Figure 1. Extensive physical associations between components of cell-matrix adhesion sites in the cytosol.

(A) Pairwise physical associations between proteins tagged with meGFP and TDmKate2 were measured in the cytosol of REF52 and NIH3T3 cells using FCCS (schematized). From these measurements the apparent association constants (Ka) and the association scores were derived as described. (B) Top, area-normalized distributions of association scores between meGFP and TDmKate2 alone (i.e., negative control, n = 126 cells, red line) and between the different analyzed components of adhesion sites in all individual valid measurements performed in REF52 cells (n = 1914 cells; green line) with their corresponding medians (vertical lines). Middle, the distribution of median association score of the 91 protein pairs (60 ≥ n ≥ 9 cells per each pair). Red line indicates the median association score of the negative control. Bottom, the total number of pairs with a median association score bigger than that of the negative control at different statistical confidences (Supplementary file 1). The p-values indicate the probability that the observed median association score of a given pair is bigger than that of the negative control by coincidence. Thus a higher −log(p-value threshold) value means a higher statistical confidence for physical association. (C) A heatmap indicating the p-value of each protein pair in REF52 cells. (D) A bar plot showing the median ± median absolute deviation (MAD) Ka for protein pairs having p-value <0.0001 (n ≥ 13 cells per pair). (E) The network of physical associations between the analyzed proteins. Shown edges are those having p-value <0.0001 in REF52 and p-value <0.02 in NIH3T3 (Supplementary file 1). Edges color and width indicate p-value categories as in (C) and proportionally Ka in REF52, respectively. (F) Based on the network shown in (E), two potential ternary complexes are indicated. (G) Mutually exclusive physical associations inferred from (D) and (E) as cases in which two or more proteins exhibit pairwise associations with another protein but not between themselves.

DOI: http://dx.doi.org/10.7554/eLife.02257.003

Figure 1—figure supplement 1. Concentrations of the ectopically expressed proteins in the FCCS measured cells.

The area plot shows the distribution of concentrations of the TDmKate2 and meGFP tagged proteins within the FCCS analyzed cells (n = 1914 cells co-expressing pairwise the analyzed integrin adhesome protein and 126 cells co-expressing the negative control TDmKate2 and meGFP). These concentrations were derived by fitting the red and green autocorrelation curves to a single component diffusion model and dividing the obtained number of particles by the red and green confocal volumes (‘Materials and methods’). For comparison, the range of typical endogenous concentrations of signalling proteins (Moran et al., 2010) is indicated on the top. Since each integrin adhesome protein typically has multiple different interactions (Zaidel-Bar et al., 2007), many of them mutually exclusive, the total concentration of all endogenous proteins competing on a given measured A-B association can be approximated as the average of typical endogenous concentrations in cells multiplied by the number of the different competing proteins. This total concentration is therefore considerably larger than the concentrations of the ectopically expressed proteins, as the later ones are within the typical range of single endogenous proteins. Therefore, a given increase in the concentrations of A and B will be distributed over the multiple different competing endogenous proteins. This considerably reduces the sensitivity of the complex concentration to a given elevation of the total levels of its components. The combination of this buffering effect and the low ectopic expression levels minimizes the alteration of the levels of the measured complexes in comparison to non-transfected cells.

DOI: http://dx.doi.org/10.7554/eLife.02257.004