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.
SUMMARY For nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g. Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long axis orientations of their adjacent mitotic neighbors. Strikingly, analysis of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage plane orientation, and that cleavage plane bias may be a widespread property of polygonal cell sheets in plants and animals.
Mechanical forces play a key role in a wide range of biological processes, from embryogenesis to cancer metastasis, and there is considerable interest in the intuitive question, “Can cellular forces be inferred from cell shapes?” Although several groups have posited affirmative answers to this stimulating question, nagging issues remained regarding equation structure, solution uniqueness and noise sensitivity. Here we show that the mechanical and mathematical factors behind these issues can be resolved by using curved cell edges rather than straight ones. We present a new package of force-inference equations and assessment tools and denote this new package CellFIT, the Cellular Force Inference Toolkit. In this approach, cells in an image are segmented and equilibrium equations are constructed for each triple junction based solely on edge tensions and the limiting angles at which edges approach each junction. The resulting system of tension equations is generally overdetermined. As a result, solutions can be obtained even when a modest number of edges need to be removed from the analysis due to short length, poor definition, image clarity or other factors. Solving these equations yields a set of relative edge tensions whose scaling must be determined from data external to the image. In cases where intracellular pressures are also of interest, Laplace equations are constructed to relate the edge tensions, curvatures and cellular pressure differences. That system is also generally overdetermined and its solution yields a set of pressures whose offset requires reference to the surrounding medium, an open wound, or information external to the image. We show that condition numbers, residual analyses and standard errors can provide confidence information about the inferred forces and pressures. Application of CellFIT to several live and fixed biological tissues reveals considerable force variability within a cell population, significant differences between populations and elevated tensions along heterotypic boundaries.
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.embryo morphogenesis | tissue mechanics | biomechanics | cinemechanometry
Laser microsurgery and finite element modeling are used to determine the cell-level mechanics of the amnioserosa-a morphogenetically crucial epithelium on the dorsal surface of fruit fly embryos (Drosophila melanogaster). In the experiments, a tightly focused laser ablates a subcellular hole (1 microm in diameter) that passes clean through the epithelium. The surrounding cells recoil from the wound site with a large range of initial recoil velocities. These depend on the embryo's developmental stage and the subcellular wound site. The initial recoil (up to 0.1 s) is well reproduced by a base finite element model, which assumes a uniform effective viscosity inside the cells, a constant tension along each cell-cell boundary, and a large, potentially anisotropic, far-field stress--one that far exceeds the stress equivalent of the cell-edge tensions. After 0.1 s, the experimental recoils slow dramatically. This observation can be reproduced by adding viscoelastic rods along cell edges or as a fine prestressed mesh parallel to the apical and basal membranes of the cell. The mesh also reproduces a number of double-wounding experiments in which successive holes are drilled in a single cell.
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