- Email: [email protected]
Adenovirus adsorption and sterol redistribution in KB cell plasma membrane
Preliminary notes nucleolar organizers and contain proteins (probably ribosomal proteins) and DNA; (2) to the accumulation of tibrillar and granular material. The fibrillar component corresponds to the nascent RNA chains (with associated proteins) spreading perpendicular to the DNA axis, and the granular component corresponds to RNP precursor particles at various stages of maturation (3) to an enlargement of the total volume of the ‘vacuoles’ which contain nuclear chromatin-like material. This investigation was supported in part by grants from the Fonds de la Recherche Scientificme Medicale (no. 3.4527.81).
References 1. Amaldi, F, Giacomoni, D & Zito-Bignami, R, Eurj biochem 11 (1969) 419. 2. Balazs, I & Schildkraut, C L, Exp cell res 101 (1976) 307. 3. Bassleer, R, Arch bio179 (1968) 181. 4. Bucher, 0 & Klbti, R, Z Zellforsch 42 (1955) 193. 5. Fingerhut, M A & Nardone, R M, In vitro 9 (1973) 167: 6. Gani, R, Exp cell res 97 (1976) 249. 7. Goessens, G, Thesis, University of Liege (1975). 8. Gonzalez, S P & Nardone, R M, Exp cell res 50 (1968) 599. 9. Lepoint, A, Virchow’s Arch b cell path01 2.5(1977)
459
Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/82/02M59-05$02.00/0
Adenovirus adsorption and sterol redistribution in KB cell plasma membrane BERNADETTE HENNACHE,’ GERARD TORPIER’ and PIERRE BOULANGER.’ ‘Laborutoire de Virologie Mokulaire (U.233) de I’INSERM, and ‘Unit& de Recherches d’lmmunologie Purasitaire (U.167) de I’INSERM. 59045-Lille, France
Filipin was used as a chemical probe for localization of sterols in freeze-fractured plasma membrane of KB cells. After adenovirus particle adsorption, marked changes occurred in the number and planar distribution of sterols and of intramembranous particles (IMPS). Filipin-sterol complexes became more abundant and both sterols and IMPS aggregated in a network pattern. It was suggested that redistribution of sterols and rearrangement of IMPS were interconnected phenomena, which represented an early cellular response to adenovirus attachment.
Summary.
In a previous report, we have described the morphological changes occurring in KB cell plasma membranes in response to the adsorption of adenovirus particles, adenovirus capsid components, and other viruses (poliovirus) or virus-like structures [IO]. After attachment of adenovirions, the characteristic 7-nm intramembranous particles 10. Lord, A & Lafontaine, J G, J cell sci 21 (1976) 193. 11. Noel, J S, Dewey, W C, Abel, J H & Thompson, R (IMPS) were no longer evenly distributed in P, J cell biol 49 (1971) 830. a random pattern on both leaflets of freeze12. Parmlev. R T. Dow. L W & Mauer. A M. Cancer fractured plasma membrane. The memres 37 (1977) 4313. 13. Potmesil, M & Goldfeder, A, Cancer res 37 (1977) brane alterations, which seemed to be spe857. 14. Sacristan-Garate, A, Navarrette, M H & De La cific for the virus particles used, was Torre, C, J cell sci 16 (1974) 333. termed ‘recognition pattern’ and consisted 15. Schnedl, W & Schnedl, M, Z Zellforsch 126(1972) of a rearrangement and clustering of IMPS 374. 16. Smetana, K, Gyorkey, F, Gyorkey, P&Busch, H, at the periphery of bare areas clear of Exp cell res 58 (1969) 303. IMPS and protruding outwards. All the 17. - Ibid 60 (1970) 175. 18. Stambrook; P J,‘J cell biol47 (1970) 200. modifications occurred at 0°C and were 19. Tokuyasu, K, Madden, S C & Zeldis, L J, J cell transient. They disappeared upon warming biol39 (1968) 630. 20. Weibel, E R, Int rev cvtol26 (1969) 235. to 37”C, a temperature at which adenovirus 21. Weissenfels, N, Z Zellforsch 62 (1964) 667. engulfment is rapid [ 151. Received July 14, 1981 Polyene antibiotic, filipin, has been Revised version received September 17, 1981 shown to form complexes with cholesterol Accepted September 22, 1981 and other related 3-@hydroxysterols [l, 4, 11, 201. Where sterols are abundant, the Printed
in Sweden
E.rp Ccl/ Rrs 137 11982)
460
Preliminary
notes
complexes are visible in freeze-fracture electron micrographs. Filipin appears therefore as a useful probe to detect a microheterogeneity in sterol distribution [6, 7, 191. In the present study, we have used freeze-fracturing after filipin treatment to analyse the topography of sterols in the plasma membrane of KB cells maintained at 37”C, or at 0°C in the presence or absence of adenovirus particles. The aim of the work was to obtain information on the modifications of the hydrophobic portion of the membrane and their relationship with the IMP rearrangement previously described [lo]. Material
and Methods
Adenovirus particles were adsorbed onto destabilized KB cells at low temperature, in order to inhibit the viral penetration [9], and at 3-5~105 virus particles/ cell, in order to saturate the KB cell receptors. The number of receptor sites for adenovirus on KB cell surface has been estimated to be 10Vcell [18]. The mixture virus+cells was incubated with stirring for 3 h at WC [lo]. Destabilization of KB cell plasma membrane by osmotic shock was performed in 0.03 M sodium citrate buffer, pH 7.0, for 10 min at 37”C, as previously described [lo]. The adsorption reaction was arrested by addition of a 12.5% glutaraldehyde solution in phosphate-buffered saline (PBS) up to 5 % final concentration, followed by immediate centrifugation for 10 min at 1000 g and 4°C. The supernatant was removed and the cell pellet overlaid with 2.5% glutaraldehyde in PBS and stored at 4°C until filipin treatment and freeze-fracturing. Filipin was obtained from Upjohn Co. (Kalamazoo, Mich.). Stock solution was prepared just before use, at 30 mM in dimethvlformamide (DMF). Samoles of packed KB cells were thoroughly’mixed with’20 vol of 0.3 mM tilipin in 0.1 M Na cacodylate containing 2.5 % glutaraldehyde, and incubated at room temperature for 30 min. Control KB cells were: (i) cells maintained at 37°C in F13 culture medium, and treated with tilipin without citrate shock (citrate control); (ii) cells treated only with hypotonic citrate prior to tilipin treatment (low-temperature control); (iii) cells treated with citrate, incubated at low temperature for 3 h and treated with DMF at 1% without tilipin (solvent control); (iv) cells treated with citrate, and incubated with PBS without adenovirus particles for 3 h at 0°C prior to tilipin treatment (virus control). The cell samples were freeze-fractured in a Balzers BAF 300 apparatus, and membrane unidirectional replicas were obtained by standard method at 45°C [IO]. The replicas were examined in a Hitachi HU-12 electron microscope. Counting of IMPS and surface measurements were performed with a MOP/AM 01 quantitative image Exp Cd Res 137 (1982)
analyser (Kontron Messgerate) on at least 20 pictures, taken at random, of each cell preparation.
Results and Discussion
In control KB cell plasma membrane, filipin treatment revealed that the sterol-rich patches were distributed at random, or irregularly clustered (fig. 1c). The lesions appeared as doughnut-shaped protuberances and craters on unidirectional replicas (fig. lc, d). Protuberances were 18.8k1.8 nm, and craters 17.1+ 1.5 nm in diameter, as determined by direct measurement. Protuberances and craters represented the same type of lesion and were due to a difference of partition of the filipin-sterol complexes between the outer and inner membrane leaflets. As shown in table 1, the density of protuberances on the P face corresponded to that of craters on the E face and vice versa. Control KB cells showed a roughly symmetrical repartition of sterol-rich patches in both plasma membrane leaflets. However, the protuberances were slightly more abundant on the internal than on the external leaflets. This slightly asymmetric repartition of the protuberances persisted after the hypotonic shock, but disappeared after incubation of the citrateshocked cells in culture medium for 3 h at 0°C. On KB cells which underwent adenovirus adsorption under saturating conditions of their surface receptors, filipin treatment showed both quantitative and qualitative I. Protoplasmic fracture face (face P) of plasma membrane ofKB cells incubated (b, d) with or (a, c) without adenovirus 2. (a, c) Uninfected KB cells (a) untreated or (c) treated with filipin, showing a random pattern of intramembranous particles (IMPS) and of (c) filipin lesions. (6, d) KB cells incubated with adenovirus particles, showing the roughly hexagonal array of (h) aggregated IMPS and (d) tilipin lesions. The characteristic aspects of the doughnut-like tilipin lesions are indicated by an arrow (pit), and by an arrow-head (protuberance). ~60000.
Fin.
Preliminury notes
30-811803
461
462
Preliminary
notes
Table 1. Filipin-induced infected KB cells
lesions in plasma
membrane
of uninfected
Protuberancesb
Cratersb
KB cell treatment before filipin”
Filipin treatment
P face
P face
(i) No shock (ii) Citrate shock (iii) Citrate+cold +solvent (iv) Citrate+cold + buffer (v) Citrate+cold + virus
+ +
91?16 116+15
-
0
E face 56k 13 68+20 0
70f18 62fll 0
+
107+75
120?71
98&59
+
245k61
126k 15
114+67
and adenovirus-
E face
Distribution pattern=
104f 18 108+23
R R
0 lOOk
R
216+31
A
0 KB cells were subjected to the following treatments. (i) Cells maintained in culture medium at 37”C, and treated with filipin immediately after harvest (citrate control). (ii) Cells treated with 0.03 M citrate buffer, pH 7.0, for 10 min at 37”C, then with filipin (cold control). (iii) Citrate-shocked cells maintained in culture medium at 0°C for 3 h, then treated with dimethylformamide without tilipin (solvent control). (iv) Citrateshocked cells, resuspended in culture medium, mixed with 0.5 vol of phosphate-buffered saline (PBS) without virus and incubated at 0°C for 3 h (virus control). (v) Cells treated the same way as in (iv), but incubated with adenovirus particles at 1Or3particles/ml in PBS. b Number of lesions per pm2 + SD. Each figure corresponds to the counting of at least 20 fields of 10 cm’. r Type of distribution. R, random pattern; A, aggregated in a roughly regular array.
changes in the membrane sterol distribution. The density of the filipin-sterol complexes was markedly higher than in control uninfected cells, and an asymmetric distribution of filipin-induced protuberances was evident (table 1). Moreover, the lesions were rearranged in a regular array and were located within the IMP-denuded zones delineated by clustered IMPS (fig. Id). The redistribution of IMPS in a roughly hexagonal network has never been found with inert particles, such as latex beads, but has been obtained with adenovirions or latex particles coated with adenovirus penton capsomers [lo]. The IMP rearrangement has therefore been suggested to be specific for adenovirus particles and has been termed ‘recognition pattern’ [lo]. Adenovirus particle adsorption onto KB cell surface receptors thus induced two types of early structural alterations in plasma membrane. (i) Rearrangement of IMPS, from a random to a regular pattern, consisting of IMP-denuded zones limited by Exp Cd
Res 137 11982)
clustered IMPS. (ii) Modification of the distribution of sterols which concentrated in the IMP-denuded zones. Both filipin complexes and IMPS adopted therefore a nearhexagonal distribution. The effects observed here might be due to some alteration of the membrane structure resulting from the osmotic shock. However, such structural modifications, if any, were not evidenced by the technique used: there was no visible change in the distribution of IMPS [lo] and sterols (table 1) after citrate treatment of the cells. Moreover: the brief osmotic shock did not alter me viability of the cells [lo]. Aggregation of IMPS has already been observed when cells undergo fusion induced by chemicals [5, 17, 221, or biological agents such as viruses [2, 8, 12, 14, 21, 231. However, membrane alterations induced by adenovirus, a non-enveloped virus, are significantly different from those previously described. (i) The IMPS and sterols cluster in a regular, roughly hexa-
Preliminary notes gonal array and (ii) the alterations can occur at low temperature, whereas for other enveloped viruses they appear only when cells are transferred to 37°C [3]. It has been proposed that binding of a virus at its receptor site modifies in some way the anchorage of IMPS on the cytoskeleton [13]. The citrate shock might amplify this phenomenon by membrane destabilization, as previously suggested [lo]. The IMPS would therefore be free to move in the membrane plane and to cluster in a network pattern inducing a redistribution of phospholipids and sterols. The sterols influence the permeability and fluidity of the cell membrane [6]. The sterol redistribution occurring in response to adenovirus-cell attachment might contribute to virus penetration. The IMP rearrangement might, on the other hand, contribute to the tightness of bonding of virus particles on the cell surface
[161. This work was supported by the CNRS (ERA 070225) the INSERM (CRL 79.5.081.1) and the Universite du Droit et de la Sante de Lille. We acknowledge with thanks the excellent technical assistance of AIain Jacob and Didier Petite, and the expert secretarial aid of Virginie Milleville and Sophie Wojcik. We are grateful to Dr J. P. Paturaud for providing us with filipin.
References 1. Andrews, L D & Cohen, A I, J cell biol 81 (1979) 215. 2. BHchi, T, Aguet, M & Howe, C, J virol 11 (1973) 1004. 3. Bachi, T, Deas, J E & Howe, C, Cell surface reviews. Virus infection and the cell surface (ed G Poste & G L Nicolson) vol. 2, pp. 83-127. North-Holland, Amsterdam (1977). 4. De Kruijff, B & Demel, R A, Biochim biophys acta 339 (1974) 57. 5. Elgsaeter, A & Branton, D, J cell biol 63 (1974) 1018. 6. Elias, PM, Friend, D S & Goerke, J, J cell biol 79 (1979) 232a. 7. Friend, D S & Elias, PM, J cell biol79 (1979) 216a. 8. Gazitt, Y, Loyter, A & Ohad, I, Biochim biophys acta 471 (1977) 361. 9. Hennache, B & Boulanger, P, Biochem j 166(1977) 237. IO. Hennache, B, Torpier, G & Boulanger, P, Exp cell res 124 (1979) 139. Printed
m Sweden
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
463
Kinsky, S C, Ann rev pharmacol 10 (1970) 119. Knutton, S, Nature 264 (1976) 672. Kohn, A, Adv virus res 24 (1979) 223. Lemay, P, Collyn-d’Hooghe, M & Torpier, G, Exp cell res 109(1977) 79. Lonberg-Helm, K & Philipson, L, J viral 4 (1969) 323. - Monographs in virology (ed J L Melnick & S Karger) vol. 9. Base1(1974). Lucy, J A, Nature 227 (1970) 815. Philipson, L, Lonberg-Helm, K & Pettersson, U, J virol 2 (1968) 1064. Tillack, T W & Kinsky, S, Biochim biophys acta 323 (1973) 43. Verkleij, A J, de Kruijff, B, Gerritsen, W F, Demel, R A, van Deenen, L L M & Ververgaert, P H J, Biochim biophys acta 291 (1973) 577. Volsky, D & Loyter, A, Biochim biophys acta 471 (1977) 243. Vos, J, Ahkong, Q F, Bothan, G M, Quirk, S J & Lucv. J A. Biochem i 158 (1976) 651. Zakai; N, Kulka, R d & Lbyter, A, Proc natl acad sci US 74 (1977) 2417.
Received July 14, 1981 Revised version received September 28, 1981 Accepted October 13, 1981
Copyright @ 1982 by Academic Press. Inc. All rights of reproduction m any form rexwed 0014.48271821020463-07$02.~X~/O
Localization of geneson fractionated rat chromosomes by molecular hybridization J. G. COLLARD, J. SCHIJVEN, A. TULP and M. MEULENBROEK, The Netherlands Cancer Institute, Division of Cell Biology. 1066 CX Amsterdam, The Netherlands Summary. Chromosomes derived from rat kidney cells were separated in specially designed sedimentation chambers by velocity sedimentation at 30 g. The DNA of the chromosomal fractions was used in molecular hybridization experiments to localize single-copy genes on the fractionated rat chromosomes. By crosshybridization with a mouse immunoglobulin light chain kappa c-DNA probe, the rat immunogiobuhn genes were detected only on the DNA of chromosomal fractions highly enriched for chromosomes 3, 4 and 5. The rat albumin gene was detected on fractions greatly enriched for chromosomes 11, 13 and 14. The described method allows the localization of structural genes or introduced DNA sequences on the chromosomal level especially in those cell systems in which no suitable somatic cell hybrids are available.
Cloning of genes by recombinant DNA technology opens extensive possibilities for Exp
Cdl
Rrs
137 (1982)
References 1. Amaldi, F, Giacomoni, D & Zito-Bignami, R, Eurj biochem 11 (1969) 419. 2. Balazs, I & Schildkraut, C L, Exp cell res 101 (1976) 307. 3. Bassleer, R, Arch bio179 (1968) 181. 4. Bucher, 0 & Klbti, R, Z Zellforsch 42 (1955) 193. 5. Fingerhut, M A & Nardone, R M, In vitro 9 (1973) 167: 6. Gani, R, Exp cell res 97 (1976) 249. 7. Goessens, G, Thesis, University of Liege (1975). 8. Gonzalez, S P & Nardone, R M, Exp cell res 50 (1968) 599. 9. Lepoint, A, Virchow’s Arch b cell path01 2.5(1977)
459
Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/82/02M59-05$02.00/0
Adenovirus adsorption and sterol redistribution in KB cell plasma membrane BERNADETTE HENNACHE,’ GERARD TORPIER’ and PIERRE BOULANGER.’ ‘Laborutoire de Virologie Mokulaire (U.233) de I’INSERM, and ‘Unit& de Recherches d’lmmunologie Purasitaire (U.167) de I’INSERM. 59045-Lille, France
Filipin was used as a chemical probe for localization of sterols in freeze-fractured plasma membrane of KB cells. After adenovirus particle adsorption, marked changes occurred in the number and planar distribution of sterols and of intramembranous particles (IMPS). Filipin-sterol complexes became more abundant and both sterols and IMPS aggregated in a network pattern. It was suggested that redistribution of sterols and rearrangement of IMPS were interconnected phenomena, which represented an early cellular response to adenovirus attachment.
Summary.
In a previous report, we have described the morphological changes occurring in KB cell plasma membranes in response to the adsorption of adenovirus particles, adenovirus capsid components, and other viruses (poliovirus) or virus-like structures [IO]. After attachment of adenovirions, the characteristic 7-nm intramembranous particles 10. Lord, A & Lafontaine, J G, J cell sci 21 (1976) 193. 11. Noel, J S, Dewey, W C, Abel, J H & Thompson, R (IMPS) were no longer evenly distributed in P, J cell biol 49 (1971) 830. a random pattern on both leaflets of freeze12. Parmlev. R T. Dow. L W & Mauer. A M. Cancer fractured plasma membrane. The memres 37 (1977) 4313. 13. Potmesil, M & Goldfeder, A, Cancer res 37 (1977) brane alterations, which seemed to be spe857. 14. Sacristan-Garate, A, Navarrette, M H & De La cific for the virus particles used, was Torre, C, J cell sci 16 (1974) 333. termed ‘recognition pattern’ and consisted 15. Schnedl, W & Schnedl, M, Z Zellforsch 126(1972) of a rearrangement and clustering of IMPS 374. 16. Smetana, K, Gyorkey, F, Gyorkey, P&Busch, H, at the periphery of bare areas clear of Exp cell res 58 (1969) 303. IMPS and protruding outwards. All the 17. - Ibid 60 (1970) 175. 18. Stambrook; P J,‘J cell biol47 (1970) 200. modifications occurred at 0°C and were 19. Tokuyasu, K, Madden, S C & Zeldis, L J, J cell transient. They disappeared upon warming biol39 (1968) 630. 20. Weibel, E R, Int rev cvtol26 (1969) 235. to 37”C, a temperature at which adenovirus 21. Weissenfels, N, Z Zellforsch 62 (1964) 667. engulfment is rapid [ 151. Received July 14, 1981 Polyene antibiotic, filipin, has been Revised version received September 17, 1981 shown to form complexes with cholesterol Accepted September 22, 1981 and other related 3-@hydroxysterols [l, 4, 11, 201. Where sterols are abundant, the Printed
in Sweden
E.rp Ccl/ Rrs 137 11982)
460
Preliminary
notes
complexes are visible in freeze-fracture electron micrographs. Filipin appears therefore as a useful probe to detect a microheterogeneity in sterol distribution [6, 7, 191. In the present study, we have used freeze-fracturing after filipin treatment to analyse the topography of sterols in the plasma membrane of KB cells maintained at 37”C, or at 0°C in the presence or absence of adenovirus particles. The aim of the work was to obtain information on the modifications of the hydrophobic portion of the membrane and their relationship with the IMP rearrangement previously described [lo]. Material
and Methods
Adenovirus particles were adsorbed onto destabilized KB cells at low temperature, in order to inhibit the viral penetration [9], and at 3-5~105 virus particles/ cell, in order to saturate the KB cell receptors. The number of receptor sites for adenovirus on KB cell surface has been estimated to be 10Vcell [18]. The mixture virus+cells was incubated with stirring for 3 h at WC [lo]. Destabilization of KB cell plasma membrane by osmotic shock was performed in 0.03 M sodium citrate buffer, pH 7.0, for 10 min at 37”C, as previously described [lo]. The adsorption reaction was arrested by addition of a 12.5% glutaraldehyde solution in phosphate-buffered saline (PBS) up to 5 % final concentration, followed by immediate centrifugation for 10 min at 1000 g and 4°C. The supernatant was removed and the cell pellet overlaid with 2.5% glutaraldehyde in PBS and stored at 4°C until filipin treatment and freeze-fracturing. Filipin was obtained from Upjohn Co. (Kalamazoo, Mich.). Stock solution was prepared just before use, at 30 mM in dimethvlformamide (DMF). Samoles of packed KB cells were thoroughly’mixed with’20 vol of 0.3 mM tilipin in 0.1 M Na cacodylate containing 2.5 % glutaraldehyde, and incubated at room temperature for 30 min. Control KB cells were: (i) cells maintained at 37°C in F13 culture medium, and treated with tilipin without citrate shock (citrate control); (ii) cells treated only with hypotonic citrate prior to tilipin treatment (low-temperature control); (iii) cells treated with citrate, incubated at low temperature for 3 h and treated with DMF at 1% without tilipin (solvent control); (iv) cells treated with citrate, and incubated with PBS without adenovirus particles for 3 h at 0°C prior to tilipin treatment (virus control). The cell samples were freeze-fractured in a Balzers BAF 300 apparatus, and membrane unidirectional replicas were obtained by standard method at 45°C [IO]. The replicas were examined in a Hitachi HU-12 electron microscope. Counting of IMPS and surface measurements were performed with a MOP/AM 01 quantitative image Exp Cd Res 137 (1982)
analyser (Kontron Messgerate) on at least 20 pictures, taken at random, of each cell preparation.
Results and Discussion
In control KB cell plasma membrane, filipin treatment revealed that the sterol-rich patches were distributed at random, or irregularly clustered (fig. 1c). The lesions appeared as doughnut-shaped protuberances and craters on unidirectional replicas (fig. lc, d). Protuberances were 18.8k1.8 nm, and craters 17.1+ 1.5 nm in diameter, as determined by direct measurement. Protuberances and craters represented the same type of lesion and were due to a difference of partition of the filipin-sterol complexes between the outer and inner membrane leaflets. As shown in table 1, the density of protuberances on the P face corresponded to that of craters on the E face and vice versa. Control KB cells showed a roughly symmetrical repartition of sterol-rich patches in both plasma membrane leaflets. However, the protuberances were slightly more abundant on the internal than on the external leaflets. This slightly asymmetric repartition of the protuberances persisted after the hypotonic shock, but disappeared after incubation of the citrateshocked cells in culture medium for 3 h at 0°C. On KB cells which underwent adenovirus adsorption under saturating conditions of their surface receptors, filipin treatment showed both quantitative and qualitative I. Protoplasmic fracture face (face P) of plasma membrane ofKB cells incubated (b, d) with or (a, c) without adenovirus 2. (a, c) Uninfected KB cells (a) untreated or (c) treated with filipin, showing a random pattern of intramembranous particles (IMPS) and of (c) filipin lesions. (6, d) KB cells incubated with adenovirus particles, showing the roughly hexagonal array of (h) aggregated IMPS and (d) tilipin lesions. The characteristic aspects of the doughnut-like tilipin lesions are indicated by an arrow (pit), and by an arrow-head (protuberance). ~60000.
Fin.
Preliminury notes
30-811803
461
462
Preliminary
notes
Table 1. Filipin-induced infected KB cells
lesions in plasma
membrane
of uninfected
Protuberancesb
Cratersb
KB cell treatment before filipin”
Filipin treatment
P face
P face
(i) No shock (ii) Citrate shock (iii) Citrate+cold +solvent (iv) Citrate+cold + buffer (v) Citrate+cold + virus
+ +
91?16 116+15
-
0
E face 56k 13 68+20 0
70f18 62fll 0
+
107+75
120?71
98&59
+
245k61
126k 15
114+67
and adenovirus-
E face
Distribution pattern=
104f 18 108+23
R R
0 lOOk
R
216+31
A
0 KB cells were subjected to the following treatments. (i) Cells maintained in culture medium at 37”C, and treated with filipin immediately after harvest (citrate control). (ii) Cells treated with 0.03 M citrate buffer, pH 7.0, for 10 min at 37”C, then with filipin (cold control). (iii) Citrate-shocked cells maintained in culture medium at 0°C for 3 h, then treated with dimethylformamide without tilipin (solvent control). (iv) Citrateshocked cells, resuspended in culture medium, mixed with 0.5 vol of phosphate-buffered saline (PBS) without virus and incubated at 0°C for 3 h (virus control). (v) Cells treated the same way as in (iv), but incubated with adenovirus particles at 1Or3particles/ml in PBS. b Number of lesions per pm2 + SD. Each figure corresponds to the counting of at least 20 fields of 10 cm’. r Type of distribution. R, random pattern; A, aggregated in a roughly regular array.
changes in the membrane sterol distribution. The density of the filipin-sterol complexes was markedly higher than in control uninfected cells, and an asymmetric distribution of filipin-induced protuberances was evident (table 1). Moreover, the lesions were rearranged in a regular array and were located within the IMP-denuded zones delineated by clustered IMPS (fig. Id). The redistribution of IMPS in a roughly hexagonal network has never been found with inert particles, such as latex beads, but has been obtained with adenovirions or latex particles coated with adenovirus penton capsomers [lo]. The IMP rearrangement has therefore been suggested to be specific for adenovirus particles and has been termed ‘recognition pattern’ [lo]. Adenovirus particle adsorption onto KB cell surface receptors thus induced two types of early structural alterations in plasma membrane. (i) Rearrangement of IMPS, from a random to a regular pattern, consisting of IMP-denuded zones limited by Exp Cd
Res 137 11982)
clustered IMPS. (ii) Modification of the distribution of sterols which concentrated in the IMP-denuded zones. Both filipin complexes and IMPS adopted therefore a nearhexagonal distribution. The effects observed here might be due to some alteration of the membrane structure resulting from the osmotic shock. However, such structural modifications, if any, were not evidenced by the technique used: there was no visible change in the distribution of IMPS [lo] and sterols (table 1) after citrate treatment of the cells. Moreover: the brief osmotic shock did not alter me viability of the cells [lo]. Aggregation of IMPS has already been observed when cells undergo fusion induced by chemicals [5, 17, 221, or biological agents such as viruses [2, 8, 12, 14, 21, 231. However, membrane alterations induced by adenovirus, a non-enveloped virus, are significantly different from those previously described. (i) The IMPS and sterols cluster in a regular, roughly hexa-
Preliminary notes gonal array and (ii) the alterations can occur at low temperature, whereas for other enveloped viruses they appear only when cells are transferred to 37°C [3]. It has been proposed that binding of a virus at its receptor site modifies in some way the anchorage of IMPS on the cytoskeleton [13]. The citrate shock might amplify this phenomenon by membrane destabilization, as previously suggested [lo]. The IMPS would therefore be free to move in the membrane plane and to cluster in a network pattern inducing a redistribution of phospholipids and sterols. The sterols influence the permeability and fluidity of the cell membrane [6]. The sterol redistribution occurring in response to adenovirus-cell attachment might contribute to virus penetration. The IMP rearrangement might, on the other hand, contribute to the tightness of bonding of virus particles on the cell surface
[161. This work was supported by the CNRS (ERA 070225) the INSERM (CRL 79.5.081.1) and the Universite du Droit et de la Sante de Lille. We acknowledge with thanks the excellent technical assistance of AIain Jacob and Didier Petite, and the expert secretarial aid of Virginie Milleville and Sophie Wojcik. We are grateful to Dr J. P. Paturaud for providing us with filipin.
References 1. Andrews, L D & Cohen, A I, J cell biol 81 (1979) 215. 2. BHchi, T, Aguet, M & Howe, C, J virol 11 (1973) 1004. 3. Bachi, T, Deas, J E & Howe, C, Cell surface reviews. Virus infection and the cell surface (ed G Poste & G L Nicolson) vol. 2, pp. 83-127. North-Holland, Amsterdam (1977). 4. De Kruijff, B & Demel, R A, Biochim biophys acta 339 (1974) 57. 5. Elgsaeter, A & Branton, D, J cell biol 63 (1974) 1018. 6. Elias, PM, Friend, D S & Goerke, J, J cell biol 79 (1979) 232a. 7. Friend, D S & Elias, PM, J cell biol79 (1979) 216a. 8. Gazitt, Y, Loyter, A & Ohad, I, Biochim biophys acta 471 (1977) 361. 9. Hennache, B & Boulanger, P, Biochem j 166(1977) 237. IO. Hennache, B, Torpier, G & Boulanger, P, Exp cell res 124 (1979) 139. Printed
m Sweden
11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.
463
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Received July 14, 1981 Revised version received September 28, 1981 Accepted October 13, 1981
Copyright @ 1982 by Academic Press. Inc. All rights of reproduction m any form rexwed 0014.48271821020463-07$02.~X~/O
Localization of geneson fractionated rat chromosomes by molecular hybridization J. G. COLLARD, J. SCHIJVEN, A. TULP and M. MEULENBROEK, The Netherlands Cancer Institute, Division of Cell Biology. 1066 CX Amsterdam, The Netherlands Summary. Chromosomes derived from rat kidney cells were separated in specially designed sedimentation chambers by velocity sedimentation at 30 g. The DNA of the chromosomal fractions was used in molecular hybridization experiments to localize single-copy genes on the fractionated rat chromosomes. By crosshybridization with a mouse immunoglobulin light chain kappa c-DNA probe, the rat immunogiobuhn genes were detected only on the DNA of chromosomal fractions highly enriched for chromosomes 3, 4 and 5. The rat albumin gene was detected on fractions greatly enriched for chromosomes 11, 13 and 14. The described method allows the localization of structural genes or introduced DNA sequences on the chromosomal level especially in those cell systems in which no suitable somatic cell hybrids are available.
Cloning of genes by recombinant DNA technology opens extensive possibilities for Exp
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137 (1982)