Collagen Symposium， VII (1968)

Collagen and Elastin Fibrogenesis and the Fiber Forming Cell

Russell Ross

(Departments of Pathology and Oral Biology， Schools of Medicine and Dentistry， University of Washington， Seatt1e， Washington 98105， U. S. A. )

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Collagen forming cells from a number of different systems wi11 be discussed

in this report. These cells inc1ude fibroblasts from a number of connective tissues

inc1uding healing skin wounds， developing fetal bovine ligamentum nuchae， deve-loping embryonic and fetal digital flexor tendon， tissue culture， cartilage， bone，

and forming dentine. All of these cells have a simi1ar development of two of

their organelle systems， i. e.， a highly developed rough endoplasmic reticulum

and Golgi complex1，2，3l. Prior to differentiation， embryonic mesenchymal cells

contain numerous free cytoplasmic aggregates of ribosomes. During differentia-

tion， the ribosomes become attached to the membranes of the rough endoplasmic

reticulum and by the time the cells have matured to fibroblasts， osteoblasts， odontoblasts， etc.， most of the ribosomes are attached to rough endoplasmic

reticulum membrane (Figs. 1， 2， 3).

The fibroblast has been the most thoroughly investigated of these cells. In

addition 10 the extensive rough endoplasmic reticulum and Golgi complex， fibro・blasts can be recognized by the presence of aggregates of fine filaments within

the cel1 cortex (Fig. 1). Numerous regions where the rough endoplasmic reticulum

cisternae are free of attached ribosomes and the cisternal membranes approximate， and in some instances appear to merge with the plasma membrane， are also evident. Many small vesic1es and caveoli line the surface of the cell and numerous

mitochondria can also be seen (Figs. 1， 2， 3).

An additional observation of interest can be seen during the development of

many embryonic and fetal structures. These consist of focal sites of attachment

between the connective tissue cells4l， similar in appearance to the intermediate

junction of the junctional complex described in various epithelial cells by Palade

and Farquhar5l • At these sites the membranes of two adjacent cells approximate

to within a distance of approximately 200 angstroms. Both the cytoplasm imme-

diately subjacent to the plasmalemma in these regions and the intercゅUularmaterial

are markedly increased in density. Such sites have commonly been seen between

fibroblasts in developing tendons， in developing ligamentum nuchae， and between -odontoblasts (Fig. 3). These attachment sites appear to play an important role

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in the formation of discrete structural entities in connective tissue structures such

as tendon， dentine， and ligamentum nuchae4，6).

Studies using proline-H3 to examine the pathway of this amino acid as it is

incorporated into collagen have been completed jn this laboratory. A typical

example of an electron microscope autoradiograph taken two hours after the

administration of proline-H3 can be seen in Figure 4. In this micrograph the

silver grains can be seen to overlay profiles of both the endoplasmic reticulum

and the Golgi complex. A kinetic study of the incorporation of the labeled

proline into protein in the fibroblast has demonstrated what appear to be two

different possible pathways for this amino acid2，71. We have suggested that one

of these pathways may represent the one taken by collagen precursors that may

be secreted directly from the endoplasmic reticulum yisternae astheymergewith

the membrane either at the surface of the cell or via vesicles which pinch off

from the cisternae and subsequently merge with the plasma membrane. A second

pathway may exist for noncollagenous proteins that are complexed with polysac-

charides. The latter involves passage of the amino acid from the rough endo聞

plasmic .reticulum to the Golgi complex and then outside the cell via vesicle trans-

port. These two postulated pathways are presented in Figure 5. Further analysis of

Legend of Electron Micrographs

Fig. 1 This electron micrograph contains part of a fibroblast from a ten day hea1ing guinea pig skin wound. The dominant feature of this cell is the extensive development of the cisternae of rough endoplasmic reticulum (er) and the well developed Golgi ∞mpl巴x(G). Two aggr巴gatesof fine fibrils near the cell peripheryωn be seen (f) as well as regions where the membranes of the c¥sternae of the rough endoplasmic reticulum approximate the plasma membrane (arrow). Num巴rouscollag巴n抽 rils(c) can b巴 notedin the extracellular regions. Magnification x 21，500.

Fig. 2 This micrograph contains part of a fibroblast from a d巴velopingfive month fetal calf ligam印刷mnuchae. The outstanding feature seen in this cell is the巴xt巴nsivedevelopment of th巴 roughendoplasmic reticulum with its numerous attached ribosom巴s. Two fibrous compo. nents can be seen in the extracellular space; th巴s巴arethe numerous ∞llagen fibrils (c) and several elastic fibers (el). The elastic fibers contain two morphologic compon巴nts，even at this ear匂stage. The first consists of tubular fibrils， approximately 100 A in diameter， that surround th巴second， a central semi-amorphous structure， that does not stain with lead or uranyl acetate. Aggregates of the 100 A fibrils are characteristic of the immature elastic fiber. Magnification x 19，000.

Fig. 3 This micrograph' contains parts of two odontoblasts from a developing rat molar tooth bud. Nuc1ei with prominent nuc1eoli are apparent as well as num巴rouscisternae of rough endoplasmic reticulum. The two ce11s are joined near their base by focal junctional sites that ∞nsist of areas of increased density both beneath the plasma membrane and between th巴 cells(arrows). These are typical of the junctional sites seen b巴twe巴nconnective tissu巴 cellsin this system as w巴11ωintendon and ligamentum nuchae. Magnification x 10，000.

Fig. 4 An autoradiograph two hours after proline-H' administration. Parts of two fibro. 制astsare pres己批 inwhich the rough endoplasmic reticulum (er) and Golgi zon己s(G) appear to ∞ntain the amino acid. Magnification x 16，200. Reproduced from Ross (1966) with the per咽

mission of the Editors of THE USE OF RADIOAUTOGRAPHY IN INVESTIGATING PROTEIN SYNTHESIS.

117

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←ーヨー Aminoocids

ーー多 Collogenprecursors

園田今 Protein -polysocchoride complexes

Fig. 5 The cell in this figure repres巴ntsan idealiz巴ddiagram of a fibroblast. Two postu-

lated pathways of amino acid in∞rporation into protein in this cell are shown by the thre巴 typ巴S

of arrows. The arrow attached to th巴 blackdot represents the entrance of amino acids， presumably through the cell membran巴， to the aggregates of [ribosomes attached to th巴 roughendoplasmic

reticulum. There the amino acids will be incorporated into the various proteins that are seques-tered in the cisternae of the rough endoplasmic reticulum. D巴pendentupon wh巴th巴rthese proteins

are collagen preωrsors， or proteins to be complexed with polysaccharide， they may follow at least two different routes through the cell. It is suggested that∞llagen precursors are secreted directiy from the cisternae of the rough endoplasmic reticulum (small black arrow) either via direct， inter-

mittent cisternal communications with the plasma membrane or via the form呂tionof vesicies that eventually fuse with th巴 plasmamembrane and rel巴aseth巴irmaterial. It is proposed that proteins

to be∞mplexed with polysaccharide are also sequ巴steredin the cisternae of the rough endoplasmic

reticulum， but that these proteins separate by vesicle formation from the rough endoplasmic reticulum in regions adjacent to the saccules and vesicles of the Golgi compl巴x. These vesicles are presumed to merge with th巴 Golgicisternae where they are complexed with polysaccharide and subsequently

secreted from the cell， again by the process of vesicle formation， migration， and日ilsionwith the

plasma membrane.

Fig. 6 This electron micrograph contains part of the extracellular space of an eight month

old fetal bovine ligamentum nucha巴 Herethe elastic fiber can be seen 10 be markedly enlarged

in comparison to those seen in Fig. 2. The elastic fiber now ∞nsists largely of the central amor-

phous component which contains embedded tubular fibrils and is surrounded by similar fibrils on the periphery. Collagen fibrilsωn also be seen in both longitudinal and cross-sections in this

micrograph. Magnification x 50， 000.

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this situation to determine if two pathways exist is being performed on tissue

culture fibroblasts. Pulse labeling of the fibroblasts will be used in the hope of

separating these two pathways if they actual1y exist.

Ligamentum nuchae has been utilized in experiments examining the formation of elastin as wel1 as collagen. Studies of the development of ~lastic fibers in

fetal bovine ligamentum nuchae have disclosed that the immature elastic fibers

develop as aggregates of fine tubular fibrils， approximately 100 A in diameter.

that with increasing fetal age appear to surround semi-amorphous components

(Fig. 2)8，9). The centrally located semi-amorphous component eventually becomes

the dominant structure in the elastic fiber as can be seen in larger elastic fibers

from older fetuses (Fig. 6). The nature of these two components in the elastic

fiber has not yet been identified and is being pursued in this laboratory.

ACKNOWLEDGMENTS

This investigation was supported by grants from the Untied States Public

Health Service DE-01703-05 and GM-13543-01. Dr. Ross is the holder of a

Career Development Award from the United States Public Health Service No.

DE-09053-05.

The author would like to acknowledge the technical assistance of Janet

Demorest， Leslie Caldwell， Franque Remington， Rene Col1man， and Dawn

Bockus. The author would also like to acknowledge the assistance of Mr. Johsel

Namkung in the preparation of the prints for publication and the secretarial

services of Mrs. Dorris Knibb.

REFERENCES

1. Ross， R. and Benditt， E. P. : Wound Hea¥ing and Collagen Formation. 1. Sequential Changes in Components of Guinea Pig Skin Wounds Observed in the El巴ctronMicroscope. J. Bioρhysical and Biochemical Cytology， 1961， 11: 677-700.

2. Ross， R. and Benditt， E. P. : Wound Hea¥ing and Collagen Formation. V. Quantitative Electron Microscope Radioautographic Observations of Pro¥ine-H' Utilization by Fibroblasts. J. Cell Biology， 1965， 27: 83-106.

3. Movat， H. Z. and Fernando， N. V. P. : The Fine Structure of Connective Tissue. 1. The Fibro-blast. Exρerimental and Molecular Pathology， 1962， 1: 509.

4. Ross， R. and Greenlee， T. K. ， Jr. : Attachment Sites Between Connective Tissu巴 Cells.Science， 1966， In Press.

5. Farquhar， M. G. and Palade， G. E. : Junctional Complex巴sin Various Epithelia. J Cell Biology， 1963， 17: 375.

6. Greenlee， T. K. ，Jr. and Ross， R. : The D巴velopmentof the Rat Flexor Digital Tendon， A Fine Structure Study. J. Ultrastructure Research， In Press.

7. Ross， R. : Synthesis and Secretion of Collagen by Fibroblasts in Healing Wounds. In THE USE OF RADlOAUTOGRAPHY IN INVESTIGATING PROTEIN SYNTHESIS (c. Leblond and K. B. Warren， eds.)， 1966， p.273， Academic Press， New York.

124

:8. Greenl巴巴，T. K. ， Jr.， Ross， R.， and Hartman， J. L. : The Fine Structure of Elastic Fibers. J. Cell Biology， 1966， 30: 59-72.

9. Hartman， J. L. and Ross， R. : The Fine Structure of Developing Fetal Bovine Ligamentum Nuchae. IADR Abstracts， 1966， p.42.

DISCUSSION

Dr. Rubin Borasky

、 Whatstain did you use for the ribosomes?

Dr. Russel Ross

Either 1.5 % uranylacetate or uranylacetate fol1owed by lead tartrate. Dr. Mitio Niizima

1 should like to draw your attention concerning the junction between the

odontoblasts or fibroblasts in the tendon. Dr. Watanabe in my laboratory found

also the same type of junction between mesenchymal cel1s in the subcutaneous

space in the amphibian tadpoles.

Dr. Russel Ross

Yes， such attachment sites appeared to be quite common between cells of

mesenchymal origin and undoubtedly will be found in many connective tissues.

Certainly in tendon and dentin these attachment sites enable the cells to form

integrated morphologic structures.

Dr. Osamu Kawase

(1) What is the significance of intracytoplasmic filaments?

(2) How do you think about the nature of two components of the elastic fiberつ

Dr. Russel Ross

(1) 1 believe they are r巴latedto cel1 motility and contractility and not to

col1agen synthesis.

(2) 1 believe they may possibly be two different proteins. The evidence is

not yet complete. However， the 100 A tubular fibrils clearly are not collagen， because they are resistant to collagenase digestion.

Anonymous

(1) How do you think about the cel1 attachments in slime molds?

(2) What protein is complexed in Golgi apparatus?

Dr. Russel Ross

(1) They do represent different physico-chemical sit巴s.

(2) A non-collagenous protein.

A Comment Added by Dr. Frank N. Low

lhe interstitial elements in hepatoma look very much like early elastic fibrils

in the normally developing chick embryo.