SummaryThe contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division [1–3], migration [4], and tissue morphogenesis [5]. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells [6] and during division, where its misregulation can lead to aneuploidy [7]. Despite its importance, our knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence [8–15]. Here, we used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 and the Arp2/3 complex. Each nucleator contributed a similar amount of F-actin to the cortex but had very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. Our findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator.

Epithelial monolayers are one-cell thick tissue sheets that separate internal and external environments. As part of their function, they have to withstand extrinsic mechanical stresses applied at high strain rates. However, little is known about how monolayers respond to mechanical deformations. Here, by subjecting suspended epithelial monolayers to stretch, we find that they dissipate stresses on a minute timescale in a process that involves an increase in monolayer length, pointing to active remodelling of cell architecture during relaxation. Strikingly, monolayers consisting of tens of thousands of cells relax stress with similar dynamics to single rounded cells and both respond similarly to perturbations of actomyosin. By contrast, cell-cell junctional complexes and intermediate filaments do not relax tissue stress, but form stable connections between cells, allowing monolayers to behave rheologically as single cells. Taken together our data show that actomyosin dynamics governs the rheological properties of epithelial monolayers, dissipating applied stresses, and enabling changes in monolayer length. the Rosetrees Trust, the UCL Graduate School, the EPSRC funded doctoral training program CoMPLEX, and the European Research Council (ERC-CoG MolCellTissMech, agreement 647186 to GC). N.K. was in receipt of a UCL Overseas Research Scholarship. N.K. was supported by the Prof Rob Seymour Travel Bursary Fund for research visits to Barcelona. J.F. and A.B. were funded by BBSRC grant (BB/M003280 and BB/M002578) to G.

Cellular shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here, we investigate how nucleation-promoting factors (NPFs) mediate interaction between nucleators. In vitro, the NPF SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our experiments in vitro reveal this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.