- Email: [email protected]
Dynamic organization of the axonal cytoskeleton: 1. Neurofilament proteins
$27
6. Axoplasmic transport DYNAMIC
ORGANIZATION
OF T H E A X O N A L
CYTOSKELETON;
I. N e u r o f i l a m e n t
proteins.
Y O S H I A K I K O M I Y A & T O M O K O T A S H I R O , D e p a r t m e n t of B i o c h e m i s t r y , I n s t i t u t e of B r a i n R e s e a r c h , T h e U n i v e r s i t y of T o k y o F a c u l t y of M e d i c i n e , H o n g o , T o k y o 113, J a p a n . A x o n a l t r a n s p o r t of p h o s p h o r y l a t e d neurofilament proteins in t h e s c i a t i c n e r v e of rats w a s i n v e s t i g a t e d b y i n j e c t i n g [ 3 2 p ] - o r t h o p h o s p h a t e ( 3 2 p i ) i n t o the fourth lumbar dorsal ganglion. Major phosphorylated axonal proteins migrating s l o w l y w e r e i d e n t i f i e d as 2 0 0 K a n d 160K s u b u n i t s of the n e u r o f i l a m e n t t r i p l e t by o n e - a n d t w o - d i m e n s i o n a l electrophoresis. When labelled with 32Pi, a small portion of neurofilament p r o t e i n s w a s f o u n d to be m i g r a t i n g at a r a t e c o n s i d e r a b l y f a s t e r t h a n t h a t of ordinary neurofilaments labelled with [3sS]-methionine. The faster moving portion s e e m e d to be t r a n s p o r t e d at an a l m o s t e q u a l r a t e b e t w e e n two b r a n c h e s of b i f u r c a t i n g a x o n s of d o r s a l r o o t g a n g l i o n c e l l s , t h o u g h the m a j o r p o r t i o n w i t h the o r d i n a r y s l o w r a t e w a s f o u n d to m i g r a t e m u c h s l o w e r in the c e n t r a l b r a n c h t h a n in the p e r i p h e r a l one. Two subpopulations of a x o n a l n e u r o f i l a m e n t p r o t e i n s w e r e s e p a r a t e d b y I% T r i t o n t r e a t m e n t , s o l u b l e a n d i n s o l u b l e . T h e f o r m e r f r a c t i o n was n e g r i g i b l y obtained when labelled with [35S]-methionine. In c o n t r a s t a c o m p a r a b l e a m o u n t of the s o l u b l e f r a c t i o n to the i n s o l u b l e one w a s r e c o v e r e d w h e n l a b e l l e d w i t h 32pi. ]'he s o l u b l e s u b p o p u l a t i o n s e e m e d to h a v e a s l i g h t l y f a s t e r r a t e of t r a n s p o r t t h a n the i n s o l u b l e one. W h e n 32pi w a s i n j e c t e d s u b - e p i n e u r a l l y into the s c i a t i c n e r v e b u n d l e , 2 0 0 K a n d 160K s u b u n i t s of n e u r o f i l a m e n t p r o t e i n s w e r e p h o s p h o r y l a t e d in situ, a n d s u b j e c t e d to m o v e o n l y d i s t a l l y f r o m t h e s i t e of i n j e c t i o n at t h e o r d i n a r y slow rate. F r o m the r e s u l t s m e n t i o n e d a b o v e , a x o n a ] n e u r o f i l a m e n t s are s u g g e s t e d to be m i g r a t i n g in the d e p o l y m e r i z e d form, which may correspond to a T r i t o n - s o l u b l e subpopulation, a n d to u n d e r g o d y n a m i c interconversion between polymerized and depolymerized forms, t h o u g h the l a t t e r o n e s e e m s to be s m a l l in a m o u n t , which m a y be r e g u l a t e d b y p h o s p h o r y l a t i o n a n d d e p h o s p h o r y l a t i o n of t h e i r s u b u n i t s .
DYNAMIC
ORGANIZATION
OF T H E A X O N A L
CYTOSKELETON
: 2.Microtubule
T O M O K O T A S H I R O & Y O S H I A K I K O M I Y A , D e p a r t m e n t of B i o c h e m i s t r y , R e s e a r c h , T h e U n i v e r s i t y of T o k y o F a c u l t y of M e d i c i n e , H o n g o ,
proteins.
I n s t i t u t e of B r a i n T o k y o 113, J a p a n .
Characteristic organization of t h e a x o n a l m i c r o t u b u l e p r o t e i n s w a s i n v e s t i g a t e d b y a n a l y z i n g the s l o w a x o n a l t r a n s p o r t in the rat s c i a t i c n e r v e a f t e r inj e c t i o n of L - [ 3 5 S ] m e t h i o n i n e i n t o t h e L4 d o r s a l r o o t g a n g l i o n or L 3 - L s l u m b a r spinal cord. A large proportion ( 4 0 - ? 0 % ) of r a d i o l a b e l e d t u b u l i n t r a n s p o r t e d in the a x o n was f o u n d to be i n s o l u b l e w h e n e x t r a c t e d w i t h l% T r i t o n at 4°C . T h i s c o l d - s t a b l e population was also not solubilized under other microtubule-depolymerizing condit i o n s s u c h as in the p r e s e n c e of c a l c i u m , c o l c h i c i n e or n o c o d a z o l e , suggesting t h a t it is in a f o r m d i f f e r e n t f r o m the d y n a m i c m i c r o t u b u l e s in e q u i l i b r i u m w i t h the s o l u b l e d i m e r . T h e c o l d - s t a b i l i t y d o e s n o t a r i s e s o l e l y f r o m the i n t e r a c t i o n with neurofilaments (NF) s i n c e s u b s t a n t i a l a m o u n t s of t u b u l i n r e m a i n e d i n s o l u b l e even after extraction of NF proteins from the cold-insoluble fraction with 4M urea. F r o m the c o l d - l a b i l e f r a c t i o n , m i c r o t u b u l e s w e r e o b t a i n e d w h i c h c o n t a i n e d a d i s t i n c t set of m i c r o t u b u l e - a s s o c i a t e d p r o t e i n s (MAPs). Analysis of t h e t r a n s p o r t pattern of both the cold-stable and -labile t u b u l i n I-4 w e e k s a f t e r i s o t o p e i n j e c t i o n r e v e a l e d the t i m e - d e p e n d e n t b r o a d e n i n g of t h e m a i n w a v e ( l a b i l e / s t a b l e ratio;0.5-0.8) a n d t h e s e p a r a t i o n of a f a s t e r m i g r a t i n g w a v e e n r i c h e d in the c o l d - l a b i l e t u b u l i n ( l a b i l e / s t a b l e r a t i o ; Z - 3 ) . The microtubule-stabilizing alkaloid Taxol, when applied l o c a l l y to t h e axons by injecting into the sciatic nerve bundle, inhibited the transport of t u b u l i n . T a x o l h a d no e f f e c t on the f a s t t r a n s p o r t . F r o m t h e s e r e s u l t s it is c o n c l u d e d t h a t a x o n a l m i c r o t u b u l e s e x i s t in t h e two forms, the s t a b l e f o r m a n d the d y n a m i c form. W h i l e v e s i c l e t r a n s p o r t o c c u r s a l o n g p o l y m e r i z e d m i c r o t u b u l e s , t u b u l i n i t s e l f is t r a n s p o r t e d t h r o u g h t h e d y n a m i c form, p o s s i b l y in the d e p o l y m e r i z e d s t a t e . As the s t a b l e p o p u l a t i o n a l s o a p p a r e n t l y und e r g o e s t r a n s p o r t , a r e v e r s i b l e e x c h a n g e b e t w e e n the s t a b l e a n d the d y n a m i c f o r m s is s u g g e s t e d .
6. Axoplasmic transport DYNAMIC
ORGANIZATION
OF T H E A X O N A L
CYTOSKELETON;
I. N e u r o f i l a m e n t
proteins.
Y O S H I A K I K O M I Y A & T O M O K O T A S H I R O , D e p a r t m e n t of B i o c h e m i s t r y , I n s t i t u t e of B r a i n R e s e a r c h , T h e U n i v e r s i t y of T o k y o F a c u l t y of M e d i c i n e , H o n g o , T o k y o 113, J a p a n . A x o n a l t r a n s p o r t of p h o s p h o r y l a t e d neurofilament proteins in t h e s c i a t i c n e r v e of rats w a s i n v e s t i g a t e d b y i n j e c t i n g [ 3 2 p ] - o r t h o p h o s p h a t e ( 3 2 p i ) i n t o the fourth lumbar dorsal ganglion. Major phosphorylated axonal proteins migrating s l o w l y w e r e i d e n t i f i e d as 2 0 0 K a n d 160K s u b u n i t s of the n e u r o f i l a m e n t t r i p l e t by o n e - a n d t w o - d i m e n s i o n a l electrophoresis. When labelled with 32Pi, a small portion of neurofilament p r o t e i n s w a s f o u n d to be m i g r a t i n g at a r a t e c o n s i d e r a b l y f a s t e r t h a n t h a t of ordinary neurofilaments labelled with [3sS]-methionine. The faster moving portion s e e m e d to be t r a n s p o r t e d at an a l m o s t e q u a l r a t e b e t w e e n two b r a n c h e s of b i f u r c a t i n g a x o n s of d o r s a l r o o t g a n g l i o n c e l l s , t h o u g h the m a j o r p o r t i o n w i t h the o r d i n a r y s l o w r a t e w a s f o u n d to m i g r a t e m u c h s l o w e r in the c e n t r a l b r a n c h t h a n in the p e r i p h e r a l one. Two subpopulations of a x o n a l n e u r o f i l a m e n t p r o t e i n s w e r e s e p a r a t e d b y I% T r i t o n t r e a t m e n t , s o l u b l e a n d i n s o l u b l e . T h e f o r m e r f r a c t i o n was n e g r i g i b l y obtained when labelled with [35S]-methionine. In c o n t r a s t a c o m p a r a b l e a m o u n t of the s o l u b l e f r a c t i o n to the i n s o l u b l e one w a s r e c o v e r e d w h e n l a b e l l e d w i t h 32pi. ]'he s o l u b l e s u b p o p u l a t i o n s e e m e d to h a v e a s l i g h t l y f a s t e r r a t e of t r a n s p o r t t h a n the i n s o l u b l e one. W h e n 32pi w a s i n j e c t e d s u b - e p i n e u r a l l y into the s c i a t i c n e r v e b u n d l e , 2 0 0 K a n d 160K s u b u n i t s of n e u r o f i l a m e n t p r o t e i n s w e r e p h o s p h o r y l a t e d in situ, a n d s u b j e c t e d to m o v e o n l y d i s t a l l y f r o m t h e s i t e of i n j e c t i o n at t h e o r d i n a r y slow rate. F r o m the r e s u l t s m e n t i o n e d a b o v e , a x o n a ] n e u r o f i l a m e n t s are s u g g e s t e d to be m i g r a t i n g in the d e p o l y m e r i z e d form, which may correspond to a T r i t o n - s o l u b l e subpopulation, a n d to u n d e r g o d y n a m i c interconversion between polymerized and depolymerized forms, t h o u g h the l a t t e r o n e s e e m s to be s m a l l in a m o u n t , which m a y be r e g u l a t e d b y p h o s p h o r y l a t i o n a n d d e p h o s p h o r y l a t i o n of t h e i r s u b u n i t s .
DYNAMIC
ORGANIZATION
OF T H E A X O N A L
CYTOSKELETON
: 2.Microtubule
T O M O K O T A S H I R O & Y O S H I A K I K O M I Y A , D e p a r t m e n t of B i o c h e m i s t r y , R e s e a r c h , T h e U n i v e r s i t y of T o k y o F a c u l t y of M e d i c i n e , H o n g o ,
proteins.
I n s t i t u t e of B r a i n T o k y o 113, J a p a n .
Characteristic organization of t h e a x o n a l m i c r o t u b u l e p r o t e i n s w a s i n v e s t i g a t e d b y a n a l y z i n g the s l o w a x o n a l t r a n s p o r t in the rat s c i a t i c n e r v e a f t e r inj e c t i o n of L - [ 3 5 S ] m e t h i o n i n e i n t o t h e L4 d o r s a l r o o t g a n g l i o n or L 3 - L s l u m b a r spinal cord. A large proportion ( 4 0 - ? 0 % ) of r a d i o l a b e l e d t u b u l i n t r a n s p o r t e d in the a x o n was f o u n d to be i n s o l u b l e w h e n e x t r a c t e d w i t h l% T r i t o n at 4°C . T h i s c o l d - s t a b l e population was also not solubilized under other microtubule-depolymerizing condit i o n s s u c h as in the p r e s e n c e of c a l c i u m , c o l c h i c i n e or n o c o d a z o l e , suggesting t h a t it is in a f o r m d i f f e r e n t f r o m the d y n a m i c m i c r o t u b u l e s in e q u i l i b r i u m w i t h the s o l u b l e d i m e r . T h e c o l d - s t a b i l i t y d o e s n o t a r i s e s o l e l y f r o m the i n t e r a c t i o n with neurofilaments (NF) s i n c e s u b s t a n t i a l a m o u n t s of t u b u l i n r e m a i n e d i n s o l u b l e even after extraction of NF proteins from the cold-insoluble fraction with 4M urea. F r o m the c o l d - l a b i l e f r a c t i o n , m i c r o t u b u l e s w e r e o b t a i n e d w h i c h c o n t a i n e d a d i s t i n c t set of m i c r o t u b u l e - a s s o c i a t e d p r o t e i n s (MAPs). Analysis of t h e t r a n s p o r t pattern of both the cold-stable and -labile t u b u l i n I-4 w e e k s a f t e r i s o t o p e i n j e c t i o n r e v e a l e d the t i m e - d e p e n d e n t b r o a d e n i n g of t h e m a i n w a v e ( l a b i l e / s t a b l e ratio;0.5-0.8) a n d t h e s e p a r a t i o n of a f a s t e r m i g r a t i n g w a v e e n r i c h e d in the c o l d - l a b i l e t u b u l i n ( l a b i l e / s t a b l e r a t i o ; Z - 3 ) . The microtubule-stabilizing alkaloid Taxol, when applied l o c a l l y to t h e axons by injecting into the sciatic nerve bundle, inhibited the transport of t u b u l i n . T a x o l h a d no e f f e c t on the f a s t t r a n s p o r t . F r o m t h e s e r e s u l t s it is c o n c l u d e d t h a t a x o n a l m i c r o t u b u l e s e x i s t in t h e two forms, the s t a b l e f o r m a n d the d y n a m i c form. W h i l e v e s i c l e t r a n s p o r t o c c u r s a l o n g p o l y m e r i z e d m i c r o t u b u l e s , t u b u l i n i t s e l f is t r a n s p o r t e d t h r o u g h t h e d y n a m i c form, p o s s i b l y in the d e p o l y m e r i z e d s t a t e . As the s t a b l e p o p u l a t i o n a l s o a p p a r e n t l y und e r g o e s t r a n s p o r t , a r e v e r s i b l e e x c h a n g e b e t w e e n the s t a b l e a n d the d y n a m i c f o r m s is s u g g e s t e d .