Danton H. O'Day

CaMBOT: profiling and characterizing calmodulin-binding proteins

Calmodulin (CaM) is an essential calcium-binding protein that binds to and activates a diverse population of downstream targets (calmodulin-binding proteins; CaMBPs) that carry out its critical signalling functions. In spite of the central importance of CaM in Ca(2+)-mediated signal transduction pathways in all eukaryotes, many CaMBPs remain to be identified and characterized. SDS-PAGE followed by gel overlay with recombinant, metabolically radiolabelled CaM (Calmodulin-binding Overlay Technique, CaMBOT) is a valuable method for following behavioural, developmental, forensic and physiological changes in total CaMBP populations and to identify candidate CaMBPs for further study. CaMBOT has also been adapted to isolate cDNAs encoding novel CaMBPs in various organisms. Recently, the method was used to examine the CaMBP complement encoded by the Arabidopsis genome and to identify a new family of transcription activators. To add to its diversity, CaMBOT may be useful for finding target proteins for work on phytoremediation and for the screening of pharmaceuticals and toxic agents that, directly or indirectly, affect CaM and its target proteins. This review discusses all of these topics and the role of CaMBOT in characterizing a functional unit of the proteome-proteins regulated by calmodulin.

Cell fusion, nuclear fusion, and zygote differentiation during sexual development of Dictyostelium discoideum

Abstract The fluorescent nuclear stain Hoechst 33258 was used to study the nuclear events during mating of Dictyostelium discoideum in liquid culture. These studies revealed that cell fusion begins about 11 hr after the sexually compatible cultures are mixed and continues until 26 hr. Approximately 37% of the cells fuse during this 15-hr period. At first the fused cells are relatively small, but by 20 hr the fusion products become evident as morphologically distinct giant cells. Starting at 22 hr these giant cells are transformed into true zygotes as nuclear fusion begins. Both the fusion of amebae and the differentiation of zygote giant cells are Ca 2+ -dependent events as revealed by studies using EGTA. The nuclear events of zygote differentiation involve nuclear swelling, migration, and fusion. The precise timing of these events has been detailed. Of particular interest for genetic analyses via the macrocyst is the presence of a small population of multinucleate cells (maximum level is 1.67% of the cell population) which usually possess 3 or 4 nuclei but may have as many as 10 or more. Although these multinucleate cells contain many nuclei, our evidence suggests that only one is a zygote nucleus. The genetic implications of these data and the potential value of using the mating system for the analysis of cell fusion are discussed.

Ca2+ and cell fusion during sexual development in liquid cultures of Dictyostelium discoideum☆

Abstract Cell fusion resulting in zygote giant cell formation is the first observable event of sexual development in D. discoideum . The results reported here show that this process is Ca 2+ -dependent and that by increasing the level of Ca 2+ in the medium the number of cell fusions can be increased 57-fold over control cultures. The data also suggest that Ca 2+ has both an early and late function in the development of zygotes and these functions are mediated at the cell surface. These results plus the availability of a liquid culture for generating large volumes of cells make sexual development in D. discoideum an excellent system for the analysis of membrane fusion in eukaryotes.

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.

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.

Endogenous Autoinhibitors Regulate Changes in Actin Tyrosine Phosphorylation During Dictyostelium Spore Germination

Phosphorylation of proteins on tyrosine residues has been shown to govern many cellular processes, but little work has focused on the role of tyrosine phosphorylation during germination. Under optimal conditions, D. discoideum spores synchronously germinate each liberating a single amoeba. The total amount of phosphotyrosine containing proteins observed in spores was greatest during quiescence with a gradual decline during spore activation and emergence of nascent amoeba. During dormancy, tyrosine residues of actin were heavily phosphorylated, but they gradually underwent dephosphorylation upon spore activation and this process continued through emergence. Interestingly, an endogenous autoinhibitor(s), which blocks germination, induces tyrosine phosphorylation of actin. Conversely, the removal of the autoinhibitor(s) was followed by a decrease in phosphorylation. Thus, during germination of Dictyostelium spores, actin is dephosphorylated, with the level of phosphorylation regulated by the autoinhibitor(s) and/or the autoactivator. This change in actin phosphorylation appears to play a direct role since actin dephosphorylation and reorganization is a necessary prelude to germination.

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.

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.

EGF-like peptide-enhanced cell motility in Dictyostelium functions independently of the cAMP-mediated pathway and requires active Ca2+/calmodulin signaling.

Current knowledge suggests that cell movement in the eukaryotic slime mold Dictyostelium discoideum is mediated by different signaling pathways involving a number of redundant components. Our previous research has identified a specific motility-enhancing function for epidermal growth factor-like (EGFL) repeats in Dictyostelium, specifically for the EGFL repeats of cyrA, a matricellular, calmodulin (CaM)-binding protein in Dictyostelium. Using mutants of cAMP signaling (carA(-), carC(-), gpaB(-), gpbA(-)), the endogenous calcium (Ca(2+)) release inhibitor TMB-8, the CaM antagonist W-7, and a radial motility bioassay, we show that DdEGFL1, a synthetic peptide whose sequence is obtained from the first EGFL repeat of cyrA, functions independently of the cAMP-mediated signaling pathways to enhance cell motility through a mechanism involving Ca(2+) signaling, CaM, and RasG. We show that DdEGFL1 increases the amounts of polymeric myosin II heavy chain and actin in the cytoskeleton by 24.1±10.7% and 25.9±2.1% respectively and demonstrate a link between Ca(2+)/CaM signaling and cytoskeletal dynamics. Finally, our findings suggest that carA and carC mediate a brake mechanism during chemotaxis since DdEGFL1 enhanced the movement of carA(-)/carC(-) cells by 844±136% compared to only 106±6% for parental DH1 cells. Based on our data, this signaling pathway also appears to involve the G-protein β subunit, RasC, RasGEFA, and protein kinase B. Together, our research provides insight into the functionality of EGFL repeats in Dictyostelium and the signaling pathways regulating cell movement in this model organism. It also identifies several mechanistic components of DdEGFL1-enhanced cell movement, which may ultimately provide a model system for understanding EGFL repeat function in higher organisms.

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.