Affiliation:
1. Biochemistry Program
2. Biology Departments, Vassar College, 124 Raymond Ave., Poughkeepsie, New York 12604
3. Chemistry
Abstract
ABSTRACT
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
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,General Medicine,Microbiology
Cited by
42 articles.
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