Keratin intermediate filaments make up the main intracellular cytoskeletal network of epithelia and provide, together with their associated desmosomal cell-cell adhesions, mechanical resilience. Remarkable differences in keratin network topology have been noted in different epithelial cell types ranging from a well-defined subapical network in enterocytes to pancytoplasmic networks in keratinocytes. In addition, functional states and biophysical, biochemical, and microbial stress have been shown to affect network organization. To gain insight into the importance of network topology for cellular function and resilience, quantification of 3D keratin network topology is needed.
We used Airyscan superresolution microscopy to record image stacks with an x/y resolution of 120 nm and axial resolution of 350 nm in canine kidney-derived MDCK cells, human epidermal keratinocytes, and murine retinal pigment epithelium (RPE) cells. Established segmentation algorithms (TSOAX) were implemented in combination with additional analysis tools to create a numerical representation of the keratin network topology in the different cell types. The resulting representation contains the XYZ position of all filament segment vertices together with data on filament thickness and information on the connecting nodes. This allows the statistical analysis of network parameters such as length, density, orientation, and mesh size. Furthermore, the network can be rendered in standard 3D software, which makes it accessible at hitherto unattained quality in 3D. Comparison of the three analyzed cell types reveals significant numerical differences in various parameters.