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PubMed1991

Elastin-associated microfibrils (10 nm) in a three-dimensional fibroblast culture.

The purpose of this study is to present a three-dimensional dermal fibroblast model. Skin fibroblasts cultured in this system deposit large amounts of collagen and microfibrils. Fibroblasts were seeded onto a nylon filtration mesh and incubated in the presence or absence of ascorbic acid. Collagen fibril formation was found in the presence of ascorbic acid whereas microfibril formation was seen independent of ascorbic acid supplementation. Immunoelectron microscopy revealed that microfibrils were labeled with fibrillin at 67 nm periodicity. Isolated microfibrils studied by rotary shadowing had a beaded appearance consisting of beads linked to each other by a filamentous structure. The spaces between the beads ranged from 10.00-33.33 nm, suggesting that these microfibrils may have an extension-contraction mechanism. Furthermore, the size and spacing of the beads were similar to that seen in microfibrils from tissues (measured after rotary shadowing). Fibroblasts cultured in a three-dimensional mesh represent an effective in vitro model with which to study microfibril formation.

Cells, Cultured↗
PubMed1994

Fibrillin immunoreactivity is associated with normal or fragmented elastic microfibrils at the dermal-epidermal junction in recessive dystrophic epidermolysis bullosa.

Elastic microfibrils containing fibrillin are a part of the fibroreticular network of normal epidermal basement membrane. In dystrophic epidermolysis bullosa (DEB) at least one other fibroreticular component, anchoring fibrils, which contain type VII collagen, is known to be abnormal. We therefore questioned whether elastic microfibrils and fibrillin expression might also be abnormal in DEB. By indirect immunofluorescence, and pre-embedding immunogold electron microscopy using a monoclonal antifibrillin antibody, we found no difference from control samples in either the quantity of the labelling or in the ultrastructural appearances of the immunolabelled fibrils in intact DEB skin. In areas of dermal-epidermal separation, however, we observed a number of thin, fragmented sublamina densa wisp-like structures, which still labelled for fibrillin despite lacking the typical ultrastructural features of normal elastic microfibril bundles. As a consequence of blistering in DEB, elastic microfibril bundles are disrupted, and fragmented microfibrils may still remain attached to the blister roofs. Many of these elastic microfibrils cannot be distinguished from rudimentary or altered anchoring fibrils on morphology alone, and might therefore account for misinterpretation of ultrastructural disorders of the dermal-epidermal junction. We postulate that, in intact skin, elastic microfibrils might contribute to dermal-epidermal adherence, in the absence of normal-functioning anchoring fibrils.

Adolescent↗
PubMed1997

Microfibrils provide non-linear elastic behaviour in the abdominal artery of the lobster Homarus americanus.

1. Microfibrils are becoming increasingly recognized as an important component of the extra-cellular matrix. However, almost nothing is known about their mechanical role in the diversity of tissues in which they are found. 2. Microfibrils form the principal structural component in the wall of the abdominal artery of the lobster Homarus americanus. We have used previous estimates of the mechanical properties of these microfibrils, estimates of the fraction of the aorta wall volume occupied by the microfibrils, and their angular distribution as a function of strain in a numerical model that predicts the macroscopic mechanical properties of the whole tissue. 3. Microfibrils alone, when their reorientation and deformation are accounted for, characterize the stress-strain behaviour of the vessel. Evidence of the evolutionary conservation of fibrillin between medusans, echinoderms and vertebrates implies that the mechanical properties of lobster microfibrils may apply to microfibrillar function in other taxa. This will have profound implications on the perceived roles of microfibrils in development, physiology and disease.

Animals↗
PubMed1986

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts.

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.

Animals↗
PubMed1998

Fibrillin containing elastic microfibrils support platelet adhesion under dynamic shear conditions.

The vascular subendothelium contains macromolecular structures called microfibrils. Type VI collagen is one protein found in microfibrils that supports platelet adhesion and aggregation and we have previously evaluated the roles of platelet receptors and vWf involved in these processes under physiological shear conditions. Here we investigate the ability of fibrillin containing elastic microfibrils to support mural thrombus formation. Our results show that elastic microfibril surfaces support platelet adhesion under low shear conditions at a level similar to collagen VI tetramers. However, the degree of aggregation on the elastic microfibril surface is much higher. Both adhesion and aggregation were shown to be mediated by the GPIIb-IIIa platelet receptor. Elastic microfibrils do not support the formation of mural thrombi under high shear conditions. These results suggest roles for both collagen VI and fibrillin containing elastic microfibrils in modulating the platelet response to blood vessel injury.

Actin Cytoskeleton↗
PubMed2000

Cellulose microfibrils in plants: biosynthesis, deposition, and integration into the cell wall.

Cellulose occurs in all higher plants and some algae, fungi, bacteria, and animals. It forms microfibrils containing the crystalline allomorphs, cellulose I alpha and I beta. Cellulose molecules are 500-15,000 glucose units long. What controls molecular size is unknown. Microfibrils are elongated by particle rosettes in the plasma membrane (cellulose synthase complexes). The precursor, UDP-glucose, may be generated from sucrose at the site of synthesis. The biosynthetic mechanism may involve lipid-linked intermediates. Cellulose synthase has been purified from bacteria, but not from plants. In plants, disrupted cellulose synthase may form callose. Cellulose synthase genes have been isolated from bacteria and plants. Cellulose-deficient mutants have been characterised. The deduced amino acid sequence suggests possible catalytic mechanisms. It is not known whether synthesis occurs at the reducing or nonreducing end. Endoglucanase may play a role in synthesis. Nascent cellulose molecules associate by Van der Waals and hydrogen bonds to form microfibrils. Cortical microtubules control microfibril orientation, thus determining the direction of cell growth. Self-assembly mechanisms may operate. Microfibril integration into the wall occurs by interactions with matrix polymers during microfibril formation.

Arabidopsis Proteins↗
PubMed2001

Function-structure relationship of elastic arteries in evolution: from microfibrils to elastin and elastic fibres.

Evolution of species has led to the appearance of circulatory systems including blood vessels and one or more pulsatile pumps, typically resulting in a low-pressurised open circulation in most invertebrates and a high-pressurised closed circulation in vertebrates. In both open and closed circulations, the large elastic arteries proximal to the heart damp out the pulsatile flow and blood pressure delivered by the heart, in order to limit distal shear stress and to allow regular irrigation of downstream organs. To achieve this goal, networks of resilient and stiff proteins adapted to each situation--i.e. low or high blood pressure--have been developed in the arterial wall to provide it with non-linear elasticity. In the low-pressurised circulation of some invertebrates, the mechanical properties of arteries can almost be entirely microfibril-based, whereas, in high-pressurised circulations, they are due to an interplay between a highly resilient protein, an elastomer in the octopus and elastin in most vertebrates, and the rather stiff protein collagen. In vertebrate development, elastin is incorporated in elastic fibres, on a earlier deposited scaffold of microfibrils. The elastic fibres are then arranged in functional concentric elastic lamellae and, with the smooth muscle cells, lamellar units. The microfibrils may also play a direct functional role in all mature arteries of high- and low-pressurised circulations. Finally, since blood pressure regularly increases with developmental stages, it appears possible that the early deposition of microfibrils, which are highly-conserved in evolution, corresponds, at least in part, to an early microfibril-driven elasticity in low-pressurised arteries, present across species. In vertebrates, when pressure developmentally rises above a threshold value, the vascular wall stress may turn on the expression of other resilient protein genes, including the elastin gene. Elastin would then be deposited on microfibrils and resulting in the elastic fibre network and elastic lamellae whose mechanical properties are adapted to allow for proper arterial work at higher pressures.

Animals↗
PubMed2002

In vitro versus in vivo cellulose microfibrils from plant primary wall synthases: structural differences.

Detergent extracts of microsomal fractions from suspension cultured cells of Rubus fruticosus (blackberry) were tested for their ability to synthesize in vitro sizable quantities of cellulose from UDP-glucose. Both Brij 58 and taurocholate were effective and yielded a substantial percentage of cellulose microfibrils together with (1-->3)-beta-d-glucan (callose). The taurocholate extracts, which did not require the addition of Mg(2+), were the most efficient, yielding roughly 20% of cellulose. This cellulose was characterized after callose removal by methylation analysis, electron microscopy, and electron and x-ray synchrotron diffractions; its resistance toward the acid Updegraff reagent was also evaluated. The cellulose microfibrils synthesized in vitro had the same diameter as the endogenous microfibrils isolated from primary cell walls. Both polymers diffracted as cellulose IV(I), a disorganized form of cellulose I. Besides these similarities, the in vitro microfibrils had a higher perfection and crystallinity as well as a better resistance toward the Updegraff reagent. These differences can be attributed to the mode of synthesis of the in vitro microfibrils that are able to grow independently in a neighbor-free environment, as opposed to the cellulose in the parent cell walls where new microfibrils have to interweave with the already laid polymers, with the result of a number of structural defects.

Bacteria↗
PubMed2004

Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis.

To test the role of cortical microtubules in aligning cellulose microfibrils and controlling anisotropic expansion, we exposed Arabidopsis thaliana roots to moderate levels of the microtubule inhibitor, oryzalin. After 2 d of treatment, roots grow at approximately steady state. At that time, the spatial profiles of relative expansion rate in length and diameter were quantified, and roots were cryofixed, freeze-substituted, embedded in plastic, and sectioned. The angular distribution of microtubules as a function of distance from the tip was quantified from antitubulin immunofluorescence images. In alternate sections, the overall amount of alignment among microfibrils and their mean orientation as a function of position was quantified with polarized-light microscopy. The spatial profiles of relative expansion show that the drug affects relative elongation and tangential expansion rates independently. The microtubule distributions averaged to transverse in the growth zone for all treatments, but on oryzalin the distributions became broad, indicating poorly organized arrays. At a subcellular scale, cellulose microfibrils in oryzalin-treated roots were as well aligned as in controls; however, the mean alignment direction, while consistently transverse in the controls, was increasingly variable with oryzalin concentration, meaning that microfibril orientation in one location tended to differ from that of a neighboring location. This conclusion was confirmed by direct observations of microfibrils with field-emission scanning electron microscopy. Taken together, these results suggest that cortical microtubules ensure microfibrils are aligned consistently across the organ, thereby endowing the organ with a uniform mechanical structure.

Arabidopsis↗
PubMed2005

Cell wall extension results in the coordinate separation of parallel microfibrils: evidence from scanning electron microscopy and atomic force microscopy.

Enlargement of the cell wall requires separation of cellulose microfibrils, mediated by proteins such as expansin; according to the multi-net growth hypothesis, enlargement passively reorients microfibrils. However, at the molecular scale, little is known about the specific movement of microfibrils. To find out, we examined directly changes in microfibril orientation when walls were extended slowly in vitro under constant load (creep). Frozen-thawed cucumber hypocotyl segments were strained by 20-30% by incubation in pH 4.5 buffer or by incubation of heat-inactivated segments in alpha-expansin or a fungal endoglucanase (Cel12A). Subsequently, the innermost layer of the cell wall was imaged, with neither extraction nor homogenization, by field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). AFM images revealed that sample preparation for FESEM did not appreciably alter cell wall ultrastructure. In both FESEM and AFM, images from extended and non-extended samples appeared indistinguishable. To quantify orientational order, we used a novel algorithm to characterize the fast Fourier transform of the image as a function of spatial frequency. For both FESEM and AFM images, the transforms of non-extended samples were indistinguishable from those of samples extended by alpha-expansin or Cel12A, as were AFM images of samples extended by acidic buffer. We conclude that cell walls in vitro can extend slowly by a creep mechanism without passive reorientation of innermost microfibrils, implying that wall loosening agents act selectively on the cross-linking polymers between parallel microfibrils, rather than more generally on the wall matrix.

Cell Wall↗
PubMed1986

The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component.

Immunohistochemical methods were used for the detection of the amyloid P component in the microfibrils of two regions: the zonule of the eye and the connective tissue of the foot pad in 20- to 50-gm mice. Following fixation by immersion in 4% formaldehyde, the eyes and foot pads were embedded in paraffin, and sections were immunostained for light microscopy by using antiamyloid P component antiserum followed by peroxidase-antiperoxidase procedure. For electron microscopy, formaldehyde-fixed tissues were immunostained for the amyloid P component with protein A-gold by using either thin Lowicryl sections or frozen sections which were then embedded in Epon for thin sectioning. In the zonule of the eye, the light microscope showed that zonular fibers were strongly immunostained for the amyloid P component; there was also weak staining of the nonpigmented ciliary epithelium at the distal end of the fibers and of the zonular lamella at their proximal end. The electron microscope revealed clear-cut immunolabeling of the microfibrils making up zonular fibers as well as of individual microfibrils. In the foot pad, the light microscope detected a weak diffuse staining of connective tissue, whereas the electron microscope showed immunolabeling restricted to microfibrils. It was concluded that the amyloid P component was present in, or associated with, microfibrils. Purified amyloid P component was prepared and examined in the electron microscope after either negative staining or routine processing. After negative staining, it appeared as flat pentagonal units, frequently associated into columns. After routine processing, the units looked like cross sections of microfibrillar tubules. The dimensions of the units matched those of the hypothetical segments of the tubules. It was concluded that this tubule consisted of a column of amyloid P units. The cohesion of the units within the column was likely to be reinforced by the bands present at the surface of microfibrils.

Amyloid↗
PubMed1989

Association of fibronectin with the microfibrils of connective tissue.

The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase-antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion. Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves. Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective-tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.

Actin Cytoskeleton↗
PubMed1989

Ultrastructural cytochemistry of aortic microfibrils in the Arctic lamprey, Lampetra japonica.

In the ventral aorta of lamprey, microfibrils are major components of the extracellular matrix. With special reference to these microfibrils, we have cytochemically examined the lamprey ventral aorta, utilizing the tannic acid (pH 7.0)-uranyl acetate (TA-UA) method, elastase en bloc digestion, Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method, and ferritin- or horseradish peroxidase-labeled concanavalin A (Con A) methods. The lamprey microfibrils were strongly stained with PA-TCH-SP and both Con A methods, but did not show TA-UA staining nor elastase sensitivity. These cytochemical properties of lamprey microfibrils are identical with those of mammalian elastin-associated microfibrils. On the other hand, in spite of extensive examination, TA-UA positive and elastase-sensitive extracellular components were not found, so that lamprey ventral aorta does not appear to contain elastin. These results indicate that lamprey aortic connective tissue contains microfibrils as elastic components, but deposition of amorphous elastin does not occur.

Actin Cytoskeleton↗
PubMed1996

The modulus of elasticity of lobster aorta microfibrils.

The presence of elastic fibres in the extracellular matrix (ECM) provides physiologically important elastic properties for many tissues. Until recently, microfibrils, one component of the ECM, were thought primarily to serve as a scaffolding on which elastin is deposited during development to form elaunin fibres [1]. The most prominent protein that forms mammalian microfibrils is fibrillin. It is known that mutations in the fibrillin gene cause a heterogenous connective tissue disease called Marfan syndrome [2], so information on mechanical properties of microfibrils or their role in tissue function would be useful. Microfibrils are also found in the ECM of some invertebrate tissues, and there is growing evidence that the protein forming the structure is homologous to mammalian fibrillin [3,4]. It has been shown that the microfibril-based arterial wall of the lobster has viscoelastic properties [5], and we have now utilized this primitive artery to measure the modulus of elasticity of microfibrils. It is similar to that of the rubber-like protein elastin.

Animals↗
PubMed1984

Demonstration of microfibrils in Bruch's membrane of the eye.

The cationic dyes ruthenium red and alcian blue were used to visualize a population of microfibrils in Bruch's membrane, a compound basement membrane located in the uveal tract of the eye between the retinal pigment epithelium and choriocapillaris. Microfibrils were tubular structures, 10-12 nm in diameter, that showed a characteristic beaded pattern. The majority of microfibrils appeared as a dense mantle around the layer of amorphous elastin. Microfibrils and collagen fibers were also present as a loosely organized meshwork in the collagenous zone of the membrane. Microfibrils were also seen along the basal surface of the retinal pigment epithelium where they appeared to insert into the substance of the basal lamina. Ruthenium red staining of microfibrils was not abolished by prior exposure of tissue to several kinds of degradative enzymes. The findings suggest that the elastic properties of Bruch's membrane may depend on both the elastin and microfibrillar components.

Alcian Blue↗
PubMed1985

Microfibrils, elastic anchoring components of the extracellular matrix, are associated with fibronectin in the zonule of Zinn and aorta.

Microfibrils are striated tubules that play a role in the formation of elastin fibers by providing a scaffold upon which newly synthesized elastin is deposited. Ultrastructural and staining studies also demonstrate microfibrils that terminate where elastin is sparse or absent in basal laminae, plasma membranes, and the collagenous matrix. The most striking accumulation of microfibrils is found in the zonule of Zinn, the transparent and elastic suspensory ligament of the lens, which contains no elastin. Application of immunocytochemical staining with a peroxidase-antiperoxidase (PAP) procedure demonstrates that fibronectin is associated with the microfibrils of the zonule and aorta. Aggregates of microfibrils are identical to oxytalan ('acid enduring') fibers that have been described in peridontal membranes and other sites subject to mechanical stress and they can be found in sites as disparate as the rabbit zonule, rat hepatic stroma and human cardiac papillary muscle, indicating that microfibrils are a widely distributed connective tissue element with a function that extends beyond elastogenesis; their association with fibronectin and localization suggests that they serve as an elastic anchoring component of the extracellular matrix.

Animals↗
PubMed2003

The supramolecular organization of collagen VI microfibrils.

Collagen VI has a ubiquitous distribution throughout connective tissues, and has key roles in linking cells and matrix macromolecules. We have generated three-dimensional reconstructions of collagen VI microfibrils using automated electron tomography (AET) in order to obtain new insights into the organisation of collagen VI in assembled microfibrils. Analysis of the reconstruction data has allowed the resolution of the double-beaded structure into smaller subunits. Volume calculations from the tomography data indicate that ten and six A-domains could be packed into the N and C-terminal regions from each monomer, respectively. A putative location for the globular N-terminal regions of the alpha3 chain, important for microfibril assembly and function, has been identified. Some surfaces of the alpha3 chain N-terminal domains appear to be exposed on the surface of a microfibril, where they may provide an interactive surface for molecules. Analysis of the interbead region provides evidence for complex triple helical supercoiling in microfibrils. Frequently, two strands were visualised emerging from the beaded region and merging into a single interbead region. Measurements taken from the AET data show that there is a decrease in periodicity from dimer/tetramer to microfibrils. Molecular combing reverses this effect by mechanically increasing periodicity to give measurements similar to the component dimers/tetramers. Together, these data have provided important new insights into the organisation and function of these large macromolecular assemblies.

Animals↗
PubMed2003

Fibrillin microfibrils are stiff reinforcing fibres in compliant tissues.

Fibrillin-rich microfibrils have endowed tissues with elasticity throughout multicellular evolution. We have used molecular combing techniques to determine Young's modulus for individual microfibrils and X-ray diffraction of zonular filaments of the eye to establish the linearity of microfibril periodic extension. Microfibril periodicity is not altered at physiological zonular tissue extensions and Young's modulus is between 78 MPa and 96 MPa, which is two orders of magnitude stiffer than elastin. We conclude that elasticity in microfibril-containing tissues arises primarily from reversible alterations in supra-microfibrillar arrangements rather than from intrinsic elastic properties of individual microfibrils which, instead, act as reinforcing fibres in fibrous composite tissues.

Aged↗