Hydrogel-based microchannels to measure confinement- and stiffness-sensitive Yes-associated-protein activity in epitheli
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Biomaterials for 3D Cell Biology Research Letter
Hydrogel-based microchannels to measure confinement- and stiffness-sensitive Yes-associated-protein activity in epithelial clusters Samila Nasrollahi and Amit Pathak, Department of Mechanical Engineering and Materials Science, Washington University, Saint Louis, MO 63130, USA Address all correspondence to Amit Pathak at [email protected] (Received 29 May 2017; accepted 22 August 2017)
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.
Introduction Cells sense their microenvironment not only through biochemical signals, but also through physical and mechanical cues present in the extracellular matrix (ECM). These mechanical cues are mainly attributed to ECM stiffness, topography, dimensionality, and porosity,[1,2] all of which are known to regulate fundamental cellular functions such as proliferation, migration, and differentiation.[3–8] In recent years, growing evidence suggests that there is a direct link between various nuclear transcription factors and mechano-regulated cell activities.[9,10] In particular, Yes-associated-protein (YAP) is recognized as the classical sensor that transduces mechanical signals, including ECM stiffness and cell shape, into biologic outcomes.[11] These mechanosensitive functions of subcellular YAP localization are processed through the Hippo signaling pathway, which acts upstream of YAP. Upon activation of the Hippo pathway, large tumor suppressor homolog (LATS) gets phosphorylated, resulting into YAP phosphorylation.[12] The inactivated (phosphorylated) YAP is excluded from the nucleus and aggregated within the cytoplasm.[13] However, the inactivated form of Hippo pathway leads to de-phosphorylation of YAP and re-localization to nucleus to induce gene expression.[14,15] Therefore, the shuttling of YAP between the nucleus and cytoplasm serves as a key mechanism through which mechanical cues are transduced to gene expression.[16] Several recent studies have demonstrated a direct relationship between ECM stiffness and the nuclear YAP activation in various cells types when cultured singly on two-dimensional (2D) substrates of variable stiffness.[17–20] On stiff fibronectincoated polyacrylamide (PA) gels, mammary epithelial cells (MECs) and human mesenchymal stem cells (hMSCs) showed
predominantly nuclear YAP localization. However, on soft substr
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