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A kinked molecular model for the collagen-containing fibrils in the egg case of the dogfish Scyliorhinus caniculus
TISSUE & CELL 1986 18 (2) 201-208 © 1986 Longman Group Ltd
DAVID P. KNIGHT* and STEPHEN H U N T t
A KINKED MOLECULAR MODEL FOR THE COLLAGEN-CONTAINING FIBRILS IN THE EGG CASE OF THE DOGFISH SCYLIORHINUS CANICUL US Keywords: Collagen, fibrils, kinked molecules, model, egg case, Scyliorhinus ABSTRACT. A model for the organization of fibrils in the collagenous egg case of the dogfish Scyliorhinus caniculus is proposed on the basis of observations on ultrathin sections tilted in a goniometer stage. The centrosymmetric transve/se banding pattern consists of an A band flanked immediately by two dense C bands which are in turn flanked by broad B bands. In the model, the B bands contain molecular segments packed in a square lattice forming a rectangular prismatic cell parallel to the fibril long axis. The molecules kink in the C band or gradually bend at the ends of the B band to tilt up or down at 25° to the fibril axis in the C and A bands. The tilt is such that the corner of one B band unit is body centred on the face of the next B band unit. Our model has similarities to models for the arrangement of type IV collagen in basement membrane, a-actinin in the Z-discs of striated muscle and the intercellular material in spot desmosomes.
Introduction
T h e longitudinal s t r u c t u r e of rat tail t e n d o n a n d o t h e r collagen fibrils which show t h e classical positive a n d n e g a t i v e b a n d i n g patt e r n s is well u n d e r s t o o d . T h e r e is g o o d e v i d e n c e t h a t t h e m o l e c u l e s are a r r a n g e d with a longitudinal stagger of 234 a m i n o acid residues ( C h a p m a n a n d H u l m e s , 1984). T h e t r a n s v e r s e s t r u c t u r e of collagen fibrils is, h o w e v e r , less well u n d e r s t o o d . W e t rat tail t e n d o n u n d e r c e r t a i n conditions gives a low angle e q u a t o r i a l X - r a y diffraction p a t t e r n i n d i c a t i n g a high degree of t r a n s v e r s e o r d e r at least in some parts of t h e structure (Miller a n d W r a y , 1971; W o o d h e a d - G a l l o w a y a n d M a c h i n , 1976; F o l k h a r d et al., 1984). This o r d e r is n o t r e t a i n e d in dry t e n d o n specimens and, p r o b a b l y for this r e a s o n , long-range transverse o r d e r c a n n o t b e d e m o n s t r a t e d in e l e c t r o n m i c r o g r a p h s of t e n d o n collagen * Department of Biological Sciences, King Alfred's College, Sparkford Road, Winchester SO22 4NR, UK. "~Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LA1 4YQ, UK. Received 6 November 1985 201 14
fibrils. T r a n s v e r s e paracrystalline s t r u c t u r e has, h o w e v e r , b e e n d e m o n s t r a t e d b y us i n , e l e c t r o n m i c r o g r a p h s o f the c o l l a g e n c o n t a i n i n g fibrils of t h e egg case of the dogfish (Knight a n d H u n t , 1974, 1976). T h e s e fibrils h a v e a c e n t r o s y m m e t r i c a l t r a n s v e r s e b a n d i n g p a t t e r n with a periodicity D of a p p r o x i m a t e l y 37 n m c o m p o s e d of a broad B band and a narrower A band f l a n k e d by two d e n s e C b a n d s ( K n i g h t and H u n t , 1974). A n l l x l l n m s q u a r e - p a c k e d transverse arrangement forms a rectangular prismatic unit cell in t h e B bands. T h e r e c t a n g u l a r prisms a p p e a r to b e a r r a n g e d in a b o d y - c e n t r e d fashion relative to the prisms in the a d j a c e n t B b a n d s b u t are s e p a r a t e d by two C a n d o n e A b a n d . In t h e s e earlier studies we w e r e only able to build a m o d e l for t h e B b a n d ; the structure of the C a n d A b a n d s r e m a i n e d u n c e r t a i n a l t h o u g h we did suggest t h a t t h e molecules m i g h t b e tilted in this r e g i o n ( K n i g h t a n d H u n t , 1976). T h e p r e s e n t c o m m u n i c a t i o n gives evid e n c e for a m o d e l of t h e fibril in which the molecules are tilted t h r o u g h + 2 5 ° in t h e C a n d A bands. T h e m o l e c u l e s a p p e a r to
KNIGHT AND HUNT
202 b e n d at the ends of the B b a n d to lie in the diagonal plane of the B b a n d rectangular prisms. This D periodically kinked molecular arrangement somewhat resembles the structure of the Z-line of m a m m a l i a n striated muscle and of fibrillar material in desmosomes, but does not appear to have b e e n described in other biological fibrils. Materials and Methods
The general methods used for preparing ultrathin sections of the egg case of the dogfish Scyliorhinus caniculus have already b e e n described (Knight and Hunt, 1976). The thinnest sections which could be cut proved to have insufficient contrast even after prolonged staining. The most useful sections had a thickness of 40-60 n m judging by their grey interference colour and the feed setting on the ultramicrotome. A Philips E M 300 T E M fitted with a high resolution, eucentric, rotating, goniometer stage was used to examine the fibrils in ultrathin sections at defined angles of tilt. T h e material is particularly well suited to this type of investigation because the bulk of the thickness of the egg case is built up from an orthogonal arrangement of fibrils. This means that a vertical longitudinal section of the egg case shows parallel-faced laminae composed of alternating layers of fibrils sectioned longitudinally, at 45 ° to their long axis and transversely. Sections were first rotated until the plane of goniometric tilt was exactly parallel or at right angles to the laminae. This made it possible to accurately define the plane of goniometric tilt, even at high magnification. Initially, sections were tilted progressively
and micrographs taken of the same area at every 5 ° of tilt between 0 and 45 °. Eater, pairs of micrographs were taken with a tilt of 25 ° between them. Morphological features and lumps of stain were used to identify small areas unambiguously at different angles of tilt. It proved necessary to correct astigmatism on the grain of the specimen at each angle of tilt; the spring holding the grid in the specimen holder is magnetic and induces astigmatism which varies as the specimen is tilted. A n instrumental magnification of 54,000× was used throughout. Results
Two projections of the paracrystalline square lattice can be seen within the B bands in longitudinal sections of the fibrils (Knight and Hunt, 1976). In the first projection (see Fig. 1), hereafter referred to as projection 1, longitudinal filaments with a regular side to side spacing of 11 n m can be seen r u n n i n g through the B b a n d and lying approximately parallel to the long axis of the fibril. The filaments within one B band are laterally displaced by 5-5 n m relative to filaments in the adjacent B b a n d so that filaments in every other B b a n d appear in line. In projection 1, obliquely orientated filaments can often be seen traversing the A and C bands at a m e a n angle of 19-4 ° to the C b a n d (s.d._+7°; n = 4 5 ) . In some instances, a n d over short distances, the spacing (approximately 11 rim) and angle to the C b a n d (approximately 20 °) and the direction of tilt all appear constant. Filaments within the A and C bands appear to be tilted up or down with an equal frequency. They always
Fig. 1. Longitudinal section of a fibril with regions showing projection 1 (see text). Filamentous densities with a side-to-side spacing of 11 nm and which sometimes appear double, are seen running longitudinallythrough the B band. The A band contains obliquely orientated filaments (see arrows) which sometimescan be traced through the C bands to the ends of the B band filaments, x390,000. Fig. 2. As above with a region showing projection 2 (see text) and an adjacent region showing projection 1 (boxed). The A bands in projection 1 contain filamentous densities (arrowed) which appear to be fainter continuationsof those in the adjacent B bands. Some of the B band filaments in projection 1 (boxed) appear dumb-bell-shaped,x390,000.
204
KNIGHT AND HUNT
appear less dense than their counterparts in the B bands. The tilted filaments in the A and C bands sometimes appear to join a longitudinal filament in the B band to give an appearance of two V's joined by their apices: > - < . In projection 1, the filaments within the B band often appear to broaden out where they approach the C bands to give a dumb-bell-shaped appearance (see Fig. 1 and the boxed area in Fig. 2). This effect is not seen in projection 2 described below. The second projection (projection 2) of the square lattice seen in the B band in longitudinal sections is less frequently encountered than projection 1 described above. It is characterized by filamentous densities within the B band, which run more or less parallel to the long axis of the fibril with a regular side to side spacing of 8 nm (see Fig. 2). Unlike projection 1, the filaments are in lateral register in adjacent B bands. The filamentous densities often appear to run straight through the intervening C and A bands although they are often thinner and not as dense a s those in the B bands. They sometimes appear incomplete as they traverse the A band. The small diameter (approximately 1.5 nm) of the filaments in the thinnest and best orientated longitudinal and transverse sections is compatible with the suggestion that they represent the collagen molecules themselves.
We suggested earlier (Knight and Hunt, 1976) that projection 1 is derived from a plane of section parallel to sides of the B band square lattice while projection 2 represents a plane of section along the diagonal plane of the B band square lattice. This has been confirmed by taking a micrograph of a region of the fibril showing projection 1 and then tilting the section through 45 ° about an axis parallel to the long axis of the fibril before taking a second micrograph. When this is done, tilting converts the image of projection 1 into an image of projection 2. We reported that only 11 nm square lattices are seen in the thinnest transverse sections approximately 30 nm thick while only 8 nm square lattices are seen in the thickest sections approximately 80 nm thick (Knight and Hunt, 1976). Sections of intermediate thickness show both 8 and 11 nm square lattices. The 8 nm lattice in the thicker sections is thought to result from the bodycentred superposition of two 11 nm lattices in sections sufficiently thick (approximately 80 nm) to contain approximately two D periods, 2x37 nm (Knight and Hunt, 1976). The appearance of the filamentous densities in the A and C bands in projections 1 and 2 are compatible with a simple model for the fibril in which molecular segments are parallel to the long axis in the B band but are inclined in the A and C bands in the diagonal plane of the B band square lattice. Faint. filamentous densities seen running in
Figs 3a, b. Tilted pair of electron micrographs; (a) taken at +12-5 ° and (b) at - 1 2 . 5 °. ×530,000. a. The main domain (top right to centre left) shows an 8 nm square lattice with the side of the square parallel to the axis of flit. Several examples of filaments running m the diagonals of an i1 nm square lattice can be seen at the bottom left (arrowed). These are thought to represent A band molecular segments tilted in the diagonal plane of the B band square lattice. (b) The image of the main domain (top right to centre left) has been converted into an 11 nm square lattice with the diagonal parallel to the axis of tilt. Tilting an 11 nm lattice about an axis parallel to its sides (bottom right (a)) does not produce a punctate lattice but streaks out the points into filaments running perpendicular to the axis of tilt (arrows). Figs 4, 5. Photographs of a steel rod and plastic tubing model of the proposed molecular arrangement within the fibril whose long axis runs right to left. The rods are held in place by lucite plates in the B bands. Fig. 4: when the model is viewed in a plane parallel to the side of the B band, square lattice (projection 1), filaments appear to cross the A bands obliquely at an angle of about 20°. Fig. 5: When the same model is viewed in a plane parallel to the diagonal of the B band square lattice, the filaments appear to run straight through the A band but are, in fact, tilted about an axis running in the plane of the paper.
KNIGHT AND HUNT
206 the diagonals of the 11 n m square lattice in transverse sections of the fibril may be indications of the tilted molecular segments (see Fig. 3a). Projections 1 and 2 of this model are illustrated in Figs 4 and 5 respectively. If it is assumed that the A b a n d has a width of 7 n m, the C b a n d a width of 9 n m , that the sides of the square lattice are 11 n m and, for ease of calculation, that the molecules kink abruptly in the middle of the C band, the angle through which the molecules tilt (8) is calculated at 25.9 ° from the expression 11/v2
Tan 0= - 16
(see Fig. 6). The angle (01) at which the molecules would appear to be tilted in projection 1 is calculated at 19° from the expression 11/2 T a n 8 1 - 16 (see Fig. 7). This value is in good agreem e n t with the m e a n observed value of 19.5 ° (see above). The faintness in projection i of the filamentous densities in the C and A bands compared with the B band could be
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-
explained in the following way: in a section of given thickness, twice the n u m b e r of filaments will appear superimposed in the B b a n d compared with the C and A bands if equal n u m b e r s of filaments tilt up and down. The dumb-bell appearance of the filaments in projection 1 may result from a gradual bending of the filaments at the ends of the B band rather than the abrupt kinking assumed for ease of calculation. The tilting of the molecules through an angle of approximately 25 ° in the diagonal plane of the B band square lattice was confirmed by tilting transverse sections of, the fibrils. Paracrystalline domains, showing an 8 n m punctate square lattice with sides parallel to the axis of goniometric tilt, were identified in micrographs taken at - 1 2 . 5 ° of goniometric tilt. W h e n the same domain was identified in micrographs t a k e n at +25 ° of goniometric tilt, the image had changed to an 11 n m punctate square lattice with diagonal axis parallel to the plane of goniometric tilt (see Figs 3a, b). This phen o m e n o n was observed m a n y times in tilted pairs of micrographs, in one case in four separate domains within an area of less than 0 . 5 / z m 2. It was only seen where the axis of goniometric tilt was parallel to the plane of
___i6_ . . . . . .
Fig. 6. Isometric drawing of the molecularmodel assumingan abrupt kinkingin the middle of the C band for ease of calculation. The angle of tilt (8) is calculatedfrom the dimensions (in nm) obtained from electron micrographs. Only one of the tilted molecular segments is illustrated.
11
16
I. . . . . . . . . . . . . . . . . .
,
Fig. 7. The apparent angle of tilt (01) seen in projection 1) (see text) is calculated from dimensions (in nm) obtained from electron micrographs. Only one of the tilted molecular segments is illustrated.
MOLECULAR MODEL FOR EGG CASE COLLAGEN the sides of the 8 nm lattice and was most sharply defined when a goniometric tilt of 25 ° was used between images but could still be observed at angles of up to 35 °. A tilt of 15° only converted the point lattice into faint streaks perpendicular to the axis of tilt. A goniometric tilt of 25 ° about an axis parallel to the sides of the 11 nm square lattice did not produce a second punctate lattice but merely streaked out the image of lattice points into filaments running perpendicular to the axis tilt (see Figs 3a, b). All these observations are compatible with the model illustrated in Figs 4 and 5. The transformation of the 11 nm square lattice into an 8 nm lattice on tilting through 25 ° is thought to originate in the following way: The section thickness of approximately 40 nm would include two B and one A band. A view of the section perpendicular to the B band lattice would show two 11 nm lattices superimposed in a body-centred fashion to give an 8 nm lattice (see above and Knight and Hunt, 1976). Tilting the section through 25 ° in the diagonal plane of the B band lattice would give rise to an 11 nm square lattice as tilted filaments in the A band would now be viewed end on. The fact that the image is a complete 11 nm punctate square lattice after tilting through 25 ° might suggest that, within small domains, the filaments are all tilted the same way in the A bands. A n alternative explanation would be that each B band filament divides into two A band filaments, one which tilts up and the other down. The observation, however, that negative stain penetrates equally into the A and B bands (Knight and Hunt, 1974) makes it unlikely that the molecular density in the A band is twice that of the B band. B and A bands would have equal density if B band filaments are dimeric associations of two parallel segments which divide into two monomers one tilting up and the other down in the A band. The thinness of A band filaments relative to the B ones is compatible with this suggestion. The observation that the transformation of the 8 nm lattice into the 11 nm lattice is best seen with a goniometric tilt of 25 ° is in agreement with the value for the angle of tilt (25.9 °) calculated from the dimension of the lattice and the widths of the A and C bands (see above).
207 Discussion
The observations reported here on longitudinal, and on tilted transverse, sections suggest a model of the general type shown in Figs 4 and 5 in which the molecules undergo D periodic tilting or kinking through an angle of approximately 25 ° . The filaments are thought to tilt in the diagonal plane of the B band square lattice. Several variants of the model shown in Figs 4 and 5 cannot be ruled out at present. For example, the filaments seen in the A band may not represent a direct continuation of the molecular segments in the B band but result from overlap in the C band. In this connection it must be remembered that the high density and exclusion of negative stain by the C band is compatible with the suggestion that this represents a region of molecular overlap (Knight and Hunt, 1974). This, however, is not the only possible explanation of stain exclusion and density; alternatively the C band may represent a region in which accessory proteins are bound and/or where there is extensive cross-linking. Another variant, discussed above, is that each B band filament gives rise to two A band ones. Dogfish egg-case collagen shows similari: ties with type IV collagen obtained from basement membrane: both are secreted by epithelial cells rather than fibroblasts. Both form composite structures containing large quantities of accessory proteins (Knight and Hunt, 1974; Timpl et al., 1984). Another similarity is that both molecules may contain a flexible, non-helical molecular segment which permits two stiff and straight, triple-helical segments to be held at an angle to each other. Enzymatic degradation studies combined with rotary shadowing of isolated molecular fragments suggest that type IV collagen m o l e c u l e s contain nonhelical segments which m a y provide a flexible hinge between two stiff, straight helical segments (Hoffman et al., 1984; Timpl et al., 1984; Martin et al., ]985). A model has been proposed for the ultrastructural organization of collagen in Descemet's membrane in which the helical segments form the sides of regular, layered, planar hexagonal arrays and the non-helical segments kink to form the angles of the hexagons (Madri et al., 1984). It is interestiing to note that a
208 similar, stacked, h e x a g o n a l a p p p e a r a n c e is s e e n in t h e p r e c u r s o r g r a n u l e s in t h e oviducal gland cells w h i c h f o r m the fibrils of t h e egg case b u t h e r e t h e side to side spacing is 38 n m , similar to t h e D p e r i o d of t h e fibrils a n d in c o n t r a s t to t h e spacing of a p p r o x i m a t e l y 100 n m s e e n in D e s c e m e t ' s m e m b r a n e (Knight a n d H u n t , u n p u b lished). It is an intriguing s p e c u l a t i o n that dogfish egg-case collagen a n d m a m m a l i a n type I V collagen are closely r e l a t e d proteins. If this proves to b e t h e case, t h e very h i g h rate of secretion, a p p r o x i m a t e l y 1 g of collagen p e r oviducal g l a n d p e r day for the g r e a t e r s p o t t e d dogfish, w o u l d m a k e this excellent m a t e r i a l for s t u d y i n g the process of secretion of these i m p o r t a n t proteins. It is interesting to n o t e t h a t a k i n k e d molecular model has been proposed ( W o o d h e a d - G a l l o w a y , 1980) to account for t h e low angle X-ray diffraction p a t t e r n of stretched mammalian tendon.
KNIGHT AND HUNT O u r m o d e l for the dogfish egg-case fibrils show s o m e similarity to t h e s t r u c t u r e of t h e Z line of v e r t e b r a t e striated muscle ( F r a n z i n i - A r m s t r o n g , 1973; Ullrick et al., 1977; Y a m a g u c h i et al., 1985) a n d to t h e fibrilar m a t e r i a l traversing the i n t e r c e l l u l a r space in d e s m o s o m e s ( A r n n a n d Staehelin, 1981; Staehelin, 1974; S t a e h e l i n a n d Hull, 1978). W e can only guess at t h e biophysical significance of this unusual p a t t e r n of construction. P e r h a p s the a r r a n g e m e n t in all t h r e e situations adds s t r e n g t h to t h e fibrils b y p e r m i t t i n g the dissipation of e n e r g y w h e n t h e kinks straighten during stretching.
Acknowledgements W e t h a n k the M R C for financial s u p p o r t (project grant number G8418317CB); Mr R a y Griffin a n d t h e staff of S o u t h a m p t o n G e n e r a l H o s p i t a l E M U n i t for technical h e l p a n d D r J o h n G a l l o w a y for advice.
References Arnn, J. and Staehelin, L. A. 1981. The structure and function of spot desmosomes. Dermatology, 20, 330-339. Chapman, J. A. and Hulmes, D. J. S. 1984. Electron microscopy of the collagen fibril. In Ultrastructure of the Connective Tissue Matrix (eds A. Ruggeri and P. M. Motta). Martinus Nijhoff, Boston. Folkhard, W., Knorzer, E., Mosler, E. and Nemetschek, T. 1984. Packing of collagen molecules modified with 2-propanol. J. molec. BioL, 177, 841-844. Franzini-Armstrong, C. 1973. The structure of a simple Z-line. J. Cell Biol., 58, 630-642. Hoffman, H., Voss, T., Kuhn, K. and Engel, J. 1984. Localization of flexible sites in threadlike molecules from electron micrographs: comparison of interstitial, basement membrane and intima collagens. J. molec. Biol., 172, 325--343. Knight, D. P. and Hunt, S. 1974. Fibril structure of collagen in egg capsule of dogfish. Nature, Lond., 249, 379--380. Knight, D, P. and Hunt, S. 1976. Fine structure of the dogfish egg case: a unique collagenousmaterial. Tissue & Cell, 8, 183-193. Madri, J. A., Pratt, B. M., Yurchenko, P. D. and Furthmayr, H. 1984. The ultrastructural organization and architecture of basement membrane. Ciba Foundation Symp., 108, 6-18. Martin, G. R., Timpl, R., Muller, P. K. and Kuhn, K. 1985. The genetically distinct collagens. Trends Biochem. Sci., 10, 285--287. Miller, A. and Wray, J. S. 1971. Molecular packing in collagen. Nature, Lond., 230, 437-439. Staehelin, L. A. 1974. Structure and function of intercellular junctions, lnt. Rev. Cytol., 39, 191-283. Staehelin, L. A. and Hull, B. E. 1978. Junctions between living cells. Sci. Am., 238, 141-152. Timpl, R., Fujiwara, S., Dziadek, M., Aumaulley, M., Weber, S. and Engel, J. 1984. Laminin, proteoglycan, nidogen and collagen IV: structural models and molecular interactions. Ciba Foundation Syrup., 108, 25-37. Ulrick, W. C., Toselli, P. A., Saide, J. D. and Phear, W. P. C. 1977. Fine structure of the vertebrate Z-disc. J. rnolec. Biol., 115, 61-74. Woodhead-Galloway, J. 1980. Structure of the collagen fibril: some variations on a theme of tetragonally packed dimers. Proc. R. Soc. Lond., B209, 275-297. Woodhead-Galloway, J. and Machin, P. A. 19761 Modern theories of liquids and the diffuse equatorial X-ray scattering from collagen. Acta Crystallogr. Sect, A, 32, 368-372. Yamaguchi, M., Izumimoto, M., Robson, R. M. and Stromer, M. H. 1985. Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture. J. molec. Biol., 184, 621-644.
DAVID P. KNIGHT* and STEPHEN H U N T t
A KINKED MOLECULAR MODEL FOR THE COLLAGEN-CONTAINING FIBRILS IN THE EGG CASE OF THE DOGFISH SCYLIORHINUS CANICUL US Keywords: Collagen, fibrils, kinked molecules, model, egg case, Scyliorhinus ABSTRACT. A model for the organization of fibrils in the collagenous egg case of the dogfish Scyliorhinus caniculus is proposed on the basis of observations on ultrathin sections tilted in a goniometer stage. The centrosymmetric transve/se banding pattern consists of an A band flanked immediately by two dense C bands which are in turn flanked by broad B bands. In the model, the B bands contain molecular segments packed in a square lattice forming a rectangular prismatic cell parallel to the fibril long axis. The molecules kink in the C band or gradually bend at the ends of the B band to tilt up or down at 25° to the fibril axis in the C and A bands. The tilt is such that the corner of one B band unit is body centred on the face of the next B band unit. Our model has similarities to models for the arrangement of type IV collagen in basement membrane, a-actinin in the Z-discs of striated muscle and the intercellular material in spot desmosomes.
Introduction
T h e longitudinal s t r u c t u r e of rat tail t e n d o n a n d o t h e r collagen fibrils which show t h e classical positive a n d n e g a t i v e b a n d i n g patt e r n s is well u n d e r s t o o d . T h e r e is g o o d e v i d e n c e t h a t t h e m o l e c u l e s are a r r a n g e d with a longitudinal stagger of 234 a m i n o acid residues ( C h a p m a n a n d H u l m e s , 1984). T h e t r a n s v e r s e s t r u c t u r e of collagen fibrils is, h o w e v e r , less well u n d e r s t o o d . W e t rat tail t e n d o n u n d e r c e r t a i n conditions gives a low angle e q u a t o r i a l X - r a y diffraction p a t t e r n i n d i c a t i n g a high degree of t r a n s v e r s e o r d e r at least in some parts of t h e structure (Miller a n d W r a y , 1971; W o o d h e a d - G a l l o w a y a n d M a c h i n , 1976; F o l k h a r d et al., 1984). This o r d e r is n o t r e t a i n e d in dry t e n d o n specimens and, p r o b a b l y for this r e a s o n , long-range transverse o r d e r c a n n o t b e d e m o n s t r a t e d in e l e c t r o n m i c r o g r a p h s of t e n d o n collagen * Department of Biological Sciences, King Alfred's College, Sparkford Road, Winchester SO22 4NR, UK. "~Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LA1 4YQ, UK. Received 6 November 1985 201 14
fibrils. T r a n s v e r s e paracrystalline s t r u c t u r e has, h o w e v e r , b e e n d e m o n s t r a t e d b y us i n , e l e c t r o n m i c r o g r a p h s o f the c o l l a g e n c o n t a i n i n g fibrils of t h e egg case of the dogfish (Knight a n d H u n t , 1974, 1976). T h e s e fibrils h a v e a c e n t r o s y m m e t r i c a l t r a n s v e r s e b a n d i n g p a t t e r n with a periodicity D of a p p r o x i m a t e l y 37 n m c o m p o s e d of a broad B band and a narrower A band f l a n k e d by two d e n s e C b a n d s ( K n i g h t and H u n t , 1974). A n l l x l l n m s q u a r e - p a c k e d transverse arrangement forms a rectangular prismatic unit cell in t h e B bands. T h e r e c t a n g u l a r prisms a p p e a r to b e a r r a n g e d in a b o d y - c e n t r e d fashion relative to the prisms in the a d j a c e n t B b a n d s b u t are s e p a r a t e d by two C a n d o n e A b a n d . In t h e s e earlier studies we w e r e only able to build a m o d e l for t h e B b a n d ; the structure of the C a n d A b a n d s r e m a i n e d u n c e r t a i n a l t h o u g h we did suggest t h a t t h e molecules m i g h t b e tilted in this r e g i o n ( K n i g h t a n d H u n t , 1976). T h e p r e s e n t c o m m u n i c a t i o n gives evid e n c e for a m o d e l of t h e fibril in which the molecules are tilted t h r o u g h + 2 5 ° in t h e C a n d A bands. T h e m o l e c u l e s a p p e a r to
KNIGHT AND HUNT
202 b e n d at the ends of the B b a n d to lie in the diagonal plane of the B b a n d rectangular prisms. This D periodically kinked molecular arrangement somewhat resembles the structure of the Z-line of m a m m a l i a n striated muscle and of fibrillar material in desmosomes, but does not appear to have b e e n described in other biological fibrils. Materials and Methods
The general methods used for preparing ultrathin sections of the egg case of the dogfish Scyliorhinus caniculus have already b e e n described (Knight and Hunt, 1976). The thinnest sections which could be cut proved to have insufficient contrast even after prolonged staining. The most useful sections had a thickness of 40-60 n m judging by their grey interference colour and the feed setting on the ultramicrotome. A Philips E M 300 T E M fitted with a high resolution, eucentric, rotating, goniometer stage was used to examine the fibrils in ultrathin sections at defined angles of tilt. T h e material is particularly well suited to this type of investigation because the bulk of the thickness of the egg case is built up from an orthogonal arrangement of fibrils. This means that a vertical longitudinal section of the egg case shows parallel-faced laminae composed of alternating layers of fibrils sectioned longitudinally, at 45 ° to their long axis and transversely. Sections were first rotated until the plane of goniometric tilt was exactly parallel or at right angles to the laminae. This made it possible to accurately define the plane of goniometric tilt, even at high magnification. Initially, sections were tilted progressively
and micrographs taken of the same area at every 5 ° of tilt between 0 and 45 °. Eater, pairs of micrographs were taken with a tilt of 25 ° between them. Morphological features and lumps of stain were used to identify small areas unambiguously at different angles of tilt. It proved necessary to correct astigmatism on the grain of the specimen at each angle of tilt; the spring holding the grid in the specimen holder is magnetic and induces astigmatism which varies as the specimen is tilted. A n instrumental magnification of 54,000× was used throughout. Results
Two projections of the paracrystalline square lattice can be seen within the B bands in longitudinal sections of the fibrils (Knight and Hunt, 1976). In the first projection (see Fig. 1), hereafter referred to as projection 1, longitudinal filaments with a regular side to side spacing of 11 n m can be seen r u n n i n g through the B b a n d and lying approximately parallel to the long axis of the fibril. The filaments within one B band are laterally displaced by 5-5 n m relative to filaments in the adjacent B b a n d so that filaments in every other B b a n d appear in line. In projection 1, obliquely orientated filaments can often be seen traversing the A and C bands at a m e a n angle of 19-4 ° to the C b a n d (s.d._+7°; n = 4 5 ) . In some instances, a n d over short distances, the spacing (approximately 11 rim) and angle to the C b a n d (approximately 20 °) and the direction of tilt all appear constant. Filaments within the A and C bands appear to be tilted up or down with an equal frequency. They always
Fig. 1. Longitudinal section of a fibril with regions showing projection 1 (see text). Filamentous densities with a side-to-side spacing of 11 nm and which sometimes appear double, are seen running longitudinallythrough the B band. The A band contains obliquely orientated filaments (see arrows) which sometimescan be traced through the C bands to the ends of the B band filaments, x390,000. Fig. 2. As above with a region showing projection 2 (see text) and an adjacent region showing projection 1 (boxed). The A bands in projection 1 contain filamentous densities (arrowed) which appear to be fainter continuationsof those in the adjacent B bands. Some of the B band filaments in projection 1 (boxed) appear dumb-bell-shaped,x390,000.
204
KNIGHT AND HUNT
appear less dense than their counterparts in the B bands. The tilted filaments in the A and C bands sometimes appear to join a longitudinal filament in the B band to give an appearance of two V's joined by their apices: > - < . In projection 1, the filaments within the B band often appear to broaden out where they approach the C bands to give a dumb-bell-shaped appearance (see Fig. 1 and the boxed area in Fig. 2). This effect is not seen in projection 2 described below. The second projection (projection 2) of the square lattice seen in the B band in longitudinal sections is less frequently encountered than projection 1 described above. It is characterized by filamentous densities within the B band, which run more or less parallel to the long axis of the fibril with a regular side to side spacing of 8 nm (see Fig. 2). Unlike projection 1, the filaments are in lateral register in adjacent B bands. The filamentous densities often appear to run straight through the intervening C and A bands although they are often thinner and not as dense a s those in the B bands. They sometimes appear incomplete as they traverse the A band. The small diameter (approximately 1.5 nm) of the filaments in the thinnest and best orientated longitudinal and transverse sections is compatible with the suggestion that they represent the collagen molecules themselves.
We suggested earlier (Knight and Hunt, 1976) that projection 1 is derived from a plane of section parallel to sides of the B band square lattice while projection 2 represents a plane of section along the diagonal plane of the B band square lattice. This has been confirmed by taking a micrograph of a region of the fibril showing projection 1 and then tilting the section through 45 ° about an axis parallel to the long axis of the fibril before taking a second micrograph. When this is done, tilting converts the image of projection 1 into an image of projection 2. We reported that only 11 nm square lattices are seen in the thinnest transverse sections approximately 30 nm thick while only 8 nm square lattices are seen in the thickest sections approximately 80 nm thick (Knight and Hunt, 1976). Sections of intermediate thickness show both 8 and 11 nm square lattices. The 8 nm lattice in the thicker sections is thought to result from the bodycentred superposition of two 11 nm lattices in sections sufficiently thick (approximately 80 nm) to contain approximately two D periods, 2x37 nm (Knight and Hunt, 1976). The appearance of the filamentous densities in the A and C bands in projections 1 and 2 are compatible with a simple model for the fibril in which molecular segments are parallel to the long axis in the B band but are inclined in the A and C bands in the diagonal plane of the B band square lattice. Faint. filamentous densities seen running in
Figs 3a, b. Tilted pair of electron micrographs; (a) taken at +12-5 ° and (b) at - 1 2 . 5 °. ×530,000. a. The main domain (top right to centre left) shows an 8 nm square lattice with the side of the square parallel to the axis of flit. Several examples of filaments running m the diagonals of an i1 nm square lattice can be seen at the bottom left (arrowed). These are thought to represent A band molecular segments tilted in the diagonal plane of the B band square lattice. (b) The image of the main domain (top right to centre left) has been converted into an 11 nm square lattice with the diagonal parallel to the axis of tilt. Tilting an 11 nm lattice about an axis parallel to its sides (bottom right (a)) does not produce a punctate lattice but streaks out the points into filaments running perpendicular to the axis of tilt (arrows). Figs 4, 5. Photographs of a steel rod and plastic tubing model of the proposed molecular arrangement within the fibril whose long axis runs right to left. The rods are held in place by lucite plates in the B bands. Fig. 4: when the model is viewed in a plane parallel to the side of the B band, square lattice (projection 1), filaments appear to cross the A bands obliquely at an angle of about 20°. Fig. 5: When the same model is viewed in a plane parallel to the diagonal of the B band square lattice, the filaments appear to run straight through the A band but are, in fact, tilted about an axis running in the plane of the paper.
KNIGHT AND HUNT
206 the diagonals of the 11 n m square lattice in transverse sections of the fibril may be indications of the tilted molecular segments (see Fig. 3a). Projections 1 and 2 of this model are illustrated in Figs 4 and 5 respectively. If it is assumed that the A b a n d has a width of 7 n m, the C b a n d a width of 9 n m , that the sides of the square lattice are 11 n m and, for ease of calculation, that the molecules kink abruptly in the middle of the C band, the angle through which the molecules tilt (8) is calculated at 25.9 ° from the expression 11/v2
Tan 0= - 16
(see Fig. 6). The angle (01) at which the molecules would appear to be tilted in projection 1 is calculated at 19° from the expression 11/2 T a n 8 1 - 16 (see Fig. 7). This value is in good agreem e n t with the m e a n observed value of 19.5 ° (see above). The faintness in projection i of the filamentous densities in the C and A bands compared with the B band could be
-
-
explained in the following way: in a section of given thickness, twice the n u m b e r of filaments will appear superimposed in the B b a n d compared with the C and A bands if equal n u m b e r s of filaments tilt up and down. The dumb-bell appearance of the filaments in projection 1 may result from a gradual bending of the filaments at the ends of the B band rather than the abrupt kinking assumed for ease of calculation. The tilting of the molecules through an angle of approximately 25 ° in the diagonal plane of the B band square lattice was confirmed by tilting transverse sections of, the fibrils. Paracrystalline domains, showing an 8 n m punctate square lattice with sides parallel to the axis of goniometric tilt, were identified in micrographs taken at - 1 2 . 5 ° of goniometric tilt. W h e n the same domain was identified in micrographs t a k e n at +25 ° of goniometric tilt, the image had changed to an 11 n m punctate square lattice with diagonal axis parallel to the plane of goniometric tilt (see Figs 3a, b). This phen o m e n o n was observed m a n y times in tilted pairs of micrographs, in one case in four separate domains within an area of less than 0 . 5 / z m 2. It was only seen where the axis of goniometric tilt was parallel to the plane of
___i6_ . . . . . .
Fig. 6. Isometric drawing of the molecularmodel assumingan abrupt kinkingin the middle of the C band for ease of calculation. The angle of tilt (8) is calculatedfrom the dimensions (in nm) obtained from electron micrographs. Only one of the tilted molecular segments is illustrated.
11
16
I. . . . . . . . . . . . . . . . . .
,
Fig. 7. The apparent angle of tilt (01) seen in projection 1) (see text) is calculated from dimensions (in nm) obtained from electron micrographs. Only one of the tilted molecular segments is illustrated.
MOLECULAR MODEL FOR EGG CASE COLLAGEN the sides of the 8 nm lattice and was most sharply defined when a goniometric tilt of 25 ° was used between images but could still be observed at angles of up to 35 °. A tilt of 15° only converted the point lattice into faint streaks perpendicular to the axis of tilt. A goniometric tilt of 25 ° about an axis parallel to the sides of the 11 nm square lattice did not produce a second punctate lattice but merely streaked out the image of lattice points into filaments running perpendicular to the axis tilt (see Figs 3a, b). All these observations are compatible with the model illustrated in Figs 4 and 5. The transformation of the 11 nm square lattice into an 8 nm lattice on tilting through 25 ° is thought to originate in the following way: The section thickness of approximately 40 nm would include two B and one A band. A view of the section perpendicular to the B band lattice would show two 11 nm lattices superimposed in a body-centred fashion to give an 8 nm lattice (see above and Knight and Hunt, 1976). Tilting the section through 25 ° in the diagonal plane of the B band lattice would give rise to an 11 nm square lattice as tilted filaments in the A band would now be viewed end on. The fact that the image is a complete 11 nm punctate square lattice after tilting through 25 ° might suggest that, within small domains, the filaments are all tilted the same way in the A bands. A n alternative explanation would be that each B band filament divides into two A band filaments, one which tilts up and the other down. The observation, however, that negative stain penetrates equally into the A and B bands (Knight and Hunt, 1974) makes it unlikely that the molecular density in the A band is twice that of the B band. B and A bands would have equal density if B band filaments are dimeric associations of two parallel segments which divide into two monomers one tilting up and the other down in the A band. The thinness of A band filaments relative to the B ones is compatible with this suggestion. The observation that the transformation of the 8 nm lattice into the 11 nm lattice is best seen with a goniometric tilt of 25 ° is in agreement with the value for the angle of tilt (25.9 °) calculated from the dimension of the lattice and the widths of the A and C bands (see above).
207 Discussion
The observations reported here on longitudinal, and on tilted transverse, sections suggest a model of the general type shown in Figs 4 and 5 in which the molecules undergo D periodic tilting or kinking through an angle of approximately 25 ° . The filaments are thought to tilt in the diagonal plane of the B band square lattice. Several variants of the model shown in Figs 4 and 5 cannot be ruled out at present. For example, the filaments seen in the A band may not represent a direct continuation of the molecular segments in the B band but result from overlap in the C band. In this connection it must be remembered that the high density and exclusion of negative stain by the C band is compatible with the suggestion that this represents a region of molecular overlap (Knight and Hunt, 1974). This, however, is not the only possible explanation of stain exclusion and density; alternatively the C band may represent a region in which accessory proteins are bound and/or where there is extensive cross-linking. Another variant, discussed above, is that each B band filament gives rise to two A band ones. Dogfish egg-case collagen shows similari: ties with type IV collagen obtained from basement membrane: both are secreted by epithelial cells rather than fibroblasts. Both form composite structures containing large quantities of accessory proteins (Knight and Hunt, 1974; Timpl et al., 1984). Another similarity is that both molecules may contain a flexible, non-helical molecular segment which permits two stiff and straight, triple-helical segments to be held at an angle to each other. Enzymatic degradation studies combined with rotary shadowing of isolated molecular fragments suggest that type IV collagen m o l e c u l e s contain nonhelical segments which m a y provide a flexible hinge between two stiff, straight helical segments (Hoffman et al., 1984; Timpl et al., 1984; Martin et al., ]985). A model has been proposed for the ultrastructural organization of collagen in Descemet's membrane in which the helical segments form the sides of regular, layered, planar hexagonal arrays and the non-helical segments kink to form the angles of the hexagons (Madri et al., 1984). It is interestiing to note that a
208 similar, stacked, h e x a g o n a l a p p p e a r a n c e is s e e n in t h e p r e c u r s o r g r a n u l e s in t h e oviducal gland cells w h i c h f o r m the fibrils of t h e egg case b u t h e r e t h e side to side spacing is 38 n m , similar to t h e D p e r i o d of t h e fibrils a n d in c o n t r a s t to t h e spacing of a p p r o x i m a t e l y 100 n m s e e n in D e s c e m e t ' s m e m b r a n e (Knight a n d H u n t , u n p u b lished). It is an intriguing s p e c u l a t i o n that dogfish egg-case collagen a n d m a m m a l i a n type I V collagen are closely r e l a t e d proteins. If this proves to b e t h e case, t h e very h i g h rate of secretion, a p p r o x i m a t e l y 1 g of collagen p e r oviducal g l a n d p e r day for the g r e a t e r s p o t t e d dogfish, w o u l d m a k e this excellent m a t e r i a l for s t u d y i n g the process of secretion of these i m p o r t a n t proteins. It is interesting to n o t e t h a t a k i n k e d molecular model has been proposed ( W o o d h e a d - G a l l o w a y , 1980) to account for t h e low angle X-ray diffraction p a t t e r n of stretched mammalian tendon.
KNIGHT AND HUNT O u r m o d e l for the dogfish egg-case fibrils show s o m e similarity to t h e s t r u c t u r e of t h e Z line of v e r t e b r a t e striated muscle ( F r a n z i n i - A r m s t r o n g , 1973; Ullrick et al., 1977; Y a m a g u c h i et al., 1985) a n d to t h e fibrilar m a t e r i a l traversing the i n t e r c e l l u l a r space in d e s m o s o m e s ( A r n n a n d Staehelin, 1981; Staehelin, 1974; S t a e h e l i n a n d Hull, 1978). W e can only guess at t h e biophysical significance of this unusual p a t t e r n of construction. P e r h a p s the a r r a n g e m e n t in all t h r e e situations adds s t r e n g t h to t h e fibrils b y p e r m i t t i n g the dissipation of e n e r g y w h e n t h e kinks straighten during stretching.
Acknowledgements W e t h a n k the M R C for financial s u p p o r t (project grant number G8418317CB); Mr R a y Griffin a n d t h e staff of S o u t h a m p t o n G e n e r a l H o s p i t a l E M U n i t for technical h e l p a n d D r J o h n G a l l o w a y for advice.
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