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Studying brain diseases and developing therapies requires versatile in vitro systems for long-term neuronal cultures. SH-SY5Y neuroblastoma cells are ideal for modeling neurodegenerative diseases. Although SH-SY5Y cells are commonly used in 2D cultures, 3D systems offer more physiologically relevant models. Studies have shown 3D culturing up to 7 days, but a simple, reproducible, and tunable system has yet to be identified. Cellulose holds potential to fulfill these needs. Cellulose and its derivatives are sustainable, cytocompatible, and ideal for synthesizing biocompatible hydrogels. Its abundance and ease of chemical modification make it a highly attractive biomaterial. This study explored nanocellulose-based hydrogels for promoting neuronal growth and morphogenesis. To enhance cell adhesion, a small amount of collagen was added to the hydrogel, and the resulting cell morphologies were analyzed and compared with those cultured in collagen and Matrigel. By chemically oxidizing cellulose and adjusting the blend, we developed composites that maintained neuronal viability for over 14 days in 3D cultures. Our findings show that nanocellulose-collagen composites offer superior cytocompatibility, promoting neuronal viability and neurite outgrowth more effectively than Matrigel and collagen. These tunable biomaterials support long-term 3D neuronal cultures, making them valuable for creating standardized models for disease research and drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Mechanical signals from the extracellular matrix are crucial in guiding cellular behavior. Two-dimensional hydrogel substrates for cell cultures serve as exceptional tools for mechanobiology studies because they mimic the biomechanical and adhesive characteristics of natural environments. However, the interdisciplinary knowledge required to synthetize and manipulate these biomaterials typically restricts their widespread use in biological laboratories, which may not have the material science expertise or specialized instrumentation. To address this, we propose a scalable method that requires minimal setup to produce 2D hydrogel substrates with independent modulation of the rigidity and adhesiveness within the range typical of natural tissues. In this method, norbornene-terminated 8-arm polyethylene glycol is stoichiometrically functionalized with RGD peptides and crosslinked with a di-cysteine terminated peptide via a thiol-ene click reaction. Since the synthesis process significantly influences the final properties of the hydrogels, we provide a detailed description of the chemical procedure to ensure reproducibility and high throughput results. We demonstrate examples of cell mechanosignaling by monitoring the activation state of the mechanoeffector proteins YAP/TAZ. This method effectively dissects the influence of biophysical and adhesive cues on cell behavior. We believe that our procedure will be easily adopted by other cell biology laboratories, improving its accessibility and practical application.

Microenvironmental mechanical signals are fundamental regulators of cell behavior both in physiological and in pathological context, particularly in the induction and maintenance of tumorigenic properties. It is thus of utmost importance to experimentally recreate conditions that mimic the physical attributes of real tissues to study their impact on cell behavior and in particular on the induction of cancer stem cell (CSC) properties. Here we present protocols to investigate the role of mechanical stiffness on reprogramming of primary mammary gland cells into CSCs, including the synthesis of hydrogel substrates of the desired stiffness, the isolation and culture of primary differentiated normal cells derived from the human mammary gland, and the assessment of their CSC attributes after oncogene-mediated transformation., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Ageing is intimately connected to the induction of cell senescence 1,2 , but why this is so remains poorly understood. A key challenge is the identification of pathways that normally suppress senescence, are lost during ageing and are functionally relevant to oppose ageing 3 . Here we connected the structural and functional decline of ageing tissues to attenuated function of the master effectors of cellular mechanosignalling YAP and TAZ. YAP/TAZ activity declines during physiological ageing in stromal cells, and mimicking such decline through genetic inactivation of YAP/TAZ in these cells leads to accelerated ageing. Conversely, sustaining YAP function rejuvenates old cells and opposes the emergence of ageing-related traits associated with either physiological ageing or accelerated ageing triggered by a mechano-defective extracellular matrix. Ageing traits induced by inactivation of YAP/TAZ are preceded by induction of tissue senescence. This occurs because YAP/TAZ mechanotransduction suppresses cGAS-STING signalling, to the extent that inhibition of STING prevents tissue senescence and premature ageing-related tissue degeneration after YAP/TAZ inactivation. Mechanistically, YAP/TAZ-mediated control of cGAS-STING signalling relies on the unexpected role of YAP/TAZ in preserving nuclear envelope integrity, at least in part through direct transcriptional regulation of lamin B1 and ACTR2, the latter of which is involved in building the peri-nuclear actin cap. The findings demonstrate that declining YAP/TAZ mechanotransduction drives ageing by unleashing cGAS-STING signalling, a pillar of innate immunity. Thus, sustaining YAP/TAZ mechanosignalling or inhibiting STING may represent promising approaches for limiting senescence-associated inflammation and improving healthy ageing., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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) deliver to cells. Here, protocols are streamlined 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 >10 kPa.  The mechanosignaling properties of these hydrogels are investigated in distinct cell types by monitoring the activation state of YAP/TAZ. By independently modulating substrate stiffness and adhesiveness, it is 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, it is found that YAP/TAZ nuclear accumulation occurs when the projected area of the nucleus surpasses a critical threshold of approximatively 150 µm 2 . This work suggests the existence of distinct checkpoints for cellular mechanosensing., (© 2021 Wiley-VCH GmbH.)

In spite of tremendous advances made in the comprehension of mechanotransduction, implementation of mechanobiology assays remains challenging for the broad community of cell biologists. Hydrogel substrates with tunable stiffness are essential tool in mechanobiology, allowing to investigate the effects of mechanical signals on cell behavior. A bottleneck that slows down the popularization of hydrogel formulations for mechanobiology is the assessment of their stiffness, typically requiring expensive and sophisticated methodologies in the domain of material science. Here we overcome such barriers offering the reader protocols to set-up and interpret two straightforward, low cost and high-throughput tools to measure hydrogel stiffness: static macroindentation and micropipette aspiration. We advanced on how to build up these tools and on the underlying theoretical modeling. Specifically, we validated our tools by comparing them with leading techniques used for measuring hydrogel stiffness (atomic force microscopy, uniaxial compression and rheometric analysis) with consistent results on PAA hydrogels or their modification. In so doing, we also took advantage of YAP/TAZ nuclear localization as biologically validated and sensitive readers of mechanosensing, all in all presenting a suite of biologically and theoretically proven protocols to be implemented in most biological laboratories to approach mechanobiology., (© 2021. The Author(s).)

Defining the interplay between the genetic events and microenvironmental contexts necessary to initiate tumorigenesis in normal cells is a central endeavour in cancer biology. We found that receptor tyrosine kinase (RTK)-Ras oncogenes reprogram normal, freshly explanted primary mouse and human cells into tumour precursors, in a process requiring increased force transmission between oncogene-expressing cells and their surrounding extracellular matrix. Microenvironments approximating the normal softness of healthy tissues, or blunting cellular mechanotransduction, prevent oncogene-mediated cell reprogramming and tumour emergence. However, RTK-Ras oncogenes empower a disproportional cellular response to the mechanical properties of the cell's environment, such that when cells experience even subtle supra-physiological extracellular-matrix rigidity they are converted into tumour-initiating cells. These regulations rely on YAP/TAZ mechanotransduction, and YAP/TAZ target genes account for a large fraction of the transcriptional responses downstream of oncogenic signalling. This work lays the groundwork for exploiting oncogenic mechanosignalling as a vulnerability at the onset of tumorigenesis, including tumour prevention strategies.

Autophagy, besides ensuring energy metabolism and organelle renewal, is crucial for the biology of adult normal and cancer stem cells. However, it remains incompletely understood how autophagy connects to stemness factors and the nature of the microenvironmental signals that pattern autophagy in different cell types. Here we advance in these directions by reporting that YAP/TAZ transcriptionally control autophagy, being critical for autophagosomal degradation into autolysosomes. YAP/TAZ are downstream effectors of cellular mechanotransduction and indeed we found that cell mechanics, dictated by the physical property of the ECM and cytoskeletal tension, profoundly impact on autophagic flux in a YAP/TAZ-mediated manner. Functionally, by using pancreatic and mammary organoid cultures, we found that YAP/TAZ-regulated autophagy is essential in normal cells for YAP/TAZ-mediated dedifferentiation and acquisition of self-renewing properties. In tumor cells, the YAP/TAZ-autophagy connection is key to sustain transformed traits and for acquisition of a cancer stem cell state by otherwise more benign cells. Mechanistically, YAP/TAZ promote autophagic flux by directly promoting the expression of Armus, a RAB7-GAP required for autophagosome turnover and whose add-back rescues autophagy in YAP/TAZ-depleted cells. These findings expand the influence of YAP/TAZ mechanotransduction to the control of autophagy and, vice versa, the role of autophagy in YAP/TAZ biology, and suggest a mechanism to coordinate transcriptional rewiring with cytoplasmic restructuring during cell reprogramming., Competing Interests: The authors declare no conflict of interest.

Mechanical signals are increasingly recognized as overarching regulators of cell behaviour, controlling stemness, organoid biology, tissue development and regeneration. Moreover, aberrant mechanotransduction is a driver of disease, including cancer, fibrosis and cardiovascular defects. A central question remains how cells compute a host of biomechanical signals into meaningful biological behaviours. Biomaterials and microfabrication technologies are essential to address this issue. Here we review a large body of evidence that connects diverse biomaterial-based systems to the functions of YAP/TAZ, two highly related mechanosensitive transcriptional regulators. YAP/TAZ orchestrate the response to a suite of engineered microenviroments, emerging as a universal control system for cells in two and three dimensions, in static or dynamic fashions, over a range of elastic and viscoelastic stimuli, from solid to fluid states. This approach may guide the rational design of technological and material-based platforms with dramatically improved functionalities and inform the generation of new biomaterials for regenerative medicine applications.

Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression 1 . However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A-SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ 2 . Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A-SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A-SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A-SWI/SNF, thereby preventing the formation of the ARID1A-SWI/SNF-YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A-SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis.

Shaping ceramic materials at the nanoscale in 3D is a phenomenal engineering challenge, that can offer new opportunities in a number of industrial applications, including metamaterials, nano-electromechanical systems, photonic crystals, and damage-tolerant lightweight materials. 3D fabrication of sub-micrometer ceramic structures can be performed by two-photon laser writing of a preceramic polymer. However, polymer conversion to a fully ceramic material has proven so far unfeasible, due to lack of suitable precursors, printing complexity, and high shrinkage during ceramic conversion. Here, it is shown that this goal can be achieved through an appropriate engineering of both the material and the printing process, enabling the fabrication of preceramic 3D shapes and their transformation into dense and crack-free SiOC ceramic components with highly complex, 3D sub-micrometer architectures. This method allows for the manufacturing of components with any 3D specific geometry with fine details down to 450 nm, rapidly printing structures up to 100 µm in height that can be converted into ceramic objects possessing sub-micrometer features, offering unprecedented opportunities in different application fields.