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

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.