Description:
<jats:title>ABSTRACT</jats:title>
<jats:p>
Copines 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
<jats:italic>Dictyostelium discoideum</jats:italic>
genome and focused our studies on
<jats:italic>cpnA</jats:italic>
. Our previous localization studies of green fluorescent protein-tagged CpnA in
<jats:italic>Dictyostelium</jats:italic>
suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a
<jats:italic>cpnA</jats:italic>
<jats:sup>
<jats:italic>−</jats:italic>
</jats:sup>
knockout strain, and here we report the initial characterization of the mutant phenotype. The
<jats:italic>cpnA</jats:italic>
<jats:sup>−</jats:sup>
cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions,
<jats:italic>cpnA</jats:italic>
<jats:sup>
<jats:italic>−</jats:italic>
</jats:sup>
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,
<jats:italic>cpnA</jats:italic>
<jats:sup>
<jats:italic>−</jats:italic>
</jats:sup>
cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the
<jats:italic>cpnA</jats:italic>
<jats:sup>
<jats:italic>−</jats:italic>
</jats:sup>
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
<jats:italic>cpnF</jats:italic>
showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of
<jats:italic>Dictyostelium</jats:italic>
development.
</jats:p>