Molecular Mechanisms of Membrane Fusion

Edited by Shinpei Ohki Darrell Doyle and Thomas D. Flanagan State University of New York at Buffalo Buffalo, Mew York

and Sek Wen Hui and Eric Mayhew Roswell Park Memorial Institute Buffalo, New York

Plenum Press · New York and London

CONTENTS

Calcium-Induced Membrane Fusion: From Liposomes to C e l l u l a r Membranes 1

D. Papahadjopoulos, P.R. Meers, Κ. Hong, J.D. Ernst, I.M. Goldstein, and N. DUzglines,

Gel Phase Fusion of Dipalmitoyl Phosphatidylcholine Small Unilamellar V e s i c l e s 17

T.E. Thompson and D. Lichtenberg 2+

S t r u c t u r a l Characterization of Lamellar Mg Complexes of Dilauroylphosphatidic Acid Using 3 1 P and ^ C NMR 25

Μ.P. Murari, M.P. O'Brien, and J.H. Prestegard L i p i d Polymorphism, L i p i d Asymmetry and Membrane Fusion 37

P.R. C u l l i s and M.J. Hope

Membrane Fusion Via Intermediates i n La/Ηχι Phase Transitions... 53 D. P. Siegel, Η. E l l e n s , and J . Bentz

The Influence of Polar Group Identity on the Interactions Between Phospholipid B i l a y e r s 73

R.P. Rand and V.A. Parsegian

I n t r i n s i c C o l l o i d a l Attraction/Repulsion Between L i p i d B i l a y e r s and Strong Attraction Induced by Non-Adsorbing Polymers 83

E. Evans and D. Needham

Mechanisms of Membrane Fusion i n Acidic Lipid-Cation Systems.... 101 R.C. MacDonald

Polar I n t e r f a c i a l I n t e r a c t i o n s , Hydration Pressure and Membrane Fusion 113

C. J . van Oss, M.K. Chaudhury, and R.J. Good

Surface Tension, Hydration Energy and Membrane Fusion 123 S. Ohki

Interactions i n Liposomal-Drug Delivery I n Vivo and In Vi t r o . . . . 139 E. Mayhew

Drug Delivery By Immunoliposomes 149 D. C o l l i n s and L. Huang

Osmotic Forces and the Fusion of Biomembranes 163 J.A. Lucy and Q.F. Ahkong

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Fusion in B i o l o g i c a l and Model Membranes: S i m i l a r i t i e s and Differences 181

J . Zimmerberg

E a r l y Steps i n the Exocytosis of Secretory V e s i c l e s i n Mast C e l l s 197

W. Aimers and L . J . Breckenridge

Ele c t r o f u s i o n and E l e c t r o t r a n s f e c t i o n of C e l l s 209 U. Zimmermann

Pre-Selection of B-Lymphocytes by Antigen for Fusion to Myeloma C e l l s by Pulsed E l e c t r i c F i e l d (PEF) Method 223

T.Y. Tsong, M. Tornita, and M.M.S. Lo

The Mechanism of Erythrocyte Ghost Fusion by E l e c t r i c F i e l d Pulses 237

A.E. Sowers and V. Kapoor

Water-Mediated E f f e c t s of PEG on Membrane Properties and Fusion 255

K. Arnold, A. Herrmann, K. Gawrisch, and L. Pratsch

Control of C e l l Membrane Fusion by L i p i d Composition 273 D.S. Roos

Role of Phospholipid Asymmetry i n C e l l u l a r Membrane Fusion 289 R.A. Schlegel and P. Williamson

U l t r a s t r u c t u r a l Studies of the K i n e t i c s of Fusion 303 S.W. Hui, D.A. Stenger, and S.K. Huang

pH Triggered Synthetic Peptides: Models for V i r a l Fusion Sequences 317

F.C. Szoka, J r . , N.K. Subbarao, and R.A. Parente

Membrane Fusion i n Model Systems for Exocytosis: Characterization of Chromaffin Granule Fusion Mediated By Synexin and C a l e l e c t r i n 325

W.J. Zaks and C.E. Creutz

Synexin, Calcium and the Hydrophobic Bridge Hypothesis for Membrane Fusion 341

H.B. Pollard, E. Rojas, A.L. Burns, and C. Parra

Molecular Mechanism of Protein-Mediated Low pH-Induced Membrane Fusions 357

S - I . Ohnishi and M. Murata

pH-Dependent Fusion of Vesicular Stomatitis Virus with C e l l s : Studies of Mechanism Based on an A l l o s t e r i c Model 367

R. Blumenthal, A. Pu r i , A. Walter, and 0. Eidelman

Properties of a V i r a l Fusion Protein 385 R.W. Doms and A. Helenius

Parameters Affecting the Fusion of Viruses with A r t i f i c i a l and B i o l o g i c a l Membranes 399

D. Hoekstra, K. Klappe, Τ. Stegmann, and S. Nir

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Active Function of Membrane Receptors in Fusion of Enveloped Viruses with C e l l Plasma Membranes 413

A. Loyter, 0. Nussbaum, and V, Citovsky 1 Entry 1 of Enveloped Viruses into Liposomes 427

A. M. Haywood

Fusion A c t i v i t y of Influenza Virus and Reconstituted V i r a l Envelopes: Direct Evidence for Fusion i n an I n t r a c e l l u l a r Acidic Compartment 441

J . Wilschut and T. Stegmann

K i n e t i c s and Extent of Fusion of Influenza Virus and Sendai Virus with Liposomes 451

S. Nir, T. Stegmann, D. Hoekstra, and J . Wilschut

Strategies for the Investigation of Exocytotic Membrane Fusion 467

M. G r a t z l

Enzymatic Regulation of Membrane Fusion During Synchronous Exocytosis i n Paramecium C e l l s 477

H. P l a t t n e r , C.J. Lumpert, U. Gras, J . Vilmart-Seuwen, B. Stecher, Β. Höhne, Μ. Momayezi, R. Pape, and Η. Kersken

Studies on the Structure and Function of the Asialoglycoprotein Receptor i n the C e l l , In Vitro and i n Reconstituted Membranes 495

D. Doyle, J . P e t e l l , and J . Sawyer

Phosphorylation Events i n Regulation of Exocytosis 513 B. H. S a t i r and T.J. Murtaugh

Geometric Topology of Membrane Fusion: From Secretion to I n t e r c e l l u l a r Junctions 521

P.P. da S i l v a fSlow A r t i f a c t s * in Assays of L i p i d Mixing Between Membranes 531

J.R. S i l v i u s , R. Leventis, and P.M. Brown

Why Fusion Assays Disagree 543 N. Düzgünes, T.M. Allen, J . Fedor, and D. Papahadjopoulos

Concentration Dependence of DPHpPC Fluorescence Lifetime: Photophysics and U t i l i t y for Monitoring Membrane Fusion 557

B.R. Lentz, S.W. Burgess, and E. Gratton

Contributors 567

Attendees 573

Index 579

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STRATEGIES FOR THE INVESTIGATION OF EXOCYTOTIC MEMBRANE FUSION

Manfred G r a t z l

A b t e i l u n g Anatomie und Z e l l b i o l o g i e Universität Ulm, B u n d e s r e p u b l i k D e u t s c h l a n d

INTRODUCTION

The t r a n s f e r o f hormones from t h e c e l l i n t o t h e e x t r a c e l l u l a r s p a c e d u r i n g s e c r e t i o n by e x o c y t o s i s i s a c c o m p l i s h e d by t h e f u s i o n of t h e s e c r e t o r y v e s i c l e membrane w i t h t h e p l a s m a membrane. As opposed t o t h e s t u d y o f a r t i f i c i a l membranes o r i s o l a t e d n a t u r a l membranes a s models, s u i t a b l e m o d i f i e d s e c r e t o r y c e l l s have been i n t r o d u c e d i n t h e p a s t few y e a r s t o i n v e s t i g a t e e x o c y t o t i c membrane f u s i o n . The g roups w o r k i n g i n t h e l a t t e r f i e l d a r e a t t e m p t i n g t o l e a r n a s much a s p o s s i b l e from n a t u r e w i t h t h e aim o f c h a r a c t e r i z i n g t h e p r o p e r t i e s of membrane f u s i o n a s i t o c c u r s i n t h e i n t a c t c e l l .

The i n h e r e n t p r o b l e m i n s u c h i n v e s t i g a t i o n s i s t h a t t h e i n t e r a c t i n g membranes a r e not a c c e s s i b l e from o u t s i d e t h e c e l l s . M a n i p u l a t i o n of t h e c y t o p l a s m i n a r a t h e r l i m i t e d way c a n be c a r r i e d o u t u s i n g i o n o p h o r e s . D i r e c t a c c e s s t o t h e c y t o p l a s m and i t s c o m p l e t e c o n t r o l r e q u i r e s t h e breakdown o f t h e p l a s m a membrane a s a p e r m e a b i l i t y b a r r i e r . With s i n g l e c e l l s t h i s c a n be done u s i n g c e l l s a t t a c h e d t o p i p e t t e s ( c . f . F e r n a n d e z e t a l . , 1 9 8 4 ) . C e l l s u s p e n s i o n s have been p e r m e a b i l i z e d w i t h d i f f e r e n t t e c h n i q u e s w i t h o u t i m p a i r i n g t h e p r o c e s s of e x o c y t o s i s . With h i g h - v o l t a g e d i s c h a r g e s and d e t e r g e n t s , i t h a s been p o s s i b l e t o p e r m a n e n t l y p e r m e a b i l i z e c e l l s f o r s e c r e t i o n s t u d i e s . I n b o t h ways, i f c a r e f u l l y c o n t r o l l e d , t h e c e l l s seem n o t t o be a l t e r e d g r e a t l y i n so f a r a s t h e r e l e a s e p r o c e s s i s c o n c e r n e d ( c . f . K n i g h t and B a k e r , 1982; W i l s o n and K i r s h n e r , 1983; Dunn and H o l z , 1 9 8 3 ) . The l e s i o n s e v o k e d i n t h e c e l l membrane w i t h t h e s e methods d i f f e r i n s i z e . W i t h d e t e r g e n t s , c e l l u l a r m a c r o m o l e c u l e s ( l i k e c y t o p l a s m i c l a c t a t e d e h y d r o g e n a s e ) a r e l o s t , w h i l e w i t h h i g h v o l t a g e d i s c h a r g e s few s u c h m o l e c u l e s e s c a p e . I n c o n t r a s t t o t h e t e c h n i q u e s m e n t i o n e d above, d e f i n e d p o r e s c a n be i n s e r t e d i n t o t h e c e l l membrane o f s e c r e t o r y c e l l s u s i n g s t a p h y l o c o c c a l a l p h a - t o x i n . T h i s a l l o w s s m a l l s o l u t e s t o be e x c h a n g e d and k e e p s m a c r o m o l e c u l e s n e c e s s a r y f o r s e c r e t i o n w i t h i n t h e c e l l s . The a p p l i c a t i o n of a l p h a - t o x i n f o r t h e a n a l y s i s of e x o c y t o t i c membrane f u s i o n ( A h n e r t - H i l g e r e t a l . , 1985a,b, 1987;

467

Bader e t a l . , 1986; L i n d e t a l . , 1987; A h n e r t - H i l g e r and G r a t z l , 1987) w i l l be d e s c r i b e d i n t h i s c h a p t e r . The c h a r a c t e r i s t i c s o f hormone r e l e a s e from p e r m e a b i l i z e d c e l l s w i l l be compared w i t h s e c r e t i o n by i n t a c t c e l l s and 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 membranes and o f l i p o s o m e s .

PERMEABILIZATION OF SECRETORY C E L L S WITH ALPHA-TOXIN

A l p h a - t o x i n i s a w a t e r s o l u b l e p r o t e i n p r o d u c e d by most s t r a i n s o f S t a p h y l o c o c c u s a u r e u s . The mechanism of t h e t o x i n s a t t a c k o f mammalian c e l l s h a s been e s t a b l i s h e d i n a s e r i e s o f i n v e s t i g a t i o n s (Füssle e t a l . , 1981; B h a k d i e t a l . , 1981, 1984; f o r r e v i e w s e e B h a k d i and T r anum-Jensen, 1 9 8 7 ) . F i r s t t h e t o x i n ( m o l e c u l a r w e i g h t 34 KD) i s bound t o t h e c e l l s , t h e n i t h e x a m e r i z e s i n t h e p l a s m a membrane and forms s t a b l e transmembrane p o r e s , w h i c h do n o t p e r m i t t h e p a s s a g e o f m a c r o m o l e c u l e s w i t h a m o l e c u l a r w e i g h t o f 4 KD and l a r g e r b u t a l l o w s f o r t h e r e l e a s e o f s m a l l m o l e c u l e s from e r y t h r o c y t e s .

S e c r e t o r y c e l l s c a n a l s o be p e r m e a b i l i z e d by a l p h a - t o x i n . B o v i n e c h r o m a f f i n c e l l s , r a t pheochromocytoma o r i n s u l i n o m a c e l l s i n c u l t u r e , upon a d d i t i o n o f a l p h a - t o x i n , r e l e a s e s m a l l s o l u t e s l i k e m o n o v a l e n t c a t i o n s o r ATP and t a k e up t r y p a n b l u e w h i l e l a r g e p r o t e i n s s u c h a s c y t o p l a s m i c l a c t a t e d e h y d r o g e n a s e a r e r e t a i n e d by t h e c e l l s ( A h n e r t - H i l g e r e t a l . , 1985; Bader e t a l . , 1986; L i n d e t a l . , 1 9 8 7 ) . I n o t h e r words t h e s i z e o f t h e a l p h a - t o x i n p o r e a l l o w s f o r t h e r e m o v a l of a l l s m a l l m o l e c u l e s from t h e c y t o p l a s m and t h e i r r e p l a c e m e n t by o t h e r o n e s . T h e r e f o r e t h i s t e c h n i q u e c a n s e r v e t o add b u f f e r s u b s t a n c e s f o r t h e c o n t r o l o f pH and d i v a l e n t c a t i o n s . I n a d d i t i o n , d r u g s known t o i n t e r f e r e w i t h d e f i n e d i n t r a c e l l u l a r r e g u l a t o r s i t e s , b u t i n e f f e c t i v e i n i n t a c t c e l l s due t o l i m i t e d a c c e s s , c a n e a s i l y be i n t r o d u c e d i n t o s e c r e t o r y c e l l s . F i n a l l y , t h e r e m a r k a b l e i n s e n s i t i v i t y o f s e c r e t o r y v e s i c l e f u s i o n w i t h t h e p l a s m a membrane t o t h e i n c o r p o r a t i o n of a l p h a - t o x i n a l l o w s s u f f i c i e n t t i m e f o r t h e e x p e r i m e n t a l m a n i p u l a t i o n o f t h e p e r m e a b i l i z e d c e l l s .

As a c o n s e q u e n c e o f t h e s m a l l s i z e of t h e a l p h a - t o x i n p o r e , t h e t o x i n monomer i t s e l f i s e x c l u d e d from t h e c y t o p l a s m . Thus t h e t o x i n ' s a t t a c k i s r e s t r i c t e d t o t h e p l a s m a membrane w h i c h i s an a d v a n t a g e when compared t o t h e a c t i o n o f d e t e r g e n t s u s e d f o r c e l l p e r m e a b i l i z a t i o n . A l s o d e t e r g e n t s l i k e d i g i t o n i n h a r b o r t h e h a z a r d s o f m o d i f y i n g t h e c o m p o s i t i o n of t h e i n t e r a c t i n g membrane and t h u s d i s t o r t i n g t h e d a t a o b t a i n e d .

As e v i d e n c e f o r e x o c y t o s i s i n a l p h a - t o x i n p e r m e a b i l i z e d c e l l p r e p a r a t i o n s , t h e same c r i t e r i a c a n be u s e d w h i c h were a p p l i e d t o i n t a c t c e l l s : Ε. g. i n o r d e r t o e x c l u d e s i m p l e l e a k a g e o f hormones from s e c r e t o r y v e s i c l e s t h e p a r a l l e l r e l e a s e o f s m a l l and l a r g e m o l e c u l a r w e i g h t s e c r e t o r y p r o d u c t s p r e s e n t w i t h i n t h e v e s i c l e s c a n be m easured. T h i s c o - r e l e a s e , i n c o n j u n c t i o n w i t h t h e o b s e r v a t i o n t h a t l a r g e m o l e c u l e s r e s i d i n g i n t h e c y t o p l a s m r e m a i n w i t h i n t h e c e l l s c l e a r l y i n d i c a t e s t h a t hormone r e l e a s e o c c u r s v i a e x o c y t o s i s ( A h n e r t - H i l g e r e t a l . , 1985b; B a d e r e t a l . , 1 9 8 6 ) . A n o t h e r a p p r o a c h t o d e m o n s t r a t e e x o c y t o s i s makes u s e o f t h e c o m p a r t m e n t a t i o n of enzymes m e t a b o l i z i n g c a t e c h o l a m i n e s ( A h n e r t - H i l g e r e t a l . , 1 9 8 7 ) . C a t e c h o l a m i n e s a r e r e l e a s e d

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unchanged from i n t a c t pheochromocytoma c e l l s because t h e cyto p l a s m c o n t a i n i n g t h e o x i d i z i n g enzymes i s c i r c u m v e n t e d i f s e c r e t i o n occurs by e x o c y t o s i s . When t r a n s f e r r e d i n t o t h e cyt o p l a s m (by r e v e r s i n g t h e d r i v i n g f o r c e f o r v e s i c u l a r uptake o f catecholamines) o x i d i z e d c a t e c h o l a m i n e s are formed. Since t h e same s i t u a t i o n i s seen i n a l p h a - t o x i n p e r m e a b i l i z e d c e l l s , i t i s safe t o conclude t h a t t h e s e c r e t o r y v e s i c l e c o n t e n t s are d i r e c t l y t r a n s p o r t e d by e x o c y t o s i s i n t o t h e e x t r a c e l l u l a r space.

R e c e n t l y , i n a d d i t i o n t o t h e o t h e r documented procedures ( c . f . r e f e r e n c e s Bhakdi and Tranum-Jensen, 1987) a r a p i d and e f f e c t i v e p u r i f i c a t i o n p rocedure f o r a l p h a - t o x i n from t h e c u l t u r e medium o f Staphylococcus aureus ( s t r a i n Wood 46) has been d e s c r i b e d ( L i n d e t a l . , 1987). T h i s t e c h n i q u e f a c i l i t a t e s a g a i n t h e study o f membrane f u s i o n i n p e r m e a b i l i z e d s e c r e t o r y c e l l s .

CHARACTERIZATION OF EXOCYTOSIS IN ALPHA-TOXIN PERMEABILIZED CELLS

I n t h e f i r s t s e r i e s o f ex p e r i m e n t s t h e m i n i m a l ( c y t o p l a s m i c ) r e q u i r e m e n t s f o r s e c r e t o r y v e s i c l e / p l a s m a membrane f u s i o n have been e s t a b l i s h e d i n a l p h a - t o x i n p e r m e a b i l i z e d PC12 c e l l s , a r a t pheochromocytoma c e l l l i n e . I t was demonstrated t h a t t h e r e l e a s e o f dopamine from these c e l l s upon a d d i t i o n o f a l p h a - t o x i n was reduced from " e x t r a c e l l u l a r " (mM) t o i n t r a c e l l u l a r (μΜ) c a l c i u m c o n c e n t r a t i o n s ( A h n e r t - H i l g e r e t a l . , 1985a). I n media c o n t a i n i n g d e f i n e d f r e e c o n c e n t r a t i o n s o f c a l c i u m ( a d j u s t e d w i t h c h e l a t o r s and c o n t r o l l e d w i t h a c a l c i u m - s p e c i f i c e l e c t r o d e ) a c h a r a c t e r i s t i c b i p h a s i c response as a f u n c t i o n o f t h e f r e e c a l c i u m c o n c e n t r a t i o n c o u l d be observed ( F i g . 1 and A h n e r t - H i l g e r e t a l . , 1985a,b; A h n e r t - H i l g e r and G r a t z l , 1987). One phase a l r e a d y a c t i v a t e d below 1 μΜ f r e e c a l c i u m , p l a t e a u e d a t about 1-5 μΜ f r e e c a l c i u m and a second o c c u r r e d i n t h e presence of l a r g e r amounts o f f r e e c a l c i u m (10-100 μΜ) . Calcium-induced dopamine r e l e a s e c o u l d be observed a t pH 6.6 as w e l l as a t pH 7.2, i t was i n s e n s i t i v e t o changes i n t h e n a t u r e o f monovalent c a t i o n s or a n i o n s . Magnesium was unable t o r e p l a c e c a l c i u m . However, when p r e s e n t s i m u l t a n e o u s l y w i t h c a l c i u m i n 1-5 μΜ c o n c e n t r a t i o n s i t augmented hormone r e l e a s e ( F i g . 2 and A h n e r t - H i l g e r and G r a t z l , 1987). I t i s i n t e r e s t i n g t o note t h a t t h e e f f e c t o f magnesium was o n l y seen a t t h e low c a l c i u m c o n c e n t r a t i o n s which are l i k e l y t o occur i n s t i m u l a t e d c e l l s (see n e x t c h a p t e r ) . F u r t h e r a t t e m p t s t o c h a r a c t e r i z e or i d e n t i f y t h e t a r g e t s ( r e c e p t o r s ) o f c a l c i u m w i t h i n t h e pheochromocytoma c e l l s u s i n g g r o u p - s p e c i f i c r e a g e n t s or drugs i n t e r f e r i n g w i t h c a l m o d u l i n ( t r i f l u o p e r a z i n e ) have n o t y e t been s u c c e s s f u l .

One reason f o r t h a t may be t h a t a d d i t i o n a l m o d u l a t o r y r e a c t i o n s i n v o l v e d i n t h e r e g u l a t i o n o f e x o c y t o s i s are w o r k i n g i n a l p h a - t o x i n p e r m e a b i l i z e d c e l l s . From t h e i n h i b i t o r y a c t i o n of GTP-gamma-S on c a l c i u m - i n d u c e d e x o c y t o s i s , a mo d u l a t o r y r o l e o f GTP b i n d i n g p r o t e i n s has been i n f e r r e d as f o r t h e a c t i v a t i o n by TPA an i n v o l v e m e n t o f t h e p r o t e i n k i n a s e C cascade ( A h n e r t - H i l g e r e t a l . , 1987). The l a t t e r e f f e c t r e q u i r e s t h e presence o f ATP w h i l e t h e c a l c i u m - i n d u c e d e x o c y t o s i s i n t h e pheochromocytoma c e l l s used i s e n t i r e l y

469

i n d e p e n d e n t on t h i s n u c l e o t i d e ( A h n e r t - H i l g e r e t a l . , 1985a,b; A h n e r t - H i l g e r and G r a t z l , 1 9 8 7 ) .

I n t e r e s t i n g l y , c h r o m a f f i n c e l l s i n p r i m a r y c u l t u r e a l s o i n t h e a b s e n c e o f TPA r e q u i r e a d d i t i o n a l ATP f o r c a l c i u m - i n d u c e d e x o c y t o s i s ( B a d e r e t a l . , 1 9 8 6 ) . O t h e r n u c l e o t i d e s c o u l d n o t r e p l a c e ATP s u g g e s t i n g a v e r y s p e c i f i c r o l e i n t h e s e c e l l s . The ATP r e q u i r e m e n t o f s e c r e t i o n i n i n t a c t c e l l s w i l l be d i s c u s s e d i n d e t a i l i n t h e n e x t s e c t i o n of t h i s a r t i c l e .

COMPARISON OF EXOCYTOSIS IN ALPHA-TOXIN PERMEABILIZED C E L L S WITH MEMBRANE FUSION OF BIOLOGICAL MEMBRANES AND OF LIPOSOMES

The i m p o r t a n c e o f e x t r a c e l l u l a r c a l c i u m 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 was r e a l i z e d more t h a n 25 y e a r s ago ( D o u g l a s and R u b i n , 1 9 6 1 ) . I n j e c t i o n s t u d i e s w i t h mast c e l l s and t h e g i a n t s y n a p s e i n d i c a t e d t h a t i n t r a c e l l u l a r c a l c i u m may t r i g g e r s e c r e t i o n (Kanno e t a l . , 1973; M i l e d i , 1 9 7 3 ) . A f t e r i t was o b s e r v e d t h a t t r a n s m i t t e r r e l e a s e i s a c t u a l l y p a r a l l e l e d by an i n c r e a s e i n i n t r a c e l l u l a r f r e e c a l c i u m (Llinäs and N i c h o l s o n ) t h i s i s s u e was f u r t h e r i n v e s t i g a t e d i n a v a r i e t y o f s e c r e t o r y c e l l s . T h e s e s t u d i e s soon became e x p e r i m e n t a l l y e a s i e r t o be c a r r i e d o u t b e c a u s e new t e c h n i q u e s became a v a i l a b l e . R e s t i n g pheochromocytoma c e l l s o r a d r e n a l m e d u l l a r y c h r o m a f f i n c e l l s i n p r i m a r y c u l t u r e , i . e. t h e c e l l s u s e d up t o now f o r p e r m e a b i l i z a t i o n w i t h a l p h a - t o x i n , c o n t a i n about 0.1 μΜ f r e e c a l c i u m .

F i g . 1. C a l c i u m r e q u i r e m e n t o f dopamine r e l e a s e by i n t a c t and by a l p h a - t o x i n p e r m e a b i l i z e d pheochromocytoma c e l l s ( f r om A h n e r t - H i l g e r e t a l . , 1985a, by p e r m i s s i o n ) .

470

When s t i m u l a t e d , t h e f r e e c a l c i u m i n c r e a s e s t o about 0.5 μΜ ( K n i g h t and K e s t e v e n , 1983; M e l d o l e s i e t a l . , 1 9 8 4 ) . I t i s w o r t h m e n t i o n i n g , however, t h a t an a v e r a g e i n t r a c e l l u l a r f r e e c a l c i u m c o n c e n t r a t i o n has been measured i n t h e s e s t u d i e s and t h e t r u e c o n c e n t r a t i o n s j u s t b e n e a t h t h e p l a s m a membrane may be h i g h e r as a c o n s e q u e n c e o f t h e c a l c i u m i n f l u x d u r i n g s t i m u l a t i o n . I n a n o t h e r s t u d y (Kao and S c h n e i d e r , 1986) v a l u e s o f about 10 μΜ f r e e c a l c i u m have been o b t a i n e d i n c h r o m a f f i n c e l l s s t i m u l a t e d w i t h n i c o t i n e .

I n any c a s e , t h e c a l c i u m dependence of c a t e c h o l a m i n e s e c r e t i o n by i n t a c t c e l l s i s i n a c c o r d a n c e w i t h t h e s i t u a t i o n s e e n i n a l p h a - t o x i n p e r m e a b i l i z e d pheochromocytoma c e l l s and a d r e n a l m e d u l l a r y c h r o m a f f i n c e l l s i n p r i m a r y c u l t u r e ( s e e p r e v i o u s c h a p t e r ) . I n t e r e s t i n g l y enough, a l s o t h e dependence on ATP i n a l p h a - t o x i n p e r m e a b i l i z e d c h r o m a f f i n c e l l s ( B a d e r e t a l . , 1986) i s p a r a l l e l e d by a s i m i l a r o b s e r v a t i o n i n i n t a c t c e l l s ( R u b i n , 1969; K i r s h n e r and S m i t h , 1 9 6 5 ) . I n t a c t pheochromocytoma c e l l s , on t h e o t h e r hand, do not depend on c y t o p l a s m i c ATP d u r i n g s e c r e t i o n ( R e y n o l d s e t a l . , 1 9 8 2 ) , a f a c t w h i c h i s s u p p o r t e d by t h e i n s e n s i t i v i t y o f c a l c i u m - i n d u c e d dopamine r e l e a s e t o ATP i n t h e same c e l l s p e r m e a b i l i z e d w i t h a l p h a - t o x i n ( A h n e r t - H i l g e r e t a l . , 1985a,b; A h n e r t - H i l g e r and G r a t z l , 1 9 8 7 ) .

25

no addition Mg 2 ATP5 Mg 2 Ί Mg 2ATP1

F i g . 2. E f f e c t o f magnesium w i t h o r w i t h o u t ATP on c a l c i u m - i n d u c e d dopamine r e l e a s e by p e r m e a b i l i z e d pheochromocytoma c e l l s . The open b a r s r e p r e s e n t c o n t r o l s , t h e h a t c h e d b a r s a l p h a - t o x i n t r e a t e d c e l l s . The medium c o n t a i n e d 10 μΜ f r e e c a l c i u m and t h e i n d i c a t e d amounts o f f r e e magnesium (fr o m A h n e r t - H i l g e r and G r a t z l , 1987, by p e r m i s s i o n ) .

471

The e x a c t r e a s o n f o r t h e n e c e s s i t y o f ATP i n p e r m e a b i l i z e d c h r o m a f f i n c e l l s i n p r i m a r y c u l t u r e i s not known. One p o s s i b i l i t y i s t h a t r e g u l a t o r y s e q u e n c e s ( l i k e t h e P r o t e i n k i n a s e C s y s t e m ) have a l l components a c t i v a t e d b u t one key p r o t e i n i s d e p h o s p h o r y l a t e d and r e q u i r e s ATP f o r r e p h o s p h o r y l a t i o n . I n o r d e r t o i d e n t i f y s u c h s i t e s p h o s p h o r y l a t i o n s t u d i e s have been c a r r i e d o u t u s i n g i n t a c t c e l l s , p e r m e a b i l i z e d c e l l s , o r s u b c e l l u l a r f r a c t i o n s (Schulman and G r e e n g a r d , 1978; Amy and K i r s h n e r , 1981; T r e i m a n e t a l . , 1983; N i g g l i e t a l . , 1 9 8 4 ) . However, a l s o t h e t o p o g r a p h y of c h r o m a f f i n v e s i c l e d i s t r i b u t i o n i n t h e d i f f e r e n t c e l l s i n v e s t i g a t e d must be c o n s i d e r e d . I n c u l t u r e d pheochromocytoma c e l l s s e c r e t o r y v e s i c l e s a r e c o n c e n t r a t e d n e a r t h e p l a s m a membrane (Watanabe e t a l . , 1983) and t h u s a r e c l o s e t o t h e s i t e o f e x o c y t o s i s . On t h e o t h e r hand w i t h i n a d r e n a l m e d u l l a r y c h r o m a f f i n c e l l s s e c r e t o r y v e s i c l e s a r e more randomly d i s t r i b u t e d ( c . f . B a d er e t a l . , 1981) and ATP dependent v e s i c l e movement ( A l l e n e t a l . , 1985; V a l e e t a l . , 1985) c o u l d be n e c e s s a r y .

The s t e p r e q u i r i n g ATP i s not t h e p r o c e s s o f membrane f u s i o n i t s e l f b u t a p r e c e d i n g one, s i n c e 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 i s o l a t e d from c h r o m a f f i n and o t h e r s e c r e t o r y c e l l s f u s e w i t h o u t ATP added. T h e s e b i o l o g i c a l membranes f u s e h a l f maximal w i t h about 1-10 μΜ f r e e c a l c i u m , i . e. t h e same c o n c e n t r a t i o n o b s e r v e d i n s t i m u l a t e d i n t a c t c e l l s o r t h e amount o f c a l c i u m e f f e c t i v e i n a l p h a - t o x i n p e r m e a b i l i z e d c e l l s ( D a h l and G r a t z l , 1976; E k e r d t e t a l . , 1981; G r a t z l and D a h l , 1976, 1978; G r a t z l e t a l . , 1977, 1 9 8 0 ) . When t h e membranes i n v e s t i g a t e d were f u r t h e r s i m p l i f i e d by t h e p r e p a r a t i o n o f l i p o s o m e s from t h e membrane l i p i d s o f c h r o m a f f i n v e s i c l e s , c h a r a c t e r i s t i c p r o p e r t i e s l i k e t h e s u s c e p t i b i l i t y t o low c o n c e n t r a t i o n s o f c a l c i u m f o r t h e f u s i o n p r o c e s s were l o s t ( E k e r d t e t a l . , 1 9 8 1 ) . I n c o n j u n c t i o n w i t h t h e o b s e r v a t i o n t h a t t r e a t m e n t w i t h c h e m i c a l s o r enzymes of t h e s e c r e t o r y v e s i c l e membranes l e d t o t h e same c h a n g e s i n membrane f u s i o n p r o p e r t i e s , i t was c o n c l u d e d t h a t membrane p r o t e i n s a r e r e s p o n s i b l e f o r t h e s e p r o p e r t i e s ( E k e r d t e t a l . , 1 9 8 1 ) . A c t u a l l y l i p o s o m e s c o n t a i n i n g a c i d i c p h o s p h o l i p i d s e x h i b i t v e r y s i m i l a r f u s i o n p r o p e r t i e s as do t h e l i p o s o m e s p r e p a r e d from t h e membrane l i p i d s 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 ( E k e r d t e t a l . , 1981; c . f . f o r r e f . Düzgünes, 1 9 8 5 ) . I n a t t e m p t s t o r e d u c e t h e c a l c i u m r e q u i r e m e n t s o f t h e s e membranes from mM t o more p h y s i o l o g i c a l (μΜ) c o n c e n t r a t i o n s t h e e f f e c t o f s y n e x i n , a c y t o p l a s m i c p r o t e i n p r e s e n t i n a v a r i e t y of s e c r e t o r y c e l l s h a s been i n v e s t i g a t e d (Hong e t a l . , 1 9 8 2 a , b ) . I t t u r n e d o u t t h a t w i t h t h i s p r o t e i n , i n t h e p r e s e n c e o f mM magnesium, t h e t h r e s h o l d o f t h e l i p o s o m e s t o undergo f u s i o n c o u l d be r e d u c e d t o about 10 μΜ c a l c i u m . T h i s i n d i c a t e s t h a t f u s i o n o f l i p o s o m e s c a n be mo d u l a t e d i n a way t o a p p r o a c h t h e m i l i e u e x i s t i n g i n r e s t i n g o r s t i m u l a t e d c e l l s .

CONCLUSION

P e r m e a b i l i z a t i o n o f s e c r e t o r y c e l l s by s t a p h y l o c o c c a l a l p h a - t o x i n i n d e e d i s a v a l u a b l e t e c h n i q u e t o i n v e s t i g a t e membrane f u s i o n d u r i n g e x o c y t o s i s b e c a u s e i t a l l o w s t h e permanent c o n t r o l o f t h e c o m p o s i t i o n o f t h e c y t o p l a s m w i t h r e s p e c t t o s m a l l m o l e c u l e s (MW 1 KD) w i t h o u t p e r t u r b i n g t h e f u s i o n p r o c e s s i t s e l f . The s e c r e t i o n o b s e r v e d i n s u c h

472

p e r m e a b i l i z e d c e l l s i s c o m p a t i b l e w i t h s e c r e t i o n by i n t a c t c e l l s and c l o s e l y r e s e m b l e s membrane f u s i o n s t u d i e d w i t h i s o l a t e d s e c r e t o r y v e s i c l e membranes.

A f t e r e x t e n s i v e l y s t u d y i n g t h e e f f e c t s o f s m a l l s o l u t e s , i n h i b i t o r s o r a c t i v a t o r s , t h e c o n c e p t o f u s i n g n a t u r a l o c c u r r i n g p o r e f o r m i n g s u b s t a n c e s f o r c e l l p e r m e a b i l i z a t i o n opens f u r t h e r p o s s i b i l i t i e s . Permanent p o r e s w h i c h a r e b i g enough t o r e l e a s e e v e n l a r g e m o l e c u l a r w e i g h t s u b s t a n c e s from t h e c e l l - s o r t o i n t r o d u c e a n t i b o d i e s d i r e c t e d a g a i n s t membrane components s t i l l r e s i d i n g i n p e r m e a b i l i z e d c e l l s c a n be u s e d t o i d e n t i f y t h e r e l e v a n t membrane p r o t e i n s i n v o l v e d i n e x o c y t o t i c membrane f u s i o n . Then t h e y c a n be i s o l a t e d and i n c o r p o r a t e d i n t o l i p o s o m e s w i t h t h e aim o f m o d i f y i n g t h e i r p r o p e r t i e s i n a way t h a t t h e f u s i o n p r o c e s s i n t h i s s i m p l i f i e d s y s t e m i s c o m p a t i b l e w i t h t h e s i t u a t i o n s e e n i n i n t a c t s e c r e t o r y c e l l s . T h i s would t h e n e l u c i d a t e t h e m o l e c u l a r mechanism o f membrane f u s i o n d u r i n g e x o c y t o s i s .

ACKNOWLEDGEMENTS

The work o f t h e a u t h o r r e f e r r e d t o i n t h i s a r t i c l e was s u p p o r t e d by t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t (Gr 681) and t h e F o r s c h u n g s s c h w e r p u n k t No. 24 of t h e S t a t e o f Baden-Württemberg. The a u t h o r t h a n k s Mrs. M. Henning f o r h e r h e l p f u l a d v i c e on how t o word t h i s m a n u s c r i p t and Mrs. B. Mader f o r t y p i n g i t .

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