Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

Tensional homeostasis in dermal fibroblasts: Mechanical responses to mechanical loading in three-dimensional substrates

Many soft connective tissues are under endogenous tension, and their resident cells generate considerable contractile forces on the extracellular matrix. The present work was aimed to determine quantitatively how fibroblasts, grown within three-dimensional collagen lattices, respond mechanically to precisely defined tensional loads. Forces generated in response to changes in applied load were measured using a tensional culture force monitor. In a number of variant systems, resident cells consistently reacted to modify the endogenous matrix tension in the opposite direction to externally applied loads. That is, increased external loading was followed immediately by a reduction in cell-mediated contraction whilst decreased external loading elicited increased contraction. Responses were cell-mediated and not a result of material properties of the matrices. This is the first detailed characterisation of a tensional homeostatic response in cells. The maintained force, after 8 h in culture, was typically around 40–60 dynes/million cells. Maintenance of an active tensional homeostasis has widespread implications for cells in culture and forwhole tissue function. J. Cell. Physiol. 175:323–332, 1998. © 1998 Wiley-Liss, Inc.     

Eigenstrain as a mechanical set-point of cells

Cell contraction regulates how cells sense their mechanical environment. We sought to identify the set-point of cell contraction, also referred to as tensional homeostasis. In this work, bovine aortic endothelial cells (BAECs), cultured on substrates with different stiffness, were characterized using traction force microscopy (TFM). Numerical models were developed to provide insights into the mechanics of cell–substrate interactions. Cell contraction was modeled as eigenstrain which could induce isometric cell contraction without external forces. The predicted traction stresses matched well with TFM measurements. Furthermore, our numerical model provided cell stress and displacement maps for inspecting the fundamental regulating mechanism of cell mechanosensing. We showed that cell spread area, traction force on a substrate, as well as the average stress of a cell were increased in response to a stiffer substrate. However, the cell average strain, which is cell type-specific, was kept at the same level regardless of the substrate stiffness. This indicated that the cell average strain is the tensional homeostasis that each type of cell tries to maintain. Furthermore, cell contraction in terms of eigenstrain was found to be the same for both BAECs and fibroblast cells in different mechanical environments. This implied a potential mechanical set-point across different cell types. Our results suggest that additional measurements of contractility might be useful for monitoring cell mechanosensing as well as dynamic remodeling of the extracellular matrix (ECM). This work could help to advance the understanding of the cell-ECM relationship, leading to better regenerative strategies.     

The contractile properties and responses to tensional loading of Dupuytren's disease--derived fibroblasts are altered: a cause of the contracture?

Dupuytren's disease causes disability because of the development of finger flexion deformities, with distinct nodule and cord formation. This results in physical shortening of the diseased fascial tissue through a combination of cell-mediated contraction and matrix remodeling. It is this fixed tissue fabric shortening that prevents finger extension. In this experimental study, the relative contractile properties of Dupuytren nodule- and cord-derived fibroblasts were quantified in a culture force monitor model, in comparison with normal carpal ligament fibroblasts. Nine nodule, 10 cord, and four carpal ligament fibroblast cell lines were studied; each cell line was derived from a separate patient. The contractile forces generated by nodule and cord fibroblasts were significantly greater than the force generated by carpal ligament fibroblasts. There were also significant differences between nodule- and cord-derived fibroblasts, with the nodule cells demonstrating the greatest contractile force generation. The contraction profiles of both cord and nodule Dupuytren fibroblasts demonstrated delays in the attainment of tensional homeostasis, with an absence of a plateau phase by 20 hours. After the contraction phase, cell-seeded constructs were subjected to a series of four uniaxial mechanical overloads and cellular responses were monitored during each subsequent 30-minute period. Dupuytren nodule and cord fibroblast responses were significantly altered, compared with carpal ligament fibroblasts, exhibiting an increased and opposite response. Dupuytren fibroblasts, particularly nodule fibroblasts, exhibited increased force generation and a delay in reaching tensional homeostasis. The data suggest that these cells have an inherently higher basal tension and contractile ability. This results in increased shortening of the matrix, and the delay in reaching tensional homeostasis might exacerbate this response. These results represent a theoretical framework regarding the fundamental processes involved in the pathogenesis and progression of clinical flexion deformities in Dupuytren disease.     

Model-based Traction Force Microscopy Reveals Differential Tension in Cellular Actin Bundles

Adherent cells use forces at the cell-substrate interface to sense and respond to the physical properties of their environment. These cell forces can be measured with traction force microscopy which inverts the equations of elasticity theory to calculate them from the deformations of soft polymer substrates. We introduce a new type of traction force microscopy that in contrast to traditional methods uses additional image data for cytoskeleton and adhesion structures and a biophysical model to improve the robustness of the inverse procedure and abolishes the need for regularization. We use this method to demonstrate that ventral stress fibers of U2OS-cells are typically under higher mechanical tension than dorsal stress fibers or transverse arcs.     

Contribution of tree structures in the lung to lung elastic recoil

The direct contribution of forces in tree structures in the lung to lung recoil pressure and changes in recoil pressure induced by alterations of the forces are analyzed. The analysis distinguishes the contributions of axial and circumferential tensions in the trees and indicates that only axial tensions directly contribute to static recoil. This contribution is derived from analysis of the axial forces transmitted across a random plane transecting the lung. The change in recoil pressure induced by changes in axial tension is similarly derived. Alterations of circumferential tensions in the trees indirectly change recoil by causing nonuniform deformations of the surrounding lung parenchyma, and a continuum elasticity solution for the stress induced by the deformations is derived. Sample calculations are presented for the airway tree based on available data on airway morphometric and mechanical properties. The increase in recoil pressure accompanying increases in axial and circumferential tensions with contraction of airway smooth muscle is also analyzed. The calculations indicate that axial stresses in the airway tree out to bronchioles directly contribute only a small fraction of the static recoil pressure. However, it is found that contraction of smooth muscle in these airways can increase recoil pressure appreciably (10-20%), mainly by the deformation of the parenchyma with increases in circumferential tension in smaller airways. The results indicate that the geometric and mechanical properties of the airway tree are such that only peripheral elements of the tree can substantially affect the elastic properties of the lung. The possible contributions of vascular trees for which data on mechanical and morphometric properties are more limited are also discussed.     

An array of microfabricated pillars to study cell migration

Mechanical forces play an important role in various cellular functions, such as tumor metastasis, embryonic development or tissue formation. Cell migration involves dynamics of adhesive processes and cytoskeleton remodelling, leading to traction forces between the cells and their surrounding extracellular medium. To study these mechanical forces, a number of methods have been developed to calculate tractions at the interface between the cell and the substrate by tracking the displacements of beads or microfabricated markers embedded in continuous deformable gels. These studies have provided the first reliable estimation of the traction forces under individual migrating cells. We have developed a new force sensor made of a dense array of soft micron-size pillars microfabricated using microelectronics techniques. This approach uses elastomeric substrates that are micropatterned by using a combination of hard and soft lithography. Traction forces are determined in real time by analyzing the deflections of each micropillar with an optical microscope. Indeed, the deflection is directly proportional to the force in the linear regime of small deformations. Epithelial cells are cultured on our substrates coated with extracellular matrix protein. First, we have characterized temporal and spatial distributions of traction forces of a cellular assembly. Forces are found to depend on their relative position in the monolayer : the strongest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. Consequently, these forces are quantified and correlated with the adhesion/scattering processes of the cells.     

Relative microelastic mapping of living cells by atomic force microscopy.

The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample.     

Direct detection of cellular adaptation to local cyclic stretching at the single cell level by atomic force microscopy

The cellular response to external mechanical forces has important effects on numerous biological phenomena. The sequences of molecular events that underlie the observed changes in cellular properties have yet to be elucidated in detail. Here we have detected the responses of a cultured cell against locally applied cyclic stretching and compressive forces, after creating an artificial focal adhesion under a glass bead attached to the cantilever of an atomic force microscope. The cell tension initially increased in response to the tensile stress and then decreased within ∼1 min as a result of viscoelastic properties of the cell. This relaxation was followed by a gradual increase in tension extending over several minutes. The slow recovery of tension ceased after several cycles of force application. This tension-recovering activity was inhibited when cells were treated with cytochalasin D, an inhibitor of actin polymerization, or with (−)-blebbistatin, an inhibitor of myosin II ATPase activity, suggesting that the activity was driven by actin-myosin interaction. To our knowledge, this is the first quantitative analysis of cellular mechanical properties during the process of adaptation to locally applied cyclic external force.     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

Mechanics of spreading cells probed by atomic force microscopy

Cellular adhesion and motility are fundamental processes in biological systems such as morphogenesis and tissue homeostasis. During these processes, cells heavily rely on the ability to deform and supply plasma membrane from pre-existing membrane reservoirs, allowing the cell to cope with substantial morphological changes. While morphological changes during single cell adhesion and spreading are well characterized, the accompanying alterations in cellular mechanics are scarcely addressed. Using the atomic force microscope, we measured changes in cortical and plasma membrane mechanics during the transition from early adhesion to a fully spread cell. During the initial adhesion step, we found that tremendous changes occur in cortical and membrane tension as well as in membrane area. Monitoring the spreading progress by means of force measurements over 2.5 h reveals that cortical and membrane tension become constant at the expense of excess membrane area. This was confirmed by fluorescence microscopy, which shows a rougher plasma membrane of cells in suspension compared with spread ones, allowing the cell to draw excess membrane from reservoirs such as invaginations or protrusions while attaching to the substrate and forming a first contact zone. Concretely, we found that cell spreading is initiated by a transient drop in tension, which is compensated by a decrease in excess area. Finally, all mechanical parameters become almost constant although morphological changes continue. Our study shows how a single cell responds to alterations in membrane tension by adjusting its overall membrane area. Interference with cytoskeletal integrity, membrane tension and excess surface area by administration of corresponding small molecular inhibitors leads to perturbations of the spreading process.     

Cell contraction caused by microtubule disruption is accompanied by shape changes and an increased elasticity measured by scanning acoustic microscopy

The state of crosslinking of microfilaments and the state of myosin-driven contraction are the main determinants of the mechanical properties of the cell cortex underneath the membrane, which is significant for the mechanism of shaping cells. Therefore, any change in the contractile state of the actomyosin network would alter the mechanical properties and finally result in shape changes. The relationship of microtubules to the mechanical properties of cells is still obscure. The main problem arises because disruption of microtubules enhances acto-myosin-driven contraction. This reaction and its impact on cell shape and elasticity have been investigated in single XTH-2 cells. Microtubule disruption was induced by colcemid, a polymerization inhibitor. The reaction was biphasic: a change in cell shape from a fried egg shape to a convex surface topography was accompanied by an increase in elastic stiffness of the cytoplasm, measured as longitudinal sound velocity revealed by scanning acoustic microscope. Elasticity increases in the cell periphery and reaches its peak after 30 min. Subsequently while the cytoplasm retracts from the periphery, longitudinal sound velocity (elasticity) decreases. Simultaneously, a two- to threefold increase of F-actin and alignment of stress fibers from the cell center to cell-cell junctions in dense cultures are induced, supposedly a consequence of the increased tension.     

A Highlights from MBoC Selection: Myosin II Is Essential for the Spatiotemporal Organization of Traction Forces during Cell Motility

Amoeboid motility requires spatiotemporal coordination of biochemical pathways regulating force generation and consists of the quasi-periodic repetition of a motility cycle driven by actin polymerization and actomyosin contraction. Using new analytical tools and statistical methods, we provide, for the first time, a statistically significant quantification of the spatial distribution of the traction forces generated at each phase of the cycle (protrusion, contraction, retraction, and relaxation). We show that cells are constantly under tensional stress and that wild-type cells develop two opposing “pole” forces pulling the front and back toward the center whose strength is modulated up and down periodically in each cycle. We demonstrate that nonmuscular myosin II complex (MyoII) cross-linking and motor functions have different roles in controlling the spatiotemporal distribution of traction forces, the changes in cell shape, and the duration of all the phases. We show that the time required to complete each phase is dramatically increased in cells with altered MyoII motor function, demonstrating that it is required not only for contraction but also for protrusion. Concomitant loss of MyoII actin cross-linking leads to a force redistribution throughout the cell perimeter pulling inward toward the center. However, it does not reduce significantly the magnitude of the traction forces, uncovering a non–MyoII-mediated mechanism for the contractility of the cell.     

Myosin II and mechanotransduction: a balancing act

Adherent cells respond to mechanical properties of the surrounding extracellular matrix. Mechanical forces, sensed at specialized cell-matrix adhesion sites, promote actomyosin-based contraction within the cell. By manipulating matrix rigidity and adhesion strength, new roles for actomyosin contractility in the regulation of basic cellular functions, including cell proliferation, migration and stem cell differentiation, have recently been discovered. These investigations demonstrate that a balance of forces between cell adhesion on the outside and myosin II-based contractility on the inside of the cell controls many aspects of cell behavior. Disturbing this balance contributes to the pathogenesis of various human diseases. Therefore, elaborate signaling networks have evolved that modulate myosin II activity to maintain tensional homeostasis. These include signaling pathways that regulate myosin light chain phosphorylation as well as myosin II heavy chain interactions.     

Protrusive and Contractile Forces of Spreading Human Neutrophils

Human neutrophils are mediators of innate immunity and undergo dramatic shape changes at all stages of their functional life cycle. In this work, we quantified the forces associated with a neutrophil’s morphological transition from a nonadherent, quiescent sphere to its adherent and spread state. We did this by tracking, with high spatial and temporal resolution, the cell’s mechanical behavior during spreading on microfabricated post-array detectors printed with the extracellular matrix protein fibronectin. Two dominant mechanical regimes were observed: transient protrusion and steady-state contraction. During spreading, a wave of protrusive force (75 ± 8 pN/post) propagates radially outward from the cell center at a speed of 206 ± 28 nm/s. Once completed, the cells enter a sustained contractile state. Although post engagement during contraction was continuously varying, posts within the core of the contact zone were less contractile (−20 ± 10 pN/post) than those residing at the geometric perimeter (−106 ± 10 pN/post). The magnitude of the protrusive force was found to be unchanged in response to cytoskeletal inhibitors of lamellipodium formation and myosin II-mediated contractility. However, cytochalasin B, known to reduce cortical tension in neutrophils, slowed spreading velocity (61 ± 37 nm/s) without significantly reducing protrusive force. Relaxation of the actin cortical shell was a prerequisite for spreading on post arrays as demonstrated by stiffening in response to jasplakinolide and the abrogation of spreading. ROCK and myosin II inhibition reduced long-term contractility. Function blocking antibody studies revealed haptokinetic spreading was induced by β₂ integrin ligation. Neutrophils were found to moderately invaginate the post arrays to a depth of ∼1 μm as measured from spinning disk confocal microscopy. Our work suggests a competition of adhesion energy, cortical tension, and the relaxation of cortical tension is at play at the onset of neutrophil spreading.     

Mechanical forces alter zyxin unbinding kinetics within focal adhesions of living cells

The formation of focal adhesions that mediate alterations of cell shape and movement is controlled by a mechanochemical mechanism in which cytoskeletal tensional forces drive changes in molecular assembly; however, little is known about the molecular biophysical basis of this response. Here, we describe a method to measure the unbinding rate constant k(OFF) of individual GFP-labeled focal adhesion molecules in living cells by modifying the fluorescence recovery after photobleaching (FRAP) technique and combining it with mathematical modeling. Using this method, we show that decreasing cellular traction forces on focal adhesions by three different techniques--chemical inhibition of cytoskeletal tension generation, laser incision of an associated actin stress fiber, or use of compliant extracellular matrices--increases the k(OFF) of the focal adhesion protein zyxin. In contrast, the k(OFF) of another adhesion protein, vinculin, remains unchanged after tension dissipation. Mathematical models also demonstrate that these force-dependent increases in zyxin's k(OFF) that occur over seconds are sufficient to quantitatively predict large-scale focal adhesion disassembly that occurs physiologically over many minutes. These findings demonstrate that the molecular binding kinetics of some, but not all, focal adhesion proteins are sensitive to mechanical force, and suggest that force-dependent changes in this biophysical parameter may govern the supramolecular events that underlie focal adhesion remodeling in living cells.     

Mechanosensitive Adaptation of E-Cadherin Turnover across adherens Junctions

In the natural and technological world, multi-agent systems strongly depend on how the interactions are ruled between their individual components, and the proper control of time-scales and synchronization is a key issue. This certainly applies to living tissues when multicellular assemblies such as epithelial cells achieve complex morphogenetic processes. In epithelia, because cells are known to individually generate actomyosin contractile stress, each individual intercellular adhesive junction line is subjected to the opposed stresses independently generated by its two partner cells. Contact lines should thus move unless their two partner cells mechanically match. The geometric homeostasis of mature epithelia observed at short enough time-scale thus raises the problem to understand how cells, if considered as noisy individual actuators, do adapt across individual intercellular contacts to locally balance their time-average contractile stress. Structural components of adherens junctions, cytoskeleton (F-actin) and homophilic bonds (E-cadherin) are quickly renewed at steady-state. These turnovers, if they depend on forces exerted at contacts, may play a key role in the mechanical adaptation of epithelia. Here we focus on E-cadherin as a force transducer, and we study the local regulation and the mechanosensitivity of its turnover in junctions. We show that E-cadherin turnover rates match remarkably well on either side of mature intercellular contacts, despite the fact that they exhibit large fluctuations in time and variations from one junction to another. Using local mechanical and biochemical perturbations, we find faster turnover rates with increased tension, and asymmetric rates at unbalanced junctions. Together, the observations that E-cadherin turnover, and its local symmetry or asymmetry at each side of the junction, are mechanosensitive, support the hypothesis that E-cadherin turnover could be involved in mechanical homeostasis of epithelia.     

Elastic Anisotropy Governs the Range of Cell-Induced Displacements

The unique nonlinear mechanics of the fibrous extracellular matrix (ECM) facilitates long-range cell-cell mechanical communications that would be impossible for linear elastic substrates. Past research has described the contribution of two separated effects on the range of force transmission, including ECM elastic nonlinearity and fiber alignment. However, the relation between these different effects is unclear, and how they combine to dictate force transmission range is still elusive. Here, we combine discrete fiber simulations with continuum modeling to study the decay of displacements induced by a contractile cell in fibrous networks. We demonstrate that fiber nonlinearity and fiber reorientation both contribute to the strain-induced elastic anisotropy of the cell’s local environment. This elastic anisotropy is a “lumped” parameter that governs the slow decay of displacements, and it depends on the magnitude of applied strain, either an external tension or an internal contraction, as a model of the cell. Furthermore, we show that accounting for artificially prescribed elastic anisotropy dictates the decay of displacements induced by a contracting cell. Our findings unify previous single effects into a mechanical theory that explains force transmission in fibrous networks. This work may provide insights into biological processes that involve communication of distant cells mediated by the ECM, such as those occurring in morphogenesis, wound healing, angiogenesis, and cancer metastasis. It may also provide design parameters for biomaterials to control force transmission between cells as a way to guide morphogenesis in tissue engineering.     

Contractile equilibration of single cells to step changes in extracellular stiffness

Extracellular stiffness has been shown to alter long timescale cell behaviors such as growth and differentiation, but the cellular response to changes in stiffness on short timescales is poorly understood. By studying the contractile response of cells to dynamic stiffness conditions using an atomic force microscope, we observe a seconds-timescale response to a step change in extracellular stiffness. Specifically, we observe acceleration in contraction velocity (μm/min) and force rate (nN/min) upon a step decrease in stiffness and deceleration upon a step increase in stiffness. Interestingly, this seconds-timescale response to a change in extracellular stiffness is not altered by inhibiting focal adhesion signaling or stretch-activated ion channels and is independent of cell height and contraction force. Rather, the response timescale is altered only by disrupting cytoskeletal mechanics and is well described by a simple mechanical model of a constant velocity actuator pulling against an internal cellular viscoelastic network. Consistent with the predictions of this model, we find that an osmotically expanding hydrogel responds to step changes in extracellular stiffness in a similar manner to cells. We therefore propose that an initial event in stiffness sensing is establishment of a mechanical equilibrium that balances contraction of the viscoelastic cytoskeleton with deformation of the extracellular matrix.     

Nano-mechanical exploration of the surface and sub-surface of hydrated cells of Staphylococcus epidermidis

The surface of hydrated cells of Staphylococcus epidermidis has been probed using an atomic force microscope. While local force measurements over the surface of bacteria reveal a heterogeneous chemical surface, with heterogeneous mechanical properties, different kinds of force curves appear with high frequency, and are thought to provide information on features contributing strongly to the overall mechanical and surface behaviour of the cell. Force curves often present two different mechanical regimes, being the first one (outer) of about 48 nm thick, and presenting a local relative elasticity of about 0.08 N/m, which is about a third of the relative elasticity of the inner part of the cell wall, harder, with a relative elasticity of about 0.24 N/m, in water. Both regimes appears as straight lines in the force versus distance curves (the ‘corresponding’ stress–strain curves in contact mechanics), but hysteresis is observed between the approach and the retraction line in the inner regime, indicating a degree of viscoelasticity. No viscoelasticity is observed in the outer regime, however, which presents quite linear and juxtaposed approach-retraction lines. These kinds of force curves do not present measurable pull-off forces nor snap-in forces, which indicates an almost null interaction between tip and bacterial surface, which could be in agreement with the measured very high hydrophobicity of this strain. Another kind of force curve has been observed recurrently, showing peaks in the retraction curves. Adhesive pull-off forces were measured giving an average of about 2 nN. Interestingly, however, these force curves appear only when quite irregular and wavy retraction curves are present, from the very beginning of its trace (maximum indentation). This leads us to think that these pull-off forces measured by our AFM do not give information on surface forces-unbinding events at the surface of the bacteria, but could be related to events at the sub-surface of the cell surface. Oscillations seen in the retraction curve in the portion corresponding to the contact with the bacteria surface could be due to rupture phenomena within the multilayered cell wall architecture expected in Gram-positive bacteria as Staphylococcus epidermidis, which could result in local irreversible deformations of the cell surface. Imaging with a sharp tip in contact mode sometimes leads to surface damage. Force curves recorded over damaged parts of the cell surface showed a completely different behaviour, in many cases with two well-defined high-adhesion peaks, and also interestingly, with snap-in forces of about 0–2 nN, which seems to indicate a completely different electrical/hydrophobicity state only a few nanometers down from the surface. Similar indentation effects can occur in the contact of a bacterial cell with a solid surface, even when showing only atomic-molecular-scale roughness, thus interacting not only with the very surface of the cell, especially when soft layers are present in the outer. Our results highlight the importance of the cell surface mechanical properties and their interplay with purely surface properties when analyzing cell–material interaction, and show the AFM as a useful method for investigating this.