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C L OS E T O P O G R A P H I C A L R E L A T I O N S H I P B E T W E E N M I T O C H O N -D R I A A N D E R G A S T O P L A S M O F L I V E R C E L L S I N A D E F I N I T E

P H A S E O F C E L L U L A R A C T I V I T Y *BY W. BERNHARD, M.D., A~D C. ROUILLER, M.D.

(From the Institut de Recherches sur le Cancer du Centre Nation al de la RechercheScientifique, Vill eju if (Sein e), France)

PLATES 22 TO 25In n ormal r a t l i ve r cel ls , t hough one ma y som et i mes f i nd a de f i n i te g roup-

i ng of mi t ocho ndr i a l an d e rgas t op l asm i c s t ruc t u r es , t hese t wo organe ll e susua l l y do no t show any spec i a l t opograph i ca l r e l a t i onsh i p . T h i s i s t r ue o fo t he r t ypes o f ce ll s w i t h l i t tl e e rgas t op l asm where mi t och ondr i a an d e rgas t o -p l asmi c s t ruc t u r e s a r e no t i n e spec i a l ly c l ose con t ac t , and a l so o f ce il s wheret he whol e cy t op l asm i s e rgas t op l asmi c i n na t u r e , such as g l and o r p l a smace il s. T he mi t oc hond r i a a r e t hen appare n t l y s ca t t e r ed i n a haphaz a rd ma nner .

As ea r l y a s 1952 , i t was shown t ha t t he basophi l i c "c l um ps" o f Berg ( I ,2 ) p roduced i n l ive r a f t e r p ro l onged f as t i ng and r e f eed i ng cons i s ted o f densec l o t s o f e rgas t op l asm and m i t ocho ndr i a (3 , 4 ) . T h i s s t udy h as been t aken u paga i n b y F a wce t t who w i t h a s e r ie s o f exce l len t mi c rograp hs has conf i rmedt h i s obse rva t i on (5 ) .

T he p resen t work was p l anned i n o rde r t o s t udy t he f i r s t s t ages i n t he de -ve l op men t o f t hese e rgas t op l asm -mi t ochon dr i a l un i t s . T he i dea was t ha tf u r t h e r i n f o r m a t i o n o n t h e m o d e o f fo r m a t i o n o f t h e e r g a s t o p la s m i n h e p a t icce l l s cou l d t hus be ga t he red . I n o rde r t o de t e rmi ne how genera l t he phenome-non i s , hepa t i c ce l l s have been s t ud i ed no t o n l y a f t e r f a s t i ng and r e f eed i ngbut a l so in t he cour se o f i n t ense r egenera t i on fo l l owi ng pa r t i a l h epa t ec t o myor i n t ox i ca t i on wi t h ca rbon t e t r ach l or i de .

Mat er i a l and Met hodsAll the experiments were carried out on rats. Regeneration after fasting was secured byLage rstedt's method (6). In all, 26 animals were fasted for 5 days and refed after this periodon a protein-rich diet. They were killed I, 6, 15, 24, 48 hours after the first meal. In anotherseries of experiments 16 rats were partially hepatectomized (about two-thirds of the livervolume) following the usual m ethod (7). Hepatic regeneration was studied 6, 18, 24, 30, 36hours after the operation.Lastly, 39 animals were injected intraperitoneally with carbon tetrachloride (60 mg. foreach 100 gm. of weight) dissolved in olive oil (0.2 gin. for 1 rnl.). Different stages from 30minutes to 6 days after the injection were studied.* This investigation was supported by a gra nt from the Mutuelle C_~n6rale de l'Edu cationNationale.

73~. BIOPHY SIC. AND BIOCH~ZM.CYTOL., 1956, Vol. 2, No . 4, Sup pl.


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74 M I T O C H O N D R I A A N D E R G A S T O P L A S M O F L I V E R C E L L SAll the fragments were fixed in 1 per ce nt buffered osmium tetroxide (Palade), dehy-drated in alcohol, and embedde d in butyl-methacrylate. Sections we re cut with the Porter-Blum m icrotome and examined under the RCA EM U-2E electron microscope.

R E S U L T S

(a) Ergastoplasmic Regenerat ion in L ivers of An im als Refed af ter Sta rva t io n. -As p rev io u s s t u d i es h av e sh o wn , c y to p l asm ic b aso p h i l i a d ec reases sh arp ly i fn o t t o t a l l y a f t e r a f e w d a y s ' s t a r v a t io n . T h i s is a c c o m p a n i e d b y a d i m i n u t i o no r a d i sap p earan ce o f th e e rg as to p l asm (3 , 4 , 5 ) . Th e p ro m in en ce o f t h ese cy to -l o g i ca l a l t e r a t i o n s v a r i es g rea t l y fo r t h e same p er io d o f s t a rv a t i o n wi th t h es t r a in , t h e ag e , t h e p reced in g n u t r i t i o n a l s t a t e o f t h e an imal . S o me ma y d i ea f t e r 4 d a y s o f s t a r v a t i o n o r s u r v iv e u p t o 2 8 d a y s w i t h o u t s h o w i n g a n y m o d -i f i ca t i o n i n t h e i r v i t a l i t y . Th e an imal s i n o u r ex p er imen t s were i n a b ad s t a t ea f t e r 5 d ay s o f s t a rv a t i o n an d mo s t o f t h e i r h ep a t i c ce l l s h ad l o s t t h e i r e rg as -t o p l asm. Al l t h e i r mi to ch o n d r i a were swo l l en , sh o win g t h e t y p i ca l a sp ec t o ft h e " c lo u d y swel li n g " d esc r i b ed b y Gan s l e r an d Ro u i l l e r (8 ) . Du r in g t h ef i r s t h o u r s a f t e r r e f e e d i n g t h e m a j o r i t y o f t h e m i t o c h o n d r i a c a m e b a c k t on o rm al ; so me r emain e d swo l l en fo r 1 2 t o 1 8 h o u r s . W h ate v er t h e s t a t e o f t h em i t o c h o n d r i a , e r g a s t o p l a s m i c s t r u c t u r e s c a n b e o b s e r v e d t o r e t u r n a r o u n dt h e 6 t h h o u r , b u t t h e b e s t e x a m p l e s o f e r g a st o p l a sm i n t h e m i d s t o f r e g e n e r a -t i o n a r e t o b e fo u n d i n an imal s h av in g b een r e f ed fo r 1 6 t o 2 4 h o u r s (F ig s .1 an d 2 ) . A t t h i s s t ag e o f i n t en se sy n th es i s , t h e e rg as to p l asmic s t ru c tu resf o r m c o m p a c t g r o u p s e n c l o s i n g t h e m i t o c h o n d r i a . T h e y a r e d i s t r i b u t e d i na reas a lo n g t h e n u c l ea r memb ran e (F ig . 1 ) o r i n t h e mid s t o f t h e h y a lo p l asmo r ag a in a lo n g th e ce l l me m b ran e (F ig . 2 ). Tw o f ac t s mu s t b e s t r es sed : ( a )t h e e r g a s t o p l a s m i c m e m b r a n e s r e a p p e a r simultaneously a t t h e p e r i p h e r y o ft h e ce l l an d a lo n g t h e n u c l ea r en v e lo p e ; (b ) all o f t h e m i t o c h o n d r i a a r e i nc lo se co n t ac t w i th t h e e rg as to p l asm.

F r o m t h e s e o b s e r v a t i o n s i t c a n b e i n f e r r e d t h a t ( a ) n o d e n s i t y g r a d i e n t o fb aso p h i l i a , d ec reas in g f ro m n u c l eu s t o ce l l su r f ace , is d e t ec t a b l e ; ( b) t h e ch o n -d r i o m a p l a y s a n i m p o r t a n t p a r t i n t h e e l a b o r a t i o n o f b a s o p h i l i c s u b s t a n c ea s L u d f o r d a n d o t h e r s h a d a l r e a d y s u s p e c t e d ( 1 0 ) .

I t m i g h t b e s u p p o s e d t h a t t h e d e v e l o p m e n t o f e rg a s t o pl a s m i c m e m b r a n e sa lo n g o th e r memb ran es (n u c l ea r , ce l l u l a r , o r mi to ch o n d r i a l ) i s p u re ly a p h y s i -ca l p h en o men o n , t h e p re -ex i s t i n g su r f aces ac t i n g mere ly as a mech an i ca lsu p p o r t . H o we v er , t h ese e rg as to p l asmic lamel l ae n ev er ap p e ar i n a co n cen t r i ca n d r e g u l a r p a t t e r n a r o u n d t h e m e m b r a n e s a n d a r e r a t h e r g r o u p e d e s s en t i al l ywh ere t h e c o n cen t r a t i o n o f mi to ch o n d r i a ( r i ch , a s we k n o w , i n en zy m es) i sg rea t es t . F u r th e r , i t is t o b e n o t ed t h a t t h e su r f ace o f l i p id e i n c lu s io n s i s n e v erth u s en c i r c l ed b y e rg as to p l asm.D u r i n g t h e f i r st p e r i o d o f e r g a s to p l a s m f o r m a t i o n a r o u n d m i t o c h o n d r ia , o n ec a n f o l l o w t h e d e t a i l s o f i t s d e v e l o p m e n t . T h e m e m b r a n e s g e n e r a l l y p r e c e d eth e ap p earan ce o f t h e g ran u l es d esc r i b ed b y P a l ad e (1 1 , 1 2 ) . F l a t t en ed v es i c l es


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76 M I T O C H O N D R I A A N D E R G A S T O P L A S M O F L I V E R C E L L Sp h y s io lo g i ca l b eh av io r o f t h e o rg an e l l es s t u d i ed an d p erh ap s ab o u t t h e i ror ig in .

Af t e r t h r ee d i f f e r en t i n i t i a l i n ju r i es , t h e l i v e r ce l l s r esp o n d ed i n a s imi l a rm a n n e r . T h e e r g a s t o p l a s m r e a p p e a r e d i n c l o s e c o n t a c t w i t h t h e m i t o c h o n d r i aw h e r e v e r t h e y w e r e l o c a t e d , e i t h e r c l o s e t o t h e n u c l e a r m e m b r a n e o r i n t h ep er ip h ery o f t h e ce l l . Th i s t o p o g rap h i ca l r e l a t i o n sh ip b e tween mi to ch o n d r i aan d cy to p l asmic b aso p h i l i a i n l i v e r ce l l s i n d i ca t es wi th o u t an y d o u b t a phys io-logical relationship between thes e two cell organelles. Th e ro l e o f t h e n u c l eu s i nth e sy n th es i s o f cy to p l asmic n u c l eo p ro t e in s i s ce r t a i n b u t i t s eems t o u s t h a tm i t o c h o n d r i a a l s o p l a y a n i m p o r t a n t r o t e , a t l e a s t a t c e r t a i n s t a g e s o f t h ep ro d u c t i o n o f e rg as to p l asm. I t i s p ro b ab l e t h a t t h i s i n t im at e r e l a t i o n sh ipb e t w e e n m i t o c h o n d r i a a n d e r g a s t o p l a s m s t i ll e x i st s w h e n t h e l a t t e r h a s r e a c h e di t s n o rmal d ev e lo p men t , wh en , mo rp h o lo g i ca l l y , t h e as so c i a t i o n b e tween t h etwo o rg an e l l es i s l e s s o b v io u s t h an a t t h e d e f i n i t e s t ag e o f ce l l r eg en era t i o nh ere s t u d i ed . Th e r easo n t h i s a s so c i a t i o n i s d i f f i cu l t t o d e t ec t i n s t a t i c imag esm i g h t w e l l b e t h a t c o n t i n u a l p r o t o p l a s m i c m o v e m e n t s a l l o w o n l y t r a n s i t o r yco n t ac t s .

T h e b e h a v i o r o f m i t o c h o n d r i a a n d e r g a s t o p l a s m i n h e p a t i c c e l l s u n d e ro t h e r e x p e r i m e n t a l c o n d i t i o n s r e m a i n s t o b e s t u d i e d . N o t h i n g i s y e t k n o w no f t h e e rg as to p l asm-mi to ch o n d r i a r e l a t i o n sh ip i n o th e r ce l l s .

S U M M A R Y

1 . In mo s t r a t l i v e r ce l l s , n o sp ec i a l t o p o g rap h i ca l r e l a t i o n sh ip b e tweenmi to ch o n d r i a an d e rg as to p l asmic l amel l ae i s t o b e o b se rv ed . In so me ce l l s ,n ev er th e l es s , t h e two o rg an e l l es a r e g ro u p ed t o g e th er i n d en se zo n es c l ea r l ys e p a r a t e d f r o m t h e h y a l o p la s m .2 . S u ch an as so c i a t i o n can b e p ro d u ced a t w i l l i n t h e l i v e r s o f an imal s r e f edaf t e r p ro lo n g ed fas t i n g , o r i n t h e r eg en era t i v e p h ases a f t e r p a r t i a l h ep a t ec -t o m y an d i n to x i ca t i o n wi th ca rb o n t e t r ach lo r i d e . I n a l l t h ese ce ll s, t h e e rg as to -p l a s m , a f t e r h a v i n g d i s a pp e a r e d , s u d d e n l y re a p p e a r s i n th e c y t o p l a s m , e i t h e ra l o n g t h e n u c l e u s o r c e ll m e m b r a n e s , w h e r e t h e m i t o c h o n d r i a a r e g r o u p e d .

3 . I t m a y b e s u p p o s e d t h a t t h e s e t o p o g ra p h i c a l r e la t i o ns h i p s b e t w e e n m i t o -ch o n d r i a an d e rg as to p l asm d u r in g a d e f i n i t e p e r i o d o f ce l l u l a r ac t i v i t y i n d i -ca t e a c l o se fu n c t i o n a l l i n k b e tween ch o n d r io ma an d b aso p h i l i c s t ru c tu res .M i t o c h o n d r i a s e e m t o p l a y a n i m p o r t a n t p a r t i n t h e e l a b o r a t i o n o f h e p a t i ce rg as to p l asm.

B I B L I O G R A P H Y

1. Berg, W., Biochem. Z. , 1914, 61, 428.2. Berg, W., Arch. mikr. Anat., 1920, 94, 518.3. Bernhard, W., Haguenau, F., Gautier, A., and Oberling, C., Z. ZeUforsch., 1952,87, 281.


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W. BERNHARD AND C . ROUILLER 774 . B e r n h a r d , W . , G a u t i e r , A . , a n d R o u i U e r , C . , A rch . ana t . micr . e t morpho l , exp . ,1954, 43~ 236.5 . F a w c e t t , D . W . , J . N a t . C a n c e r I n s t . , 1955 , 15 , No . 5 , supp l .6 . L a g e r s t e d t , S . , A c t a A n a t . , 1949, 1, suppl. , 9 , 1 .7 . D r o c h m a n s , P . , Arch. biol . , 1950, 61, 475.8 . Gan s le r , I -I ., and Rou i l le r , C . , Schwei z . Z . P a tho l . u . B ak t . , 1956 , in p ress .9 . C a s p e r s s o n , T . , C e l l G r o w t h a n d C e l l F u n c t i o n , N e w Y o r k , W . W : N o r t o n & C o . ,

1950.10 . Ludford , R . J . , Smi les , J . , and Welch , F . V. , N a t u r e , 1948, 162, 650.11 . Pa lade , G. E . , J . B iophys i c . and B iochem. Cy to l . , 1955, 1, 59.12 . Pa lade , G. E . , J . B iophys i c . and B iochem. Cy to l . , 1955 , 6 , 567 .13 . Pa lade , G. E . , J . B iophys i c . and B iochem. Cy to l . , 1956 , 2 , No . 4 , supp l . , 85 .


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78 MITOCHONDRIA AND ERGASTOPLASM OF LIVER C E L L S

E X P L A N A T I O N O F P L A T E SPLATE 22

F I 6 . 1 . L i v e r o f r a t s t a r v e d f o r 5 d a y s a n d r e f e d a f t e r 2 4 h o u r s . E r g a s t o p l a s m ( e r )r e a p p e a r s i n l i m i t e d a re a s a l o n g t h e n u c l e a r m e m b r a n e a n d a l so a r o u n d t h e p e r i p h e r ie so f t h e m i t o c h o n d r i a . N o t e t h e h y a l o p l a s m ( b y ) f r e e o f b o t h o r g a n d i e s , n u , nuc leo lus .X 25,000.


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THE JOURNAL OFBIOPHYSICAL AND B~CHEMIC~CYTOLOGY

PLATE 22VOL. 2

(Bernhard and Rouiller: Mitochondria and ergastoplasm of liver cells)


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PraTE 23FIG. 2. Liver of rat starv ed for 5 days and refed after 15 hours. Peripheral portio n

of two parenchymatous cells (cl and c2) in order to show the reappearance of the er-gastoplasmic structures along both the cell membrane (M) and the mitochondrial sur-faces. Arrows point to show init iat ing ergastoplasm. Memb ranes outl ining clefts orcavit ies appear first . They are later co vered with ergastoplasmic granules, by, hy -aloplasm; er, ergastoplasm. X 54,000.


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T H E J O U R N A L O FBI OP HYSI CAL AND BI OCHEMI CALCY TO LO G Y

PLA TE 2 3VOL. 2

(Bernhard and Rouiller: Mitochondria and ergastoplasm of liver cells)


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PLATE 24FIG. 3. Liver of rat starved for 5 days and refed after 15 hours. Developing er-

gastoplasmic formations st i l l almost entirely free of nudeoprotein granules. N,nucleus; er , ergastoplasm; m, mitochondria; by , hyaloplasm. X 56,000.

FIG. 4. Rat l iver 24 hours after partial hepatectomy. Reappearance of ergasto-plasmic structures (er) in contact with and close to mitochondria, rob, microbodies.X 34,000.


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T H E J O U R N A L O FBIOPHYSICAL AND BIOCHEMICALC Y T O L O G Y

P L A T E 2 4VOL. 2

(Bernhard and Rouil ler : Mitochondria and ergastoplasm of l iver cells)


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PLATE 25FIG. 5. Ra t liver 6 days a fter carbon tetrachloride poisoning. I n the hy aloplasm,ergastoplasm (er) appears first around scattered mitochondria. X 35,000.


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THE JOURNAL OFBIOPHYSICAL AND BIOCHEMICALCYTOLOGY

PLATE 2 5VOL. 2

(Bernhard and Roui]ler: Mitochondria and ergastoplasm of liver cells)

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