Genetic mutations in fibrillin microfibrils cause serious inherited diseases, such as Marfan syndrome and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. Here we describe a detailed structural analysis of native fibrillin microfibrils from mammalian tissue by cryogenic electron microscopy. The major bead region showed pseudo eightfold symmetry where the amino and carboxy termini reside. On the basis of this structure, we show that a WMS deletion mutation leads to the induction of a structural rearrangement that blocks interaction with latent TGFβ-binding protein-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin αvβ3-binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as fibrillin microfibrils, mutations may have long-range structural consequences leading to the disruption of growth factor signaling and the development of disease.

Collagen fibrils are essential for metazoan life. They are the largest, most abundant, and most versatile protein polymers in animals, where they occur in the extracellular matrix to form the structural basis of tissues and organs. Collagen fibrils were first observed at the turn of the 20th century. During the last 40 years, the genes that encode the family of collagens have been identified, the structure of the collagen triple helix has been solved, the many enzymes involved in the post-translational modifications of collagens have been identified, mutations in the genes encoding collagen and collagen-associated proteins have been linked to heritable disorders, and changes in collagen levels have been associated with a wide range of diseases, including cancer. Yet despite extensive research, a full understanding of how cells assemble collagen fibrils remains elusive. Here, we review current models of collagen fibril self-assembly, and how cells might exert control over the self-assembly process to define the number, length and organisation of fibrils in tissues.

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formed in vitro from collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly, in vitro fibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.

Collagen is the most abundant secreted protein in vertebrates and persists throughout life without renewal. The permanency of collagen networks contrasts with both the continued synthesis of collagen throughout adulthood and the conventional transcriptional/translational homeostatic mechanisms that replace damaged proteins with new copies. Here, we show circadian clock regulation of endoplasmic reticulum-to-plasma membrane procollagen transport by the sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis and degradation affects a pool of newly synthesized collagen, while maintaining the persistent collagen network. Disabling the circadian clock causes abnormal collagen fibrils and collagen accumulation, which are reduced in vitro by the NR1D1 and CRY1/2 agonists SR9009 and KL001, respectively. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis wherein a sacrificial pool of collagen maintains tissue function. Here, Chang et al. show that the circadian clock regulates secretion resulting in nocturnal procollagen synthesis and daytime collagen fibril assembly in mice to maintain the homeostasis of the collagen network.

Remodelling of the dermal extracellular matrix makes a major contribution to skin fragility in the elderly. The peri-menopausal period in females is also associated with an age-like phenotype which can be reversed by hormone replacement therapy. This suggests a direct link between circulating hormone levels and tissue ageing. Despite work investigating the role of estrogen as a regulator of collagen fibril abundance and structure, the influence of estrogen on the elastic fibre system remains poorly defined. Here we used an ovariectomised (Ovx) mouse surgical menopause model to show that just 7 weeks of acute hormone deficiency significantly decreased skin tensile strength and elasticity. Systemic replacement of 17β-estradiol to physiological levels protected against these changes to the skin mechanical properties. Moreover, acute hormone deficiency differentially influenced dermal structural networks, significantly decreasing dermal elastic fibre abundance without discernible effect on collagen fibril organisation or abundance. We suggest that this specific elastic fibre proteolysis may be driven by extracellular protease activity, or be a consequence of significant adipocyte hypertrophy. 17β-estradiol supplementation in Ovx mice in vivo protected the elastic fibre system. Treatment of human dermal fibroblasts with 17β-estradiol in vitro induced the selective upregulation of tropoelastin, fibrillin-1 and associated elastic fibre-associated proteins (including EMILINs and fibulins). In summary, these data show that the elastic fibre system is significantly perturbed by estrogen deprivation. Thus, pharmacological intervention may slow the acute effects of menopause and potentially the chronic effects of ageing in skin.

ABSTRACT Hormone deficiency has been widely linked to accelerated tissue ageing, and increased incidence of chronic degenerative disease. Furthermore, age-associated hormonal dysregulation is thought to be a major contributing factor to the increased fragility of aged skin. The ageing process is driven by an aggregation of damage to cells and extracellular matrix, which can directly influence the mechanical properties of the tissue. Here we report on the correlation between mechanical properties and composition of skin from ovariectomised and aged mice, to assess the extent to which estrogen deprivation drives dermal ageing. We found that age and estrogen abrogation affected skin mechanical properties in contrasting ways: ageing led to increased tensile strength and stiffness while estrogen deprivation had the opposite effect. Mass spectrometry proteomics showed that the quantity of extractable fibrillar collagen-I decreased with ageing, but no change was observed in ovariectomised mice. This observation, in combination with measurements of tensile strength, was interpreted to reflect changes to the extent of extracellular matrix crosslinking, supported by a significant increase in the staining of advanced glycation endpoints in aged skin. Loss of mechanical strength in the skin following ovariectomy was consistent with a loss of elastic fibres. Other changes in extracellular matrix composition broadly correlated between aged and ovariectomised mice, confirming the important role of estrogen-related pathways in ageing. This study offers new insight into the relationship between tissue composition and mechanics, and suggests that the deleterious effects of intrinsic skin ageing are compounded by factors beyond hormonal dysregulation.

Tendons transmit contractile muscular force to bone to produce movement, and it is believed cells can generate endogenous forces on the extracellular matrix to maintain tissue homeostasis. However, little is known about the direct mechanical measurement of cell-matrix interaction in cell-generated human tendon constructs. In this study we examined if cell-generated force could be detected and quantified in engineered human tendon constructs, and if glycosaminoglycans (GAGs) contribute to tendon force transmission. Following de-tensioning of the tendon constructs it was possible to quantify an endogenous re-tensioning. Further, it was demonstrated that the endogenous re-tensioning response was markedly blunted after interference with the cytoskeleton (inhibiting non-muscle myosin-dependent cell contraction by blebbistatin), which confirmed that re-tensioning was cell generated. When the constructs were elongated and held at a constant length a stress relaxation response was quantified, and removing 27% of the GAG content of tendon did not alter the relaxation behavior, which indicates that GAGs do not play a meaningful role in force transmission within this system.

Connective tissues such as tendon, ligament and skin are biological fibre composites comprising collagen fibrils reinforcing the weak proteoglycan-rich ground substance in extracellular matrix (ECM). One of the hallmarks of ageing of connective tissues is the progressive and irreversible change in the tissue mechanical properties; this is often attributed to the underlying changes to the collagen fibril structure. This dataset represents a comprehensive screen of the mechanical properties and collagen fibril structure of tendon from the tails of young to old (i.e. 1.6–35.3 month-old) C57BL6/B mice. The mechanical portion consists of the load-displacement data, as well as the derived tensile properties; the structure data consists of transmission electron micrographs of collagen fibril cross section, as well as the derived cross-sectional parameters. This dataset will allow other researchers to develop and demonstrate the utility of innovative multiscale models and approaches of the extra-cellular and physiological events of ageing of current interest to ageing research, by reducing the current reliance on conducting new mammalian experiments.

The formation of uniaxial fibrous tissues with defined viscoelastic properties implies the existence of an orchestrated mechanical interaction between the cytoskeleton and the extracellular matrix. This study addresses the nature of this interaction. The hypothesis is that this mechanical interplay underpins the mechanical development of the tissue. In embryonic tendon tissue, an early event in the development of a mechanically robust tissue is the interaction of the pointed tips of extracellular collagen fibrils with the fibroblast plasma membrane to form stable interface structures (fibripositors). Here, we used a fibroblast-generated tissue that is structurally and mechanically matched to embryonic tendon to demonstrate homeostasis of cell-derived and external strain-derived tension over repeated cycles of strain and relaxation. A cell-derived oscillatory tension component is evident in this matrix construct. This oscillatory tension involves synchronization of individual cell forces across the construct and is induced in each strain cycle by transient relaxation and transient tensioning of the tissue. The cell-derived tension along with the oscillatory component is absent in the presence of blebbistatin, which disrupts actinomyosin force generation of the cell. The time period of this oscillation (60-90 s) is well-defined in each tissue sample and matches a primary viscoelastic relaxation time. We hypothesize that this mechanical oscillation of fibroblasts with plasma membrane anchored collagen fibrils is a key factor in mechanical sensing and feedback regulation in the formation of tensile tissues.

Collagen fibrils are the major mechanical component in the extracellular matrix of a broad range of multicellular animals from echinoderms to vertebrates where they provide a stable framework for tissues. They form the key tension-resisting element of a complex fiber-composite system that has a tissue-specific hierarchical structure linked to mechanical demands. Remarkably, these tissues are self-maintaining and avoid fatigue failure over the lifetime of the animal. Collagen fibrils can assemble spontaneously from purified solutions of collagen molecules. In developing tissues, however, in addition to the intrinsic self-assembly properties, there is cellular machinery that regulates fibril nucleation, spatial orientation, and fibril size, according to the tissue and stage of development. The intricate mechanisms underlying the generation of a collagen fibril network of defined architecture and mechanical properties are now becoming apparent. Impairment of this system leads ultimately to mechanical failure or tissue fibrosis.

Collagen is the most abundant secreted protein in vertebrates that persists throughout life without renewal. The unchanging nature of collagen contrasts with observed continued collagen synthesis throughout adulthood and with conventional transcriptional and translational homeostatic mechanisms that replace damaged proteins with new copies. Here we show circadian clock regulation of procollagen transport from ER-to-Golgi and Golgi-to-plasma membrane by sequential rhythmic expression of SEC61, TANGO1, PDE4D and VPS33B. The result is nocturnal procollagen synthesis and daytime collagen fibril assembly in mice. Rhythmic collagen degradation by CTSK maintains collagen homeostasis. This circadian cycle of collagen synthesis, assembly and degradation affects only a pool of newly-synthesized collagen whilst maintaining the persistent collagen network. Disabling the circadian clock causes collagen accumulation and abnormal fibrilsin vivo. In conclusion, our study has identified a circadian clock mechanism of protein homeostasis in which a sacrificial pool of collagen is synthesized and removed to maintain tissue function.

Damage to the retina has so far proved irreversible, but stem-cell therapies could hold the key to restoring sight. Damage to the retina has so far proved irreversible, but stem-cell therapies could hold the key to restoring sight.

Can tissue engineering provide a cheap and convenient alternative to surgery for eardrum repair?

Scaffolds for tissue engineering application may be made from a collagenous extracellular matrix (ECM) of connective tissues because the ECM can mimic the functions of the target tissue. The primary sources of collagenous ECM material are calf skin and bone. However, these sources are associated with the risk of having bovine spongiform encephalopathy or transmissible spongiform encephalopathy. Alternative sources for collagenous ECM materials may be derived from livestock, e.g., pigs, and from marine animals, e.g., sea urchins. Collagenous ECM of the sea urchin possesses structural features and mechanical properties that are similar to those of mammalian ones. However, even more intriguing is that some tissues such as the ligamentous catch apparatus can exhibit mutability, namely rapid reversible changes in the tissue mechanical properties. These tissues are known as mutable collagenous tissues (MCTs). The mutability of these tissues has been the subject of on-going investigations, covering the biochemistry, structural biology and mechanical properties of the collagenous components. Recent studies point to a nerve-control system for regulating the ECM macromolecules that are involved in the sliding action of collagen fibrils in the MCT. This review discusses the key attributes of the structure and function of the ECM of the sea urchin ligaments that are related to the fibril-fibril sliding action—the focus is on the respective components within the hierarchical architecture of the tissue. In this context, structure refers to size, shape and separation distance of the ECM components while function is associated with mechanical properties e.g., strength and stiffness. For simplicity, the components that address the different length scale from the largest to the smallest are as follows: collagen fibres, collagen fibrils, interfibrillar matrix and collagen molecules. Application of recent theories of stress transfer and fracture mechanisms in fibre reinforced composites to a wide variety of collagen reinforcing (non-mutable) connective tissue, has allowed us to draw general conclusions concerning the mechanical response of the MCT at specific mechanical states, namely the stiff and complaint states. The intent of this review is to provide the latest insights, as well as identify technical challenges and opportunities, that may be useful for developing methods for effective mechanical support when adapting decellularised connective tissues from the sea urchin for tissue engineering or for the design of a synthetic analogue.

The spinal cord rarely repairs itself when damaged, but ways of encouraging nerves to regrow are on the horizon. The spinal cord rarely repairs itself when damaged, but ways of encouraging nerves to regrow are on the horizon.

Pressure ulcers are complex wounds caused by pressure- and shear-induced trauma to skin and underlying tissues. Pressure-reducing devices, such as dressings, have been shown to successfully reduce pressure ulcer incidence, when used in adjunct to pressure ulcer preventative care. While pressure-reducing devices are available in a range of materials, with differing mechanical properties, understanding of how a material's mechanical properties will influence clinical efficacy remains limited. The aim of this study was to establish a standardized ex vivo model to allow comparison of the cell protection potential of two gel-like pressure-reducing devices with differing mechanical properties (elastic moduli of 77 vs. 35 kPa). The devices also displayed differing energy dissipation under compressive loading, and resisted strain differently under constant load in compressive creep tests. To evaluate biological efficacy we employed a new ex vivo porcine skin model, with a confirmed elastic moduli closely matching that of human skin (113 vs. 119 kPa, respectively). Static loads up to 20 kPa were applied to porcine skin ex vivo with subsequent evaluation of pressure-induced cell death and cytokine release. Pressure application alone increased the percentage of epidermal apoptotic cells from less than 2% to over 40%, and increased cellular secretion of the pro-inflammatory cytokine TNF-alpha. Co-application of a pressure-reducing device significantly reduced both cellular apoptosis and cytokine production, protecting against cellular damage. These data reveal new insight into the relationship between mechanical properties of pressure-reducing devices and their biological effects. After appropriate validation of these results in clinical pressure ulcer prevention with all tissue layers present between the bony prominence and external surface, this ex vivo porcine skin model could be widely employed to optimize design and evaluation of devices aimed at reducing pressure-induced skin damage.

3D domain-swapping proteins form multimers by unfolding and then sharing of secondary structure elements, often with native-like interactions. Runaway domain swapping is proposed as a mechanism for folded proteins to form amyloid fibres, with examples including serpins and cystatins. Cystatin C amyloids cause a hereditary form of cerebral amyloid angiopathy whilst cystatin B aggregates are found in cases of Unverricht-Lundborg Syndrome, a progressive form of myoclonic epilepsy. Under conditions that favour fibrillisation, cystatins populate stable 3D domain-swapped dimers both in vitro and in vivo that represent intermediates on route to the formation of fibrils. Previous work on cystatin B amyloid fibrils revealed that the α-helical region of the protein becomes disordered and identified the conservation of a continuous 20-residue elongated β-strand (residues 39-58), the latter being a salient feature of the dimeric 3D domain-swapped structure. Here we apply limited proteolysis to cystatin B amyloid fibrils and show that not only the α-helical N-terminal of the protein (residues 1-35) but also the C-terminal of the protein (residues 80-98) can be removed without disturbing the underlying fibril structure. This observation is incompatible with previous models of cystatin amyloid fibrils where the β-sheet is assumed to retain its native antiparallel arrangement. We conclude that our data favour a more generic, at least partially parallel, arrangement for cystatin β-sheet structure in mature amyloids and propose a model that remains consistent with available data for amyloids from either cystatin B or cystatin C.

Embryonic growth occurs predominately by an increase in cell number; little is known about growth mechanisms later in development when fibrous tissues account for the bulk of adult vertebrate mass. We present a model for fibrous tissue growth based on 3D-electron microscopy of mouse tendon. We show that the number of collagen fibrils increases during embryonic development and then remains constant during postnatal growth. Embryonic growth was explained predominately by increases in fibril number and length. Postnatal growth arose predominately from increases in fibril length and diameter. A helical crimp structure was established in embryogenesis, and persisted postnatally. The data support a model where the shape and size of tendon is determined by the number and position of embryonic fibroblasts. The collagen fibrils that these cells synthesise provide a template for postnatal growth by structure-based matrix expansion. The model has important implications for growth of other fibrous tissues and fibrosis.