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

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).

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

Structure and dynamics of actin network at the leading edge of migrating cells

Sébastien Schaub

Crawling motion is characteristic of most animal cells and is principally based on actin-myosin II cytoskeletal system. The major components and reactions contributing to motility have been identified, but the overall picture of how these molecular events are orchestrated to result in the motion of the cell is still missing. We used a combination of experimental imaging, image analysis, and numerical modeling to analyze at a quantitative level how the elements of the actin-myosin machinery are coordinated in several important aspects of the crawling motion: the assembly of the characteristic pattern of actin network in the leading lamellipodium, the interaction between retrograde flow of the actin network and substrate adhesions, and the coordination of motion and assembly of actin and myosin II polymers over the entire cell during steady-state migration. First, we used enhanced phase contrast microscopy correlated with fluorescence and electron microscopy to investigate the supra-filament organization of actin in the lamellipodium of motile fish epidermal keratocytes. We showed a close correspondence between individual filament orientations and optical criss-cross network pattern by quantifying the orientational distribution of features in electron microscopy and optical microscopy images. We tested the hypothesis that the criss-cross pattern observed by optical microscopy results from variation of actin density, which is due to stochastic events of branching and capping of filaments. Based on this hypothesis, we produced simulated images of lamellipodium similar to experimental images. These simulations provided quantitative tools to estimate structural and dynamical parameters of the actin network in lamellipodia, notably the filament length, the actin concentration, the curvature of filaments and capping rate. Protrusion at the edge of the cell is coupled to the substrate adhesion, but at the same time is associated with the retrograde flow of the actin network away from the edge. We observed that focal adhesions were mostly localized at the boundary between the fast retrograde flow zone (the lamellipodium) and the slower flow zone (the lamellum). We analyzed simultaneously the actin flow and the formation of focal adhesion. Based on these results, we proposed a multi-stage model for the advance of the leading edge of migrating cell: advance of the lamellipodium is followed by the formation of the nascent adhesion complexes, which results in the reduction of the flow rate, advance of the lamellum, and then protrusion of the lamellipodium from a new base. We finally focused on the dynamics of the acto-myosin system in entire migrating cell. For this purpose, we developed a tracking program which allowed quantifying motion, contraction and assembly of cytoskeletal components. We measured the dynamics of actin, myosin II and their relative motion in migrating fish keratocytes. These results supported the dynamic network contraction model suggesting that the actin network deformed by myosin II drives the translocation of cell body. Focusing on the different aspects of the crawling motion, and balancing experimental and theoretical approaches, we obtained original results, which contributed to a better understanding of the dynamics of the lamellipodium and crawling motion in general.

EPFL2006

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

Lara Buscemi Estefanell, Chiara Gabella, Jean-Jacques Meister, Benoît Vianay

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.