opines 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.
|State||Published - Mar 2007|