The regulation of membrane fusion during sexual development in Dictyostelium discoideum

During the first 24 h of sexual development in Dictyostelium discoideum, three sequential events of membrane fusion occur: gamete fusion, pronuclear fusion, and phagocytosis. The early events of sexual development are regulated by a diverse group of endogenous molecules: (i) a volatile sexual pheromone, (ii) a protein cell fusion inducing factor (CFIF), (iii) a low molecular weight autoinhibitor, (iv) and cyclic AMP. CFIC enhances cell fusion while the autoinhibitor and cyclic AMP both inhibit the event. Both extracellular and intracellular calcium ions are essential for cell and pronuclear fusion. Pharmacological analyses show that the intracellular functions of the divalent cation in these processes are mediated by calmodulin. The autoinhibitor appears to function by inhibiting calmodulin activity. Glucose, mannose, and N-acetylglucosamine containing glycoproteins have been shown to function in both cell fusion and phagocytosis. The interplay between all of these diverse molecules is examined and a review of all of the recent literature is presented.     

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.     

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.     

Inhibition of calmodulin-dependent and independent cardiac sarcoplasmic reticulum activities by R24571

R24571 a derivative of the antimycotic miconazole, appears to be 5 to 8 times more potent than trifluoperazine in its ability to inhibit the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum vesicles. The cAMP-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum was not affected by R24571. Sarcoplasmic reticulum Ca-dependent ATPase phosphoprotein intermediate formation was inhibited by R24571 concentrations that were 20 to 30 times greater than those required to inhibit calmodulin-dependent phosphorylation. However, both Ca-dependent and independent ATPase activities, as well as calcium uptake, were inhibited by R24571 concentrations that were similar to, or less than, those concentrations required to inhibit calmodulin-dependent sarcoplasmic reticulum phosphorylation. These results indicate the caution that should be exercised in using this new compound in assessing the possible involvement of calmodulin in other membrane processes.     

Gamete fusion and cytokinesis preceding zygote establishment in the sexual process of Dictyostelium discoideum

Cells of Dictyostelium discoideum become sexually mature under submerged and dark conditions, and fuse with opposite mating-type cells to form zygote giant cells, which gather surrounding cells and finally develop into dormant structures called macrocysts. In the present study, we found that the multinuclear fused cells formed during this process frequently underwent cytokinesis driven by random local movements. The split cells were capable of re-fusion, and repeated cytokinesis. These radical behaviors continued until the extensive cell aggregation started around the giant cells. Thus, gamete fusion and initiation of zygote development do not coincide in the mating of D. discoideum. Analyses by confocal microscopy and flow cytometry indicated that the cessation of the random movement followed pronuclear fusion, and that microtubule organizing centers (MTOC), abundant in the fused cells at the beginning, gradually decreased and only one of them remained within the developed macrocyst. Some of the genes known to control cell movement, such as rasGEFB and rasS, increased shortly before the cessation of repeated fusion-cytokinesis and initiation of phagocytosis. These results suggest that the sequential molecular events are necessary in D. discoideum after gamete fusion to establish a new individuality of zygotes.     

Sources of calcium and the involvement of calmodulin during steroidogenesis and oocyte maturation in follicles of Rana pipiens

In the amphibian ovarian follicle, progesterone production is thought to induce maturation of the enclosed oocyte. Intracellular mechanisms regulating these events in the somatic and germ cells are incompletely understood. However, calcium appears to play a role in the production and action of progesterone. Experiments using calcium antagonists were carried out to delineate the role of extra- and intracellular calcium during in vitro stimulation of follicular steroidogenesis and oocyte maturation. Calcium-free medium, verapamil, and La3+ were used to block Ca2+ influx and inhibited follicular progesterone accumulation in response to frog pituitary homogenate (FPH) or exogenous cAMP + IBMX. Progesterone accumulation was not impaired under identical conditions when pregnenolone was added to cultured follicles. TMB-8, an inhibitor of intracellular Ca2+ mobilization, partially inhibited progesterone levels stimulated by FPH at low doses but not higher doses of the inhibitor. However, TMB-8 inhibited FPH-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner, as well as maturation due to exogenous progesterone or La3+. Calmodulin antagonists, W-7, R24571, and trifluoperazine, were used to assess the involvement of calmodulin in the responses of these two cell types. All three antagonists inhibited progesterone accumulation induced by FPH with the apparent order of potency being R24571 greater than W-7 greater than TFP. W-7 inhibited cAMP-induced progesterone elevation, but had no effect on conversion of pregnenolone to progesterone. Of these three calmodulin antagonists, only R24571 exhibited a dramatic ability to inhibit GVBD induced by exogenous progesterone and was associated with morphologic alterations in the oocytes. These data suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+. In the oocyte, mobilization of Ca2+ from intracellular stores appears to be of primary importance to maturation while extracellular Ca2+ is not. These data provide further evidence that Ca2+ mediates the hormonally provoked responses in both cell types in the intact follicle, but that the source of Ca2+ may differ. Using intact follicles it seems apparent that exploiting this difference with selective inhibitors provides a means for differential modulation and functional uncoupling of these cells with regard to steroidogenesis and steroid action.     

Calcium and calmodulin in cellular intoxication with Clostridium difficile toxin B

In cultured human lung fibroblasts treated with Clostridium difficile toxin B, the development of the cytopathogenic effect was inhibited by the proton ionophore monensin but was not affected by some other ionophores. The calcium channel blockers verapamil and LaCl3 protected the cells against intoxication, as did the calmodulin antagonists trifluoperazine, amitriptyline, R 24571, and dansylcadaverine. Since these agents could not prevent intoxication when added after the toxin internalization was completed, we suggest that calmodulin and uptake of extracellular calcium are needed for the internalization but not for the cytosolic action of the toxin.     

The Calmodulin Antagonists, Trifluoperazine and R24571, Depolarize the Mitochondria Within Guinea Pig Cerebral Cortical Synaptosomes

Abstract: The effects of trifluoperazine and l-[bis( p -chlorophenyl)methyl] - 3 - [2,4 - dichloro - 3 - (2,4 - dichloroben-zyloxy)phenethyl]imidazolium chloride (R24571) upon synaptosomal calcium transport, plasma membrane potential, in situ mitrochondrial membrane potential, and ATP levels are investigated in order to assess the suitability of these calmodulin antagonists for investigating calmodulin-dependent processes in the nerve terminal. Both agents appear to act selectively at the mitochondrial membrane, causing extensive depolarization at concentrations in excess of 10 μ M (trifluoperazine) or 0.5 μ M (R24571). The extent of Ca uptake into the synaptosomes is decreased, consistent with the loss of the mitochondrial compartment. There is no inhibition of the efflux of Ca from the synaptosomes. Depolarization-dependent Ca uptake is not prevented by R24571. Synaptosomal ATP levels decrease to an extent consistent with the collapse of the mitochondrial potential. It is concluded that the uncoupling effect of these agents on the in situ mitochondria prevents their being used to investigate the role of calmodulin in intact synaptosomes.     

Regulation of goby intestinal ion absorption by the calcium messenger system

The role of the calcium messenger system in the regulation of ion absorption across the teleost intestine was studied using pharmacological intervention. Radiochloride transport was independent of external Ca2+ over the range 10 microM to 2.5 mM. Treatment with the Ca2+ ionophore A23187 (to hyperpolarization of the apical membrane potential of intestinal epithelial cells. The Ca2+-calmodulin antagonists trifluoperazine (TFP) and calmidazolium (R24571) produced opposite effects, i.e., stimulation of Cl- absorption and cellular depolarization. Treatment with TFP or R24571 will block or override the inhibitory action of A23187. These data suggest a regulatory role for Ca2+ in the control of intestinal NaCl absorption and mediation via calmodulin.     

Regulation of fusion of the nucleolar precursor bodies following activation of mouse oocytes: roles of the maturation-promoting factors and mitogen-activated protein kinases

Fusion of nucleoli or nucleolus precursor bodies (NPBs) has been observed during somatic cell interphase and pronuclear development of human zygotes; however, the underlying mechanism is unknown. NPB fusion and its regulation by mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) were studied in activated mouse oocytes. Small NPBs appeared about 4 h after ethanol activation, and took about 1.5 h to fuse into a large NPB, which persisted for about 10 h before disappearance. Analysis of the temporal windows for kinase action indicated that a high MAPK activity during the first 2 h and a low MPF activity during the first 3-4 h after activation were essential for subsequent NPB fusion. A preactivation decline in MAPK activity was associated with decreased NPB fusion following activation of aged oocytes. While MAPK inactivation by regulator U0126 prevented NPB fusion in oocytes activated by ethanol or 5 min Sr2+ treatments, it had no effect on oocytes fertilized or activated by 6 h Sr2+ treatment. In most cases, while rates of pronuclear formation did not differ, rates of NPB fusion differed significantly between different treatments. Our results suggest that: (i) the MAPK and MPF activities at the initial stage of activation regulate NPB fusion after pronuclear formation; (ii) pronuclear assembly and NPB fusion are two separable events that might be controlled by different mechanisms; and (iii) high MAPK activity and low MPF activity at the initial stage of activation is essential for NPB fusion when only one calcium rise is induced by ethanol, while inhibition of MAPK activity does not affect NPB fusion when the repetitive intracellular Ca2+ rises are induced after fertilization.     

Spore swelling in Dictyostelium is a dynamic process mediated by calmodulin

Abstract Spore swelling is a necessary prelude to the emergence of amoebae during spore germination in Dictyostelium discoideum . Previous work has shown that the initiation of this event requires the activity of the calcium-dependent regulatory protein calmodulin. In this study, the use of trifluoperazine, an inhibitor of calmodulin function, has shown that calmodulin activity is required throughout the swelling phase. When fully swollen spores were treated with trifluoperazine they rapidly returned to the same size and shape observed prior to swelling. These data suggest that spore swelling in D. discoideum is a dynamic process which is mediated by calmodulin.     

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.     

Does calmodulin participate in the regulation of the Ca-pump of erythrocytes in vivo?

Using a highly effective chelator of Ca2+ and 45Ca, the concentration of Cai2+ in human and rat erythrocytes was measured both at normal and accelerated Ca2+ influx into the cells. No effect of the calmodulin-dependent reaction inhibitor R24571 was observed. The Ca-ATPase from saponin-treated erythrocytes was characterized by a high affinity for Ca2+ (K 0.5-0.7 microM). This value is 2-3 times as low as that for Ca2+ concentration causing a 50% increase of the Ca-ATPase activity in erythrocyte ghosts obtained during hypoosmotic hemolysis. The Ca-ATPase activity in saponin-treated erythrocytes did not change either under the effect of calmodulin or by R24571. It was assumed that calmodulin did not participate in the regulation of the Ca2+-pump operation in erythrocytes in vivo.     

Functional and genetic characterization of calmodulin from the dimorphic and pathogenic fungus Paracoccidioides brasiliensis

Calmodulin (CaM) modulates intracellular calcium signalling and acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including human fungal pathogens, such as Candida albicans and Histoplasma capsulatum. The temperature-dependent dimorphic fungus Paracoccidioides brasiliensis is the aetiological agent of paracoccidioidomycosis (PCM). The mycelium (M) to yeast (Y) transition has been shown to be essential for establishment of the infection, although the precise molecular mechanisms of dimorphism in P. brasiliensis are still unknown. In this work, several inhibitory drugs of the Ca(2+)/calmodulin signalling pathway were tested to verify the role of this pathway in the cellular differentiation process of P. brasiliensis. EGTA and the drugs calmidazolium (R24571), trifluoperazine (TFP), and W7 were able to inhibit the M-Y transition. We have cloned and characterized the calmodulin gene from P. brasiliensis, which comprises 924 nucleotides and five introns that are in a conserved position among calmodulin genes.     

Calmodulin‐mediated events during the life cycle of the amoebozoan Dictyostelium discoideum

This review focusses on the functions of intracellular and extracellular calmodulin, its target proteins and their binding proteins during the asexual life cycle of Dictyostelium discoideum. Calmodulin is a primary regulatory protein of calcium signal transduction that functions throughout all stages. During growth, it mediates autophagy, the cell cycle, folic acid chemotaxis, phagocytosis, and other functions. During mitosis, specific calmodulin‐binding proteins translocate to alternative locations. Translocation of at least one cell adhesion protein is calmodulin dependent. When starved, cells undergo calmodulin‐dependent chemotaxis to cyclic AMP generating a multicellular pseudoplasmodium. Calmodulin‐dependent signalling within the slug sets up a defined pattern and polarity that sets the stage for the final events of morphogenesis and cell differentiation. Transected slugs undergo calmodulin‐dependent transdifferentiation to re‐establish the disrupted pattern and polarity. Calmodulin function is critical for stalk cell differentiation but also functions in spore formation, events that begin in the pseudoplasmodium. The asexual life cycle restarts with the calmodulin‐dependent germination of spores. Specific calmodulin‐binding proteins as well as some of their binding partners have been linked to each of these events. The functions of extracellular calmodulin during growth and development are also discussed. This overview brings to the forefront the central role of calmodulin, working through its numerous binding proteins, as a primary downstream regulator of the critical calcium signalling pathways that have been well established in this model eukaryote. This is the first time the function of calmodulin and its target proteins have been documented through the complete life cycle of any eukaryote.     

Zygote giant cell differentiation in Dictyostelium discoideum: biochemical markers of specific stages of sexual development.

Sexual development in Dictyostelium discoideum has many unique features making it an attractive eukaryotic model system for the study of biomembrane fusion and intercellular communication. The work presented here provides primary biochemical evidence for two distinct phases during early sexual development that appear to be defined by calcium-dependent gamete cell fusion. In addition, we introduce a novel procedure for the enrichment of zygote giant cells and use this method to define certain wheat-germ agglutinin binding glycoproteins which are specifically located in zygote giant cells and others which are markers for surrounding amoebae in the second phase of development. In addition, a G protein which is present in high amounts early in development is unique to giant cells in the second phase, suggesting a role in phagocytosis. Finally, alkaline phosphatase activity was found to mark the first phase of sexual development, suggesting a role in cell fusion. This contrasts with the patterns of alpha-mannosidase and beta-glucosidase activity that increase late in the second developmental phase, where they likely function in endocyte digestion during the cytophagic period. The developmental significance of these findings is discussed.     

Calmodulin-binding proteins in the model organism Dictyostelium: a complete & critical review

Calmodulin is an essential protein in the model organism Dictyostelium discoideum. As in other organisms, this small, calcium-regulated protein mediates a diversity of cellular events including chemotaxis, spore germination, and fertilization. Calmodulin works in a calcium-dependent or -independent manner by binding to and regulating the activity of target proteins called calmodulin-binding proteins. Profiling suggests that Dictyostelium has 60 or more calmodulin-binding proteins with specific subcellular localizations. In spite of the central importance of calmodulin, the study of these target proteins is still in its infancy. Here we critically review the history and state of the art of research into all of the identified and presumptive calmodulin-binding proteins of Dictyostelium detailing what is known about each one with suggestions for future research. Two individual calmodulin-binding proteins, the classic enzyme calcineurin A (CNA; protein phosphatase 2B) and the nuclear protein nucleomorphin (NumA), which is a regulator of nuclear number, have been particularly well studied. Research on the role of calmodulin in the function and regulation of the various myosins of Dictyostelium, especially during motility and chemotaxis, suggests that this is an area in which future active study would be particularly valuable. A general, hypothetical model for the role of calmodulin in myosin regulation is proposed.     

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.     

Signalling and sex in the social amoebozoans

The social amoebozoans have a life tricycle consisting of asexual multicellular development leading to fruiting bodies, sexual multicellular development resulting in macrocysts, and unicellular development generating microcysts. This review covers the events of sexual development in the best‐studied heterothallic (Dictyostelium discoideum) and homothallic (D. mucoroides) mating systems. Sexual development begins with pheromonal interactions that produce fusion‐competent cells (gametes) which undergo cell and pronuclear fusion. Calcium‐ and calmodulin‐mediated signalling mediates these early events. As they initiate chemotactic signalling, each zygote increases in size becoming a zygote giant cell. Using cyclic AMP (cAMP), the zygote chemotactically lures in amoebae and engulfs them in an act of cannibalistic phagocytosis. Chemotaxis proceeds more quickly than endocytosis because the breakdown products of cAMP (5‐AMP, adenosine) bind to a presumptive adenosine receptor to inhibit sexual phagocytosis. This slowing of phagocytosis allows amoebae to accumulate around the zygote to form a precyst aggregate. Zygote giant cells also produce several other signalling molecules that feed back to regulate early events. The amoebae surrounding the zygote seal their fate as zygotic foodstuff by secreting a primary cellulose wall, the extracellular sheath, around the zygote and aggregated amoebae, which prevents their escape. Phagocytosis within this precyst continues until all peripheral amoebae are internalized as endocytes and the final macrocyst wall is formed. Endocyte digestion results in a mature macrocyst with a uniform cytoplasm containing a diploid nucleus. After detailing the morphological events of heterothallic and homothallic mating, we review the various intercellular signalling events and other mechanisms involved in each stage. This complete and comprehensive review sets the stage for future research on the unique events that characterize sex in the social amoebozoans.     

Properties of the 23,000-Da phosphoproteins in cardiac sarcolemma and sarcoplasmic reticulum

The calmodulin- and cAMP-dependent protein kinase-mediated phosphorylations of isolated sarcolemma and sarcoplasmic reticulum vesicles have been compared. Similarities in the calmodulin-mediated phosphorylation of the sarcolemma and sarcoplasmic reticulum 23,000-Da phosphoproteins included their Mg2+, Na+, Ca2+, and calmodulin sensitivities, as well as the size of their dissociated subunits. In contrast, a number of differences between these phosphoproteins were indicated in their sensitivity to detergents (Triton X-100 and sodium dodecyl sulfate) and calmodulin antagonists (R24571 and trifluoperazine). Furthermore, in contrast to the sarcoplasmic reticulum phosphoprotein, the sarcolemma phosphoprotein could not be affinity labeled with 125I-calmodulin. While these results indicate the probable chemical similarity of the sarcolemma and sarcoplasmic reticulum 23,000-Da phosphoproteins, they also indicate there are differences in the lipid/phosphoprotein interactions in these two membranes.