Wellcome Centre for Cell-Matrix Research

Several algorithms have been described in the literature for protein identification by searching a sequence database using mass spectrometry data. In some approaches, the experimental data are peptide molecular weights from the digestion of a protein by an enzyme. Other approaches use tandem mass spectrometry (MS/MS) data from one or more peptides. Still others combine mass data with amino acid sequence data. We present results from a new computer program, Mascot, which integrates all three types of search. The scoring algorithm is probability based, which has a number of advantages: (i) A simple rule can be used to judge whether a result is significant or not. This is particularly useful in guarding against false positives. (ii) Scores can be compared with those from other types of search, such as sequence homology. (iii) Search parameters can be readily optimised by iteration. The strengths and limitations of probability-based scoring are discussed, particularly in the context of high throughput, fully automated protein identification.

The local microenvironment, or niche, of a cancer cell plays important roles in cancer development. A major component of the niche is the extracellular matrix (ECM), a complex network of macromolecules with distinctive physical, biochemical, and biomechanical properties. Although tightly controlled during embryonic development and organ homeostasis, the ECM is commonly deregulated and becomes disorganized in diseases such as cancer. Abnormal ECM affects cancer progression by directly promoting cellular transformation and metastasis. Importantly, however, ECM anomalies also deregulate behavior of stromal cells, facilitate tumor-associated angiogenesis and inflammation, and thus lead to generation of a tumorigenic microenvironment. Understanding how ECM composition and topography are maintained and how their deregulation influences cancer progression may help develop new therapeutic interventions by targeting the tumor niche.

The extracellular matrix (ECM) serves diverse functions and is a major component of the cellular microenvironment. The ECM is a highly dynamic structure, constantly undergoing a remodeling process where ECM components are deposited, degraded, or otherwise modified. ECM dynamics are indispensible during restructuring of tissue architecture. ECM remodeling is an important mechanism whereby cell differentiation can be regulated, including processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. In contrast, abnormal ECM dynamics lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer. Understanding the mechanisms of ECM remodeling and its regulation, therefore, is essential for developing new therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine.

Integrins are one of the major families of cell adhesion receptors (Humphries, 2000; Hynes, 2002). All integrins are non-covalently linked, heterodimeric molecules containing an α and a β subunit. Both subunits are type I transmembrane proteins, containing large extracellular domains and mostly short cytoplasmic domains (Springer and Wang, 2004; Arnaout et al., 2005). Mammalian genomes contain 18 α subunit and 8 β subunit genes, and to date 24 different α-β combinations have been identified at the protein level. Although some subunits appear only in a single heterodimer, 12 integrins contain the β1 subunit, and five contain αV.Integrin function has been determined through a combination of cell biological and genetic analyses. On the cytoplasmic face of the plasma membrane, integrin occupancy coordinates the assembly of cytoskeletal polymers and signalling complexes; on the extracellular face, integrins engage either extracellular matrix macromolecules or counter-receptors on adjacent cell surfaces. These bidirectional linkages impose spatial restrictions on signalling and extracellular matrix assembly, and thereby integrate cells with their microenvironment. In turn, membrane-proximal interactions initiate more distal functions such as tissue patterning (extracellularly) and cell fate determination (intracellularly). Genetic analyses of engineered or natural mutations have confirmed key roles for integrins in tissue integrity, cell trafficking, and differentiation (Bouvard et al., 2001; Bokel and Brown, 2002).A characteristic feature of most integrin receptors is their ability to bind a wide variety of ligands. Moreover, many extracellular matrix and cell surface adhesion proteins bind to multiple integrin receptors (Humphries, 1990; Plow et al., 2000; van der Flier and Sonnenberg, 2001). In recent years, structure-function analyses of both integrins and their ligands have revealed a similar mode of molecular interaction that explains this promiscuity. Nonetheless, the integrin literature is replete with studies describing different integrin-ligand pairs, and the major aim of this article is to provide a clarification of this picture.The poster shows the major integrin-ligand combinations, using hypothetical cell surfaces. We have not attempted a comprehensive cataloguing, but instead we have consulted with a number of colleagues and reached a consensus view on the best-validated integrin ligands. There are many other ligands for different integrins, the inclusion of which would overly complicate the poster. By citing the best studied receptor-ligand combinations, we are aware that some reports and low-affinity interactions (which are nonetheless functionally relevant) may be discriminated against, and for this we apologise. Some of the interactions that are supported by convincing data are nonetheless included below.Historically, most integrin-ligand pairs have been identified either by affinity chromatography or through the ability of subunit-specific monoclonal antibodies to block adhesion of cells to specific ligands. In some cases, direct protein-protein binding assays have been used to support biochemical or cell biological data. Despite their wide variety, it is possible to cluster integrin-ligand combinations into four main classes, reflecting the structural basis of the molecular interaction. These classes do not necessarily reflect evolutionary relationships.All five αV integrins, two β1 integrins (α5, α8) and αIIbβ3 share the ability to recognise ligands containing an RGD tripeptide active site. Crystal structures of αVβ3 and αIIbβ3 complexed with RGD ligands have revealed an identical atomic basis for this interaction (Xiong et al., 2002; Xiao et al., 2004). RGD binds at an interface between the α and β subunits, the R residue fitting into a cleft in a β-propeller module in the α subunit, and the D coordinating a cation bound in a von Willebrand factor A-domain in the β subunit. The RGD-binding integrins are among the most promiscuous in the family, with β3 integrins in particular binding to a large number of extracellular matrix and soluble vascular ligands. Although many ligands are shared by this subset of integrins, the rank order of ligand affinity varies, presumably reflecting the preciseness of the fit of the ligand RGD conformation with the specific α-β active site pockets.α4β1, α4β7, α9β1, the four members of the β2 subfamily and αEβ7 recognise related sequences in their ligands. α4β1, α4β7 and α9β1 bind to an acidic motif, termed `LDV', that is functionally related to RGD. Fibronectin contains the prototype LDV ligand in its type III connecting segment region, but other ligands (such as VCAM-1 and MAdCAM-1) employ related sequences. Although definitive structural information is lacking, it is highly likely that LDV peptides bind similarly to RGD at the junction between the α and β subunits. Osteopontin also interacts with α4β1, α4β7 and α9β1, but this apparently involves a different peptide motif, SVVYGLR, and the location of the ligand-binding site has not been identified.The β2 family employ a different mode of ligand binding, the major interaction taking place through an inserted A-domain in the α subunit (see Shimaoka et al., 2003 for the structure of a complex between the αL A-domain and ICAM-1). However, despite this fundamental mechanistic difference, the characterised sites within ligands that bind β2 integrins are structurally similar to the LDV motif. The major difference is that β1/β7 ligands employ an aspartate residue for cation coordination whereas β2 integrins use glutamate. Collectively, therefore, the LDV motif can be described by the consensus sequence L/I-D/E-V/S/T-P/S.Four α subunits containing an αA-domain (α1, α2, α10 and α11) combine with β1 and form a distinct laminin/collagen-binding subfamily. Few other validated ligands have been identified for these integrins. A crystal structure of a complex between the α2 A-domain and a triple-helical collagenous peptide has revealed the structural basis of the interaction, a critical glutamate within a collagenous GFOGER motif providing the key cation-coordinating residue (Emsley et al., 2000). Currently, the mechanism of laminin binding is unknown.Three β1 integrins (α3, α6 and α7), plus α6β4, are highly selective laminin receptors. Analysis of laminin fragments indicates that these receptors and the A-domain-containing β1 integrins bind to different regions of the ligands. In neither case has the active site been narrowed down to a particular sequence or residue.As discussed above, additional integrin ligands exist that, for the sake of clarity, we do not include in the poster, even though credible evidence exists for them. These ligands, along with their respective integrin partners, are therefore listed here: ADAM family members interact with α4β1, α5β1, α6β1, α9β1, αVβ3 and αVβ6; COMP interacts with α5β1 and αvβ3; connective tissue growth factor interacts with αVβ3 and αIIbβ3; Cyr61 interacts with α6β1, αIIbβ3, αVβ3 and αDβ2; E-cadherin interacts with α2β1; ESM-1 interacts with αLβ2; fibrillin interacts with α5β1; fibrinogen interacts with αDβ2; fibronectin interacts with αDβ2; ICAM-4 interacts with α4β1, αLβ2, αMβ2, αXβ2, αVβ3 and αIIbβ3; LAP-TGFβ interacts with α8β1 and αVβ5; MMP-2 interacts with αVβ3; nephronectin interacts with α8β1; L1 interacts with α5β1, αVβ1, αVβ3 and αIIbβ3; plasminogen interacts with αDβ2; POEM interacts with α8β1; tenascin interacts with α2β1; thrombospondin interacts with α5β1 and α6β1; VEGF-C and VEGF-D interact with α9β1; and vitronectin interacts with αDβ2. Note also that both αMβ2 and αXβ2 interact with heparin and negative charges in denatured proteins.The model invertebrates Drosophila melanogaster and Caenorhabditis elegans have a much smaller complement of integrins than vertebrates (Hynes and Zhao, 2000). Drosophila has two β subunits (βPS and βν) and five α subunits. βν has no known α subunit partner, but βPS combines with subunits that cluster with the laminin-binding and RGD-binding integrins. The remaining α chains form a Drosophila-specific clade. A similar complement of integrins is found in Caenorhabditis elegans, which suggests that the earliest metazoans possessed two primordial integrins: one laminin-specific and one RGD-ligand-specific.The genome of the early chordate Ciona intestinalis encodes eleven α and five β chain genes (Ewan et al., 2005). Two Ciona α chains cluster with laminin-binding subunits and a third clusters with RGD-binding subunits. Surprisingly, eight α chains contain an αA-domain that is related to but, distinct from, the vertebrate αA-domains. Since these subunits are expressed predominantly in blood cells, they may play a role in innate immunity. It therefore seems that collagen-binding capabilities appeared in the chordate lineage after the divergence of ascidians. Of the five Ciona β chains, one clusters with β1, one clusters with β4, and three form an ascidian-specific clade.Work performed in the authors' laboratory that is related to the topic of this manuscript was supported by the Wellcome Trust. A.B. is supported by a BBSRC CASE PhD studentship, sponsored by GlaxoSmithKline. We thank Dean Sheppard, Nancy Hogg, Tim Springer, Mark Ginsberg and Steve Ludbrook for their comments on ligand specificities of different integrin subsets.

Abstract The unambiguous identification of Central Valley spring‐run chinook salmon has become imperative since their proposed listing in 1998. The accuracy of methods used to assign individuals to their stock of origin is critical for understanding juvenile migration patterns and determining the success of protection measures. Existing microsatellites discriminate between the endangered winter‐run and other chinook but are insufficient to characterize phylogenetically less distinct runs. Here, we isolated and developed highly variable tetranucleotide microsatellites for the specific goal of increasing discriminatory power among closely related populations, providing a new power towards the reliable differentiation of nonwinter runs

Collagen is most abundant in animal tissues as very long fibrils with a characteristic axial periodic structure. The fibrils provide the major biomechanical scaffold for cell attachment and anchorage of macromolecules, allowing the shape and form of tissues to be defined and maintained. How the fibrils are formed from their monomeric precursors is the primary concern of this review. Collagen fibril formation is basically a self-assembly process (i.e. one which is to a large extent determined by the intrinsic properties of the collagen molecules themselves) but it is also sensitive to cell-mediated regulation, particularly in young or healing tissues. Recent attention has been focused on "early fibrils' or "fibril segments' of approximately 10 microns in length which appear to be intermediates in the formation of mature fibrils that can grow to be hundreds of micrometers in length. Data from several laboratories indicate that these early fibrils can be unipolar (with all molecules pointing in the same direction) or bipolar (in which the orientation of collagen molecules reverses at a single location along the fibril). The occurrence of such early fibrils has major implications for tissue morphogenesis and repair. In this article we review the current understanding of the origin of unipolar and bipolar fibrils, and how mature fibrils are assembled from early fibrils. We include preliminary evidence from invertebrates which suggests that the principles for bipolar fibril assembly were established at least 500 million years ago.

Decorin is a member of the expanding group of widely distributed small leucine-rich proteoglycans that are expected to play important functions in tissue assembly. We report that mice harboring a targeted disruption of the decorin gene are viable but have fragile skin with markedly reduced tensile strength. Ultrastructural analysis revealed abnormal collagen morphology in skin and tendon, with coarser and irregular fiber outlines. Quantitative scanning transmission EM of individual collagen fibrils showed abrupt increases and decreases in mass along their axes. thereby accounting for the irregular outlines and size variability observed in cross-sections. The data indicate uncontrolled lateral fusion of collagen fibrils in the decorindeficient mice and provide an explanation for the reduced tensile strength of the skin. These findings demonstrate a fundamental role for decorin in regulating collagen fiber formation in vivo.

Integrins are large, membrane-spanning, heterodimeric proteins that are essential for a metazoan existence. All members of the integrin family adopt a shape that resembles a large "head" on two "legs," with the head containing the sites for ligand binding and subunit association. Most of the receptor dimer is extracellular, but both subunits traverse the plasma membrane and terminate in short cytoplasmic domains. These domains initiate the assembly of large signaling complexes and thereby bridge the extracellular matrix to the intracellular cytoskeleton. To allow cells to sample and respond to a dynamic pericellular environment, integrins have evolved a highly responsive receptor activation mechanism that is regulated primarily by changes in tertiary and quaternary structure. This review summarizes recent progress in the structural and molecular functional studies of this important class of adhesion receptor.

Focal adhesions (FAs) regulate cell migration. Vinculin, with its many potential binding partners, can interconnect signals in FAs. Despite the well-characterized structure of vinculin, the molecular mechanisms underlying its action have remained unclear. Here, using vinculin mutants, we separate the vinculin head and tail regions into distinct functional domains. We show that the vinculin head regulates integrin dynamics and clustering and the tail regulates the link to the mechanotransduction force machinery. The expression of vinculin constructs with unmasked binding sites in the head and tail regions induces dramatic FA growth, which is mediated by their direct interaction with talin. This interaction leads to clustering of activated integrin and an increase in integrin residency time in FAs. Surprisingly, paxillin recruitment, induced by active vinculin constructs, occurs independently of its potential binding site in the vinculin tail. The vinculin tail, however, is responsible for the functional link of FAs to the actin cytoskeleton. We propose a new model that explains how vinculin orchestrates FAs.

The airways mucus gel performs a critical function in defending the respiratory tract against pathogenic and environmental challenges. In normal physiology, the secreted mucins, in particular the polymeric mucins MUC5AC and MUC5B, provide the organizing framework of the airways mucus gel and are major contributors to its rheological properties. However, overproduction of mucins is an important factor in the morbidity and mortality of chronic airways disease (e.g., asthma, cystic fibrosis, and chronic obstructive pulmonary disease). The roles of these enormous, multifunctional, O-linked glycoproteins in health and disease are discussed.

The cytokine TGF-β plays an integral role in regulating immune responses. TGF-β has pleiotropic effects on adaptive immunity, especially in the regulation of effector and regulatory CD4(+) T cell responses. Many immune and nonimmune cells can produce TGF-β, but it is always produced as an inactive complex that must be activated to exert functional effects. Thus, activation of latent TGF-β provides a crucial layer of regulation that controls TGF-β function. In this review, we highlight some of the important functional roles for TGF-β in immunity, focusing on its context-specific roles in either dampening or promoting T cell responses. We also describe how activation of TGF-β controls its function in the immune system, with a focus on the key roles for members of the integrin family in this process.

Basement membranes are specialized extracellular matrices consisting of tissue-specific organizations of multiple matrix molecules and serve as structural barriers as well as substrates for cellular interactions. The network of collagen IV is thought to define the scaffold integrating other components such as, laminins, nidogens or perlecan, into highly organized supramolecular architectures. To analyze the functional roles of the major collagen IV isoform alpha1(IV)(2)alpha2(IV) for basement membrane assembly and embryonic development, we generated a null allele of the Col4a1/2 locus in mice, thereby ablating both alpha-chains. Unexpectedly, embryos developed up to E9.5 at the expected Mendelian ratio and showed a variable degree of growth retardation. Basement membrane proteins were deposited and assembled at expected sites in mutant embryos, indicating that this isoform is dispensable for matrix deposition and assembly during early development. However, lethality occurred between E10.5-E11.5, because of structural deficiencies in the basement membranes and finally by failure of the integrity of Reichert's membrane. These data demonstrate for the first time that collagen IV is fundamental for the maintenance of integrity and function of basement membranes under conditions of increasing mechanical demands, but dispensable for deposition and initial assembly of components. Taken together with other basement membrane protein knockouts, these data suggest that laminin is sufficient for basement membrane-like matrices during early development, but at later stages the specific composition of components including collagen IV defines integrity, stability and functionality.

Collagens are a large family of triple helical proteins that are widespread throughout the body and are important for a broad range of functions, including tissue scaffolding, cell adhesion, cell migration, cancer, angiogenesis, tissue morphogenesis and tissue repair. Collagen is best known as the principal tensile element of vertebrate tissues such as tendon, cartilage, bone and skin, where it occurs in the extracellular matrix as elongated fibrils. Collagen is also well known for its location in basement membranes – for example, in the kidney glomerulus, where it functions in molecular filtration. However, the identification of transmembrane collagens on the surfaces of a wide variety of cells and collagens that are precursors of bioactive peptides that have paracrine functions has resulted in a revival of interest in collagen. Moreover, new developments in 3D reconstruction electron microscopy have led to new opportunities for studying intracellular trafficking of collagen. Newcomers to the field face the daunting task of sifting through 100,000 research papers that span 40 years. Here, we provide `the collagen basics'. Several excellent reviews are cited that are sources of more detailed descriptions and discussions.Collagens contain three polypeptide (α) chains, displaying an extended polyproline-II conformation, a right-handed supercoil and a one-residue stagger between adjacent chains (Brodsky and Persikov, 2005). Each polypeptide chain has a repeating Gly-X-Y triplet in which glycyl residues occupy every third position and the X and Y positions are frequently occupied by proline and 4-hydroxyproline, respectively. The three α chains are held together by interchain hydrogen bonds. Highly ordered hydration networks surround the triple helices. The significance of these interactions to collagen stability remains a matter of debate. Some collagens have interruptions (containing numerous residues) and imperfections (one to three residues) in the triple helix. The conformational changes derived from some simple imperfections have been visualised in crystal structures of model peptides (Bella et al., 2006). Integrin adhesion sequences [e.g. Arg-Gly-Asp (RGD) and Gly-Phe-O-Gly-Arg, where O is hydroxyproline] occur in the triple helical domain of several collagens and contribute to integrin-ligand-binding specificity (Bella and Humphries, 2005).There is no agreed definition for a collagen; there are triple helical proteins that are called collagens and there are proteins that have triple helical domains that are not regarded as collagens. In general, collagens are regarded as triple helical proteins that have functions in tissue assembly or maintenance. Inevitably, the line between `collagens' and `collagen-like' proteins is blurred. Vertebrate collagens are given a Roman numeral.Collagen I is the archetypal collagen in that it is trimeric, it is triple helical, its triple helix has no imperfections, it assembles into fibrils, and it has a predominately structural role in the tissue. However, most collagens differ from collagen I in one or more respects. For example, other collagens can have interruptions in the triple helix and do not necessarily assemble (in their own right) into fibrils. Furthermore, transmembrane collagens have numerous interruptions in the triple helix, do not self-assemble into fibrils, and have roles in cell adhesion and signaling.At least 28 different collagens occur in vertebrates (numbered I-XXVIII; some with common names), together with a large group of collagen-like proteins (e.g. acetyl cholinesterase, adiponectin, C1q, ficolin, macrophage receptor and surfactant protein) (for a review, see Myllyharju and Kivirikko, 2001). In general, invertebrates have far fewer collagen genes but most have examples of fibrillar and basement membrane collagen (Huxley-Jones et al., 2007). Most collagens have evolved from orthologues present in invertebrates but little is known about the molecular composition of these molecules and no systematic nomenclature exists. Notably, ∼200 cuticle-forming collagens are found in Caenorhabditis elegans (Page and Winter, 2003).Collagens can be heterotrimeric – for example, type I collagen, which contains two identical α chains and a third chain that differs, [α1(I)]2α2(I). However, the majority of collagens are homotrimers – for example, collagen II, which contains three identical α chains [α1(II)]3. Note that the α1 chain of one type of collagen (e.g. collagen I) has a primary structure different from that of the α1 chain of another type of collagen (e.g. collagen II). Collagens have non-triple helical domains at their N- and C-termini. These domains are called `non collagenous' (NC) domains and are numbered from the C-terminus (NC1, NC2, etc.).Fibril-forming collagens occur as 67-nm D-periodic fibrils that are the principal source of tensile strength in animal tissues (Kadler et al., 1996). The fibrils are indeterminate in length and range in diameter from 12 nm to >500 nm, depending on the stage of development and tissue. The periodic structure of the fibril is due to regular staggering of triple helical collagen molecules. Mammals have 11 fibrillar collagen genes, which cluster phylogenetically into three distinct subclasses (Huxley-Jones et al., 2007). The Gly-X-Y domain of fibril-forming collagens contains ∼1000 residues and is uninterrupted, with the notable exception of collagen XXIV and collagen XXVII.Fibril-forming collagens are synthesised as procollagens containing N- and C-propeptides at each end of the triple helical domain. Cleavage of the C-propeptides is required for fibrillogenesis. The C-propeptides are cleaved by procollagen C-proteinases, which are identical to the BMP-1/tolloid proteinases (Greenspan, 2005). In the case of collagen V, the proα1(V) chain is cleaved by furin to release the C-propeptide. The N-propeptides are cleaved by procollagen N-proteinases, which are identical to the ADAMTS 2, ADAMTS 3 and ADAMTS 14 proteinases (Colige et al., 2005). The proα1(V) chain of collagen V is the exception: it is cleaved by BMP-1 (for details, see Greenspan, 2005). Cleavage of the propeptides exposes telopeptide sequences that are short non-triple helical extensions of the polypeptide chains. The telopeptides contain binding sites for fibrillogenesis (Prockop and Fertala, 1998). The fibrillar collagens are stabilised by non-reducible covalent crosslinks that involve residues in the triple helix and in telopeptides (Eyre et al., 1984). The crosslinks are essential for the normal mechanical properties of collagen-containing tissues.FACITs are relatively short collagens, have interruptions in the triple helical domain and can be found at the surfaces of collagen fibrils. Collagen IX is the archetypal FACIT; it is covalently crosslinked to collagen II (Wu et al., 1992), and is post-translationally modified to carry a glycosaminoglycan side chain.Collagen IV is the prototypical network-forming collagen. It forms an interlaced network in basement membranes, where it has an important molecular filtration function. The network is generated by head-to-head interactions of two trimeric NC1 domains. The resultant hexamer is stabilised by covalent Met-Lys crosslinks (Than et al., 2002). N-to-N interactions between four collagen IV molecules establish the crosslinked `7S domain', which is an important interaction node in the extended network. Collagen VIII is a major component of Descemet's membrane and vascular subendothelial matrices, where it occurs as polygonal superstructures. The related collagen X occurs in the hypertrophic zone of growth plate cartilage and is thought to form a network similar to that of collagen VIII (Stephan et al., 2004).These collagens are type II transmembrane proteins that have a short cytosolic N-terminal domain and long interrupted triple helical extracellular (ecto) domains. They include collagens XIII and XXV, which have cell adhesive properties and occur on numerous cell types, including malignant cells. The ectodomains can be proteolytically shed by furin-like proprotein convertases. Collagen XVII is cleaved by ADAM family proteinases. A growing number of collagen-like transmembrane proteins that have triple-helical ecto domains are being identified in vertebrates and invertebrates. These have not been assigned to a specific class but have important roles in neural function and neural tube dorsalisation, eye development, modulation of growth factor activity, and have cell adhesive functions. These un-adopted collagens include ectodysplasin, gliomedin and other members of the colmedin subfamily of transmembrane collagens. The ectodomain of gliomedin is shed by BMP-1/tolloid proteinases (Maertens et al., 2007).Collagen XV is found bridging adjacent collagen fibrils near basement membranes and can form a variety of oligomeric assemblies (Myers et al., 2007). Collagen XVIII is found in some basement membranes. Cleavage of part of the NC1 domains of collagens XV and XVIII releases endostatins, which are inhibitors of endothelial cell migration and angiogenesis, reduce tumour growth in animals, and control neuronal guidance in C. elegans (Marneros and Olsen, 2005).Collagen VII is the major component of the anchoring fibrils beneath the lamina densa of epithelia. The NC1 domain of collagen VII is cleaved by BMP-1/tolloid proteinases.Collagen VI is the archetypal beaded-filament-forming collagen. It is found in most tissues where it forms structural links with cells. Collagen VI monomers crosslink into tetramers that assemble into long molecular chains known as microfibrils, which have a beaded repeat of 105 nm.The importance of collagen is exemplified in a wide spectrum of diseases, which are caused by >1000 mutations; see OMIM (Online Mendelian Inheritance of Man) for a comprehensive listing (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM). These diseases include Alport syndrome (collagen IV), certain arterial aneurysms (collagen III), Bethlem myopathy and Ullrich muscular dystrophy (collagen VI) (Baker et al., 2005), certain chondrodysplasias (collagen IX and XI), some subtypes of the Ehlers-Danlos syndrome (collagen I and V), specific subtypes of epidermolysis bullosa (collagen VII), Kniest dysplasia (collagen II), Knobloch syndrome (collagen XVIII), osteogenesis imperfecta (collagen I) (Marini et al., 2007), some instances of osteoporosis and osteoarthrosis, and Stickler syndrome (collagen II, IX and XI) (Van Camp et al., 2006). Mice with genetically engineered collagen mutations have been produced. Collagen accumulates or is ectopically expressed in adhesions, fibrosis, cirrhosis, cardiovascular disease and scars.Collagens undergo extensive post-translational modification in the endoplasmic reticulum prior to triple helix formation. A number of enzymes and molecular chaperones assist in their correct folding and trimerisation. These include several hydroxylases, two collagen glycosyltransferases and peptidyl cis-trans isomerase, in addition to protein disulphide isomerase (PDI) (for a review, see Myllyharju and Kivirikko, 2004). HSP47 is a collagen-specific molecular chaperone that is essential for the normal synthesis of collagen (Nagata, 2003). Fibril-forming collagen molecules fold in a C- to N-terminal direction. The correct trimerisation of the NC1 domains is crucial for collagen assembly and has precluded the use of antigenic tags or fluorescent proteins at the C-terminus of the chains. Folding of the trimeric NC1 domain involves the formation of intra- and inter-chain disulphide bonds (Lees and Bulleid, 1994).Collagens are relatively large proteins and are not accommodated in conventional small-diameter transport vesicles (Bonfanti et al., 1998; Trucco et al., 2004). Procollagen I can be cleaved to collagen inside the cell and intracellular collagen fibrils can occur in plasma membrane protrusions called fibripositors (see review by Canty and Kadler, 2005) (see also Canty et al., 2004). In embryonic stages, collagen fibrils are closely associated with the plasma membrane of tendon and corneal fibroblasts (Birk and Trelstad, 1986). Collagen V is needed for the nucleation of collagen-I-containing fibrils in vivo (Wenstrup et al., 2004).The triple helix is resistant to proteolytic cleavage by pepsin, trypsin and papain. Clostridium histolyticum produces collagenases that cleave triple helices at numerous sites. The ability of collagens to resist cleavage by pepsin and trypsin, and their sensitivity to cleavage by bacterial collagenase, are used as research tools to identify and characterise collagens. Degradation of collagen and gelatin (unfolded or denatured collagen) in vivo can be mediated by MMPs, cysteine proteinases (e.g. cathepsins B, K and L), and serine proteinases (e.g. plasmin and plasminogen activator) (for reviews, see Everts et al., 1996; Sabeh et al., 2004).

Collagen fibrils in the extracellular matrix allow connective tissues such as tendon, skin and bone to withstand tensile forces. The fibrils are indeterminate in length, insoluble and form elaborate three-dimensional arrays that extend over numerous cell lengths. Studies of the molecular basis of collagen fibrillogenesis have provided insight into the trafficking of procollagen (the precursor of collagen) through the cellular secretory pathway, the conversion of procollagen to collagen by the procollagen metalloproteinases, and the directional deposition of fibrils involving the plasma membrane and late secretory pathway. Fibril-associated molecules are targeted to the surface of collagen fibrils, and these molecules play an important role in regulating the diameter and interactions between the fibrils.

Collagens are triple helical proteins that occur in the extracellular matrix (ECM) and at the cell-ECM interface. There are more than 30 collagens and collagen-related proteins but the most abundant are collagens I and II that exist as D-periodic (where D = 67 nm) fibrils. The fibrils are of broad biomedical importance and have central roles in embryogenesis, arthritis, tissue repair, fibrosis, tumor invasion, and cardiovascular disease. Collagens I and II spontaneously form fibrils in vitro, which shows that collagen fibrillogenesis is a selfassembly process. However, the situation in vivo is not that simple; collagen I-containing fibrils do not form in the absence of fibronectin, fibronectin-binding and collagen-binding integrins, and collagen V. Likewise, the thin collagen II-containing fibrils in cartilage do not form in the absence of collagen XI. Thus, in vivo, cellular mechanisms are in place to control what is otherwise a protein self-assembly process. This review puts forward a working hypothesis for how fibronectin and integrins (the organizers) determine the site of fibril assembly, and collagens V and XI (the nucleators) initiate collagen fibrillogenesis.

Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin-ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin alpha(5)beta(1)-Fc fusion protein or membrane alpha(5)beta(1). Force prolonged bond lifetimes in the 10-30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca(2+)/Mg(2+) to Mg(2+)/EGTA and to Mn(2+) caused longer lifetime in the same 10-30-pN catch bond region. A truncated alpha(5)beta(1) construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.

BACKGROUND: MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis. METHODS AND FINDINGS: By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis. CONCLUSIONS: Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.

At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three-dimensional nodule-like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage-specific expression of markers of primitive clonogenic marrow stromal cells (STRO-1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.

A role for Notch signaling in human breast cancer has been suggested by both the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, a negative regulator of the Notch pathway, in a large proportion of breast carcinomas. However, it is not clear currently whether Notch signaling is frequently activated in breast tumors, and how it causes cellular transformation. Here, we show accumulation of the intracellular domain of Notch1 and hence increased Notch signaling in a wide variety of human breast carcinomas. In addition, we show that increased RBP-Jkappa-dependent Notch signaling is sufficient to transform normal breast epithelial cells and that the mechanism of transformation is most likely through the suppression of apoptosis. More significantly, we show that attenuation of Notch signaling reverts the transformed phenotype of human breast cancer cell lines, suggesting that inhibition of Notch signaling may be a therapeutic strategy for this disease.

Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA(+)/CD44(+)/CD24(low). Using these breast cancer stem cell populations, we compared the activation status of Notch receptors with the status in luminally differentiated cells, and we evaluated the consequences of pathway inhibition in vitro and in vivo. We found that Notch4 signaling activity was 8-fold higher in stem cell-enriched cell populations compared with differentiated cells, whereas Notch1 signaling activity was 4-fold lower in the stem cell-enriched cell populations. Pharmacologic or genetic inhibition of Notch1 or Notch4 reduced stem cell activity in vitro and reduced tumor formation in vivo, but Notch4 inhibition produced a more robust effect with a complete inhibition of tumor initiation observed. Our findings suggest that Notch4-targeted therapies will be more effective than targeting Notch1 in suppressing breast cancer recurrence, as it is initiated by breast cancer stem cells.