Sequential events in the formation of collagen secretion granules with special reference to the development of segment-long-spacing-like aggregates.код для вставкиСкачать
THE ANATOMICAL RECORD 199:309-320 (1981) Sequential Events in the Formation of Collagen Secretion Granules With Special Reference to the Development of Segment-Long-Spacing-Like Aggregates MOON-IL CHO AND PHILIAS R. GARANT Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, New York 11 794 ABSTRACT The formation and maturation of collagen secretion granules in periodontal ligament (PDL) fibroblasts of young male Balb-C mice were studied by electron microscopy and cytochemistry. The Golgi apparatus was composed of several dictyosomes, each consisting of a stack of five smooth-walled cisternae. Each cisterna was connected a t both ends to a dilated saccule. The cisternae, with their associated pairs of saccules, underwent progressive maturational changes from the immature to mature face of each dictyosome.The most immature saccules (type 1) were large, spherical, and filled with loosely arranged short filamentous structures. These saccules were continuous with the first and second Golgi cisternae (counting from the immature side). Golgi saccules type 2 were ellipsoidal, associated with the third and fourth cisternae, and contained parallel, elongate filaments. The most mature saccules, type 3, were more rectangular and connected by a short fifth cisterna. They contained aggregated filamentous material in the form of eight segment-long-spacing (SLS)-like crystallites, one in the center and seven at the periphery, to form a basic secretory unit. As type 3 saccules matured, the interconnecting cisterna progressively shortened until the two saccules were nearly juxtaposed. At this time the shortened fifth cisterna split to give rise to two independent presecretory granules. By progressive condensation, presecretory granules matured into secretion granules that contained a densely packed SLSlike aggregate, within which individual crystallites were no longer discernable. Maturation of cisternae and saccules involved removal of membrane, apparently by the formation and detachment of coated vesicles. The staining reaction with silver methenamine and phosphotungstic acid increased over the procollagen as the saccules matured, indicating addition of carbohydrate moieties and possible crosslinkages. It is concluded that the formation and maturation of collagen secretion granules in PDL fibroblasts involves the packaging and further modification of eight SLS-like crystallites, which are secreted as a basic unit. A number of ultrastructural and electron microscopic radioautographic studies have been made to determine the mode of collagen precursor discharge from various types of collagen-producing cells. It has been generally agreed that collagen precursors are packaged in the Golgi apparatus and released from the cell by a merocrine-type secretion involving the fusion of secretory granules with the cell membranes of chondroblasts (Revel and Hay, '63), corneal epithelial cells (Trelstad, '70, '71; 0003-276X/81/19931)309$03.50 0 1981 ALAN R. LISS. INC. Trelstad and Coulombre, '71), fibroblasts (Olsen and Prockop, '74; Trelstad, '751, odontoblasts (Weinstock and Leblond, '741, and osteoblasts (Ehrlich et al., '74; Frank and Frank, '69; Weinstock, '75; Weinstock et al., '75). The current concept of collagen fibrillogenesis suggests that secreted collagen molecules undergo a series of biochemical modifiReceived June 10, 1980 accepted August 5 , 1980 310 M.-I. CHO AND P.R. GAFlANT cations in the extracellular space prior to their assembly into 640 A" banded fibrils. The modification includes the cleavage of extensions a t both amino and carboxyl terminal ends of the procollagen molecules, during or imediately following secretion (Bornstein et al., '72; Kohn et al., '74; Morris et al., '75). It has been described previously how procollagen molecules become condensed and aggregated a s an intermediate form in the Golgi apparatus before they are secreted (Trelstad, '71; Weinstock and Leblond, '74; Ehrlich et al., '74; Scherft and Heersche, '75; Weinstock, '77; Trelstad and Hayashi, '79). Recently, Bruns et al. ('79) identified extracellular segment-longspacing (SLS)-like structures in the tissue homogenates and culture media of a variety of fibroblast cell types grown in vitro. We have routinely observed aggregates of procollagen within Golgi saccules of PDL fibroblasts. From careful examination of numerous Golgi complexes,we have been able to visualize a maturation of Golgi saccules involving the formation of SLS-like crystallites in Golgi saccules. In this report we describe the sequential steps in the development of collagen secretory granules. In previous studies of the PDL fibroblast, we described its high degree of cytoplasmic polarity and presented indirect evidence that fibroblast migration might be related to collagenous fiber formation within the ligament (Garant and Cho, 79a, b). MATERIALS AND METHODS Tissue processing for electron microscopy Male Balb-C mice weighing 17-19 gm were anesthetized by IP injection of Nembutal (1mg/lO gm body weight) prior to intracardiac perfusion of 70-80 ml of 2% glutaraldehyde in a modified Tyrode solution, pH 7.4 (Sjostrand, '67). The maxillae were then dissected free of the surrounding tissues in a dish containing 4% glutaraldehyde and 5% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 (Karnovsky, '65). The cleaned maxillae were fixed in Karnovsky's fixative for a n additional 3-4 hr, rinsed in 0.1 M cacodylate buffer, pH 7.4, for 15 min, and decalcified in 0.1 M EDTA containing 3%glutaraldehyde, pH 7.4, for about 2-3 weeks at 4°C. The first interdental areas were then dissected out and cut into two identical pieces in a dish containing 0.1 M cadodylate buffer, pH 7.4. After washing in the same buffer with several changes for 45-60 min, the pieces of tissue were postfixed in 1% Osmium tetroxide in S-collidinebuffer, pH 7.4, for 1%hr. En-block staining was carried out for 1hr a t room temperature in 1%uranyl acetate in 0.1 M maleate buffer, pH 6.2, after rinsing in the same buffer, pH 5.2, for 15 min. Tissues were then dehydrated in a graduated series of cold ethanols and propylene oxide prior to infiltration with Epon mixture at room temperature for 4hr and placed in flat embedding molds containing fresh Epon mixture. Polymerization was accomplished at 60°C for 48 hr. For light microscopic studies, 1-Fm sections were cut on a Porter-Blum MT2 ultra-microtome and stained with 1%toluidine blue in veronal acetate buffer. For ultrastructural study, ultrathin sections (60-80 nm in thickness) were cut on a PorterBlum MT2 ultramicrotome equipped with a DuPont diamond knife. The sections were doubly stained for 25 min with freshly prepared saturated uranyl acetate in 5Wo ethanol and for 7 min with lead citrate (Reynolds, '63).The stained sections were then examined and photographed with a JEM-100B electron microscope. For analysis of Golgi structure, micrographs were taken of polarized fibroblasts sectioned through the mid-point as determined by the presence of a centriole. Cytochemical studies Silver methenumine staining. Ultrathin sections (pale yellow) were treated according to the method introduced by Rambourg ('67). Upon completion of sectioning, ribbons were immediately transferred with a platinum loop to 1%periodic acid solution in distilled water to oxidize for 20 min. After several brief rinses in distilled water, the ribbons were kept on distilled water overnight and then transferred to the filtered staining solution (5 ml of 5% silver nitrate solution, 45ml of 3% methenamine, and 5 ml of 2% sodium borate) in a petri dish that was preincubated in the oven at 60°C. After staining for 30 min, the ribbons were washed and transferred to a second bath of staining solution and stained for another 30 min, a t which point the ribbons were washed by floating on distilled water for 5 min and transferred to 5% sodium thiosulphate solution in distilled water for 5-10 min. After rinsing three times, the ribbons were then picked up on formvarcoated 300-mesh grids. Phosphotungstic acid staining. Decalcified tissues were washed in several changes of 0.1 M cacodylate buffer for 1-2 h r at 4°C. The washed tissues were then dehydrated i n unpolymerized 800/0 and 97% glycol methacrylate embedding mixture (consisting of 7 parts FORMATION OF COLLAGEN SECRETORY GRANULES glycol methacrylate, 3 parts butylmethacrylate, WO2.4 dichlorobenzol peroxide paste, and 3% distilled water) for 20 min, respectively, a t 3°C. Polymerization was accomplished a t 3°C with ultraviolet light for 1-3 days (Leduc and Bernhard, '67). Ultrathin sections (70-80 nm in thickness) were stained for 5-10 min by floating on a freshly prepared, filtered solution of 1%phosphotungstic acid in 10%chromic acid in distilled water. Upon completion of staining, the sections were floated on distilled water for a few seconds and rapidly transferred to formvar-coated grids. RESULTS The fibroblasts of the periodontal ligament were elongate and highly polarized cells (Fig. 1).They contained a large juxtanuclear Golgi complex, seen as a pale staining zone in toluidine blue-stained 1- pm sections (Fig. 1).At the ultrastructural level, the Golgi complex was cylindrical in shape, occupying a large expanse of cytoplasm from the juxtanuclear area to the furcation area, from which major distal cytoplasmic processes originate. The Golgi ccjmplex, surrounded peripherally by parallel cisternae of rough endoplasmic reticulum (RER), consisted of numerous dictyosomes, each of which was composed of stacked Golgi cisternae and associated Golgi saccules (Fig. 2). A small number of short RER cisternae and mitochondria were also present within the confined area of the Golgi complex (Fig. 2). Each dictyosome contained five cisternae, approximately 2 pm in length, terminating a t each end in a n expanded saccule (Figs. 3-6). Immature cisternae were slightly dilated and devoid of any opaque material, while those on the mature side contained dense material (Figs. 3-6). Golgi saccules were classified as types 1,2, or 3 on the basis of the configuration and state of aggregation of their content. Types 1-3 represented progressive stages in the formation of presecretory granules (Figs. S 7 ) . Golgi saccule type 1 (GS1) was the largest, being some 400-530 nm in diameter, spherical, and characterized by a content of short, loosely arranged filaments 1.0-1.3 nm in thickness (Fig. 7a). GS1 appeared to be extensions of the first and second Golgi cisternae from the immature side, indicating that they were newly formed (Fig. 3). Coated vesicles were noted in contact with GS1 in greater frequency than with any of the other more mature saccules (Figs. 2, 10a, b). Golgi saccules type 2 (GS2) were approximately 280nm in diameter and 500nm in 311 length. Their content consisted of fine filaments arranged parallel to the long axis of the saccule and apparently more densely packed when compared to that of GS1 (Figs. 7b, c). GS2 were continuous with the third or fourth Golgi cisternae (Fig. 4). Coated vesicles were noted to be in continuity with GS2 membranes. Golgi saccule type 3 (GS3)extended from the ends of the most mature Golgi cisternae (Fig. 5). Three major distinguishable subtypes of GS3, representing different maturation stages, were observed. They measured approximately 430-480 nm in length and 150-200 nm in diameter (Figs. 7cLf'). The first type of GS3 was characterized by an elongate shape and the presence of a loose globular aggregate of fine threadlike material at each end (Fig. 7d). Procollagen filaments, in a parallel arrangement, bridged the two globular aggregates (Fig. 7d). A short Golgi cisterna was still present between this type of GS3 (Figs. 4, 5). The second type of GS3 had a more rectangular shape and also contained globular aggregates at each end. The filaments that bridged these aggregates now appeared thicker and more rod-like (Fig. 7e). The third type of GS3 was dumbell-shaped and contained well-organized rod-like structures with globular extensions (Fig. 7D resembling the SLS-like aggregates described by Goldberg ('74) and by Bruns et al. ('79). The average length of these SLS-like crystallites, including their globular extensions, ranged from 354-37Onm. The rod-like structures of the SLS-like crystallites, excluding both globular extensions, measured approximately 312 nm long and 20 nm wide. Cross sections through the rod-like structures of this third type of GS3 revealed eight loosely organized SLS-like crystallites; one in the center encircled by seven a t the periphery (Fig. 7g). Cross sections through the globular extensions revealed a tangled threadlike material with no discernible substructure (Fig. 7h). Presecretory granules (PSG) were approximately 470 nm long and 100 nm wide (Fig. 7i). Although the globular extensions a t each side were still present and recognizable, the identification of individual rod-like structures in a longitudinal profile was no longer possible. The total length of the SLS-like aggregates remained unchanged (Fig. 7i). Cross sections of PSG through their midpoint demonstrated that the rod-like structures were so densely aggregated that the identification of individual SLS-like crystallites was difficult but possible, and that the globular extensions were also more densely packed (Fig. 7j). 312 CHO AND P.R. GARANT FORMATION OF COLLAGEN SECRETORY GRAMJLES Secretory granules (SG) were characterized by further reduction in diameter and increased density of their content. They were 430 nm long and 70nm wide and contained a uniformly dense material, within which it was no longer possible to detect the arrangement of individual SLS-like crystallites. The terminal extensions seemed to be denser, especially in those secretory granules observed near the Golgi complex (Fig. 7k). In cross section, SG showed the presence of densely aggregated fine materials (Fig. 71). Small coated vesicles (100 nm in diameter) were concentrated in the Golgi complex (Fig. 1Oa-c). The coating material consisted of about 12 T-shaped projections attached to the outer unit membrane of the vesicle (Fig. 10c).These vesicles were almost exclusively associated with Golgi saccules 1, 2, and 3, and in lesser numbers with presecretory granules. Apparently, coated vesicles developed from a coated segment of Golgi saccule membrane by bulging into the adjacent cytoplasm until they were eventually detached (Figs. 1Oa-c). None were observed on the RER cisternae, secretory granules, or cell processes. From a n examination of numerous Golgi complexes, it was possible to construct a diagram of the structure of a typical Golgi dictyosome (Fig. 11). Although our concept is presented only as a two-dimensional figure, it is nevertheless of value in depicting the progressive maturation of cisterna and saccules and in demonstrating that the saccules approach each other during cisternal shortening and maturation. Furthermore, two presecretory granules are formed when two GS3 split away from a reduced fifth cisterna. The association of microtubules with the immature and mature faces of the Golgi complex dictyosomes depicted in Figure 11 will be described in greater detail in a subsequent communication. 313 over procollagen molecules, increased with the degree of aggregation of the content of Golgi saccules and secretory granules. The cisternae of the RER, Golgi cisternae, and Golgi saccule types 1 and 2 were relatively negative, while GS3 showed a weak positive reaction over their content of rod-like structures (Fig. 8). A more intense staining was observed in presecretory granules (Fig. 81, and a very intense reaction was observed over secretory granules (Fig. 8). In general, the staining reaction with phosphotungstic acid was more intense but similar to that obtained with silver methenamine. GS1 and GS2 were poorly stained except along their periphery, while GS3, PSG, and SG demonstrated increasingly intense reactions (Fig. 9). DISCUSSION The observed ultrastructural changes in fibroblast Golgi components and additional evidence obtained from electron microscopic autoradiographic studies of the time course of 3Hproline utilization (manuscript in preparation) demonstrates that individual Golgi cisternae and their associated saccules progress through a series of maturational events, occurring over approximately 20 min, during which time the cisternae are shortened and moved from the immature to mature face of individual dictyosomes. The maturation process also involves aggregation of procollagen molecules and a decrease in the size of the saccules. Removal of excess membrane appears to be accomplished by coated vesicle formation, a s previously suggested by Weinstock and Leblond ('74) in their study of collagen secretion by odontoblasts. A possible role for coated vesicles in cell membrane recycling has been previously proposed (Farquhar, '78; Heuser and Reese, '73; Pearse, '76). Previous studies have indicated that the triple helix conformationof procollagen molecules occurs in the RER (Brownell and Veis, '76; Hanvood et al., '73). Although theoretically Cytochemical studies possible, individual pro a chains have yet to be Silver methenamine. The intensity of silver visualized during triple helix formation in the methenamine staining, representing the pre- electron microscope. Nevertheless, the loosely cipitation of electron-opaque metallic silver and apparently randomly distributed fil- Fig. l. High-power light micrograph of PDL fibroblasts. The cells are elongate and polarized. Note large juxtanuclear clear zone caused by lighter-staining Golgi complex X 750. Fig. 2. Juxtanuclear cytoplasm of PDL fibroblast depicting large &I@ complex containing several stacks of Golgi cisternae (dictyosomes),a variety of Golgi saccules (GSI, GS2, and GS3),presecretory granules (PSG), and secretory granules (SG).Profiles of rough endoplasmic reticulum (rER), mitochondria(MI, and microtubules(Mt) are also present. C, centriole; N, nucleus. X 22,000. 314 M.-I. CHO AND P.R. GARANT FORMATION OF COLLAGEN SECRETORY GRANULES amentous material contained within GS1 are believed to be completed procollagen molecules. The progressive rearrangement of procollagen molecules into a parallel configuration within GS2 may represent the formation of intermolecular or covalent crosslinkages between individual procollagen molecules. This would be in agreement with the biochemical observation that glycosylation of hydroxylysyl residues by collagen UDP-galactosyl and UDP-glycosyl transferases occurs in the RER and Golgi apparatus (Harwood et al., '74). The removal of water, as suggested for condensing vacuoles of pancreatic acinar cells (Jamieson and Palade, '671, may help to mediate this alignment process. The well-organizedSLS-like aggregates seen in this study are similar in shape and size to those found in fibroblast culture media (Bruns et al., '79) and homogenates of tendon fibroblasts (Goldberg, '74). These were seen only in the most mature type of GS3 prior to their separation from the shortened cisternal membrane to form presecretory granules. The globular extensions a t both ends of the parallel procollagen molecules aggear to signify changes within or interactions between the non-helical portions of the procollagen molecules. Globular extensions are first observed in the early phase of GS3 formation, suggesting that they may have a role in initiating the formation of the individual SLS-like crystallites. 315 The observation that GS3 contain eight loosely arranged SLS-like crystallites and that they become more tightly organized within presecretory and secretory granules suggests that the SLS-like crystallites may undergo further modification prior to secretion. The progressive staining of more mature granules by the silver methenamine technique, confirming what was previously reported in odontoblasts (Weinstock and Leblond, '74), suggests a n increased availability of aldehyde groups, probably generated during crosslinkaging of procollagen molecules. In addition, the intense staining reaction with both silver methenamine and phosphotungstic acid, first noted in GS3, indicates that the major glycosylation of the procollagen molecule occurs a t that stage of development. The aggregative process that procollagen molecules undergo during maturation of Golgi saccules and secretory granules is especially interesting in terms of a possible relationship to fibrillogenesis. Our results suggest that the procollagen aggregate present within the secretory granule is an intermediate form between individual dispersed procollagen molecules and the polymerized fibril. In a similar vein, Bruns et al. ('79) suggested that SLSlike aggregates obtained from both tissue homogenates and culture media may be secreted into the extracellular compartment and assembled in linear arrays after removal of the non-helical extensions. Since the condensed Figs. %6. Golgi cisternae and their associated saccules. Fig. 3. Golgi saccule type 1 (GS1) characterized by its nearly spherical shape and loosely organized filamentous content. Portions of four Golgi cisternae (1-4)can he noted. GS1 appears continuous with cisterna 2. X 38,000. Fig. 4. Golgi saccule type 2 (GS2) is continuous with Golgi cisterna 4,which is still quite long in comparison with Golgi cisterna 5, connecting Golgi saccules type 3 C). Above Golgi cisternae 1 is an intracellular collagen fibril (ICC). x 44,000. Fig. 5. Two Golgi saccules type 3 (GS3) (*)nolonger demonstrate the presence of a Golgi cisternae between the two saccules. The arrow from 5 indicates the presumptive location of the fifth cisterna. Also, two dense areas at both ends of one Golgi saccule type 3 (GS3)can be seen (arrowheads). M, mitochondrion. x 51,000. Inset: Golgi saccules type 3 demonstrating dense aggregates at both ends (arrowheads). The thick filamentous material (*) connects the two dense areas. X 50,000. Fig. 6. Two presecrebry granules (PSG) appear as the first procollagen-containingstructures to become separated from the dictyosome at a position occupied by the fifth cisterna (5).Presecretory granules show a somewhat similar morphology to Golgi saccule type 3 seen in inset of Figure 5. However, the inner materials are more condensed in the PSG. The dense polar aggregates are clearly visible in both PSG and secretory granule (SG)(arrowheads). Mt, microtubule X 74,000. 316 M.-I. CHO AND P.R. GARANT FORMATION OF COLLAGEN SECRETORY GRANULES Fig. 7. Changes in shape, size, and contents of Golgi sacculesduring secretorygranule formation. x 80,000. a) Golgi saccule type 1(GS1)is characterized by its large, spherical shape and its content of loosely and randomly distributed short filamentous materials. b) Golgi saccule type 2 (GS2) is ellipsoidal in shape, and its contents of long filamentous materials are organized parallel to the long axis of the saccule. c) More advanced GS2 demonstrating a denser arrangement of its parallel filaments. d) Golgi saccule type 3 (GS3) at its earliest stage of development, demonstrating a rectangular shape and a smaller size compared with GS2. The content begins to show condensation at both ends (short arrow), connected by thickened filamentous structures (arrowheads). e) GS3 at a more advanced stage, demonstrating the presence of SLS-likecrystallites (arrowheads) extending between the condensed materials at both ends (short arrows). 0 GS3 at its most advanced stage is characterized by the presence of densely organized SLS-like crystallites (arrowheads) as well as condensed polar aggregates (short arrows). The individual unit composed of a long rod and two globular structures resembles the typical SLS-likeaggregate reported by Bruns et al. (‘79). g) Cross section of GS3 at its most advanced stage, cut at the level of the long arrow with circle seen in Figure 7f. This micrograph demonstrates that the GS3 contains seven individual SLS-like crystallites (arrowhead) at its periphery and one in its center. h) Cross section of GS3 at its most advanced stage, cut at the level of the long arrow seen in Figure 7f, showing filamentous nature of the condensed polar aggregate. i) Presecretory granule (PSG)is an elongate structure with a much smaller diameter in comparison with GS3. They have a much denser content in both the areas of the connectingrods and the globular extensions (short arrows). Identification of individual SLS-like crystallites is difficult. j) Cross section of presecretory granules. This micrograph shows two cross-sectioned presecretory granules, one cut at the level of the arrow with circle (dark arrow) and the second cut at the level of the long arrow (open arrow) seen in Figure 7i. Individual rod shapes are sometimes still observed (arrowhead). The cross section cut through the polar aggregate (open arrow) also demonstrates a much denser packing of the filamentous materials. k) Mature secretory granule (SG) characterized by an elongate shape and content of much denser material. Greater density of materials at both ends is still present (short arrows). 1) Cross section of a secretory granule demonstrating its densely packed collagen precursors. 317 318 M.-I. CHO AND P.R. GARANT Fig. 8. Golgi area stained with phosphotungstic acid. Golgi saccule type 1 (GSlt are weakly stained except for a slight increase in reaction product adjacent tothe inner surface of their limitingmembrane. Golgi saccules types 2 (GSZ)and 3 1GS3) show a stronger staining reaction, which coincideswith their greater maturity. Seeretory granules (SG) are darkly stained. Mitochondria (M) and RER (rER) remain free from staining. x 33,000. Fig. 9. Golgi area stained with silver methenamine. Secretory granules (SG)are heavily stained, while Golgi sacculestype 3 (GS3)show a weak reaction. Golgi saccules types 1 and 2, mitochondria (MI, and rough endoplasmic reticulum (rER) demonstrate a negative reaction. x 36,000. FORMATION OF COLLAGEN SECRETORY GRANULES 319 m Fig. 10. Formation of coated vesicles on Golgi saccules. a) Formation of coated vesicles begins with the thickening of a portion of the Golgi saccule membrane. x 84,000. b) Thickened portion of Golgi saccule membrane bulges out in the form of a future coated vesicle (*I. x 71,000. c) Finally, coated vesicle (arrowhead) is detached from the surface membrane. x 78,000. Inset: High-magnificationmicrograph showingthat about 12 T-shaped structures (arrowheads) are involved in forming the coat of these vesicles along their mid-plane of section. x 94,000. Fig. 11. Schematicview of a Golgi apparatus demonstrating the presumptive sequence of events involved in the formation of secretory granules. Newly synthesized collagen precursors are transported to the Golgi either by way of transitional elements (TE) or intermediate vesicles (IV). These form new Golgi cisternae, probably by fusion. Microtubules(*MT)at the immature side of Golgi apparatus are probably involved in transporting intermediate vesicles(IV) and other elongate vesicles (EV)to the Golgi. Collagen precursors are subsequentlycollected in Golgi sacculestype 1 (a, b connected with Golgi cisternae 1and 2, respectively),where they appear as randomly distributed short filamentous structures. Subsequently,they appear as long thread-like structures parallel to the long axis of Golgi saccules type 2 (a, b: connected with Golgi cisternae 3 and 4, respectively). These saccules become shorter and narrower by removal of surface membrane through formation of coated vesicles(CV). SLS-like aggregates are formed after the appearance of globular extensions (open arrows)in Golgi sacculestype 3, which are connected by a short Golgi cisterna (5).Two presecretory granules (4) with more highly condensedprecursors are formed after separation at their midpoint. Secretorygranules (5) are then transported by microtubules ( MT) at the mature side of Golgi apparatus. Bridge-like structures (dark arrows) connect secretory granules to microtubules. M.-I. CHO AND P.R. GARANT 320 mature secretory granule contains a dense core of secretory material that is of the Same diameteras the finally polymerized collagenous fibril, We Suggest that polymerization may require only slight lateral shifting of adjacent collagen molecules once the terminal peptides have been cleaved. These results indicate that the size and content of collagen secretion granules of PDL fibroblasts are determined by the assembly of a basic supramolecular unit ofprocollagen.Thus, the uniform diameter Of the great majority of collagenous fibrils in the PDL may be a determinant of cell function rather than due t , con~ ditions of the extracellular environment. LITERATURE CITED Bornstein, P., H.P. Ehrlich, and A.W. Wyke (1972) Procollagen: Conversion of the precursor to collagen by a neutral protease. Science, 175544546. Brownell, A.G., and A. Veis (1976) Intracellular location of triple helix formation of collagen. J. Biol. Chem., 251 :7137-7143. Bruns, R.R., D.J.S. Hulmes, S.F. Therrien, and J. Gross (1979) Procollagen segment-long-spacing crystallites: Their role in collagen fibrillogenesis. Proc. Nat. Acad. Sci. USA, 76:31%317. Ehrlich, H.P., R. Ross, and P. Bornstein (1974) Effect of antimicrotubular agents on the secretion of collagen. A biochemical and morphological study. J. 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