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Where no hand has gone before: probing mechanobiology at the cellular level Carlos Matellan, and Armando del Rio Hernandez ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01206 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018
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ACS Biomaterials Science & Engineering
Where no hand has gone before: probing mechanobiology at the cellular level Carlos Matellan, Armando E. del Río Hernández*
Cellular and Molecular Biomechanics Laboratory, Department of Bioengineering, Imperial College London, London SW7 2AZ, UK
* Corresponding Author email:
[email protected] Abstract Physical forces and other mechanical stimuli are fundamental regulators of cell behaviour and function. Cells are also biomechanically competent: they generate forces to migrate, contract, remodel and sense their environment. As the knowledge of the mechanisms of mechanobiology increases, the need to resolve and probe increasingly small scales calls for novel technologies to mechanically manipulate cells, to examine forces exerted by cells and to characterise cellular biomechanics. Here, we review novel methods to quantify cellular force generation, to measure cell mechanical properties and to exert localised pico- and nanonewton forces on cells, receptors and proteins. The combination of these technologies will provide further insight on the effect of mechanical stimuli on cells and the mechanisms that convert these stimuli into biochemical and biomechanical activity. Keywords: Mechanotransduction; Mechanosensing; Cellular Biomechanics; Traction force Microscopy; Cell Compliance; Microrheology.
1. Introduction – The role of mechanobiology in health and disease In the body, cells and tissues are constantly subjected to mechanical forces. Over the last two decades, research has found that cells can sense and respond to these mechanical stimuli and to the changing mechanical properties of their local microenvironment. While these biomechanical signals and responses are not as well characterised as their biochemical counterparts, the growing body of evidence suggests that mechanical forces have a profound impact on regulating cell behaviour. Mechanobiology has emerged as a field that studies how cells sense external forces and mechanical signals from their environment (mechanosensing) and transform these biomechanical cues into biochemical and biological activity (mechanotransduction). The complex interplay between biomechanical and biochemical signals plays a regulatory role in a wide variety of cellular processes. Matrix stiffness, cell shape and nanotopography can determine stem cell differentiation.1-5 Mechanical forces orchestrate embryonic development6 and tissue morphogenesis.7 Shear forces exerted by blood flow regulate endothelial function,8 as well as vascular smooth muscle and fibroblast activity when these are exposed to fluid flow.9 Mechanical loading is fundamental to maintain an adequate equilibrium between bone deposition and resorption during bone remodelling and fracture repair.10-11 Mechanical forces also play a critical role in wound healing, 1 ACS Paragon Plus Environment
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where a combination of growth factors and mechanical signals activate myofibroblasts, enabling the remodelling of the provisional matrix.12 Mechanical forces are also fundamental in the onset and progression of disease and understanding how abnormal mechanobiology interacts with a pathology is key to develop new therapeutic strategies that target the biomechanical drivers of disease. Fibrotic diseases, which can account for ~45% of all deaths in industrialised countries, can be regarded as a process of abnormal wound healing through sustained ECM deposition and activation of myofibroblasts via biochemical and biomechanical signalling.13-14 In a similar manner, while certain blood flow patterns have been shown to be athero-protective, 15 oscillatory flow dynamics activate a variety of signalling pathways that lead to pathological phenotypes and eventually to cardiovascular disease.8, 16-17 Mechanobiology is also becoming an increasingly important factor in cancer.18-20 Biomechanical and biochemical activation of cancer associated fibroblasts (CAFs) leads to remodelling of the tumour matrix, abnormal collagen deposition and formation of a stiff, fibrotic microenvironment which increases cell proliferation, promotes epithelial to mesenchymal transition (EMT), chemoresistance and facilitates invasion and migration of the cancer cells.21-23 Solid stresses generated by the hyperproliferating tumour and high interstitial fluid pressure also play a role in the development of cancer.19, 24 Likewise, formation of a fibrotic niche in a target organ is conducive to the engraftment of metastatic seeds.25 Cells not only respond to mechanical stimuli, but also have the ability to generate forces through their actomyosin cytoskeleton to sense and interact biomechanically with their environment. It is widely accepted that cells sense the mechanical properties of their microenvironment by exerting forces on the surrounding extracellular matrix.26-27 In this way, force generation is critical to the ability of cells to sense and respond to mechanical stimuli, and inhibiting actomyosin contractility in turn abrogates their mechanosensing ability. Cellular force generation is not only critical to their mechanosensing, but also their ability to migrate,28 to remodel the extracellular matrix and reorganise tissue architecture,21 to release ECM-bound growth factors (e.g. TGF-β),29 and in general to convert different stimuli into biomechanical activity. Biomechanical properties of the cell, such as morphology and compliance are also key indicators and regulators of cellular activity. Cell morphology is intimately correlated with its cytoskeletal architecture, which in turn modulates its ability to generate force, mechanosense its microenvironment and respond to mechanical stimuli. Cell morphology through adhesion confinement can alone determine lineage commitment and differentiation of mesenchymal stem cells.4 Cell elasticity is critical to the function of certain cells (e.g. red blood cells) and changes in cellular compliance are associated with pathology (e.g. malaria increases the stiffness of red blood cells).30 The compliance of cancer cells is also critical to their metastatic potential. Cancer cells have to squeeze through the basement membrane to invade the local tissue and through the endothelium to intravasate and disseminate. Cancer cell compliance has been found to be an indicator of their malignancy and metastatic potential.31-32 A deeper understanding of mechanobiology requires dedicated technology that enables mechanical manipulation at the cellular scale and below. In the same way that microscopy enabled observation in the microscale, these technologies extend our ability to probe, manipulate and physically examine to a scale relevant to cells. Over the last two decades, a number of technologies have been developed to quantify and exert forces at the cellular level and to characterise the mechanical properties of cells. They rely on miniaturised force sensors, which can measure the small forces exerted by cells (in the range of piconewton to nanonewton) with cellular and subcellular resolution, and actuators which can 2 ACS Paragon Plus Environment
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ACS Biomaterials Science & Engineering
exert defined forces on cells and subcellular structures, from the nucleus to surface mechanoreceptors. As new technologies develop, we gain the ability to probe mechanobiology below the cellular scale with increasing resolution and accuracy, from measuring the forces exerted by the engagement of a single receptor to studying the mechanical unfolding of proteins. Here, we review recent developments in techniques available to mechanically manipulate and interrogate cells. First, we review methods to measure forces exerted by cells, highlighting state-ofthe-art approaches to improve the resolution and scale of the force quantification as well as novel platforms for force quantification. Then, we review different methods to exert localised forces on cells, to characterise cell compliance or elasticity and to study the cellular response to physical stimuli. This review focuses on mechanobiology of the cell and subcellular components. Hence, we do not discuss techniques to examine tissue level mechanobiology, which can be found in other reviews.33-34
2. Measuring forces exerted by cells In tissues, cells are surrounded by a fibrous extracellular matrix (ECM), and they interact, attach, exert forces and migrate on this matrix through the formation of focal adhesions (FA). Focal adhesions are complex macromolecular structures containing integrins—transmembrane proteins that bind to ECM components on their extracellular domain—and a variety of force-sensitive adaptor molecules that connect the intracellular domain of integrins to the actin cytoskeleton and relay biomechanical and biomechanical activity to the cell, such as Talin, focal adhesion kinase (FAK) or Src kinases. Through mechanosensing, cells can respond and adapt to the mechanical properties of this local microenvironment. Cells’ capacity to sense and respond to mechanical stimuli depends on their ability to generate forces. Force generation is essential in mechanosensing, cell migration and cell adhesion. This mechanical interaction with the microenvironment is at the centre of mechanotransduction pathways and a key regulator of cell activity and function. Measuring cellular forces is fundamental to understand the underlying principles behind these processes and to study the cross-talk between the cell and its environment. The ability to quantify forces is therefore a key tool in mechanobiology, and a number of platforms have been developed to measure traction forces exerted by single cells and cell ensembles (Table 1). 2.1 Traction Force Microscopy The first technique developed to measure the forces exerted by cells on their substrate is traction force microscopy (TFM).35-36 TFM uses soft gel matrices, such as polyacrylamide (PAA)35 or polydimethyl siloxane (PDMS),37 as cell culture substrate with fiducial markers, such as fluorescent beads, dispersed in the gel . As cells spread and migrate on these substrates, they exert traction forces that deform the gels. The deformations of the gel can be monitored by tracking the movement of the fiducial markers using conventional fluorescence microscopy techniques (Fig. 1a). Based on these deformation or displacement fields and the known mechanical properties of the substrate, the most likely distribution of traction forces can be readily reconstructed. 2D TFM has been used to study the traction forces exerted by single migrating cells,35 cancer cells38 and during collective cell migration.39 TFM is a popular technique in mechanobiology and mechanotransduction research. However, it presents a number of limitations. First, conventional 2D TFM has limited sensitivity (~1 nN) and spatial resolution (~1 µm).40 This problem is two-fold: first, the resolution of the displacement field, which depends on the density of markers and determines the smallest source of force that can be resolved. Second, the resolution or accuracy in the detection of the marker positions, which in turn determines the force resolution or sensitivity. 3 ACS Paragon Plus Environment
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Despite being a continuous substrate, the displacement field of the TFM gel can only be sampled at discrete points (the fiducial markers), which limits the spatial resolution of the technique. This becomes a sampling problem in which the spatial sampling frequency is determined by the density of markers. The spatial resolution of TFM is therefore limited by the density of beads, and in turn by their point spread function (PSF, the spread of the fluorescent spot they emit): as the density of beads increases, the spread of their emissions overlap, preventing accurate tracking of their positions. This not only poses a limitation on the spatial resolution of the technique, but also a trade-off between resolution and accuracy in the detection (or force sensitivity). Different approaches have been developed to overcome the limited spatial resolution of conventional TFM. One of these approaches, super-resolved traction force microscopy (STFM), developed by ColinYork et al. relies on stimulated emission depletion microscopy (STED) to obtain resolutions
