Rotational motion during three-dimensional morphogenesis of mammary epithelial acini relates to laminin matrix assembly

Abstract

Our understanding of the mechanisms by which ducts and lobules develop is derived from model organisms and three-dimensional (3D) cell culture models wherein mammalian epithelial cells undergo morphogenesis to form multicellular spheres with a hollow central lumen. However, the mechanophysical properties associated with epithelial morphogenesis are poorly understood. We performed multidimensional live-cell imaging analysis to track the morphogenetic process starting from a single cell to the development of a multicellular, spherical structure composed of polarized epithelial cells surrounding a hollow lumen. We report that in addition to actively maintaining apicobasal polarity, the structures underwent rotational motions at rates of 15-20 μm/h and the structures rotated 360°every 4 h during the early phase of morphogenesis. Rotational motion was independent of the cell cycle, but was blocked by loss of the epithelial polarity proteins Scribble or Pard3, or by inhibition of dynein-based microtubule motors. Interestingly, none of the structures derived from human cancer underwent rotational motion. We found a direct relationship between rotational motion and assembly of endogenous basement membrane matrix around the 3D structures, and that structures that failed to rotate were defective in weaving exogenous laminin matrix. Dissolution of basement membrane around mature, nonrotating acini restored rotational movement and the ability to assemble exogenous laminin. Thus, coordinated rotational movement is a unique mechanophysical process observed during normal 3D morphogenesis that regulates laminin matrix assembly and is lost in cancer-derived epithelial cells. extracellular matrix | tubulogenesis | plasticity T issues are composed of a community of cells that act in a coordinated manner to achieve and maintain normal architecture and perform their function. The mechanisms by which cells coordinate their behavior during tissue morphogenesis is poorly understood in part due to technical bottlenecks associated with studying the process in vivo. Three-dimensional (3D) culture systems provide an environment in which normal tissue morphogenesis can be recapitulated and thus is a powerful tool for investigating the molecular signals that specify epithelial tissue architecture (1-3). Unlike monolayer cultures, epithelial cells grown in 3D recapitulate numerous features seen in vivo, including the formation of acini-like spheroids with a hollow lumen, apicobasal polarization of cells comprising these acini, and the deposition of basement membrane components collagen IV and laminin-332 (2).The initial stages of 3D morphogenesis are characterized by the ability of epithelial cells to detect surfaces with which they come in contact. Cells use integrins and dystroglycans to contact the extracellular matrix (ECM), and cadherins and desmosomes to contact their neighbors (3). These initial contacts trigger a series of cell remodeling events, which result in polarization of cells to create a basolateral surface and an apical surfa...

An active role for basement membrane assembly and modification in tissue sculpting

“…Mammary acini growing in 3D culture also rotate while assembling basement membrane (Tanner et al, 2012;Wang et al, 2013). In addition, a corset of ECM might also be used to facilitate the anisotropic cell growth required for hypocotyl elongation in Arabidopsis (Paredez et al, 2006).…”

Cytoskeletal Symmetry Breaking and Chirality: From Reconstituted Systems to Animal Development

“…This process also requires the atypical cadherin Fat [220], which is also involved in L/R symmetry breaking in other organs of D. melanogaster (see above, [214]). Interestingly, rotational motion of mammary epithelial acini in 3D environments also leads to extracellular matrix deposition and lack of rotation leads to a loss of matric deposition [222].…”

A basal cell defect promotes budding of prostatic intraepithelial neoplasia

“…2A) and observed a dramatic cellspinning phenotype as spheroids formed from a single cell (Movies 1 and 2). The rotational motion was similar to mammary epithelial acini that coordinate rotational movement with laminin matrix assembly (Wang et al, 2013). Most (65%) of the RWPE-1 spheroids contained a lumen after 10 days, as measured by presence of β-catenin on the cell surface and absence of DAPI staining nuclei in the interstices of the spheroid (Fig.…”