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

Abstract

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.Author summaryOur novel Focal Adhesion Filament Cross-correlation Kit (FAFCK) allows for fast, reliable, unbiased, and systematic detection of focal adhesions and actin stress fibers in cells and their mutual correlation. Detailed analysis of these structures which are both key elements in mechano-sensing and force transduction will help tremendously to improve quantitative analysis of mechanocellular experiments, key to understanding the complex interplay between cells and the extracellular matrix. In particular, sophisticated analysis methods such as model-based traction force microscopy will benefit from correlating the detailed datasets of stress fibers and focal adhesions.

Merging of ventral fibers at adhesions drives the remodeling of cellular contractile systems in fibroblasts

“…To analyze the impact of merging to the subpopulation of aligned stress fibers, we calculated the angles of merged fibers with respect to the long axis of the cell. Figure 5a (Hauke, Narasimhan, Primeßnig, Kaverina, & Rehfeldt, 2021), we observed that the majority of the longest fibers (≥22 μm) were aligned within 50 of the major axis of the cell (Figure 5d). We observed a significant difference in the variance of the alignment angles, with the high Fn dataset displaying higher variance.…”

“…Both factors are necessary to facilitate the efficient transmission of force into directed migration. Investigating the alignment of contractile ventral fibers in the cell in low and high Fn datasets using the Filament Sensor module of FAFCK (Hauke, Narasimhan, Primeßnig, Kaverina, & Rehfeldt, 2021), we observed that the majority of the longest fibers (≥22 μm) were aligned within 50° of the major axis of the cell (Figure 5d). We observed a significant difference in the variance of the alignment angles, with the high Fn dataset displaying higher variance.…”

“…We used the Filament Sensor tool in the Focal Adhesion Filament Cross‐Correlation Kit (Hauke et al, 2021) to measure the contractile fibers in the ventral plane of cells expressing GFP‐Myosin Light Chain. We measured fibers for 20 cells in the low and high Fn substrate datasets.…”

“…We calculated the alignment angle of the fiber as the difference between the angle of the merged fiber and the long axis angle, for 40 events in Figure 5b. We used the Filament Sensor tool in the Focal Adhesion Filament Cross-Correlation Kit (Hauke et al, 2021) to measure the contractile fibers in the ventral plane of cells expressing GFP-Myosin Light Chain.…”

Quantitative biophysical metrics for rapid evaluation of ovarian cancer metastatic potential

“…The analysis of the fluorescent actin channel for actin filament length measurement was done using the Filament Sensor 2.0 from the SFB 755 project ( Hauke et al. , 2021 ).…”