MECHANISM OF ASSEMBLY OF ALIGNED EXTRACELLULAR MATRIX FIBRILS

Abstract

The extracellular matrix (ECM) is a complex three-dimensional network consisting of secreted extracellular macromolecules consisting of fibrous proteins, proteoglycans, and glycoproteins, that provides essential structural support and stability to cells and tissues, as well as regulates vital cell behaviors, such migration, differentiation, and cell division. The physical structure of the ECM is tissue-specific and fundamental to normal tissue function. Parallel alignment of ECM fibers is crucial for the function of a variety of tissues, such as the cornea, tendon, and vasculature. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for the formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils. Microscopic analysis of matrix assembly sites, where FN fibrillogenesis is initiated, revealed that these complexes were oriented in parallel at the cell poles. Utilizing live-cell imaging, we found that these polarized matrix assembly sites progressed into fibrillar adhesions and ultimately into aligned FN fibrils. Using cells that assemble an unaligned, meshwork matrix, we demonstrate that the distribution and orientation of matrix assembly sites can be controlled through micropatterning or mechanical stretch. While elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked since we discovered that transforming growth factor beta (TGF-B1) signals enhance matrix assembly site polarity and assembly of aligned fibrils independently of cell elongation. Our work has revealed that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites which form during the initial stages of cell-FN interactions and that mechanisms of aligned fibril assembly can be independent of cell elongation. We subsequently utilize our insights into cell and matrix organization in a tissue regeneration application, developing a novel multi-layered scaffold with distinct fiber organizations to recreate smooth muscle and connective tissue layers.