cells. Previous study of a mutant strain JC-5 had shown the stability of its PAR system (but not the AR) to be temperature sensitive. However, the phenotypic expression of this mutation termed Coh A is complicated by the presence in that strain of a preexisting mutant gene Rde A, which accelerates developmental events generally and alters the pattern of morphogenesis. Genetic evidence presented here indicates that the two mutations have been separated by parasexual recombination yielding a Coh A, Rde A⁺ segregant class of which strain JC-36 is a prototype. At the permissive temperature, JC-36 follows a morphogenetic sequence like that of the wild type in respect to timing, morphogenetic pattern, and spore appearance. At the restrictive temperature, it forms normal aggregates at the usual time but exhibits two morphogenetic aberrancies during post aggregative development. First, fruit construction is arrested at a stage approximating the 16 hr “Bottle” stage of the wild type, though more squat and blunt tipped, and then the aggregate regresses. Cytodifferentiation into spores and stalk cells is also blocked. Second, a shift of slugs migrating normally at the permissive temperature to the restrictive causes the latter to disintegrate progressively as they leave clumps of cells behind them within the flattened sheath. JC-36 cells developing at the restrictive temperature also exhibited a decrease in EDTA resistant cohesivity attributable on two grounds to the sensitivity of the PAR system. In addition, the disappearance of the AR system completed in the wild type by the Mexicanhat (18–19 hr) stage is indefinitely arrested at an intermediate level in JC-36.     

An increase of calcium/manganese binding sites in cell ghosts associated with the acquisition of aggregative competence in Dictyostelium discoideum.

In Dictyostelium discoideum, the formation of multicellular aggregates represents the first morphogenetic event that leads ultimately to the construction of fruiting bodies. The altered adhesive properties of the cells can be demonstrated in ghosts derived from them which consist of largely intact membranes containing a few empty vesicles and exploded mitochondria but lacking nuclei, RNA, soluble cytoplasm and ATP [4]. A cofactor requirement for the aggregation of the ghosts can be satisfied by the following divalent cations: Ca²⁺, Mn²⁺, Zn²⁺ and Cu²⁺. In this paper it is shown that associated with the acquisition of aggregative competence is a 15–20-fold increase in the ghosts of sites capable of binding either Ca²⁺ or Mn²⁺ with relatively high affinity.     

Proteinase-activated receptor 4 (PAR4): activation and inhibition of rat platelet aggregation by PAR4-derived peptides

We studied the actions of receptor-activating peptide analogues (PAR4APs), modeled on the proteolytically-revealed tethered ligand sequence of murine proteinase-activated receptor-4 (PAR4), in a rat platelet aggregation assay. The PAR4APs GYPGKF-NH2 (GY-NH2) and AYPGKF-NH2 (AY-NH2) were able to cause aggregation with EC50 values of about 40 microM and 15 microM, respectively. The reverse human PAR4 sequence (VQGPYG-NH2, YG-NH2) and the PAR1AP SFLLR-NH2, did not cause aggregation. In contrast, trans-cinnamoyl-YPGKF-NH2 (tcY-NH2) did not cause aggregation but blocked aggregation caused by GY-NH2, AY-NH2, and thrombin without affecting ADP-mediated aggregation. We conclude that in contrast to the PAR1AP, the PAR4APs GY-NH2 and AY-NH2 activate rat platelets via a PAR4-related receptor and that peptide analogues modeled on the PAR4 tethered activating sequence can serve as useful agonist and antagonist probes for assessing the consequence of activating PAR4 either by PAR4APs or thrombin in rat tissue preparations.     

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).     

Multiple binding components for methyltrienolone in canine prostatic epithelial cells

Using a whole cell assay system, the androgen binding capacity of canine prostatic epithelial cells was evaluated in relation to their function. Radiolabeled Methyltrienolone (R1881) was used as the ligand in the presence of an excess of Triamcinolone acetonide and the amount of [3H]R1881 bound to the cells at equilibrium was determined by either displacement or saturation studies. With immature cells in culture (3 days of attachment), displacement analysis revealed the presence of high affinity binding sites which were also present in cells cultured for 10 days. With freshly dispersed prostatic cells (mostly secretory epithelial cells) as well as with older cells in culture (17 and 24 days), only less specific binding sites were observed with both unlabeled R1881 and/or dihydrotesterone (DHT). In contrast, only the high affinity androgen receptor (AR) was present in cytosolic extracts prepared from normal glands. Displacement studies performed with cultured cells at different stages of growth also showed that the basal level as well as the degree of low affinity binding increased during the maturation of non-proliferating cells. The presence of multiple binding components was demonstrated by saturation studies performed with either cultured or freshly dispersed cells. The first component, that was saturated at 5 nM of [3H]R1881, was due to AR while the other two binding components, showing positive-cooperativity (Hill coefficients of 1.90 and 5.07, respectively), were saturated at concentrations of 15 and 30 nM of [3H]R1881. In contrast, the Hill coefficient for the AR was 0.88 indicating the presence of an independent component. It was calculated that only 11.4% of the total uptake of R1881 was attributed to AR binding, suggesting that the remainder may represent an intracellular pool of androgens. Thus, a whole cell binding assay represents a dynamic system for the detection, by saturation studies, of binding components that are not revealed using the conventional displacement studies or cell-free systems. It is proposed that these acceptor sites may play a role in differentiated prostatic function rather than in cell proliferation.     

Mapping the interaction of bradykinin 1-5 with the exodomain of human protease activated receptor 4

The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1-5 (RPPGF), inhibits thrombin-induced human or mouse platelet aggregation. RPPGF binds to the exodomain of human protease-activated receptor 1 (PAR1). Studies determined if RPPGF also binds to the exodomain of human PAR4. RPPGF binds to a peptide of the thrombin cleavage site on PAR4. Recombinant wild-type and mutated exodomain of human PAR4 was prepared. The N-terminal arginine on RPPGF binds to the P2 position or proline46 on PAR4 to block thrombin cleavage. These data indicate that RPPGF influences thrombin activity by binding to the thrombin cleavage site on both PAR4 and PAR1.     

Different cellular targets for Cu- and Fe-catalyzed oxidation observed using a Cu-compatible thiobarbituric acid assay

The widely used thiobarbituric acid (TBA) assay for oxidative damage to biomolecules fails in Cu2(+)-containing solutions due to the formation of a cloudy precipitate. The chelation of Cu2+ ions with EDTA or Chelex was investigated. Both prevented precipitate formation, but only Chelex allowed proper color development in the TBA assay. The Chelex modified assay could be adapted to a variety of systems, and was applied to the detection of Cu2+/ascorbate dependent deoxyribose breakdown and oxidative damage in erythrocyte ghosts, lysates and whole cells. Using this method, it was shown that Cu2+/ascorbate caused membrane damage in ghosts but not in whole red blood cells (RBC). Fe3+/ascorbate, on the other hand, caused formation of TBA-reactive products even in whole RBC. When Cu2+ and Fe3+ were presented to isolated hemoglobin as their 1:1 nitrilotriacetate complexes, the protein bound 10-12 cupric ions per molecule, but no ferric ions. It is suggested that oxidative damage catalyzed by copper or iron ions has different cellular targets, determined by the different binding properties of the two metals to various cellular components.     

Isolation and characterization of EMS16, a C-lectin type protein from Echis multisquamatus venom, a potent and selective inhibitor of the alpha2beta1 integrin

We have isolated and characterized EMS16, a potent and selective inhibitor of the alpha2beta1 integrin, from Echis multisquamatus venom. It belongs to the family of C-lectin type of proteins (CLPs), and its amino acid sequence is homologous with other members of this protein family occurring in snake venoms. EMS16 (M(r) approximately 33K) is a heterodimer composed of two distinct subunits linked by S-S bonds. K562 cells transfected with alpha2 integrin selectively adhere to immobilized EMS16, but not to two other snake venom-derived CLPs, echicetin and alboaggregin B. EMS16 inhibits adhesion of alpha2beta1-expressing cells to immobilized collagen I at picomolar concentrations, and the platelet/collagen I interaction in solution at nanomolar concentrations. EMS16 inhibits binding of isolated, recombinant I domain of alpha2 integrin to collagen in an ELISA assay, but not the interaction of isolated I domain of alpha1 integrin with collagen IV. Studies with monoclonal antibodies suggested that EMS16 binds to the alpha2 subunit of the integrin. EMS16 inhibits collagen-induced platelet aggregation, but has no effect on aggregation induced by other agonists such as ADP, thromboxane analogue (U46619), TRAP, or convulxin. EMS16 also inhibits collagen-induced, but not convulxin-induced, platelet cytosolic Ca(2+) mobilization. In addition, EMS16 inhibits HUVEC migration in collagen I gel. In conclusion, we report a new, potent viper venom-derived inhibitor of alpha2beta1 integrin, which does not belong to the disintegrin family.     

Control of the aggregation factor-aggregation receptor interaction in sponges by protein kinase C

By means of immunobiochemical and immunocytological techniques it was found that the aggregation factor (AF) from the sponge Geodia cydonium is stored in vesicles of spherulous cells. During the reaggregation process of dissociated cells, the AF which is present extracellularly was determined to be bound to the cell-surface-associated aggregation receptor (AR) only during the initial phase (0-5 h after addition of the AF to the single cell suspension). At later stages (20 h), the AF colocalized with extracellular structures, e.g., collagen and glycoconjugates. Immobilized to nitrocellulose, the AR, a molecule with Mr of 43.5 kDa, displayed its binding affinity to the AF only if it was isolated from early aggregates (5 h). The transition of the AF-susceptible to the AF-deficient state of the plasma membrane was mimicked in vitro by incubation of plasma membranes from early aggregates with purified protein kinase C. This conversion to the AF-deficient state could be prevented by the protein kinase C inhibitor staurosporine. Together with earlier findings, which revealed that the AR is phosphorylated by protein kinase C, we propose that in the sponge system this enzyme controls intercellular processes involved in morphogenesis.     

Enzymatic dissection of embryonic cell adhesive mechanisms

In this paper we describe a kinetic assay for cell adhesion which measures the formation of cell clusters. Cluster formation is dependent on both calcium and protein synthesis, two parameters essential for the formation of histotypic aggregates. We also describe modifications of the stndard method for trypsinization of tissues which result in populations of single cells that appear to bear intact and functional cell surface adhesive systems. These modifications involve the use of chymotrypsin and the inclusion of calcium during enzyme digestion of tissues with trypsin and chymotrypsin. Using the cluster formation assay and the modified tissue dissociation techniques, we demonstrate the presence of two functionally distinct adhesive systems operating among embryonic chick neural retina cells. These two systems differ in proteolytic sensitivity, protection by calcium against proteolysis, dependence on calcium for function and morphogenetic potential. Cells possessing one of these intact adhesive systems are capable of extensive morphogenetic interactions in the absence of protein synthesis.     

High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase

An L-arabinose isomerase of Escherichia coli was immobilized using covalent binding to agarose to produce D-tagatose, a bulking sweetener that can be economically used as a sugar substitute. The immobilized L-arabinose isomerase stably produced an average of 7.5 g-tagatose/L.day for 7 days with a productivity exceeding that of the free enzyme (0.47 vs 0.30 mg/U.day). Using a scaled-up immobilized enzyme system, 99.9 g-tagatose/L was produced from galactose with 20% equilibrium in 48 h. The process was repeated two more times with production of 104.1 and 103.5 g-tagatose/L. D-Tagatose production using an immobilized L-arabinose isomerase has a high potential for commercial application.     

A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy

A fluorescence microscopic assay for the activity of complement, perforin, and other cytolytic proteins which form transmembrane pores in cellular membranes is described. The assay was worked out and tested with red blood cell membranes (ghosts) and was then applied to intact hemoglobin-free cells. Resealed human erythrocyte ghosts were incubated with complement or perforin. A small polar fluorescent probe (fluorescein-labeled 1-kDa dextran, FD1) which permeates through complement and perforin pores but not through normal cell membranes was added to the samples. The capability of the confocal laser scanning microscope (CLSM) to generate thin optical sections was exploited to visualize and quantitate fluorescence inside single ghosts and thus determine the fraction of ghosts which had become permeable for FD1. The activity of complement or perforin was quantitated by plotting the fraction of permeable cells versus the concentration of the pore-forming protein. The results were in good agreement with those of a conventional hemolytic assay. The CLSM-based assay was then applied to intact hemoglobin-free cells for which only few alternative assays are available. Compared to conventional hemolytic assays for the activity of pore-forming proteins the assay described here can be applied to a large variety of natural and artificial membrane systems. The assay can be performed under nonlysing conditions. Furthermore, the assay is simple, relatively fast, and requires only extremely small amounts of cells and pore-forming proteins.     

Effect of synexin on aggregation and fusion of chromaffin granule ghosts at pH 6

Fusion of chromaffin granule ghosts was induced by synexin at pH 6, 37 degrees C, in the presence of 10(-7) M Ca2+. To study the kinetics and extent of this fusion process we employed two assays that monitored continuously mixing of aqueous contents or membrane mixing by fluorescence intensity increases. In both assays chromaffin granule ghosts were either labeled on the membrane or in the included aqueous phase. The ratios of blank to labeled chromaffin granule ghosts were varied from 1 to 10. The results were analyzed in terms of a mass action kinetic model, which views the overall fusion reaction as a sequence of a second-order process of aggregation followed by a first-order fusion reaction. The model calculations gave fare simulations and predictions of the experimental results. The rate constants describing membrane mixing are more than 2-fold larger than those for volume mixing. The analysis also indicated that the initial aggregation and fusion processes, up to dimer formation, were extremely fast. The rate constant of aggregation was close to the limit in diffusion-controlled processes, whereas the fusion rate constant was about the same as found in fastest virus-liposome fusion events at pH 5. A small increase in volume was found to accompany the fusion between chromaffin granule ghosts. Using ratios of synexin to chromaffin granule ghost protein of 0.13, 0.41 and 1.15 indicated that the overall fusion rate was larger for the intermediate (0.41) case. The analysis showed that the main activity of synexin was an enhancement of the rate of aggregation. At intermediate or excessive synexin concentrations it, respectively, promoted moderately, or inhibited the actual fusion step.     

Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs)

We tested the hypothesis that human tissue kallikreins (hKs) may regulate signal transduction by cleaving and activating proteinase-activated receptors (PARs). We found that hK5, 6 and 14 cleaved PAR N-terminal peptide sequences representing the cleavage/activation motifs of human PAR1 and PAR2 to yield receptor-activating peptides. hK5, 6 and 14 activated calcium signalling in rat PAR2-expressing (but not background) KNRK cells. Calcium signalling in HEK cells co-expressing human PAR1 and PAR2 was also triggered by hK14 (via PAR1 and PAR2) and hK6 (via PAR2). In isolated rat platelets that do not express PAR1, but signal via PAR4, hK14 also activated PAR-dependent calcium signalling responses and triggered aggregation. The aggregation response elicited by hK14 was in contrast to the lack of aggregation triggered by hK5 and 6. hK14 also caused vasorelaxation in a phenylephrine-preconstricted rat aorta ring assay and triggered oedema in an in vivo model of murine paw inflammation. We propose that, like thrombin and trypsin, the kallikreins must now be considered as important 'hormonal' regulators of tissue function, very likely acting in part via PARs.