Microfibrillar Assembly Research Articles

Nanoscale Structural Comparison of Fibrillin-1 Microfibrils Isolated from Marfan and Non-Marfan Syndrome Human Aorta.

Fibrillin-1 microfibrils are essential elements of the extracellular matrix serving as a scaffold for the deposition of elastin and endowing connective tissues with tensile strength and elasticity. Mutations in the fibrillin-1 gene (FBN1) are linked to Marfan syndrome (MFS), a systemic connective tissue disorder that, besides other heterogeneous symptoms, usually manifests in life-threatening aortic complications. The aortic involvement may be explained by a dysregulation of microfibrillar function and, conceivably, alterations in the microfibrils' supramolecular structure. Here, we present a nanoscale structural characterization of fibrillin-1 microfibrils isolated from two human aortic samples with different FBN1 gene mutations by using atomic force microscopy, and their comparison with microfibrillar assemblies purified from four non-MFS human aortic samples. Fibrillin-1 microfibrils displayed a characteristic "beads-on-a-string" appearance. The microfibrillar assemblies were investigated for bead geometry (height, length, and width), interbead region height, and periodicity. MFS fibrillin-1 microfibrils had a slightly higher mean bead height, but the bead length and width, as well as the interbead height, were significantly smaller in the MFS group. The mean periodicity varied around 50-52 nm among samples. The data suggest an overall thinner and presumably more frail structure for the MFS fibrillin-1 microfibrils, which may play a role in the development of MFS-related aortic symptomatology.

Authentication of collagen VI antibodies

BackgroundCollagen VI is a ubiquitously-expressed macromolecule that forms unique microfibrillar assemblies in the extracellular matrix. Mutations in the COL6A1, COL6A2 and COL6A3 genes result in congenital muscular dystrophy, arguing that collagen is critical for skeletal muscle development and function. Antibodies against collagen VI are important clinical and diagnostic tools in muscular dystrophy. They are used to confirm genetic findings by detecting abnormalities in the distribution, organization and overall levels of collagen VI in cells and tissues isolated from patients.MethodsMany antibodies have been raised against tissue-purified collagen VI and individual collagen VI chains, however few have been properly validated for sensitivity and chain specificity. To address this deficiency, we compared the ability of 23 commercially-available antibodies to detect extracellular collagen VI by immunohistochemistry on frozen tissue sections. To determine chain specificity, immunoblot analyses were conducted on cell lysates isolated from cells transfected with cDNAs for each individual chain and cells expressing all three chains together.ResultsOur analyses identified 15 antibodies that recognized tissue collagen VI by immunohistochemistry at varying intensities and 20 that successfully detected collagen VI by immunoblotting. Three antibodies failed to recognize collagen VI by either method under the conditions tested. All chain-specific antibodies that worked by immunoblotting specifically recognized their correct chain, and no other chains.ConclusionsThis series of side-by-side comparisons reveal at least two antibodies specific for each chain that work well for immunohistochemistry on frozen sections. This validation study expands the repertoire of antibodies available for muscular dystrophy studies caused by defects in collagen VI.

A Mouse Model for Dominant Collagen VI Disorders: HETEROZYGOUS DELETION OF Col6a3 EXON 16

Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the Col6a3 gene. The resulting heterozygous mouse, Col6a3(+/d16), produced comparable amounts of normal Col6a3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The Col6a3(+/d16) mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The Col6a3(+/d16) mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.

ELLAGIC ACID DIRECTED GROWTH OF Au–Pt BIMETALLIC NANOPARTICLES AND THEIR CATALYTIC APPLICATIONS

In this work, we report the facile formation of bimetallic nanoparticles of Au–Pt in the presence of the plant polyphenol ellagic acid (EA). It was found that EA formed micro-fibrillar assemblies, which aggregated into micro-bundles under aqueous conditions. Those micro-bundles acted as templates for the growth of Au nanoparticles, as well as bimetallic Au–Pt nanoparticles biomimetically. At higher concentrations of EA, it was observed that in addition to forming fibrous micro-bundles, columnar assemblies of EA were formed in the presence of the metal nanoparticles. The formation of the assemblies was found to be concentration dependent. It appears that upon binding to metal ions and subsequent formation of the nanoparticles, morphological changes occur in the case of EA assemblies. The morphological changes observed were probed by electron microscopy. Further, the ability of the materials to degrade the toxic aromatic nitro compound 2-methoxy-4-nitroaniline was explored, where 50% degradation was observed within 15 min, indicating that such hybrid materials may have potential applications in environmental remediation.

Deposition of apatite in mineralizing vertebrate extracellular matrices: A model of possible nucleation sites on type I collagen

The positions of charged residues in the primary sequence of amino acids comprising the molecular model of type I collagen, the major extracellular protein found in vertebrate tissues, have been earlier characterized by Chapman and Hardcastle [Chapman, J.A., and Hardcastle, R.A. (1974). The staining pattern of collagen fibrils. II. A comparison with patterns computer-generated from the amino acid sequence. Connect. Tissue Res. 2:151–159]. When the sequence of residues is packed in the quarter-staggered arrangement described originally by Hodge and Petruska [Hodge, A.J., and Petruska, J.A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In Aspects of Protein Structure, G.N. Ramachandran (ed.) pp. 289–300. New York: Academic Press] in two dimensions and in the quasi-hexagonal model of microfibrillar assembly and molecular packing structure in three dimensions detailed recently by Orgel et al. (Orgel, J.P.R.O., Miller, A., Irving, T.C., Fischetti, R.F., Hammersley, A.P., and Wess, T.J. (2001). The in situ supermolecular structure of type I collagen. Structure 9:1061–1069; Orgel, J.P.R.O., Irving, T.C., Miller, A., and Wess, T.J. (2006). Microfibrillar structure of type I collagen in situ. Proc. Natl. Acad. Sci. U.S.A. 103: 9001–9005], the common sites of charged amino acids, specifically glutamic and aspartic acid, lysine and arginine, and hydroxylysine and histidine, of type I collagen have been examined in the present study and their locations determined in relation to one another. The respective positions of these amino acid residues are notable in several features in two dimensions within a single collagen triple helix as well as in adjacent helices. There are, first, numerous sites in which the same amino acid is adjacent in each of the three collagen helices. Second, many sites exist in which two of the same amino acids and one of the same charge are adjacent in the three helices. Third, the same two or three glutamic and/or aspartic amino acids are found in close proximity to amino acids with their counterparts, aspartic and glutamic acid, respectively. Fourth, several sites occur in which the same two or three amino acids of one charge are present in close proximity to the same two or three amino acids of opposite charge (glutamic acid and lysine or arginine residues or aspartic acid and lysine or arginine residues). Fifth, there are several sites where hydroxylysine contributes charged groups in place of one of the three lysine or arginine residues common in adjacent collagen helices. The strikingly repetitive and close nature of these specific charged groups in two dimensions is even more apparent when the molecular packing structure is investigated in three dimensions. In this instance, the most recent model of Orgel et al. [Orgel, J.P.R.O., Irving, T.C., Miller, A., and Wess, T.J. (2006). Microfibrillar structure of type I collagen in situ. Proc. Natl. Acad. Sci. U.S.A. 103: 9001–9005] has been correlated for the first time with the model of Landis et al. [Landis, W.J., Song, M.J., Leith, A., McEwen, L., and McEwen, B. (1993). Mineral and organic matrix interaction in normally calcifying tendon visualized in three dimensions by high voltage electron microscopic tomography and graphic image reconstruction. J. Struct. Biol. 110: 39–54] showing channels traversing molecular arrays of collagen. Here, many of the charged amino acid sites correspond to the known type I collagen hole zones defined by Hodge and Petruska [Hodge, A.J., and Petruska, J.A. (1963). Recent studies with the electron microscope on ordered aggregates of the tropocollagen macromolecule. In Aspects of Protein Structure, G.N. Ramachandran (ed.) pp. 289–300. New York: Academic Press]. As such, these residues present the locations highly likely to bind Ca2+ and ions in stereochemical configurations that could serve directly as nucleation centers for the subsequent growth and development of apatite crystals representing initial events in vertebrate mineralization. Based on these results, type I collagen appears to provide a molecular framework for direct formation of apatite without the necessary intervention or mediation of other molecules in extracellular matrices of vertebrate calcifying tissues.

Marfan's syndrome

1978 www.thelancet.com Vol 366 December 3, 2005 Marfan’s syndrome is a multisystem connective tissue disorder of autosomal dominant inheritance. Approximately one in every 5000 individuals is affected, though this figure is probably an underestimate. The condition shows no predilection for any particular race or geographical background. It exhibits complete penetrance but variable expression and age dependency. About a quarter of affected individuals have no family history of the disease—ie, they are new mutations. Marfan’s syndrome demonstrates pleiotropy with diverse manifestations in different organ systems. Clinical diagnosis depends on a combination of major and minor signs defined in the revised 1996 Ghent criteria. The hallmark features are noted in the cardiovascular, ocular, and skeletal systems (table). The diagnosis of Marfan’s syndrome can be challenging because of phenotypic variability even between affected individuals of the same family, low specificity of many of the clinical signs, and multiple Marfan-like microfibrillar disorders with overlapping features. The gene for Marfan’s syndrome was localised to chromosome 15q21 in 1990 and cloned in 1991. FBN1 is a huge gene, spanning 110 kb of genomic DNA and comprising 65 exons. It encodes the microfibrillar protein fibrillin 1. Mutational analysis is difficult and impractical at times, in part because of the size of the gene, the heterogeneity of mutations discovered, and the lack of mutational hot spots. Moreover, there are currently no molecular techniques that offer a highly sensitive mutation detection rate of FBN1. Fibrillin 1 is a major component of extracellular microfibrils. It consists of cysteine-rich repeats interspersed by latent transforming growth factor binding protein-like and epidermal growth factor-like motifs. The microfibrils are thought to confer important biomechanical properties in connecting, anchoring, and maintaining tissues and organs. Many mutations associated with fibrillin 1 appear to confer a dominant negative mechanism of pathogenesis, but some associated with fibrillin-1 haploinsufficiency have also been described. These mutations can lead to defects in the synthesis, secretion, modification, folding, stabilisation, incorporation, and assembly of the FBN1 protein. Severe neonatal Marfan’s syndrome is associated with a cluster of mutations in the middle portion of FBN1, but there are no other established correlations between genotype and phenotype. Currently, our understanding of fibrillin biochemistry and microfibrillar assembly is limited. Experiments in mice could provide insight into fibrillin pathogenesis and microfibrillar organisation, leading to a better understanding of the pleiotropic manifestations of Marfan’s syndrome and perhaps development of a cure. Lancet 2005; 366: 1978–81

Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome

Marfan syndrome is a connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1). A dominant-negative mechanism has been inferred based upon dominant inheritance, mulitimerization of monomers to form microfibrils, and the dramatic paucity of matrix-incorporated fibrillin-1 seen in heterozygous patient samples. Yeast artificial chromosome–based transgenesis was used to overexpress a disease-associated mutant form of human fibrillin-1 (C1663R) on a normal mouse background. Remarkably, these mice failed to show any abnormalities of cellular or clinical phenotype despite regulated overexpression of mutant protein in relevant tissues and developmental stages and direct evidence that mouse and human fibrillin-1 interact with high efficiency. Immunostaining with a human-specific mAb provides what we believe to be the first demonstration that mutant fibrillin-1 can participate in productive microfibrillar assembly. Informatively, use of homologous recombination to generate mice heterozygous for a comparable missense mutation (C1039G) revealed impaired microfibrillar deposition, skeletal deformity, and progressive deterioration of aortic wall architecture, comparable to characteristics of the human condition. These data are consistent with a model that invokes haploinsufficiency for WT fibrillin-1, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. In keeping with this model, introduction of a WT FBN1 transgene on a heterozygous C1039G background rescues aortic phenotype.

Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome.

Marfan syndrome is a connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1). A dominant-negative mechanism has been inferred based upon dominant inheritance, mulitimerization of monomers to form microfibrils, and the dramatic paucity of matrix-incorporated fibrillin-1 seen in heterozygous patient samples. Yeast artificial chromosome-based transgenesis was used to overexpress a disease-associated mutant form of human fibrillin-1 (C1663R) on a normal mouse background. Remarkably, these mice failed to show any abnormalities of cellular or clinical phenotype despite regulated overexpression of mutant protein in relevant tissues and developmental stages and direct evidence that mouse and human fibrillin-1 interact with high efficiency. Immunostaining with a human-specific mAb provides what we believe to be the first demonstration that mutant fibrillin-1 can participate in productive microfibrillar assembly. Informatively, use of homologous recombination to generate mice heterozygous for a comparable missense mutation (C1039G) revealed impaired microfibrillar deposition, skeletal deformity, and progressive deterioration of aortic wall architecture, comparable to characteristics of the human condition. These data are consistent with a model that invokes haploinsufficiency for WT fibrillin-1, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. In keeping with this model, introduction of a WT FBN1 transgene on a heterozygous C1039G background rescues aortic phenotype.

New Molecular Mechanism for Ullrich Congenital Muscular Dystrophy: A Heterozygous In-Frame Deletion in the COL6A1 Gene Causes a Severe Phenotype

Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have recently been shown to cause Ullrich congenital muscular dystrophy (UCMD), a frequently severe disorder characterized by congenital muscle weakness with joint contractures and coexisting distal joint hyperlaxity. Dominant mutations in all three collagen VI genes had previously been associated with the considerably milder Bethlem myopathy. Here we report that a de novo heterozygous deletion of the COL6A1 gene can also result in a severe phenotype of classical UCMD precluding ambulation. The internal gene deletion occurs near a minisatellite DNA sequence in intron 8 that removes 1.1 kb of genomic DNA encompassing exons 9 and 10. The resulting mutant chain contains a 33-amino acid deletion near the amino-terminus of the triple-helical domain but preserves a unique cysteine in the triple-helical domain important for dimer formation prior to secretion. Thus, dimer formation and secretion of abnormal tetramers can occur and exert a strong dominant negative effect on microfibrillar assembly, leading to a loss of normal localization of collagen VI in the basement membrane surrounding muscle fibers. Consistent with this mechanism was our analysis of a patient with a much milder phenotype, in whom we identified a previously described Bethlem myopathy heterozygous in-frame deletion of 18 amino acids somewhat downstream in the triple-helical domain, a result of exon 14 skipping in the COL6A1 gene. This deletion removes the crucial cysteine, so that dimer formation cannot occur and the abnormal molecule is not secreted, preventing the strong dominant negative effect. Our studies provide a biochemical insight into genotype-phenotype correlations in this group of disorders and establish that UCMD can be caused by dominantly acting mutations.

Effects on Collagen VI mRNA Stability and Microfibrillar Assembly of Three COL6A2Mutations in Two Families with Ullrich Congenital Muscular Dystrophy

We recently reported a severe deficiency in collagen type VI, resulting from recessive mutations of the COL6A2 gene, in patients with Ullrich congenital muscular dystrophy. Their parents, who are all carriers of one mutant allele, are unaffected, although heterozygous mutations in collagen VI caused Bethlem myopathy. Here we investigated the consequences of three COL6A2 mutations in fibroblasts from patients and their parents in two Ullrich families. All three mutations lead to nonsense-mediated mRNA decay. However, very low levels of undegraded mutant mRNA remained in patient B with compound heterozygous mutations at the distal part of the triple-helical domain, resulting in deposition of abnormal microfibrils that cannot form extensive networks. This observation suggests that the C-terminal globular domain is not essential for triple-helix formation but is critical for microfibrillar assembly. In all parents, the COL6A2 mRNA levels are reduced to 57-73% of the control, but long term collagen VI matrix depositions are comparable with that of the control. The almost complete absence of abnormal protein and near-normal accumulation of microfibrils in the parents may account for their lack of myopathic symptoms.

Microfibril-associated Glycoprotein-2 Interacts with Fibrillin-1 and Fibrillin-2 Suggesting a Role for MAGP-2 in Elastic Fiber Assembly

Elastic fibers are composed of the protein elastin and a network of 10-12 nm microfibrils. The microfibrillar proteins include, among others, the fibrillins and microfibril-associated glycoproteins-1 and -2 (MAGP-1 and MAGP-2). Little is known about how microfibrillar proteins interact to support fiber assembly. We used the C-terminal half of MAGP-2 in a yeast two-hybrid library screen to identify relevant ligands. Six of 13 positive clones encoded known microfibrillar proteins, including fibrillin-1 and -2. Deletion analysis of partial fibrillin-1 and -2 clones revealed a calcium-binding epidermal growth factor repeat-containing region near the C terminus responsible for binding. This region is distinct from the region of fibrillin-1 reported by others to bind MAGP-1. The MAGP-2 bait was unable to interact productively with other epidermal growth factor repeats in fibrillin-1, demonstrating specificity of the interaction. Deletion analysis of the MAGP-2 bait demonstrated that binding occurred in a core region containing 48% identity and 7 conserved cysteine residues with MAGP-1. Immunoprecipitation of MAGP-2 from transfected COS-7 cells resulted in the coprecipitation of fibrillin. These results demonstrate that MAGP-2 specifically interacts with fibrillin-1 and -2 and suggest that MAGP-2 may help regulate microfibrillar assembly. The results also demonstrate the utility of the yeast two-hybrid system to study protein-protein interactions of the extracellular matrix.

Interactions of Fibrillin-1 with Heparin/Heparan Sulfate, Implications for Microfibrillar Assembly

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.

Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1.

Dissecting aortic aneurysm is the hallmark of Marfan syndrome (MFS) and the result of mutations in fibrillin-1, the major constituent of elastin-associated extracellular microfibrils. It is yet to be established whether dysfunction of fibrillin-1 perturbs the ability of the elastic vessel wall to sustain hemodynamic stress by disrupting microfibrillar assembly, by impairing the homeostasis of established elastic fibers, or by a combination of both mechanisms. The pathogenic sequence responsible for the mechanical collapse of the elastic lamellae in the aortic wall is also unknown. Targeted mutation of the mouse fibrillin-1 gene has recently suggested that deficiency of fibrillin-1 reduces tissue homeostasis rather than elastic fiber formation. Here we describe another gene-targeting mutation, mgR, which shows that underexpression of fibrillin-1 similarly leads to MFS-like manifestations. Histopathological analysis of mgR/mgR specimens implicates medial calcification, the inflammatory-fibroproliferative response, and inflammation-mediated elastolysis in the natural history of dissecting aneurysm. More generally, the phenotypic severity associated with various combinations of normal and mutant fibrillin-1 alleles suggests a threshold phenomenon for the functional collapse of the vessel wall that is based on the level and the integrity of microfibrils.

Marfan Syndrome: New Clues to Genotype—Phenotype Correlations

Fibrillin 1 is the main constituent of extracellular microfibrils. Microfibrils can exist as inividual structures or associate with elastin to form elastic fibres. Fibrillin 1 mutations are the cause of the pleiotropic manifestations of the Marfan syndrome (MFS) which principally involve the musculoskeletal, ocular and cardiovascular systems. MFS pathogenesis requires high levels of mutant fibrillin 1 molecules with dominant-negative activity on microfibrillar assembly and function. Gene-targeting experiments in the mouse have shed new light on fibrillin 1 function, genotype-phenotype correlations and aneurysm progression. These experiments have documented the involvement of fibrillin 1 in mantaining tissue homeostasis, suggested the existence of a critical threshold of functional microfibrils for tissue biomechanics, and outlined novel contributors to the pathogenic sequence of vascular wall collapse.

Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome.

Aortic aneurysm and dissection account for about 2% of all deaths in industrialized countries; they are also components of several genetic diseases, including Marfan syndrome (MFS). The vascular phenotype of MFS results from mutations in fibrillin-1 (FBN1), the major constituent of extracellular microfibrils. Microfibrils, either associated with or devoid of elastin, give rise to a variety of extracellular networks in elastic and non-elastic tissues. It is believed that microfibrils regulate elastic fibre formation by guiding tropo-elastin deposition during embryogenesis and early post-natal life. Hence, vascular disease in MFS is thought to result when FBN1 mutations preclude elastic fibre maturation by disrupting microfibrillar assembly. Here we report a gene-targetting experiment in mice that indicates that fibrillin-1 microfibrils are predominantly engaged in tissue homeostasis rather than elastic matrix assembly. This finding, in turn, suggests that aortic dilation is due primarily to the failure by the microfibrillar array of the adventitia to sustain physiological haemodynamic stress, and that disruption of the elastic network of the media is a secondary event.

Scanning transmission electron microscopy mass analysis of fibrillin-containing microfibrils from foetal elastic tissues

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.

Expression of a mutant human fibrillin allele upon a normal human or murine genetic background recapitulates a Marfan cellular phenotype.

The Marfan syndrome (MFS) is a connective tissue disorder inherited as an autosomal dominant trait and caused by mutations in the gene encoding fibrillin, a 350-kD glycoprotein that multimerizes to form extracellular microfibrils. It has been unclear whether disease results from a relative deficiency of wild-type fibrillin; from a dominant-negative effect, in which mutant fibrillin monomers disrupt the function of the wild-type protein encoded by the normal allele; or from a dynamic and variable interplay between these two pathogenetic mechanisms. We have now addressed this issue in a cell culture system. A mutant fibrillin allele from a patient with severe MFS was expressed in normal human and murine fibroblasts by stable transfection. Immunohistochemical analysis of the resultant cell lines revealed markedly diminished fibrillin deposition and disorganized microfibrillar architecture. Pulse-chase studies demonstrated normal levels of fibrillin synthesis but substantially reduced deposition into the extracellular matrix. These data illustrate that expression of a mutant fibrillin allele, on a background of two normal alleles, is sufficient to disrupt normal microfibrillar assembly and reproduce the MFS cellular phenotype. This underscores the importance of the fibrillin amino-terminus in normal microfibrillar assembly and suggests that expression of the human extreme 5' fibrillin coding sequence may be sufficient, in isolation, to produce an animal model of MFS. Lastly, this substantiation of a dominant-negative effect offers mutant allele knockout as a potential strategy for gene therapy.

Microfibrillar components in dental pulp: Presence of both type VI collagen- and fibrillin-containing microfibrils

Microfibrillar elements were isolated from developing and formed bovine dental pulp by a procedure involving bacterial collagenase tissue digestion and chromatography on Sepharose CL-2B. Two microfibrillar assemblies could be demonstrated. Type VI collagen microfibrils with a characteristic periodicity of about 100 nm appeared as long, thin, flexible filaments. In a number of cases these structures aggregated by lateral association. Microfibrils of 10–14 nm dia were identified as containing fibrillin on the basis of their distinctive, periodic, beaded morphology. In addition to long, single strands there were instances of chains coalescing to give amorphous aggregates. No differences in the type of microfibrillar assemblies were evident between developing and formed pulp, although fibrillin-containing microfibrils were more abundant in formed pulp.

Attachment of human vascular smooth muscles cells to intact microfibrillar assemblies of collagen VI and fibrillin.

Human vascular smooth muscle cells have been used to assess the implied role of connective tissue microfibrils as cellular ligands. Preparations of intact high-M(r) microfibrillar assemblies of collagen VI and of fibrillin, respectively, were isolated from foetal bovine skin and used as ligands in cell attachment and spreading assays. Intact collagen VI microfibrils were capable of mediating cell attachment and partial spreading. Cell attachment assays using ligands composed of defined collagen VI fragments generated by pepsin or bacterial collagenase digestions demonstrated that both the triple-helical and non-collagenous domains of collagen VI had cell adhesion activity, although at reduced levels relative to intact microfibrils. Fibronectin was identified as a modulator of intact collagen VI microfibril-mediated cell attachment. These observations are indicative of complex multiple interactions between collagen VI microfibrils and smooth muscle cells. Purified fibrillin-containing microfibrils were also shown to support smooth muscle cell adhesion. Both pepsin-resistant and pepsin-sensitive domains of fibrillin exhibited some cell attachment activity, but at reduced levels relative to the intact fibrillin microfibrils. These data provide the first direct evidence of a physiological role for intact microfibrillar assemblies in cell-matrix interactions, and the involvement of integrin cell surface receptors containing the beta 1 subunit.