Epithelial repair is a two-stage process driven first by dying cells and then by their neighbours.

Epithelial cells maintain an essential barrier despite continuously undergoing mitosis and apoptosis. Biological and biophysical mechanisms have evolved to remove dying cells while maintaining that barrier. Cell extrusion is thought to be driven by a multicellular filamentous actin ring formed by neighbouring cells, the contraction of which provides the mechanical force for extrusion, with little or no contribution from the dying cell. Here, we use live confocal imaging, providing time-resolved three-dimensional observations of actomyosin dynamics, to reveal new mechanical roles for dying cells in their own extrusion from monolayers. Based on our observations, the clearance of dying cells can be subdivided into two stages. The first, previously unidentified, stage is driven by the dying cell, which exerts tension on its neighbours through the action of a cortical contractile F-actin and myosin ring at the cell apex. The second stage, consistent with previous studies, is driven by a multicellular F-actin ring in the neighbouring cells that moves from the apical to the basal plane to extrude the dying cell. Crucially, these data reinstate the dying cell as an active physical participant in cell extrusion.

A kinetic mechanism for cell sorting based on local variations in cell motility.

Our current understanding of cell sorting relies on physical difference, either in the interfacial properties or motile force, between cell types. But is such asymmetry a prerequisite for cell sorting? We test this using a minimal model in which the two cell populations are identical with respect to their physical properties and differences in motility arise solely from how cells interact with their surroundings. The model resembles the Schelling model used in social sciences to study segregation phenomena at the scale of societies. Our results demonstrate that segregation can emerge solely from cell motility being a dynamic property that changes in response to the local environment of the cell, but that additional mechanisms are necessary to reproduce the envelopment behaviour observed in vitro. The time course of segregation follows a power law, in agreement with the scaling reported from experiment and in other models of motility-driven segregation.

A phenomenological density-scaling approach to lamellipodial actin dynamics(†).

The integration of protein function studied in vitro in a dynamic system like the cell lamellipodium remains a significant challenge. One reason is the apparent contradictory effect that perturbations of some proteins can have on the overall lamellipodium dynamics, depending on exact conditions. Theoretical modelling offers one approach for understanding the balance between the mechanisms that drive and regulate actin network growth and decay. Most models use a 'bottom-up' approach, involving explicitly assembling biochemical components to simulate observable behaviour. Their correctness therefore relies on both the accurate characterization of all the components and the completeness of the relevant processes involved. To avoid potential pitfalls due to this uncertainty, we used an alternative 'top-down' approach, in which measurable features of lamellipodium behaviour, here observed in two different cell types (HL60 and B16-F1), directly inform the development of a simple phenomenological model of lamellipodium dynamics. We show that the kinetics of F-actin association and dissociation scales with the local F-actin density, with no explicit location dependence. This justifies the use of a simplified kinetic model of lamellipodium dynamics that yields predictions testable by pharmacological or genetic intervention. A length-scale parameter (the lamellipodium width) emerges from this analysis as an experimentally accessible probe of network regulatory processes.

Separation of blood cells with differing deformability using deterministic lateral displacement(†).

Determining cell mechanical properties is increasingly recognized as a marker-free way to characterize and separate biological cells. This emerging realization has led to the development of a plethora of appropriate measurement techniques. Here, we use a fairly novel approach, deterministic lateral displacement (DLD), to separate blood cells based on their mechanical phenotype with high throughput. Human red blood cells were treated chemically to alter their membrane deformability and the effect of this alteration on the hydrodynamic behaviour of the cells in a DLD device was investigated. Cells of defined stiffness (glutaraldehyde cross-linked erythrocytes) were used to test the performance of the DLD device across a range of cell stiffness and applied shear rates. Optical stretching was used as an independent method for quantifying the variation in stiffness of the cells. Lateral displacement of cells flowing within the device, and their subsequent exit position from the device were shown to correlate with cell stiffness. Data showing how the isolation of leucocytes from whole blood varies with applied shear rate are also presented. The ability to sort leucocyte sub-populations (T-lymphocytes and neutrophils), based on a combination of cell size and deformability, demonstrates the potential for using DLD devices to perform continuous fractionation and/or enrichment of leucocyte sub-populations from whole blood.

Analysis of turnover dynamics of the submembranous actin cortex.

The cell cortex is a thin network of actin, myosin motors, and associated proteins that underlies the plasma membrane in most eukaryotic cells. It enables cells to resist extracellular stresses, perform mechanical work, and change shape. Cortical structural and mechanical properties depend strongly on the relative turnover rates of its constituents, but quantitative data on these rates remain elusive. Using photobleaching experiments, we analyzed the dynamics of three classes of proteins within the cortex of living cells: a scaffold protein (actin), a cross-linker (α-actinin), and a motor (myosin). We found that two filament subpopulations with very different turnover rates composed the actin cortex: one with fast turnover dynamics and polymerization resulting from addition of monomers to free barbed ends, and one with slow turnover dynamics with polymerization resulting from formin-mediated filament growth. Our data suggest that filaments in the second subpopulation are on average longer than those in the first and that cofilin-mediated severing of formin-capped filaments contributes to replenishing the filament subpopulation with free barbed ends. Furthermore, α-actinin and myosin minifilaments turned over significantly faster than F-actin. Surprisingly, only one-fourth of α-actinin dimers were bound to two actin filaments. Taken together, our results provide a quantitative characterization of essential mechanisms under-lying actin cortex homeostasis.

Mechanisms of leading edge protrusion in interstitial migration.

While the molecular and biophysical mechanisms underlying cell protrusion on two-dimensional substrates are well understood, our knowledge of the actin structures driving protrusion in three-dimensional environments is poor, despite relevance to inflammation, development and cancer. Here we report that, during chemotactic migration through microchannels with 5 µm × 5 µm cross-sections, HL60 neutrophil-like cells assemble an actin-rich slab filling the whole channel cross-section at their front. This leading edge comprises two distinct F-actin networks: an adherent network that polymerizes perpendicular to cell-wall interfaces and a 'free' network that grows from the free membrane at the cell front. Each network is polymerized by a distinct nucleator and, due to their geometrical arrangement, the networks interact mechanically. On the basis of our experimental data, we propose that, during interstitial migration, medial growth of the adherent network compresses the free network preventing its retrograde movement and enabling new polymerization to be converted into forward protrusion.