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Sequential events in the formation of collagen secretion granules with special reference to the development of segment-long-spacing-like aggregates.

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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.
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Bruns, R.R., D.J.S. Hulmes, S.F. Therrien, and J. Gross
(1979) Procollagen segment-long-spacing crystallites:
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Goldberg, B. (1974) Electron microscopic studies of procollagen from cultured human fibroblasts. Cell, 1 :165-191.
Harwood, R., M.E. Grant, and D.S. Jackson (1973) The subcellular localization of interchain disulfide bond formation
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Hanvood, R., M.E. Grant, and D.S. Jackson (1974)Secretion
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cells. Biochim. Biophys. Res. Comm., 59:947.
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development, formation, long, granules, references, spacing, sequential, segmento, special, event, like, aggregates, secretion, collagen
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