Quantifying Focal Adhesion Dynamics Figure 7. The S178A mutation in Paxillin alters adhesion assembly and disassembly. (A and B) The rate of adhesion assembly and disassembly are significantly decreased by the S178A mutation. The S178A median FA assembly rate is decreased by 42% compared to the wild-type cells, while the median disassembly rate is decreased by 36%. (C and D) The S178A mutation shifts the median adhesion birth location away from the cell edge, but has no effect on the location of cell death. The S178A median adhesion birth position is 31% greater than wild-type median birth position. The median position at adhesion death is decreased by 4% between the S178A and wild-type cells. P-values were calculated in the same manner as in Figure 5. doi:10.1371/journal.pone.0022025.g007 cell edge. In comparison to WT cells, we find that the median distance from the edge at birth is greater by 30% in S178A mutants (Figure 7C). There is no significant difference between WT and mutant cells with regard to where an adhesion dies, suggesting that spatial aspects of the disassembly process (i.e. where disassembly occurs) is not dependent and/or sensitive to JNK phosphorylation (Figure 7D). Finally, we compared the length of time spent in the assembly, stationary, and disassembly phases for cells expressing either WT or S178A EGFP-Paxillin. Results suggest that the S178A mutation causes adhesions to be longer-lived, spending a greater amount of time in the assembly phase than WT cells and lesser time in the disassembly phase (Figure 8). There is no difference in time spent in the stability phase. As a whole, our results demonstrate the most pronounced effects of the S178A mutation occur in the assembly phase: position at birth, assembly rate, and time spent assembling. Discussion We have described the development of a set of computational tools suitable for the global characterization of FA spatiotemporal dynamics and assessing the results of network perturbation on adhesion properties and behavior. The S178A mutation was presented as a proof-of-concept perturbation study for the application of these tools to the analysis of complex FA phenotypes. Through this analysis, we were able to show that adhesion dynamics fall into distinct behavioral subtypes occurring in different regions of the cell, and that the S178A Paxillin mutant causes significant changes in FA assembly and disassembly. While requiring further investigation, these observations suggest a potential mechanism for the previously observed migration defects [20] and suggest that JNK, via Paxillin, may play a significant role in the control of the FA lifecycle. PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22025