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CYTOBIOLOGIE European Journal of Cell Biqljpgy ;

Volume 13 • Numbe^ 1/ jiine 19||

C O N T E N T S V O L U M E 13 III

AMELUNXEN, F., E. THIO TIANG NIO , E. SPIESS Untersuchungen zur Zellwandbildung. I. Elektronenmikroskopische Beobachtungen zur Feinstruktur der Ranken von Cucurbita maxima L. I n v e s t i g a t i o n s on cell w a l l f o r m a t i o n . 1. E l e c t r o n microscopical observations on the f ' m e structure o f the t e n d r i l s of C u c u r b i t a m a x i m a L .

233

AMELUNXEN, F., siehe SPIESS, E. 251

AMELUNXEN, F., E. SPIESS, E. THIO TIANG NIO Untersuchungen zur Zellwandbildung. III. Autoradiographie von Dünnschnitten und biochemische Analyse isolierter Dictyosomen der Ranken von Cucurbita maxima L. 260

I n v e s t i g a t i o n s on cell w a l l f o r m a t i o n . I I I . I n v e s t i g a t i o n s on t h i n sections by a u t o r a d i o g r a p h y and biochemical a n a l y s i s o f i s o l a t e d dictyosomes o f t e n d r i l s of C u c u r b i t a m a x i m a L .

ANDRIES, J. C.

Variations ultrastructurales au sein des cellules epitheliales mesenteriques d'Aeshna cyanea (Insecte, Odonate) en fonction de la prise de nourriture 451 M i d g u t cells u l t r a s t r u c t u r a l m o d i f i c a t i o n s d u r i n g d i g e s t i o n i n Aesbna cyanea (Insecte, O d o n a t a )

BARANOWSKI, Z .

Three-dimensional analysis of movement in Physarum polycephalum plasmodia 118 D r e i d i m e n s i o n a l e A n a l y s e der Bewegung bei P h y s a r u m p o l y c e p h a l u m - P l a s m o d i e n

BRUNK, U . T., siehe SCHELLENS, J. P. M . 93

CHESCOE, D. , siehe DUCKETT, J. G . 322

COOKE, P., A. M . POISNER

Microfilaments in bovine adrenal chromaffin cells 442 M i k r o f i l a m e n t e i n den c h r o m a f f i n e n Z e l l e n der Kinder-Nebenniere

DAHL, G . , siehe SCHUDT, C H . 211

DEICHGRÄBER, G. , siehe SCHNEPF, E. 341

DEUMLING, B., J. SINCLAIR, J. N . TIMMIS, J. INGLE

Demonstration of satellite D N A components in several plant species with the Ag+-Cs2S04 gradient technique 224 V o r k o m m e n v o n S a t e l l i t e n - D N S i n einigen P f l a n z e n a r t e n bei A n w e n d u n g der Ag+-Cs2SÖ4 G r a d i e n t e n t e c h n i k

DEXHEIMER, J.

Etüde de la secretion de mucilage par les cellules des glandes digestives de Drosera (D. rotundifolia L.; et D. capensis L.). Application de quelques techniques cytochimiques 307 Study o f the secretion of mucilage i n digesting gland cells o f D r o s e r a

D'HAESE, J., siehe HINSSEN, H . 132

DIETERICH, C. E., siehe DIETERICH, H . J. 57

IV C O N T E N T S V O L U M E 13

DIETERICH, H . J., K. A. ROSENBAUER, C. E . DIETERICH Das Blutgefäßsystem des Pecten oculi beim Haussperling (Passer domesticus). Architektonik und Ultrastruktur nach licht-, transmissions- und rasterelektronen-mikroskopischen Untersuchungen 57 The blood vessel System o f the pecten o c u l i i n the house s p a r r o w (Passer domesticus). l t s a r c h i t e c t u r e and f i n e structure studied by l i g h t , t r a n s m i s s i o n , and scanning electron microscopy

DUCKETT, J. G. , D. CHESCOE

A combined ultrastructural and X-ray microanalytical study of spermatogenesis in the bryophyte, Phaeoceros laevis (L.) Prosk. 322 K o m b i n i e r t e u l t r a s t r u k t u r e l l e und röntgenmikroanaly tische Untersuchung der Spermatogenese i m Lebermoos Phaeoceros l a e v i s ( L . ) Prosk.

ELDER, J. H . , J. MORRE, T H . W . KEENAN (Short Communication)

Distribution of glycoproteins among subcellular fractions from rat liver 279 G l y c o p r o t e i n - M u s t e r v o n Z e l l f r a k t i o n e n aus R a t t e n l e b e r

ELLIS, R. A. , C. C. GOERTEMILLER JR.

Scanning electron microscopy of intercellular Channels and the localization of ouabain sensitive p-nitrophenyl Phosphatase activity in the salt-secreting lacrymal glands of the marine turtle Chelonia mydas 1 R a s t e r e l e k t r o n e n m i k r o s k o p i s c h e D a r s t e l l u n g der I n t e r z e l l u l a r s p a l t e n und L o k a l i s a t i o n v o n O u a b a i n - s e n s i t i v e r p-Nitrophenyl-phosphatase-Aktivität i n den l a c r i m a l e n S a l z drüsen der Meeresschildkröte C h e l o n i a mydas

FRANKE, W. W., T H . W. KEENAN, J. STADLER, R. GENZ, E . -D. JARASCH, J. KARTENBECK Nuclear membranes from mammalian liver. VII. Characteristics of highly purified nuclear membranes in comparison with other membranes 28 Zusammensetzung hochgereinigter K e r n m e m b r a n f r a k t i o n e n aus der R a t t e n l e b e r i m Vergleich m i t anderen M e m b r a n e n

FRANKE, W. W., U. SCHEER, M . F. TRENDELENBURG, H . SPRING, H . ZENTGRAF

Absence of nucleosomes in transcriptionally active chromatin 401 C h r o m a t i n während der T r a n s k r i p t i o n : das Fehlen v o n Nucleosomen

FRIEDLANDER, M . , J. GERSHON, A. REINHARTZ

Atypical cycle of the nucleolus in spermatocytes of the insect Locusta migratoria 171 Besonderheiten des N u c l e o l u s - Z y k l u s i n S p e r m a t o c y t e n der Wanderheuschrecke L o c u s t a m i g r a t o r i a

GENZ, R., siehe FRANKE, W. W. 28

GERSHON, J., siehe FRIEDLANDER, M . 171

GHOSH, S. (Short Communication) Nucleolar development in induced binucleate cells of Allium cepa L. 163 N u c l e o l u s - E n t w i c k l u n g i n künstlich i n d u z i e r t e n z w e i k e r n i g e n Z w i e b e l z e l l e n

GOERTENMILLER JR., C. C , siehe ELLIS, R. A. 1

GRATZL, M . , siehe SCHUDT, C H . 211

C O N T E N T S V O L U M E 13 V

GRATZL, M . , D. SCHWAB The effect of microtubular inhibitors on secretion from liver into blood plasma and bile 199 D i e W i r k u n g mikrotubulärer H e m m s t o f f e a u f die S e k r e t i o n der Leber i n das B l u t p l a s m a und die G a l l e

GUNNING, B. E . S., siehe ROBARDS, A . W . 85

HAUSMANN, K. , K . - H . MOCIKAT

Direct evidence for a surface coat on the plasma membrane of the ciliate Litonotus duplostriatus 469 D i r e k t e r N a c h w e i s eines »surface coat« a u f der P l a s m a m e m b r a n des C i l i a t e n L i t o n o t u s d u p l o s t r i a t u s

HEBANT, C , R. P. C. JOHNSON

Ultrastructural features of freeze-etched water-conducting cells in Polytrichum (Polytrichales, Musci) 354 U l t r a s t r u k t u r der wasserleitenden Z e l l e n v o n P o l y t r i c h u m nach Gefrierätzung

HERLAN, G. , F. WUNDERLICH (Short Communication)

Isolation of a nuclear protein matrix from Tetrahymena macronuclei 291 I s o l a t i o n einer K e r n p r o t e i n m a t r i x aus T e t r a h y m e n a macronuclei

HINSSEN, H . , J. D'HAESE

Synthetic fibrils from Physarum actomyosin - seif assembly, Organization and contraction 132 Synthetische F i b r i l l e n aus i s o l i e r t e m S c h l e i m p i l z a c t o m y o s i n - Entstehung, S t r u k t u r

und K o n t r a k t i o n

INGLE, J., siehe DEUMLING, B. 224

JARASCH, E.-D., siehe FRANKE, W . W . 28

JOHNSON, R. P. C , siehe HEBANT, C. 354

KARTENBECK, J., siehe FRANKE, W . W . 28

KEENAN, T H . W. , siehe ELDER, J . H . 279

KEENAN, T H . W. , siehe FRANKE, W . W . 28

KLOPPSTECH, K. , H . G . SCHWEIGER

In vitro translation of poly (A) R N A from Acetabularia 394 I n v i t r o T r a n s l a t i o n v o n P o l y ( A ) R N A aus A c e t a b u l a r i a

LINDGREN, A . , siehe SCHELLENS, J. P. M . 93

LJUBESIC, N . , siehe SCHNEPF, E . 341

MEYER-ROCHOW, V. B. (Short Communication) A tri-directional microvillus orientation in the monocellular, distal rhabdom of a nocturnal beetle 476 I n drei verschiedenen Richtungen angeordnete M i k r o v i l l i i m monozellulären, d i s t a l e n R h a b d o m eines n a c h t a k t i v e n Käfers

MOCIKAT, K . - H . , siehe HAUSMANN, K. 469

VI C O N T E N T S V O L U M E 13

MOLLENHAUER, H . H . , D. J. MORRE Transition elements between endoplasmic reticulum and Golgi apparatus in plant cells 297 Übergangsstrukturen z w i s c h e n endoplasmatischem R e t i k u l u m und G o l g i - A p p a r a t

i n p f l a n z l i c h e n Z e l l e n

MOOR, H . , siehe NIEDERMEYER, W. 364

MORRE, D. J., siehe ELDER, J. H . 279

MORRE, D. J., siehe MOLLENHAUER, H . H . 297

NIEDERMEYER, W., G. R. PARISH, H . MOOR

The elasticity of the yeast cell tonoplast related to its ultrastructure and chemical composition. I. Induced swelling and shrinking; a freeze-etch membrane study 364 D i e Elastizität des H e f e t o n o p l a s t e n i n bezug a u f seine U l t r a s t r u k t u r und chemische Zusammensetzung. I . I n d u z i e r t e s Dehnen und Schrumpfen; Membranuntersuchungen m i t der Gefrierätztechnik

NIEDERMEYER, W.

The elasticity of the yeast cell tonoplast related to its ultrastructure and chemical composition. II. Chemical and cytochemical investigations 380 D i e Elastizität des H e f e t o n o p l a s t e n i n bezug a u f seine U l t r a s t r u k t u r und chemische Zusammensetzung. I I . Chemische und cytochemische Untersuchungen

NEHLS, R., G . SCHAFFNER (Short Communication)

Specific negative staining of proteins in situ with iron tannin 285 P r o t e i n s p e z i f i s c h e r N e g a t i v k o n t r a s t i n s i t u m i t E i s e n - T a n n i n

PARISH, G. R., siehe NIEDERMEYER, W. 364

PAYNE, H . L. , siehe ROBARDS, A. W. 85

PETTE, D. , siehe SCHUDT, C H . 74

PIGON, A.

Cell surface and medium conditioning in Acanthamoeba culture 107 Zelloberfläche und M e d i u m - c o n d i t i o n i n g i n A c a n t h a m o e b a - K u l t u r e n

PIPAN, N .

Death and phagocytosis of epithelial cells in developing mouse kidney 435 Tod und P h a g o z y t o s e der E p i t h e l z e l l e n während der E n t w i c k l u n g der Mäuseniere

POISNER, A. M . , siehe COOKE, P. 442

PRZELECKA, A . , A. SOBOTA

Calcium dependent deposits at the plasma membrane during development of the oocyte of Galleria mellonella 182 Verändertes Ca-Bindungsvermögen des P l a s m a l e m m a s während der O o c y t e n -E n t w i c k l u n g v o n G a l l e r i a m e l l o n e l l a

REINHARTZ, A. , siehe FRIEDLANDER, M . 171

C O N T E N T S V O L U M E 13 VII

ROBARDS, A . W., H . L. PAYNE, B. E. S. GUNNING

Isolation of the endodermis using wall-degrading enzymes 85 I s o l i e r u n g der Endodermis m i t H i l f e Z e l l w a n d - a b b a u e n d e r Enzyme

ROSENBAUER, K. A . , siehe DIETERICH, H . J. 57

SCHAFFNER, G . , siehe NEHLS, R. 285

SCHEER, U . , siehe FRANKE, W. W. 401

SCHELLENS, J. P. M . , U. T. BRUNK, A . LINDGREN

Influence of serum on ruffling activity, pinocytosis and proliferation of in vitro cultivated human glia cells 93 D e r Einfluß des Serums a u f Ruffling-Aktivität, P i n o c y t o s e und Z e l l v e r m e h r u n g v o n k u l t i v i e r t e n menschlichen G l i a - Z e l l e n

SCHNEPF, E., G . DEICHGRÄBER, N . LJUBESIC

The effects of colchicine, ethionine, and deuterium oxide on microtubules in young Sphagnum leaflets. A quantitative study 341 D i e W i r k u n g v o n C o l c h i c i n , Äthionin und D e u t e r i u m o x i d a u f die M i k r o t u b u l i i n jungen Sphagnum-Blättchen

SCHUDT, CH. , G . DAHL, M . GRATZL

Calcium-induced fusion of plasma membranes isolated from myoblasts grown in culture 211 C a l c i u m - i n d u z i e r t e Fusion v o n P l a s m a m e m b r a n e n , i s o l i e r t aus M y o b l a s t e n i n K u l t u r

SCHUDT, CH. , D . PETTE

Influence of monosaccharides, medium factors and enzymatic modification on fusion of myoblasts in vitro 74 Einflüsse v o n M o n o s a c c h a r i d e n , M e d i u m f a k t o r e n und enzymatischer M o d i f i k a t i o n

a u f die Fusion v o n M y o b l a s t e n i n v i t r o

SCHWAB, D . , siehe GRATZL, M . 199

SCHWEIGER, H . G. , siehe KLOPPSTECH, K. 394

SINCLAIR, J., siehe DEUMLING, B. 224

SOBOTA, A . , siehe PRZELECKA, A . 182

SPIESS, E., siehe AMELUNXEN, F. 233

SPIESS, E., siehe AMELUNXEN, F. 260

SPIESS, E., E. THIO TIANG NIO, F. AMELUNXEN Untersuchungen zur Zellwandbildung. II. Nachweis der Zellwandsynthese und der Synthese von Cellulose, Hemicellulose und Pektin in den Ranken von Cucurbita maxima L . 251 I n v e s t i g a t i o n s on cell w a l l f o r m a t i o n . 11. P r o v e for cell w a l l synthesis and synthesis o f cellulose, hemicellulose and pectin i n t e n d r i l s o f C u c u r b i t a m a x i m a L .

SPRING, H . , siehe FRANKE, W. W. 401

STADLER, J., siehe FRANKE, W . W. 28

STIEMERLING, R., siehe STOCKEM, W. 158

VIII C O N T E N T S V O L U M E 13

STOCKEM, W., R. STIEMERLING

Intracellular segregation of endocytotically ingested substances 158 Intrazelluläre Segregation endocytotisch aufgenommener Substanzen

STOCKERT, J. C.

Metachromatic staining of nucleolar differentiations by toluidine blue-molybdate in Chironomus salivary glands 191 M e t a c h r o m a t i s c h e Färbung v o n N u c l e o l u s - D i f f e r e n z i e r u n g e n i n C h i r o n o m u s -Speicheldrüsen durch T o l u i d i n b l a u - M o l y b d a t

THIO TIANG NIO, E., siehe AMELUNXEN, F. 233

THIO TIANG NIO , E., siehe AMELUNXEN, F. 260

THIO TIANG NIO , E., siehe SPIESS, E. 251

TIMMIS, J. N . , siehe DEUMLING, B. 224

TRENDELENBURG, M . F., siehe FRANKE, W. W. 401

WOLBURG, H .

Differences in distributions of 3 H-proline and 3 H-uridine-labelled Compounds in the fish visual System during ouabain-induced Wallerian degeneration 13 U n t e r s c h i e d l i c h e V e r t e i l u n g v o n 3 H - P r o l i n - und 3 H - U r i d i n - m a r k i e r t e n Verbindungen i m Seh-System der Karausche während der durch O u a b a i n i n d u z i e r t e n W a l l e r s c h e n D e g e n e r a t i o n

WUNDERLICH, F., siehe HERLAN, G. 291

ZENTGRAF, H . , siehe FRANKE, W. W. 401

B O O K R E V I E W S

BOSCHKE, F. (Hrsg.): Photochemistry 169

DOSE, K. , H . RAUCHFUSS: Chemische Evolution und der Ursprung lebender Systeme 170

DREWS, U . : Cholinesterase in Embryonic Development 482

FLORKIN, M . , E. H . STOTZ (Hrsg.): Comprehensive Biochemistry 168

HOLT , D. B., M . D. MUIR, P. R. GRANT, I. M . BOSWARVA (Hrsg.): Quantitative Scanning

Electron Microscopy 168

MODIS, L.: Topo-optical Investigations of Mucopolysaccharides (Acid Glycosaminoglycans) 482

ORCI, L., A. PERRELET: Freeze-Etch Histology 170

C Y T O B I O L O G I E 13, 211-223 (1976) @ Wissenschaftliche Verlagsges. m b H • Stuttgart

Calcium-induced fusion of plasma membranes isolated from myoblasts grown in culture

Calcium-induzierte Fusion von Plasmamembranen, isoliert aus Myoblasten in Kultur

CHRISTIAN S C H U D T 1 ) , GERHARD D A H L , and M A N F R E D G R A T Z L

Fachbereich Biologie der Universität Konstanz und Fachbereich Theoretische Medizin der Universität des Saarlandes, Homburg/Saar

Received September 20, 1975 in revised form February 26, 1976

and April 28, 1976

Abstract

M e m b r a n e f u s i o n - c a l c i u m - i s o l a t e d p l a s m a membranes - m y o b l a s t s 1. Plasma membrane vesicles isolated from myoblasts grown in vitro were found to fuse in

the presence of calcium ions. 2. As revealed by freeze cleaving techniques, addition of 1.4 mM CaCl* or 1.4 IHM MgClo

resulted in attachment of vesicles and aggregation of intramembranous particles. 3. Plasma membrane vesicles fuse solely in the presence of calcium, and fusion is identified

by the appearance of twinned vesicles. 4. Pore formation was preceded by an aggregation of intramembranous particles in the area

of membrane contact. .5. 20 m M MgCfe oder 10"6 M lysolecithin both decreased the amount of vesicle fusion in a

similar manner to the inhibition of the fusion of myoblasts.

Introduction

Membrane fusion appears to be essential in several biological processes taking place at the cellular and subcellular level [28, 31]. One of these is the spontaneous cell fusion which occurs during the development of striated skeletal muscle [24]. Muscle cell fusion can be inhibited in vitro if the cells are grown in media containing low calcium concentrations [37]. After 50 hours growth and proliferation, fusion can be triggered by raising the calcium concentration in the medium to about 2 m M . Under these conditions myoblasts form polynuclear myotubes in a monolayer within 6 to 8 hours [7, 37].

*) DR. CHR. SCHUDT, Fachbereich Biologie der Universität Konstanz, 7750 Konstanz, Germany.

212 CHRISTIAN SCHUDT, GERHARD DAHL, and MANFRED GRATZL

Although it has been suggested that calcium ions act on sites of plasma membranes accessible to ions of the extracellular fluid [36], no Information about the site of specific calcium binding is available. Furthermore, the question of the involvement of certain proteins, phospholipids or intracellular messengers in the mechanism of cell fusion remains to be resolved.

Morphological observations and studies by RASH et al. [29, 30] concerning the electrical coupling of fusing myogenic cells in vitro as well as in vivo indicate that intramembranous particles (IMPs) play an important role in myoblast fusion. The particles aggregate in the area of contact, form gap junctions and thus may mediate the initial events of fusion.

Since methods for the isolation of plasma membranes from muscle cells are now available [34], the interaction of these membranes can be studied in the absence of cytoplasmic components. It is of particular interest to show whether plasma membranes isolated from myoblasts are still able to fuse in a cell-free System. Moreover, it is important to establish whether a fusion of isolated plasma membrane vesicles depends on parameters similar to those affecting myoblast fusion [7, 32, 37] and whether a rearrangement of IMPs is related to the fusion process.

Materials and methods

Cell cultures of chick embryo breast muscle were prepared as described previously [6]. Cells were grown in a medium containing low calcium concentrations (10~5 M CaCl») to keep fusion below 5 % [6, 37]. After 52 hours myoblasts are competent for fusion, and on increasing the calcium concentration to 1.4 mM the cells form myotubes within 6 to 8 hours [7, 37]. Plasma membranes were prepared from myoblasts competent for fusion according to the method described by KENT et al. [19]. The membrane fraction floating to the interphase between 1 3 % and 27 °/o sucrose was collected by centrifugation at 100 000 g for 30 minutes and resuspended in 200 ul of a 0.25 M sucrose medium buffered by 20 mM HEPES at p H 7.3. Phase contrast microscopy showed that the membrane fraction consisted of vesicles as well as membrane sheets. To transform sheets into vesicles, the preparation was passed 5 times through a syringe needle, inner diameter 0.2 mm.

Plasma membrane Suspension (0.01 ml, protein concentration 2.0 mg/ml) was incubated at 37° C with 0.01 ml HEPES-buffered sucrose Solution (0.25 M) containing different concentrations of CaCl2, MgCl'2 or synthetic lysolecithin. Incubations were carried out in 0.5 ml Polyethylene reaction vessels. After 30 minutes, 0.01 ml 2 % glutaraldehyde containing the components used in the experiment was added. After 5 minutes of fixation at 37° C, 0.01 ml glycerol was added for cryoprotection.

Freeze cleaving: Small droplets (less than 1 ul) of the Suspension were frozen on gold specimen holders in freon 22 which was cooled in liquid nitrogen and were then stored in liquid nitrogen. The cleaving process was performed according to the method of MOOR and MÜHLETHALER [22] in a Balzers freeze-etch unit BAF 300. Fracturing and replication were performed at —100° C. Organic material was removed from the replicas using sodium hypochlorite. After washing in distilled water, replicas were picked up on formvar- and carbon-coated single hole grids and examined in a Siemens Elmiskop 101 at 80 or 100 K V .

Photographs were taken as positives (platinum deposition: black). Direction of shadowing is indicated by an encircled arrowhead.

For identification of fracture faces we have used the nomenclature introduced recently by BRANTON etal. [8]: The membrane faces are denoted as P and E, where P corresponds to the earlier A-face and E to the earlier B-face.

Aggregation of intramembranous particles was quantitatively evaluated as described by DUPPEL and DAHL [11]: Vesicles containing particle-free membrane areas of 0.01 um 2 (cor-

Fusion of plasma membranes 213

responding to 12 X 12 mm in micrographs with total magnification of 120 000 X ) , with an unchanged total number of particles, were designated as vesicles exhibiting particle aggregation. An aggregation index was obtained from the ratio of vesicles with and without particle aggregation. As a control the maximum number of particles within 0.01 um 2 was determined. In addition, the distribution of intramembranous particles was checked using Standard error of mean (SEM) of the mean density of particles (Tab. II).

Protein concentration was determined fluorometrically according to FAIRBANKS [13]. Glycerol-3-phosphate dehydrogenase (mitochondrial) activity was determined according to

GARDENER [16]. a-neurotoxin isolated from the venom of Naja naja siamensis was a kind gift from DR. F.

HUCHO, Fachbereich Biologie der Universität Konstanz, and was labelled with iodine-125 according to MARCHALONIS [20]. Its binding properties and competition with decamethonium was determined according to PATERSON [27]. Synthetic lysolecithin (ETiß-H) was a generous gift from DR. U . WELTZIEN, Max-Planck-Institut für Immunbiologie, Freiburg. Unspecified chemicals were of analytical grade.

Results

Plasma membranes of cultured chick embryo muscle cells were purified according to the method described by SCHIMMEL et al. [34] and slightly modified by K E N T et al. [19]. After 52 hours growth in a medium containing low calcium concentration, cells were removed from the culture dishes by scraping with a rubber policeman. Enzymatic

Fig. 1. Electron micrograph of a thin sectioned pellet from a preparation of plasma membranes. Membranes have been incubated in a Solution with 1.4 m\i CaCU. The shape of the membrane profiles varies and fused vesicles may be observed ( a r r o w ) . - 50 000 X . - Scale 0.2 u m .

214 CHRISTIAN SCHUDT, GERHARD DAHL , and MANFRED GRATZL

Fig. 2. Electron micrograph of a freeze fractured Suspension of plasma membranes in low calcium concentration. Spherical vesicles are dispersed in the medium (a). The intramembranous particles are distributed randomly and adhere more to the convex P-faces (b) than to the concave E-faces (c). - a. 35 000 X . - Scale 0.5 um. - b and c. 100 000 X . - Scale 0.2 um.


detachment was avoided in order to prevent any modification of the cell surface. Cells were homogenized and plasma membranes isolated according to [19]. The purity of plasma membranes was determined by the distribution of acetylcholine receptor-bound a-neurotoxin in the various membrane fractions. This has been reported to be a reliable marker of myoblast plasma membrane [21, 28]. Table I shows the protein content and distribution of two markers in several fractions taken from different stages of the Separation procedure. Thin sections of the isolated membrane fraction collected from the interphase between 13 °/o and 27 %> sucrose show smooth-surfaced membranes containing no electron dense material. Nuclei, mitochondria or membranes originating from rough endoplasmic reticulum were rarely observed (Fig. 1).

Tab. I. Distribution and relative specific activities of subcellular markers in the fractionation of fusion-competent myoblasts.

Glycerolphosphate a-neurotoxin binding Protein dehydrogenase

Fraction relative relative (%) (%) speci f ic (%) speci f ic

activity activity

Homogenste, particulate 100 100 1 100 1 1700 g pellet 86 80 0.93 36 0.42

27 % gradient fraction 1.3 0.2 0.15 8 6.2 32 % gradient fraction 2.3 1.7 0.74 5 2.2

Freeze cleaving of the isolated vesicular preparation (Fig. 2) showed that the intramembranous particles (IMPs) adhere more to the convex P-faces (1000/u,m2) than to the concave E-faces (200/um2) (Tab. II). A few concave vesicles with high particle density may represent inside-out plasma membranes or vesicles derived from endoplasmic reticulum. In agreement with the low glycerol-3-phosphate dehydrogenase activity, mitochondria were rarely observed. Contamination by nuclear membranes, which can easily be detected by their pores, was negligible. From the biochemical as well as the morphological data it can be concluded that most of the isolated vesicles represented plasma membranes orientated right-side-out.

Tab. II. Aggregation of intramembranous particles and total number of intramembranous particles of isolated myoblast plasma membranes under different conditions.

Addit ives in Aggregation Maximum number of Number of the incubation index *) particles within a Mean density particles per medium (P-face) defined area of P-face per 0.01 j im 2 um 2 in

a b c (0.01 um 2) P-face E-face

none 7 32 0.22 12.6 ± 5 . 2 (n = 28) 10.3 ± 3 . 6 (n = 39) 1031 235

1.4 M C a C I 2 78 35 2.03 18.5 ± 5 . 9 (n = 58) 10.5 ± 9 . 3 (n = 55) 1050 275

1.4 M M g C I 2 37 20 1.85 17.3 ± 4 . 9 (n = 24) 9.9 ± 8.2 (n = 47) 996 293

*) Number of vesic les with (a) and without (b) particle-free areas of 0.01 um 2 . Ratio a : b = c. A total area of 0.4 to 0.6 u m 2 was counted of each preparation. For details see Methods.


When plasma membranes were incubated in a medium to which no calcium was added, vesicles were dispersed (Fig. 2 a) and IMPs were distributed randomly in the convex P-face (Fig. 2 b). Increasing the concentration of calcium in the incubation medium to 1.4 m M resulted in a clustering of vesicles (Fig. 3). Concomitantly, IMPs were found to be aggregated in a large proportion of the vesicle preparation (Tab. II).

Fig. 3. Freeze-fractured vesicles of myoblast plasma membranes incubated in 1.4 IHM CaCl>. Vesicles are clustered and some are fused. - 30 000 X . - Scale 0.5 um.

Fig. 4. Higher magnification of plasma membrane vesicles incubated in 1.4 I H M CaCl->. Intramembranous particles are aggregated in zones of contact between vesicles (small arrows) and in zones where the fracturing procedure has removed the overlying material. Twinned vesicles and tripled vesicles indicate vesicle fusion (large a r r o w s ) . - 100 000 X . - Scale 0.2 um.

Fusion of plasma membranes


Vesicles in tight apposition were observed frequently and in the areas of contact, aggregations of IMPs appeared in membrane P-faces (Fig. 4). In addition to an attachment of vesicles and the aggregation of IMPs, twinned vesicles were observed in the presence of 1.4 m M calcium (Fig. 4). Within the resolution of freeze fracture electron

Fig. 5. Plasma membrane vesicles incubated in a Solution containing 1.4 mM CaCl2. -a. Convex P-face of fused vesicles with a seamline occupied by an aggregation of IMPs ( a r r o w ) . - b. Concave E-face of fused vesicles with an aggregation of IMPs along the waists (arrows). - 100 000 X . - Scale 0.2 u m .


microscopy (30 A) no ridge in the membrane which might eventually separate the two vesicles could be detected. The cleavage plane in these structures is continuous in the P-face as well as in the E-face (Fig. 4 and 5), and in addition, thin sections (Fig. 1) reveal that twinned vesicles were not separated by a septum. This indicates that two vesicles have formed a common lumen by fusion. Only a very low percentage of fused vesicles were observed after incubation in the absence of calcium (Tab. III).

Fig. 6. Fused plasma membrane vesicles exhibiting a softened waist. IMPs are randomly distributed in membrane P-faces (a) and E-faces (b). - 100 000 X . - Scale 0.2 um.


Tab. III. Percentage of vesicle fusion under different incubation conditions.

Addi t ives in the incubation medium

Percentage of vesic le fusion

(%)

Number of counted ves ic les

none 1.1 1000

1.4 X 1 0 " 3 M C a C I 2 15.3 1200

1.4 X 1 0 " 3 M M g C I 2 1.8 1000

1.4 X 1 0 " 3 M C a C I 2

2 X 1 0 " 2 M M g C I 2

6.2 1000

1.4 X 1 0 ~ 3 M C a C I 2

10" G M lysolecithin 7.4 400

Among the fused vesicles two populations could be distinguished. One of them was characterized by an accumulation of IMPs which followed a seamline and were found in both P- and E-faces of the membrane (Fig. 5 a and 5 b). In the second population this seamline was not detectable and fused vesicles exhibited soft waists (Fig. 6 a and 6 b). From these figures it can be seen that IMPs were distributed in a manner similar to their array before fusion took place. Incubation of vesicles in the presence of 1.4 mM MgCb instead of CaCb resulted in a clustering of vesicles and an aggregation of IMPs. However, in contrast to calcium, magnesium did not induce fusion (Tab. III).

Comparable to monolayer myoblast fusion [26, 35], isolated membrane vesicles fused at 1.4 mM CaCb and were effectively inhibited from fusing by 20 mM MgCb and 10 -6 M lysolecithin (Tab. III).

Discussion

The membrane fraction used in this study consisted mainly of plasma membranes as shown by biochemical analysis, thin section and freeze fracture electron microscopy. The presence of membranes originating from sarcoplasmic reticulum (which might be also present in this preparation) cannot be ruled out in view of the methods used. However, BASKIN [4] found intramembranous particles at a density of 86/um2 in sarcoplasmic membranes prepared from 14-day old chick embryos. The high average particle density of 1000/u.m2 found in the present investigation makes it unlikely that sarcoplasmic membranes are present to a significant extent.

Furthermore, ultrastructural studies of FISCHMAN [14] showed that sarcoplasmic reticulum was not observed in unfused myoblasts.

Addition of divalent cations such as magnesium or calcium to the isolated plasma membranes resulted in an attachment of vesicles to form Clusters. A concomitant aggregation of IMPs was also often observed in the zone of contact between adjacent vesicles. In contrast to vesicle attachment and particle aggregation - both of which can be evoked by either calcium or magnesium - the fusion of vesicles observed as twin structures can only be demonstrated after incubation with calcium. Therefore the pore formation resulting in a common lumen between two vesicles seems to be calcium-specific, as is the fusion of isolated secretory vesicles [10, 17], mast cell secretion [18], and transmitter release [21].


The calcium-induced fusion of vesicles was inhibited by 20 mM MgCl2. This calcium-magnesium antagonism has already been shown in myoblast fusion [35] and secretion [9, 15,21].

Calcium-magnesium antagonism and lysolecithin inhibition suggest that the mechanism of myoblast fusion differs from that of artificially induced fusion processes. Virally induced erythrocyte fusion [38], fusion of nascent membranes of E c h i n o s p h a e r i u m n u c l e o f i l u m [39] and fusion induced by fusogenic lipids [2] do not show these ion selectivities.

Aggregation of IMPs has been shown in many Systems and has been correlated with cell to cell contact [12], agglutination of erythrocytes [23], fusion processes in mucocyst secretion [33], and to myoblast fusion [30]. Furthermore, aggregation of IMPs preceeds the fusion of secretory vesicles with plasma membrane in intact pancreatic islet cells [5] as well as calcium-specific fusion of isolated secretory vesicles [10, 17]. However, from experiments described in this paper, it cannot be determined whether the aggregation of IMPs is due to intramembranous bridge formation (induced by divalent cations) between components in one vesicular membrane, or whether it is a consequence of intermembranous bridge formation between neighbouring vesicles. It can however be concluded that aggregation of IMPs is related to membrane attachment and may be a necessary prerequisite for fusion. This is indicated by the appearance of IMPs in the waists of fused vesicles. These IMPs may represent remnants of aggregates observed in the zones of vesicular contact.

Low molecular weight Compounds such as cyclic nucleotides thought to participate in the mechanism of fusion [31], are not present in our System. In addition, the amount of cytoplasmic enzymes should also have been greatly reduced by the membrane isola-tion procedure. Therefore it seems likely that components other than those fixed on the membrane do not contribute to the basic process of fusion.

A H K O N G et al. [1] suggested (without morphological evidence) that aggregation of membrane proteins and fusion may be induced by a micellising agent such as lysolecithin. In our experiments lysolecithin produced just the opposite effect; a decrease in the amount of fused vesicles was observed.

In conclusion, we suggest that the following Steps occur in the course of fusion: 1. Dependent on divalent cations, vesicles contact each other and IMPs aggregate. 2. Under the specific influence of calcium ions, vesicles fuse at the zones of particle

aggregation forming twin structures. 3. This early stage of fusion is characterized by a seamline which may be stabilized

by the presence of IMPs. 4. Subsequently, IMPs redistribute randomly and the waist softens.

A c k n o w l e d g e m e n t s . We thank Mrs. I. KÜMMEL, Mrs. E. JARDEL, and M r . R. WEISS for excellent technical assistance. We are grateful to DR . M . A . CAHILL and DR. C . R. WULF for reading the manuscript. - This work was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 138 "Biologische Grenzflächen und Spezifität" and Sonderforschungsbereich 38 " M e m branforschung".

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