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.

Interplay between cytoskeletal forces, membrane tension and shape in rapidly migrating cells

Chiara Gabella

Cell migration is a fundamental process in all animal cells. The ability of cell to migrate is crucial for many pathological and physiological conditions such as morphogenesis, the immune response to inflammatory or vascular diseases and cancer metastasis. Actin polymerization pressure and membrane tension are two major forces defining the dynamics of cell leading edge during migration: actin polymerization pushes the membrane forward to drive the motion, while membrane tension is believed to counteract and control protrusion. Recent studies implicated membrane tension in control of cell spreading, limiting lateral extension of the cell, confining protrusion to a single leading edge, and crushing actin network during retraction at the cell rear; however, direct experimental evidence is conflicting. Moreover, actin polymerization and membrane tension are not directly opposing each other, since membrane tension is oriented along the membrane surface, while actin polymerization is directed generally parallel to the substrate. Thus the shape of the leading edge may have important, albeit as yet unknown effect on the force balance. To understand how these forces are orchestrated at the cell’s leading edge, we employed the model system of fish epithelial keratocytes which are spontaneously highly motile and are characterized by a remarkably stable shape and constant protrusion rate, making any changes due to experimental treatments traceable and accessible for quantitative analysis. We set out to measure actin protrusion rate, membrane tension, and three dimensional shape of these cells subjected to manipulation of osmotic pressure, cytoskeletal contractility and drastic shape perturbations (cell wounding). To accomplish this goal, we first developed a simple and reliable technique to measure cell height and volume. This method is based on exclusion of fluorescent dye from the volume of the cell: the decrease of the fluorescent signal with respect to the background is proportional to the height and the volume of the object. We calibrated the technique using a glass microfabricated pattern with steps of defined heights and validated it by comparing our measurements with the ones obtained by atomic force microscopy (AFM). The method is fast and allows precise monitoring of height and volume of rapidly migrating cells, and do not require any modification to the standard epifluorescence setup. We applied our method to measure the real-time volume dynamics of migrating fish epidermal keratocytes subjected to osmotic stress and cytoskeletal perturbations. We then analyzed the relationship between shape of the cell, protrusion rate and membrane tension. We find that protrusion rate does not correlate with membrane tension, but, instead, is strongly correlated with cell roundness. We rationalized the relationship between cell roundness and protrusion velocity using the framework developed for wetting phenomena. The contact angle that the cell forms with the substrate defines the contribution of membrane tension to the force balance at the leading edge: the higher the contact angle, the less load is applied from the membrane to the actin network and therefore the faster is actin protrusion. We validated this concept by manufacturing topographically modified surfaces and showing that the leading edge of the cell exhibits pinning on substrate ridges - a phenomenon characteristic of spreading of liquid drops. These results demonstrate the role of contact angle at the leading edge in control of protrusion, which has important implications for cell shape dynamics and cell migration in 3D. We have also analyzed protrusion velocity in the case of high planar curvature of the advancing edge which was accomplished by making small circular wounds in keratocyte’s lamellipodia. The fact that these wounds presenting negative planar curvature close faster than the protrusion rate at the edge of the cell, is consistent with the important role of membrane curvature in the control of protrusion velocity. Our study underscores the fundamental importance of membrane configuration for cellular force balance and edge dynamics. It provides a novel framework for understanding the relationship between global cell shape and its local dynamics, of the balance between lamellipodia protrusion and blebbing, and for modulation of cell shape and motion by substrate topology.

EPFL2013

Mechanical properties of peripheral stress fibers in adherent cells spreading on patterned substrates

Céline Jeanne Labouesse

Many cellular processes require or depend on mechanical feedback from the cell's micro-environment. For example, the local organization of the actin cytoskeleton is known to adapt to the physical properties of the extracellular matrix. To gain better understanding of the mechanisms controlling this process, we have studied the particular case of peripheral stress fibers, thick concave contractile bundles of actin filaments. We have employed micropatterned adhesive substrates to constrain cell shape and adhesion layout. We have then used fluorescence microscopy, traction force microscopy and cantilever-based force measurements to experimentally characterize the assembly and mechanical tension of these peripheral stress fibers. We combined these experimental data with numerical simulations of cell shape and cell tension to propose a general mechanical model of stress fiber behavior. We first identified the formation of peripheral stress fibers as significant of a behavioral switch triggered by non-adhesive gaps in the substrate geometry. These bundles indeed form above a threshold distance of 3-4µm, necessary to stimulate adhesion maturation, and lead to a redirection of cellular tension parallel to the cell edge. Additional analysis of cells on rigid and soft substrates showed that the radius and tension of peripheral bundles weakly increase with spanning distance. Thus both the cytoskeleton local organization and its mechanical properties can be controlled to some extent by non-adhesive features in substrate geometry. The peripheral fibers' concave curvature results from the balance of the line and surface tensions. However, the nature of these forces, and in particular the contribution of myosin-dependent contractility, are not clear. To get insight into this force balance, we inhibited myosin activity and monitored the shape and tension responses. We found that myosin inhibition led to a decrease in the traction forces and an increase in arc radius, indicating that line tension dropped to a lesser extent than surface tension. These results suggest a myosin-independent component in the tension of peripheral arcs. We propose a simple physical model in which the line tension is the sum of a myosin-dependent active component and of a passive elastic component. Numerical simulations of this model reproduce well the measured shape dynamics and allow estimating the relative contributions of elasticity and contractility to the arc line tension. We then developed an alternative setup to directly probe the elasticity of semi-isolated peripheral fibers, using micromanipulation techniques. A soft cantilever was used to measure the force with which the fiber resisted a transverse deformation. The overall mechanical response of the fiber can be represented by a 3-parameter-solid model, i.e. a spring in series with a Kelvin-Voigt unit. The response was separated into two regimes: first a fast traction during which elastic response dominated, followed by a viscoelastic relaxation. The elastic and viscoelastic coefficients were extracted from the force-elongation curves and from the elongation velocity during relaxation. Inhibiting myosin activity decreased the active pre-tension, but had no clear effect on the elasticity. Our results show that myosin is an important actor, but not the only one, in the response of peripheral fibers to external mechanical constraints (active deformation) or geometrical constraints (limited adhesive area).

EPFL2015

Dynamics of actin and myosin II during cell polarization and directional locomotion

Sophie Bohnet

Crawling cell motility is characteristic of most animal cells and is involved in many important biological processes such as embryogenesis, immune response, and wound healing. It involves several steps: protrusion (dynamic surface extension) at the front of the cell, attachment to the substratum, and forward translocation of the cell body accompanied by the detachment and retraction of the rear portion of the cell. Crawling motion is believed to be based on actin, a major component of the cytoskeleton, and myosin II, a motor protein that is believed to produce contraction force by moving along actin filaments. Actin and myosin II are the two major structural and force-generating components of the cell's motile machinery. Protrusion is thought to be driven by the assembly of a dense actin network that is attached to the substrate through integrin-containing adhesions. Previous studies estimated the forces generated by actin polymerization and the adhesion forces, but the net force developed by the protruding leading edge has not been yet determined. The forward translocation of the cell body is believed to depend on the interaction of myosin with the actin network, but the exact mechanisms behind this interaction are still unclear. Moreover, it is still poorly understood how the molecular reactions involved in each step of the crawling motion are coordinated to result in the integrated cellular response. In this thesis work, we have quantitatively characterized the assembly and motion of actin and myosin II in the stationary and migratory states of the cell, and during the transition between these two steady-states. We have also measured the force developed by the protruding leading edge. The experimental system used is the fish epidermal keratocytes, one of the most spectacular models of cell locomotion. These cells are characterized by fast and persistent migration, and a stable and simple cytoskeletal organization. Enhanced phase contrast, interference reflection, classical fluorescence and fluorescent speckles microscopy were used as imaging techniques along with computer-based tracking approaches. We have first studied actin movement and assembly in the lamellipodium of keratocyte (thin, sheet-like extension at the leading edge of the cell). Actin dynamics in the lamellipodia of many cell types are characterized by retrograde flow of the actin network away from the leading edge. This flow is believed to result from membrane resistance to actin assembly and contractile forces in the network, and was speculated to play a role in controlling the cell shape and motion. Retrograde flow was previously observed in every crawling cell with the exception of keratocytes. We have for the first time detected actin retrograde flow throughout the keratocyte lamellipodium at velocities of 1–3 µm/min and analyzed its organization and relation to the cell motion during both unobstructed persistent migration and events of cell collision. We have found that freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium, whereas in colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of the flow in other lamellipodial regions. Our findings support the universality of the flow phenomenon and indicate that maintenance of the keratocyte shape during locomotion depends on the regulation of retrograde flow and actin polymerization. We have subsequently quantitatively characterized the assembly and movement of actin and myosin II throughout the entire keratocyte. Actin exhibited a slow backward motion in the lamellipodium and a faster forward motion in the trailing cell body. The change of direction occurred along the line parallel to the leading edge of the cell just in front of the front boundary of the cell body. Myosin II also moved forward in the cell body, but no motion with respect to the substratum was detected in the lamellipodium. At the cell sides, actin and myosin II exhibited motion towards the cell center with velocities increasing with the distance from the center. Net assembly of actin occurred locally at the leading edge of the lamellipodium, and disassembly at the back of the lamellipodium. Myosin II assembled in a distributed manner in the lamellipodium and disassembled in the cell body. Eventually, a simultaneous tracking of actin and myosin II revealed that myosin II moved forward relatively to actin throughout the cell. Taken together, these results suggest a longitudinal and transverse contraction along the lamellipodium/cell body junction. To understand how the polarized patterns of the organization of actin/myosin machinery were first established, we have studied in the next part of the work random cell polarization, i.e. the spontaneous transition between the stationary and the migratory state of a cell in the absence of external directional stimuli. Cell polarization and directional locomotion are believed to involve asymmetry in actin assembly, contractility, and substrate adhesion. We have analyzed the distribution and dynamics of actin and myosin II and the dynamics of substrate adhesions during cell polarization. In the stationary state, the cells exhibited lamellipodia with a uniform rate of actin retrograde flow all around the cell perimeter. Upon initiation of directional movement, retrograde flow accelerated at the prospective rear of the cell, but the lamellipodia initially persisted all around the perimeter. The assembly of actin network also continued all around the cell. Local areas of detachment from the substrate were observed at the base of rear lamellipodia. Treatment with myosin inhibitor blebbistatin nearly completely blocked the polarization. Depolymerization of microtubules with nocodazole resulted in attenuated polarization. Taken together, these results suggest that initial polarization results from asymmetry in actin retrograde flow, which is induced by myosin-dependent contraction and stabilized by microtubules. Finally, we have estimated the force necessary to stall the protruding leading edge of the keratocytes. We arrested the leading edge of a moving cell with a hydrodynamic load generated by a fluid flow from a micropipette. The flow arrested the protrusion locally, as the cell approached the pipette, causing an arc-shaped indentation and upward folding of the leading edge. Modeling of the fluid flow gave a surprisingly low value for the arresting force of just a few piconewtons per micrometer. This small force did not abolish actin polymerization nor disrupt the adhesions formed before the arrest, but rather interfered with the weak nascent adhesions at the very front of the cell. We conclude that a weak external force is sufficient to reorient the growing actin network at the leading edge and to stall the cell protrusion. Our study of the dynamics of actin and myosin II in polarizing and migrating cells provides novel and important insights into the cell motility mechanism and establishes a fundamental quantitative data set necessary to test and improve theoretical models of cell motility.

EPFL2006