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Microfibrillar Assemblies of Foetal Bovine Skin
Matrix Vol. 1311993,pp. 103-112 © 1993 by Gustav Fischer Verlag, Stuttgart· Jena . New York
Microfibrillar Assemblies of Foetal Bovine Skin
Developmental Expression and Relative Abundance of Type VI Collagen and Fibrillin
CAY M. KIELTY, LINDA BERRY, STEPHEN P. WHITTAKER, MICHAEL E. GRANT and C. ADRIAN SHUTTLEWORTH Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
Abstract Intact type VI collagen micro fibrils and fibrillin-containing microfibrils were isolated from foetal bovine skin and investigated immunochemically and ultrastructurally. Substantial variations were detected in the abundance and macromolecular assembly of these structures at progressive stages of gestation. Microfibrils of collagen VI were increasingly abundant in skin through foetal development from late first trimester to term. The pattern of expression of fibrillin-containing microfibrils in foetal skin differed from that of collagen VI. Fibrillin-containing micro fibrils were particularly sparse in first trimester skin, and present only as short assemblies. However, by early second trimester there had been a sharp increase in the abundance and length of these fibrillin-containing microfibrils. These observations are consistent with early second trimester being a key phase of fibrillin assembly. In the third trimester, fibrillin-containing microfibrils were frequently isolated in association with amorphous material. This information on the pattern of expression and assembly of collagen VI micro fibrils and fibrillin-containing microfibrils in foetal skin implies temporally and functionally distinct contributions of these two components to the establishment of the fibrous dermal matrix. Key words: collagen VI, fibrillin, microfibrils.
Introduction Connective tissue microfibrils are a group of apparently unrelated, thin filamentous structural macromolecules which have historically been loosely classified on the basis of their gross morphological characteristics (Low, 1962; Kobayashi, 1977). The primary biological purpose of such structures apparently lies in the provision of connecting links between the major elements of connective tissues, cells and the basal lamina, and as such they make a substantial contribution to the maintenance of tissue integrity (Bruns et aI., 1986; Sakai et aI., 1986; Keene et aI., 1988; Bonaldo et aI., 1990; Bray et aI., 1990; Dahlback et aI., 1990). This class of matrix macromolecules includes at least two distinct microfibrillar assemblies, type VI collagen micro fibrils and fibrillin-containing microfibrils. Both of these structures have widespread distributions in elastic and non-
elastic tissues, and are major structural elements of specific tissues such as skin, blood vessel walls and lung (Cleary and Gibson, 1983; Sakai et aI., 1986; Timpl and Engel, 1987; Kielty et aI., 1992a). Many details of the complex macromolecular organizations of these connective tissue microfibrils, and of their contributions to the accumulation and maintenance of connective tissue matrices remain to be clearly defined. Type VI collagen is essentially a glycoprotein with a short central collagenous core (Timpl and Engel, 1987; Chu et aI., 1987; Kielty et aI., 1990; 1991). It is assembled via a well established and complex multistage mechanism into extensive and highly flexible micro fibrils (Engvall et aI., 1986; Colombatti and Bonaldo, 1987), which can exist in association with hyaluronan (Kielty et aI., 1992 b; McDevitt et aI., 1992). The micro fibrils form a filamentous network which extends throughout the connective tissue
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and anchors the large interstitial structures such as collagen fibrils, blood vessels and nerves in the surrounding matrix. Collagen VI has been implicated as a cell adhesion macromolecule (Aumailley et aI., 1990), and we have recently demonstrated that intact type VI collagen microfibrils can bind human vascular smooth muscle cells in vitro (Kielty et aI., 1992c). Collagen VI may therefore exert a potent influence on the organization of matrix both in normal development and tissue maintenance, and in connective tissue diseases involving major tissue reorganization (Keene et aI., 1988; Bonaldo et aI., 1990). Fibrillin is a large non-collagenous glycoprotein (Mr 350,000) and the major structural component of the beaded 10-14nm microfibrils which are integral components of almost all tissues and frequently seen in association with elastin (Cleary and Gibson, 1983; Sakai et aI., 1986; 1991; Wright and Mayne, 1988; Gibson et aI., 1989; Kumaritilake et aI., 1989; Maddox et aI., 1989; Dahlback et aI., 1990; Keene et aI., 1991). In skin, fibrillin forms a unique and extensive dermal network which stretches as a continuum from the periphery of elastic fibres to thin microfibril bundles close to the dermal-epithelial junction. This distribution suggests a primary role for fibrillin microfibrils in anchoring the dermal elastic fibres in the extracellular matrix and to the lamina densa (Dahlback et aI., 1990), and correlates well with its proposed role in directing the deposition of elastin during elastic fibrillogenesis (Ross et aI., 1977; Mecham et aI., 1991)! We have investigated the pattern of expression, relative abundance and macromolecular dimensions of intact collagen VI microfibrils and fibrillin-containing microfibrils isolated from developing foetal skin from the initial stages of fibrous matrix accumulation to term, using a recently developed protocol for the extraction of intact microfibrillar arrays from foetal and adult elastic tissues (Kielty et aI., 1991). This immunohistochemical and ultrastructural study was initiated to define the temporal pattern of expression and assembly of collagen VI and fibrillin, as an approach to clarifying their involvement in the ordered laying down of the complex fibrous dermal matrix during foetal development.
Materials and Methods Materials
Foetal calves were obtained from the local abattoir within 1 h of death. Pepsin (EC 3.4.23.1) from pig stomach mucosa, bacterial collagenase (type 1 A), deoxyribonuclease (DNase), phenylmethanesulphonyl fluoride, N-ethylmaleimide, diaminobenzidine, dithiothreitol and prestained non-collagenous molecular weight markers were obtained from the Sigma Chemical Company, Poole, Dorset, UK. Enhanced chemiluminescence (ECl) Western blotting detection kits were obtained from Amersham
International, Amersham, Bucks., UK. Tween-20 was obtained from BDH chemicals, Poole, Dorset, UK. Sepharose Cl-2B and PDI0 columns were supplied by PharmacialKB, Milton Keynes, Bucks, UK. Peroxidase-conjugated swine IgGs to rabbit immunoglobulins were supplied by Dakopatt ltd., High Wycombe, Bucks, UK. Monoclonal antibodies to elastin were obtained from Sigma Immunochemicals, Poole, Dorset, UK. Polyclonal antiserum to human tropoelastin was a gift from Professor R.P. Mecham (St. louis, USA). Mica sheets were obtained from T AAB laboratory Equipment ltd., Reading, Berks, UK. Solubilization and size fractionation ofmatrix macromolecules
Samples of skin (2 g wet weight) were dissected and homogenized in lO ml of 0.05 M-TrisIHCI,pH7.4 containing 0.4 M-NaCl, 0.01 M-CaCh, 0.01 M-MgCI2 , 2 mMphenylmethanesulphonyl fluoride (PMSF) and 10 mM-Nethylmaleimide (NEM). Bacterial collagenase (type 1 A) and DNase were added to final concentrations of 0.2 mglml and 0.1 mglml respectively and the digestions allowed to proceed at 4°C for 6 h or 18 h with gentle stirring. The digestions were terminated by addition of EDTA to a final concentration of 20 mM, and the digests centrifuged at lO,OOO g for 30 min. Aliquots of soluble extracts were desalted by passing through PD~10 desalting columns equilibrated in distilled water, and freeze-dried prior to analysis by SDS-polyacrylamide-gel electrophoresis (SDSPAGE). The remainder of each extract was chromatographed directly without concentration under non-reducing, non-denaturing conditions on a gel filtration column (1.5 x 90 cm) of Sepharose Cl-2B. The column was equilibrated and eluted at room temperature with 0.05 M-TrisIHCI, pH7.4, containing O.4M-NaCl. Column runs were constantly monitored at 280 nm and fractions corresponding to the different peaks pooled, and prepared for electrophoretic and ultrastructural analyses. Rotary shadowing electron microscopy
Aliquots from the excluded peaks of chromatographed extracts (6 hand 18 h digests) were analysed by rotary shadowing electron microscopy for their content of intact high-Mr extracellular matrix macromolecules using the mica sandwich technique (Kielty et aI., 1991). In some experiments, samples were incubated for 3 h at room temperature in the presence of 10 mM-cysteine or lO mMdithiothreitol prior to grid preparation and rotary shadowing. Fractions were diluted directly into 0.2 M-ammonium acetate, pH 6.0 to a final concentration of approximately 100 ""glml, and 5!J.l droplets were sandwiched between two sheets of freshly cleaved mica. The high-Mr aggregates were allowed to adsorb onto the mica surfaces for 5 min. The
Microfibrils of Developing Skin mica sandwiches were washed in 0.2 M-ammonium acetate and then plunged into liquid nitrogen. The sandwiches were split open under nitrogen, dried in vacuo, rotary shadowed with platinum wire on a tungsten filament at an angle of 4 and then coated with carbon. The carbon replicas were floated off onto distilled water, and picked up on uncoated 200-mesh copper grids. In some experiments, protein-anti body-gold complexes were prepared from high-M r fractions. Briefly, a 250 f,ll aliquot was incubated with diluted antiserum. After 1 h, excess antibody was removed by chromatography on Sepharose CL-2B (150 mm x 10 mm) equilibrated in 20 mM-magnesium acetate and run at 0.5 mllmin. The protein-antibody complex was collected from the void volume peak, diluted with 10 ml of 20 mM-magnesium acetate and prepared for electron microscopy as described (Sheehan et aI., 1987). Prior to rotary shadowing, grids were washed in 50 f,ll droplets of 5 mM-magnesium acetate, pH 6.0/0.1 % Tween 20 (4 x 1 min) and incubated for 1 h on 50 f,ll droplets of diluted protein A-gold. Excess protein A-gold was removed by washing the grids 4x with 50 f,ll droplets of 5 mM-magnesium acetate, pH 6.0/0.1 % Tween 20, once with 5 mMmagnesium acetate, pH 6.0, and finally with 95% ethanol. After air-drying, the grids were rotary shadowed with platinumltungsten at an angle of 8 0 and examined in a ]EOL 1200 EX electron microscope at 120 kV. Length measurements of the type VI collagen and fibrillin aggregates were carried out on micrographs using a modified Microsemper software package (Synoptics, Cambridge, UK) on an Olivetti M28/Matrox PIPI024 frame store system using a line grating replica of 2160 lines/mm. 0
Electrophoresis and western blotting
The salt extracts and the pooled column fractions were analysed by discontinuous SDS-PAGE on 6% gels (LaemmIi, 1970) in the presence or absence of 10 mM-dithiothreitol. Molecular weights were determined by reference to standards, which were collagenous (types I and V collagens) and non-collagenous. The prestained non-collagenous standards used were fumarase (M r 48,500), pyruvate kinase (M r 58,000), fructose-6-phosphate kinase (M r 84,000) ~-galactosidase (M r 116,000) and a2-macroglobulin (M r 180,000). Gels were stained for protein using Coomassie Brilliant Blue. Western blotting was carried out as described by Kielty et al. (1990). Proteins were electrophoretically transferred to nitrocellulose filters, and incubated at room temperature with antiserum at appropriate dilution. For the identification of collagen VI chains, a 1: 1000 dilution of either/both of two polyclonal antisera raised in rabbits to type VI collagen were used. One of these is known to recognize specifically the a3(VI) chain (Ayad et aI., 1989) and the other the al (VI)/a2(VI) component (Kielty et aI., 1991). A polyclonal antibody raised in a rabbit to intact fibrillin-containing micro fibrils was used
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for the identification of fibrillin microfibrillar components. This antibody recognizes a glycoprotein of Mr 290,000 (unreduced) and Mr 350,000 (reduced) by globular standards, molecular masses similar to those recorded for radio labelled fibrillin from tissue culture medium by Sakai et al. (1986). The molecule recognized by our antibody is therefore probably fibrillin, which is the only confirmed structural component of these microfibrils. No other candidate microfibrillar components exhibit comparable electrophoretic mobilities. The shift in electrophoretic mobility in the presence of reducing agent may reflect unfolding of internally disulphide-bonded epidermal growth factor(EGF)-like domains within the primary structure of fibrillin (Maslen et aI., 1991; Dietz et aI., 1992). Elastin components were identified using both a commercially available monoclonal antibody to a-elastin, and a polyclonal antiserum to tropoelastin which was provided by Dr. R. P. Mecham (St. Louis, USA). Positive reactions were identified after incubation with second antibody - peroxidase-labelled swine IgGs to rabbit immunoglobulins, using either diaminobenzidine as substrate or enhanced chemilluminescence (ECL).
Results Isolation of high-Mraggregates from foetal bovine skin
The experiments presented in this section relate to tissue obtained from individual bovine foetuses from the first (80d), second (140d) and third (240d) trimesters of gestation. Idendical results were obtained with a number of animals of similar ages. We have previously shown that digestion of foetal bovine skin with bacterial collagenase under non-reducing conditions facilitates the efficient release of abundant soluble intact microfibrils which can be isolated in the void volume of a Sepharose CL-2B column (Kielty et aI., 1991). Here we have made use of this protocol to study the developmental expression and assembly of type VI collagen microfibrils and fibrillin-containing micro fibrils. Bacterial collagenase-digested tissues were extracted in 0.05M-Tris/HCI, pH7A containing OAMNaCI and subjected to digestion with DNase prior to the isolation of solubilized microfibrillar assemblies by gel filtration. It can be seen that the relative proportions of highMr aggregates isolated in this way from foetal skin at progressive stages of gestation increased with the age of the foetus from 10-15% of the total solubilized material at 80d gestation to almost 30% by late-third trimester (Fig. 1). In one experiment where tissues were extracted without bacterial collagenase digestion, very few high-Mr aggregates were solubilized. Column fractions from each separation were pooled as indicated.
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Fig. I. Isolation of high-Mr assemblies by gel-filtration chromatography. Foetal bovine skin extracts after bacterial collagenase digestion (A-C) or without treatment with bacterial collagenase (D) were chromatographed at 20 DC on a column of Sepharose (CL-2B under non-reducing, non-denaturing conditions. The column buffer was OAM-NaCI, 0.05 M-Tris/HCI, pH7A. (A) Foetal bovine skin, SOd gestation. (B) Foetal bovine skin, 140d gestation. (C) Foetal bovine skin, 240d gestation. (D) Foetal bovine skin, 140d gestation. Fractions were pooled as indicated.
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Fig. 2. Electron micrographs after rotary shadowing of intact type VI collagen microfibrils isolated from foetal bovine skin (140d gestation) after bacterial collagenase digestion in the presence of protease inhibitors, and fractionated by gel-filtration chromatography on Sepharose CL-2B. (A) Extensive intact microfibrils isolated after 18 h bacterial collagenase digestion. (B) Lateral aggregates of intact collagen VI micro fibrils isolated after 6 h bacterial collagenase digestion. Bars = 100 nm.
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The initial identification and characterization of type VI collagen microfibrils and fibrillin-containing microfibrils present in the high-M r excluded fraction of each skin extract was carried out by rotary shadowing electron microscopic analysis. (A) Type VI Collagen Abundant type VI collagen microfibrils with characteristic periodicity of 102 nm and cross-sectional diameter of 3-5 nm (Kielty et al., 1991) were present in the high-Mr fraction at each foetal age examined (Fig. 2A). The identity of these microfibrils was confirmed by immunogold rotary shadowing electron microscopy using two well-charac-
Fig. 3. Electron micrographs after rotary shadowing of fibrillincontaining micro fibrils isolated from foetal bovine skin after digestion by bacterial collagenase in the presence of protease inhibitors, and fractionated by gel-filtration chromatography on Sepharose CL-2B. (A) Incompletely assembled fibrillin-containing microfibril isolated from foetal bovine skin at 80d gestation, with characteristic beaded domains indicated by large arrowheads. (B) Long intact fibrillin-containing microfibril isolated from foetal bovine skin (140d gestation). (C) Intact fibrillin-containing microfibril isolated from foetal bovine skin (240d gestation) which is associated with amorphous material (small arrowhead). Bars = 100 nm
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terized anti-collagen VI sera. Collagen VI microfibrils of between 10 and 20 tetramer units were particularly abundant in the first trimester skin extracts. More extensive collagen VI microfibrillar arrays (commonly between 50 and 80 tetra mer units) predominated by mid-second trimester. No further increase in microfibril length was apparent in the third trimester. The skin extracts which had been solubilized without bacterial collagenase digestion, contained virtually no solubilized collagen VI. Substantial conformational variations were identified in the largely non-helical beaded domains of all preparations, from partially disaggregated to compact and bead-like structures. The occurrence of these conformations appeared not to be agerelated. The length distributions and the frequency of conformational variations within the beaded domains of the collagen VI microfibrils isolated either by the 6 h or 18 h extraction protocols were indistinguishable at each stage of development, indicating that the individual micro fibrils are resistant to degradation under these conditions. The intact collagen VI microfibrils isolated from foetal skin had a capacity to form lateral aggregates of parallel bundles of micro fibrils which were usually but not always in periodic register (Fig. 2B). These aggregates were most abundant in preparations obtained after a brief 6 h bacterial collagenase digestion of second and third trimester skin. Exposure to EDTA during preparation failed to disrupt these higher ordered structures, but incubation with reducing agents (10 mM -cysteine and 10 mM -dithiothreitol) largely abolished the lateral association of microfibrils. (B) Fibrillin Isolated intact fibrillin-containing microfibrils were identified on the basis of their distinctive periodic beaded morphology and dimensions (Wright and Mayne, 1988; Fleischmajer et aI., 1991; Kielty et aI., 1991) (Fig. 3). Analysis by rotary shadowing highlighted striking variations not
only in the abundance of these micro fibrils in skin at progressive stages of gestation, but also in their degree of polymerization. Fibrillin-containing micro fibrils were particularly scarse in first trimester skin extracts. Assembled micro fibrils were identified in only a few fields, where they were present either as short microfibrillar sections of less than 5-beaded periods (Fig. 3 A), or in a state of incomplete polymerization as judged by the loose conformations of beaded and inter-bead domains. By mid-second trimester, however, there had been a dramatic increase both in the abundance and in the observed lengths of the fibrillincontaining assemblies, with extensive structures (up to 10 flm in length) now identifiable in virtually every field (Fig. 3 B). Similar extensive fibrillin-containing micro fibrils were isolated from third trimester skin, and in some cases lateral association of micro fibrils was observed. Microfibrils isolated from third trimester extracts were frequently associated with large clumps of amorphous material (Fig. 3 C). It has previously been observed that fibrillin monomers are not dissociated from extracted microfibrillar assemblies in reducing and denaturing conditions (Sakai et aI., 1991; C.M. Kielty, unpublished observations). Analysis by rotary shadowing has thus highlighted variations in the degree of polymerization of the fibrillin-containing microfibrils isolated from skin at progressive stages of gestation (Fig. 4). There was no evidence for an association of fibrillin with collagen VI microfibrils. Immunogold localization of the antigenic determinants of a polyclonal antibody raised against intact fibrillin micro fibrils revealed these antibodies to bind to the periodic compact 'beaded' domains (Fig.5). In some cases, two or more gold particles were associated with a single beaded structure, suggesting repeating epitopes on the surfaces of the beaded domains. Sample preparation for immunogold rotary shadowing differed from the normal protocol, and included a detergent treatment. In these electron micrographs, the fibrillin-contain-
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Fig. 4. Size distribution of fibrillin-containing micro fibrils isolated from foetal bovine skin from first (Sad), second (140d) and third (240d) trimesters. First trimester skin contained very short, incompletely assembled microfibrils. In contrast, mid-second trimester and third trimester skin extracts contained very extensive fibrillin-containing microfibrils. Bars represent the range of microfibril lengths isolated at each foetal age.
Microfibrils of Developing Skin
109
Fig.S. Long intact fibrillin-containing microfibrils after treatment with primary antibody/immunogold-conjugated second antibody, demonstrating that the anti-fibrillin serum recognizes surface epitope(s) within the beaded domains. Bar = 100 nm.
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...220 ...180 Fig. 6. Electrophoretic analysis of the isolated high-Mr material (F1) from foetal bovine skin which eluted in the void volume of a Sepharose CL-2B column. Samples were analysed by SDSPAGE on 6% gels under reducing (tracks 1- 3) or non-reducing (track 4) conditions. The electrophoretic mobilities of collagenous and non-collagenous standards are indicated by arrows. The prominent band with Mr 140,000 in tracks 1- 3 is the u1(VI)/u2(VI) component of type VI collagen. Track 1, 80d skin; track 2, 140d skin; track 3, 240d skin. Track 4, identification of fibrillin (Mr290,000) in the high-Mr fraction of 240d skin extracts by Western blotting using anti-fibrillin serum.
ing microfibrils had undergone conformational changes within the thin interbead filamentous domains, and were apparently more flexible structures as a consequence.
Electrophoretic analysis of solubilized proteins The elution profiles of type VI collagen and fibrillin from the various skin extracts (Fig. 1) were monitored by SDS-
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+ PAGE under reducing and non-reducing conditions in combination with Western blotting (Fig. 6). Protein staining revealed that under reducing conditions the major protein component of the excluded volume (Fl) of the collagenase-digested soluble extracts was a prominent high-M r component of Mr 140,000 which has previously been identified as the a1(VI)/a2(VI) component of type VI collagen (Kielty et al., 1991) (Fig. 6). F1 is also likely
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to contain the glycosaminoglycan hyaluronan which occurs in structural association with collagen VI microfibrils, and this may account for the low level of Coomassie Blue staining seen in this track (Kielty et aI., 1992b). In the third trimester extract, additional components with molecular masses of 74,000, 170,000, 280,000 and 350,000 were also present. Interestingly, Western blotting using anti-elastin serum identified the Mr 74,000 component which eluted both in the void volume (F1) and the included volume (F3) of the third trimester skin extracts (data not shown). When column fractions were analysed on SDS/PAGE gels under non-reducing conditions, the polyclonal antibody shown by rotary shadowing electron microscopy to recognize surface epitopes of the beaded domains of the fibrillincontaining microfibrils, recognized a single component of M r 290,000 in the high-Mr fraction of the solubilized skin extracts (Fig. 6). This component was also present in the included volume (F3). The proportions of this component eluting in low- and high-Mr fractions varied with the foetal age of the skin extract. In first trimester extracts, it was present predominantly as low-Mr material, whilst in second and third trimester skin extracts it eluted mainly in the highMr fraction.
Discussion The pattern of expression of connective tissue microfibrils in foetal bovine skin has been investigated using a procedure recently developed for the selective isolation of intact micro fibrils of type VI collagen and fibrillin respectively (Kielty et aI., 1991). The efficiency of the protocol as a means of extracting intact micro fibrils depends on a bacterial collagenase digestion step under non-reducing conditions, which destroys the collagenous framework of the tissue and releases virtually the entire tissue content of collagen VI and fibrillin from foetal skin. In this paper, we demonstrate variations in the abundance and macromolecular organizations of these distinct microfibrillar structures of skin through development. Mid-first trimester is a stage of foetal skin development associated with stratification of the epidermis, establishment of the reticular lamina network underlying the dermal-epithelial junction, and the onset of fibrous matrix accumulation in presumptive dermis (Dahlback et aI., 1990). The presence of abundant and increasing levels of collagen VI microfibrils from this developmental stage towards term implicate collagen VI as an intrinsic structural element of the developing dermal matrix. Laterally-associated collagen VI microfibrillar aggregates were also identified in second and third trimester skin extracts. These aggregates closely resemble the collagen VI-containing filamentous structures previously detected in adult skin by immunohistochemistry (Bruns et aI., 1984; 1986; Keene et aI., 1988; Bray et aI., 1991) and they may represent the
major tissue form of collagen VI. The chemical basis of the lateral association is unclear. It is possible to exclude the involvement of interactions requiring divalent cations, but disulphide bonds may playa role in stabilizing these higherordered structures. However, other potential mechanisms of collagen VI microfibrillar association, such as hydrophobic, ionic interactions, covalent cross-links, or the mediation of a distinct matrix macromolecule such as hyaluronan, cannot be excluded. The identification of the fibrillin-containing microfibrils in this investigation has relied both on morphological characteristics and immunochemical analyses. Our polyclonal antibody recognizes a protein of Mr 290,000 under non-reducing conditions, whilst after reduction there is an apparent increase in molecular mass to 350,000. These mobilities correspond to those observed for radiolabelled fibrillin monomers isolated from tissue culture medium by Sakai et ai. (1986), and are therefore consistent with our antibody recognizing fibrillin. The shift in electrophoretic mobility on reduction probably reflects the unfolding of the internally disulphide-bonded precursor EGF-like domains present within the primary structure of fibrillin (Maslen et aI., 1991). The abundance and pattern of assembly of fibrillin in foetal bovine skin through gestation was strikingly different from that of type VI collagen. We have identified early second trimester as the major developmental stage in the assembly of the fibrillin-containing microfibrils of skin. Thus, the deposition of fibrillin and the arrangement of developing elastic fibres appears to be superimposed within a pre-existing, immature matrix but preceding the synthesis of elastin in the third trimester. These observations are consistent with the proposed role of the fibrillin-containing micro fibrils in directing elastic fibrillogenesis. The laterally-associated fibrillin-containing microfibrils isolated here from third trimester skin extracts were reminiscent of the microfibrillar bundles lacking elastin cores that have been observed histochemically and described as oxytalan fibres (Fullmer et aI., 1974; Goldfischer et aI., 1983). This observation highlights the possibility that the fibrillin-containing microfibrils may have a physiological role independent of elastin in foetal bovine skin, which may be to endow developing skin with a connecting framework between the dermis, basal lamina and epithelium. The application of this novel experimental approach to the study of matrix has provided insights into the assembly, macromolecular organization and biological roles of the microfibrillar components of developing skin. We have been able to establish distinct developmental patterns for the expression and assembly of collagen VI and fibrillin. These data demonstrate that the ordered accumulation of fibrous material in foetal skin morphogenesis depends in part on a precisely ordered spatiotemporal timetable for the expression of these and other major matrix macromolecules.
Microfibrils of Developing Skin Acknowledgements This work was supported by the Wellcome Trust. References Ayad, S., Marriott, A., Morgan,K. and Grant, M.E.: Bovine cartilage types VI and IX collagens. Biochem. J. 262: 753-761, 1989. Aumailley,M., Mann,K., von der Mark,H. and Timpl,R.: Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its a2(VI) and a3(VI) chains. Exp. Cell Res. 181: 463-474, 1989. Bonaldo, P., Russo, V., Buccioti, F., Doliana, R. and Colombatti, A.: Structural and functional features of the a3 (VI) chain indicates a bridging function for collagen VI in connective tissues. Biochemistry 29: 1245 -1254, 1990. Bray,D.F., Frank,C.B. and Bray, R. c.: Cytochemical evidence for a proteoglycan-associated filamentous network in ligament extracellular matrix. J. Orthopaed. Res. 8: 1-12, 1990. Bruns, R.: Beaded filaments and long-spacing fibrils. Relation to type VI collagen. J. Ultrastr. Res. 89: 136-145, 1984. Bruns, R., Press, W., Engvall, E., Timpl, R. and Gross,J.: Type VI collagen in extacellular, lOO-nm periodic filaments and fibrils: identification by immunoelectron microscopy. J. Cell Bioi. 103: 393-404,1986. Chu, M.-L., Mann, K., Deutzmann, R., Pribula-Conway, D., Hsu-Chen,C.-C., Bernard,M.P. and Timpl,R.: Characterization of three constituent chains of type VI collagen by peptide sequences and cDNA clones. Eur. J. Biochem. 168: 309-317, 1987. Cleary, E.G. and Gibson,M.A.: Elastin-associated micro fibrils and microfibrillar proteins. Int. Rev. Connect. Tiss. Res. 10: 97-209,1983. Colombatti, A. and Bonaldo, P.: Biosynthesis of chick type VI collagen. II. Processing and secretion in fibroblasts and smooth muscle cells. J. Bioi. Chem. 262: 14461-14466, 1987. Dahlback, K., Ljungquist, A., Lofberg, H., Dahlback, B., Engvall, E. and Sakai, L. Y.: Fibrillin immunoreactive fibers constitute a unique network in the human dermis: immunohistochemical comparison of the distributions of fibrillin, vitronectin, amyloid P component, and orcein stainable structures in normal skin and elastosis. J. Invest. Dermatol. 94: 284-291, 1990. Dietz, H. c., Pyeritz, R. E., Puffenberger, E. G., Kendzior, R.]., Corson, G. M., Maslen, C. L., Sakai, L. Y., Francomano, C. A. and Cutting, G. R. Marfan phenotype variability in a family segregating a missense mutation in the epidermal growth factorlike motif of the fibrillin gene. J. Clin. Invest. 89: 1674-1680, 1992. Fleischmajer, R., Timpl, R. and Faraggiani, T.: Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis. J. Histochem. Cytochem. 39: 51-58, 1991. Fullmer,H.M., Sheetz,].H. and Narkates, A.].: Oxytalan connective tissue fibers: a review. J. Oral Pathology 3: 291-316, 1974. Gibson, M.A., Kumaratilake,].A. and Cleary,E.G.: The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues. J. Bioi. Chem. 264: 4590-4598,1989. Goldfischer, S., Coltoff-Schiller, B., Schwartz, E. and Blumenfeld, 0.0.: Ultrastructure and staining properties of aortic microfibrils (oxytalan).J. Histochem. Cytochem. 31: 382-390, 1983. Keene, D. R., Engvall, E. and Glanville, R. W.: Ultrastructure of type VI collagen in human skin and cartilage suggests an
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anchoring function for this filamentous network. J. Cell Bioi. 107: 1995-2006, 1988. Keene, D. R., Sakai, L. Y. and Burgeson, R. E.: Human bone contains type III collagen, type VI collagen, and fibrillin: Type III collagen is present on specific fibres that may mediate attachment of tendons, ligaments and periosteum to calcified bone cortex. J. Histochem. Cytochem. 39: 59-69, 1991. Kielty, C. M., Boot-Handford, R. P., Ayad, S., Shuttleworth, C. A. and Grant, M. E.: Molecular composition of type VI collagen. Evidence for chain heterogeneity in mammalian tissues and cultured cells. Biochem.J. 272: 787-795, 1990. Kielty, C. M., Cummings, c., Whittaker, S. P., Shuttleworth, C. A. and Grant, M. E.: Isolation and ultrastructural analysis of microfibrillar structures from foetal bovine elastic tissues. J. Cell Sci. 99: 797-807, 1991. Kielty, C. M., Hopkinson, I. and Grant, M. E.: The collagen family: structure, assembly and organization in the extracellular matrix. In: Connective Tissue and its Heritable Disorders: Molecular, Genetic and Medical Aspects, ed. by Royce, P.M. and Steinmann, B., Wiley-Liss, New York, 1992a (in press). Kielty, C. M., Whittaker, S. P., Grant, M. E. and Shuttleworth, C. A.: Type VI collagen microfibrils: evidence or a structural association with hyaluronan. J. Cell Bioi. 118: 979-990, 1992b. Kielty, C. M., Whittaker, S. P., Grant, M. E. and Shuttleworth, C. A.: Attachment of human smooth muscle cells to intact microfibrillar assemblies of collagen VI and fibrillin. J. Cell Sci. 1992c (in press). Kobayashi, T.: Anchoring of basal lamina to elastic fibres by elasticfibrils. J. Invest. Dermatol. 68: 389-390, 1977. Kumaratilake,].A., Gibson, M.A., Fanning,].C. and Cleary, E. G.: The tissue distribution of microfibrils reacting with a monospecific antibody to MAGP, the major glycoprotein antigen of elastin-associated microfibrils. Eur. J. Cell BioI. 50: 117-127,1989. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680-685, 1970. Low,F.N.: Microfibrils: fine filamentous components of the tissue space. Anat. Rec. 142: 131-137,1962. Maddox, B. K., Sakai, L. Y., Keene, D. R. and Glanville, R. W.: Connective tissue microfibrils. Isolation and characterization of three large pepsin-resistant domains of fibrillin. J. BioI. Chem. 264: 21381-21385, 1989. Maslen, C. L., Corson, G. M., Maddox, K., Glanville, R. W. and Sakai, L. Y.: Partial sequence of a candidate gene for the Marfan syndrome. Nature 352: 334-337, 1991. McDevitt, C. A., Marcelino,]. and Tucker, L.: Interaction of intact type VI collagen with hyaluronan. FEBS Lett. 294: 167-170, 1992. Mecham, R.P. and Heuser,]. E.: The elastic fiber. In: Cell Biology of Extracellular Matrix, ed. by Hay, E. D., Plenum, New York, 1991, pp. 1-48. Ross, R., Fialkow, P.]. and Altman, K.: The morphogenesis of elastic fibers. Adv. Exp. Med. BioI. 79: 7 -17, 1977. Sakai, L. Y., Keene, D. R. and Engvall, E.: Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J. Cell Bioi. 103: 2499-2509,1986. Sakai,L.Y., Keene,D.R., Glanville,R.W. and Bachinger,H.P.: Purification and partial characterization of fibrillin, a cysteinerich structural component of connective tissue microfibrils. J. BioI. Chem. 266: 14763-14770,1991. Sheehan, J. K., Ratcliffe, A., Oates, K. and Hardingham, T. E.: The
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detection of substructures within proteoglycan molecules. Biochem.J. 247: 267-276, 1987. Timpl, R. and Engel,].: Type VI collagen. In: Structure and Function of Collagen Types, ed. by Mayne, R. and Burgeson, R., Academic Press, Orlando, FL, 1987, pp.10S-143. Wright, D. W. and Mayne, R.: Vitreous humor of chicken contains
two fibrillar systems: an analysis of their structures. J. Ultrastr. Malec. Str. Res. 100: 224-234, 1988. Dr. C. M. Kielty, Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester M13 9PT, UK.
Microfibrillar Assemblies of Foetal Bovine Skin
Developmental Expression and Relative Abundance of Type VI Collagen and Fibrillin
CAY M. KIELTY, LINDA BERRY, STEPHEN P. WHITTAKER, MICHAEL E. GRANT and C. ADRIAN SHUTTLEWORTH Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
Abstract Intact type VI collagen micro fibrils and fibrillin-containing microfibrils were isolated from foetal bovine skin and investigated immunochemically and ultrastructurally. Substantial variations were detected in the abundance and macromolecular assembly of these structures at progressive stages of gestation. Microfibrils of collagen VI were increasingly abundant in skin through foetal development from late first trimester to term. The pattern of expression of fibrillin-containing microfibrils in foetal skin differed from that of collagen VI. Fibrillin-containing micro fibrils were particularly sparse in first trimester skin, and present only as short assemblies. However, by early second trimester there had been a sharp increase in the abundance and length of these fibrillin-containing microfibrils. These observations are consistent with early second trimester being a key phase of fibrillin assembly. In the third trimester, fibrillin-containing microfibrils were frequently isolated in association with amorphous material. This information on the pattern of expression and assembly of collagen VI micro fibrils and fibrillin-containing microfibrils in foetal skin implies temporally and functionally distinct contributions of these two components to the establishment of the fibrous dermal matrix. Key words: collagen VI, fibrillin, microfibrils.
Introduction Connective tissue microfibrils are a group of apparently unrelated, thin filamentous structural macromolecules which have historically been loosely classified on the basis of their gross morphological characteristics (Low, 1962; Kobayashi, 1977). The primary biological purpose of such structures apparently lies in the provision of connecting links between the major elements of connective tissues, cells and the basal lamina, and as such they make a substantial contribution to the maintenance of tissue integrity (Bruns et aI., 1986; Sakai et aI., 1986; Keene et aI., 1988; Bonaldo et aI., 1990; Bray et aI., 1990; Dahlback et aI., 1990). This class of matrix macromolecules includes at least two distinct microfibrillar assemblies, type VI collagen micro fibrils and fibrillin-containing microfibrils. Both of these structures have widespread distributions in elastic and non-
elastic tissues, and are major structural elements of specific tissues such as skin, blood vessel walls and lung (Cleary and Gibson, 1983; Sakai et aI., 1986; Timpl and Engel, 1987; Kielty et aI., 1992a). Many details of the complex macromolecular organizations of these connective tissue microfibrils, and of their contributions to the accumulation and maintenance of connective tissue matrices remain to be clearly defined. Type VI collagen is essentially a glycoprotein with a short central collagenous core (Timpl and Engel, 1987; Chu et aI., 1987; Kielty et aI., 1990; 1991). It is assembled via a well established and complex multistage mechanism into extensive and highly flexible micro fibrils (Engvall et aI., 1986; Colombatti and Bonaldo, 1987), which can exist in association with hyaluronan (Kielty et aI., 1992 b; McDevitt et aI., 1992). The micro fibrils form a filamentous network which extends throughout the connective tissue
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and anchors the large interstitial structures such as collagen fibrils, blood vessels and nerves in the surrounding matrix. Collagen VI has been implicated as a cell adhesion macromolecule (Aumailley et aI., 1990), and we have recently demonstrated that intact type VI collagen microfibrils can bind human vascular smooth muscle cells in vitro (Kielty et aI., 1992c). Collagen VI may therefore exert a potent influence on the organization of matrix both in normal development and tissue maintenance, and in connective tissue diseases involving major tissue reorganization (Keene et aI., 1988; Bonaldo et aI., 1990). Fibrillin is a large non-collagenous glycoprotein (Mr 350,000) and the major structural component of the beaded 10-14nm microfibrils which are integral components of almost all tissues and frequently seen in association with elastin (Cleary and Gibson, 1983; Sakai et aI., 1986; 1991; Wright and Mayne, 1988; Gibson et aI., 1989; Kumaritilake et aI., 1989; Maddox et aI., 1989; Dahlback et aI., 1990; Keene et aI., 1991). In skin, fibrillin forms a unique and extensive dermal network which stretches as a continuum from the periphery of elastic fibres to thin microfibril bundles close to the dermal-epithelial junction. This distribution suggests a primary role for fibrillin microfibrils in anchoring the dermal elastic fibres in the extracellular matrix and to the lamina densa (Dahlback et aI., 1990), and correlates well with its proposed role in directing the deposition of elastin during elastic fibrillogenesis (Ross et aI., 1977; Mecham et aI., 1991)! We have investigated the pattern of expression, relative abundance and macromolecular dimensions of intact collagen VI microfibrils and fibrillin-containing microfibrils isolated from developing foetal skin from the initial stages of fibrous matrix accumulation to term, using a recently developed protocol for the extraction of intact microfibrillar arrays from foetal and adult elastic tissues (Kielty et aI., 1991). This immunohistochemical and ultrastructural study was initiated to define the temporal pattern of expression and assembly of collagen VI and fibrillin, as an approach to clarifying their involvement in the ordered laying down of the complex fibrous dermal matrix during foetal development.
Materials and Methods Materials
Foetal calves were obtained from the local abattoir within 1 h of death. Pepsin (EC 3.4.23.1) from pig stomach mucosa, bacterial collagenase (type 1 A), deoxyribonuclease (DNase), phenylmethanesulphonyl fluoride, N-ethylmaleimide, diaminobenzidine, dithiothreitol and prestained non-collagenous molecular weight markers were obtained from the Sigma Chemical Company, Poole, Dorset, UK. Enhanced chemiluminescence (ECl) Western blotting detection kits were obtained from Amersham
International, Amersham, Bucks., UK. Tween-20 was obtained from BDH chemicals, Poole, Dorset, UK. Sepharose Cl-2B and PDI0 columns were supplied by PharmacialKB, Milton Keynes, Bucks, UK. Peroxidase-conjugated swine IgGs to rabbit immunoglobulins were supplied by Dakopatt ltd., High Wycombe, Bucks, UK. Monoclonal antibodies to elastin were obtained from Sigma Immunochemicals, Poole, Dorset, UK. Polyclonal antiserum to human tropoelastin was a gift from Professor R.P. Mecham (St. louis, USA). Mica sheets were obtained from T AAB laboratory Equipment ltd., Reading, Berks, UK. Solubilization and size fractionation ofmatrix macromolecules
Samples of skin (2 g wet weight) were dissected and homogenized in lO ml of 0.05 M-TrisIHCI,pH7.4 containing 0.4 M-NaCl, 0.01 M-CaCh, 0.01 M-MgCI2 , 2 mMphenylmethanesulphonyl fluoride (PMSF) and 10 mM-Nethylmaleimide (NEM). Bacterial collagenase (type 1 A) and DNase were added to final concentrations of 0.2 mglml and 0.1 mglml respectively and the digestions allowed to proceed at 4°C for 6 h or 18 h with gentle stirring. The digestions were terminated by addition of EDTA to a final concentration of 20 mM, and the digests centrifuged at lO,OOO g for 30 min. Aliquots of soluble extracts were desalted by passing through PD~10 desalting columns equilibrated in distilled water, and freeze-dried prior to analysis by SDS-polyacrylamide-gel electrophoresis (SDSPAGE). The remainder of each extract was chromatographed directly without concentration under non-reducing, non-denaturing conditions on a gel filtration column (1.5 x 90 cm) of Sepharose Cl-2B. The column was equilibrated and eluted at room temperature with 0.05 M-TrisIHCI, pH7.4, containing O.4M-NaCl. Column runs were constantly monitored at 280 nm and fractions corresponding to the different peaks pooled, and prepared for electrophoretic and ultrastructural analyses. Rotary shadowing electron microscopy
Aliquots from the excluded peaks of chromatographed extracts (6 hand 18 h digests) were analysed by rotary shadowing electron microscopy for their content of intact high-Mr extracellular matrix macromolecules using the mica sandwich technique (Kielty et aI., 1991). In some experiments, samples were incubated for 3 h at room temperature in the presence of 10 mM-cysteine or lO mMdithiothreitol prior to grid preparation and rotary shadowing. Fractions were diluted directly into 0.2 M-ammonium acetate, pH 6.0 to a final concentration of approximately 100 ""glml, and 5!J.l droplets were sandwiched between two sheets of freshly cleaved mica. The high-Mr aggregates were allowed to adsorb onto the mica surfaces for 5 min. The
Microfibrils of Developing Skin mica sandwiches were washed in 0.2 M-ammonium acetate and then plunged into liquid nitrogen. The sandwiches were split open under nitrogen, dried in vacuo, rotary shadowed with platinum wire on a tungsten filament at an angle of 4 and then coated with carbon. The carbon replicas were floated off onto distilled water, and picked up on uncoated 200-mesh copper grids. In some experiments, protein-anti body-gold complexes were prepared from high-M r fractions. Briefly, a 250 f,ll aliquot was incubated with diluted antiserum. After 1 h, excess antibody was removed by chromatography on Sepharose CL-2B (150 mm x 10 mm) equilibrated in 20 mM-magnesium acetate and run at 0.5 mllmin. The protein-antibody complex was collected from the void volume peak, diluted with 10 ml of 20 mM-magnesium acetate and prepared for electron microscopy as described (Sheehan et aI., 1987). Prior to rotary shadowing, grids were washed in 50 f,ll droplets of 5 mM-magnesium acetate, pH 6.0/0.1 % Tween 20 (4 x 1 min) and incubated for 1 h on 50 f,ll droplets of diluted protein A-gold. Excess protein A-gold was removed by washing the grids 4x with 50 f,ll droplets of 5 mM-magnesium acetate, pH 6.0/0.1 % Tween 20, once with 5 mMmagnesium acetate, pH 6.0, and finally with 95% ethanol. After air-drying, the grids were rotary shadowed with platinumltungsten at an angle of 8 0 and examined in a ]EOL 1200 EX electron microscope at 120 kV. Length measurements of the type VI collagen and fibrillin aggregates were carried out on micrographs using a modified Microsemper software package (Synoptics, Cambridge, UK) on an Olivetti M28/Matrox PIPI024 frame store system using a line grating replica of 2160 lines/mm. 0
Electrophoresis and western blotting
The salt extracts and the pooled column fractions were analysed by discontinuous SDS-PAGE on 6% gels (LaemmIi, 1970) in the presence or absence of 10 mM-dithiothreitol. Molecular weights were determined by reference to standards, which were collagenous (types I and V collagens) and non-collagenous. The prestained non-collagenous standards used were fumarase (M r 48,500), pyruvate kinase (M r 58,000), fructose-6-phosphate kinase (M r 84,000) ~-galactosidase (M r 116,000) and a2-macroglobulin (M r 180,000). Gels were stained for protein using Coomassie Brilliant Blue. Western blotting was carried out as described by Kielty et al. (1990). Proteins were electrophoretically transferred to nitrocellulose filters, and incubated at room temperature with antiserum at appropriate dilution. For the identification of collagen VI chains, a 1: 1000 dilution of either/both of two polyclonal antisera raised in rabbits to type VI collagen were used. One of these is known to recognize specifically the a3(VI) chain (Ayad et aI., 1989) and the other the al (VI)/a2(VI) component (Kielty et aI., 1991). A polyclonal antibody raised in a rabbit to intact fibrillin-containing micro fibrils was used
105
for the identification of fibrillin microfibrillar components. This antibody recognizes a glycoprotein of Mr 290,000 (unreduced) and Mr 350,000 (reduced) by globular standards, molecular masses similar to those recorded for radio labelled fibrillin from tissue culture medium by Sakai et al. (1986). The molecule recognized by our antibody is therefore probably fibrillin, which is the only confirmed structural component of these microfibrils. No other candidate microfibrillar components exhibit comparable electrophoretic mobilities. The shift in electrophoretic mobility in the presence of reducing agent may reflect unfolding of internally disulphide-bonded epidermal growth factor(EGF)-like domains within the primary structure of fibrillin (Maslen et aI., 1991; Dietz et aI., 1992). Elastin components were identified using both a commercially available monoclonal antibody to a-elastin, and a polyclonal antiserum to tropoelastin which was provided by Dr. R. P. Mecham (St. Louis, USA). Positive reactions were identified after incubation with second antibody - peroxidase-labelled swine IgGs to rabbit immunoglobulins, using either diaminobenzidine as substrate or enhanced chemilluminescence (ECL).
Results Isolation of high-Mraggregates from foetal bovine skin
The experiments presented in this section relate to tissue obtained from individual bovine foetuses from the first (80d), second (140d) and third (240d) trimesters of gestation. Idendical results were obtained with a number of animals of similar ages. We have previously shown that digestion of foetal bovine skin with bacterial collagenase under non-reducing conditions facilitates the efficient release of abundant soluble intact microfibrils which can be isolated in the void volume of a Sepharose CL-2B column (Kielty et aI., 1991). Here we have made use of this protocol to study the developmental expression and assembly of type VI collagen microfibrils and fibrillin-containing micro fibrils. Bacterial collagenase-digested tissues were extracted in 0.05M-Tris/HCI, pH7A containing OAMNaCI and subjected to digestion with DNase prior to the isolation of solubilized microfibrillar assemblies by gel filtration. It can be seen that the relative proportions of highMr aggregates isolated in this way from foetal skin at progressive stages of gestation increased with the age of the foetus from 10-15% of the total solubilized material at 80d gestation to almost 30% by late-third trimester (Fig. 1). In one experiment where tissues were extracted without bacterial collagenase digestion, very few high-Mr aggregates were solubilized. Column fractions from each separation were pooled as indicated.
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Fig. I. Isolation of high-Mr assemblies by gel-filtration chromatography. Foetal bovine skin extracts after bacterial collagenase digestion (A-C) or without treatment with bacterial collagenase (D) were chromatographed at 20 DC on a column of Sepharose (CL-2B under non-reducing, non-denaturing conditions. The column buffer was OAM-NaCI, 0.05 M-Tris/HCI, pH7A. (A) Foetal bovine skin, SOd gestation. (B) Foetal bovine skin, 140d gestation. (C) Foetal bovine skin, 240d gestation. (D) Foetal bovine skin, 140d gestation. Fractions were pooled as indicated.
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Fig. 2. Electron micrographs after rotary shadowing of intact type VI collagen microfibrils isolated from foetal bovine skin (140d gestation) after bacterial collagenase digestion in the presence of protease inhibitors, and fractionated by gel-filtration chromatography on Sepharose CL-2B. (A) Extensive intact microfibrils isolated after 18 h bacterial collagenase digestion. (B) Lateral aggregates of intact collagen VI micro fibrils isolated after 6 h bacterial collagenase digestion. Bars = 100 nm.
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The initial identification and characterization of type VI collagen microfibrils and fibrillin-containing microfibrils present in the high-M r excluded fraction of each skin extract was carried out by rotary shadowing electron microscopic analysis. (A) Type VI Collagen Abundant type VI collagen microfibrils with characteristic periodicity of 102 nm and cross-sectional diameter of 3-5 nm (Kielty et al., 1991) were present in the high-Mr fraction at each foetal age examined (Fig. 2A). The identity of these microfibrils was confirmed by immunogold rotary shadowing electron microscopy using two well-charac-
Fig. 3. Electron micrographs after rotary shadowing of fibrillincontaining micro fibrils isolated from foetal bovine skin after digestion by bacterial collagenase in the presence of protease inhibitors, and fractionated by gel-filtration chromatography on Sepharose CL-2B. (A) Incompletely assembled fibrillin-containing microfibril isolated from foetal bovine skin at 80d gestation, with characteristic beaded domains indicated by large arrowheads. (B) Long intact fibrillin-containing microfibril isolated from foetal bovine skin (140d gestation). (C) Intact fibrillin-containing microfibril isolated from foetal bovine skin (240d gestation) which is associated with amorphous material (small arrowhead). Bars = 100 nm
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terized anti-collagen VI sera. Collagen VI microfibrils of between 10 and 20 tetramer units were particularly abundant in the first trimester skin extracts. More extensive collagen VI microfibrillar arrays (commonly between 50 and 80 tetra mer units) predominated by mid-second trimester. No further increase in microfibril length was apparent in the third trimester. The skin extracts which had been solubilized without bacterial collagenase digestion, contained virtually no solubilized collagen VI. Substantial conformational variations were identified in the largely non-helical beaded domains of all preparations, from partially disaggregated to compact and bead-like structures. The occurrence of these conformations appeared not to be agerelated. The length distributions and the frequency of conformational variations within the beaded domains of the collagen VI microfibrils isolated either by the 6 h or 18 h extraction protocols were indistinguishable at each stage of development, indicating that the individual micro fibrils are resistant to degradation under these conditions. The intact collagen VI microfibrils isolated from foetal skin had a capacity to form lateral aggregates of parallel bundles of micro fibrils which were usually but not always in periodic register (Fig. 2B). These aggregates were most abundant in preparations obtained after a brief 6 h bacterial collagenase digestion of second and third trimester skin. Exposure to EDTA during preparation failed to disrupt these higher ordered structures, but incubation with reducing agents (10 mM -cysteine and 10 mM -dithiothreitol) largely abolished the lateral association of microfibrils. (B) Fibrillin Isolated intact fibrillin-containing microfibrils were identified on the basis of their distinctive periodic beaded morphology and dimensions (Wright and Mayne, 1988; Fleischmajer et aI., 1991; Kielty et aI., 1991) (Fig. 3). Analysis by rotary shadowing highlighted striking variations not
only in the abundance of these micro fibrils in skin at progressive stages of gestation, but also in their degree of polymerization. Fibrillin-containing micro fibrils were particularly scarse in first trimester skin extracts. Assembled micro fibrils were identified in only a few fields, where they were present either as short microfibrillar sections of less than 5-beaded periods (Fig. 3 A), or in a state of incomplete polymerization as judged by the loose conformations of beaded and inter-bead domains. By mid-second trimester, however, there had been a dramatic increase both in the abundance and in the observed lengths of the fibrillincontaining assemblies, with extensive structures (up to 10 flm in length) now identifiable in virtually every field (Fig. 3 B). Similar extensive fibrillin-containing micro fibrils were isolated from third trimester skin, and in some cases lateral association of micro fibrils was observed. Microfibrils isolated from third trimester extracts were frequently associated with large clumps of amorphous material (Fig. 3 C). It has previously been observed that fibrillin monomers are not dissociated from extracted microfibrillar assemblies in reducing and denaturing conditions (Sakai et aI., 1991; C.M. Kielty, unpublished observations). Analysis by rotary shadowing has thus highlighted variations in the degree of polymerization of the fibrillin-containing microfibrils isolated from skin at progressive stages of gestation (Fig. 4). There was no evidence for an association of fibrillin with collagen VI microfibrils. Immunogold localization of the antigenic determinants of a polyclonal antibody raised against intact fibrillin micro fibrils revealed these antibodies to bind to the periodic compact 'beaded' domains (Fig.5). In some cases, two or more gold particles were associated with a single beaded structure, suggesting repeating epitopes on the surfaces of the beaded domains. Sample preparation for immunogold rotary shadowing differed from the normal protocol, and included a detergent treatment. In these electron micrographs, the fibrillin-contain-
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Fig. 4. Size distribution of fibrillin-containing micro fibrils isolated from foetal bovine skin from first (Sad), second (140d) and third (240d) trimesters. First trimester skin contained very short, incompletely assembled microfibrils. In contrast, mid-second trimester and third trimester skin extracts contained very extensive fibrillin-containing microfibrils. Bars represent the range of microfibril lengths isolated at each foetal age.
Microfibrils of Developing Skin
109
Fig.S. Long intact fibrillin-containing microfibrils after treatment with primary antibody/immunogold-conjugated second antibody, demonstrating that the anti-fibrillin serum recognizes surface epitope(s) within the beaded domains. Bar = 100 nm.
-
1
2
3
4
... 290
...220 ...180 Fig. 6. Electrophoretic analysis of the isolated high-Mr material (F1) from foetal bovine skin which eluted in the void volume of a Sepharose CL-2B column. Samples were analysed by SDSPAGE on 6% gels under reducing (tracks 1- 3) or non-reducing (track 4) conditions. The electrophoretic mobilities of collagenous and non-collagenous standards are indicated by arrows. The prominent band with Mr 140,000 in tracks 1- 3 is the u1(VI)/u2(VI) component of type VI collagen. Track 1, 80d skin; track 2, 140d skin; track 3, 240d skin. Track 4, identification of fibrillin (Mr290,000) in the high-Mr fraction of 240d skin extracts by Western blotting using anti-fibrillin serum.
ing microfibrils had undergone conformational changes within the thin interbead filamentous domains, and were apparently more flexible structures as a consequence.
Electrophoretic analysis of solubilized proteins The elution profiles of type VI collagen and fibrillin from the various skin extracts (Fig. 1) were monitored by SDS-
140~
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+ PAGE under reducing and non-reducing conditions in combination with Western blotting (Fig. 6). Protein staining revealed that under reducing conditions the major protein component of the excluded volume (Fl) of the collagenase-digested soluble extracts was a prominent high-M r component of Mr 140,000 which has previously been identified as the a1(VI)/a2(VI) component of type VI collagen (Kielty et al., 1991) (Fig. 6). F1 is also likely
110
C. M. Kielty et ai.
to contain the glycosaminoglycan hyaluronan which occurs in structural association with collagen VI microfibrils, and this may account for the low level of Coomassie Blue staining seen in this track (Kielty et aI., 1992b). In the third trimester extract, additional components with molecular masses of 74,000, 170,000, 280,000 and 350,000 were also present. Interestingly, Western blotting using anti-elastin serum identified the Mr 74,000 component which eluted both in the void volume (F1) and the included volume (F3) of the third trimester skin extracts (data not shown). When column fractions were analysed on SDS/PAGE gels under non-reducing conditions, the polyclonal antibody shown by rotary shadowing electron microscopy to recognize surface epitopes of the beaded domains of the fibrillincontaining microfibrils, recognized a single component of M r 290,000 in the high-Mr fraction of the solubilized skin extracts (Fig. 6). This component was also present in the included volume (F3). The proportions of this component eluting in low- and high-Mr fractions varied with the foetal age of the skin extract. In first trimester extracts, it was present predominantly as low-Mr material, whilst in second and third trimester skin extracts it eluted mainly in the highMr fraction.
Discussion The pattern of expression of connective tissue microfibrils in foetal bovine skin has been investigated using a procedure recently developed for the selective isolation of intact micro fibrils of type VI collagen and fibrillin respectively (Kielty et aI., 1991). The efficiency of the protocol as a means of extracting intact micro fibrils depends on a bacterial collagenase digestion step under non-reducing conditions, which destroys the collagenous framework of the tissue and releases virtually the entire tissue content of collagen VI and fibrillin from foetal skin. In this paper, we demonstrate variations in the abundance and macromolecular organizations of these distinct microfibrillar structures of skin through development. Mid-first trimester is a stage of foetal skin development associated with stratification of the epidermis, establishment of the reticular lamina network underlying the dermal-epithelial junction, and the onset of fibrous matrix accumulation in presumptive dermis (Dahlback et aI., 1990). The presence of abundant and increasing levels of collagen VI microfibrils from this developmental stage towards term implicate collagen VI as an intrinsic structural element of the developing dermal matrix. Laterally-associated collagen VI microfibrillar aggregates were also identified in second and third trimester skin extracts. These aggregates closely resemble the collagen VI-containing filamentous structures previously detected in adult skin by immunohistochemistry (Bruns et aI., 1984; 1986; Keene et aI., 1988; Bray et aI., 1991) and they may represent the
major tissue form of collagen VI. The chemical basis of the lateral association is unclear. It is possible to exclude the involvement of interactions requiring divalent cations, but disulphide bonds may playa role in stabilizing these higherordered structures. However, other potential mechanisms of collagen VI microfibrillar association, such as hydrophobic, ionic interactions, covalent cross-links, or the mediation of a distinct matrix macromolecule such as hyaluronan, cannot be excluded. The identification of the fibrillin-containing microfibrils in this investigation has relied both on morphological characteristics and immunochemical analyses. Our polyclonal antibody recognizes a protein of Mr 290,000 under non-reducing conditions, whilst after reduction there is an apparent increase in molecular mass to 350,000. These mobilities correspond to those observed for radiolabelled fibrillin monomers isolated from tissue culture medium by Sakai et ai. (1986), and are therefore consistent with our antibody recognizing fibrillin. The shift in electrophoretic mobility on reduction probably reflects the unfolding of the internally disulphide-bonded precursor EGF-like domains present within the primary structure of fibrillin (Maslen et aI., 1991). The abundance and pattern of assembly of fibrillin in foetal bovine skin through gestation was strikingly different from that of type VI collagen. We have identified early second trimester as the major developmental stage in the assembly of the fibrillin-containing microfibrils of skin. Thus, the deposition of fibrillin and the arrangement of developing elastic fibres appears to be superimposed within a pre-existing, immature matrix but preceding the synthesis of elastin in the third trimester. These observations are consistent with the proposed role of the fibrillin-containing micro fibrils in directing elastic fibrillogenesis. The laterally-associated fibrillin-containing microfibrils isolated here from third trimester skin extracts were reminiscent of the microfibrillar bundles lacking elastin cores that have been observed histochemically and described as oxytalan fibres (Fullmer et aI., 1974; Goldfischer et aI., 1983). This observation highlights the possibility that the fibrillin-containing microfibrils may have a physiological role independent of elastin in foetal bovine skin, which may be to endow developing skin with a connecting framework between the dermis, basal lamina and epithelium. The application of this novel experimental approach to the study of matrix has provided insights into the assembly, macromolecular organization and biological roles of the microfibrillar components of developing skin. We have been able to establish distinct developmental patterns for the expression and assembly of collagen VI and fibrillin. These data demonstrate that the ordered accumulation of fibrous material in foetal skin morphogenesis depends in part on a precisely ordered spatiotemporal timetable for the expression of these and other major matrix macromolecules.
Microfibrils of Developing Skin Acknowledgements This work was supported by the Wellcome Trust. References Ayad, S., Marriott, A., Morgan,K. and Grant, M.E.: Bovine cartilage types VI and IX collagens. Biochem. J. 262: 753-761, 1989. Aumailley,M., Mann,K., von der Mark,H. and Timpl,R.: Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its a2(VI) and a3(VI) chains. Exp. Cell Res. 181: 463-474, 1989. Bonaldo, P., Russo, V., Buccioti, F., Doliana, R. and Colombatti, A.: Structural and functional features of the a3 (VI) chain indicates a bridging function for collagen VI in connective tissues. Biochemistry 29: 1245 -1254, 1990. Bray,D.F., Frank,C.B. and Bray, R. c.: Cytochemical evidence for a proteoglycan-associated filamentous network in ligament extracellular matrix. J. Orthopaed. Res. 8: 1-12, 1990. Bruns, R.: Beaded filaments and long-spacing fibrils. Relation to type VI collagen. J. Ultrastr. Res. 89: 136-145, 1984. Bruns, R., Press, W., Engvall, E., Timpl, R. and Gross,J.: Type VI collagen in extacellular, lOO-nm periodic filaments and fibrils: identification by immunoelectron microscopy. J. Cell Bioi. 103: 393-404,1986. Chu, M.-L., Mann, K., Deutzmann, R., Pribula-Conway, D., Hsu-Chen,C.-C., Bernard,M.P. and Timpl,R.: Characterization of three constituent chains of type VI collagen by peptide sequences and cDNA clones. Eur. J. Biochem. 168: 309-317, 1987. Cleary, E.G. and Gibson,M.A.: Elastin-associated micro fibrils and microfibrillar proteins. Int. Rev. Connect. Tiss. Res. 10: 97-209,1983. Colombatti, A. and Bonaldo, P.: Biosynthesis of chick type VI collagen. II. Processing and secretion in fibroblasts and smooth muscle cells. J. Bioi. Chem. 262: 14461-14466, 1987. Dahlback, K., Ljungquist, A., Lofberg, H., Dahlback, B., Engvall, E. and Sakai, L. Y.: Fibrillin immunoreactive fibers constitute a unique network in the human dermis: immunohistochemical comparison of the distributions of fibrillin, vitronectin, amyloid P component, and orcein stainable structures in normal skin and elastosis. J. Invest. Dermatol. 94: 284-291, 1990. Dietz, H. c., Pyeritz, R. E., Puffenberger, E. G., Kendzior, R.]., Corson, G. M., Maslen, C. L., Sakai, L. Y., Francomano, C. A. and Cutting, G. R. Marfan phenotype variability in a family segregating a missense mutation in the epidermal growth factorlike motif of the fibrillin gene. J. Clin. Invest. 89: 1674-1680, 1992. Fleischmajer, R., Timpl, R. and Faraggiani, T.: Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis. J. Histochem. Cytochem. 39: 51-58, 1991. Fullmer,H.M., Sheetz,].H. and Narkates, A.].: Oxytalan connective tissue fibers: a review. J. Oral Pathology 3: 291-316, 1974. Gibson, M.A., Kumaratilake,].A. and Cleary,E.G.: The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues. J. Bioi. Chem. 264: 4590-4598,1989. Goldfischer, S., Coltoff-Schiller, B., Schwartz, E. and Blumenfeld, 0.0.: Ultrastructure and staining properties of aortic microfibrils (oxytalan).J. Histochem. Cytochem. 31: 382-390, 1983. Keene, D. R., Engvall, E. and Glanville, R. W.: Ultrastructure of type VI collagen in human skin and cartilage suggests an
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anchoring function for this filamentous network. J. Cell Bioi. 107: 1995-2006, 1988. Keene, D. R., Sakai, L. Y. and Burgeson, R. E.: Human bone contains type III collagen, type VI collagen, and fibrillin: Type III collagen is present on specific fibres that may mediate attachment of tendons, ligaments and periosteum to calcified bone cortex. J. Histochem. Cytochem. 39: 59-69, 1991. Kielty, C. M., Boot-Handford, R. P., Ayad, S., Shuttleworth, C. A. and Grant, M. E.: Molecular composition of type VI collagen. Evidence for chain heterogeneity in mammalian tissues and cultured cells. Biochem.J. 272: 787-795, 1990. Kielty, C. M., Cummings, c., Whittaker, S. P., Shuttleworth, C. A. and Grant, M. E.: Isolation and ultrastructural analysis of microfibrillar structures from foetal bovine elastic tissues. J. Cell Sci. 99: 797-807, 1991. Kielty, C. M., Hopkinson, I. and Grant, M. E.: The collagen family: structure, assembly and organization in the extracellular matrix. In: Connective Tissue and its Heritable Disorders: Molecular, Genetic and Medical Aspects, ed. by Royce, P.M. and Steinmann, B., Wiley-Liss, New York, 1992a (in press). Kielty, C. M., Whittaker, S. P., Grant, M. E. and Shuttleworth, C. A.: Type VI collagen microfibrils: evidence or a structural association with hyaluronan. J. Cell Bioi. 118: 979-990, 1992b. Kielty, C. M., Whittaker, S. P., Grant, M. E. and Shuttleworth, C. A.: Attachment of human smooth muscle cells to intact microfibrillar assemblies of collagen VI and fibrillin. J. Cell Sci. 1992c (in press). Kobayashi, T.: Anchoring of basal lamina to elastic fibres by elasticfibrils. J. Invest. Dermatol. 68: 389-390, 1977. Kumaratilake,].A., Gibson, M.A., Fanning,].C. and Cleary, E. G.: The tissue distribution of microfibrils reacting with a monospecific antibody to MAGP, the major glycoprotein antigen of elastin-associated microfibrils. Eur. J. Cell BioI. 50: 117-127,1989. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227: 680-685, 1970. Low,F.N.: Microfibrils: fine filamentous components of the tissue space. Anat. Rec. 142: 131-137,1962. Maddox, B. K., Sakai, L. Y., Keene, D. R. and Glanville, R. W.: Connective tissue microfibrils. Isolation and characterization of three large pepsin-resistant domains of fibrillin. J. BioI. Chem. 264: 21381-21385, 1989. Maslen, C. L., Corson, G. M., Maddox, K., Glanville, R. W. and Sakai, L. Y.: Partial sequence of a candidate gene for the Marfan syndrome. Nature 352: 334-337, 1991. McDevitt, C. A., Marcelino,]. and Tucker, L.: Interaction of intact type VI collagen with hyaluronan. FEBS Lett. 294: 167-170, 1992. Mecham, R.P. and Heuser,]. E.: The elastic fiber. In: Cell Biology of Extracellular Matrix, ed. by Hay, E. D., Plenum, New York, 1991, pp. 1-48. Ross, R., Fialkow, P.]. and Altman, K.: The morphogenesis of elastic fibers. Adv. Exp. Med. BioI. 79: 7 -17, 1977. Sakai, L. Y., Keene, D. R. and Engvall, E.: Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils. J. Cell Bioi. 103: 2499-2509,1986. Sakai,L.Y., Keene,D.R., Glanville,R.W. and Bachinger,H.P.: Purification and partial characterization of fibrillin, a cysteinerich structural component of connective tissue microfibrils. J. BioI. Chem. 266: 14763-14770,1991. Sheehan, J. K., Ratcliffe, A., Oates, K. and Hardingham, T. E.: The
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two fibrillar systems: an analysis of their structures. J. Ultrastr. Malec. Str. Res. 100: 224-234, 1988. Dr. C. M. Kielty, Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester M13 9PT, UK.