Three-dimensional morphogenesis of MDCK cells induced by cellular contractile forces on a viscous substrate

Misako Imai,Takeomi Mizutani,Kazushige Kawabata,Hisashi Haga,Kazuya Furusawa

doi:10.1038/srep14208

Abstract

Substrate physical properties are essential for many physiological events such as embryonic development and 3D tissue formation. Physical properties of the extracellular matrix such as viscoelasticity and geometrical constraints are understood as factors that affect cell behaviour. In this study, we focused on the relationship between epithelial cell 3D morphogenesis and the substrate viscosity. We observed that Madin-Darby Canine Kidney (MDCK) cells formed 3D structures on a viscous substrate (Matrigel). The structures appear as a tulip hat. We then changed the substrate viscosity by genipin (GP) treatment. GP is a cross-linker of amino groups. Cells cultured on GP-treated-matrigel changed their 3D morphology in a substrate viscosity-dependent manner. Furthermore, to elucidate the spatial distribution of the cellular contractile force, localization of mono-phosphorylated and di-phosphorylated myosin regulatory light chain (P-MRLCs) was visualized by immunofluorescence. P-MRLCs localized along the periphery of epithelial sheets. Treatment with Y-27632, a Rho-kinase inhibitor, blocked the P-MRLCs localization at the edge of epithelial sheets and halted 3D morphogenesis. Our results indicate that the substrate viscosity, the substrate deformation, and the cellular contractile forces induced by P-MRLCs play crucial roles in 3D morphogenesis.

Highlights

Enhances the degree of elasticity and the traction force of a single cell[20]
In NT and dimethyl sulfoxide (DMSO) treated samples, we found that mono- and di-phosphorylated MRLC (1P-MRLC and 2P-MRLC) localized along the periphery of the tulip hat-like structure (Fig. 5a,b, arrowheads). 1P-MRLC and 2P-MRLC localization toward the outer edge of the epithelial sheet structure disappeared when the cells were treated with 10 μ M Y-27632 (Fig. 5c)
We detected 1P-MRLC and 2P-MRLC phosphorylation in cells treated with Y-27632 and ML-7 by western blotting. Both 1P-MRLC and 2P-MRLC significantly decreased after treatment with 10 μ M Y-27632 (**p < 0 .01), and 1P-MRLC decreased after treatment with 2 μ M ML-7 (*p < 0 .05, Fig. 5e,f). These results indicate that 2P-MRLC localization toward the outer edge of the epithelial sheet is essential for the 3D tulip hat-like morphogenesis, though MRLC mono-phosphorylation affects it

Summary

Introduction

Enhances the degree of elasticity and the traction force of a single cell[20]. The signalling pathways involved in MRLC phosphorylation have been previously described. The small G protein, RhoA, activates the Rho-associated protein kinase (Rho-kinase), which phosphorylates MRLC21. During tissue invagination and furrow formation during the development process, cellular contractile forces support the proper morphogenesis. In the development of a mouse lens, the balanced activities of small G proteins, Rac[1] and RhoA, were shown to control cell shape and support proper lens morphogenesis[24]. The inhibition of RhoA, ROCK, and myosin II activities in MDCK cells led to the inversion of the orientation of epithelial cell polarity, resulting in abnormal cyst formation[12]. The cells presented a tulip hat-like 3D morphology induced by the deformation of the peripheral substrate via cellular contractile forces. The cellular contractile forces generated in the edge of the cell sheets were required for the tulip hat-like morphogenesis

Methods

Results

Conclusion