Environmental stiffness restores mechanical homeostasis in vimentin-depleted cells.

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cell's microenvironment and the cellular morphological and mechanical state-this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.

SFAlab: image-based quantification of mechano-active ventral actin stress fibers in adherent cells.

Ventral actin stress fibers (SFs) are a subset of actin SFs that begin and terminate at focal adhesion (FA) complexes. Ventral SFs can transmit forces from and to the extracellular matrix and serve as a prominent mechanosensing and mechanotransduction machinery for cells. Therefore, quantitative analysis of ventral SFs can lead to deeper understanding of the dynamic mechanical interplay between cells and their extracellular matrix (mechanoreciprocity). However, the dynamic nature and organization of ventral SFs challenge their quantification, and current quantification tools mainly focus on all SFs present in cells and cannot discriminate between subsets. Here we present an image analysis-based computational toolbox, called SFAlab, to quantify the number of ventral SFs and the number of ventral SFs per FA, and provide spatial information about the locations of the identified ventral SFs. SFAlab is built as an all-in-one toolbox that besides analyzing ventral SFs also enables the identification and quantification of (the shape descriptors of) nuclei, cells, and FAs. We validated SFAlab for the quantification of ventral SFs in human fetal cardiac fibroblasts and demonstrated that SFAlab analysis i) yields accurate ventral SF detection in the presence of image imperfections often found in typical fluorescence microscopy images, and ii) is robust against user subjectivity and potential experimental artifacts. To demonstrate the usefulness of SFAlab in mechanobiology research, we modulated actin polymerization and showed that inhibition of Rho kinase led to a significant decrease in ventral SF formation and the number of ventral SFs per FA, shedding light on the importance of the RhoA pathway specifically in ventral SF formation. We present SFAlab as a powerful open source, easy to use image-based analytical tool to increase our understanding of mechanoreciprocity in adherent cells.