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Copine A, a calcium-dependent membrane-binding protein, transiently localizes to the plasma membrane and intracellular vacuoles in Dictyostelium.

ABSTRACT: BACKGROUND: Copines are soluble, calcium-dependent membrane binding proteins found in a variety of organisms. Copines are characterized as having two C2 domains at the N-terminal region followed by an "A domain" at the C-terminal region. The "A domain" is similar in sequence to the von Willebrand A (VWA) domain found in integrins. The presence of C2 domains suggests that copines may be involved in cell signaling and/or membrane trafficking pathways. RESULTS: We have identified six copines genes in the Dictyostelium discoideum genome, cpnA-cpnF, and have focused our studies on cpnA. CpnA is expressed throughout development and was shown to be capable of binding to membranes in a calcium-dependent manner in vitro. A GFP-tagged CpnA was also capable of binding to membranes in a calcium-dependent manner in vitro. In live wildtype Dictyostelium cells expressing GFP-CpnA, GFP-CpnA was typically found in the cytoplasm without any specific localization to membranes. However, in a small subset of starved cells, GFP-CpnA was observed to bind transiently (typically approximately 1-10 s) to the plasma membrane and intracellular vacuoles. In some cells, the transient membrane localization of GFP-CpnA was observed to occur multiple times in an oscillatory manner over several minutes. In plasma membrane disrupted cells, GFP-CpnA was observed to associate with the plasma membrane and intracellular vacuoles in a calcium-dependent manner. In fixed cells, GFP-CpnA labeled the plasma membrane and intracellular vacuoles, including contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes. CONCLUSION: Our results show that Dictyostelium has multiple copine homologs and provides an excellent system in which to study copine function. The localization of a GFP-tagged CpnA to the plasma membrane, contractile vacuoles, organelles of the endolysosomal pathway, and phagosomes suggests that CpnA may have a role in the function of these organelles or the trafficking to and from them. In addition, we hypothesize that the observed transient oscillatory membrane localization of GFP-CpnA in a small subset of starved cells is caused by fast calcium waves and speculate that CpnA may have a role in development, particularly in the differentiation of stalk cells.

SUBMITTER: Damer CK

PROVIDER: S-EPMC1327671 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Copine A, a calcium-dependent membrane-binding protein, transiently localizes to the plasma membrane and intracellular vacuoles in Dictyostelium.

Damer Cynthia K CK Bayeva Marina M Hahn Emily S ES Rivera Javier J Socec Catherine I CI

BMC cell biology 20051212

<h4>Background</h4>Copines are soluble, calcium-dependent membrane binding proteins found in a variety of organisms. Copines are characterized as having two C2 domains at the N-terminal region followed by an "A domain" at the C-terminal region. The "A domain" is similar in sequence to the von Willebrand A (VWA) domain found in integrins. The presence of C2 domains suggests that copines may be involved in cell signaling and/or membrane trafficking pathways.<h4>Results</h4>We have identified six c ...[more]

PMID: 16343335

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Project description:BackgroundCopines are calcium-dependent phospholipid-binding proteins found in many eukaryotic organisms and are thought to be involved in signaling pathways that regulate a wide variety of cellular processes. Copines are characterized by having two C2 domains at the N-terminus accompanied by an A domain at the C-terminus. Six copine genes have been identified in the Dictyostelium genome, cpnA - cpnF.ResultsIndependent cell lines expressing CpnA, CpnB, CpnC, CpnE, or CpnF tagged with green fluorescent protein (GFP) were created as tools to study copine protein membrane-binding and localization. In general, the GFP-tagged copine proteins appeared to localize to the cytoplasm in live cells. GFP-tagged CpnB, CpnC, and CpnF were also found in the nucleus. When cells were fixed or when live cells were treated with calcium ionophore, the GFP-tagged copine proteins were found associated with the plasma membrane and vesicular organelles. When starved Dictyostelium cells were stimulated with cAMP, which causes a transitory increase in calcium concentration, all of the copines translocated to the plasma membrane, but with varying magnitudes and on and off times, suggesting each of the copines has distinct calcium-sensitivities and/or membrane-binding properties. In vitro membrane binding assays showed that all of the GFP-tagged copines pelleted with cellular membranes in the presence of calcium; yet, each copine displayed distinct calcium-independent membrane-binding in the absence of calcium. A lipid overlay assay with purified GFP-tagged copine proteins was used to screen for specific phospholipid-binding targets. Similar to other proteins that contain C2 domains, GFP-tagged copines bound to a variety of acidic phospholipids. CpnA, CpnB, and CpnE bound strongly to PS, PI(4)P, and PI(4,5)P2, while CpnC and CpnF bound strongly to PI(4)P.ConclusionsOur studies show that the Dictyostelium copines are soluble cytoplasmic and nuclear proteins that have the ability to bind intracellular membranes. Moreover, copines display different membrane-binding properties suggesting they play distinct roles in the cell. The transient translocation of copines to the plasma membrane in response to cAMP suggests copines may play a specific role in chemotaxis signaling.

Dataset Information

Copine A, a calcium-dependent membrane-binding protein, transiently localizes to the plasma membrane and intracellular vacuoles in Dictyostelium.

Publications

Copine A, a calcium-dependent membrane-binding protein, transiently localizes to the plasma membrane and intracellular vacuoles in Dictyostelium.

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