Emergence of Homeostatic Epithelial Packing and Stress Dissipation Through Divisions Oriented Along the Long Cell Axis
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Emergence of homeostatic epithelial packing and stress dissipation through divisions oriented along the long cell axis Tom P. J. Wyatta,b,c, Andrew R. Harrisc,d,1, Maxine Lamb,1, Qian Chenge, Julien Bellisb,f, Andrea Dimitracopoulosa,b, Alexandre J. Kablae, Guillaume T. Charrasc,g,h,2,3, and Buzz Baumb,h,2,3 aCenter for Mathematics, Physics, and Engineering in the Life Sciences and Experimental Biology, bMedical Research Council’s Laboratory for Molecular Cell Biology, gDepartment of Cell and Developmental Biology, and hInstitute for the Physics of Living Systems, University College London, London WC1E 6BT, United Kingdom; cLondon Centre for Nanotechnology, University College London, London, WC1H 0AH, United Kingdom; dBioengineering, University of California, Berkeley, CA 94720; eDepartment of Engineering, University of Cambridge, Cambridge CB2 1PZ, United Kingdom; and fCentre de Recherche de Biochimie Macromoléculaire, 34293 Montpellier, France Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved March 31, 2015 (received for review November 3, 2014) Cell division plays an important role in animal tissue morphogen- At the cellular level, relatively simple rules appear to govern esis, which depends, critically, on the orientation of divisions. In division orientation. These rules were first explored by Hertwig isolated adherent cells, the orientation of mitotic spindles is (14), who showed that cells from early embryos divide along their sensitive to interphase cell shape and the direction of extrinsic long axis, and were further refined using microfabricated chambers mechanical forces. In epithelia, the relative importance of these (15). However, by following division orientation in cells adhering two factors is challenging to assess. To do this, we used suspended to micropatterned substrates, more recent studies identified additional roles for both the geometrical arrangement of integ- monolayers devoid of ECM, where divisions become oriented – following a stretch, allowing the regulation and function of rin-mediated cell substrate adhesions (16) and extrinsic me- epithelial division orientation in stress relaxation to be character- chanical forces in orienting divisions (17). Consistent with this, adhesive and mechanical cues have been reported to guide di- ized. Using this system, we found that divisions align better with vision orientation in vivo (18) and in epithelial monolayers in the long, interphase cell axis than with the monolayer stress axis. developing embryos (12, 19). Nevertheless, the respective roles Nevertheless, because the application of stretch induces a global of cell shape and mechanical tension in guiding division orien- realignment of interphase long axes along the direction of exten- tation in epithelia remain poorly defined, as does the contribu- sion, this is sufficient to bias the orientation of divisions in the tion of oriented division to mechanical feedback control. direction of stretch. Each division redistributes the mother cell mass Previously, we established suspended epithelial monolayers along the axis of division. Thus, the global bias in division orientation lacking ECM as a minimal model system in which to study epithelial enables cells to act collectively to redistribute mass along the axis of biology. Because cell divisions in these monolayers become oriented stretch, helping to return the monolayer to its resting state. Further, following a stretch, we were able to explore the regulation and this behavior could be quantitatively reproduced using a model function of division orientation. We found that divisions align better designed to assess the impact of autonomous changes in mitotic cell with the long, interphase cell axis than with the monolayer stress mechanics within a stretched monolayer. In summary, the propensity axis. This phenomenon, combined with the alignment of cellular of cells to divide along their long axis preserves epithelial homeo- long axes induced by stretch, results in a global bias in the orien- stasis by facilitating both stress relaxation and isotropic growth tation of divisions in the direction of extension. Each division without the need for cells to read or transduce mechanical signals. Significance morphogenesis | cell division | mechanical feedback | quantitative biology | mitotic rounding Animal cells undergo a remarkable series of shape changes as they pass through mitosis and divide. In an epithelial tissue, the he morphogenesis of animal tissues results from coordinated impact of these morphogenetic processes depends strongly on Tchanges in the shape, size, and packing of their constituent the orientation of division. However, the cues orienting divisions cells (1–3). These include autonomous cell shape changes (4), remain poorly understood. Here, we combine live imaging and the response of cells to extrinsic stresses, and the effects of mechanical perturbations with computational modeling to in- passive tissue deformation (5). When coordinated across a tissue, vestigate the effects of shape changes accompanying mitosis these active cellular processes and passive responses enable ep- and division in stretched monolayers in the absence of neighbor exchange. We show that divisions orient with the long cell axis ithelial sheets to undergo shape changes while retaining rela- rather than with the stress direction, and show how oriented tively normal cell packing (6) and help return tissues to their divisions contribute to the restoration of cell packing and stress resting state following a perturbation (7). Although the molec- relaxation. In doing so, we identify a clear role for oriented cell ular basis of this cooperation is not understood, several studies division in morphogenetically active tissues. have suggested a role for mechanical feedback (8, 9). Cell di- vision has been suggested to participate in this feedback (10) Author contributions: T.P.J.W., A.J.K., G.T.C., and B.B. designed research; T.P.J.W., A.R.H., because the rate of animal cell proliferation responds to changes Q.C., and J.B. performed research; T.P.J.W., M.L., Q.C., J.B., and A.D. analyzed data; and T.P.J.W., A.J.K., G.T.C., and B.B. wrote the paper. in extrinsic forces in several experimental settings (9). Further, The authors declare no conflict of interest. division makes an important contribution to tissue morphogen- This article is a PNAS Direct Submission. esis in animals (11, 12), accounts for much of the topological 1A.R.H. and M.L. contributed equally to this work. disorder observed in epithelia (13), can drive tissue elongation 2G.T.C. and B.B. contributed equally to this work. (10), and can facilitate the return to homeostatic cell packing 3To whom correspondence may be addressed. Email: [email protected] or b.baum@ following a deformation (2). Importantly, for each of these ucl.ac.uk. functions, the impact of cell division depends critically on the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. orientation of divisions. 1073/pnas.1420585112/-/DCSupplemental. 5726–5731 | PNAS | May 5, 2015 | vol. 112 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1420585112 Downloaded by guest on September 29, 2021 redistributes cell mass along the axis of division. Thus, when ori- A No stretch Stretch B Stretch ented across a monolayer, divisions act collectively to redistribute 2.5 No stretch mass along the axis of stretch, helping to return the monolayer to its 2.0 resting state. In summary, this analysis shows that the propensity of cells to divide along their long axis preserves epithelial homeostasis 1.5 1.0 by facilitating both stress relaxation and isotropic growth without -90 -45 0 45 90 the need for cells to read or transduce mechanical signals. Stretch axis Interphase aspect ratio Cell long axis direction w.r.t stretch direction (degrees) Results C 40 D 40 400 400 Strain 30 Strain 30 Uniaxial Monolayer Extension Results in Sustained Cell Elongation 300 Force 300 Force and Tension Oriented Along the Axis of Stretch. Previously, we used 20 – 200 200 20 suspended Madin Darby canine kidney II (MDCK) monolayers as 10 a model system with which to study epithelial mechanics (20). For 100 100 10 Tissue stretch (%) 0 Tissue stretch (%) 0 Force per cell (nN) the study of cell division in monolayers, we modified the device (21) 017030 10051 Force per cell (nN) 0 50 100 150 200 to allow imaging over several hours (Fig. S1). Following a stretch, Time (sec) Time (min) Before perturbation After cells in suspended monolayers did not change neighbors (Fig. S2). n = 25 n = 20 cells E F 90 Instead, they responded by elongating in the direction of stretch by not significant an amount roughly equal to the amount applied at the monolayer stretch axis aligned with 60 level (20). Extension was accompanied by a decrease in monolayer Cell long axis thickness (20) and a small decrease in width. Cells then remained Stretch axis A B Before perturbation elongated until they divided (Fig. 1 and and Fig. S3). After 30 We confirmed that suspended monolayers generated through collagenase treatment (20) were devoid of a continuous load- 0 stretch orientation (degrees) bearing ECM but retained apicobasal polarization over the course Local stress orientation w.r.t. 0˚ 90˚ stretch axis of our experiments (Fig. S4). Hence, the transmission of tension Cell long axis Cell long axis orientation perpendicular to across suspended monolayers depends entirely on intercellular Stretch axis w.r.t. stretch direction junctions, which remained stable over the