Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane.

BACKGROUND: Copines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA - cpnF. RESULTS: Independent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P. CONCLUSIONS: Our studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.

Copine C plays a role in adhesion and streaming in Dictyostelium.

Copines are a family of calcium-dependent membrane-binding proteins. To study these proteins, anull mutant for cpnC was created in Dictyostelium, which has six copines genes (cpnA-cpnF). During development, cpnC- cells were able to aggregate, but did not form streams. Once aggregated into mounds, they formed large ring structures. cpnC- cells were less adherent to plastic substrates, but more adherent to other cells. These phenotypes correlated with changes in adhesion protein expression with decreased expression of SibA and increased expression of CsaA in developing cpnC- cells. We also measured the expression of RegA, a cAMP phosphodiesterase, and found that cpnC- cells have reduced RegA expression. The reduced RegA expression in cpnC- cells is most likely responsible for the observed phenotypes.

Regulation of Hip1r by epsin controls the temporal and spatial coupling of actin filaments to clathrin-coated pits.

Recently, it has become clear that the actin cytoskeleton is involved in clathrin-mediated endocytosis. During clathrin-mediated endocytosis, clathrin triskelions and adaptor proteins assemble into lattices, forming clathrin-coated pits. These coated pits invaginate and detach from the membrane, a process that requires dynamic actin polymerization. We found an unexpected role for the clathrin adaptor epsin in regulating actin dynamics during this late stage of coated vesicle formation. In Dictyostelium cells, epsin is required for both the membrane recruitment and phosphorylation of the actin- and clathrin-binding protein Hip1r. Epsin-null and Hip1r-null cells exhibit deficiencies in the timing and organization of actin filaments at clathrin-coated pits. Consequently, clathrin structures persist on the membranes of epsin and Hip1r mutants and the internalization of clathrin structures is delayed. We conclude that epsin works with Hip1r to regulate actin dynamics by controlling the spatial and temporal coupling of actin filaments to clathrin-coated pits. Specific residues in the ENTH domain of epsin that are required for the membrane recruitment and phosphorylation of Hip1r are also required for normal actin and clathrin dynamics at the plasma membrane. We propose that epsin promotes the membrane recruitment and phosphorylation of Hip1r, which in turn regulates actin polymerization at clathrin-coated pits.

Copine A is expressed in prestalk cells and regulates slug phototaxis and thermotaxis in developing Dictyostelium.

Copines are calcium-dependent membrane-binding proteins found in many eukaryotic organisms. We are studying the function of copines using the model organism, Dictyostelium discoideum. When under starvation conditions, Dictyostelium cells aggregate into mounds that become migrating slugs, which can move toward light and heat before culminating into a fruiting body. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to form fruiting bodies and instead arrest at the slug stage. In this study, we compared the slug behavior of cells lacking the cpnA gene to the slug behavior of wild-type cells. The slugs formed by cpnA- cells were much larger than wild-type slugs and exhibited no phototaxis and negative thermotaxis in the same conditions that wild-type slugs exhibited positive phototaxis and thermotaxis. Mixing as little as 5% wild-type cells with cpnA- cells rescued the phototaxis and thermotaxis defects, suggesting that CpnA plays a specific role in the regulation of the production and/or release of a signaling molecule. Reducing extracellular levels of ammonia also partially rescued the phototaxis and thermotaxis defects of cpnA- slugs, suggesting that CpnA may have a specific role in regulating ammonia signaling. Expressing the lacZ gene under the cpnA promoter in wild-type cells indicated cpnA is preferentially expressed in the prestalk cells found in the anterior part of the slug, which include the cells at the tip of the slug that regulate phototaxis, thermotaxis, and the initiation of culmination into fruiting bodies. Our results suggest that CpnA plays a role in the regulation of the signaling pathways, including ammonia signaling, necessary for sensing and/or orienting toward light and heat in the prestalk cells of the Dictyostelium slug.

Phosphatidylserine exposure promotes increased adhesion in Dictyostelium Copine A mutants.

The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of the cell membrane, but PS can be flipped to the outer surface in a process called PS exposure. PS exposure is important in many cell functions, yet the mechanisms that control PS exposure have not been extensively studied. Copines (Cpn), found in most eukaryotic organisms, make up a family of calcium-dependent phospholipid binding proteins. In Dictyostelium, which has six copine genes, CpnA strongly binds to PS and translocates from the cytosol to the plasma membrane in response to a rise in calcium. Cells lacking the cpnA gene (cpnA-) have defects in adhesion, chemotaxis, membrane trafficking, and cytokinesis. In this study we used both flow cytometry and fluorescent microscopy to show that cpnA- cells have increased adhesion to beads and bacteria and that the increased adhesion was not due to changes in the actin cytoskeleton or cell surface proteins. We found that cpnA- cells bound higher amounts of Annexin V, a PS binding protein, than parental cells and showed that unlabeled Annexin V reduced the increased cell adhesion property of cpnA- cells. We also found that cpnA- cells were more sensitive to Polybia-MP1, which binds to external PS and induces cell lysis. Overall, this suggests that cpnA- cells have increased PS exposure and this property contributes to the increased cell adhesion of cpnA- cells. We conclude that CpnA has a role in the regulation of plasma membrane lipid composition and may act as a negative regulator of PS exposure.

Copine A plays a role in the differentiation of stalk cells and the initiation of culmination in Dictyostelium development.

BACKGROUND: Copines are calcium-dependent phospholipid-binding proteins found in diverse eukaryotic organisms. We are studying the function of copines in Dictyostelium discoideum, a single-celled amoeba that undergoes cell differentiation and morphogenesis to form multicellular fruiting bodies when placed in starvation conditions. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to complete the developmental cycle, arresting at the slug stage. The aim of this study is to further characterize the developmental defect of the cpnA- cells. RESULTS: Time-lapse imaging revealed that cpnA- cells exhibited delayed aggregation and made large mounds that formed one large slug as compared to the smaller slugs of the wild-type cells. While the prespore cell patterning appeared to be normal within the cpnA- slugs, the prestalk cell patterning was different from wild-type. When cpnA- cells were mixed with a small percentage of wild-type cells, chimeric fruiting bodies with short stalks formed. When a small percentage of cpnA- cells was mixed with wild-type cells, the cpnA- cells labeled with GFP were found located throughout the chimeric slug and in both the stalk and sporehead of the fruiting bodies. However, there appeared to be a small bias towards cpnA- cells becoming spore cells. When cpnA- cells were developed in buffer containing EGTA, they were also able to differentiate into either stalk or spore cells to form fruiting bodies with short stalks. CONCLUSIONS: Our results indicate that CpnA is involved in the regulation of aggregation, slug size, and culmination during Dictyostelium development. More specifically, CpnA appears to be involved in the function and differentiation of prestalk cells and plays a role in a calcium-regulated signaling pathway critical to triggering the initiation of culmination.

Copine A regulates the size and exocytosis of contractile vacuoles and postlysosomes in Dictyostelium.

Copines are a family of cytosolic proteins that associate with membranes in a calcium-dependent manner and are found in many eukaryotic organisms. Dictyostelium discoideum has six copine genes (cpnA-cpnF), and cells lacking cpnA(cpnA- ) have defects in cytokinesis, chemotaxis, adhesion, and development. CpnA has also been shown to associate with the plasma membrane, contractile vacuoles (CV), and organelles of the endolysosomal pathway. Here, we use cpnA- cells to investigate the role of CpnA in CV function and endocytosis. When placed in water, cpnA- cells made abnormally large CVs that took longer to expel. Visualization of CVs with the marker protein GFP-dajumin indicated that cpnA- cells had fewer CVs that sometimes refilled before complete emptying. In endocytosis assays, cpnA- cells took up small fluorescent beads by macropinocytosis at rates similar to parental cells. However, cpnA- cells reached a plateau sooner than parental cells and had less fluorescence at later time points. p80 antibody labeling of postlysosomes (PL) indicated that there were fewer and smaller PLs in cpnA- cells. In dextran pulse-chase experiments, the number of PLs peaked earlier in cpnA- cells, and the PLs did not become as large and disappeared sooner as compared to parental cells. PLs in cpnA- cells were also shown to have more actin coats, suggesting CpnA may play a role in actin filament disassembly on PL membranes. Overall, these results indicate that CpnA is involved in the regulation of CV size and expulsion, and the maturation, size, and exocytosis of PLs.

Copine A Interacts with Actin Filaments and Plays a Role in Chemotaxis and Adhesion.

Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA- cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.

Copine A, a calcium-dependent membrane-binding protein, transiently localizes to the plasma membrane and intracellular vacuoles in Dictyostelium.

BACKGROUND: Copines are soluble, calcium-dependent membrane binding proteins found in a variety of organisms. Copines are characterized as having two C2 domains at the N-terminal region followed by an "A domain" at the C-terminal region. The "A domain" is similar in sequence to the von Willebrand A (VWA) domain found in integrins. The presence of C2 domains suggests that copines may be involved in cell signaling and/or membrane trafficking pathways. RESULTS: We have identified six copines genes in the Dictyostelium discoideum genome, cpnA-cpnF, and have focused our studies on cpnA. CpnA is expressed throughout development and was shown to be capable of binding to membranes in a calcium-dependent manner in vitro. A GFP-tagged CpnA was also capable of binding to membranes in a calcium-dependent manner in vitro. In live wildtype Dictyostelium cells expressing GFP-CpnA, GFP-CpnA was typically found in the cytoplasm without any specific localization to membranes. However, in a small subset of starved cells, GFP-CpnA was observed to bind transiently (typically approximately 1-10 s) to the plasma membrane and intracellular vacuoles. In some cells, the transient membrane localization of GFP-CpnA was observed to occur multiple times in an oscillatory manner over several minutes. In plasma membrane disrupted cells, GFP-CpnA was observed to associate with the plasma membrane and intracellular vacuoles in a calcium-dependent manner. In fixed cells, GFP-CpnA labeled the plasma membrane and intracellular vacuoles, including contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes. CONCLUSION: Our results show that Dictyostelium has multiple copine homologs and provides an excellent system in which to study copine function. The localization of a GFP-tagged CpnA to the plasma membrane, contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes suggests that CpnA may have a role in the function of these organelles or the trafficking to and from them. In addition, we hypothesize that the observed transient oscillatory membrane localization of GFP-CpnA in a small subset of starved cells is caused by fast calcium waves and speculate that CpnA may have a role in development, particularly in the differentiation of stalk cells.

Copine A is required for cytokinesis, contractile vacuole function, and development in Dictyostelium.

Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA. Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA- knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA- cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA- cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA- cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA- cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes except cpnF showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of Dictyostelium development.