\ o l . 2 5 ? ,N . l ! ) ,I s s u e , )ol , , , , b " .

r 0 , , ' ] i , " i i ] ; t ; i l l t j lls:,

Prnledn It S A

Extracellular Matrix Synthesis by Skeletal Muscle in Culture

PRO'I'EINS AND I]FI.'IIC]]' OI.' F]NZY]I1I.I I )I-](; ItA I )A'I'Io N'

(fteceivedlirr publicatitin,March 19, 1982)

Robert L. Beach, Willis V. Burton, William J. llendricks, and Barry W. Festoff{

From the Neurobiology Reseo.rch Lahorrt.tar.y',Kansos Cit1, l'aterans Artnti.nistration Medical Center ancl Deportment of
Neuroktg;-, Uniuersit! of Kansus (';tllege rtf Htullh Screrrccsun,:l IlosTtital

Extracellular matrix proteins produced by a mouse elit'ar:ellular, prestrmably mlscle-elaborated protein, acetvl-
skeletal muscle cell line, G8-1, were isolated and char- cholinesterase, from the neuromuscular .iunction (Hall and
acterized. Cultures were incubated with ['r5S]methio- Kelly, l97l; Betz and Sakman, l97ll). Thus, at least some
nine or [3H]glycine and [;rH]proline, and t]re labeled. rnusr:leproteins interact directlv with the basal lanrina at the
substrate-attached proteins were obtained zrfter cellu- neutomuscular jrrnction.
lar proteins were extracted by deoxycholate in neutral Recent elegant experiments (Saneset al.,1978; Nlarshall e1
salt. The labeled rnatrix was analyzed lry gel electro- ol., 7977\ have demonstrated that the basal lamina of the
phoresis and fluorography before and after enzymatic neuromuscular .junction directs the site of reinnervation by
digestion. Of the nine major bands present iin the ma-
the nerve. F urther studies showed that acetylcholine receptors
trix, four were identified. Fibronectin and collagen
in regenerating nrusclefibers accumulate at sites whiirh <ror-
were detected on the bases of thcir relative rnobilities,
restrrond to regions of the basal lamina where the original
differential labeling with :rH-uersus 3us-labeled amino
svnal)se was present (Burden et ttl., 1979). In-addilgll:-i
acids and their solubilization by proteasc free collagen-
ase. High molecular weight material which w:rs prr-'scnt i n n e u r o n t u s t t t l 4 rd. i s e a s e , s
ryrvalions
in the matrix was also sensitive to collagcnase and h a vr' sifige1t qd -ririiT-' n-i;TlJFlail o ns-r n I he I.](' N'l nrav I rt'
probably included cross-linked collagcn and laminin. tl )) rr lrlrtflrlirr\r' \ ' (uleelleCr rI sl s rI n tl l) ii li lt [hr(()ugsi:l.l]egs: l ss \ D l s ( r r r (et / u
ar1. ,. , IL9J to{fl ;, fl l/uu a alf
d rnr ( i'se u

Proteirrs co-migrating with actin and myosin were also I 1.,Tos0a;-i*Rr\scffi ,'1 /., I 982).'l'aken together, rhese
"
present in the extracellular matrix. 'I'hese results art: reports scrve t.o tlerrronstrate the irnportanct' of stuclving the
novel in that they demonstrate that thc skeletal muscle metabolism ancl production of IiCIM bv skeletal nruscle cells.
phenotype, not contaminated with fibroblastic cle- ln this regard, sel'eral studics have shown that smooth tnus<rle
ments, is able to synthesize basal lamina-type macro- r:ells protlu<'e an ext.ensivelXlM (Rurke and lloss, I!)7!);'lones
molecules and incorporate them into :rn insolublc, ex- et ol., 19791.Howevcr, one of the pritrtarv difficulties experi-
tracellular matrix. Since this cell linc is ablc to form e n c e c l i n s t u r l v i r r g s h e l e t a l r r r t r s tl e F ) ( ' M p r o d u c t i o n i n t i s s u e
functional synaptic contacts rr-ith neuron:rl cells, the 'l'he
t:ulture is the lrresencc ol rntrltiple t'ellular tvpes. G-8
influence of nerve on basal lamina production bv rnus- t r r t ) r r s eI t r v o i r i z r s l t t , l i l i n e t ( ' h r i s t i i n e t a l . . l f l 7 7 ) o f T e r s n
cle in t,itro is oossible. potential s\sl i,jll lrr u hir h to slrrrlv the spt'tilic skeletal rnust:le
tt.ll phenotr'pr. iirr. nirrstlt'tell line is tapable of nit:otinit,
Lholrnergir' s\nAI)sf lrirrrrzrtion(('hristian c1 o/., lf)77) ancl
The importance of the ECM' both in mus< le cell rlevrl,,p- e x h i l , i t s i r v a n e t \ , r l s k t : l r t a l n r u s c l e t e l l - s 1 l e c i f i cp' r o t e i t r s a t i r l
ment and mainlenance of the differentiated state of the -.kel I ) r o l ) e r t i e s i n c l u d i n g a <t , t r , l t h o l i n e r e ( ' e l ) t o f s ( N o b l e c 1 c / . ,
etal muscle fiber and its neuromuscular iunction is n'ell estab- 1 9 7 1 : S u g i v a n r a , l f ) 7 7 t , l i j S a s . r ' m n r e t r i c .b a s a l l a n i i n a - a s s o c i -
lished. Earlier *ork (Hauschka and Konigsberg, l.9fi6: Kon- a t e d a c e t v k : h o l i n e s t e r a s e ( S L r g i t ' a n r a .I 9 7 7 ) , c o n l r a c ' t i l e p r o -
igsberg, 1970t established a requirenrent lirr collagen as a t e i n s ( M o < r r c e r 1a / . , l 9 8 l ) , n n d s t l i a t i o n s { N o b l t : e t o l . , l t ) 7 8 \ .
substrate in the developnrent ol nrvoblasts into m.\'otrrbes in We felt this cell line rvoulcl bt'p:rrticulallv rrsef\rl in exploring
primary cultures. in othel studies using adult, innervated the components o[ the Fl(]N{ svnthesized bv mtrscle cells. A
muscle, morphologic specralization of the nrus<:lelibcr base- prelirninarv report ol E(lM Jrrotein produclion bv G-8 nruscle
ment membrane in svnaptit legrons u'as rlenrr.instr-ated bv cells during nt1'ggencsis htrs appt'lrerl ([i]t'a1lt 31 rrl.. 1981b).
Z a c k s e t 4 1 . ( 1 9 6 1 . 1 9 7 ; l a . 1 ! l l l l r r . S o n r t , r , r i r l t . r r c ei s a v a i l a b l c
suggesting that muscle cells. ur ()t'.lr-r 1() ploduce a highlv I{ATF]RIALS ANI) NIIi]'THoI)S
s p e c i a l i z e da n d d e v e l o p e d l l C \ 1 r l u : t . r i i e l i l r t r v i t h o t h e r c e l l s
i n t h e e n v i r o n m e n t ( l , i p t o n . 1 q i l , a t l , t r ' . l i i d i e s h a v e rs h o r v n
that treatment with coliagenast, {,r' I)r'()teilsef'an release an

' This uork was

s u p p o r t e db 1 \ a t i L l t a i l n . : r : . r : e . r ' : i { r . , l ' l r ( i r r r :
1 R 0 1 N S 1 7 1 9 ;a. g r a n t f r o m t h e M u s c u l a rl ) r . t r , ' p h r . { s : ( ) (: i 1 : r ( , rr.nr . 1
-l
t h e \ l e d i c a l R e s e a r c h S e r v i c e o f t h e V e t . , r a r ' , .A r l r : , i r r . : r r . : i ' r n . h r
c o - c t so f p u b l i c a t i o n o f t h i s a r t i c l e \ \ ' e r e d e r r a \ e i i t n p a r l b \ i h r
pavment of page charges. This article must theret,)re he herebr
m a r k e d " a d L e r t i s e m e n l "i n a c c o r d a n c es i t h l S [ ' . . : . ( . : e , t ) l , n : - ] - 1
solelv to indicate this fact.
i T o s h o n . r c o r r e s o o n d e n c es h o u l d b e a d d r e s s e c l .
I
The abbreviations used arc: IICM. extracellular matrrr: I)\1F..
I ) u l b e c c o ' sm o d i f i e d E a g l e ' s m e d i u m ; F B S , l e t a l b o v i n e . e r u n r : l ' B : .
phosphate-bufferedsaline (137 mu NaCl, 2.; mv KCl, l.;l nrrr
KHrPO,, 8.0 mu Na,HPOq, 0.7 mv CaCl:. 0.5 mrr \IsCl,t.

11 4 3 7
I 1.1rlE S /; t' I t' I t t I XI tt s c I t"l[ rt t ri.t l' r'rtt t trt l i t tt t i.tt ): i tt.tt

l t t r t t r ; r i tl , l r t s n i : rr t s t l t ' s tt i l r t ' r 1t ' l s t ' n h t ' r t ' ( l " r ' s l o { f t ' l r r / 1 1 ) 8 2 1a' l l ( l w a s
i t t g a p t o t o t o l r l e s t r i l r c r li r v W i r l s l r l t t r r lI ' l t i l l r p s i l l l s ( i l : \ l { ( ' l l l r \ r t l ) l i i : 1 s
tLrpplt^ l l s { ' ( ii l l I / r { / i l 1 1 .
r ' " , i . 1 . " r it o n l l t t t ' I r t t ' ( 7 1 h ) l t r t ' t t t t ' t l t t t t t l s l t s t l l ' r t t g t ' t l t o I ) N l l ' l ttr
No . l r t t r / r s r s o l ( ' r ' l l s t r t t t ! , l 4 u t r i t t ' s * l ' ) ( ' ! t s a n l J t l t ' sw t ' r c a J r J r l i e r l
I r r e n l e ( lu ' i t h i r ' i h t t r s e s t ' r t l t l t ( [ i . ( ' . I ] i o l r i g i l r i l s i ' t ' l l " r i t I S ' L o t
2 4 h . t r r i r l i n t ' i t n r i . r l l t t 0 r o t i t ' o x r t t t i t littI gt'ls rts is,rlrttt'tlrrt sitlttplt'lrtrlli'r. Silrttplt's ol telltll;rt l)litteills in
2 ( i ( x x ) )a n r l 0 . 5 , i F ' l l s . A f t r : r '
I '' (1'c lr tltorltlr,rl;tlI r)r ('1t'' illl(l Mg 1 ' X ' r ' l ' I l S $ l ' t c s o l r l l r i l i z t ' r li t r s a r l t p l t '
(Sigrla) were adde(l (10 i t n t l 1 i ) r 1 . r t ' s 1 t t ' iti r r " i v t { ) l r r i : i r 1 j
inottl l r t r l l i ' r ( r ' i , / i I t i t ' r t r t l t t r r t ' l l t i r l i o l 'I t i ' i g l r c t ' t o l , 1 0 " s o ( l i l l l l l r l o d e c v l
later) tho nrediunr was chlrngetlbaek to l)MI'l *lllt lr" I"IJS l'r
s r r l l r r t I u r r r i { i - l l ) n r N { ' l ' r . i s} I ( l l , p l l r ; . , r 1 S o l r r b l e s a t t l p l o s l v h i c h r e -
onset of fusion in cultures whith wcre lo tro irrlrt'lt,rl lts Itlloltlrtsts.
1tt r l r r i L e r it . 0 r r t . r , n l r . a t i o nr v e l t , l l r t , c i l t i t l t e i l l t r , a r l d i t i 0 l r 0 f : r t r t ' r 1 u : r lv t l l u n l e
these cultures s,ere plated lir x l0'' cells/{;0,llin) rilr;}i). itilo$t'rl 'l'hese
labeled o1'l0'i trichloroai'erir' :rtrid (r'/r'l precipitates were then rinsed
attach for 6 h, and then lerl I)MFl u'ith 10'; I"IIS arrri
a1 0 "Cl s'rth a(etolle jl tintes, tlried, antl solubilized in saniple butlt'r'
compounds. of
svnlireslztrd [r1 tht' (i8- I ttttts AII gel saniJrles rvert' he:rleti to l)ir "(-- for 3 nriti after adtiition
Cetl-labeltng Procedures-Proteins ' 'S
a f t e r m e t a b o l i c l a b t ' l i n g r ' " i th r ' ' [ Iniet hio rtilre sarrrplc btrller.
cle cells w e r e d e t e c t c d
( l ' 1 ( ' i , / r r i r n o l ) r u r ( l L - [ 2 ;t'1.i)-'ll lpro S o l r r h i l i z e t l s : L n t l t l e sw t ' r c : r t r a l t ' z e r l o n ' 1 - l i r i l i t r c a r g r a d i e n t s o d i u n r
(9ilir Cli/mniol), or [2-"H]glvciner
all fronr Neu l'ingllntl Nttt Ltrtr :\ 'tr r|rilecr'I sLrllirte-lrirlvrtct'vlantide sltrb gels ttsirlg the distotrlinuous
lrne (lll9 or 160 Ci/mmol),
'Hlabeled irrrlli't- stslt:nt ol l;retnntli (19?0). After electrophorcsis' gels wercr
amino acids s'ere added ar 101i('i, ntl rrtt d:N 7 til rlrrr-1l lor (Bi<t-Rad
lixt'cl:rrrrl st;tuit'd tn 0.f i Coomassie brilliant blue It-2i-r0
labeling nryotubes or after att.achment on dav I firr [abeling nllottlilsts.
l , a b s , l l . i c h r i r o t r ' 1 ,( ' A ) i I r ' 1 1 > ' lr n e t h a n o l , l O c i a c e t i c a c i d A f t e r d e s t a i r t -
Media'weie not changed until cells sefe 1)ro(css('(l:J dii\s lalef 'l{
( N t ' r v I ' ) n g l r r l r t l N t t t l t ' r rt ) irri;, gt'ls ,-:oirtitittittg or "'S rvcrt' impt'e'gnated u'ith I'INHANCI'l
Radioactive sanrples were t ounterl in Aqu:rsol -80 ''C for 1-5 wercks
packard M0dcl ;lJ5rt litluitl si inrill:rliorr iliew linglantl Nut:leat), dried,:rnd t'xprtsed at
at 30_35?, efficilncv in a
( : 0 uu t e r . N{olt't ular wt'ight nrarliers :rttrl protein stand:rrrls u'cre visrl:rlized aftt'r
('oontassie blttt'stililting atrd thc posilions ol these bands rvere nlarketl
ECMtrsolation-Matriceswelreistll:rterlastll'sct.illt'tlllrer,itrrtslr'ttr 'l'he
rvilh l)IlS in ink on the f'luorographs. folkrwing rnolet:ttlar weighl ntarkcrs
He<lman el o1. (1979). Rricflv' culturt's were titlsctl ll tinrt's -
(Ilio-ltart) u't'rt: rtserl: Invosin (,[1, - 2fi].(XX))' f galactosirlase ('ll'
( p H 7 . a ) , e x t r a c t e d l l t i m e s w i t h 0 . 5 1 1s o d i u r n r l t ' o x Y i , h o l a t t . ( S i g r n a ) i n ('V'
rttrst'rl 1 1 6 , 2 i , 0 ) , p h r , s p h o r v l a s e t l \ M , . - 9 2 . , r - n x ) )b' o v i n e s t ' r u r t r a l b u m i n
1b mvr Tris-HCl buffer, pH 8.0. with 150 rtrr't Na(ll,:rnd again
- fli':](X)), ltttd ovallrttrnin (M' - '1ir'lX)0)'
l] times s'ith 1() nrM.I.riS bu|filr, lllI 8.()- All sltlls wt,rtl 1lt'tlilrtttt.tl
At titr was purif it'd tirrnr rabhit leg an<i b:rr:k ntust les bv thc rnethori
gentlvtrnice(()-4"C])inthepresencetll.llrtltlrht'trr'lrlrt'thr'lsrrllilnvI
tif sllutlich atirl Wlrlt (l{)71). !'ibronet:tiu w:rs purifierrl 1'rortlhuntalt
ilro.ia" ( S i g m a ) . I n s o l u b l e n r a t e r i : r l r t ' n t a i t t i t r g 1 r 1 1 : rh({ ' ( l 1 o t h e l l i s h
scr:tlrittg p l a s n r i r l r v t h < ' I t r ei h o t l o l l l t r o s l n h t i r ' 1 a 1 . ( l l ) 7 8 ) M l o s i r t u ' : r s p u I i f i t ' t i
w a s s o l u b i l u e d a t 3 7 . c ] w i t h s a r n p l e l r r r l i i ' r ( s t ' r ,l r t ' l , r r v l h r
'l'eflOn
s(.ral)('f. Flxlt'actiott at fto
'('
rlr<l li'ottt thickt'tr gizzrtrrls its lrreviottslv desct'ilrt'ci (Be:rch 11 41, l9ftia)
the bottont 0f the clish with a
o r I h r r t t g t ' t h c t o l l t l t r t siti,ttt A l l o l h t r r ( ' r t l t ( ' r r t sr v e r t ' t r l t h t h i g h t : s t l l t t r i l l t o n t r t t t ' r c i a l l l ' a v a i l a l l l t ' '
not int.rease the rariioactivit\.exlra(1ori
ollserve<i on polvacrylanride gels
Digt'stion of llCM-ht erltcritrtelrls rlt'rsigttt'rl1o (i('1('rllrlll{ lh{' It Fls (l L'fs
s L r s c e p t i b i l i t v o { t h e } , 1 ( ' N { r 0 r r e r r l r la) lr o t { ' 'ol l \ 'i1, r i l 1 i t ( k . l n i i l t i , r ' - r r , t , '
I rrrE" r1l-'rrgott'srs ol ()B t ('t'lls in l/i/r'r.r-Strbt<)n{luent IX)l)tlla
preparr:d as aboVt'. Ir<illo$ilrg th(' l0 lrl\1 Ii' riri't's r;rti,tt-
,t,rr, wert arlrlcd rlt (l ('ill () 1 \1'l rls pll r'() r otltrtittlllg l1t tions oi ()8-l cr'ilsgrowlr in l0 or'20fi l'tls did not fuse''l'hrrs'
"nru.t"*
n t N l L l a ( l l ! . 1 ' h C i n c r r l t : r t i o n s$ r r t , i n i l r ; i l I r l l r r I i t t l . i r . t l i l i ( I l l t
(1151r(-5 ( ' u l l u r ( ' s I ) l r I t ( ' ( li t t ) x l 0 ' c c l l s / ( i 0 - n r r n r l i s h c o l t s i s l e c lt o t : r l l !
r'l r1'r'
t . i l 7 " ( 1 . ' l ' h e f i r l l o u i l r g o l l z ' t l l { ' s \ \ ' t ' l - { ' r l : ( 'i (t li l L ) i r { I t l l t t r p - i l r , , 1r r r r 1 \ r s e n r lr v o l r l r r s l so t t < l i ' t t ' i l i, r t t h t ' ( ' I l ( l o l t h e l i - d a ' " ' l a b e l i n g
. i g n t a ) i t t i i l t l i i s l i t s ( ' ( l r ( r r l i I ) ( ) l (l l i ( ' l r l L l l tI . r i - I I
'l'ht'st'
I I I , b c l v i n e p a n ( ' r e 2 l sS 1rt.r'totl. t t ' l l s l : I r l g ( ' ( ll l ' o n r a f l a l t e n e d . s l e l l a t e n ) o t ' l ) h o l -
( r r
u n i t s / r n g o i p r o t e i r t ; ( ' a l b i o c h t ' n i ' B t ' l l l i l l ! , : ' i t 1 )I ) i t ' g t ' - 1 ' l ' 1 . r 1 r ' r o g r ' ( F ' t g . l . ' 1t t o a t t t o r t ' b i p o l : t r s h a p e u ' h e r e ( ' e l l d e n s i t ) ' \ \ ' i l s
'rii'
f r e c c , r l l a g e n a s e( I l r t r n t I I l , A r l r ' : L I r t t I J i ( ) l i t (t I l l ( ' s l ' t l t l r t ' r o k \ \ '
( J { i L r i ] 1 1 :l l l g ' h i g h t , r .r l , . i c . I / l ) . A l t e l t r e a t l l r e r r t $ i t h r l r i d i l t e a n { l f ' l u o r ( x l e o x -
ust'cl itt l{) units/nll. ITrokilurst'1fl'oIli ilttlltrtrl Ltlilit" ()n rllrr' (i'
S i g l l l i t ) $ : r s u s t : d a t 0 l m 5 , ' t l r l l ' l r r s t l t i t l i l A r ' l \r\ ; t s i s ' r l r r t I t l l r ( r r l l 1 f ( ' s l l
V r . r r i d i n t ': t t l r i t r k ( ' { l i t t t r t ' l t s e i t r f i r s i o t l \ \ ' i l s a l ) l ) i l r ( l n t

ti
.'-

.'{\

! i,. - Phast' contra5t ml( r()'

g r a p h s o i ( i E - 1 c e l l s . " { . . r ,I 1 j , , - . ' -
Iabeieo -'. n,robl:t.1.. ( cl,'nlr,ll':tr.','-
lht presetttt (,:\\tll (le\elr)l)ttiI:1"' :Lrirt''
:ht drrr belore labeltnq o1 nI\ otLll)r': \\ d:
irll:l.r:eai IJ. a nl\'otube cultuLt rt1 lht
:lnlr ,)l l)repal.alioll of nrVotrrbt Fl('11
\lajnt:lra:lrlt. X I,J.
 Sheleto I Muscle Mrtrix Proclu tI iort in L)ttrr.r 11 4 3 9

the fusion index was 0.ir and nratrlre nrvottrbrs nt'rr seen (l"rg. M,.x l0- 3
lC).'l'he fusion index rt'rrrainedhigh through thc 1()thdav of
culture and m-yotubesrnaturerdfurther (l'ig. l/)i.'[*ir:s corre-
:400 ''ditg
#

lated with morphological observationstirrrt <ells labelcrl fronr

'l'hese
dav 7 to dav l0 were predonrinantl.r rnyotubes. cells r t - - e
were mature-appearing,multinucleaterl mr'otrrbt,s,often se"'- 200
eral hundred micrometers long, with infrequent branches (['ig.
lD).
["'SJ Methionine Labeling-N{r'otube r:ult.ureslatreledwith
ir6-
['t"S]methionine from dav 7 to da.r, l0 or from dav ll to 14 92
incorporated approximatel.,'10,000cpni/60-rnm dish into ma-
66-
terial extracted as ECM. Characterizatir-.rt of molet'ular c,rrrt-
l)osition of the labeled ECM was acc,rrnpiishld ftrllowing
separation bv 4-151?SDS-polvar:rylamidegel elcctrophoresis & tP
45
and detection by fluorograilhv. Fig. 2 shows the tontp,sitlon
'"'S-labeled
of material found in the r:ell extrircts and the
matrix produced by nl,otube cultures.'l'he <'omltositionol the
labeled ECM was relativelv simple (Fig. 2, lane 5); r.,nlv8-10
bands were seen. This gel photograph was intentionally r-rn- P:r,'
derexposed as not t.o ribscure the nttnreror.rsllands in the
,":,;,':.,:ii
'I'he -
deoxl,cholate cell extract. bands in the ll(lM a.t M,
240,000,200,000,45,000,and 3C,000were also cvident ir.rthe
deoxl,cholatecell extra<rts(F'ig.2, lanr:s 2:rnd.J) and in cells 1 2 3 4 5 6
r e l e a s e df r o m t h e d i s h b v C a ' ' - a n d M g ' ' - f r e e P R S ( F i g . 2 , Flr;. iJ. Fluorograph of ['"'Slmethionine- or ['rH]glycine- and
Iane 11.High molectrlar weight nraterial (>,1(!0,()00) barelv [ ' t H l p r o l i n c - l a h e l e d < ' t . l l u l a r : r n d c x t r a c e l l u l a r m a t r i x f r a c t i o n s
able to penetrate the gel was enrichcrl in thc I1(l\{ relativerto isolatcd from mvotubc anrl mvotrl:rst cultures. Lane l. ['"Sl
the deoxvcholateextrat t. r n c t h i o t r i t r ,L r l r c l e rfli ( ' M 1 l ' l r t i o r ri i ' o r t r1 ( )r l a v n r r , o t r r b te: u l t u r e( 1 . ( X ) 0
'II
r ' 1 r n r )1. , r r r r lJ . n r o L ' tr r l ; r rw c i g h t n r i r l k e r l r r o t e i n s .L u n e ; ) , [ lgivcine
i r r r r l[ ' L i ] p r o l i r r t ' - l a b r l t ' It ,l l ( ' \ l l r i r L t i o nI l o n r i 0 - r l a y r n v o t r r b t , c u l t u r e
( ( ; , ( X X()p n l ) . L r r nt ' ' i . l r r l 1 r ' i r r s l a n r l : r r r l s .( l r o m a s s i t , t r l u e b : r n r : i su e r e
M. x lO-3
n r a r k e < lo r i t h t ' l l r r o r o g r r r p l :l r r s r r l l l r l r o s i l i o n ( i n r l e s c e t r r l i r r g orrler:
>4 0 0 h u n r : n l r l a s n r af i h r o r r t c t i r r( r L r L r l r k ,. 1t 1 ,- - 2 . 1 0 , ( X Xr)')h. i c k o ng i z z a r r l
1t r n v r r s i r(r, 1 / ,- . : ( X i . l X X )i)r.u r lr r r ' l i r r( , / t 1.,' , 1 5 , ( X ) 0 )L) .r t n e 5 , " I I I a b e l e d
r l c o x v c h < l l a toex l r : r c i o l j i t l i r v t r r r , o l r l ; r (s ul l t l r r e ( ( i , 0 { Xc)p n r ) .] , o n e 6 ,
*,*{5 ' I l l ; r l r t ' l c dI ' l ( ' M { j r r r r rj i r l ; r v
r r r v o l r l : r st lr r l t L r r t ' ( { i , ( t ( cXp) r r r ) . ' [ ' h eg e l
w i r s e x l l o s t ' r ll i r r . i 0 r l r r v s .
- :
-; .-
t

j':,,1tnt, Lrtht'ltrtg-\1r'otube lltlM

E /'Hl()h,citte art,I la-
irelerl br itrtorp,,liilr,n i)f I II ]lrlolirie anrl I Lllgl.vcine was
"il . i r n r I r l l J 1i n i l ) \ i t . 5 l ) r ' (t : t o t h r | . S l n r e t h i o n i r r e - l a l l e l e dn r i l -
il lt6 trrr.'l it,', r,rIilll:ili(rlt,rt ll ialttleclI.l('\1 rias lrlsolelativelv
- s i n r p l t . r r l r , r i r lr - i ' t l r i r i i r l . \ \ ( , r ( , a p l ) i t r e t i r ) .N l o s t o f t h e s e t r a n d s
- 92 'tS-E('\,1 '}{-ECM
-) co-nrigratecl rrith those seen in {l'ig. :l).

*
q>
nt
T
66
w a s i t l s ( ) e n r i c h e d i n h i g h n r o l e t r r l a r w c i g h t t r ) a t e l ' i a l ,r ' e l a t i v e
t.r ['li]tle<.rxvcholate extracts. I{oq'ervel in'[I-labeled
t l - r c b a n d a t - 1 1 ,- 2 4 ( ) , 0 ( X )\ \ ' a s r l i e n r o s t p r o n t i n e n t b a n c l , r v h i l e
F](lNI,

* C
r- i r l " S - ! l L l N { . l h e b a n < l s a r : l ( X ) . ( X Xa) n d . l l r , ( X X ) \ \ , e r ( ' r l r ( ) r e
{- - A <
l r r r r t r r i n e n t( r ' f F i g l J ,l o r t t , s , ' la n t l 6 r ' r , , , ' s r l1so r t r ,1 ) . I n a c l d i t i o n ,
5-
''FI-
a p r o t e i n b a n r l i r t I , l , - l i ( ) . ( i ( ( ) r , " : r sc ' l e ' a r l v v i s u a l i z e d i n

!r
.fl

'l'his
--l
labeled I,lCM. b a n r l r , , ' i r sn ( ) t s ( ' ( ' n i n " S - l i ( ' M (r'f !'ig. ;1,
L o l t e s , Ja n d 6 r ' t , , r ' s r lzost r L ' ! 1 . A 1 ; r i r r tb a n r l u t t h i s . l / , . a p l r e a l e d
i n [ ' F l l d e o x r t ' h o l a t e r , x t r a ( ' 1 si l . ' i C . ; ] . l t t r t r . .' 5 ) . A l t h o u g h r t t o s t
.* o { t h e b a n t l s r i b 5 , ' 111, 1 i1n i s o l a t t . r iI , . ( ' \ 1 L o L r k ll r t , i c l e n t i i i e r il n

il
-
r l e r t r v ch o l a t . r - \ t l i r (1 s . i t ( ' ( ) n ) l r . ) ] l \ , )or l l t t t t t ; . i a n d 5 i n F - i g .
l l r l e n t o t t s t r a l r n l l r ' k ( - { i r l i l l ' t , r ' , . r(' ., -, i , r r l l e p r o t t i r r r ( , t - n l ) r ) s i t i ( ) I r
r t t f i r s t i l a ! r i , ) r - i ' , ) r , - \ , 1 n r l r L t t. i. r , i , r r n c la t - 1 1 - 5 ; J . ( ) ( Xr )l , a s
1 2 3 4 5 e n | r . h t . d r n H - l ' . t ' , \ 1 r , ' t l r l r i r r t - ,t 1, , i H l d e r i r r ' < h 0 l a t e e x t r a ( ' t s .
- { d c i i t i r , n a l l r .h r j : r : : , , , i . ,i l i i r f \ \ r - l ! h r r r ) i l t e r i a l( > - 1 1 ) i ) . o i )w( )a)s
F I c ; l . F l u o r o g r a p h o f [ r ] ' r S l m e t h i o n i n eI n b t , l e d c e l l u l a r a n d
e n h a n c r d i n H - l - t \ l l e i a t r r c t , , I l L a l r r l t ' r(l ( ' l l c \ t r a ( t s ( f i g .
extrace llular matrix fractions isolated I'rom ml otube cultures.
L a b e l i n g a n d e x i r a c t i o n p r o c e r l r r r eas l e d t , s c n h e cui n c l e r" \ l a l e r i a l . \ . l r t t t ' ,r i i ' , ' \ i r \ ! r t t t , . j t . S i n r r l l ' rr l r t i t r t , r r c e si r e t u e e n I . l ( ' \ 1 a n d
and }lethods. Lone l. tells leLeased eL r l ' . i i r f ] t - | r r I h , r l r l t I r . \ I l ' l t (i s \ \ r - l r 1 , l r : r . r ' \ r r lr i h t , t f t t l t t s t l e c t , l l s l r e t ' C
f ' r o n ta L r ) - r i a vn r \ ' ( ) r u l ) L r
'-
br La ann \lg l f i . e l ' B S t t r . t - t i,xgt , n r r 1: , r , , ,- ; . . l , t . . r ri . , r v . t . - l , , i ' . 1 - , \1\ l ' n ,-lnrerhr,,rrirr"
e x t r a c t e d i u t o d e o x Y c h o l a t ef r o m a 1 - 1 - d a ri . ] t \ ( ) l u i r ( , , , , 1 . , , yr e1 . { H i 1 ) I n F r g . ' i . l a r t e - 1 ,t h e ( - o r i r r r r r . s r ei r l u e b a n r l s t i ' o m u n l a b r , l e c l
c p m ) : 1 r r n e , Jd, e o x v t h o l a t ee x t r a c t { r o n ) t h e rs a n t r i . L t l r r t - a . i n l l t n e
hun.ril-tplasnra fibronecrtin. chicken gizzard m\'osin, antl rnirbit
1 ( 2 5 . ( X Xci p m t : l a n e 4 . n t o l e c u l a r u ' e r g h t n t a r k e r l ) r ( , t r u r \ r C r r o n r a s s r e
b l u e b a n d s u e r e n t a r k e d o n t h e f l u o r o g r a p h b r . s u l ; e r p o s i t r o na r t d r h e s k e l e t a l m u s c i e a c t i n w e r e r e p r e s e n t e d b . " -t h e r k r u b l t , t a t J 1 ,
- 1 . 1 0 , 0 0 0 ,t h e b a n d a t . 1 / , - : 1 ) 0 . 0 0 0 , a n r l t h t . b a n c l a t - 1 1 , . -
s t a n d a r d s w e r e m v o s i n , p - g a l a c t o s i d a s e ,p h o s p h o n l a s e B . b o r . i n e
s e r u m a l b u m i n , a n d o v a l b u n i n ) ; I a n e 5 , e x t r a c e l l u l a rn t a t r i x p r e p a r e d 1 5 , 0 0 0 . T h e s e p r o t e i r l s \ \ ' r l ' e s e c n t { } t . - 1 1 1 i r 1 3 1 .n ' t t h t h e p r e -
f r o m l 4 ' d a v m v o t u b e s ( 1 , 0 0 0c p n r ) . I ' h e g i : l r v a s e x p o s e dl o r 2 l d a v s . 'FI- 'sS,labeled
r.iouslv mentioned bands in the end ECM.
I 1.1,10 -\ltt'lL'ltt I Xluscle Xlutrix Prrtdut'tion in Vitrr,
'l'hese 'I'altt,n I
t h t ' e e l l r o t t ' i n s , i l ( ' l i l l . l l l ' " i ) : i l r r ' r t r l l ' i i r t ' o t t e t ' l i l tl t i i v t '
pleviortslt lteelt lirttlltl to ltt'assoii:riri. rritll tlt'telgent-ttrslrl- F'rotliotr,t1'I!-or " ' S I u h c l . e tIlI ( ' M r e l c u s u ! t l t t r i n g { ' h i n c u h u t i t t n
u'i t h neutrrt I Pr')l('ascs
t t b l e r t t a t l i x p r o r l t t c ' t ' t ll r l ' l r v l t t ' i l ' l r l l t r ' l i l v t l i ' s ( [ l t ' r l r r r a n c 1 'l'r.v1rsit,
: t i r r ie l a s l a s t w e r e u s e t l a l l 0 1 g l n i l , l t r o t e a s e - f r e ec o l l a g e n '
a / - , l 1 ) 7 1 )I;l i r r l * ' t ' i i c / ( t 1 . . i i i T t i ( ' r r l 1 : , ' 1 / r i i i : A { r l .
n s e a t i ( ) r t n i t s l ' n r i ,u r o k i r r a s ea t 0 . 0 t i u n i t s / m l , a l r d p l a s m i n o g e na t 4
C o m p a r i s o n c i t h t ' I ' l ( ' ] l ' l p r o r l r r ' t ' t l l ) \ ' , r t l ' r f i ; 1 5 1 .: t t l r l n l t - -l'ris,
rg/nrl. IncLrbat!1n was in 1) I rrr ttt C1Cl1, qfl
otubes undel these (lxl)eriIn('I1t.1l r:lrtrtiitiotrs t evr';rlt'd ntl 1u !-,rl
'tll$li;j'"' '''s
t [ ]Met labelecl
clearll' discelnillle diflbretrce-t. Irr Fr1. 'i !utt' ti (l()-t'lav tnv- r'rz1r'e
o t u b e E C I M ) ( ' o l l t a i l i e ( l r e l a t i v e l v l e i s i t r ' i ' * i t t i i n ( 1n ) o l t ' a ( ' t i l l
N<ine 0.241+ 0.044 0 . 1 9 0+ 0.016
t h a n l a r r c , ' 3( ; ] - d a v m v t i b l a s t I ' l ( ' 1 , 1i h o w t ' r ' e t ' t h i s r t a s s o t i l t ' - Collagenase 0 . 1 9 3+ 0 . 0 1 2 0 . 1 8 5+ 0.012
wh:rt variabie in other pt'eparatiotts. l,ikt'rl'isc' "S-lalti'letl Llrokinase 0 . 1 9 2+ 0 . 0 2 8 0 . i l 1 8+ 0.027
n r a t . r i c e s o f n t r ' o b l a s t s a t t d t r t v t l t t l i t i : s . 1 ' ( ' (l ' s i l r l i l a l ( t t o t s h o w t r ) ' L.lrokinaso* plasminogen 0.530+ 0.060" 0.565 + 0.030"
I . I C M t r o m i 0 - d a v r n v o t r r l r e s ( [ " i g . l . l t t t r b " ) ! 1 ' : - 1nso t d i s t i r r - Fllastase 0.1177 + 0.012" 0.586 + 0.040"
'i /rrrr: '5)' 1 rr psiit 0.719+ 0.036" 0 . 6 5 5+ 0.109"
guishable from FICM {r'onr l'l-rial'rtrvoirtlte:'(l'-ig
t r ) n z y m a t i c l ) i g e s t i o r t o l ' I I C M ' - \ ' \ ' u n f x i ; l s s e s - s t ' rti i t i ' s r r " " /, .- ('l.(-li.

ceptibilitv ol (iti-l rnt'otube Fl(lNl to s( ir'iirl ex{fa.elltil:rrh'

a c t i v e c n z . , , u t e s .F ' i g . . 1 s h o w s t h e { ! ' i l c i i o ' r l l " S - l a l r r l l e r i l ' l C M M , .x 1 0 - 3
which is solubilizcd drrring a 4 li ii:t rtlltilion r,vith:l t'xlt'ittel-
Iui:rr proteases at Ireutral p11.'l'n'trlsirr atrcl llt'tiv:ttetl lrlasrrtirr- z 400
o g e n ( p l a s m i n ) r e l e a s e d l l 0 ( i o r r t t o r t ' t t { t i r e i n s o l r r } r l ' - rm i L t r i x
b v 2 h . ' l ' h e r e r l e t t s e : r p p r , , , r c h ert tl 1 r l : t l e : r rllt t , t r t t l c l4 h . I i l i l s t a s e
rt'leased radioactivitv with a silnilal tittte coltrse t() lrvl'sin
'Fl- -i*t .l
(not show'n) and b.v 4 h relcasccl nt'arlv ir(l'ri o1' or "S-
'i'iris 200
labeled rrtatrix ntatt'r'ial ('l':rbleIi. i r l l r l r ' : r l s t ts h o w ' s t h a l
t h e f i i i c ' t i < i no f r ' o t t n t s r e l c r t s e r j I r t t t r o l i i l t l t s t ' - a t l i v a l e r l p l : r s -
r n i n u g e n a n d t r l l t s i n i l r e i n t l e p t ' r t < l t ' t to 1l l h i : p l r r t i ct i l l l l l a b e i . il6
I ' r o t e a s e - 1 l t ' e l r a t ' t e l i ; r l t ' o l i r t g t ' t t : t s .i l i d r r o t ( l l l l t r l t i t a t i v e l v l ( ' - 92
l e a s e r a t l i r r a t ' it v i t v : r i r o v t ' l , r r f 1 i ' ti ' o t i t l o l : . { ' l ' e l l l e rl i .
'l'he , 6 6
i n s o l t r b l e r n a t t ' r i a i t ' t ' r t t a i n i t l gi t i l , ' t t ' n z v n l i t t i r ' t l i g c s l i o n
w r t s s tt b s e t 1 1 t t e n t a . lnv : ri v z c r l l l l s o t l tI u t , r l l I l t ' i r ! s rl l l a t t ' - g e l t ' l t ' r ' fi,j
'l
t r r i p h o r e s i si t n r l f l t i o r o g t ' i r p h r ' ( l " i r :i ,. t l i i r s t t l l l r r r r r ' 2 )t r l i l r t i t *> 45
n i t t c ( l n r o s t o l t l t t ' l r : t t t t i sl ) r ' ( ' s { ' I it lt r t l r t l ' l l i g l r c i n t ' l r n r l [ ' l l l
plolint'-laltelt'rllttattir..\llrirrrtItlii,tiJit,l'llisllrlrt'lt'rlrtt;ttt'ti:tl
f u t l n i n g a l l h e ( i \ t ' f l o l l t i t l r ( l s i , l l r t ' l r t \ \ t l t r l l t ' i t t l l t tr i t ' t g l r t
l t a n r l s . l ) t o t t ' a s e - i l t ' t ,' , r l l ; t ; 1 , ' t i l t s. ,l t i t : ( ' . ' i t ' l i t t t i n l r t t : rtll r i ' l l -
l a b e l c d l r : r r t r il a r . r r r t r ,a) t , 1 1 - l ; 1 r i r I t r i n r l I t , r l r r r . t ,1r l ) r ,l r ) t I l t *ff,[.'.
s i t i t ' s o t ' t h t ' l i t r l o r t t ' t t i nh i i t l r l ; t i t r l , r il i r t ' i i r . l rt t i o l e c t t l l t ri i t ' t g l t 1 1 2 3
n r a t e r i a l t h : r t b a t e l \ e n t e t ' t ' r lt h t ' ; i e 1 . Irlr;. i. i,'luolrlgraph of :r gcl containing the'rH-labeled Ii('M
:'SlMt'thionine-labelt'rl
[ t t t a t r i s , , r . ' : tl sr l s o t ' 1 fi , i t ' r r t l v d i g e s t e t l rt'mitining lround to thc tissut'culturer dish follox-ing 4 h ot
b r t t r ' p s i n a l t h < > u g hs o r r l e l l l a l e l i i t l o l . 1 / , ' = 4 ; i . ( X X a ) ncl lowet- digcstion at l]7 (' with prott'ases or buffcr control. Lune l.
nrolt,r'lrlltl u t'ight rernaine'tl. ('ollagr:rtrriie f('(ltlced tht' filrro- i r r l r r l r r r l i o rsri t i r l r r l r s i r r r ) ( ) r r g r r i l ) . L t t n e 2 . i t r c t t l l a t t t t twt i t . h b t l f l e r
l r k r r , ' . / - a r l r ', 1 .i r . r r t r r t l i . r t* i l i i ; t r o t c a s e - f r e tt t' l l l i l g e t l a s c( l 0 u t r t t s T
I t e ( t i I l l ) t t t l ( lh r r t h r t r l t t o t l t ' l t t e l l t ' r ' t , r n t h e I n a t e r i a l e t t h t t o p
Inlr
ot rhe gel rvirt,n "S-labt,lt'rl rnlrlr'ilr's\\('r(' rlnal]'z('dllv S])S
g e l e l e l t t o J r h o l e s i si t i t t ' t e r t z t t t t n l i r r l i g c s ti o r t s { t l o t s h t t w n } . 'i
rnr.r,: II
\ \ ' e h i r r e i t s s i g r t t ' r:1t l ) J ) i r , ' r )trr l ( ) i ( ' r l l l i itlt ' i g h t s , r l l l t e l : t l ' t ' l e r i
']l . \ r a n t n r i r r , t 1 'n t a . j r t r l a h e l e d E ( ' t r [ b a n t l s
s l ) t ' (i e : r r t t r r r i l S ' l l i l , , ' 1 , ' rri k , ' l t ' t r L l l r : l : r l e [ ' ] ( ' \ ' l l r l i r , l r l -
i.rrlrt,ltrt lrrnLl. loun.l in I'l('11 pleparations 1r'ont(i8-l ntuscle t'ells
I)aris.)It r l i t f i r t t , r l t ' r ' l t l t \l l\ f i g i l r . t r r : : l , ' l l r ' ' ; 1 - 1 ' l " , i l l g l r l i t h l t l i r '}{
{ r ' , r \ \ n L n Tn ( . I ) r e s r r i ( t , r r 1 lgl!cine,/prolinc or ["'S ]rncthionine i:Ls
r i t ' . r r i l r e r l L r n r i t , r" \ 1 r r 1 t , r r r L -a r r d M e t h o r l s . " I ) l | a w e r e o b t a i n e d f t o r r l
I ) r r ' (r . ( i l n { I r s L r l l : .
a. . llltih. l'r,' I slNI.l
\1 ' 1r 1, . | . j r rl \ , . r , l , . Dl t r , . l i l ( ' l l lrl,tjlrrl l:,l,el.rl

! . O >l(lrl + +
L;
(l
::JI\ I " i i r tl t t r : t l i n +
:1Xl \l\,1)\lll + I

I;o ( r'liagen +
U o4 ;E +
a
irll +
Ji '1ir . , \ i TI T t I +
j J.1 f +
2I + +
a2
1ir +
z
:
plots. Table II shot's the molecular weights and tentativc
-i
.t, 3 identification of several F)CN{ polvpeptides, based on data
v i 'a
Dir aN (h) presentedabove.

F r t ; . . 1 .l - r a c t i o n r ; f { ' r s l n r r t h i o n r n e - i a b e l e d F l ( ' } 1 r t ' l e a s e d DIS(]LSSION

during incubation of miitrix rvith crvvmcs, bu1{t'r 0.1 rt Tris,
(
Hedman et al. 1979t described a simple procedure for
1 0 m r t C a C l z ,p H 8 . 0 . : . . r ) ( ,r 1 ( l ( l i t i o r. i-: i . u r ' ( ) k i n a s(tr' ) ( ) { j
unitsrml) anci p l a s m i n o g t , n
r - i : rnlt 1r\l):ul 1 -1) r 1 g lllll isolatron of the extt'acelltrlarmatrix produced bv fibroblasts
 <J;t'!elrtl. tr{u*l.e Xlotrix I'r<tdut'ttori m I'itro 1T441

i n c u l t u r t ' . F l v c o n i h i n e r l n l o r J r h o l o g i t ' a l a n r l l r ; o , i r q ' r r i i c a lt e t ' h - as lirrntrtirr.rer:t'nfll' shown to Lret'o-distributed with acetyl-

t t i r l r r l s , t h e s e a t t l h t t r s i r l t ' n ti l i i : r l f i b r o r t t ' t t i r ' . . , l r i l a g c n ' a t l t i t'irolitrtrr'*'('.r[)1.{)r t'lrrsterson mvotube surfaces (Daniels e/ al.,
o t . h e r f a l v c o p x ) t e i n s i n I i ( - - M p r o r l l r c e d i r v l J ' [ ' ; : ,i e i l s . \ \ ' r ' l i a r , ' t : l08 l). l'resr,rnrairlv. thr: releaseof labeled fibronectin bv pro-
c h a r a c t e r i z e d t h e e x t . r a ( l e l l u l a t ; r r - t i t r ' i t : si r t i ' ' , i l " ; l l i " r i i ; l t o a trlarie-frlrtcollagt'nasr.is tlue to t.lie solubilization of collagen
s i m i l a r d e o x y c h o l a t e i n s o l u b l e n r a t r i x l ) r ( x l I l r : e r li r ' , ' i o i l a l s l i e l - t.o r,.,htchit u'its horrnd in the IICM.
etal rrtuscle ceLlsln r,ilro. Analvsrs ol thr'l'l(--lNl li', lltl t,lc'i'tnr- -Sev+:rai<:eli1r'pesin addit,ion to llbroblasts (Hedman el a/.,
phoresis and enzvntatic digestiol has ctrabler'l rls io identti'r' l{}79i have bei'n i,howrt to prt-rduceECM in culture. Smooth
some of the proteins which are dnnositerl rrn the stt!rqtttttuln' mrrst'ie cells produced an extensive ECM which has been
isolated antl c'haract,t'rizecl (Jones et al.,1979; Burke and Ross'
A s a n i n d i r - : a t i o r ro f p o s s i b l e d e v e ' l r i p m e i l t ; l l r r ' g r t l a t l o n ' r f t h e s e
p r o t e i n s w e h a v e a l s o i ' o m p a r t ' d t i l c l l C M 1 1 1 ' ' ; 1 l x l r : thl v r ' i ' o - 19?9).Other authors have examined the collagencomponents
blast and nr1'r,rtube lr,rltrtres. of rnatrix Jtroducedbv endothelial cells (Tseng et al', l98l )' In
P r e v u t u s s t u d i e s c m p l o y i n g i t i 0 r ' h i r r r r i t . l : .r1i r i l r n ) i r i l t r ) ( ) l { ) fi r .i i i iidriition, L-'ulpr:1a/. (1980)have studied some of the extracel-
probes have e:itablistred tiitrt fibront'r't ilt ,r- r::'' r*'llt ;l' tiit' irrlir' yrrriteirtswirich were deposited by neurons in culture.
'i
detergent-iusolliirle rrtatrix prodtrorrl L'"' ,' ','"1i1';"' lll gells lrc i,fi iitl- shel .li,i nlus('ie cell line has also been shown to
(Ilirdwell el tt., 1978; Fledman r'1 o1 , llt'i'!l {lr;i;' 1{il}0! Iiat pLl,'iLtcesi)lr']rr t::.i r,,'ll, ilar proteins or substrate-attachedma-
'.t,riailnt'lririii]f{r.t:ri..il.i!'bandat M. - 55,000(Schubert 1977)'
ntusclt' cell lint's exI)rcss fibroliccllrl 'lttrtl;! ti" "l-eilt-':;r\ i''?
l r l x r ( C l r e n , 1 9 7 ? : F u r c h t t : t a l - , l i i t 8 t a r t r l t h c ( . , 8 -I r r t c r i : ; e< r c i ! We f,.r,rnda sinri;.r tjCM band ^t M, - 53,000. However,
l i n e t k - , e sa s w e l l ( W a l s h n n d I ' l r i l l i l r s , l l r l r i i ' ! V r ' i r " i t t - ' d e t t t - c()liiigon or t:rlilagettous proteins were not identified in the
o n s t r a l o d h e r e t h z r t G 8 - l s k e i e t l l m t t s < ' i t ' 1 ' 1 ' r l '';: . 1i , l l l t ' s l z t ' a
( ziflrrt ritltd\' {Si:hLrbertl9?7). ln another previous study using
p r o t t ' i t t s i n r i l i l r t o f i l r r o n e r : i i t ' t: l t l r l i n r ' o r l r o l a t l ' i i ' l t o a s r t b - clonai rat trru,,111b;,rS it was suggested that muscle cells were
stratt,-attached I'l(lNt. Orrr ldt:ntilit'atiorl ll 1l,i:; Irrrltrin as cap:rtri,.. ol synthcsizingsoluble collagenousproteins but were
fibrottectitr is based on (rz) it:i t o-tlligf:,tii,', ililh prrrifietl unable to tnt'nrporatc thcnr into an organizedinsoluble matrix
I'ibrone'ctin antl {l} its releast' tr<rtii tl;i ritrtlrl.'- iir'ri coliaplt n {l,i;rton, ll)i?i. In 1.1'rr lrrt'sentsttrdy, we have used gel electro-
"',
i s t l i g c s t e c lt r i t h l l r o t e a s e - { r c c t r t t l l l l g t t t l t s t ' l " ' : t l i t i ' t , t t t l l ' i L r n z t ;rhorcsis arld r':rzvrniltic degradat.ionto identify the maior
t i < l n0 f t h t ' i d e n t i t r o { t h e r l f , - j ' 1 1 ) . t X X r
h r t r t t i' t , rr ' 1 r r ' } t l f i ' t i rIls ('r)n-rl)oneilt:, in|orltoraled into ECM by myoblasts and mv-
r'i till: i';;t"ri otubt's 'lt i ullut,-s t'ree of nonntvoflenic cells. These ECM
1 l r < l l i r l e r ll l t t ' r ' t t t l i a t i s r t t t< l l t h t ' r l i a t i l i ' i l ) i c I \ : i ) i i
in
'll-
r r n r l : i l a i r e l t . r Il ' l C M . f i l r l i ' r ( ' { t l : r ( { ) l } { r l l i ; ,. t' i r o r ' t i i i l t prrlrtralir.'tis ir;lVefeatlrit's sinrilar to those described in pre-
g l v t : i r t t ' s: t n r l P t ' r i l i t t r " ,l l t t t < . r t l l l i I l l l t ' 1 1 ' r)ir l t t : { ' ' . ; / r l ) l )l t ' s l t i r t e : l r'iolrsst trrlils wil ir lonr','.'tive tissttecells,such as the presence
( Y a n r : , r r l cat r t l . . 1 ( ) | t : l l ' t r r r s l, i t , , i n r r l k t . , l l , , , , ; i 1 1 i 1 1 r lrtti'irrit ' l i r r 1 , r { i i i r r , r t r e t t i r tt,r , i l i t g t ' r ta, n < lh i g h m o l e c u l a rw e i g h t m a t e r i a l .
o l l . h i s l l r r n rsi t ' t ' r t i r t ' i l - i r r l r t ' i ,l r) ri 'l r i ) i l l t l t i ( ) ' l s i s ' t : t , l t s t :'itri t: ti !' ln a.rlrlition.!v.r rii!{rrl'r.'vtrlt'nce that in the absence of fibro-
i t s i r l r . n t i l i r ' ; r t i , rr -r ti i l , t , , t r , t, i : , blasttt: elenlcnt.qskt'lr'1alnrrlscle (rells produce one or more
l ) t o t e i t t s r v i - r l < itr, r - n i i g r l l t l r . i t l r : 1 , 1 i r l; t ' ) ( l l l i \ i ) s l i r : r 1 ei i l s { ) genetit'f1,pesol r"ollagen.F)xllerirnentsto exanline further the
( o n t l ) { ) I t e t I t so f t h i ' I t ) a t ! ' l \ . N r t l l - i r t ' ,i 1 l i ; l l ! s ( ) t l ) 1 t , i ,r rl t t l t t ' s t : t'ollag,-'ntylr's, tltt' pr<tt'ollagenssynthesized and released,
c r t n t t t t t r . i l tlrl r t l t t ' i n s i s r . r r r l i k e ' ll r( ': i 1 ' l . \ (lr, i l : i / ) 1 1 'l ." i l s l . g l t t i t ' el1i'<'tso1'tret" r' t'xtLii<ls, vitalttin (), and neuronal co-cttlture
, ' r ' 1 . , ( ' N ll , t c i j t ' , , 1 i, l r r ti l t g ! r i t t l t l t 1 1 1 1 , r g l n e sai sr e i l t l l r o g -
t ' r ' l l u l i i r a c t i n a n t l t t l \ ' o s i l t t l l ' ( ' 5 \ i r l i r t s i z t '1r l: i l ' r t , ' ' t . r i r e l r l r v
l - r ' " - ' ' | ' r l ' ; , r ; s .S u < ' h s 1t i c l : , ' - i , i i r \ ' l u r t h ( ' r t ' l L r < ' t d a t m e echanisnls of inter-
t l < r r r i t ln t t t s c ' l e c e l i r t r v o t r . t l r t ' st h i t i l t l i r r ' i , i ) ' ' i ,
'lr ' .i,1rl't-l--('-\1 a( tr()nt)!l$een : r' l r t t t lt n t t s t ' i e a n d h e l l ) i d e n t i l ' r ' a b e r r a t i o n s
I l ) f i l . ( ] ; r l l e l s . i 9 7 8 1 .t h e v a t ' c n ( , 1 e r t t ' t t h , ' r i
r c l a t i r t ' t o r t t ' r ' r r b l a si t C M ( [ i ' i g .; i t . S t ^ r ' , , i t u-. . t ' . t - ] . t lg ] ( ) l 1 l ) : t i l l I l i I i t I \ { ' i ' . r i lr( Lll,,ltrtlS, tllirJ dist'ases

t('uip r ' 1 r r l . . l 9 8 r ) l L e t t t u r n e : i u . 1 9 , " ' i :5 t i t t t l r e t t . l ( i - r i r h t ; i r

i t . r : : , t , i r ; r , / i r . - . l r t t t r i { , r s e r l t r o v i r l e dt e t h n i t ' a l i l s s t s l i l n c ei n
s l i O $ r t t i t l t t t h t , s c I r r o t e i n s a r r ' [ , r e s e l l l ; r l ; l l t , i ,i l I ] l t ' I l t i ; o i t t 1 .
' , , [ , , . : - , \ l f ' l i i ] " r i t ' . r t r j l \ 1 l | h a c l l ) i l t t e r s o l l t s s i s t e di n t h e t : e l l
t i r l c e l l s i n l L r l t i r r r ' ( ' \ ' e na l i e r d e r t e r g e n l s o i t i l t t l i z : r t i o t r( l J i r d i . r ' l l , L r : l r i - \ \ r i l r r r n k( i , r , L ( ; f i i t l l l f o r 1 \ l ) l n go f t h e n r a n u s < r i p t .
'l'ht'r't'firre,
t,l cl.. lgisr we Ieel these tt'ri prott'itr:' rr'l)resent
l r ' 1 o - c k t . l t . t t r 'l l t ' r ' n ( ' r ) tisn r ' < . ' l v e ri ln t h e a t l a l h r r l t ' r t t o f ( i 8 - l t ' c i i : : It FlI.'flI'tliNclFls
'l
tri the Fl('\1. h,' ri.'tt'tnrinrtti{)o I rl l t t t i P r ' t t i r rl r n ( l i s o e l { : { t } i (
] J t , r rl ,r . l t . L . , K e l l r ' , I ' . ' t ' . . F t a b i t c h J. . A . , a n d C o t m a n , C . W ( l 9 8 l a )
I ) r ' o l ) ( ' ] 1 j r : , ) ll i l ( ' s ( ' l ) r ( ) t e i n sw i l l b t o l g I e a l i r t t t ' r e s t i n t h i : r I Jr t r n R t ' s .2 2 5 , l i r - 9 ; i
t ' e g a r i l .a : l r u : ! l t ' , r l l s s v r r t h t ' s i z rn r r t l t i i i l t ' f i r r r n st ; l i i r i i i r a l t < l B e a c h . t l . i , . , I ) n l l e r s o n , M . I t . . I l e n d r i c k s , W . J . . i { o s ' e r , J A a n d
n l \ t ) s i r ) t t r LL t i l i r t t ' r ( ' h t l l t t l . , 1 ( . t - , i\;\.: h ; r i t ' r ' r t t t l . , l ' : i ' i i ) . ' , ( i i l l t . !estr;lf. l]. W. tl98lb) "L ('tll Btology 91, 13a
\ \ ' r l r : r . , ' t t t , i i l t ' ] i l r -l .t t t l ) t t \ t ' t . 1 . ' n t i i i , t , r i it.,ttt l l l l l ' i 1 l 1 r l ' r , - B r t z , W . , a n r l S : r k r r r a nI,l . ( 1 9 ; ; l ) , 1 . P h v s i o l . ( l ' t t n d . t 2 3 0 , 6 7 3 - 6 8 8
l i n e { H l * L . 1 r n { - } 3 i r f i e i l l , r t t r l r - 1 1 - : . i l l t ! ) ( )r r s :: t g e l l e t l ( I l r r d r r e l l .( ' . | i . , i ) o s J x r d a l o r v i t zI ). . . t r n < N i i t o l s o t l .( i t , . ( 1 9 7 8 )P r o c .
N a t l . A t a d . . S Lr i ' . S . A . i 5 , : 1 2 7 ' l . - 3 2 i 1
1 \ ' p e ( ) t t l i L r t g r[ n' t.i t t r r r i t l l . r r . 1 , ' ] 1 , I t , j ' . l ; ' l l ' l : , ' i l ' l a b e l e r l
B r r r r r s t e t lIr).. . a u r l S u 1 1 H c . ( 1 9 8 0 1' \ n n u . I l t t B t t t c h e n . 4 9 ' 9 5 7 - 1 m 3
E C j \ l a n d t s a n l . t . i { ) lr)' r / i i f t , l i t r i i - l 1 l I i ' i l l r ; ' l r . i s i l g g e s t sa ('ell Bittl'
f J u r r l r : rS r .. . l : ; r 1 r g o r l[1' . f J . ,t r n r l] 1 ' \ 1 a h a n . t ' J ( 1 9 7 9 )' l
h i g h l e r e l r , 1g l r l i n e l ) r L , . r r . ., r r .) ,'l irlethionine
8 2 , . !l l - + l ;
in the p|otern. lJoth r,i rF--. 1,r , *. , i r . t r ' t e ' r i s t i 0t 'f ( r 1 7 1 :f l , ' ( ' r t n n e L ' t? ) s , r r z1e1 e sE . ,
i l L r r k e . I \ 1 . . r r r i lh , , - , , .I l t l i )
c ' o l l : r g e n l l l i n s t t ' t n r r t r r l> l g t . , ' - i' . -: . r'' ,'t * altltough il11-l''
-
i r l r t , l i ' : t ' l € ' (-
l i t t l e , r r r r ( )t o t a l r a d r o a t t i v i t r t : i ' t i r , : t ' 1 . ' i . t : t'itt il l- li .',: / . " I { 1 . 1 ( r -I '

p i - 1 , 1 1 , 1 i t ' l l a . t - t i r r .. I ( ! ' , . . l n r : . r . : , 1 i l , , r ' . : : r . i { . 1 1 9 ; 5 )P r o t . N a t l . A c o d . S c i .

t i v e l r d e g r a d e d i r . ,a h i g h l . r p u l i l i t r l . t r l L i l r ' ; r : f it
i : I l.) 1rrLr,,,-Lr
l i x p e t ' i r t t e n t . i i r e ( i . r I T e t l t h ' i n I ) 1 ' r ' s ] ] ' . :lsl ) i l r - r r ' f l l , i i l r - l t t h l s
-l'hl: t : . : r . r ; ; i r .r ' \ \t,-,,rt I) (, l'rat,r.k,,l., and Nirenberg.M (197;)
c o l l a g e n i s o n t ' o f t h e h a s e m e n i n t t t t t i , tl r l r ' ' . i . 1 i r t t :
'l'r'1re t'
\ r''r,r' 1\'r.i: 1i ( 196. 'q )-:198
seenls likel\ bet luse sol,.rbilizerl .l , ,,jirtge:, ii:plil\: ii t l . . i 1 l , ( iI ) ( ) l l l e n , C ( 1 9 8 0 ) I ) i l t c h e m t s l r y ' 1 9 ,
' l - i : r . r - ',rl i. ! r iu- - .r. , A : r : i : r : , f , . ' r .
b a n d a t t h i s n r r r l e ' r ' i i l aur' e r g h t r a n g e ', ' l ' i r r r i ' i' ' r : i ' l ' , ' l '
c o l l a g e n r , L rl sr a n i l n r i g r a t e si t i t r t ' t n e r l i a t i ' 1 i, r. . t \ \ r ' r t r t h e , i a : t , l t).,.::t,-. \1 '' \'tgnr \1 . lJrrrr.r.I{.. Sondercgger, I). ancl \iogel, A'
'!'
. \ , : r , , , . , r - - 1 b s t r 7 , i - 1l
B s u b u n i t s o f i n t e r - q t i t i a l c o l l a g e n i r . I n , r , l , l t t r r , r ri h c h i g h n l o - (' . Bla.k. tl \1.. Duhoqitz, \:. Hughes, G. R V. and
i)r,..n,. \
l e c : u l a ru e i g h t n r i i t e l i e l * ' h i c h a i r p e a l e t l i r - r i . i l i i r i : r ' b a t r r l a t
i J a L I e r , A . . i . ( 1 9 t i ) a ) J l u s c l e o n d ^ V e r u e3 , 4 8 7 - 4 9 0
t h e t o p o f t h e g e l s e e m e d t o c o n t a t r rt o l l a i l " r l i , ) : I , i , , t , ' l i l \ . r - i t l)uante. \'. (-.. Stephens, H. R., I)unn, M., Bailev. A. J., and l)uiroq'itz,
is also degraded bv collagenase. Silliirrr'1.. ,i tLr t: ttt,rre \'. ri9,90b) Nctture (l'rtntl.t 284, 4i0-472
'H -l'hi,"
heavil1'labeled in than'rf'S prelrarations. n)ate rial ma\' F l l s o n , t L F . . B e : g n r a n . . 1 .F i . . a r r r i F l l - s o n ,i ' l C l 1 9 E 1 ) J C e l l B i t t l ' 9 1 ,
represent cross-linked ('ollagen a:rrl ass;rrtiatcd 1ttoteins suc'h 3-19a
l1-112 Sheletol XIusc'leMatrix Procluction in Vitro

[ , ' e s t o l l ,l ] . \ \ ' . . I ' a t t e r s o n .M . l l . i r i l t l l l o n l s t t r l l t i ( l 1 f S 2 )J . ( ' e l l S a n e s , , l . l t . , M a r s h a l , 1 , . M . , a n d M c M a h a n , t l J . ( 1 9 7 8 )J . C e l I B i o l '

I ' h . t s u t l .I 1 0 , l 1 ( ) l 9 i r 7tt, l7(;--198
'l'., S c l r u l r e r t ,l ) . ( 1 9 ? ( j ) E x p . ( ' c t l 1 ? e s .1 0 2 , 3 2 9 - 3 4 0
!'urclrt. 1,. l v l r r s h e r ,l ) . F . , a n d W t ' I l r l c l s th a i t ' t ( ' r a b b ' ( 1 . ( l 9 7 t l )
( ' r l l l i l . 2 t ; ; J - 2 7t S r l r i r i r t . r l |, ) . { l 1 ; ; t l ) r , t i n R c s . 1 3 2 ,i t j l T - j } . l t i
( l a l r c l s , , r . L ( 1 9 ; 1 r I)) e t ' l l i o l 7 3 . i ; 1 4 - 1 5 2 Strrrrdith, ,J. A. ancl Watt, S. ( l97l ) ./. Iliol. Chent. 246, 4866-487|
Ilalt, Z. \V..anrlKeth.ll. lJ. (1971)Nc/.Nlrl lltttl.232'6'2 t)il Stephotrs,I{. Il.. I)uanr:e. V. C., I)unn, M. J., Bailev, A. J , and
[ { a r r s c h k a ,S . F . , a n c l K o i t i g s b e r g ,I . R . ( 1 g ( i l r )P n t c . N a t l ' 4 c a d S o I ) r r t r t r w i l z .V . ( 1 9 8 2 i J . N e u r o l . S c i . 5 3 , 4 5 - 6 2
I , : .S . A . 5 5 , I i 9 1 2 6 S u g i v a n i a ,H . ( 1 9 7 7 ) F ' t r ) B SL e t t . 8 4 , 2 5 7 - 2 6 0
[ { t ' t l m a n , K . . K u r k i n e n , M . , A l i t a l o , K . . \ ' a h e r i . A ' J o h a n s s o n ,a n < l Timpl. R., Wierleman. FI., Van I)elden, V., Furthmaver, H., and
H o o k . M . ( 1 9 ? 9 ). / . ( ' , e l lR b l . 8 1 , 8 3 - 9 1 Kuhn, K. (1981) tr'ur. J. Biochem. l2O,20:l-211
L : r e n r m l i ,L j . K . ( 1 9 7 0 ) N a t t r r e \ L o n t l . ) 2 2 7 , 6 8 0 - 6 t J 5 T s e n g , S . C . G . , S a v i o n , N . , G o s p o d a r o w i c z ,D . , a n d S t e r n , R ( 1 9 8 1 )
'l'.,
Joncs, I). A., Scott-Rurdc'n, and (;evers. W. (1979) I)nx' Natl' J. Biol. Chent. 256, 3361-3365
Atad. Sci. L'. S. A. 76' 353-1157 Walsh, F., and I']hillips, Il (1981) Dett. Biol. 81,229-237
K o r r i g s l r e r g I. . I i . ( 1 9 7 0 )i n O h e n t s t r l ' a n , l t r f t t l p t u l a r B i t t l i 4 t o f t h e W h a l e r r , R . ( ' i . , I i u t l e r - B r o w n e , G - S . , a n d G r o s F . ( 1 9 7 6 )P r o c N a t l
I n t r a t e l l u l t t r l l l u t r i : : ( P : ] r a z s .I ' 1 . A . . e t l ) V o i . ; 1 , p p . l i 7 1 ) - 1 8 1 0 ' Acrrd. Sci. tj. S. A. 73.2018-2022
A c a d e n t i c I ' r e s s .N r : w Y o r k W h a l e r n ,R . ( i . , S r : h i v a r t z .K . , B o u r v e r e t , I ' . , S e l l , S . M . , a n d G r o s G
l , t ' t o u r n e a uI,' . C l .( 1 9 8 1 )D e u . I l i o l . 8 5 ' i l i | - 1 2 : ( i 9 7 9 ) I ' r r ' . ; \ , ; t i 1 .A c o d . S r : i . [ . 1 .S . A . 7 6 , 5 1 9 7 - 5 2 0 ) '
l , i 1 r t o nR , . F L ( 1 9 7 7 )D e L ' .B k t l . 6 1 , 1 i r ; l - l { i i Y a n r i r d a , K . I l l . , O i r l e n , K . , a n d I ' a s t a n , I . ( 1 9 7 8 )A n n . N . Y A c a d
M a r s h a l l ,1 . ,M . . , S a n e s ,J . R . , a n d M t : M a h a n , \ ' . ' 1 . ( 1 0 7 7 )F r o c N o l l ' Sci.312,256--'li7
A c a d . S t i . I ' . , S .A . 7 4 ' : 1 0 7 : t ] 0 1 1 Z a c k s . S . L , a r r d R l u m b c r s , J . M . ( . l 9 6 1 ) . , f .B k t p h y s . B i o c h e n t C y t o l
M o o r e , S . F ) . ,I ' h i l l i p s , F l . ,a n d W a l . h , I " . S . ( 1 9 8 1 \ N e u r r t c h c r n l n t S ' 10, 5r7-52rJ
't;-36 7\:rcks,S. I., Vandenburgh, H., and Sheff, M. F. (i97;la) J Histochem'
'l'. ( ' v l o c h e n t .2 l , 8 9 5 - 9 0 1
Nrrhlt', M. l).. }]rown. IL, anti I'eacock, J. Il. (l97ll) I'roc Nntl'
Atarl. Sti. ti. S. A. 75, jl'188-3492 Z a t k s , S . t . , S h e l f , M . F . , a n d S a i t o , A . ( 1 9 7 3 b )J . H i s t o c h e m C y t o
l l u o s l a h t i . F l . , \ ' u e n t o , M . , a n d F ) n g v a l l ,l ' 1 .( 1 1 ) ; 8 )I l i o c h i . m . I l u t p h | s ' L'h<:n2 t .7 , 7 0 ; l - 7 . I 4
A r ' 1 oS l i . { , 2 1 0 - 2 1 8