Cyclic AMP signaling in Dictyostelium promotes the translocation of the copine family of calcium-binding proteins to the plasma membrane

Abstract

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.Electronic supplementary materialThe online version of this article (10.1186/s12860-018-0160-5) contains supplementary material, which is available to authorized users.

Differential expression and subcellular localization of Copines in mouse retina

“…In contrast, Cpne6, originally known as N‐copine, is specifically expressed in the hippocampus and the olfactory bulbs postnatally and has recently been shown to be required for late, mature functions such as long‐term potentiation (LTP) in the hippocampus (Burk et al, ; Reinhard et al, ). Although Copines have a high sequence homology, they differ in their sensitivities to intracellular Ca 2+ concentration (Ilacqua et al, ; P. V. Perestenko et al, ). This might lead to nuanced functional differences, despite cellular overlap of expression.…”

“…These are related to cytoskeletal rearrangements such as activitydependent alteration in spine morphology during long-term potentiation in mouse hippocampus (Reinhard et al, 2016), myofilament stability in C. elegans (Warner et al, 2013), and plant growth in Arabidopsis (Hua, Grisafi, Cheng, & Fink, 2001). Copines were also associated with signaling pathways and trafficking such as contractile vacuole function and cytokinesis in Dictyostelium in response to cAMP signaling (Damer et al, 2007;Ilacqua et al, 2018) and recycling TrkB receptors to promote BDNF-TrkB signaling in mouse hippocampus (Burk et al, 2018). From this study, we conclude that Copines exhibit differential cellular expression in the mouse retina.…”

Copine A Interacts with Actin Filaments and Plays a Role in Chemotaxis and Adhesion

“…Previous studies in our lab have shown that GFP-tagged CpnA localized to the cytosol in live Dictyostelium cells [10,15]. However, when cells were treated with a calcium ionophore in the presence of calcium, GFP-tagged CpnA was found associated with the plasma membrane and intracellular organelles.…”

Copine A regulates the size and exocytosis of contractile vacuoles and postlysosomes in Dictyostelium

“…Our studies on cpnA knockout (KO) mutants ( cpnA − ) in Dictyostelium suggest that CpnA is involved in many cellular functions including cytokinesis, adhesion, and chemotaxis, and developmental functions including aggregation, slug phototaxis and thermotaxis, culmination, and stalk cell formation [12–15]. CpnA binds to membranes in a calcium‐dependent manner and specifically binds to acidic phospholipids with strongest binding to phosphatidylserine and phosphatidylinositol phosphate [11,16]. GFP‐tagged CpnA was found in the cytoplasm in live cells but translocated to the plasma membrane in response to cAMP stimulation [16].…”

“…CpnA binds to membranes in a calcium‐dependent manner and specifically binds to acidic phospholipids with strongest binding to phosphatidylserine and phosphatidylinositol phosphate [11,16]. GFP‐tagged CpnA was found in the cytoplasm in live cells but translocated to the plasma membrane in response to cAMP stimulation [16]. In fixed cells, CpnA localized to the plasma membrane, endosomes, lysosomes, phagosomes, and contractile vacuoles (CV) [11].…”