ABSTRACT
Fibrillin-rich microfibrils are evolutionarily ancient macromolecular assemblies of the extracellular matrix. They have unique extensible properties that endow vascular and other tissues with long-range elasticity. Microfibril extensibility supports the low pressure closed circulations of lower organisms such as crustaceans. In higher vertebrates, microfibrils act as a template for elastin deposition and are components of mature elastic fibres. In man, the importance of microfibrils is highlighted by the linkage of mutations in their principal structural component, fibrillin-1, to the heritable disease Marfan syndrome which is characterised by severe cardiovascular, skeletal and ocular defects. When isolated from tissues, fibrillin-rich microfibrils have a complex ultrastructural organisation with a characteristic 'beads-on-a-strong' appearance. X-ray fibre diffraction studies and biomechanical testing have shown that microfibrils are reversibly extensible at tissue extensions of 100%. Ultrastructural analysis and 3D reconstructions of isolated microfibrils using automated electron tomography have revealed new details of how fibrillin molecules are aligned within microfibrils in untensioned and extended states, and delineated the role of calcium in regulating microfibril beaded periodicity, rest length and molecular organisation. The molecular basis of how fibrillin molecules assemble into microfibrils, the central role of cells in regulating this process, and the identity of other molecules that may coassemble into microfibrils are now being elucidated. This information will enhance our understanding of the elastic mechanism of these unique extracellular matrix polymers, and may lead to new microfibril-based strategies for repairing elastic tissues in ageing and disease.
Subject(s)
Extracellular Matrix Proteins/metabolism , Microfibrils/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Animals , Biopolymers , Elasticity , Extracellular Matrix Proteins/ultrastructure , Fibrillin-1 , Fibrillins , Forecasting , Humans , Microfibrils/chemistry , Microfibrils/ultrastructure , Microfilament Proteins/ultrastructure , Microscopy, Atomic Force , Protein FoldingABSTRACT
Type VIII collagen is upregulated after vessel injury, and this collagen has been implicated in both smooth muscle cell migration and angiogenesis. This study examines the temporal and spatial pattern of expression of type VIII collagen in porcine coronary vessels at specific time points after balloon angioplasty. In situ hybridization studies demonstrated that collagen VIII messenger ribonucleic acid (mRNA) was markedly elevated in the neoadventitia at 3 days post-angioplasty. By 14 days, elevated collagen VIII message was seen mainly in the neointima and this expression decreased to background levels by 90 days. The distribution of collagen VIII protein, detected using immunohistochemistry, was similar but the up-regulation lagged behind the mRNA increase by a few days. Pre-treatment of sections with pepsin highlighted variations in the organization and appearance of extracellular collagen VIII containing structures in both injured and normal vessels. New vessel formation was evident in the neoadventitia after 3 days, but there was no colocalization of type VIII collagen immunostaining with that of von Willebrand factor (a marker of endothelial cells) in the neoadventitia. These data show that up-regulation of collagen VIII in the neoadventitia is an important early marker of the coronary arterial response to injury, and is not associated with new vessel formation.
Subject(s)
Angioplasty, Balloon, Coronary , Collagen Type VIII/metabolism , Muscle, Smooth, Vascular/physiology , Neovascularization, Physiologic/physiology , Up-Regulation/physiology , Animals , Cell Movement , Coronary Vessels , Female , In Situ Hybridization , RNA, Messenger , Swine , von Willebrand Factor/metabolismABSTRACT
BACKGROUND: Several of the characteristic clinical features of photoaged skin, including wrinkling, are thought to be dependent on changes in the dermal matrix brought about by chronic sun exposure. Such changes include reductions in collagens I, III and VII, an increase in elastotic material in the reticular dermis and a marked reduction in the microfibrillar glycoprotein fibrillin. OBJECTIVES: To examine whether type VI collagen, a microfibrillar collagen necessary for cell-cell and cell-matrix communication, is affected by the photoageing process. METHODS: Six healthy volunteers with moderate to severe photoageing were enrolled into the study. Immunohistochemistry and in situ hybridization histochemistry were used to examine the levels of type VI collagen in photoprotected and photoaged sites. RESULTS: In photoprotected skin, type VI collagen was concentrated in the papillary dermis immediately below the dermal-epidermal junction, around blood vessels, hair follicles and glandular structures. The distribution of type VI collagen was unchanged in photoaged skin, although we observed an increase in the abundance of the alpha3 chain of collagen VI in the upper papillary dermis, at its junction with the dermal-epidermal junction (P < 0.05). No alterations were observed for any alpha chain at the mRNA level. CONCLUSIONS: These studies suggest that chronic sun exposure (photoageing) has little or no effect on either the distribution, abundance or levels of expression of type VI collagen in human skin. Thus, type VI collagen, unlike other matrix components so far studied, appears to be relatively unaffected by the photoageing process.
Subject(s)
Collagen/metabolism , Skin Aging/physiology , Skin/metabolism , Aged , Collagen/genetics , Female , Fluorescent Antibody Technique , Forearm , Humans , Immune Sera , In Situ Hybridization , Male , Middle Aged , RNA, Messenger/geneticsABSTRACT
We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.
Subject(s)
Microfibrils/chemistry , Microfibrils/ultrastructure , Microfilament Proteins/ultrastructure , Amino Acid Sequence , Animals , Antibodies/immunology , Automation , Binding Sites, Antibody , Biopolymers/chemistry , Biopolymers/immunology , Biopolymers/metabolism , Cattle , Cells, Cultured , Epidermal Growth Factor/chemistry , Fibrillins , Fibroblasts , Fluorescent Antibody Technique , Gold Colloid/metabolism , Humans , Image Processing, Computer-Assisted , Microfibrils/immunology , Microfibrils/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/immunology , Microfilament Proteins/metabolism , Microscopy, Electron, Scanning Transmission , Models, Molecular , Molecular Sequence Data , Muscle Tonus , Protein Structure, Quaternary , Protein Structure, Tertiary , Tomography/methodsABSTRACT
Constructs of each of the three chains of type VI collagen were generated and examined in an in vitro transcription/translation assay supplemented with semipermeabilized cells. Each of the constructs when used in the in vitro system was shown to be glycosylated and to undergo intracellular assembly, the extent of which was determined by the nature of the C-terminal globular domains. All three chains containing the C1 domain formed monomers; however, the C2 domain was required for dimer and tetramer formation. In the case of the full-length alpha2(VI) chain, monomers, dimers, and tetramers formed in a time-dependent manner. Although the splice variant alpha2(VI)C2a could form monomers, it was unable to form dimers and tetramers. Similar results to the alpha2(VI) chain were found for the full-length alpha1(VI) chain, although assembly was at a slower rate. In the case of the alpha3(VI) chain containing both C1 and C2 domains only monomers were observed. Addition of the C3, C4, and C5 did not change this pattern. Homology modeling suggested that a 10-amino acid insertion in the C2 domain of the alpha3(VI) chain may interfere with dimer formation. A near full-length construct of the alpha3(VI) chain only formed monomers but was shown to facilitate tetramer formation in cotranslation experiments.
Subject(s)
Collagen/chemistry , Collagen/metabolism , Alternative Splicing , Amino Acid Sequence , DNA, Complementary/metabolism , Dimerization , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Glycoside Hydrolases/metabolism , Glycosylation , Humans , Models, Molecular , Molecular Sequence Data , Pepsin A/metabolism , Precipitin Tests , Protein Binding , Protein Biosynthesis , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Time Factors , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
Fibrillin-rich microfibrils are a unique class of extensible connective tissue macromolecules. Their critical contribution to the establishment and maintenance of diverse extracellular matrices was underlined by the linkage of their principal structural component fibrillin to Marfan syndrome, a heritable connective tissue disorder with pleiotropic manifestations. Microscopy and preparative techniques have contributed substantially to the understanding of microfibril structure and function. The supramolecular organisation of microfibrillar assemblies in tissues has been examined by tissue sectioning and X-ray diffraction methods. Published findings are discussed and new information reported on the organisation of microfibrils in the ciliary zonular fibrils by environmental scanning electron microscopy. This review summarises microscopy and X-ray diffraction studies that are informing current understanding of the ultrastructure of fibrillin-rich microfibrils.
Subject(s)
Extracellular Matrix Proteins/ultrastructure , Microfibrils/ultrastructure , Microfilament Proteins/ultrastructure , Ectopia Lentis/genetics , Elasticity , Extracellular Matrix Proteins/genetics , Fibrillins , Humans , Marfan Syndrome/genetics , Microfilament Proteins/genetics , Models, StructuralABSTRACT
The molecular mechanisms of fibrillin assembly into microfibrils are poorly understood. In this study, we investigated human fibrillin-1 carboxy-terminal processing and assembly using a recombinant approach. Processing of carboxy-terminal fibrillin-1 was strongly influenced by N-glycosylation at the site immediately downstream of the furin site, and by association with calreticulin. The carboxy terminus of fibrillin-2 underwent less efficient processing than carboxy-terminal fibrillin-1 under identical conditions. Size fractionation of the amino-terminal region of fibrillin-1, and of unprocessed and furin-processed carboxy-terminal region of fibrillin-1, revealed that the amino terminus formed abundant disulphide-bonded aggregates. Some association of unprocessed carboxy-terminal fibrillin-1 was also apparent, but processed carboxy-terminal sequences remained monomeric unless amino-terminal sequences encoded by exons 12-15 were present. These data indicate the presence of fibrillin-1 molecular recognition sequences within the amino terminus and the extreme carboxy-terminal sequence downstream of the furin site, and a specific amino- and carboxy-terminal association which could drive overlapping linear accretion of furin-processed fibrillin molecules in the extracellular space. Differences in processing of the two fibrillin isoforms may reflect differential abilities to assemble in the extracellular space.
Subject(s)
Microfilament Proteins/metabolism , Subtilisins/metabolism , Endoplasmic Reticulum/metabolism , Fibrillin-1 , Fibrillin-2 , Fibrillins , Furin , Glycosylation , Humans , Microfilament Proteins/chemistry , Molecular Chaperones/metabolism , Molecular Chaperones/pharmacology , Protein Conformation , Protein Processing, Post-Translational/drug effects , Tumor Cells, CulturedABSTRACT
We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9-11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin. Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.
Subject(s)
Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Amino Acid Sequence , Animals , COS Cells , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Calnexin , Calreticulin , Cell Line , Dimerization , Exons , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Fibrillin-1 , Fibrillin-2 , Fibrillins , Glycine/analysis , Humans , Microfilament Proteins/genetics , Proline/analysis , Protein Biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleoproteins/metabolism , Transcription, Genetic , TransfectionABSTRACT
Fibrillin molecules form the structural framework of elastic fibrillin-rich microfibrils of the extracellular matrix. We have investigated the proteolysis of recombinant fibrillin molecules by five matrix metalloproteinases. Cleavage sites were defined at the carboxy-terminal end of the fibrillin-1 proline-rich region and the corresponding fibrillin-2 glycine-rich region (exon 10), and within exon 49 towards the carboxy-terminus of fibrillin-1. Cleavage at these sites is predicted to disrupt the structure and function of the fibrillin-rich microfibrils.
Subject(s)
Metalloendopeptidases/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Cloning, Molecular , DNA, Complementary , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Fibrillins , Microfilament Proteins/genetics , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transcription, Genetic , TransfectionABSTRACT
Chronic sun exposure results in photoaged skin with deep coarse wrinkles and loss of elasticity. We have examined the distribution and abundance of fibrillin-rich microfibrils, key structural components of the elastic fiber network, in photoaged and photoprotected skin. Punch biopsies taken from photoaged forearm and from photoprotected hip and upper inner arm of 16 subjects with a clinical range of photoaging were examined for fibrillin-1 and fibrillin-2 expression and microfibril distribution. In situ hybridization revealed decreased fibrillin-1 mRNA but unchanged fibrillin-2 mRNA levels in severely photoaged forearm biopsies relative to photoprotected dermal sites. An immunohistochemical approach demonstrated that microfibrils at the dermal-epidermal junction were significantly reduced in moderate to severely photoaged forearm skin. Confocal microscopy revealed that the papillary dermal microfibrillar network was truncated and depleted in photoaged skin. These studies highlight that the fibrillin-rich microfibrillar network associated with the upper dermis undergoes extensive remodeling following solar irradiation. These changes may contribute to the clinical features of photoaging, such as wrinkle formation and loss of elasticity.
Subject(s)
Dermis/metabolism , Epidermis/metabolism , Microfilament Proteins/metabolism , Skin Aging/pathology , Adult , Aged , Aged, 80 and over , Dermis/pathology , Epidermis/pathology , Extracellular Matrix Proteins/metabolism , Female , Fibrillin-1 , Fibrillin-2 , Fibrillins , Humans , Immunohistochemistry , In Situ Hybridization , Male , Microfilament Proteins/genetics , Microscopy, Confocal , Middle Aged , RNA, Messenger/metabolism , Sunlight/adverse effectsABSTRACT
Fibrillin is the principal structural component of the 10-12 nm diameter elastic microfibrils of the extracellular matrix. We have previously shown that both fibrillin molecules and assembled microfibrils are susceptible to degradation by serine proteases. In this study, we have investigated the potential catabolic effects of six matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-12, MMP-13 and MMP-14) on fibrillin molecules and on intact fibrillin-rich microfibrils isolated from ciliary zonules. Using newly synthesized recombinant fibrillin molecules, major cleavage sites within fibrillin-1 were identified. In particular, the six different MMPs generated a major degradation product of approximately 45 kDa from the N-terminal region of the molecule, whereas treatment of truncated, unprocessed and furin-processed C-termini also generated large degradation products. Introduction of a single ectopia lentis-causing amino acid substitution (E2447K; one-letter symbols for amino acids) in a calcium-binding epidermal growth factor-like domain, predicted to disrupt calcium binding, markedly altered the pattern of C-terminal fibrillin-1 degradation. However, the fragmentation pattern of a mutant fibrillin-1 with a comparable E-->K substitution in an upstream calcium-binding epidermal growth factor-like domain was indistinguishable from wild-type molecules. Ultrastructural examination highlighted that fibrillin-rich microfibrils isolated from ciliary zonules were grossly disrupted by MMPs. This is the first demonstration that fibrillin molecules and fibrillin-rich microfibrils are degraded by MMPs and that certain amino acid substitutions change the fragmentation patterns. These studies have important implications for physiological and pathological fibrillin catabolism and for loss of connective tissue elasticity in ageing and disease.
Subject(s)
Connective Tissue/metabolism , Metalloendopeptidases/metabolism , Microfilament Proteins/metabolism , Aging , Amino Acid Substitution , Binding Sites , Calcium/metabolism , Ectopia Lentis/genetics , Endoplasmic Reticulum/metabolism , Exons/genetics , Extracellular Matrix Proteins/biosynthesis , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/ultrastructure , Fibrillin-1 , Fibrillins , Humans , Microfilament Proteins/biosynthesis , Microfilament Proteins/genetics , Microfilament Proteins/ultrastructure , Microscopy, Electron , Molecular Weight , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/ultrastructure , Polymers/metabolism , Proline/genetics , Proline/metabolism , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructureABSTRACT
Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of approximately 56 nm. A Ca2+-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca2+. In the presence of Ca2+, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca2+ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca2+ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca2+-containing, staggered microfibrillar arrays were used to interpret the effects of Ca2+. These observations highlight the importance of Ca2+ to microfibrils and microfibrillar arrays in vivo.
Subject(s)
Calcium/metabolism , Extracellular Matrix Proteins/chemistry , Microfilament Proteins/chemistry , Animals , Biopolymers , Cattle , Ciliary Body/chemistry , Ciliary Body/metabolism , Ciliary Body/ultrastructure , Computer Simulation , Extracellular Matrix/ultrastructure , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/ultrastructure , Fibrillins , Microfilament Proteins/metabolism , Microfilament Proteins/ultrastructure , Models, Molecular , X-Ray DiffractionABSTRACT
Mice carrying the Tight skin (Tsk) mutation harbor a genomic duplication within the fibrillin-1 (Fbn 1) gene that results in a larger than normal in-frame Fbn 1 transcript. In this study, the consequences of the Tsk mutation for fibrillin-containing microfibrils have been examined. Dermal fibroblasts from Tsk/+ mice synthesized and secreted both normal fibrillin (approximately 330 kD) and the mutant oversized Tsk fibrillin-1 (approximately 450 kD) in comparable amounts, and Tsk fibrillin-1 was stably incorporated into cell layers. Immunohistochemical and ultrastructural analyses of normal and Tsk/+ mouse skin highlighted differences in the gross organization and distribution of microfibrillar arrays. Rotary shadowing of high Mr preparations from Tsk/+ skin demonstrated the presence of abundant beaded microfibrils. Some of these had normal morphology and periodicity, but others were distinguished by diffuse interbeads, longer periodicity, and tendency to aggregate. The presence of a structurally abnormal population of microfibrils in Tsk/+ skin was unequivocally demonstrated after calcium chelation and in denaturating conditions. Scanning transmission electron microscopy highlighted the presence of more mass in Tsk/+ skin microfibrils than in normal mice skin microfibrils. These data indicate that Tsk fibrillin-1 polymerizes and becomes incorporated into a discrete population of beaded microfibrils with altered molecular organization.
Subject(s)
Connective Tissue/metabolism , Microfilament Proteins/metabolism , Mutation , Animals , Chelating Agents/pharmacology , Connective Tissue/ultrastructure , Edetic Acid/pharmacology , Fibrillin-1 , Fibrillins , Fibroblasts/metabolism , Guanidines/pharmacology , Leukocyte Elastase/metabolism , Mice , Mice, Inbred C57BL , Microfilament Proteins/biosynthesis , Microfilament Proteins/genetics , Microscopy, Confocal , Microscopy, Electron , Skin/metabolism , Skin/ultrastructure , Trypsin/metabolismABSTRACT
A method is described for the purification of collagen VI microfibrils and fibrillin-containing microfibrils, respectively. High M(r) microfibril-rich preparations isolated from nuchal ligament by bacterial collagenase digestion and size fractionation were purified by CsCl density gradient centrifugation. Localization of collagen VI and fibrillin within the gradient was achieved by SDS-PAGE/Western blotting. Large collagen VI microfibrillar aggregates were present at the top of the gradient. Hyaluronidase pretreatment dissociated these aggregates and enabled purification of collagen VI microfibrils at a density of 1.33 g/ml. Fibrillin-containing microfibrils separated at 1.37 g/ml and copurified with MAGP1, but not LTBP1, LTBP2, or fibronectin. Confirmation of the intact status of the purified microfibrils was obtained by rotary shadowing. The ability to separate and purify these complex macromolecules provides a powerful means of addressing their molecular composition, organization, and structure:function relationships.
Subject(s)
Collagen/isolation & purification , Microfilament Proteins/isolation & purification , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/ultrastructure , Animals , Cattle , Centrifugation, Density Gradient , Collagen/chemistry , Connective Tissue/chemistry , Connective Tissue/ultrastructure , Electrophoresis, Polyacrylamide Gel , Fibrillins , Immunoblotting , Microfilament Proteins/chemistry , Microscopy, Electron , Sodium Dodecyl Sulfate , Structure-Activity RelationshipABSTRACT
The latent transforming growth factor-beta binding proteins (LTBP) are a recently identified family of widely expressed multidomain glycoproteins that range in size from 125 kDa to 240 kDa. Four LTBP genes have been described, and the homology of latent transforming growth factor-beta binding proteins molecules to the fibrillins has resulted in their inclusion in the so-called 'fibrillin superfamily'. They form intracellular covalent complexes with latent transforming growth factor-beta and target these growth factors to the extracellular matrix. This review describes their structure, summarizes current understanding of their dual roles as growth factor binding proteins and components of the extracellular matrix, and highlights their significance in tissue development and disease.
Subject(s)
Carrier Proteins/physiology , Intracellular Signaling Peptides and Proteins , Transforming Growth Factor beta , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Extracellular Matrix/metabolism , Humans , Latent TGF-beta Binding Proteins , Molecular Sequence DataSubject(s)
Collagen/metabolism , Animals , Biopolymers , Cattle , Collagen/chemistry , Collagen/genetics , DNA, Complementary , Endothelium, Vascular/metabolism , HumansSubject(s)
Cysteine/chemistry , Microfilament Proteins/chemistry , Animals , COS Cells , Circular Dichroism , DNA, Complementary , Fibrillins , Microfilament Proteins/genetics , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Connective tissue microfibrils are key structural elements of the dermal matrix which play major roles in establishing and maintaining the structural and mechanical integrity of this complex tissue. Type VI collagen microfibrils form extensive microfibrillar networks which intercalate between the major collagen fibrils and are juxtaposed to cellular basement membranes, blood vessels and other interstitial structures. Fibrillin microfibrils define the continuous elastic network of skin, and are present in dermis as microfibril bundles devoid of measureable elastin extending from the dermal-epithelial junction and as components of the thick elastic fibres present in the deep reticular dermis. Electron microscopic analyses have revealed both classes of microfibrils to have complex ultrastructures. The ability to isolate intact native microfibrils from skin has enabled a combination of high resolution and biochemical techniques to be applied to elucidate their structure:function relationships. These approaches have generated new information about their molecular organisation and physiological interactions in health and disease.
Subject(s)
Actin Cytoskeleton/physiology , Actin Cytoskeleton/ultrastructure , Connective Tissue/ultrastructure , Skin Physiological Phenomena , Skin/ultrastructure , Actin Cytoskeleton/metabolism , Animals , Basement Membrane/metabolism , Basement Membrane/ultrastructure , Blood Vessels/metabolism , Blood Vessels/ultrastructure , Cattle , Collagen/metabolism , Collagen/physiology , Collagen/ultrastructure , Connective Tissue/metabolism , Connective Tissue/physiology , Elastin/metabolism , Elastin/physiology , Elastin/ultrastructure , Humans , Microscopy, Electron , Microscopy, Electron, Scanning Transmission , Skin/growth & development , Skin Diseases/metabolism , Structure-Activity RelationshipABSTRACT
We have applied scanning transmission electron microscopy to intact native fibrillin-containing microfibrils isolated from foetal bovine elastic tissues in order to derive new insights into microfibril organisation. This technique provides quantitative data on the mass per unit length and axial mass distribution of unstained, unshadowed macromolecules. Scanning transmission electron microscopy of microfibrils from aorta, skin and nuchal ligament revealed that the beads corresponded to peaks of mass and the interbead regions to troughs of mass. These major features of axial mass distribution were characteristic of all microfibrils examined. Tissue-specific and age-dependent variations in mass were identified in microfibrils that were structurally comparable by rotary shadowing electron microscopy. Increased microfibril mass correlated with increasing gestational age. The additional mass was associated predominantly at, or close to, the bead. Some microfibril populations exhibited pronounced assymetry in their axial mass distribution. These data indicate that intact native microfibrillar assemblies from developing elastic tissues are heterogeneous in composition. Loss of mass following chondroitinase ABC or AC lyase treatment confirmed the presence of chondroitin sulphate in nuchal ligament microfibrillar assemblies.
Subject(s)
Actin Cytoskeleton/ultrastructure , Elastic Tissue/embryology , Microfilament Proteins/analysis , Microscopy, Electron, Scanning Transmission , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/metabolism , Animals , Aorta/embryology , Aorta/ultrastructure , Cattle , Chondroitin ABC Lyase/metabolism , Chondroitin Lyases/metabolism , Elastic Tissue/chemistry , Elastic Tissue/ultrastructure , Fibrillins , Ligaments/embryology , Ligaments/ultrastructure , Neck , Skin/embryology , Skin/ultrastructureABSTRACT
Fibrillin-containing microfibrils are key architectural structures of the upper dermis and integral components of the dermal elastic fibre network. Microfibril bundles intercalate into the dermal-epithelial junction and provide an elastic connection between the dermal elastic fibre network and the epidermis. Immunohistochemical studies have suggested that they are laid down both at the dermal-epithelial junction and in the deep dermis. While dermal fibroblasts are responsible for deposition of the elastin and microfibrillar components that comprise the elastic fibres of the deep dermis, the cellular origin of the microfibril bundles that extrude from the dermal-epithelial junction is not well defined. We have used fresh tissues, freshly isolated epidermis and primary human and porcine keratinocyte cultures to investigate the possibility that keratinocytes are responsible for deposition of these microfibrils. We have shown that keratinocytes in vivo and in vitro synthesize both fibrillin-1 and fibrillin-2, and assemble beaded microfibrils concurrently with expression of basement membrane collagen. These observations suggest that keratinocytes co-ordinate the secretion, deposition and assembly of these distinct structural elements of the dermal matrix, and have important implications for skin remodelling.