Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 94
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The cell-binding region offibronectin contains an integrin-binding heptapeptide sequence,GRDSPC in the single-letter amino acid code (see Figure 2-13).This heptapeptide and several variants were synthesizedchemically. Different concentrations of each synthetic peptidewere added to polystyrene dishes that had the proteinimmunoglobulin G (IgG) firmly attached to their surfaces; thepeptides were then chemically cross-linked to the IgG.Subsequently, cultured normal rat kidney cells were added to thedishes and incubated for 30 minutes to allow adhesion.
After thenonbound cells were washed away, the relative amounts of cellsthat had adhered firmly were determined by staining the boundcells with a dye and measuring the intensity of the staining witha spectrophotometer. The plots shown here indicate that celladhesion increased above the background level with increasingpeptide concentration for those peptides containing the RGDsequence but not for the variants lacking this sequence(modification underlined). [From M. D.
Pierschbacher and E. Ruoslahti,1984, Proc. Nat’l. Acad. Sci. USA 81:5985.]222CHAPTER 6 • Integrating Cells into Tissues(a)Fibrin,heparansulfatebinding(b)CollagenbindingEIIIBEIIIAIIICSRGDSSNH2COOHHeparansulfatebindingType I repeatFibrinbindingSynergyregionRGDsequenceType II repeatType III repeatIntegrin▲ FIGURE 6-25 Model of fibronectin binding to integrinthrough its RGD-containing type III repeat. (a) Scale modelof fibronectin is shown docked by two type III repeats to theextracellular domains of integrin. Structures of fibronectin’sdomains were determined from fragments of the molecule.The EIIIA, EIIIB, and IIICS domains (not shown; see Figure 6-23)are variably spliced into the structure at locations indicated byA three-dimensional model of fibronectin binding tointegrin based on structures of parts of both fibronectinand integrin has been assembled (Figure 6-25a).
In a highresolution structure of the integrin-binding fibronectin typeIII repeat and its neighboring type III domain, the RGD sequence is at the apex of a loop that protrudes outward fromthe molecule, in a position facilitating binding to integrins(Figure 6-25a, b). Although the RGD sequence is requiredfor binding to several integrins, its affinity for integrins issubstantially less than that of intact fibronectin or of the entire cell-binding region in fibronectin.
Thus structural features near to the RGD sequence in fibronectins (e.g., partsof adjacent repeats, such as the synergy region; see Figurearrows. (b) A high-resolution structure shows that the RGDbinding sequence (red) extends outward in a loop from itscompact type III domain on the same side of fibronectin as thesynergy region (blue), which also contributes to high-affinitybinding to integrins. [Adapted from D.
J. Leahy et al., 1996, Cell84:161.]6-25b) and in other RGD-containing proteins enhance theirbinding to certain integrins. Moreover, the simple solubledimeric forms of fibronectin produced by the liver or fibroblasts are initially in a nonfunctional closed conformationthat binds poorly to integrins because the RGD sequence isnot readily accessible. The adsorption of fibronectin to a col(a) EXPERIMENTAL FIGURE 6-26 Integrins mediate linkagebetween fibronectin in the extracellular matrix and thecytoskeleton. (a) Immunofluorescent micrograph of a fixedcultured fibroblast showing colocalization of the 51 integrinand actin-containing stress fibers.
The cell was incubated withtwo types of monoclonal antibody: an integrin-specific antibodylinked to a green fluorescing dye and an actin-specific antibodylinked to a red fluorescing dye. Stress fibers are long bundles ofactin microfilaments that radiate inward from points where thecell contacts a substratum. At the distal end of these fibers, nearthe plasma membrane, the coincidence of actin (red) andfibronectin-binding integrin (green) produces a yellowfluorescence. (b) Electron micrograph of the junction offibronectin and actin fibers in a cultured fibroblast. Individualactin-containing 7-nm microfilaments, components of a stressfiber, end at the obliquely sectioned cell membrane. Themicrofilaments appear in close proximity to the thicker, denselystained fibronectin fibrils on the outside of the cell.
[Part (a) fromJ. Duband et al., 1988, J. Cell Biol. 107:1385. Part (b) from I. J. Singer,1979, Cell 16:675; courtesy of I. J. Singer; copyright 1979, MIT.](b)FibronectinfibrilsCellexteriorPlasmamembraneActin-containingmicrofilamentsCell interior0.5 m6.5 • Adhesive Interactions and Nonepithelial Cellslagen matrix or the basal lamina or, experimentally, to a plastic tissue-culture dish results in a conformational change thatenhances its ability to bind to cells. Most likely, this conformational change increases the accessibility of the RGD sequence for integrin binding.Microscopy and other experimental approaches (e.g., biochemical binding experiments) have demonstrated the role ofintegrins in cross-linking fibronectin and other ECM components to the cytoskeleton. For example, the colocalization ofcytoskeletal actin filaments and integrins within cells can bevisualized by fluorescence microscopy (Figure 6-26a).
Thebinding of cell-surface integrins to fibronectin in the matrixinduces the actin cytoskeleton–dependent movement of someintegrin molecules in the plane of the membrane. The ensuing mechanical tension due to the relative movement of different integrins bound to a single fibronectin dimer stretchesthe fibronectin.
This stretching promotes self-association ofthe fibronectin into multimeric fibrils.The force needed to unfold and expose functional selfassociation sites in fibronectin is much less than thatneeded to disrupt fibronectin–integrin binding. Thus fibronectin molecules remain bound to integrin while cellgenerated mechanical forces induce fibril formation.
In effect, the integrins through adapter proteins transmit the intracellular forces generated by the actin cytoskeleton toextracellular fibronectin. Gradually, the initially formedfibronectin fibrils mature into highly stable matrix components by covalent cross-linking. In some electron micrographic images, exterior fibronectin fibrils appear to bealigned in a seemingly continuous line with bundles of actinfibers within the cell (Figure 6-26b).
These observations andthe results from other studies provided the first example ofa molecularly well defined adhesion receptor (i.e., an integrin) forming a bridge between the intracellular cytoskeletonand the extracellular matrix components—a phenomenonnow known to be widespread.223Hyaluronan, a highly hydrated GAG, is a major component of the ECM of migrating and proliferating cells.Certain cell-surface adhesion receptors bind hyaluronan tocells.■Large proteoglycan aggregates containing a centralhyaluronan molecule noncovalently bound to the core protein of multiple proteoglycan molecules (e.g., aggrecan)contribute to the distinctive mechanical properties of thematrix (see Figure 6-22).■Fibronectins are abundant multiadhesive matrix proteinsthat play a key role in migration and cellular differentiation. They contain binding sites for integrins and ECMcomponents (collagens, proteoglycans) and can thus attachcells to the matrix (see Figure 6-23).■The tripeptide RGD sequence (Arg-Gly-Asp), found infibronectins and some other matrix proteins, is recognizedby several integrins.■6.5 Adhesive Interactionsand Nonepithelial CellsAfter adhesive interactions in epithelia form during differentiation, they often are very stable and can last throughout thelife span of epithelial cells or until the cells undergo differentiation into loosely associated nonpolarized mesenchymalcells, the epithelial–mesenchymal transition.
Although suchlong-lasting (nonmotile) adhesion also exists in nonepithelialtissues, some nonepithelial cells must be able to crawl acrossor through a layer of extracellular matrix or other cells. Inthis section, we describe various cell-surface structures innonepithelial cells that mediate long-lasting adhesion andtransient adhesive interactions that are especially adapted forthe movement of cells. The detailed intracellular mechanismsused to generate the mechanical forces that propel cells andmodify their shapes are covered in Chapter 19.KEY CONCEPTS OF SECTION 6.4The Extracellular Matrix of Nonepithelial TissuesConnective tissue, such as tendon and cartilage, differsfrom other solid tissues in that most of its volume is madeup of extracellular matrix (ECM) rather than cells.■The synthesis of fibrillar collagen (e.g., types I, II, andIII) begins inside the cell with the chemical modificationof newly made chains and their assembly into triplehelical procollagen within the endoplasmic reticulum.
After secretion, procollagen molecules are cleaved, associatelaterally, and are covalently cross-linked into bundles calledfibrils, which can form larger assemblies called fibers (seeFigure 6-20).■The various collagens are distinguished by the ability oftheir helical and nonhelical regions to associate into fibrils, to form sheets, or to cross-link other collagen types(see Table 6-1).■Integrin-Containing Adhesive StructuresPhysically and Functionally Connect the ECMand Cytoskeleton in Nonepithelial CellsAs already discussed in regard to epithelia, integrin-containinghemidesmosomes connect epithelial cells to the basal laminaand, through adapter proteins, to intermediate filaments of thecytoskeleton (see Figure 6-1).
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