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Changes in phospholipid composition during the development of Dictyostelium discoideum
ARCHIVES OF BIOCHEMISTRY Vol. 219, No. 1, November,
Changes
AND BIOPHYSICS pp. 21-29, 1982
in Phospholipid Composition during Dictyostelium discoideum’ AKIRA
Biological
Laboratory,
Hakodate
Received
College,
March
the Development
of
HASE
Hokkaido
University
30, 1982, and in revised
of form
Education,
July
Hakodate
040,
Japan
2, 1982
The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. dkcoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45-58 and lo-24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the l-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [3H]ethanolamine and [‘4C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.
The cellular slime mold, Dictyostelium dkcoideum is an organism suitable for studying cell differentiation at the molecular level because of its simple morphology and life cycle. When exponentially growing cells are deprived of their bacterial food source, they commence a developmental sequence: they aggregate into multicellular organisms, transform into two different cell types, and form fruiting bodies consisting of a mass of spores supported by a stalk.
Recently the importance of cell membranes in the differentiation of D. discoideum has been reported by many authors and compositional changes in membrane proteins and glycoproteins during the development have been extensively studied (see the reviews by Newell (1) and McMahon and Hoffman (2)). Despite the importance of lipids in the maintenance of the structure and function of the membranes, however, relatively less effort has been made for studies of membrane lipids of D. discoideum (3-14). For phospholipids, Ellingson (5) has reported drastic qualitative and quantitative compositional changes during the differ-
’ This work was carried out while the author was at the Department of Botany, Faculty of Science, Hokkaido University, Sapporo, Japan. 21
0003-9861/82/130021-09$02.00/O Copyriabt 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
22
AKIRA
entiation of this organism and suggested that these changes are related, in some way, to the cell adhesion and spore formation. Using lipids from purified plasma membranes as starting materials, Weeks and Herring (10) and De Silva and Siu (11) reported only minor changes in the amounts of major phospholipid constituents of plasma membranes during the early stages of development. Thus, some information on phospholipids of D. discoideum has accumulated, but an important problem remains disregarded. As reported by Ferber et al. (15, 16), D. discoideum cells have relatively high phospholipase activities and these activities drastically change during the aggregation and differentiation. If the endogenous phospholipase(s) degrade some phospholipids during the process of cell fractionation and/or lipid extraction, artifical changes in the phospholipid composition should occur. In fact, the degradation of phospholipids by endogenous phospholipase(s) occurred during the preparation of plasma membranes and the amounts of lyso forms of ethanolamine phosphoglyceride and choline phosphoglyceride and those of other degradation products increased markedly (unpublished observation). Therefore, developmentally regulated changes in phospholipid composition of the plasma membrane itself would not be examined accurately. In my studies, the harvested cells were immediately frozen in liquid N, and lyophilized, and then lipids were extracted from the dried whole cells under anhydrous conditions to prevent degradation by endogenous phospholipase(s). By adopting these procedures, highly reproducible data could be obtained. There were no drastic, qualitative and quantitative changes in phospholipid composition in association with the cell aggregation and cell differentiation; but gradual quantitative changes occurred. Metabolic pathways which led to the changes in phospholipid composition were also examined in this work. MATERIALS
AND
METHODS
Organism and culture conditions. Cells of D. di.coideum, strain NC4, were grown in association with
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Escherichia coli on SM agar plates (17). Development of D. discoideum cells was initiated by harvesting vegetative cells (O-h development) from the growing medium, washing them several times in cold SM buffer and once in Bonner’s salt solution (18) containing 0.2 mg/ml streptomycin sulfate, and then depositing them onto Whatman No. 50 filters which were placed on sponge pads saturated with the Bonner’s solution containing streptomycin sulfate, as described previously (13, 14). The cells at 0, 4, 8, 12, 16, 20, and 24 h of development were harvested from the filters, washed with cold distilled water, immediately frozen in liquid Nx, and then lyophilized. The lyophilized cells were weighed and stored at -28°C in tubes free from moisture. Extraction of lipids. Lipids were extracted from the dried cells by a modification of the method of Folch et al. (19) as described previously (13, 14). Characterization of individual phospholipids. Twothirds of the total lipids extracted from whole cells at each stage of development was applied to a silicic acid column and phospholipids were separated from neutral lipids by the method of Borgstrom (20). Each phospholipid fraction and neutral lipid fraction was evaporated completely and weighed. Then the phospholipid fractions of various stages of development were combined and separated into each constituent by silicic acid column chromatography according to the method of Hanahan et al. (21) and preparative, thin-layer chromatography on silica gel H (E. Merck, Darmstadt) plates which were developed with chloroform/methanol/28% NHs (65/35/5, v/v). The individual phospholipids were then characterized by infrared spectrometry (22), color reactions on thin-layer plates, and/or cochromatography with purchased standard phospholipids (Sigma Chem. Co. Ltd). The spray reagents for color reactions were as follows: molybdenum blue reagent for phosphorus (23), a ninhydrin reagent for free amino groups (24), Dragendorfs reagent for choline (25), Schiffs reagent for plasmalogen (26), and a periodate-Schiff s reagent for glycol groups (27). Two-dimensional, thin-layer chromatography for quantitative analysis ofphospholipids. The other onethird of the total lipids extracted from cells at various stages of development was used. One milligram of the total lipids was applied onto a TLC*-silica gel 60G/ silica gel H (E. Merck; l/l, w/w) plate which was developed in the first direction with chloroform/methanol/28% NH3 (65/35/5, v/v) and in the second direction with chloroform/acetone/methanol/acetic acid/water (100/40/20/20/10, v/v) as described by ’ Abbreviations used: EPG, ethanolamine phosphoglyceride; CPG, choline phosphoglyceride; IPG, inositol phosphoglyceride; SPG, serine phosphoglyceride; CL, cardiolipin; PG, phosphatidylglycerol; PA, phosphatidic acid; TLC, thin-layer chromatography.
Dictyostelium
discoideum
PHOSPHOLIPID
Singh and Privett (28). The areas containing the individual phospholipids were detected by exposure to I2 vapor, scraped from the plate, and analyzed for phosphate as described by Bartlett (29). Reaction thin-layer chromatography for quantitative analysis of plosmatogen. The plasmalogen forms of ethanolamine phosphoglyceride (EPG) and choline phosphoglyceride (CPG) were separated from their diacyl and alkyl-ether forms by reaction thin-layer chromatography on the plate mentioned above according to the method of Vaskovsky and Demhitzky (30) and analyzed for phosphate. That is, 1 mg of the total lipids was spotted in one corner of the plate and developed in the first direction with chloroform/ methanol/28% NH3 (65/35/5, v/v). The chromatogram was dried in a current of cold air for 10 min, and a l-cm-wide zone from the start to the front was sprayed with 1 N HCl in methanol. The plate was dried again in a current of air for 30 min and developed in the second direction with chloroform/ acetone/methanol/acetic acid/water (100/40/20/ 20/10, v/v). Labeling of phospholipids with [3H]ethanolamine atzd [‘4C]choline. Vegetative cells were harvested and washed several times with cold SM buffer, then the cell density was adjusted to 2 X 10s cells/ml with the same buffer containing 20 &i/ml [l-3H]ethan-l-ol2-amine-HCl (24 Ci/mmol, Amersham Japan Ltd.), 2 FCi/ml [methyl-‘4C]choline-C1 (58 mCi/mmol, Amersham Japan Ltd.), and 0.25 mg/ml streptomycin sulfate, and the cells were incubated for 1 h at 22°C with continuous shaking. Next, the cells were harvested, washed with cold SM buffer, and placed on Whatman No. 50 filter papers. After incubation for 0.5, 8, 16, and 24 h on the filters, the cells were harvested, frozen in liquid NB, and then lyophilized. Unlabeled vegetative cells (0 h), aggregating cells (8 h), and tip-forming aggregates (12 h) were also harvested from filters and labeled simultaneously with [3H]ethanolamine and [‘4C]choline for 30 min as mentioned above, then again harvested and washed. Aliquots of the cells from the three groups were dissolved in NCS tissue solubilizer (Amersham/Searle Corp.) and the radioactivities were measured. The remaining cells in each group were lyophilized. Lipids were extracted from the lyophilized cells and each phospholipid constituent was separated by thinlayer chromatography and analyzed for phosphate content and radioactivity. Radioactivity was measured in a Beckman liquid scintillation spectrometer using a toluene-based scintillator.
Phospholipid Contents Stages of Development
tive changes during the development. Changes in the levels of phospholipids and neutral lipids per total lipids are shown in Fig. 1, from which it can be seen that the levels of the two lipid fractions are relatively constant during the development. These results were different from the results of Long and Coe (6) and De Silva and Siu (ll), who reported developmental changes in lipid and phospholipid contents. Phospholipids
in Cells at Various
The lipid content was 10-12'S of dry cell weight and there were no gross quantita-
of D. discoideum
Cells
Figure 2 shows that ten classes of phospholipid were detected at all stages of development. The results of identification of each phospholipid are also included in Fig. 2. The major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride (EPG) and choline phosphoglyceride (CPG) which accounted for 44-60 and 27-48 mol-% of the phospholipid phosphorus, respectively. EPG and CPG included 45-58 and lo-24% of their plasmalogen forms, respectively. There were also alkyl-ether compounds but these were not quantified. The other phospholipid of some significance was inositol phosphoglyceride (IPG) which contained 4-6 mol-% of the phospholipid phosphorus. Serine phosphoglyceride (SPG), phosphatidylglycerol (PG), cardiolipin (CL), and lyso forms of EPG and CPG, phosphatidic acid (PA), and an unidentified phosphorus-containing lipid, which might correspond to the (N-acyl)ethanolamine phosphoglycerides or acylphosphatidylglycerol as reported by Ellingson (12), were minor constituents, each of which contained 0.2-2.7% of the phospholipid phosphorus, respectively. IPG and other minor phospholipids also contained trace amounts of the plasmalogen forms but their amounts were not measured. Changes during
RESULTS
23
COMPOSITION
in Phospholipid the Development
Composition
As shown in Fig. 2, there were no marked qualitative changes in phospholipid composition during the development, but certainly gradual quantitative changes
24
AKIRA
’
I.
0
I1 4
I. 6 12 16 Time (hours)
I 20
II 24
FIG. 1. Changes in phospholipid and neutral lipid contents during the development of D. discoideum. Total lipids prepared from cells at various stages of development were fractionated into neutral lipids and phospholipids by silicic acid column chromatography. Each fraction was evaporated to complete dryness and weighed. Each value is the mean with the SD of four experiments. The indicated times correspond to the following stages of development: 0 h, vegetative stage; 4 h, interphase; 8 h, early aggregation stage; 12 h, late aggregation stage; 16 h, preculmination stage; 20 h, culmination stage; 24 h, l-day-sorocarp stage. 0, Phospholipids; 0, neutral lipids.
occurred (see Fig. 3). Notably, the contents of the two major phospholipids (EPG and CPG) changed in a reciprocal fashion during the development. In vegetative-stage cells (O-h development), the major phospholipid was EPG which contained 60% of the phospholipid phosphorus. During the development, the level of EPG grad-
a
b
c
d
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ually decreased to about 44% in l-day sorocarps (24-h development). In contrast, CPG gradually increased from 27% in vegetative-stage cells to 48% in preculmination-stage cells (16-h development), then slightly decreased to 43% in l-day sorocarps. The decrease of EPG content during the earlier developmental stages (O-4 h) was due mainly to decreases of components other than plasmalogen, which consisted of the diacyl form of EPG (3-sn-phosphatidylethanolamine) and its alkyl-ether form (plasmanylethanolamine), but, in the middle and late stages, a prominent decrease of the plasmalogen form (plasmenylethanolamine) occurred (Fig. 3). In contrast, the changes in amount of CPG were due mainly to the marked increase of the diacyl form (3-en-phosphatidylcholine) and/or the alkyl-ether form (plasmanylcholine) during all the processes of the development. The increase of IPG in early aggregation-stage cells (8-h development) was also reproducible (Fig. 3). Other minor phospholipids including the lyso forms of EPG and CPG did not show significant changes in their levels during the development (Fig. 3).
FIG. 2. Two-dimensional, thin-layer chromatograms of phospholipids from D. discoideum cells at various stages of development. One milligram of the total lipid fraction from (a) vegetative-stage cells (O-h development), (b) early aggregation-stage cells (8 h), (c) preculmination-stage cells (16 h), or (d) l-day sorocarps (24 h) was spotted at the origin on a silica gel plate and developed as described under Materials and Methods. The separated phospholipids were visualized by spraying with molybdenum blue reagent (23). The results of identification of individual phospholipids are as follows: 0, origin; 1, ethanolamine phosphoglyceride (EPG); 2, choline phosphoglyceride (CPG); 3, inositol phosphoglyceride (IPG); 4, serine phosphoglyceride (SPG); 5, cardiolipin (CL); 6, phosphatidylglycerol (PG); 7, lyso form of EPG; 8, lyso form of CPG; 9, phosphatidic acid (PA); 10, an unidentified phosphorus-containing lipid.
Dictyostelium
discoideum
PHOSPHOLIPID
14
i
lo-
0
4
a
12 I6 Time I hours)
20
25
COMPOSITION
to the compositional changes in phospholipids. Figure 4 shows the changes in 3H radioactivity due to labeled ethanolamine and 14C radioactivity due to labeled methyl groups of choline in EPG and CPG during the development. As the decrease of 3H radioactivity in EPG and the antipodal increase in CPG showed (Fig. 4a), the conversion of EPG to CPG by stepwise methylation seemed to occur. However, 14C counts in CPG also increased (Fig. 4b),
24
FIG. 3. Changes in each phospholipid constituent during the development. Each phospholipid constituent was separated by two-dimensional, thin-layer chromatography or reaction thin-layer chromatography on a silica gel plate and analyzed for phosphate as described under Materials and Methods. Each value is the mean with the SD of four determinations for each of three independent experiments. 0, EPG; 0, CPG; A, IPG; A, SPG; H, CL; 0, PG; w, lyso form of EPG; V, lyso form of CPG, X, PA, o, an unidentified phosphorus-containing lipid; -, total of each phospholipid; - - -, diacyl and alkyl-ether forms; -. -, plasmalogen form.
I------i 05
Changes in 3H and 14C Radioactivities in Phospholipids during the Development The causes of these changes in EPG and CPG contents can be interpreted easily if we postulate the activation of stepwise methylation of EPG during the development. However, the decrease in EPG during the middle and late development was due mainly to the decreased amount of its plasmalogen form, while the increase of CPG was independent of the quantitative changes in its plasmalogen form (Fig. 3). Furthermore, the fatty acid compositions of EPG and CPG were different from each other (3). These facts strongly suggested the presence of other pathways which led
6 16 Time k~~rs)
24
FIG. 4. Changes in (a) 3H and (b) 14C distributions among phospholipids during the development. Vegetative-stage cells were harvested, labeled with 20 &i/ml [3H]ethanolamine and 2 &i/ml [‘4C]choline for 1 h, replaced on Whatman No. 50 filters, and then incubated at 22°C as described under Materials and Methods. At the indicated times, cells were harvested and lyophilized for lipid extraction. Total lipids containing 100 rmol phospholipid-phosphorus were applied to the silica gel plate and each phospholipid component was separated by two-dimensional, thinlayer chromatography or reaction thin-layer chromatography as described under Materials and Methods. Then the spot corresponding to each phospholipid constituent was scraped off and analyzed for radioactivity. 0, EPG; 0, CPG, X, other phospholipids; -, total; - - -, diacyl and alkyl-ether forms; -. -, plasmalogen form.
26
AKIRA
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CPG was due to the activation of stepwise methylation of EPG but the other twothirds were due to de nouo synthesis of CPG from CDP-choline and diglyceride. Uptake and Incorporation of [3H]EthanoZumine and [ z4C]Choline at the Early and Late Aggregation Stages
05
8 16 Time (tours)
24
FIG. 5. Changes in the specific radioactivities of (a) 3H and (b) i4C in EPG and CPG during the development. The specific radioactivities of 3H and “C in EPG and CPG were calculated by dividing the total counts shown in Fig. 4 by the amounts of individual lipids which were expressed as pmol phospholipid phosphorus. l , EPG; 0, CPG; p, total; - - -, diacyl and alkyl-ether forms; * -, plasmalogen form.
which showed the occurrence of synthesis of CPG from CDP-choline and diglyceride. Figure 5 shows the changes in specific radioactivity of 3H and 14C counts in EPG and CPG. The same pattern of changes as those shown in Fig. 4 was observed. If we assume that the rate of synthesis of each phospholipid is reflected in the increased rate of its specific radioactivity and that the degradation rates of CPGs of different origins are the same as each other, the rate of the production of CPG by stepwise methylation of EPG was the same as that of the synthesis of CPG from CDP-choline and diglyceride (Fig. 5). Especially, during the middle stages of development (8-16 h), the rate of increase of 14Cradioactivity in CPG was 2 times higher than that of 3H radioactivity in CPG (Fig. 5), which showed that one-third of the increased amount of
As Table I shows, the uptake of radioactive precursors of EPG and CPG by cells was the highest at the vegetative stage, but the rate of uptake did not decrease so much during the aggregation of cells. However, a drastic decrease of incorporation into phospholipids was observed with aggregation-stage cells. Especially, the decrease of incorporation of 14C radioactivity into phospholipids was higher than that of incorporation of 3H radioactivity. These facts showed that the rate of synthesis of EPG and CPG diminished markedly during the development of D. discoideum. But, the decrease of incorporated 14C radioactivity observed with aggregation-stage cells disagreed with the increase of the total radioactivity of 14Ccounts of CPG (Fig. 4) and also with that of its specific activity (Fig. 5). This disagreement between the two results may be solved when we postulate as follows: D. discoideum cells have a large pool of choline, in which [‘4C]choline taken up was stocked temporarily and then gradually used for CPG synthesis. And, as the size of the choline pool grew bigger during the development, the [14C]choline taken up became harder to be incorporated into CPG. Preliminary pulse and chase experiments supported the above assumption (data not shown). DISCUSSION
Highly reproducible data showing quantitative changes in phospholipid composition during the development of D. discoideum could be obtained in the present study in which much attention was given to avoid the degradation of phospholipids by endogenous phospholipase(s). The contents of both EPG and CPG changed, in contrast with the results of Weeks and Herring (10) and De Silva and Siu (11)
Dictyostelium
discoideum
PHOSPHOLIPID
which were obtained with purified plasma membranes. However, there was a suspicion in the early stage of the present study that the components present in vegetativestage cells were of bacterial origin. More than 80% of the phospholipids in E. coli is EPG. Therefore, I examined the fatty acid compositions of phospholipids from vegetative-stage cells and lipids from E. coli cells (13). E. coli lipids showed high contents of palmitate, stearate, and C19cyclopropanate, but D. discoideum phospholipids showed a relatively low content of palmitate and only trace amounts of stearate and cyclopropanate, while their contents in neutral lipids were high (13). This shows that lipids of E. coli cells which have been taken up into D. discoideum cells by phagocytosis were rapidly digested. On the other hand, it might be possible that the presence of ethanolamine, one of the digestion products of phospholipids, affected the phospholipid composition of vegetative-stage cells. However, the observations that the gradual changes in EPG and CPG contents continued after the interphase and that the decrease in the EPG content was due mainly to the decrease of plasmalogen-form EPG clearly demonstrated the true occurrence of developmental changes in phospholipid composition, which were independent of the food SUPPlY.
Whether or not the phospholipid composition of plasma membranes is directly reflected in that of whole cells is obscure at present. But, because the development of the intracellular membranous components is shown to be poor in D. discoideum cells, changes in phospholipid composition in plasma membranes may somewhat affect the compositional changes in phospholipids prepared from whole cells. Thus, by comparison with the present results, we can reconsider the results obtained by using lipids from plasma membranes. How the compositional changes occur is a fundamental problem to be solved. Using a labeling procedure with L- [ methyl3H]methionine, Mato and his co-workers reported the occurrence of phospholipid methylation in D. discoideum cells during the aggregation (31, 32). Therefore, as the
COMPOSITION
28
AKIRA
most probable cause of the changes seemed to be the activation of stepwise methylation of EPG, an experiment in which radioactive precursors of phospholipids were supplied to cells at the early stage and the fate of the radioisotopes was traced during further development was carried out. As Figs. 4 and 5 show, I could not interpret the cause of the decrease of EPG and the increase of CPG as the stepwise methylation of EPG only. It seems that a regulatory mechanism of the metabolic pathway which leads to the changes in phospholipid composition during the development of D. discoideum is rather complex. It might include the reduction of EPG synthesis, the activation of selective degradation of EPG, especially of its plasmalogen form, and de nouo synthesis of CPG from CDP-choline and diglyceride, in addition to the stepwise methylation of EPG molecules. Furthermore, the possibility that the activation of de nouo synthesis of CPG through the CDP-choline pathway was due to the increased size of the choline pool in cells at the later stages is suggested. The biological significance of the changes in phospholipid composition is unknown. But there are two possibilities as follow: [l] The activities of many membranebound enzymes are supported by phospholipids and also affected by the chemical structure of phospholipid head groups (reviewed in Ref. (33)). If the phospholipid composition varies, some membrane-bound enzymes may change their activities. Since membrane-bound enzymes play a major part in various functions of the membranes, the changes in the enzyme activities then may affect the total membrane functions. The activities of some developmentally regulated membrane-bound enzymes may be regulated in such manner. [2] Recently, Cullis and De Kruijff (34,35) reported interesting results from 31P-NMR analysis of the polymorphic behavior of aqueous dispersions of phosphatidylethanolamines of natural and synthetic origins. In mammalian membranes such as the erythrocyte membrane, phosphatidylethanolamine had a tendency to destabilize the bilayer structure at a physiological
HASE
temperature. In contrast, phosphatidylcholine and sphingomyelin showed a tendency to stabilize the bilayer configuration of biological membranes. With these results, they discussed that this tendency of phosphatidylethanolamine may be related directly to many important properties of biological membranes, such as the fusion of membranes and flip-flop of phospholipids. And, during the preparation of this paper, Boggs et al. (36) reported that plasmalogen-form EPG further destabilized the lamellar structure of biological membranes. According to their observations, my results can be interpreted as follows: The high concentration of EPG in vegetative-stage cells may destabilize the bilayer structure, and this may facilitate the following functions of the cell membrane: phagocytosis, exocytosis, and cell division which should be accompanied by membrane fusion. However, for cell differentiation in this organism, which may be supported by stable and tight cell-cell contact (37), this property of membrane lipids may become inconvenient. Therefore, the levels of EPG, especially of its plasmalogen form, decrease and those of CPG increase and thus the bilayer structure may be stabilized. These possibilities need further investigation. Although there are no changes in membrane fluidity during the development (10, 38, 39), the changes in phospholipid composition may contribute to some roles of membrane lipids in the regulation of membrane functions. To clarify the importance of these changes in phospholipid composition, I have been studying the effects of modification of the phospholipid composition on the growth and development of D. discoideum. ACKNOWLEDGMENT I wish to thank Emeritus Professor I. Harada of Hokkaido University for his encouragement and Professor S. Tanifuji for his advice and critical reading of the manuscript. REFERENCES 1. NEWELL, nition
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692-697.
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25,
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SKIPSKI, CLAY,
48,
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HERRING,
S., AND
A. (1981) 12, 1833194.
DITTMER,
WAGNER,
Res. 21,681-686. 11.
Scar&.
ROUSER, G., KRITCHEVSKY, G., HELLER, LIEBER, E. (1963) J. Amer. Oil Chem.
25.
15. 9. CREAN,
D. J., DITTMER, E. (1957)
Lipid
337.60-67.
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425-454.
AND KORN, 3210-3215.
238,
5. ELLINGSON, LONG,
Acta
B. (1952)
HANAHAN, INA,
238, 3199-3209.
Chem.
6.
BORGSTR~M, 101-110.
2. MCMAHON,
4.
29
COMPOSITION
Press,
18.
BONNER,
J. T. (1947)
19.
FOLCH, J., LEES, M., G. A. (1957) J. Biol.
New
J. Exp. AND
Chem.
in Vol.
Cell Physi2, pp. 397-
York.
ZOOS.
38.
497-509.
ALTON,
T.
Biol.
106, i-26.
SLOANE-STANLEY,
226,
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VON
60,
DREELE,
Biochim. 39.
KAWAI,
Funct.
H.,
AND
LODISH,
H.
180-206. P. H., AND
Biophys. S., AND
3.31-37.
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TANAKA,
WILLIAMS,
464,
K. L. (1977)
378-388. K. (1978) Cell Struct.
Changes
AND BIOPHYSICS pp. 21-29, 1982
in Phospholipid Composition during Dictyostelium discoideum’ AKIRA
Biological
Laboratory,
Hakodate
Received
College,
March
the Development
of
HASE
Hokkaido
University
30, 1982, and in revised
of form
Education,
July
Hakodate
040,
Japan
2, 1982
The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. dkcoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45-58 and lo-24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the l-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [3H]ethanolamine and [‘4C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.
The cellular slime mold, Dictyostelium dkcoideum is an organism suitable for studying cell differentiation at the molecular level because of its simple morphology and life cycle. When exponentially growing cells are deprived of their bacterial food source, they commence a developmental sequence: they aggregate into multicellular organisms, transform into two different cell types, and form fruiting bodies consisting of a mass of spores supported by a stalk.
Recently the importance of cell membranes in the differentiation of D. discoideum has been reported by many authors and compositional changes in membrane proteins and glycoproteins during the development have been extensively studied (see the reviews by Newell (1) and McMahon and Hoffman (2)). Despite the importance of lipids in the maintenance of the structure and function of the membranes, however, relatively less effort has been made for studies of membrane lipids of D. discoideum (3-14). For phospholipids, Ellingson (5) has reported drastic qualitative and quantitative compositional changes during the differ-
’ This work was carried out while the author was at the Department of Botany, Faculty of Science, Hokkaido University, Sapporo, Japan. 21
0003-9861/82/130021-09$02.00/O Copyriabt 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
22
AKIRA
entiation of this organism and suggested that these changes are related, in some way, to the cell adhesion and spore formation. Using lipids from purified plasma membranes as starting materials, Weeks and Herring (10) and De Silva and Siu (11) reported only minor changes in the amounts of major phospholipid constituents of plasma membranes during the early stages of development. Thus, some information on phospholipids of D. discoideum has accumulated, but an important problem remains disregarded. As reported by Ferber et al. (15, 16), D. discoideum cells have relatively high phospholipase activities and these activities drastically change during the aggregation and differentiation. If the endogenous phospholipase(s) degrade some phospholipids during the process of cell fractionation and/or lipid extraction, artifical changes in the phospholipid composition should occur. In fact, the degradation of phospholipids by endogenous phospholipase(s) occurred during the preparation of plasma membranes and the amounts of lyso forms of ethanolamine phosphoglyceride and choline phosphoglyceride and those of other degradation products increased markedly (unpublished observation). Therefore, developmentally regulated changes in phospholipid composition of the plasma membrane itself would not be examined accurately. In my studies, the harvested cells were immediately frozen in liquid N, and lyophilized, and then lipids were extracted from the dried whole cells under anhydrous conditions to prevent degradation by endogenous phospholipase(s). By adopting these procedures, highly reproducible data could be obtained. There were no drastic, qualitative and quantitative changes in phospholipid composition in association with the cell aggregation and cell differentiation; but gradual quantitative changes occurred. Metabolic pathways which led to the changes in phospholipid composition were also examined in this work. MATERIALS
AND
METHODS
Organism and culture conditions. Cells of D. di.coideum, strain NC4, were grown in association with
HASE
Escherichia coli on SM agar plates (17). Development of D. discoideum cells was initiated by harvesting vegetative cells (O-h development) from the growing medium, washing them several times in cold SM buffer and once in Bonner’s salt solution (18) containing 0.2 mg/ml streptomycin sulfate, and then depositing them onto Whatman No. 50 filters which were placed on sponge pads saturated with the Bonner’s solution containing streptomycin sulfate, as described previously (13, 14). The cells at 0, 4, 8, 12, 16, 20, and 24 h of development were harvested from the filters, washed with cold distilled water, immediately frozen in liquid Nx, and then lyophilized. The lyophilized cells were weighed and stored at -28°C in tubes free from moisture. Extraction of lipids. Lipids were extracted from the dried cells by a modification of the method of Folch et al. (19) as described previously (13, 14). Characterization of individual phospholipids. Twothirds of the total lipids extracted from whole cells at each stage of development was applied to a silicic acid column and phospholipids were separated from neutral lipids by the method of Borgstrom (20). Each phospholipid fraction and neutral lipid fraction was evaporated completely and weighed. Then the phospholipid fractions of various stages of development were combined and separated into each constituent by silicic acid column chromatography according to the method of Hanahan et al. (21) and preparative, thin-layer chromatography on silica gel H (E. Merck, Darmstadt) plates which were developed with chloroform/methanol/28% NHs (65/35/5, v/v). The individual phospholipids were then characterized by infrared spectrometry (22), color reactions on thin-layer plates, and/or cochromatography with purchased standard phospholipids (Sigma Chem. Co. Ltd). The spray reagents for color reactions were as follows: molybdenum blue reagent for phosphorus (23), a ninhydrin reagent for free amino groups (24), Dragendorfs reagent for choline (25), Schiffs reagent for plasmalogen (26), and a periodate-Schiff s reagent for glycol groups (27). Two-dimensional, thin-layer chromatography for quantitative analysis ofphospholipids. The other onethird of the total lipids extracted from cells at various stages of development was used. One milligram of the total lipids was applied onto a TLC*-silica gel 60G/ silica gel H (E. Merck; l/l, w/w) plate which was developed in the first direction with chloroform/methanol/28% NH3 (65/35/5, v/v) and in the second direction with chloroform/acetone/methanol/acetic acid/water (100/40/20/20/10, v/v) as described by ’ Abbreviations used: EPG, ethanolamine phosphoglyceride; CPG, choline phosphoglyceride; IPG, inositol phosphoglyceride; SPG, serine phosphoglyceride; CL, cardiolipin; PG, phosphatidylglycerol; PA, phosphatidic acid; TLC, thin-layer chromatography.
Dictyostelium
discoideum
PHOSPHOLIPID
Singh and Privett (28). The areas containing the individual phospholipids were detected by exposure to I2 vapor, scraped from the plate, and analyzed for phosphate as described by Bartlett (29). Reaction thin-layer chromatography for quantitative analysis of plosmatogen. The plasmalogen forms of ethanolamine phosphoglyceride (EPG) and choline phosphoglyceride (CPG) were separated from their diacyl and alkyl-ether forms by reaction thin-layer chromatography on the plate mentioned above according to the method of Vaskovsky and Demhitzky (30) and analyzed for phosphate. That is, 1 mg of the total lipids was spotted in one corner of the plate and developed in the first direction with chloroform/ methanol/28% NH3 (65/35/5, v/v). The chromatogram was dried in a current of cold air for 10 min, and a l-cm-wide zone from the start to the front was sprayed with 1 N HCl in methanol. The plate was dried again in a current of air for 30 min and developed in the second direction with chloroform/ acetone/methanol/acetic acid/water (100/40/20/ 20/10, v/v). Labeling of phospholipids with [3H]ethanolamine atzd [‘4C]choline. Vegetative cells were harvested and washed several times with cold SM buffer, then the cell density was adjusted to 2 X 10s cells/ml with the same buffer containing 20 &i/ml [l-3H]ethan-l-ol2-amine-HCl (24 Ci/mmol, Amersham Japan Ltd.), 2 FCi/ml [methyl-‘4C]choline-C1 (58 mCi/mmol, Amersham Japan Ltd.), and 0.25 mg/ml streptomycin sulfate, and the cells were incubated for 1 h at 22°C with continuous shaking. Next, the cells were harvested, washed with cold SM buffer, and placed on Whatman No. 50 filter papers. After incubation for 0.5, 8, 16, and 24 h on the filters, the cells were harvested, frozen in liquid NB, and then lyophilized. Unlabeled vegetative cells (0 h), aggregating cells (8 h), and tip-forming aggregates (12 h) were also harvested from filters and labeled simultaneously with [3H]ethanolamine and [‘4C]choline for 30 min as mentioned above, then again harvested and washed. Aliquots of the cells from the three groups were dissolved in NCS tissue solubilizer (Amersham/Searle Corp.) and the radioactivities were measured. The remaining cells in each group were lyophilized. Lipids were extracted from the lyophilized cells and each phospholipid constituent was separated by thinlayer chromatography and analyzed for phosphate content and radioactivity. Radioactivity was measured in a Beckman liquid scintillation spectrometer using a toluene-based scintillator.
Phospholipid Contents Stages of Development
tive changes during the development. Changes in the levels of phospholipids and neutral lipids per total lipids are shown in Fig. 1, from which it can be seen that the levels of the two lipid fractions are relatively constant during the development. These results were different from the results of Long and Coe (6) and De Silva and Siu (ll), who reported developmental changes in lipid and phospholipid contents. Phospholipids
in Cells at Various
The lipid content was 10-12'S of dry cell weight and there were no gross quantita-
of D. discoideum
Cells
Figure 2 shows that ten classes of phospholipid were detected at all stages of development. The results of identification of each phospholipid are also included in Fig. 2. The major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride (EPG) and choline phosphoglyceride (CPG) which accounted for 44-60 and 27-48 mol-% of the phospholipid phosphorus, respectively. EPG and CPG included 45-58 and lo-24% of their plasmalogen forms, respectively. There were also alkyl-ether compounds but these were not quantified. The other phospholipid of some significance was inositol phosphoglyceride (IPG) which contained 4-6 mol-% of the phospholipid phosphorus. Serine phosphoglyceride (SPG), phosphatidylglycerol (PG), cardiolipin (CL), and lyso forms of EPG and CPG, phosphatidic acid (PA), and an unidentified phosphorus-containing lipid, which might correspond to the (N-acyl)ethanolamine phosphoglycerides or acylphosphatidylglycerol as reported by Ellingson (12), were minor constituents, each of which contained 0.2-2.7% of the phospholipid phosphorus, respectively. IPG and other minor phospholipids also contained trace amounts of the plasmalogen forms but their amounts were not measured. Changes during
RESULTS
23
COMPOSITION
in Phospholipid the Development
Composition
As shown in Fig. 2, there were no marked qualitative changes in phospholipid composition during the development, but certainly gradual quantitative changes
24
AKIRA
’
I.
0
I1 4
I. 6 12 16 Time (hours)
I 20
II 24
FIG. 1. Changes in phospholipid and neutral lipid contents during the development of D. discoideum. Total lipids prepared from cells at various stages of development were fractionated into neutral lipids and phospholipids by silicic acid column chromatography. Each fraction was evaporated to complete dryness and weighed. Each value is the mean with the SD of four experiments. The indicated times correspond to the following stages of development: 0 h, vegetative stage; 4 h, interphase; 8 h, early aggregation stage; 12 h, late aggregation stage; 16 h, preculmination stage; 20 h, culmination stage; 24 h, l-day-sorocarp stage. 0, Phospholipids; 0, neutral lipids.
occurred (see Fig. 3). Notably, the contents of the two major phospholipids (EPG and CPG) changed in a reciprocal fashion during the development. In vegetative-stage cells (O-h development), the major phospholipid was EPG which contained 60% of the phospholipid phosphorus. During the development, the level of EPG grad-
a
b
c
d
HASE
ually decreased to about 44% in l-day sorocarps (24-h development). In contrast, CPG gradually increased from 27% in vegetative-stage cells to 48% in preculmination-stage cells (16-h development), then slightly decreased to 43% in l-day sorocarps. The decrease of EPG content during the earlier developmental stages (O-4 h) was due mainly to decreases of components other than plasmalogen, which consisted of the diacyl form of EPG (3-sn-phosphatidylethanolamine) and its alkyl-ether form (plasmanylethanolamine), but, in the middle and late stages, a prominent decrease of the plasmalogen form (plasmenylethanolamine) occurred (Fig. 3). In contrast, the changes in amount of CPG were due mainly to the marked increase of the diacyl form (3-en-phosphatidylcholine) and/or the alkyl-ether form (plasmanylcholine) during all the processes of the development. The increase of IPG in early aggregation-stage cells (8-h development) was also reproducible (Fig. 3). Other minor phospholipids including the lyso forms of EPG and CPG did not show significant changes in their levels during the development (Fig. 3).
FIG. 2. Two-dimensional, thin-layer chromatograms of phospholipids from D. discoideum cells at various stages of development. One milligram of the total lipid fraction from (a) vegetative-stage cells (O-h development), (b) early aggregation-stage cells (8 h), (c) preculmination-stage cells (16 h), or (d) l-day sorocarps (24 h) was spotted at the origin on a silica gel plate and developed as described under Materials and Methods. The separated phospholipids were visualized by spraying with molybdenum blue reagent (23). The results of identification of individual phospholipids are as follows: 0, origin; 1, ethanolamine phosphoglyceride (EPG); 2, choline phosphoglyceride (CPG); 3, inositol phosphoglyceride (IPG); 4, serine phosphoglyceride (SPG); 5, cardiolipin (CL); 6, phosphatidylglycerol (PG); 7, lyso form of EPG; 8, lyso form of CPG; 9, phosphatidic acid (PA); 10, an unidentified phosphorus-containing lipid.
Dictyostelium
discoideum
PHOSPHOLIPID
14
i
lo-
0
4
a
12 I6 Time I hours)
20
25
COMPOSITION
to the compositional changes in phospholipids. Figure 4 shows the changes in 3H radioactivity due to labeled ethanolamine and 14C radioactivity due to labeled methyl groups of choline in EPG and CPG during the development. As the decrease of 3H radioactivity in EPG and the antipodal increase in CPG showed (Fig. 4a), the conversion of EPG to CPG by stepwise methylation seemed to occur. However, 14C counts in CPG also increased (Fig. 4b),
24
FIG. 3. Changes in each phospholipid constituent during the development. Each phospholipid constituent was separated by two-dimensional, thin-layer chromatography or reaction thin-layer chromatography on a silica gel plate and analyzed for phosphate as described under Materials and Methods. Each value is the mean with the SD of four determinations for each of three independent experiments. 0, EPG; 0, CPG; A, IPG; A, SPG; H, CL; 0, PG; w, lyso form of EPG; V, lyso form of CPG, X, PA, o, an unidentified phosphorus-containing lipid; -, total of each phospholipid; - - -, diacyl and alkyl-ether forms; -. -, plasmalogen form.
I------i 05
Changes in 3H and 14C Radioactivities in Phospholipids during the Development The causes of these changes in EPG and CPG contents can be interpreted easily if we postulate the activation of stepwise methylation of EPG during the development. However, the decrease in EPG during the middle and late development was due mainly to the decreased amount of its plasmalogen form, while the increase of CPG was independent of the quantitative changes in its plasmalogen form (Fig. 3). Furthermore, the fatty acid compositions of EPG and CPG were different from each other (3). These facts strongly suggested the presence of other pathways which led
6 16 Time k~~rs)
24
FIG. 4. Changes in (a) 3H and (b) 14C distributions among phospholipids during the development. Vegetative-stage cells were harvested, labeled with 20 &i/ml [3H]ethanolamine and 2 &i/ml [‘4C]choline for 1 h, replaced on Whatman No. 50 filters, and then incubated at 22°C as described under Materials and Methods. At the indicated times, cells were harvested and lyophilized for lipid extraction. Total lipids containing 100 rmol phospholipid-phosphorus were applied to the silica gel plate and each phospholipid component was separated by two-dimensional, thinlayer chromatography or reaction thin-layer chromatography as described under Materials and Methods. Then the spot corresponding to each phospholipid constituent was scraped off and analyzed for radioactivity. 0, EPG; 0, CPG, X, other phospholipids; -, total; - - -, diacyl and alkyl-ether forms; -. -, plasmalogen form.
26
AKIRA
HASE
CPG was due to the activation of stepwise methylation of EPG but the other twothirds were due to de nouo synthesis of CPG from CDP-choline and diglyceride. Uptake and Incorporation of [3H]EthanoZumine and [ z4C]Choline at the Early and Late Aggregation Stages
05
8 16 Time (tours)
24
FIG. 5. Changes in the specific radioactivities of (a) 3H and (b) i4C in EPG and CPG during the development. The specific radioactivities of 3H and “C in EPG and CPG were calculated by dividing the total counts shown in Fig. 4 by the amounts of individual lipids which were expressed as pmol phospholipid phosphorus. l , EPG; 0, CPG; p, total; - - -, diacyl and alkyl-ether forms; * -, plasmalogen form.
which showed the occurrence of synthesis of CPG from CDP-choline and diglyceride. Figure 5 shows the changes in specific radioactivity of 3H and 14C counts in EPG and CPG. The same pattern of changes as those shown in Fig. 4 was observed. If we assume that the rate of synthesis of each phospholipid is reflected in the increased rate of its specific radioactivity and that the degradation rates of CPGs of different origins are the same as each other, the rate of the production of CPG by stepwise methylation of EPG was the same as that of the synthesis of CPG from CDP-choline and diglyceride (Fig. 5). Especially, during the middle stages of development (8-16 h), the rate of increase of 14Cradioactivity in CPG was 2 times higher than that of 3H radioactivity in CPG (Fig. 5), which showed that one-third of the increased amount of
As Table I shows, the uptake of radioactive precursors of EPG and CPG by cells was the highest at the vegetative stage, but the rate of uptake did not decrease so much during the aggregation of cells. However, a drastic decrease of incorporation into phospholipids was observed with aggregation-stage cells. Especially, the decrease of incorporation of 14C radioactivity into phospholipids was higher than that of incorporation of 3H radioactivity. These facts showed that the rate of synthesis of EPG and CPG diminished markedly during the development of D. discoideum. But, the decrease of incorporated 14C radioactivity observed with aggregation-stage cells disagreed with the increase of the total radioactivity of 14Ccounts of CPG (Fig. 4) and also with that of its specific activity (Fig. 5). This disagreement between the two results may be solved when we postulate as follows: D. discoideum cells have a large pool of choline, in which [‘4C]choline taken up was stocked temporarily and then gradually used for CPG synthesis. And, as the size of the choline pool grew bigger during the development, the [14C]choline taken up became harder to be incorporated into CPG. Preliminary pulse and chase experiments supported the above assumption (data not shown). DISCUSSION
Highly reproducible data showing quantitative changes in phospholipid composition during the development of D. discoideum could be obtained in the present study in which much attention was given to avoid the degradation of phospholipids by endogenous phospholipase(s). The contents of both EPG and CPG changed, in contrast with the results of Weeks and Herring (10) and De Silva and Siu (11)
Dictyostelium
discoideum
PHOSPHOLIPID
which were obtained with purified plasma membranes. However, there was a suspicion in the early stage of the present study that the components present in vegetativestage cells were of bacterial origin. More than 80% of the phospholipids in E. coli is EPG. Therefore, I examined the fatty acid compositions of phospholipids from vegetative-stage cells and lipids from E. coli cells (13). E. coli lipids showed high contents of palmitate, stearate, and C19cyclopropanate, but D. discoideum phospholipids showed a relatively low content of palmitate and only trace amounts of stearate and cyclopropanate, while their contents in neutral lipids were high (13). This shows that lipids of E. coli cells which have been taken up into D. discoideum cells by phagocytosis were rapidly digested. On the other hand, it might be possible that the presence of ethanolamine, one of the digestion products of phospholipids, affected the phospholipid composition of vegetative-stage cells. However, the observations that the gradual changes in EPG and CPG contents continued after the interphase and that the decrease in the EPG content was due mainly to the decrease of plasmalogen-form EPG clearly demonstrated the true occurrence of developmental changes in phospholipid composition, which were independent of the food SUPPlY.
Whether or not the phospholipid composition of plasma membranes is directly reflected in that of whole cells is obscure at present. But, because the development of the intracellular membranous components is shown to be poor in D. discoideum cells, changes in phospholipid composition in plasma membranes may somewhat affect the compositional changes in phospholipids prepared from whole cells. Thus, by comparison with the present results, we can reconsider the results obtained by using lipids from plasma membranes. How the compositional changes occur is a fundamental problem to be solved. Using a labeling procedure with L- [ methyl3H]methionine, Mato and his co-workers reported the occurrence of phospholipid methylation in D. discoideum cells during the aggregation (31, 32). Therefore, as the
COMPOSITION
28
AKIRA
most probable cause of the changes seemed to be the activation of stepwise methylation of EPG, an experiment in which radioactive precursors of phospholipids were supplied to cells at the early stage and the fate of the radioisotopes was traced during further development was carried out. As Figs. 4 and 5 show, I could not interpret the cause of the decrease of EPG and the increase of CPG as the stepwise methylation of EPG only. It seems that a regulatory mechanism of the metabolic pathway which leads to the changes in phospholipid composition during the development of D. discoideum is rather complex. It might include the reduction of EPG synthesis, the activation of selective degradation of EPG, especially of its plasmalogen form, and de nouo synthesis of CPG from CDP-choline and diglyceride, in addition to the stepwise methylation of EPG molecules. Furthermore, the possibility that the activation of de nouo synthesis of CPG through the CDP-choline pathway was due to the increased size of the choline pool in cells at the later stages is suggested. The biological significance of the changes in phospholipid composition is unknown. But there are two possibilities as follow: [l] The activities of many membranebound enzymes are supported by phospholipids and also affected by the chemical structure of phospholipid head groups (reviewed in Ref. (33)). If the phospholipid composition varies, some membrane-bound enzymes may change their activities. Since membrane-bound enzymes play a major part in various functions of the membranes, the changes in the enzyme activities then may affect the total membrane functions. The activities of some developmentally regulated membrane-bound enzymes may be regulated in such manner. [2] Recently, Cullis and De Kruijff (34,35) reported interesting results from 31P-NMR analysis of the polymorphic behavior of aqueous dispersions of phosphatidylethanolamines of natural and synthetic origins. In mammalian membranes such as the erythrocyte membrane, phosphatidylethanolamine had a tendency to destabilize the bilayer structure at a physiological
HASE
temperature. In contrast, phosphatidylcholine and sphingomyelin showed a tendency to stabilize the bilayer configuration of biological membranes. With these results, they discussed that this tendency of phosphatidylethanolamine may be related directly to many important properties of biological membranes, such as the fusion of membranes and flip-flop of phospholipids. And, during the preparation of this paper, Boggs et al. (36) reported that plasmalogen-form EPG further destabilized the lamellar structure of biological membranes. According to their observations, my results can be interpreted as follows: The high concentration of EPG in vegetative-stage cells may destabilize the bilayer structure, and this may facilitate the following functions of the cell membrane: phagocytosis, exocytosis, and cell division which should be accompanied by membrane fusion. However, for cell differentiation in this organism, which may be supported by stable and tight cell-cell contact (37), this property of membrane lipids may become inconvenient. Therefore, the levels of EPG, especially of its plasmalogen form, decrease and those of CPG increase and thus the bilayer structure may be stabilized. These possibilities need further investigation. Although there are no changes in membrane fluidity during the development (10, 38, 39), the changes in phospholipid composition may contribute to some roles of membrane lipids in the regulation of membrane functions. To clarify the importance of these changes in phospholipid composition, I have been studying the effects of modification of the phospholipid composition on the growth and development of D. discoideum. ACKNOWLEDGMENT I wish to thank Emeritus Professor I. Harada of Hokkaido University for his encouragement and Professor S. Tanifuji for his advice and critical reading of the manuscript. REFERENCES 1. NEWELL, nition
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