Plant Cell Reports
Plant Cell Reports (1988) 7:341-343
© Springer-Verlag 1988
Changes in the integrity of large unilamellar vesicles due to their interaction with tobacco cell suspensions Alexander E. Gad, Carmi Lubitz-Omero, Nurit Rosenberg, and Arie Altman Department of Horticulture, Faculty of Agriculture, Otto Warburg Center for Biotechnology in Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel Received March 8, 1988 - Communicated by J. Schell
ABSTRACT Negatively charged large unilamellar vesicles (LUV) were incubated with tobacco (Nicotiana tabacum var. xanthi) cell suspensions and with the cell-free medium of the cell suspensions. The extent of cell-LUV interaction was determined by the leakage of the LUV contents. Cells enhanced the leakage of LUV contents and this effect increased with cell age. Addition of polylysine to the reaction mixture increased even further the leakage of the LUV contents. The cell-free medium of the cell suspension also affected the integrity of the LUV. Cell-free medium, by itself, promoted leakage of LUV contents and caused a reduction in the leakage exerted by polylysine. Centrifugation (8000g) of the cell-free medium decreased its effect, heat treatment (122°C) did not alter its effect and sonication enhanced it. The effects of the cell-free medium are attributed to the presence of cell wall debris of disintegrated cells.
INTRODUCTION The efficient transfer of nucleic acids into plant protoplasts is an important goal for the development of transformation techniques. Liposomes have been successfully used to infect protopolasts with viral R N A (Fukanaga 1981; Nagata, 1981; Fraley, 1982; Watanabe, 1982; Rouze, 1983; Houssain et al, 1985) and to transfer plasmid DNA into plant protoplasts. (Deshayes, 1985; Rosenberg et al, 1987). In most of these reports the transfection was obtained by using polyethyleneglycol (PEG) which is a known to transform m a m m a l i a n cells, However, PEG is often detrimental to protoplasts (Kao 1974). The viability of the cells was also affected by their incubation with vesicles and by the vesicle composition (Fraley et al, 1982). In contrast to the extensive study of the state of the cells, insufficient attention was given to the integrity of the vesicles during their interaction with the cells. The leakage of vesicles should be minimal, in order to ensure satisfactory delivery of their content into the cells. In the present work the changes in the rate of leakage of vesicle contents were measured during their incubation with cell suspensions and cell-free media of cell suspen-
Offprint requests to: A. E. Gad
sions. The effect of culture age and of the cell-free medium were also monitored. Our data show that senescing cell cultures, as well as the media in which they are cultured, decrease vesicle stability. Vesicle-cell interaction takes place in the presence of polylysine. MATERIALS AND METHODS
Materlal$ Phosphatidylcholine (PC), cardiolipin (CL), polylysine (37000 M.W.) and polyglutamate (60000 M.W.) were purchased from Sigma Chemicals Co. Phosphatidylethanolamine (PE) was purchased from Avanti Polar Lipids. 5(6)-carboxyfluorescein (CF) was purchased from Eastman and was further purified according to Ralston et al (1981). The detergent A m m o n y x LO was purchased from Onyx Corp. The poly amino acids were suspended in buffer and kept in small aliquots at -18°C. Each aliquot was never refrozen since degradation occurred during repetitive thawing. Phosphlipids concentration was determined by the concentration of phosphate (Bartlett 1959), thereafter liposome concentration was given as lipid phosphorous content.
Cell SuspenMons cell suspensions of Nicotiana tabaccum vat. xanthi were grown at 25°C (16 h light, 120 rpm) on Murashige & Skoog medium (1962) also containing 3% sucrose, 1 mg/L 2,4-D, 10 mg/L thiamine HC1, 0.5 mg/L folic acid and 0.5 mg/L biotin (pH=5.7). Cells were regularly subcultured, unless otherwise mentioned, every 7 days to give a suspension of 20% (V/V) packed cell volume. Cells were separated, at various times during their subculture, from the culture medium by centrifugation (5 rain., 200rpm) The cells were resuspended in 0.4 M mannitol, 0.1 M NaC1, 0.1 mM EDTA, 5 mM Hepes (pH=7.4), and the supernatant (from here on referred to as - cell-free medium) was collected and stored at -18°C for further use. For specific experiments, the cell-free medium was either centrifuged (5 min, 8000g), or autoclaved (122% 1.2 arm. for 20 min.) or sonicated for 2 min in a bath type sonicator and autoclaved (122°C, 1.2 atm, 20 minutes). Several cell fractions were prepared as follows, in order to separate membranes from cell walls. Cell-free medium was mixed with diethyl ether (1:1; V/V), spun for 10 min. at 2000g and resuspended in fresh culture medium. This pro-
342 cedure was repeated 3 times. Subsequently, the suspension in culture medium was left overnight at room temperature to remove traces of ether. Lipid-depleted cell walls were prepared by grinding cells with equal volume of growing medium and spinning for 30 rain at 7000g. The pellet was resuspended in 1% cholate (in culture medium) and spun 3 times at 2500g (resuspended each time in culture medium) to remove the detergent. The resulting pellet was treated as above for extraction of lipids from cell walls.
L U V Preparation Large unilamellar vesicles (LUV) were prepared by the reverse phase evaporation technique (Szoka & Papahadjopoulos 1978) followed by extrusion through 0.4 and 0.2 gm polycarbonate membranes (Olson et al, 1979). LUV consisted of CL:PC:PE (3:2:1 molar ratio) or CL:PC:FE (3:2:I) + 30% mol cholesterol, prepared in 0.4M mannito], 0.1M NaC1, 0.1mM EDTA and 5mM Hepes (pH----7.4) with 13 mM CF. Vesicles were separated from non-encapsulated CF by gel chromatography on Sephadex G-75.
probe CE Polylysine induced a marked increase in the leakage and its i~clusion in the mixture of LUV and cells increased the rate of leakage even more. The extent of leakage in the presence of cells and polylysine was greater than their additive effects, when applied separately (Fig. 1). Even preincubation, for 3 s, of the cells with polylysine resulted in an increase, though much smaller than observed without preincubation, in the leakage of LUV contents. The extent of leakage was affected also by the age of the culture (counted from the last subculture). Older cell suspensions (14 days vs. 7 days) induced greater changes in the LUV stability. 25
i
I
I
i
l
I
20~ . I.iJ Ii...
~ . _ 15
,,,g
Determination of L U V 8tabilitll
Q::
LUV were incubated with cells and various cell fractions. LUV stability was monitored using the dequenching of CF fluorescence, initially trapped in the LUV at high concentrations (Weinstein et a1,1977). The increase in the fluorescence of the liposome-entrapped CF results from the dilution of the probe leaking from the LUV to the medium, thus being indicative of changes in LUV stability. All measurements were carried out in an SLM 4800 spectrofluorometer at 25°C (493nm ex., 516nm era. with a 510nm cut-off filter).
3°
RESULTS PE-containing LUV exhibited only slight leakage of contents on standing;
Plant Cell Reports (1988) 7:341-343
© Springer-Verlag 1988
Changes in the integrity of large unilamellar vesicles due to their interaction with tobacco cell suspensions Alexander E. Gad, Carmi Lubitz-Omero, Nurit Rosenberg, and Arie Altman Department of Horticulture, Faculty of Agriculture, Otto Warburg Center for Biotechnology in Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel Received March 8, 1988 - Communicated by J. Schell
ABSTRACT Negatively charged large unilamellar vesicles (LUV) were incubated with tobacco (Nicotiana tabacum var. xanthi) cell suspensions and with the cell-free medium of the cell suspensions. The extent of cell-LUV interaction was determined by the leakage of the LUV contents. Cells enhanced the leakage of LUV contents and this effect increased with cell age. Addition of polylysine to the reaction mixture increased even further the leakage of the LUV contents. The cell-free medium of the cell suspension also affected the integrity of the LUV. Cell-free medium, by itself, promoted leakage of LUV contents and caused a reduction in the leakage exerted by polylysine. Centrifugation (8000g) of the cell-free medium decreased its effect, heat treatment (122°C) did not alter its effect and sonication enhanced it. The effects of the cell-free medium are attributed to the presence of cell wall debris of disintegrated cells.
INTRODUCTION The efficient transfer of nucleic acids into plant protoplasts is an important goal for the development of transformation techniques. Liposomes have been successfully used to infect protopolasts with viral R N A (Fukanaga 1981; Nagata, 1981; Fraley, 1982; Watanabe, 1982; Rouze, 1983; Houssain et al, 1985) and to transfer plasmid DNA into plant protoplasts. (Deshayes, 1985; Rosenberg et al, 1987). In most of these reports the transfection was obtained by using polyethyleneglycol (PEG) which is a known to transform m a m m a l i a n cells, However, PEG is often detrimental to protoplasts (Kao 1974). The viability of the cells was also affected by their incubation with vesicles and by the vesicle composition (Fraley et al, 1982). In contrast to the extensive study of the state of the cells, insufficient attention was given to the integrity of the vesicles during their interaction with the cells. The leakage of vesicles should be minimal, in order to ensure satisfactory delivery of their content into the cells. In the present work the changes in the rate of leakage of vesicle contents were measured during their incubation with cell suspensions and cell-free media of cell suspen-
Offprint requests to: A. E. Gad
sions. The effect of culture age and of the cell-free medium were also monitored. Our data show that senescing cell cultures, as well as the media in which they are cultured, decrease vesicle stability. Vesicle-cell interaction takes place in the presence of polylysine. MATERIALS AND METHODS
Materlal$ Phosphatidylcholine (PC), cardiolipin (CL), polylysine (37000 M.W.) and polyglutamate (60000 M.W.) were purchased from Sigma Chemicals Co. Phosphatidylethanolamine (PE) was purchased from Avanti Polar Lipids. 5(6)-carboxyfluorescein (CF) was purchased from Eastman and was further purified according to Ralston et al (1981). The detergent A m m o n y x LO was purchased from Onyx Corp. The poly amino acids were suspended in buffer and kept in small aliquots at -18°C. Each aliquot was never refrozen since degradation occurred during repetitive thawing. Phosphlipids concentration was determined by the concentration of phosphate (Bartlett 1959), thereafter liposome concentration was given as lipid phosphorous content.
Cell SuspenMons cell suspensions of Nicotiana tabaccum vat. xanthi were grown at 25°C (16 h light, 120 rpm) on Murashige & Skoog medium (1962) also containing 3% sucrose, 1 mg/L 2,4-D, 10 mg/L thiamine HC1, 0.5 mg/L folic acid and 0.5 mg/L biotin (pH=5.7). Cells were regularly subcultured, unless otherwise mentioned, every 7 days to give a suspension of 20% (V/V) packed cell volume. Cells were separated, at various times during their subculture, from the culture medium by centrifugation (5 rain., 200rpm) The cells were resuspended in 0.4 M mannitol, 0.1 M NaC1, 0.1 mM EDTA, 5 mM Hepes (pH=7.4), and the supernatant (from here on referred to as - cell-free medium) was collected and stored at -18°C for further use. For specific experiments, the cell-free medium was either centrifuged (5 min, 8000g), or autoclaved (122% 1.2 arm. for 20 min.) or sonicated for 2 min in a bath type sonicator and autoclaved (122°C, 1.2 atm, 20 minutes). Several cell fractions were prepared as follows, in order to separate membranes from cell walls. Cell-free medium was mixed with diethyl ether (1:1; V/V), spun for 10 min. at 2000g and resuspended in fresh culture medium. This pro-
342 cedure was repeated 3 times. Subsequently, the suspension in culture medium was left overnight at room temperature to remove traces of ether. Lipid-depleted cell walls were prepared by grinding cells with equal volume of growing medium and spinning for 30 rain at 7000g. The pellet was resuspended in 1% cholate (in culture medium) and spun 3 times at 2500g (resuspended each time in culture medium) to remove the detergent. The resulting pellet was treated as above for extraction of lipids from cell walls.
L U V Preparation Large unilamellar vesicles (LUV) were prepared by the reverse phase evaporation technique (Szoka & Papahadjopoulos 1978) followed by extrusion through 0.4 and 0.2 gm polycarbonate membranes (Olson et al, 1979). LUV consisted of CL:PC:PE (3:2:1 molar ratio) or CL:PC:FE (3:2:I) + 30% mol cholesterol, prepared in 0.4M mannito], 0.1M NaC1, 0.1mM EDTA and 5mM Hepes (pH----7.4) with 13 mM CF. Vesicles were separated from non-encapsulated CF by gel chromatography on Sephadex G-75.
probe CE Polylysine induced a marked increase in the leakage and its i~clusion in the mixture of LUV and cells increased the rate of leakage even more. The extent of leakage in the presence of cells and polylysine was greater than their additive effects, when applied separately (Fig. 1). Even preincubation, for 3 s, of the cells with polylysine resulted in an increase, though much smaller than observed without preincubation, in the leakage of LUV contents. The extent of leakage was affected also by the age of the culture (counted from the last subculture). Older cell suspensions (14 days vs. 7 days) induced greater changes in the LUV stability. 25
i
I
I
i
l
I
20~ . I.iJ Ii...
~ . _ 15
,,,g
Determination of L U V 8tabilitll
Q::
LUV were incubated with cells and various cell fractions. LUV stability was monitored using the dequenching of CF fluorescence, initially trapped in the LUV at high concentrations (Weinstein et a1,1977). The increase in the fluorescence of the liposome-entrapped CF results from the dilution of the probe leaking from the LUV to the medium, thus being indicative of changes in LUV stability. All measurements were carried out in an SLM 4800 spectrofluorometer at 25°C (493nm ex., 516nm era. with a 510nm cut-off filter).
3°
RESULTS PE-containing LUV exhibited only slight leakage of contents on standing;
