Purpose and Structure of the Extracellular Matrix
Katie Ballek
What is the ECM?
Embryonic cells produce and secrete the extracellular matrix (ECM) during the early stages of development. The ECM sets up a microenvironment that is critical for many developmental processes. (Rozario et al.,2010) Dynamic reciprocity is the term used to describe the interactions between the cell and its surroundings in this microenvironment and is predicted to play a role in tissue function via crosstalk between the ECM and the cell membrane. (Nelson et al., 2006)
Rovario et al. referred to the ECM as a "morphogenetic language" that can be interpreted by cells in close proximity. Specialized receptors on cells have the capability to sense information from the composition of the surrounding ECM that can have an effect on cellular behavior. For example, the ECM has effects on adhesion, cell polarity, migration, cell survival, proliferation, cell growth, and differentiation. (Rozario et al., 2010)
Cell behaviors can be altered by numerous methods utilizing the ECM. Density, composition, and architecture of the ECM can change cell signaling, cell shape, cell adhesivity, and cell motility. Thus, how and when these differences in the ECM emerge can provide insight into the development of an organism. (Rozario et al.,2010)
Cell behaviors can be altered by numerous methods utilizing the ECM. Density, composition, and architecture of the ECM can change cell signaling, cell shape, cell adhesivity, and cell motility. Thus, how and when these differences in the ECM emerge can provide insight into the development of an organism. (Rozario et al.,2010)
Components of the ECM
The two main classes of macromolecules composing the ECM are PGs and fibrous proteins which include collagens, elastins, fibronectins, and laminins. (Frantz et al., 2010) Another major component are integrins, which are transmembrane proteins which act as receptors for the ECM.
Collagens
Collagens are currently thought to be the most abundant proteins in the animal kingdom. About 30% of the total protein mass of most multicellular organisms are made up of collagen. (Frantz et al., 2010) Collagens can be either fibrillary or non-fibrillary and are composed of pro-α-chains arranged in a triple helix. (Rozarioet al., 2010) They can have an effect on structure, adhesion, chemotaxis, migration, and development.
Collagen fibrils are transcribed, secreted, and organized by fibroblasts. (Frantz et al., 2010) These fibrils have a great tensile strength which contributes to the flexibility and structure of tissues. The location and density of these fibrous proteins vary greatly by tissue type and can be modified to fit the needs of the tissue when it comes to what forces the tissue has become exposed to via trauma, exercise, or other means.
One of the first insertion mutations discovered involving collagen is the α1(I) collagen gene in mice which is lethal to embryos in late development due to rupture of the aorta. This confirms the role of collagen fibrils in flexibility and structure of the vessel. (Rozario et al.,2010)
Fibronectin
Fibronectin (FN) is another fibrous protein which affects cell function and attachment. This attachment from cell surface receptors to other ECM molecules such as focal adhesions and collagen. (Halper et al., 2014) FN can undergo forces as a mechanoregulator which allow it to stretch and unfold thus exposing integrin binding sites. (Frantz et al., 2010) Also, alternative splicing of FN plays a role in tissue-specific and developmental stage-specific expression of transcripts. (Rozario et al.,2010)
Laminins
The basement membrane is a type of ECM which contains a high density of a fibrous protein called laminin. In cells of the mammary gland, laminin-1 is essential in normal structure of the gland and the loss of laminin-1 promotes tumor progression. ECM breakdown and remodeling is also observed to be essential in differentiation of functional luminal epithelial cells.
Laminins have been used for in vitro manipulation of cells to induce differentiation. Mammary epithelial cells in culture can be induced to differentiate when the proper microenvironment is recreated. For example, milk producing cells of the mammary gland require rounded cells and laminin to express the hormone lactoferrin. (Nelson et al., 2006)
Integrins
Integrins play a role in adhesion, resistance to mechanical stress, and cell signaling. (Rozario et al., 2010) Transmembrane PGs can function as co-receptors for molecules of the ECM, however, integrins are the main receptor for binding ECM proteins such as collagens, fibronectin, and laminins. These receptors have low affinities for their ligands which allow the frequent remodeling of the ECM. Also, integrins are capable of activating signaling pathways within the cell in response to the ECM environment.
Two noncovalent glycoproteins termed α and β associate to form the integrin heterodimer. Integrins vary greatly with multiple types of α and β subunits. The functions of these integrins may overlap, but are often specific to the cell or tissue. The affinity and specificity of integrins for their ligands is also in part determined by divalent cations outside the cell. Integrins differ from cell to cell to interact in specific ways with the ECM and induce intracellular signaling.
A closer look at the heterodimer structure of integrins.
An example of how the ECM can interact with and bind integrins.
For more information on integrins click here.
Elastin, Fibulin, and Fibrillin
Not only is rigidity a necessary characteristic for tissues, but elasticity is also important for the maintenance and protection of the cell. Certain tissues need to have the capability to respond to the forces that act upon them by shifting, bending, and stretching to maintain function. These properties are enhanced and made possible by the ECM component elastin as well as microfibrils that provide structure for these elastin proteins.
Collagen fibrils and layers of smooth muscle cells associate with concentric sheets of elastin in developing arteries. This association prevents obstruction of vessels as seen in the elastin null knockout mice. The smooth muscle in the vessel wall proliferates beyond organization, becomes rigid, and infiltrates into the lumen. These mice died shortly after birth. (Li et al., 1998)
For example, loss of fibulin-4 or fibulin-5 can create malformations in the skin and aorta, as well as reduce pliability in the lungs and vasculature. (Rozario et al.,2010)
Fibrillin is the core of both elastic and non-elastic microfibrils. In elastic fibrils, fibrillin acts as protection by covering the elastin fibrils. (Frantz et al., 2010)
Fibronectin helps arrange fibrillin-1. (Halper et al., 2014)
For example, loss of fibulin-4 or fibulin-5 can create malformations in the skin and aorta, as well as reduce pliability in the lungs and vasculature. (Rozario et al.,2010)
Fibrillin is the core of both elastic and non-elastic microfibrils. In elastic fibrils, fibrillin acts as protection by covering the elastin fibrils. (Frantz et al., 2010)
Fibronectin helps arrange fibrillin-1. (Halper et al., 2014)
Glycosaminoglycans and Proteoglycans
Glycosaminoglycans (GAGs) are highly electrophilic molecules composed of repetitive linkages of disaccharides, each containing a hexosamine and a uronic acid, to form linear unbranched polymers. They are polyanionic molecules which attract Na2+ to the area. GAGs reside in the interstitial spaces which when take on water and swell when Na2+ concentrations are high. This swelling can open up pathways such as those relating to invasion and migration of cells.
When core proteins are linked to GAGs they form proteoglycans (PGs). Hyaluronan (HA) is the only known GAG to not link up with a protein to form a PG. However, HA is important in the formation of pericellular matrices by assembling with aggregating PGs to affect diffusion of growth factors and morphogens. (Rozario et al., 2010)
PGs take a gel-like form in the interstitial spaces of tissue and act to buffer, hydrate, bind, and resist force. For example, the PG perlecan plays a role in glomerular filtration within the kidney. Also, PGs within the basement membrane (BM) can either promote or inhibit angiogenesis and PGs on the cell surface can facilitate the association of ligands and receptors. (Frantz et al., 2010)
Functions of the ECM
The aforementioned components of the ECM work together to accomplish a variety of functions such as regulation of cell movements, morphogen diffusion, and provision of binding sites for cell surface receptors. (Rozario et al., 2010) The ECM is capable of regulating biochemical and biomechanical cues involved in many processes such as differentiation, homeostasis, and morphology. (Frantz et al., 2010)
Migration
The ECM is known to be involved in the binding of integrins to allow the initiation and discontinuation of adhesion. When this interaction is combined with the cytoskeleton's ability to contract and generate traction, cell migration is made possible. Migratory cells show varying affinities for particular components of the ECM based upon their variety of integrins. This specificity allows the integrins of the migratory cell to migrate in the correct direction based on the surrounding ECM.
During the formation of the vertebrate heart myocardial precursors differentiate in the lateral regions of the organism and then migrate towards the midline to fuse with endocardial cells to form the inner endothelial layer and the outer muscular layer of the heart tube. When this migration is hindered two hearts form on either side of the embryo, a condition known as cardio bifida. A mutation in fibronectin expression in zebrafish is known to cause this malformation. (Rozario et al., 2010)
Branching Morphogenesis
Many ECM components such as PGs, GAGs, and collagens, have been shown to have a role in branched organ development. They have been shown to be regulators of gland, kidney, gut, and lung formation via the invasion of epithelial buds and tubes into ECM-rich mesenchyme. The dynamic composition of the ECM is able to change to suit the needs of the cell and create a microenvironment adequate for development.
Changes in the deposition of ECM molecules, both increase and decrease in function, can inhibit this branching. The increase of collagen expression via TGFβ can reduce budding and ductal growth in mammary gland cultures. On the other hand, an antibody directed against G-domain peptides of laminin α1 also inhibits branching in salivary glands.
The concentration, function, and location of the ECM components provide a variation of regulation that can either promote or inhibit branching. This created microenvironment makes it difficult to analyze the particular function of each component, but does make it evident that the ECM plays an important role in the branching of developing organs. (Rozario et al., 2010)
Microenvironment Modification
Different molecules among the components of the ECM can impact expression of other ECM molecules within the microenvironment. One example of this is the reduction of fibronectin expression via laminin in mammary epithelial cells. The concentration of fibronectin normally varies throughout development being more abundant during proliferative stages and downregulated preceding certain stages of differentiation when growth is arrested. (Rozario et al., 2010)
Physical Forces
When fibronectin is stretched, access to integrin binding sites is provided leading to ligation and clustering of integrins. These interactions affect both adhesivity and differentiation within the cell. For example, a fragment of fibronectin that has a mutation in the cell binding region in which the amino acid in position 1048 is altered from Leu to Pro changes the affinity for α5β1.
The morphology of a cell can alter the fate of differentiation and the ECM may play an indirect role in cell shape. For example, mesenchymal stem cells cultured in laminin have more elongated cells that differentiate into smooth muscle. However, if these cells were cultured without laminin, they were more rounded in shape and smooth muscle marker expression was inhibited. (Rozario et al., 2010)
Structural Support
Collagens in particular play major roles in the rigidity and solidarity of tissues such as skin, connective tissue, vasculature, and even the eyes. Loss of collagen I fibers and fibrils can lead to skin blisters and ruptures in the blood vessels.
However, other ECM components can also play a role in structural integrity. Some laminin chains are critical in the structure of skin. Loss of laminin β3 and γ2 function leads to defects in desmosomes and severe blistering of the skin. (Rozario et al., 2010)
The ECM can also affect nucleus structure. For example, addition of laminin induces histone deacetylation in mammary epithelial cell lines. Histone acetylation and deacetylation can also be altered by changes to cell morphology such as cell rounding previously discussed. (Nelson et al., 2006)
Growth Factor Signaling
ECM components are suspected of modulating distribution of morphogens, sequestering and releasing factors, as well as promoting or restricting access to receptors for ligands. (Rozario et al., 2010) For example, the ECM can regulate the expression of transcription factors, such as mammary gland factor (MGF), which can then bind milk protein genes in the promoter region. (Nelson et al., 2006)
TGFβ may be the best understood example of the ECM interacting with growth factors. Latent TGFβ binding proteins (LTBPs) can interact with ECM proteins like fibrillin and fibronectin and multiple integrins. Regulation of protease expression and secretion by ECM components also affects the timing and location of TGFβ secretion.
The presence of specific integrins has an affect on proteolysis in some systems perhaps functioning on a location for docking of proteases. As the spatio-temporal distribution of integrins is altered so is the affinity for these proteases. This alteration affects the amount of TGFβ signaling.
Syndecan is an ECM receptor that is also essential in growth factor signaling. They bind ligands via GAG side chains. Their isoforms are adjusted throughout development in space and time which may indicate a role in signaling.
Syndecans can bind numerous growth factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) to activate numerous pathways possibly by increasing affinity for the receptors or sequestering ligands. (Rozario et al., 2010)
Differentiation
ECM molecules can affect differentiation signals as well. Expression changes and levels of varying and often opposing ECM molecules play a role in the determination of cell fate. For example, ECM proteins laminin and collagen IV increase while fibronectin decreases in the limb bud of mice to encourage muscular differentiation.
Under regular in vivo conditions, embryonic stem cells from the inner cell mass of mouse blastocysts are not competent to make trophoblastic cells to implant and to establish the placenta. However, when the microenvironment is altered this differentiation is made possible. When these inner mass cells are placed on a feeder layer containing the ECM molecules collagen IV, but lacking other ECM molecules such as collagen I, laminin, and fibronectin they differentiate into trophoblastic cells. (Rozario et al., 2010)
ECM Movement
It is predicted that most movements of cells and tissues are somehow affected by the ECM. The microenvironment formed by the components of the ECM may be a signal to cells to move towards or away from certain arrangements.
In cell culture the ECM may be fixed in position. This type of in vitro study allows the observation of the translocation of cells independent from the movement and reorganization of the ECM. When the matrix is unable to be remodeled, cell motility is increased. (Rozario et al., 2010)
Zamir and colleagues (Czirok et al., 2006; Zamir et al., 2005; Zamir et al., 2008) observed epiblastic cell movements in the primitive streak of the avian embryo via fibronectin with a fluorescent tag. It was noted that with particular conditions migratory cells are able to carry components of their ECM with them. Studies of these ECM displacements and how they correlate to certain stages of development could be enlightening in regards to signaling the movement of cells and tissues.
For example, when the functionality of fibronectin or α5β1 is inhibited changes in cell motility are observed. Instead of extending along the axis the tissue thickens as cells overlap. Perhaps the ECM plays a role in crosstalk among integrins and cadherins affecting adhesion and traction. (Rozario et al., 2010)
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