Solvent specific persistence length of molecular type I collagen

Type I collagen is a fibril‐forming protein largely responsible for the mechanical stability of body tissues. The tissue level properties of collagen have been studied for decades, and an increasing number of studies have been performed at the fibril scale. However, the mechanical properties of collagen at the molecular scale are not well established. In the study presented herein, the persistence length of pepsin digested bovine type I collagen is extracted from the conformations assumed when deposited from solution onto two‐dimensional surfaces. This persistence length is a measure of the flexibility of the molecule. Comparison of the results for molecules deposited from different solvents allows for the study of the effect of the solutions on the flexibility of the molecule and provides insight into the molecule's behavior in situ. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 329–335, 2014.     

Matrix loading: assembly of extracellular matrix collagen fibrils during embryogenesis

Nothing in biology stimulates the imagination like the development of a single fertilized egg into a newborn child. Consequently, a major focus of biomedical research is aimed at understanding cell differentiation, proliferation, and specialization during child health and human development. However, the fact that the increase in size and shape of the growing embryo has as much to do with the extracellular matrix (ECM) as with the cells themselves, is largely overlooked. Cells in developing tissues are surrounded by a fiber-composite ECM that transmits mechanical stimuli, maintains the shape of developing tissues, and functions as a scaffold for cell migration and attachment. The major structural element of the ECM is the collagen fibril. The fibrils, which are indeterminate in length, are arranged in different tissues in exquisite supramolecular architectures, including parallel bundles, orthogonal lamellae, and concentric weaves. This article reviews our current understanding of the synthesis and assembly of collagen fibrils, and discusses challenging questions about how cells assemble an organized ECM during embryogenesis.     

Nanoscale structure of type I collagen fibrils: Quantitative measurement of D-spacing

This article details a quantitative method to measure the D-periodic spacing of type I collagen fibrils using atomic force microscopy coupled with analysis using a two-dimensional fast fourier transform approach. Instrument calibration, data sampling and data analysis are discussed and comparisons of the data to the complementary methods of electron microscopy and X-ray scattering are made. Examples of the application of this new approach to the analysis of type I collagen morphology in disease models of estrogen depletion and osteogenesis imperfecta (OI) are provided. We demonstrate that it is the D-spacing distribution, not the D-spacing mean, that showed statistically significant differences in estrogen depletion associated with early stage osteoporosis and OI. The ability to quantitatively characterize nanoscale morphological features of type I collagen fibrils will provide important structural information regarding type I collagen in many research areas, including tissue aging and disease, tissue engineering, and gene knockout studies. Furthermore, we also envision potential clinical applications including evaluation of tissue collagen integrity under the impact of diseases or drug treatments.     

Fibrous long spacing type collagen fibrils have a hierarchical internal structure

Nanodissection of single fibrous long spacing (FLS) type collagen fibrils by atomic force microscopy (AFM) reveals hierarchical internal structure: Fibrillar subcomponents with diameters of approximately 10 to 20 nm were observed to be running parallel to the long axis of the fibril in which they are found. The fibrillar subcomponent displayed protrusions with characteristic approximately 270 nm periodicity, such that protrusions on neighboring subfibrils were aligned in register. Hence, the banding pattern of mature FLS-type collagen fibrils arises from the in-register alignment of these fibrillar subcomponents. This hierarchical organization observed in FLS-type collagen fibrils is different from that previously reported for native-type collagen fibrils, displaying no supercoiling at the level of organization observed.     

Characterization of adhesion properties of the cardiomyocyte integrins and extracellular matrix proteins using atomic force microscopy

Integrins are transmembrane adhesion receptors that play important roles in the cardiovascular system by interacting with the extracellular matrix (ECM). However, direct quantitative measurements of the adhesion properties of the integrins on cardiomyocyte (CM) and their ECM ligands are lacking. In this study, we used atomic force microscopy (AFM) to quantify the adhesion force (peak force and mean force) and binding probability between CM integrins and three main heart tissue ECM proteins, ie, collagen (CN), fibronectin (FN), and laminin (LN). Functionalizing the AFM probes with ECM proteins, we found that the peak force (mean force) was 61.69 ± 5.5 pN (76.54 ± 4.0 pN), 39.26 ± 4.4 pN (59.84 ± 3.6 pN), and 108.31 ± 4.2 pN (129.63 ± 6.0 pN), respectively, for the bond of CN‐integrin, FN‐integrin, and LN‐integrin. The binding specificity between CM integrins and ECM proteins was verified by using monoclonal antibodies, where α₁₀‐ and α₁₁‐integrin bind to CN, α₃‐ and α₅‐integrin bind to FN, and α₃‐ and α₇‐integrin bind to LN. Furthermore, adhesion properties of CM integrins under physiologically high concentrations of extracellular Ca²⁺ and Mg²⁺ were tested. Additional Ca²⁺ reduced the adhesion mean force to 68.81 ± 4.0 pN, 49.84 ± 3.3 pN, and 119.21 ± 5.8 pN and binding probability to 0.31, 0.34, 0.40 for CN, FN, and LN, respectively, whereas Mg²⁺ caused very minor changes to adhesion properties of CM integrins. Thus, adhesion properties between adult murine CM integrins and its main ECM proteins were characterized, paving the way for an improved understanding of CM mechanobiology.     

Deposition of type X collagen in the cartilage extracellular matrix

In cultured chick embryo chondrocytes, type X collagen is preferentially deposited in the extracellular matrix, the ratio between type II and type X collagen being about 5 times higher in the culture medium than in the cell layer. When the newly synthesized collagens deposited in slices from the epiphyseal cartilage of 17-day-old embryo tibiae were isolated, type X collagen was always the major species. In agreement with this result the mRNA for type X collagen was the predominant mRNA species purified from the same tissue. When the total collagen (unlabeled) deposited in the epiphyseal cartilage was analyzed, it was observed that type X collagen represented only 1/15 of the type II collagen recovered in the same preparation. The possible explanations for these differences are discussed.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Atomic force microscopy study of the collagen fibre structure

Observations of intact reconstituted and native collagen fibres were performed with the atomic force microscope. The results are compared between the two types of fibres and with those obtained previously with the electron microscope on freeze-etched or negative stained samples. Some of the findings presented here indicate that the specimens observed in air with the atomic force microscope were still in a hydrated state.     

Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.     

Role of the extracellular matrix in prostate carcinogenesis

This review summarizes the current state of knowledge regarding the proteins composing the extracellular matrix in the human prostate. The normal expression as well as the changes which occur in PIN and carcinoma are described for the lamins, collagens, and glycosaminoglycans.     

The precision of lateral size control in the assembly of corneal collagen fibrils

Collagen fibrils in the corneal stroma have been recognised to have a high degree of uniformity of diameter and spatial arrangement compared with those in other mature connective tissues. The precision of this lateral size control has been determined in this study by mass per unit length measurements on fibrils isolated from adult bovine corneal stroma. At the molecular level, however, there are substantial variations in lateral size, both between fibrils and along individual fibrils. The mean mass per unit length was measured to be 304 kDa nm(-1), equivalent to 347 collagen molecules in transverse section and had a standard deviation of 8.3%. The variation of lateral size along individual fibrils was measured as a mass slope over approximately 7 microm lengths (100 D-periods) and had a mean mass slope equivalent to 0.56 molecules per D-period. Smoothly tapered tips of length approximately 7 microm were also observed with a mass slope of about approximately three molecules per D-period. The frequency of these tips was used to estimate a mean fibril length of approximately 940 microm in the sample tissue. Observations of molecular polarity within the fibril shafts and tips were used to consider possible models of fibril assembly.     

The roles of types XII and XIV collagen in fibrillogenesis and matrix assembly in the developing cornea

Corneal transparency depends on the architecture of the stromal extracellular matrix, including fibril diameter, packing, and lamellar organization. The roles of collagen types XII and XIV in regulation of corneal fibrillogenesis and development were examined. The temporal and spatial expression patterns were analyzed using semi-quantitative RT-PCR, in situ hybridization, Western analysis, and immunohistochemistry. Expression of types XII and XIV collagens in cornea development demonstrated that type XII collagen mRNA levels are constant throughout development (10D-adult) while type XIV mRNA is highest in early embryonic stages (10D-14D), decreasing significantly by hatching. The spatial expression patterns of types XII and XIV collagens demonstrated a homogeneous signal in the stroma for type XIV collagen, while type XII collagen shows segregation to the sub-epithelial and sub-endothelial stroma during embryonic stages. The type XII collagen in the anterior stroma was an epithelial product during development while fibroblasts contributed in the adult. Type XIV collagen expression was highest early in development and was absent by hatching. Both types XII and type XIV collagen have different isoforms generated by alternative splicing that may alter specific interactions important in fibrillogenesis, fibril-fibril interactions, and higher order matrix assembly. Analysis of these splice variants demonstrated that the long XII mRNA levels were constant throughout development, while the short XII NC3 mRNA levels peaked early (12D) followed by a decrease. Both type XIV collagen NC1 splice variants are highest during early stages (12D-14D) decreasing by 17D of development. These data suggest type XII collagen may have a role in development of stromal architecture and maintenance of fibril organization, while type XIV collagen may have a role in regulation of fibrillogenesis.     

Role of decorin on in vitro fibrillogenesis of type I collagen

Tendon and corneal decorins are differently iduronated dermatan sulphate/proteoglycan (DS/PG) and the biochemical parameter that differentiates type I collagens is the hydroxylysine glycoside content. We have examined the effect of tendon and corneal decorins on the individual phases (tlag, dA/dt) of differently glycosylated type I collagens fibril formation, at molar ratios PG:collagen monomer ranging from 0.15 : 1 to 0.45 : 1. The results obtained indicate that decorins exert a different effect on the individual phases of fibril formation, correlated to the degree of glycosylation of collagen: at the same PG:collagen ratio the fibril formation of highly glycosylated corneal collagen is more efficiently inhibited than that of the poorly glycosylated one (tendon). Moreover tendon and corneal decorins exert a higher control on the fibrillogenesis of homologous collagen with respect to the heterologous one. These data suggest a possible tissue-specificity of the interaction decorin/type I collagen correlated to the structure of the PG and collagen present in extracellular matrices. This revised version was published online in November 2006 with corrections to the Cover Date.     

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16^Ink4a or p21^Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces.     

Three-dimensional organization of the extracellular matrix secreted by cultured rat smooth muscle cells

Summary Specific interactions between cells and the extracellular matrix (ECM) in which they are embedded play a vital role in tissue organization. In recent years, many of the individual components of the extracellular matrix have been isolated and their molecular structures elucidated, but the detailed topography of most extracellular matrices, as they are deposited by cells, is still largely unknown. In this study, the insoluble extracellular matrix produced by cultured rat vascular smooth muscle cells has been characterized morphologically using high-resolution electron microscopy of rotary platinum replicas. These cells grew as flat sheets in culture, secreting their matrix laterally and basally. The matrix was composed of a cross-linked fibrillar meshwork. Some fine fibers (10 to 15 nm in diameter) were naked, but most of the filamentous mesh was covered with coarse granular material. Limited digestion with trypsin or pancreatic elastase removed most of this coating, indicating that the granules were glycoproteins and proteoglycans. Another subset of matrix fibrils (20 to 40 nm in diameter) was identified as type I collagen by direct comparison with purified bovine skin collagen. In addition to exposing the underlying filamentous substructure of the matrix, protease treatment also revealed large, straight fiber bundles and globules of amorphous material suspended in the filamentous web. This novel view of a complex matrix promises to provide spatial information that will be useful in future studies of cell interactions with the ECM. These studies were supported in part by NIH Biomedical Research Support grant S07-RR-05684.     

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster–specific decrease in mitochondrial DNA–encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.     

Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman

Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as ‘the Iceman’. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 ± 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young''s modulus of single mummified fibrils was 4.1 ± 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 ± 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years.     

Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM

The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin alpha(2)beta(1) polarized strongly in the fibril direction. In contrast, alpha(2)beta(1)-deficient cells adhered but polarized less well, suggesting a role of integrin alpha(2)beta(1) in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.     

The fibrous system in the extracellular matrix of hydromedusae

The ultrastructure and the histochemistry of the fibrous system in the mesogloeal extracellular matrix (ECM) of two hydromedusae (Polyorchis penicillatus and Aglanlha digitale) has been examined. There is a fundamental difference in the architecture of the fibrous system between the two species. In Polyorchis, 60-150 A thick, striated fibrils with periodicities of 60-65 A form a three-dimensional network which fills in the entire ECM of outer and inner mesogloea. In the outer mesogloea vertical fibres (up to 1.8 mum in diameter) penetrate the threedimensional network and branch near the exumbrellar and subumbrellar side. These branches impinge on a dense matrix covering the exumbrellar and subumbrellar surface. In Aglantha the branches of thick vertical fibres anchor at the subumbrellar side in a dense plexus (0.2-0.3 mum in thickness) which consists of two types of fibrils (35-40 and 80-100 nm in diameter). Towards the exumbrellar side the vertical fibres branch and intermingle with a meshwork of non-striated fibrils with uniform diameter (35-40 nm). These fibrils form a laminated structure (about 1 mum in thickness) so that fibrils of each layer course in the same direction but fibrils of adjacent layers run perpendicularly to each other. The banded pattern with periodicities of 600-640 A observed in the electron microscope and by histochemical methods confirm the thick vertical fibres and their branches to be a collagen. There is also strong evidence that the laminated structure in Aglantha represents layers of collagen fibrils.     

Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptors

Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the β₁ integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or β₁ integrin antibody (agonist for β₁ integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by β₁ integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor. J. Cell. Biochem. 67:166–175, 1997. Published 1997 Wiley-Liss, Inc.