title: High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells.
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.
authors.affiliation: Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Mathew E
authors.initials: ME
authors.lastname: Berginski
authors.forename: Eric A
authors.initials: EA
authors.lastname: Vitriol
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Hahn
authors.forename: Shawn M
authors.initials: SM
authors.lastname: Gomez
mesh_terms: D000818:Animals; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D051419:Paxillin / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation
chemical_list: D051419:Paxillin; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse; D048031:JNK Mitogen-Activated Protein Kinases