Figure 1.
PKA is activated at the leading edge of migrating SKOV3 cells.
SKOV-3 cells were transfected with pmAKAR3, plated onto fibronectin-coated dishes overnight, stimulated with 10 ng/ml EGF for 4-6 h, and then imaged by FRET microscopy. Images were captured every 30 sec, then pseudocolored according to FRET ratio (color scale shown in frame 0′): this montage depicts frames 2 min apart. For the whole sequence, see Video S1. Scale bar = 5 µm.
Figure 2.
A modified radial migration assay – the Donut assay.
(A) A schematic representation of the donut migration assay in which cells are seeded at confluence on an extracellular matrix-coated glass coverslip within a donut-shaped silicone gasket. After the cells have stably adhered, the gasket is removed, allowing the cells to migrate radially. Images of the monolayer are captured immediately after donut removal and at any time point thereafter that is suitable to allow migration of a given cell line. A custom ImageJ macro uses the initial image as a subtractive mask to determine the number of migrated cells in the final image taken at the end of the assay. Details are given in Materials and Methods. (B) A picture of five donut gaskets on a single 25 mm coverslip; this allows facile setup of replicate assays to increase throughput and generate statistically significant data. (C) COS-7 cells were subject to the donut migration assay in the presence of 2 µM cytochalasin D (cytoD) or DMSO as a vehicle control. Representative images taken at the beginning (0 h) and end (20 h) of the assay, as well as masked (final minus initial) images depicting migrated cells are shown. (D) The number of migrated COS-7 cells was calculated using the ImageJ macro as described in Materials and Methods. Data represent the mean ± S.E. of five donuts per coverslip with three separate coverslips per treatment. (* = P<0.001).
Figure 3.
PKA activity and anchoring are required for SKOV-3 cell migration.
(A) SKOV-3 cells were transfected with either empty mCherry plasmid or mCherry-PKI, then subject to donut migration assays. Representative thresholded images of the migrated cells (Migrated, with nuclei pseudocolored green), the total population of transfected cells (Transfected), and an overlay of these two images are shown. Insets depict enlargements of the areas indicated by the squares in the top panels. Thus, the total migrated cells (‘TM’) are depicted in green, the total transfected cells (‘TX’) are depicted in red, and the yellow nuclei depict transfected cells that migrated, i.e. migrated and transfected cells (‘MX’). (B) For cells transfected with either empty mCherry (mCh) or mCherry-PKI (PKI), the average percent (± S.E.) of migrated transfected cells over the total number of migrated cells (‘MX/TM’) was calculated. To normalize for transfection efficiency, average percent (± S.E.) of migrated transfected cells over the total number of transfected cells (‘MX/TX’) was also calculated. (* = P<0.05) (C) SKOV-3 cells were transfected with plasmids encoding EGFP fused to inhibitors of type-I (RIAD) or type-II (sAis) PKA anchoring, or their respective scrambled controls (RIAD scr, sAis scr), then subject to donut migration assays and analysis as described in (A and B). (** = P<0.001)
Figure 4.
Leading edge PKA activity is mediated by RII-AKAP interactions.
(A-D) SKOV-3 cells co-expressing the indicated plasmids in conjunction with pmAKAR3 were subject to the donut migration assay for 10 hr and imaged via widefield fluorescent microscopy and FRET microscopy. Representative pseudocolored FRET images of cells co-expressing mCherry fused to (A) scrambled superAKAPis (sAKAPis scr), (B) superAKAPis (sAKAPis), (C) scrambled RIAD (RIAD scr), or (D) RIAD are shown. White arrow heads point to leading edge PKA activity. Scale bar = 10 µm. (E) To quantify the percent of cells exhibiting leading edge PKA activity, the cells were divided into leading edge, cytoplasmic, right and left quadrants, and a ratio of the FRET ratio values was generated via linescan analysis through the highest intensity pixels within the leading edge. (F) The tabulated results of cells exhibiting leading edge PKA activity is shown as the percentage of cells displaying low (≤1.19), medium (1.2<x<1.5), or high (≥1.5) LE/Cyto FRET ratios. (G) shows a graphical representation of the tabulated data where cells are scored as having leading edge PKA activity if the LE/Cyto FRET ratios are >1.2 (n = 15 from three separate experiments).
Figure 5.
A modification of the donut assay allows measurement of matrix invasion.
(A) A schematic representation depicting the donut invasion assay. After removal of the silicone gasket, the cell monolayer is covered with a Matrigel overlay, and invasion is analyzed as described for Figure 2. (B, C) Migration and Matrigel invasion of non-tumorigenic human immortalized ovarian (HIO-80) epithelial cells (B) and tumorigenic SKOV-3 (C) was assessed at 24, 36, and 48 h after donut removal, in the presence or absence of the matrix metalloprotease inhibitor GM6001 (25 µM). Graphs represent the mean ± S.E. of the number of migrated or invaded cells. (* = P<0.05)
Figure 6.
PKA is activated at the leading edge of invading SKOV-3 cells.
(A-D) SKOV-3 cells transiently expressing pmAKAR3 were subject to the donut invasion assay and images of cells on the monolayer periphery were acquired 10 h after gasket removal. Pseudocolored FRET images are shown, with insets depicting enlargements of the areas indicated by the white rectangles in each panel. White arrows indicate the direction of invasion with respect to the cell monolayer. Scale bar = 10 µm. (E, F) To quantify the number of invading cells exhibiting leading edge PKA activity, the average FRET ratio was measured by linescan in the leading edge (LE) and the cytoplasm (Cyto) of cells and a ratio of these values (FRET(LE/Cyto)) was determined. (F) shows the percentage of cells exhibiting low, medium, or high LE/Cyto FRET ratios, as described in Figure 4 (n = 28 from four separate experiments).
Figure 7.
Invading edge PKA activity is mediated by RII-AKAP interactions.
(A-D) SKOV-3 cells co-expressing the indicated plasmids in conjunction with pmAKAR3 were subject to the donut invasion assay for 18 hr and imaged via FRET microscopy. Representative pseudocolored FRET images of cells co-expressing mCherry fused to (A) scrambled superAKAPis (sAKAPis scr), (B) superAKAPis (sAKAPis), (C) scrambled RIAD (RIAD scr), or (D) RIAD are shown. White arrowheads point to leading edge PKA activity (scale bar = 10 µm). (E) The results, as the percentages of cells exhibiting PKA activity at the invasive edge (I.E.), were tabulated as in Figure 4 (n = 21 from three separate experiments).
Figure 8.
PKA activity and anchoring are required for SKOV-3 cell invasion.
(A, B) SKOV-3 cells were transiently transfected with plasmids to inhibit PKA activity or anchoring (mCh-PKI, GFP-RIAD, GFP-sAis) or their respective controls (Empty mCh, GFP-RIAD_scr, GFP-sAis_scr), then subject to invasion analysis as described in Figures 3 and 4. (* = P<0.01; ** = P<0.05). In parallel to the method described in Figure 3, invasion was calculated as the average percentage (± S.E.) of invading transfected cells over the total number of invading cells (A; ‘IX/I’) or, to normalize for transfection efficiency, the average percentage of invading transfected cells over the total number of transfected cells (B; ‘IX/X’).