8/14/2019 Extracellular Matrix0001
1/6
\o l . 25?, . l ! ) , ssue,) l , , , ,b " . r0 , , ' ] i , " i i
] ; t ; l l t j l l s : ,Prn ledn I t S A
Extracellular Matrix Synthesis by Skeletal Muscle in
CulturePRO'I'EINS AND I]FI.'IIC]]' O .' F]NZY]I1.I I )I-]( tA
I)A'I'Io N'
( f teceived i rr publicat i t in , March 19, 1982)
Robert L. Beach, Willis V. Burton, William J. llendricks, and
Barry W. Festoff{From the Neurobiology
Reseo.rchLahorrt.tar.y',Kansos Cit1, 'aterans Artnti.nistration
Medical Center ancl Deportment ofNeuroktg;-, Uniuersit! of Kansus
(';tllege rt f Htullh Screrrccsun,:l IlosTtital
Extracellular matrix proteins produced by a mouseskeletal muscle
cell line, G8-1, were isolated and char-acterized. Cultures were
incubated with ['r5S]methio-nine or [3H]glycine and [;rH]proline,
and t]re labeled.substrate-attached proteins were obtained zrfter
cellu-la r proteins were extracted by deoxycholate in neutralsalt.
The labeled rnatrix was analyzed lry ge l electro-phoresis and
fluorography before and after enzymaticdigestion. Of the nine major
bands present iin the ma-trix, four were identified. Fibronectin
and collagenwere detected on the bases of thcir relative
rnobilities,differential labeling with :rH-uersus 3us-labeled
aminoacids and their solubilization by proteasc free collagen-ase.
High molecular weight material which w:rs prr- 'scntin th e matrix
wa s also sensit ive to collagcnase an dprobably included
cross-linked collagcn and laminin.Proteirrs co-migrat ing with act
in an d myosin were alsopresent in the extracellular matrix.
'I'hese results art:novel in that they demonstrate that thc
skeletal musclephenotype, no t contaminated with f ibroblast ic
cle-ments, is able to synthesize basal lamina-type macro-molecules
and incorporate them into :rn insolublc, ex-tracellular matrix.
Since this cell l inc is ablc to formfunctional synaptic contacts
rr- ith neuron:rl cells, theinf luence of nerve on basal lamina
production bv rnus-cle in t,itro is oossible.
The importance of the ECM ' both in mus< e cel l r levr l ,
,p-ment and mainlenance of the d if ferentiated state of the
-.keleta l muscle f iber and i ts neuromuscular iunction is n 'e l
l estab-l ished. Earl ier *ork (Hauschka and Konigsberg, l .9f i6:
Kon-igsberg, 1970t establ ished a requirenrent l irr col lagen as
asubstrate in the developnrent ol nrvoblasts into m.\ 'otrrbes
inprimary cultures. in othel studies using adult, innervatedmuscle,
morphologic specral izat ion of th e nrus
8/14/2019 Extracellular Matrix0001
2/6
i t t g a p t o t o t o l r l e s t r i l r c r ir v W i r l s l
r l t t r r l ' l t i l l r p s i l l l s ( i l : \ l { ( ' l l r \
r t l ) l i i : 1 sr ' " , i . 1 . " r i 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 ls l t s t l l ' r t t
g t ' t l o I ) N l l ' l t L r p p l t ^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 N o2 ( i ( x x ) )a n r
l 0 . 5 , i F ' l l s . A f t r : r 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 l i t t I( S i g r
l a ) w e r e a d d e ( l ( 1 0 I i t n t l 1 i ) ' ' r 1 . r t ' s
1 t t ' ti r r " i v t { ) l r r i : i r 1 j ( 1 ' c rl a t e r ) t
h o n r e d i u n r w a s c h l r n g e t l b a e k t o l ) M I ' l
* l l l t l r " I " I J S l ' r i n o t t lonset o f fus ion in cu
l tu res whi th wcre lo tro i r r l r t ' l t , r l ts I t l lo l t
l r ts ts .these cu l tu res s,ere p la ted li r x l0 ' ' ce l ls /
{ ;0 , l l in ) r i l r ; } i ) . i t i lo$ t ' r l 1t ta t tach
for 6 h , and then ler l I )MFl u ' i th 10 ' ; I " I IS ar r r i
labe ledcompounds.
Cet l - labe l tng Procedures-Pro te ins svn l i res lz t rd [r1
th t ' ( i8 - I t t t t t scl e ce l ls were de tectcd af te r
metabo l ic la b t ' l ing r ' " i th r ' ' [ ' S In ie t h io r t
i l re(9 i l i r C l i /mn io l ) , or [2 - "H]g lvc iner ( l ' 1 (
' i , / r r i rno l ) ru r ( l L - [2 ; t ' 1 . i ) - ' l l lp rol
rne ( l l l9 or 160 Ci /mmol ) , al l f ronr Neu l ' ing l ln t l
Nt t t L t r t r : \ 't r'H labe led amino acids s 'e re added ar
101 i ( ' i , nt l r r t t d: N 7 t i l r l r r r -1l lo rlabe l
ing nryo tubes or a f te r a t t .achment on da v I f i r r [abe l
ing n l lo t t l i l s ts .M e d i a ' w e i e n o t c h a n g e d
u n t i l c e l l s s e f e 1 ) r o ( c s s ( ' ( l : J d i i \ s l
a l e fRad ioact ive sanrp les were t ounter l in Aqu:rso l (Nt '
rv I ' )ng l r r l r t lNt t t l t ' r r )a t 30_35?, e f f ic i
lncv in a packard M0dcl ; lJ5r t l i t lu i t l s i in r i l l : r
l io r r( :0uut e .
ECMtrso la t ion-Matr iceswel re is t l l : r te r last l l ' sc
t . i l l t ' t l l l re r , i t r r ts l r ' t t rHe
