Matrix Organization and Dimensionality

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications, trials & patents

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 major questions concerning the mechanisms of these cell-extracellular matrix interactions. For example, what unique mechanisms do different types of mammalian cells use to adhere to and to migrate through different three-dimensional (3D) extracellular matrix environments compared to flat (2D) cell culture substrates, and how do human cells generate fibronectin fibrils in 2D and 3D matrix environments? We are currently exploring the cell-matrix adhesions formed to 2D compared to 3D fibrillar extracellular matrix under dynamic conditions. An extracellular matrix molecule that has been studied in considerable depth in regular 2D cell culture is fibronectin. We and others previously described two distinct mechanisms for assembly of fibrillar fibronectin, one involving lateral translocation of the alpha5-beta1 integrin along flat tissue culture substrates, and a second mechanism involving the translocation of entire focal adhesions along a basement membrane substrate driven by an actomyosin "contractile winch" system. We are continuing these studies of the mechanisms of fibronectin matrix assembly. We are also exploring in depth our published observation that there is an unusually high level of integrin activation in a 3D fibrillar collagen environment to determine the role of actomyosin contractility in this activation state. CRISPR-Cas9 has provided powerful tools for editing genes. Our laboratory developed a straightforward, stepwise protocol to knock out any specific target gene in human fibroblasts. The approach included designing a sgRNA, construction of a single vector expressing both sgRNA and Cas9, rapid production of high-titer lentiviruses, and transduction of cells to generate efficiently a pool of cells with a high percentage of specific knockout of that gene. These studies are providing insights into the roles of dimensionality and specific genes in the interactions of cells with the extracellular matrix. They can range from a "1D" interaction as a cell interacts with a single bundle of collagen, to 2D on basement membranes and flat substrates, to 3D in native interstitial extracellular matrices. Understanding the differing nature of these interactions may provide insights useful for rational tissue engineering.