Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
129
STRUCTURAL PERTURBATIONS IN THE PLASMA MEMBRANE DURING CONCANAVALIN A ENDOCYTOSIS ff T. Hatae* and E.L. Benedetti Laboratoire de Microscopic Electronique Institut de Recherche en Biologie MolEculaire de 1'Universite de Paris VII et du C.N.R.S. 2, Place Jussieu, 75221 Paris Cedex 05, France ABSTRACT -by its interaction Endocytosis of Concanavalin A, triggered with the surface of cells from a murine plasmocytoma line, is characterised by various steps which can be visualised by cytoPlasma membrane internalisation is chemistry and freeze-fracturing. initiated by clustering of Concanavalin A receptors and by formation of intramembraneous particle necklaces, which were observed on fracture faces. Subsequent perturbation of the lipid bilayer precedes the fusion and formation of closed vesicles. INTRODUCTION Endocytosis is a process whereby materials are conveyed from the exterior into a cell. It may necessarily precede ligand participation in regulatory functions (Korn, 1975; Silverstein eta& 1977; Stockem, 1977). Currently it is thought that internalisation starts with ligand interaction with specific plasma membranereceptor sites, followed by a perturbation of the lipid bilayer and membrane protein (Scheckman and Singer, 1976). After invagination, the discrete membrane domains (at the outer edge of the nascentvesicles) fuse and the closed vesicles separate from the plasma membrane (Simionescu et al., 1974). Many membrane functions including junctional assembly (Decker and Friend, 1974; Benedetti et al., 1974), cell fusion and fertilisation (Weiss et al., 1977), and exocytosis and secretion (Satir et al., 1973; Chi et al., 1976; De Camilli et al., 1976; Pinto da Silva and Nogueira, 1977; Burwen and Satir, 1977; Lawson et 1977; Orci et al., 1977) have been characterised as involving a al,, rearrangement of the intramembraneous particles (IMPS) in the plane of the membrane, as revealed by freeze-fracture. These IMPS are believed to represent membrane-spanning amphipatic proteins (Benedetti et al., 1977). _-_- ---* Permanent address : Department of Anatomy, School of ?ledicine, Kyushu University, Fukukoa 812, Japan 2% To whom requests for reprints should be addressed.
130
Ceil Biology
International
Reports,
In this paper we describe a cytochemical study of Concanavalin A (Con A) endocytosis in cells, showing that the rearrangements of the sequestration of membrane lipid are a prelude and the formation of closed vesicles.
Vol. 2, No. 2, 1978
and freeze-fracture murine plasmocytoma IMPS, and the to membrane fusion
MATERIALS AND METHODS The cell line used in our experiments is an epithelial-like variant (ME2CR) of BALB/C mouse plasmocytoma MOPC 173 which grows normally without agglutinating in the presence of Con A (GuErin et al., 1974). Confluent cultures were treated with Con A (100 pg/m1/5x105 cells) in phosphate-buffered saline (PBS) for 30 min at 4' C. The cultures were washed in PBS and either fixed in cacodylate-buffered glutaraldehyde (0.1 M pH 7.2) for 60 min or incubated with horseradish peroxidase (HRP) (50 pg/ml) for 30 min (Gonatas et al., 1975) after which they were washed and incubated in PBS at 37" C for various periods of time between 2 and 60 min before fixation in 2.5 % glutaraldehyde for 60 min. In other samples the peroxidase treatment or the 37" C incubation in PBS were omitted. The Con A-HRP complex was revealed by treating the cultures after washing in PBS with 3-3' diaminobenzidine (DAB) (0.5 mg/ml in Tris-HCl buffer pH 7.4 containing 0.1 % H202 for 30 min. Post-fixation was with 1 % Osmium tetroxide in cacodylate buffer (0.1 M pH 7.2) for 45 min. Thin sections of EPON 812-embedded cultures were preparedusing a LKB III microtome and stained with lead citrate. The freeze fracture experiments were carried out using either the cultures incubated with Con A only at 4" C or with the cultures also incubated at 37" C for various periods of time. The cultures were immediately frozen in freon 22 and kept in liquid nitrogen, or fixed in glutaraldehyde for 20 min and infiltrated with 25 % glycerol in water. The freeze-fracture and replicas were made in a EM 300 and 400 electron BalzersBA 360 apparatus at - 150' C. Philips microscopes were used for visualisation. RESULTS Thin sections of the cells fixed immediately after Con A treatment at 4" C show that the Con A-HRP-DAB complex forms a thick layer of electron dense precipitate continuous, uniform, caveolae coating the plasma membrane. Only very few DAB-positive are observed in close association with the inner cytoplasmic surface of the plasma membrane (Fig. 1). As incubation at 37" C proceeds, the number of caveolae and endocytotic vesicles containing DAB positive material increases. Samples incubated for 15 min at 37" C show a spotty distribution of DAB positive material and various steps of plasma membrane invagination (Fig. 2). The size of the endocytotic vesicles is rather uniform (70 nm in diameter) (Fig. 2). Details of the initial steps of invagination were also clearly observed on cells incubated with Con A and fixed directly without
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
131
1 - Thin section of a plasmocytona cell after Con A-LIR? --Fig. treatment at 4" C. The Con A-HRP-DAB complex forms an uniform iaver coating the plasma membrane. Fig. 2 - Thin section of a plasmocytoma cell incubated for 15 min at 37" C. The Con A-HRP-DAB complex is no longer uniform but. rat-nelspotty. Xany endocytotic vesicles containing the complex arc pr‘qcsn.t. Fig. 3 - Thin section of a plasmocytoma ceil incubated wit:1 CO:I >I for IO min at 37" C without HRP treatment, stained with lead ci tr-ate. Note the clustered and fuzzy aspect of the glycocalyx dorlnin (arr-ows). Fig. 4 - Same preparation as Fig. 3. %ote the invagination of tl:c plasma membrane containing dense material which probably corr..es;lc::tds to the Con A-receptor complex.
132
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
In discrete plasma membrane domains the HRP-DAB treatment. glycocalyx extensions appear thicker than over the rest of the cell surface and at corresponding positions a filamentous patch adheres to the inner plasma membrane leaFlet (Fig. 3). The nascent invaginations become filled by the glycocalyx-Con A complex during incubation at 37” C (Fig. 4). All stages of Con A endocytosis can be seen in thin sections of samples incubated at 37” C for 15 min and so freeze-fracture observations were carried out on similar samples. By freeze-fracture, the cleavage of the plasma membrane produces two faces which differ with regard to the number of IMPS. The outer aspect of the inner membrane half (PF) (Fig. 5) exhibits a larger number of IMPS than that observed on the complementary inner aspect of the outer membrane half (FF) (Fig. 6). ‘i‘he plasma membrane fracture faces of samples which were fixed immediately after Con A treatment, without incubating at 3,‘” C, show on t!le fracture surface that the IMPS are random]-y distributed (Figs. 5 and 6). An overall view of fracture faces of the plasma me:-ihrane after 15 min incubation at 37” C is shown in Figs. 7 and 8. The Erscture plane follows the contours of the caveolae and produces rounded depressions on the PF and dome-like protuberances on t!le EF. Arrays of IMPS form irregular necklaces around the pronounced depressions and the oval slabs visualised on FF (Fig. 7). Occasionally on an EF, small clusters and fragmented arrays of IMPS outline the protruding fracture edges of the caveolae (Fig. Xj. Fig. 9 shows a tangential The cleavage plane has followed the view of the PF fracture face. and arrays of SW’s around the rim of the contours of the caveolae, ingrowing caveolae are clearly exposed. The EF fracture face in Figs. 10 and 11 show stages of vesicle detachment from the plasma clusters of protruding globular entities membrane . Small (representing micelle formation) displaying a more heterogeneous
Fig. 5 - Freeze-fracture replica of a plastocytoma cell incubated withon A at 4” C. The protoplasmic fracture face (PF) shows a random distribution oE IPlPs. Fig. 6 - Same samples as Fig. 5. ‘The external fracture face (RF) displays a few IMPS, randomly distributed.
Cell Biology
international
Reports,
Vol. 2, No. 2, 1978
Fir7cI . !. - Freeze-fracture replica of a plasmocytoma cell treated w:Ch Con A and incubated for 15 min at 37” C. The PF shivs a nunber cl! rouud depressions aud oval slabs marked by I?tI’ neck?sces. Fit,.~ 8 - Same preparation as Fig. 7. The EF i.s char4cterii:ed b,s ihe presence of round, dome-like, and inwardly directed craters whici have a few I>iPs associated with the outiininz riz.
almost Fig. 9 - Same preparation as Fig. 7, showing a fracture transversely through the plasma membrane. A number of vesicular openings are shown limited by a circular array of IMPS. of an EF showing Fig. 10 - Same preparation as Fig. 7. Micrograph various steps of vesicles detachment characterized by small craters and domes of various sizes associated with patches of globular material. structures Fig. I1 - Same preparation as Fig. 7. On an EF, dome-like are associated with clusters of protruding globular, aggregated entities larger than IMPS (arrows).
Cell Biology
shape than necks still
International
Reports,
Vol. 2, No. 2, 1978
135
that of IMPS may represent the fracture aspect of narrow connecting the caveolae with the plasma membrane.
DISCUSSION The present study shows that in a Con A-resistant variant of the Con A is rapidly internalised by MOPC 173 murine plasmocytoma, endocytosis when the cells are incubated at 37" C. Other instances have been reported where either cell surface immunoglobulins labelled with anti-immunoglobulin antibodies or ricin-HRP induce a similar process and after endocytosis the complexes are rapidly conveyed to the Golgi apparatus (Gonatas et al., 1975) . Our observations on thin sections suggest that the initial step of endocytosis consists of a rapid clustering of the exposed receptors in discrete domains following Con A binding. Similar conclusions have been reached by Scheckman and Singer (1976) studying the endocytosis of -Ferritin-Con A complexes in neonatal erythrocytes. These authors state that small and discretely separated Ferritin-Con A clusters are found before internalization, whereas extensive aggregation of receptors over larger areas of the cell membrane is usually associated with "patches" and "cap" formation in other types oE cells. The most striking freeze-fracture image in our experiments is that which shows the formation of the IMP circular array, which serves as a landmark in identifying the initial internalization sites of Con A. The formation of a particulate necklace around the initial sites of plasma membrane invaginations may represent a relatively stable protein border favouring the segregation of a membrane domain in which the lipid accumulates and from which the IMP-associated protein is excluded. It has also been postulated (Van der Bosch and McConnell, 1975) that Con A may act directly on the membrane phospholipid, enhancing the structural fluctuation of domain boundaries when the phospholipid molecules are in a favourable state Ear membrane Eusion. This bilayer perturbation may result in micelle formation in a discrete lipid domain, triggering the fusion process and the "pinching off" of the vesicles. If our freeze-fracture study leads us to formulate the hypothesis that rearrangement of the IMP protein characterises the initial stage of endocytosis, it also provides some indications that the miscellisation of the lipid domain may coincide with the final stage in the fusion of the vesicular membrane (Pinto da Silva and Nogueira, 1977). This view reconciles the model of Poste and Allison (1973) with that of Ahkong et al. (1975) by postulating that both the Eormation of lipid-protein segregated domains and lipid miscellisation are necessary for internalisation of endosomes. ACKNOWLEDGEMENTS !ie wish to thank Mrs. C. Guerin-Marchand for kindly supplying the cell cultures used in our experiments. We would like to express our deep appreciation to Mrs. J.M. Oliver-Berlin and Mr. R.D. Berlin
136
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
for their careful and critical reading of the manuscript. The secretarial assistance of Mrs. Emmanuel is gratefully acknowledged. This work has been supported by DGRST contract no 74.7.0173 and by the Service de la Cooperation Scientifique et Technique du Ministsre des Affaires Etrangeres. REFERENCES Ahkong, A.F., Fisher, D., Tampion, W. and Lucy, J.A. (1975) Mechanism of cell fusion. Nature, 253, 194-195. Benedetti, E.L., Dunia, I. and Bloemendal, H. (1974) Development of junctions during differentiation of lens fibers. Proceedings of the National Academy of Science, 71, 5073-5077. Benedetti, E.L., Dunia, I., Olive, Jrand Cartaud, J. (1977) Modulation of plasma membrane architecture in animal cells. In : C. Nicolau and A. Paraf (eds.). Structural and Kinetic Approach to Plasma Membrane Functions, pp. 60-76. Springer Verlag, Berlin. Burwen, S.J. and Satir, B.H. (1977) A freeze-fracture study of early membrane events during mast cell secretion. Journal of Cell Biology, 73, 660-671. Chi, E.Y., Lagunoff, D. and Koehler, J.K. (1976) Freeze-fracture study of mast cell secretion. Proceedings of the National Academy of Sciences, 73, 2823-2827. De Camilli, P., Peluchetx, D. and Meldolesi, J. (1976) Dynamic changes of the luminal plasmalemna in stimulated parotid acinar cells. A freeze-fracture study. Journal of Cell Biology, -70 ' 59-74. Decker, R.S. and Friend, D.S. (1974) Assembly of gap junctions during amphibian neurulation. Journal of Cell Biology, 62, 32-47. Gonatas, N.K., Steiber, A., Kim, S.U., Graham, D.I. and Avrameas, S. (1975) Internalisation of neuronal plasma membrane ricin receptors into the Golgi apparatus. Experimental Cell Research, 94, 426-431. Cue%, C., Zachowski, A., Prigent, B., Paraf, A., Dunia, I., Diawara, M.A. and Benedetti, E.L. (1974) Correlation between the mobility of the inner plasma membrane structure and agglutination by Concanavalin A in two cell lines of MOPC 173 plasmocytoma cells. Proceedings of the National Academy of Sciences, 71, 114-117. Korn, E.D. (1975) Biochemistry of endocytosis. In : C.F. Fox (eds.) Biochemistry of Cell Walls and Membranes. Series One Volume 2, University Park Press Baltimore. PP. l-26. Butterworths-London C. and Gilula, N.B. Lawson, D., Raff, M.C., Gomperts, B., Fewtrell, (1977) Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells. Journal of Cell Biology, 72, 242-259. Orcc L., Perrelet, A. and Friend, D. (1977) Freeze-fracture of membrane fusion during exocytosis in pancreatic B-cells. Journal of Cell Biology, 75, 23-30. Pinto da Silva, P. a2 Nogueira, M.L. (1977) Membrane fusion during secretion. An hypothesis based on electron microscope observation of Phytophthora Palmivora zoospores during encystment. Journal
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
137
of Cell Biology, 73, 161-181. Poste, G. and Allison, A.C. (1973) Membrane fusion. Biochimica and Biophysics Acta, 300, 421-465. P. (1973) Membrane fusion in a Satir, B., Schooley, C. and Satir, model system. Mucocyst secretion in Tetrahymena. Journal of Cell Biology, 56, 153-178. Scheckman, RTand Singer, S.J. (1976) Clustering and endocytosis of membrane receptors can be induced in mature erythrocytes of neonatal but not adult humans. Proceedings of the National Academy of Sciences, 73, 4075-4079. Silverstein S.C., Steinman, R.M. and Cohn, Z.A. (1977) Endocytosis. Annual Review of Biochemistry, 46, 669-722. Simionescu, M., Simionescu, N. and?alade, G.E. (1974) Morphometric data on the endothelium of blood capillaries. Journal of Cell Biology, 60, 128-152. Stockem, W. (1977) Endocytosis. In : G.A. Jamison and D.M. Robinson (eds.) Mammalian Cell Membranes. Volume 5, pp. 151-189. Butterworths-London-Boston. Van der Bosch, J. and McConnell, H.M. (1975) Fusion of dipalmitoylphosphatidylcholine vesicle membranes induced by Concanavalin A. Proceedings of the National Academy of Sciences, 72, 4409-4413. Weiss, R.L., Goodenough, D.A. and Goodenough, U.A. (1977) Membrane differentiations at sites specialized for cell fusion. Journal of Cell Biology, 72, 144-160.
International
Reports,
Vol. 2, No. 2, 1978
129
STRUCTURAL PERTURBATIONS IN THE PLASMA MEMBRANE DURING CONCANAVALIN A ENDOCYTOSIS ff T. Hatae* and E.L. Benedetti Laboratoire de Microscopic Electronique Institut de Recherche en Biologie MolEculaire de 1'Universite de Paris VII et du C.N.R.S. 2, Place Jussieu, 75221 Paris Cedex 05, France ABSTRACT -by its interaction Endocytosis of Concanavalin A, triggered with the surface of cells from a murine plasmocytoma line, is characterised by various steps which can be visualised by cytoPlasma membrane internalisation is chemistry and freeze-fracturing. initiated by clustering of Concanavalin A receptors and by formation of intramembraneous particle necklaces, which were observed on fracture faces. Subsequent perturbation of the lipid bilayer precedes the fusion and formation of closed vesicles. INTRODUCTION Endocytosis is a process whereby materials are conveyed from the exterior into a cell. It may necessarily precede ligand participation in regulatory functions (Korn, 1975; Silverstein eta& 1977; Stockem, 1977). Currently it is thought that internalisation starts with ligand interaction with specific plasma membranereceptor sites, followed by a perturbation of the lipid bilayer and membrane protein (Scheckman and Singer, 1976). After invagination, the discrete membrane domains (at the outer edge of the nascentvesicles) fuse and the closed vesicles separate from the plasma membrane (Simionescu et al., 1974). Many membrane functions including junctional assembly (Decker and Friend, 1974; Benedetti et al., 1974), cell fusion and fertilisation (Weiss et al., 1977), and exocytosis and secretion (Satir et al., 1973; Chi et al., 1976; De Camilli et al., 1976; Pinto da Silva and Nogueira, 1977; Burwen and Satir, 1977; Lawson et 1977; Orci et al., 1977) have been characterised as involving a al,, rearrangement of the intramembraneous particles (IMPS) in the plane of the membrane, as revealed by freeze-fracture. These IMPS are believed to represent membrane-spanning amphipatic proteins (Benedetti et al., 1977). _-_- ---* Permanent address : Department of Anatomy, School of ?ledicine, Kyushu University, Fukukoa 812, Japan 2% To whom requests for reprints should be addressed.
130
Ceil Biology
International
Reports,
In this paper we describe a cytochemical study of Concanavalin A (Con A) endocytosis in cells, showing that the rearrangements of the sequestration of membrane lipid are a prelude and the formation of closed vesicles.
Vol. 2, No. 2, 1978
and freeze-fracture murine plasmocytoma IMPS, and the to membrane fusion
MATERIALS AND METHODS The cell line used in our experiments is an epithelial-like variant (ME2CR) of BALB/C mouse plasmocytoma MOPC 173 which grows normally without agglutinating in the presence of Con A (GuErin et al., 1974). Confluent cultures were treated with Con A (100 pg/m1/5x105 cells) in phosphate-buffered saline (PBS) for 30 min at 4' C. The cultures were washed in PBS and either fixed in cacodylate-buffered glutaraldehyde (0.1 M pH 7.2) for 60 min or incubated with horseradish peroxidase (HRP) (50 pg/ml) for 30 min (Gonatas et al., 1975) after which they were washed and incubated in PBS at 37" C for various periods of time between 2 and 60 min before fixation in 2.5 % glutaraldehyde for 60 min. In other samples the peroxidase treatment or the 37" C incubation in PBS were omitted. The Con A-HRP complex was revealed by treating the cultures after washing in PBS with 3-3' diaminobenzidine (DAB) (0.5 mg/ml in Tris-HCl buffer pH 7.4 containing 0.1 % H202 for 30 min. Post-fixation was with 1 % Osmium tetroxide in cacodylate buffer (0.1 M pH 7.2) for 45 min. Thin sections of EPON 812-embedded cultures were preparedusing a LKB III microtome and stained with lead citrate. The freeze fracture experiments were carried out using either the cultures incubated with Con A only at 4" C or with the cultures also incubated at 37" C for various periods of time. The cultures were immediately frozen in freon 22 and kept in liquid nitrogen, or fixed in glutaraldehyde for 20 min and infiltrated with 25 % glycerol in water. The freeze-fracture and replicas were made in a EM 300 and 400 electron BalzersBA 360 apparatus at - 150' C. Philips microscopes were used for visualisation. RESULTS Thin sections of the cells fixed immediately after Con A treatment at 4" C show that the Con A-HRP-DAB complex forms a thick layer of electron dense precipitate continuous, uniform, caveolae coating the plasma membrane. Only very few DAB-positive are observed in close association with the inner cytoplasmic surface of the plasma membrane (Fig. 1). As incubation at 37" C proceeds, the number of caveolae and endocytotic vesicles containing DAB positive material increases. Samples incubated for 15 min at 37" C show a spotty distribution of DAB positive material and various steps of plasma membrane invagination (Fig. 2). The size of the endocytotic vesicles is rather uniform (70 nm in diameter) (Fig. 2). Details of the initial steps of invagination were also clearly observed on cells incubated with Con A and fixed directly without
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
131
1 - Thin section of a plasmocytona cell after Con A-LIR? --Fig. treatment at 4" C. The Con A-HRP-DAB complex forms an uniform iaver coating the plasma membrane. Fig. 2 - Thin section of a plasmocytoma cell incubated for 15 min at 37" C. The Con A-HRP-DAB complex is no longer uniform but. rat-nelspotty. Xany endocytotic vesicles containing the complex arc pr‘qcsn.t. Fig. 3 - Thin section of a plasmocytoma ceil incubated wit:1 CO:I >I for IO min at 37" C without HRP treatment, stained with lead ci tr-ate. Note the clustered and fuzzy aspect of the glycocalyx dorlnin (arr-ows). Fig. 4 - Same preparation as Fig. 3. %ote the invagination of tl:c plasma membrane containing dense material which probably corr..es;lc::tds to the Con A-receptor complex.
132
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
In discrete plasma membrane domains the HRP-DAB treatment. glycocalyx extensions appear thicker than over the rest of the cell surface and at corresponding positions a filamentous patch adheres to the inner plasma membrane leaFlet (Fig. 3). The nascent invaginations become filled by the glycocalyx-Con A complex during incubation at 37” C (Fig. 4). All stages of Con A endocytosis can be seen in thin sections of samples incubated at 37” C for 15 min and so freeze-fracture observations were carried out on similar samples. By freeze-fracture, the cleavage of the plasma membrane produces two faces which differ with regard to the number of IMPS. The outer aspect of the inner membrane half (PF) (Fig. 5) exhibits a larger number of IMPS than that observed on the complementary inner aspect of the outer membrane half (FF) (Fig. 6). ‘i‘he plasma membrane fracture faces of samples which were fixed immediately after Con A treatment, without incubating at 3,‘” C, show on t!le fracture surface that the IMPS are random]-y distributed (Figs. 5 and 6). An overall view of fracture faces of the plasma me:-ihrane after 15 min incubation at 37” C is shown in Figs. 7 and 8. The Erscture plane follows the contours of the caveolae and produces rounded depressions on the PF and dome-like protuberances on t!le EF. Arrays of IMPS form irregular necklaces around the pronounced depressions and the oval slabs visualised on FF (Fig. 7). Occasionally on an EF, small clusters and fragmented arrays of IMPS outline the protruding fracture edges of the caveolae (Fig. Xj. Fig. 9 shows a tangential The cleavage plane has followed the view of the PF fracture face. and arrays of SW’s around the rim of the contours of the caveolae, ingrowing caveolae are clearly exposed. The EF fracture face in Figs. 10 and 11 show stages of vesicle detachment from the plasma clusters of protruding globular entities membrane . Small (representing micelle formation) displaying a more heterogeneous
Fig. 5 - Freeze-fracture replica of a plastocytoma cell incubated withon A at 4” C. The protoplasmic fracture face (PF) shows a random distribution oE IPlPs. Fig. 6 - Same samples as Fig. 5. ‘The external fracture face (RF) displays a few IMPS, randomly distributed.
Cell Biology
international
Reports,
Vol. 2, No. 2, 1978
Fir7cI . !. - Freeze-fracture replica of a plasmocytoma cell treated w:Ch Con A and incubated for 15 min at 37” C. The PF shivs a nunber cl! rouud depressions aud oval slabs marked by I?tI’ neck?sces. Fit,.~ 8 - Same preparation as Fig. 7. The EF i.s char4cterii:ed b,s ihe presence of round, dome-like, and inwardly directed craters whici have a few I>iPs associated with the outiininz riz.
almost Fig. 9 - Same preparation as Fig. 7, showing a fracture transversely through the plasma membrane. A number of vesicular openings are shown limited by a circular array of IMPS. of an EF showing Fig. 10 - Same preparation as Fig. 7. Micrograph various steps of vesicles detachment characterized by small craters and domes of various sizes associated with patches of globular material. structures Fig. I1 - Same preparation as Fig. 7. On an EF, dome-like are associated with clusters of protruding globular, aggregated entities larger than IMPS (arrows).
Cell Biology
shape than necks still
International
Reports,
Vol. 2, No. 2, 1978
135
that of IMPS may represent the fracture aspect of narrow connecting the caveolae with the plasma membrane.
DISCUSSION The present study shows that in a Con A-resistant variant of the Con A is rapidly internalised by MOPC 173 murine plasmocytoma, endocytosis when the cells are incubated at 37" C. Other instances have been reported where either cell surface immunoglobulins labelled with anti-immunoglobulin antibodies or ricin-HRP induce a similar process and after endocytosis the complexes are rapidly conveyed to the Golgi apparatus (Gonatas et al., 1975) . Our observations on thin sections suggest that the initial step of endocytosis consists of a rapid clustering of the exposed receptors in discrete domains following Con A binding. Similar conclusions have been reached by Scheckman and Singer (1976) studying the endocytosis of -Ferritin-Con A complexes in neonatal erythrocytes. These authors state that small and discretely separated Ferritin-Con A clusters are found before internalization, whereas extensive aggregation of receptors over larger areas of the cell membrane is usually associated with "patches" and "cap" formation in other types oE cells. The most striking freeze-fracture image in our experiments is that which shows the formation of the IMP circular array, which serves as a landmark in identifying the initial internalization sites of Con A. The formation of a particulate necklace around the initial sites of plasma membrane invaginations may represent a relatively stable protein border favouring the segregation of a membrane domain in which the lipid accumulates and from which the IMP-associated protein is excluded. It has also been postulated (Van der Bosch and McConnell, 1975) that Con A may act directly on the membrane phospholipid, enhancing the structural fluctuation of domain boundaries when the phospholipid molecules are in a favourable state Ear membrane Eusion. This bilayer perturbation may result in micelle formation in a discrete lipid domain, triggering the fusion process and the "pinching off" of the vesicles. If our freeze-fracture study leads us to formulate the hypothesis that rearrangement of the IMP protein characterises the initial stage of endocytosis, it also provides some indications that the miscellisation of the lipid domain may coincide with the final stage in the fusion of the vesicular membrane (Pinto da Silva and Nogueira, 1977). This view reconciles the model of Poste and Allison (1973) with that of Ahkong et al. (1975) by postulating that both the Eormation of lipid-protein segregated domains and lipid miscellisation are necessary for internalisation of endosomes. ACKNOWLEDGEMENTS !ie wish to thank Mrs. C. Guerin-Marchand for kindly supplying the cell cultures used in our experiments. We would like to express our deep appreciation to Mrs. J.M. Oliver-Berlin and Mr. R.D. Berlin
136
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
for their careful and critical reading of the manuscript. The secretarial assistance of Mrs. Emmanuel is gratefully acknowledged. This work has been supported by DGRST contract no 74.7.0173 and by the Service de la Cooperation Scientifique et Technique du Ministsre des Affaires Etrangeres. REFERENCES Ahkong, A.F., Fisher, D., Tampion, W. and Lucy, J.A. (1975) Mechanism of cell fusion. Nature, 253, 194-195. Benedetti, E.L., Dunia, I. and Bloemendal, H. (1974) Development of junctions during differentiation of lens fibers. Proceedings of the National Academy of Science, 71, 5073-5077. Benedetti, E.L., Dunia, I., Olive, Jrand Cartaud, J. (1977) Modulation of plasma membrane architecture in animal cells. In : C. Nicolau and A. Paraf (eds.). Structural and Kinetic Approach to Plasma Membrane Functions, pp. 60-76. Springer Verlag, Berlin. Burwen, S.J. and Satir, B.H. (1977) A freeze-fracture study of early membrane events during mast cell secretion. Journal of Cell Biology, 73, 660-671. Chi, E.Y., Lagunoff, D. and Koehler, J.K. (1976) Freeze-fracture study of mast cell secretion. Proceedings of the National Academy of Sciences, 73, 2823-2827. De Camilli, P., Peluchetx, D. and Meldolesi, J. (1976) Dynamic changes of the luminal plasmalemna in stimulated parotid acinar cells. A freeze-fracture study. Journal of Cell Biology, -70 ' 59-74. Decker, R.S. and Friend, D.S. (1974) Assembly of gap junctions during amphibian neurulation. Journal of Cell Biology, 62, 32-47. Gonatas, N.K., Steiber, A., Kim, S.U., Graham, D.I. and Avrameas, S. (1975) Internalisation of neuronal plasma membrane ricin receptors into the Golgi apparatus. Experimental Cell Research, 94, 426-431. Cue%, C., Zachowski, A., Prigent, B., Paraf, A., Dunia, I., Diawara, M.A. and Benedetti, E.L. (1974) Correlation between the mobility of the inner plasma membrane structure and agglutination by Concanavalin A in two cell lines of MOPC 173 plasmocytoma cells. Proceedings of the National Academy of Sciences, 71, 114-117. Korn, E.D. (1975) Biochemistry of endocytosis. In : C.F. Fox (eds.) Biochemistry of Cell Walls and Membranes. Series One Volume 2, University Park Press Baltimore. PP. l-26. Butterworths-London C. and Gilula, N.B. Lawson, D., Raff, M.C., Gomperts, B., Fewtrell, (1977) Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells. Journal of Cell Biology, 72, 242-259. Orcc L., Perrelet, A. and Friend, D. (1977) Freeze-fracture of membrane fusion during exocytosis in pancreatic B-cells. Journal of Cell Biology, 75, 23-30. Pinto da Silva, P. a2 Nogueira, M.L. (1977) Membrane fusion during secretion. An hypothesis based on electron microscope observation of Phytophthora Palmivora zoospores during encystment. Journal
Cell Biology
International
Reports,
Vol. 2, No. 2, 1978
137
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