High-Resolution Quantification of Focal Adhesion Spatiotemporal Dynamics in Living Cells Mathew E. Berginski1., Eric A. Vitriol2., Klaus M. Hahn4,5*, Shawn M. Gomez1,3,4* 1 Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina, United States of America, 2 Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina, United States of America, 3 Department of Computer Science, University of North Carolina, Chapel Hill, North Carolina, United States of America, 4 Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina, United States of America, 5 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America Abstract Focal adhesions (FAs) are macromolecular complexes that provide a linkage between the cell and its external environment. In a motile cell, focal adhesions change size and position to govern cell migration, through the dynamic processes of assembly and disassembly. To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones. As a proof-of-concept, we show how a single point mutation in Paxillin at the Jun-kinase phosphorylation site Serine 178 changes FA size, distribution, and rate of assembly. This study provides a detailed, quantitative picture of FA spatiotemporal dynamics as well as a set of tools and methodologies for advancing our understanding of how focal adhesions are dynamically regulated in living cells. A full, open-source software implementation of this pipeline is provided at http://gomezlab.bme.unc.edu/tools. Citation: Berginski ME, Vitriol EA, Hahn KM, Gomez SM (2011) High-Resolution Quantification of Focal Adhesion Spatiotemporal Dynamics in Living Cells. PLoS ONE 6(7): e22025. doi:10.1371/journal.pone.0022025 Editor: Maddy Parsons, Kings College London, United Kingdom Received March 24, 2011; Accepted June 12, 2011; Published July 14, 2011 Copyright: 2011 Berginski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants GM57464,P01 HL080166 (KMH), and F31 NS062487 (EAV), startup funds from the UNC School of Medicine (SMG) and the NSF Graduate Research Fellowship Program (MEB). Financial support for these studies was also provided, in part, by the United States Environmental Protection Agency grant (RD833825). However, the research described in this article has not been subjected to the Agency’s peer review and policy review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: khahn@med.unc.edu (KMH); smgomez@unc.edu (SMG) . These authors contributed equally to this work. Introduction Focal adhesions (FAs) are dynamic, multi-component protein complexes that serve as points of integration for both mechanical and chemical signaling, while playing a central role in a variety of processes including cancer metastasis, atherosclerosis and wound healing [1,2,3]. Characterizing how these structures dynamically change is essential for understanding cell migration, which requires that adhesions are continuously remodeled as the cell moves forward. During motility, new adhesions are born at the leading edge of a protruding lamellipodia. They then enlarge and are either disassembled at the base of the protrusion in a process known as adhesion turnover, or become longer-lived structures that are eventually dismantled in the retracting tail at the rear of the cell [4,5,6]. In this cycle as well as other FA-mediated processes, FA dynamics are highly regulated by structural and signaling molecules [7,8,9]. Alterations in the balance of these regulating factors plays a key role in adhesion turnover and thus in adhesion signaling and normal cell function. Microscope imaging of FAs underlies a significant portion of our current understanding of adhesion dynamics, with methods such as total internal reflection fluorescence microscopy (TIRF) providing high-resolution images suitable for quantitative analy- sis[10]. However, challenges in image capture and downstream analysis have generally led to the characterization of only a relatively small number of hand-picked adhesions within any given cell [7,8,11,12,13]. Recent technical and methodological improve- ments have allowed for the automated detection and character- ization of focal adhesions for high-throughput screening studies. For instance, Paran and colleagues [14] have reported on the use of a high-throughput high-resolution imaging system to screen a plant extract library for effects on adhesion morphology and distribution. The same high-throughput imaging system was used to perform multicolor analysis on various adhesion components [15] and this system was used in an siRNA screen against adhesion related genes [16]. In these studies, researchers were able to obtain molecular signatures of protein components within focal adhe- sions, resolve sub-domains within adhesions, and identify clusters of genes that had similar effects on focal adhesion morphology and placement. These studies demonstrate the power of identifying and characterizing large numbers of adhesions within a cell. However, as the approaches used in these studies relied on cell fixation, critical aspects of focal adhesion biology, including their spatiotemporal dynamics, were lost. PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22025