546
Specialia
a t e d l i v e r cells receive a ' n o r m a l ' e n z y m e c o n t e n t . B e c o m i n g older a n d m o v i n g t o t h e v e n a centralis, t h i s e n z y m e c o n t e n t c h a n g e s , d e p e n d i n g o n age a n d diet.
EXPERIENTIA 29/5
p o r t s c h e n L e b e r a c i n u s a b g e l a g e r t wird, was d u r c h d e n A b b a u y o n E n z y m e n , die in d e n Z o n e n 2 u n d 3 a m G l y k o g e n a u f b a u b e t e i l i g t sind u n d d u t c h die physiologische R e g e n e r a t i o n i n d e r Zone 1 v e r u r s a c h t wird.
Zusammen/assung. Nachweis, dass n a c h f e t t r e i c h e r D i ~ t G l y k o g e n h a u p t s $ c h l i c h in der Zone 1 des R a p p a -
W.
DEN
OTTER,
L. F. BLIKKENDAAL-LIEFTINCK a n d
J. W. I~OTEN 8 8 Acknowledgments. The authors are grateful to Prof. A. DF MINJER and Prof. L. W. J. HOLLE~AN for support and stimulating discussions. We acknowledge Mr. G. TUIT and Miss T. DEIJS for skilful technical assistance and Mr. A. J. H. HOSE~ANS for preparing photomicrographs.
Department o/Pathology, State University, Pasteurstraat 2, Utrecht (The Netherlands), 19 October 7972.
T h e Effect of T e m p e r a t u r e on N u c l e a r P e r m e a b i l i t y I t h a s b e e n s h o w n t h a t t h e t r a n s f e r of R N A f r o m t h e n u c l e u s to t h e c y t o p l a s m is a t e m p e r a t u r e d e p e n d e n t process 1-3. T h e s e studies, h o w e v e r , do n o t d i s t i n g u i s h b e t w e e n t h e effect of t e m p e r a t u r e o n t h e p r o c e s s i n g of R N A w i t h i n t h e n u c l e u s a n d its effect o n t h e p e r m e a b i l i t y of t h e n u c l e a r e n v e l o p e t o m a c r o m o l e c u l e s . I n t h i s invest i g a t i o n t h e a c t i o n of t e m p e r a t u r e specifically on t h e e x c h a n g e process w a s s t u d i e d b y i n j e c t i n g colloidal gold particles, c o a t e d w i t h p o l y v i n y l p y r r o l i d o n e , i n t o t h e cytop l a s m of t h e m u l t i n u c l e a t e d a m o e b a Chaos chaos. T h e i n j e c t e d cells were i n c u b a t e d a t d i f f e r e n t t e m p e r a t u r e s a n d t h e i n t r a c e l l u l a r d i s t r i b u t i o n of t h e p a r t i c l e s d e t e r m i -
An electron micrograph of a permeable nucleus foliowing incubation at 2~ The gold particles (smalI arrows) are concentrated in the nucleoplasm (N). Breaks in the nuclear envelope are apparent (large arrows). Small vesicles (V), possibly fragments of the envelope, are frequently associated with permeable nuclei. In this instance the vesicles are restricted to the cytoplasm, but they have also been seen in the nueleoplasm.
n e d w i t h t h e e l e c t r o n microscope. Since t h e s e particles are inert, a n d n o t a l t e r e d b y t e m p e r a t u r e d e p e n d e n t c h a n g e s in cell m e t a b o l i s m , t h e i r a b i l i t y t o e n t e r t h e n u c l e u s s h o u l d d e p e n d p r i m a r i l y on t h e c h a r a c t e r i s t i c s of t h e n u c l e a r envelope, a s s u m i n g t h a t diffusion w i t h i n t h e c y t o p l a s m is n o t r a t e l i m i t i n g . F u r t h e r m o r e , b o t h colloidal p a r t i c l e s a n d r i b o n u c l e o p r o t e i n s cross t h e n u c l e a r e n v e l o p e t h r o u g h c e n t r a l c h a n n e l s w i t h i n t h e n u c l e a r p o r e s 4, 5. T h u s , v a r i a t i o n s in t h e u p t a k e of gold p a r t i c l e s s h o u l d reflect c h a n g e s in t h e p r o p e r t i e s of t h e p a t h w a y s used for n a t u r a l l y occurring substances. T h e e x p e r i m e n t s were p e r f o r m e d o n well-fed, i n t e r p h a s e a m o e b a e . T h e p r o d e c u r e s for c u l t u r i n g t h e ceils, p r e p a r i n g colloidal gold, m i c r o i n j e c t i o n , a n d e l e c t r o n m i c r o s c o p y h a v e b e e n d e s c r i b e d in p r e v i o u s reports4, s. T w o gold f r a c t i o n s were used; one c o n t a i n e d p a r t i c l e s r a n g i n g f r o m 30-170 A in d i a m e t e r (L-fraction), a n d t h e second c o n t a i n ed 25-55 ~ p a r t i c l e s (S-fraction). T h e a m o e b a e were i n j e c t e d a t r o o m t e m p e r a t u r e ( a p p r o x i m a t e l y 25 ~ a n d left a t t h a t t e m p e r a t u r e or r a p i d l y t r a n s f e r r e d t o a n i n c u b a t o r set a t 34 ~ 10 ~ or 2~ T h e cells were fixed in OsOa, 30 or 50 m i n a f t e r injection, a n d s u b s e q u e n t l y s e c t i o n e d a n d e x a m i n e d w i t h t h e e l e c t r o n microscope, Gold p a r t i c l e s were c o u n t e d a n d m e a s u r e d in a d j a c e n t regions of n u c l e o p l a s m a n d c y t o p l a s m a c c o r d i n g t o t h e methods described earlierL T h e r e s u l t s of t h e 50 m i n e x p e r i m e n t s p e r f o r m e d w i t h t h e L - f r a c t i o n are s h o w n in T a b l e I, A. T h e p e r c e n t of t h e t o t a l p a r t i c l e c o u n t p r e s e n t in t h e n u c l e o p l a s m , d e c r e a s e d as t h e t e m p e r a t u r e was lowered f r o m 34 ~ to 10~ The d e c r e a s e f r o m 34 ~ to 25~ is s t a t i s t i c a l l y s i g n i f i c a n t ( P < 0.025), as i s t h e decrease f r o m 25 ~to 10 ~ ( P < 0.001). Surprisingly, w h e n t h e t e m p e r a t u r e was d r o p p e d t o 2 ~ t h e r e was a n i n c r e a s e in t h e c o n c e n t r a t i o n of p a r t i c l e s in t h e n u c l e o p l a s m , a n d t h e r e s u l t s are n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h o s e o b t a i n e d a t 25~ ( P > 0.5). Closer e x a m i n a t i o n of t h e cells i n c u b a t e d a t 2 ~ s h o w e d t h a t t h e r e were 2 s e p a r a t e p o p u l a t i o n s of nuclei. T h i s w a s n o t t h e case a t h i g h e r t e m p e r a t u r e s . I n one p o p u l a t i o n (nonp e r m e a b l e nuclei), c o n s i s t i n g of a p p r o x i m a t e l y 70~o of
1 K. BIER, Chromosoma 76, 58 (1965). K. ISHIKAWA,C. KURODAand K. OGATA, Biochim. biophys. Acts 179, 316 (1969). 3 M. HORISBERGERand H. AMos, Bioehem. J. 717, 347 (1970). C. FEm)~ERR, J. Cell Biol. 25, 43 (1965). B. J. STFVENS and SWIFT, J. Cell Biol. 31, 55 (1965). 6 C. FELDHFRR, J. Cell Biol. 31, 199 (1966). 7 This investigation was supported, ill part, by the Damon Runyon Memorial Fund (Grant DRG-1152).
15.5. 1973
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547
Table I. Particle distribution Temperature No. of (~ amoebae
No. of nuclei
Totalvolume examined (~zm~) "
No. of particles in cytoplasm
No. of particIes innucleoplasm
Percent of total count in nucleoplasm
High and low value for individual amoebae (%)
A) 50 min experiments - L-fraction 34 5 44 25 11 64 10 7 63 2 5 50
71 78 100 82
8,171 11,058 10,226 6,347
5,997 4,959 586 3,856
46 32 8 32
51-35 47-16 15-2 37-10
B) 30 min experiments - L-fraction 25 6 37 2 6 52
59 84
4,676 5,217
1,176 217
21 3
27-13 7-1
C) 50 min experiments - S-Fraction 25 5 40 10 5 23
64 35
10,544 4,211
11,175 2,037
51 32
63-48 41-26
Half of each value represents nueleoplasm and half cytoplasm.
Table II. Size distribution of particles in nuclei (%) Temp.(~
Number ofparticles measured
0-35 k
35-70 X
70-105 X
105-140 A
>140 X
34 10
1000 600
1 2
52 56
42 41
5 1
0 0
t h e nuclei, t h e c o n c e n t r a t i o n of g o l d p a r t i c l e s in t h e nucleop l a s m was o n l y 3 ~o of t h a t in t h e c y t o p l a s m . T h e r e m a i n d e r of t h e nuclei were p e r m e a b l e , a n d a c t u a l l y c o n t a i n e d h i g h e r c o n c e n t r a t i o n s of gold t h a n t h e c y t o p l a s m . T h e l a t t e r f i n d i n g c a n be e x p l a i n e d b y t h e f a c t t h a t t h e e n v e lopes of t h e p e r m e a b l e nuclei c o n t a i n e d localized regions ill w h i c h b r e a k d o w n of m e m b r a n e s could b e o b s e r v e d (Figure). N o n - p e r m e a b l e a n d p e r m e a b l e nuclei were f r e q u e n t l y f o u n d a d j a c e n t t o o n e a n o t h e r in tile s a m e cell, , i n d i c a t i n g t h a t t h e t e m p e r a t u r e d e p e n d e n t v a r i a t i o n s in gold u p t a k e are l i m i t e d b y t h e n u c l e a r "envelope, r a t h e r t h a n b y v a r i a t i o n in t h e m i g r a t i o n r a t e s of t h e colloidal p a r t i c l e s w i t h i n t h e c y t o p l a s m . T a b l e I , B gives t h e r e s u l t s of e x p e r i m e n t s in w h i c h t h e cells were i n c u b a t e d a t 25~a n d 2 ~ for 30 rain. A t t h i s t i m e i n t e r v a l t h e differences in n u c l e a r u p t a k e are s i g n i f i c a n t ( P < 0.001), a n d a t 2~ o n l y a b o u t 2~o of t h e nuclei were p e r m e a b l e , d e m o n s t r a t ing t h a t t h e r e is e s s e n t i a l l y n o m e m b r a n e b r e a k d o w n d u r i n g t h e first 30 r a i n a t low t e m p e r a t u r e . One e x p l a n a t i o n for t h e o b s e r v e d decreases in n u c l e a r p e r m e a b i l i t y is t h a t l o w e r i n g t h e t e m p e r a t u r e effectively r e d u c e s t h e size of t h e c e n t r a l c h a n n e l s w i t h i n t h e pores. To t e s t t h i s h y p o t h e s i s t h e size distribut_ions of t h e L-fract i o n p a r t i c l e s p r e s e n t in t h e nuclei following i n c u b a t i o n a t 10 ~ a n d 34~ for 50 m i n were d e t e r m i n e d (see T a b l e II). T h e r e is a difference in t h e n u m b e r of p a r t i c l e s a t t h e u p p e r l i m i t of t h e p e r m e a b i l i t y range, b u t i t is u n l i k e l y t h a t t h i s could a c c o u n t for t h e differences in t h e u p t a k e r a t e s since o n l y a s m a l l p e r c e n t a g e of p a r t i c l e s are i n v o l v e d . F u r t h e r e v i d e n c e t h a t l o w e r i n g t h e t e m p e r a t u r e does n o t s i m p l y e x c l u d e l a r g e r particles, b u t also a l t e r s t h e e x c h a n g e r a t e s of s m a l l p a r t i c l e s was o b t a i n e d b y s t u d y i n g t h e u p t a k e of S - f r a c t i o n p a r t i c l e s a t 25 ~ a n d 10~ (see T a b l e I, C). A l t h o u g h t h i s f r a c t i o n c o n t a i n s p a r t i c l e s w h i c h are well b e l o w t h e size r a n g e (105-140 ~ ) in w h i c h v a r i a t i o n s
in exclusion were d e t e c t e d , t h e r e is still a h i g h l y s i g n i f i c a n t difference in n u c l e a r u p t a k e ( P < 0.001). Ill c o n c l u s i o n i t h a s b e e n s h o w n t h a t t h e r e l a t i v e r a t e s of e x c h a n g e of p a r t i c u l a t e m a t e r i a l across t h e n u c l e a r e n v e l o p e decreases as t h e t e m p e r a t u r e is lowered f r o m 34 ~ to 10~ One m i g h t e x p e c t s i m i l a r t e m p e r a t u r e s effects for t h e e x c h a n g e of r i b o n u c l e o p r o t e i n , since t h i s s u b s t a n c e utilizes t h e s a m e p a t h w a y s as colloidal gold. W h e n t h e cells were i n c u b a t e d a t 2 ~ t h e r e were indicat i o n s of n u c l e a r e n v e l o p e b r e a k d o w n . A l t h o u g h i t c o u l d b e shown that the nuclear envelope represents the temperature d e p e n d e n t b a r r i e r , i t could n o t b e d e t e r m i n e d f r o m t h e a v a i l a b l e d a t a w h e t h e r p h y s i c a l or c h e m i c a l processes are effected. I t was e v i d e n t h o w e v e r , t h a t t h e o b s e r v e d differences in t h e c o n c e n t r a t i o n of gold w i t h i n t h e n u c l e o p l a s m were n o t d u e s i m p l y t o c h a n g e s in t h e d i m e n sions of t h e c e n t r a l c h a n n e l s .
Zusammenjassung. Die r e l a t i v e T r a n s p o r t g e s c h w i n d i g k e i t v o n k o l l o i d a l e m Gold d u r c h die M e n l b r a n des Zellk e r n s v e r r i n g e r t sich, w e n n die T e m p e r a t u r y o n 34 ~ a u f 10 ~ h e r a b g e s e t z t wird. E i n ~ihnlicher T e m p e r a t u r e f f e k t d a r t fiir d e n T r a n s p o r t v o n R i b o n u k l e o p r o t e i n e n a n g e n o m m e n werden, d a diese S u b s t a n z e n d e n s e l b e n W e g b e n i i t z e n wie kolloidale Teilctlen. W e r d e n die Zellen bei 2 ~ i n k u b i e r t , so m a c h e n sich A n z e i c h e n e i n e r Z e r s t 6 r u n g der K e r n m e m b r a n e b e m e r k b a r . Die b e o b a c h t e t e n K o n z e n t r a t i o n s u n t e r s c h i e d e y o n k o l l o i d a l e m Gold i n n e r h a l b des K e r n p l a s m a s k o n n t e n n i c h t d u r c h G r 6 s s e n ~ i n d e r u n g e n der Zentralkan~ile in d e n K e r n p o r e n erkl~irt werden. CARL M. FELDHERR
Department o/ Pathology University o/Florida, College o/Medicine Gainesville, Florida 32601 (USA), 25 September 7972.