Abstract
Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical, and cell mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in canine, murine, and human epithelial cells both, in vitro and in vivo. Numerical models are derived from confocal airyscan high-resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature, and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral, and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.
Highlights
Quantification of cellular properties is a central task of cell biology (Howard, 2014; Liberali and Pelkmans, 2012; Lidke, 2017; Lippincott-Schwartz, 2014; Piston, 2014; Roca-Cusachs et al, 2017).It is necessary for scientific analysis, testing and model building
We present a novel approach to create the complete digital representation of keratin intermediate filament networks in three dimensions (3D), providing maps and quantitative measurements of the entire keratin network in cultured cells and in cells within their native tissue context
We wanted to generate high-resolution fluorescence data that would be suitable for image analysis of single filaments and filament bundles and would represent keratin filament networks in different epithelial cell types
Summary
Quantification of cellular properties is a central task of cell biology (Howard, 2014; Liberali and Pelkmans, 2012; Lidke, 2017; Lippincott-Schwartz, 2014; Piston, 2014; Roca-Cusachs et al, 2017). A tool was described that is suitable for segmentation of cytoskeletal networks including keratin networks using data obtained by electron microscopy and super-resolution fluorescence microscopy (Flormann et al, 2021). This tool, lacks 3D capability and does not capture information on filament thickness. TSOAX has been applied for 3D segmentation of actin networks using fluorescence images of yeast cells (Xu et al, 2019) It is an implementation of SOAX (Xu et al, 2015, 2011), which was developed from the Stretching Open. The developed image analysis tools were applied to confocal airyscan high-resolution fluorescence image stacks of keratin networks in single cells either in cultured epithelial monolayers or within their native tissue context. Cinematic rendering and immersive visualization make analysis of network heterogeneity accessible at a hitherto unknown quality and precision of 3D reconstruction
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