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

Abstract

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.

Phosphatidylserine exposure promotes increased adhesion in Dictyostelium Copine A mutants

“…Actin filament dynamics are necessary for the phagocytic engulfment of particles and are important in cell adhesion [ 23 ]. We previously showed that CpnA is able to interact with actin filaments in a calcium-dependent manner and may play a role in actin depolymerization at the surface of membranes [ 13 , 17 ]. To further understand CpnA’s role in adhesion, we used the actin depolymerizing drug, Latrunculin A (LatA) to both inhibit phagocytosis [ 24 ] and to see if actin filaments play a role in the increased adhesion observed in cpnA - cells.…”

“…Altered membrane lipid composition and/or calcium concentrations in cpnA - cells may also contribute to the increased postlysosome exocytosis phenotype we previously reported [ 17 ]. Other studies have reported a link between PS exposure and membrane trafficking.…”

“…Mutant studies showed that cpnA - cells had no defects in growth or viability, but did have defects in development [ 14 – 16 ]. cpnA - cells also exhibited defects in processes related to membrane trafficking and the actin cytoskeleton including contractile vacuole exocytosis, postlysosome maturation and exocytosis, chemotaxis, and adhesion [ 13 , 17 ]…”

“…Mutant studies showed that cpnAcells had no defects in growth or viability, but did have defects in development [14][15][16]. cpnAcells also exhibited defects in processes related to membrane trafficking and the actin cytoskeleton including contractile vacuole exocytosis, postlysosome maturation and exocytosis, chemotaxis, and adhesion [13,17] Dictyostelium cells feed on bacteria and yeast by phagocytosis. Phagocytosis requires the attachment of particles to the outside surface of the cell and actin polymerization to remodel the plasma membrane to allow for engulfment of the particles.…”

Copines, a Family of Calcium Sensor Proteins and Their Role in Brain Function

“…In round worm C. elegans, Copine controls synaptic expression of nicotinic acetylcholine receptors [ 4 ] and TRP channel GON-2 that stimulates gonadal cell division [ 5 ] and is required for myofilament stability [ 6 ]. In protist, social amoeba Dictiostellium, Copine A is important for cell differentiation, cytokinesis, contractile vacuole function, phospholipid distribution, and slug formation [ 7 , 8 , 9 , 10 , 11 ]. In Copine A mutants, slug formation deficits could be rescued by introducing a small number of wild-type cells into the amoeba population suggesting that Copine regulates the secretion of unknown factors essential for slug formation [ 12 ].…”