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PubMed1981

Possible roles of microfibrils in elastogenesis.

On examination in the electron microscope elastic tissue is seen to consist of an amorphous component surrounded by microfibrillar components. The exact relationship between these components is unknown, although during development the microfibrils appear before the amorphous material. In this report we summarize our recent observations on the microfibrillar material. At high magnification the microfibrils are seen to have a poorly staining central core around the periphery of which are arranged more densely staining filaments which appear to wind around the microfibrils in a spiral fashion. Careful measurements of microfibrillar diameters from the aorta of four large species show that there are significant differences in the mean diameter and population distributions with species. The mean diameter of the microfibrils changes with age during fetal and postnatal development. The results of immunoelectronmicroscopic localization of an antibody to a microfibrillar component are reported and the possible roles of microfibrils in elastic tissue formation are examined.

Adolescent↗
PubMed1997

Partial biochemical and immunologic characterization of fibrillin microfibrils from sea cucumber dermis.

The dermis of the sea cucumber Cucumaria frondosa is a mutable collagenous tissue composed of collagen fibrils, microfibrils, proteoglycans, and other soluble and insoluble components. A major constituent of the dermis is a network of 10-14 nm microfibrils which surrounds and penetrates bundles of collagen fibrils. These microfibrils, which are morphologically very similar to the fibrillin microfibrils of vertebrates, were found to be insoluble in protein denaturants, including chaotropic agents and ionic and nonionic detergents, regardless of the reduction of disulfide bonds. The microfibrils are covalently crosslinked by epsilon-(gamma-glutamyl)lysine at a concentration of 3.725 nmol/mg dry weight of purified insoluble material. The network is susceptible to proteolysis by trypsin, chymotrypsin, and pancreatic elastase, but not by bacterial collagenase. Amino acid compositional analysis of the network shows it to be composed of 25% ASX and GLX residues. Comparison with the proteins in the SwissProt database gives the network protein a high probability of being related to the mammalian protein fibrillin. The network is glycosylated: approximately 7% of the mass is constituted by neutral and amino sugars. The intact microfibrillar network cross-reacted with a well-characterized antiserum to mammalian fibrillin.

Amino Acids↗
PubMed1990

Binding of plasma von Willebrand factor by arterial microfibrils.

We developed an ELISA test to measure the binding of plasma von Willebrand factor (vWF) to arterial microfibrils and compared this interaction to the binding of vWF to collagen under the same conditions. We found that vWF binds to microfibrils in a similar manner as it binds to collagen: the binding was independent of the presence of cations, and temperature of incubation and was displaced by 1 M NaCl. Using purified 125I-vWF we showed that the binding was saturable and could be displaced by cold vWF in excess. Using immunoblotting we showed that vWF binds to a 97 kDa protein present in the microfibrils different from the 128 kDa thrombospondin-like structure (GP 128) which in microfibrils is known to interact with blood platelets. These results indicate that in the subendothelium, microfibrils bind plasma vWF and this reinforces the thrombogenic role of these structures.

Cations, Divalent↗
PubMed1989

The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues.

A procedure has been developed which is much more specific for the solubilization of the elastin-associated microfibrils from fetal bovine nuchal ligament using treatment with reductive saline in place of reductive guanidine hydrochloride buffer. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reductive saline extracts were shown to contain only five major protein bands with Mrs of 340,000, 78,000, 70,000, 31,000, and 25,000. The 31-kDa species was identified immunologically as the previously described macromolecule named microfibril-associated glycoprotein (MAGP) (Gibson, M. A., Hughes, J. L., Fanning, J. C., and Cleary, E. G. (1986) J. Biol. Chem. 261, 11429-11436). The proteins were purified by gel permeation, ion exchange, and affinity chromatography. Amino acid analyses showed that each protein had a profile which was distinct from that of MAGP although each was also high in acidic amino acids and cystine. The 340- and 78-kDa species were each demonstrated by immunoelectron microscopy with affinity-purified antibodies to be derived from the elastin-associated microfibris, and these were provisionally named microfibrillar protein 340 (MP340) and microfibrillar protein 78 (MP78), respectively. Each of the above antibodies gave a tissue distribution identical to that of anti-MAGP antibodies, and thus MP340 and MP78 also were identified with the 12-nm microfibrils of nonelastic tissues. MP340 was shown to absorb out completely the microfibrillar immunoreactivity of anti-(reductive guanidine hydrochloride extract) antibodies, indicating that MP340 was (a) the major microfibrillar constituent in these extracts and (b) the second unidentified microfibrillar antigen described previously. The relationship of the 70- and 25-kDa proteins to microfibrils is yet to be established. Immunoblot and immunoabsorption studies showed that MAGP and MP78 were immunologically related to MP340 but not to each other. Cyanogen bromide peptide mapping indicated that MAGP was structurally related to MP340. It is postulated that MAGP and MP78 are constituents of MP340 which in turn is the subunit of which the 12-nm microfibrils are composed.

Amino Acids↗
PubMed1982

In vitro chitin synthesis in an insect: formation and structure of microfibrils.

Chitin microfibril synthesis in Tribolium castaneum chitin synthetase (CS) assays results in the formation of a considerable precipitate. Such precipitate does not appear in the absence of the CS substrate (uridine diphospho N-acetyl-D-glucosamine) or in the presence of polyoxin-D, a potent CS inhibitor. Diflubenzuron which blocks in vivo chitin synthesis in insects has no effect on the precipitate formation at a relatively high concentration (3 x 10(-4) M). Electron microscopic examination of the CS product reveals a network of long, parallel oriented microfibrils. The microfibrils vary in thickness from 10 to 80 nm. Thinner elements merge to form larger and thicker microfibrils which are packed in distinct bundles. Particles associated with the microfibrils are largely spheroidal and range from about 50 to 250 nm in diameter. Coiled fibroid structures enclosed inside particles were observed. Similar coiled structures or extended fibroids attached to the outside (shell) of particles were also found. The nature of the particles and their possible role in chitin synthesis and fibrillogenesis in insects are discussed.

Animals↗
PubMed1997

Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization.

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.

Animals↗
PubMed1999

Variation of cellulose microfibril angles in softwoods and hardwoods-a possible strategy of mechanical optimization.

Position-resolved small-angle X-ray scattering was used to investigate the nanostructure of the wood cell wall in two softwood species (Norwegian spruce and Scots pine) and two hardwood species (pedunculate oak and copper beech). The tilt angle of the cellulose fibrils in the wood cell wall versus the longitudinal cell axis (microfibril angle) was systematically studied over a wide range of annual rings in each tree. The measured angles were correlated with the distance from the pith and the results were compared. The microfibril angle was found to decrease from pith to bark in all four trees, but was generally higher in the softwood than in the hardwood. In Norwegian spruce, the microfibril angles were higher in late wood than in early wood; in Scots pine the opposite was observed. In pedunculate oak and copper beech, low angles were found in the major part of the stem, except for the very first annual rings in pedunculate oak. The results are interpreted in terms of mechanical optimization. An attempt was made to give a quantitative estimation for the mechanical constraints imposed on a tree of given dimensions and to establish a model that could explain the general decrease of microfibril angles from pith to bark.

Cell Wall↗
PubMed2004

Roles of microtubules and cellulose microfibril assembly in the localization of secondary-cell-wall deposition in developing tracheary elements.

The roles of cellulose microfibrils and cortical microtubules in establishing and maintaining the pattern of secondary-cell-wall deposition in tracheary elements were investigated with direct dyes to inhibit cellulose microfibril assembly and amiprophosmethyl to inhibit microtubule polymerization. When direct dyes were added to xylogenic cultures of Zinnia elegans L. mesophyll cells just before the onset of differentiation, the secondary cell wall was initially secreted as bands composed of discrete masses of stained material, consistent with immobilized sites of cellulose synthesis. The masses coalesced, forming truncated, sinuous or smeared thickenings, as secondary cell wall deposition continued. The absence of ordered cellulose microfibrils was confirmed by polarization microscopy and a lack of fluorescence dichroism as determined by laser scanning microscopy. Indirect immunofluorescence showed that cortical microtubules initially subtended the masses of dye-altered secondary cell wall material but soon became disorganized and disappeared. Although most of the secondary cell wall was deposited in the absence of subtending cortical microtubules in dye-treated cells, secretion remained confined to discrete regions of the plasma membrane. Examination of non-dye-treated cultures following application of microtubule inhibitors during various stages of secondary-cell-wall deposition revealed that the pattern became fixed at an early stage such that deposition remained localized in the absence of cortical microtubules. These observations indicate that cortical microtubules are required to establish, but not to maintain, patterned secondary-cell-wall deposition. Furthermore, cellulose microfibrils play a role in maintaining microtubule arrays and the integrity of the secondary-cell-wall bands during deposition.

Asteraceae↗
PubMed2005

Microfibril-associated glycoprotein-1 and fibrillin-2 are associated with tropoelastin deposition in vitro.

Elastic system fibers consist of microfibrils and tropoelastin. During development, microfibrils act as a template on which tropoelastin is deposited. Microfibril-associated glycoprotein-1 (MAGP-1) and fibrillin-2, the major components of microfibrils, provide the likely template for tropoelastin deposition. In this study, we used the RNA interference (RNAi) technique to establish MAGP-1 and fibrillin-2 gene-specific knock-downs individually in elastin-producing cells (human gingival fibroblasts). We then examined the extracellular deposition of tropoelastin by western blotting. These two genes were specifically suppressed to < 30% of the control level, and this was responsible for the diminution of tropoelastin deposition. An immunofluorescence study also confirmed that RNAi-mediated down-regulation of MAGP-1 or fibrillin-2 led to the loss of tropoelastin immunoreactivity. These results suggest that MAGP-1 and fibrillin-2 are, directly or indirectly, associated with the extracellular deposition of tropoelastin during elastic fiber formation in human gingival fibroblasts in vitro.

Cells, Cultured↗
PubMed2005

Fibrillin microfibrils.

Fibrillin microfibrils are widely distributed extracellular matrix assemblies that endow elastic and nonelastic connective tissues with long-range elasticity. They direct tropoelastin deposition during elastic fibrillogenesis and form an outer mantle for mature elastic fibers. Microfibril arrays are also abundant in dynamic tissues that do not express elastin, such as the ciliary zonules of the eye. Mutations in fibrillin-1-the principal structural component of microfibrils-cause Marfan syndrome, a heritable disease with severe aortic, ocular, and skeletal defects. Isolated fibrillin-rich microfibrils have a complex 56 nm "beads-on-a-string" appearance; the molecular basis of their assembly and elastic properties, and their role in higher-order elastic fiber formation, remain incompletely understood.

Aging↗
PubMed2006

Fibrillins: from biogenesis of microfibrils to signaling functions.

Fibrillins are large proteins that form extracellular microfibril suprastructures ubiquitously found in elastic and nonelastic tissues. Mutations in fibrillin-1 and -2 lead to a number of heritable connective tissue disorders generally termed fibrillinopathies. Clinical symptoms in fibrillinopathies manifest in the skeletal, ocular, and cardiovascular systems and highlight the importance of fibrillins in development and homeostasis of tissues and organs, including blood vessels, bone, and eye. Microfibrils appear to have dual roles in (1) conferring mechanical stability and limited elasticity to tissues, and (2) modulating the activity of growth factors of the transforming growth factor beta (TGF-beta) superfamily. This chapter's focus is on the biogenesis of microfibrils, developmental expression patterns of fibrillins, signaling functions of microfibrils, and mouse models deficient in fibrillins.

Animals↗
PubMed2001

Fibrillin-rich microfibrils of the extracellular matrix: ultrastructure and assembly.

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.

Ectopia Lentis↗
PubMed2004

Cellulose microfibril angle in the cell wall of wood fibres.

The term microfibril angle (MFA) in wood science refers to the angle between the direction of the helical windings of cellulose microfibrils in the secondary cell wall of fibres and tracheids and the long axis of cell. Technologically, it is usually applied to the orientation of cellulose microfibrils in the S2 layer that makes up the greatest proportion of the wall thickness, since it is this which most affects the physical properties of wood. This review describes the organisation of the cellulose component of the secondary wall of fibres and tracheids and the various methods that have been used for the measurement of MFA. It considers the variation of MFA within the tree and the biological reason for the large differences found between juvenile (or core) wood and mature (or outer) wood. The ability of the tree to vary MFA in response to environmental stress, particularly in reaction wood, is also described. Differences in MFA have a profound effect on the properties of wood, in particular its stiffness. The large MFA in juvenile wood confers low stiffness and gives the sapling the flexibility it needs to survive high winds without breaking. It also means, however, that timber containing a high proportion of juvenile wood is unsuitable for use as high-grade structural timber. This fact has taken on increasing importance in view of the trend in forestry towards short rotation cropping of fast grown species. These trees at harvest may contain 50% or more of timber with low stiffness and therefore, low economic value. Although they are presently grown mainly for pulp, pressure for increased timber production means that ways will be sought to improve the quality of their timber by reducing juvenile wood MFA. The mechanism by which the orientation of microfibril deposition is controlled is still a matter of debate. However, the application of molecular techniques is likely to enable modification of this process. The extent to which these techniques should be used to improve timber quality by reducing MFA in juvenile wood is, however, uncertain, since care must be taken to avoid compromising the safety of the tree.

Cell Wall↗
PubMed2006

Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose.

Never-dried native celluloses (bleached sulfite wood pulp, cotton, tunicin, and bacterial cellulose) were disintegrated into individual microfibrils after oxidation mediated by the 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radical followed by a homogenizing mechanical treatment. When oxidized with 3.6 mmol of NaClO per gram of cellulose, almost the totality of sulfite wood pulp and cotton were readily disintegrated into long individual microfibrils by a treatment with a Waring Blendor, yielding transparent and highly viscous suspensions. When observed by transmission electron microscopy, the wood pulp and cotton microfibrils exhibited a regular width of 3-5 nm. Tunicin and bacterial cellulose could be disintegrated by sonication. A bulk degree of oxidation of about 0.2 per one anhydroglucose unit of cellulose was necessary for a smooth disintegration of sulfite wood pulp, whereas only small amounts of independent microfibrils were obtained at lower oxidation levels. This limiting degree of oxidation decreased in the following order: sulfite wood pulp > cotton > bacterial cellulose, tunicin.

Catalysis↗
PubMed2001

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation.

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.

Collagen↗
PubMed2002

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils.

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.

Amino Acid Sequence↗
PubMed2003

Covalent and non-covalent interactions of betaig-h3 with collagen VI. Beta ig-h3 is covalently attached to the amino-terminal region of collagen VI in tissue microfibrils.

Transforming growth factor-beta induced gene-h3 (betaig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that betaig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 x 10(-8) m. Rotary shadowing of immunogold-labeled complexes of recombinant betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.

Animals↗
PubMed2006

The C5 domain of the collagen VI alpha3(VI) chain is critical for extracellular microfibril formation and is present in the extracellular matrix of cultured cells.

Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha3(VI) N6-C5 chains, tetramers containing truncated alpha3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha3(VI) chain is not essential for microfibril formation.

Adolescent↗