Extreme Mechanics Letters 20 (2018) 125–139

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Extreme Mechanics Letters

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Techniques to stimulate and interrogate cell–cell adhesion mechanics Ruiguo Yang a,b , Joshua A. Broussard c,d , Kathleen J. Green c,d , Horacio D. Espinosa e,f,g, * a Department of Mechanical and Materials Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, United States b Nebraska Center for Integrated Biomolecular Communication, University of Nebraska-Lincoln, Lincoln, NE 68588, United States c Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, United States d Department of Dermatology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, United States e Department of Mechanical Engineering, Northwestern University, Evanston, IL 60208, United States f Theoretical and Applied Mechanics Program, Northwestern University, Evanston, IL 60208, United States g Institute for Cellular Engineering Technologies, Northwestern University, Evanston, IL 60208, United States article info a b s t r a c t

Article history: Cell–cell adhesions maintain the mechanical integrity of multicellular tissues and have recently been Received 28 October 2017 found to act as mechanotransducers, translating mechanical cues into biochemical signals. Mechan- Received in revised form 3 December 2017 otransduction studies have primarily focused on focal adhesions, sites of cell-substrate attachment. These Accepted 4 December 2017 studies leverage technical advances in devices and systems interfacing with living cells through cell– Available online 7 December 2017 extracellular matrix adhesions. As reports of aberrant signal transduction originating from mutations in Keywords: cell–cell adhesion molecules are being increasingly associated with disease states, growing attention is Mechanobiology being paid to this intercellular signaling hub. Along with this renewed focus, new requirements arise for Cell–cell adhesion the interrogation and stimulation of cell–cell adhesive junctions. This review covers established experi- Cell mechanics mental techniques for stimulation and interrogation of cell–cell adhesion from cell pairs to monolayers. BioMEMS © 2017 Elsevier Ltd. All rights reserved. FRET

Contents

1. Introduction...... 126 2. Cell–cell adhesion complexes...... 126 2.1. Adherens junctions...... 126 2.2. Desmosomes...... 126 3. Techniques to study cell–cell adhesion...... 127 3.1. Cell monolayer studies...... 127 3.1.1. Deformable substrates...... 127 3.1.2. Fluid flow...... 128 3.2. Single cell level stimulation and interrogation...... 129 3.2.1. Traction force microscopy...... 129 3.2.2. Substrates with flexible microposts...... 129 3.2.3. Micropipette aspiration...... 130 3.2.4. Optical traps and stretchers...... 131 3.2.5. Magnetic beads...... 131 3.2.6. Atomic force microscopy...... 132 3.2.7. Laser ablation...... 132 4. MEMS and beyond for parallel stimulation and interrogation...... 132 4.1. Moveable structures...... 133 4.2. 3D Nanofabrication...... 134 5. Sensing intra -cellular forces with FRET imaging...... 134 6. Outlook...... 134 Acknowledgments...... 135

* Corresponding author at: Department of Mechanical Engineering, Northwestern University, Evanston, IL 60208, United States. E-mail address: [email protected] (H.D. Espinosa). https://doi.org/10.1016/j.eml.2017.12.002 2352-4316/© 2017 Elsevier Ltd. All rights reserved. 126 R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139

References...... 135

1. Introduction

Our bodies experience a wide variety of mechanical inputs on a continuous basis. Whether from opening a door, noticing a tap on the shoulder, or perceiving the acceleration of an elevator, our brains translate physical forces into information that we use to understand and interact with our environment. In a similar way, cells within our body’s tissues generate, sense, and respond to mechanical cues within their local environment to direct normal physiological processes, and when things go awry, aberrant me- chanical signaling can lead to the development and progression of Fig. 1. Cell–cell adhesion in epithelial cells. a. Adherens junctions (AJs) and desmo- disease. somes are cadherin-based intercellular junctions, which, along with adhesions at the cell–ECM (HD: hemidesmosome; FA: focal adhesion), are responsible for main- In the body, physical forces are generated by and transmit- tenance of the epithelial phenotype. b. The major components of the desmosome ted through the musculoskeletal system comprising bones and junction are desmocollin (Dsc), desmoglein (Dsg), plakoglobin (PG), plakophilin muscles as well as the structures that hold them together: joints, (PKP), and desmoplakin (DP), which connect to intermediate filaments (IFs). c. The major components of classical AJs are the transmembrane protein E-cadherin, p120, ligaments and tendons. In cells, this function is performed by α-, and β-catenin. cytoskeletal filaments and their associated adhesive organelles [1]. Cell–cell and cell–extracellular matrix (ECM) adhesive organelles are macromolecular structures that integrate individual cells into 2. Cell–cell adhesion complexes complex tissues by anchoring cytoskeletal components [2]. They have been the subject of intensive scientific investigation due to There are four main types of specialized cell–cell junctions their importance in cell and tissue mechanics, bridging the gap in mammalian cells. These include tight junctions, gap junctions, between interactions at the molecular level to forces at the cellular adherens junctions, and desmosomes [9,10]. Tight junctions seal scale. In recent years, attention has shifted toward understanding the paracellular space, limiting the passage of molecules and ions the integral roles cell–cell adhesions play in transducing mechan- through the space between cells, and stopping the movement of ical cues into biochemical signals, a process often referred to as membrane proteins between the upper and lower portions of the mechanotransduction [3]. Under normal homeostatic conditions, cell [11]. Gap junctions function as pores between adjoining cells, the adhesion/cytoskeleton systems form a highly integrated net- allowing molecules, ions, and electrical currents to pass directly work that regulates tissue morphogenesis, collective cell migra- between cells [9]. This review will focus on adherens junctions tion, cell proliferation, and differentiation [4]. However, a number and desmosomes, which are cadherin-based intercellular junctions of pathological conditions and developmental disorders, including that link to the actin and intermediate filament (IF) cytoskeletons, skin disorders [5], arthritis [6], atherosclerosis [7], and cancer [8], respectively (Fig. 1a). may emerge from aberrant cell–cell adhesion or mechanical cues. 2.1. Adherens junctions Hence, the study of cell–cell adhesion has taken center stage aim- ing to understand its capacity in maintaining tissue- and cell-level Adherens junctions (AJs) are multiprotein complexes whose mechanical integrity and more importantly, in converting forces core components comprise transmembrane classical cadherins and stresses at the cell–cell junction into regulatory pathways that and intracellular armadillo family members (Fig. 1b). The extra- dictate cell behavior. cellular domains of the classical cadherins, including E-cadherin, Over the past two decades, we have witnessed a wide vari- N-cadherin, VE-cadherin, and P-cadherin, form both hetero- and ety of techniques to stimulate and probe cells in vitro. With the homophilic interactions and through calcium-dependent trans in- advancement and wide adoption of these techniques, we have teractions link neighboring cells together [12]. On the intracellular established a large body of literature about how cells move, dif- side, they provide a platform for the recruitment of armadillo ferentiate, connect with other cells and probe their environment. proteins p120 catenin and β-catenin. α-catenin interacts with β- The techniques were not invented for the sole purpose of studying catenin and provides the linkage to the actin cytoskeleton. Many of cell–cell adhesion; rather they were designed to probe mechanical the classical cadherins have been shown to be mechanically sen- responses and/or to stimulate biochemical responses in cells inter- sitive [13], some capable of forming catch-bonds that strengthen acting physically in their suspended or adherent states. The con- and become longer lived in the presence of mechanical force [14]. sequence was that investigators gained many insights about how Moreover, both extracellular and intracellular mechanical stimuli can promote force-mediated stabilization of the AJ/actin linkage adhesive organelles give rise to cell and tissue level architecture. through recruitment of the actin binding protein vinculin, which The majority of the techniques were designed to stimulate and occurs through a conformational change in α-catenin that reveals probe cell–ECM interactions, which serve at the forefront of the a vinculin binding site [15]. physical interaction between cells and their external environment. During the course of such studies, we have learned that cell–cell 2.2. Desmosomes interactions work together with and even regulate cell–ECM adhe- sions. Some of the probing and stimulation techniques require the Desmosomes are also calcium-dependent adhesive junctions presence of robust cell–cell junctions. Here, we present techniques and have a molecular organization similar to that of the AJ that have been widely used to explore cell mechanics, and then (Fig. 1c) [16]. Desmosomal cadherins, which in humans include how they can be applied for use in cell–cell adhesion studies. desmogleins (Dsg1-4) and desmocollins (Dsc1-3), are expressed R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139 127 in a tissue-type and differentiation-dependent manner [17,18]. They can form hetero- and/or homophilic trans interactions be- tween their extracellular domains, though compared with classical cadherins these interactions are not as well understood [16,19]. The intracellular portions of desmosomal cadherins recruit the armadillo proteins plakoglobin and plakophilins. These interact with the plakin protein family member desmoplakin (DP), which in turn links the desmosomal core to the IF cytoskeleton [16]. The roles of the desmosome/IF network in mechanical signaling are not well understood. However, removing the keratin IF sys- tem was shown to affect the mechanical properties of cells as well as their ability to migrate [20]. Moreover, manipulating the strength of the desmosome/IF linkage using DP mutants regulates both intercellular forces in cell pairs and cell stiffness in cell pairs and larger groups of cells, through a process involving the actin cytoskeleton [21].

3. Techniques to study cell–cell adhesion

A wide variety of techniques have been used to study the mechanics of living cells. Early studies were done on groups of cells, using techniques such as substrate deformation, in which cells are cultured on a deformable substrate, and then subjected to a uniform monotonic or cyclic strain. To recapitulate the effects of fluid flow on cell layers, flow-induced shear experiments have also been performed, in which a fluid flowing over a cell layer imposes shear stress on the cells. To investigate cell-substrate and cell–cell interactions, traction force microscopy and micropost substrates were developed to enable the measurement of forces exerted by cells while stationary or in motion. In many instances, the mechan- ical properties of individual cells or cell pairs is of interest because Fig. 2. Mechanical stretching of a monolayer of cells. a. Uniaxial stretching; b: they provide insight into how cells physically adapt to extracellular Biaxial stretching; c: Substrate flection; d: Stretching with curved template; e: stimuli and their microenvironment. In such cases, methods with Equiaxial stretching with vacuum suction. single cell resolution are employed. They include atomic force microscopy (AFM), optical traps/optical tweezers, magnetic beads, micropipette aspiration, and laser ablation. Another method to other method is substrate flection, in which the substrate is bent, probe cell mechanical responses employs micro-electro mechani- usually using four-point bending [49](Fig. 2c). This method offers cal systems (MEMS). These devices take advantage of actuators and the ability to achieve low strains, and has also been demonstrated sensors, with tunable displacement and force measurement reso- to impose uniform longitudinal stresses on cells. Many studies lution, by either directly attaching to the cell or moving a structure have also used circular substrates with out-of-plane and in-plane that the cell is attached to (resolutions for force and displacement deformation [50]. An issue with out-of-plane deformation is the are listed in Table 1). Some examples of microfabricated devices heterogeneity of the strain imposed on the substrate. The radial include uniaxial and biaxial pullers, micropillars, and cantilever component of strain is usually uniform, but the circumferential beams. component varies from zero at the ends to some maximum in the center. In-plane deformation has been used to avoid problems of 3.1. Cell monolayer studies heterogeneous strain distributions that result from out-of-plane deformation. One in-plane deformation method is stretching the 3.1.1. Deformable substrates substrate around a circular ring (not shown, similar to Fig. 2e). This By culturing cells on the surface of a deformable substrate, method is similar to the out-of-plane technique of stretching the strains can easily be imposed on the cells by manipulating the substrate around a curved template (Fig. 2d), but with this method, substrate. Uniaxial and biaxial strains can be achieved, depending the portion of the substrate inside the ring remains on a single on how the substrate is deformed. There have been many studies plane, resulting in uniform strain distributions [51–53]. Similarly, on a variety of cell types, including bone tissue [22,39,40], lung a vacuum can be used to stretch a circular substrate around the cells [41,42], and neurons [43]. In addition, this method has been perimeter of a raised central cylindrical platform (Fig. 2e). The used to study cyclic loading on cell groups [23,44,45]. The me- portion of the substrate on the platform remains on a single plane, chanical properties of the substrate can be controlled by altering resulting in uniform strain distributions. This method has found its thickness or its chemical composition [46], which in turn will wide success in commercialized systems such as Flexcell. affect the stiffness of the substrate, allowing for finer control over Early attempts to stimulate a group of cells with mechanical strain levels imposed on cells. Overall, this technique is simple strain aimed to understand their change in material properties in to implement, but it does not offer high displacement resolution response to the stimulation [41,42], while underlying mechanisms compared to other techniques. of the biochemical and subsequently the biophysical responses are Substrate deformation can be achieved by two modalities. The limited. This is mainly due to the nature of the technique but also first is by stretching the substrate longitudinally. This method has the lack of tools to follow cellular responses at a small scale. In been demonstrated to impose uniform longitudinal stresses on recent years, cyclic stretching with equiaxial strain generated most cells on the substrate uniaxially (Fig. 2a) or biaxially (Fig. 2b) and of the significant findings in how different cell types respond to os- can also be used to impose oscillating stresses on cells [47,48]. The cillating strain. Commercialized instrumentation makes it a readily 128 R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139

Table 1 Techniques to study cell–cell adhesion with resolutions in force and displacements. Technique Force/Stress range (Resolution) Displacement range (Resolution) Deformable Substrates 50–1000 Pa [22] 0–100 µm [22] 0%–70% [23] (0.04%) Fluid Flow 0–22 Pa [24,25](0.05 Pa) 0–50% [26] Micropipette Aspiration 0–700 Pa (0.1 Pa) [27,28] 0–100 µm (25 nm) [29] Optical Traps/stretcher 0–300 pN (5 pN) [30,31] 0–5 µm (1 nm) [32] Magnetic Beads 0–120 pN (1 pN) [33] N/A Atomic Force Microscopy 0–20 nN (1 pN) [34] 0–100 µm (1–5 nm) [34] Uniaxial Puller 0–1.5 µN (1 nN) [35] 0–100 µm (40 nm) [35] Biaxial Puller 0–60 µN (1 nN) [36] 0–3.4 µm (10 nm) [36] Microposts 0–100 nN (10 pN) [37] 0–1000 nm (10 nm) [37] Cantilever Beam Deflection 0–1 µN (50 pN) [38] 0–50 µm (10 nm) [38] available technique in many biological labs; the capacity to work within the physiological cell culture condition propels its wide acceptance. The effects of cyclic stretching on the reorganization of cytoskeleton, the proliferation of different types of cells and the differentiation of stem cells have all been reported [54–57]. Stretch can be used to study mechanotransduction, as the pro- cess, in general, involves force-induced conformational changes in a mechanosensor protein that trigger additional protein–protein interactions. It is widely accepted that applied strains from stretch- ing unfolds adhesion molecules in focal adhesion (FA) sites. For example, stretching induces conformational changes in the FA protein talin, which activates vinculin recruitment and ultimately leads to actin filament clustering and FA enhancement in a force- dependent manner [58]. Therefore, stretch can be used as a tool to examine force-induced alterations in mechanosensitive protein localization and recruitment, and when combined with molecular Fig. 3. Fluid shear stimulation of a monolayer of cells. a. Fluid shear in a two-plate tension sensors (discussed below) can be used to observe force- based flow chamber; b. Fluid shear with a plate and rotating cone system. induced protein conformational changes. These alterations in the FAs lead to other intracellular biochemical signals which result in a host of cellular behaviors. Two types of fluid flow systems are commonly used. The first Recent studies have found that the stretching stimulation may not only strain mechanosensors at the FA but also at cell–cell ad- is a parallel plate system (Fig. 3a), in which fluid flow is driven hesive junctions [59,60]. Beneath the complex mechanotransduc- through a small rectangular chamber using a pressure differential, tion signaling network lies the coordinated crosstalk of integrins normally a syringe pump. A variety of parallel plate systems have and cadherins that regulates cell signals and forces [61,62]. The been developed [71–77], and technological advances allowed for force transmission can potentially be facilitated by the AJ protein smaller microscopic parallel plate systems [24,25,78]. In these α-catenin, which reinforces intercellular tension by tightening studies, the dimensions of the channel can be varied to control the linkage between AJs and F-actin when the cell–cell junction the flow characteristics and thus shear stress, and are kept small experiences strain that exposes the vinculin binding site in α- to ensure a low Reynolds number and thus laminar flow. In one catenin [63]. It has also been reported that intercellular tension study, the width of the fluid flow channel was varied between 0.25 is increased when adherent cells are stretched, as measured by and 1 mm, which changed the shear stress imposed on a culture a Förster resonance energy transfer (FRET)-based tension sensor of fibroblasts [79]. To generate easily-controlled uniform shear embedded in E-cadherin [59], pointing towards a direct impact of stress, a cone and plate system filled with cell medium is employed stretch stimulation at cell–cell junctions. Further, transcriptional (Fig. 3b), in which a cone is rotated along its axis above the surface activities of YAP and β-catenin upon mechanical strain function of a circular plate, creating a fluid flow [80–84]. The tangential in a E-cadherin dependent manner [64]. Besides mechanosen- speed of the cone increases with increased distance from the axis sor molecules at junctions, mechanical stretch also stimulates of rotation, but the distance between the cone and flat plate also mechanosensitive ion channels, such as piezo1 [65]. increases, resulting in uniform shear stress distributions on the cell monolayer along the plate and the cone surface. By varying the 3.1.2. Fluid flow angle of the cone and the speed of rotation, shear stresses of differ- Fluid flow can be used to impose shear stress onto a group of ent magnitudes can be achieved. Specialized fluid flow profiles and cells, another type of mechanical stimulus. To this end, cells are flow chamber geometries are often designed to generate different cultured on the surface of a fluid flow channel. As fluid flows over temporal and spatial shear stress gradients [26,85]. the culture, shear stress is imposed on the cells from the boundary Fluid flow is best studied in the endothelial cell system to layer between the cells and the fluid. Fluid shear stress has been mimic the effect of blood flow on the vessel wall, particularly the used in a variety of studies, including investigating the influence of endothelium, aiming to understand the process of atherosclerosis. fluid shear on proliferation of bovine aortic endothelial cells [66] In general, fluid flow produces two principal stress components: and investigating rolling adhesion of white blood cells in shear the stress perpendicular to the vessel wall and the stress parallel flow [67]. The primary advantage of this method is the natural to the monolayer of endothelial cells. The former is the tensile environment this study takes place in. Cells commonly interact stress exerting a dilating force on the vessel wall. The latter, fluid with fluid flow in vivo, so this setup allows for a natural testing shear stress, represents the frictional force the fluid flow exerts on environment, thus fluid flow methods have been widely adopted to the endothelial cells, and it activates a cascade of mechanotrans- study the effect of blood flow on the physiology of endothelia [68– duction pathways that leads to endothelial cell proliferation [86], 70]. reorganization of the cytoskeleton [87] and even cell death [88]. R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139 129

Although the molecular basis for mechanosensing of fluid shear stress and for cells’ ability to discriminate flow patterns remains to be fully revealed, studies have shown a mechanotransduction cas- cade coordinates the redistribution of forces at cell–cell junctions and cell–ECM adhesions [89]. The endothelial AJ contains VE-cadherin and platelet endothe- lial cell adhesion molecule (PECAM)-1, both serving as anchors to the cell cytoskeleton and sustaining tension across the cell– cell junction. When a monolayer of endothelial cells is subject to fluid shear stress, though the cell layer tends to re-align itself in the direction of the flow to minimize drag resistance, the tension gradient at the apical surface of the cell membrane is eventually transmitted to junctional complexes. To maintain mechanical in- tegrity, significant cytoskeletal remodeling takes place upon the onset of fluid flow [70]. Specifically, it has been reported that shear flow triggers the enhanced association of PECAM-1 with vimentin, which facilitates the transfer of actomyosin-generated tension within the actin network, thus relieving the stress at VE- cadherin adhesion complex [90,91]. This sequence of events leads to the rapid increase of tension at junctional PECAM-1 and a si- multaneous drop of tension at junctional VE-cadherin, measured by FRET sensors in both adhesion molecules [90]. It is not surprising that PECAM-1 was identified as a mechanosensor that is associated with vimentin, an IF protein within endothelial cells. Similar roles for the IF-anchoring complex, the desmosome, are also reported in epithelial cells [92] with evidence of abundant crosstalk between desmosomes and AJs.

3.2. Single cell level stimulation and interrogation

Techniques used to quantify forces and stresses imposed on cells were developed to more fully understand biological responses to external mechanical stimuli. By studying isolated single cells or individual cells within a large group, complex responses to exter- nal mechanical stimuli can be recorded. However, these techniques Fig. 4. Cell force interrogation techniques and their use in cell–cell adhesion studies. offer new challenges to manipulate and position cells individually, a. TFM (a1) and micropost arrays (a2); b. Micropipette aspiration for single cells with single cell control. Among the most common approaches (b1) and cell–cell adhesion (b2); c. Optical trapping and stretcher for single cells are traction force microscopy (TFM), substrates with deformable (c1) and for cell–cell adhesion (c2); d. MTC for single cells (d1) and for cell–cell adhesion (d2); e. AFM based single mechanical interrogation (e1), single molecule microposts, micropipette aspiration, optical tweezers, magnetic force spectroscopy (SMFS) (e2) and single cell force spectroscopy (SCFS) for cell–cell twist cytometry (MTC), atomic force spectroscopy (AFM), laser adhesion (e3). ablation, and cantilever based spectroscopy. A description of each technique follows. In the study of cell–cell adhesion, TFM has shown direct ev- 3.2.1. Traction force microscopy idence of crosstalk between cell–cell and cell–ECM adhesions TFM is the first mature technique to be employed to measure by indirectly quantifying cell–cell tugging forces through their cell generated forces [93]. It consists of a flexible substrate con- force balance with cell–ECM traction forces [102–104]. These taining embedded fluorescent beads over which adherent cells findings showed that integrin-mediated adhesions regulate ten- are cultured. In 2D TFM (Fig. 4a1), measurements of the substrate sion and composition of cell–cell junctions [105–109]; conversely, deformation together with the application of an inverse method cadherin-based cell–cell adhesions in epithelial cells modulate using half-space elasticity solutions enables the construction of a cell–ECM traction forces [102]. For example, inhibiting cell–cell ad- map of the in-plane component of the traction vectors. As such hesion in small colonies of keratinocytes by reducing calcium lev- the technique is capable of measuring forces at the single cell els or genetically depleting cadherins (E-, and P-cadherin) results level. The displacements of beads due to substrate deformation are in remarkably different traction force patterns. Under control con- calculated by means of digital image correlation techniques [94]. ditions, cooperative traction forces with maximum stress are local- Common substrates used for TFM include polyacrylamide or silicon ized to the periphery of cell colonies, and inhibiting cell–cell adhe- based gels. They are selected due to their linear elasticity, optical sion results in independent and significant traction forces evenly transparency, and tunability of elastic moduli through polymer distributed throughout the colonies [102]. Similar cooperative ac- chain crosslinking, over several orders of magnitude [95]. Knowing tivities of cell–cell and cell–ECM adhesion were also observed in the elastic moduli of the substrates, the displacement field can be heart muscle cells [110]. Studies using TFM also revealed that converted to a traction force field using an inverse method [93, the force balance as well as molecular tension in E-cadherin are 96,97]. These forces are often on the order of nanonewton or modulated by spatial distributions of FA sites [111]. TFM was also tens of nanonewtons. To measure both the normal and in-plane used advantageously to study mechanisms of cell migration [112]. components of traction vectors, 3D TFM has been developed in recent years [94,98–100]. Potential sources of inaccuracy in com- 3.2.2. Substrates with flexible microposts puting the displacement field include polymer degradation and A simple and elegant version of TFM was achieved by micro- nonlinearity of local deformation among others [101]. fabrication of arrays of polydimethylsiloxane (PDMS) posts using 130 R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139 micromolding [113](Fig. 4a2). When cells are cultured over the substrate, in-plane components of adherent forces are revealed by the deflection of the microposts. Their cantilever geometry and chemical composition of the PDMS determine their stiffness. Hence, by using optical microscopy, the deflection amplitude and direction can be measured, from which the forces the cell is ex- erting on the microposts can be identified. The primary advan- tage of this technique is the large number of independent force measurements easily achieved as a function of position and time, which when combined, provide a vector map of traction forces. One embodiment of this technique used microposts with varying stiffness by changing the geometry of individual microposts. This allows for control of the sensitivity of force measurements on certain regions of the cell [113]. Further, by combining micropost fabrication with microcontact printing of ECM proteins, a cell pair can be confined to a predefined matrix pattern [114]. Relying on the zero net-force relationship in equilibrium condition, the forces being transmitted through the cell pair interface can be obtained from the vector sum of the forces acting on each set of microposts under the cell pair. Limitations of this technique are the lack of quantification of the component of the force vector normal to the substrate and the inability to use the microposts to apply forces on cells. By embedding a magnetic cobalt nanowire inside a subset of the microposts, application of forces to cells is possible using an externally applied magnetic field [37]. This approach was used to study the relationship between external mechanical forces and cytoskeleton induced forces. The applied forces were correlated to FAs and traction forces measured on non-magnetic posts near the magnetic posts [37]. Force stimulation has also been achieved by stretching the substrate containing micropost arrays [115]. In the study of cell–cell adhesion, an early attempt using micro- Fig. 5. Micropost arrays reveal that desmosome/IF linkage regulates cell–cell post arrays combined with microcontact printing showed that ac- adhesion forces. a. Micropost arrays 10 µm in height and 2 µm in diameter are tomyosin generated contractile force regulates the size of AJs [114], fabricated using micromolding of PDMS. b. Force balance established between cell pairs are used to calculate cell–cell tugging force. c, d, e. Distribution of intercellular indicating that cell contractility affects not only the composition at forces in nN as measured by micropost arrays for DPNTP, S2849G DP and WtDP, cell–ECM adhesions but also the composition of cell–cell junctions. respectively. c, d and e are recreated from Fig. 2 in [21], reprinted with permission.). In a recent study, the desmosome/IF linkage at cell–cell junctions has also been shown to regulate not only traction forces at cell– ECM adhesions, but also modulate forces at cell–cell junctions [21]. 3.2.3. Micropipette aspiration Using a combined approach of micropost arrays and AFM, epithe- Micropipette aspiration is a simple technique in which a lial cells with genetic mutation of a desmosome/IF linker molecule, single cell is partially or completely aspirated into the tip of DP, which lacks the IF-binding domain, exhibited significantly a micropipette (Fig. 4b1). In an aspiration experiment, a mi- reduced traction forces and potentially increased tension or con- cropipette with inner diameter smaller than that of a suspended formational changes in α-catenin of AJs [21,116]. This evidence cell, equipped with a micromanipulator positioning system, is lends support to the notion that abundant molecular commu- brought into contact with a cell while a negative pressure is ap- nication exists between the two mechanically active junctional plied. The negative pressure forces the suspended cell to attach to complexes [117]. The same study showed that the desmosome/IF the tip of the micropipette as a microscope imaging system records linkage has a significant impact on global cell mechanics as well cell deformation during the process [118]. This simple procedure as tension within the cell–cell interface [21]. Different forms of has been used to investigate membrane elasticity [119,120], to DP were expressed in A431 cell lines to investigate the role of quantify mechanical and material properties of single cells and this linkage in regulating cell mechanics. These include: DPNTP, cell nuclei [121,122], to measure molecular adhesions between a truncated form of DP that lacks the IF binding domain, S2849G pairs of cells [123,124], and to study mechanotransduction within DP, which contains a serine to glycine mutation that enhances single cells [125,126]. The technique imposes large strains on cells, the desmosome/IF linkage, and wildtype DP (WtDP). Micropost and as a result is incompatible with some cell types [27]. High arrays of different dimensions and elasticities were fabricated us- deformation gradients along the edge of the pipette and friction ing micromolding (Fig. 5a, shown is micropost arrays with height between the pipette surface and the cell membrane often impact of 10 µm and diameter of 2 µm and elastic modulus of 2.41 measurement results. Mathematical models have been used to MPa). Cell–cell adhesion forces were derived by the zero-sum force convert resulting strains into stresses in erythrocytes [127,128] balance between the two cells (Fig. 5b). Measurement results from and chondrocytes [129], but they have limited accuracy due to the the micropost arrays show that DPNTP expression results in a model assumptions. Finite element analyses with large deforma- significant reduction of average intercellular forces while S2849G tions have been performed to determine mechanical properties of DP expression increases these forces significantly, and expression suspended cells, such as chondrocytes, with different viscoelastic of WtDP only increases the intercellular forces slightly (Fig. 5c, models [28,130]. d, e). These experimental data measured using micropost arrays To study cell–cell adhesion, two cells are brought into contact suggest that the desmosome/IF linkage also modulates cell–ECM with each other using two micropipettes in a process called dual interactions, similar to AJs [102]. micropipette aspiration (DPA) (Fig. 4b2). The micropipettes use a R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139 131 slight vacuum that can securely hold the cell on the micropipette cells [138] and infected blood cells [31,142] were stretched using without significant aspiration so as to avoid large strains. Cells form an optical trap to quantify their elastic moduli. The diseased cells a cadherin-based cell–cell adhesion complex after a certain contact were tested in various stages of the infection, revealing the shear time. While one micropipette maintains a sufficient amount of modulus steadily increased throughout the duration of the infec- pressure to hold the cells in place, the pressure in the other mi- tion, increasing by about an order of magnitude at the final stage cropipette is increased while the pipette retracts until the two cells of the disease. are separated. The forces required to break the cadherin adhesions In the study of cell adhesion, optically trapped beads, a few can be calculated from the aspiration pressure required to separate micrometers in diameter, were tethered to matrix proteins such as the two cells [131,132], which is in the nanonewton range. When functional peptides [143] or fibronectins [144] to measure adhe- separating cell doublets with cadherin bonds, due to the short sion forces (Fig. 4c2). The technique enabled real-time monitoring separation time of less than 1 s, the process can be regarded as sep- of traction forces, during the formation of cell adhesion sites, aration of two elastic solids where the separation force depends on functioning as a high resolution (∼pN) force sensor. However, due the density and dissociation rate of adhesion bonds [59]. We note to the complexity of the optical setup, only a few beads could be that the micropipette aspiration technique can be easily integrated tracked simultaneously, limiting its spatial resolution. The tech- with fluorescence microscopy, which allows the imaging of the nique has also been deployed to investigate the interactions of aggregation of adhesion molecules as well as the measurement of AJs with filamentous actin (F-actin) [145]. To this end, two optical cell cortex deformation in real-time. Contact time can be controlled trapped beads attach to both ends of a single actin filament sus- to ensure the maturity of the cell–cell adhesion complexes. In this pended above a purified cadherin–catenin complex. The objective context, the technique has been used to study cadherin binding was to mimic the parallel spatial arrangement of F-actin filaments forces between CHO cells and red blood cells [133] and to quantify with the cell membrane at the cell–cell junction. To apply tension, cadherin-dependent cell–cell adhesion in E-cadherin expressing the cadherin–catenin complex was anchored onto a microsphere cell doublets [29]. attached to a movable stage. During the cyclic motion of the stage, Studies using micropipette aspiration have only been con- when the immobilized complex bound to the suspended filament, ducted on suspended cells until a recent report extended its utility the interaction force can be obtained from the optically-displaced to cells in adherent states. In the study, a single endothelial cell beads. The study revealed that α-catenin is required to form the adherent on a substrate was aspirated from the apical surface AJ/actin filament tether and that the bond between the complex using a micropipette tip, placed perpendicular to the substrate, and the actin filament is tension-enhanced and catch-bond like. while an inclined mirror was employed to measure the cell strains A variation on optical traps, known as optical stretchers, uses during aspiration [134]. The experiments revealed that the in- divergent lasers that interact directly with the cell without the teraction between cytoskeletal actin and ECM, in adherent cells, need for microbead handles [146–149]. This technique was most increases the contractile forces within the cytoskeleton and thus recently employed to probe the mechanics of cell–cell inter- the resistance to aspiration. This critical element was missing in faces [149]. In this study, mature cell–cell junctions of a drosophila studies on suspended cells. In addition, similar to micropipette embryo were interrogated by optically imposing a deformation aspiration in suspended cells, the nucleus was shown to be highly (<1 µm) on the cell–cell interface. Observation of the retraction deformable under the considerably large strains resulting from of the cell–cell junction to the original shape and the restoration aspiration. These experimental results were interpreted employing of force balance provides information about the mechanics of cell– a viscoelastic finite element model. cell junctions during early tissue morphogenesis. The study shows that tension at cell–cell junctions, on the order of 100 pN, can equi- 3.2.4. Optical traps and stretchers librate over a few seconds and that the time-dependent properties Optical traps use momentum conservation of diffracting pho- of the junction can be reproduced by a simple viscoelastic model. tons to impart small forces on dielectric objects [135]. This method has been used to study many molecules [136], including the ki- 3.2.5. Magnetic beads netics of RNA unfolding [137]. When used to study cells, dielectric Magnetic beads can be used as handles to apply forces to a microbeads are adhered to the cell and act as handles for the optical cell, in a process often referred to as magnetic twisting cytometry trap (Fig. 4c1). The adhesion strength limits the maximum force (MTC) (Fig. 4d1). The beads are coated with a group of molecules an optical trap can exert on a cell [30,138]. Once the microbeads that allow them to bind to specific cell surface receptors [23]. Once are adhered to the cell, a laser is directed through one of the they are attached, they can be manipulated through control of an microbeads, refracting the laser. The refraction changes the mo- external magnetic field. This method was developed in 1950 [150] mentum of the photons, and thus changes the momentum of the and has been used in a variety of studies, including applying cyclic microbead, inducing a force. The microbead is attracted towards loadings to cells [33,151–154]. The technique has been mostly the focal point of the laser, and therefore the force can be controlled applied to investigate the mechanical properties, especially vis- by altering how the laser is focused on the microbead [139]. For coelasticity, of cells [151], but was also applied to fundamental this to work, the refractive index of the bead must be larger than studies of the role of membrane forces in gene regulation [155]. the refractive index of the medium surrounding the cell. Generally, Two main advantages of MTC are the ability to apply torque to cells this technique is used to apply static loading to cells, but can also and the ability to easily apply cyclic loadings through control of the be used for cyclic loading, using an acousto-optic modulator [140]. magnetic field. However, due to unfavorable scaling of magnetic The two main advantages of these techniques are high force reso- forces with size, applying large forces with this technique requires lution and lack of physical contact between the actuation and the large beads relative to cell size [156]. cell. Using this technique, sub pN forces were achieved, and cells Similar to the optical trapping method, MTC can apply and mea- were studied in their natural environments as they do not need to sure forces with pN resolution. To this end, magnetic beads are nor- be physically attached to an instrument. However, the maximum mally coated with RGD sequences that link them to integrin recep- achievable force is limited to a few hundred pN, as high laser power tors, which led to the discovery of integrin-based mechanotrans- may impart radiation damage onto the cell. duction [153]. Recent studies on mechanotransduction at cell–cell A study that took full advantage of the resolution achieved with junctions have revealed that applied forces at E-cadherin have optical traps examined the impact malaria has on the mechanical profound impact on cell contractility and cell mechanics [157]. properties of diseased red blood cells [141]. Healthy red blood A combination of MTC for force loading at the cell–cell adhesion 132 R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139 complex and traction force microscopy (TFM) to quantify traction (illustrated Fig. 6a). AFM studies on A431 cells expressing vari- forces at cell–ECM interactions was employed (Fig. 4d2). Beads ous forms of DP have shown that removing the desmosome/IF coated with E-cadherin ligands were employed to induce an os- linkage reduces cellular stiffness significantly compared with con- cillating shear stress (∼10 Pa) through modulation of the applied trols, conversely, enhancing the linkage increases cellular stiffness. magnetic field. The study revealed that shear stress induced at cell– These results suggest that changes at the cell–cell adhesion com- cell junctions can trigger a significant increase in traction forces. plex have a major impact on global cell mechanics. In addition, This also demonstrates a new mechanotransduction pathway from the stiffness changes observed on cells with DP mutations can cell–cell junctions to FAs, potentially through EGFR-PI3K mediated be abrogated by depolymerizing actin filaments, indicating a po- pathways. It is yet to be determined the extent to which the α- tential crosstalk between the desmosome/IF and AJ/actin filament catenin-actin filament linkages participate in the force sensing and networks [21]. Furthermore, considering the significant difference transducing process, despite the many previous reports confirming in the mechanical properties of the cytoskeleton and the cytosol, their tension-maintaining and conformation-altering roles [145, AFM images obtained by applying a small force to the cell can 158]. reveal the cytoskeleton structures at the cell–cell adhesion junc- tion as shown in Fig. 6b. The imaged structure can be compared 3.2.6. Atomic force microscopy to a correlative immunofluorescence image to identify the type Atomic force microscopy (AFM) is a technique that was origi- of filaments (similar to the labeling of IF cytoskeleton in Fig. 6c). nally developed to map surfaces of any material with nanometer Mechanical measurements can be performed on individual fila- resolution. A microfabricated silicon cantilever beam, having a ments to reveal their tensional states when the junction complexes sharp tip at its end, is brought into contact with a surface. As are modulated. These mechanical characterizations will inform our the tip interacts with the surface, the cantilever beam bends. By understanding from a global cell mechanics perspective, where the tracking its deflection, using a laser beam deflected off the back cell cytoskeleton is considered as a tensegrity structure [177]. of the cantilever beam, the tip–surface interaction force can be Cell–cell adhesion studies also take advantage of the binding measured when the cantilever stiffness is known. The technique force measurement capability of AFM. SMFS experiments on the has sub-pN force resolution and sub-nm displacement resolution, desmosomal cadherin, Dsg3, have revealed the distinctive bind- but has a limited maximum force and displacement. It has been ing affinities of Dsg3 molecules at different regions of the cell adopted to the study of cell mechanics by relating the amount surface [178]. Using a Dsg3 functionalized AFM probe, the study the cantilever beam deflects to the force the probe exerts. AFM showed that homophilic Dsg3 binding forces are stronger at cell– has been successfully employed to study cell elastic [159–163] cell contacts than at other cell surface areas. Further, this binding and viscoelastic properties [164], as well as nuclei stiffness [165] event can be effectively blocked by calcium depletion and Dsg3 (Fig. 4e1). In addition to compressive forces, AFM can also be antibodies [178]. In a separate study, the same group identified used to apply tensile forces to a cell. One way this can be done that inhibition of Dsg3 binding, which occurs in the autoimmune is by culturing cells directly on the AFM tip. This allows for easy disease pemphigus vulgaris, does not lead to cell–cell adhesion manipulation of cells, and has been used to study cell–cell and cell- loss, rather it alters downstream signaling events that may con- substrate interactions [166]. In addition, the AFM tip can be func- tribute to that effect, such as p38 mitogen-activated protein ki- tionalized to bind to cell surface receptors in a technique widely nase (p38 MAPK) [179]. Studies using SMFS also revealed the known as single molecule force spectroscopy (SMFS) (Fig. 4e2). mechanisms of bond formation between desmosomal cadherins: Once functionalized, the probe can be retracted to apply tensile Dsc forms calcium-dependent homophilic bonds and Dsg forms forces and unwind or break molecular bonds [167]. SMFS has been calcium-independent heterophilic bonds with Dsc. [180]. Similar used extensively to study binding affinities for a host of molecules, approaches have been taken in the study of E-cadherin based including the unbinding of DNA protein pairs [168,169]. cell–cell adhesions, and the mechanism of catch bond formation In the study of cell–cell adhesion, AFM has been used in the between E-cadherin molecules has been demonstrated [181,182]. context of single cell force spectroscopy (SCFS) [170,171](Fig. 4e3). The technique is similar in principle with micropipette based cell– 3.2.7. Laser ablation cell adhesion studies using DPA, where two cells are brought into The contractile forces present within cell–cell contacts of living contact and pulled apart to quantify their interaction. Compared cells can be directly measured using laser ablation [183]. The with the DPA method, SCFS offers higher resolution (pN compared cell cortex at sites of cell–cell contact is physically cut using a to nN for DPA) and less native strain to the cells before contact. high-power laser (e.g. two-photon laser). A fiduciary fluorescently In SCFS adhesion measurements, a living cell is first attached onto tagged membrane marker is used to track the displacement of the a tip-less AFM cantilever, normally by means of matrix protein vertices at either side of an ablated cell–cell junction. The rate of coating, and the cell is brought into contact with another cell on vertex recoil can then be used to extract force-related parameters the substrate by lowering the cantilever using a z-piezo stage. including the contractile force that was present within the junction During the retraction phase, the interactions can be measured as well as the ratio between junction elasticity and the viscosity of by recording the cantilever deflection. The sensitivity in force the cytoplasm [183]. Tension within cell–cell junctions can then measurement and the fine position control, by the z-piezo stage, be compared across experimental conditions by comparing initial enables quantification of subtle cell–cell interactions during the recoil velocities. This approach has been used to determine the initial phase of adhesion. These forces are often retraction rate- contributions of Src kinase [184], actin regulating proteins like N- dependent and dwelling time-dependent [170,172]. A prolonged WASP [185] and the Rho guanosine nucleotide exchange factor dwelling time often leads to the study of cooperative binding. SCFS Ect2 [186], and myosin II [187] to the generation of tension at sites studies have led to the conclusion that levels of E-cadherin deter- of cell–cell contact. Finally, this method is capable of measuring mine the adhesion strength between different types of zebrafish cell–cell forces both in traditional cell culture models as well as in progenitor cells during development [173]. Destructive methods vivo models like zebrafish [188]. are also used to probe cellular responses including the use of AFM probes [162,163] or laser pulses [174–176] to dissect cytoskeleton 4. MEMS and beyond for parallel stimulation and interrogation components. Performed on the center of individual cells, AFM nanomechan- The aforementioned techniques, while effective, present some ical studies can reveal subtle changes in global cell mechanics limitations in terms of force and displacement resolutions, and R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139 133

Fig. 6. AFM imaging reveals cytoskeletal bundles. a. AFM nanomechanical measurements reveal global cell mechanics. b. AFM image is scanned at the cell–cell junction. Scale bar: 2 µm. c. To identify the filaments in the AFM image, an immunofluorescence image is taken at the junction to visualize IF (Red: IF; Blue: plakoglobin; Green: DP). Scale bar: 5 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

the linkage causes the three mobile portions of the platform to move in mutually orthogonal directions from each other. If small displacements are assumed, the relative motion away from each other is at the same speed. This results in uniform biaxial strain on the cell. It is worth mentioning that electrostatic actuation is not shown in the diagrams of Fig. 7. Electrostatic actuation in a liquid environment for biological applications has been developed using a differential actuation design [191]. The system is operated at high frequencies to compensate for the attenuation due to impedance loss in conductive media [192]. In sum, the MEMS-based stretching technique has not been reliably deployed to study cell–cell adhe- sion mainly because of the difficulty in aligning the cell doublets in Fig. 7. MEMS based single cell interrogation and stimulation systems. a. Uniaxial a spatial arrangement where the strain direction is perpendicular single cell stretching; b: Biaxial single cell stretching. (Reprinted with permission from Fig. 4 of [189].). to the cell–cell junction. In addition, the difficulty also lies in the compatibility of MEMS materials for cell attachment and growth. To investigate cell–cell junctions and meet the challenges men- tioned, we fabricated and integrated a microdevice capable of imaging modalities. To overcome such limitations, researchers re- parallel stretching and sensing of forces (Fig. 8). The device is sorted to the design flexibility offered by MEMS through creation of floating above a window through a silicon wafer and consists of specialized platforms for cell–cell adhesion studies. Parallel stim- folded beams (load sensor) and shuttles to support rafts on which ulation and measurement of forces were achieved by employing cell pairs are cultured. It is designed to apply tension to a pair of compliant mechanisms embodied in various configurations [189]. cells placed between the two shuttles. The stretching is achieved by means of an external manipulator and a metallic needle that 4.1. Moveable structures interfaces with the device through an aperture made on the right shuttle (Fig. 8a). Considering the large deformability of cells and In a moveable platform MEMS device, a cell is adhered to a the high load resolution needed to capture breaking of desmosome platform that is split into two or more parts. The cell is adhered to cell–cell adhesions, we use a set of folded beams over a length of the platform while the parts are together, and then the parts of the 500 µm possessing a stiffness of 12 pN/nm, which allows measure- platform are separated using an external actuator, e.g., piezoelec- ment of forces as low as 250 pN using digital image correlation tric actuator, and mechanical linkages. As the parts of the platform ∼ separate, the cell is stretched, and the degree to which the cell is (DIC). This force is equivalent to the strength of 3–6 cadherin deformed can be controlled by the separation distance between the bonds [193]. DIC enables correlation of features in a pair of images parts of the platform. Two variations of this technique have been using software analysis. A mathematical correlation algorithm is implemented, a uniaxial puller and a biaxial puller. applied to track the position of features (here we track features An example uniaxial puller consists of two platforms, one of included in the surface of the shuttles shown in Fig. 8b) from which is fixed while the other is moveable (Fig. 7a). The moveable one image to the next relative to an initial reference image. The platform is attached to an external piezoelectric actuator, which accuracy of this method is approximately ± 0.1 pixels (resolutions can control the displacement of the platform. In one study, a uni- of 20 nm have been achieved [194] when a 100X objective, 1.40 axial puller was used to study mechanical properties of hydrated N.A., is used). collagen fibrils [190]. An electrostatic comb drive actuator was To contain the live cells, a microfabricated parylene C raft employed to actuate one of the platforms, while the other was in the shape of a bowtie, which is patterned with extracellular held rigidly in place. The main advantages of using an electrostatic matrix (ECM) proteins, is placed on the device in a gap between comb-drive actuator include low power consumption using mod- the shuttles (Fig. 8c–e). The raft is fabricated by patterning the erate driving voltages, and high speed and accuracy. Also, use of deposited parylene C layer on top of a sacrificial layer, PNIPA. The an electrostatic comb drive actuator allowed for cyclic loading of patterned raft can be released by increasing the media temperature the cell. A biaxial puller was developed that used an electrostatic to above 37 degrees, which dissolves the PNIPA (Fig. 8f). Cells are comb-drive actuator and a cleverly designed kinematic linkage then plated over the microdevice platform, and a pair of cells is that allowed for controlled actuation of four segments of a platform placed across the two half rafts using micropipette aspiration. The at the same time [36](Fig. 7b). In this setup, one part of the stage cell pair is confined in position by the physical well in the parylene was fixed, while the other three were connected to a kinematic layer, which is approximately 8 µm in depth. The shuttles bear an linkage and an electrostatic actuator. When the actuator moves, opening that corresponds to the location of the cell pair on the 134 R. Yang et al. / Extreme Mechanics Letters 20 (2018) 125–139

Fig. 8. Parallel stimulation and interrogation of cell–cell adhesion with bioMEMS system. a. SEM image of the microfabricated device, composed of folded beams (load sensor), and shuttles to mount the rafts in which cells are cultured (b). c–e. The parylene C rafts are successfully released from the substrate by dissolving a sacrificial layer (PNIPA) in 37 ◦C water, manipulated using a micropipette and positioned on a device. f. Raft fabrication and release. Top view of the rafts with stamped ECM proteins. rafts such that the cells can be observed by an inverted fluores- change and the resulting tension within the sensor can be revealed cence microscope. Simultaneous imaging by confocal fluorescence qualitatively, possibly quantitatively with calibration [216], by microscopy would enable tracking of the evolution of intercellular monitoring the FRET ratio. Surprisingly, use of an E-cadherin ten- adhesions and cytoskeletal networks. sion sensor demonstrated that membrane-associated E-cadherin is under constitutive actomyosin generated tension, irrespective 4.2. 3D Nanofabrication of whether or not it is present in cell–cell contacts; however, E- cadherin tension is increased at cell–cell contacts when adhering Advances in nanofabrication techniques promise to enable cells are stretched [59]. Moreover, introducing an α-catenin FRET unique cell adhesion studies. For example, two-photon polymer- sensor into AJs revealed a rapid and reversible conformational ization (TPP) has enabled the fabrication of devices at nanoscale change when activated by mechanical strain. Further, it confirms and more importantly using biocompatible fabrication materi- that force dependent conformational change is followed by recruit- als [195]. A new class of micro-scaffold with nanometer scale ment of vinculin to its α-catenin binding site [215]. An α-actinin features has been developed for cell attachment and growth [196– FRET sensor was used to demonstrate that fluid shear stresses are 199], for force measurement from cell adhesion induced interac- transmitted to AJs through force redistribution within cytoskeletal tions [200], and for stimulation of FAs [201]. This method is still in binding proteins, and changes in cytoskeletal tension and reor- its early stages, but it is very promising because it offers biocom- ganization are upstream in the response of cells to flow [217]. patibility and the potential to probe cells within a 3D environment. Finally, other types of cell–cell junctions, including tight junctions, can affect the forces within AJs. Depletion of the tight junction 5. Sensing intra -cellular forces with FRET imaging component ZO1 in endothelial cells resulted in a redistribution of actomyosin and a decrease in AJ tension as assessed using a VE- To gain insights into the conformational changes of cadherin tension sensor [218]. mechanosensor molecules and to reveal the mechanisms of mechanosensing, genetically encoded tension sensors were 6. Outlook developed to introduce fluorophores with compatible emis- sion/absorption spectra [202–205]. FRET is based on the nonra- This review covers the most widely used techniques in stim- diative energy transfer between one fluorophore, the donor, and ulating and probing cell generated forces, focusing on cell–cell another fluorophore, the acceptor. The efficiency of this transfer adhesion studies. While effective, these techniques are mostly depends highly on the relative distance between the fluorophores. restricted to in vitro studies, and it is still rather difficult to probe The Förster distance, a characteristic length at which efficiency of physical interactions in vivo. These techniques generally require energy transfer equals 50%, is a few nanometers [206]. Thus, FRET specialized instrumentation and detailed calibration to realize de- works with a length scale that is comparable with conformation sirable signal-to-noise ratio and resolution. Thus, improvements changes of molecules within cells. Tremendous progress has been should be made to facilitate the adoption of these techniques in made in using FRET for the study of cell generated forces and routine laboratory protocols. Researchers need also to consider mechanosensing of external forces. There have been reports of a variety of issues in deciding which method to adopt. Besides FRET measurements for detecting force-activated RhoA [207,208], displacement resolution (micrometer or nanometer) and force res- Rac [209], Focal adhesion kinase (FAK) [210] and Src kinase [211]. olution (piconewton or nanonewton), the in vivo or in vitro obser- Cell–cell junction molecules, E-cadherin [59,111,212], VE- vation conditions, the dimensionality of measurements (2D or 3D), cadherin [213], PECAM-1 [90], as well as linker molecules α- the temporal resolution of the mechanotransduction pathways are catenin [214] have been reported as effective mechanosensors. all factors that influence inter- and intra-cellular processes. Specif- FRET sensors are most effective when used in parallel with other ically, for cell–cell adhesion studies, it is not yet possible to develop mechanical stimulation techniques, such as MTC [157,215]. 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