Pheromones do regulate sexual development in Dictyostelium discoideum.

Based on negative data, Douglas et al refuted earlier work by the O'Day laboratory on the role of pheromones in the sexual development of Dictyostelium discoideum. This letter addresses some of the issues with their study.

Cannibalistic sexual phagocytosis in Dictyostelium discoideum is modulated by adenosine via an A2-like receptor.

A unique aspect of phagocytosis during the sexual cycle of Dictyostelium discoideum is the ability of the zygote giant cell (ZGC) to attract and engulf hundreds of amoebae of the same species. The work presented here is one of two initial attempts to understand the signal transduction pathways that are involved during this event. Our data indicate that the uptake of amoebae by the ZGCs is negatively modulated by 5'-AMP and adenosine (ADO). The hierarchical inhibition of phagocytosis by the ADO analogues, N-ethylcarboxyadiadenosine and phenyl isopropyl adenosine, argue that the phagocytosis of amoebae by ZGCs is mediated by an ADO receptor, which is similar to the purinogenic class of receptor, A2. As with the A2 receptor, stimulation of cells by ADO binding causes an up-regulation of cAMP synthesis thereby increasing the intracellular levels of cAMP. During the phagocytic phase of sexual development, 5'-AMP and ADO undergo marked and successive increases in amounts supporting their natural role in modulating phagocytosis. This negative modulation of phagocytosis by ADO is similar to that found in mammalian macrophages and may represent an evolutionary precursor to that regulatory process. Furthermore, the slowing of phagocytosis has important implications to sexual development of D. discoideum.

Detection of calmodulin-binding proteins and calmodulin-dependent phosphorylation linked to calmodulin-dependent chemotaxis to folic and cAMP in Dictyostelium.

Calmodulin (CaM) antagonists, trifluoperazine (TFP) or calmidazolium (R24571), dose-dependently inhibited cAMP and folic acid (FA) chemotaxis in Dictyostelium. Developing, starved, and refed cells were compared to determine if certain CaM-binding proteins (CaMBPs) and CaM-dependent phosphorylation events could be identified as potential downstream effectors. Recombinant CaM ([35S]VU-1-CaM) gel overlays coupled with cell fractionation revealed at least three dozen Ca(2+)-dependent and around 12 Ca(2+)-independent CaMBPs in Dictyostelium. The CaMBPs associated with early development were also found in experimentally starved cells (cAMP chemotaxis), but were different for the CaMBP population linked to growth-phase cells (FA chemotaxis). Probing Western blots with phosphoserine antibodies revealed several phosphoprotein bands that displayed increases when cAMP-responsive cells were treated with TFP. In FA-responsive cells, several but distinct phosphoproteins decreased when treated with TFP. These data show that unique CaMBPs are present in growing, FA-chemosensitive cells vs. starved cAMP-chemoresponsive cells that may be important for mediating CaM-dependent events during chemotaxis.

Signal transduction during cannibalistic sexual phagocytosis: calcium is not the trigger but GTP-binding protein function is essential.

After fertilization, the zygote giant cell of Dictyostelium discoideum chemoattracts and subsequently engulfs hundreds of amoebae of the same species and strains from which it was derived. A pharmacological approach indicates that, while it may have some role, calcium is not the trigger for this cannibalistic phagocytic process. Of several agents that perturb intracellular calcium levels [A23187, LaCl, 8-diethylamino-octyl-3,4,5-trimethoxylbenzoate (TMB-8), and chlorotetracycline], only A23187 had an effect in reducing amoebal ingestion. In keeping with this, agents which interfered with downstream effectors of calcium function did not alter sexual phagocytosis. Calmidazolium and trifluoperazine, which inhibit calmodulin function, were ineffective, as were a protein kinase C inhibitor (staurosporine) and activator (phorbol 12-myristate 13-acetate). On the other hand, the nucleotide analogues GTP gamma S and GDP beta S both inhibited sexual phagocytosis indicating a role for GTP-binding protein activity at some stage in the process. Sub-fractionation of cells from non-phagocytic and phagocytic stage cell cultures followed by immunolocalization after SDS-PAGE and western blotting revealed a number of GTP-binding proteins in both the cell membrane and intracellular membrane fractions that might function during the events of sexual phagocytosis.

Comparative analysis of chemotaxis in Dictyostelium using a radial bioassay method: protein tyrosine kinase activity is required for chemotaxis to folate but not to cAMP.

The role of signal transduction during chemotaxis of Dictyostelium discoideum cells to cAMP and folic acid was investigated using a radial bioassay technique. The effects of signalling agonists were assessed by measuring the diameters of visible rings formed by the outward migration of amoebae up radial gradients of chemoattractant. This rapid and simple bioassay method yields chemotactic rates equivalent to more complex assay systems. In support of previous studies, chemotaxis toward both cAMP and folic acid was inhibited in a dose-dependent manner by LaCl3, EDTA, chlorotetracycline and A1F3, supporting the importance of calcium ions and G protein-mediated signalling in both chemotactic events. The work was extended by examining the effects of the protein tyrosine kinase inhibitor genistein. This agent inhibited chemotaxis to folate in a dose-dependent manner but had no observable effect on chemotaxis toward cAMP. The notion that phosphorylation of proteins on tyrosine residues is critical for chemotaxis to folic acid was supported by Western blotting experiments with monoclonal anti-phosphotyrosine antibodies which detected two candidate proteins of M(r) 52,000 and 38,000 in the membranes of folate-responsive amoebae. These two bands disappeared with starvation which leads to the loss of responsiveness of folic acid and the acquisition of responsiveness to cAMP. Time-lapse videomicrography also revealed some unique differences in chemotactic response. Starved cells responded to cAMP as individuals but feeding cells chemoattracted to folic acid on a populational basis. The ability to compare two different types of chemotaxis using a simple, rapid and accurate bioassay system should enhance future studies of chemotaxis in wild-type and mutant strains of D. discoideum.

Calmodulin function and calmodulin-binding proteins during autoactivation and spore germination in Dictyostelium discoideum.

Dictyostelium discoideum spores can be activated to initiate germination either endogenously via a diffusible autoactivator, or exogenously via heat. Following activation, three successive stages of germination occur, the lag stage, spore swelling and amoebal emergence. A previous study [Lydan M. A. and Cotter D. A. (1994) FEBS Lett. 115, 137-142] has shown that spore swelling is dependent on the activity of calmodulin. In this study, the calmodulin antagonists trifluoperazine and calmidazolium inhibited autoactivation, but had no effect upon heat activation. These agents also inhibited amoebal emergence following either form of activation. The effects caused by the anti-calmodulin agents were specific to an inhibition of calmodulin function since agents which modulate the activity of protein kinase C had no effect upon spore germination. A calcium-dependent calmodulin-binding protein of about 64,000 M(r) may be associated with the process of autoactivation since it was only seen in those spores which respond to the autoactivator. Overall, this study provides evidence to show that calmodulin plays a regulatory role during autoactivation and amoebal emergence during spore germination in D. discoideum and provides evidence for the calmodulin-dependent mechanisms which mediate each of these phases of germination.

Neurobiology of mother-infant interactions: experience and central nervous system plasticity across development and generations.

The optimal coordination between the new mammalian mother and her young involves a sequence of behaviors on the part of each that ensures that the young will be adequately cared for and show healthy physical, emotional, and social development. This coordination is accomplished by each member of the relationship having the appropriate sensitivities and responses to cues that characterize the other. Among many mammalian species, new mothers are attracted to their infants' odors and some recognize them based on their odors; they also respond to their infants' vocalizations, thermal properties, and touch qualities. Together these cues ensure that the mother will nurse and protect the offspring and provide them with the appropriate physical and stimulus environment in which to develop. The young, in turn, orient to the mother and show a suckling pattern that reflects a sensitivity to the mothers odor, touch, and temperature characteristics. This article explores the sensory, endocrine, and neural mechanisms that underlie this early mother-young relationship, from the perspective of, first, the mother and, then, the young, noting the parallels between them. It emphasizes the importance of learning and plasticity in the formation and maintenance of the mother-young relationship and mediation of these experience effects by the brain and its neurochemistry. Finally, it discusses ways in which the infants' early experiences with their mothers (or the absence of these experiences) may come to influence how they respond to their own infants when they grow up, providing a psychobiological mechanism for the inter-generational transmission of parenting styles and responsiveness.

Aggregation during sexual development in Dictyostelium discoideum.

A microcinematographic analysis of the behaviour and movements of cells and cell masses in mated cultures (NC4 X VI2) of Dictyostelium discoideum indicates that a chemotactic process directs cell aggregation during macrocyst development. Zygote giant cells form before aggregation begins and act as the aggregation centres. Young multicellular macrocyst stages are sources of cyclic AMP, and amoebae from macrocyst cultures orient chemotactically to cyclic AMP. The data, coupled with other characteristics such as pulsatile streaming, suggest that the aggregation process leading to macrycyst development is the same as that occurring during fruit construction. Other aspects of sexual development are also discussed. Based upon these data, we propose a model for the sequence of events leading to macrocyst development in D. discoideum.

Cell differentiation during fruiting body formation in polysphondylium pallidum.

The spatial pattern of cellular differentiation was studied during fruiting body formation in Polysphondylium pallidum using 3 different staining methods: Calcofluor fluorescence (cellulose accumulation), neutral red (prestalk cells) and immunofluorescence (prespore cells). Neutral-red staining revealed the existence of a clear prestalk region which becomes evident during aggregation and continues throughout culmination. Immunofluorescent staining demonstrated that cells in the prestalk region gradually lose their presporeness (fluorescence) as they are transformed into differentiated stalk cells. Calcofluor staining revealed that stalk cell differentiation begins during the mid-aggregation phase and that the mode of formation of the main stalk and the side branches differs slightly in morphology. Calcofluor staining also demonstrated the development, during aggregation, of a thick cellulosic girdle with lateral tubular extensions which surround the aggregation streams. The above results are discussed in terms of our present knowledge about differentiation and morphogenesis in cellular slime moulds.

Acid protease activity during germination of microcysts of the cellular slime mold Polysphondylium pallidum.

Extracts of dormant microcysts of Polysphondylium pallidum demonstrate pH optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular pH 6.0 caseinolytic specific activity does not change significantly. The pH 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the pH 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the pH 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study.

Alpha-mannosidase and microcyst differentiation in the cellular slime mold Polysphondylium pallidum.

The intracellular and extracellular pattern of alpha-mannosidase (EC 3.2.1.24) activity was studied during microcyst differentiation in the cellular slime mold, Polysphondylium pallidum. The evidence suggests that microcyst differentiation requires continuous protein synthesis. alpha-Mannosidase activity is present in amoebae and increases with differentiation, and the data indicate that this increase in activity requires concurrent protein synthesis. The enzyme is excreted during the differentiation process, and the release of the enzyme is not stopped by cycloheximide. A cystless mutant does not show the normal intracellular pattern of alpha-mannosidase but does excrete the enzyme. Microcyst differentiation is proposed as an alternative system to multicellular slime mold development for the biochemical analysis of certain aspects of cellular differentiation.

Lectin binding and inhibition studies reveal the importance of D-glucose, D-mannose and N-acetylglucosamine during early sexual development of Dictyostelium discoideum.

Fluorescein-conjugated and non-conjugated lectins were used to determine which surface sugars are involved in the early events of sexual (macrocyst) development in Dictyostelium discoideum. Only zygote giant cells showed unique binding of FITC-WGA and FITC-PNA while all cell types (amoebae, gametes, binucleates, giant cells) showed identical patterns of FITC-Con A, -Gorse and -RCA II binding. In spite of its non-selective labelling of all cell types, Con A inhibited macrocyst formation. The temporal addition of Con A with and without specific hapten sugars indicates the importance of both D-mannose and D-glucose in phagocytosis and, possibly, cell fusion. WGA also inhibited macrocyst formation. Varying the time of addition of the lectin plus/minus its primary hapten sugar implicates N-acetylglucosamine as being important in cell fusion. Neither Gorse, RCA II nor PNA had any detectable inhibitory effects on macrocyst development leaving the appearance of increased PNA receptors at the giant cell surface as an enigma.

Concanavalin A and wheat germ agglutinin binding glycoproteins associated with cell fusion and zygote differentiation in Dictyostelium discoideum: effects of calcium ions and tunicamycin on glycoprotein profiles.

To determine which glycoproteins may be critical to sexual development in Dictyostelium discoideum, cell samples from different developmental stages were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and blotted to nitrocellulose. Concanavalin A (ConA) and wheat germ agglutinin (WGA) binding proteins were visualized on the blots using an immunochemical procedure employing peroxidase-antiperoxidase. ConA labelled at least 28 proteins, but only one band showed calcium-dependent changes in its expression. WGA bound at least 30 proteins and changes in several bands were observed that did not occur in calcium-deficient controls. Two WGA-binding glycoproteins which migrated at 200 and 166 kilodaltons (kDa), respectively, showed developmental changes associated with the time of cell fusion. One WGA-binding and one ConA-binding glycoprotein migrating at 130 and 126 kDa, respectively, appeared later during sexual development, in association with the phase of zygote differentiation. Several WGA- and ConA-binding glycoproteins decreased during sexual development, but were not affected by the absence of calcium ions. Tunicamycin (1 microgram/mL) inhibited cell fusion when added to sexual cultures prior to the appearance of the 166-kDa glycoprotein gp166. The effects of this inhibitor on development support the importance of glycoproteins to cell fusion during sexual development in D. discoideum.

The immediate induction of extensive cell fusion by Ca2+ addition in Dictyostelium discoideum.

In mated cultures (NC4 X V12) of Dictyostelium discoideum containing 1.0 mM CaCl2, cell fusion generates large numbers of binucleate cells which develop into zygote giant cells. In the absence of Ca2+, binucleate formation does not occur. When 1.0 mM CaCl2 is added to Ca2+-deficient cultures at 18 h, 50% of the cells fuse within 45 min producing large multinucleate syncytia. Small, presumptive gametes appear in Ca2+-deficient cultures and reach a peak of about 20% of the cell population by 10 h, but they maintain this plateau and do not fuse. Upon the addition of CaCl2, the presumptive gametes immediately fuse, producing binucleate cells which develop rapidly into morphologically distinct giant cells. Cell fusion continues, resulting in the formation of extremely large (40-80 microns diameter) multinucleate syncytia by 45 min. The induction of this extensive, synchronous cell fusion does not occur in the presence of other chloride salts and EGTA inhibits it, revealing that Ca+ is the regulatory ion.

Phagocytic specificity during sexual development in Dictyostelium discoideum.

During the sexual cycle of Dictyostelium discoideum, zygote giant cells develop and serve as foci for further development by chemoattracting and cannibalizing hundreds of local amoebae. Previous work has shown that the phagocytic process bears similarities to and differences from asexual endocytosis. In the present study, sexual phagocytosis in D. discoideum was found to be species and developmental stage specific. It was inhibited selectively by glucose and concanavalin A. Although a partial, inhibitory effect of mannose on phagocytosis was not statistically significant, alpha-methylmannosamine, like alpha-methyl-glucose, significantly restored the phagocytic competence of giant cells treated with concanavalin A. Other sugars (N-acetyl-glucosamine, N-acetylgalactosamine, and galactose) and lectins (wheat germ agglutinin, Ulex europus type I, and Ricinis communis agglutinin type I) had no significant effect on sexual phagocytosis. Together these data indicate that a glucose-type receptor is involved in selective uptake of D. discoideum amoebae by giant cells.

Calmodulin-dependent phosphorylation and dephosphorylation during fertilization in Dictyostelium discoideum.

Many calmodulin-dependent biological phenomena are regulated by calmodulin-dependent phosphorylation and dephosphorylation. In Dictyostelium discoideum, cell and pronuclear fusion during fertilization are both calmodulin-dependent biomembrane fusion events. Calmodulin-dependent phosphorylation and dephosphorylation activity was present in sexually developing D. discoideum cells suggesting it has a role during fertilization. In support of this, a discrete calmodulin-independent kinase was present only during cell and pronuclear fusion.

Calmodulin and calmodulin-binding proteins during cell fusion in Dictyostelium discoideum: developmental regulation by calcium ions.

The calcium-dependent regulatory protein calmodulin (CaM) mediates diverse cellular functions via a large number of calmodulin-binding and -dependent proteins (CaMBPs). The use of [35S]calmodulin, labeled during its expression (VU-1-CaM) in Escherichia coli, visualized over 25 CaMBPs in Dictyostelium discoideum. Seven, with M(r)s of 155,000, 91,000, 85,000, 48,000, 46,000, 38,000, and 28,000, were present only during sexual development. In addition, intracellular calmodulin levels were low during gamete formation but rose during cell fusion in response to the presence of extracellular calcium. Thus, calmodulin appears to mediate gamete formation and fusion through two distinct mechanisms: first, via unique developmentally regulated CaMBPs, and, second, via the regulation of intracellular calmodulin levels. The identification of the CaMBP spectrin in sexually developing Dictyostelium cells suggests that this cytoskeleton/plasma membrane, crosslinking protein may function during biomembrane fusion in D. discoideum as it does in other organisms.

The autoinhibitor of cell fusion in Dictyostelium inhibits calmodulin.

During early sexual development in Dictyostelium discoideum cell and pronuclear fusion are negatively regulated by an endogenous autoinhibitor. Here, the autoinhibitor was partially purified from the culture medium and found to inhibit both cell and pronuclear fusion while augmenting gamete numbers. These developmental effects suggested that calmodulin might be an intracellular target for the autoinhibitor. In support of this data, the partially purified autoinhibitor inhibited the calmodulin-dependent activation of phosphodiesterase in a dose-dependent manner, but had no effect on either a calmodulin-insensitive form of phosphodiesterase or the calmodulin-independent enzymes acid and alkaline phosphatase. Thus, the autoinhibitor of sexual development in Dictyostelium discoideum appears to regulate cell and pronuclear fusion at least in part by a direct effect on calmodulin.

Different developmental functions for calmodulin in Dictyostelium: trifluoperazine and R24571 both inhibit cell and pronuclear fusion but enhance gamete formation.

The calmodulin antagonists trifluoperazine and compound R24571 were used to study the function of calmodulin during sexual development in Dictyostelium discoideum. Calmodulin activity is required for both cell fusion and pronuclear fusion. However, cell fusion and pronuclear fusion were each maximally inhibited at different concentrations of the same inhibitor suggesting differential calmodulin activity during these events. In contrast, trifluoperazine and R24571 were both found to enhance rather than inhibit the formation of gametes. This suggests an additional role for calmodulin as a negative regulator of gamete development. These results provide evidence of a role for calmodulin as both a positive (biomembrane fusion) and a negative (gamete development) regulator of developmental events in Dictyostelium. They also reveal calmodulin as a mediator of pronuclear fusion for zygote development in this eukaryote.