Hydrogel-based microchannels to measure confinement- and stiffness-sensitive Yes-associated-protein activity in epithelial clusters

Abstract

Nuclear translocation of Yes-associated-protein (YAP) in single cells serves as a key sensor of matrix stiffness. On two-dimensional (2D) polyacrylamide (PA) hydrogels, we found that nuclear YAP localization in epithelial clusters increases with gel stiffness and reduces with cell density. To measure YAP activity in 3D-like confinement of tunable stiffness, we fabricated PA-based microchannels. Here, narrower channels enhanced nuclear YAP localization even in softer extracellular matrix and denser epithelial clusters, both of which reduced YAP activation in 2D. Thus, the presented hydrogel microchannel-based platform may reveal new mechanosensitive cellular signatures in 3D-like settings, which cannot be captured on standard 2D hydrogels.

Molecular Mechanism of Hippo–YAP1/TAZ Pathway in Heart Development, Disease, and Regeneration

“…The subcellular localization and activity of YAP1/TAZ varies in cells with different matrix stiffness. In a stiff matrix, there is nuclear translocation and higher transcriptional activity of YAP1/TAZ; in contrast, a soft matrix is characterized by cytoplasmic sequestration and reduced activity of YAP1/TAZ (Nasrollahi and Pathak, 2017). The above studies potentially support the hypothesis that, in the context of cardiac ECM remodeling and/or fibrosis, the ECM stiffness and/or cytoskeletal tension may change and affect YAP1 activity; furthermore, activated YAP1 modulates the progression of ECM remodeling and fibrosis, which further exacerbates cardiac injury and even contributes to the development of chronic heart failure.…”

Photocrosslinked Alginate-Methacrylate Hydrogels with Modulable Mechanical Properties: Effect of the Molecular Conformation and Electron Density of the Methacrylate Reactive Group

“…Hydrogels for biomedical applications can be produced from natural polysaccharides [6], such as cellulose [7], chitosan (CH) [3,4], sodium alginate (ALG) [5,8], and dextran [9], and also from some synthetic polymers such as poly(acrylamide) (PAM) [10], poly(ethylene glycol) (PEG) [11,12], and poly(vinyl alcohol) (PVA) [13], among others. With respect to the synthesis methodologies, they can be produced by either physical or chemical crosslinking or even by the combination of both [14].…”

Solvent-Induced Nanotopographies of Single Microfibers Regulate Cell Mechanotransduction

“…When comparing the YAP nuclear translocation with observations seen depending on the 2D substrate stiffness, most previous studies defined the ∼100 kPa regime as the substrate elastic modulus (E-modulus), above which cells exhibit high ratios of YAP shuttling from the cytoplasm to the nucleus . For example, cells grown on stiff elastic substrates of 120 and 150 kPa demonstrate YAP signal ratios of ∼2.5 and ∼3.5, respectively. , Based on atomic force microscopy (AFM) measurements performed on the fibers presented here, their stiffness appears to be in the MPa range without any significant differences between surface topographies (Figure S1E, Supporting Information). However, AFM measurements on single fibers are very challenging due to the curvature, mobility, and heterogeneity of the fibers, which together with the micro- and nanoscale surface topographies of fibers lead to a large distribution of the data.…”

“…71 For example, cells grown on stiff elastic substrates of 120 and 150 kPa demonstrate YAP signal ratios of ∼2.5 and ∼3.5, respectively. 69,72 Based on atomic force microscopy (AFM) measurements performed on the fibers presented here, their stiffness appears to be in the MPa range without any significant differences between surface topographies (Figure S1E, Supporting Information). However, AFM measurements on single fibers are very challenging due to the curvature, mobility, and heterogeneity of the fibers, 73 which together with the micro-and nanoscale surface topographies of fibers lead to a large distribution of the data.…”