CYTOBIOLOGIE
Zeitschrift für experimentelle Zellforschung
Organ
der Deutschen
Gesellschaft
für E l e k t r o n e n m i k r o s k o p i e e . V .
Band/Vol. 12 • 1975/76
VOOC*
W I S S E N S C H A F T L I C H E
V E R L A G S G E S E L L S C H A F T
M
B
H
S T U T T G A R T
I N H A L T B A N D 12 • C O N T E N T S V O L U M E 12
III
A U E L J R . , J . H . , siehe M A S K E N , J . F .
363
A K E D O , H . , siehe M O R I , Y .
397
A L L E N , R. D .
Freeze-fracture evidence f o r intramembrane changes accompanying membrane
recycling i n Paramecium
D a r s t e l l u n g intramembranöser
Veränderungen
während
bei P a r a m e c i u m m i t H i l f e der
Gefrierätz-Technik
der
254
Nahrungsvakuolenzyklose
ALLENSPACH, A . L.
A c i d Phosphatase activity i n e m b r y o n i c chick esophagus lacking
" p r o g r a m m e d cell d e a t h " (crooked neck dwarf mutant)
Saure
Phosphatase-Aktivität
(crooked
neck
des Hühnerembryo-Oesophagus
d w a r f ) m i t V e r l u s t des »programmierten
356
bei Z w e r g m u t a n t e n
embryonalen
Zelltods«
A M M E R E R , G . , siehe S C H N E D L , W .
140
BEINBRECH, G , H . J . K U H N , J . W .HERZIG, J . C. RÜEGG
Evidence f o r t w o attached m y o s i n cross-bridge states of different potential energy
A n h e f t e n v o n Myosin-Querbrücken
i n z w e i energetisch
verschiedenen
385
Zuständen
BEREITER-HAHN, J.
Beziehungen v o n Feinstruktur u n d mitochondrialer F o r m g e b u n g
F i n e s t r u c t u r a l and d y n a m i c shape
429
relations o f mitochondria
BERGER, W . , G . D A H L , H . - P . MEISSNER
Structural and f u n c t i o n a l alterations i n fused membranes of secretory granules
during exocytosis i n pancreatic islet cells of the mouse
S t r u k t u r e l l e u n d f u n k t i o n e l l e Vorgänge
bei M e m b r a n f u s i o n sekretorischer
Granula
während der Exocytose
i n I n s e l z e l l e n des Pankreas
der M a u s
119
BOUVIER, D., P H . CHEVAILLIER
A correlation between histone acetylation and chromatin changes
in spermatids of the locust (Locusta migratoria)
H i s t o n a c e t y l i e r u n g u n d Spermiogenese
i n der Heuschrecke
287
(Locusta
migratoria)
B R A A T Z , R.
Differential histochemical l o c a l i z a t i o n of calcium and its relation to Shuttle
Streaming i n Physarum
Histochemischer Nachweis unterschiedlicher
z u r Pendelströmung
i n Schleimpilzen
Calciumverteilung u n d ihre
B R A N D I S , H . , siehe S E T H I , K . K .
74
Beziehung
407
B R A S E L T O N , J . P . (Short C o m m u n i c a t i o n )
R i b o n u c l e o p r o t e i n staining of A l l i u m cepa kinetochores
R i b o n u c l e o p r o t e i n - K o n t r a s t i e r u n g der K i n e t o c h o r e n v o n A l l i u m
148
cepa
BRUN, A . , U. BRUNK
Iron Stimulation of the g r o w t h of i n vitro cultivated fibroblasts
D e r Einfluß
v o n Eisen
a u f das W a c h s t u m
417
v o n Fibroblastenkulturen
B R U N K , U . , siehe B R U N , A .
417
C H E V A I L L I E R , P H . , siehe B O U V I E R , D .
287
IV
INHALT BAND 12 • CONTENTS V O L U M E 12
C Z A K E R , R . , siehe S C H N E D L , W .
140
D A H L , G . , siehe B E R G E R , W .
119
DAHL, G , M . GRATZL
C a l c i u m - i n d u c e d fusion of isolated secretory vesicles f r o m the islet of Langerhans
C a l c i u m - i n d u z i e r t e F u s i o n i s o l i e r t e r sekretorischer
Vesikel
der Langerhans'sehen
344
Insel
D A V I D S O N , M . , siehe R O T H , L . E .
79
F A L K , H . , siehe L I E D V O G E L , B .
155
FORER, A . , W M . T . JACKSON
A c t i n filaments i n the endosperm mitotic spindles i n a higher plant,
H a e m a n t h u s katherinae Baker
A c t i n - F i l a m e n t e i n M i t o s e s p i n d e l n des Endosperms
Haemanthus katherinae
Baker
einer
höheren
199
Pflanze,
F R A N K E , H . , siehe G O E T Z E , T .
274
F R A N K E , H . , R. T Ö N J E S , T . G O E T Z E , E. G O E T Z E
(Short C o m m u n i c a t i o n )
Evidence f o r the occurrence of microtubules i n the visceral rat yolk-sac epithelium
476
N a c h w e i s v o n M i k r o t u b u l i i m v i s z e r a l e n D o t t e r s a c k e p i t h e l der R a t t e
GILLOTEAUX, J .
Les specialisations membranaires des fibres musculaires lisses d'un mollusque:
Ultrastructure d u muscle retracteur anterieur du byssus ( A B R M )
de M y t i l u s edulis L . ( M o l l u s c a Pelecypoda)
M e m b r a n e s p e c i a l i z a t i o n s i n smooth
m u s c l e fibres
of a mollusc: Ultrastructure
o f t h e a n t e r i o r byssus
retractor muscle ( A B R M ) i nM y t i l u s edulis L .
(Mollusca
Pelecypoda)
440
GILLOTEAUX, J.
Les connexions intercellulaires d'un muscle lisse: Ultrastructure du muscle retracteur
anterieur d u byssus ( A B R M ) de M y t i l u s edulis L . ( M o l l u s c a Pelecypoda)
b i t e r c e l l u l a r c o n n e c t i o n s o f a smooth
muscle: Ultrastructure of the anterior
byssal
retractor muscle ( A B R M ) o f M y t i l u s edulis L .( M o l l u s c a
Pelecypoda)
457
G O E T Z E , E . , siehe F R A N K E , H .
476
G O E T Z E , E . , siehe G O E T Z E , T .
274
G O E T Z E , T . , H . F R A N K E , W . H U H N , R. T Ö N J E S , B. S C H L A G , E. G O E T Z E
Density gradient Separation, submicroscopic, and enzymatic characterization
of cell organelles i n v o l v e d i n the protein uptake and degradation i n the rat yolk-sac
after c u l t u r i n g w i t h I - l a b e l e d h u m a n serum a l b u m i n
123
274
D i c h t e g r a d i e n t e n t r e n n u n g , submikroskopische
und enzymatische Charakterisierung
v o n Z e l l o r g a n e l l e n , die a n der P r o t e i n a u f n a h m e u n d am P r o t e i n a b b a u i m D o t t e r s a c k
v o n R a t t e n nach I n k u b a t i o n m i t ] - m a r k i e r t e m h u m a n e m S e r u m a l b u m i n
beteiligt sind
I U
G O E T Z E , T . , siehe F R A N K E , H .
476
G R A T Z L , M . , siehe D A H L , G .
344
G R E N I E R , J . F . , siehe S T O C K , C .
111
I N H A L T B A N D 12 • C O N T E N T S V O L U M E 12
V
H A A S , K . , I. R E N T S C H L E R
Struktur u n d Zusammensetzung des Oberflächenwachses v o n T r i f o l i u m h y b r i d u m L .
215
S t r u c t u r e and c o m p o s i t i o n o f t h e c u t i c u l a r w a x o f T r i f o l i u m h y b r i d u m L .
H E F F R O N , J . J . A . , siehe H E P B U R N , H . R .
HEPBURN, H . R . ,J. J. A. HEFFRON
On
481
(Short C o m m u n i c a t i o n )
the skeletal collagen of an O n y c h o p h o r a n , Peripatopsis mosleyi
Über das Collagengerüst
bei O n y c h o p h o r a , P e r i p a t o p s i s
481
mosleyi
H E R Z I G , J . W . , siehe B E I N B R E C H , G .
385
HÖWELING, K . , K .ZAAR, H . KLEINIG
Spherulation of Physarum p o l y c e p h a l u m . III. Induction w i t h m a n n i t o l
Sphärulation
244
von Physarum polycephalum. III.Induktion mit Mannit
H Ü H N , W . , siehe G O E T Z E , T .
274
JACKSON, W M .
T . , siehe F O R E R , A .
199
K L E I N I G , H . , siehe H Ö W E L I N G , K .
224
KLOPPSTECH, K . , H . - G . SCHWEIGER
N o n r i b o s o m a l ribonucleoprotein-particles i n Acetabularia mediterranea
N i c h t ribosomale
Kibonucleoprotein-Partikeln i nAcetabularia
1
mediterranea
KOROHODA, W . , W . STOCKEM
Experimentally induced destabilization of the cell membrane and cell surface
activity i n A m o e b a proteus
E x p e r i m e n t e l l i n d u z i e r t e Veränderungen
u n d Zelloberflächen-Aktivität
bei Amoeba
der
proteus
K U H N , H . J . , siehe B E I N B R E C H , G .
LAI,
93
Zellmembran-Stabilität
385
V . , L . M . SRIVASTAVA
N u c l e a r changes during differentiation of x y l e m vessel elements
Kernveränderungen
während
der D i f f e r e n z i e r u n g v o n
220
Xylem-Bahnen
LIEBRICH, W., N . PAWELETZ
Scanning electron microscopic observations o n cells g r o w n i n vitro.
III. T h e underside of H e L a cells
Rasterelektronenmikroskopische
U n t e r s u c h u n g e n a n i n v i t r o gezüchteten
321
Zellen.
III. D i e Unterseite von H e L a Zellen
LIEBRICH, W., N . PAWELETZ
(Short C o m m u n i c a t i o n )
Aggregates of H e L a cells: A source f o r annulate lamellae
HeLa-Zellen-Aggregate
als Reservoir
473
für R i n g l a m e l l e n
L I E D V O G E L , B . , P. S I T T E , H . F A L K
C h r o m o p l a s t s i n the daffodil: fine structure a n d chemistry
C h r o m o p l a s t e n der Gelben
Narzisse: Feinbau und
Chemie
155
I N H A L T B A N D 12 • C O N T E N T S V O L U M E 12
VI
M A S K E N , J . F . , J . H . A B E L JR., G . D . N I S W E N D E R
T h e effect of synthetic gonadotropin-releasing h o r m o n e ( G n - R H ) o n pituitary
h o r m o n e release i n urethane-blocked rats
D i e W i r k u n g von synthetischem
363
Gonadotropin-releasing-Hormon (Gn-RH)
a u f die Hormonausschüttung
der Hypophyse
Urethan-blockierter
Ratten
M C D O N A L D , K . L . , siehe T I P P I T , D . H .
52
M E I S S N E R , H . - P . , siehe B E R G E R , W .
119
M O R E N O D l A Z D E L A E S P I N A , S., M . C . RlSUENO
Effect of a-amanitine o n the nucleolus of meristematic cells of A l l i u m cepa
i n interphase a n d mitosis: an ultrastructural analysis
W i r k u n g von a - A m a n i t i n a u f den Nucleolus
Z w i e b e l w u r z e l n während
Interphase
175
der meristematischen
u n d M i t o s e : Eine
Z e l l e n von
u l t r a s t r u k t u r e l l eUntersuchung
M O R I , Y., H . AKEDO, Y. TANIGAKI, M . OKADA
D i f f e r e n t i a l agglutinability of rat red b l o o d cells d u r i n g fetal development
by concanavalin A
Unterschiedliche
während
397
A g g l u t i n i e r b a r k e i t von R a t t e n e r y t h r o c y t e n d u r c h Concanavalin
der fetalen
A
Entwicklung
NICKERSON, P. A .
Effect of colchicine and estradiol o n m a m m o t r o p h s and chromophobes
i n the M o n g o l i a n gerbil anterior pituitary g l a n d
D i e W i r k u n g von C o l c h i c i n u n d östradiol
Z e l l e n des Hypophysenvorderlappens
377
a u f die mammotrophen
i m mongolischen
und
chromophoben
Gerbil
N I S W E N D E R , G . D . , siehe M A S K E N , J . F .
363
O K A D A , M . , siehe M O R I , Y .
397
P A W E L E T Z , N . , siehe L I E B R I C H , W .
321
P A W E L E T Z , N . , siehe L I E B R I C H , W .
473
P I C K E T T - H E A P S , J. D .
C e l l division and evolution i n Bulbochaete. I I I . Sexual reproduetion and
e v o l u t i o n of the branched habit
Z e l l t e i l u n g u n d E n t w i c k l u n g von Bulbochaete.
E n t w i c k l u n g des
JH. Sexuelle
28
Vermehrung und
Verzweigungsmusters
P I C K E T T - H E A P S , J . D . , siehe T I P P I T , D . H .
52
R E N T S C H L E R , I., siehe H A A S , K .
215
R I S U E N O , M . C , siehe M O R E N O D I A Z D E L A E S P I N A , S.
175
R O T H , L . E., J. J . V O L L E T , M . D A V I D S O N
M i c r o t u b u l e s i n the heliozoan a x o p o d i u m . I V . Cell-surface regulation
by using hyaluronate
M i k r o t u b u l i i m H e l i o z o e n - A x o p o d i u m . I V . K o n t r o l l e der
79
Zelloberfläche
durch Hyaluronate
R Ü E G G , J . C , siehe B E I N B R E C H , G .
385
S C H L A G , B . , siehe G O E T Z E , T .
274
VII
I N H A L T B A N D 12 • C O N T E N T S V O L U M E 12
SCHNEDL, W . , R . CzAKER, G . ÄMMERER, H . G . SCHWARZACHER
Induction of premature chromosome condensation ( P C C ) depending
on the cell cycle phase
Abhängigkeit
140
der p r a e m a t u r e n C h r o m o s o m e n k o n d e n s a t i o n
(PCC) vom Zellzyklus
S C H W A R Z A C H E R , H . G . , siehe S C H N E D L , W .
140
S C H W E I G E R , H . - G . , siehe K L O P P S T E C H , K .
1
S E N T E I N , P.
A c t i o n de plusieurs amides sur l'appareil achromatique et les chromosomes
dans des oeufs d'Urodeles en segmentation
A c t i o n o f several
amides
o n t h e a c h r o m a t i c a p p a r a t u s and t h e
i n c l e a v i n g U r o d e l e eggs
SETHI, K . K . , H . BRANDIS
305
chromosomes
(Short C o m m u n i c a t i o n )
Construction of novel m a m m a l i a n cell types by fusing enucleated portions and
heterologous nuclear components i n presence of inactivated Sendai virus
H e r s t e l l u n g neuer
Säuger-Zelltypen
durch Fusion entkernten Zellmaterials und
heterologer
Kernkomponenten i nGegenwart von inaktiviertem Sendai-Virus
407
S I T T E , P., siehe L I E D V O G E L , B .
155
S R I V A S T A V A , L . M . , siehe L A I , V .
220
STOCK, C , J . F . GRENIER
Intra and extracellular myelin bodies i n the exocrine pancreas
of the obese hyperglycaemic mouse
I n t r a - u n d extrazelluläre
der
obetisch
Myelinkörper
hyperglykämischen
i mexokrinen
111
Pankreas
Maus
S T O C K E M , W . , siehe K O R O H O D A , W .
93
T A N I G A K I , Y . , siehe M O R I , Y .
397
TIPPIT, D .H . , K . L . M C D O N A L D , J . D . PICKETT-HEAPS
C e l l division i n the centric d i a t o m M e l o s i r a varians
52
Z e l l t e i l u n g bei der z e n t r i s c h e n D i a t o m e e M e l o s i r a v a r i a n s
T Ö N J E S , R . , siehe F R A N K E , H .
476
T Ö N J E S , R . , siehe G O E T Z E , T .
274
T S C H E R M A K - W O E S S , E. (Short C o m m u n i c a t i o n )
Effect of hypoxia o n A l l i u m cepa chromosomes: N o detection of half-chromatids
W i r k u n g von Sauerstoffmangel
a u f die Chromosomen
145
von Allium:
Kein Nachweis von Halbchromatiden
V O L L E T , J . J . , siehe R O T H , L . E .
79
W A G N E R , R . C.
Cytochemical localization o f a-glycerophosphate acyltransferase i n the uropygial gland
Cytochemische
Lokalisation von a-Glycerophosphat-Acyltransferase
W E R Z , G . , siehe Z E R B A N , H .
i n der
332
Bürzeldrüse
13
VIII
I N H A L T B A N D 12 • C O N T E N T S V O L U M E 12
W l M A L A R A T N A , S. D .
N u c l e o l a r size differences of the roots of O r y z a sativa
Größenunterschiede
189
der N u c l e o l e n i n der W u r z e l v o n O r y z a s a t i v a
Z A A R , K . , siehe H Ö W E L I N G , K .
224
ZERBAN, H . , G . WERZ
Localization
stages of the
Acetabularia
Lokalisation
verschiedenen
Acetabularia
of nucleoside diphosphatases and thiamine pyrophosphatase in various
life cycle of the green algae Acetabularia cliftonii and
mediterranea
von Nucleosiddiphosphatasen und Thiaminpyrophosphatase i n
E n t w i c k l u n g s - S t a d i e n der Grünalgen
Acetabularia cliftonii u n d
mediterranea
13
BUCHBESPRECHUNGEN
C L A R A , M . , K . H E R S C H E L , H . F E R N E R : Atlas der normalen mikroskopischen Anatomie
des Menschen
PRECHT, H . , J . CHRISTOPHERSEN, H . H E N S E L , W . LARCHER:
T e m p e r a t u r e and L i f e
416
152
CYTOBIOLOGIE
V O L U M E 12 • N O . 2
Pages 344-355 • 1976
Calcium-induced fusion of isolated secretory vesicles
from the islet of Langerhans
Calcium-induzierte F u s i o n isolierter sekretorischer V e s i k e l
der L a n g e r h a n s ' s c h e n Insel
GERHARD D A H L ) a n d M A N F R E D G R A T Z L
1
Fachrichtung Physiologie u n d Fachrichtung Physiologische C h e m i e
im Fachbereich Theoretische M e d i z i n der Universität des Saarlandes, H o m b u r g / S a a r
Received September 1, 1975
Abstract
C a l c i u m - membrane
f u s i o n - secretory
vesicles
-
pancreas
(1) Isolated endocrine pancreatic secretory vesicles were exposed to series of cations. A s
revealed by freeze cleaving only C a
was able to cause f u s i o n of these vesicles f o r m i n g a
common lumen.
(2) C a - s p e c i f i c fusion was preceded by an aggregation of m e m b r a n e associated particles.
Particle aggregation also occured i n vesicles incubated w i t h the other cations ( B a , S r , M n ,
M g , L a ) tested i n 10" M concentrations.
(3) F u s i o n occured i n the assumed p h y s i o l o g i c a l ränge of free intracellular C a
concentrat i o n . Fusion of secretory vesicles can already be observed at 10" M C a
and is m a x i m u m at
10" M C a , i n d i c a t i n g that small variations of intracellular free c a l c i u m concentration m a y
trigger fusion of secretory vesicles.
(4) A s the C a - s p e c i f i c fusion of isolated secretory vesicles takes place w i t h the same
m o r p h o l o g i c a l changes observed i n intact cells d u r i n g f u s i o n of the vesicle membrane w i t h
the plasma membrane i n the exocytotic process, it is c o n c l u d e d that C a
may act as f i n a l
intracellular trigger i n stimulus-secretion c o u p l i n g .
2 +
2+
2+
2 +
3+
2+
2 +
4
2 +
6
5
2 +
2 +
2+
2 +
Introduction
Glucose
is k n o w n
to
be
the
physiological Stimulus to
release
insulin
from
p a n c r e a t i c B - c e l l . I n t h e s e q u e n c e o f events l e a d i n g t o e x o e y t o s i s c a l c i u m i o n s are
the
of
critical i m p o r t a n c e . T h i s has been c o n c l u d e d f r o m the r e q u i r e m e n t of c a l c i u m i n the
e x t r a c e l l u l a r m e d i u m f o r t h e release o f i n s u l i n f r o m t h e islets o f L a n g e r h a n s [ 1 1 , 19, 2 5 ] .
Since glucose stimulates the net u p t a k e of c a l c i u m b y i s o l a t e d p a n c r e a t i c islets it has
*) D R . G . D A H L , I. Physiologisches Institut, Fachbereich Theoretische M e d i z i n , der Universität
des Saarlandes, D 665 H o m b u r g / S a a r .
Fusion of secretory vesicles
been
suggested
that
stimulus-secretion
means
an increase
c o u p l i n g [12,
of an i o n o p h o r e -
345
of i n t r a c e l l u l a r c a l c i u m plays an i m p o r t a n t role
22].
has been
Recently
calcium -
i n t r o d u c e d i n t o the cells
s h o w n to i n d u c e i n s u l i n release f r o m
in
by
pancreatic
B - c e l l s even i n t h e a b s e n c e o f g l u c o s e [37]. T h e r e f o r e , a n i n c r e a s e o f i n t r a c e l l u l a r C a
concentration
seems t o
secretion,
to
but
be
an
be
not
only
essential
link
a concomitant
i n the
chain
occurrence
of
of
Stimulus
events b e t w e e n
the
2 +
induced
raise
of
e x t r a c e l l u l a r g l u c o s e c o n c e n t r a t i o n a n d t h e release o f i n s u l i n .
In
a p r e v i o u s p a p e r [3] f u s i o n o f s e c r e t o r y vesicles a m o n g t h e m s e l v e s
plasma membrane
and with
the
has been s h o w n t o o c c u r i n g l u c o s e s t i m u l a t e d B - c e l l s . B o t h events
e x h i b i t the s a m e u l t r a s t r u c t u r a l aspects. It t h e r e f o r e seemed u s e f u l t o v e r i f y , w h e t h e r b y
i n t e r a c t i o n o f c a l c i u m w i t h a S u s p e n s i o n o f i s o l a t e d s e c r e t o r y vesicles f u s i o n s c o m p a r a b l e
to those observed
i n i n t a c t cells c a n be i n d u c e d i n the absence o f o t h e r
intracellular
components.
Materials and methods
By microdissection under a binocular microscope about 100 islets were obtained f r o m one
pancreas of a mouse ( N M R I / H a n / 2 0 ) . W e used about 400 islets for each experiment. T o remove
remnanis of exocrine tissue the islets were incubated at 3 7 ° C w i t h 2 m g / m l collagenase
(type III, fraction A , obtained f r o m Sigma C h e m i c a l C o . , St. L o u i s , U S A ) i n 10 m M cacodylate
buffer ( p H 7.0) containing 0.25 M sucrose. A f t e r about 10 m i n microscopic observations revealed
that most of the exocrine tissue had been removed. T h e enzymatic treatment was then stopped
by a d d i t i o n of an excess of icecold buffer.
Secretory granules were isolated by differential centrifugation as described by H O W E L L et al.
[14] w i t h the f o l l o w i n g modifications: T h e islets were homogenized m a n u a l l y i n 0.4 m l of
10 m M cacodylate buffer ( p H 7.0) containing 0.25 M sucrose and 1 m M E G T A (CSE-solution)
in a glass homogenizer w i t h a loosely fitting teflon pistil. R e m a i n i n g large fragments were
rehomogenized i n 0.2 m l C S E , and these homogenates were pooled before centrifugation. T h i s
and the subsequent procedure were performed at 4 ° C .
N u c l e i , cell debris and the m i t o c h o n d r i a l fraction were removed by centrifugation at 5500 g
for 5 m i n . T h e supernatant yielded secretory granules after centrifugation at 24 000 g for
10 m i n as a y e l l o w i s h fluffy pellet. T h e islets of Langerhans contain t w o major types of cells,
about 80 % B-cells containing insulin and about 20 % A-cells containing glucagon [9, 27].
Therefore the isolated mixture of granules represented m a i n l y B-granules and w i l l be called
secretory vesicles i n this p u b l i c a t i o n . T h e vesicle fraction was washed once in C S E , homogenized
gently by hand in a small volume of C S E to get a protein concentration of about 0.05 m g / m l
and used immediately for the experiments. Purity of secretory granule fraction was checked by
thin section electron microscopy.
h t c u b a t i o n p r o c e d u r e : In 1.5 m l Polyethylene reaction vessels 0.3 m l of the vesicle Suspension
were m i x e d w i t h 0.3 m l of C S E containing different concentrations of C a
(the concentration
of w h i c h was adjusted according to P O R T Z E H L et al. [28]), M g , M n , B a , S r
or L a
and
incubated for 5 m i n at 3 7 ° C . T h e n 0.3 m l 2 % glutaraldehyde in C S E containing the corresponding concentration of ions used in the experiment were added for f i x a t i o n . 5 m i n later
0.3 m l glycerol was added for cryoprotection and after 10 m i n at r o o m temperature the vessels
were centrifuged 4 m i n i n a M o d e l 3200 Eppendorff centrifuge at 12 000 r p m . T h e f i x e d vesicle
fraction appeared as a small pellet w h i c h was resuspended i n one d r o p of its supernatant.
Freeze-cleaving:
S m a l l dropletts (less than 1 ul) of the Suspension were frozen o n golden
specimens holders i n F r e o n 22 cooled by l i q u i d nitrogen, and afterwards stored i n l i q u i d nitrogen.
T h e cleaving was performed according to the method of M O O R and M Ü H L E T H A L E R [26] i n a
Balzers freeze-etch unit B A F 3 0 0 . Fracturing and replication was performed at — 1 0 0 ° C .
Replicas were cleaned f r o m organic material by s o d i u m hypochloride. A f t e r w a s h i n g i n
destilled water, replicas were picked up o n f o r m v a r and carbon-coated single hole grids.
2 +
2 +
2 +
2 +
2+
3 +
G E R H A R D D A H L and
346
MANFRED GRATZL
R e p l i c a s were examined i n a Siemens-Elmiskop 1 0 1 at 8 0 or 1 0 0 k V . Photographs were taken
as positives (platinum deposition: black). F o r identification of fracture faces the nomenclaturc
of M C N U T T and W E I N S T E I N [ 2 3 ] has been used.
T h i n sections:
Pellets were fixed w i t h 2 °/o glutaraldehyde at room temperature and postfixed
in a 1 °/o Solution of osmium tetroxide. After dehydration in a graded series of alcohols and
p r o p y l e n d i o x i d e the pellet was embedded in E p o n . T h i n sections were cut o n a Reichertm i c r o t o m e O M U 3 , and stained w i t h lead citrate and uranylacetate. Protein concentration was
determined according to L O W R Y et al. [ 2 1 ] . N o n specified chemicals were of the purest grade
c o m m e r c i a l l y available.
Results
A n e x a m p l e o f s e c r e t o r y vesicles p r e p a r e d a c c o r d i n g t o the m e t h o d d e s c r i b e d is s h o w n
i n F i g u r e 1 a a n d b . I n t h i n s e c t i o n s ( F i g . 1 a) the e l e c t r o n - d e n s e c o r e s o f the
vesicles
can
be
observed
which
are
believed to
contain
insulin.
Some
secretory
microsomal
c o n t a m i n a t i o n is p r e s e n t . O n l y o c c a s i o n a l l y m i t o c h o n d r i a c a n be o b s e r v e d (not s h o w n
in
F i g . 1 a).
The
purity
of
the
isolated
vesicles
appeared
to
be
sufficient
for
the
e x p e r i m e n t s a n d a d e n s i t y g r a d i e n t step w a s o m i t t e d b e c a u s e o f the r e p o r t e d l o w y i e l d
o f v e s i c l e s [14]. R e p l i c a s o f the f r e e z e - c l e a v e d p r e p a r a t i o n s ( F i g . 1 b) i n d i c a t e that
the
f r a c t u r e p l a n e f o l l o w s p r e f e r e n t i a l l y the m e m b r a n e s . T h e s p l i t m e m b r a n e s o f i s o l a t e d
secretory
vesicles e x h i b i t the s a m e features
as s e c r e t o r y
vesicles i n i n t a c t B - c e l l s
[3]:
R a n d o m l y d i s t r i b u t e d m e m b r a n e a s s o c i a t e d p a r t i c l e s ( M A P s ) stick m o r e t o the c o n c a v e
A - f a c e t h a n t o the c o n v e x B - f a c e . T h i s m a y i n d i c a t e p r e s e r v a t i o n o f i n t a c t
For
our
investigations we
have
considered
o n l y vesicles
with
large
membranes.
diameter.
The
v e s i c l e s o f s m a l l e r size m a y represent vesicles f r a c t u r e d i n the a p i c a l p a r t o r m i c r o s o m e s .
T h e presence
of
membranes
o f p l a s m a m e m b r a n e s c a n be e x c l u d e d , p r o v i d e d they a p p e a r as sheets
o r as
r i g h t s i d e - o u t vesicles
(convex
A - f a c e ) . If t h e y
f o r m inside-out
v e s i c l e s t h e y m a y s t i l l be d i s t i n g u i s h a b l e f r o m s e c r e t o r y vesicles b y the s m a l l e r d i a m e t e r
o f M A P s [3]. H o w e v e r , as i n d i c a t e d b y the t h i n s e c t i o n ( F i g . 1 a) a g r o s s c o n t a m i n a t i o n
w i t h v e s i c l e s o t h e r t h a n s e c r e t o r y vesicles is u n l i k e l y .
T h e d i s t r i b u t i o n of M A P s
for
calcium concentrations
i n vesicle m e m b r a n e s
below
as s h o w n i n F i g u r e 1 b is t y p i c a l
1 0 " M . D e s p i t e the
8
close
vicinity
of
the
vesicles
n e i t h e r a n a g g r e g a t i o n o f vesicles o r o f M A P s , n o r f u s i o n c a n be o b s e r v e d u n d e r these
c o n d i t i o n s . In c o n t r a s t b y r a i s i n g the C a
2 +
concentrations to 10" M o r higher particle6
r i c h B - f a c e s b e c o m e v i s i b l e ( F i g . 2). In t h i s case M A P s are a g g r e g a t e d . T h e y are o b s e r v e d
i n r e g i o n s w h e r e n e i g h b o u r i n g vesicles are a t t a c h e d t o each o t h e r as w e l l as i n vesicles
w h e r e a c o n t a c t w i t h a n e i g h b o u r i n g o n e is n o t v i s i b l e ( F i g . 2 a, b ) . B e s i d e s the s t r u c tural
(Fig.
changes
concerning
the
array
of
MAPs
siamese
twin
vesicles
are
observed
2 a, b). I n the w a i s t o f these t w i n s t r u c t u r e s o c c a s i o n a l l y a r i n g o f p a r t i c l e s is
v i s i b l e i n m e m b r a n e B - f a c e s ( F i g . 3 a) a n d A - f a c e s ( F i g . 3 b ) . B o t h h a l v e s o f the
twin
v e s i c l e s h a v e the size of n o r m a l s e c r e t o r y vesicles. T h i s f o r m a t i o n w a s n e v e r f o u n d at
low
Ca
2 +
c o n c e n t r a t i o n . T h e cleveage p l a n e f r o m o n e t o the o t h e r v e s i c l e is c o n t i n u o u s
i n t h e m e m b r a n e B - f a c e ( F i g . 2 a) as w e l l as i n the A - f a c e ( F i g . 3 b ) . T h i s i n d i c a t e s t h a t
these t w i n s t r u c t u r e s r e p r e s e n t f u s e d s e c r e t o r y vesicles w i t h o n e c o m m o n l u m e n .
In
s o m e t w i n vesicles the w a i s t s d o n o t s h o w the a g g r e g a t i o n o f M A P s
described
a b o v e ( F i g . 4). T h e s e s t r u c t u r e s m a y r e p r e s e n t the c o r r e s p o n d i n g m e m b r a n e faces t o the
f u s e d vesicles s h o w n i n F i g u r e 3 o r a l a t e r stage o f f u s i o n d e v e l o p m e n t w e r e p a r t i c l e s
are r a n d o m l y r e d i s t r i b u t e d . T h e d i f f e r e n t features o f f u s i o n d e s c r i b e d a b o v e h a v e b e e n
a l s o o b s e r v e d i n c o n c e n t r a t i o n s as l o w as 10" M C a
6
2 +
b u t the f r e q u e n c y o f these events
Fusion of secretory vesicles
347
F i g . 1 a. Electron m i c r o g r a p h of a thin sectioned pellet f r o m the vesicle preparation. It consists
mostly of B-granules characterized by the dense core surrounded by a halo and the l i m i t i n g
membrane. Some m i c r o s o m a l c o n t a m i n a t i o n is present. - 45 000 X . - Scale 0.5 u m . - b . Electron
micrograph of a freeze-fractured vesicle preparation incubated in l o w C a
concentration
( < 10 M ) . R a n d o m l y distributed membrane associated particles ( M A P s ) stick more to the
coneave A-faces (A) than to the convex B-faces (B) of the l i m i t i n g membranes. E n c i r c l e d
arrowhcad indicates direction of shadovving. - (SO 000 X . - Scale 0.2 u m .
2 +
s
348
G E R H A R D D A H L and
MANFRED GRATZL
F i g . 2. Frecze-fracturcd vesicles incubated in high C a " concentration. M A P s are frcquent in
membrane B-faces and are accumulatcd. T h i s accumulation is present in zones of contact
betvveen neighbouring vesicles ( l a r g e a r r o w ) but is also visible on top of convex vesicles. T w i n
vesicles with a waist ( s m a l l a r r o w ) are visible and the continuous cleavage plane of the mem2
Fusion of secretory vesicles
w a s h i g h e s t at C a
2 +
concentrations
o f 10
M
3
349
a n d d i d n o t c h a n g e u p t o 10
3
M
Ca
2 +
( T a b . 1).
To
d e t e r m i n e w h e t h e r the p r o c e s s e s o b s e r v e d are s p e c i f i c f o r C a
w e tested v a r i o u s
2 +
o t h e r c a t i o n s . A s s h o w n i n T a b l e 1 n o f u s i o n s o f v e s i c l e s c o u l d be i n d u c e d b y
cations,
although aggregation
of M A P s
-
less p r o n o u n c e d -
occured in
other
membrane
B - f a c e s . A t t a c h m e n t s o f d i s t i n c t v e s i c l e s w e r e a l s o p r e s e n t u n d e r these c o n d i t i o n s .
Discussion
T h e i s o l a t i o n o f s e c r e t o r y v e s i c l e s f r o m e n d o c r i n e p a n c r e a t i c tissue is r a t h e r l a b o r i o u s
and the d i f f i c u l t i e s a r i s i n g d u r i n g their i s o l a t i o n have been r e p o r t e d i n v a r i o u s p u b l i c a t i o n s [5, 14,
15, 17]. A s d e s c r i b e d a b o v e , the p o p u l a t i o n o f s e c r e t o r y vesicles o b t a i n e d
are d e r i v e d m a i n l y f r o m B - c e l l s a n d the effects o b s e r v e d s h o u l d be a t t r i b u t e d t o
granules rather t h a n to A - g r a n u l e s . T h e presence
B-
of m i c r o s o m e s does not affect
the
e x p e r i m e n t s d e s c r i b e d , s i n c e m i c r o s o m e s w e r e s h o w n t o be u n a b l e t o fuse u n d e r
the
c o n d i t i o n s d e s c r i b e d i n t h i s i n v e s t i g a t i o n (GRATZL
and DAHL,
1975
-
manuscript in
preparation).
A g g r e g a t i o n o f M A P s is a p r o m i n e n t f e a t u r e o f s e c r e t o r y v e s i c l e m e m b r a n e s , e x p o s e d
to i n c r e a s i n g C a
2 +
c o n c e n t r a t i o n s . T h i s c h a n g e i n the a r r a y o f i n t r a m e m b r a n o u s p a r t i c l e s
w a s f o u n d t o be i n d u c e d a l s o b y o t h e r c a t i o n s , t h o u g h t o a l o w e r e x t e n t . T h e o b s e r v e d
aggregation
of M A P s
devoid of M A P s
appears
at l o w C a
2 +
on
the B - f a c e o f
the
membrane,
which
c o n c e n t r a t i o n s . A g g r e g a t i o n of M A P s
is p r a c t i c a l l y
m a y arise f r o m a
l a t e r a l m o v e m e n t a n d i n t r a m e m b r a n o u s p a r t i c l e s i n t h i s case m a y a d h e r e m o r e t o
B - f a c e t h a n t o t h e A - f a c e o f the m e m b r a n e u p o n e x p o s u r e t o C a
2 +
the
. H o w e v e r , the h i g h
p a r t i c l e d e n s i t y o n the B - f a c e c o u l d a l s o be d u e t o a n Integration o f p e r i p h e r a l p a r t i c l e s
i n the m e m b r a n e w h i c h are u s u a l l y n o t v i s i b l e i n f r e e z e - c l e a v i n g .
Particle aggregation was observed i n zones of contact between n e i g h b o u r i n g vesicles
as w e l l as i n o t h e r m e m b r a n e a r e a s . I n the l a t t e r case the m a t e r i a l a b o v e the
exposed
m e m b r a n e faces h a d b e e n r e m o v e d b y the c l e a v a g e p r o c e s s . T h e r e f o r e , it c a n n o t
decided
whether
the
aggregation
of
MAPs
occurs
only in contact
regions
or
be
also
i n d e p e n d e n t l y o f m e m b r a n e c o n t a c t . T h i s i m p l i c a t e s the i n a b i l i t y t o c o n c l u d e w h e t h e r
an aggregation of M A P s favours a m e m b r a n e contact or whether a contact induces a n
aggregation of M A P s .
In f u s e d v e s i c l e s a b a n d o f M A P s w a s f o u n d i n the r e g i o n o f the r e m a i n i n g w a i s t .
This
may
be
a
relict of
the
aggregation
of
MAPs
in contacting
vesicles
and
e x i s t e n c e i n B - a n d A - f a c e s m a y be a n i n d i c a t i o n f o r a d i f f e r e n t d i s t r i b u t i o n o f
their
MAPs
at v a r i o u s stages o f f u s i o n d e v e l o p m e n t .
T h e r e is a c c u m u l a t i v e e v i d e n c e f o r t h e k e y r o l e o f M A P s
biological membranes
(cf.
ref.
[29]): T h e
i n the f u s i o n p r o c e s s
aggregation of M A P s
in the B - f a c e o f
of
the
m e m b r a n e o f v e s i c l e s c o n t a c t i n g each o t h e r as w e l l as n e i g h b o u r i n g the p l a s m a m e m b r a n e w a s a l r e a d y d e s c r i b e d i n g l u c o s e s t i m u l a t e d B - c e l l s b y BERGER, D A H L a n d M E I S S NER
[3]
as
an event
preceding membrane
fusion. The
results
of
these a u t h o r s
also
brane. E n c i r c l e d a r r o w h e a d indicates direction of s h a d o w i n g . - a. C a - c o n c e n t r a t i o n : 2 X
10-* M . - 150 000 X . - Scale 0.2 u m . - b. C a - c o n c e n t r a t i o n : 2 X 10" M . A n aggregation of
M A P s ( l a r g e a r r o w ) is visible on the B-face of a vesicle attaching t w o fused vesicles ( s m a l l
a r r o w ) . - 100 000 X . - Scale 0.2 u m .
2+
2+
5
Ci Kit H A HO D A H L and
350
MANFRED
GKATZL
F i g . 3 . Freeze-fractured vesicles incubated in a Solution containing 2 X 10~ M C a . Encircled
arrovvheads indicate direction of s h a d o w i n g . - a. B-face of fused vesicles w i t h a ring-like
aggregation of M A P s ( a r r o w ) . - b. Aggregation of M A P s on the A-face of fused vesicles
( a r r o w ) . - 100 000 X . - Scale 0.2 u m .
5
2 +
i n d i c a t e d that the a g g r e g a t i o n of M A P s m a y r e p r e s e n t a z o n e of i n c r e a s e d p e r m e a b i l i t y .
A s i m i l a r b e h a v i o u r w a s s h o w n f o r the f u s i o n o f m y o b l a s t p l a s m a m e m b r a n e s ,
where
f u s i o n is p r e c e d e d b y the d e v e l o p m e n t of g a p j u n e t i o n s c o u p l i n g m y o b l a s t s e l e c t r i c a l l y
[31,
32].
Vesicles
p r e s e n c e of C a ~
+
of
plasma
membranes
isolated
f r o m myoblasts
also
w i t h t r a n s i t o r y f o r m a t i o n of a n a g g r e g a t i o n of M A P s
fused
(SCHUDT,
in
the
DAHL
Fusion of secretory vesicles
351
F i g . 4. Fused vesicles w i t h o u t aggregation of M A P s neither on A-face (a) nor on B-face
Fncircled arrovvheads indicate direction of s h a d o w i n g . - 100 000 X . - Scale 0.2 u m .
(b).
and G R A T Z L , 1 9 7 5 - m a n u s c r i p t i n p r e p a r a t i o n ) . A p a r t i c u l a r f o r m a t i o n of M A P s is a l s o
described by S A T I R
et al.
[ 3 4 ] for m e m b r a n e fusion d u r i n g mueoeyst secretion in tetra-
hymena. F i n a l l y , aggregations of M A P s
w e r e f o u n d to a e c o m p a n y the v i r u s i n d u c e d
f u s i o n of h e n e r y t h r o c y t e s [ 2 J .
G l y c e r o l w a s suggested b y S C H V V A B - S T E Y et al.
hom
tetrahymena pyriformis.
This was
[ 3 6 ] to cause f u s i o n of m i t o c h o n d r i a
also reported by A H K O N G
et al.
[1] for
the
352
G E R H A R D D A H L and
fusion
of hen erythrocytes. W e
MANFRED
GRATZL
used i n o u r experiments
glycerol for cryoprotection
d u r i n g t h e f r e e z e - f r a c t u r e p r o c e s s . H o w e v e r , g l y c e r o l w a s a d d e d after p r e f i x a t i o n w i t h
glutaraldehyde. Therefore, in o u r experiments glycerol should not have induced fusion.
F u r t h e r m o r e C a - s p e c i f i c f u s i o n of G o l g i vesicles w a s o b s e r v e d e v e n i n the a b s e n c e of
2 +
g l y c e r o l ( G R A T Z L a n d D A H L , 1975 - m a n u s c r i p t i n p r e p a r a t i o n ) .
T a b . 1.
C a - d e p e n d e n c e and specificity of the f u s i o n of pancreatic endocrine secretory granules.
2+
Cation
CQ
+
2
Concentration
(M)
Particle
Aggregation
-
<
10-8
-
2 x
10-6
+
2 x
10 " 5
2 x
I0"4
+•+
+++
•
++
2 x
I0
Mg 2 *
ix
10 ""^
Sr
2*
1 x
I0"
A
+
Ba *
1 x
I0"
A
+
Mn
+
1 x
10"*
+
1x
IQ"*
2
La
+
++
++•
2
3
Fusion
- 3
++
-
_
+
-
< I 7o
I - 5 % I These f igures were evaluated by counting
5 -10 % f 200 vesicles for each cation concentration
>
10% J
in a scanned replica.
D ü r i n g t h e Stimulation o f p a n c r e a t i c B - c e l l s w i t h g l u c o s e t w o p r o c e s s e s
are
t o t a k e p l a c e : F u s i o n o f s e c r e t o r y vesicles a m o n g e a c h o t h e r a n d f u s i o n o f
known
secretory
vesicles w i t h the p l a s m a m e m b r a n e . B o t h m e m b r a n e f u s i o n s e x h i b i t t h e s a m e m o r p h o logy
[3].
The
coincidence of
b o t h events
is d e s c r i b e d f o r d i f f e r e n t c e l l s a n d c a l l e d
" C o m p o u n d e x o c y t o s i s " [7] o r " t a n d e m e x o c y t o s i s " [18]. T h e s i m i l a r i t y o f i n t e r v e s i c u l a r
f u s i o n a n d t h e f u s i o n o f s e c r e t o r y vesicles w i t h t h e p l a s m a m e m b r a n e i n i n t a c t B - c e l l s [3]
l e d u s t o the c o n c l u s i o n t h a t the s t u d y o f i n t e r v e s i c u l a r f u s i o n m a y g i v e i n d i c a t i o n s
a b o u t the m e c h a n i s m o f m e m b r a n e f u s i o n d u r i n g t h e e x o c y t o t i c p r o c e s s .
T h e results presented s h o w , that C a
2 +
is a b l e t o i n d u c e f u s i o n o f i s o l a t e d s e c r e t o r y
v e s i c l e s w i t h t h e s a m e m o r p h o l o g i c a l changes o b s e r v e d d u r i n g m e m b r a n e f u s i o n i n i n t a c t
cells [3]. E x p o s u r e o f vesicles t o o t h e r c a t i o n s l e d a l s o t o a c e r t a i n e x t e n t t o a g g r e g a t i o n
of M A P s a n d attachment of vesicles. H o w e v e r , other cations w e r e inefficient to trigger
the f u s i o n o f these v e s i c l e s .
Specificity of C a
2 +
t o i n d u c e e x o c y t o s i s w a s o b s e r v e d b y W O L L H E I M et al.
these s t u d i e s e x t e r n a l l y a d d e d C a
2 +
i n s u l i n release f r o m m o n o l a y e r c u l t u r e s o f e n d o c r i n e p a n c r e a s . M g
replace
Ca
2 +
.
Though Ba
authors* conditions B a
Specificity for C a
2 +
2 +
2 +
[37].
w i t h the a i d o f a n i o n o p h o r e w a s a b l e t o
2 +
and Sr
a l o n e h a d a s t i m u l a t o r y effect o n i n s u l i n
2 +
In
cause
could not
release,
in
the
w a s n o t s h o w n t o be e f f e c t i v e i n t h e p r e s e n c e o f t h e i o n o p h o r e .
a n d lack of response to other cations applied i n t r a c e l l u l a r l y were
r e p o r t e d a l s o u p o n o t h e r Systems i n c l u d i n g m a s t c e l l s [16]
a n d f r o m the g i a n t Synapse
o f t h e s q u i d [24]. S i m i l a r i l y t h e use o f i o n o p h o r e s i n d i f f e r e n t Systems suggest t h a t
is a l i n k b e t w e e n Stimulus a n d s e c r e t i o n [4, 10, 30,
38].
Ca
2 +
Fusion of secretory vesicles
In
o u r studies f u s i o n of secretory
tions of C a
2 +
353
v e s i c l e s c a n be o b s e r v e d at v e r y l o w c o n c e n t r a -
(10~ M ) . T h e c o n c e n t r a t i o n o f free C a
6
2 +
i n d i f f e r e n t cells w a s r e p o r t e d t o
be i n the s a m e r ä n g e (cf. ref. [33]). T h e r e f o r e , s m a l l v a r i a t i o n s i n t h e c o n c e n t r a t i o n o f
free i n t r a c e l l u l a r C a
2 +
m a y trigger exocytosis i n pancreatic B-cells. Structural changes
o f i s o l a t e d c h r o m a f f i n g r a n u l e s a s s o c i a t e d w i t h a r e v e r s i b l e a g g r e g a t i o n o f vesicles h a v e
been d e s c r i b e d [8] u s i n g v e r y h i g h c o n c e n t r a t i o n o f C a
(4 m M ) . T h i s
2 +
concentration
is f a r f r o m p h y s i o l o g i c a l c o n d i t i o n s . T h e s e effects h a v e b e e n o b t a i n e d a l s o w i t h
We
Mg
2 +
.
t h e r e f o r e h e s i t a t e t o c o m p a r e these results w i t h t h e C a - s p e c i f i c f u s i o n o f s e c r e t o r y
2+
vesicles r e p o r t e d i n t h i s p a p e r .
From
o u r experiments
it becomes evident that f u s i o n of the pancreatic
secretory vesicles requires the i n t e r a c t i o n of C a
of
Ca
2 +
to pancreatic
secretory
vesicles
2 +
endocrine
w i t h the vesicular m e m b r a n e . B i n d i n g
has been
shown by DEAN
[6]
using m i c r o -
electrophoresis. T h e b i n d i n g of c a l c i u m t o secretory vesicles i n s t i m u l a t e d B-cells w a s
a l s o f o u n d i n h i s t o c h e m i c a l s t u d i e s a n d e l e m e n t a l X - r a y a n a l y s i s [13, 3 5 ] . P r e l i m i n a r y
results r e c e n t l y d e s c r i b e d b y LAZARUS a n d DAVIES [20] suggest a C a - i n d u c e d i n s u l i n
2 +
release o c c u r i n g d u r i n g i n c u b a t i o n o f i s o l a t e d B - g r a n u l e s w i t h p u r i f i e d p l a s m a m e m branes i n the presence of C a
2 +
and A T P .
In c o n c l u s i o n t h e e x p e r i m e n t s d e s c r i b e d i n t h i s p u b l i c a t i o n i n d i c a t e t h a t C a
2 +
in the
p a n c r e a t i c B - c e l l is a b l e t o act as f i n a l i n t r a c e l l u l a r t r i g g e r i n s t i m u l u s - s e c r e t i o n c o u p l i n g
c a u s i n g t h e f u s i o n o f m e m b r a n e s . It w i l l b e a t e m p t i n g t a s k f o r t h e f u t u r e t o e l u c i d a t e
the n a t u r e a n d i n t e r a c t i o n o f t h e m i s s i n g l i n k s b e t w e e n
glucose
Stimulus a n d C a
2 +
-
i n d u c e d e x o c y t o s i s i n t h e p a n c r e a t i c islet.
Acknowledgements.
T h e authors are indebted to Prof. D R . W . B E R G E R a n d D R . H . P. M E I S S N E R
for valuable criticisms a n d suggestions d u r i n g the progress of this w o r k . W e thank M r s . M . E L I S ,
M r s . I. K Ü M M E L a n d M r . R . W E I S S f o r excellent technical assistence. - T h i s w o r k was s u p p o r t e d
by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 38 " M e m b r a n f o r s c h u n g " .
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}
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