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. 2019 Jul 21;8(7):758.
doi: 10.3390/cells8070758.

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

Matthew J Buccilli  1 , April N Ilacqua  1 , Mingxi Han  1 , Andrew A Banas  1 , Elise M Wight  1 , Hanqian Mao  1 , Samantha P Perry  1 , Tasha S Salter  1 , David R Loiselle  2 , Timothy A J Haystead  2 , Cynthia K Damer  3
Affiliations

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

Matthew J Buccilli et al. Cells. .

Abstract

Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA- cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.

Keywords: Dictyostelium; actin; adhesion; cAMP; calcium; chemotaxis; copine.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Identification of potential CpnA binding partners by column chromatography. (A) Eluates from a control column (Control) and a CpnA-linked column (CpnA) lysate were analyzed by SDS-PAGE. Protein standards (MW) are in kDa. (B) Protein bands (1–5) eluted from the CpnA-linked column were identified by mass spectrometry.
Figure 2
Figure 2
Identification of potential CpnA binding partners by immunoprecipitation. (A) Immunoprecipitations performed with an antibody to GFP using cells expressing GFP and GFP-CpnA were analyzed by SDS-PAGE. Protein standards (MW) are in kDa. (B) Protein bands (1–11) were identified by mass spectrometry.
Figure 3
Figure 3
CpnA binds actin filaments in a calcium-dependent manner. Purified GST-CpnA was incubated with F-actin in the presence and absence of calcium. F-actin was pelleted in an ultracentrifuge, and both the supernatant (S) and pellet (P) were analyzed by SDS-PAGE (Actin) and western blot (GST-CpnA) with an anti-GST antibody. GST-CpnA was also centrifuged in the absence of F-actin (No actin control) and both the supernatant (S) and pellet (P) were analyzed by western blot with an anti-GST antibody. The F-actin binding experiments were performed at least three times, and representative blots are shown.
Figure 4
Figure 4
CpnA binds F-actin, but not G-actin. Immunoprecipitations with an antibody to GFP were performed with cells expressing GFP, GFP-Ado, and GFP-CpnA. Precipitated proteins were incubated with F-actin or G-actin and precipitated again. IPs were analyzed using a western blot with an antibody to actin. Actin alone (actin) was also run on the gel as a positive control. The experiment with each cell type was performed at least three times, and representative blots are shown.
Figure 5
Figure 5
cpnA KO cells exhibit chemotaxis defects. cAMP and folate chemotaxis assays were performed with parental, cpnA−, and cpnA−cre cells. Cells were plated on agar plates 5 mm away from wells containing either buffer or 100 μM cAMP (A) or 100 μM folate (B). Images (A,B) were taken 3 h later. Wells were directly to the right of each cell drop in each image. Scale bar =1mm. (C) Distances cells moved towards and away from wells with cAMP. (D) Distances cells moved towards and away from wells with folate. Three replicates within the same trial for each cell type and chemoattractant were averaged, and then the means from 2–3 trials were averaged. Error bars = standard error. Replicate data were analyzed by a 2-way ANOVA, with post-hoc comparisons using the Tukey method, p < 0.05. Means with different letters are significantly different.
Figure 6
Figure 6
cpnA KO cells exhibit defects in directional sensing, motility, and polarity. Parental, cpnA, and cpnA−cre cells were starved and placed in a Dunn chamber within a cAMP gradient (cAMP) or no gradient (buffer). cpnA− cells were also pulsed with cAMP every six minutes for six hours (cpnA− pulsed) before being placed in the Dunn chamber. Images were taken every 10 s for 10 min using DIC microscopy and individual cells were tracked using ImageJ. (A) Representative cell trajectories from one trial. (B) Representative DIC images of cells within a cAMP gradient. (C) Mean changes in X and Y between 10s images of individual cells: parental (triangles); cpnA−cre (squares); cpnA− (circles); solid symbols are for cAMP gradient; open symbols are for buffer control. Mean changes in X and Y were analyzed by t-tests, * p < 0.05. (D) Mean velocities of individual cells within the Dunn chamber. (E) Mean circularity of cells within the Dunn chamber at a single timepoint. Black bars indicate cAMP gradient; Gray bars indicate buffer control; n = 94 for parental in buffer; n = 128 for parental in cAMP; n = 53 for cpnA−cre in buffer; n = 60 for cpnA−cre in cAMP gradient; n = 74 for cpnA− in buffer; n = 57 for cpnA− in cAMP gradient; n = 53 for cpnA− pulsed in buffer; n = 47 for cpnA− pulsed in cAMP gradient. Mean velocities and circularities were analyzed by a 2-way ANOVA with post-hoc comparisons with the Tukey method, p < 0.05. Means with different letters are significantly different.
Figure 7
Figure 7
cpnA KO cells exhibit reduced actin polymerization in response to cAMP stimulation. Parental and cpnA KO cells (cpnA− and cpnA−cre) cells were pulsed with cAMP every 6 min for 5 h to induce development. Cells were stimulated with cAMP. At various timepoints after stimulation, cells were treated with Triton X-100, and the insoluble actin cytoskeleton was pelleted. Pellets and supernatants were analyzed by SDS-PAGE. The percent of actin in the pellet (F-actin) was determined using densitometry. The densitometry data were normalized to the 0 s timepoint, and three experiments from the same pulsed cell population were averaged. The means from two of cpnA KO or three parental pulsed cell populations were averaged. Error bars = standard error. The % of F-actin in the parental strain at 5 s was significantly different from the 0 s timepoint, and significantly different from the relative % F-actin in the cpnA− and cpnA−cre cells at the 5 s timepoint. (t-tests, * p < 0.05).
Figure 8
Figure 8
cpnA KO cells exhibit increased adhesion. Parental and cpnA− KO cells were plated on 35x10 mm Petri dishes and imaged with phase-contrast microscopy before and after rotation on a benchtop orbital shaker at 50, 75, and 100 RPM. The number of cells in each image were counted using the Cell Counter plugin in ImageJ and averaged for each RPM for three trials. (A) Images before and after rotation at 75 RPM. Scale bar represents 25 µm. (B) Percent difference of cells remaining adhered to the bottom of Petri dishes after rotation. Error bars represent standard error. Means for each rotation speed with different letters are significantly different (t-tests, p < 0.05).
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