A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells.

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.