Fast amoeboid migration requires cells to apply mechanical forces on their surroundings via transient adhesions. However, the role these forces play in controlling cell migration speed remains largely unknown. We use three- dimensional force microscopy to examine the mechanics underlying the chemotactic migration of wild-type Dictyostelium cells, as well as mutant strains with defects in contractility, internal F-actin cross-linking and cortical integrity. We show that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. The three-dimensional pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate downward without adhering to it. These compressive pressure-induced forces are not associated to adhesion sites, and may allow amoeboid cells to push off surrounding structures when migrating in complex three- dimensional environments. We find a relationship between the strength of these three-dimensional forces and the migration speed and we show that the cell migration speed increases with the ratio of the tangential to normal forces. This finding indicates that the migration speed increases when axial contractility balances cortical tension, allowing the cells to modulate their three- dimensional shape and move faster. Additionally, we develop a new methodology for the calculation of the three -dimensional forces exerted by migrating cells improved by a Lagrange multipliers optimization that provides a stress field in equilibrium and equal to zero outside the region in which the cell is localized. Furthermore, we design a novel elastometry technique based on the exact solution of the elastic equation of equilibrium, the measurement of the deformation exerted by cells when moving and the application of an optimization algorithm for solving a non -linear least-squares problem. This novel method enables the characterization of the Poisson ratio of polymer-based substrates on real time, which is essential for a precise calculation of the traction forces. The value of the Poisson ratio that we obtain for the polyacrylamide gels used in our experiments is 0.45. A similar methodology could be applied to calculate the mechanical properties and constitutive equations for other extracellular environments, which are not perfectly elastic

Streams of migratory cells are initiated by the formation of tandem pairs of cells connected head to tail to which other cells subsequently adhere. The mechanisms regulating the transition from single to streaming cell migration remain elusive, although several molecules have been suggested to be involved. In this work, we investigate the mechanics of the locomotion ofDictyosteliumtandem pairs by analyzing the spatiotemporal evolution of their traction adhesions (TAs). We find that in migrating wild-type tandem pairs, each cell exerts traction forces on stationary sites (∼80% of the time), and the trailing cell reuses the location of the TAs of the leading cell. Both leading and trailing cells form contractile dipoles and synchronize the formation of new frontal TAs with ∼54-s time delay. Cells not expressing the lectin discoidin I or moving on discoidin I-coated substrata form fewer tandems, but the trailing cell still reuses the locations of the TAs of the leading cell, suggesting that discoidin I is not responsible for a possible chemically driven synchronization process. The migration dynamics of the tandems indicate that their TAs' reuse results from the mechanical synchronization of the leading and trailing cells' protrusions and retractions (motility cycles) aided by the cell-cell adhesions.

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.