studies in the same cell line indicate that this 5 mm range coincides with the end of the lamellipodia and the beginning of the lamella, where the structure of the actin cytoskeletal network changes significantly. Recently published data indicate that this transition, where stable actin structures differentiate into branched structures that exert force on the leading edge for protrusion, is determined by interactions between the cytoskeleton and adhesion proteins [23]. Further investigation will be required to more fully interpret this observation and its relation to the lamella-lamellipodium interface [24]. Our analysis enabled us to quantify differences in FA dynamics caused by mutation of Paxillin at a JNK phosphorylation site. Both adhesion assembly and disassembly were affected. In addition to these strong perturbations, more subtle changes in FA dynamics and localization were also detected, including a decrease in adhesion size. In agreement with our results, a recent siRNA screen of FA proteins within fixed cells that included JNK knockdown also measured decreases in adhesion size [16]. Based on our results, a summary model of the FA lifecycle in both wild-type and S178A cells is depicted in Figure 9. Shown to scale, the S178A mutation shows distinct effects on both the assembly and disassembly phases of FA development, but these effects are different in magnitude. Determining what FA development signals are involved in perturbing assembly, stability and disassembly is an ongoing process, but these proof of principle TIRF experiments demonstrate the capabilities of the software analysis system to make biologically significant new observations. Development of new and/or improved analysis modules is ongoing. In prior studies, analysis of the cell edge velocity has proven to be a robust phenotype that can be used to quantify the effect of many different perturbations to the signaling networks that control cellular motility [25]. Integration of this type of data will allow the rates of cell edge movement to be analyzed in terms of FA phenotypes. Such studies will help to bridge the gap between FA dynamics and the well-developed fields of cell edge and cytoskeletal dynamics. The data sets collected using the software also provide information about the specific properties of the adhesions during each phase of their lifecycle. There are also several types of measurements that we plan on adding to the analysis system to help quantify polarized cells behaviors, such as adhesion sliding. We also expect to continue to develop the spatial analysis methods beyond the ‘‘distance from the cell edge’’ Quantifying Focal Adhesion Dynamics measure used here. Such spatial methods will also be important in understanding polarized cell behaviors. In summary, we have described a system for quantifying the spatiotemporal dynamics of FAs, generating highly-detailed descriptions of FA behavior based on large populations, and further enabling high-content screening methods to be applied to understanding the perturbation of FA signaling networks. The system was applied to quantifying the differences in FA development generated through a single amino acid mutation of the FA scaffolding protein Paxillin. Future studies of other FA perturbation methods with high-content analysis methods should provide a comprehensive picture of the role of FA signaling proteins in the control of FA development and localization. Methods Cell Culture NIH 3T3 fibroblasts and 293 LinXE ecotropic packaging cells were cultured in 5% CO2 at 37uC in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech) supplemented with 10% fetal calf serum, 1% L-Glutamine, and 1% penicillin-streptomycin. Fibro- blasts were imaged in Ham’s F-12K medium without phenol red (SAFC Biosciences) with 2% fetal bovine serum, 15 mM HEPES, 1% L-Glutamine, and 1% penicillin-streptomycin. To make stable cell lines, retroviral vectors were transfected into 293 LinXE cells plated in 6 cm dishes with Fugene 6 (Roche) according to the manufacturer’s protocol (using 18 mL of Fugene 6 and 4.5 mg of DNA). The media was replaced after 12 hours. Viral supernatant was harvested 48 hours after media replacement, passed through a .45 mm syringe filter and then added to NIH 3T3s plated at subconfluent densities at a ratio of 1:3 (viral supernatant/normal media). Cells were infected for several rounds until they reached expression levels sufficient for live cell imaging. All of the constructs used in this study have been verified to colocalize with endogenous proteins [20,26,27]. No differences were detectable in the expression levels of the EGFP-Paxillin and EGFP-PaxillinS178A constructs (Figure S3). Microscopy Prior to imaging, NIH 3T3s were plated onto coverslips coated with 5 mg/mL Fibronectin (Sigma) for 30 min. Fibroblasts expressing EGFP-PaxS187A required 2–3 hours to adhere to the Figure 9. Summary of results and conceptual model of how the S178A mutant affects the adhesion life cycle. Durations and slopes are shown to scale. doi:10.1371/journal.pone.0022025.g009 PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22025