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Membrane internalisation into the Golgi apparatus by Concanavalin A induces a compensatory membrane biogenesis and membrane exocytosis
Exp. Path. Bd. 14, S. 351-354 (1977) Institute of Pathology (Head: Prof. Dr. F. BOLeK), Friedrich-Schiller University J ena Short communication
Membrane internalisation into the Golgi apparatus by Concanavalin A induces a compensatory membrane biogenesis and membrane exocytosis By J. ROTH With 2 figures (Received August 25, 1977) Key words: electron microscopy; Concanavalin A; membrane recycling; membrane flow; membrane biogenesis; Golgi apparatus From studies of cellular secretion and synthesis of membrane proteins the concept of membrane flow for the process of membrane biogenesis has been deduced (BENNETT 1956, NOVIKOFF et al. 1962, SIEKEVITZ et al. 1967, FRANKE et al. 1971). According to the membrane flow concept, the membrane biogenesis is accomplished by a transfer from one cell compartment to another. Obviously, the Golgi apparatus occupies a central position in this process as well as in membrane recycling and related processes of membrane reutilisation. On the other hand, the binding of a multivalent ligand such as antibodies or lectins results in some changes concerning the arrangement of the corresponding cell surface receptors or lectin-binding sites caused by their lateral movement in the plane of the membrane. TAYLOR et al. (1971) described for the first time such a ligand-induced redistribution of cell surface receptors into clusters and patches and in a polar cap. This redistribution is followed by an internalisation process (DE PETRIS and RAFF 1972, LOOR 1972, UNANUE et al. 1972, ANTOINE et al. 1974, ROTH 1974, GONATAS et aI. 1976). Furthermore, there exists some evidence for the recycling of internalised plasma membranes (JAMIESON and PALADE 1971a, b, HOLTZMAN et al. 1971). In the present study the events following the redistribution of the Concanavalin Abinding sites were examined electron microscopically. It was of particular interest to follow the way of the returning membranes in the cells. Furthermore, it was searched for morphological manifestations of compensatory mechanisms for the membrane removal in the course of the membrane internalisation. The experiments were performed on monolayer cultures of an established epithelial cell line from rat liver (KARSTEN et al. 1976). For visualisation of the Concanavalin A-binding sites the Concanavalin A-peroxidase technique according to BERNHARD and AVRAMEAS(1971) was used. The cell surface labeling was performed on the unfixed cells in the culture flasks in situ at 37°C. After incubation of the cells with Concanavalin A (100 ,ug!ml, 15 min) and horseradish peroxidase (100 /lg!ml, 15 min) the thoroughly washed cell cultures were reincubated with phosphate buffered isotonic saline for internalisation of the labeled cell surface for periods of time ranging between 5 min and 180 min. The peroxidase activity was demollstrated after the method of GRAHAM and KARNOVSKY (1966) after fixation of the cell cultures with 1.6 % glutaraldehyde in phosphate buffered isotonic saline. Then the cells were postfixed with 2 % OS04 in 0.1 M cacodylate buffer (pH 7.2) for 2 hours at 4°C and were embedded in situ in V.estopal W. The cytochemical controls for specificity of the labeling technique included the preincubation of the Con A and peroxidase solutions with the appropriate inhibitory sugar 1 hour before cell labeling. Further controls were run by omitting the Concanavalin A incubation step. Following different periods of reincubation of the cell cultures after cell surface labeling with Concanavalin A and peroxidase redistribution into clusters with simultaneous inter-
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nalisation occurred as already reported earlier (ROTH 1975, Roth et al. 1975). The internalisation led to a staining of the elements of the Golgi apparatus (fi~. 1). Within the same cell the unstained Golgi apparatus showed hypertrophy with a transition from the lamellar cisternal form to a vacuolar form (fig. 2). Furthermore, vesicles derived irom hypertrophied Golgi apparatus could be observed in close vicinity to the plasma memInane and fusion of the vesicle limiting membrane and the plasma membrane occurred. The internalisation of labeled membrane areas into the Golgi apparatus is an expression of a membrane recycling as also observed for other cells in vitro (ANTOINE et al. 1974, GONATAS et al. 1975, 1977)..-\ new observation made in the present study is the hypertrophy of the Golgi apparatus which does not participate in the membrane recycling. The hypertrophy of these Golgi apparatus and the subsequent formation of vesicles and their movement to and fusion with the plasma membrane may be an indication of a compensatory membrane biogenesis following the extensive membrane internalisation. The results will be discussed in detail elsewhere (ROTH 1977).
Literature ANTOINE, J. C., S. AVR.HIEAS, N. K GONATAS, A. STIEBER and J. O. GONATAS, Plasma membrane and internalized immunoglobulins of lymph node cells studied with conjugates of antibody or its Fab fragments with horseradish peroxidase. J. Cell BioI. 63, 12-23 (1974). BENNETT, M. S., The concept of membrane flow and membrane vesiculation as mechanisms for active transport and ion pumping. J. Biophys. Biochem. Cytol. 2, 99-103 (1956). BERNHARD, W., and S. AVRAMEAS, Ultrastructural visualization of cellular carbohydrate components by means of Concanavalin A. Exp. Cell Res. 64, 232-236 (1971). DE PETRIS, S., and M. C. R\FF, Distribution of immunoglobulin on the surface of mouse lymphoid cells as determined by immunoferritin electron microscopy. Antibody-induced, temperaturedependent redistribution and its implications for membrane structure. Eur. J. Immunol. 2, 523 to 535 (1972). FRANKE, W. W., D. J. ;\:[ORRE, B. DEUllLING, R D. CHEETHAM, J. KARTENBECK, E.-D. JARRASCH and H.- W. ZENTGRAF, Synthesis and structure and turnover of membrane proteins in rat liver: An examination of the membrane flow hypothesis. Z. Naturforsch. 26b, 1031-1039 (1971). GONATAS, N. K., A. STIEBER, S. U. KIM, D. I. GRAHAM and S. AVRAMEAS, Internalization of nellronal plasma membrane ricin receptors into the Golgi apparatus. Exp. Cell Res. 94, 426-431 (1975). - J. O. GmuTAs, A. STIEBER, J.-C. AXTOINE and S. AVRAMEAS, Quantitative ultrastructural autoradiographic studies of iodinated plasma membranes during segregation and internalization of surface immunoglobulins. J. Cell BioI. 70, 477-493 (1976). - S. U. KUI, A. STIEBER and S. AVR.-DIEAS, Internalization of lectins in neuronal gerl. J. Cell BioI. 73, 1-13 (1977). GRAHAM, R C., and M. J. K\RNOVSKY, The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302 (1966). HOLTZMAN, E., A. R. FREE)UN and L. A. KASHNER, Stimulation dependent alterations in peroxidase uptake by lobster neuro-muscular junctions. Science 173,733-766 (1971). J.-DIIESON, J. D., and G. E. PALADE, Condensing vacuole conversion and zymogen granule discharge in pancreatic exocrine cells: metabolic studies. J. Cell BioI. 48, 503-522 (1971 a). - - Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells. J. Cell BioI. iiO, 135-158 (1971 b). K\RSTEN, U., G. NEUPERT and R. THUST, Characteristics of an established epithelial cell line (RL-19) from rat liver. Exp. Path. 12, 88-99 (1976). LooR, F., L. FORNI and B. PERNIS, The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulins. Eur. J. Immunol. 2, 203-212 (19/2). NOVIKOFF, A. B., E. ESSNER, S. GOLDFISHER and M. HEUS, The Interpretation of Ultrastructure (ed. R. J. H. HARRIS). Academic Press, New York-London 1962, p.149.
Fig. 1. The membrane recycling is indicated by the staining of a Golgi apparatus after internalisation of plasma membrane areas labeled with Concanavalin A and peroxidase. X 39,000. Fig. 2. The membrane recycling into Golgi apparatus (G I ) led to hypertophy of another Golgi apparatus (G 2 ) in the same cell. Many unstained vesicles de'tived from hypertrophied Golgi apparatus are localised in close vicinity of the plasma membrane. X 18,000.
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ROTH, J., Distribution of Concanavalin A receptors on normal liver cells and on Zajdela ascites hepatoma cells. Int. J. Cancer 14, 762-770 (1974). - Concanavalin A receptors on normal rat liver cells, on in vivo by diethylnitrosamine transformed rat liver cells and on Zajdela ascites hepatoma cells: morphokinetic analysis on cell surface dynamics. Exp. Path. 11, 291-304 (1975). - Compensatory membrane biogenesis and exocytosis as a result of Concanavalin A-induced membrane internalization. Exp. Cell Res., in press (1977). - NEUPERT, G., and F. BOLeK Concanavalin A receptors in the plasma membrane of rat liver cells: comparative electron microscopic investigations on normal cells and on cells in vivo transformed by diethylnitrosamine. Exp. Path. 10, 143-155 (1975). SIEKEVITZ, P., G. E. PALADE, G. E. CHAD and T. OMURA, Organrzational Biosynthesis (ed. M. VOGEL et 301.). Academic Press, New York 1967, p. 331. TAYLOR, R B., W. P. H. DUFFUS, M. C. RAFF and S. DE PETRIS, Redistribution and pinocytosis of I ymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody. Nature New BioI. 233, 225-229 (1971). UNAKUE, E. R, W. D. PERKINS and M. J. KARNOVSKY, Ligand-induced movement of lymphocyte membrane macromolecules. 1. Analysis by immunofluorescence and ultrastructural autoradiography. J. Exp. Med. 136, 885-906 (1973). Author's address: Dr. sc. med. J. ROTH, Pathologisches Institut der Friedrich-Schiller- Universitat, Ziegelmiihlenweg 1, DDR - 69 Jena.
Verantwortlich fUr die Redaktion: Prof. Dr. F. Bclck, 69 Jena. Verlag: VEB Gustav Fischer Verlag, 69 Jena, Villengang 2, Telefon 27332. Satz und Druck: Druckerei "Magnus Poser", 69 Jena. - VerijUentlicht unter der Lizenznummer 1546 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Aile Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Verlsges und des Verfassers sowie mit Angabe der Quelle gestattet. Printed in German Democratic Republic. Artikel-Nr. (EDV) 56519
Membrane internalisation into the Golgi apparatus by Concanavalin A induces a compensatory membrane biogenesis and membrane exocytosis By J. ROTH With 2 figures (Received August 25, 1977) Key words: electron microscopy; Concanavalin A; membrane recycling; membrane flow; membrane biogenesis; Golgi apparatus From studies of cellular secretion and synthesis of membrane proteins the concept of membrane flow for the process of membrane biogenesis has been deduced (BENNETT 1956, NOVIKOFF et al. 1962, SIEKEVITZ et al. 1967, FRANKE et al. 1971). According to the membrane flow concept, the membrane biogenesis is accomplished by a transfer from one cell compartment to another. Obviously, the Golgi apparatus occupies a central position in this process as well as in membrane recycling and related processes of membrane reutilisation. On the other hand, the binding of a multivalent ligand such as antibodies or lectins results in some changes concerning the arrangement of the corresponding cell surface receptors or lectin-binding sites caused by their lateral movement in the plane of the membrane. TAYLOR et al. (1971) described for the first time such a ligand-induced redistribution of cell surface receptors into clusters and patches and in a polar cap. This redistribution is followed by an internalisation process (DE PETRIS and RAFF 1972, LOOR 1972, UNANUE et al. 1972, ANTOINE et al. 1974, ROTH 1974, GONATAS et aI. 1976). Furthermore, there exists some evidence for the recycling of internalised plasma membranes (JAMIESON and PALADE 1971a, b, HOLTZMAN et al. 1971). In the present study the events following the redistribution of the Concanavalin Abinding sites were examined electron microscopically. It was of particular interest to follow the way of the returning membranes in the cells. Furthermore, it was searched for morphological manifestations of compensatory mechanisms for the membrane removal in the course of the membrane internalisation. The experiments were performed on monolayer cultures of an established epithelial cell line from rat liver (KARSTEN et al. 1976). For visualisation of the Concanavalin A-binding sites the Concanavalin A-peroxidase technique according to BERNHARD and AVRAMEAS(1971) was used. The cell surface labeling was performed on the unfixed cells in the culture flasks in situ at 37°C. After incubation of the cells with Concanavalin A (100 ,ug!ml, 15 min) and horseradish peroxidase (100 /lg!ml, 15 min) the thoroughly washed cell cultures were reincubated with phosphate buffered isotonic saline for internalisation of the labeled cell surface for periods of time ranging between 5 min and 180 min. The peroxidase activity was demollstrated after the method of GRAHAM and KARNOVSKY (1966) after fixation of the cell cultures with 1.6 % glutaraldehyde in phosphate buffered isotonic saline. Then the cells were postfixed with 2 % OS04 in 0.1 M cacodylate buffer (pH 7.2) for 2 hours at 4°C and were embedded in situ in V.estopal W. The cytochemical controls for specificity of the labeling technique included the preincubation of the Con A and peroxidase solutions with the appropriate inhibitory sugar 1 hour before cell labeling. Further controls were run by omitting the Concanavalin A incubation step. Following different periods of reincubation of the cell cultures after cell surface labeling with Concanavalin A and peroxidase redistribution into clusters with simultaneous inter-
351
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nalisation occurred as already reported earlier (ROTH 1975, Roth et al. 1975). The internalisation led to a staining of the elements of the Golgi apparatus (fi~. 1). Within the same cell the unstained Golgi apparatus showed hypertrophy with a transition from the lamellar cisternal form to a vacuolar form (fig. 2). Furthermore, vesicles derived irom hypertrophied Golgi apparatus could be observed in close vicinity to the plasma memInane and fusion of the vesicle limiting membrane and the plasma membrane occurred. The internalisation of labeled membrane areas into the Golgi apparatus is an expression of a membrane recycling as also observed for other cells in vitro (ANTOINE et al. 1974, GONATAS et al. 1975, 1977)..-\ new observation made in the present study is the hypertrophy of the Golgi apparatus which does not participate in the membrane recycling. The hypertrophy of these Golgi apparatus and the subsequent formation of vesicles and their movement to and fusion with the plasma membrane may be an indication of a compensatory membrane biogenesis following the extensive membrane internalisation. The results will be discussed in detail elsewhere (ROTH 1977).
Literature ANTOINE, J. C., S. AVR.HIEAS, N. K GONATAS, A. STIEBER and J. O. GONATAS, Plasma membrane and internalized immunoglobulins of lymph node cells studied with conjugates of antibody or its Fab fragments with horseradish peroxidase. J. Cell BioI. 63, 12-23 (1974). BENNETT, M. S., The concept of membrane flow and membrane vesiculation as mechanisms for active transport and ion pumping. J. Biophys. Biochem. Cytol. 2, 99-103 (1956). BERNHARD, W., and S. AVRAMEAS, Ultrastructural visualization of cellular carbohydrate components by means of Concanavalin A. Exp. Cell Res. 64, 232-236 (1971). DE PETRIS, S., and M. C. R\FF, Distribution of immunoglobulin on the surface of mouse lymphoid cells as determined by immunoferritin electron microscopy. Antibody-induced, temperaturedependent redistribution and its implications for membrane structure. Eur. J. Immunol. 2, 523 to 535 (1972). FRANKE, W. W., D. J. ;\:[ORRE, B. DEUllLING, R D. CHEETHAM, J. KARTENBECK, E.-D. JARRASCH and H.- W. ZENTGRAF, Synthesis and structure and turnover of membrane proteins in rat liver: An examination of the membrane flow hypothesis. Z. Naturforsch. 26b, 1031-1039 (1971). GONATAS, N. K., A. STIEBER, S. U. KIM, D. I. GRAHAM and S. AVRAMEAS, Internalization of nellronal plasma membrane ricin receptors into the Golgi apparatus. Exp. Cell Res. 94, 426-431 (1975). - J. O. GmuTAs, A. STIEBER, J.-C. AXTOINE and S. AVRAMEAS, Quantitative ultrastructural autoradiographic studies of iodinated plasma membranes during segregation and internalization of surface immunoglobulins. J. Cell BioI. 70, 477-493 (1976). - S. U. KUI, A. STIEBER and S. AVR.-DIEAS, Internalization of lectins in neuronal gerl. J. Cell BioI. 73, 1-13 (1977). GRAHAM, R C., and M. J. K\RNOVSKY, The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302 (1966). HOLTZMAN, E., A. R. FREE)UN and L. A. KASHNER, Stimulation dependent alterations in peroxidase uptake by lobster neuro-muscular junctions. Science 173,733-766 (1971). J.-DIIESON, J. D., and G. E. PALADE, Condensing vacuole conversion and zymogen granule discharge in pancreatic exocrine cells: metabolic studies. J. Cell BioI. 48, 503-522 (1971 a). - - Synthesis, intracellular transport, and discharge of secretory proteins in stimulated pancreatic exocrine cells. J. Cell BioI. iiO, 135-158 (1971 b). K\RSTEN, U., G. NEUPERT and R. THUST, Characteristics of an established epithelial cell line (RL-19) from rat liver. Exp. Path. 12, 88-99 (1976). LooR, F., L. FORNI and B. PERNIS, The dynamic state of the lymphocyte membrane. Factors affecting the distribution and turnover of surface immunoglobulins. Eur. J. Immunol. 2, 203-212 (19/2). NOVIKOFF, A. B., E. ESSNER, S. GOLDFISHER and M. HEUS, The Interpretation of Ultrastructure (ed. R. J. H. HARRIS). Academic Press, New York-London 1962, p.149.
Fig. 1. The membrane recycling is indicated by the staining of a Golgi apparatus after internalisation of plasma membrane areas labeled with Concanavalin A and peroxidase. X 39,000. Fig. 2. The membrane recycling into Golgi apparatus (G I ) led to hypertophy of another Golgi apparatus (G 2 ) in the same cell. Many unstained vesicles de'tived from hypertrophied Golgi apparatus are localised in close vicinity of the plasma membrane. X 18,000.
353
ROTH, J., Distribution of Concanavalin A receptors on normal liver cells and on Zajdela ascites hepatoma cells. Int. J. Cancer 14, 762-770 (1974). - Concanavalin A receptors on normal rat liver cells, on in vivo by diethylnitrosamine transformed rat liver cells and on Zajdela ascites hepatoma cells: morphokinetic analysis on cell surface dynamics. Exp. Path. 11, 291-304 (1975). - Compensatory membrane biogenesis and exocytosis as a result of Concanavalin A-induced membrane internalization. Exp. Cell Res., in press (1977). - NEUPERT, G., and F. BOLeK Concanavalin A receptors in the plasma membrane of rat liver cells: comparative electron microscopic investigations on normal cells and on cells in vivo transformed by diethylnitrosamine. Exp. Path. 10, 143-155 (1975). SIEKEVITZ, P., G. E. PALADE, G. E. CHAD and T. OMURA, Organrzational Biosynthesis (ed. M. VOGEL et 301.). Academic Press, New York 1967, p. 331. TAYLOR, R B., W. P. H. DUFFUS, M. C. RAFF and S. DE PETRIS, Redistribution and pinocytosis of I ymphocyte surface immunoglobulin molecules induced by anti-immunoglobulin antibody. Nature New BioI. 233, 225-229 (1971). UNAKUE, E. R, W. D. PERKINS and M. J. KARNOVSKY, Ligand-induced movement of lymphocyte membrane macromolecules. 1. Analysis by immunofluorescence and ultrastructural autoradiography. J. Exp. Med. 136, 885-906 (1973). Author's address: Dr. sc. med. J. ROTH, Pathologisches Institut der Friedrich-Schiller- Universitat, Ziegelmiihlenweg 1, DDR - 69 Jena.
Verantwortlich fUr die Redaktion: Prof. Dr. F. Bclck, 69 Jena. Verlag: VEB Gustav Fischer Verlag, 69 Jena, Villengang 2, Telefon 27332. Satz und Druck: Druckerei "Magnus Poser", 69 Jena. - VerijUentlicht unter der Lizenznummer 1546 des Presseamtes beim Vorsitzenden des Ministerrates der Deutschen Demokratischen Republik. Aile Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Verlsges und des Verfassers sowie mit Angabe der Quelle gestattet. Printed in German Democratic Republic. Artikel-Nr. (EDV) 56519