Vito Conte

Control of cell–cell forces and collective cell dynamics by the intercellular adhesome

Dynamics of epithelial tissues determine key processes in development, tissue healing and cancer invasion. These processes are critically influenced by cell–cell adhesion forces. However, the identity of the proteins that resist and transmit forces at cell–cell junctions remains unclear, and how these proteins control tissue dynamics is largely unknown. Here we provide a systematic study of the interplay between cell–cell adhesion proteins, intercellular forces and epithelial tissue dynamics. We show that collective cellular responses to selective perturbations of the intercellular adhesome conform to three mechanical phenotypes. These phenotypes are controlled by different molecular modules and characterized by distinct relationships between cellular kinematics and intercellular forces. We show that these forces and their rates can be predicted by the concentrations of cadherins and catenins. Unexpectedly, we identified different mechanical roles for P-cadherin and E-cadherin; whereas P-cadherin predicts levels of intercellular force, E-cadherin predicts the rate at which intercellular force builds up.

Forces driving epithelial wound healing

A fundamental feature of multicellular organisms is their ability to self-repair wounds through the movement of epithelial cells into the damaged area. This collective cellular movement is commonly attributed to a combination of cell crawling and “purse-string” contraction of a supracellular actomyosin ring. Here we show by direct experimental measurement that these two mechanisms are insufficient to explain force patterns observed during wound closure. At early stages of the process, leading actin protrusions generate traction forces that point away from the wound, showing that wound closure is initially driven by cell crawling. At later stages, we observed unanticipated patterns of traction forces pointing towards the wound. Such patterns have strong force components that are both radial and tangential to the wound. We show that these force components arise from tensions transmitted by a heterogeneous actomyosin ring to the underlying substrate through focal adhesions. The structural and mechanical organization reported here provides cells with a mechanism to close the wound by cooperatively compressing the underlying substrate.

Video force microscopy reveals the mechanics of ventral furrow invagination in Drosophila

The absence of tools for mapping the forces that drive morphogenetic movements in embryos has impeded our understanding of animal development. Here we describe a unique approach, video force microscopy (VFM), that allows detailed, dynamic force maps to be produced from time-lapse images. The forces at work in an embryo are considered to be decomposed into active and passive elements, where active forces originate from contributions (e.g., actomyosin contraction) that do mechanical work to the system and passive ones (e.g., viscous cytoplasm) that dissipate energy. In the present analysis, the effects of all passive components are considered to be subsumed by an effective cytoplasmic viscosity, and the driving forces are resolved into equivalent forces along the edges of the polygonal boundaries into which the region of interest is divided. Advanced mathematical inverse methods are used to determine these driving forces. When applied to multiphoton sections of wild-type and mutant Drosophila melanogaster embryos, VFM is able to calculate the equivalent driving forces acting along individual cell edges and to do so with subminute temporal resolution. In the wild type, forces along the apical surface of the presumptive mesoderm are found to be large and to vary parabolically with time and angular position, whereas forces along the basal surface of the ectoderm, for example, are found to be smaller and nearly uniform with position. VFM shows that in mutants with reduced junction integrity and myosin II activity, the driving forces are reduced, thus accounting for ventral furrow failure.

Collective cell durotaxis emerges from long-range intercellular force transmission

The ability of cells to follow gradients of extracellular matrix stiffness—durotaxis—has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion.

A 3D finite element model of ventral furrow invagination in the Drosophila melanogaster embryo.

The paper describes a mechanical model of epithelial tissue development in Drosophila embryos to investigate a buckling phenomenon called invagination. The finite element method is used to model this ventral furrow formation in 3D by decomposing the total deformation into two parts: an imposed active deformation, and an elastic passive deformation superimposed onto the latter. The model imposes as boundary conditions (i) a constant yolk volume and (ii) a sliding contact condition of the cells against the vitelline membrane, which is interpolated as a B-Spline surface. The active deformation simulates the effects of apical constriction and apico-basal elongation of cells. This set of local cellular mechanisms leads to global shape changes of the embryo which are associated with known gene expressions. Using the model we have tested different plausible hypotheses postulated to account for the mechanical behaviour of epithelial tissues. In particular, we conclude that only certain combinations of local cell shape change can successfully reproduce the invagination process. We have quantitatively compared the model with a 2D model and shown that it exhibits a more robust invagination phenomenon. The 3D model has also revealed that invagination causes a yolk flow from the central region to the anterior and posterior ends of the embryo, causing an accordion-like global compression and expansion wave to move through the embryo. Such a phenomenon cannot be described by 2D models.

Quantifying forces in cell biology

Cells exert, sense, and respond to physical forces through an astounding diversity of mechanisms. Here we review recently developed tools to quantify the forces generated by cells. We first review technologies based on sensors of known or assumed mechanical properties, and discuss their applicability and limitations. We then proceed to draw an analogy between these human-made sensors and force sensing in the cell. As mechanics is increasingly revealed to play a fundamental role in cell function we envisage that tools to quantify physical forces may soon become widely applied in life-sciences laboratories.

Robust mechanisms of ventral furrow invagination require the combination of cellular shape changes

Ventral furrow formation in Drosophila is the first large-scale morphogenetic movement during the life of the embryo, and is driven by co-ordinated changes in the shape of individual epithelial cells within the cellular blastoderm. Although many of the genes involved have been identified, the details of the mechanical processes that convert local changes in gene expression into whole-scale changes in embryonic form remain to be fully understood. Biologists have identified two main cell deformation modes responsible for ventral furrow invagination: constriction of the apical ends of the cells (apical wedging) and deformation along their apical-basal axes (radial lengthening/shortening). In this work, we used a computer 2D finite element model of ventral furrow formation to investigate the ability of different combinations of three plausible elementary active cell shape changes to bring about epithelial invagination: ectodermal apical-basal shortening, mesodermal apical-basal lengthening/shortening and mesodermal apical constriction. We undertook a systems analysis of the biomechanical system, which revealed many different combinations of active forces (invagination mechanisms) were able to generate a ventral furrow. Two important general features were revealed. First that combinations of shape changes are the most robust to environmental and mutational perturbation, in particular those combining ectodermal pushing and mesodermal wedging. Second, that ectodermal pushing plays a big part in all of the robust mechanisms (mesodermal forces alone do not close the furrow), and this provides evidence that it may be an important element in the mechanics of invagination in Drosophila.

A biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo

The article provides a biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo. Ventral furrow formation is the first large-scale morphogenetic movement in the fly embryo. It involves deformation of a uniform cellular monolayer formed following cellularisation, and has therefore long been used as a simple system in which to explore the role of mechanics in force generation. Here we use a quantitative framework to carry out a systematic perturbation analysis to determine the role of each of the active forces observed. The analysis confirms that ventral furrow invagination arises from a combination of apical constriction and apical–basal shortening forces in the mesoderm, together with a combination of ectodermal forces. We show that the mesodermal forces are crucial for invagination: the loss of apical constriction leads to a loss of the furrow, while the mesodermal radial shortening forces are the primary cause of the internalisation of the future mesoderm as the furrow rises. Ectodermal forces play a minor but significant role in furrow formation: without ectodermal forces the furrow is slower to form, does not close properly and has an aberrant morphology. Nevertheless, despite changes in the active mesodermal and ectodermal forces lead to changes in the timing and extent of furrow, invagination is eventually achieved in most cases, implying that the system is robust to perturbation and therefore over-determined.

Stress-dependent morphogenesis: continuum mechanics and truss systems

A set of equilibrium equations is derived for the stress-controlled shape change of cells due to the remodelling and growth of their internal architecture. The approach involves the decomposition of the deformation gradient into an active and a passive component; the former is allowed to include a growth process, while the latter is assumed to be hyperelastic and mass-preserving. The two components are coupled with a control function that provides the required feedback mechanism. The balance equations for general continua are derived and, using a variational approach, we deduce the equilibrium equations and study the effects of the control function on these equations. The results are applied to a truss system whose function is to simulate the cytoskeletal network constituted by myosin microfilaments and microtubules, which are found experimentally to control shape change in cells. Special attention is paid to the conditions that a thermodynamically consistent formulation should satisfy. The model is used to simulate the multicellular shape changes observed during ventral furrow invagination of the Drosophila melanogaster embryo. The results confirm that ventral furrow invagination can be achieved through stress control alone, without the need for other regulatory or signalling mechanisms. The model also reveals that the yolk plays a distinct role in the process, which is different to its role during invagination with externally imposed strains. In stress control, the incompressibility constraint of the yolk leads, via feedback, to the generation of a pressure in the ventral zone of the epithelium that eventually eases its rise and internalisation.

Mapping forces and kinematics during collective cell migration

Fundamental biological processes including morphogenesis and tissue repair require cells to migrate collectively. In these processes, epithelial or endothelial cells move in a cooperative manner coupled by intercellular junctions. Ultimately, the movement of these multicellular systems occurs through the generation of cellular forces, exerted either on the substrate via focal adhesions (cell-substrate forces) or on neighboring cells through cell-cell junctions (cell-cell forces). Quantitative measurements of multicellular forces and kinematics with cellular or subcellular resolution have become possible only in recent years. In this chapter, we describe some of these techniques, which include particle image velocimetry to map cell velocities, traction force microscopy to map forces exerted by cells on the substrate, and monolayer stress microscopy to map forces within and between cells. We also describe experimental protocols to perform these measurements. The combination of these techniques with high-resolution imaging tools and molecular perturbations will lead to a better understanding of the mechanisms underlying collective cell migration in health and disease.