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.

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 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.