Quantifying Focal Adhesion Dynamics Figure 5. The assembly and disassembly rates of EGFP-Paxillin and EGFP-FAK adhesions are the same. The blue numbers in each plot are the p-values of the difference in median values between the EGFP-Paxillin and EGFP-FAK adhesions. P-values were calculated using the bootstrapped confidence intervals with 50000 replicates. Data from 10 cells are included. doi:10.1371/journal.pone.0022025.g005 Paxillin S178A Mutant Perturbation The previous results establish the ability of our approach to quantify various adhesion properties and behaviors. Furthermore, the ability to identify and characterize very large numbers of adhesions provides the potential to detect changes in adhesion phenotype that are difficult or impossible to characterize manually and/or with small numbers of measurements. As a proof-of-principle, we utilized our system to investigate the effect of a Paxillin mutation (Serine 178 to Alanine) on several aspects of FA behavior. Specifically, a principal regulatory mechanism of Paxillin is phosphorylation, with over 40 sites of phosphorylation currently identified [9]. The roles of many of these phosphorylation sites have yet to be characterized, but many of those that have been studied demonstrate strong effects on cell migration. Of particular interest is the c-Jun N-terminal kinase (JNK) phosphorylation site Serine 178 (S178). Preventing JNK phosphorylation through mutation of this Serine to Alanine, or by inhibition of JNK signaling, inhibits cell motility [20,21]. More recently, it has been shown that phosphorylation of S178 enhances Paxillin’s interaction with FAK, resulting in tyrosine phosphory- lation at residues 31 and 118 [22]. Furthermore, expression of the phosphomimetic Y31D/Y118D Paxillin can rescue the S178A mutant phenotype. This and related work suggests that JNK phosphorylation of Paxillin may be an important early step in adhesion formation. However, the effects of this mutation on adhesion dynamics have not been well characterized. Using our analysis system we found that the S178A mutation induced a number of significant effects on the morphological, dynamic and spatial properties of adhesions. The mean area of the S178A mutant adhesions decreased by 23%, while the mean axial ratio decreased by 5% in the S178A mutants (Figure 6). Perhaps most relevant to the observed alterations in cell motility, there is an approximately 42% reduction in the median rate of adhesion assembly (Figure 7A). We also observe a smaller (30%) but statistically significant decrease in median disassembly rate (Figure 7B). Thus, the kinetics of FA assembly and disassembly are strongly affected by this mutation, but in a non-symmetric manner. We previously observed that adhesions in wild-type cells have different distributions of birth and death positions relative to the Figure 6. The S178A mutation in Paxillin decreases adhesion size and axial ratio. There are 44685 adhesions in the wild-type and 73305 adhesions in the S178A data sets. The p-values were calculated using the Wilcox Rank Sum test. Data from 9 cells are included in the S178A data set. doi:10.1371/journal.pone.0022025.g006 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22025