Gradient of Rigidity in the Lamellipodia of Migrating Cells Revealed by Atomic Force Microscopy

Changes in mechanical properties of the cytoplasm have been implicated in cell motility, but there is little information about these properties in specific regions of the cell at specific stages of the cell migration process. Fish epidermal keratocytes with their stable shape and steady motion represent an ideal system to elucidate temporal and spatial dynamics of the mechanical state of the cytoplasm. As the shape of the cell does not change during motion and actin network in the lamellipodia is nearly stationary with respect to the substrate, the spatial changes in the direction from the front to the rear of the cell reflect temporal changes in the actin network after its assembly at the leading edge. We have utilized atomic force microscopy to determine the rigidity of fish keratocyte lamellipodia as a function of time/distance from the leading edge. Although vertical thickness remained nearly constant throughout the lamellipodia, the rigidity exhibited a gradual but significant decrease from the front to the rear of the lamellipodia. The rigidity pro. le resembled closely the actin density pro. le, suggesting that the dynamics of rigidity are due to actin depolymerization. The decrease of rigidity may play a role in facilitating the contraction of the actin-myosin network at the lamellipodium/cell body transition zone.

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

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

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.