Quantifying Focal Adhesion Dynamics Figure 3. Automated measurement of focal adhesion dynamics. (A) Each of the adhesions in the cells is tracked, allowing the position and properties of single adhesions and populations to be assessed. Here a single adhesion (in green), the surrounding adhesions (in blue) and the cell edge (in red) are followed for 49 minutes. The cell edge is only outlined in the first frame. The scale bar is 10 mm. (B) The intensity of EGFP- Paxillin in the tracked adhesion in (A) through time. The green, yellow and red lines are smoothed using the Lowess algorithm and correspond to the assembly, stable and disassembly phases, respectively. (C) The normalized log-linear fit of the Paxillin intensity through time during the assembly phase of the adhesion in part (B). The inset depicts several of the images from which the Paxillin intensity was gathered. (D) The normalized log-linear fit of the Paxillin intensity through time during the disassembly phase of the adhesion in part (B). The inset depicts several of the images from which the Paxillin intensity was gathered. (E) The assembly and disassembly rates for adhesions whose Paxillin intensity curve fits have R2 values of 0.9 or greater. The top and bottom lines of the boxes indicate the 3rd and 1st quartiles respectively, while the bold central lines indicate the median values. The whiskers extend up to 1.5 times the interquartile range. doi:10.1371/journal.pone.0022025.g003 a stationary/mature phase where intensity remains relatively stable (Figure 3B). We used our system to characterize the rates of FA assembly and disassembly by repeating the analysis detailed in Figure 3A–D on all adhesions identified in the EGFP-Paxillin data set by our software (n = 21 cells). Results were focused on FAs having lifetimes of at least 20 minutes, where the detected assembly or disassembly rate is positive and the p-value of the rate model is below 0.05 (Figure 3E). The mean rate of assembly of 0.03160.023 min21 is 55% greater than that of disassembly (0.02060.014 min21). While these average rates are slower than earlier published reports, the values determined in previous studies were estimated from far fewer measurements (typically dozens of adhesions) and can be found within the variance of our data set. Thus, this automated computational approach provides a comprehensive picture of the breadth of adhesion assembly and disassembly dynamics without biasing analysis toward any particular subset of adhesions. Spatial Properties of FA Assembly and Disassembly In addition to estimation of assembly and disassembly rates, the analysis pipeline also collects spatial properties of FAs, allowing spatial aspects of FA behavior and dynamics to be similarly studied. Using the same set of experiments used to determine the kinetics of assembly and disassembly, we asked where, relative to the cell edge, adhesions tend to be born/die (Figure 4). The majority (63%) of adhesions are born less than 5 mm from the cell edge, with a mean distance from the edge at birth of 6.34 mm (Figure 4A). In contrast, adhesions tend to die further from the edge with only 27% of adhesions dying within 5 mm of the edge (Figure 4B). The mean distance from the edge at death was 9.5 mm. This suggests the existence of two distinct, but partially overlapping ‘‘zones’’ within which preferential birth or death of FAs occurs. When looking at both FA birth/death location and assembly/disassembly rate simultaneously, we find that higher assembly rates are observed in births that occur near the edge while no obvious effect of spatial location on the rate of disassembly is apparent (Figure 4). Analysis of EGFP-labeled FAK adhesions To support the use of these methods in the study other FA proteins, we expressed FAK labeled with EGFP. After gathering time-lapse movies of 10 cells tracking the position of FAK in NIH 3T3s using TIRFM, we applied the same set of algorithms to determine the assembly and disassembly rate of the FAs. The rates of assembly and disassembly of FAs were found to be statistically indistinguishable when comparing labeled Paxillin to labeled FAK in live cells (Figure 5). In contrast, subtle but statistically significant differences in adhesion areas and axial size were found when comparing EGFP-Paxillin vs EGFP-FAK labeled adhesions (Figure S2). This result is not unexpected as different spatial and/or stoichiometric relationships are expected for both Paxillin and FAK within FAs [18,19]. These results further indicate the capability of this system to be generally applicable to the measurement of other adhesion components besides Paxillin. Figure 4. Spatial properties of FA positions at birth and death. (A) The majority of adhesions are born within 5 mm of the cell edge and the greatest variance in assembly rates are also observed in this 5 mm band. (B) The distribution of the distance of death location from the cell edge indicates that adhesion disassembly typically occurs along a broader band from the cell edge as compared to the position at adhesion birth. Also, the variance in disassembly rate is roughly the same regardless of the position at adhesion death. This data was collected from 21 EGFP-Paxillin cells. doi:10.1371/journal.pone.0022025.g004 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22025