Broadly Applicable Hydrogel Fabrication Procedures Guided by Yap/Taz-Activity Reveal Stiffness, Adhesiveness and Nuclear Projected Area as Checkpoints for Mechanosensing.

Mechanical signals are pivotal ingredients in how cells perceive and respond to their microenvironments, and to synthetic biomaterials that mimic them. In spite of increasing interest in mechanobiology, probing the effects of physical cues on cell behavior remains challenging for a cell biology laboratory without experience in fabrication of biocompatible materials. Hydrogels are ideal biomaterials recapitulating the physical cues that natural Extracellular Matrices (ECM) delivers to cells. Here we streamlined protocols for the synthesis and functionalization of cell adhesive Polyacrylamide-based (PAA-OH) and fully-defined Polyethyleneglycol-based (PEG-RGD) hydrogels tuned at various rigidities for mechanobiology experiments, from 0.3 to >10kPa. We investigated the mechanosignaling properties of these hydrogels in distinct cell types by monitoring the activation state of YAP/TAZ. By independently modulating substrate stiffness and adhesiveness, we found that although ECM stiffness represents an overarching mechanical signal, the density of adhesive sites does impact on cellular mechanosignaling at least at intermediate rigidity values, corresponding to normal and pathological states of living tissues. Using these tools, we found that YAP/TAZ nuclear accumulation occurs when the projected area of the nucleus surpasses a critical threshold of approximatively 150 μm2 . This work suggests the existence of distinct checkpoints for cellular mechanosensing. This article is protected by copyright. All rights reserved.