Matrix Organization and Dimensionality

National Institute Of Dental & Craniofacial Research

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Abstract

Cells interact with biochemically and structurally distinct forms of extracellular matrix in different tissues, at different stages of embryonic development, and during adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions: 1. What unique mechanisms do different types of mammalian cells use to migrate through different three-dimensional (3D) extracellular matrix environments compared to flat (2D) cell culture substrates? 2. Are there mechanisms for regulatory crosstalk between different types of extracellular matrix? We have been exploring which features of the classical models of cell motility and signaling established using regular 2D cell culture are valid in the structurally complex 3D environments found in tissues. The two major classes of extracellular matrix in mammals are the 3D fibrillar extracellular matrix and the 2D basement membrane. Although basement membranes can be three-dimensional on a macroscopic scale as they surround tissues, from the more microscopic point of view of individual cells adhering to a basement membrane, that substrate is basically two-dimensional. We recently reported that a variety of cell types interact with the components of 2D basement membranes -- i.e., laminin or collagen IV, or both together, or in the form of commercially available Matrigel extract -- by inducing exceptionally rapid assembly of a fibrillar fibronectin matrix. This form of regulatory cross-talk between the basement membrane type of extracellular matrix and fibrillar matrix assembly reveals a cell-based functional relationship linking these two very different types of matrix. We have initiated examination of cell interactions by other cell types with in vivo-derived basement membranes, which suggested that use of peritoneal basement membrane might provide a good model for examining invasion across basement membranes by tumor cells. Although studies of cell migration in 3D have become widespread, there are often differences in the types of 3D matrix used by different investigators for drawing conclusions about the mechanisms of 3D cell migration. We carefully compared the responses of cells to multiple different model systems of 3D extracellular matrix. We first compared two different types of 3D collagen hydrogel, rat tail collagen and bovine collagen, which differ in their extent of cross-linking and fibril arrangement. This commonly used type of 3D matrix is molecularly simple, since it lacks the other protein and proteoglycan components of in vivo extracellular matrix. Consequently, we compared the behavior of cells in these collagen matrices to the same cell type migrating within 'tissue matrix gel' derived from intact porcine mammary tissue, as well as with aligned cell-derived 3D matrices. The fibrillar matrix architecture of these systems differed substantially. 3D reconstituted rat tail collagen and tissue matrix gel matrices had short, randomly oriented collagen fibrils. In contrast, bovine collagen had long, larger fibril bundles. A particularly major difference was that the cell-derived matrices had strongly oriented parallel arrays of fibrils; the matrix generated by tumor-associated fibroblasts was particularly dense and anisotropic. We have observed a variety of differences in cellular responses, including differences in morphology, speed of motility, and directionality of migration. We conclude from this study that one cannot merely study cells in 3D matrix but must instead consider carefully the properties of the 3D environment. For example, there can be very different migration parameters depending on whether a matrix environment is oriented or not, and there are also clear differences depending on the composition of the 3D matrix that should ideally be similar to the in vivo system being studied. Consequently, modeling these processes in cell, organoid, or organ culture will require careful consideration in advance of experimentation concerning the composition and structural organization of the 3D matrix to be used. The assembly of basement membranes occurs at the basal surface of epithelial cells in embryos. In Drosophila, the machinery for synthesis and secretion of basement membrane collagen is at the basal end of the cell close to the site of assembly. In embryonic mouse salivary glands, however, mRNA for collagen IV and laminin is at the opposite, apical end of epithelial cells, along with the endoplasmic reticulum. We are investigating the mechanisms by which these important basement membrane components are transported, secreted, and assembled appropriately at the basal ends of embryonic mammalian organs. Focusing on the mechanisms of single-cell migration, we have been characterizing the local choreographed movements of specific cellular features of cells migrating in a 3D matrix. They include the local movements of cell-matrix adhesions, the leading edge, nucleus, and trailing edge of cells compared to the associated deformations of the local adjacent collagenous matrix. These studies have established a novel 3D cell migration cycle that differs from the classical 2D cell migration cycle studied in hundreds of studies on flat tissue culture dishes. Specifically, it involves a distinctive sequence initiated by an anterior cell contraction requiring myosin IIA, which then results in a striking anterior matrix deformation/prestrain, followed by protrusion of the leading edge (which in 2D instead starts first), then formation of new cell adhesions to add to an exceptional number of integrin-based cell surface adhesions, followed at the end of the cycle by a slow, relatively passive retraction of the cell rear. This mechanism is a form of front wheel drive for cells migrating in a 3D matrix, which contrasts with the classical retraction-coupled spreading of cells on 2D surfaces.