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The effect of polyunsaturated fatty acids and growth temperature on the differentiation of the cellular… Mohan Das, D.V. 1978

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THE .EFFECT OF POLYUNSATURATED FATTY ACIDS AND GROWTH TEMPERATURE ON THE DIFFERENTIATION OF THE CELLULAR SLIME MOLD DICYOSTELIUM DISCOIDEUM M.Sc, M.S. Univ e r s i t y of Baroda, India, 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming by D. V./Mohan Das to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1978 (c) D.V. Mohan Das In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of H I C f l o S IOIOG7 The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date c3 f O o v ' e . i ^ e ^ \0.1rt ABSTRACT C e l l s of Dictyostelium discoideum grown on media containing poly-unsaturated f a t t y acids (PUFA) exhibit impaired d i f f e r e n t i a t i o n when placed on a s o l i d surface i n the absence of a l l n u t r i e n t s . The temper-rature dependence studies on the rates of growth and d i f f e r e n t i a t i o n were performed to te s t i f t h i s was due to increased plasma membrane f l u i d i t y . PUFA had no adverse e f f e c t on c e l l growth at temperatures at or below the optimum growth temperature, 22°C. At 25°C however, there was considerable i n h i b i t i o n and at 27°C growth was completely eliminated i n the presence of PUFA. However, d i f f e r e n t i a t i o n i n PUFA c e l l s was i n h i b i t e d at a l l temperatures and although the i n h i b i t i o n was somewhat less pronounced at low temperatures there was no decrease i n the optimum temperature of d i f f e r e n t i a t i o n . Furthermore, although the i n v i t r o aggregation of vegetative c e l l s and the reaggregation of dispersed aggregation - phase c e l l s were markedly temperature dependent, PUFA supplementation did not markedly influence t h i s dependence. These data are not consistent with the hypothesis that impaired d i f f e r e n t i a t i o n i s due to increased plasma membrane f l u i d i t y . The d i f f e r e n t i a t i o n of Dictyostelium discoideum, Ax-2, an axenic s t r a i n i s markedly influenced by the temperature at which the c e l l s are grown. This temperature adaptation was not accomplished by an a l t e r a t i o n i n the f a t t y acid composition of the organism. In contrast, the wild type s t r a i n Dictyostelium discoideum NC-4, d i f f e r e n t i a t e d optimally at 25°C - 27°C, regardless of the temperature of growth. i i i TABLE OF CONTENTS Page Abstract i i L i s t of Tables v L i s t of Figures v i L i s t of Abbreviations v i i SECTION I - INTRODUCTION 1. Properties of aqueous suspension of phospholipids 1 2. Manipulation of membrane l i p i d composition 5 3. E f f e c t s of f a t t y acid manipulation on growth 9 4. E f f e c t s of l i p i d manipulation on c e l l surface properties.... 12 5. E f f e c t s of f a t t y acid manipulation on other membrane functions 14 6. L i f e cycle of Dictyostelium discoideum 16 SECTION II - MATERIALS AND METHODS Materials 21 Organisms and c u l t u r a l conditions 21 Fatty acid manipulation 21 Growth experiments 22 D i f f e r e n t i a t i o n experiments... 22 Aggregation of vegetative c e l l s 23 Reaggregation of aggregation-phase c e l l s 23 Determination of f a t t y acid composition 24 SECTION I I I - RESULTS AND DISCUSSION - Section I - The E f f e c t of Poly-unsaturated Fatty Acids on the Physiology of Dictyostelium discoideum Results Growth of p_. discoideum 26 D i f f e r e n t i a t i o n of p_. discoideum 26 Aggregation of vegetative-phase c e l l s 31 Reaggregation of aggregation-phase c e l l s . . 36 Discussion 39 i v SECTION IV - RESULTS AND DISCUSSION - Section I - The E f f e c t of Page of Growth Temperature on the D i f f e r e n t i a t i o n of Dictyostelium  discoideum Results 45 Discussion 50 SECTION V - GENERAL CONCLUSIONS AND DISCUSSION 55 BIBLIOGRAPHY 59 V LIST OF TABLES Page Table 1 Fatty acid composition of JJ. discoideum, Ax-2 grown at d i f f e r e n t temperatures 51 v i LIST OF FIGURES Page Figure 1 Schematic presentation of a polar l i p i d and l i p i d b i l a y e r s 3 Figure 2 L i f e cycle of D. discoideum.. 19 Figure 3 The e f f e c t of temperature and PUFA suplementation on the growth rate of JJ. discoideum, Ax-2 28 Figure 4 The e f f e c t of temperature and PUFA supplementation on the d i f f e r e n t i a t i o n of D_. discoideum, Ax-2 30 Figure 5 Time course of vegetative JJ. discoideum, Ax-2 aggregation at three d i f f e r e n t temperatures 33 Figure 6 E f f e c t of temperature and PUFA supplementation on the aggregation of vegetative D_. discoideum, Ax-2 c e l l s 35 Figure 7 Time course of reaggregation of dispersed aggregation-phase c e l l s of JJ. discoideum, Ax-2 at three d i f f e r e n t temperatures 38 Figure 8 E f f e c t of temperature and PUFA supplementation on the reaggregation of aggregation-phase c e l l s of JJ. discoideum, Ax-2 41 Figure 9 The e f f e c t of growth temperature on the d i f f e r e n t i a t i o n of JJ. discoideum, Ax-2 c e l l s at various temperatures 47 Figure 10 The e f f e c t of growth temperature on the d i f f e r e n t i a t i o n of D. discoideum, Ax-2 c e l l s grown without bovine serum albumin i n the medium 49 Figure 11 The e f f e c t of growth temperature on the time taken for f r u i t i n g body formation i n JJ. discoideum, NC-4 53 LIST OF ABBREVIATIONS Bovine serum albumin Ethylene diamine tetraacetate Polyunsaturated f a t t y acid Unsaturated f a t t y acid v i i i ACKNOWLEDGEMENTS I wish to thank Dr. Gerald Weeks f o r h i s h e l p f u l suggestions and constant encouragement during a l l stages of t h i s work. I a l s o wish to thank Dr. J.J.R. Campbell and other members of my committee f o r t h e i r c r i t i c a l e v a l u a t i o n of t h i s work and h e l p f u l suggestions. Thanks are due to Mrs. Kathy LaRoy, and other members of our l a b o r a t o r y f o r t h e i r help. I thank a l l my f r i e n d s who made l i f e bearable during v a r i o u s stages of t h i s work. I a l s o thank Mrs. Rosario Bauzon f o r t y p i n g t h i s t h e s i s . INTRODUCTION Properties of Aqueous Suspensions of Phospholipids. Phospholipids are the major constituents of b i o l o g i c a l membranes Considerable i n s i g h t into the physical state of these molecules i n b i o l o g i c a l membranes has come from studies on the aqueous suspensions of phospholipids, since these molecules form b i l a y e r s under these conditions. L i p i d b i l a y e r s consist of two layers of polar l i p i d s oriented i n such a way that the polar "heads" of the l i p i d s face the aqueous phase and the nonpolar " t a i l s " form an inner hydrophobic core. I t has been shown that a l l b i o l o g i c a l membranes are i n part comprised of l i p i d b i l a y e r s . A t y p i c a l polar l i p i d and a l i p i d b i l a y e r are schematised i n Figure 1. These b i l a y e r s undergo a temperature dependent phase t r a n s i t i o n from a gel state where the acyl chains are c l o s e l y packed to a l i q u i d c r y s t a l l i n e state where the a c y l chains are i n a more random array (Figure 1). These t r a n s i t i o n s have been detected by a number of d i f f e r e n t techniques such as X-ray d i f f r a c t i o n (1), d i f f e r e n t i a l thermal analysis (2), dilatometry (3), electron spin (4) or nuclear magnetic resonance (5), l a s e r Raman investigations (6) and fluo-. rescence probes (7). This phase t r a n s i t i o n occurs over a very narrow range (At) for b i l a y e r s of a s i n g l e homogenous phospholipid, and the t r a n s i t i o n temperature (Tt) i s e a s i l y defined. For example, for d i p a l m i t o y l phosphatidyl choline the temperature range over which the phase t r a n s i t i o n occurs i s only of the order of 2-3° C and the mid-point 2 Figure 1. Schematised presentation of (a) a phospholipid, (b) a l i p i d bilayer in the gel state-and (c) a l i p i d bilayer in the liquid-crystalline state. P-polar 'head' group; N-nonpolar fatty acyl ' t a i l s ' . n A A M X A A / Q Q OQQQQQQ QQCXIIQ 0 0 0 0 0 b b b d 8 4 of the t r a n s i t i o n i s c l e a r l y defined at 41° C (2). In more complex mixtures, however, such as the l i p i d extracts of b i o l o g i c a l membranes, the At i s much broader and the Tt i s usually defined as the temper-ature corresponding to the mid-point of the t r a n s i t i o n . For example, for the l i p i d s of E_. c o l i grown under f a i r l y t y p i c a l conditions the At was found to be 11 to 13° C and Tt was between 14 and 17° C (8). It has been suggested by McConnell and coworkers that during the broad phase t r a n s i t i o n exhibited by at l e a s t some l i p i d mixtures l a t e r a l phase separations occur, such that there are regions of more s o l i d and more f l u i d l i p i d within the b i l a y e r (9). The Tt i s markedly dependent upon the nature of the l i p i d compo-nents as summarized below. (i) The Tt of phospholipid acylated with saturated f a t t y acids i s higher than those with mono-unsaturated f a t t y acids. Further desaturation further decreases the Tt. ( i i ) Tt of phospholipid i s dependent upon chain length. Increasing the chain length increases the t r a n s i t i o n temperature. ( i i i ) Phospholipids containing trans double bonds have a higher Tt than those containing the homologous c i s double bonds. (iv) Phospholipids with f a t t y acids containing branched, cyclopropane or other bulky side groups have lower Tt than the corresponding saturated f a t t y acids. (v) Heterogeneity of e i t h e r chain length or saturation broadens the range of the t r a n s i t i o n (see above). 5 (vi) The Tt i s also dependent on the polar head groups of the phospholipids i n the bilayer. For example, the Tt for vesicles of phosphatidyl choline i s lower than that for phosphatidyl ethanolamine. A pure 1,2-dimyristoyl phosphatidyl choline v e s i c l e has a Tt at 27° C, while the corresponding 1,2-dimyristoyl phosphatidyl ethanol-amine v e s i c l e exhibits a Tt at 55° C. Studied by d i f f e r e n t i a l scanning calorimetry these transitions give sharp peaks (At =4 to 6° C), while a 1:1 mixture of the above phospholipids shows a broad peak (At = 16° C) the mid-point occurring at 43° C (10). One other important point that has emerged from studies of t h i s type i s the influence of s t e r o l s , ubiquitous constituents of eucar-yotic c e l l membranes, on bilayer structure. The addition of cholesterol to phospholipid bilayers abolishes the phase t r a n s i t i o n , presumably by ' f l u i d i z i n g ' the gel phase and ' s o l i d i f y i n g ' the l i q u i d c r y s t a l l i n e phase (10). 2. The Manipulation of Membrane L i p i d Composition. Some of the most d e f i n i t i v e studies that confirm the basic rules summarized i n the l a s t section r e l a t i n g f a t t y acid composition to membrane f l u i d i t y have come from studies on the bacteria Acholeplasma  l a i d a w i i (11) and.Escherichia c o l i (12). A. l a i d l a w i i r e a d ily incorporates fa t t y acids and sterols from the growth media into i t s membranes and the membranes can be extensively modified. In E_. c o l i , f a t t y acid requiring mutants have been isolated (13,14) and 6 manipulation of fatty acid content has been achieved by varying the supplemented fatty acid. The membrane fatty acid composition of E_. c o l i has also been modified by the use of specific inhibitors in the fatty acid bio-synthetic pathway. 3-Decynoyl-N-acetylcysteamine (3-DNAC) is a specific inhibitor of unsaturated fatty acid (UFA) synthesis (15), and the growth inhibition can be reversed by supplementing an appropriate UFA. Similarly, the antibiotic cerulenin inhibits both unsaturated and saturated fatty acid synthesis in E_. c o l i and supplementation of a saturated and an unsaturated fatty acid is required to reverse this inhibition (16). By feeding the inhibited cells with known fatty acids, the membrane fatty acid composition of E_. c o l i may be manipulated. Fatty acid manipulation has since been extended to other more complex biological organisms than the bacteria. Fatty acid requiring mutants have proved useful in manipulating the fatty acid composition of Yeast and Neurospora (17,18). In one study, however, i t was found that wild type yeast took up supplemented fatty acid (19) resulting in a modified fatty acid composition of the mitochondrial membrane. In addition, considerable modification of the membrane fatty acids of the cili a t e d protozoan Tetrahymena pyriformis may be accomplished by growing them in suitable fatty acid or by manipulating the growth temperature (20). Direct fatty acid incorporation into cultured animal c e l l membranes has also been achieved, but the fatty acid had to be complexed in some way because of the toxicity of the free fatty acids. In one study, the fatty acids were supplemented as sorbitan esters 7 (Tweens) to serum-free minimal e s s e n t i a l media that contained no a d d i t i o n a l l i p i d , and both BHK and LM c e l l s incorporated the f a t t y acids during growth (21,22). The endogenous f a t t y acid synthesis was minimized by the i n c l u s i o n of a biotin-antagonist i n the medium. Ferguson and coworkers (23) however, found that LM c e l l s could be grown i n a serum-free medium by simply supplementing the media with f a t t y acids complexed to bovine serum albumin. Considerable incorp-oration of the exogenous f a t t y acids was obtained. Horwitz and co-workers (24) found that the membrane f a t t y acids of 3T3 and SV 101-3T3 c e l l s could be altered by growth in medium containing exogenous f a t t y acids, l i p i d - d e p l e t e d serum and av i d i n to block endogenous f a t t y acid synthesis. C u r t i s and coworkers (25,26) used the c e l l s ' own a c y l transferase system to incorporate selected f a t t y acids into phosphatidyl l i p i d s of the plasma membranes of chick neural r e t i n a c e l l s and mouse L-929 c e l l s . The c e l l s were supplemented with the chosen f a t t y a c i d s , ATP and CoA to f a c i l i t a t e the acyl transferase reaction. More recently, animal c e l l mutants defective i n f a t t y acids biosynthesis have been i s o l a t e d and should prove u s e f u l i n l i p i d modification studies. Chang and Vagelos (27) i s o l a t e d a mutant from cultured Chinese hamster ovary c e l l s r e q u i r i n g un-saturated f a t t y acids. The enzymatic defect i n the mutant was found to be l o c a l i z e d i n the microsomal stearoyl-CoA desaturase. Fatty acid manipulation i n whole animals has also been accomplished by modifying the l i p i d compositions of t h e i r d i e t (28-32). In general, the animals compensate the modifications i n f a t t y acid content to maintain a s u i t a b l e f l u i d i t y of the c e l l membranes. 8 From the above discussion i t i s cl e a r that f a t t y acid composit-ions of the membranes of a v a r i e t y of b i o l o g i c a l systems are amenable to manipulation for studies on the function of these membrane components i n c e l l s . I t i s more d i f f i c u l t to manipulate phospholipid head group compositions since phospholipids tend to be degraded by c e l l s . Nonetheless, there has been considerable progress i n t h i s area. Glaser and coworkers (33) altered the phospholipid composition i n LM-cells by growth i n serum-free tissue culture medium containing either natural (N-methyl ethanolamine or N,N-dimethylethanolamine) or unnatural (1-2-amino-I-butanol or 3-amino-I-propanol) constituents; up to 50% of the c e l l u l a r phospholipids contained the analogue supplied. In addition when the c i l i a t e d protozoan, Tetrahymena  pyriformis was grown in, medium supplemented with 1-0-hexadecyl g l y -c e r o l , a remarkable a l t e r a t i o n i n the polar headgroup composition of plasma membrane was seen (20). There was a large increase i n 2-amino ethyl phosphonolipid with a compensatory decrease i n the ethanol-amine phosphoglyceride content of the plasma membranes. It has also been observed by some workers that phospholipids can be incorporated d i r e c t l y into the membranes of cultured animals c e l l s by incubating the c e l l s with phospholipid v e s i c l e s of desired composition (34,35). In t h i s case, the phospholipid v e s i c l e s appear to be taken up in t a c t as i f by fusion with the plasma membrane l i p i d b i l a y e r . A number of mutants of E_. c o l i defective i n the phospholipid biosynthetic pathways have been recently i s o l a t e d . One such mutant i s auxotrophic for g l y c e r o l as a r e s u l t of defective glycerol-3-P 9 a c y l transferase, the f i r s t enzyme of phospholipid biosynthesis i n E_. c o l i (36). Phospholipid synthesis could be stopped and r e -i n i t i a t e d by deprivation of g l y c e r o l followed by i t s readdition. This mutant does not allow manipulation of the phospholipid species since t o t a l phospholipid synthesis i s blocked. However, other mutants defective i n phosphatidyl serine synthetase (37) and phosphatidyl serine decarboxylase (38) have been i s o l a t e d and characterized. These mutants have proved u s e f u l i n manipulating the phospholipid composition of E_. c o l i membrane. Such mutants i f a v a i l a b l e i n higher organisms would provide a convenient system to study the part played by the d i f f e r e n t phospholipids i n the structure and function of b i o l o g i c a l membranes, but t h e i r i s o l a t i o n may be d i f f i c u l t . 3. E f f e c t s of Fatty Acid Manipulation on Growth. The i s o l a t i o n of an UFA auxotroph of E_. c o l i (13) proved that UFAs were e s s e n t i a l for normal c e l l u l a r physiology. The UFA auxotrophs of E_. c o l i could be supplemented with a wide v a r i e t y of f a t t y acids. Most long-chain f a t t y acids having a hydrocarbon chain with a s t e r i c disorder i n the c e n t r a l portion of the hydrocarbon chain supported growths , Cis-monounsaturated .andccis-polyunsaturated f a t t y acids, trans-unsaturated f a t t y acids, various cyclopropane f a t t y acids, brominated f a t t y acids and some branch chain f a t t y acids, a l l reversed the defect i n unsaturated f a t t y acid synthesis. Some unsaturated and branched chain f a t t y acids w i l l not support growth, the physio-l o g i c a l reason for which i s unknown. Overath and coworkers (39) studied the temperature dependence of growth of a double mutant of E_. c o l i which could not synthesize or degrade UFA, i n the presence of d i f f e r e n t UFA's. They found that the * 9 growth properties of the c e l l s supplemented with cis-18:1(A ) and c i s 18:1 (A"'""'") were i d e n t i c a l to those of wild type of E_. c o l i . 9 12 9 12 15 Supplementation with c i s 18:2 (A ' ) and c i s 18:3 (A ' ' ) did not a l t e r the minimum temperature of growth but the maximum temperature was lowered from 45° C to 40° C. In the case of cyclopropane f a t t y acid and trans-18:1, the minimal temperature of growth was r a i s e d , but the maximum temperature was unaffected. The r e s u l t s of t h i s study suggested that when the membrane becomes too f l u i d (cis-18:3 at high temperature) or too r i g i d (cyclopropane and trans-18:1 at low temperature) growth ceases. The high minimum temperature for trans-18:1 grown c e l l s (30° C) i s very dramatic and has been confirmed by a number of workers (40,41). Furthermore, the b a c t e r i a grown on trans-18:1 at 37° C l y s e when trans-ferred to low temperatures (40). Several l a b o r a t o r i e s have shown that UFA auxotrophs l y s e upon prolonged starvation for UFA (12). S i l b e r t (42) suggested that the l y s i s was due to the synthesis of abnormal phospholipids containing two saturated f a t t y acids and thus changing the phase;properties of the membrane. Cronan and Gelman (43) studying a s i m i l a r mutant showed that t h i s organism needed at l e a s t 15 to 20% unsaturated f a t t y acid to grow at 37° C. Saturated f a t t y acids are also an e s s e n t i a l component for normal membrane function. This was shown by the i s o l a t i o n of mutants of E_. c o l i that required both saturated and unsaturated f a t t y acids for growth (44) and by the need for both saturated and unsaturated f a t t y acids to reverse the i n h i b i t o r y e f f e c t of cerulenin (16). Studies on the mutant defective i n both saturated and unsaturated Please refer to footnote (a) on Table 1. 11 f a t t y acid synthesis (14) showed that the maximum l e v e l s for the saturated and unsaturated f a t t y acids for normal growth of E_. c o l i were 74% and 82% r e s p e c t i v e l y . Studies on the e f f e c t of membrane-lipid phase t r a n s i t i o n s on membrane structure and the growth of Acholeplasma l a i d l a w i i (45) confirm the idea that f o r normal membrane function, the membrane should not be too r i g i d or too f l u i d . Both the absolute rates and the temperature c o e f f i c i e n t s of c e l l growth are s i m i l a r for c e l l s whose membrane l i p i d s are e n t i r e l y or predominantly i n the l i q u i d c r y s t a l l i n e state, but absolute growth rates decline r a p i d l y and temperature c o e f f i c i e n t s increase when most.of :the membrane l i p i d s become s o l i d i f i e d . Some c e l l growth does continue even at temper-atures at which l e s s than 10% of the t o t a l l i p i d remains i n the f l u i d s t ate. The minimum growth temperature under c e r t a i n conditions i s c l e a r l y defined by the lower boundary of the gel to l i q u i d - c r y s t a l -l i n e phase t r a n s i t i o n of the membrane l i p i d s . The physical state of membrane l i p i d s can also influence the optimum and maximum growth temperature. Wisnieski and Kiyamoto (46) studying a mutant Saccharomyces 9 c e r i v i s a e defective i n the A -desaturase enzyme found that a wide 9 range of f a t t y acids could replace the n a t u r a l l y occurring 16:1(A ) c i s and 18:1(A ) c i s f a t t y acids. The supplemented f a t t y acids were incorporated into the membranes of t h i s yeast i r r e s p e c t i v e of t h e i r double-bond p o s i t i o n , s t e r i c c onfiguration, chain length and degree of unsaturation. However, they found that the trans-18:1(A ) f a t t y acid could not support growth. This can be explained by trans-18:1 containing membranes being too r i g i d to allow normal membrane functions at 30° C, the optimum growth temperature f o r yeast. Some studies have been reported recently on the e f f e c t of membrane l i p i d a l t e r a t i o n on animal c e l l p r o l i f e r a t i o n . Hatten and coworkers (47) found that the growth of 3T3 c e l l s i n a l i p i d - d e p l e t e d medium with a biotin-antagonist was arrested while that of the transformed SV 101-3T3 c e l l s was not. This i n h i b i t i o n could be overcome by exogenous addition of b i o t i n or f a t t y acids. They also found that the saturation density of 3T3 and SV 101-3T3 c e l l s grown i n such a medium supplemented with exogenous f a t t y acids was a function of the p a r t i c u l a r f a t t y acid supplemented and the c e l l type. S i m i l a r l y , Fox and coworkers (48) found that LM c e l l s grown at 28° C showed a marked decrease i n the maximum, c e l l density achieved compared with that at normal temperatures. This was reversed by feeding the c e l l s with f a t t y acids which might be expected to 9 increase the f l u i d i t y of the membrane b i l a y e r (cis-18:1 (A ) and 9 12 15 'N cis-18:3 (A ' ' )J The supplementation of f a t t y acids having a 9 higher t r a n s i t i o n temperature (trans'-18: i ( A ) or 19:0) ' did not enhance the growth of these c e l l s . The above r e s u l t s suggest that the p r o l i f e r a t i o n of animal c e l l s i s dependent on membrane l i p i d composition and hence the f l u i d i t y of the membrane. Ef f e c t s of L i p i d Manipulation on C e l l Surface Properties. Studies have also been performed on the surface properties of c e l l s to see the e f f e c t s of f a t t y acid manipulation. A l t e r a t i o n of the f a t t y a c i d composition of mouse LM c e l l l i p i d s dramatically affected the Concanavalin A binding and Concanavalin A-mediated 13 hemadsorption properties of these c e l l s . The marked increase i n the binding and hemadsorption that was observed at 15-19° C i n normal c e l l s was s h i f t e d to 22-28° C for c e l l s containing a higher proportion of saturated f a t t y acids and lowered to 7-11° C for c e l l s containing polyunsaturated f a t t y acids (49). S i m i l a r l y , Horwitz and coworkers (24) found a s i g n i f i c a n t e f f e c t on the temperature dependence of agglutination of 3T3 and SV 101 3T3 c e l l s by wheat germ agg l u t i n a t i n and Concanavalin A, when the c e l l membranes were manipulated by f a t t y a c i d incorporation. The e f f e c t of temperature on the agglutination by the two l e c t i n s was somewhat d i f f e r e n t and the temperature dependency was i n both cases modified by the nature of the incorporated f a t t y acid. These experiments suggested that the l i p i d phase i n the plasma membranes of these c e l l s i s heterogeneous, and that l e c t i n agglutination i s dependent upon the f a t t y acid composition of the membrane. In a l l studies the temperature dependence of agglutination was i d e n t i c a l f o r both normal (3T3) and the transformed (SV 101 3T3) c e l l types. Curt i s and coworkers (25,26,50,51) studied the e f f e c t of f a t t y a c i d manipulation on c e l l adhesion of chick neural r e t i n a c e l l s and mouse L929 c e l l s . Such a l t e r a t i o n s were found to cause changes i n c e l l adhesion i n these c e l l s . For example, when mouse L929 c e l l s were incubated i n the presence of s t e a r i c (18:0) or ara c h i d i c 20:0) acid c e l l adhesion l e v e l s increased while s i m i l a r incubations with cis-18:2 ( A 9 ' 1 2 ) (jinolelc) and cis-18:3 ( A 9 ' U ' 1 5 ) ( L i n o l e n i c ) acids decreased c e l l adhesion. They also found s i g n i f i c a n t changes i n the plasmalemmal f l u i d i t y , using an assay based on patching of fluorescent antibody (26). Here again they 14 found that the unsaturated f a t t y acids have a f l u i d i z i n g e f f e c t on the membrane while saturated f a t t y acids exert a s o l i d i f y i n g e f f e c t , when substituted into the membrane. They suggested that membrane f l u i d i t y affected c e l l adhesion by a l t e r i n g the association of the membrane s i t e s involved i n adhesion. An a d d i t i o n a l study that indicates a c o r r e l a t i o n between that of membrane f l u i d i t y and c e l l surface properties i s that of Cohn and coworkers (-52). They incorporated saturated and trans-unsaturated f a t t y acids complexed to bovine serum albumin into mouse peritoneal macrophages and found that the incorporation of both acids resulted i n considerably lower l e v e l s of phagocytosis and pinocytosis. These studies a l l i n d i c a t e that the c e l l surface properties of mammalian c e l l s are altered by changes i n membrane l i p i d composition. 5. E f f e c t s of Fatty Acid Manipulation of Other Membrane Functions. The e f f e c t of temperature on sugar transport and on the a c t i v i t y of many membrane bound enzymes have been studied i n both native and f a t t y acid manipulated c e l l s . The r e s u l t s i n d i c a t e that membrane f l u i d i t y markedly e f f e c t s these functions. (a) Sugar transport The e f f e c t of temperature on the rate of transport of lactose and B-glucoside i n unsaturated f a t t y acid (UFA) auxotrophs of E_. c o l i was found to depend on the f a t t y acids supplemented. The breaks i n the Arrhenius plots for transport occurred at r e l a t i v e l y high temperatures for the c e l l s supplement-9 ed with the more ordered f a t t y acid (trans-18:1A ) and at low 15 temperatures for the more disordered f a t t y acids (cis-18:2(A ' ) and cis-18:3 ( A 9 ' 1 2 ' 1 5 ) ) (39,53). Overath and coworkers (8,54) found good agreement between the temperature t r a n s i t i o n s for B-galactoside transport and the temperature t r a n s i t i o n of the membrane l i p i d s as detected by fluorescence electron spin resonance, l i g h t s c a t t e r i n g and densitometry. Fox and coworkers (55 ,57 ) found multiple breaks i n the Arrhenius plots for 6-galactoside and 3-glucoside transport i n E_. c o l i , which coincided with the d i s c o n t i n u i t i e s i n temperature plots of spin probe p a r t i t i o n i n g i n the membranes. These experiments suggest a c o r r e l a t i o n between the membrane l i p i d composition and the rate of transport of sugars across the membranes. (b) Membrane bound enzymes A number of enzymes are known to be t i g h t l y or loo s e l y associated with c e l l u l a r membranes. There i s much in t e r e s t i n the necessity and influence of membrane l i p i d on these enzymes. A c e r t a i n amount of evidence suggests that the t r a n s i t i o n i n an Arrhenius plot for a membrane bound enzyme may be a r e f l e c t i o n of the i n t e r a c t i o n of the protein with i t s associated l i p i d (58). Recombination experiments of membrane enzymes and t h e i r associated l i p i d s have been done i n some systems, proving the e s s e n t i a l r o l e of these l i p i d s i n the enzyme action. One representative example i s the study of Kimelberg and Papahadjo-poulos (59) who found that the a c t i v i t y of d e l i p i d i z e d and p u r i f i e d (Na , K ) stimulated ATP-ase could be stimulated 20-f o l d by the addition of either phosphatidyl g l y c e r o l or phos-phatidyl serine. They studied the temperature-dependence of the reconstituted a c t i v i t y using known f a t t y a c y l chains i n the phosphatidyl g l y c e r o l s and found that the temperature trans-i t i o n s i n the Arrhenius plots varied depending upon the nature of the supplemented a c y l chains. In addition, they found good agreement between the temperatures of d i s c o n t i n u i t i e s i n Arrhenius plots and the phase t r a n s i t i o n s of the phosphatidyl glycerols as measured by a d i f f e r e n t i a l scanning calorimeter. Studies on the temperature dependence of a number of d i f f e r e n t membrane bound enzymes i n UFA auxotrophs of E_. c o l i supplemented with various f a t t y acids also indicate that the breaks i n Arrhenius plots correspond to f l u i d i t y of the membrane l i p i d s . In addition i t has been shown that d i f f e r e n t enzymes i n the same E_. c o l i membrane have d i f f e r e n t l i p i d s associated with them, r e s u l t i n g i n d i s t i n c t temperature dependence p r o f i l e s . L i f e Cycle of Dictyostelium discoideum. Dictyostelium discoideum i s found i n nature as a s o i l amoeba i n forest d e t r i t u s . In i t s natural habitat the c e l l s feed on b a c t e r i a of decaying matter and d i v i d e by binary f i s s i o n , l i k e many other amoeboid organisms. However, when the l o c a l environment i s depleted of food, the c e l l s undergo a process of d i f f e r e n t i a t i o n . In the laboratory d i f f e r e n t i a t i o n may be induced by depositon of the harvested c e l l s on to f i l t e r papers i n the absence of nutrients. The i n d i v i d u a l c e l l s now c o l l e c t i n large sreaming patterns to form aggregates containing up to 10 c e l l s . The c e l l s which form aggregates are c a l l e d aggregation-competent c e l l s . In due course, the aggregates form a migratory slug or pseudoplasmodium which i s covered by a c e l l u o s i c sheath (60). Af t e r a v a r i a b l e period of migration the pseudoplasmodium stops and develops into a f r u i t i n g body, comprised of s t a l k c e l l s and spore c e l l s (Figure 2). A number of a x e n i c a l l y growing mutants of t h i s organism has been is o l a t e d which can be grown i n the laboratory i n a r i c h l i q u i d medium i n the absence of b a c t e r i a . The d i f f e r e n t i a t i o n of the i d e n t i c a l u n i c e l l u l a r amoebae into two d i s t i n c t types of c e l l s provides a simple eucaryotic model for studies on c e l l d i f f e r e n t i a t i o n . During the u n i c e l l u l a r amoeboid stage of growth the plasma membranes have no s p e c i f i c c e l l i n t e r a c t i o n r o l e , but t h i s s i t u a t i o n changes during d i f f e r e n t i a t i o n . I t i s known that the l i p i d composition of Dictyostelium  discoideum changes during the d i f f e r e n t i a t i o n process (61,62), but i t i s not known i f these changes are e s s e n t i a l for c e l l - c e l l i n t e r -faction. One possible approach to determine the r o l e for the l i p i d b i l a y e r i n c e l l - c e l l i n t e r a c t i o n i s to modify the l i p i d compositon of the plasma membrane and study the influence of t h i s change on c e l l d i f f e r e n t i a t i o n . In an e a r l i e r study from t h i s laboratory (63) i t was shown that the axenic mutant, Ax-2 could be grown i n the presence of PUFA and incorporate large quantities of these acids into the c e l l u l a r l i p i d s . These c e l l s exhibited normal growth, but impaired d i f f e r e n t i a t i o n and i t was suggested that the impaired d i f f e r e n t i a t i o n was due to an increase i n plasma membrane f l u i d i t y . To test t h i s hypothesis the temperature dependence of some c e l l u l a r 18 Figure 2. L i f e Cycle of D_. discoideum The numbers indic a t e the number of hours elapsed a f t e r the amoebae were deposited on f i l t e r pads for d i f f e r e n t i a t i o n . Between 0 hrs to 14 hrs-aggregation stage. Between 14 hrs and 16 hrs - the pseudoplasmodial stage. Between 16 hrs to 24 hrs - culmination stage. 20 functions has been studied and these studies are described i n the f i r s t part of t h i s t h e s i s . An alternate method of manipulating the membrane f l u i d i t y of plasma membranes of microorganisms i s the growth of the c e l l s at d i f f e r e n t temperatures. Bacteria (12), Yeast (64) and Tetrahymena (20) a l l appear to modify t h e i r f a t t y acid compositions under these conditions to maintain constant membrane f l u i d i t y . In an attempt to modify the l i p i d composition of D_. discoideum, the axenic s t r a i n , Ax-2, was grown i n l i q u i d medium and the wild type s t r a i n , NC-4 was grown i n ass o c i a t i o n with E_. aerogenes at d i f f e r e n t temperatures. The e f f e c t of growth-temperature on the f a t t y acid composition and the d i f f e r e n t i a t i o n of the organism was studied and these studies are described i n the second part of t h i s t h e s i s . 21 MATERIALS AND METHODS Materials B a c t e r i o l o g i c a l peptone and yeast extract were obtained from Oxoid. Bovine serum albumin (BSA) was the f a t t y acid poor grade from Calbiochem. Fatty acids were obtained from Sigma and a l l other reagents were the best a v a i l a b l e from Fisher S c i e n t i f i c Co. Organisms and c u l t u r a l conditions. Dictyostelium discoideum s t r a i n s NC-4 and Ax-2 were obtained from Dr. J.M. Ashworth some years ago and have since been maintained i n t h i s laboratory.- NC-4 i s a !wild type' s t r a i n and capable of growing only on b a c t e r i a l food source while the•axenicmutant Ax-2 grows well i n a nutrient l i q u i d medium. NC-4 c e l l s were grown i n a s s o c i a t i o n with Enterobacter aerogenes on nutrient agar plates as described by Sussman (65). The Ax-2 s t r a i n was grown on HL-5 medium, a r i c h nutrient medium containing Bacto-peptone (1.54%), yeast extract (1.54%), Na 2H P0 4 (0.1%), K H 2P0 4 (0.5%) and glucose at a concentration of 86 mM. The inoculated media were i n -cubated on gyratory shakers at 200 rpm. Both NC-4 and Ax-2 s t r a i n s gave optimal growth when incubated at 22°C. Fatty acid manipulation. For f a t t y acid incorporation the HL-5 medium was supplemented with f i l t e r - s t e r i l i z e d bovine serum albumin to a f i n a l concentration of 0.4%. To t h i s were added s t e r i l e ethanol solutions of the three polyunsaturated f a t t y acids, l i n o l e i c , l i n o l e n i c and arachidonic. to give f i n a l 22 concentrations of 100 \M for each acid i n the polyunsaturated f a t t y acid (PUFA) supplemented medium. Inocula for the PUFA medium and for bovine serum albumin supplemented controls were exponentially growing c e l l s adapted to growth i n the presence of bovine serum albumin. Growth was monitored by determining c e l l numbers i n a hemocytometer counting chamber. In a l l comparisons between normal and polyunsaturated f a t t y acid incorporated (PUFA) c e l l s , the normal c e l l s were grown i n HL-5 medium containing 0.4% bovine serum albumin. Growth experiments. Inoculum was added to 65 ml of unsupplemented or PUFA supplemented medium i n 250 ml Erlenmeyer f l a s k s to give an i n i t i a l c e l l density of 2-5 x 10^ c e l l s / m l . These were gyrated at 200 rpm at the indicated temperatures. Samples were a s e p t i c a l l y withdrawn at i n t e r v a l s of 12 hours and counted i n a hemocytometer counting chamber to monitor the c e l l d e n s i t i e s . The generation times were calculated from the growth curves thus obtained. D i f f e r e n t i a t i o n experiments. NC-4 c e l l s were harvested from the plates when confluent l y s i s of the b a c t e r i a l lawns appeared and washed four times by gentle suspension i n cold deionized water followed by c e n t r i f u g a t i o n at 700 x g for two minutes. The f i n a l b a c t e r i a - f r e e c e l l suspension was resuspended i n cold deionized water at 1.5 x 10 c e l l s / m l . Ax-2 c e l l s were grown to a density of approximately 5 x 10 c e l l s / ml and harvested by c e n t r i f u g a t i o n at 700 x g for 2 minutes. The c e l l p e l l e t s were washed i n cold deionized water as above and the f i n a l 23 p e l l e t was suspended i n cold deionized water at 1.5 x 10 c e l l s / m l . Aliquots of 0.3 ml each of the above c e l l suspensions were spread onto Whatman No. 50 f i l t e r papers (4.25 cm) soaked i n lower pad -2 -3 -2 so l u t i o n ( K c l , 2 x 10 M; Mg Cl- 2, 3 x 10 M; phosphate, pH 6.5, 5 x 10 M and streptomycin sulphate, 500 mg/1) ( 6 6 ) . The f i l t e r s were maintained i n 60 mm p l a s t i c p e t r i dishes, incubated at the desired temperatures and observed p e r i o d i c a l l y i n order to determine the times required for pseudoplasmodium and f i n a l f r u i t i n g body formation. Aggregation of vegetative c e l l s . Exponentially growing c e l l s were harvested at a density of approx-imately 5 x 10 c e l l s / m l and washed twice with cold deionized water. The washed c e l l s were resuspended i n 2.0 ml of 0.001 m T r i s - C l buffer (pH 7.4) -2 -2 -3 containing 10 M NaCI, 10 M KCl and 1.5 x 10 M Ca C l 2 (Bonner s a l t s -T r i s ) at a c e l l density of 2.0 - 2.5 x 10 c e l l s / m l i n 20 ml glass v i a l s and gyrated at 120 rpm at the indicated temperature. At the end of the incubation period the number of unaggregated c e l l s (single c e l l s ) was determined using a hemocytometer counting chamber and the percentage of aggregated c e l l s was calculated from the d i f f e r e n c e between t h i s value and the t o t a l c e l l number. Reaggregation of aggregation - phase c e l l s . Aggregation-phase c e l l s were prepared by spreading washed exponential 8 phase c e l l s at a density of approximately 2 x 10 c e l l s / m l on to a 2% agar plate containing Bonner's s a l t s - T r i s at 1/10 normal concentration. The plates were incubated at 22°C for 10 hours. The aggregates were harvested from the plates using 16.7 mM sodium-potassium phosphate, pH 24 6.8, 10 mM EDTA (phosphate-EDTA) and washed twice with the same buffer. The c e l l s were resuspended i n 2.0 ml phosphate-EDTA at a density of 2-2.5::x 10 c e l l s / m l and any remaining aggregates were dispersed by f i v e passages through a 20 guage needle attached to a 2.0 ml disposable syringe. The c e l l suspension was i n a 20 ml glass v i a l gyrated at 120 rpm at the indicated temperatures. At the end of the incubation period unaggregated c e l l s (present as singles or doubles) were counted using a hemocytometer counting chamber and the percentage of aggregated c e l l s was calculated from the d i f f e r e n c e between t h i s value and the t o t a l number of c e l l s . Determination of f a t t y acid composition. Harvested and washed c e l l s were resuspended i n d i s t i l l e d water and extracted with choloroform/methanol by a modified Bligh and Dyer procedure for c e l l f r a c t i o n s (67) as described by Kates (68) . L i p i d extracts were assayed for f a t t y acids using the following scheme. A su i t a b l e aliquot was evaporated to dryness under nitrogen and 1.0 ml d i s t i l l e d water and 1.0 ml of 15% KOH i n methanol added. Saponification was achieved by incubation at 70°C for 1 hour under r e f l e x . The so l u t i o n was evaporated to a small volume (approximately 0.3 ml) and 1.0 ml of d i s t i l l e d water added. Non-saponif.iable material was removed by extraction three times with n-pentane. Following a c i d i f i c a t i o n of the sample with 0.25 ml of 24 N H^SO^ the saponifiable f r a c t i o n was removed by pentane extraction. The saponifiable f r a c t i o n was evaporated to dryness and f a t t y acids methylated by addition of 1.0 ml of BF^ i n methanol and incubation at 37° C overnight. Samples were analyzed by g a s - l i q u i d chromatography on a 12% diethylene g l y c o l succinate column at 150 C. The component methylated acids were i d e n t i f i e d by comparison with the retention times of authentic standards, and by comparison with the previously published f a t t y acid composition of Dictyostelium discoideum ( 6 9 ) . 26 RESULTS AND DISCUSSION Section I The E f f e c t of Polyunsaturated Fatty Acids on the Physiology of Dictyostelium discoideum Results. Growth of p_. discoideum. The growth of p_. discoideum, Ax-2 i n HL5, BSA-supplemented media i s markedly temperature dependent (Fig. 3). Optimum growth rates were obtained between 20° C - 22° C, either i n the presence or absence of PUFA. The i n c l u s i o n of PUFA had no adverse e f f e c t at a l l temperatures below 22° C, i n fact growth was s l i g h t l y stimulated. At 25° C, however, PUFA caused considerable i n h i b i t i o n and at 27° C there was no growth at a l l ( F i g. 3). D i f f e r e n t i a t i o n of D_. discoideum. The data i n F i g . 4 shows that temperature also a f f e c t s the time required for both slug and f i n a l f r u i t i n g body formation. The optimum temperature for the d i f f e r e n t i a t i o n of c e l l s grown i n the absence of PUFA was 22° C and d i f f e r e n t i a t i o n was more rapid above the optimum temperature than below i t . The proportion of s t a l k and spore c e l l s and the o v e r a l l f r u i t i n g body s i z e was also influenced by temperature, as has been described i n d e t a i l e a r l i e r by other workers ( 7 0 ) . Growth i n the presence of PUFA caused an i n h i b i t i o n of d i f f e r e n t i a t i o n . Fewer f r u t i n g bodies were produced i n comparison to controls. No attempt was made to quantitate t h i s reduction, however, since the sizes of the f i n a l f r u i t i n g bodies i n PUFA supplemented c e l l s was extremely v a r i a b l e . In addition, the time taken for the formation of slugs and f r u i t i n g bodies 27 Figure 3 Abscissa: temperature (°C); ordinate 1/generation time (hrs X) The e f f e c t of temperature and PUFA supplementation on the growth rate of JJ. discoideum, Ax-2. The exponential growth rate of D. discoideum grown i n the presence ( A ) or the absence (0) of PUFA was measured at each of the indicated temperatures as described under Methods. 28 29 Figure 4. Abscissa: temperature (°C); ordinate 1 / d i f f e r e n t i a t i o n time (hrs ) The e f f e c t of temperature and PUFA supplementation on the d i f f e r e n t -i a t i o n of p_. discoideum, Ax-2. C e l l s were grown at 22°C either i n the presence, (A) or the absence, (0) of PUFA and then set up to d i f f e r e n t -i a t e on Whatman f i l t e r papers i n the absence of a l l n u t r i e n t s , as described under Methods. F i g . 2(a) shows the times required for 90% of the population to reach the pseudoplasmodial stage. F i g . 2(b) shows the time required f or 90% of the population to reach the f i n a l f r u i t i n g body stage. The data p l o t t e d are the means of four independent experiments. The error bars represent the standard errors of the means. was considerably longer i n the case of the PUFA supplemented c e l l s (Fig. 4). The optimum temperature for, the d i f f e r e n t i a t i o n of the PUFA supplemented c e l l s was i d e n t i c a l to that for the controls (22° C) but there was a s l i g h t change i n the shape of the plot of the r e c i p r o c a l of the time required for d i f f e r e n t i a t i o n against temperature (Fig. 4). In PUFA supplemented c e l l s d i f f e r e n t i a t i o n was more rapid below the optimum temperature than above, whereas for unsupplemented c e l l s the converse was true. Thus the i n h i b i t i o n produced by PUFA supplementation was somewhat l e s s pronounced at low temperatures. Aggregation of vegetative-phase c e l l s . Garrod and Born (71) showed that the aggregation of vegetative c e l l of p_. discoideum was markedly i n h i b i t e d at low temperatures. The e f f e c t of temperature on aggregation has been studied here i n somewhat more d e t a i l . Figure 5 shows the time course of aggregation at 3 temperatures Aggregation was rapid at 22° C and was e s s e n t i a l l y complete a f t e r 30 minutes incubation. In contrast, at 6° C the rate of aggregation was much slower and aggregation was s t i l l not complete a f t e r 60 minutes incubation. When the c e l l s were switched to 22° C, however, aggregation was r a p i d l y completed (Fig. 5). At 40° C only low l e v e l s of aggregation were observed and there was no increase i n aggregation upon prolonged incubation. Furthermore, when these c e l l s were switched to 22° C there was s t i l l no increase i n the amount of aggregation. Thus, while at low temperatures the i n h i b i t i o n of c e l l aggregation-is r e v e r s i b l e , at elevated temperatures the i n h i b i t i o n i s i r r e v e r s i b l e . The data i n F i g . 6 show the i n i t i a l rate of c e l l aggregation, over Figure 5 . Abscissa: time (min); ordinate: c e l l aggregation (%) Time course of vegetative JJ. discoideum aggregation at three d i f f e r e n t temperatures. C e l l s were grown i n the absence of PUFA, washed and resuspended i n Bonner's s a l t s - T r i s at a c e l l density of 2.2 x 10 c e l l s / m l . They were allowed to aggregate, while being gyrated (120 rpm) at 6°C, ( A ) ; 22°C, ( 0 ) or 40°C, ( • ). Per cent aggregation was determined as described under Methods. At the arrows the 6°C and 40°C incubations were transferred to 22°C. 33 —I L_ L_ 2 0 4 0 6 0 T i m e (m in ) Figure 6. Abscissa: temperature (°C); ordinate: c e l l aggregation (%) E f f e c t of temperature and PUFA supplementation on the aggregation of vegetative JJ. discoideum c e l l s . C e l l s were grown eit h e r i n the presence, ( A ) ; or the absence, ( 0 ) of PUFA. Washed c e l l s were resuspended at c e l l d ensities of 2.0 - 2.5 x 10 c e l l s / m l i n Bonner's s a l t s - T r i s and allowed to aggregate for 7.5 min. The per cent aggregation, observable at t h i s time, was determined as described under Methods. The data shown are the means of three independent experiments The standard errors of the means have not been shown i n the i n t e r e s t s c l a r i t y but i n no instance were they i n excess of ± 7.0%. 36 a r e l a t i v e l y wide range of temperature. The rate of c e l l aggregation increased markedly to a maximum o f 18 . C - 23 C and then d ecreased with further increase i n temperature. The aggregation of PUFA supplemented c e l l s showed a s i m i l a r dependence upon temperature. There was no change i n the optimum temperature range, although at most assay temperatures the aggregation of PUFA supplemented c e l l s was s l i g h t l y lower than that of the unsupplemented c e l l s . Thus, s l i g h t l y higher temperatures were required to produce l e v e l s of aggregation that were comparable to those of c e l l s grown i n the absence of PUFA. The aggregation of vegetative c e l l s shown i n F i g . 5 and 6 i s abolished by the i n c l u s i o n of EDTA i n the suspension medium (data not shown) and probably r e s u l t s from i n t e r a c t i o n s of contact s i t e ( s ) B (72,73). Reaggregation of aggregation-phase c e l l s . C e l l s of p_. discoideum, dispersed from stable aggregates r e a d i l y reassociate i n the presence of EDTA (74,75). This EDTA stable aggreg-ati o n i s not expressed i n vegetative c e l l s and the c e l l surface molecules responsible for t h i s aggregation have been termed contact A s i t e s (72 ,73). Figure 7 shows the time course for reaggregation of dispersed aggregation-phase c e l l s at 3 d i f f e r e n t temperatures. At 22° C reaggreg-ation was rapid and e s s e n t i a l l y complete by 10 minutes. At 39° C the rate of reaggregation was slow and there was no increase when c e l l s were transferred to 22° C ( F i g . 7). At 9° C the rate of reaggregation was again slow, but rapid reaggregation was observed when the c e l l s u l t i m a t e l y transferred to 22° C. Figure 7. Abscissa: time (min); ordinate: c e l l aggregation (%) Time course of reaggregation of dispersed aggregation-phase c e l l s of D_. discoideum at three d i f f e r e n t temperatures. C e l l s were allowed to aggregate on an agar surface for 10 hours, harvested, washed and resuspended at a; c e l l density of 2.2 x 10 c e l l s / m l i n phosphate-EDTA. Aggregates were disrupted by 5 passages through a 20 gauge hypodermic needle and the sin g l e c e l l s were swirled at 120 rpm for the indicated times. Assays were conducted at 22° C, ( 0 ); 9° C, ( A ) and 39° C ( • ). Per cent aggregation was determined as described under Methods. At the arrows the 15°C and 39°C incubations were transferred to 22°C. 39 Figure 8 shows a more de t a i l e d study of the e f f e c t of temperature on the reaggregation of aggregation phase c e l l s . The reaggregation of PUFA supplemented c e l l s exhibited a s l i g h t l y d i f f e r e n t temperature dependence than that of unsupplemented c e l l s . The aggregation of the l a t t e r was optimal over a r e l a t i v e l y broad temperature range (18° C -33° C) , while, i n contrast, the aggregation of the PUFA supplemented c e l l s continued to increase up to a maximum of 33° C. In addition the reaggregation of the PUFA supplemented c e l l s was s l i g h t l y lower than that of the unsupplemented c e l l s at a l l temperatures below 30° C. Discussion. It i s possible that the ph y s i c a l state of the plasma membrane l i p i d might influence c e l l - c e l l i n t e r a c t i o n . S c h a e f f e r . and C u r t i s showed that a l t e r a t i o n of the l i p i d composition of mouse L929 c e l l s by an i n v i t r o f a t t y acid supplementation procedure markedly altered the aggregation of these c e l l s ; unsaturated f a t t y acids i n h i b i t e d aggregation, whereas saturated f a t t y acids enhanced the process (26) . I t was suggested that c l u s t e r i n g of cell-adhesion s p e c i f i c molecules within the plane of the membrane might be a necessary p r e r e q u i s i t e for the formation of stable c e l l - c e l l i n t e r a c t i o n s , and that a l t e r a t i o n s in membrane f l u i d i t y might profoundly influence t h i s molecular c l u s t e r i n g (26,74). C e r t a i n l y i t would appear that c l u s t e r i n g of l e c t i n binding s i t e s within the plane of the membrane i s e s s e n t i a l for lectin-mediated agglutination of mammalian c e l l s , since agglutination does not occur under conditions that abolish t h i s c l u s t e r i n g (75). Both the aggregation of vegetative c e l l s and the reaggregation of dispersed aggregation-phase c e l l s were markedly temperature dependent extending the e a r l i e r observations of Garrod and Figure 8. Abscissa; temperature (°C); ordinate: c e l l aggregation (%) E f f e c t of temperature and PUFA supplementation on the reaggregation of dispersed aggregation-phase D_. discoideum. C e l l s were grown either i n the presence, ( A ); or the absence, ( 0 ) of PUFA and allowed to aggregate for 10 hours on an agar surface. They were then harvested, washed and resuspended at a c e l l density of 2.0 - 2.5 x 10 c e l l s / m l i n phosphate-EDTA. The aggregates were disrupted by 5 passages through a 20 gauge needle and the c e l l s were reaggregated by s w i r l i n g at 120 rpm on a gyratory incubator at the indicated temperatures for 7.5 mins. Per cent aggregation was determined as described under Methods. The data shown are the means of four independent experiments. The standard errors of the means have not been shown i n the i n t e r e s t of c l a r i t y , but i n no instance d i d they exceed ± 7%. 41 _J 1 I 1 o o o CD C\J (%) u o i j e 6 e j 6 6 e ||33 42 Born (73) (Figure 5 and 7). The i n h i b i t i o n of both types of aggregation at low temperatures was r e v e r s i b l e , suggesting that a f l u i d membrane might be e s s e n t i a l f o r c e l l aggregation ( F i g . 5 and 7); reduced membrane f l u i d i t y might i n h i b i t aggregation by preventing the c o r r e c t o r i e n t a t i o n or a s s o c i a t i o n i n the membrane of the molecules r e s p o n s i b l e f o r c e l l c o ntact. I t i s u n l i k e l y that the low r a t e s of aggregation observed at low temperatures i s due to an inadequate supply of energy because metabolic poisons such as d i n i t r o p h e n o l and azide have no e f f e c t on the reaggregation of dispersed aggregation-phase c e l l s as measured i n t h i s i n v i t r o assay (Weeks, G., unpublished o b s e r v a t i o n s ) . I t i s a l s o u n l i k e l y that the reduced r a t e s of aggregation at low temperature are due to decreased r a t e s of c e l l u l a r c o l l i s i o n . The i n h i b i t i o n of i n v i t r o aggregation at high temperatures i s i r r e v e r s i b l e and probably due to p r o t e i n denaturation. To f u r t h e r study the r e l a t i o n s h i p between f l u i d i t y and c e l l - c e l l i n t e r a c t i o n , attempts were made to .modify membrane f l u i d i t y by growth of D_. discoideum i n the presence of PUFA. Large q u a n t i t i e s of PUFA were incorporated i n t o c e l l u l a r l i p i d and, although there was no e f f e c t on growth, d i f f e r e n t i a t i o n was markedly impaired (63). This l e d to the hypothesis that increased f l u i d i t y of the plasma membrane was r e s p o n s i b l e f o r the impaired c e l l - c e l l i n t e r a c t i o n d u r i ng d i f f e r e n t i a t i o n (63). However, a more d e t a i l e d a n a l y s i s of the d i f f e r e n t i a t i o n and i n v i t r o aggregation of PUFA supplemented c e l l s has f a i l e d to s u b s t a n t i a t e the increased plasma membrane f l u i d i t y hypothesis. I f PUFA i n c o r p o r a t i o n does increase membrane f l u i d i t y then optimal r a t e s of d i f f e r e n t i a t i o n and aggregation might be expected to occur at lower temperatures than 43 those for unsupplemented c e l l s . In f a c t , when d i f f e r e n t i a t i o n was analyzed at d i f f e r e n t temperatures, i t was found that the optimum temperature for the d i f f e r e n t i a t i o n of PUFA supplemented and unsupplemented c e l l s was i d e n t i c a l . I n h i b i t i o n of d i f f e r e n t i a t i o n was apparent at a l l temperatures and there was only a s l i g h t r e v e r s a l of the i n h i b i t i o n at low temperature (Fig. 4). Furthermore, both the rates of aggregation of vegetative c e l l s and the rates of reaggregation of dispersed aggregation-phase c e l l s were generally lower for PUFA supplemented c e l l s than for unsupplemented c e l l s at temperatures below 30° C (Fig. 6 and 8 ) , data inconsistent with the concept of increased plasma membrane f l u i d i t y i n PUFA supplemented c e l l s . Recent measurements of plasma membrane f l u i d i t y by electron spin resonance and fluorescence dep o l a r i z a t i o n techniques have found no increase i n the f l u i d i t y of PUFA grown c e l l s (F.G. Herring, I. T a t i s c h e f f and G. Weeks, unpublished observations). At the present time, i t i s not known whether the increased unsaturation of the acyl chains is. i n -s u f f i c i e n t to produce a measurable increase i n membrane f l u i d i t y , or whether there i s some compensating change i n membrane l i p i d composition that keeps the membrane f l u i d i t y constant. The only evidence that i s consistent with the idea that PUFA grown c e l l s exhibit increased membrane f l u i d i t y comes from .the growth experiments ( F i g . 3). In the o r i g i n a l study (63) i t was shown that growth at 22° C was unaffected by PUFA supplementation and t h i s has been confirmed i n the present study. In addition, there was no i n h i b i t i o n of growth at lower temperatures, as a r e s u l t of PUFA supplementation, but at higher temperatures there was marked i n h i b i t i o n ( F ig. 3). Since there i s no detectable increase i n plasma membrane f l u i d i t y , i t i s possible that PUFA supplementation a f f e c t s i n t e r n a l membrane functions, which i n h i b i t 0 d i f f e r e n t i a t i o n at a l l temperatures and growth only at elevated temperatures. U n t i l the mechanism of i n h i b i t i o n i s elucidated, i t would appear wise to withhold judgement on the meaning of experiments attempt-ing to a l t e r surface membrane f l u i d i t y by supplementation with PUFA. 45 RESULTS AND DISCUSSION Section I I The Effect of Growth Temperature on the Dif f e r e n t i a t i o n of Dictyostelium discoideum Results. Cells grown i n HL5 media containing bovine serum albumin at 15° C, 22° C, or 26° C were set up to d i f f e r e n t i a t e at various temperatures within the range of 15° C - 27° C. The times required for pseudo-plasmodium and f r u i t i n g body formation were measured and the results are shown i n Fig. 9. While there i s no pronounced s h i f t i n the optimum temperature for d i f f e r e n t i a t i o n , there i s , nonetheless, evidence that d i f f e r e n t i a t i o n i s influenced by the temperature of growth. Cells grown at 15° C differentiated faster at 15° C and 19° C, than c e l l s grown at 22° C and 27° C. Conversely, c e l l s grown at 27° C differentiated faster at 25° C and 27° C than c e l l s grown at 15° C or 22° C. Cells grown at 22° C were intermediate i n their rates of d i f f e r e n t i a t i o n at the sub-optimal temperatures. In this experiment c e l l s were grown i n the presence of BSA since consistent growth at 27° C could not be obtained i n i t s absence. In order to eliminate the p o s s i b i l i t y that BSA was i n some way responsible for these results the experiment was repeated with c e l l s grown at 15° C and 26° C i n the absence of bovine serum albumin. The results shown i n Fig. 10 reveal a s i m i l a r , although somewhat less pronounced effect of growth temperature on d i f f e r e n t i a t i o n . One possible explanation for t h i s temperature adaptation might be an a l t e r a t i o n i n membrane l i p i d composition during growth to maintain constant membrane f l u i d i t y , which might have an adverse effect on the subsequent d i f f e r e n t i a t i o n of the organism following a temperature s h i f t . Since many microorganisms adapt t h e i r membrane f l u i d i t y i n response to 46 Figure 9. The e f f e c t of growth temperature on the d i f f e r e n t i a t i o n of D. discoideum at. various temperatures. C e l l s of D. discoideum, Ax-2 were grown at 26° C, ( A ); 22° C, ( • ); or 15° C, ( 0 ); i n HL5 media containing bovine serum albumin. The c e l l s were allowed to d i f f e r e n t i a t e on Whatman No. 50 f i l t e r papers at the indicated temperatures as described under Methods, and were observed p e r i o d i c a l l y . Figure 9a shows the time required for 90% of the population to reach the pseudoplasmodium stage and Figure 9b shows the time required for 90% of the population to reach the f r u i t i n g body stage. Figure 10. The e f f e c t of growth temperature on the d i f f e r e n t i a t i o n of p_. discoideum grown without bovine serum albumin i n the medium. C e l l s of D_. discoideum Ax-2 were grown at 26°C ( A ) or 15°C ( 0 ) i n HL5 media containing no bovine serum albumin. D i f f e r e n t i a t i o n conditions and assessment were as described for F i g . 9. 49 T e m p e r a t u r e ( °C ) 50 environmental temperature by manipulating t h e i r f a t t y acid compositions (12,64,20) the f a t t y acid compositions of c e l l s grown at 15° C and 26° C were analyzed. The r e s u l t s , shown i n Table 1, reveal l i t t l e change i n the f a t t y acid composition due to a l t e r a t i o n i n the growth temperature. The e f f e c t of growth temperature on the d i f f e r e n t i a t i o n of the wild type s t r a i n , NC4 was also analyzed. The r e s u l t s shown i n Figure 11 indic a t e that t h i s s t r a i n does not exhibit the temperature adaptation shown by the axenic mutant. Discussion. Two i n t e r e s t i n g e f f e c t s of temperature on the d i f f e r e n t i a t i o n of D_. discoideum have been reported previously. Poff and Skokut (-76) showed that the temperature range of pseudoplasmodial thermotaxis was modified by growth of the organism at a d i f f e r e n t temperature. These authors suggested that the adaptation might be due to a modification of membrane l i p i d f l u i d i t y which markedly affected the thermotactic response. Bonner and S i l f k i n (70) and more recently other workers (77,78) have shown that the d i f f e r e n t i a t i o n temperature markedly influences the proportion of s t a l k and spore c e l l s i n the f i n a l f r u i t i n g body. The mechanism involved i h t h i s phenomenon has not been explained, but i t i s conceivable that an a l t e r a t i o n i n membrane f l u i d i t y might influence c e l l - c e l l i n t e r a c t i o n , and hence the morphological fate of c e r t a i n c e l l s . In t h i s study evidence i s presented for a temperature adaptation i n s t r a i n Ax-2. The growth temperature c l e a r l y influences the rate of subsequent d i f f e r e n t i a t i o n following a temperature s h i f t . While there are only s l i g h t changes i n the optimum temperature for d i f f e r e n t i a t i o n , 51 Table 1. Fatty acid composition of D. discoideum, Ax-2 grown at different temperatures. Fatty Acid Growth Temperature 15° C % 26° C 14:Oa 1.25 0.65 palmitaldehyde 6.75 6.0 16:0 10.5 6.0 16:1 (A 9) 3.75 2.25 16:2 ( A 5 ' 9 ) or 17:0b 2.8 1.25 18:0 2.65 3.7 18:1 (A 9) and ( A J 1 ) b 19.75 24.8 18:2 ( A 5 ' 9 ) and ( A 5 ' 1 1 ) b 44.5 53.1 Others 1.8 1.75 The figures are averages of values from fatty acid analyses of membranes from three individual experiments. In a l l fatty aicd abbreviations the number preceding the colon is the chain length; the number following the colon is the number of double bonds and the numbers following A denote the positions of the double bonds. Thus 5,9-octadecadienoic acid i s abbreviated to 18:2 ( A 5 ' 9 ) , etc. The methyl esters of these two fatty acids are not separated under the conditions used in the present study. Several unidentified minor components. 52 Figure 11. The e f f e c t of growth temperature on the time taken for f r u i t i n g body formation i n p_. discoideum, NC-4. C e l l s grown at 27° C, ( A ); 22° C, ( • ); or 15° C, ( 0 ), i n association with Enterobacter aerogenes, and then allowed to d i f f e r e n t - ..: i a t e i n the absence of b a c t e r i a at the indicated temperatures as described under Methods. The time required for 90% of the population to form mature f r u i t i n g bodies i s shown. 15 19 2 3 2 7 Temperature ( °C ) 54 there are marked e f f e c t s at the temperature extremes (Fig. 9 and .Fig. 10). Again a possible explanation for these r e s u l t s i s that the organism modifies i t s membrane l i p i d composition during growth, and that t h i s then influences the subsequent d i f f e r e n t i a t i o n of the organism, following the s h i f t i n temperature. Other microorganisms modify f a t t y acid composition, and hence membrane f l u i d i t y , to compensate for changes i n growth temperature (12,64,20). However, only a s l i g h t d i f f e r e n c e i n the f a t t y acid composition of D. discoideum grown at 15° C and 26° C (Table 1) was found i n the present study and no evidence consistent with the idea that the membrane f l u i d i t y adjusts to the environmental temperature was observed. I f altered membrane f l u i d i t y i s the basis of the temperature adaptation reported i n t h i s study, then i t i s not accomplished by an a l t e r a t i o n i n f a t t y acid composition. The wild type s t r a i n , NC-4 does not exhibit the temperature adapt-ati o n behaviour demonstrated by the axenic mutant. This does not r u l e out altered membrane f l u i d i t y as the cause of the modified d i f f e r e n t i a t i o n of the axenic mutant, since the wild type s t r a i n might readapt i t s l i p i d composition r a p i d l y during the ea r l y stages of d i f f e r e n t i a t i o n . There i s evidence that continued f a t t y acid synthesis i s necessary for the d i f f e r e n t i a t i o n process, as d i f f e r e n t i a t i o n i s i n h i b i t e d by the a n t i -b i o t i c cerulenin, and t h i s i n h i b i t i o n can be reversed by the addi t i o n of a palmitic acid supplement (79). 55 GENERAL CONCLUSIONS AND DISCUSSION The experiments de t a i l e d i n t h i s thesis were designed to test ( i ) whether the impairment i n d i f f e r e n t i a t i o n of PUFA supplemented c e l l s i s due to a change i n the f l u i d i t y of t h e i r plasma membranes, and ( i i ) whether the f l u i d i t y of the plasma membranes of p_. discoideum can be modified by growth of these c e l l s at extremes of temperatures. The important r e s u l t s are summarized below. (i) The impairment i n the d i f f e r e n t i a t i o n of PUFA supplemented c e l l s observed previously (63) was confirmed. ( i i ) The addition of PUFA to vegetative c e l l s resulted i n an i n h i b i t -ion of growth only at temperatures above the optimum growth temperature, suggesting that increased membrane f l u i d i t y could be the reason for the i n h i b i t i o n of growth (Fig. 3). ( i i i ) The i n h i b i t i o n of c e l l aggregation at lower incubation temper-atures could be reversed by changing the incubation temperatures to optimal l e v e l s ( F ig. 5 and 7) suggesting that a c e r t a i n l e v e l of plasma membrane f l u i d i t y i s e s s e n t i a l for the normal aggregation process. (iv) Increased f l u i d i t y i n the PUFA supplemented c e l l membranes compared to those of normal c e l l s was not indicated by i n v i t r o assays of vegetative c e l l aggregation or reaggregation of dispersed aggregation competent c e l l s . (v) Varying the growth temperature markedly influenced the e f f e c t of temperature on d i f f e r e n t i a t i o n ( F i g . 9 and 10), which suggested a possible a l t e r a t i o n i n plasma membrane f l u i d i t y r e s u l t i n g from adaptation to the new growth temperature. (vi) The f a t t y acid composition of c e l l s grown at the two extreme temperatures of growth i s not appreciably d i f f e r e n t . 56 ( v i i ) The wild type s t r a i n NC-4 does not exhibit the adaptation to growth temperature observed i n the axenic mutant. There are a few precedents f o r f a t t y acid manipulation inf l u e n c i n g c e l l - c o n t a c t s . C u r t i s and coworkers (25,26,50,51) reported predictable changes i n c e l l - c e l l and c e l l - s u b s t r a t e adhesion i n chick neural r e t i n a c e l l s and mouse L929 c e l l s r e s u l t i n g from the manipulation of the f a t t y acid composition of the plasma membrane phospholipids. Experiments of Horwitz and coworkers (24) on l e c t i n agglutination of 3T3 and SV 101 3T3 c e l l s suggest the importance of the l i p i d phase of the plasma membranes. I t was suggested that d i f f e r e n t l e c t i n receptors were located i n d i f f e r e n t l i p i d environments. Unfortunately, there was no physical data a v a i l a b l e for either of the two systems to support the idea of f l u i d i t y changes i n the f a t t y acid manipulated c e l l s . The a v a i l a b l e data suggest that despite PUFA supplementation, c e l l s manage to conserve the f l u i d i t y of the l i p i d environments required for the cohesion of vegetative c e l l s and the aggregation of aggregation competent c e l l s and hence show responses s i m i l a r to those of normal c e l l s . In f a c t , the f l u i d i t y of plasma membranes of D_. discoideum c e l l s grown i n presence of PUFA i s no greater than that of normal c e l l membranes as determined by electron spin resonance and fluorescence de p o l a r i z a t i o n techniques (F.G. Herring et_ al_. , unpublished observations). The observed impairment i n d i f f e r e n t i a t i o n i n PUFA supplemented c e l l s must therefore be due to factors other than increased plasma membrane f l u i d i t y . The causes of the impairment of d i f f e r e n t i a t i o n of PUFA grown c e l l s remain unclear. I t i s possible that an increase i n the f l u i d i t y of the i n t e r n a l membranes might a f f e c t the enzymes important i n the process of 57 d i f f e r e n t i a t i o n rather than growth e s p e c i a l l y i f they have a s p e c i f i c l i p i d requirement. However, as yet no d i f f e r e n t i a t i o n s p e c i f i c i n t e r n a l membrane enzymes have been described. It i s equally possible that impaired d i f f e r -e n t i a t i o n of PUFA supplemented c e l l s has nothing to do with altered membrane f l u i d i t y . At present there i s no evidence to d i s t i n g u i s h these p o s s i b i l i t i e s . Adaptation of membrane f l u i d i t y to growth temperature appears to be quite common i n microorganisms. Sinensky (80) found that E. c o l i incorporated long-chain saturated f a t t y acids into phospholipids when the growth temperature was increased. The f l u i d i t y ' o f the membranes was analyzed by e l e c t r o n spin resonance which gave a phase t r a n s i t i o n corresponding to the increased amount of saturated f a t t y a c y l chains i n the membrane phospholipids. S i m i l a r l y , Kates and Baxter (64) found that the mesophilic yeast Candida l i p o l y t i c a when grown at 10°C accumulates more l i n o l e i c acid and l e s s o l e i c acid compared to the same c e l l s grown at 25°C. They also found that only those c e l l s grown at 10°C contained l i n o l e n i c acid which was not detected i n the c e l l s grown at the higher temperature. Similar r e s u l t s were reported i n Tetrahymena pyriformis by Thompson and Nozawa (20) and they suggested that the f a t t y acid desaturase a c t i v i t y i s regulated by the f l u i d i t y of the enzyme's immediate l i p i d surroundings. In the experiments described here, p_. discoideum c e l l s grown at d i f f e r e n t temperatures exhibited an altered temperature-differentiation r e l a t i o n s h i p which suggested a possible f l u i d i t y change i n the membrane l i p i d s . As shown i n Table 1, however, there seems to be no appreciable di f f e r e n c e i n the f a t t y acid compositions of t h e i r plasma membranes. 58 Hence they do not compensate for the change i n growth temperature by a l t e r i n g t h e i r f a t t y acid composition. McElhaney and Souza (81) reported a B_. stereothermophilus mutant s t r a i n which lacks the a c t i v i t y for temperature adaptation and i s unable to grow at higher temperatures at which the wild type s t r a i n grows. It i s possible that the c e l l s of JJ. discoideum also do not require t h i s a b i l i t y i n the small temperature range i n which i t i s capable of growing. It i s i n t e r e s t i n g to note that a f t e r PUFA incorporation the same c e l l s i n some way compensate for the change i n the f a t t y acid composition and present the c e l l with a membrane of normal f l u i d i t y . It should be noted, however, that the increased unsaturation of the membrane f a t t y acids might not be s u f f i c i e n t to cause a d r a s t i c change i n the o v e r a l l f l u i d i t y of the plasma membranes. This p o s s i b i l i t y i s currently being investigated. 59 BIBLIOGRAPHY 1. L u z z a t i , V. 1968. 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