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The plasma membrane lipid composition affects fusion between cells and model membranes
Chemico-Biological Interactions 164 (2006) 167–173
The plasma membrane lipid composition affects fusion between cells and model membranes Roumen Pankov a,∗ , Tania Markovska b , Peter Antonov c , Lidia Ivanova d , Albena Momchilova b a
Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University, 8, Dragan Tzankov Str., Sofia 1164, Bulgaria b Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria c Department of Physics and Biophysics, Medical University, 1431 Sofia, Bulgaria d Cytos Biotechnology AG, Wagistrasse 25, Postfach, CH-8952 Zurich, Switzerland
Received 13 June 2006; received in revised form 20 September 2006; accepted 26 September 2006
Abstract Investigations were carried out on the effect of plasma membrane lipid modifications on the fusogenic capacity of control and ras-transformed fibroblasts. The plasma membrane lipid composition was modified by treatment of cells with exogenous phospholipases C and D, sphingomyelinase and cyclodextrin. The used enzymes hydrolyzed definite membrane lipids thus inducing specific modifications of the lipid composition while cyclodextrin treatment reduced significantly the level of cholesterol. The cells with modified membranes were used for assessment of their fusogenic capacity with model membranes with a constant lipid composition. Treatment with phospholipases C and D stimulated the fusogenic potential of both cell lines whereas the specific reduction of either sphingomyelin or cholesterol induced the opposite effect. The results showed that all modifications of the plasma membrane lipid composition affected the fusogenic capacity irrespective of the initial differences in the membrane lipid composition of the two cell lines. These results support the notion that the lipid composition plays a significant role in the processes of membrane–membrane fusion. This role could be either direct or through modulation of the activity of specific proteins which regulate membrane fusion. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Fusion; Membrane lipids; Sphingomyelin; Cholesterol
1. Introduction Membrane fusion is a fundamental biological process, occurring when two separate lipid membranes merge into a single lipid bilayer. Membrane–membrane fusion is essential for development and functioning of multicellular organisms. Yet the intimate mechanisms
∗
Corresponding author. Tel.: +359 2 8167 274. E-mail address: [email protected] (R. Pankov).
underlying the fusion between membranes are still unresolved. Most of the reports are devoted to the role of specific proteins which actively participate in the processes of interaction and fusion between biological membranes [1]. Hence, little attention is paid to the role of the lipid composition of membranes undergoing fusion. Several recent articles have reported substantial evidence concerning the effect of the lipid composition of the membranes on their capacity to fuse [2,3]. In the present work, we have studied the fusogenic potential of two cell lines with different lipid compo-
0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.09.010
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sition [4] and plasma membrane asymmetry [5]. We have analyzed the influence of specific alterations of the plasma membrane lipid composition on the cellular fusogenic capacity with liposomes with constant composition. The alterations of the plasma membrane lipids were induced by treatment of the cells with exogenous phospholipases C and D, sphingomyelinase as well as cyclodextrin. Each one of the used lipases hydrolyzes definite membrane lipids, thus inducing partial delipidation of the cellular plasma membranes. Cyclodextrin treatment reduces the cellular content of cholesterol. The results showed that all modulations of the plasma membrane lipids affected the fusogenic capacity of both cell lines. These data imply that the lipid composition of the cellular plasma membranes is also an important factor which influences at a different degree the capacity of cells to interact and fuse with membranes. 2. Materials and methods 2.1. Materials 1-Oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]dodecanoyl]-sn-glycero-3-phosphoethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl) dioleoyl phosphatidylethanolamine (N-Rh-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). Culture medium, antibiotic/antimycotic solution and serum were from GIBCO BRL Life Technologies. Phospholipases C and D, sphingomyelinase, cyclodextrin, phospholipids and other chemicals (except where otherwise stated) were from Sigma Chemicals Co. (St. Louis, MO). 2.2. Cell culture NIH 3T3 fibroblasts transfected with activated human Ha-ras gene (Der 1986) and the parental cell line were a kind gift from Dr. Channing J. Der (The University of North Carolina at Chapel Hill, Chapel Hill, NC). The cells were cultured as described elsewhere [6]. In brief, fibroblasts were grown in Dulbecco’s modified Eagles medium containing 400 g/ml G 418 (Geneticin), supplemented with 9% (v/v) fetal calf serum at 37 ◦ C in a humidified 5% CO2 atmosphere. To obtain cell suspension, the monolayers were harvested by treatment with 0.05% trypsin and resuspended by pipetting in Hepes-buffered saline (HBS), 136 mM NaCl, 2 mM KCl, 0.5 mM MgCl2, 5 mM glucose, 10 mM Hepes (pH 7.4). Plasma membranes were isolated from control and rastransformed cells as described elsewhere [7]. This pro-
cedure included loading of the post-nuclear supernatant on a discontinous sucrose gradient and centrifugation at 100,000 × g for 2.5 h. The plasma membrane fraction was collected at a density of approximately 45% (w/v), suspended in ice-cold 100 mM buffer and used immediately for lipid extraction as described below. 2.3. Incubation of control and ras-transformed fibroblasts with model membranes (liposomes) Control and ras-transformed fibroblasts (106 cells/ml) were incubated with liposomes prepared by sonication of phosphatidylcholine-1-palmitoyl-2-oleoyl (POPC)/NBD-PE/N-Rh-PE (98:1:1) (0.5 mg/ml) in the presence of 10 mM calcium as fusion promoter at 37 ◦ C in a total volume of 1 ml [8,9]. Incubations were terminated by washing the cells with PBS containing 10 mM EDTA. 2.4. Fluorescence dequenching assay Cell-vesicle fusion was assessed by measuring the dequenching of fluorescence of the NBD-lipid analogues at 450 nm (excitation) and 530 nm (emission) wavelength on a Jobin Yvon JY 3D fluorescence spectrophotometer. The fluorescence dequenching was measured after mixing the liposomes containing fluorescent probes with the cells. The percent of fluorescence dequenching was calculated using the following equation: %fluorescence dequenching =
Fi − Fo × 100 Ft − F o
where Fi is the fluorescence intensity after the incubation, Fo the initial fluorescence before incubation and Ft is the fluorescence intensity measured after treatment with 1% Triton X-100. 2.5. Treatment of cells with exogenous phospholipases C and D and sphingomyelinase [10] Incubations with the two phospholipases were performed for 1 h at 37 ◦ C. The reaction was started by addition of 5 U ml−1 of the corresponding enzyme to the incubation medium. Degradation of sphingomyelin was performed using 1.0 U ml−1 sphingomyelinase added to the incubation medium for 1 h at 37 ◦ C. Cell permeability to trypan blue was not altered significantly as a result of enzyme treatment of cells. The cells were washed twice with ice-cold PBS and collected for isolation of the plasma membranes as described above. The isolated plasma membrane fractions were used for lipid analysis.
R. Pankov et al. / Chemico-Biological Interactions 164 (2006) 167–173
2.6. Lipid extraction and phospholipid analysis Extraction of the membrane lipids was performed with chloroform/methanol according to the method of Bligh and Dyer [11]. The organic phase obtained after extraction was concentrated and analyzed by thin layer chromatography. The individual phospholipid fractions were separated on silica gel G 60 plates in a solvent system containing chloroform/methanol/2propanol/triethylamine/0.25% KCl (30:9:25:18:6, v/v) [12]. The location of the separate fractions was visualized by fluorescence or iodine staining. The spots were scraped and quantified by determination of the inorganic phosphorus [13].
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Table 1 Lipid composition of plasma membranes from control and rastransformed fibroblasts (mol%) Lipids
Control
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
12.24 43.20 6.35 7.62 1.45 16.12 13.02
± ± ± ± ± ± ±
ras-transformed 1.12 2.76 0.87 0.63 0.15 1.18 0.78
15.33 35.17 5.21 4.15 3.07 21.31 15.76
± ± ± ± ± ± ±
1.02a 2.95a 0.75 0.48b 0.26a 1.12a 0.83b
Results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing control to ras-transformed cells. a P < 0.01. b P < 0.05.
2.7. Incubation of cells with cyclodextrins [14] The cells (2 × 106 ) were rinsed twice with PBS and incubated with 10 mM methyl--cyclodextrin for 30 min at 37 ◦ C under constant agitation. After the incubation the cells were rinsed three times with PBS and used for plasma membrane isolation. 2.8. Cholesterol determination The cholesterol content of the membranes was assayed by gas chromatography–mass spectrometry of trimethylsilyl ether. The cholesterol derivative was separated by gas chromatography on a medium polarity RTX-65 capillary column (0.32 mm internal diameter, length 30 m and film thickness 0.25 m). Calibration was achieved by a weighted standard of cholesterol. 2.9. Other procedures Differences between the means were analyzed by Student’s t-test.
to analyze the influence of the lipid composition on the membrane fusogenic capacity we modified the lipid composition of the plasma membranes of control and ras-cells by treatment with exogenous phospholipases C and D as well as with sphingomyelinase. Treatment of cells with exogenous phospholipase C (PLC) partially delipidated the cellular plasma membranes by reducing the level of definite membrane lipids. The lipid/protein ratio was reduced as a result of phospholipase C treatment from 313 to 201 nmol lipid/mg protein in plasma membranes from control cells and from 381 to 280 nmol lipid/mg protein in membranes from ras-cells. The percentage participation of phosphatidylcholine (PC) in the total lipids was reduced most significantly followed by phosphatidylethanolamine (PE) in the plasma membranes of PLC-treated control and ras-cells (compare Tables 1 and 2). The percentage of the lipids which were not hydrolyzed by PLC was elevated due to the decrease of two major membrane fractions such as
3. Results The cellular fusogenic capacity was investigated using control and ras-transformed fibroblasts as well as model membranes containing two fluorophores—NBDPE and N-Rh-PE. The lipid composition (expressed as percent of the total lipids) of the plasma membranes isolated from control and ras-transformed cells is shown in Table 1. The fusogenic potential of the cells was assessed by measuring the fluorescence dequenching [2,8]. The results showed that the two cell lines exhibited a different ability to fuse with the model membranes (Fig. 1). Apparently, the ras-transformed cells demonstrated a higher capacity to fuse with the fluorescent liposomes compared to control cells (Fig. 1). In order
Fig. 1. Fusogenic capacity of control (white bars) and ras-transformed fibroblasts, treated with phospholipase C (PLC), sphingomyelinase (SMase), phospholipase D (PLD) and cyclodextrin (CD). The fusogenic capacity of the cells was assessed by their ability to fuse with model membranes containing quenched fluorescent probes and was expressed as % fluorescence dequenching which was calculated as explained under Materials and Methods.
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Table 2 Lipid composition of plasma membranes from control and rastransformed fibroblasts treated with phospholipase C (mol%)
Table 3 Lipid composition of plasma membranes from control and rastransformed cells treated with sphingomyelinase (mol%)
Lipids
Control
Lipids
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
20.37 ± 29.20 ± 2.12a 7.26 ± 0.72 8.32 ± 0.81 2.85 ± 0.14a 12.05 ± 1.17 19.96 ± 1.10a
ras-transformed 1.66a
20.96 ± 23.57 ± 1.53a 6.61 ± 0.54 6.87 ± 0.77 5.21 ± 0.29 16.35 ± 1.05 20.43 ± 1.21a 1.38a
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
Control 3.85 46.65 6.63 8.95 1.54 17.97 14.42
± ± ± ± ± ± ±
ras-transformed 0.23a 3.37 0.51 0.76 0.22 1.53 1.27
7.16 40.79 4.43 4.58 3.32 21.77 17.95
± ± ± ± ± ± ±
0.44a 2.16 0.29 0.31 0.25 0.17 1.03
Results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment with phospholipase C, shown in this table. a P < 0.01.
The results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment with sphingomyelinase, shown in this table. a P < 0.001.
PC and PE (Tables 1 and 2). The cells with modified lipid composition were used for estimation of their fusion capacity. The results showed that PLC treatment of cells increased the efficiency of both cell lines to undergo fusion with model membranes (Fig. 1). Significantly stimulated fusion was observed between liposomes and ras-transformed cells treated with PLC (Fig. 1). Since the lipid alterations induced by PLC treatment enhanced the fusogenic capacity of the cells, we further studied the eventual effect of some of the lipid fractions, which were augmented as a result of PLC treatment, on the membrane fusogenic capacity. As the relative content of SM was augmented most markedly in PLC treated membranes (from 12.24 to 20.37% in control cells and from 15.33 to 20.96% in ras-cells), we analyzed the effect of SM manipulation on fusion effectiveness by treatment of cells with exogenous sphingomyelinase. In sphingomyelinase-treated membranes SM was the only reduced lipid—from 38.35 to 10.87 nmol/mg protein in control cells and from 57.45 to 22.94 nmol/mg protein in ras cells. This treatment reduced the level of sphingomyelin in the plasma membranes of both control and ras-transformed fibroblasts (compare Tables 1 and 3). The cells thus obtained were used for measurement of their fusogenic potential (Fig. 1). The results showed that sphingomyelinase treatment reduced the capacity of the modified cells to fuse with the fluorescent model membranes. Another series of cells with modified plasma membrane lipids was obtained by treatment with phospholipase D (PLD). This phospholipase hydrolyses the headgroups of definite membrane lipids turning them into phosphatidic acid (PA). The lipid/protein ratio in membranes from phospholipase D-treated cells was reduced from 313 to 251 nmol lipid/mg protein for control cells
and from 381 to 334 nmol lipid/mg protein in ras cells. The results showed that most substantially was decreased the percentage of PC in plasma membranes from PLDtreated cells (Tables 1 and 4). This treatment increased most significantly the fusogenic capacity of both control and ras-cells (Fig. 1). Further studies were focused on the effect of cholesterol (CH) depletion on the cellular fusogenic capacity because fusion is often related to membrane fluidity and cholesterol affects the membrane structural organization. For this purpose the cells were incubated with cyclodextrin, which reduced the cholesterol content from 13.02 to 3.85 mol% in control cells and from 15.76 to 4.12 mol% in ras-transformed cells. Cholesterol was the only lipid that was reduced in membranes from cyclodextrin-treated cells (Tables 1 and 5). The results showed that cholesterol depletion reduced the fusogenic capacity of both treated cell lines (Fig. 1). Table 4 Lipid composition of plasma membranes from control and rastransformed cells treated with phospholipase D (mol%) Lipids
Control
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
15.23 25.34 6.93 7.72 13.15 16.31 15.32
± ± ± ± ± ± ±
ras-transformed 1.22 2.02a 0.54 0.69 1.27a 1.16 1.28
16.04 19.35 5.91 6.71 15.27 20.43 16.29
± ± ± ± ± ± ±
0.97 1.35a 0.44 0.51 1.26a 1.72 1.63
The results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment of cells with phospholipase D, shown in this table. a P < 0.001.
R. Pankov et al. / Chemico-Biological Interactions 164 (2006) 167–173 Table 5 Lipid composition of plasma membranes from control and rastransformed cells treated with cyclodextrin (mol%) Lipids
Control
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
14.27 46.79 6.95 7.93 1.76 18.45 3.85
± ± ± ± ± ± ±
ras-transformed 1.28 3.36 0.54 0.67 0.15 1.42 0.28a
16.86 41.05 5.87 5.35 3.49 23.26 4.12
± ± ± ± ± ± ±
1.22 3.15 0.43 0.49 0.26 1.64 0.36a
The results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment of cells with cyclodextrin, shown in this table. a P < 0.001.
4. Discussion Membrane fusion is a ubiquitous process involved in different cellular events like secretion, endocytosis, fertilization, etc. Major attention in the investigation of fusion is focused on the role of specific proteins which regulate the processes of interaction and fusion between membranes [15,16]. There are quite a few studies devoted to the influence of the lipid composition of membranes on their fusogenic capacity [2,3]. In the present paper we report some intriguing observations on the effect of definite lipid modifications on the fusogenic capacity of membranes. To monitor the influence of the membrane lipid composition on the cellular fusogenic potential, we chose two cell lines with different lipid metabolism and composition—nontransformed and ras-transformed fibroblasts [6]. These two cell lines served as an experimental model for assessment of the effect of certain lipid modifications on the capacity of the cells to fuse with model membranes with a constant lipid composition. Control and ras-transformed cells had a different fusogenic potential before any modifications of their membrane lipid composition have been induced (Fig. 1). In our previous work, we reported scramblase activation which could increase the level of aminophospholipids in the external plasma membrane monolayer of ras-cells [5]. Since PE is an aminophospholipid that induces formation of hexagonal structures in membranes [17] its accumulation in the outer membrane monolayer could create sites with reduced stability. The presence of such sites could be responsible for the higher fusogeneity of ras-cells. To analyze the effect of PE on cell-vesicle fusion, we treated the two cell lines with PLC which was expected to reduce the level of PE in the membranes. PLC selectively hydrolyzes definite
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membrane lipids, including PE, turning them into diacylglycerols. However, the results showed that the level of PE could hardly be responsible for the higher fusogenic capacity of ras-cells because the decrease of PE due to PLC-treatment was accompanied by elevation of the cellular fusogenic effectiveness (Fig. 1). Further, we focused our attention on the role of other lipid fractions which were augmented as a result of PLC treatment of cells. As mentioned above, PLC induced a reduction of the major membrane lipids—PC and PE, as well as an increase of the relative content of these membrane lipids, which were not hydrolyzed by PLC (Table 2). These lipids were mainly SM and CH, followed by PA, which, although a minor membrane component, was increased substantially in membranes of PLC-treated cells. To analyze whether alterations in the SM level could affect the fusogenic capacity of the cellular membranes, we treated the two cell lines with exogenous sphingomyelinase. Such treatment decreases specifically the quantity of SM and induces accumulation of ceramide in the plasma membrane. The content of the rest of the membrane phospholipids and cholesterol, calculated per mg membrane protein, remained unchanged for both sphingomyelinase treated cell lines (data not shown). Interestingly, cells with a lower level of SM exhibited a lower ability to fuse with liposomes, compared to sphingomyelinase-untreated cells (Fig. 1). These results implied that the increase of the relative content of SM could be one possible reason for the elevated fusogenic capacity of PLC treated cells. However, Deeba et al. [2] reported that changes in the SM level do not affect the process of fusion between liposomes and erythrocytes. It is also possible that the elevated ceramide level in sphingomyelinase-treated cells perturbs the fusion process due to its membrane-rigidifying effect [18]. Further, we analyzed whether alterations in the level of other lipids could underlie the observed changes in the membranes fusugeneity. There is evidence that some acidic phospholipids stimulate fusion between model membranes [19]. However, the observed alterations of the acidic phospholipids phosphatidylinositol and PS were quite insignificant (1–2%) and could hardly induce any detectable effect on the fusion process. The only phospholipid which was elevated significantly in PLC–treated membranes, besides SM, was PA which is a minor membrane fraction with a significant role in cell signaling [20]. So further experiments were devoted to modulation of the PA level by treatment with exogenous PLD. This treatment induced a dramatic increase of PA, as well as a decrease only of PC in plasma membranes of both cell lines (Tables 1 and 4). The fusogenic capacity of these cells was elevated most substantially compared
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to all other modified cells (Fig. 1). It is possible that PA affects the membrane fusogenic capacity due to its specific molecular shape [21], which perturbs the bilayer structure of the membrane lipids, thus inducing local disorders. It is also possible that the reduction of PC which is a “fusion-incompetent” lipid [2] could contribute to the elevation of the fusogenic capacity of cells treated with PLC and especially with PLD. As mentioned above PLD treatment induced a significant degradation mainly of PC, which was accompanied by PA accumulation. In addition, membrane-perturbing effect could also be ascribed to diacylglycerols, which are products of PLC activity and are known to disturb the structural organization of the membrane bilayer [22,23]. Finally, we modified the membrane content of cholesterol, which was one of the increased lipids in PLCtreated cells (from 13.02 to 19.96 mol% in control cells and from 15.76 to 20.43 mol% in ras-cells). Another reason for focusing our attention on cholesterol was that this lipid, besides SM, is one of the two basic components of membrane raft domains which are presumed to participate in the fusion between cells [24]. Our observation that cholesterol reduction was accompanied by lowering of the fusogenic capacity of both cell lines confirmed the presumption that this raft component is likely to play a certain role in cell–cell fusion [3]. Thus, it seems likely that SM and CH, which are the two basic lipid components of membrane raft domains, might play a definite role in the processes of cell–cell interaction and fusion possibly by affecting the “fusion competent” proteins, which are localized predominantly in the raft domains [25]. One can speculate that the higher fusogenic potential of ras-cells could be related to the higher level of SM and cholesterol in the membranes of these cells, which is a prerequisite for raft formation [26]. It is also possible that the higher capacity of ras-cells to undergo fusion is a result of the stimulated processes of endocytosis reported for these cells [27]. Thus the higher ability of ras-cells to fuse with other membranes could as well be a mechanism by which these cells compensate the loss of membrane lipid components due to stimulated endocytosis. In conclusion, our studies showed that modifications of the plasma membrane lipid composition can affect at a different degree the capacity of the cells to fuse with model membranes. Possibly, the varying of the membrane lipid composition might turn out to be a factor which modulates membrane fusogenic capacity and is also related to membrane and cellular pathology. Recent evidence suggests that rafts can serve as docking sites of a broad range of pathogens including viruses, bacteria, etc. [28]. For example, HIV-1 is an enveloped virus that fuses with the plasma membrane of the host cells and
this process can be modulated by the lipid components of raft domains [25,28]. Thus, besides the leading role of the specific proteins, which regulate fusion, the alterations of the membrane lipid composition also seem to affect the cellular fusogenic potential. It is possible that the lipid composition of the membranes plays an indirect role in the cellular fusogenic capacity by modulating the active conformation of the membrane-bound “fusioncompetent” proteins. At present, more studies are needed for elucidation of the fine mechanisms underlying the processes of cell–cell interactions and fusion. Acknowledgments This work was supported by the Bulgarian National Fund for Scientific Research (Grant 1404/04 and partially by Grant BY-Б-1/05). References [1] R. Jahn, T. Lang, T. Sudhof, Membrane fusion, Cell 112 (2003) 519–533. [2] F. Deeba, H.N. Tahseen, K. Sharma Sharad, N. Ahmad, S. Akhtar, M. Saleemuddin, O. Mohammad, Phospholipid diversity: correlation with membrane–membrane fusion events, Biochim. Biophys. Acta 1669 (2005) 170–181. [3] A. Baily, M. Zhukovsky, A. Gliozzi, L. Chernomordik, Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes, Arch. Biochem. Biophys. 439 (2005) 211–221. [4] A. Momchilova, T. Markovska, R. Pankov, Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts, Biochimie 80 (1998) 1053–1062. [5] A. Momchilova, L. Ivanova, T. Markovska, R. Pankov, Stimulated nonspecific transport of phospholipids results in elevated external appearance of phosphatidylserine in ras-transformed fibroblasts, Arch. Biochem. Biophys. 381 (2000) 295–301. [6] A. Momchilova, T. Markovska, Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in rastransformed fibroblasts, Int. J. Biochem. Cell Biol. 31 (1999) 311–318. [7] R. Pankov, T. Markovska, P. Antonov, L. Ivanova, A. Momchilova, Cholesterol distribution in plasma membranes of 1 integrin expressing and 1 integrin deficient fibroblasts, Arch. Biochem. Biophys. 442 (2005) 160–168. [8] D. Struck, D. Hoekstra, R. Pagano, Use of resonance energy transfer to monitor membrane fusion, Biochemistry 20 (1981) 4093–4099. [9] H. Dao, C. McIntyre, R. Sleight, Large-scale preparation of asymmetrically fluorescent lipid vesicles, Anal. Biochem. 196 (1991) 46–53. [10] A. Momchilova, D. Petkova, K. Koumanov, Phospholipid composition modifications influence phospholipase A2 activity in rat liver plasma membranes, Int. J. Biochem. 18 (1986) 945–952. [11] E. Bligh, W. Dyer, A rapid method of total lipid extraction and purification, Can. J. Physiol. 37 (1959) 911–917. [12] J. Touchstone, J. Chen, K. Beaver, Improved separation of phospholipids by thin-layer chromatography, Lipids 15 (1980) 61–62.
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The plasma membrane lipid composition affects fusion between cells and model membranes Roumen Pankov a,∗ , Tania Markovska b , Peter Antonov c , Lidia Ivanova d , Albena Momchilova b a
Department of Cytology, Histology and Embryology, Biological Faculty, Sofia University, 8, Dragan Tzankov Str., Sofia 1164, Bulgaria b Institute of Biophysics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria c Department of Physics and Biophysics, Medical University, 1431 Sofia, Bulgaria d Cytos Biotechnology AG, Wagistrasse 25, Postfach, CH-8952 Zurich, Switzerland
Received 13 June 2006; received in revised form 20 September 2006; accepted 26 September 2006
Abstract Investigations were carried out on the effect of plasma membrane lipid modifications on the fusogenic capacity of control and ras-transformed fibroblasts. The plasma membrane lipid composition was modified by treatment of cells with exogenous phospholipases C and D, sphingomyelinase and cyclodextrin. The used enzymes hydrolyzed definite membrane lipids thus inducing specific modifications of the lipid composition while cyclodextrin treatment reduced significantly the level of cholesterol. The cells with modified membranes were used for assessment of their fusogenic capacity with model membranes with a constant lipid composition. Treatment with phospholipases C and D stimulated the fusogenic potential of both cell lines whereas the specific reduction of either sphingomyelin or cholesterol induced the opposite effect. The results showed that all modifications of the plasma membrane lipid composition affected the fusogenic capacity irrespective of the initial differences in the membrane lipid composition of the two cell lines. These results support the notion that the lipid composition plays a significant role in the processes of membrane–membrane fusion. This role could be either direct or through modulation of the activity of specific proteins which regulate membrane fusion. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Fusion; Membrane lipids; Sphingomyelin; Cholesterol
1. Introduction Membrane fusion is a fundamental biological process, occurring when two separate lipid membranes merge into a single lipid bilayer. Membrane–membrane fusion is essential for development and functioning of multicellular organisms. Yet the intimate mechanisms
∗
Corresponding author. Tel.: +359 2 8167 274. E-mail address: [email protected] (R. Pankov).
underlying the fusion between membranes are still unresolved. Most of the reports are devoted to the role of specific proteins which actively participate in the processes of interaction and fusion between biological membranes [1]. Hence, little attention is paid to the role of the lipid composition of membranes undergoing fusion. Several recent articles have reported substantial evidence concerning the effect of the lipid composition of the membranes on their capacity to fuse [2,3]. In the present work, we have studied the fusogenic potential of two cell lines with different lipid compo-
0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.09.010
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sition [4] and plasma membrane asymmetry [5]. We have analyzed the influence of specific alterations of the plasma membrane lipid composition on the cellular fusogenic capacity with liposomes with constant composition. The alterations of the plasma membrane lipids were induced by treatment of the cells with exogenous phospholipases C and D, sphingomyelinase as well as cyclodextrin. Each one of the used lipases hydrolyzes definite membrane lipids, thus inducing partial delipidation of the cellular plasma membranes. Cyclodextrin treatment reduces the cellular content of cholesterol. The results showed that all modulations of the plasma membrane lipids affected the fusogenic capacity of both cell lines. These data imply that the lipid composition of the cellular plasma membranes is also an important factor which influences at a different degree the capacity of cells to interact and fuse with membranes. 2. Materials and methods 2.1. Materials 1-Oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]dodecanoyl]-sn-glycero-3-phosphoethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl) dioleoyl phosphatidylethanolamine (N-Rh-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). Culture medium, antibiotic/antimycotic solution and serum were from GIBCO BRL Life Technologies. Phospholipases C and D, sphingomyelinase, cyclodextrin, phospholipids and other chemicals (except where otherwise stated) were from Sigma Chemicals Co. (St. Louis, MO). 2.2. Cell culture NIH 3T3 fibroblasts transfected with activated human Ha-ras gene (Der 1986) and the parental cell line were a kind gift from Dr. Channing J. Der (The University of North Carolina at Chapel Hill, Chapel Hill, NC). The cells were cultured as described elsewhere [6]. In brief, fibroblasts were grown in Dulbecco’s modified Eagles medium containing 400 g/ml G 418 (Geneticin), supplemented with 9% (v/v) fetal calf serum at 37 ◦ C in a humidified 5% CO2 atmosphere. To obtain cell suspension, the monolayers were harvested by treatment with 0.05% trypsin and resuspended by pipetting in Hepes-buffered saline (HBS), 136 mM NaCl, 2 mM KCl, 0.5 mM MgCl2, 5 mM glucose, 10 mM Hepes (pH 7.4). Plasma membranes were isolated from control and rastransformed cells as described elsewhere [7]. This pro-
cedure included loading of the post-nuclear supernatant on a discontinous sucrose gradient and centrifugation at 100,000 × g for 2.5 h. The plasma membrane fraction was collected at a density of approximately 45% (w/v), suspended in ice-cold 100 mM buffer and used immediately for lipid extraction as described below. 2.3. Incubation of control and ras-transformed fibroblasts with model membranes (liposomes) Control and ras-transformed fibroblasts (106 cells/ml) were incubated with liposomes prepared by sonication of phosphatidylcholine-1-palmitoyl-2-oleoyl (POPC)/NBD-PE/N-Rh-PE (98:1:1) (0.5 mg/ml) in the presence of 10 mM calcium as fusion promoter at 37 ◦ C in a total volume of 1 ml [8,9]. Incubations were terminated by washing the cells with PBS containing 10 mM EDTA. 2.4. Fluorescence dequenching assay Cell-vesicle fusion was assessed by measuring the dequenching of fluorescence of the NBD-lipid analogues at 450 nm (excitation) and 530 nm (emission) wavelength on a Jobin Yvon JY 3D fluorescence spectrophotometer. The fluorescence dequenching was measured after mixing the liposomes containing fluorescent probes with the cells. The percent of fluorescence dequenching was calculated using the following equation: %fluorescence dequenching =
Fi − Fo × 100 Ft − F o
where Fi is the fluorescence intensity after the incubation, Fo the initial fluorescence before incubation and Ft is the fluorescence intensity measured after treatment with 1% Triton X-100. 2.5. Treatment of cells with exogenous phospholipases C and D and sphingomyelinase [10] Incubations with the two phospholipases were performed for 1 h at 37 ◦ C. The reaction was started by addition of 5 U ml−1 of the corresponding enzyme to the incubation medium. Degradation of sphingomyelin was performed using 1.0 U ml−1 sphingomyelinase added to the incubation medium for 1 h at 37 ◦ C. Cell permeability to trypan blue was not altered significantly as a result of enzyme treatment of cells. The cells were washed twice with ice-cold PBS and collected for isolation of the plasma membranes as described above. The isolated plasma membrane fractions were used for lipid analysis.
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2.6. Lipid extraction and phospholipid analysis Extraction of the membrane lipids was performed with chloroform/methanol according to the method of Bligh and Dyer [11]. The organic phase obtained after extraction was concentrated and analyzed by thin layer chromatography. The individual phospholipid fractions were separated on silica gel G 60 plates in a solvent system containing chloroform/methanol/2propanol/triethylamine/0.25% KCl (30:9:25:18:6, v/v) [12]. The location of the separate fractions was visualized by fluorescence or iodine staining. The spots were scraped and quantified by determination of the inorganic phosphorus [13].
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Table 1 Lipid composition of plasma membranes from control and rastransformed fibroblasts (mol%) Lipids
Control
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
12.24 43.20 6.35 7.62 1.45 16.12 13.02
± ± ± ± ± ± ±
ras-transformed 1.12 2.76 0.87 0.63 0.15 1.18 0.78
15.33 35.17 5.21 4.15 3.07 21.31 15.76
± ± ± ± ± ± ±
1.02a 2.95a 0.75 0.48b 0.26a 1.12a 0.83b
Results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing control to ras-transformed cells. a P < 0.01. b P < 0.05.
2.7. Incubation of cells with cyclodextrins [14] The cells (2 × 106 ) were rinsed twice with PBS and incubated with 10 mM methyl--cyclodextrin for 30 min at 37 ◦ C under constant agitation. After the incubation the cells were rinsed three times with PBS and used for plasma membrane isolation. 2.8. Cholesterol determination The cholesterol content of the membranes was assayed by gas chromatography–mass spectrometry of trimethylsilyl ether. The cholesterol derivative was separated by gas chromatography on a medium polarity RTX-65 capillary column (0.32 mm internal diameter, length 30 m and film thickness 0.25 m). Calibration was achieved by a weighted standard of cholesterol. 2.9. Other procedures Differences between the means were analyzed by Student’s t-test.
to analyze the influence of the lipid composition on the membrane fusogenic capacity we modified the lipid composition of the plasma membranes of control and ras-cells by treatment with exogenous phospholipases C and D as well as with sphingomyelinase. Treatment of cells with exogenous phospholipase C (PLC) partially delipidated the cellular plasma membranes by reducing the level of definite membrane lipids. The lipid/protein ratio was reduced as a result of phospholipase C treatment from 313 to 201 nmol lipid/mg protein in plasma membranes from control cells and from 381 to 280 nmol lipid/mg protein in membranes from ras-cells. The percentage participation of phosphatidylcholine (PC) in the total lipids was reduced most significantly followed by phosphatidylethanolamine (PE) in the plasma membranes of PLC-treated control and ras-cells (compare Tables 1 and 2). The percentage of the lipids which were not hydrolyzed by PLC was elevated due to the decrease of two major membrane fractions such as
3. Results The cellular fusogenic capacity was investigated using control and ras-transformed fibroblasts as well as model membranes containing two fluorophores—NBDPE and N-Rh-PE. The lipid composition (expressed as percent of the total lipids) of the plasma membranes isolated from control and ras-transformed cells is shown in Table 1. The fusogenic potential of the cells was assessed by measuring the fluorescence dequenching [2,8]. The results showed that the two cell lines exhibited a different ability to fuse with the model membranes (Fig. 1). Apparently, the ras-transformed cells demonstrated a higher capacity to fuse with the fluorescent liposomes compared to control cells (Fig. 1). In order
Fig. 1. Fusogenic capacity of control (white bars) and ras-transformed fibroblasts, treated with phospholipase C (PLC), sphingomyelinase (SMase), phospholipase D (PLD) and cyclodextrin (CD). The fusogenic capacity of the cells was assessed by their ability to fuse with model membranes containing quenched fluorescent probes and was expressed as % fluorescence dequenching which was calculated as explained under Materials and Methods.
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Table 2 Lipid composition of plasma membranes from control and rastransformed fibroblasts treated with phospholipase C (mol%)
Table 3 Lipid composition of plasma membranes from control and rastransformed cells treated with sphingomyelinase (mol%)
Lipids
Control
Lipids
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
20.37 ± 29.20 ± 2.12a 7.26 ± 0.72 8.32 ± 0.81 2.85 ± 0.14a 12.05 ± 1.17 19.96 ± 1.10a
ras-transformed 1.66a
20.96 ± 23.57 ± 1.53a 6.61 ± 0.54 6.87 ± 0.77 5.21 ± 0.29 16.35 ± 1.05 20.43 ± 1.21a 1.38a
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
Control 3.85 46.65 6.63 8.95 1.54 17.97 14.42
± ± ± ± ± ± ±
ras-transformed 0.23a 3.37 0.51 0.76 0.22 1.53 1.27
7.16 40.79 4.43 4.58 3.32 21.77 17.95
± ± ± ± ± ± ±
0.44a 2.16 0.29 0.31 0.25 0.17 1.03
Results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment with phospholipase C, shown in this table. a P < 0.01.
The results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment with sphingomyelinase, shown in this table. a P < 0.001.
PC and PE (Tables 1 and 2). The cells with modified lipid composition were used for estimation of their fusion capacity. The results showed that PLC treatment of cells increased the efficiency of both cell lines to undergo fusion with model membranes (Fig. 1). Significantly stimulated fusion was observed between liposomes and ras-transformed cells treated with PLC (Fig. 1). Since the lipid alterations induced by PLC treatment enhanced the fusogenic capacity of the cells, we further studied the eventual effect of some of the lipid fractions, which were augmented as a result of PLC treatment, on the membrane fusogenic capacity. As the relative content of SM was augmented most markedly in PLC treated membranes (from 12.24 to 20.37% in control cells and from 15.33 to 20.96% in ras-cells), we analyzed the effect of SM manipulation on fusion effectiveness by treatment of cells with exogenous sphingomyelinase. In sphingomyelinase-treated membranes SM was the only reduced lipid—from 38.35 to 10.87 nmol/mg protein in control cells and from 57.45 to 22.94 nmol/mg protein in ras cells. This treatment reduced the level of sphingomyelin in the plasma membranes of both control and ras-transformed fibroblasts (compare Tables 1 and 3). The cells thus obtained were used for measurement of their fusogenic potential (Fig. 1). The results showed that sphingomyelinase treatment reduced the capacity of the modified cells to fuse with the fluorescent model membranes. Another series of cells with modified plasma membrane lipids was obtained by treatment with phospholipase D (PLD). This phospholipase hydrolyses the headgroups of definite membrane lipids turning them into phosphatidic acid (PA). The lipid/protein ratio in membranes from phospholipase D-treated cells was reduced from 313 to 251 nmol lipid/mg protein for control cells
and from 381 to 334 nmol lipid/mg protein in ras cells. The results showed that most substantially was decreased the percentage of PC in plasma membranes from PLDtreated cells (Tables 1 and 4). This treatment increased most significantly the fusogenic capacity of both control and ras-cells (Fig. 1). Further studies were focused on the effect of cholesterol (CH) depletion on the cellular fusogenic capacity because fusion is often related to membrane fluidity and cholesterol affects the membrane structural organization. For this purpose the cells were incubated with cyclodextrin, which reduced the cholesterol content from 13.02 to 3.85 mol% in control cells and from 15.76 to 4.12 mol% in ras-transformed cells. Cholesterol was the only lipid that was reduced in membranes from cyclodextrin-treated cells (Tables 1 and 5). The results showed that cholesterol depletion reduced the fusogenic capacity of both treated cell lines (Fig. 1). Table 4 Lipid composition of plasma membranes from control and rastransformed cells treated with phospholipase D (mol%) Lipids
Control
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
15.23 25.34 6.93 7.72 13.15 16.31 15.32
± ± ± ± ± ± ±
ras-transformed 1.22 2.02a 0.54 0.69 1.27a 1.16 1.28
16.04 19.35 5.91 6.71 15.27 20.43 16.29
± ± ± ± ± ± ±
0.97 1.35a 0.44 0.51 1.26a 1.72 1.63
The results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment of cells with phospholipase D, shown in this table. a P < 0.001.
R. Pankov et al. / Chemico-Biological Interactions 164 (2006) 167–173 Table 5 Lipid composition of plasma membranes from control and rastransformed cells treated with cyclodextrin (mol%) Lipids
Control
Sphingomyelin Phosphatidylcholine Phosphatidylserine Phosphatidylinositol Phosphatidic acid Phosphatidylethanolamine Cholesterol
14.27 46.79 6.95 7.93 1.76 18.45 3.85
± ± ± ± ± ± ±
ras-transformed 1.28 3.36 0.54 0.67 0.15 1.42 0.28a
16.86 41.05 5.87 5.35 3.49 23.26 4.12
± ± ± ± ± ± ±
1.22 3.15 0.43 0.49 0.26 1.64 0.36a
The results are means ± S.D. of three separate experiments performed in triplicates. Statistical significance was assessed by comparing the mean value of each lipid fraction shown in Table 1 (which was used as untreated control) with the corresponding mean value obtained after treatment of cells with cyclodextrin, shown in this table. a P < 0.001.
4. Discussion Membrane fusion is a ubiquitous process involved in different cellular events like secretion, endocytosis, fertilization, etc. Major attention in the investigation of fusion is focused on the role of specific proteins which regulate the processes of interaction and fusion between membranes [15,16]. There are quite a few studies devoted to the influence of the lipid composition of membranes on their fusogenic capacity [2,3]. In the present paper we report some intriguing observations on the effect of definite lipid modifications on the fusogenic capacity of membranes. To monitor the influence of the membrane lipid composition on the cellular fusogenic potential, we chose two cell lines with different lipid metabolism and composition—nontransformed and ras-transformed fibroblasts [6]. These two cell lines served as an experimental model for assessment of the effect of certain lipid modifications on the capacity of the cells to fuse with model membranes with a constant lipid composition. Control and ras-transformed cells had a different fusogenic potential before any modifications of their membrane lipid composition have been induced (Fig. 1). In our previous work, we reported scramblase activation which could increase the level of aminophospholipids in the external plasma membrane monolayer of ras-cells [5]. Since PE is an aminophospholipid that induces formation of hexagonal structures in membranes [17] its accumulation in the outer membrane monolayer could create sites with reduced stability. The presence of such sites could be responsible for the higher fusogeneity of ras-cells. To analyze the effect of PE on cell-vesicle fusion, we treated the two cell lines with PLC which was expected to reduce the level of PE in the membranes. PLC selectively hydrolyzes definite
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membrane lipids, including PE, turning them into diacylglycerols. However, the results showed that the level of PE could hardly be responsible for the higher fusogenic capacity of ras-cells because the decrease of PE due to PLC-treatment was accompanied by elevation of the cellular fusogenic effectiveness (Fig. 1). Further, we focused our attention on the role of other lipid fractions which were augmented as a result of PLC treatment of cells. As mentioned above, PLC induced a reduction of the major membrane lipids—PC and PE, as well as an increase of the relative content of these membrane lipids, which were not hydrolyzed by PLC (Table 2). These lipids were mainly SM and CH, followed by PA, which, although a minor membrane component, was increased substantially in membranes of PLC-treated cells. To analyze whether alterations in the SM level could affect the fusogenic capacity of the cellular membranes, we treated the two cell lines with exogenous sphingomyelinase. Such treatment decreases specifically the quantity of SM and induces accumulation of ceramide in the plasma membrane. The content of the rest of the membrane phospholipids and cholesterol, calculated per mg membrane protein, remained unchanged for both sphingomyelinase treated cell lines (data not shown). Interestingly, cells with a lower level of SM exhibited a lower ability to fuse with liposomes, compared to sphingomyelinase-untreated cells (Fig. 1). These results implied that the increase of the relative content of SM could be one possible reason for the elevated fusogenic capacity of PLC treated cells. However, Deeba et al. [2] reported that changes in the SM level do not affect the process of fusion between liposomes and erythrocytes. It is also possible that the elevated ceramide level in sphingomyelinase-treated cells perturbs the fusion process due to its membrane-rigidifying effect [18]. Further, we analyzed whether alterations in the level of other lipids could underlie the observed changes in the membranes fusugeneity. There is evidence that some acidic phospholipids stimulate fusion between model membranes [19]. However, the observed alterations of the acidic phospholipids phosphatidylinositol and PS were quite insignificant (1–2%) and could hardly induce any detectable effect on the fusion process. The only phospholipid which was elevated significantly in PLC–treated membranes, besides SM, was PA which is a minor membrane fraction with a significant role in cell signaling [20]. So further experiments were devoted to modulation of the PA level by treatment with exogenous PLD. This treatment induced a dramatic increase of PA, as well as a decrease only of PC in plasma membranes of both cell lines (Tables 1 and 4). The fusogenic capacity of these cells was elevated most substantially compared
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to all other modified cells (Fig. 1). It is possible that PA affects the membrane fusogenic capacity due to its specific molecular shape [21], which perturbs the bilayer structure of the membrane lipids, thus inducing local disorders. It is also possible that the reduction of PC which is a “fusion-incompetent” lipid [2] could contribute to the elevation of the fusogenic capacity of cells treated with PLC and especially with PLD. As mentioned above PLD treatment induced a significant degradation mainly of PC, which was accompanied by PA accumulation. In addition, membrane-perturbing effect could also be ascribed to diacylglycerols, which are products of PLC activity and are known to disturb the structural organization of the membrane bilayer [22,23]. Finally, we modified the membrane content of cholesterol, which was one of the increased lipids in PLCtreated cells (from 13.02 to 19.96 mol% in control cells and from 15.76 to 20.43 mol% in ras-cells). Another reason for focusing our attention on cholesterol was that this lipid, besides SM, is one of the two basic components of membrane raft domains which are presumed to participate in the fusion between cells [24]. Our observation that cholesterol reduction was accompanied by lowering of the fusogenic capacity of both cell lines confirmed the presumption that this raft component is likely to play a certain role in cell–cell fusion [3]. Thus, it seems likely that SM and CH, which are the two basic lipid components of membrane raft domains, might play a definite role in the processes of cell–cell interaction and fusion possibly by affecting the “fusion competent” proteins, which are localized predominantly in the raft domains [25]. One can speculate that the higher fusogenic potential of ras-cells could be related to the higher level of SM and cholesterol in the membranes of these cells, which is a prerequisite for raft formation [26]. It is also possible that the higher capacity of ras-cells to undergo fusion is a result of the stimulated processes of endocytosis reported for these cells [27]. Thus the higher ability of ras-cells to fuse with other membranes could as well be a mechanism by which these cells compensate the loss of membrane lipid components due to stimulated endocytosis. In conclusion, our studies showed that modifications of the plasma membrane lipid composition can affect at a different degree the capacity of the cells to fuse with model membranes. Possibly, the varying of the membrane lipid composition might turn out to be a factor which modulates membrane fusogenic capacity and is also related to membrane and cellular pathology. Recent evidence suggests that rafts can serve as docking sites of a broad range of pathogens including viruses, bacteria, etc. [28]. For example, HIV-1 is an enveloped virus that fuses with the plasma membrane of the host cells and
this process can be modulated by the lipid components of raft domains [25,28]. Thus, besides the leading role of the specific proteins, which regulate fusion, the alterations of the membrane lipid composition also seem to affect the cellular fusogenic potential. It is possible that the lipid composition of the membranes plays an indirect role in the cellular fusogenic capacity by modulating the active conformation of the membrane-bound “fusioncompetent” proteins. At present, more studies are needed for elucidation of the fine mechanisms underlying the processes of cell–cell interactions and fusion. Acknowledgments This work was supported by the Bulgarian National Fund for Scientific Research (Grant 1404/04 and partially by Grant BY-Б-1/05). References [1] R. Jahn, T. Lang, T. Sudhof, Membrane fusion, Cell 112 (2003) 519–533. [2] F. Deeba, H.N. Tahseen, K. Sharma Sharad, N. Ahmad, S. Akhtar, M. Saleemuddin, O. Mohammad, Phospholipid diversity: correlation with membrane–membrane fusion events, Biochim. Biophys. Acta 1669 (2005) 170–181. [3] A. Baily, M. Zhukovsky, A. Gliozzi, L. Chernomordik, Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes, Arch. Biochem. Biophys. 439 (2005) 211–221. [4] A. Momchilova, T. Markovska, R. Pankov, Phospholipid dependence of membrane-bound phospholipase A2 in ras-transformed NIH 3T3 fibroblasts, Biochimie 80 (1998) 1053–1062. [5] A. Momchilova, L. Ivanova, T. Markovska, R. Pankov, Stimulated nonspecific transport of phospholipids results in elevated external appearance of phosphatidylserine in ras-transformed fibroblasts, Arch. Biochem. Biophys. 381 (2000) 295–301. [6] A. Momchilova, T. Markovska, Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in rastransformed fibroblasts, Int. J. Biochem. Cell Biol. 31 (1999) 311–318. [7] R. Pankov, T. Markovska, P. Antonov, L. Ivanova, A. Momchilova, Cholesterol distribution in plasma membranes of 1 integrin expressing and 1 integrin deficient fibroblasts, Arch. Biochem. Biophys. 442 (2005) 160–168. [8] D. Struck, D. Hoekstra, R. Pagano, Use of resonance energy transfer to monitor membrane fusion, Biochemistry 20 (1981) 4093–4099. [9] H. Dao, C. McIntyre, R. Sleight, Large-scale preparation of asymmetrically fluorescent lipid vesicles, Anal. Biochem. 196 (1991) 46–53. [10] A. Momchilova, D. Petkova, K. Koumanov, Phospholipid composition modifications influence phospholipase A2 activity in rat liver plasma membranes, Int. J. Biochem. 18 (1986) 945–952. [11] E. Bligh, W. Dyer, A rapid method of total lipid extraction and purification, Can. J. Physiol. 37 (1959) 911–917. [12] J. Touchstone, J. Chen, K. Beaver, Improved separation of phospholipids by thin-layer chromatography, Lipids 15 (1980) 61–62.
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