Summary of the effects of siRNAs on the analyzed features. Single images of representative sample wells, showing significantly high or low z scores in FA morphological features or significantly high or low manual scores in FA distribution or cell shape features. The images in the “Low Z-score” or “Low score” columns are those with significantly low z scores or manual scores, respectively. The images in the “High Z-score” or “High score” columns are those with significantly high z scores or manual scores, respectively. (a) Untransfected and Risc-free–transfected HeLa cells expressing YFP-paxillin (controls). (b) Examples of hits that varied widely from control cells in their FA morphological features: FA area, FA intensity, FA length, and small and round FAs. (c) Examples of hits that varied significantly from control cells in FA distribution features: FA abundance, and peripheral FA. (d) Examples of hits that varied significantly from control cells in cell shape features: cell elongation and cell spreading. The siRNAs are designated by their Entrez gene symbol (National Center for Biotechnology Information). For full information concerning the hits, see Table S2. Bar (applies to all images), 10 µm.

Phenotypic signatures defined by clustering of the siRNA effects, and the biological features enriched in each cluster. Graphic representation of the mean scores of recorded features, showing the “phenotypic signature” of each cluster. The number of siRNAs in each cluster is indicated below the cluster name. Clusters with similar effects but with varying intensities are framed together. Tables on the right indicate biological features that are enriched in each cluster, with P values < 0.01. NE, no effect. Error bars represent standard deviation. (a) Clusters of FA morphology features. (b) Clusters of FA distribution features. (c) Clusters of cell shape features.

Proposed hierarchy of FA morphology regulation. A proposed model for the hierarchy of genes regulating FA morphology. FA growth was found to be regulated by a common molecular pathway, leading to a coordinated increase in the FA's area, length, and paxillin concentration (indicated by the shape and green color intensity). This pathway was inhibited by siRNAs from clusters MC1–4 (the list of siRNAs in this figure is from MC1, which displayed the most striking effect). The concerted regulation of this pathway could be uncoupled by siRNA perturbations, which suggests additional, independent control of individual FA morphology features, as depicted. MC5 siRNAs inhibited the increase in the area and intensity of FA, without disturbing the increase in FA length. MC6 siRNAs affected the pathway, causing an increase in FA area, and MC7 siRNAs increased the percentage of small and round FAs, without affecting paxillin intensity. The siRNAs in the clusters that uncoupled the correlated features are shown together with their “uncoupling factor” in parentheses.

Workflow of the screen. Schematic description of the screen, including the cellular assay, automatic image acquisition, scoring of the images, statistical evaluation of the data (including data validation), and bioinformatics analysis.

Correlations between FA morphological features in control cells. (a) Scatter plots of FA morphological parameters from two different control wells (one high-resolution image per well) showing the correlation between the following FA morphological parameters: FA area, FA mean intensity (A.U., arbitrary units), and FA length. The mean correlation value (R) is shown. (b) A typical image of control cells (RISC Free), segmented in order to define FA objects and calculate their parameters. The centers of mass of the FAs are marked by crosses, color-coded according to the magnitude of the designated feature (area, mean intensity, and length). Red, high; yellow, medium; blue, low. Bar, 10 µm.

Cross-correlations between all pairs of FA features, cell shape features, and cell coverage, in the significant “hits” of the siRNA screen. Spearman correlation values (see Materials and methods) between all pairs of measured features were calculated for all the screen hits (483 siRNAs). Correlation values are color-coded (see scale bar on the right), and the corresponding correlation (R) value is designated in each square. P-values of the correlations are indicated by the numbers in parentheses. The images shown exemplify significant correlations (P < 0.0005; marked by an asterisk), and refer to the square or squares enclosed by the red line, and connected with an arrow from the red dot. For example, the negative correlation between FA abundance and FA area, mean intensity, and length is indicated by the low abundance of big, bright, and long FAs in P101-PI3K, and high abundance of small, faint and short FAs in AMFR. An image of control (RF) cells is shown for comparison. The siRNAs are designated by the siRNA-targeted Entrez gene symbol. Perc., percentile. Bars, 10 µm.

FA adhesion dynamics of TLN1, EGFR, and CAV2. Time-lapse videos (Videos 1–8) of HeLa cells transfected with TLN1, EGFR, CAV2, or Risc-free (RF) siRNA were used to create temporal ratio images (a) and for autocorrelation analysis (b). For each siRNA, the ratio between two time points, 10 min apart, is presented in color code: new pixels are blue, pixels that disappeared are red, and unchanged pixels are represented by variable colors, depending on the specific local intensity ratio. Yellow indicates identical intensities in the two time points. Note the high predominance of yellow pixels in TLN1-transfected cells, and note the high proportion of red and blue pixels in EGFR-transfected cells. For each siRNA, four different videos were used to perform autocorrelation analysis. Autocorrelation is calculated by comparing the adhesions at each time point with those at time zero. All points are plotted, as well as the single exponential decay fitting curve. The extracted decay time is: RF = 37.81 ± 2.07 min, TLN1 = 94.25 ± 12.71 min, EGFR = 24.45 ± 1.32 min, and CAV2 = 37.50 ± 0.89 min. The differences between RF, TLN1, and EGFR are highly significant (P < 0.0001). The knockdown of the three siRNAs is shown in Fig. S4 b. Bar, 10 µm.