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.