Jean E. Schwarzbauer

Assembly of Fibronectin Extracellular Matrix

In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to α5ß1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation.

The ins and outs of fibronectin matrix assembly

Cell phenotype is specified by environmental cues embedded in the architecture and composition of the extracellular matrix (ECM). Much has been learned about matrix organization and assembly through analyses of the ECM protein fibronectin (FN). FN matrix assembly is a cell-mediated process in which soluble dimeric FN is converted into a fibrillar network. Binding of cell surface integrin receptors to FN converts it to an active form, which promotes fibril formation through interactions with other cell-associated FN dimers. As FN fibrils form on the outside of the cell, cytoplasmic domains of integrin receptors organize cytoplasmic proteins into functional complexes inside. Intracellular connections to the actin cytoskeletal network and stimulation of certain key intracellular signaling pathways are essential for FN–integrin interactions and propagation of FN fibril formation. Thus, assembly of native functional ECM depends on exquisite coordination between extracellular events and intracellular pathways.

Fibronectin fibrillogenesis: a paradigm for extracellular matrix assembly.

Fibronectin matrix assembly is a regulated stepwise process. In the past year, analyses of fibronectin domains, integrin and cytoskeletal contributions, and fibril architecture have provided new insights into assembly mechanisms and matrix control of cell functions. Like fibronectin, laminin polymerization is cell-mediated. Thus a common pathway for extracellular matrix assembly is emerging.

Fibronectins, Their Fibrillogenesis, and In Vivo Functions

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN-FN and cell-FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.

Fibronectin: from gene to protein

Recent advances in several key areas of fibronectin biology are discussed. These include its expression, from transcription to secretion of dimers, the structural requirements for several of the binding activities, potential roles for alternatively spliced segments in cell adhesion, and the assembly of a fibronectin matrix.

Control of Cell Cycle Progression by Fibronectin Matrix Architecture

Developmental patterning and differentiation, maintenance of parenchymal cell function, and the size, shape, and invasiveness of tumors are all orchestrated by cell interactions with the extracellular matrix. Here we show that the fibrillar structure of fibronectin (FN) matrix encodes essential regulatory cues and controls cell proliferation and signaling through changes in matrix architecture. A matrix assembled from native FN stimulated cell growth. In contrast, a mutant FN (FNΔIII1–7) that contains all known cell binding motifs but forms a structurally distinct matrix inhibited progression from G0/G1 into S phase. Furthermore, FNΔIII1–7 suppressed the stimulatory capacity of native FN and induced different levels of tyrosine phosphorylation of pp125FAK. The differential effects on cell growth were ablated by blocking formation of matrix fibrils. Thus, modification of matrix architecture provides a novel approach to control cell proliferation.

Fibronectin Expression Modulates Mammary Epithelial Cell Proliferation during Acinar Differentiation

The mammary gland consists of a polarized epithelium surrounded by a basement membrane matrix that forms a series of branching ducts ending in hollow, sphere-like acini. Essential roles for the epithelial basement membrane during acinar differentiation, in particular laminin and its integrin receptors, have been identified using mammary epithelial cells cultured on a reconstituted basement membrane. Contributions from fibronectin, which is abundant in the mammary gland during development and tumorigenesis, have not been fully examined. Here, we show that fibronectin expression by mammary epithelial cells is dynamically regulated during the morphogenic process. Experiments with synthetic polyacrylamide gel substrates implicate both specific extracellular matrix components, including fibronectin itself, and matrix rigidity in this regulation. Alterations in fibronectin levels perturbed acinar organization. During acinar development, increased fibronectin levels resulted in overproliferation of mammary epithelial cells and increased acinar size. Addition of fibronectin to differentiated acini stimulated proliferation and reversed growth arrest of mammary epithelial cells negatively affecting maintenance of proper acinar morphology. These results show that expression of fibronectin creates a permissive environment for cell growth that antagonizes the differentiation signals from the basement membrane. These effects suggest a link between fibronectin expression and epithelial cell growth during development and oncogenesis in the mammary gland.

Fibronectin and stem cell differentiation - lessons from chondrogenesis.

Summary The extracellular matrix (ECM) is an intricate network of proteins that surrounds cells and has a central role in establishing an environment that is conducive to tissue-specific cell functions. In the case of stem cells, this environment is the stem cell niche, where ECM signals participate in cell fate decisions. In this Commentary, we describe how changes in ECM composition and mechanical properties can affect cell shape and stem cell differentiation. Using chondrogenic differentiation as a model, we examine the changes in the ECM that occur before and during mesenchymal stem cell differentiation. In particular, we focus on the main ECM protein fibronectin, its temporal expression pattern during chondrogenic differentiation, its potential effects on functions of differentiating chondrocytes, and how its interactions with other ECM components might affect cartilage development. Finally, we discuss data that support the possibility that the fibronectin matrix has an instructive role in directing cells through the condensation, proliferation and/or differentiation stages of cartilage formation.

Stimulatory effects of a three-dimensional microenvironment on cell-mediated fibronectin fibrillogenesis

The assembly of fibronectin into a fibrillar matrix is a regulated step-wise process that involves binding to integrin receptors and interactions between fibronectin molecules. This process has been studied extensively using cells in two-dimensional (2D) monolayer culture. In most situations in vivo, however, matrix assembly occurs within existing three-dimensional (3D) extracellular matrix networks. In an attempt to mimic this environment, we analyzed matrix assembly by fibroblasts cultured on a pre-assembled 3D fibronectin matrix and found significant stimulation of fibronectin fibril assembly compared to cells in 2D culture. Lower amounts of fibronectin were needed to initiate the assembly process, fibrils accumulated to higher density, and the 3D fibril organization played a key role in the stimulatory effect. Moreover, cells expressing activation-dependent integrins were able to assemble fibronectin matrix without exogenous stimulation, suggesting regulatory effects of the 3D fibronectin matrix on integrin activity. These results provide evidence for an additional level of control of fibronectin deposition through cell interactions with the local microenvironment.

A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly

Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1–7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNΔIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9–10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4–7, had no effect on assembly. In contrast, two deletions that included repeat III2, ΔIII1–2 and ΔIII2–5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN–FN interactions during fibril growth.