Extracellular matrix assembly: a multiscale deconstruction

Abstract

The biochemical and biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. The molecules that are associated with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled determine the structure and the organization of the resultant ECM. The product is a specific ECM signature that is comprised of unique compositional and topographical features that both reflect and facilitate the functional requirements of the tissue.

The extracellular matrix (ECM) has important roles in regulating the development, function and homeostasis of all eukaryotic cells^1–3. In addition to providing physical support for cells, the ECM actively participates in the establishment, separation and maintenance of differentiated tissues and organs by regulating the abundance of growth factors and receptors, the level of hydration and the pH of the local environment^1,4. These diverse functions are achieved through its complex chemical composition and organization. Although it is primarily composed of water, proteins and polysaccharides, the ECM shows exquisite tissue specificity as a result of the unique ECM compositions and topographies that are generated through a dynamic biochemical and biophysical interplay between the various cells in each tissue and the evolving microenvironment⁴. The mature ECM can also undergo dynamic remodelling in response to environmental stimuli, such as applied force or injury, which enables the tissue to maintain homeostasis and to respond to physiological challenges and stresses, including disease^2,5–8.

Understanding cellular differentiation, tissue morphogenesis and physiological remodelling requires an understanding of the ECM components that are produced by cells, as well as the assembly of those macromolecules into a functional three-dimensional structure^2,3. The macromolecules that constitute the ECM have evolved structural and chemical properties that are particularly suited to their specific biological functions in their respective tissues. Small, modular subunits form homopolymers and heteropolymers that become supramolecular assemblies with highly specialized organizations^1–3. These assemblies contain binding domains for growth factors and chemokines, establish complex adhesion surfaces and form diffusion barriers between different cellular layers; thus, ECM molecules can function both as support for cells and as active participants in signalling to control cell behaviour^9,10. In all cases, each class of ECM molecule has evolved the ability to interact with another class to produce unique physical and signalling properties that support tissue structure, growth and function¹. Growing insight into the biological importance of the ECM in development, pathophysiology and the normal function of tissues throughout the body has confirmed the importance of elucidating the mechanisms of ECM assembly.

This Review provides a general overview of our current understanding of the ‘structural principles’ that underlie the assembly of the ECM at several levels, highlighting key molecular components and their organization into an interlocking network. We also consider the biochemical and mechanical cues that affect ECM assembly and how its organization directly relates to its tissue-specific functions, including as a structural support, as a barrier between different tissues and as a source of both chemical and physical cues. We highlight unanswered questions that require further investigation and the techniques and model systems that may facilitate this research.

Key molecular players in the ECM

Our understanding of the composition, structure and function of the ECM is continuously evolving, aided by the discovery of novel ECM molecules, the mapping of internal sites within ECM proteins that are crucial for self-assembly and for interactions with other ECM components, the characterization of the proteases and the protease inhibitors that are responsible for ECM degradation and turnover, and the identification of novel receptors and signalling mechanisms that mediate cellular responses to the ECM^1–3. The architecture of the ECM is highly organized as a result of the innate properties of its constituent molecules and their interactions, as well as the activities of resident cells. The interactions between cells and the environment that are created by the ECM in turn have important roles in directing processes in development, homeostasis and pathogenesis. In this Review, we define the ECM as all secreted molecules that are immobilized outside a cell, including growth factors, cytokines and cell adhesion molecules, although we primarily focus on the macromolecules that are mainly responsible for tissue-type specific extracellular architecture.

The protein and non-protein constituents of the ECM vary not only in terms of their functional roles but also in terms of their structure. The polypeptide chains of ECM macromolecules often consist of many individual domains, and homologous domains between macromolecules can have sequence and structural differences that impart different functions. Individual ECM macromolecules rarely exist in isolation but instead often integrate into supramolecular structures containing different molecular species that differ in both their identity and their relative abundance. The ECM is primarily composed of two main classes of macromolecules: fibrous proteins (including collagens and elastin) and glycoproteins (including fibronectin, proteoglycans (PGs) and laminin)¹.

Fibrillar collagens

Collagens are major proteins of the ECM and are arguably the most dominant. Decades of research have uncovered 28 different collagen types, and each type is comprised of homotrimers and heterotrimers that are formed by three polypeptide chains (known as α-chains) (FIG. 1; TABLE 1). More than 40 distinct α-chains have been identified in humans as well as multiple other proteins containing collagen-like domains^11,12. The structural hallmark of all collagens is their triple helix — a tight right-handed helix of three α-chains, each of which contains one or more regions characterized by the repeating amino acid motif Gly-X-Y, where X and Y can be any amino acid¹³ (FIG. 1). Some collagen molecules assemble as homotrimers, whereas others assemble as heterotrimers that are comprised of two or three distinguishable α-chain types. The large family of collagens and proteins with collagenous domains has revealed that the collagen triple helix is a basic motif that can be adopted by proteins that have different functions. The rod-like domain has the potential to engage in various modes of self-association and the capacity to bind to cell surface receptors, other proteins, glycosaminoglycans (GAGs) and nucleic acids. A detailed description of the structural organizations of the different types of collagens can be found in REF. 14. For the purposes of this Review, we specifically focus on the assembly of fibrillar collagen, which comprises multiple complex intracellular and extracellular post-translational processes from the translational product to a fibrillar structure that is capable of withstanding tensile forces. The unique mechanical properties of fibrillar collagen are mainly controlled by its structure, which shows the important relationship between three-dimensional protein structure and the function of the resultant ECM⁶.

Figure 1. Collagen structure.

a | The standard fibrillar collagen molecule is characterized by amino- and carboxy-terminal propeptide sequences, which flank a series of Gly-X-Y repeats (where X and Y represent any amino acids but are frequently proline and hydroxyproline) (step 1). These form the central triple helical structure of procollagen and collagen. Three α-chains (the illustration shows two α1-chains and one α2-chain, which is representative of type I collagen) are intracellularly assembled into the triple helix following initiation of this process by the C-terminal domain (step 2). Procollagen is secreted by cells into the extracellular space (step 3) and converted into collagen by the removal of the N- and C-propeptides via metalloproteinase enzymes (step 4).

b | Fibril-associated collagens with interrupted triple helices (FACIT) and related collagens have a different structure to standard fibrillar collagen; they contain non-collagenous regions — that is, non-triple helical sequences. These lead to kinks in the resulting macromolecular structure that straighten under small strains. Figure part a is modified, with permission, from REF 135 © 2012 Fan et al.; licensee BioMed Central Ltd, and from REF. 136, Klug, William S.; Cummings, Michael R., Concepts of Genetics, 5th Edition, © 1997. Reprinted by permission of Pearson Education, Inc., Upper Saddle River, NJ. Figure part b: this figure was originally published in Biochem. J. Jäälinoja, J., Ylöstalo, J., Beckett, W., Hulmes, D. J. S. & Ala-Kokko, L., Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains. Biochem. J. 2008; 409: 545–554 © the Biochemical Society.

Table 1.

Primary types of collagens^*

Classes	Type	Genes encoding proteins typical of collagen composition	Description
Fibrillar	I	COL1A1 and COL1A2	Quarter-stagger packed fibrils that are primarily found in fibrous stromal matrices, such as skin, bone, tendons and ligaments
	II	COL2A1
	III	COL3A1
	V	COL5A1, COL5A2 and COL5A3
	XI	COL11A1, COL11A2 and COL11A3
	XXIV	COL24A1
	XXVII	COL27A1
FACIT	IX	COL9A1, COL9A2 and COL9A3	Fibril-associated molecular bridges that are associated with type I (XII, XVI, XIX, XXI) and type II (IX, XVI, XIX) collagen fibrils
	XII	COL12A1
	XIV	COL14A1
	XVI	COL16A1
	XIX	COL19A1
	XX	COL20A1
	XXI	COL21A1
	XXII	COL22A1
Basement membrane	IV	COL4A1, COL4A2, COL4A3, COL4A4, COL4A5 and COL4A6	Network structure comprised of laminins and basement membrane proteins
Long chain	VII	COL7A13	Anchoring fibrils that are associated with the basement membrane
Filamentous	VI	COL6A1, COL6A2, COL6A3, COL6A5 and COL6A6	Beaded microfibrils
Short chain	VIII	COL8A1	Hexagonal lattice structure
	X	COL10A1	These collagens both regulate, and are regulated by, hypertrophy in cartilage
Multiplexins	XV	COL15A1	Multiple triple helix domains with interruptions containing chondroitin sulphate and heparin sulphate glycosaminoglycans
	XVIII	COL18A1
MACIT (transmembrane domain)	XIII	COL13A1	Cell surface molecules with extracellular, membrane-spanning and intracellular domains
	XVII	COL17A1
	XXIII	COL23A1

^*Collagen types are classified on the basis of domain structure homology and suprastructural assembly.

FACIT, fibril-associated collagens with interrupted triple helices; MACIT, membrane-associated collagens with interrupted triple helices.

Four distinct stages define collagen fibril assembly after the transcription and translation of the procollagen α-chains: import into the rough endoplasmic reticulum, where the α-chains are modified so that they can form triple-helical procollagen; modification of procollagen in the Golgi apparatus and its packaging into secretory vesicles; cleavage of the procollagen to form the collagen molecule in the extracellular space; and lysyl oxidase-catalysed crosslinking between collagen molecules to stabilize the supramolecular collagen structure^{6,11,12,15,16} (FIGS 1,2a). All fibrillar collagens are initially synthesized as precursor molecules that contain large amino- and carboxy-terminal propeptides and a signal recognition sequence, which promotes targeting to the rough endoplasmic reticulum, where post-translational modifications take place, leading to the assembly of procollagen molecules¹⁷. Within the endoplasmic reticulum, particular lysine and proline residues within the propeptides are hydroxylated by lysyl and prolyl hydroxylase enzymes to promote the stability and glycosylation of the triple helices following formation¹⁷. At the same time, a limited number of lysine hydroxyl groups are glycosylated. These proline and hydroxyproline amino acids can comprise 20% of the molecule, contributing to triple helix stabilization. Although the exact mechanism that controls the stabilization of the triple helix has generated much controversy, the current consensus suggests that increased stabilization is partly due to its high content of amino acids, increased interchain hydrogen bonds through hydration networks and electrostatic interactions between lysine residues^13,18,19. Following lysyl and prolyl hydroxylation and O-linked glycosylation, α-chains associate to form procollagen, and triple helix trimerization is initiated by the association of the C-propeptide domains through both specific chain recognition and the formation of a stable nucleus, which favours triple-helical assembly^15,20–23. Recent work elucidating the crystal structure of the C-propeptide domain from procollagen III suggests that there is a structural mechanism by which α-chains are selectively recognized, with the chain recognition sequence (CRS) of one chain interacting with the CRS of a neighbouring chain during intracellular trimerization of procollagen to ensure correct homotrimeric and heterotrimeric chain stoichiometry²⁰. In addition, protein disulphide isomerase catalyses the formation and the rearrangement of intramolecular and intermolecular disulphide bonds²⁴. Many other proteins influence and guide triple helix formation during procollagen assembly, including prolyl 4-hydroxylase, 78 kDa glucose-regulated protein (GRP78) and GRP94 (REF. 15). Importantly, the tight packing of the three α-chains near the common axis places steric constraints on every third amino acid position, with only the smallest amino acid, glycine, accommodated without chain distortion; this generates the Gly-X-Y repeating motif and imparts further stability to the triple helix¹³. Detailed reviews discussing the collagen triple helix structure and assembly can be found in REFS 13,15,25,26.

Figure 2. Fibrillar collagen assembly.

a | After procollagen is secreted into the extracellular space, collagen type-specific metalloproteinase enzymes remove the amino- and carboxy-propeptides. Collagen is assembled into cross-striated microfibrils that occur in the extracellular matrix of connective tissues. This can be observed via electron microscopy as regularly spaced bands, as represented by the grey, striped bar. The position of non-fibrillar collagens in this structure is shown in similar diagrams in part b. Short microfibrils merge into mature fibrils through longitudinal and axial growth. To form mature fibres, lysyl oxidase catalyses the formation of intramolecular and intermolecular covalent crosslinks between collagen molecules. Fibres are bundled together within a connective tissue and stabilized via interactions with fibril-associated collagens with interrupted triple helices (FACIT) collagens and small leucine-rich repeat proteoglycans (SLRPs) into linear structures capable of transmitting tensile forces. Specifically in the case of tendons, fibres ranging in size from 1 μm to 20 μm are bundled into 15 400 μm diameter triangular subfascicles, subfascicles are bundled into 150 1000 μm fascicles and fascicles are grouped into tertiary bundles ranging from 1000 μm to 3000 μm. Finally, multiple tertiary bundles are enclosed by the epitenon connective tissue, which contains the vascular, lymphatic and nerve sources for the tendon tissue.

b | In addition to fibrillar collagen, other collagen subtypes, such as type V and FACIT collagens, are incorporated into the fibril structure. Type V collagen is inserted between strands of the microfibril, and FACIT collagens cling to the surface of the microfibril and work with SLRPs to stabilize higher order structures. Figure part a is modified, with permission, from REF. 135 © 2012 Fan et al.; licensee BioMed Central Ltd, from Klug, William S.; Cummings, Michael R., Concepts of Genetics, 5th Edition, © 1997 (REF. 136). Reprinted by permission of Pearson Education, Inc., Upper Saddle River, NJ, and from REF. 138, reprinted from Trends Biotechnol., 26, Liu, Y., Ramanath, H. S. & Wang, D.-A., Tendon tissue engineering using scaffold enhancing strategies, 201–209, Copyright (2008), with permission from Elsevier. Figure part b: this figure was originally published in Biochem. J. Jäälinoja, J., Ylöstalo, J., Beckett, W., Hulmes, D. J. S. & Ala-Kokko, L., Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains. Biochem. J. 2008; 409: 545–554 © the Biochemical Society.

In the second step, after assembly in the endoplasmic reticulum, procollagen is packaged in the Golgi apparatus for export into the extracellular matrix. For the third step of collagen fibril synthesis, evidence suggests that procollagen processing, exocytosis and initial fibril assembly are closely associated with membrane-enclosed compartments¹⁵. Early models proposed that cells exert considerable spatial control over this pericellular space, with procollagen excreted by exocytosis and modified within deep, narrow recesses between cellular protrusions close to the cell body^27,28. Although procollagen processing was originally viewed as a solely extracellular event (with the second and third steps of collagen fibril synthesis being distinct and mutually exclusive), later work provided evidence that procollagen processing and collagen fibril assembly are initiated in compartments between the Golgi apparatus and the plasma membrane that are targeted to plasma membrane extrusions (known as fibripositors) that project from the cell surface. Recent electron microscopy studies confirmed the presence of collagen fibrils in cell surface fibripositors^29–32. Following or during secretion of procollagen into the ECM, proteolytic processing of the large procollagen NH₂- and COOH-propeptide domains leads to the production of mature collagen molecules that then self-assemble into fibrils. Enzymatic removal of the propeptide domains is carried out by collagen type-specific metalloproteinases from the a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) and bone morphogenetic protein 1 (BMP1) and tolloid-like families, as well as by furin-like proprotein convertases^16,33–36 (FIG. 1). If the C-propeptide remains attached, procollagen solubility in the extracellular space remains high, which inhibits premature fibril assembly; by contrast, the persistence of the N-propeptide influences fibril shape and diameter, possibly by increasing the surface area to volume ratio, without affecting fibril formation^16,37. In collagens I, II and III, the N-propeptides are completely removed, leaving short N-propeptides¹⁵. More detailed information on the roles and processing of the C- and N-propeptides can be found in REF. 16.

After cleavage of procollagen into collagen, initial fibril formation events occur at the cell surface³⁸. Collagen organization in fibrils has been intensely studied by electron microscopy and X-ray diffraction^39–44. Collagen self-assembles to form microfibrils with a quarter stagger axial D periodicity of approximately 67 nm to create the characteristic ‘striation’ that was recognized in initial electron microscopy observations of collagen-containing tissues^16,45 (FIG. 2a). Intermediate-sized microfibrils are thought to form via a multistage process that includes the nucleation, organization and unilateral elongation of short primary fibrils. Current models suggest that microfibril assembly is nucleated and modulated by the inclusion of collagens V and/or XI, which, as discussed above, retain portions of their N-terminal propeptide to co-assemble into fibrils with types I, II and III (FIGS 1,2b). The N-terminal propeptides of collagens V and/or XI are thought to project outwards through the gap between adjacent collagen molecules to interact with the fibril surface, which limits lateral growth via steric hindrance and charge interactions³⁸ (FIG. 2b). Although collagen V is a quantitatively minor component relative to collagen I and comprises less than 5% of the total collagen content in most tissues, it is considered to be a dominant regulator of fibrillogenesis, as deletion of collagen V in the mouse is associated with a lack of fibril assembly³⁸.

After collagen microfibrils have been assembled, short microfibrils may merge or grow into fibrils, increasing both longitudinally and axially; these transitions are regulated by various molecules^38,46 (FIG. 2). For example, the large body of evidence gained using in vitro techniques and in vivo knockout transgenic experiments shows that small leucine-rich repeat proteoglycans (SLRPs) with collagen-binding properties, including decorin, biglycan, fibromodulin and lumican, can affect fibril growth rate, size, morphology and content^15,47,48. Most heterotypic collagen fibrils are composed of a mixture of fibrillar collagens, with the FACIT (fibril-associated collagens with interrupted triple helices) collagens integrating into the fibrils, selectively altering the surface properties of the collagen fibril and its interactions with additional ECM molecules such as the SLRPs^37,49. Throughout the process of fibril assembly and growth, interactions among the assembling fibrils, accessory macromolecules and cell adhesion receptors actively organize the maturing fibril.

To form mature collagen fibres, further modifications are made during the assembly of collagen fibril aggregates. In the final step of collagen biosynthesis, covalent crosslinks are introduced into the supramolecular assembly to provide stability and enhanced mechanical properties^50–52. Extracellular lysyl oxidases catalyse and facilitate the oxidative deamination of targeted peptidyl lysine residues, which results in the formation of aldols or β-ketoamines by reacting with aldehydes or amino groups on lysines between collagen monomers to form intramolecular and intermolecular covalent crosslinks^49,51–53. The N- and C-terminals of neighbouring collagen monomers interact with each other and are covalently crosslinked by lysyl oxidase, both within and between microfibres^52–54. Overall, interactions between the fibrillar and the FACIT collagens, as well as among collagens, proteoglycans and crosslinking enzymes, can function as key regulators of fibrillar collagen organization, resulting in immense tissue-, stage- and age-specific differences in both fibril composition and structure. These variations are finely tuned to fulfil bespoke biochemical, structural and biomechanical roles²⁵. Importantly, the assembly of collagen fibrils is mainly driven by the unique chemical and physical interactions of the interacting components. This cell-driven self-assembly results in a multi-hierarchical collagen structure, which provides binding sites for other proteins and cells and is crucial for the mechanical integrity of the ECM⁵⁵.

Proteoglycans

In contrast to the predominantly fibrillar structure of collagens, proteoglycans form the basis of higher order ECM structures around cells. The primary biological function of proteoglycans derives from the biochemical and hydrodynamic characteristics of the GAG components of the molecules, which bind water to provide hydration and compressive resistance. As such, proteoglycans are found in abundance in cartilage and neural ECM^56,57. Proteoglycans are characterized by a core protein that is covalently linked to GAGs, which are long, negatively charged, linear chains of disaccharide repeats. They are classified into subtypes on the basis of the structure of these GAG carbohydrate chains, as well as on the distribution and the density of these chains along the core protein^58,59. Major GAGs include heparin sulphate, chondroitin sulphate, dermatan sulphate, hyaluronan and keratin sulphate^58,60–62 (the structures of heparin sulphate and chondroitin sulphate are shown in FIG. 3a).

Figure 3. Non-collagenous molecules of the ECM.

Lecticans (aggrecan is shown; part a) and small leucine-rich repeat proteoglycans (SLRPs; decorin, biglycan and lumican are shown; part b) are major proteoglycans (PGs). The SLRPs are characterized by differences in their amino terminus structure and highly conserved leucine rich repeats (LRR) in the core molecule. Lecticans have a core protein with binding domains for glycosaminoglycan (GAG) chains flanked by globular domains that interact with hyaluronic acid (at the N terminus) and tenascin R (at the carboxy terminus). Common GAGs include chondroitin sulphate and heparan sulphate, the chemical structures of which are depicted. The assembled bottle-brush-like aggrecan link protein hyaluronic acid structure is shown. Laminins are formed by the incorporation of α, β and γ chains into a cruciform, Y-shaped or rod-like structure (part c). These chains are characterized by different domains, as shown. The domain structures depicted represent only one isoform for each chain type, but major differences between the basic composition of the chains (such as the lack of globular regions in β and γ chains) are also present in the other isoforms. Laminins interact with cell surface receptors, such as integrins, primarily through globular domains in the chain. Fibronectin domain structure and the domains to which extracellular matrix (ECM) molecules and cell surface receptors bind are indicated (part d). The fibronectin molecule forms a dimer through disulphide bonds on its C terminus. The folded fibronectin molecule forms via ionic interactions between type III domains of neighbouring molecules and is deformed by mechanical force to reveal cryptic binding sites for other fibronectin molecules and cell surface receptors when interacting with cells. Figure part a: this image originally appeared in Diamond Light Source Ltd’s Annual Report 2010 (p72–73) and has been reproduced with permission from Diamond Light Source Ltd, the University of Liverpool, T. R. Rudd, M. A. Skidmore, S. E. Guimond, R. Xu, R. Hussain, D. G. Fernig, G. Siligardi and E. A. Yates http://www.diamond.ac.uk/Science/Research/Highlights/study2.html. Figure part b is modified from REF. 139, Nature Publishing Group. Figure part c is modified, with permission, from Yurchenco, P. D. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb. Perspect. Biol. 3, a004911 (2011), Cold Spring Harbour Laboratory Press, and REF. 141, Nature Publishing Group. Figure part d is modified, with permission, from Schwarzbauer, J. E. & DeSimone, D. W. Fibronectins, their fibrillogenesis, and in vivo functions. Cold Spring Harb. Perspect. Biol. 3, a005041 (2011), Cold Spring Harbour Laboratory Press, and REF. 82, modified with permission from Annual Review of Cell Dev. Biol., Volume 26 by Annual Reviews, http://www.annualreviews.org. EGF, epidermal growth factor; LM domain, laminin domain.

Heparan sulphate proteoglycans (HSPGs) are a major part of the basement membrane, which is a type of ECM structure (see below). Their GAG chains are comprised of highly sulphated chains of hexuronic acid and -glucosamine disaccharide repeats. HSPGs can be cell surface-bound, such as the syndecans and CD44v3, glycophosphatidylinositol (GPI)-linked, such as glypicans, or secreted ECM proteins, such as agrin, collagen XVIII and perlecan⁶³. Variety in HSPGs is introduced through splice isoforms that control GAG chain lengths, the spacing of GAG chain attachments along the protein core and the extent of sulphation and epimerization of the sugars within the GAG chains^62,63. Resulting proteoglycans show different binding affinities for molecules within the ECM. For example, fibroblast growth factors (FGFs) are particularly sensitive to the spacing and sulphation of GAG chains in their interactions with HSPGs⁶⁴. HSPGs have high negative charges that enable them to easily bind to other proteins, including growth factors such as FGF, chemokines, other ECM proteins, such as laminin and fibronectin, and cell surface receptors^63,65,66. As such, they have been implicated in regulating many cellular processes, including cell growth and migration^63,67,68.

Chondroitin sulphate proteoglycans (CSPGs) can be found in cartilage and neural ECM. CSPGs are functionalized with sulphated polysaccharides with repeating disaccharides of glucuronic acid and N-acetylgalactosamine⁶⁸. Lecticans (also known as hyalectins) are the most common CSPGs in the neural ECM^68,69 (FIG. 3a). This family consists of four members: aggrecan, neurocan, veriscan and brevican^61,69. The core protein is highly homologous across lecticans, which are characterized by globular domains at both the N and the C termini. Lecticans contain binding domains for hyaluronic acid (which is a common glycosaminoglycan in cartilage and the nervous system), lectins and growth factors^56,69,70. As with HSPGs, alternative splicing and different densities of GAG chains on the core protein lead to considerable variations in the properties of these proteins⁵⁶.

In addition to being dominant components of the ECM, proteoglycans can also function as accessory proteins in tissues rich in other matrix proteins. For example, the SLRPs are a family of proteoglycans that have been implicated in fibrillar collagen assembly⁴⁸. This family includes well-known members such as decorin, biglycan and lumican, with new members still being discovered and characterized. All SLRPs have two regions — a variable N-terminal domain that contains sulphated tyrosine or acidic amino acid residues and a conserved carboxy terminus that contains the leucine-rich repeats (LRRs) for which the family was named^48,71 (FIG. 3b). GAG chains can then be added to these core proteins⁴⁸. The LRRs contain collagen-binding regions and have been well characterized as a result of this property; the function of the N-terminal domains is less extensively understood⁴⁸. Elucidation of the crystal structure of decorin suggests that these N-terminal domains function as a capping mechanism, whereby cysteine residues within this domain form disulphide bonds that link back to the C terminus via leucines in the consensus sequence⁷².

Five classes of SLRPs have so far been established, and each class is distinguished by their homology on the protein and genomic levels, by the presence of N-terminal cysteine-rich clusters and by chromosomal placement. Classes I–III are so-called ‘canonical’ classes, which are distinguished by the relative abundance of data describing their function and localization within the body, whereas classes IV–V have only been recently described and there are many unanswered questions about their function. A full listing and description of SLRPs is beyond the scope of this Review, but such information can be found in REFS 73,74. The Class I SLRPs decorin and biglycan have been extensively studied and participate in collagen fibrillogenesis^57,75. Not only do these proteins facilitate the formation of collagen fibrils but they also regulate fibril width and growth through mechanisms that have not been fully elucidated⁷⁴. In addition to their roles as structural proteins in ECM assembly, SLRPs are considered to be ‘active’ members of the ECM and to participate in cell signalling. Binding sites for growth factors, cytokines and ECM proteins other than collagen have recently been found on SLRPs⁷¹.

Connectors in the ECM

Collagen, proteoglycans, and hyaluronic acid represent major structural components within the ECM. They provide much of the supportive framework within which other ECM components and cells interact. Additional ECM components, such as laminin or fibronectin, function as bridges between structural ECM molecules to reinforce this network, as well as to connect the ECM to cells and to soluble molecules within the extracellular space. These connectors tend to be multidomain glycoproteins that contain binding motifs for other ECM proteins, growth factors and cell surface receptors¹. In this Review, we focus on laminin and fibronectin as examples and point to others in TABLE 2 for further reference.

Table 2.

Non-collagenous ECM proteins

Protein	Location	Description	Refs
Agrin	Skeletal muscles	Large PG that is crucial in the development of the neuromuscular junction; contains laminin G domains, Kazal-type serine protease inhibitor domains and epidermal growth factor domains	142–144
Integrin-binding sialoprotein (also known as bone sialoprotein)	Bone	Phosphorylated and sulphated glycoprotein that is crucial for the structure of mineralized matrix; binds to calcium and hydroxyapatite and mediates cell attachment through an RGD sequence that recognizes the vitronectin receptor	145–147
Matrilins	Cartilage and connective tissues	Non-collagenous, disulphide-bonded proteins that are involved in the formation of filamentous networks; contains von Willebrand factor A domains	148–151
Elastin	Arteries, connective tissue, lungs, skin and bladder	One of two components of elastic fibres that provide structural integrity along with reversible extensibility and deformability; forms covalently crosslinked polymers	152–155
Fibrinogen	Blood	Soluble glycoprotein, which, after cleavage by thrombin, is converted to fibrin monomer molecules that self-associate to form insoluble, homopolymeric ‘clots’	153,156, 157
Fibrillin	Connective tissues and cardiovascular tissues	Predominant component of microfibrils, which forms a sheath around elastin in the formation of elastic fibres	158,159
Fibulins	Basement membranes, blood and arteries	Calcium-binding glycoprotein that is found in fibrillar matrices and blood	153,160, 161
Netrins	Basement membranes	Laminin-related protein that is involved in axon guidance and the development of the vasculature, lung, pancreas, muscle and the mammary gland	162,163
SPARC (also known as osteonectin)	Bone	Cysteine-rich, acidic, calcium-binding glycoprotein that is required for collagen calcification, mineralization initiation and mineral crystal formation	164,165
Secreted phosphoprotein 1 (also known as osteopontin)	Bone and kidneys	Negatively-charged glycoprotein that is involved in the attachment of cells to mineralized bone matrix	146,166, 167
Reelin	Brain	Large glycoprotein that is involved in guiding neuron and glial cell positioning in the developing brain	168,169
Tenascins	Connective tissues	Glycoproteins that are involved in mediating inflammatory and fibrotic processes	119,153, 170,171
Vitronectin	Blood	Adhesive glycoprotein that is involved in blood coagulation and wound healing	172,173

ECM, extracellular matrix; PG, proteoglycan.

Laminins

Laminins are a family of large, mosaic glycoproteins composed of globular, laminin-type epidermal growth factor (EGF)-like repeats and α-helical domains (FIG. 3c). They are primarily located in the basal lamina and some mesenchymal compartments. Interactions between modular domains within the laminin molecule and other proteins enable laminins to mediate interactions between cells via cell surface receptors (such as integrins and dystroglycan) and other components of the ECM (such as nidogens (also known as entactins), perlecans and collagens)⁷⁶ (FIG. 3c).

Laminins consist of α, β and γ chains that combine via the triple-helical coiled-coil domain in the centre of each chain to form structures with a cruciform (cross-shaped), Y-shaped (three arms) or rod-shaped (single arm) structure⁷⁶ (FIG. 3c). Different chain isoforms combine to generate distinct assembled heterotrimers⁷⁶. These laminins are described by their combined chain numbers; for example, α1β1γ1 is known as laminin 111. This nomenclature was preceded by single number assignments when there were fewer known isoforms. The original designations remain preferred in some settings so, for example, it is possible to find references to laminin 5 instead of α3Aβ3γ2. For a useful table that cross-references these different systems, see REF. 77. So far, five α-chains (α1–α5), four β-chains (β1–β4) and three γ-chains (γ1–γ3) are known, and they combine to form 16 different laminin heterotrimers^77,78 Although laminin 111 has been shown to self-assemble into aggregates, laminins primarily function as bridges between other molecules^79,80.

Fibronectin

Many of the ECM proteins described above interact with cells through crucial connections with the multidomain protein fibronectin to regulate cell adhesion, migration and differentiation⁸¹. Fibronectin is secreted as a large ECM glycoprotein that assembles via cell-mediated processes into fibrillar structures around cells⁸². Each fibronectin subunit consists of three modules of repeating units, each of which has distinct structures: type I, type II and type III structures^81,82 (FIG. 3d). These modules contain binding motifs that are important in facilitating the interaction of fibronectin with cell surface receptors, such as integrins, collagen and gelatin, and intramolecular units that enable self-assembly of the molecule^82,83. Intramolecular disulphide bonds between each type I and type II module stabilize the folded tertiary structure of the fibronectin subunit, and fibronectin dimers form via antiparallel disulphide bonds at the C terminus⁸².

Fibronectin fibril assembly involves interactions between its RGD and synergy sequences with corresponding binding sites within cell surface receptors such as integrins^81,82,84. Initial binding between fibronectin dimers and integrins leads to the activation and clustering of these receptors⁸². Integrin clustering is thought to promote further fibronectin–fibronectin intermolecular interactions, and tethering of fibronectin molecules to the cell surface enables cell-mediated contractility to exert force on the fibronectin molecule and to change its conformation⁸¹. This change in conformation exposes cryptic binding sites within the fibronectin molecule and also enables it to take a form that is more conducive to self-assembly into fibrils. The maturation and the movement of fibrillar adhesions within the cell is thought to guide the formation of fibronectin fibrils^82,85. Generation of force along these adhesions further controls the assembly of the fibronectin network and the maturation of multiple clusters of integrin-based adhesions brings nascent fibronectin bundles together to form a larger structure^82,85.

In addition to the key molecular players we have described in this Review, the ECM is composed of many other classes of macromolecules that we have not included owing to space constraints. For brief descriptions of their chemical composition and where in the body they may be found, as well as excellent references that may enrich understanding of these important components, see TABLE 2.

Organizing the ECM

The ECM is not simply a collection of proteins. Different tissues have unique and specialized ECM components and ECM organization, which enables each ECM to carry out tissue-specific roles, including structural support, the transmission of forces and macromolecular filtration^1,86. Three ECM landscapes, spanning diverse structures and functions, are discussed in this section, with a focus on how each supramolecular ECM is fabricated and organized. With a specialized axial and longitudinal organization, the first ECM assembly example consists of the fibrillar collagen-rich matrix that comprises mechanical load-bearing tendons. To provide an example of a non-fibrillar but protein-rich ECM, the basal lamina is discussed as the second example. With a much more amorphous, GAG-rich structure, the final example discussed is the perineuronal net (PNN), which is found in brain ECM. The unique structure–function relationships exhibited by these ECMs are highlighted. This section describes the ECMs of two specific tissues (tendon and brain) as well as the basal lamina, which is found in many tissues.

Fibrillar collagen-rich tendons

Fibrillar ECM that consists of substantial amounts of collagens I, II and III, such as the ECM found in tendons, ligaments and cartilage, has distinct structural and biomechanical properties that facilitate the effective transmission and absorption of cyclical tensile forces while avoiding injury^87–89. These properties arise from the specialized axial and longitudinal structural organization of the collagen fibre hierarchy, which provides a scaffold for cell and macromolecular attachment^89,90. The tendon is primarily a uniaxial force-transmitting connective tissue consisting of bundles of parallel fibrillar ECM and resident tendon fibroblasts (known as tenoblasts) that connect bone to muscle. Both the structure and the function of this fibrillar ECM provide biochemical and mechanical signals that cooperate in the integrated regulation of tenoblast proliferation, survival, differentiation and migration, which ultimately feedforward to maintain physiological ECM synthesis and assembly⁹¹.

The ECM that constitutes the tendon consists of collagen (65–80% of its dry mass, primarily collagen I) and elastin (1–2% of its dry mass) embedded in a well-hydrated, proteoglycan-rich matrix synthesized by tenoblasts⁹⁰. In the mature tendon, bundles of aligned collagen fibrils are organized into a functionally continuous fibrous ECM, which enables them to guide motion and to transmit forces^89,92. Throughout tendon development, multiple independent steps of collagen fibrillogenesis and ECM assembly take place and eventually contribute to a functionally mature tissue that has a complex ECM hierarchical structure.

In the first of three distinct steps in tendon collagen fibrillogenesis, initial collagen fibril assembly occurs near the surface of tendon fibroblasts, with collagen incorporating into microfibrils, which are the lowest order filamentous structures (approximately 4 nm in diameter), having an axial D periodicity^{16,88–90,93} (FIG. 2a). Through spatially regulated protein secretion, tenoblasts help to guide this initial assembly step; evidence suggests that fibripositors of tenoblasts contiguously present these microfibrils from one cell to another along the tendon axis through side-to-side and end-to-end tenoblast alignment^88,94,95. Procollagen processing, homotypic and heterotypic collagen interactions and other matrix macromolecules influence the nucleation and the growth of these microfibril intermediates (see above). Importantly, these micrfibrils remain semi-flexible, which enables the repositioning of these structures within the tendon tissue throughout tissue maturation and force transmission⁸⁸.

In the second step, longitudinal fibril growth occurs with microfibril intermediates fusing tip-to-tip as a result of lateral associations between adjacent tapered ends, and thus incorporating into continuous, mechanically functional fibrils^87,96,97 (FIG. 2a). Intermediate microfibrils are present in two structural forms — unipolar fibrils that have C and N termini and N,N-bipolar fibrils, in which the molecules have their N termini pointed towards one of the tips¹⁵. End-to-end fusion specifically requires the C terminus of unipolar fibrils⁹⁷. SLRPs often coat the body of microfibril intermediates, which promotes the fusion of the proteoglycan-free ends and inhibits premature side-to-side fusions⁹⁷.

The third step involves the axial growth of the microfibrils via incorporation of, and interactions with, other microfibrils, as well as with microdomains of extracellular macromolecules such as FACIT collagens and proteoglycans (FIG. 2b). These collagen-interacting molecules regulate and restrict this axial growth and limit fibril diameter, which provides resident tendon fibroblasts with a mechanism for regulating this ECM assembly, resulting in fibril structures ranging in size from 10 nm to 500nm^88,89 (FIG. 2a).

Fibres ranging in size from 1 μm to 20 μm form the next level of structure, with multiple fibrils grouped together by an endotenon connective tissue containing nerves, as well as blood and lymphatic vessels⁸⁹. Fibres are bundled into 15–400 μm diameter triangular subfascicles, at which point they are compressed by surrounding structures and stabilized via interactions with FACIT collagens and SLRPs^89,90. Subfascicles are bundled into 150–1000 μm fascicles, which are further bundled into tertiary bundles ranging from 1000 μm to 3000 μm^89,90. Finally, multiple tertiary bundles are enclosed by the epitenon connective tissue, which, similar to the endotenon, contains the vascular, lymphatic and nerve sources for the tendon tissue^89,90 (FIG. 2a).

Throughout the tissue, non-collagenous matrix, which consists of glycoproteins, proteoglycans, GAGs and other small molecules, surrounds and stabilizes collagen fibrils and fibres. The negatively charged groups of the sulphated proteoglycans and GAGs bind water, which improves the mechanical properties of the tissue under both shear and compressive stresses⁹⁰. The stability and mechanical properties of this tissue are further strengthened and stabilized by collagen crosslinking, which is carried out by enzymes such as lysyl oxidase, as well as by non-enzymatic glycation⁸⁹. Taken together, this hierarchical fibrillar structure results in tendon tissue that is capable of transmitting forces from muscle to bone without sustaining injury⁹¹.

Although collagen fibrillogenesis has been intensively studied for multiple tissues, many outstanding questions and areas of active research remain. Further elucidation of the biochemical mechanisms that underlie collagen fibril assembly will enable the development of rational therapies and tissue-engineering strategies for tissue repair and/or for the regeneration of adult tissues with mechanical properties that are similar to those that are observed in normal development and function. In addition, through the continued investigation and clarification of the structures and functions of the various domains along the collagen fibril, along with how these domains affect supramolecular assembly, novel insights into the roles of collagen at a tissue level in fibrotic diseases may highlight new targets to limit or to inhibit fibrosis. Improvements in high-resolution imaging, the establishment of in vivo genetic knockout (and overexpression) models and the introduction of novel in vitro self-assembling ECMs add to the available range of tools that investigators can use to continue elucidating the underlying mechanisms responsible for the different levels of supramolecular collagen assembly¹⁵.

Basal lamina

The basal lamina is a set of cell-adherent, organized layers of ECM molecules that form the supporting structure for epithelial, endothelial, muscle and fat cells, the central nervous system and the Schwann cells of peripheral nerves^98–100. These ‘sheet-like’ layers appear early in development, segregating tissues, functioning as macromolecular filters and providing sites for cell adhesion^86,101,102. The basal lamina shields tissues from disruptive biochemical and biophysical stresses and mediates communication among cells in the tissue and between cells and their external environment¹⁰¹. Although ‘basal lamina’ and ‘basement membrane’ are often used interchangeably in some tissues, such as skeletal muscle, the basement membrane is comprised of two distinct structures: the basal lamina and an external, fibrillar reticular lamina, which is directly linked to the cell membrane by the basal lamina¹⁰³.

The basal lamina predominately assembles at or near cell surfaces¹⁰⁴. The resulting ECM architecture consists of interconnected polymers of collagen IV and VII (FIG. 4a,b), laminins bound to nidogen glycoproteins and HSPGs⁹⁹. Laminin deposits on the surface of cells self-assemble into sheets through terminal domain interactions that are organized by cell surface molecules, such as integrins and dystroglycan, and by binding to nidogen through a single globular domain^102,105 (FIG. 4c). Collagen IV networks formed through interactions between its N-and C-terminal domains (FIG. 4a) then provide a three-dimensional scaffold for the association of laminin–nidogen complexes into less ordered aggregates, which interact with the collagen IV structure through binding sites on the nidogen molecule^102,106. These collagen IV networks are thought to stabilize the assembling basal lamina through covalent crosslinks¹⁰¹. In addition, sulphated proteoglycans, such as agrin, perlecan and collagen XVIII, can incorporate into the lamina, which provides areas of negative charge and tethers growth factors¹⁰⁷. These layers then recruit other basal lamina proteins, many of which have binding sites for one another, which leads to the formation of a mature basal lamina. Collagen VII can further stabilize this structure, in addition to connecting the basal lamina to the reticular lamina to form the basement membrane, via interactions with collagen IV (FIG. 4b,c). The inclusion of tissue-specific isoforms of laminin and collagen IV with different proteoglycan species and accessory proteins provides specific properties to the basal lamina in different locations. The basal lamina seems to be predominantly attached through laminins to cell surface receptors and sulphated glycoproteins¹⁰⁴. These interactions are responsible for the activation of intracellular signalling, such as that involved in establishing cell polarity.

Figure 4. Basal lamina assembly.

Two major collagens participate in the assembly of the basal lamina: collagen IV and collagen VII.

a | Collagen IV is characterized by an amino-terminal 7S domain and a carboxy-terminal NC1 domain these are globular domains that facilitate the formation of hexamers and dimers, respectively. These then form highly ordered lattice structures, as shown.

b | Collagen VII forms long dimers that then bend and connect with the globular regions of collagen IV lattices.

c | Components of the basal lamina, and how they interact, are shown on the left-hand side. Within the basal lamina, laminins form a sheet on the cell surface by recruiting soluble laminin. This sheet is reinforced via interactions among chains of different laminin molecules and by binding between laminin globular regions and cell surface receptors (for example, integrins and dystroglycan). Collagen IV binds to laminins via nidogens and forms a scaffold on top of the laminins. This scaffold is further secured by collagen VII, which, in its unbent form (sometimes termed anchoring fibrils), can link the basal lamina to the reticular lamina to form the basement membrane. Assembled basal lamina is shown on the right-hand side. Figure parts a and b are modified from REF. 100, Nature Publishing Group. Figure part c is modified from REF. 100, Nature Publishing Group, with permission, from Yurchenco, P. D. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb. Perspect. Biol. 3, a004911 (2011), Cold Spring Harbor Laboratory Press and from REF. 141, Nature Publishing Group. DG, dystroglycan.

The structure and composition of the basal lamina directly contributes to its functions within tissues: to anchor the epithelium to the dermis, to guide development and differentiation and to function as a mechanical barrier to cells and macromolecules. The basement membrane is deposited early in development (soon after implantation) and helps to establish polarity in the epiblast¹⁰⁸. Expression of ECM components within the basal lamina is tightly controlled in order to regulate intracellular signalling via activation of different receptors for these proteins and, in turn, to regulate cell fate determination at different stages of development^109–111. The scaffold structure of the basal lamina, along with its secure connections to the dermis, means it provides excellent support for the epithelium. It is highly crosslinked and functions as an efficient barrier to both cellular and molecular traffic between distinct layers of tissue. However, it is also thin enough to permit transit in certain conditions, such as when immune cells infiltrate during inflammation¹¹². Many of its components contain binding domains that allow the basement membrane to bind and to retain growth factors and chemokines, which have crucial roles in directing differentiation and guiding tissue repair and regeneration^103,113,114.

Although much has been elucidated about the basal lamina, our understanding of its functions and interactions with cells is by no means complete. There remain interesting unanswered questions regarding the regulatory processes in place for the production of various components of the basal lamina, how the basal lamina imposes cell polarity and, through this, influences cell behaviour, the many roles of the proteoglycans in directing cell fate, and more. Research to address these questions uses a combination of cell biology, novel approaches in synthetic chemistry and glycobiology, and cutting-edge imaging techniques to visualize large-scale ECM structures.

Proteoglycan-rich matrices of the brain

Proteoglycan-rich ECM can be found in cartilage and in the central nervous system and lacks the distinctive superstructural organization that characterizes ECM surrounding other tissues. Instead, it is characterized by amorphous aggregates of HSPGs and CSPGs, crosslinked by other small proteoglycans. However, that is not to say that these ECMs lack organization altogether. In this section, we focus on the brain ECM as an example.

ECM in the brain contains fibrous components such as collagens, fibronectins and other molecules discussed above. However, these proteins represent a small percentage of the brain ECM, which predominantly consists of hyaluronic acid, proteoglycans and GAGs. Thus, it is a useful tissue in which to highlight proteoglycan-rich ECM properties and functions. For a full discussion of neural ECM and its various components, see REFS 56,115,116.

Early in development, the neural ECM is produced and secreted by both neurons and glial cells¹¹⁷. The composition and organization of neural ECM is highly dynamic throughout development and varies in different parts of the adult brain^56,59,118. It is characterized by amorphous protein aggregates and is similar in organization to the proteoglycan component of cartilage. In both developing and adult brains, hyaluronic acid, which is a large, linear polysaccharide, functions as a central organizing molecule around which the other components of the ECM aggregate^61,117. Glycoproteins within the tenascin family have an oligomeric structure that facilitates this assembly, and proteoglycans with hyaluronic acid-binding domains on their N termini and glycoprotein-binding domains on their C termini connect these proteins^69,70,119. In contrast to the highly regulated structures in collagen-rich matrices or in the basal lamina, neural ECM aggregates form a latticed network that varies in its composition and organization throughout development^120–122. Depending on the stage of development, this network can be mostly made up of neurocans, versicans, and tenascin C, as is found in the first few weeks of life, or brevican and aggrecan, as is the case in the adult brain^122–124. These different proteoglycan profiles correspond with a ‘loose’ network that is made up of larger proteoglycans in early development, which is replaced and tightened with smaller proteoglycans as the brain develops^69,122. These loose networks provide space for the developing brain to grow and to extend the neural network; tight networks in later life restrict growth and reinforce existing connections.

An exception to the mainly unorganized nature of the brain ECM are PNNs, so-called because they form a regular structure around the cell bodies of neurons, dendrites and the initial segments of axons¹²². The formation of PNNs is initiated by the production of hyaluronan by hyaluronan synthases on the inner surface of the plasma membrane¹²⁵. Linear unsulphated hyaluronic acid chains deposited on the cell surface bind to extracellular CSPGs; these interactions are reinforced by link proteins (which are poorly characterized glycoproteins known to bind to both hyaluronic acid and proteoglycans) and tenascin R¹²⁵ (FIG. 5). In this network, the N terminus of CSPG binds to hyaluronic acid and the C terminus of CSPG binds to tenascin R, forming trimeric crosslinkers that set up a stoichiometric relationship that determines the organization of the PNN¹²⁵.

Figure 5. Perineuronal nets.

Perineuronal nets (PNNs) surround cells of the brain and act as inhibitors of both growth and migration. PNNs are lattices of hyaluronic acid, proteoglycans and tenascin molecules. The basic structure of a PNN is depicted here. Hyaluronic acid chains coated with proteoglycans are linked by trimeric tenascin R molecules to form a fairly organized structure around neurons. The chondroitin sulphate proteoglycans (CSPGs) within the net bind to cell surface CSPGs and can thus control cell behaviour. Figure modified from REF. 115, Nature Publishing Group.

The brain ECM, as with all other ECMs, provides structural support for cells within the organ. It also actively participates in the function of the brain; for example, the formation of PNNs is perfectly timed with the termination of plasticity in the developing brain, termed the ‘critical period’ (REFS 125,126). Conversely, delay of PNN formation or degradation of the PNN is associated with prolonged, or a return of, plasticity¹²⁷. Furthermore, the degradation of CSPGs within the PNN is associated with a restoration of neural plasticity^124,127. The density and ‘tightness’ of the ECM around neurons can inhibit their growth at the end of development⁶⁹. Deposition of proteoglycans provides an adhesive surface for growth factors and then cells during axon regeneration after injury or in neurite growth^{125,128–130}. The differential expression of proteoglycans has been implicated in various neurological conditions, including Alzheimer’s disease and epilepsy⁶⁵. In particular, it has been suggested that increased secretion of CSPGs after injury inhibits regeneration and repair, which may exacerbate an initially small injury^131,132. The manner in which ECM deposition and degeneration is related to neural pathogenesis is an active area of research. As researchers unravel the complexities that are responsible for the context-dependent effects of ECM alteration in the nervous system, they are also making progress towards understanding how to mitigate long-term effects of damage to the neural ECM.

Conclusions and future perspectives

The ECM has important roles in regulating cell and tissue development and function^1,86. The ECM acts with specificity in different tissues of the body by taking advantage of the wide variety of ECM proteins that exist, as well as unique structures formed by interactions among these ECM components⁴. This diversity results in highly distinctive ECMs that can be directly linked to the particular roles they have in their respective tissues. As such, the production and assembly of the ECM is an important aspect of maintaining cell and tissue homeostasis. These processes follow different temporal and spatial rules in various tissues, with load-bearing tissues such as tendons exhibiting highly ordered, fibrillar structures and the continually evolving brain showing a less organized, GAG-rich ECM^28,61,117. Therefore, disruption of the relative abundance of ECM proteins or their interactions with one another has important consequences for the behaviour and the fate of cells within that tissue^5,133,134. In order to study how perturbations to ECM remodelling influence pathological conditions such as cancer and cardiovascular disease, it is necessary to understand its ground state. Researchers have made great progress in characterizing the many components of the ECM and the manners in which these components interact with one another. Although this remains an ongoing area of investigation, the next step is to more thoroughly describe the temporal and spatial mechanisms by which ECM production and assembly are regulated in cells and tissues. Genetic modifications offer a way to perturb these factors in a controlled manner, and advances in in vivo imaging techniques will facilitate observations of the resulting effects. Not only would this understanding benefit tissue engineers seeking to build organs in vitro, but pinpointing such details may also identify more nuanced approaches to therapy in cancer, inflammatory diseases and more.

Glossary

Topographies

The three-dimensional qualities of surfaces or structures, including contours and relief. In the context of the ECM this includes features such as peaks and valleys, changes in roughness and geometric features.

Morphogenesis

The process of cell movement during embryonic development that controls the size, the shape and the patterning of tissues and organs.

Proteoglycans

(PGs). Glycoproteins that consist of a core protein to which one or more glycosaminoglycan chain is attached.

Glycosaminoglycans

(GAGs). Long, linear, charged polysaccharides that comprise repeating pairs of sugars, of which one is an amino sugar.

Fibrillar collagen

Polymerized, supramolecular collagen that has been organized into fibrils; collagen types I, II and III form fibrils.

Tensile forces

The forces required to exert a certain amount of tension (which is a type of normal stress) on a one-dimensional object. In this context, a pulling force on a cable tending to cause extension of the cable.

Procollagen

A trimeric collagen precursor molecule containing large amino- and carboxy-terminal propeptide domains.

Lysyl oxidase

An enzyme that participates in the formation of collagen polymers by facilitating oxidative deamination of peptidyl lysine residues on collagen monomers.

Pericellular space

The space surrounding a cell.

Fibripositors

(Also known as fibropositors). Plasma membrane protrusions projecting from the cell surface, where procollagen can potentially be processed, assembled and organized by individual cells.

Fibrillogenesis

The development or formation of fibrils, used to refer to collagen- or fibronectin-rich structures.

Small leucine-rich repeat proteoglycans

(SLRPs). A family of proteoglycans that share a common leucine-rich-repeat motif in their conserved carboxy termini; they have been strongly implicated in modulating fibrillar collagen assembly.

FACIT

(Fibril-associated collagens with interrupted triple helices). A type of collagen that does not form fibrils by itself but that is associated with the surface of fibrillar collagen.

Basement membrane

A thin, complex extracellular matrix that separates endothelial and epithelial cells from their subjacent connective tissues. It is composed of various collagens, proteoglycans and adhesive glycoproteins.

Syndecans

One of the two major families of heparin sulphate proteoglycans.

Glypicans

One of the two major families of heparin sulphate proteoglycans.

Epimerization

The formation of stereoisomers.

Integrins

A large family of heterodimeric transmembrane proteins, which are present in the plasma membrane as heterodimers of α- and β-subunits and function as receptors for cell adhesion molecules.

Nidogens

(Also known as entactins). Glycoproteins and key parts of the basement membrane.

Fibrillar adhesions

Cell–matrix connections that are typified by their location near the centre of the cell and their higher length to width ratio compared with focal adhesions at the periphery of cells.

Fascicles

Bundles of fibres.

Reticular lamina

A collagen-rich layer that is often found below the basal lamina that connects the combined basement membrane to the connective tissue.