BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1345-1352
Vol. 186, No. 3,1992
August 14, 1992
AT
L O C A L I Z A T I O N OF stag p 2 5 A / r a b 3 A p25, A SMALL GTP-BINDING PROTEIN, A C T I V E ZONE OF THE R A T N E U R O M U S C U L A R JUNCTION 1
THE
Akira Mizoguchi2~, Masahiko Arakawa*, Motomaru Masutani*, Akira Tamekane*, Hideyuki Yamaguchi**, Naoki Minami**, Yoshimi Takai***,
and Chizuka Ide*
Departments of *Anatomy and *** Biochemistry Kobe University School of Medicine, Kobe 650, Japan **Nippon Bio-Rad Laboratories, Tokyo 104, Japan Received June 30, 1992
Summary:
s m g p 2 5 A is a small G p r o t e i n w h i c h has b e e n s u g g e s t e d to r e g u l a t e n e u r o t r a n s m i t t e r r e l e a s e from the synapses. We i n v e s t i g a t e d here the u l t r a s t r u c t u r a l l o c a l i z a t i o n of this small G p r o t e i n in the rat n e u r o m u s c u l a r junction by an i m m u n o p e r o x i d a s e method. The results s h o w e d that s m g p 2 5 A was d i s t r i b u t e d n o n - u n i f o r m l y on the p r e s y n a p t i c p l a s m a m e m b r a n e and a m o n g the s y n a p t i c v e s i c l e s w i t h the focal accumul a t i o n on the d i s c r e t e p r e s y n a p t i c sites w h i c h c o r r e s p o n d e d to the a c t i v e zones, the r e g i o n s of the p r e s y n a p t i c p l a s m a m e m b r a n e specialized for the e x o c y t o s i s of the s y n a p t i c vesicles. This u n i q u e distrib u t i o n of s m g p 2 5 A suggests that it plays an i m p o r t a n t role in the a t t a c h m e n t and fusion of the s y n a p t i c v e s i c l e s w i t h the a c t i v e zones. @ 1992 A c a d e m i c
Press,
There more than
is the
vesicle
and has b e e n synapses
(for reviews,
transport
is a small
suggested
(3,4)
cells
proteins
of small G p r o t e i n s see Refs.
of m o r e t h a n t w e n t y m e m b e r s
p 2 5 A / r a b 3 A p25
crine
superfamily
forty m e m b e r s
consisting cellular
Inc.
as well
(5,6).
have t w o
such as e n d o c y t o s i s G protein
to r e g u l a t e as s e c r e t i o n
Like
has b e e n
1,2).
to this
in intra-
the
GTP-bound
f r o m the
cells
tab f a m i l y
active
smg
rab f a m i l y
release
from the e n d o c r i n e
of
The tab family
and e x o c y t o s i s ,
neurotransmitter
forms,
consists
implicated
which belongs
all other G proteins,
interconvertible
which
and exosmall
G
and GDP-
I T h i s w o r k was s u p p o r t e d b y g r a n t s - i n - a i d for s c i e n t i f i c r e s e a r c h f r o m the M i n i s t r y of Education, Science, and Culture, Japan, and by a grant f r o m O S A K A C a n c e r R e s e a r c h F u n d to A. M i z o g u c h i a n d C. Ide. 2 To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .
0006-291X/92 $4.00 1345
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
bound
inactive
proteins
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
forms.
The m o d e of a c t i o n of the t a b
in i n t r a c e l l u l a r v e s i c l e t r a n s p o r t
GTP-bound active
f a m i l y small G
is s p e c u l a t e d t h a t the
f o r m l o c a t e d on a d o n o r v e s i c l e
f u n c t i o n s to c o n f i r m
the c o r r e c t a t t a c h m e n t a n d f u s i o n of the d o n o r v e s i c l e tot m e m b r a n e . membrane the
cytosol.
active
After
and the
fusion,
resultant
h y d r o l y s i s of GTP o c c u r s
GDP-bound inactive
After GDP/GTP
exchange
in the
a c t i o n of the
small G p r o t e i n s
In the c a s e of s m g p25A, on s y n a p t i c v e s i c l e s
cytosol,
the G T P - b o u n d
However,
inhibitor
and the r e g u l a t o r y p r o t e i n w h i c h i n d u c e s
is i d e n t i f i e d and n a m e d
(smg p25 GDI)
On the o t h e r hand, synaptic vesicles
9,10) .
f r o m the
s m g p25 GDP
synap-
dissociation
(7,8). it is w e l l k n o w n t h a t
in the s y n a p s e s ,
are a r r a n g e d not at r a n d o m but
m a n n e r to p e r f o r m the e x o - e n d o c y t o t i c see Refs.
this
is p r o v e d to be p r e s e n t
the d i s s o c i a t i o n of the G D P - b o u n d f o r m of t h i s p r o t e i n tic p l a s m a m e m b r a n e
into
has b e e n p o o r l y e l u c i d a t e d .
indeed this protein
(3,4)
on the a c c e p t o r
form dissociates
form reassoeiats with a new donor vesicle.
cyclical
w i t h an a c c e p -
Particularly,
cycles
exocytosis
in a h i g h l y o r g a n i z e d
effectively
of the
the
(for reviews,
synaptic vesicles
is
k n o w n to be a c c o m p l i s h e d o n l y at the v e r y r e s t r i c t e d sites of the presynaptic plasma membrane,
the a c t i v e
acteristics
synapses have been e s t a b l i s h e d
However,
in the m a m m a l i a n
the u l t r a s t r u c t u r a l
has not b e e n
zones,
whose
structural char(11,12).
l o c a l i z a t i o n of s m g p 2 5 A in the s y n a p s e s
fully understood.
In the p r e s e n t
study,
we h a v e
i n v e s t i g a t e d the u l t r a s t r u c t u r a l
l o c a l i z a t i o n of s m g p 2 5 A by an i m m u n o p e r o x i d a s e m e t h o d in the neuromuscular
junction.
on the a c t i v e
zones and on the g r o u p s
just a b o v e the a c t i v e
We r e p o r t h e r e that of the
rat
s m g p 2 5 A is a c c u m u l a t e d synaptic vesicles
located
zones. Materials
and
Methods
Materials F e m a l e S p r a g u e - D a w l e y rats (200-250 g) w e r e a n e s t h e t i z e d w i t h e t h e r a n d p e r f u s e d t h r o u g h the a s c e n d i n g a o r t a w i t h a f i x a t i v e c o n t a i n i n g 2% p a r a f o r m a l d e h y d e and 8% s u c r o s e in 50 m M p h o s p h a t e - b u f f e r e d s a l i n e at pH 7.4 (PBS) . All the f o l l o w i n g p r o c e d u r e s w e r e d o n e at 4oC. A f t e r the p e r f u s i o n , the l u m b r i c a l m u s c l e s of h i n d feet w e r e d i s s e c t e d out and i m m e r s e d in the same f i x a t i v e as d e s c r i b e d a b o v e s u p p l e m e n t e d w i t h i0 ~_M ( p - a m i d i n o p h e n y l ) m e t h a n e s u l f o n y l fluoride, 5 m M EGTA, and 1 m M MgCI2 for 4 h. These t h r e e a d d i t i v e s were s u p p l e m e n t e d to all the s o l u t i o n s u s e d in the h i s t o c h e m i c a l p r o c e d u r e s up to the final w a s h i n g s o l u t i o n s a f t e r the i n c u b a t i o n w i t h the secondary antibody. The p u r p o s e for s u p p l e m e n t i n g the t h r e e a d d i t i v e s was to p r e v e n t p o s s i b l e p r o t e o l y t i c d e g r a d a t i o n of s m g p25A. The f i x e d t i s s u e was c r y o p r o t e c t e d t h r o u g h a r a n g e of i n c r e a s i n g s u c r o s e c o n c e n t r a t i o n s up to 30%, q u i c k frozen, a n d cut on a c r y o s t a t at 8 Bm. Immunohistochemistry s m g p 2 5 A on the s e c t i o n s was d e t e c t e d by use of the p r e - e m b e d d i n g i m m u n o p e r o x i d a s e m e t h o d w i t h a m o u s e m o n o 1346
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c l o n a l a n t i b o d y against smg p25A, SG-II-7 (IgGl), w h o s e s p e c i f i c i t y was p r e v i o u s l y e s t a b l i s h e d (3). A f t e r i n c u b a t i o n w i t h 50 m M T r i s - H C l b u f f e r e d saline at pH 7.4 (TBS) c o n t a i n i n g 5% b o v i n e s e r u m a l b u m i n (BSA) for 30 min, sections were i n c u b a t e d w i t h the a n t i - s m g p 2 5 A monoc l o n a l a n t i b o d y at a c o n c e n t r a t i o n of 5 ~g/ml in TBS c o n t a i n i n g 1% B S A or w i t h the a n t i - s y n a p t o p h y s i n m o n o c l o n a l antibody, SY38 (13) (Boehringer M a n n h e i m ) , at a c o n c e n t r a t i o n of 1 ~g/ml for 48 h. The antis y n a p t o p h y s i n a n t i b o d y was u s e d as a p o s i t i v e i m m u n o r e a c t i v e control for the s y n a p t i c v e s i c l e - a s s o c i a t e d protein. N o n - i m m u n e m o u s e IgG at a c o n c e n t r a t i o n of 5 ~g/ml was u s e d as a n e g a t i v e control. After w a s h i n g w i t h TBS for 15 min four times, the s e c t i o n s were i n c u b a t e d w i t h a sheep a n t i - m o u s e i m m u n o g l o b u l i n F(ab')2 f r a g m e n t l a b e l e d w i t h h o r s e r a d i s h p e r o x i d a s e (Amersham) at a d i l u t i o n of 1:25 in TBS cont a i n i n g 1% BSA. A f t e r washing, t i s s u e - b o u n d p e r o x i d a s e a c t i v i t y was v i s u a l i z e d by i n c u b a t i o n w i t h 0.05% 3 , 3 ' - d i a m i n o b e n z i d i n e (DAB) in 50 m M T r i s - H C I b u f f e r at pH 7.5 for 30 min, and then w i t h the DAB solut i o n c o n t a i n i n g 0.006% h y d r o g e n p e r o x i d e for 5 min. The s e c t i o n s were p o s t f i x e d w i t h 2% o s m i u m t e t r o x i d e for 1 h, d e h y d r a t e d t h r o u g h ethanol and e m b e d d e d in Epon. U l t r a t h i n serial s e c t i o n s were e x a m i n e d by an e l e c t r o n m i c r o s c o p e (100SX, JEM) . Double-immunofluorescence S t u d y The s e c t i o n s were s e q u e n t i a l l y s t a i n e d w i t h the a n t i - s y n a p t o p h y s i n a n t i b o d y at a c o n c e n t r a t i o n of 1 ~g/ml for 24 h, w i t h the a n t i - m o u s e i m m u n o g l o b u l i n l a b e l e d w i t h Texas R e d (Amersham) at a d i l u t i o n of 1:25 for 24 h, a n d t h e n w i t h the antis m g p 2 5 A a n t i b o d y w h i c h h a d been l a b e l e d w i t h F I T C u s i n g a c o m m e r c i a l kit ( B o e h r i n g e r Mannheim) at a c o n c e n t r a t i o n of 5 ~g/ml for 24 h. The s e c t i o n s were e x a m i n e d by a c o n f o c a l laser s c a n n i n g i m a g i n g s y s t e m (MRC-600, B i o - R a d L a b o r a t o r i e s ) t h r o u g h a m i c r o s c o p e (Axiovert, Zeiss) u s i n g a 1 0 0 X / I . 4 N A objective. Results
By light m i c r o s c o p y , in the axon t e r m i n a l branches
p 2 5 A was v e r y
axons,
axons
similar
preterminal
axons,
of s m g p 2 5 A
(Fig.
rescence
expansions
of the m o t o r
in the p r e t e r m i n a l
pattern
strong
arising
IA).
By laser
to that
of s y n a p t o p h y s i n
optical
section
(Fig.
microscopy,
in the a x o n t e r m i n a l
found
in the p r o c e s s e s (Fig.
immunoreactivity sites
(Fig.
2B).
folds,
focally
smg p25A
was
pattern that
in the
was w e a k e r the
than that
immunofluo-
expansions same
found of smg
was very
0.5 ~m thick
immunoreactivity
sites
cells
or the p o s t s y n a p t i c
of the p r e s y n a p t i c
1347
intense
on the d i s c r e t e
contained
a n d a group of s y n a p t i c to the
was
but no i m m u n o r e a c t i v i t y
identified
was cut p e r p e n d i c u l a r
the r e g i o n s
f r o m the
At h i g h e r m a g n i f i c a t i o n ,
E a c h of t h e s e
tic p l a s m a m e m b r a n e terminal
strong
expansions,
of the S c h w a n n
2A).
was
obtained
found
i, C a n d D) .
found
of the m u s c l e
immunoreactivity
scanning microscopy,
in the axon t e r m i n a l
similar
By e l e c t r o n
except
was
of t e r m i n a l
immunoreactivity
The d i s t r i b u t i o n
of s y n a p t o p h y s i n
synaptophysin
of s m g p 2 5 A
immunoreactivity
from ramification
and m o d e r a t e
(Fig.
to that
IB) .
smg p25A
long axis plasma
folds
smg p25A
presynaptic
a region
vesicles.
was
of p r e s y n a p -
W h e n the axon
of the p o s t s y n a p t i c
membrane
with
intense
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fi~. I. Light microscopy micrographs showing distributions of s m g p25A (A) and synaptophysin (B) with the immunoperoxidase method, and confocal laser scanning micrographs showing the distributions of s m g p25A (C) and synaptophysin (D) obtained from the same 0.5 bm thick optical section in the neuromuscular junctions of rat lumbrical muscles. The axon terminals of en f a c e view (large arrows) and lateral view (arrowheads) are strongly stained in (A) and (B) by the immunoperoxidase method. The preterminal axons (small arrows) are moderately stained in (A) and weakly stained in (B) . The immunofluorescence pattern of s m g p25A (C) in the axon terminal expansions is very similar to that of synaptophysin (D) . Bars: i0 ~m in (A) to (D) .
immunoreactivity
were
located
just
folds.
The
groups
of
tivity
were
always
found
proximity tense
above
shown
to t h e
activity
were
found
(Fig.
of t h e
less
the
neighboring distribution
as
ones
reported
(Fig.
2D) .
found
on
expansions
p25A In
reported
synaptic but
groups as
by
on of
were
an
of t h e
contrast, all
2E),
very
similar
(11,12).
In t h e
strong
the which
(13,14).
0.4
was
to
vesicles
stained
Synaptophysin
1348
were
the
found
with
The
active
myelinated in t h e
immunoreactivity
almost
axon
by
from and
smg
zones axons, axoplasm was
terminal
uniformly
immunoreactivity
intense
size
intense
of t h e
diffusely
sur-
surrounded
2C) .
at t h e
inon
separated
with
preterminal found
in-
immunore-
presynaptic
vesicles
(Fig.
with
p25A
was
usually
those
in c l o s e
between
of t h e
sites
synaptophysin
synaptic were
~m
presynaptic
or
smg
which
and
immunoreac-
membrane
of
synaptic dots
vesicles
average
the
with
located
sections
usually
postsynaptic
immunoreactivity
discrete
synaptic
and
intense
plasma
levels
vesicles
no
immunoreactivity
virtually (Fig.
moderate
to
adjacent with
in c o n t a c t
presynaptic
shown
previously
smg
located
In g l a n c i n g
pattern
two
vesicles
Low
the
were
immunoreactivity
moderate
ously
2B).
the
~m in w i d t h
of t h e
the
immunoreactive
the
between
sites,
terminal,
immunoreactivity
p25A
on
0.1-0.2
synaptic to be
regions
immunoreactive
mitochondria
the
gaps the
immunoreactivity.
tensely
face
the
to be
as p r e v i -
was
also
found
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Fis. 2. E l e c t r o n m i c r o g r a p h s showing the distributions of s m g p 2 5 A (A to D) a n d s y n a p t o p h y s i n (E a n d F) in t h e a x o n t e r m i n a l e x p a n s i o n s (A to C a n d E) a n d t h e p r e t e r m i n a l a x o n s (D a n d F) . In (A), t h e a x o n terminal expansions (asterisks) are strongly stained, but the proc e s s e s of t h e S c h w a n n c e l l s (arrows) a n d t h e p o s t s y n a p t i c f o l d s of t h e muscle (PF) a r e u n s t a i n e d . The discrete presynaptic s i t e s w i t h intense smg p25A immunoreactivity (arrowheads) a r e s e e n in a c r o s s s e c t i o n (B) a n d in a g l a n c i n g s e c t i o n (C) of t h e a x o n t e r m i n a l e x p a n sions, but the mitochondria (M) a r e u n s t a i n e d . The axon terminal exp a n s i o n in (C) is p r e s u m e d to be a g l a n c i n g s e c t i o n of t h e p r e s y n a p t i c s u r f a c e , f r o m t h e f a c t t h a t no m i t o c h o n d r i o n is s e e n i n s i d e . In (E), t h e r e a r e no d i s c r e t e s i t e s w i t h a c c u m u l a t i o n s of s y n a p t o p h y s i n immunoreactivity. The myelin sheathes (MY) s u r r o u n d i n g t h e p r e t e r m i n a l a x o n s (AX) a r e f a i n t l y s e e n in (D) a n d (F) . T h e t u b u l o v e s i c u l a r membranes (arrows) w i t h m o d e r a t e s y n a p t o p h y s i n immunoreactivity are seen in t h e p r e t e r m i n a l a x o n s (AX) in (F) . B a r s : 0.5 ~/n in (A) to (F) .
1349
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on t u b u l o v e s i c u l a r 2F) .
The
membranes
control
no s i g n i f i c a n t
sections
signal
in the p r e t e r m i n a l
myelinated
stained with non-immune
(data not
mouse
axons
(Fig.
IgG e x h i b i t e d
shown).
Discussion In the p r e s e n t following
two findings:
ity on the d i s c r e t e the g r o u p s zones
of the
synaptic
expansions
artifact
derived
is that
expansions;
is not
uniformly lation
on s y n a p t i c
unique
all other
vesicles
in the
among
synaptic
the
synaptic
synaptotagmin
reported
located uniformly
to be
synapses
(for reviews,
The r e a s o n tense
smg p25A
lowing three zones
active
immunoreactivity
morphological
intramembrane imately
frequency
which
icles
and
data
vesicles
smg p25A-poor
synapses,
(for reviews, synaptic cles
see Refs.
sites since
synapsin
vesicles
I,
have b e e n in the
2) The
zones
sites w i t h
is that the
of the m a m m a l i a n
method
(11,12)
sites:
plasma
of the
postsynaptic
well
size of the
rows
of i0 nm
membrane,
localization
fol-
coincide
i) The
parallel
in-
active
is a p p r o x active
folds;
indicate synapses,
the
zone
3) The
that
the
synapses
heterogeneity
smg p25A-rich
F r o m the v i e w p o i n t
in the
9,10).
the m o l e c u l a r
of the
synaptic
synaptic
as i l l u s t r a t e d
ves-
perform
in Fig.
In the e x o - e n d o c y t o t i c
cycle
that the
smg p25A-rich
synaptic
to the s y n a p t i c
vesicles
just b e f o r e
1350
of
cell b i o l o g y
vesicles
we s p e c u l a t e
vesicles,
correspond
fracture
two a d j a c e n t
ones.
cycles
proteins,
presynaptic
as the a c t i v e
it is e s t a b l i s h e d
the e x o - e n d o c y t o t i c
accumu-
is 2-3 p e r ~m 2.
clearly in the
This
presynaptic
9,10).
on the p r e s y n a p t i c
zones
a n d that
f o u n d almost
including
synaptic
of the d o u b l e
~m;
was
for
and dif-
axons,
control
immunoreactive
the gaps b e t w e e n
of the a c t i v e
synaptic
of the
freeze
is c o m p o s e d
particles
Our p r e s e n t the
by the
0.08 ~m x 0 . 1 2 - 0 . 2 0
is just a b o v e
on the
in the
and s y n a p t o b r e v i n ,
characteristics
of the p r e s y n a p t i c
zone,
myelinated
why we r e g a r d the d i s c r e t e
established
with those
found uniformly
proteins
myeli-
of a t e c h n i c a l
same p r e p a r a t i o n .
SV2,
distribution
The e v i d e n c e
vesicle-associated
(p65),
see Refs.
result
on the d i s c r e t e
vesicle-associated
synaptophysin,
zones a n d
the a c t i v e
and d i f f u s e
procedures.
as a p o s i t i v e
immunoreactivity
immunoreactiv-
the a c t i v e
immunoreactivity
was
the
of the p r e t e r m i n a l
to be the
of the p r e t e r m i n a l
immunoreactivity
of s m g p 2 5 A
is quite
2) U n i f o r m
of s m g p 2 5 A
obtained
just above
in the a x o p l a s m
considered
time
of s m g p 2 5 A
located
immunoreactivity
in the a x o p l a s m
first
containing
f r o m the h i s t o c h e m i c a l
smg p25A
synaptophysin
sites
vesicles
The a c c u m u l a t i o n
terminal
for the
I) The a c c u m u l a t i o n
immunoreactivity
h a t e d axons.
fusely
we have
presynaptic
in axon t e r m i n a l
of s m g p 2 5 A
this
study,
3
of the
exocytosis
vesiand
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
/ I [El
~
• Synaptophysin , Neurotransmitter
[~]
~~ , ~ ~~~ ~ ~ / /
........ [C] I ..."- Presynaptic SynapticVesicle
~ :¢~
~
plasmamembrane Activezone
" ........................
Fia. 3. Diagrammatic summary of the distributions of smg p25A and synaptophysin in the exo-endocytotic cycle of the synaptic vesicles in the synapses. A precursor of the synaptic vesicles is transported through the axons as tubulovesicular membranes to the synapses A) . In the synapses, the synaptic vesicles move toward the active zones, the regions of the presynaptic plasma membrane specialized for exocytosis, as the synaptic vesicles accumulate the neurotransmitters B) . Subsequently, the synaptic vesicles attach to the active zones (Stage C) . Finally, exocytosis of the synaptic vesicles with neurotransmitter release is accomplished by the Ca 2+ influx through the voltage-operated Ca 2+ channels clustered at the active zones D) . After the exocytosis, the empty synaptic vesicles are endocytosed E) and recycled, smg p25A is accumulated on the synaptic vesicles in the stages B, C, and D, while synaptophysin is distributed uniformly on the synaptic vesicles in all the stages in this cycle.
(Stage
(Stage
(Stage
(Stage
those
in t h e
facts
that
and
in
act
the
contact
isolated
of
with
from
the
contain
more
We
speculate
also
the
active
(Fig. to
3,
stage
of
the
of t h e action
inactive
mode
that
3,
zones
and
specific
the
the
the
after
synaptic
can
of t h e
C,
are
and
the
above
vesicles
stimulation
the
stimulation
vesicles
used
from
just
synaptic
vesicles
be
D),
located
exocytotic
synaptic
smg p25A
stages
B,
that
isolated
smg p25A-poor
to
stages
vesicles
before
those
In turn,
to
ment
and
the
axons
fusion
Ca 2+ i n f l u x
of
the
small
as
(15).
located
near
between
endocytosis
a useful
exo-endocytotic
findings
would
axoplasm
smg p25
GDI.
smg p25A
synaptic
cycle
as
to
general
marker of t h e
in t h e
synapses, from
The
with
the of
the
when
smg p25
and
active attach-
zones.
synaptic
a
GDI
correct
active
is
GDP-bound
GTP-bound the
mode
smg p25A
In t h e
confirm
princi-
a possible
follows:
probably
exocytosis
1351
(1,2),
be
vesicles.
vesicles
subsequent
the
dissociates
functions
synaptic
and
G proteins
synapses
in t h e
with
vesicles
of t h e and
of
complex,
a group
synaptic
present
in t h e
complexed
with
on t h e
action
through
comes
form
of
of t h e s e
smg p25A
form
associates
the
than
correspond
basis
of
transported
signal
active
synaptosomes
E).
the
(Fig. synaptic
vesicles.
On ple
the
smg p25A
zones
identify
synaptic
exocytosis
smg p25A-rich
After
vesi-
Vol. 186, No. 3, 1992
cles,
the
GTP b o u n d
GDP-bound
inactive
dissociates finding
is h y d r o l y z e d
which becomes
f r o m the active
bound
to s m g p 2 5 A
was p r e v i o u s l y
speculated
tion
(0.04/min)
tein
(17).
of s m g p 2 5 A
zones
(16),
by the
from the
speculate
even
with
active
zones,
active
that
f r o m the
diffusion
away
with
synaptic
may
reflect
slowly
after
turnover
that
number
of GTPase reflect
may
zones by s m g p 2 5 A performs
acceptor
fact that
the
protein
smg p25A
from the active
GDI
which remains
the GTP hywhich
of this
reac-
activating
that
pro-
dissociation
continues
above
the
GDI and Our
exocytosis,
in the p r e s e n c e finding
s m g p25
cytosol.
the
smg p25A
some p u t a t i v e
zones
small
to GDP to p r o d u c e
complexed
into the
continues
Alternatively,
plexed
without
to s m g p 2 5 A form,
of s m g p 2 5 A on the active
drolysis
We also
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
slowly.
two r e a c t i o n s
are a n c h o r e d
com-
on the
on the active
zones
zones.
Acknowledgment We thank
Mr.
Y. Kikui
for his e x c e l l e n t
photographic
assistance.
References i. Bourne, H.R., Sanders, D.A., and McCormick, F. (1990) N a t u r e 348, 125-132. 2. Takai, Y., Kaibuchi, K., Kikuchi, A., and Kawata, M. (1992) Int. Rev. Cytol. 133, 187-230. 3. Mizoguchi, A., Kim, S., Ueda, T., Kikuchi, A., Yorifuji, H., Hirokawa, N., and Takai, Y. (1990) J. Biol. Chem. 265, 1187211879. 4. F i s h e r v. Mollard, G., Mignery, G.A., Baumert, M., Perin, M.S., Hanson, T.J., Burger, P.M., Jahn, R., and S~dhof, T.C. (1990) Proc. Natl. Aead. Sci. U S A 87, 1988-1992. 5. Mizoguchi, A., Kim, S., Ueda, T., and Takai, Y. (1989) Biochem. Biophys. Res. Commun. 162, 1438-1445. 6. Darchen, F., Zahraoui, A., Hammel, F., Monteils, M.P., Tavitian, A., a n d Scherman, D. (1990) Proc. Natl. Acad. Sci. U S A 87, 56925696. 7. Sasaki, T., Kikuchi, A., Araki, S., Hata, Y., Isomura, M., Kuroda, S., and Takai, Y. (1990) J. Biol. Chem. 265, 2333-2337. 8. Araki, S., Kikuchi, A., Hata, Y., Isomura, M., and Takai, Y. (1990) J. Biol. Chem. 265, 13007-13015. 9. Kelly, R. B. (1988) N e u r o n I, 431-438. I0. SOdhof, T.C. and Jahn, R. (1991) N e u r o n 6, 665-677. ii. Ellisman, M.H., Rash, J.E., Staehelin, L.A., a n d Porter, K.R. (1976) J. Cell Biol. 68, 752-774. 12. Fukunaga, H., Engel, A.G., Lang, B., N e w s o m - D a v i s , J., and Vincent, A. (1983) Proc. Natl. Acad. Sci. U S A 80, 7636-7640. 13. Wiedenmann, B. and Franke, W.W. (1985) Cell 41, 1017-1028. 14. N a v o n e F., Jahn, R., Di Gioia, G., Stukenbrok, H., Greengard, P., and De Camilli, P. (1986) J. Cell Biol. 103, 2511-2527. 15. F i s h e r v. Mollard, G., S~dhof, T.C., and Jahn, R. (1991) Nature 349, 79-81. 16. Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904. 17. Burstein, E.S., Linko-Stentz, K., Lu, Z., and Macara, I.G. (1990) J. Biol. Chem. 266, 2689-2692.
1352
Vol. 186, No. 3,1992
August 14, 1992
AT
L O C A L I Z A T I O N OF stag p 2 5 A / r a b 3 A p25, A SMALL GTP-BINDING PROTEIN, A C T I V E ZONE OF THE R A T N E U R O M U S C U L A R JUNCTION 1
THE
Akira Mizoguchi2~, Masahiko Arakawa*, Motomaru Masutani*, Akira Tamekane*, Hideyuki Yamaguchi**, Naoki Minami**, Yoshimi Takai***,
and Chizuka Ide*
Departments of *Anatomy and *** Biochemistry Kobe University School of Medicine, Kobe 650, Japan **Nippon Bio-Rad Laboratories, Tokyo 104, Japan Received June 30, 1992
Summary:
s m g p 2 5 A is a small G p r o t e i n w h i c h has b e e n s u g g e s t e d to r e g u l a t e n e u r o t r a n s m i t t e r r e l e a s e from the synapses. We i n v e s t i g a t e d here the u l t r a s t r u c t u r a l l o c a l i z a t i o n of this small G p r o t e i n in the rat n e u r o m u s c u l a r junction by an i m m u n o p e r o x i d a s e method. The results s h o w e d that s m g p 2 5 A was d i s t r i b u t e d n o n - u n i f o r m l y on the p r e s y n a p t i c p l a s m a m e m b r a n e and a m o n g the s y n a p t i c v e s i c l e s w i t h the focal accumul a t i o n on the d i s c r e t e p r e s y n a p t i c sites w h i c h c o r r e s p o n d e d to the a c t i v e zones, the r e g i o n s of the p r e s y n a p t i c p l a s m a m e m b r a n e specialized for the e x o c y t o s i s of the s y n a p t i c vesicles. This u n i q u e distrib u t i o n of s m g p 2 5 A suggests that it plays an i m p o r t a n t role in the a t t a c h m e n t and fusion of the s y n a p t i c v e s i c l e s w i t h the a c t i v e zones. @ 1992 A c a d e m i c
Press,
There more than
is the
vesicle
and has b e e n synapses
(for reviews,
transport
is a small
suggested
(3,4)
cells
proteins
of small G p r o t e i n s see Refs.
of m o r e t h a n t w e n t y m e m b e r s
p 2 5 A / r a b 3 A p25
crine
superfamily
forty m e m b e r s
consisting cellular
Inc.
as well
(5,6).
have t w o
such as e n d o c y t o s i s G protein
to r e g u l a t e as s e c r e t i o n
Like
has b e e n
1,2).
to this
in intra-
the
GTP-bound
f r o m the
cells
tab f a m i l y
active
smg
rab f a m i l y
release
from the e n d o c r i n e
of
The tab family
and e x o c y t o s i s ,
neurotransmitter
forms,
consists
implicated
which belongs
all other G proteins,
interconvertible
which
and exosmall
G
and GDP-
I T h i s w o r k was s u p p o r t e d b y g r a n t s - i n - a i d for s c i e n t i f i c r e s e a r c h f r o m the M i n i s t r y of Education, Science, and Culture, Japan, and by a grant f r o m O S A K A C a n c e r R e s e a r c h F u n d to A. M i z o g u c h i a n d C. Ide. 2 To w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .
0006-291X/92 $4.00 1345
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
bound
inactive
proteins
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
forms.
The m o d e of a c t i o n of the t a b
in i n t r a c e l l u l a r v e s i c l e t r a n s p o r t
GTP-bound active
f a m i l y small G
is s p e c u l a t e d t h a t the
f o r m l o c a t e d on a d o n o r v e s i c l e
f u n c t i o n s to c o n f i r m
the c o r r e c t a t t a c h m e n t a n d f u s i o n of the d o n o r v e s i c l e tot m e m b r a n e . membrane the
cytosol.
active
After
and the
fusion,
resultant
h y d r o l y s i s of GTP o c c u r s
GDP-bound inactive
After GDP/GTP
exchange
in the
a c t i o n of the
small G p r o t e i n s
In the c a s e of s m g p25A, on s y n a p t i c v e s i c l e s
cytosol,
the G T P - b o u n d
However,
inhibitor
and the r e g u l a t o r y p r o t e i n w h i c h i n d u c e s
is i d e n t i f i e d and n a m e d
(smg p25 GDI)
On the o t h e r hand, synaptic vesicles
9,10) .
f r o m the
s m g p25 GDP
synap-
dissociation
(7,8). it is w e l l k n o w n t h a t
in the s y n a p s e s ,
are a r r a n g e d not at r a n d o m but
m a n n e r to p e r f o r m the e x o - e n d o c y t o t i c see Refs.
this
is p r o v e d to be p r e s e n t
the d i s s o c i a t i o n of the G D P - b o u n d f o r m of t h i s p r o t e i n tic p l a s m a m e m b r a n e
into
has b e e n p o o r l y e l u c i d a t e d .
indeed this protein
(3,4)
on the a c c e p t o r
form dissociates
form reassoeiats with a new donor vesicle.
cyclical
w i t h an a c c e p -
Particularly,
cycles
exocytosis
in a h i g h l y o r g a n i z e d
effectively
of the
the
(for reviews,
synaptic vesicles
is
k n o w n to be a c c o m p l i s h e d o n l y at the v e r y r e s t r i c t e d sites of the presynaptic plasma membrane,
the a c t i v e
acteristics
synapses have been e s t a b l i s h e d
However,
in the m a m m a l i a n
the u l t r a s t r u c t u r a l
has not b e e n
zones,
whose
structural char(11,12).
l o c a l i z a t i o n of s m g p 2 5 A in the s y n a p s e s
fully understood.
In the p r e s e n t
study,
we h a v e
i n v e s t i g a t e d the u l t r a s t r u c t u r a l
l o c a l i z a t i o n of s m g p 2 5 A by an i m m u n o p e r o x i d a s e m e t h o d in the neuromuscular
junction.
on the a c t i v e
zones and on the g r o u p s
just a b o v e the a c t i v e
We r e p o r t h e r e that of the
rat
s m g p 2 5 A is a c c u m u l a t e d synaptic vesicles
located
zones. Materials
and
Methods
Materials F e m a l e S p r a g u e - D a w l e y rats (200-250 g) w e r e a n e s t h e t i z e d w i t h e t h e r a n d p e r f u s e d t h r o u g h the a s c e n d i n g a o r t a w i t h a f i x a t i v e c o n t a i n i n g 2% p a r a f o r m a l d e h y d e and 8% s u c r o s e in 50 m M p h o s p h a t e - b u f f e r e d s a l i n e at pH 7.4 (PBS) . All the f o l l o w i n g p r o c e d u r e s w e r e d o n e at 4oC. A f t e r the p e r f u s i o n , the l u m b r i c a l m u s c l e s of h i n d feet w e r e d i s s e c t e d out and i m m e r s e d in the same f i x a t i v e as d e s c r i b e d a b o v e s u p p l e m e n t e d w i t h i0 ~_M ( p - a m i d i n o p h e n y l ) m e t h a n e s u l f o n y l fluoride, 5 m M EGTA, and 1 m M MgCI2 for 4 h. These t h r e e a d d i t i v e s were s u p p l e m e n t e d to all the s o l u t i o n s u s e d in the h i s t o c h e m i c a l p r o c e d u r e s up to the final w a s h i n g s o l u t i o n s a f t e r the i n c u b a t i o n w i t h the secondary antibody. The p u r p o s e for s u p p l e m e n t i n g the t h r e e a d d i t i v e s was to p r e v e n t p o s s i b l e p r o t e o l y t i c d e g r a d a t i o n of s m g p25A. The f i x e d t i s s u e was c r y o p r o t e c t e d t h r o u g h a r a n g e of i n c r e a s i n g s u c r o s e c o n c e n t r a t i o n s up to 30%, q u i c k frozen, a n d cut on a c r y o s t a t at 8 Bm. Immunohistochemistry s m g p 2 5 A on the s e c t i o n s was d e t e c t e d by use of the p r e - e m b e d d i n g i m m u n o p e r o x i d a s e m e t h o d w i t h a m o u s e m o n o 1346
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
c l o n a l a n t i b o d y against smg p25A, SG-II-7 (IgGl), w h o s e s p e c i f i c i t y was p r e v i o u s l y e s t a b l i s h e d (3). A f t e r i n c u b a t i o n w i t h 50 m M T r i s - H C l b u f f e r e d saline at pH 7.4 (TBS) c o n t a i n i n g 5% b o v i n e s e r u m a l b u m i n (BSA) for 30 min, sections were i n c u b a t e d w i t h the a n t i - s m g p 2 5 A monoc l o n a l a n t i b o d y at a c o n c e n t r a t i o n of 5 ~g/ml in TBS c o n t a i n i n g 1% B S A or w i t h the a n t i - s y n a p t o p h y s i n m o n o c l o n a l antibody, SY38 (13) (Boehringer M a n n h e i m ) , at a c o n c e n t r a t i o n of 1 ~g/ml for 48 h. The antis y n a p t o p h y s i n a n t i b o d y was u s e d as a p o s i t i v e i m m u n o r e a c t i v e control for the s y n a p t i c v e s i c l e - a s s o c i a t e d protein. N o n - i m m u n e m o u s e IgG at a c o n c e n t r a t i o n of 5 ~g/ml was u s e d as a n e g a t i v e control. After w a s h i n g w i t h TBS for 15 min four times, the s e c t i o n s were i n c u b a t e d w i t h a sheep a n t i - m o u s e i m m u n o g l o b u l i n F(ab')2 f r a g m e n t l a b e l e d w i t h h o r s e r a d i s h p e r o x i d a s e (Amersham) at a d i l u t i o n of 1:25 in TBS cont a i n i n g 1% BSA. A f t e r washing, t i s s u e - b o u n d p e r o x i d a s e a c t i v i t y was v i s u a l i z e d by i n c u b a t i o n w i t h 0.05% 3 , 3 ' - d i a m i n o b e n z i d i n e (DAB) in 50 m M T r i s - H C I b u f f e r at pH 7.5 for 30 min, and then w i t h the DAB solut i o n c o n t a i n i n g 0.006% h y d r o g e n p e r o x i d e for 5 min. The s e c t i o n s were p o s t f i x e d w i t h 2% o s m i u m t e t r o x i d e for 1 h, d e h y d r a t e d t h r o u g h ethanol and e m b e d d e d in Epon. U l t r a t h i n serial s e c t i o n s were e x a m i n e d by an e l e c t r o n m i c r o s c o p e (100SX, JEM) . Double-immunofluorescence S t u d y The s e c t i o n s were s e q u e n t i a l l y s t a i n e d w i t h the a n t i - s y n a p t o p h y s i n a n t i b o d y at a c o n c e n t r a t i o n of 1 ~g/ml for 24 h, w i t h the a n t i - m o u s e i m m u n o g l o b u l i n l a b e l e d w i t h Texas R e d (Amersham) at a d i l u t i o n of 1:25 for 24 h, a n d t h e n w i t h the antis m g p 2 5 A a n t i b o d y w h i c h h a d been l a b e l e d w i t h F I T C u s i n g a c o m m e r c i a l kit ( B o e h r i n g e r Mannheim) at a c o n c e n t r a t i o n of 5 ~g/ml for 24 h. The s e c t i o n s were e x a m i n e d by a c o n f o c a l laser s c a n n i n g i m a g i n g s y s t e m (MRC-600, B i o - R a d L a b o r a t o r i e s ) t h r o u g h a m i c r o s c o p e (Axiovert, Zeiss) u s i n g a 1 0 0 X / I . 4 N A objective. Results
By light m i c r o s c o p y , in the axon t e r m i n a l branches
p 2 5 A was v e r y
axons,
axons
similar
preterminal
axons,
of s m g p 2 5 A
(Fig.
rescence
expansions
of the m o t o r
in the p r e t e r m i n a l
pattern
strong
arising
IA).
By laser
to that
of s y n a p t o p h y s i n
optical
section
(Fig.
microscopy,
in the a x o n t e r m i n a l
found
in the p r o c e s s e s (Fig.
immunoreactivity sites
(Fig.
2B).
folds,
focally
smg p25A
was
pattern that
in the
was w e a k e r the
than that
immunofluo-
expansions same
found of smg
was very
0.5 ~m thick
immunoreactivity
sites
cells
or the p o s t s y n a p t i c
of the p r e s y n a p t i c
1347
intense
on the d i s c r e t e
contained
a n d a group of s y n a p t i c to the
was
but no i m m u n o r e a c t i v i t y
identified
was cut p e r p e n d i c u l a r
the r e g i o n s
f r o m the
At h i g h e r m a g n i f i c a t i o n ,
E a c h of t h e s e
tic p l a s m a m e m b r a n e terminal
strong
expansions,
of the S c h w a n n
2A).
was
obtained
found
i, C a n d D) .
found
of the m u s c l e
immunoreactivity
scanning microscopy,
in the axon t e r m i n a l
similar
By e l e c t r o n
except
was
of t e r m i n a l
immunoreactivity
The d i s t r i b u t i o n
of s y n a p t o p h y s i n
synaptophysin
of s m g p 2 5 A
immunoreactivity
from ramification
and m o d e r a t e
(Fig.
to that
IB) .
smg p25A
long axis plasma
folds
smg p25A
presynaptic
a region
vesicles.
was
of p r e s y n a p -
W h e n the axon
of the p o s t s y n a p t i c
membrane
with
intense
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fi~. I. Light microscopy micrographs showing distributions of s m g p25A (A) and synaptophysin (B) with the immunoperoxidase method, and confocal laser scanning micrographs showing the distributions of s m g p25A (C) and synaptophysin (D) obtained from the same 0.5 bm thick optical section in the neuromuscular junctions of rat lumbrical muscles. The axon terminals of en f a c e view (large arrows) and lateral view (arrowheads) are strongly stained in (A) and (B) by the immunoperoxidase method. The preterminal axons (small arrows) are moderately stained in (A) and weakly stained in (B) . The immunofluorescence pattern of s m g p25A (C) in the axon terminal expansions is very similar to that of synaptophysin (D) . Bars: i0 ~m in (A) to (D) .
immunoreactivity
were
located
just
folds.
The
groups
of
tivity
were
always
found
proximity tense
above
shown
to t h e
activity
were
found
(Fig.
of t h e
less
the
neighboring distribution
as
ones
reported
(Fig.
2D) .
found
on
expansions
p25A In
reported
synaptic but
groups as
by
on of
were
an
of t h e
contrast, all
2E),
very
similar
(11,12).
In t h e
strong
the which
(13,14).
0.4
was
to
vesicles
stained
Synaptophysin
1348
were
the
found
with
The
active
myelinated in t h e
immunoreactivity
almost
axon
by
from and
smg
zones axons, axoplasm was
terminal
uniformly
immunoreactivity
intense
size
intense
of t h e
diffusely
sur-
surrounded
2C) .
at t h e
inon
separated
with
preterminal found
in-
immunore-
presynaptic
vesicles
(Fig.
with
p25A
was
usually
those
in c l o s e
between
of t h e
sites
synaptophysin
synaptic were
~m
presynaptic
or
smg
which
and
immunoreac-
membrane
of
synaptic dots
vesicles
average
the
with
located
sections
usually
postsynaptic
immunoreactivity
discrete
synaptic
and
intense
plasma
levels
vesicles
no
immunoreactivity
virtually (Fig.
moderate
to
adjacent with
in c o n t a c t
presynaptic
shown
previously
smg
located
In g l a n c i n g
pattern
two
vesicles
Low
the
were
immunoreactivity
moderate
ously
2B).
the
~m in w i d t h
of t h e
the
immunoreactive
the
between
sites,
terminal,
immunoreactivity
p25A
on
0.1-0.2
synaptic to be
regions
immunoreactive
mitochondria
the
gaps the
immunoreactivity.
tensely
face
the
to be
as p r e v i -
was
also
found
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
Fis. 2. E l e c t r o n m i c r o g r a p h s showing the distributions of s m g p 2 5 A (A to D) a n d s y n a p t o p h y s i n (E a n d F) in t h e a x o n t e r m i n a l e x p a n s i o n s (A to C a n d E) a n d t h e p r e t e r m i n a l a x o n s (D a n d F) . In (A), t h e a x o n terminal expansions (asterisks) are strongly stained, but the proc e s s e s of t h e S c h w a n n c e l l s (arrows) a n d t h e p o s t s y n a p t i c f o l d s of t h e muscle (PF) a r e u n s t a i n e d . The discrete presynaptic s i t e s w i t h intense smg p25A immunoreactivity (arrowheads) a r e s e e n in a c r o s s s e c t i o n (B) a n d in a g l a n c i n g s e c t i o n (C) of t h e a x o n t e r m i n a l e x p a n sions, but the mitochondria (M) a r e u n s t a i n e d . The axon terminal exp a n s i o n in (C) is p r e s u m e d to be a g l a n c i n g s e c t i o n of t h e p r e s y n a p t i c s u r f a c e , f r o m t h e f a c t t h a t no m i t o c h o n d r i o n is s e e n i n s i d e . In (E), t h e r e a r e no d i s c r e t e s i t e s w i t h a c c u m u l a t i o n s of s y n a p t o p h y s i n immunoreactivity. The myelin sheathes (MY) s u r r o u n d i n g t h e p r e t e r m i n a l a x o n s (AX) a r e f a i n t l y s e e n in (D) a n d (F) . T h e t u b u l o v e s i c u l a r membranes (arrows) w i t h m o d e r a t e s y n a p t o p h y s i n immunoreactivity are seen in t h e p r e t e r m i n a l a x o n s (AX) in (F) . B a r s : 0.5 ~/n in (A) to (F) .
1349
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
on t u b u l o v e s i c u l a r 2F) .
The
membranes
control
no s i g n i f i c a n t
sections
signal
in the p r e t e r m i n a l
myelinated
stained with non-immune
(data not
mouse
axons
(Fig.
IgG e x h i b i t e d
shown).
Discussion In the p r e s e n t following
two findings:
ity on the d i s c r e t e the g r o u p s zones
of the
synaptic
expansions
artifact
derived
is that
expansions;
is not
uniformly lation
on s y n a p t i c
unique
all other
vesicles
in the
among
synaptic
the
synaptic
synaptotagmin
reported
located uniformly
to be
synapses
(for reviews,
The r e a s o n tense
smg p25A
lowing three zones
active
immunoreactivity
morphological
intramembrane imately
frequency
which
icles
and
data
vesicles
smg p25A-poor
synapses,
(for reviews, synaptic cles
see Refs.
sites since
synapsin
vesicles
I,
have b e e n in the
2) The
zones
sites w i t h
is that the
of the m a m m a l i a n
method
(11,12)
sites:
plasma
of the
postsynaptic
well
size of the
rows
of i0 nm
membrane,
localization
fol-
coincide
i) The
parallel
in-
active
is a p p r o x active
folds;
indicate synapses,
the
zone
3) The
that
the
synapses
heterogeneity
smg p25A-rich
F r o m the v i e w p o i n t
in the
9,10).
the m o l e c u l a r
of the
synaptic
synaptic
as i l l u s t r a t e d
ves-
perform
in Fig.
In the e x o - e n d o c y t o t i c
cycle
that the
smg p25A-rich
synaptic
to the s y n a p t i c
vesicles
just b e f o r e
1350
of
cell b i o l o g y
vesicles
we s p e c u l a t e
vesicles,
correspond
fracture
two a d j a c e n t
ones.
cycles
proteins,
presynaptic
as the a c t i v e
it is e s t a b l i s h e d
the e x o - e n d o c y t o t i c
accumu-
is 2-3 p e r ~m 2.
clearly in the
This
presynaptic
9,10).
on the p r e s y n a p t i c
zones
a n d that
f o u n d almost
including
synaptic
of the d o u b l e
~m;
was
for
and dif-
axons,
control
immunoreactive
the gaps b e t w e e n
of the a c t i v e
synaptic
of the
freeze
is c o m p o s e d
particles
Our p r e s e n t the
by the
0.08 ~m x 0 . 1 2 - 0 . 2 0
is just a b o v e
on the
in the
and s y n a p t o b r e v i n ,
characteristics
of the p r e s y n a p t i c
zone,
myelinated
why we r e g a r d the d i s c r e t e
established
with those
found uniformly
proteins
myeli-
of a t e c h n i c a l
same p r e p a r a t i o n .
SV2,
distribution
The e v i d e n c e
vesicle-associated
(p65),
see Refs.
result
on the d i s c r e t e
vesicle-associated
synaptophysin,
zones a n d
the a c t i v e
and d i f f u s e
procedures.
as a p o s i t i v e
immunoreactivity
immunoreactiv-
the a c t i v e
immunoreactivity
was
the
of the p r e t e r m i n a l
to be the
of the p r e t e r m i n a l
immunoreactivity
of s m g p 2 5 A
is quite
2) U n i f o r m
of s m g p 2 5 A
obtained
just above
in the a x o p l a s m
considered
time
of s m g p 2 5 A
located
immunoreactivity
in the a x o p l a s m
first
containing
f r o m the h i s t o c h e m i c a l
smg p25A
synaptophysin
sites
vesicles
The a c c u m u l a t i o n
terminal
for the
I) The a c c u m u l a t i o n
immunoreactivity
h a t e d axons.
fusely
we have
presynaptic
in axon t e r m i n a l
of s m g p 2 5 A
this
study,
3
of the
exocytosis
vesiand
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
/ I [El
~
• Synaptophysin , Neurotransmitter
[~]
~~ , ~ ~~~ ~ ~ / /
........ [C] I ..."- Presynaptic SynapticVesicle
~ :¢~
~
plasmamembrane Activezone
" ........................
Fia. 3. Diagrammatic summary of the distributions of smg p25A and synaptophysin in the exo-endocytotic cycle of the synaptic vesicles in the synapses. A precursor of the synaptic vesicles is transported through the axons as tubulovesicular membranes to the synapses A) . In the synapses, the synaptic vesicles move toward the active zones, the regions of the presynaptic plasma membrane specialized for exocytosis, as the synaptic vesicles accumulate the neurotransmitters B) . Subsequently, the synaptic vesicles attach to the active zones (Stage C) . Finally, exocytosis of the synaptic vesicles with neurotransmitter release is accomplished by the Ca 2+ influx through the voltage-operated Ca 2+ channels clustered at the active zones D) . After the exocytosis, the empty synaptic vesicles are endocytosed E) and recycled, smg p25A is accumulated on the synaptic vesicles in the stages B, C, and D, while synaptophysin is distributed uniformly on the synaptic vesicles in all the stages in this cycle.
(Stage
(Stage
(Stage
(Stage
those
in t h e
facts
that
and
in
act
the
contact
isolated
of
with
from
the
contain
more
We
speculate
also
the
active
(Fig. to
3,
stage
of
the
of t h e action
inactive
mode
that
3,
zones
and
specific
the
the
the
after
synaptic
can
of t h e
C,
are
and
the
above
vesicles
stimulation
the
stimulation
vesicles
used
from
just
synaptic
vesicles
be
D),
located
exocytotic
synaptic
smg p25A
stages
B,
that
isolated
smg p25A-poor
to
stages
vesicles
before
those
In turn,
to
ment
and
the
axons
fusion
Ca 2+ i n f l u x
of
the
small
as
(15).
located
near
between
endocytosis
a useful
exo-endocytotic
findings
would
axoplasm
smg p25
GDI.
smg p25A
synaptic
cycle
as
to
general
marker of t h e
in t h e
synapses, from
The
with
the of
the
when
smg p25
and
active attach-
zones.
synaptic
a
GDI
correct
active
is
GDP-bound
GTP-bound the
mode
smg p25A
In t h e
confirm
princi-
a possible
follows:
probably
exocytosis
1351
(1,2),
be
vesicles.
vesicles
subsequent
the
dissociates
functions
synaptic
and
G proteins
synapses
in t h e
with
vesicles
of t h e and
of
complex,
a group
synaptic
present
in t h e
complexed
with
on t h e
action
through
comes
form
of
of t h e s e
smg p25A
form
associates
the
than
correspond
basis
of
transported
signal
active
synaptosomes
E).
the
(Fig. synaptic
vesicles.
On ple
the
smg p25A
zones
identify
synaptic
exocytosis
smg p25A-rich
After
vesi-
Vol. 186, No. 3, 1992
cles,
the
GTP b o u n d
GDP-bound
inactive
dissociates finding
is h y d r o l y z e d
which becomes
f r o m the active
bound
to s m g p 2 5 A
was p r e v i o u s l y
speculated
tion
(0.04/min)
tein
(17).
of s m g p 2 5 A
zones
(16),
by the
from the
speculate
even
with
active
zones,
active
that
f r o m the
diffusion
away
with
synaptic
may
reflect
slowly
after
turnover
that
number
of GTPase reflect
may
zones by s m g p 2 5 A performs
acceptor
fact that
the
protein
smg p25A
from the active
GDI
which remains
the GTP hywhich
of this
reac-
activating
that
pro-
dissociation
continues
above
the
GDI and Our
exocytosis,
in the p r e s e n c e finding
s m g p25
cytosol.
the
smg p25A
some p u t a t i v e
zones
small
to GDP to p r o d u c e
complexed
into the
continues
Alternatively,
plexed
without
to s m g p 2 5 A form,
of s m g p 2 5 A on the active
drolysis
We also
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
slowly.
two r e a c t i o n s
are a n c h o r e d
com-
on the
on the active
zones
zones.
Acknowledgment We thank
Mr.
Y. Kikui
for his e x c e l l e n t
photographic
assistance.
References i. Bourne, H.R., Sanders, D.A., and McCormick, F. (1990) N a t u r e 348, 125-132. 2. Takai, Y., Kaibuchi, K., Kikuchi, A., and Kawata, M. (1992) Int. Rev. Cytol. 133, 187-230. 3. Mizoguchi, A., Kim, S., Ueda, T., Kikuchi, A., Yorifuji, H., Hirokawa, N., and Takai, Y. (1990) J. Biol. Chem. 265, 1187211879. 4. F i s h e r v. Mollard, G., Mignery, G.A., Baumert, M., Perin, M.S., Hanson, T.J., Burger, P.M., Jahn, R., and S~dhof, T.C. (1990) Proc. Natl. Aead. Sci. U S A 87, 1988-1992. 5. Mizoguchi, A., Kim, S., Ueda, T., and Takai, Y. (1989) Biochem. Biophys. Res. Commun. 162, 1438-1445. 6. Darchen, F., Zahraoui, A., Hammel, F., Monteils, M.P., Tavitian, A., a n d Scherman, D. (1990) Proc. Natl. Acad. Sci. U S A 87, 56925696. 7. Sasaki, T., Kikuchi, A., Araki, S., Hata, Y., Isomura, M., Kuroda, S., and Takai, Y. (1990) J. Biol. Chem. 265, 2333-2337. 8. Araki, S., Kikuchi, A., Hata, Y., Isomura, M., and Takai, Y. (1990) J. Biol. Chem. 265, 13007-13015. 9. Kelly, R. B. (1988) N e u r o n I, 431-438. I0. SOdhof, T.C. and Jahn, R. (1991) N e u r o n 6, 665-677. ii. Ellisman, M.H., Rash, J.E., Staehelin, L.A., a n d Porter, K.R. (1976) J. Cell Biol. 68, 752-774. 12. Fukunaga, H., Engel, A.G., Lang, B., N e w s o m - D a v i s , J., and Vincent, A. (1983) Proc. Natl. Acad. Sci. U S A 80, 7636-7640. 13. Wiedenmann, B. and Franke, W.W. (1985) Cell 41, 1017-1028. 14. N a v o n e F., Jahn, R., Di Gioia, G., Stukenbrok, H., Greengard, P., and De Camilli, P. (1986) J. Cell Biol. 103, 2511-2527. 15. F i s h e r v. Mollard, G., S~dhof, T.C., and Jahn, R. (1991) Nature 349, 79-81. 16. Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904. 17. Burstein, E.S., Linko-Stentz, K., Lu, Z., and Macara, I.G. (1990) J. Biol. Chem. 266, 2689-2692.
1352
