Protrusion-retraction switches and traction forces in spontaneous cell polarization

Cell migration is a fundamental property of all animal cells which is involved in processes such as embryogenesis, immune response, tissue regeneration and cancer metastasis. Directional migration requires cell polarization which could happen in response to external signals but also spontaneously. Polarization involves the change in the cell edge activities from random distribution of protrusion and retraction to their separation into two large regions corresponding to the leading and trailing edges of the cell. Many studies investigated cytoskeletal mechanisms of protrusions and retractions but it is still not well understood how the cell orchestrates protrusion and retraction along its edge and what triggers transitions between these two types of activity. These questions are essential to understand both cell polarization and cycles of protrusion and retraction which characterize exploratory edge dynamics before polarization. This thesis focuses on the analysis of cell edge dynamics during the initiation of motion and on the mechanisms of transition (switches) between protrusion and retraction. We considered the cell polarization and protrusion-retraction cycles as two related phenomena and aimed to identify common mechanisms. Live cell imaging, cytoskeletal inhibitors, traction force microscopy, patterned substrates, micromanipulation, and computational analysis and modeling were used to examine cell polarization in the experimental model of fish epithelial keratocytes that are characterized by simple and regular shape and robust polarization and motion. We found that protrusion-retraction switches happened at maximal distance from the cell center both during cell edge fluctuation and directional motion and did not depend on the edge orientation with respect to the cell motion direction. Computational model demonstrated that switches at a threshold distance were sufficient for self-organization of edge activity leading to spontaneous polarization and stable cell shape and motion suggesting that distance-sensing is a fundamental mechanism of cell symmetry breaking. Next we investigated the mechanisms of distance-sensing focusing on two hypotheses: traction forces and tridimensional cell shape. Traction force may increase with the distance from the cell center (e.g., due to a build-up of actomyosin network) leading to detachment of the edge and initiation of retraction. We discovered that traction forces indeed increased with the distance and that protrusion-retraction switches occurred near maximal forces. Local external force also induced protrusion-retraction switch. However, inhibition of contractility reduced traction forces and abolished the dependence of force on the distance, but did not prevent cell polarization. Taken together, these results suggest that traction forces are sufficient, but not necessary mediators of distance-dependent switch and cell polarization. In an alternative mechanism, tridimensional shape of the cell edge may depend on the distance from the cell center and affect the balance of forces at the edge, inducing switches. We have locally modified this tridimensional force balance by using substrates with topographic features and showed that switches preferentially happen near these features. These results provide a novel framework to understand cell edge dynamics and symmetry breaking in terms of protrusion-retraction switches and physical force balance at the cell edge.

Analysis and Modeling of the Dynamics of Traction Forces during Cell Polarization and their Role in the Organization of Edge Activity

Zeno Messi

The capacity to break symmetry and organize activity to move directionally is a fundamental property of eukaryotic cells. To explain the organization of cell-edge activity, models commonly rely on front-to-back gradients of functional components or regulatory factors, but they do not explain how the front-back axis is defined in the first place. Recently, a novel and successful principle for self-organization of cell-edge activity was proposed, in which local cell-edge dynamics depends on the distance from the cell center, but not on the orientation with respect to the front-back axis. In this thesis, we test the hypothesis that edge motion is controlled by distance sensing via traction forces.\par We show that traction forces exerted on the substrate by polarizing cells are highly dynamical and follow motion of the cell-edge, that stress increases with the distance from the cell center and that maximal forces are located at a fixed distance from the edge near the sites of protrusion-retraction switches. We observe that traction forces correlate with edge extension in cell populations under different experimental conditions. We demonstrate with a fully mechanical model that distance dependence of the force is an emergent property of elastic fiber networks. We next show that dynamics of traction forces and cell-edge motion are correlated during protrusion-retraction cycles, indicating that traction forces trigger the switch from protrusion to retraction. Actin retrograde flow correlates with stress during retraction but not during protrusion suggesting that increase of stress during protrusion is independent of the motion of the actin network. Finally, incorporating a mechanism of cytoskeletal turnover in our model of fiber network we produce systems that display oscillatory fluctuations of their edge and other experimental behaviors.\par Our results provide strong evidence that traction forces play a major role in the organization of cell-edge activity and polarization.

EPFL2021

Traction Forces Control Cell-Edge Dynamics and Mediate Distance Sensitivity during Cell Polarization

Alicia Carine Bornert, Zeno Messi, Franck Claude Raynaud

Traction forces are generated by cellular actin-myosin system and transmitted to the environment through adhesions. They are believed to drive cell motion, shape changes, and extracellular matrix remodeling [1 -3] . However, most of the traction force analysis has been performed on stationary cells, investigating forces at the level of individual focal adhesions or linking them to static cell parameters, such as area and edge curvature [4-10] . It is not well understood how traction forces are related to shape changes and motion, e.g., forces were reported to either increase or drop prior to cell retraction [11-15]. Here, we analyze the dynamics of traction forces during the protrusionretraction cycle of polarizing fish epidermal keratocytes and find that forces fluctuate together with the cycle, increasing during protrusion and reaching maximum at the beginning of retraction. We relate force dynamics to the recently discovered phenomenological rule [16] that governs cell-edge behavior during keratocyte polarization: both traction forces and probability of switch from protrusion to retraction increase with the distance from the cell center. Diminishing forces with cell contractility inhibitor leads to decreased edge fluctuations and abnormal polarization, although externally applied force can induce protrusion-retraction switch. These results suggest that forces mediate distance sensitivity of the edge dynamics and organize cell-edge behavior, leading to spontaneous polarization. Actin flow rate did not exhibit the same distance dependence as traction stress, arguing against its role in organizing edge dynamics. Finally, using a simple model of actin-myosin network, we show that force-distance relationship might be an emergent feature of such networks.

2020

Force Transmission in Migrating Cells

Maxime Fred Maurice Fournier

Ability to migrate is a fundamental property of all animal cells. Cell migration underlies such important physiological and pathological processes as embryonic development, immune response, tissue regeneration and cancer metastasis. As cell migration involves forces and motion, it is ultimately a physical phenomenon that should be understood in terms of fundamental physical laws. Cell migration mechanisms are based on the activity of the cytoskeleton, a system of intracellular filaments and motor proteins. During cell migration, forces developed by the cytoskeleton are transmitted, through specific adhesions complexes, to the substrate. Composition and dynamics of the cytoskeleton has been subject of many studies, but it remains poorly understood how the cytoskeleton reactions and its interaction with extra cellular matrix are orchestrated to result in coordinated migration behavior. The main part of this thesis consists of the experiments and analysis of force transmission during cell migration in the experimental system of fish epidermal keratocytes. Our experimental approaches involved microinjection for proteins dynamics quantification, micromanipulation, use of compliant substrate, simultaneous imaging of cytoskeleton and substrate deformation, and the use of cytoskeletal inhibitors. In parallel with the experiments, significant computational work has been performed to program and adapt existing algorithms to analyze experimental data. Thanks to the combination of our experimental and computational approaches, we were able to correlate actin dynamics and traction forces over the whole cell, to map the efficiency of force transmission, and to reveal slipping and gripping mechanisms differentially involved in stress transmission in different parts of the cell. We also investigated contributions of actin assembly and myosin-dependent contractility to force generation and provided evidence that cell translocation could be powered by two different engines. Additionally, we performed micromanipulation experiments to identify the cell polarity cues associated with specific cytoskelatal reactions and regions of the cell. We have also collaborated with the laboratory of one of the leading modellers in the field of cell biophysics to validate a new mathematical model of cell migration and force transmission.