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.
Highlights
Copines are a family of cytosolic proteins that associate with membranes in a calcium-dependent manner and are found in many eukaryotic organisms
We obtained four independent cpnA KOs cell lines, one with and one without the loxP sites flanking the incorporated bsr gene, in both NC4A2 and AX4 parental axenic laboratory strains. Both the NC4A2 and AX4 strains were used in the contractile vacuoles (CV) assays; only the data for NC4A2 strains are reported for the endocytosis assays
This is because we found that various AX4 strains assayed behaved quite differently from each other and from the NC4A2 strains in the endocytosis assays, making it difficult to make conclusions with respect to the AX4 cpnA KO strains
Summary
Copines are a family of cytosolic proteins that associate with membranes in a calcium-dependent manner and are found in many eukaryotic organisms. PLs in cpnAÀ cells were 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. Copines are highly conserved calcium-dependent membrane-binding proteins characterized by having two C2 domains at the N terminus followed by an A domain at the C terminus [1]. Tomsig et al [4] hypothesized that the molecular function of copines was to bind to proteins and shuttle them to membranes in response to a rise in intracellular calcium concentration. The brain-specific Copine-6 was found to be involved in changes to dendritic spine morphology in hippocampal neurons in response to neuronal activity [9]
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