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Article

Loss of Copine D Leads to Ras Activation in Dictyostelium discoideum

by
Cody T. Morrison
1,2,
Sela K. Damer-Daigle
2,
Allison G. Maillette
2 and
Cynthia K. Damer
1,2,*
1
Biochemistry, Cell & Molecular Biology Program, Central Michigan University, Mount Pleasant, MI 48859, USA
2
Department of Biology, Central Michigan University, Mount Pleasant, MI 48859, USA
*
Author to whom correspondence should be addressed.
Cells 2026, 15(15), 1389; https://doi.org/10.3390/cells15151389
Submission received: 6 July 2026 / Revised: 28 July 2026 / Accepted: 28 July 2026 / Published: 31 July 2026
(This article belongs to the Special Issue Dictyostelium as a Model in Cell Dynamics and Disease)
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Abstract

Copines are a family of calcium-dependent phospholipid-binding proteins conserved across numerous eukaryotes. The expression of multiple copine genes is dysregulated in several human cancers, yet their molecular functions remain poorly understood. We are investigating copine genes (cpnA-cpnF) in the amoeba Dictyostelium discoideum, a well-established model for studying conserved signaling pathways that regulate chemotaxis. During development, individual amoebae release and move toward cAMP. cAMP chemotaxis utilizes Ras/PI3K signaling, a pathway that is highly conserved in mammalian cells and often dysregulated in cancer. Using two cpnD mutants that were generated via restriction enzyme-mediated integration (REMI), we found that cpnD mutants exhibited increased cellular proliferation, precocious development, and larger fruiting bodies. Additionally, we found that cpnD mutants had increased cell spreading, producing a flattened morphology and larger cell area. Because activated Ras has been shown to promote this phenotype, we measured Ras activity and found that cpnD mutants exhibited increased Ras activation. cpnD mutants also formed significantly smaller contractile vacuoles during osmotic stress. Inhibition of PI3K suppressed both the enlarged cell area and reduced contractile vacuole phenotypes, indicating that these defects result from increased Ras/PI3K signaling. Finally, we found that GFP-tagged CpnD transiently localized to the plasma membrane following cAMP stimulation, suggesting CpnD may have a role in cAMP signaling. Together, these findings identify CpnD as a potential negative regulator of Ras signaling and provide the first evidence of copine involvement in the Ras/PI3K pathway, suggesting a conserved mechanism that may help explain how altered copine expression contributes to cancer progression.

1. Introduction

Copines are a family of evolutionarily conserved, calcium-dependent membrane-binding proteins found in a variety of eukaryotes ranging from unicellular organisms to humans [1]. Copines are characterized by a conserved domain structure consisting of two N-terminal C2 domains and a C-terminal A domain that is similar to the von Willebrand factor A (VWA) domain found in integrins [1]. The C2 domains function to facilitate calcium-dependent lipid binding [1,2], whereas the A domain serves as a protein-binding motif [2]. Together, these domains suggest that copines act as calcium-responsive sensors that recruit or regulate proteins at membrane surfaces in response to intracellular calcium signals [2]. Humans have nine copines encoded in their genome that exhibit differential expression throughout various tissue types. Dysregulated copine expression has been reported in numerous cancers and human diseases [3,4,5,6,7,8]. Additionally, functional studies suggest that human copines play a role in promoting cell proliferation, migration, and metastasis [8,9,10]. Despite these associations, the molecular and cellular mechanisms by which copines regulate signaling pathways remain incompletely understood, highlighting the need for further investigations of copine function in vivo.
To address this, our lab aims to define the mechanistic function of copine proteins using the model organism Dictyostelium discoideum. Dictyostelium has six copine genes, while other model organisms either have no copine genes, like yeast and Drosophila, or only a few copine genes, like C. elegans [11]. In their vegetative state, Dictyostelium exists as single-celled amoebae that feed on bacteria through phagocytosis; however, upon nutrient depletion, changes in gene expression initiate Dictyostelium to undergo development. Development begins when starving amoebae release cyclic adenosine monophosphate (cAMP) pulses, which act as a chemoattractant to initiate aggregation. Cells aggregate into mounds, which transform into migrating slugs. The slugs culminate into fruiting bodies that consist of a stalk supporting a spore head [12]. Early developmental aggregation involves changes in cell migration and adhesion, both of which are processes important in cancer cell metastasis. Therefore, by leveraging Dictyostelium’s unique biology, we aim to uncover the conserved copine functions relevant to both normal cellular processes and cancer progression.
Dictyostelium aggregation is a well-studied model of chemotaxis, where individual starving amoebae detect, relay, and move toward periodic pulses of cAMP. Chemotaxis is driven by a complex signal transduction network that converts the external cAMP signal into localized actin polymerization at the leading edge of the cell. Extracellular cAMP binds to G-protein-coupled receptors, which activate heterotrimeric G-proteins. This leads to the rapid activation of Ras proteins at the leading edge of migrating cells [13]. Ras signals to downstream effectors like phosphoinositide 3-kinase (PI3K) to drive signaling cascades that are involved in the regulation of cell polarity, chemotaxis, F-actin dynamics, phagocytosis, and gene expression [14,15,16,17]. Studies have shown that constitutively active RasC or RasG leads to hyperactivation of signaling pathways, resulting in increased cell spreading, cytoskeletal activity, and altered migration [18,19,20,21,22]. Importantly, the mechanisms of Ras-mediated chemotaxis are conserved between Dictyostelium and mammalian cells. In mammalian cells, Ras signaling pathways also regulate cellular proliferation, growth, survival, and metabolism, and are frequently activated by mutations in cancer [23,24]. As such, the Ras signaling pathway is often targeted for therapeutic interventions in cancer models [25].
The six copine genes (cpnA-cpnF) that encode proteins CpnA-CpnF in Dictyostelium exhibit 28–60% amino acid identity and have distinct expression patterns throughout development, suggesting that they perform nonredundant functions [11,26]. Studies with cpnA null (cpnA-) cells have revealed numerous cellular phenotypes, including defects in chemotaxis, adhesion, cytokinesis, contractile vacuole (CV) function, development, and phosphatidylserine (PS) exposure [26,27,28,29,30]. cpnA- cells also showed increased cytosolic calcium concentrations, indicating a role for CpnA in calcium homeostasis [30]. cpnC- cells exhibited different cellular phenotypes, including distinct defects in chemotaxis, adhesion, and development. cpnC- cells also exhibited significantly reduced expression of RegA, a cAMP phosphodiesterase, important in the regulation of intracellular cAMP levels [31]. In this study, we describe the phenotypic analysis of cpnD mutants. Here, we show that cpnD mutants display distinct defects in morphology, growth, development, and CV size, along with increased Ras activation. Furthermore, we show that GFP-tagged CpnD localizes to the cytosol and transiently translocates to and from the plasma membrane in response to cAMP stimulation. Together, all three copine proteins appear to serve as regulators of key cell signaling pathways.

2. Materials and Methods

2.1. Dictyostelium Cell Strains and Culture

The parental axenic strain (AX4) and the cpnD mutants were obtained from the Dictyostelium Stock Center [32]. AX4 (DBS0351471) and cpnD mutants were grown on plastic Petri dishes at 18 °C in VL-6 media (VL60102; Formedium, Norfolk, UK) supplemented with penicillin-streptomycin (60 U/mL, P4333, MilliporeSigma, Burlington, MA, USA). cpnD mutants were cultured in media containing blasticidin (30 µg/mL, ant-bl-1; InvivoGen, San Diego, CA, USA). The cpnD mutant cell lines were generated via restriction enzyme-mediated integration (REMI) and are part of the genome-wide Dictyostelium insertion (GWDI) bank [33]. cpnD mutant cell lines consist of a 1589 bp REMI insert containing a blasticidin-resistant gene (bsr) inserted into the first (291 bp) or second (459 bp) exon of the endogenous cpnD gene to create the cpnD(i291) (DBS0388750) and cpnD(i459) (DBS0383124) cells, respectively. The cpnD mutant cell lines were clonally isolated, and the location of the insertion was verified by PCR and sequencing. The cDNA for cpnD was obtained using RT-PCR and subcloned into the SacI site of the pTX-GFP plasmid [34,35]. The plasmid was electroporated into the AX4 parental cell strain. Cells were pulsed twice with a 5-s interval at 0.85 kV. Cells were placed on ice for 5 min and then plated in VL-6 media. Transformants were selected using G418 (ant-gn-2, InvivoGen, San Diego, CA, USA) at 60 µg/mL after 24 h.

2.2. Growth Assay

AX4 and cpnD mutant cell lines were harvested from plates and counted. Cells (5 × 104 cells/mL) in 12.5 mL of VL-6 media with 60 U/mL penicillin-streptomycin were shaken (125 RPM) at 18 °C. Cell densities of three small samples were estimated each day and averaged. The mean cell densities on each day for each cell strain from three trials were averaged. To determine significant differences between cell growth in AX4 and cpnD mutant cells, a repeated measures ANOVA with Tukey’s post hoc multiple comparisons test was performed.

2.3. Developmental Assays

AX4 and cpnD mutant cells were harvested from plates, counted, and spun at 437× g for 5 min at 4 °C. Cell pellets were washed twice with ice-cold developmental buffer ((DB), 5 mM Na2HPO4, 5 mM KH2PO4, 1 mM CaCl2, 2 mM MgCl2, pH 6.5), and resuspended to obtain 5 × 107 cells/mL. Cells (2.5 × 107) were plated on black filters (HABP04700; MilliporeSigma, Burlington, MA, USA), atop DB-soaked pads in dishes (09-753-53C; Fisher Scientific, Pittsburgh, PA, USA), and allowed to develop for 48 h. Images were taken using a Nikon SMZ800N dissecting microscope every two hours for 28 h, followed by a 48-h image to observe mature fruiting bodies. Two developmental assays were performed, and representative images for each cell line were chosen. The area of the spore heads from each cell line (n > 700) across two trials was measured using ImageJ version 2. Individual fruiting body measurements were graphed for each cell line after removing outliers. A one-way ANOVA with Tukey’s post hoc multiple comparisons test was performed to determine statistical significance between the AX4 and cpnD mutant cell lines.
For developmental assays on bacterial lawns, E. coli B/R were grown in 2 mL of VL-6 without penicillin-streptomycin shaking (220 RPM) at 37 °C for 16 h. AX4 and cpnD mutant cell lines were harvested, counted, and serially diluted to obtain a final concentration of 500 cells/mL. E. coli B/R (500 µL) and Dictyostelium cells (100 µL) were plated on SM/5 agar plates (0.2% proteose peptone 2, 0.2% yeast extract, 0.2% glucose, 0.2% MgSO4 × 7 H2O, 0.19% KH2PO4, 1% K2HPO4, 1.5% agar, pH 6.4) and incubated at 18 °C. Plates were imaged with a Nikon SMZ800N dissecting microscope on days 3, 4, and 5.

2.4. Cell Area Measurements

AX4 and cpnD mutant cells were harvested, counted, and spun at 437× g for 5 min at 4 °C. The cell pellet was resuspended in VL-6 media at 2 × 106 cells/mL. On glass-bottom dishes, 200 µL of the cell suspension was plated and allowed to adhere for 20 min. Cells were then imaged using a TE2000-S Nikon Eclipse microscope with a 60× oil objective and differential interference contrast (DIC) microscopy. The cross-sectional area of individual cells from each cell type (n > 700) across two trials was measured using ImageJ version 2, and individual cell measurements were graphed for each cell type after removing outliers. A one-way ANOVA with Tukey’s post hoc multiple comparisons test was performed to determine statistical significance between the AX4 and cpnD mutant cell lines.

2.5. Adhesion Assay

AX4 and cpnD mutant cells were harvested, counted, and centrifuged at 437× g for 5 min at 4 °C. The cell pellets were resuspended in VL-6 media. Cells (2 × 106 cells) were plated on 60 mm Petri dishes. A Nikon TE2000 microscope with a 20× phase-contrast objective was used to image cells at three fixed locations on the Petri dish. The plates were placed on a rotator (50 RPM) for 15 min. The media was removed along with any cells that had become unattached, and new media was added. Images were captured at the same locations. Cells were rotated again at 75 and 100 RPM, and images were taken. The ImageJ version 2 Cell Counter plugin was used to count the number of cells in each image and cell counts were averaged. The percentage of detached cells after each rotation was determined by subtracting the average number of cells adhered after rotation from the average number of cells present before rotation and dividing this difference by the average number of cells present before rotation. Data from five trials were averaged and analyzed for significant differences between AX4 and cpnD mutant cells by a two-way ANOVA with uncorrected Fisher’s LSD post hoc test.

2.6. Ras Activation Assay

AX4 and cpnD mutant cells were harvested, counted, and spun at 437× g for 5 min at 4 °C. The cell pellets were resuspended in VL-6 media at 4 × 106 cells/mL. To assess Ras activity in the AX4 and cpnD mutant cells, we used an activated Ras pull-down assay kit (BK008-S; Cytoskeleton Inc., Denver, CO, USA) following the manufacturer’s protocol, with the exception that 4 × 106 cells/mL were used for the pull-down assays. Total Ras whole cell samples and activated Ras samples were analyzed by Western blot with an antibody to Ras (see Section 2.7). Data from three and four trials were averaged for the activated Ras and total Ras, respectively. The data were analyzed for significant differences by a one-way ANOVA with Tukey’s post hoc multiple comparisons test.

2.7. Western Blot

AX4 and cpnD mutant cells were harvested, counted, and spun at 437× g for 5 min at 4 °C. The cell pellets were resuspended in sample buffer (0.2 M Tris-HCl, 0.4 M DTT, 277 mM SDS, 6 mM Bromophenol blue, 4.3 M Glycerol), and 2 × 106 cells were loaded into each well of a stain-free gel (4568034; BIO-RAD, Hercules, CA, USA) and run for 2 h at 100 volts. Stain-free gels were imaged using a BIO-RAD ChemiDoc Touch Imaging System and then transferred to a polyvinylidene difluoride (PVDF) membrane (88518; ThermoFisher Scientific, Waltham, MA, USA) for 1 h at 100 volts. The PVDF membranes were incubated in Blotto (5% dried milk in 1% (v/v) Tween-20 in phosphate-buffered saline (PBS-T)) for 30 min, and then incubated with α-SibA (1:1000) polyclonal antibody (Genèva Antibody Facility, Genèva, Switzerland) for 2 h at room temperature, or α-Pan Ras (1:250) mouse monoclonal antibody for Ras pulldown samples (BK008-S; Cytoskeleton, Denver, CO, USA) for 3 h at room temperature. The membranes were washed three times for 5 min with PBS-T. After washing, the membranes were incubated with an α-rabbit HRP-conjugated secondary antibody (1:15,000) or an α-mouse HRP-conjugated secondary antibody (1:15,000) for 1 h at room temperature in Blotto. The membranes were washed three times for 10 min with PBS-T. Membranes were imaged on the BIO-RAD ChemiDoc Touch Imaging system using Femtoglow HRP substrate (FWPS02; Michigan Diagnostics, Royal Oak, MI, USA).

2.8. Western Blot Densitometry

For densitometry analysis of SibA Western blots, band densities were measured using Image Lab v6.1 from BIO-RAD. Each band density on the Western blot was normalized to the density of all proteins in the corresponding lane from the stain-free gel image. After normalization to the stain-free gel, the band densities were normalized to the average band densities across all lanes. Western blot and gel images from six trials were analyzed, and normalized band densities were averaged and analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons test.
For densitometry analysis of Ras Western blots, band densities of active Ras and total Ras were measured using Image Lab v6.1 from BIO-RAD. Each band density was normalized to the density of all proteins in the corresponding lane from the stain-free gel image. The normalized densities of the active Ras bands were further normalized to the average band densities across all active Ras lanes, and the same was done for total Ras as described above. Normalized active Ras band densities were further normalized to the normalized band densities of their respective total Ras samples. Western blot images from three trials (active Ras) or four trials (total Ras) were analyzed, and normalized band densities were averaged and analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons test.

2.9. Contractile Vacuole Assay

AX4 and cpnD mutant cells in VL-6 media (2 × 106 cells/mL) were plated on 35 mm glass-bottom dishes and allowed to adhere for 20 min. The media was removed and replaced with water or water with 25 µM LY294002 (9901S; Cell Signaling Technologies, Danvers, MA, USA). After a two-hour incubation period, the cells were imaged using a TE2000-S Nikon Eclipse microscope with a 60× oil objective and differential interference contrast (DIC) microscopy. The cross-sectional area of each contractile vacuole (n > 209 CVs/cell type) was measured using ImageJ version 2, outliers were removed, and a one-way ANOVA with Tukey’s post hoc multiple comparisons test was performed to analyze significant differences between AX4 and each of the cpnD mutant cells, with and without LY294002 treatment.

2.10. GFP-Tagged CpnD Assays

Parental cells containing the GFP-CpnD expression plasmid were harvested from plates, counted using a hemocytometer, and centrifuged at 437× g for 5 min. The cells were resuspended in DB at 5 × 106 cells/mL. In 35 mm glass-bottom dishes, 2 mL of cell suspension was plated and allowed to starve for 8 h. After 8 h, the DB was removed, and 200 µL of fresh DB with 2 mM caffeine was added to the central well of the glass-bottom dish and allowed to incubate for 30 min. After incubation, 100 µL of the cell suspension was removed from the plates before confocal time-lapse imaging began (images taken every 3 s). After 10 s of imaging, 100 µL of 10 µM cAMP in DB was added. Images were obtained using a Nikon A1R or AX-R confocal microscope with a 60× oil objective. Images were processed in Adobe Photoshop, and levels of brightness and contrast were adjusted. The membrane on and off times were estimated from 7 cells across 3 trials, and average times were calculated.

3. Results

3.1. cpnD Mutants Have Increased Proliferation in Both Axenic and Bacterial Cultures

To study the function of CpnD in Dictyostelium, we obtained two cpnD mutant cell lines and the parental (AX4) strain from the Dictyostelium Stock Center [32]. The cpnD mutants were created as part of a REMI-seq project to generate a genome-wide mutant resource for Dictyostelium [33]. The cpnD mutants contain a 1589 bp insert in the first (cpnD(i291)) or second (cpnD(i459)) exon of the endogenous cpnD gene, respectively (Figure 1A). We isolated clonal populations of each cell strain and confirmed the presence and location of the REMI insertions in each mutant via PCR (Supplementary Figure S1) and Sanger sequencing.
To determine if the mutants exhibited normal proliferation, we performed growth assays with both the parental and cpnD mutant cell lines. Cells were grown in a shaking suspension, and cell samples were collected and counted using a hemocytometer each day for 7 days. Both cpnD(i291) and cpnD(i459) cells appeared to proliferate faster than the parental cell line, but only the cpnD(i291) cells were significantly different from the parental AX4 cells (Figure 1B). We also examined cell proliferation on bacterial lawns. AX4 cells and cpnD mutants were plated on agar with E. coli B/R. As Dictyostelium feeds on the E. coli B/R, clear plaques arise within the bacterial lawn. Images were taken of the plaques on days 3, 4, and 5 after plating. The plaques created by the cpnD mutants appeared on day 2, while AX4 plaques appeared on day 3. cpnD mutant plaques were also noticeably larger than the parental AX4 plaque on day 3 (Figure 1C). As cells consume the bacteria at the edge of the plaque, cells within the plaque starve and go through development to form fruiting bodies. On day 4, both cpnD mutants had begun development with the formation of mounds and slugs, while AX4 cells had not yet formed multicellular structures. On day 5, AX4 cells had formed mounds and a few fruiting bodies, while the cpnD mutants had formed mostly fruiting bodies (Figure 1C). These data indicate that cpnD mutants proliferate faster in both axenic and bacterial cultures.

3.2. cpnD Mutants Exhibit Precocious Development and Have Larger Fruiting Bodies than Parental Cells

We next investigated whether cpnD mutants exhibited normal developmental morphology and timing by monitoring the synchronized development of both the cpnD mutants and parental AX4 cells. Parental and cpnD mutants in starvation buffer were plated on nitrocellulose filters, and images were captured throughout a 48-h timeframe. Representative images of all three cell lines are shown at 4 different time points in Figure 2A. At 6 h, none of the three cell lines displayed multicellular structures. At 18 h, AX4 cells had formed small mounds. In contrast, cpnD(i459) cells had progressed beyond the mound stage and formed large slugs, while the cpnD(i291) cells were at an intermediate stage of development, forming large mounds with some beginning to tip and transition into slugs. These observations indicate that both cpnD mutants develop more rapidly than AX4 cells, with cpnD(i459) cells developing faster than cpnD(i291) cells. Additionally, both cpnD mutants appeared to form larger mounds, slugs, and fruiting bodies compared to AX4 cells (Figure 2A). To quantify these differences, we measured the area of fruiting body spore heads (n > 700 across two trials) using ImageJ. Both cpnD mutants produced significantly larger spore heads than AX4, with cpnD(i459) forming significantly larger spore heads than cpnD(i291) (Figure 2B).

3.3. cpnD Mutant Cells Display a Flattened Morphology and Are Significantly Larger than the Parental Cells

Upon morphological inspection of the cpnD mutant cells, we noticed that some of the mutant cells appeared flatter and “pancake”-shaped compared to the parental AX4 cell line. To quantify these observations, we imaged cells using differential interference contrast (DIC) microscopy and measured the cell area. On average, both the cpnD mutants had a significantly larger mean cell area than the parental cells. Interestingly, cpnD(i459) cells had the largest area and significantly differed in size from the cpnD(i291) cells (Figure 3A,B).

3.4. cpnD Mutants Have Decreased Cell-Substrate Adhesion

The flatter, pancake-like morphology of the cpnD mutant cells suggested that cpnD mutants may have altered substratum adhesion; increased and decreased adhesion have both been shown to be associated with a flattened cellular morphology [36,37]. To test this, parental and cpnD mutants were allowed to adhere to Petri dishes, then rotated at 3 speeds (50, 75, and 100 RPM). After each rotation, the media was replaced to remove detached cells. Three marked locations on the dish were imaged using phase contrast microscopy to assess the number of cells that remained adhered to the dish. We found that both cpnD mutants showed significantly reduced adhesion to the Petri dish at 50 and 75 RPM compared to the parental cell line (Figure 4A). There were no significant differences in cell-substrate adhesion between the parental and cpnD mutant cell lines at 100 RPM, suggesting that the forces inflicted on the cells at high RPM are greater than the adhesion strength of Dictyostelium.
SibA is a transmembrane protein essential for cell-substrate adhesion in Dictyostelium and has been shown to affect cellular adhesion based on its expression [38]. We have previously shown that cpnC- cells have significantly decreased cell-substrate adhesion and reduced SibA expression [31]. To determine if the decreased cell-substrate adhesion in cpnD mutants may be due to changes in SibA expression, whole-cell samples of parental and cpnD mutant cells were analyzed by Western blot to determine levels of SibA expression. SibA expression was reduced in both cpnD mutants compared to parental cells (Figure 4B). Because the α-SibA antibody recognizes an additional protein, we have previously performed Western blotting with sibA null cells to verify that the higher molecular weight band is SibA [31].

3.5. cpnD Mutant Cells Have Increased Levels of Activated Ras

The flatter, pancake-like cellular morphology has been observed in cells that have increased activation of RasC [21], possibly due to the activation of the actin cytoskeleton, causing cells to create protrusions and spread out [39]. Additionally, research has shown that global activation of Ras by recruitment of the RasGef, GefA, to the cellular membrane promotes cell spreading [40]. Because our cpnD mutants were significantly larger and flatter than the parental cell line, we wanted to test whether the cpnD mutants had increased activated Ras. To test this, we performed an activated Ras pulldown assay. Normally, vegetative cells that have not been starved and have not been stimulated with cAMP show very little Ras activation [14]. However, our vegetative cpnD mutants did have significantly higher levels of active Ras compared to the parental strain, while the levels of total Ras between all cell lines were not significantly different (Figure 5).

3.6. cpnD Mutants Have a Reduced CV Area

Contractile vacuoles are intracellular organelles involved in the regulation of water and various ions in numerous cells, including Dictyostelium [41]. We have previously shown that cpnA- cells have larger and more persistent CVs [26,28]. Because of the role CpnA plays in CV function, we wanted to determine if cpnD mutants exhibited similar CV defects. To test this, we incubated parental and cpnD mutant cells on glass-bottom dishes in water for 2 hours and then captured images of cells using DIC microscopy. The cross-sectional area of individual CVs in each cell in the images was measured (n > 1450 CVs per cell line across three trials). We found that cpnD mutants exhibited significantly smaller CVs compared to the parental cell line (Figure 6A, B). Interestingly, the cpnD(i459) mutants had the smallest average CV area, while the average CV area of cpnD(i291) cells was significantly larger than cpnD(i459) mutants, but still significantly smaller than the parental cells.

3.7. PI3K Inhibition Suppresses the Large Cell Area and Small CV Defects Observed in cpnD Mutants

To determine if the increased Ras activation observed in cpnD mutants is responsible for their flatter morphology, we used the PI3K inhibitor LY294002 to inhibit PI3K, which is activated by Ras. We performed DIC microscopy to image cells in the absence or presence of LY294002 treatment (n > 180 cells per cell line across two trials) and measured the mean cell area. As we previously observed (Figure 3), the mean cell area of the cpnD mutants was significantly larger than that of the parental cell line. However, upon LY294002 treatment, the mean cell area of both cpnD mutant cell lines was significantly reduced, so much so that the cpnD(i291) cells were no longer significantly larger than the parental cell line (Figure 7A).
To determine whether the small CV phenotype observed in cpnD mutants was associated with Ras/PI3K overactivation, we searched for similar phenotypes reported in the literature. One study identified sodC, which encodes a superoxide dismutase, in a screen for mutants with constitutively active PI3K [42]. Further studies showed that sodC- cells exhibited smaller than normal CVs that could be rescued by the PI3K inhibitor, LY294002 [43]. To determine whether LY294002 could suppress the small CV defect observed in the cpnD mutants, we performed CV assays with LY294002-treated and untreated parental AX4 and cpnD mutant cell lines. Cells were allowed to adhere to glass-bottom dishes and then incubated in water with or without LY294002 before performing DIC microscopy to measure the individual area of CVs (n > 200 CVs per cell line across two trials). As we previously observed, the mean CV area of the cpnD mutants was smaller compared to AX4 (Figure 7B). However, when we treated the cells with LY294002, the mean CV area of the cpnD mutant cell lines significantly increased, while the CV area of the parental AX4 strain did not change (Figure 7B). These experiments indicate that the flat morphology and small CV defects observed in cpnD mutants are due to increased activation of the Ras/PI3K signaling pathway.

3.8. GFP-Tagged CpnD Translocates from the Cytosol to the Plasma Membrane in Response to cAMP Stimulation

Ras proteins are uniformly distributed along the plasma membrane of Dictyostelium cells, and it has been shown that cAMP stimulation results in the activation of Ras (Ras-GTP) from its inactive state (Ras-GDP) [44,45]. Ras and subsequently PI3K are activated at the plasma membrane in response to cAMP stimulation [13,15,21,22]. We hypothesized that if CpnD is involved in this pathway, CpnD will translocate to the plasma membrane in response to cAMP stimulation. To test this hypothesis, we subcloned the cDNA for cpnD into the pTX-GFP vector at the SacI site for the overexpression of CpnD tagged with GFP at the N-terminus (GFP-CpnD) in the parental cell line. Cells were starved for 8 h to induce aggregation and then incubated with caffeine for 30 min to suppress the release of endogenous cAMP. Time-lapse confocal microscopy was used to image cells before and after cAMP stimulation. In some cells, we captured GFP-tagged CpnD translocating from the cytosol to the plasma membrane and then returning to the cytosol (Figure 8, Supplementary Video S1). The average time of translocation after cAMP stimulation was 24 ± 4 s, while the average time on the membrane was 18 ± 4 s (n = 7 cells, across 3 trials). These experiments indicate that CpnD translocates to the plasma membrane in response to cAMP and could potentially play a role in the regulation of Ras at the plasma membrane.

4. Discussion

To study the function of CpnD, we obtained two REMI mutants from the Dictyostelium Stock Center that differed in the location of the insert within the cpnD gene [33]. The cpnD(i291) cell line contains the REMI insert within the first exon of the endogenous cpnD gene, while the cpnD(i459) cell line contains the REMI insert in the second exon. We initially hypothesized that the phenotypic defects of the cpnD(i459) mutant with the REMI insert after the first C2 domain might be less severe because the allele may encode a partially functional protein. However, we observed the opposite; the phenotypic defects seen in the cpnD(i459) mutant were more severe than those of the cpnD(i291) mutant. One possible explanation is that the cpnD(i459) mutant is making a truncated CpnD protein containing the C2 domain that exerts a dominant-negative effect. The truncated protein may retain the ability to bind some target proteins but lack normal function, thereby blocking other proteins from accessing those same targets.
The Ras activation assays we performed did not allow us to identify which of the different Ras isoforms were overactivated in the cpnD mutants. However, both RasC and RasG have been shown to be involved in the activation of PI3 kinases that control early developmental cAMP signaling and various actin-mediated functions like chemotaxis, macropinocytosis, and phagocytosis [15,46,47]. We hypothesize that all the phenotypes observed in the cpnD mutants can be attributed to the overactivation of Ras. Ras activity is regulated by RasGEFs and RasGAPs [48], and the Dictyostelium genome has at least 25 putative RasGEFs [49] and 14 putative RasGAPs [40], and previous studies with mutations that cause the overactivation of Ras have resulted in similar phenotypes. For example, expression of constitutively active RasC in pten- cells leads to cell spreading and a flattened morphology [21]. Likewise, recruitment of RasGEF to the plasma membrane also causes cell spreading [40].
Although the increased growth and precocious development may seem contradictory given that cells cease to divide during development, Ras proteins are involved in regulating both the cytoskeletal changes that occur necessary for chemotaxis during development and the cytoskeletal changes necessary for macropinocytosis and phagocytosis during growth. Therefore, it is possible that the loss of cpnD could increase both growth-related behaviors in vegetative cells and prime cells for development when starved. Previous studies have shown that loss of the RasGAP NF1 allows for axenic growth by increasing macropinocytosis and results in increased phagocytosis [50]. Similarly, loss of the RasGAP C2GAP2 results in increased macropinocytosis and phagocytosis, which leads to increased growth rate [51]. While we did not directly assess macropinocytosis and phagocytosis in the cpnD mutants, we speculate that the slightly faster growth in axenic culture and increased plaque size on bacterial lawns could be attributed to increased macropinocytosis and phagocytosis, respectively.
Ras is also activated in response to increased superoxide species, and Dictyostelium lacking superoxide dismutase (sodC-) have increased activated RasG [42,52]. sodC- cells also have significantly smaller contractile vacuoles (CVs), a phenotype that could be suppressed upon inhibition of PI3K with LY294002 [43,52]. The smaller CV phenotype observed in the cpnD mutants could also be suppressed with LY294002 treatment, indicating that increased Ras activation in these mutants also causes the small CV phenotype. cpnD mutants also exhibited precocious development and reduced cell-substratum adhesion, as well as decreased SibA expression. These phenotypes are also likely due to the higher levels of activated Ras in the cpnD mutants. Ras is activated in response to cAMP signaling, which regulates early development and changes in gene expression during development. sibA is a gene that is downregulated during early Dictyostelium development [31]; therefore, we hypothesize that the increased Ras activation in cpnD mutants results in precocious development, which is dependent on early developmental gene expression changes, like reduced sibA expression.
Higher levels of active Ras suggest that CpnD acts as a negative regulator of Ras. CpnD could act directly on Ras, or CpnD could indirectly regulate Ras, perhaps through the regulation of a Ras-GAP or Ras-GEF. If CpnD acts upstream of Ras in response to cAMP stimulation, then we would expect CpnD to be located at the plasma membrane or to translocate to the plasma membrane in response to cAMP. We previously showed that the other 5 copines in Dictyostelium are cytosolic proteins that bind to specific acidic phospholipids in a calcium-dependent manner and that all five copines transiently translocate to the plasma membrane in response to cAMP stimulation. Each of the five copine proteins translocated from the cytosol to the plasma membrane with different timing and magnitude [35]. Here, we show that CpnD also translocates from the cytosol to the plasma membrane in response to cAMP stimulation. The timing is most similar to GFP-tagged CpnC data [35], with the GFP-tagged CpnD showing an average “ON” time from cAMP stimulation of 24 ± 4 s and lasting around 18 ± 4 s total.
cAMP triggers a rapid rise in the cytosolic concentration of calcium within seconds, with published peak estimates typically in the range of 10–20 s [53,54,55,56]. However, a more recent paper showed calcium levels staying higher for longer and peaking around 40 s after cAMP stimulation [57]. The transient rise in calcium concentration in response to cAMP creates periodic oscillations of intracellular calcium concentrations during aggregation with similar timing of cAMP waves. CpnD translocation to the plasma membrane could be in response to changes in calcium concentrations and may also follow a periodic pattern of translocation to and from the plasma membrane during aggregation.
Previous work has demonstrated that Ras activation has three distinct phases, with maximal activation occurring 6 s after cAMP stimulation, followed by symmetry breaking, and then confinement of Ras activity to the leading edge of the cell 20–30 s later [13]. Because our data suggest that GFP-CpnD is transiently recruited to the membrane after the initial activation of Ras, we suspect CpnD may function during the symmetry breaking or confinement stages of Ras activation, possibly to facilitate negative feedback mechanisms that restrict Ras signaling during cellular polarization. The RasGAP, C2GAP1, which contains one C2 domain, is rapidly (~6 s) translocated to the plasma membrane in response to cAMP and plays an important role in the regulation of the adaptation to cAMP signals [58,59]. The targeting of C2GAP1 is sensitive to calcium, and the timing changes depending on the cytosolic calcium concentration [57]. The rapid translocation of C2GAP1 to the membrane suggests that CpnD is not involved in the recruitment of C2GAP1 to the membrane; however, CpnD could be translocated to the membrane in response to higher levels of calcium and regulating the RasGAP already at the membrane.
This study is the first to describe copine proteins as having a regulatory function in Ras activation and its subsequent downstream signaling effects. In genetic cancer screenings, it has been shown that numerous copine genes are dysregulated [3]. Additionally, studies have shown that constitutively active Ras leads to downstream signaling pathway dysregulation, and mutations that cause constitutively active Ras are highly prevalent in human cancers [22,24]. Therefore, understanding the biological role of copines could add to our understanding of how cancer progresses, as well as provide insights into new therapeutic interventions.

5. Conclusions

Loss of cpnD leads to elevated Ras activation, which likely explains multiple phenotypes, including cell spreading and altered development. CpnD appears to function as a negative regulator of Ras and transiently localizes to the plasma membrane in response to cAMP stimulation, suggesting a role in feedback control of Ras activity during early development.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells15151389/s1, Figure S1: PCR verification of REMI inserts in cpnD gene; Video S1: GFP-tagged CpnD translocates to the plasma membrane in response to cAMP stimulation.

Author Contributions

Conceptualization, C.T.M., S.K.D.-D. and C.K.D.; methodology, C.T.M., S.K.D.-D., A.G.M. and C.K.D.; software, C.T.M., S.K.D.-D. and C.K.D.; validation, C.T.M., A.G.M. and C.K.D.; formal analysis, C.T.M., S.K.D.-D. and C.K.D.; investigation, C.T.M., A.G.M. and S.K.D.-D.; resources, C.K.D.; data curation, C.T.M., and S.K.D.-D.; writing—original draft preparation, C.T.M.; writing—review and editing, C.T.M. and C.K.D.; visualization, C.T.M.; supervision, C.K.D.; project administration, C.K.D.; funding acquisition, C.K.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by NIH, grant number 2R15GM078089-03. Additional funding was provided by Central Michigan University’s Department of Biology and the College of Science and Engineering. The confocal microscopy images were taken on a Nikon AX-R purchased with funding from the NSF, grant number 2510241.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data files are available from the Dryad Repository database: http://datadryad.org/share/LINK_NOT_FOR_PUBLICATION/ADIay9eZ041lJTonV4nspYrkSIlDYxypyX2bVhV0GxI (accessed on 27 July 2026).

Acknowledgments

We thank the undergraduate students in the Damer Lab for their contributions, including Sonya Ruiz, Ashlyn Nagel, Robyn Winegarten, and Jordyn Anklam. We also thank Central Michigan University’s imaging facilities.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
REMIRestriction Enzyme-Mediated Integration
cpnCopine gene
CpnCopine protein
cAMPCyclic Adenosine Monophosphate
GWDIGenome-Wide Dictyostelium Insertion
PI3KPhosphoinositide 3-Kinase

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Cells 15 01389 g001
Figure 1. cpnD mutants have increased proliferation in both axenic and bacterial cultures. (A) Schematic showing the location of the 1589 bp REMI insert in the first (cpnD(i291)) or second (cpnDi(459)) exon of the endogenous AX4 cpnD gene in the two cpnD mutants. (B) AX4 cells and both cpnD mutants were grown in a shaking suspension starting at a cellular density of 5 × 104 cells/mL. Cell densities were counted and averaged once a day for 7 days using a hemocytometer. Mean cell densities were calculated from three trials, and a repeated measures ANOVA with Tukey’s post hoc multiple comparisons test was performed to analyze significant differences between cell lines. * indicates a significant difference between the parental AX4 cell line and cpnD(i291), p < 0.05. Error bars = standard error. (C) AX4 and cpnD mutant cells were plated with E. coli B/R on SM/5 agar. Plaques were imaged after 3, 4, and 5 days after plating, and representative images are shown. Day 3 scale bar = 1000 µm. Day 4 and 5 scale bars = 500 µm.
Figure 1. cpnD mutants have increased proliferation in both axenic and bacterial cultures. (A) Schematic showing the location of the 1589 bp REMI insert in the first (cpnD(i291)) or second (cpnDi(459)) exon of the endogenous AX4 cpnD gene in the two cpnD mutants. (B) AX4 cells and both cpnD mutants were grown in a shaking suspension starting at a cellular density of 5 × 104 cells/mL. Cell densities were counted and averaged once a day for 7 days using a hemocytometer. Mean cell densities were calculated from three trials, and a repeated measures ANOVA with Tukey’s post hoc multiple comparisons test was performed to analyze significant differences between cell lines. * indicates a significant difference between the parental AX4 cell line and cpnD(i291), p < 0.05. Error bars = standard error. (C) AX4 and cpnD mutant cells were plated with E. coli B/R on SM/5 agar. Plaques were imaged after 3, 4, and 5 days after plating, and representative images are shown. Day 3 scale bar = 1000 µm. Day 4 and 5 scale bars = 500 µm.
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Figure 2. cpnD mutants exhibit precocious development and have significantly larger fruiting bodies. (A) AX4 and cpnD mutant cells were resuspended in starvation buffer and plated on nitrocellulose filters to synchronize developmental timing. Representative images across two trials are shown throughout a 48-h imaging window. At 6 h, none of the cell lines exhibited multicellular characteristics. At 18 h, AX4 cells formed small mounds, while cpnD(i459) mutants formed large slugs and cpnD(i291) formed large mounds with some slugs. At 24 h, AX4 cells formed slugs, while both cpnD mutants formed larger slugs and showed occurrences of fruiting body formation. At 48 h, all three cell lines reached their terminal developmental time point, exhibiting fruiting bodies of various sizes. Scale bar = 1 mm. (B) Insets from white boxes shown in Figure 2A of AX4 and cpnD mutant cells at the 48 h developmental time point. The area of individual spore heads (n > 700 across two trials) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean area of fruiting body spore heads. Different lowercase letters above the bars indicate statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Error bars = standard error.
Figure 2. cpnD mutants exhibit precocious development and have significantly larger fruiting bodies. (A) AX4 and cpnD mutant cells were resuspended in starvation buffer and plated on nitrocellulose filters to synchronize developmental timing. Representative images across two trials are shown throughout a 48-h imaging window. At 6 h, none of the cell lines exhibited multicellular characteristics. At 18 h, AX4 cells formed small mounds, while cpnD(i459) mutants formed large slugs and cpnD(i291) formed large mounds with some slugs. At 24 h, AX4 cells formed slugs, while both cpnD mutants formed larger slugs and showed occurrences of fruiting body formation. At 48 h, all three cell lines reached their terminal developmental time point, exhibiting fruiting bodies of various sizes. Scale bar = 1 mm. (B) Insets from white boxes shown in Figure 2A of AX4 and cpnD mutant cells at the 48 h developmental time point. The area of individual spore heads (n > 700 across two trials) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean area of fruiting body spore heads. Different lowercase letters above the bars indicate statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Error bars = standard error.
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Figure 3. cpnD mutants have a larger cell area than parental cells. (A) The cell area of AX4 and cpnD mutant cells (n > 700 cells across two trials) was measured using ImageJ, and individual data points are plotted for each cell type with bars representing the mean cell area. Different lowercase letters above the bars indicate statistically significant differences among the cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons (p < 0.0001). (B) AX4 and cpnD mutants were plated on glass-bottom dishes and imaged using DIC microscopy. Representative images are shown with insets from white boxes. Scale bar = 25 µm.
Figure 3. cpnD mutants have a larger cell area than parental cells. (A) The cell area of AX4 and cpnD mutant cells (n > 700 cells across two trials) was measured using ImageJ, and individual data points are plotted for each cell type with bars representing the mean cell area. Different lowercase letters above the bars indicate statistically significant differences among the cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons (p < 0.0001). (B) AX4 and cpnD mutants were plated on glass-bottom dishes and imaged using DIC microscopy. Representative images are shown with insets from white boxes. Scale bar = 25 µm.
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Figure 4. cpnD mutants have reduced cell-substrate adhesion and SibA expression. (A) AX4 and cpnD mutant cells were plated on 60 mm Petri dishes. Cells were imaged with phase-contrast microscopy at three marked spots before and after rotation at 50, 75, and 100 RPM. The number of cells in each image was counted and averaged. The percentage of detached cells after rotation was calculated by subtracting the average number of cells remaining after each rotation from the average number of cells present before rotation, divided by the number of cells present before rotation. Data from five trials were averaged and analyzed for significant differences between the AX4 and cpnD mutant cells using a repeated measures two-way ANOVA with uncorrected Fisher’s LSD post hoc test. ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, ns indicates no significant difference. Error bars = standard error. (B) Densitometry analysis of the SibA band on Western blots. The mean intensity of the SibA Western blot band from AX4 cells and cpnD mutants was normalized to their respective lanes on stain-free gel images. The bar graph represents the average normalized band densities from 6 trials, and these data were analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons test. Different letters indicate a significant difference (p < 0.05), while the same letters indicate no significant difference. A representative Western blot analysis of SibA expression in AX4 and cpnD mutant cells is included below the bar graph. Error bars = standard error.
Figure 4. cpnD mutants have reduced cell-substrate adhesion and SibA expression. (A) AX4 and cpnD mutant cells were plated on 60 mm Petri dishes. Cells were imaged with phase-contrast microscopy at three marked spots before and after rotation at 50, 75, and 100 RPM. The number of cells in each image was counted and averaged. The percentage of detached cells after rotation was calculated by subtracting the average number of cells remaining after each rotation from the average number of cells present before rotation, divided by the number of cells present before rotation. Data from five trials were averaged and analyzed for significant differences between the AX4 and cpnD mutant cells using a repeated measures two-way ANOVA with uncorrected Fisher’s LSD post hoc test. ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, ns indicates no significant difference. Error bars = standard error. (B) Densitometry analysis of the SibA band on Western blots. The mean intensity of the SibA Western blot band from AX4 cells and cpnD mutants was normalized to their respective lanes on stain-free gel images. The bar graph represents the average normalized band densities from 6 trials, and these data were analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons test. Different letters indicate a significant difference (p < 0.05), while the same letters indicate no significant difference. A representative Western blot analysis of SibA expression in AX4 and cpnD mutant cells is included below the bar graph. Error bars = standard error.
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Figure 5. cpnD mutants have increased levels of activated Ras. Densitometry analysis of the activated and total Ras bands on Western blots. For active Ras, each band density was normalized to the density of all proteins in the corresponding lane from the stain-free gel image. The normalized band densities of the active Ras bands were normalized to the band densities across all active Ras lanes, and the same was done for total Ras. Normalized active Ras band densities were further normalized to the normalized band densities of their respective total Ras samples. Data from three trials were averaged. Different lowercase letters above the bars indicate statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons test (p < 0.05). Bars sharing the same letter are not significantly different. For total Ras, data from four trials were averaged and analyzed for statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons test (p < 0.05). ns indicates no significant difference. Representative Western blot analyses of active Ras and total Ras are shown below their respective bar graphs. Error bars = standard error.
Figure 5. cpnD mutants have increased levels of activated Ras. Densitometry analysis of the activated and total Ras bands on Western blots. For active Ras, each band density was normalized to the density of all proteins in the corresponding lane from the stain-free gel image. The normalized band densities of the active Ras bands were normalized to the band densities across all active Ras lanes, and the same was done for total Ras. Normalized active Ras band densities were further normalized to the normalized band densities of their respective total Ras samples. Data from three trials were averaged. Different lowercase letters above the bars indicate statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons test (p < 0.05). Bars sharing the same letter are not significantly different. For total Ras, data from four trials were averaged and analyzed for statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons test (p < 0.05). ns indicates no significant difference. Representative Western blot analyses of active Ras and total Ras are shown below their respective bar graphs. Error bars = standard error.
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Figure 6. cpnD mutant cells have a reduced CV area. AX4 and cpnD mutants were plated on glass-bottom dishes and incubated in water for two hours and then imaged using DIC microscopy. (A) The area of individual CVs in AX4 and cpnD mutants (n > 1450 cells across four trials) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean CV area. Different lowercase letters above the bars indicate statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Error bars = standard error. (B). Representative images are shown with insets from white boxes. Scale bar = 25 µm.
Figure 6. cpnD mutant cells have a reduced CV area. AX4 and cpnD mutants were plated on glass-bottom dishes and incubated in water for two hours and then imaged using DIC microscopy. (A) The area of individual CVs in AX4 and cpnD mutants (n > 1450 cells across four trials) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean CV area. Different lowercase letters above the bars indicate statistically significant differences among cell types based on a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Error bars = standard error. (B). Representative images are shown with insets from white boxes. Scale bar = 25 µm.
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Figure 7. PI3K inhibition suppresses both cell and CV size defects. (A) AX4 and cpnD mutant cells were plated on glass-bottom dishes in the absence (solid bars) or presence (striped bars) of 25 µM PI3K inhibitor (LY294002) and imaged with DIC microscopy. The cell area of AX4 and cpnD mutants (n > 180 cells across two trials) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean cell area. Data were analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Different lowercase letters above the bars indicate statistically significant differences, while bars sharing the same letter are not significantly different. Error bars = standard error. (B) AX4 and cpnD mutant cells were plated on glass-bottom dishes and incubated in water in the absence (solid bars) or presence (striped bars) of 25 µM PI3K inhibitor. The area of individual CVs (n > 209 CVs per cell line) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean CV area. Data across two trials were analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Different lowercase letters above the bars indicate statistically significant differences, while bars sharing the same letter are not significantly different. Error bars = standard error.
Figure 7. PI3K inhibition suppresses both cell and CV size defects. (A) AX4 and cpnD mutant cells were plated on glass-bottom dishes in the absence (solid bars) or presence (striped bars) of 25 µM PI3K inhibitor (LY294002) and imaged with DIC microscopy. The cell area of AX4 and cpnD mutants (n > 180 cells across two trials) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean cell area. Data were analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Different lowercase letters above the bars indicate statistically significant differences, while bars sharing the same letter are not significantly different. Error bars = standard error. (B) AX4 and cpnD mutant cells were plated on glass-bottom dishes and incubated in water in the absence (solid bars) or presence (striped bars) of 25 µM PI3K inhibitor. The area of individual CVs (n > 209 CVs per cell line) was measured using ImageJ. Individual data points are plotted for each cell type with bars representing the mean CV area. Data across two trials were analyzed for significant differences using a one-way ANOVA with Tukey’s post hoc multiple comparisons after removing outliers (p < 0.0001). Different lowercase letters above the bars indicate statistically significant differences, while bars sharing the same letter are not significantly different. Error bars = standard error.
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Figure 8. GFP-tagged CpnD translocates to the plasma membrane in response to cAMP stimulation. Parental cells expressing GFP-CpnD were resuspended in DB and plated on a 35-mm glass-bottom dish before an 8-h starvation period. After 8 h, the DB was removed from the dish, and the cells were incubated in 2 mM caffeine for 30 min. Following caffeine treatment, time-lapse confocal microscopy images were obtained every 3 s, and 5 µM cAMP was added after 10 s of imaging (+cAMP in figure). Images were obtained using a Nikon AX-R confocal microscope with a 60× oil immersion objective. Representative images are shown. Arrows indicate GFP-CpnD at the cell membrane. The average time of GFP-CpnD translocation to the membrane was 24 ± 4 s, while the average time on the membrane after cAMP stimulation was 18 ± 4 s (n = 7 cells, across 3 trials). Scale bar = 10 µm.
Figure 8. GFP-tagged CpnD translocates to the plasma membrane in response to cAMP stimulation. Parental cells expressing GFP-CpnD were resuspended in DB and plated on a 35-mm glass-bottom dish before an 8-h starvation period. After 8 h, the DB was removed from the dish, and the cells were incubated in 2 mM caffeine for 30 min. Following caffeine treatment, time-lapse confocal microscopy images were obtained every 3 s, and 5 µM cAMP was added after 10 s of imaging (+cAMP in figure). Images were obtained using a Nikon AX-R confocal microscope with a 60× oil immersion objective. Representative images are shown. Arrows indicate GFP-CpnD at the cell membrane. The average time of GFP-CpnD translocation to the membrane was 24 ± 4 s, while the average time on the membrane after cAMP stimulation was 18 ± 4 s (n = 7 cells, across 3 trials). Scale bar = 10 µm.
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Morrison, C.T.; Damer-Daigle, S.K.; Maillette, A.G.; Damer, C.K. Loss of Copine D Leads to Ras Activation in Dictyostelium discoideum. Cells 2026, 15, 1389. https://doi.org/10.3390/cells15151389

AMA Style

Morrison CT, Damer-Daigle SK, Maillette AG, Damer CK. Loss of Copine D Leads to Ras Activation in Dictyostelium discoideum. Cells. 2026; 15(15):1389. https://doi.org/10.3390/cells15151389

Chicago/Turabian Style

Morrison, Cody T., Sela K. Damer-Daigle, Allison G. Maillette, and Cynthia K. Damer. 2026. "Loss of Copine D Leads to Ras Activation in Dictyostelium discoideum" Cells 15, no. 15: 1389. https://doi.org/10.3390/cells15151389

APA Style

Morrison, C. T., Damer-Daigle, S. K., Maillette, A. G., & Damer, C. K. (2026). Loss of Copine D Leads to Ras Activation in Dictyostelium discoideum. Cells, 15(15), 1389. https://doi.org/10.3390/cells15151389

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