Jcb_201309029 1..8

Cell division is an integral part of tissue morphogenesis and homeostasis, and control of the orientation of cell division is essential to proper development and maintenance of tissue architecture (Gillies and Cabernard, 2011; Morin and Bellaïche, 2011). In 2D cultures, when a cell rounds to divide, it maintains numerous short retraction fibers that link the cell body to the substratum. The retraction fibers bear forces that direct the orientation of the mitotic spindle (Théry et al., 2005; Fink et al., 2011) and help the daughter cells to respread and separate (Cramer and Mitchison, 1993; Burton and Taylor, 1997). In vivo, mitotic cells in the developing zebrafish neural tube (Alexandre et al., 2010) or in the nervous system and retina of the mouse embryo (Saito et al., 2003; Kosodo et al., 2008) form daughter cells whose differentiation fates depend on their connections to their extracellular surroundings. It seems likely that physical interaction between cells and the extracellular matrix is crucial for proper regulation of cell division. Previous studies of cell division in culture have traditionally used glass or plastic dishes. These 2D culture systems have yielded important insights into the mechanism of cell division; however, they present environments that are rigid, uniform, and flat, and thus fail to reflect the character of cell–matrix interactions encountered in vivo. Natural fibrous matrices such as collagen or fibrin mimic more closely the physiological extracellular matrix (Cukierman et al., 2002; Pampaloni et al., 2007; Fraley et al., 2010; Hakkinen et al., 2011). However, the measurement of forces induced by cells fully embedded in 3D matrices is a challenge that requires 3D live-cell imaging and quantitative, minimally invasive tools. Thus, we have a limited understanding of how physical forces regulate cell division in 3D environments. Recent advances have extended measurements of 2D planar stresses to the third dimension by using confocal imaging combined with digital volume correlation (DVC; Maskarinec et al., 2009) or particle tracking algorithms (Legant et al., 2010; Koch et al., 2012) to resolve matrix displacements in all three spatial dimensions. Previous work has shown that external forces regulate cell division in 2D cultures (Burton and Taylor, 1997; Fink et al., 2011). Here we examine the hypothesis that forces applied by dividing cells against the extracellular matrix (“traction forces”) control the orientation of cell division in three dimensions. We use a physiologically relevant matrix that mimics the essential features of many tissue environments: soft, fibrous, and 3D. By combining 4D (x, y, z, and t) time-lapse imaging with DVC, we mapped full-field matrix displacements to identify sites at which Physical forces direct the orientation of the cell division axis for cells cultured on rigid, two-dimensional (2D) substrates. The extent to which physical forces regulate cell division in three-dimensional (3D) environments is not known. Here, we combine live-cell imaging with digital volume correlation to map 3D matrix displacements and identify sites at which cells apply contractile force to the matrix as they divide. Dividing cells embedded in fibrous matrices remained anchored to the matrix by long, thin protrusions. During cell rounding, the cells released adhesive contacts near the cell body while applying tensile forces at the tips of the protrusions to direct the orientation of the cell division axis. After cytokinesis, the daughter cells respread into matrix voids and invaded the matrix while maintaining traction forces at the tips of persistent and newly formed protrusions. Mechanical interactions between cells and the extracellular matrix constitute an important mechanism for regulation of cell division in 3D environments. Contractile forces regulate cell division in three-dimensional environments