Role in normal cellular functions
Formation of the extracellular matrix (ECM) requires cells to secrete ECM proteins. Assembly is achieved by following a strict hierarchical assembly pattern which begins with the deposition of fibronectin filaments on the cell surface, a process known as fibrillogenesis (1). Cells continue to remodel the ECM by degradation and reassembly mechanisms, the dynamic nature of the ECM being particularly apparent during development, wound healing, and certain disease states (see below) (2). It is estimated that there are over 300 proteins comprising the mammalian ECM or “core matrisome” and this does not include the large number of ECM-associated proteins (3). Cells interact with the ECM through receptors such as integrins and syndecans, resulting in the transduction of multiple signals to regulate key cellular processes such as differentiation, proliferation, survival, and motility of cells (3). The ECM has also been shown to bind growth factors such as VEGF, HGF and BMPs which are thought to create growth factor gradients that regulate pattern formation during development (4). Many of the ECM-regulated cell processes operate via reorganization of the actin and microtubule cytoskeletons (5).
Role in disease
Aberrant regulation or genetic defects of the ECM components often results in a pathogenic state.
Genetic diseases: Several diseases have been shown to be caused by mutations in ECM genes, including macular degenerative disease (Fibulin 3, ref. 6), osteoarthritis (Asporin, ref. 7) and congenital muscular dystrophy (Laminins, ref. 8).
Cancer and Metastasis: There are many examples demonstrating that altered expression of a given ECM protein is correlated with cancer (9). It is also well established that degradation of the ECM, via the actions of matrix metalloproteinases, is a prerequisite to metastatic invasion of cancer cells (10).
Atherosclerosis: This disease has been linked to a buildup of collagen plaques (11).
The field of regenerative medicine and tissue engineering is utilizing ECM components to try to generate a predictable formation of tissues and organs from a given cell type (12,13).
Assays Used to study ECM
There is a preponderance of commercially available ECM reagents and cell attachment/invasion assays. The best known of the ECM reagents is MatrigelTM from BD which is an ECM extract from the Engelbreth-Holm-Swarm mouse tumor. MatrigelTM is composed of laminin (56%), collagen type IV (31%), and enactin (8%), and several growth factors, including EGF (0.7 ng/ml), PDGF (12 ng/ml), IGF-1 (16ng/ml), and TGF-a (2.3 ng/ml) (14). Similar products include ECMatrixTM (Millipore) and ECM gel (Sigma). Most commercially available invasion assays utilize a Boyden-chamber like system (15).
Cytoskeleton offers a unique line of fluorescently-labeled and biotinylated fibronectins and laminins. Several applications of these products are listed below.
Application #1: In vitro invadopodia/podosome invasion assay (Cat.# FNR01, FNR02, LMN01, LMN02)
Cytoskeleton’s fluorescently-labeled fibronectins and laminins can be used in an in vitro invadopodia/podosome invasion assay (16). This assay allows a high resolution examination of local cell invasion on specific ECM components and can be used to assess the invasive potential of cells and to examine compounds/pathways that affect this stage of invasion. Originally described by Artym et al. for gelatin, the assay is equally applicable to fluorescent fibronectin or laminin (16).
Application #2: Signaling Pathways Involved in Fibronectin Matrix Assembly: Fibrillogenesis (Cat.# FNR01, FNR02, FNR03)
Unlike other ECM components that can self-polymerize under physiological conditions, fibronectin matrix assembly is a cell-dependent process. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic, and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes. Also, tissue engineering strongly depends on the ability to control the rate and pattern of ECM formation. Cytoskeleton’s labeled fibronectins can be used to monitor fibrillogenesis;
Fluorescent Fibronectin Substrates (FNR01 & FNR02)
This method involves fluorescent tracing of fibril formation through incorporation of fluorescent fibronectins (17).
The conversion of soluble fibronectin to insoluble fibrils on the cell surface can be visualized by feeding cell culture with media containing either TRITC-labeled fibronectin (Cat.# FNR01) or HiLyte488-labeled fibronectin (Cat.# FNR02). The level of incorporated fibronectin can be observed and quantitated by fluorescence microscopy (18).
Biotinylated Fibronectin Substrate (FNR03)
This method incorporates biotinylated fibronectin into the cells of interest and quantitates detergent soluble (cell bound) and detergent insoluble (fibrils) fibronectin. This assay has been used successfully to examine the role of Rho proteins in fibrillogenesis (19).
Application #3: Tissue Engineering Applications (FNR03 & LMN03)
It has been shown that biotinylated ECM proteins can adopt a more native conformation when bound to a streptavidin surface and that this can lead to enhanced cellular binding to the ECM (20).