Junction Mapper Download
Celina Ruffel
Junction Mapper is a semi-automated software application for analysing data from images of cells in close proximity to each other in monolayers. The focus of Junction Mapper is to measure the morphology of cell boundaries, define single junctions and quantify the length, area and intensity of the staining of different proteins localised at cell-cell contacts. The output are various unique parameters that assess the contacting interface between cells and up to two junctional markers.
We designed and validated a semi-automated system (Figure 2), Junction Mapper, that builds from our quantitative analysis of images from RNAi screens (Erasmus et al., 2016). Our previous software defines an E-cadherin mask to calculate the intensity specifically around junctions as a percentage of thresholded area of the whole original image. The E-cadherin mask is also used to subtract an ROI from an image of a distinct marker (i.e. F-actin), so that mostly the signal localized at contacts is considered. Junction Mapper implements novel quantification tools (corners, length and area) and a variety of novel primary and secondary parameters expressed per individual junction.
(A-D) Epithelial cells expressing active Rac1 (A) or controls (B) were stained for cadherin receptors. Screenshots of Junction Mapper analyses shows different dilation values (green outline of different widths). Corners delimiting each contacting interface are shown as yellow squares. Selected junctions were quantified with different dilation settings and impact on primary parameters is exemplified on length-based measurements (C, Interface Contour, Fragmented Junction Length) and area-based parameters (D, E-cadherin Area, E-cadherin intensity). Increasing the dilation values modifies the area-based (D) but not the length-based measurements (C). (E-H) Similar analyses were performed on images of endothelial cells treated with thrombin (E) or controls (F) and corresponding graphs show the primary parameters quantifications (G,H). Junction undulation caused by thrombin treatment is not captured by the edge map, which is seen as a comparatively straight line (G). Larger dilation values obtained higher VE-cadherin area measurements at the undulated contacts (H). Values obtained are from selected junctions shown and under the different conditions applied.
(A) Novel parameters were defined to normalise the quantifications with respect to the area or length of contacts. The secondary parameters assess the configuration of the contacting interface (Interface Linearity Index), how much the staining of a junction marker occupies the interface length (Coverage Index) or area (Interface Occupancy). The distribution of junction marker is measured in two ways: their intensity levels within the area occupied by the junction fragments (Cluster Density) or the contacting interface (E-cadherin intensity at interface area). Detailed information of the calculation of parameters is described in Appendix 2. (B-D) Reproducibility of quantification by Junction Mapper in independent biological replicates. (B) Keratinocytes expressing activated Rac1 (green) or controls (non-expressing cells) were stained for E-cadherin (blue). (C-D) Images obtained from two independent biological replicates (replicate 1 and replicate 2) were processed to obtain the secondary parameters Coverage Index (C) or Interface Occupancy (D). Numbers at the top inside graphs show the number of junctions quantified in each sample from two biological replicates (N = 2); ns, non-significant. Arrowheads point to residual E-cadherin staining; thick arrow shows lack of cadherin staining. Scale bar = 20 μM.
(A) Human normal keratinocytes were transfected with pRK5-myc-H-RasG12V, fixed and stained with anti-E-cadherin and anti-myc antibodies. Images are shown of E-cadherin staining and myc staining as a marker of transfected cells. Coloured rectangles mark areas shown as a zoom on the left of the images and highlight control junctions (orange), junctions between expressing and non-expressing cells (en, purple) or between two transfected cells (ee, red). Arrowheads point to E-cadherin staining. (B-F) Quantification of the primary parameters using Junction Mapper. Graphs are plotted showing values of each parameter (Y axis) versus different junction types (X-axis). The parameter name is at the top of each graph and a diagram representing the quantification is shown on the left of its corresponding graph. Data is from one experiment (technical replicate) and the number of junctions analysed for each condition is found at the bottom of the graphs, below each scatter box plot. Statistical analysis was performed using One-way ANOVA, followed by Games-Howell post-hoc test. Non-significant (ns) and significant p-values (
(A, G) Human normal keratinocytes expressing myc-Rac1Q61L or GFP-SrcY527F were fixed and stained with anti-E-cadherin antibodies. Images are shown of E-cadherin staining and GFP or myc as a marker of expressing cells. Coloured rectangles mark areas shown as a zoom on the left of the images and highlight control junctions (orange), junctions between expressing and non-expressing cells (en, purple) or between two transfected cells (ee, red). Arrowheads point to E-cadherin staining. (B-F), (H-L) Quantification of the primary parameters using Junction Mapper. Graphs are plotted showing values of each parameter (Y axis) versus different junction types (X-axis). The parameter name is at the top of each graph and a diagram representing the quantification is shown on the left of its corresponding graph. Technical replicates (Rac1) or biological replicates (Src, N = 2) were quantified. Number of junctions analysed in each condition is found at the bottom of the graphs, below each scatter plot. Statistical analysis was performed using One-way ANOVA, followed by Games-Howell post-hoc test. Non-significant (ns) and significant p-values (
Human normal keratinocytes were transfected with pRK5-myc-H-RasG12V (A), pEGFP-SrcY527F (C) or pRK5-myc-Rac1Q61L (E). Cells were fixed and stained with anti-E-cadherin and, for (A) and (E), anti-myc antibodies. Images are shown of E-cadherin and transfected cells (anti-myc or GFP). Coloured rectangles mark areas shown as a zoom on the left of the images and highlight control junctions (orange), junctions between expressing and non-expressing cells (en, purple) or between two transfected cells (ee, red). Arrowheads point to E-cadherin staining. (B, D, F) Quantification of different parameters obtained with Junction Mapper. Graphs are plotted to show values of each parameter (Y-axis) versus different junction types (X-axis) for H-RasG12V (B), SrcY527F (D) and Rac1Q61L (F). The parameter name and a diagram representing the quantification are shown on top of each graph. Technical (H-Ras, Rac1) or biological replicates (Src, N = 2) were analysed. Number of junctions quantified in each condition is shown at the bottom of the graphs, below each scatter box plot. Statistical analysis was performed using One-way ANOVA, followed by Games-Howell post-hoc test. Non-significant (ns) and significant p-values (
We decided to focus on junctions that were shared by two transfected cells (ee), where phenotypes are clearer (Frasa et al., 2010; Lozano et al., 2008). Upon transfection of activated forms of H-Ras, Src or Rac1, a progressive disappearance of E-cadherin from the interface between cells was observed in different patterns (Figure 5A,C,E). When compared to controls, the undulation of the interface was increased among cells expressing H-RasG12V or SrcY527F (higher Interface Linearity Index, Figure 5B,D), but remained unchanged for Rac1Q61L-perturbed junctions (ee, Figure 5F). These data indicate that, as E-cadherin is removed from junctions, the interface between H-RasG12V and SrcY527F expressing cells becomes less tensile. The percentage of the interface area occupied by E-cadherin receptors was decreased in all samples (Interface Occupancy, Figure 5B,F), but did not reach significance in Src-expressing cells (Figure 5D).
The intensity levels of cadherin at junctions was significantly reduced following transfection of activated H-Ras or Src when measured as raw values (E-cadherin Intensity) or corrected per contacting area between two cells (E-cadherin Intensity per Interface Area) (Figure 5B,D). In contrast, after active Rac1 expression, neither parameter was significantly changed. Consistent with the distinct phenotype of junction perturbation seen in Rac1Q61L-transfected cells, the density of cadherin clusters was decreased in H-RasG12V and SrcY527F, but slightly augmented in RacQ61L.
Different parameters are normalised to controls (junctions from non-expressing cells) arbitrarily set as 100 (orange colour). Values are represented as circles of proportional sizes for junctions between two expressing cells (red colour). Non-significant values are shown in pink colour (ns).
When analysed with Junction Mapper, CIP4, VAV2 or EEF1A siRNA did not interfere with the linearity of the contacting interface (Figure 6B,D,F), in line with the appearance of normal, linear junctions. Consistent with our previous findings (Erasmus et al., 2016), a small decrease in E-cadherin intensity was observed in both CIP4- and VAV2-depleted cells (Figure 6B,F), while EEF1A siRNA promoted the unusual phenotype of higher levels of cadherin receptors (Figure 6D). These distinct patterns were also seen when E-cadherin intensity and area were normalized to the interface area (Intensity per Interface Area and Interface Occupancy, respectively). Strikingly, despite the similar reduction in E-cadherin intensity levels following VAV2 and CIP4 depletion, receptors were removed in different ways from junctions. The clusters of cadherin receptors were less dense with lower levels of CIP4, while upon VAV2 siRNA, the density of the clusters slightly increased (Cluster density, Figure 6B,F).
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