Download Binding of Soluble Type I Collagen to Fibroblasts

Published December 1, 1982
Binding of Soluble Type I Collagen to Fibroblasts:
Specificities for Native Collagen Types, Triple Helical
Structure, Telopeptides, Propeptides, and Cyanogen
Bromide-derived Peptides
BURTON D. GOLDBERG and ROBERT E. BURGESON
Department of Pathology, New York University Medical Center, New York, New York 1001@and
Department of Pediatrics, Los Angeles County Harbor-- University of California Los Angeles Medical
Center, Torrance, California 90509
The binding of type I collagen to fibroblasts is considered to
be specific because the radiolabeled ligand is readily displaced
by unlabeled type I collagen but not by noncollagenous proteins, nor by every molecule containing some collagen structure
(1). In the present study, binding inhibition measurements have
further defined ligand specificities with respect to collagen
types, native triple helical assembly, and regions of procollagen
and collagen chains that contain binding determinants.
MATERIALS AND METHODS
Binding I n h i b i t i o n Assays
Swiss mouse 3T3 fibroblast cultures were used in standard binding assays as
described (1, 2). Unlabeled lathyritic rat skin (LRSC) and other unlabeled
collagenous proteins were dissolved in 0.05 M Tris HCI-0.15 M NaCI pH 7.5
buffer at 1-2 mg/ml. Protein concentrations of the stock solutions were computed
from the difference in absorbances at 215 and 225 nm of appropriate dilutions of
the stock (3). For each experiment, increasing amounts (0-100 #g) of unlabeled
LRSC and of putative binding proteins were each premixed with a fixed amount
of ~zr'I-LRSC (~ 130,000 cpm) in 2 ml of binding buffer (Dulbecco's modification
of Eagle's medium with half bicarbonate concentration, 0.05 M HEPES, pH 7.2
and 2.5 mg/ml bovine serum albumin). The solutions were warmed at 37°C for
30 min, cooled to room temperature (26°C) and added to washed, replicate
confluent cultures of 3T3 fibroblasts. After a 2-h incubation at 26°C, the cell
layers were rapidly washed and lysed, and the radioactivity specifically bound to
the ceils was measured. Percentage inhibition of binding at each protein concentration was computed by:
1
c
cpm bound with unlabeled protein ~ ×
~
~
protein,/
102
Limited Digestion o f LRSC with Pepsin and
a-Chymotrypsin
Pepsin was purchased from Sigma Chemical Co., St. Louis, MO and achymotrypsin from Worthington Biochemical Corp., Freehold, NJ. Pepsin digestion was done according to the procedures of Leibovich and Weiss (4) and
Helseth and Veis (5). Digestion with a-chymotrypsin was done by the procedure
of Bornstein et al. (6). Control samples without enzyme additions were carried
through all the procedures. Samples were finally dissolved in Tris buffer and
tested as inhibitors of binding as described above.
SDS PAGE
Samples were dissolved in sample buffer containing 2% SDS, 10% glycerol.
0.001% bromphenol blue in 0.0625 M Tris HCI, pH 6.8. After heating at 100°C
TttE JOURNAL OI C E i l BrOLOGY • VOLUME 95 DECEMBER 1982 7 5 2 - 7 5 6
752
© The Rockefeller University Press • (X~21-9525/82/12/0752/05 $100
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ABSTRACT Unlabeled collagenous proteins were quantified as inhibitors of binding of native,
soluble, radioiodinated type I collagen to the fibroblast surface. Collagen types IV, V, a minor
cartilage isotype (1~2c~3c~), and the collagenlike tail of acetylcholinesterase did not inhibit
binding. Collagen types II and III behaved as competitive inhibitors of type I binding.
Denaturation of native collagenous molecules exposed cryptic inhibitory determinants in the
separated constituent ~ chains. Inhibition of binding by unlabeled type I collagen was not
changed by enzymatic removal of the telopeptides. Inhibitory determinants were detected in
cyanogen bromide-derived peptides from various regions of helical ~1(I) and al(lll) chains.
The aminoterminal propeptide of chick procd(I) was inhibitory for binding, whereas the
carboxyterminal three-chain propeptide fragment of human type I procollagen was not. The
data are discussed in terms of the proposal that binding to surface receptors initiates the
assembly of periodic collagen fibrils in vivo.
Published December 1, 1982
for 2 min, the samples were applied to a 3% stacking/6%separating slab gel (7)
and, after development,the gels were stained with Coomassie Blue.
Materials
Collagenousproteinswere generouslydonated by the followinginvestigators:
Dr. Terrone L. Rosenberry,Case Western Reserve University,Cleveland,OH,
the collagenliketail of acetylcholinesterase;Dr. Jerome Seyer, Veterans Administration MedicalCenter, Memphis,TN, cyanogenbromidepeptidesof collagen;
Dr. George Martin,National Instituteof Dental Research. Bethesda, MD, LRSC
and rodent collagen types 11 and IV; Dr. Bjrrn R. Olsen, Department of
Biochemistry,Collegeof Medicineand Dentistryof New Jersey-RutgersMedical
School, Piscataway,NJ, the aminoterminalpropeptide of chick pro a l(I).
The syntheticpeptidesDNP-Pro-GIn-GIy-Ile-AIa-GIy-GIn-D-Arg
and DNPPro-Leu-Gly-lle-Ala-Gly-Arg-NH2were purchased from the Peptide Institute,
Protein Research Foundation, Osaka, Japan.
RESULTS
Specificities: Collagen Structure, Collagen Types,
and Native Helicity
100
~
g6o
if_
4o
z
~
2O
=.c_o.~-----,~
,
5
10
15
/ig PROTEIN
q
20
1(30
80
g 6c
m_ 4(
g
o
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0--0
5
10
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~g PROTEIN
20
F~GURE 1 Acetylcholinesterase as a
competitor of 12SI-LRSC binding. Increasing amounts of unlabeled LRSC
and the collagenlike tail of acetylcholinesterase were each mixed with 1.36
× 105 cpm of ~251-LRSC in binding
buffer. Binding studies were performed in replicate cultures and the
percentage inhibitions produced by
the unlabeled proteins were computed. @, LRSC. O, acetylcholinesterase.
FIGURE 2 Native collagen types
as inhibitors of '251-LRSC binding.
Composite of binding inhibition
experiments performed with the
following native collagen types:
type I, LRSC; type II from a rat
chondrosarcorna; type III, from
human placenta; type IV, from the
mouse EHS sarcoma; la2a3a cartilage isotype, from human cartilage, and type V, from human placenta.
2
0
~
t
,
Q
~
FIGURE 3 Type II inhibition: scatchard plot. In15
creasing amounts (0-100
o
"
/~g) of unlabeled LRSC
were premixed with a fixed
amount of '2%/RSC (0.16
5[
~
pmol, 118,000 cpm) in the
L
J
,
presence (O) and absence
0
0.5 to
1.5 2.0 2.5 3.0 (O) of 5 #g (17.5 pmol) of
B (PICOMOLES)
unlabeled type II collagen.
The respective mixtures were presented to replicate plates of 3T3
fibroblasts and specific binding measured after 2.5 h at 26°C. Picomoles of type I collagen (radiolabeled and unlabeled) bound (B)
and free (F) were calculated.
0
3O
"&
2O
I
0
I 040
2
I
60
L (PtCOMOLES)
I
80
FIGURE 4 Type ll i n h i b i t i o n : Hanes-Woolf plot. Data from experiment of Fig. 3. (L) Picomoles of type I collagen ligand (labeled and
unlabeled) added. (B) Picomoles of type I collagen ligand (labeled
and unlabeled) bound. (O), 5/xg (17.5 pmol) of unlabeled type II
collagen added as inhibitor. (z~), in absence of type II inhibitor.
GOLDBERG AND 6URGESON
Specificities for Collagen Binding
753
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Potential competitors for 12~I-LRSC binding sites were compared to the unlabeled LRSC in inhibition assays as described
in Materials and Methods. That general collagen structure does
not ensure binding to or blocking of type I sites is shown in
Fig. 1. In this instance the putative competitor was the collagenlike tail of acetylcholinesterase (8, 9). Additions of up to 20
t~g of this unlabeled protein did not significantly inhibit binding
of the radioactive ligand, whereas 20/xg of unlabeled LRSC
produced 78% inhibition of binding. C l q , the complement
component containing some collagen structure, also fails to
inhibit type I binding (1).
Native Collagen types II, III, IV, V, and a minor isotype
present in cartilage but distinct from type II collagen (10) were
tested in binding inhibition assays (Fig. 2). These data show
that unlabeled type II and type I collagens cause equivalent
inhibition whereas unlabeled type III collagen is a somewhat
less efficient inhibitor of binding. The much lower levels of
inhibition produced by the other collagen types are considered
to be of doubtful significance.
Graphical analyses of binding data were performed to determine whether collagen types II and III acted as competitive
or noncompetitive inhibitors of type I binding. Fig. 3 presents
Scatchard plots of type I binding with and without the addition
of 5 #g (17.5 pmol) of type II collagen. Calculation of the
slopes showed that inhibition was accompanied by an increase
in the binding dissociation constant (KD) from 7.5 X 10-9 M to
1.7 x 10-8 M. The common abscissal intercept for the two lines
indicates that the n u m b e r of cell binding sites was not changed
by the inhibition. Fig. 4 is a Hanes-Woolf plot (11, 12) of the
same data and it, like the Scatchard plot, is typical for a form
of competitive inhibition. Qualitatively similar plots were obtained for inhibition by type III collagen.
The collagen types tested in the experiments of Fig. 2 were
native triple helical molecules. It is of interest to know whether
native helicity critically determines binding specificity. If triple
helicity is critical for binding, the separated (denatured) a
chains should yield different binding inhibition data than the
assembled parent molecules. In the experiments presented in
Fig. 5, the three denatured a chains from the minor cartilage
isotype and the denatured a 1 and a2 chains of type V collagen
were individually tested in b i n d i n g inhibition assays. Whereas
the parent triple helical molecules did not inhibit binding (see
Fig. 2), each chain class of the cartilage isotype and the a2(V)
chain gave equivalent or significant inhibition relative to the
LRSC standard. Denatured reduced and alkylated type III
chains were better inhibitors of type I binding than native III,
and nondenatured reduced and alkylated type IV collagen did
not inhibit binding (data not shown). We conclude that triple
helicity can confer binding specificity and that denaturation of
a chains can expose additional binding determinants.
Published December 1, 1982
Are Telopeptides Required for Binding
Potential Binding Determinants in Cyanogen
Bromide-derived Helical Peptides
Peptides generated by cyanogen bromide cleavage of helical
I0(]
I
g~ oc
2a
g6c
aIci
. . . . • a2(~)
4c
2C
/
oi-
_~o.-.o""
I
5
I
I
[
i0
15
20
#g PROTEIN
FIGURE 5 Denatured (x chains as
competitors of '2SI-LRSC binding.
Cartilage isotype chains l(x, 2a,
and 3~x and a l and c~2 chains of
type V collagen were resolved I~y
carboxymethyl cellulose chromatography under denaturing conditions (13). The individual chain
classes and native LRSC (I) were
tested as inhibitors of '251-LRSC
binding.
~ Bc
5
10
15
20
25
#g PROTEIN
al(1) RAT
NI I I I
B
0.J6
754
THE JOURNAL OF CELL BIOLOGY,VOLUME 95, 1982
I
3
0.56
at(I} HUMAN NlZl i I
FIGURE 6 Gel electrophoresis of LRSC and
chymotrypsin-modified LRSC. Lane (A):
LRSC digested with chymotrypsin at pH 7.5,
15°C, 24 h, at enzyme/substrate ratio of 1/
10. Reaction stopped by cooling to 4°C,
addition of protease inhibitors and acidification. Collagen separated by three cycles
of precipitation with NaCI to 7.5%. Lane (B):
Control LRSC. Same protocol, enzyme omitted. 5 p,g of collagen applied to both lanes.
FIGURE 7 Binding inhibition by
LRSC and chymotrypsin-modified
LRSC. Samples of Fig. 6 used as
inhibitors of 12%LRSC binding.
Chymotrypsin-modified
LRSC
(O) and control LRSC (O), activated at 37°C, 30 min. Chymotrypsin-modified LRSC ([7) and
control LRSC (IlL w i t h o u t thermal
activation.
100
_g 6c
~ 4c
~_
20
0°
(]1 (I) CHICK
a
al(~)
HUMAN
N
I
7
I
c
t.O
3
l
i
I
6
I
I 6A I 6B c
0.50
0.05
3
I I 6 I I
8
0.039
0.27
0
I
I
4
0
c
0.4@
O. f8
S
O. f t
t
0
O.OG?
N I I4 I I
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The telopeptides are short (10-25 residues) nonhelical extensions at the ends of the collagen a chains. Collagen molecules
in solution can self-assemble to form native fibrils (14) and the
initiation ("nucleation") of such self-assembly seems to depend
upon a thermally induced conformational change in the amino
terminal telopeptides (5). Collagen binding to the fibroblast
also requires thermal activation of the ligand, and such binding
has been proposed as an initiating event for fibrillogenesis (1,
2). Accordingly, experiments were performed to determine
whether the telopeptides were necessary for binding of collagen
to the fibroblast. The tetopeptides were first removed from
unlabeled LRSC by limited digestion with a-chymotrypsin or
pepsin. Fig. 6 shows the SDS-polyacrylamide gel patterns given
by a-chymotrypsin-digested LRSC and the control preparation. The enzyme-digested collagen contains somewhat fewer
covalently cross-linked molecules (y and fl forms) and has a
major population of al and a2 chains with slightly greater
mobilities than the control preparation. This result shows that
the enzyme digested the telopeptides and did not alter the triple
helix in most of the molecules. Binding inhibition assays were
performed with enzyme-digested and control LRSC, with and
without prior thermal activation (37°C, 30 min) of the samples.
Fig. 7 shows that, with heat activation, molecules lacking
telopeptides bound as well as intact collagen. In the absence of
heat activation, neither preparation displayed significant binding. The latter result shows that the requirement for heat
activation is independent of radioiodination of the ligand.
Similar data were obtained when the telopeptides were removed by limited digestion with pepsin. We conclude that the
telopeptides are not necessary for thermal activation and binding of type I collagen to fibroblasts.
regions o f a l type I and III chains were evaluated as inhibitors
of type I binding. Fig. 8 depicts the positions in the a chains of
the peptides tested and quantifies inhibition by each peptide
relative to the type I LRSC standard. The values given below
the lines are the ratio of LRSC (#g)/peptide ~g) which produced 30% inhibition of t25I-LRSC binding in the same experiment. For all of the experiments, 1-2/~g of LRSC regularly
produced 30% inhibition of binding. Inhibition ratios <0.1 are
not considered as significant, and a ratio value of zero means
that addition of 100 ~g of the peptide produced <30% inhibition of binding.
By the test of inhibition, binding determinants were identified in more than one peptide in each species and type of a l
chain, and these peptides were variably distributed along the
chains from amino to carboxy termini. Peptides of similar size
from the same regions of type I a chains of different species
varied in binding capacities, for example, human a l(I)-CB8 as
compared to the homologous peptides of rat and chick.
The rodent a I(I)-CB7 peptide which includes residues 552822 of the al chain is notable for being equivalent on a
concentration basis to LRSC as an inhibitor of binding. The
same peptide has been reported to contain the determinants
for attachment of fibronectin to ctl (I) but the following observations indicate that ~zSI-LRSC is not binding to fibronectin
on the fibroblast surface (a) aI(I)-CB7 has been claimed to
contain the sole binding site for fibronectin (15), but the data
of Fig. 8 show that peptides positioned elsewhere in al(I)
chains can significantly inhibit binding of ~25I-LRSC. (b) Critical determinants for binding of a I(I)-CB7 to fibronectin have
been localized to the cleavage site for animal collagenase
(residues 775-776) and a chymotrypsin-sensitive site (residues
779-780) (15). However, two synthetic peptides which have
identical sequences to residues 773-780 and 775-778 of al(I),
respectively, and which serve as substrates for animal collagenase (16), did not inhibit x2~I-LRSC binding (binding inhibition
I
~
O. 10
I
0.67
9
C
0.54
FIGURE 8 Cyanogen bromide-derived helical peptides as inhibitors
of binding. Linear schematic of helical a chains of coltagen types I
and III indicating positions, standard numbering (above lines), and
relative sizes of peptides produced by cyanogen bromide cleavages.
Only the peptides tested in binding assaysare numbered. Numbers
below lines are the ratio of p.g LRSC//Lg peptide that produced 30%
inhibition of 1251-LRSC binding. N and C, amino and carboxytermini
of the chains, respectively.
Published December 1, 1982
ratios of zero, calculated as for experiments of Fig. 8). Moreover, human cd(III)-CB5, which also contains the coUagenase
cleavage site, produced minimal inhibition of binding (see Fig.
8).
Potential Binding Determinants in Propeptides
Collagen is secreted as a precursor molecule (procollagen)
that has large polypeptides (propeptides) covalently linked to
the terminal telopeptides of the three helical ot chains of
collagen. Type I procollagen could bind to the cell surface
through the demonstrated determinants in its central triple
helical region but it is possible that propeptide residues could
also contribute to procollagen binding. A reasonable candidate
for a propeptide binding region is the short, collagenlike segment ("Col 3") in the aminoterminal propeptides of type I
procollagen (see reference 17 for review). When the aminopropeptide isolated from the chick pro al(I) chain (18) was tested
as an inhibitor of 125I-LRSC binding, it gave a binding inhibition ratio of 0.19, indicating some affmity for the cell binding
site.
The carboxyterminal propeptides of type I procoUagen do
not contain any collagenlike structure and they failed to inhibit
125I-LRSC binding in the standard assay system. The protein
tested was a three-chain, disulfide-linked fragment of 100,000
daltons isolated from human type I procollagen (19).
Quantitative inhibition assays have further defined the specificity of binding of native, soluble radioiodinated type I collagen to the fibroblast surface. The binding is not inhibited by
every collagenous molecule; thus, the collagenlike tail of acetylcholinesterase and native collagen types IV, V and cartilage
lct2~t3a do not inhibit binding. Only native collagen types II
and III inhibit type I binding, and graphical analyses indicate
that they act as competitive inhibitors. These inhibitions can
be ascribed to interactions with type I cell binding sites or to
complexes formed directly with type I collagen. Radioiodinated
type II collagen binds to the fibroblast (unpublished data), so
there is independent evidence for a cell binding site for this
collagen type. In terms of receptor models, competitive inhibition can mean that collagens I, II, and III bind to the same
cell receptor site, to different sites that interact allosterically, or
to both shared and independent sites. Interactions of collagens
I, II, and III with a common receptor on the fibroblast can be
interpreted in the following context. Collagen types I and III
are synthesized by the fibroblast and the mesenchymal precursor of the differentiated chondrocyte. Although the latter cell
type synthesizes only type II collagen, it is prone to dedifferentiate and to switch to the synthesis of collagen types I and
III (20). The data suggest, therefore, that the same cell binding
complex is used in the course of these phenotype conversions.
Previously published and less complete binding inhibition
data supported the view that the primary structure of collagen
a chains determined the binding specificity of the assembled
molecule (1). We currently report that c~chains separated from
collagen types V and cartilage la2a3a inhibit type I binding
but that the triple helical molecules formed from such chains
are not inhibitory in the assays. We now conclude, therefore,
that tertiary or quartenary molecular structure can critically
modify the binding specificities of the collagens.
Enzymatic removal of telopeptides from native unlabeled
type I collagen did not alter its capacity for inhibiting 125ILRSC binding nor obviate the requirement for thermal acti-
We thank Sheila Heitner and Josa Williamson for their expert assistance.
This study was supported by the National Institutes of Health grant
HL 17551 and the Bear Foundation.
Received for publication 26 April 1982, and in revised form 10 August
198Z
REFERENCES
I. Goldberg, B. 1979. Binding of soluble type I collagen molecules to the fibroblast plasma
membrane. Cell. 16:265-275.
2. Goldberg, B. D. 1982. Binding of soluble type I collagen to fibroblasts: effects of thermal
activation of ligand, ligand concentration, pinocytosis, and cytoskeletal modifiers. J. Cell
BioL95:747-751.
GOLDBERG AND BURGESON Speoficiti~s for Collagen Binding
755
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DISCUSSION
vation of the ligand.
Potential binding sites in helical al(I) and al(III) chains
from different species were partially mapped by testing a
limited number of cyanogen bromide-derived peptides as inhibitors. Inhibitory and noninhibitory peptides were thus identified, and inhibitory activity was shown to be subject to species
variation. As all of the peptides shared the repeating collagen
triplet Gly-X-Y and were not much different in their content
of proline and hydroxyproline, these elements of collagen
primary structure must be insufficient as determinants of binding specificity. Every al(I) and txl(III) chain was shown to
contain two or more inhibitory peptides and, considered as a
group, such peptides were variously positioned in the chains.
It seems reasonable to suppose that some or all of these regions
contribute to binding determinants expressed by the assembled
triple helical molecules.
Fibronectin is a reasonable candidate as the molecule responsible for the binding of collagens to the fibroblast surface.
However, the ligand-binding specificities described above do
not correspond to those reported for collagen-fibronectin interactions. In contrast to our data, fibronectin binds to the collagenlike tail of acetylcholinesterase (21), has different binding
affinities for the various collagen types (22-24), and has been
reported to bind exclusively to a single region of the td(I)
chain (15). Because of these discordancies and other data (1),
we do not favor the view that fibronectin is responsible for the
binding of native, soluble x2SI-LRSC to the fibroblast surface.
The reported data are interpreted to mean that ligand determinants distributed over the central collagen triple helix are
sufficient to bind the interstitial procollagens and collagens
(types I, II and III) to the fibroblast plasma membrane. Such
binding is proposed as an in vivo mechanism for initiating
fibril assembly and for specifying the spatial order of that
assembly (1, 2). The essential element of the hypothesis is that
binding to a membrane component is determinate for fibrillogenesis, and so the model can include those cases where
procollagen fibrils apparently assemble within intraceUular
vacuoles (25, 26). The binding model allows for modulations
of fibril assembly by cell receptor metabolism as well as by
ligand-ligand interactions and, if there are independent binding
sites for each collagen type, the model provides a mechanism
for segregation of type-specific fibrils. The model assumes that,
upon achieving a limiting size, the soluble surface-bound aggregates detach from the cell and that they then combine to
form insoluble fibrils. Perhaps it is at this later stage of fibrillogenesis that collagen-fibronectin and proteoglycan interactions become important. The binding model as formulated is
quite compatible with proposals by others that type I collagen
fibrils form by stepwise additions of molecular aggregates (see
reference 27, for review).
Published December 1, 1982
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15. Kleinman, H. K., E. B. MeGoodwin, G. R. Martin, R. J. Klebe, P P. Fietzek, and D. E.
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16. Masui+ Y., T. Takemoto, S. Sakakibara, H. Hori. and Y. NagaL I977. Synthetic substrates
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c,l(1) chains from embryonic chick tendon procollagen. Biochemisto,. 19:2447-2454.
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20. yon der Mark, K. 1980. Immunological studies on collagen type transition in chondrogenesis. Cur. Top. Dey. BioL 14:t99-225.
21+ Emmerling, M. R., C. D. Johnson, D. F. Mosher, B. H Lipton. and J. E. Lilien 1981
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22. Dessau, W., B. C. Adelmann. R. Timpl, and G. R. Marlin. 1978. Identification of the sites
in collagen a-chains that bind serum anti-gelatin factor (cold insoluble globulin) Biochem.
J. 169:55 59.
23. Jilek, F., and H. Hormann. 1978. Cold-insoluble globulin (fibronectin), affinity to soluble
collagen of various types. Hoppe-Seyler's Z. Physiol. Chem. 359:247-250.
24. Engvall, E., E. Ruoslahti, and E. J. Miller. 1978. Affinity of fibronectln to collagens of
different genetic types and to fibrinogen. J. Exp. Meal 147:1584-1595.
25+ Trelstad, R. L. 1971. Vacuoles in the embryonic chick corneal epithelium, an epithelium
which produces collagen. J. Cell BioL 48:689~694.
26. Weinstock, M.. and C. P. LeBlond 1974. Synthesis, migration, and release of precursor
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THE JOURNAL OF CELL BIOLOGY • VOLUME 95, 1982