ARTICLE Received 28 Feb 2015 | Accepted 12 Oct 2015 | Published 25 Nov 2015 DOI: 10.1038/ncomms9872 OPEN Mitotic cells contract actomyosin cortex and generate pressure to round against or escape epithelial confinement Barbara Sorce1, Carlos Escobedo1,2, Yusuke Toyoda3, Martin P. Stewart1,4,5, Cedric J. Cattin1, Richard Newton1, Indranil Banerjee6, Alexander Stettler1, Botond Roska6, Suzanne Eaton3, Anthony A. Hyman3, Andreas Hierlemann1 & Daniel J. Mu¨ller1 Little is known about how mitotic cells round against epithelial confinement. Here, we engineer micropillar arrays that subject cells to lateral mechanical confinement similar to that experienced in epithelia. If generating sufficient force to deform the pillars, rounding epithelial (MDCK) cells can create space to divide. However, if mitotic cells cannot create sufficient space, their rounding force, which is generated by actomyosin contraction and hydrostatic pressure, pushes the cell out of confinement. After conducting mitosis in an unperturbed manner, both daughter cells return to the confinement of the pillars. Cells that cannot round against nor escape confinement cannot orient their mitotic spindles and more likely undergo apoptosis. The results highlight how spatially constrained epithelial cells prepare for mitosis: either they are strong enough to round up or they must escape. The ability to escape from confinement and reintegrate after mitosis appears to be a basic property of epithelial cells. 1 Department of Biosystems Science and Engineering, Eidgeno¨ssische Technische Hochschule (ETH) Zurich, Mattenstrasse 26, Basel 4058, Switzerland. 2 Department of Chemical Engineering, Queen’s University, 19 Division Street, Kingston, Ontario, Canada K7L 3N6. 3 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. 4 Department of Chemical Engineering, Massachusetts Institute of Technology (MIT), 500 Main Street, Cambridge, Massachusetts 02139-4307, USA. 5 The David H. Koch Institute for Integrative Cancer Research, 500 Main Street, Cambridge, Massachusetts 02139-4307, USA. 6 Neural Circuit Laboratories, Friedrich Miescher Institute (FMI) for Biomedical Research, Maulbeerstrasse 66, Basel 4058, Switzerland. Correspondence and requests for materials should be addressed to D.J.M. (email: [email protected]). NATURE COMMUNICATIONS | 6:8872 | DOI: 10.1038/ncomms9872 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9872 t the beginning of mitosis, cells markedly change their arrays of micropillars that can be used to investigate forces morphology as they round up1,2. During mitotic cell generated by cell adhesion, migration and differentiation at Arounding, the microtubule cytoskeleton forms the mitotic subcellular scales38–41. Analysing the deflection of micropillars of spindle, a central machinery that captures and organizes known geometry and dimensions in response to cell-generated chromosomes3,4. Mitotic cell rounding occurs in the vast forces allows the quantification of these forces and sheds light on majority of animal cells1,5 and plays a role in maintaining the dynamic processes of adhesion, differentiation and tissue organization2,6–10. It is now clear from studies in tissue mechanotransduction. To measure mechanical forces at the culture that cell rounding is driven by the contraction of the subcellular level in these applications, the micropillar spacing actomyosin cortex and associated proteins4,6,10–13. The cortex can must be much smaller compared with cellular dimensions. Until only produce contractile forces and mitotic cells also generate an now, however, micropillar arrays that mimic the mechanical outward force by the modulation of intracellular pressure, which constraints of the epithelia have not been introduced. is governed by plasma membrane transporters14. Together, these Here we introduce micropillar arrays with spatial and mechanisms lead to an B10-fold increase in cortex tension and mechanical properties designed to impose lateral confinement hydrostatic pressure as cells progress through mitosis14,15. Recent on epithelial cells similar to that which they would experience studies have revealed that the generation of cell cortex contraction normally in monolayers. For our studies we use Madin–Darby and tension directly correlates with the accumulation of active canine kidney (MDCK) cell lines that are commonly used as a myosin II at the cortex16. The master regulator of mitosis, cyclin- model for epithelial cells11,42. We observe that MDCK cells dependent kinase 1, balances cell cortex tension and hydrostatic deflect nearby micropillars as they round and thereby create pressure by using RhoA kinase to stimulate and p21-activated sufficient space for mitosis21,22. Optically analysing the deflection kinases to suppress myosin II recruitment to the cortex. of the individual confining micropillars enables us to quantify the While previous in vitro studies provide valuable insight into the rounding force generated by individual mitotic cells. By specific mechanism of cell rounding, they do not fully describe the chemical perturbation, we confirm that this rounding force rounding of cells in vivo. Cells in vivo are spatially confined in is generated by an inward-directed actomyosin contraction more than one dimension by other cells and surrounding tissue and an outward-directed hydrostatic (for example, osmotic) and, to round, a mitotic cell must exert force9,17–19. The pressure14,21,22. If micropillar separation does not allow sufficient mechanisms of cell rounding in the confinement of tissue are space for mitotic cell rounding, cells travel up the pillars to escape not well studied. Cell culture studies indicate that the loss of confinement, driven by actomyosin contraction and the substrate adhesion is sufficient for the rounding of isolated cells20, hydrostatic pressure generated by the rounding mitotic cell. but that actomyosin cortex contraction and the accompanying Once freed from confinement, these cells conduct mitosis in an increase in intracellular pressure are required for the generation unperturbed manner. After mitosis, the daughter cells return to of rounding forces against confining structures14,21,22. spaces between the confining micropillars. Mitotic cells unable to Cell rounding under confinement is particularly relevant to cell escape spatial confinement cannot orient their microtubule division in an epithelium. Epithelia comprise densely packed spindle properly and are more likely to undergo apoptosis. layers of cells that are organized into sheets. These sheets form tissues such as the epidermis, the surfaces of the eye and the surfaces of the hollow tubes and sacs that make up the digestive, Results respiratory, reproductive and urinary tracts. Tightly packed Mitotic cells round against adjacent cells and change height. epithelial cells secrete an extracellular matrix called the basal We wanted to characterize morphological changes of mitotic cells lamina, which anchors the epithelial tissue to the basement in the epithelium. As an established epithelial in vitro model, we membrane. This membrane acts as a scaffold on which epithelial chose MDCK cells. When cultivated on membrane supports, cells can grow and regenerate after injury. Epithelia fulfil a variety MDCK cells differentiate in a cuboidal epithelial layer28 and of functions including protection, absorption, sensory reception retain properties characteristic of kidney epithelial cells, such as and secretion. Tight junctions between cells enable epithelial tight junctions and distinct basal and apical membrane layers to act as effective mechanical barriers23,24. If epithelial domains20,29. Using confocal microscopy we monitored mitotic layers are damaged, their protective role is compromised which MDCK cells in the epithelial layer (Fig. 1). We followed the may result in problems in tissue development and regeneration or mitotic state by expressing enhanced green fluorescent protein the occurrence of diseases such as cancer25–27. It has been shown (eGFP)-labelled histones (H2B-eGFP), the actomyosin cortex by that epithelial cells rounding for mitosis regulate adhesion and expressing actin-mCherry and cell junctions by expressing eGFP- orient their spindle axes28,29. Epithelial cells that cannot round labelled E-cadherin. Confocal microscopy images show epithelial for mitosis cannot properly orient and assemble their mitotic mitotic cells rounding and increasing in height by E5 mm while spindle, which can lead to their mislocalization within the tissue retaining tight junctions. Mitotic rounding was observed for and eventually to apoptosis, cancer or other disease states7,18,30. MDCK cells plated on permeable filter supports and grown for 3 Despite our understanding of the role and importance of days (Fig. 1a) and 14 days (Supplementary Fig. 1). This epithelia, the mechanisms governing the rounding of epithelial observation is in accord with previous reports that mitotic cells for mitosis and their influence on cell division have not yet epithelial cells lose their cuboidal architecture and round towards been fully described. the apical surface of the epithelia20,28. Furthermore, the confocal Cells in vivo continually encounter and respond to a multitude images reveal that mitotic cells mechanically deform adjacent of environmental stimuli. While the role of biochemical signals cells and orient their metaphase plate perpendicular to the has