Begoña Álvarez-González

Both contractile axial and lateral traction force dynamics drive amoeboid cell motility

During chemotactic movement, D. discoideum exhibits step-wise amoeboid motility driven by both contractile axial forces and lateral forces.

Three-Dimensional Balance of Cortical Tension and Axial Contractility Enables Fast Amoeboid Migration

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 used three-dimensional force microscopy to measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examined wild-type cells as well as mutants with defects in contractility, internal F-actin crosslinking, and cortical integrity. We showed that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. We found that the migration speed increases when axial contractility overcomes cortical tension to produce the cell shape changes needed for locomotion. We demonstrated that 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 without adhering to it, and which may be relevant for amoeboid migration in complex three-dimensional environments.

Cooperative cell motility during tandem locomotion of amoeboid cells

Tandem pairs of Dictyostelium cells migrate synchronously with an ~54-s time delay between the formation of their frontal protrusions. Each cell establishes two active adhesions, with the trailing cell reusing the location of the adhesions of the leading cell. This coordinated motility is mechanically driven and aided by cell–cell adhesions.

Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion

Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during migration.

Two-Layer Elastographic 3-D Traction Force Microscopy

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.

Three-Dimensional Traction Force Distribution in Migrating Amoeboid Cells

We have developed a method which enables us to determine cellular traction forces exerted perpendicular to the substrate in addition to the in-plane forces. This solution also enables to analyze the errors associated to existing two-dimensional traction cytometry methods, which assume either that the vertical displacements or that the vertical stresses are zero on the surface of the substrate. We obtain information about the substrate deformation by imaging a small volume of the elastic substrate with embedded fluorescent marker beads. Correlation with a reference image enables us to obtain the 3D deformation of the substrate. The corresponding traction forces are obtained by solving the elastostatic equation for a linearly elastic medium using the calculated deformation of the substrate. Our studies of Dictyostelium cells moving over flat substrates are designed to reveal the importance of various cytoskeletal components for the organization of the traction stresses in all three dimensions. We are looking at different Dictyostelium mutants with crosslinking defects, such as myosin II-null cells and cortexilin-null cells, in order to study the role that these crosslinkers play in the overall distribution of the traction forces. We find that wt Dictyostelium cells push on the substrate near the center of the cell and pull at the periphery. The magnitude of these perpendicular forces is comparable to the magnitude of the forces produced in the plane of the substrate. Our initial findings show that the effects of mutations on the parallel forces do not necessarily predict the effects on the perpendicular forces. For example, myosin II-null cells show a significant reduction of the front to back organization of the parallel traction forces while the push pull distribution of forces remains unaffected.