Microsurgery-aided in-situ force probing reveals extensibility and viscoelastic properties of individual stress fibers.

Actin-myosin filament bundles (stress fibers) are critical for tension generation and cell shape, but their mechanical properties are difficult to access. Here we propose a novel approach to probe individual peripheral stress fibers in living cells through a microsurgically generated opening in the cytoplasm. By applying large deformations with a soft cantilever we were able to fully characterize the mechanical response of the fibers and evaluate their tension, extensibility, elastic and viscous properties.

Cell shape dynamics reveal balance of elasticity and contractility in peripheral arcs.

The mechanical interaction between adherent cells and their substrate relies on the formation of adhesion sites and on the stabilization of contractile acto-myosin bundles, or stress fibers. The shape of the cell and the orientation of these fibers can be controlled by adhesive patterning. On nonadhesive gaps, fibroblasts develop thick peripheral stress fibers, with a concave curvature. The radius of curvature of these arcs results from the balance of the line tension in the arc and of the surface tension in the cell bulk. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, is not clear. To get insight into the force balance, we inhibit myosin activity and simultaneously monitor the dynamics of peripheral arc radii and traction forces. We use these measurements to estimate line and surface tension. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that both line tension and surface tension dropped, but the line tension decreased to a lesser extent than surface tension. These results suggest that myosin-independent force contributes to tension in the peripheral arcs. We propose a simple physical model in which the peripheral arc line tension is due to the combination of myosin II contractility and a passive elastic component, while surface tension is largely due to active contractility. Numerical solutions of this model reproduce well the experimental data and allow estimation of the contributions of elasticity and contractility to the arc line tension.

Ultra-soft cantilevers and 3-D micro-patterned substrates for contractile bundle tension measurement in living cells.

Actin-myosin microfilament bundles or stress-fibers are the principal tension-generating structures in the cell. Their mechanical properties are critical for cell shape, motion, and interaction with other cells and extracellular matrix, but were so far difficult to access in a living cell. Here we propose a micro-fabricated two-component setup for direct tension measurement on a peripheral bundle within an intact cell. We used 3-D substrates made of silicon elastomer to elevate the cell making the filament bundle at its border accessible from the side, and employed an ultra-soft (spring constant 0.78 nN µm(-1)) epoxy-based cantilever for mechanical probing. With this setup we were able for the first time to measure the tension in peripheral actin bundles in living primary fibroblasts spread on a rigid substrate.

Contact angle at the leading edge controls cell protrusion rate.

Plasma membrane tension and the pressure generated by actin polymerization are two antagonistic forces believed to define the protrusion rate at the leading edge of migrating cells [1-5]. Quantitatively, resistance to actin protrusion is a product of membrane tension and mean local curvature (Laplace's law); thus, it depends on the local geometry of the membrane interface. However, the role of the geometry of the leading edge in protrusion control has not been yet investigated. Here, we manipulate both the cell shape and substrate topography in the model system of persistently migrating fish epidermal keratocytes. We find that the protrusion rate does not correlate with membrane tension, but, instead, strongly correlates with cell roundness, and that the leading edge of the cell exhibits pinning on substrate ridges-a phenomenon characteristic of spreading of liquid drops. These results indicate that the leading edge could be considered a triple interface between the substrate, membrane, and extracellular medium and that the contact angle between the membrane and the substrate determines the load on actin polymerization and, therefore, the protrusion rate. Our findings thus illuminate a novel relationship between the 3D shape of the cell and its dynamics, which may have implications for cell migration in 3D environments.

The nano-scale mechanical properties of the extracellular matrix regulate dermal fibroblast function.

Changes in the mechanical properties of dermis occur during skin aging or tissue remodeling and affect the activity of resident fibroblasts. With the aim to establish elastic culture substrates that reproduce the variable softness of dermis, we determined Young's elastic modulus E of human dermis at the cell perception level using atomic force microscopy. The E of dermis ranged from 0.1 to 10 kPa, varied depending on body area and dermal layer, and tended to increase with age in 26-55-year-old donors. The activation state of human dermal fibroblasts cultured on "skin-soft" E (5 kPa) silicone culture substrates was compared with stiff plastic culture (GPa), collagen gel cultures (0.1-9 kPa), and fresh human dermal tissue. Fibroblasts cultured on skin-soft silicones displayed low mRNA levels of fibrosis-associated genes and increased expression of the matrix metalloproteinases (MMPs) MMP-1 and MMP-3 as compared with collagen gel and plastic cultures. The activation profile exhibited by fibroblasts on "skin-soft" silicone culture substrates was most comparable with that of human dermis than any other tested culture condition. Hence, providing biomimetic mechanical conditions generates fibroblasts that are more suitable to investigate physiologically relevant cell processes than fibroblasts spontaneously activated by stiff conventional culture surfaces.

Ultrafast Ca2+ wave in cultured vascular smooth muscle cells aligned on a micropatterned surface.

Communication between vascular smooth muscle cells (SMCs) allows control of their contraction and so regulation of blood flow. The contractile state of SMCs is regulated by cytosolic Ca2+ concentration ([Ca2+]i) which propagates as Ca2+ waves over a significant distance along the vessel. We have characterized an intercellular ultrafast Ca2+ wave observed in cultured A7r5 cell line and in primary cultured SMCs (pSMCs) from rat mesenteric arteries. This wave, induced by local mechanical or local KCl stimulation, had a velocity around 15 mm/s. Combining of precise alignment of cells with fast Ca2+ imaging and intracellular membrane potential recording, allowed us to analyze rapid [Ca2+]i dynamics and membrane potential events along the network of cells. The rate of [Ca2+]i increase along the network decreased with distance from the stimulation site. Gap junctions or voltage-operated Ca2+ channels (VOCCs) inhibition suppressed the ultrafast Ca2+ wave. Mechanical stimulation induced a membrane depolarization that propagated and that decayed exponentially with distance. Our results demonstrate that an electrotonic spread of membrane depolarization drives a rapid Ca2+ entry from the external medium through VOCCs, modeled as an ultrafast Ca2+ wave. This wave may trigger and drive slower Ca2+ waves observed ex vivo and in vivo.

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.

The single-molecule mechanics of the latent TGF-ß1 complex.

BACKGROUND: TGF-ß1 controls many pathophysiological processes including tissue homeostasis, fibrosis, and cancer progression. Together with its latency-associated peptide (LAP), TGF-ß1 binds to the latent TGF-ß1-binding protein-1 (LTBP-1), which is part of the extracellular matrix (ECM). Transmission of cell force via integrins is one major mechanism to activate latent TGF-ß1 from ECM stores. Latent TGF-ß1 mechanical activation is more efficient with higher cell forces and ECM stiffening. However, little is known about the molecular events involved in this mechanical activation mechanism. RESULTS: By using single-molecule force spectroscopy and magnetic microbeads, we analyzed how forces exerted on the LAP lead to conformational changes in the latent complex that can ultimately result in TGF-ß1 release. We demonstrate the unfolding of two LAP key domains for mechanical TGF-ß1 activation: the α1 helix and the latency lasso, which together have been referred to as the "straitjacket" that keeps TGF-ß1 associated with LAP. The simultaneous unfolding of both domains, leading to full opening of the straitjacket at a force of ~40 pN, was achieved only when TGF-ß1 was bound to the LTBP-1 in the ECM. CONCLUSIONS: Our results directly demonstrate opening of the TGF-ß1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-ß1 to LTBP-1 is mandatory. Interfering with mechanical activation of latent TGF-ß1 by reducing integrin affinity, cell contractility, and binding of latent TGF-ß1 to the ECM provides new possibilities to therapeutically modulate TGF-ß1 actions.

Dynamic measurement of the height and volume of migrating cells by a novel fluorescence microscopy technique.

We propose a new technique to measure the volume of adherent migrating cells. The method is based on a negative staining where a fluorescent, non-cell-permeant dye is added to the extracellular medium. The specimen is observed with a conventional fluorescence microscope in a chamber of uniform height. Given that the fluorescence signal depends on the thickness of the emitting layer, the objects excluding the fluorescent dye (i.e., cells) appear dark, and the decrease of the fluorescent signal with respect to the background is expected to give information about the height and the volume of the object. Using a glass microfabricated pattern with steps of defined heights, we show that the drop in fluorescence intensity is indeed proportional to the height of the step and obtain calibration curves relating fluorescence intensity to height. The technique, termed the fluorescence displacement method, is further validated by comparing our measurements with the ones obtained by atomic force microscopy (AFM). We apply our method to measure the real-time volume dynamics of migrating fish epidermal keratocytes subjected to osmotic stress. The fluorescence displacement technique allows fast and precise monitoring of cell height and volume, thus providing a valuable tool for characterizing the three-dimensional behaviour of migrating cells.

Propagation of fast and slow intercellular Ca(2+) waves in primary cultured arterial smooth muscle cells.

Smooth muscle contraction is regulated by changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). In response to stimulation, Ca(2+) increase in a single cell can propagate to neighbouring cells through gap junctions, as intercellular Ca(2+) waves. To investigate the mechanisms underlying Ca(2+) wave propagation between smooth muscle cells, we used primary cultured rat mesenteric smooth muscle cells (pSMCs). Cells were aligned with the microcontact printing technique and a single pSMC was locally stimulated by mechanical stimulation or by microejection of KCl. Mechanical stimulation evoked two distinct Ca(2+) waves: (1) a fast wave (2mm/s) that propagated to all neighbouring cells, and (2) a slow wave (20µm/s) that was spatially limited in propagation. KCl induced only fast Ca(2+) waves of the same velocity as the mechanically induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP(3)) and ryanodine receptors, shows that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca(2+) influx through voltage-operated Ca(2+) channels, whereas, the slow wave was due to Ca(2+) release primarily through IP(3) receptors. Altogether, these results indicate that temporally and spatially distinct mechanisms allow intercellular communication between SMCs. In intact arteries this may allow fine tuning of vessel tone.

Identification and functional response of interstitial Cajal-like cells from rat mesenteric artery.

Cells with irregular shapes, numerous long thin filaments, and morphological similarities to the gastrointestinal interstitial cells of Cajal (ICCs) have been observed in the wall of some blood vessels. These ICC-like cells (ICC-LCs) do not correspond to the other cell types present in the arterial wall: smooth muscle cells (SMCs), endothelial cells, fibroblasts, inflammatory cells, or pericytes. However, no clear physiological role has as yet been determined for ICC-LCs in the vascular wall. The aim of this study has been to identify and characterize the functional response of ICC-LCs in rat mesenteric arteries. We have observed ICC-LCs and identified them morphologically and histologically in three different environments: isolated artery, freshly dispersed cells, and primary-cultured cells from the arterial wall. Like ICCs but unlike SMCs, ICC-LCs are positively stained by methylene blue. Cells morphologically resembling methylene-blue-positive cells are also positive for the ICC and ICC-LC markers α-smooth muscle actin and desmin. Furthermore, the higher expression of vimentin in ICC-LCs compared with SMCs allows a clear discrimination between these two cell types. At the functional level, the differences observed in the variations of cytosolic free calcium concentration of freshly dispersed SMCs and ICC-LCs in response to a panel of vasoactive molecules show that ICC-LCs, unlike SMCs, do not respond to exogenous ATP and [Arginine](8)-vasopressin.

Mechanisms of propagation of intercellular calcium waves in arterial smooth muscle cells.

In rat mesenteric arteries, smooth muscle cells exhibit intercellular calcium waves in response to local phenylephrine stimulation. These waves have a velocity of approximately 20 cells/s and a range of approximately 80 cells. We analyze these waves in a theoretical model of a population of coupled smooth muscle cells, based on the hypothesis that the wave results from cell membrane depolarization propagation. We study the underlying mechanisms and highlight the importance of voltage-operated channels, calcium-induced calcium release, and chloride channels. Our model is in agreement with experimental observations, and we demonstrate that calcium waves presenting a velocity of approximately 20 cells/s can be mediated by electrical coupling. The wave velocity is limited by the time needed for calcium influx through voltage-operated calcium channels and the subsequent calcium-induced calcium release, and not by the speed of the depolarization spreading. The waves are partially regenerated, but have a spatial limit in propagation. Moreover, the model predicts that a refractory period of calcium signaling may significantly affect the wave appearance.

A new lock-step mechanism of matrix remodelling based on subcellular contractile events.

Myofibroblasts promote tissue contractures during fibrotic diseases. To understand how spontaneous changes in the intracellular calcium concentration, [Ca(2+)](i), contribute to myofibroblast contraction, we analysed both [Ca(2+)](i) and subcellular contractions. Contractile events were assessed by tracking stress-fibre-linked microbeads and measured by atomic force microscopy. Myofibroblasts exhibit periodic (approximately 100 seconds) [Ca(2+)](i) oscillations that control small (approximately 400 nm) and weak (approximately 100 pN) contractions. Whereas depletion of [Ca(2+)](i) reduces these microcontractions, cell isometric tension is unaffected, as shown by growing cells on deformable substrates. Inhibition of Rho- and ROCK-mediated Ca(2+)-independent contraction has no effect on microcontractions, but abolishes cell tension. On the basis of this two-level regulation of myofibroblast contraction, we propose a single-cell lock-step model. Rho- and ROCK-dependent isometric tension generates slack in extracellular matrix fibrils, which are then accessible for the low-amplitude and high-frequency contractions mediated by [Ca(2+)](i). The joint action of both contraction modes can result in macroscopic tissue contractures of approximately 1 cm per month.

Force transmission in migrating cells.

During cell migration, forces generated by the actin cytoskeleton are transmitted through adhesion complexes to the substrate. To investigate the mechanism of force generation and transmission, we analyzed the relationship between actin network velocity and traction forces at the substrate in a model system of persistently migrating fish epidermal keratocytes. Front and lateral sides of the cell exhibited much stronger coupling between actin motion and traction forces than the trailing cell body. Further analysis of the traction-velocity relationship suggested that the force transmission mechanisms were different in different cell regions: at the front, traction was generated by a gripping of the actin network to the substrate, whereas at the sides and back, it was produced by the network's slipping over the substrate. Treatment with inhibitors of the actin-myosin system demonstrated that the cell body translocation could be powered by either of the two different processes, actomyosin contraction or actin assembly, with the former associated with significantly larger traction forces than the latter.

Intercellular calcium waves are associated with the propagation of vasomotion along arterial strips.

Vasomotion consists of cyclic arterial diameter variations induced by synchronous contractions and relaxations of smooth muscle cells. However, the arteries do not contract simultaneously on macroscopic distances, and a propagation of the contraction can be observed. In the present study, our aim was to investigate this propagation. We stimulated endothelium-denuded rat mesenteric arterial strips with phenylephrine (PE) to obtain vasomotion and observed that the contraction waves are linked to intercellular calcium waves. A velocity of approximately 100 microm/s was measured for the two kinds of waves. To investigate the calcium wave propagation mechanisms, we used a method allowing a PE stimulation of a small area of the strip. No calcium propagation could be induced by this local stimulation when the strip was in its resting state. However, if a low PE concentration was added on the whole strip, local PE stimulations induced calcium waves, spreading over finite distances. The calcium wave velocity induced by local stimulation was identical to the velocity observed during vasomotion. This suggests that the propagation mechanisms are similar in the two cases. Using inhibitors of gap junctions and of voltage-operated calcium channels, we showed that the locally induced calcium propagation likely depends on the propagation of the smooth muscle cell depolarization. Finally, we proposed a model of the propagation mechanisms underlying these intercellular calcium waves.

The covalent attachment of adhesion molecules to silicone membranes for cell stretching applications.

Strain devices with expandable polydimethylsiloxane (PDMS) culture membranes are frequently used to stretch cells in vitro, mimicking mechanically dynamic tissue environments. To immobilize cell-adhesive molecules to the otherwise non-adhesive PDMS substrate, hydrophobic, electrostatic and covalent surface coating procedures have been developed. The efficacy of different coating strategies to transmit stretches to cells however is poorly documented and has not been compared. We describe a novel and simple procedure to covalently bind extracellular matrix proteins to the surface of stretchable PDMS membranes. The method comprises PDMS oxygenation, silanization, and covalent protein cross-linking to the silane. We demonstrate improved attachment ( approximately 2-fold), spreading ( approximately 2.5-fold) and proliferation ( approximately 1.2-fold) of fibroblasts to our new coating over established coating procedures. Further, we compared the efficiency of different PDMS coating techniques to transmit stretches. After 15% stretch, the number of maximally (15 +/- 5%) stretched cells on our PDMS surface coating was approximately 7-fold higher compared with alternative coating protocols. Hence, covalent linkage of adhesive molecules is superior to non-covalent methods in providing a coating that resists large deformations and that fully transmit this stretch to cultured cells.

Myofibroblast communication is controlled by intercellular mechanical coupling.

Neoformation of intercellular adherens junctions accompanies the differentiation of fibroblasts into contractile myofibroblasts, a key event during development of fibrosis and in wound healing. We have previously shown that intercellular mechanical coupling of stress fibres via adherens junctions improves contraction of collagen gels by myofibroblasts. By assessing spontaneous intracellular Ca2+ oscillations, we here test whether adherens junctions mechanically coordinate myofibroblast activities. Periodic Ca2+ oscillations are synchronised between physically contacting myofibroblasts and become desynchronised upon dissociation of adherens junctions with function-blocking peptides. Similar uncoupling is obtained by inhibiting myofibroblast contraction using myosin inhibitors and by blocking mechanosensitive ion channels using Gd3+ and GSMTx4. By contrast, gap junction uncouplers do not affect myofibroblast coordination. We propose the following model of mechanical coupling for myofibroblasts: individual cell contraction is transmitted via adherens junctions and leads to the opening of mechanosensitive ion channels in adjacent cells. The resulting Ca2+ influx induces a contraction that can feed back on the first cell and/or stimulate other contacting cells. This mechanism could improve the remodelling of cell-dense tissue by coordinating the activity of myofibroblasts.

Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow.

Dynamic actin network at the leading edge of the cell is linked to the extracellular matrix through focal adhesions (FAs), and at the same time it undergoes retrograde flow with different dynamics in two distinct zones: the lamellipodium (peripheral zone of fast flow), and the lamellum (zone of slow flow located between the lamellipodium and the cell body). Cell migration involves expansion of both the lamellipodium and the lamellum, as well as formation of new FAs, but it is largely unknown how the position of the boundary between the two flow zones is defined, and how FAs and actin flow mutually influence each other. We investigated dynamic relationship between focal adhesions and the boundary between the two flow zones in spreading cells. Nascent FAs first appeared in the lamellipodium. Within seconds after the formation of new FAs, the rate of actin flow decreased locally, and the lamellipodium/lamellum boundary advanced towards the new FAs. Blocking fast actin flow with cytochalasin D resulted in rapid dissolution of nascent FAs. In the absence of FAs (spreading on poly-L-lysine-coated surfaces) retrograde flow was uniform and the velocity transition was not observed. We conclude that formation of FAs depends on actin dynamics, and in its turn, affects the dynamics of actin flow by triggering transition from fast to slow flow. Extension of the cell edge thus proceeds through a cycle of lamellipodium protrusion, formation of new FAs, advance of the lamellum, and protrusion of the lamellipodium from the new base.

Calcium dynamics and vasomotion in arteries subject to isometric, isobaric, and isotonic conditions.

In vitro, different techniques are used to study the smooth muscle cells' calcium dynamics and contraction/relaxation mechanisms on arteries. Most experimental studies use either an isometric or an isobaric setup. However, in vivo, a blood vessel is neither isobaric nor isometric nor isotonic, as it is continuously submitted to intraluminal pressure variations arising from heart beat. We use a theoretical model of the smooth muscle calcium and arterial radius dynamics to determine whether results may be considerably different depending on the experimental conditions (isometric, isobaric, isotonic, or cyclic pressure variations). We show that isobaric conditions appear to be more realistic than isometric or isotonic situations, as the calcium dynamics is similar under cyclic intraluminal pressure variations (in vivo-like situation) and under a constant pressure (isobaric situation). The arterial contraction is less pronounced in isotonic than in isobaric conditions, and the vasoconstrictor sensitivity higher in isometric than isobaric or isotonic conditions, in agreement with experimental observations. Interestingly, the model predicts that isometric conditions may generate artifacts like the coexistence of multiple stable states. We have verified this model prediction experimentally using rat mesenteric arteries mounted on a wire myograph and stimulated with phenylephrine.

Does the endothelium abolish or promote arterial vasomotion in rat mesenteric arteries? Explanations for the seemingly contradictory effects.

BACKGROUND AND AIMS: Vasomotion consists in cyclic oscillations of the arterial diameter induced by the synchronized activity of the smooth muscle cells. So far, contradictory results have emerged in the literature about the role of the endothelium in the onset and maintenance of vasomotion. Here our aim is to understand how the endothelium may either abolish or promote vasomotion. METHODS AND RESULTS: We investigate rat mesenteric arterial strips stimulated with phenylephrine (PE). Our results show that the endothelium is not necessary for vasomotion. However, when the endothelium is removed, the PE concentration needed to induce vasomotion is lower and the rhythmic contractions occur for a narrower range of PE concentrations. We demonstrate that endothelium-derived relaxing products may either induce or abolish vasomotion. On the one hand, when the strip is tonically contracted in a nonoscillating state, an endothelium-derived relaxation may induce vasomotion. On the other hand, if the strip displays vasomotion with a medium mean contraction, a relaxation may induce a transition to a nonoscillating state with a small contraction. CONCLUSION: Our findings clarify the role of the endothelium on vasomotion and reconcile the seemingly contradictory observations reported in the literature.