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Biological and Environmental Phenomena at the Interface
Mechanosensing at cellular interfaces. Ryan J Leiphart, Dongning Chen, Ana P Peredo, Abigail E Loneker, and Paul A Janmey Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02841 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018
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Mechanosensing at cellular interfaces. Ryan J. Leiphart1, Dongning Chen1, Ana P. Peredo1, Abigail E. Loneker1, Paul A. Janmey1,2* 1 Department of Bioengineering, University of Pennsylvania, 210 S 33rd St, Philadelphia, PA, USA 2 Institute for Medicine and Engineering, Department of Physiology, University of Pennsylvania, 3340 Smith Walk, Philadelphia, PA, USA Abstract At the plasma membrane interface, cells use various adhesions to sense their extracellular environment. These adhesions facilitate the transmission of mechanical signals that dictate cell behavior. This review discusses the mechanisms by which these mechanical signals are transduced through cell-matrix and cell-cell adhesions and how this mechanotransduction influences cell processes. Cell-matrix adhesions require the activation of and communication between various transmembrane protein complexes such as integrins. These links at the plasma membrane affect how a cell senses and responds to its matrix environment. Cells also communicate with each other through cellcell adhesions, which further regulate cell behavior on a single- and multi-cellular scale. Coordination and competition between cell-cell and call-matrix adhesions in multicellular aggregates can, to a significant extent, be modeled by differential adhesion analyses between the different interfaces even without knowing the details of cellular signaling. In addition, cell-matrix and cell-cell adhesions are connected by an intracellular cytoskeletal network that allows for direct communication between these distinct adhesions and activation of specific signaling pathways. Other membrane-embedded protein complexes, such as growth factor receptors and ion channels, play additional roles in mechanotransduction. Overall, these mechanoactive elements show the dynamic interplay between the cell, its matrix, and neighboring cells and how these relationships affect cellular function. Introduction Cells and the tissue they form are filled with a hierarchy of interfaces: from the interface between the cytosol, and nanoscale secretory vesicles or larger organelles like the nucleus, Golgi, and endoplasmic reticulum, to the interface between the plasma membrane and extracellular components. The cellular machines that drive motility, division, and differentiation are controlled by signals that originate at these interfaces. On a larger scale, cells assemble into structures like blood vessels, nerve fibers, muscles, and other tissues in which multicellular complexes are interfaced with the fibrous network of the extracellular matrix (ECM) and its associated interstitial polymers. Contacts between different interfaces are defined not only by adhesion energy due to a combination of binding between transmembrane receptors and weaker but more numerous bonds between surface-exposed molecules, but also by repulsion due to the excluded volume of macromolecules extending from the surface and the generally large negative surface charge of cell membranes and some components of extracellular matrices3-4. One of the functions of interfaces is to partition space to allow control of chemical composition, but as is increasingly recognized, these interfacial structures often have some properties of viscoelastic materials that can resist and transmit forces. The
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viscoelastic properties of lipid bilayers, chromatin, the cytoskeleton, and the ECM all create possibilities to generate physical stimuli. Transmission and transduction of forces can carry signals far from the surfaces at which they were generated5-6. The relationship between the force imposed on a tissue and its resultant deformation, often quantified as elastic modulus, is strictly regulated during development and normal physiological function 7. The response of cells to forces they experience within tissues and the resistance of their microenvironment is often as striking and molecularly well-defined as the cell's response to chemical stimuli. This review will summarize recent studies of the mechanobiology of cells in various configurations ranging from single cells in culture, to multicellular systems of cells in 2D and 3D environments. Aspects of cell biology in which the mechanical properties of cells and their extracellular environment are particularly involved include: • • • •
Mechanosensing through cell-matrix adhesions Mechanosensing through cell-cell adhesions Interplay between cell-cell and cell-matrix adhesions Mechanical effects on growth factor and transmembrane protein signaling
A recurring theme that emerges from studies of mechanotransduction at cell interfaces is that both the transmission of forces across the interface and the transduction of physical to biochemical signals require the formation of multiprotein complexes that span and adhere to the lipid bilayer of the cell membranes. The requirement for multiple proteins to assemble permits responses to physical stimuli to differ depending on which proteins are subjected to force, and so can provide as much temporal, spatial, and signaling specificity as occurs when receptors are activated by chemical ligands. Here we discuss some of the key factors involved in mechanotransductive effects and seek to find general patterns. Mechanosensing through cell-matrix adhesions Cells sense their mechanical environment through cell-cell and cell-matrix adhesions, as shown schematically in Figure 1. The stiffness of the extracellular matrix is a strong determinant of the morphology and motility of many cell types, but the specific response to different magnitudes of matrix elastic modulus is cell-type specific as well as dependent on the type of adhesion receptor by which the cell binds its substrate. Substrate stiffness can, for example, affect the growth and movement of cancer cells8, the differentiation of stem cells9, and the activation of pro-fibrotic functions in genetically normal cells10. Not all cells respond to stiffness in the same way. For example, some cancer cell types appear to have a diminished response to stiffness compared to normal cells from the same organ11, and neurons, in contrast to most other cell types, spread better and move toward soft substrates12-13. The cell’s adhesion to surrounding matrix is primarily, but not exclusively, mediated by integrins - transmembrane proteins with a large extracellular domain and variably sized intracellular domains that can form adhesion complexes. Integrins are composed of two heterodimeric subunits, α and β, each of have several variants, which can be paired in numerous combinations. Different combinations of these subunits bind specific extracellular matrix proteins, with for example α2β1 integrins primarily binding collagen while α5β1 and αV class integrins preferentially binding fibronectin (FN). Most cells express a subset of these integrin combinations, which can lead to differential responses
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Figure 1. Schematic of key mechanosensitive molecules and their interactions. Cellmatrix and cell-cell adhesions are coordinated molecular complexes. These complexes are interconnected via intracellular cytoskeletal networks. This interconnectivity allows for force transduction. This schematic highlights only a few key molecules involved in mechanotransduction. when culturing cells on different ECM components. On the intracellular side, the type of cytoskeletal network, usually actin but sometimes intermediate filaments, and the particular adapter proteins that link integrins to the cytoskeletal filaments depend on the type of activated integrin. Enzymes, including focal adhesion kinase (FAK), and GTPases are also recruited to the adhesion sites and activate downstream signaling. Due to the complex association of proteins at adhesion sites, integrin ligation can activate a number of different signaling pathways and has been implicated in a wide variety of cell behaviors, including cell migration, proliferation, survival, and differentiation 14. Much recent research has looked to more clearly elucidate the differential role of integrins in mechanosignaling. Distinct mechanisms of FAK mechanoactivation via α2 and α5 integrin ligation have been described1. Ligation of α2 integrin by collagen is stiffness-independent and is sufficient to activate FAK and increase nuclear translocation of YAP, a transcription regulator that is intimately involved in long-term cell responses to matrix mechanics 15. α5 integrin activation requires the tension-dependent unfolding of the extracellular matrix protein fibronectin (FN), which exposes a second integrin binding site. Exposure of this “synergy” site is needed to fully activate FAK signaling. Figure 2
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shows that when cells adhere to the substrate through fibronectin receptors like α5 integrins, a stiff substrate is required for maximal FAK activation. Inhibition of actin polymerization or myosin motor activity prevents full FAK activation on FN, indicating that intracellular force generation is needed for FN unfolding. In contrast, when cells bind to collagen-coated substrates by different adhesion proteins, FAK is maximally activated even when the substrate stiffness is very low. Cooperativity between FN-associated integrins has also been described, with the α5β1 integrins forming nascent adhesions and coexpression of αV integrins forming stable focal adhesions16. The β3 subunit of the αVβ3 integrin accumulates at areas of high traction force, whereas β1 integrins, are more mobile and uniformly distributed16. The retrograde transport of β1 integrin, but not β3, is required for persistent cell migration and adhesion17. Additionally, stabilization of the αVβ3 bond to FN through cell tension is required to adjust cell contractility to substrate stiffness16. These results indicate that mechanical activation by FN may be more readily mediated by αVβ3 integrins than by α5β1 integrins. Overexpression of αVβ6 integrin can induce monotonic increases in cell force generation with increasing substrate stiffness, further corroborating the importance of αV class integrins in the cellular response to mechanical signals18. Differences in the binding affinity and detachment speed of αVβ6 and α5β1 integrins have also been elucidated, and these differential integrin binding dynamics are sufficient to explain how integrins regulate force generation and integrin recruitment in response to substrate stiffness18. Ligation by the FN synergy site strengthens FN binding to α5β1 and αIIbβ3 integrins upon force application, but αV class integrins are not affected19. Additionally, in the absence of the synergy site, αV class integrins can compensate in response to increased stiffness 19. This indicates that while force generation regulates β1 integrins though FN unfolding, substrate stiffness is sufficient to directly alter cell adhesion through αV class integrins. Activation of the FN synergy site has also been shown to promote maturation of nascent adhesions to mature focal adhesions 16, 18.
Integrin-mediated adhesion and signaling relies on the recruitment of intracellular proteins to activated integrins14, and protein recruitment is altered by substrate stiffness and the resulting cell-generated tension. For example, a compact domain of talin, a protein critical for linking integrins to the cytoskeleton, is unfolded under tension. This unfolding allows talin to interact with another cytoskeletal binding protein, vinculin, and facilitates mechanotransduction20-21 through the recruitment of core focal adhesion
Figure 2. (A) FAK Activation as measured by a fluorescence energy transfer construct, in human fibrosarcoma (HT1080) cells. Pink staining indicates higher levels of FAK activation, while blue indicates low levels of FAK activation. (B) Quantification of FAK signal intensity. Reprinted with permission from Seong et al. 1. ACS Paragon Plus Environment
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proteins 22. Vinculin is a cytoskeletal scaffold protein that is localized to both focal adhesions and cell-cell adherens junctions. It directly interacts with both talin and actin and is required for responses to mechanical force such as changes in actin assembly, cell migration, cell spreading, and traction force generation23. Tension has also been shown to recruit zyxin, which further facilitates actin polymerization at focal adhesions24. Displacement of zyxin decreases force-induced actin polymerization. Recruitment of proteins to focal adhesions may also vary by integrin type. Vinculin and zyxin are preferentially recruited to α5β1 integrins over α2β1 integrins25. This is consistent with proteomic analysis of integrin-based adhesions that suggests α-subunit dependent protein recruitment in α4β1 integrins26. Other cytoplasmic proteins, such as α-actinin and filamin, can directly interfere with talin-mediated integrin activation 27. α-actinin has been shown to compete with talin binding to β3 integrins, while it cooperatively binds with talin in the activation of α5β1 integrins28. Cell-generated strain fields, patterning, and curvature on deformable interfaces. Attempts to model cell-matrix adhesions and the impact of substrate stiffness have had some success in describing experimental results. A very basic model in which a single cell is represented as a pre-strained elastic disk bound to an elastic substrate via molecular bonds suggests that cell traction forces increase with distance from the cell center. On soft substrates, force increases linearly with distance, but increases exponentially on stiff substrates29. On initially flat deformable substrates, forces generated by the cell are exerted both tangentially along the interface and orthogonally into or out of the surface, as illustrated in Figure 3. The length scales over which strain fields decay from the edge of the cell depend strongly on the material properties of the substrate and are especially long range for fibrous or strain-stiffening materials 30-32.
Figure 3. A. Schematic diagram of the strain fields produced tangential and orthogonal to the interface of a contractile cell and a linear elastic compliant substrate. Adapted with permission from He et al.29. B. isotropic spatial distribution of a collagen gel formed within a 200 x 200 µm mesh, and the reorganization of the network by a single fibroblast within it center (C). The length scale over which a single contractile cell can align a fibrous matrix like collagen matrix is on the order of a mm (D). Adapted with permission from Mohammadi et al 32.
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Maximum cell traction has been shown to have biphasic dependence on substrate stiffness33-34. A molecular clutch mechanism of integrin binding is able to model changes in focal adhesion formation in response to ligand spacing on substrates of varying stiffness, matching experimental results18, 35. This mechanism is modeled with resistive connections for integrin-matrix and integrin-integrin interactions. Integrin binding and dissociation are modeled with kon and koff rates. The model can be altered with the introduction of multiple kon and koff values to account for different integrin binding dynamics18. If the substrate is not perfectly elastic, the dissipative aspect of a viscoelastic substrate can lead to large changes in the rate at which cells spread and the area they maintain at steady state 36. The interplay between viscous and elastic moduli on the response of cells depends in a complex manner on the magnitude of the moduli and the kinetics of the bonds made from the cell to the substrate and from the molecular clutches to the cell's contractile machinery 37. In addition to effects of substrate stiffness, integrin activation and cell behavior can be modulated by other substrate surface features, including topography, orientation, and dimensionality (2D vs. 3D). Studies of cells on rigid glass surfaces have shown that integrin clustering and formation of focal adhesions are impaired on patterned substrates where integrin ligand is separated by more than a few tens of nanometers38. Further investigation of this phenomenon on polyacrylamide gels of differing stiffness showed that the optimal ligand spacing is a function of stiffness, which can be explained by force loading35. Conflicting conclusions have been reached on the centrality of surface topography to mechanosensing. While one study has suggested that surface porosity and accompanying protein tethering of polyacrylamide gels alters mechanosensing39, others have shown that alterations of protein tethering or porosity, independent of stiffness, fail to recapitulate the impact of altered elastic moduli9. Substrate curvature also has an impact on cellular morphology and differentiation. Modulation of surface topography can push mesenchymal stem cell (MSC) differentiation toward osteogenic or adipogenic lineages40. Compared to flat or concave surfaces, convex surfaces lead to nuclear deformation, slower migration, and promotion of osteogenic differentiation of MSCs 41. Osteoblast organization increases on microchannels of 10 or 250 m diameters compared to flat surfaces42. Gaussian curvature can also affect the organization of actin stress fibers and change the movement of cells. Notably, the angle between orientation of actin fibers and the direction of cell migration is different on surfaces of Gaussian curvature compared to flat substrates, on which cells migrate parallel to the direction at which they orient their
apical stress fibers 43. Most work in cellular mechanosensing has been done in 2D culture on hydrogels or other polymer matrices, where focal adhesions tend to form on stiffer environments. In 3D electrospun fibrous matrices, lower fiber stiffness enabled cell-mediated deformation and contraction of the fibers, increasing surrounding ligand density and therefore increasing focal adhesion formation44. This phenomenon can be quantitatively described with a multiscale model that balances stiffness with the ability of cells to break crosslinks and recruit fibers45.
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Mechanosensing through cell-cell adhesions In addition to matrix interactions, cells have multiple forms of attachment to neighboring cells. These attachments provide a link between the cytoskeletons of both cells, allowing for the transmission and sensing of forces between these cells. This mechanical communication is vital for the development and homeostasis of multicellular systems, including blood vessels 46, the heart 47, and dermal tissue 48. Mechanical cues between cells in these tissues help stratify cell layers and establish vital barriers. Most cell-cell adhesions are mediated by cadherins (Figure 1). Cadherins are Ca2+-dependent, transmembrane proteins that bind other cadherins on adjacent cells. Intracellularly, cadherins associate with β-catenin, α-catenin, and vinculin, among other proteins, to form adherens junctions (AJs). α-catenin serves as a link between cadherins and the actin cytoskeleton and is necessary for AJ formation49. The link to the actin cytoskeleton allows for force transduction across AJs via acto-myosin contractility. Vinculin is not required for AJ formation but is necessary for mechanosensitive stiffening of AJs. Vinculin is recruited to AJs under tension due to stretch-dependent exposure of a vinculin-binding domain on α-catenin50, demonstrating that vinculin is a mechanosensitive player in AJs. E-cadherin is a major regulator of AJ tension and function, but other cadherins are able to compensate for loss of E-cadherin. In epithelial monolayers that contain both E- and P-cadherin, the two cadherins play different roles. P-cadherin concentration predicts the magnitude of intercellular tension, while Ecadherin concentration predicts the rate at which intercellular tension increases51. However, if E-cadherin is knocked out, intercellular tension dynamics remain unchanged. This result indicates that P-cadherin is able to change its regulatory role in the absence of E-cadherin. Another important mechanotransductive aspect of AJs is their response to external stimuli. AJs dynamically remodel in response to the physical cues surrounding cells. Figure 4 shows that in response to flow-induced shear stress, epithelial AJs grow from punctate structures with radial actin fibers to expanded junctions with actin fibers parallel to the plasma membrane52. AJ remodeling under shear stress is preceded by a decrease and subsequent increase in cytoskeletal tension. This indicates that intracellular tension mediates shear stress-induced AJ remodeling. Cytoskeleton-driven AJ remodeling is also observed in endothelial cells. Fluid shear stress leads to decreased tension across VE-cadherin, while PECAM-1, another adhesive molecule, experiences increased tension that corresponds with vimentin association53. Tensiondependent changes in AJ properties are also observed at the molecular level, with cadherin-catenin-actin complexes shifting from a weak to a strong bound state when cadherin tension is increased54. These findings highlight the role of mechanical forces on cell-cell adhesion regulation and maintenance, from the molecular to the adhesive complex level. Forces sensed through AJs can directly impact cell processes such as proliferation and differentiation. E-cadherin mediates cell cycle entry and progression in response to cellular strain. After early induction of strain, YAP1 transiently localizes to the nucleus and drives cell cycle entry 55. β-catenin then localizes to the nucleus to progress the cell through S phase. These processes occur independently of each other, but cadherin-mediated adhesion is required to transit between these phases of YAP1 and β-catenin transcriptional activity. β-catenin is also involved in Wnt signaling, which
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plays an important role in organ development. E-cadherin regulates the distinct roles of β-catenin in adhesion complexes and Wnt signaling. It competitively binds β-catenin to prevent nuclear translocation and subsequent transcriptional activation56, with cadherin phosphorylation increasing its affinity for β-catenin57. While E-cadherin balances the roles of β-catenin in adhesion and Wnt signaling, cadherin activity is simultaneously being regulated. When the endocytosis-controlling factors Cdc42, Arp2/3, and dynamin are knocked out in Drosophila epithelial cells, AJs become elongated, leading to discontinuous junctions and destabilized adhesions58. This result demonstrates that AJs are constantly being recycled, a process that impacts the signaling properties of these adhesions.
Figure 4. Application of shear flow leads to AJ and cytoskeletal remodeling. Within 60 minutes of applied shear flow (arrow indicates flow direction), AJs remodel from punctate junctions with perpendicular actin stress fibers to elongated junctions with actin fibers parallel to the cell membrane. Staining for E-cadherin and α-actinin above shows the transition in the presence of shear flow from focal AJs to more “belt-like” adhesion structures along the plasma membrane. Reprinted with permission from Verma et al.48 Cell-cell adhesions have pronounced effects on multicellular system dynamics. In endothelial monolayers, cell-cell interactions create stress fields that span many cells. While these stress fields are heterogeneous on a macroscopic level, there is a high degree of alignment over distances of 10-20 cells6. Endothelial gaps tend to form at the edges of these oriented domains rather than at sites of high stresses. Changes in adhesion tension are also transmitted over long distances. Perturbing VE-cadherin adhesions in a single cell leads to aberrant AJ remodeling in cells distal to the stimulus59. The adhesions that link adjacent cells are therefore highly interconnected and can form functional units composed of multicellular aggregates. Force sensing across these multicellular systems impacts cell proliferation. For instance, increased forces in confluent epithelial monolayers can activate the membrane-cytoskeletal linker merlin60. Merlin immobilizes epidermal growth factor (GF) receptor, a transducer of cell proliferation, which inhibits proliferation. Epithelial monolayers can also become immobilized. Upon reaching confluence, epithelial sheets no longer migrate but can be reactivated via RAB5A-induced endocytosis, which activates cadherin recycling and promotes directed cell migration61. Cadherin-mediated cell-cell adhesions can directly affect monolayer dynamics and are therefore highly controlled through regulatory events, such as cadherin recycling.
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Other cell-cell adhesions, such as tight junctions (TJs), regulate cell adhesion and mechanotransduction, yet their exact role in tension regulation remains controversial. Knockout of crucial TJ proteins ZO-1 and GEF-H1 disrupts epithelial cytokinesis62. This effect on cell division is attributed to increased AJ tension, suggesting that tight junctions negatively regulate AJ tension. However, other studies have shown that TJs can positively regulate AJ tension. Knockout of ZO-1 hinders endothelial barrier formation and cell migration by reducing tension on VE-cadherin63. In this context, TJs positively regulate AJ tension through myosin IIA, as myosin was shown to be redistributed from junctional belts to stress fibers upon ZO-1 knockout. These contradicting effects of TJs on AJ tension may be due to functional differences between epithelial and endothelial cells, but the discrepancy is not fully understood. AJs also regulate TJ formation and tension. In stratified epithelia for example, E-cadherin is required for proper formation of polarized TJs64. Therefore TJs and AJs appear to have bidirectional forms of communication. While the exact mechanisms through which different cell-cell structures regulate each other remain unknown, there is abundant evidence implicating cell-cell adhesions as force-sensing signaling hubs that can regulate multiple cell processes and maintain tissue homeostasis and organization.
Interplay between cell-cell and cell-matrix adhesions Both cell-cell and cell-matrix adhesions can separately impact differentiation, proliferation, and migration, among other cell events. Their composition and modes of operation can be very distinct 65-66, yet both adhesion types are interconnected and their crosstalk through common molecules and signaling pathways affects downstream adhesion dynamics and signaling transduction. For this reason, cell-cell and cell-matrix adhesion interplay should be considered when investigating cellular adhesions and their mechanotransductive activity. Even without knowing the molecular details of the signals emanating from transmembrane adhesion receptors, the response of multicellular aggregates as they encounter an adhesive substrate can to a large extent be modeled by consideration of the relative adhesion energies between cell-cell and cell-substrate interfaces. The concept of differential adhesion between two different cell types with different affinities for each other has been highly successful in explaining how initially mixed populations of different cell types separate into different phases. By analogy with the demixing of different liquids with different surface tension the classic differential adhesion hypothesis developed by Steinberg and coworkers in the 1960s revealed the principle by which an aggregate of cells with a lower surface tension tends to separate from and surround an aggregate of cells with higher surface tension to which it adheres 67 . Such concepts have been extended to analyze how cell aggregates spread on surfaces of different adhesion energies 68. In addition to determining adhesion energies at the cell membrane, integrins and cadherins can trigger intracellular changes in response to sensed forces. These modifications are initiated by the activation of signaling pathways that are closely integrated and that at times overlap 69. Integrins and cadherins share multiple common downstream effectors such as non-receptor tyrosine kinases, scaffolding components, and small GTPases. They jointly alter cell physiology via synergistic, reciprocal, or intrinsically robust mechanisms. Other modes of crosstalk occur through the formation of macromolecular complexes that bring cadherins and integrins together, which allows for
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their interaction and collaboration with GF receptors and transmembrane proteins 70. Therefore, the changes at cell-cell adhesions can greatly impact mechanosensation, expression, and activity of cell-matrix adhesions and vice versa through their interdependence and intrinsic physical connections. A striking example of the interplay between cell-cell and cell-matrix adhesions is observed when cell spheroids, collections of hundreds of cells grown in liquid culture without an ECM, are deposited on surfaces with specific integrin ligands. Figure 5 shows two such spheroids, each composed of aggregated cells with differential Ecadherin expression, that were placed on fibronectin-coated surfaces2. When E-cadherin expression is high (top panel), the spheroid spreads as a coherent wave. In contrast, when E-cadherin expression is lowered (bottom panel) and cell-cell contact is reduced, the spreading is faster and cells emerge as single units. The degree to which cells spread also depends on the elastic moduli of the fibronectin-coated surfaces, with spreading of the spheroids increasing as substrate stiffness increased from
