Abstract
BackgroundCopines 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.ResultsTime-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.ConclusionsOur 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.
Highlights
Copines are calcium-dependent phospholipid-binding proteins found in diverse eukaryotic organisms
Results copine A (cpnA)- cells are arrested in the slug stage of development In a previous study, we showed that Dictyostelium cells lacking the cpnA gene exhibit developmental defects [22]. cpnA- cells become arrested in the slug stage of development, indicating that cpnA is necessary for normal development, in the last stage of development called culmination
Wild-type and cpnA- cells were plated on black filters and allowed to develop for 24-48 hours; Dictyostelium cells placed in starvation buffer on filters at a particular density will complete their development in a characteristic 24-hour period
Summary
Copines are calcium-dependent phospholipid-binding proteins found in diverse eukaryotic organisms. Copines are highly conserved calcium-dependent membrane binding proteins found in many eukaryotic organisms including Paramecium, Dictyostelium, Arabidopsis, C. elegans, mice, and humans [1,2,3,4,5]. The copine family is characterized as having two C2 domains in the N-terminal half of the protein followed by an A domain in the Cterminal half. The C2 domain is a calcium-dependent phospholipidbinding motif originally identified in protein kinase C. Single and multiple copies of C2 domains are found in a large number of eukaryotic proteins. Most proteins containing a single C2 domain are involved in signaling pathways; examples include protein kinases, lipid kinases, phospholipases, and GTPase activating proteins. Most proteins that have multiple C2 domains are involved in membrane trafficking and exocytosis. Some examples of multiple C2 domain proteins are synaptotagmin, rabphilin, DOC2, each of which has two C2 domains, and munc, which has three C2 domains [6]
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