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Endocytosis and membrane lipid composition in wild-type and mutant Paramecium tetraurelia Pollock, Carol 1980

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ENDOCYTOS.IS AND MEMBRANE LIPID COMPOSITION IN WILD-TYPE AND MUTANT PARAMECIUM TETRAURELIA by CAROL POLLOCK B. Sc. (Hons), The U n i v . o f Ma n i t o b a , Winnipeg, M a n i t o b a , 1972 M.Sc., The Univ. o f Ma n i t o b a , Winnipeg, Manitoba, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n \ THE FACULTY OF GRADUATE STUDIES'*, (Department of Zoology) i We a c c e p t the t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d \ . THE UNIVERSITY OF BRITISH COLUMBIA October 1980 (6) CAROL POLLOCK, 1980 In presenting th is thesis in par t ia 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 Library shal l make i t f ree ly ava i lab le for reference and study. I further agree that permission for extensive copying of th i s thesis for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It i s understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my writ ten permission. Department nf ' ^ C > 0 * ® C] The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 • E - 6 B P 75-51 1 E i i ABSTRACT E i g h t y - t w c t a m p e r a t u r e - s e n s i t i v e ' mutants of Paramecium t e t r a u r e l i a t h a t i n v o l v e some a s p e c t of e n d o c y t o s i s or v a c u o l a r p r o c e s s i n g were i s o l a t e d f o l l o w i n g n i t r o s o g u a n i d i n e t r e a t m e n t . Two of t h e s e , the r e c e s s i v e m u t a t i o n s f v c (food v a c u o l e clumping) and dmj_ ( d e f e c t i v e membrane) , were examined i n some d e t a i l . . At t h e r e s t r i c t i v e t e m p e r a t u r e , 34.5°C, t h e f o o d v a c u o l e s of f v c c e l l s clumped i n one area of t h e c e l l , u s u a l l y the p o s t e r i o r . Fewer food v a c u o l e s accumulated i n f v c c e l l s than i n w i l d - t y p e c e l l s a f t e r 20 minutes i n a food v a c u o l e marker, such as blue w a t e r c o l o r (BP)., The f v c n u t a t i o n was o r i g i n a l l y r e c o v e r e d as a double m u t a t i o n and was l i n k e d by 34 ± 3 ( s t a n d a r d d e v i a t i o n ) map u n i t s t o t h e t n d (te m p e r a t u r e -s e n s i t i v e , t r i c h o c y s t . n o n - d i s c h a r g e ) l c c u s . . The mutant, dmj, had m o r p h o l o g i c a l l y abnormal v a c u o l e s which, i n t h e l i g h t m i c r o s c o p e , appeared as a mass of f u s e d or d i s r u p t e d v a c u o l e s . . An u l t r a s t r u c t u r a l s t u d y r e v e a l e d t h a t t h e s t r u c t u r a l i n t e g r i t y of the f o o d v a c u o l e s was l o s t . Abnormal m i t o c h o n d r i a and v e s i c u l a r s t r u c t u r e s were a l s o observed, as w e l l as c e l l s i n which l a r g e p o r t i o n s o f the c y t o p l a s m were m i s s i n g , presumably h a v i n g been d i g e s t e d by t h e h y d r o l y t i c enzymes t h a t were r e l e a s e d from t h e f c o d v a c u o l e . A g a s - l i g u i d c h r o m a t o g r a p h i c a n a l y s i s o f w i l d - t y p e and mutant c e l l s i n d i c a t e d t h a t t h e major f a t t y a c i d s were: p a l m i t i c ( 1 6:0), s t e a r i c ( 1 8:0), o l e i c ( 1 8 : 1 ) , l i n o l e i c ( 18:2), l i n o l e n i c ( 18:3), a r a c h i d o n i c ( 2 0 : 4 ) , d o c o s a t e t r a e n o i c (22:4), te t r a c o s a d i e n o i c (24^2) , and t e t r a c o s a t e t r a e n o i c (24:4). With an i n c r e a s e i n temperature from 27°C t o 34.5°C the p e r c e n t c o m p o s i t i o n , i n w i l d - t y p e whole c e l l l i p i d s , of 16:0 i n c r e a s e d and t h a t of 24:4 d e c r e a s e d . . However, i n dm1_ c e l l s , the o p p o s i t e o c c u r r e d , i . e . the p e r c e n t c o m p o s i t i o n o f 16:0 d e c r e a s e d and 24:4 i n c r e a s e d . In w i l d - t y p e c e l l s these changes were r e f l e c t e d m a i n l y i n the. f a t t y a c i d s o f t h e p h o s p h o l i p i d s p h o s p h a t i d y l c h o l i n e (PC) and p h o s p h a t i d y l e t h a n o l a m i n e (PE) w i t h o n l y s l i g h t changes o c c u r r i n g i n t h e p h o s p h o n o l i p i d , 2-a m i n o e t h y l p h o s f h o n o l i p i d (AEPL). . In dm1 c e l l s , t h e f a t t y a c i d s o f PC, PE and AEPL v a r i e d w i t h temperature i n a manner s i m i l a r t o the p a t t e r n observed w i t h whole dm_1 c e l l s . The u n s a t u r a t i o n i n d e x ( 0 . 1 . = number of d o u b l e bonds per 100 a c y l groups) d e c r e a s e d i n w i l d - t y p e c e l l s , i n whcle c e l l f a t t y a c i d s and p a r t i c u l a r l y i n PE, w i t h an i n c r e a s e i n t emperature. However, the U . L . i n dm_1 whole c e l l f a t t y a c i d s and p h o s p h o l i p i d s i n c r e a s e d w i t h an i n c r e a s e i n t e m p e r a t u r e . A s u r v e y of s e v e r a l c h e m i c a l a g e n t s i n d i c a t e d t h a t d i b u c a i n e , sodium d o d e c y l s u l f a t e and c o l c h i c i n e i n h i b i t e d e n d o c y t o s i s . The mutant, f vc, was more s e n s i t i v e t o sodium d o d e c y l s u l f a t e and c o l c h i c i n e t h a n w i l d - t y p e c e l i s . . D i m e t h y l s u l f o x i d e produced p a r t i a l phenocopies o f the dmj mutant i n w i l d - t y p e c e l l s . A f t e r t r e a t m e n t «ith 6-12% d i m e t h y l s u l f o x i d e t h e food v a c u o l e s f u s e d and s e p a r a t i o n of the n a s c e n t food v a c u o l e from the g u l l e t was i n h i b i t e d . , dmj. c e l l s were more s e n s i t i v e t o d i m e t h y l s u l f o x i d e than were w i l d - t y p e c e l l s . . The r e s u l t s i n d i c a t e d t h a t t h e mutant, f v c was abnormal i n some a s p e c t of i n t r a c e l l u l a r t r a n s p o r t which may i n v o l v e i v m i c rotubules or m i c r o f i l a m e n t s . The mutant, dmj[, d i d not undergo the same changes i n f a t t y a c i d composition as wild-type c e l l s i n response to an i n c r e a s e i n temperature. The vacuolar membranes i n t h i s mutant degenerated and m a t e r i a l which was normally seguestered i n the vacuoles *as r e l e a s e d , r e s u l t i n g i n c e l l u l a r destruction.., V TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES .... , x i LIST OF FIGURES x i v LIST OF PLATES , x V i ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x v i i CHAPTER I ENDOCYTOSIS IN PARAMECIUM TETRAU RELIA 1 A.. D e s c r i p t i o n o f e a d o c y t o s i s and i n t r a c e l l u l a r d i g e s t i o n . 1 B. K i n e t i c s of a c c u m u l a t i o n and l o s s of f o o d v a c u o l e s 6 C. . Membrane r e c y c l i n g ... 8 D. Purpose o f t h i s t h e s i s 11 CHAPTER I I STANDARDIZATION OF CONDITIONS ...............15 A. . I n t r o d u c t i o n 15 B. M a t e r i a l s and Methods .17 1. . Growth and p r e p a r a t i o n o f Paramecium t e t r a u r e l i a 17 2.. S e l e c t i o n of foo d v a c u o l e markers .17 3. E f f e c t of temperature .. •. , 18 4.. E f f e c t o f t i m e i n marker ........................18 5.. E f f e c t of c u l t u r e medium .18 C. R e s u l t s 19 1.. Growth and p r e p a r a t i o n of P. t e t r a u r e l i a . . . 1 9 2.. S e l e c t i o n o f food v a c u o l e markers . . 1 9 3. E f f e c t of tem p e r a t u r e ... 19 4.. E f f e c t o f time i n marker ........................ 2C 5.. E f f e c t of c u l t u r e medium . . . 2 0 D. . D i s c u s s i o n 21 CHAPTER III MUTAGENESIS AND DESCRIPTION OF PUTATIVE MUTANTS 32 A. . Introduction 32 B. . Materials and Methods .........32 1.. Mutagenesis 32 2.. Selection procedure ..............................33 a. individual l i n e s e l e c t i o n 33 b. . mass selection ............................... 34 3.. Description of putative mutants ................. 35 4. Genetic analysis 36 C. . Results , 36 1.. Mutagenesis ..................................... 36 2.. Yield of putative mutants 37 3.. Description of putative mutants .................. 37 4. Genetic analysis 38 a. A10 x pawn A 38 b. A5 x pawn A .............................••.••38 c. A5 x spot ...39 D. . Discussion 40 CHAPTER IV FURTHER CHAR ACT ERIZATICN OF THE MUTANTS dm_1 AND fvc 56 A. . Introduction .. 56 B. Materials and Methods 56 1. . E f f e c t of time in BP 56 2.. Loss cf colored vacuoles af t e r removal from BP ...57 3.. E f f e c t of concentration of BE 58 4.. E f f e c t of time at 34.5°C .58 a., asynchronous c e l l s 58 b. synchronous c e l l s 58 5.. Down-shift t o p e r m i s s i v e t e m p e r a t u r e 59 6.. E f f e c t of i n c r e a s i n g temperature ................ 59 C. _ R e s u l t s . • 59 1.. E f f e c t of time i n BP 59 2. L o s s of c o l o r e d v a c u o l e s a f t e r removal from BP ... 60 3. . E f f e c t of c o n c e n t r a t i o n o f BP .61 4 . . E f f e c t of t i m e a t 34. 5°C 61 a. asynchronous c e l l s 61 b. synchronous c e l l s ............................ 62 5. .Down-shift t o p e r m i s s i v e temperature .............. 63 6. . E f f e c t of i n c r e a s i n g t e m p e r a t u r e 63 D. . D i s c u s s i o n .........64 CHAPTER V EFFECTS OF CHEMICAL AGENTS ON WILD-TYPE AND MUTANT CELLS 94 A. . I n t r o d u c t i o n .................................... 94 B. M a t e r i a l s and Methods 98 1. Procedure f o r d e t e r m i n i n g e f f e c t s of c h e m i c a l agents 98 a. . p r e p a r a t i o n of c e l l s 98 b. p r e p a r a t i o n of c h e m i c a l agents 99 c mixing of c e l l s and c h e m i c a l agents .....99 d. a n a l y s i s o f r e s u l t s ........100 2. , DMSO 10 1 a. , e f f e c t of c o n c e n t r a t i o n ......................101 b. o b s e r v a t i o n s o f l i v e c e l l s 101 c. e f f e c t of t r e a t m e n t d u r a t i o n .....102 d. . a d d i t i o n of RP f o l l o w e d by DMSO and a second v i i i water color . . . 102 3.. Dibucaine .........103 4. SDS 103 5.. Colchicine 103 6.. Cytochalasin B 104 C. . Results ,.. • 104 1. . DMSO 104 a. effect of concentration 104 b. observations of l i v e c e l l s 105 c. effect of treatment duration ..107 d. addition of RP followed by DMSO and a second watercolor ....107 2.. Dibucaine ...........110 3. SDS ... 11C 4. . Colchicine ......................................110 5. Cytochalsin B .....111 D. Discussion 111 CHAPTER VI 0LTRA52? RU CT U R E OF WILD-TYPE AND dm_1 CELLS ...137 A. . Introduction ....137 B. Materials and Methods ..137 C. . Results ...138 D. Discussion • .142 1.. Slight abnormalities 143 2.. Degeneration of structure .. ..........143 3.. Large vacuoles 143 4. . Total degeneration 143 CHAPTER VII FATTY ACID COMPOSITION OF WILD-TYPE AND dml. CELLS 158 i x A. Introduction 158 B. . Materials and Methods ...........................160 1.. Extraction of l i p i d s .........16C 2.. Thin layer chromatography 16C 3. . Preparation of g l y c e r y l ethers ..161 4. Methylation ...162 a. , f a t t y acids .162 b. glyceryl ethers 162 5.. Gas l i g u i d chromatography ........................ 163 C. Results 164 1. . Fatty acid composition of whcle c e l l s ........... 164 2. Fatty acid composition of phospholipids .........167 D. . Discussion ...........168 CHAPTER VIII CONCLUDING REMARKS , ..190 LITERATURE CITED 192 APPENDIX I CULTURE TECHNIQUES .......................... 215 A. . Axenic medium .................215 B. . Dryl solution 217 APPENDIX II EHENOTx PES OF PUTATIVE MUTANTS .224 A. Individual l i n e s e l e c t i o n 224 B. . Mass selection ....225 APPENDIX III ELECTRON MICROS COPY 226 APPENDIX IV DETERMINATION OF FATTY ACID COMPOSITION ....228 A. .Lipid extraction • .......228 B. Thin layer chromatography 229 C. Removal of phospholipids from s i l i c a gel ........229 D. . Preparation of g l y c e r y l ethers .................. 230 E. Methylation of f a t t y esters ....231 T r i m e t h y l s i l a t i o n of g l y c e r y l e t h e r s Gas l i q u i d chromatography LIST OF TABLES 2-1. E f f e c t of temperature on the accumulation of vacuoles •,. , 23 3-1. Phenotypes of putative mutants . .< 44 3-1, cont. Phenotypes of putative mutants ............... 45 3- 2. . A1 0 x joawn , 46 3-3. A5 x pawn 47 3-4. A5 x sjcot - ... 48 3- 5. tnd x spot ; fvc x spot 49 4- 1. E f f e c t of blue watercolor at 34.5°C on vacuolar morphology 68 4-2. Effect of removal from blue watercolor on vacuolar morphology 69 4-3. Ef f e c t of concentration cf blue watercolor on vacuolar morphology 70 4-4. Ef f e c t of time at 34 .5°C on vacuolar morphology ... 71 4-5. Effect cf time at 34.5°C on vacuolar clumping ..... 72 4-6. Vacuolar clumping i n A5 and fvc c e l l s 73 4-7. Vacuolar morphology i n synchronous c e l l s at 34.5°C 74 4-8. Vacuolar morphology after s h i f t to 27°C 75 4-9. Vacuolar clumping after s h i f t tc 27°C 76 4-10. Vacuolar clumping in fvc and A5 c e l l s a f t e r s h i f t to 27°C . , , 77 4-11..Effect of temperature on vacuolar morphology ......78 4- 12. Effect of temperature on vacuolar clumping ....... 79 5- 1..Effect of dimethyl sulfoxide at 27°C .............. 116 5-2. Effect of dimethyl sulfoxide at 34.5°C ............117 5-3. .Length of c o n t r a c t i l e vacuole cycle ..118 x i i 5-4. E f f e c t of 12% d i m e t h y l s u l f o x i d e a t 34.5°C ...119 5-5. E f f e c t c f d i m e t h y l s u l f o x i d e and r e d and b l u e w a t e r c o l o r a t 34.5°C 120 5 - 6 . . E f f e c t of d i m e t h y l s u l f o x i d e and r e d and b l a c k w a t e r c o l o r a t 34.5°C 121 5-7. . E f f e c t o f d i b u c a i n e at 27°C 122 5-8. . E f f e c t of d i b u c a i n e a t 34. 5°C .123 5-9. E f f e c t of sodium d o d e c y l s u l f a t e a t 27°C 124 5 - 1 0 . . E f f e c t of sodium d o d e c y l s u l f a t e at 34.5°C 125 5 - 1 1 . . E f f e c t of c o l c h i c i n e a t 34.5°C 126 7-1. F a t t y a c i d c o m p o s i t i o n of K h o l e c e l l s 174 7-2..Changes i n whole c e l l f a t t y a c i d c o m p o s i t i o n ......175 7-3. . D i s t r i b u t i o n of whale c e l l f a t t y a c i d s ....176 7-4* F a t t y a c i d s o f p h o s p h a t i d y l c h o l i n e .177 7 - 5 . . F a t t y a c i d s o f p h o s p h a t i d y l e t h a n o l a m i n e ........... 178 7-6. F a t t y a c i d s o f 2 - a m i n o e t h y l p h o s p h o n o l i p i d 179 7-7. Changes i n p h o s p h o l i p i d f a t t y a c i d c o m p o s i t i o n ....180 7 - 8 . . D i s t r i b u t i o n of p h o s p h a t i d y l c h o l i n e f a t t y a c i d s ...181 7 - 9 . . D i s t r i b u t i o n of p h o s p h a t i d y l e t h a n o l a m i n e f a t t y a c i d s 182 7 - 1 0 . . D i s t r i b u t i o n of 2 - a m i n o e t h y l p h o s p h o n o l i p i d f a t t y a c i d s 183 Appendix 1-1. .Glass t u b i n g r e g u i r e d f o r a d a p t a t i o n t o a x e n i c medium 219 Appendix IV-1. F a t t y a c i d c o m p o s i t i o n of whole c e l l s -experiment 1.. 233 Appendix I V - 2 . F a t t y a c i d c o m p o s i t i o n of whole c e l l s -exp e r i m e n t 2 234 Appendix IV-3. Fatty acid composition of whole c e l l s -experiment 3. • 235 x i v LIST OF FIGURES 1- 1. . M o r p h o l o g i c a l changes i n f e e d v a c u o l e s d u r i n g d i g e s t i o n . 13 2- 1.. A c c u m u l a t i o n of v a c u o l e s i n carmine p a r t i c l e s and r e d w a t e r c o l o r . 24 2-2._Accumulation o f v a c u o l e s i n r e d w a t e r c o l o r . . . . . . . . . 26 2-3. A c c u m u l a t i o n of v a c u o l e s i n b l u e w a t e r c o l o r 28 2- 4. Accumulation of v a c u o l e s i n b u f f e r and a x e n i c medi urn 30 3- 1. Accumulation o f v a c u o l e s i n 4y-21 .50 3- 2. . Schematic i l l u s t r a t i o n o f t h e dmt phenotype. .52 4-1..Accumulation of v a c u o l e s i n b l u e w a t e r c o l o r a t 27°C. 80 4- 2. A c c u m u l a t i o n of v a c u o l e s i n b l u e w a t e r c o l o r at 3 4 . 5 ° C 82 4-3. Loss of c o l o r e d v a c u o l e s a t 27°C.................. 84 4-4. Loss of c o l o r e d v a c u o l e s a t 34.5°C 86 4-5. . A c c umulation o f v a c u o l e s i n w i l d - t y p e and dm_1 c e l l s a t 34.5°C 88 4- 6. . A c c u B u l a t i o n of v a c u o l e s i n w i l d - t y p e and A5 c e l l s a t 34.5°C. 90 4- 7. . Accumulation of v a c u o l e s i n s y n c h r o n i z e d c e l l s a t 34. 5°C 92 5- 1. Schematic i l l u s t r a t i o n of m o r p h o l o g i c a l e f f e c t s of d i m e t h y l s u l f o x i d e . 127 5- 2. Frame-by-frame movement cf a food v a c u o l e 129 5-3. E n d o c y t o s i s i n 6% d i m e t h y l s u l f o x i d e . 131 5-4. F u s i o n of v a c u o l e s i n 6% d i m e t h y l s u l f o x i d e . . . . . . . . 133 7-1..w6 pathway of polunsaturated f a t t y acid biosynthesis. .184 7-2. Fatty acids of wild-type c e l l s .186 7-3. Fatty acids of dm1 c e l l s . 188 Appendix 1-1. Glass tubing reguired f o r transfer of c e l l s to axenic medium.. 220 Appendix 1-2. Transfer of c e l l s tc axenic medium........ 222 xvi LIST OF PLATES III-1. Wild-type and mutant c e l l s in watercolor at 34.5°C 54 V- 1..Morphological e f f e c t s of dimethyl sulfoxide. ....... 135 VI- 1..Food vacuoles of a wild-type c e l l . 148 VI-2. . Wild-type c e l l tangential tc the surf ace. .......... 15C VI-3. . Cortex and g u l l e t regions cf djtl c e l l s . ...152 VI-4..Food vacuoles of dml c e l l s . ........154 VI-5. . Extreme cases of the dmjt phenotype. 156 x v i i ACKNOWLEDGEMENTS I would l i k e to thank my supervisor, Dr. J.D. Berger, for his support, encouragement and guidance in preparing t h i s t h e s i s . I wculd also l i k e to thank several others who contributed graciously of t h e i r time and advise: Adrian Smith for his help with the sectioning, Dr. C.E. .Vance and the people in his laboratory, p a r t i c u l a r l y Harry Paddon, for t h e i r help with the f a t t y acid i s o l a t i o n and analysis, P h y l l i d a Morton for the beautiful figures, the students in the lab, Don Jones, Phyllida Morton and Colin Rasmussen, the faculty and students in the Genetics and C e l l Biology Programme at the University of B r i t i s h Columbia, my family and friends who have encouraged and supported me over the years, and f i n a l l y , my wonderful husband, Jim, for his constant love and understanding.. Research funds were provided by National Research Council Grant 67-6300 to Dr. . J. D.. Berger. This thesis i s dedicated to the memory of Glenn Morton. 1 CHAPTER I ENDOCxTOSIS IN PARAMECIUM TETRAURELIA A. D e s c r i p t i o n of e n d o c y t o s i s and i n t r a c e l l u l a r dijgestion E n d o c y t o s i s i s the process by which e x t r a c e l l u l a r m a t e r i a l i s i n t e r n a l i z e d via v e s i c u l a r i z a t i o n . G e n e r a l l y , e n d o c y t o s i s occurs as a conseguence of l o c a l changes i n membrane p r o p e r t i e s , such as m i c r o v i s c o s i t y ( B e r l i n and Fera, 1977) or membrane p o t e n t i a l (Reis et a l . , 1979). S p e c i a l i z e d areas of the s u r f a c e membrane of macrophages c o n t a i n r e c e p t o r s i t e s which may represent s p e c i f i c o r g a n e l l e s adapted f o r the b i n d i n g and i n t e r n a l i z a t i o n of macromolecules (Anderson e t a l . , 1977). In c i l i a t e s , the o r a l apparatus, an extremely complex s t r u c t u r e , i s h i g h l y s p e c i a l i z e d f o r food entrapment and i n t e r n a l i z a t i o n . An example of such a s p e c i a l i z a t i o n i s the rows of c i l i a that form the o r a l membranelles i n hymenostomes such as Paramecium and Tetrahymena . The f e e d i n g apparatus i n Paramecium i s a shallow c i l i a t e d groove l e a d i n g to a c i l i a t e d tube (the buccal c a v i t y , g u l l e t or cytopharynx) which extends i n t o the tody c a v i t y (Mast, 1947). The c i l i a f o r c e suspended p a r t i c l e s , such as b a c t e r i a , i n t o the cytopharynx where mucus s e c r e t i o n s coat the p a r t i c l e s , s t i c k i n g them t o g e t h e r and thereby c o n c e n t r a t i n g them (Jahn et a l . , 1965, Korn and Weisoaan, 1967, N i l s s o n , 1970, R i c k e t t s and R a p p i t t , 1975).. Food vacuoles form at the base of the cytopharynx (Figure 1 — 1 A) and c i r c u l a t e i n the cytoplasm where 2 d i g e s t i o n o c c u r s (Jurand, 1961). The changes i n food v a c u o l e morphology t h a t occur t h r o u g h o u t t h e d i g e s t i v e c y c l e i n Paramecium have been e x t e n s i v e l y d e s c r i b e d ( J u r a n d , 1961, Jurand and Selman, 1969), A l l e n , 1974,1976). Young v a c u o l e s c r phagosomes (s t a g e I) c o n t a i n d e n s e l y packed, u n d i g e s t e d t a c t e r i a ( F i g u r e 1-1B). The v a c u o l a r membrane i s very r e g u l a r and many s m a l l v e s i c l e s a r e o b s e r v e d around the growing v a c u o l e . When paramecia a r e v i t a l l y s t a i n e d w i t h n e u t r a l r e d , s m a l l g r a n u l e s appear i n c l o s e p r o x i m i t y t o young v a c u o l e s ( H a l l and Dunihue, 1930, Dunihue, 1931, Volkonsky, 1934, Eosenfcaum and W h i t t n e r , 1962). These g r a n u l e s were l a t e r i d e n t i f i e d as lysosomes, o r g a n e l l e s t h a t c o n t a i n h y d r o l y t i c enzymes ( M u l l e r and Toro, 1962, Eosenbaum and W h i t t n e r , 1962). lysosomes f u s e w i t h phagosomes f o r m i n g d i g e s t i v e v a c u o l e s or secondary lysosomes (De Duve, 1963, De Duve and W a t t i a u x , 1966, E s t e v e , 1970, R i c k e t t s , 197C) . P r i o r to t h e f u s i o n o f lysosome and phagosome, a c i d phosphatase, which i s an enzyme commonly used as a marker f o r lysosomes (Gomori, 1 952, Barka and Anderson, 1962), i s l o c a l i z e d i n t h e s m a l l v e s i c l e s s u r r o u n d i n g , but not w i t h i n , t h e young v a c u o l e s ( E s t e v e , 1970). In s t a g e I I ( F i g u r e 1-1C) , the v a c u o l e s w e l l s and t h e membrane becomes i r r e g u l a r but t a c t e r i a l morphology remains unchanged. By t h i s s t a j e a c i d phosphatase a c t i v i t y appears i n s i d e the f o o d v a c u o l e s , around t h e membranes as w e l l as around the t a c t e r i a ( E s t e v e , 1970). The pH of the f o o d v a c u o l e d r o p s g r a d u a l l y a t f i r s t and f i n a l l y may r e a c h a minimum o f around 1.4 ( S h a p i r o , 1927, Mast, 1947).. 3 In older vacuoles (stage I I I , Figure 1-1D), the bacteria are undergoing digestion and condense away from the vacuolar membrane which i s very i r r e g u l a r . Small vesicles (pinocytotic or cup-shaped vesicles) contain material of a density si m i l a r to that of food vacuoles and pinch cff from the vacuole. Acid phosphatase a c t i v i t y i s very high i n both the vacuoles and the vesicles (Esteve, 1970) and the pH legins to r i s e (Mast, 1947). The nature of the food vacuole membrane, as determined by freeze-fracture techniques, also changes as the vacuoles proceed through the cytoplasm. Changes i n pH r e s u l t i n a l t e r a t i o n s i n the intramembrane p a r t i c l e d i s t r i b u t i o n (Pinto da S i l v a , 1972) and t h i s i s r e f l e c t e d i n the reversed p o l a r i t y of the p a r t i c l e d i s t r i b u t i o n i n food vacuole membranes as compared with the plasma membrane (Allen, 1976) . In p e r i t r i c h s , tha u l t r a s t i u c t u r e of the cup-shaped vesicles has been described i n d e t a i l (Faure-Fremiet et a l . , 1962, Favard and :arasso, 1964, Carasso et a l . , 1964). McKanna (1973a,b) suggests that these cup-shaped coated ves i c l e s , which have a modified membrane consisting of a highly ordered coat similar to that observed i n Hydra (Slautterbacx, 1967), may be important i n the recognition and absorption of macromolecules. In Hydra, empty vesicles i n the cytoplasm exhibit a condensed coat consisting of 5 nm x 20 nm subunits shaped l i k e pegs with a globule near the d i s t a l end. As digestion proceeds, the coat i s transformed into the extended form with the subunits resembling extended filaments. In the extended form the coat can bind s p e c i f i c molecules (eg. f e r r i t i n ) . The membrane with bound macromolecules pinches 4 off into the cytoplasm at which time the coat resumes the condensed conformation and releases the macromolecules into the lumen of the vesicle. .Ia cultured human f i b r o b l a s t s s i m i l a r l y coated regions of the plasma membrane invaginate, pinch off from the surface membrane and migrate through the cytoplasm where they fuse with lysosomes (Anderson et a l . , 1977). The ultrastructure of the coated vesi c l e s in p e r i t r i c h s i s the same as i n Hydra, which suggests a similar function i».e. the extended coat selects and binds macromolecules from the vacuolar contents and the condensed coat represents a "clean" membrane available for recycling (McKanna, 1973a,b, see section 3) . Coated vesicles have also been implicated in the i n t r a c e l l u l a r transfer of membranes i n mammalian c e l l s (Pearse, 1976, Mollenhauer et a l . , 1977), radiolarians (Cachon and Cachon, 1977) and virus infected mammalian c e l l s (Rothman and Fine, 1980) . In the f i n a l stage (stage IV, Figure 1 - I E ) r old vacuoles fuse with the cytoproct and the undigested debris i s expelled via exocytosis,. By t h i s stage acid phosphatase a c t i v i t y i s no longer apparent i n the vacuoles (Esteve, 1970). The actual process of exocytosis involves the fusion of the food vacuole membrane with the plasma membrane at the cytoproct. Microtubules are important in guiding food vacuoles to the cytoproct as well as possibly supplying the forces reguired for fusion (Allen and Wolf, 1974).. The morphological changes which occur during the vacuolar cycle of Tetrahymena , a c i l i a t e c l o s e l y related to Paramecium are very similar ( E l l i o t t and Clemmcns, 1966, Ricketts, 1972, 5 Nilsson, 1976, 1977a), as are the exccytic events (Blum and Greenside, 1976, Allen and Wolf, 1979). Acid phosphatases have also been described and l o c a l i z e d i n Tetrah ymena (Seaman, 1961a, Muller and Toro, 1962, Allen et a 1. , 1963, Klamer and Fennell, 1963, E l l i o t t and Clemmons, 1966, Ricketts and Rappitt, 1974a), and the d i s t r i b u t i o n i s very s i m i l a r to that i n Paramecium except that i n stage II acid phosphatase i s not i n i t i a l l y limited to the vacuolar membrane but i s found throughout the food vacuole. .In p e r i t r i c h s , acid phosphatase i s not l o c a l i z e d i n membrane-bound vesicles but occurs i n high concentration within endoplasmic reticulum around the young food vacuoles (Faure-Fremiet et a l . , 1962, Goldfisher et a l . , 1963, Carasso et a l . , 1964).. Endocytosis has been described i n many organisms including other c i l i a t e s : Blepharisma (Dembitzer, 1968) , Colpoda (Rudzinska et a l . , 1966), Euplotes (Klcetzel, 1974), Hyalophysa (Bradbury, 1973), Paraciaita (Hauser, 1970), Phascolodon (Tucker, 1972), Terebrpspira (Bradbury and Goyal, 1 976), and Tokophyra (Rudzinska, 19 70^, as well as i n Pelomyxa (Roth, 1960), AiS2§_!£| (Mercer, 1959), Euglena (Sommer and Blum, 1965), Planaria (Rosenbaum and Eclon, 1960), macrophages (Essner, 1960, Cohn, 1971), and polymorphonuclear leukocytes (Conn and Hirsch, 1960). 6 B. Kinetics of accumulation and less cf food vacuoles C i l i a t e s can be fed substances which allow the number of labeled vacuoles which accumulate or are l o s t during a s p e c i f i c period to be e a s i l y determined. In t h i s manner the rates of accumulation and loss can be calculated. In Paramecium , the time required to form a single food vacuole depends on the nature of the ingested p a r t i c l e s (Mast, 1947) and varies from 0.44 minutes (Lea, 1942a) to 6.0 minutes (Metalnikov, 1912) with most values for Paramecium and Tetrahymena between 1.5 and 4.0 minutes (Muller and Tore, 1962, Berger, 1971, Ricketts, 1971a, Nilsson, 1972, Berger and Pollock, 1980). After an i n i t i a l rapid rate of accumulation of vacuoles, t h i s rate decreases (Nilsson, 1972, Berger and Pollock, 1980), but double labeling experiments i n d i c a t e that t h i s i s not due to cessation of feeding but rather attainment of an eguilibrium state between accumulation and less of labeled vacuoles (Berger and Pollock, 1980) . . In Paramecium , the exocytosis of vacuoles i s non-se l e c t i v e and independent of the age of the vacuole (Berger and Pollock, 1580). However, in Tetrahymena , a maturation period, which varies with the material ingested but i s generally around 60 minutes, i s reguired before a vacuole can be exocytosed (McBeath and Orias, personal communication). This may coincide with the 50 minute pre-digestion period during which there are no u l t r a s t r u c t u r a l changes in the ingested bacteria implying that digestion has not begun (Gebauer, 1977). This i s quite di f f e r e n t from Paramecium where r a d i o a c t i v i t y from deoxyribonucleic acid (DNA) of tritium-labeled bacteria appears 7 i n macronuclear DN& 4-5 minutes aft e r the addition of the bacteria (Berger, 1971}.. Once the maturation period has elapsed, egestion i s probably random in Tetrahymena as well (Eothstein and Blum, 1974, McEeath and Orias, personal communication) although i n some studies sequential l o s s of vacuoles was observed i n starved c e l l s (Eicketts and Eappitt, 1976) as well as i n well-fed c e l l s (Eicketts, 1 979). When starved c e l l s are used (Eicketts and Eappitt, 1976) the res u l t s obtained may not represent the true s i t u a t i o n since starved c e l l s undergo many changes, such as biochemical and ul t r a s t r u c t u r a l modifications (Levy and E l l i o t t , 1968), differences in amounts of acid phosphatase released a f t e r exposure to dig e s t i b l e p a r t i c l e s (Eicketts, 1971a), differences i n the rates of uptake of pa r t i c l e s (Eicketts, 1971b, Nilsson, 1976) , and changes i n c e l l volume (Eicketts and Eappitt, 1974b). When well-fed Tetrahymena were fed latex spheres followed by carmine p a r t i c l e s , sequential excretion of the vacuoles also occurred (Eicketts, 1979).. However, the res u l t s of the r e c i p r o c a l experiment (carmine followed by latex) are les s clear-cut and suggest the observed pattern of elimination may have been influenced by experimental design since the time between feeding the f i r s t and second markers i s 75 minutes which i s close to the maturation time reguired for food vacuoles in Tetrahymena (McBeath and Orias, personal communication) . The time that elapses between the formation of a food vacuole and i t s subseguent egestion (i.e..turnover time) i s guite variable and depends to a certain extent on the material 8 ingested (Metalnikov, 1912, Gebauer, 1977, Berger and Pollock, 1980) . around 45 minutes i s required f o r 50% of the vacuoles to turn over i n c e l l s that have been fed labeled or unlabeled bacteria (Berger, 1971). The rate of turnover of food vacuoles, and thus the e f f i c i e n c y cf feeding, may be related to the a v a i l a b i l i t y of d i g e s t i b l e materials (Berger and Pollock, 1580) . In amoebae, the rate of turnover of food vacuoles increases i n well-fed c e l l s as compared with starved c e l l s (Chapman-Andresen, 1968, Chapman-Andresen and Christensen, 1S70, Chapman-Andresen, 1977). In Paramecium , the rate of turnover of food vacuoles increases when non-nutritive p a r t i c l e s are ingested (Berger and Pollock, 1980) . . Thus, i f food i s p l e n t i f u l and the c e l l s well-fed, the h a l f - l i f e of a food vacuole would be short and the e f f i c i e n c y of feeding low but i f food i s scarce the h a l f - l i f e of vacuoles and the e f f i c i e n c y of feeding would increase (Berger and Pollock, 1S80) . In spite of differences in the turnover rates of vacuoles containing n u t r i t i v e and non-nutritive p a r t i c l e s , the k i n e t i c s of accumulation and loss of these vacuoles i s simi l a r (Berger and Pollock, 1980). C. Membrane recycling Actively phagocytizing c e l l s use amounts of membrane greater than their t o t a l surface in periods of less than ten minutes (Kloetzel, 1970,1974, Eicketts, 1971a, Goodall et a l . , 1972, McKanna, 1973a, Allen, 1974, Eicketts and Eappitt, 1974b). The source of t h i s membrane i s a pool of 9 c y t o p l a s m i c membranes ( C h l a p o w s k i and Band, 197 1, G o o d a l l e t a l . , 1972, Weidenbach and Thompson, 1974). T h i s membrane c o u l d be i n t h e form of v e s i c l e s s i n c e t h e y have been obse r v e d a t t h e base o f the b u c c a l c a v i t y and around growing f o o d v a c u o l e s (Roth, 1957, R a n d a l l and F i t t o n - J a c k s o n , 1958, M i l l e r and Stone, 1963) and a r e f r e g u e n t l y seen f u s i n g w i t h t h e plasma membrane a t the base of t h e g u l l e t , t h e o r i g i n o f n a s c e n t f o o d v a c u o l e s (Kennedy, 1965, E l l i o t t and Clemmons, 1966, McKanna, 1969,1973a, R u d z i n s k a , 1970, K l o e t z e l , 1970,1974, B a r d e l e , 1972, Bradbury, 1973, A l l e n , 1974, Howell and P a u l i n , 1 976). V e s i c l e s have a l s o been observed a r i s i n g from s p e c i a l i z e d areas o f the G o l g i a p p a r a t u s and then f u s i n g w i t h t h e plasma memlrane ( H i c k s , 1966, F a l k , 1969, C h l a p o w s k i and Band, 1971). Ey l a b e l i n g t h e c e l l s u r f a c e of P i e t y o s t e l i u m , i t i s p o s s i b l e t o f o l l o w the i n t e r n a l i z a t i o n of l a b e l e d p o r t i o n s of t h e plasma membrane and t h e i r subsequent reappearance a t t h e plasma membrane ( T h i l o and V o g e l , 1980). Coated v e s i c l e s a l s o a r i s e from t h e endoplasmic r e t i c u l u m and may then f u s e w i t h t h e G o l g i a p p a r a t u s where m o d i f i c a t i o n s o c c u r (Mollenhauer e t a l . , 1977, Morre e t a l . , 1979, Rothman and F i n e , 1980) or they may t r a n s f e r membrane p r o d u c t s d i r e c t l y w i t h o u t p r o c e e d i n g v i a t h e G o l g i (Cachon and Cachon, 1977). In Paramecium ca a da turn , t h e s e v e s i c l e s are d i s k - s h a p e d , 0.2-0 .5 micrometers (um) i n d i a m e t e r , w i t h membranes 9 nm t h i c k , s i m i l a r i n s t r u c t u r e to the plasma membrane ( A l l e n , 1974). They a r i s e by p i n o c y t c s i s from f o o d v a c u o l e s d u r i n g d i g e s t i o n and from the former f o o d v a c u o l e a f t e r i t f u s e s w i t h t h e c y t o p r o c t ( A l l e n and Wclf, 1974). The d i s k s l i e 10 along microtubules, i n single f i l e and approximately 40 nm away from them. The microtubules d i r e c t the disks to the l e f t cytostomal l i p which i s a region specialized for seguestering the disks. As new membrane i s required f o r endocytosis, the disks fuse with th=- growing vacuolar membrane (Allen, 1974). No cross-bridges between the microtubules and vesicles have been observed in the micrctubule-mediated disk recycling process (Allen, 1975).. Cross-bridges between microtubules r e s u l t in the directed movement of c e l l u l a r components, as i s the case with the mobility cf non-flagellated sperm (Eobison, 1966) , the movement of granules i n melanocytes (Green, 1968), chromosome movement during mitosis (Hepler et a l . , 1970), and synaptic vesi c l e s i n nerve axons (Smith, 1971), as well as changes i n nuclear shape (Mcintosh and Porter, 1967) , contraction and expansion of axostyles and tentacles (Giimestone and Cleveland, 1965, Eudzinska , 1967, Bannister and Tat c h e l l , 1968, Eardele, 1974), and i n the propulsion of food into the cytopharyngeal basket of the c i l i a t e , ghascoloion (Tucker, 1972). In P._ caudatum , the microtubules are firmly bound, at a distance of 30-40 nm, in a filamentous material. . The ve s i c l e s crient with the microtubules but the r e s u l t i n g movement could be due to the formation and breakage of linkages within the filamentous material or a polymerization at one end and concurrent depolymerization at the other end cf the microtubules {Allen, 1975). The study of freeze-fracture sections of vacuolar membranes provides additional evidence f o r membrane recycling. The d i s t r i b u t i o n of 8. 5 nm p a r t i c l e s on the two faces of a 11 f r a c t u r e d membrane a r e c h a r a c t e r i s t i c f o r membrane t y p e s ( B r a n t o n , 1971, Dempsey e t a l . , 1974). Changes i n pH or tempe r a t u r e can r e s u l t i n r e v e r s i b l e c lumping of t h e p a r t i c l e s ( P i n t o da S i l v a , 1972, Sp e t h and W u n d e r l i c h , 1973).. I n caudatum , the c y t o p h a r y n g e a l membrane, a t t a c h e d f o o d v a c u o l e s and d i s k - s h a p e d v e s i c l e s a l l have a h i g h l y p a r t i c u l a t e B f a c e ( A l l e n , 1976), whereas the plasma membrane has a more p a r t i c u l a t e A f a c e . C i r c u l a t i n g food v a c u o l e s have fewer p a r t i c l e s per u n i t - a r e a than d i s k - s h a p e d v e s i c l e s and t h e i r A f a c e i s more p a r t i c u l a t e than t h e i r B f a c e . . T h i s r e v e r s e d p o l a r i t y c o u l d be due t o t h e low pH of t h e v a c u o l e o r i t might i n d i c a t e a s p e c i a l i z e d f u n c t i o n o f t h e v a c u o l a r membrane. When t h e f o o d v a c u o l e f u s e s w i t h t h e c y t o p r c c t , the r e s u l t i n g d i s k s have t h e same p o l a r i t y as t h o s e a t t h e cy t o p h a r y n x i n d i c a t i n g t h a t t h e same v e s i c l e s g e n e r a t e d through e x o c y t o s i s as w e l l as t h e v e s i c l e s t h a t p i n c h o f f from t h e f o o d v a c u o l e make t h e i r way back t o the g u l l e t where they are added t o a new food v a c u o l e ( A l l e n , 1976).. Dm . Purpose of t h i s t h e s i s Much i s new known about t h e proc e s s c f food v a c u o l e f o r m a t i o n , b o t h t h e d i g e s t i v e and membrane r e c y c l i n g a s p e c t s , and the i m p l i c a t i o n s of t h i s knowledge a r e f a r - r e a c h i n g . E n d o c y t o s i s and i n t r a c e l l u l a r d i g e s t i o n are w i d e s p r e a d , i f not u n i v e r s a l , a s p e c t s of the b i o l o g y of e u k a r y o t e s . I n p r o t o z o a and l o w e r metazoa, t h i s p r o c e s s i s r e s p o n s i b l e f o r the i n g e s t i o n and d i g e s t i o n of n u t r i e n t s * I n v e r t e b r a t e s , t h e same 12 type of process i s responsible for the ingestion and degradation cf foreign substances and organisms by the phagocytic c e l l s of the immune system. P__ t e t r a u r e l i a i s an excellent organism for a study of endocytosis since i t i s easily cultivated and maintained, has a short generation time and has been extsnsively studied i n many aspects of i t s b i o l o g i c a l processes (Sonneborn, 197C). The induction and analysis of mutations i n organisms i s an excellent way to understand the nature of a given process and temperature-sensitive mutants i n particular f a c i l i t a t e the examination of events that are c r u c i a l to the s u r v i v a l of the organism. The genetics of P. t e t r a u r e l i a i s r e l a t i v e l y well-understood and many mutants have been isolated and studied (Sonneborn, 1S74)..The purpose of t h i s thesis w i l l be to examine endocytosis and the vacuolar cycle i n P. .t e t r a u r e l i a using the induction of temperature-sensitive mutants to further elucidate some of the events of t h i s process. 1 3 Figure 1 - 1 . Morphological changes in food vacuoles during digestion. A. accumulation and concentration of bacteria at the base of the gullet (g) B. .accumulation of small v e s i c l e s (1) around the newly formed food vacuole C rD. as digestion proceeds, the membrane becomes highly irr e g u l a r and pinocytotic vesicles (p) pinch off from the food vacuole E. the old food vacuole fuses vith the membrane at the cytoproct and the vacuolar membrane fragments into v e s i c l e s (v) 15 CHAPTER II STANDARDIZATION OF CONDITIONS A.. Introduction Many factors influence the formaticn of food vacoules i n c i l i a t e s : temperature (Lee,. 1942a, Nilsson, 1972), pH (Lee, 1942b, Nilsson, 1976), cent r i f u g a t i c n and media changes (Nilsson, 1972) and the n u t r i t i o n a l state of the c e l l s (Chapman-Andresen and Nilsson, 1968, Eicketts, 1971b, Nilsson, 1972). Starved c e l l s can d i s t i n g u i s h between n u t r i t i v e and non-n u t r i t i v e materials (Mast, 1947, Bcsenbaum and Whittner, 1962, Muller et a l . , 1965, Eicketts, 1971a), as long as the two types of p a r t i c l e s are not fed simultaneously (Eicketts, 1971a). C e l l u l a r age i s also important; increased clonal age i s accompanied by a decrease in phagocytic competence (Chapman-Andresen and Nilsson, 1968, Smith-Sonneborn and Eodermel, 1976). The stage in the c e l l cycle i s also s i g n i f i c a n t because dividing c e l l s do not form food vacuoles (Chapman-Andresen and Nilsson, 1968, Aufderheide, 1976)..The rate of endocytosis as well as the number of vacuoles per c e l l changes throughout the c e l l cycle (Berger, 1971, E i c k e t t s , 1971b, Nilsson, 1976, Smith-Sonneborn and Eodermel, 1976) . Tetrahymena require the presence of inducer substances such as peptone-yeast broth or the dye, trypan blue (Seaman, 1961a), proteins, polypeptides, ribonucleic acid (RNA) , polysaccharides, and glucose (Eicketts, 1972). These may 16 bind at s p e c i f i c s i t e s at the base cf the g u l l e t (Seaman, 1961b, Ricketts, 1972). The concentration of p a r t i c l e s may a f f e c t the number of food vacuoles formed... Well-fed c e l l s phagocytize p a r t i c l e s according to t h e i r concentration i n the medium (Ricketts, 197 1a, Smith-Sonneborn and Rodermel, 1976, Berger and Pollock, 1980) and l a r g e r p a r t i c l e s , such as yeast with a diameter of 4 um, r e s u l t i n the formation of larger food vacuoles (Mast, 1947).. Both n u t r i t i v e and non-nutritive materials are commonly used as markers for food vacuoles. The rate of uptake of these markers i s an i n d i c a t i o n of phagocytic rate.. Some of the n u t r i t i v e markers that have been used are: yeast stained with Congo Red (Mast, 1947), heat k i l l e d Serratia marcescens (Seaman, 196 1b), tritium-labeled Interofcacter aeroqenes (Berger, 1971) and r a d i o a c t i v e l y labeled sucrose and albumin (Ricketts and Rappitt, 1975).,There are also a wide variety of non-nutritive markers that have been u t i l i z e d : India ink (Lee, 1 942a,b), trypan blue (Seaman, 1961a,b) , c o l l o i d a l thorium oxide, f e r r i t i n and latex beads (Favard and Carasso, 1964) , c o l l o i d a l gold ( E l l i o t t and Clemmons, 1966) , carmine p a r t i c l e s (Chapman-Andresen and Nilsson, 1968), alcian blue (Nilsson, 1972) , tantalum p a r t i c l e s (Orias and Pollock, 1975), a c r y l i c colors (McEeath and Orias, personal communication) , and watercolors (Berger and Pollock, 1 980). Therefore, considering the myriad factors that influence endocytosis, procedures and conditions were investigated and standardized i n order to reduce, as much as possible, the 17 inherent v a r i a b i l i t y of the system. B. Mat e r i a l s and Methods 1 • Growth and preparation of Paramecium t e t r a u r e l i a P..tetraurelia (Sonneborn , 1975), formerly Paramecium au r e l i a , syngen 4, was grown i n autoclaved grass medium inoculated with Enterobacter aerogenes (Sonneborn, 1970). Exponentially growing c e l l s approximately 20 f i s s i o n s old were centrifuged and resuspended i n twice the o r i g i n a l volume of fresh culture f l u i d . After an e g u i l i b r a t i o n period of 5-15 minutes the c e l l s were incubated for 6 hours at the experimental temperature. _ The« c e l l s were then exposed to a food vacuole marker at the experimental temperature, fixed i n a few drops of 37% formaldehyde and the number of food vacuoles that accumulated in a given time period was counted i n 30-50 c e l l s . . 2« Select ion of food vacuole markers The a b i l i t y of P.. t e t r a u r e l i a to phagocytize watercolors (Gunter Wagner designer's colors) was compared with carmine p a r t i c l e s . . 1.0 mg/ml red watercolor (EP, Pelikan, carmine #34), 0.8 mg/ml carmine p a r t i c l e s (Fisher S c i e n t i f i c ) and 1.0 mg/ml carmine p a r t i c l e s were suspended i n culture f l u i d . _• Egual volumes of marker were mixed with c e l l s in culture f l u i d and samples were removed and fixed a f t e r 1, 5, 10, 15, 20, 30, and 40 minutes. 18 3.. Effect of temperature C e l l s were incubated at 17°C, 27°C and 34.5°C for 6 hours and then fed RP (1 mg/ml) or blue watercolor (BP, Pelikan, Ultramarine #120, 1.5 mg/ml) for 20 minutes. . The c e l l s were fi x e d and the number of colored food vacoules per c e l l was counted. 4. Effect of time i n marker C e l l s were incubated for 6 hours at 27°C or 34.5°C and fed RP or BP prepared as previously indicated. Samples were removed and fixed after 1, 3, 5, 10 , 15, 20, 30, 40, 50, and 60 minutes i n the watercolor. 5. Ef f e c t of culture medium C e l l s were adapted to axenic medium (Keenen et a l . , 1978) by a modification of the method of Allen and Nerad (1978) (Appendix I ) . They were fed black watercolor (Pelikan, Mixing Black #012, 1.0 mg/ml) i n axenic medium.. In addition, c e l l s were grown i n bacterized medium, adapted to Dryl^s solution (Dryl, 1959) (Appendix I) and fed black watercolor. Samples were removed after 1 , 3, 5, 10, 15, 20, 30, 45, and 60 minutes in watercolor, fixed and examined. 19 C . Eg s a l t s 1« Growth and preparation of P. t e t r a u r e l i a The outlined procedure was routinely followed. In spite of these precautions variations between experiments did occur. Therefore, wild-type control c e l l s were included f o r comparison in a l l experiments. . 2.. Selection of food vacuole markers Bed watercolor (EP) was re a d i l y phagocytized by Paramecium c e l l s and the food vacuoles formed were c l e a r l y delineated and easy to count (Plate III-1a)..By comparison, the food vacuoles formed after ingsstion of carmine p a r t i c l e s were less regular i n shape and they were not as c l e a r l y defined; A s i g n i f i c a n t l y higher number of colored vacuoles accumulated a f t e r 20 minutes in watercolor (Figure 2-1) and there was no difference between the number that accumulated i n 0.4 or 0.5 mg/ml carmine p a r t i c l e s . . 3.. Effect of temperature There was an increase i n the mean number of colored vacuoles that accumulated a f t e r 20 minutes in EP or BP with an increase i n temperature from 17°C to 27°C (Table 2-1).. There was no further increase at 34.5°C, indicating a wide temperature range for maximum endocytic a c t i v i t y . There was no s i g n i f i c a n t difference between the mean number of vacuoles that 20 accumulated after 20 minutes i n EP or EP. 4. Effect of time i n marker There was an i n i t i a l rapid increase in the number of colored vacuoles that accumulated i n EP or BP (Figures 2-2 and 2-3) . This increase was sharper at 34.5°C than 27°C. In a l l cases a plateau at around 14 vacuoles per c e l l was reached at which point an eguilibrium state between the formation and loss of colored vacuoles was apparent. There were no s i g n i f i c a n t differences between c e l l s incubated at 27°C or 34.5°C once the steady state was reached . _ 5» Effect of culture medium The number of food vacuoles that accumulated i n axenic medium as well as their s i z e and shape were si m i l a r to those present i n tacterized medium (Figure 2-4) . However, those colored vacuoles formed i n Dryl's solution were very small and the mean number increased greatly to 22.6, 95% confidence i n t e r v a l (c.i.) = 20. 8-24.5, a f t e r 60 minutes i n black watercolor (compared with 12.9, c . i . = 12.0-13.7, i n axenic and 14.1, c . i . = 12.9-15.2, ia bacterized medium). In both cases an eguilibrium state between formation and l o s s was reached although i t took longer to reach i n Dryl's solution. The reason for the unusual re s u l t s with black watercolor in Dryl's solution i s unknown. I t i s known that the size of the p a r t i c l e s influences the size of the food vacuoles (Mast, 1947), 21 therefore i t i s possible that black watercolor contains very small p a r t i c l e s . . When c e l l s in Dryl's solution were fed EP instead of black watercolor, the mean number of vacuoles formed a f t e r 60 minutes was 10.3 ( c . i . .= 9.1-11.6).. D. Discussion Watercolors are very useful markers for phagocytosis because they are non-toxic and the cclcred vacuoles formed are c l e a r l y v i s i b l e in the cytoplasm of the c e l l . Within 20 minutes of exposure to watercolors a steady state exists between formation and loss of colored vacuoles.. Although these watercolors are a r t i f i c i a l markers, the kinetics of formation and loss are s i m i l a r to those obtained when tritium-labeled bacteria are used (Berger and Pollock, 1980). . The temperatures of 27°C and 34.5°C are both excellent for endocytosis. This i s i n agreement with Lee (1942a) who has found that between 20°C and 35°C there i s l i t t l e increase i n the rate of endocytosis i n P..aurelia but below 20°C or above 35°C the rate decreases.. Nilsson (1972) has found that i n Tetrahymena 28°C i s optimum for endocytosis with a decrease occurring below or above that temperature. In Paramecium i t appears that a mean of around 14 vacuoles per c e l l can be maintained between 27°C and 34.5°C. When c e l l s are fed watercolor in Dryl's solution the size and number of vacuoles that accumulate varies with the watercolor used. This v a r i a b i l i t y can be decreased i f dead bacteria are added to the watercolor in Dryl's solution (Berger 22 and P o l l o c k , 1980). I n t h e presence of l i v e b a c t e r i a or p a r t i c l e s f r c m a x e n i c medium the number of f o o d v a c u o l e s t h a t accumulate i n w a t e r c o l o r and t h e i r s i z e and shape a r e c o n s i s t a n t . . Whereas the use of a x e n i c medium would seem p a r t i c u l a r l y advantageous, t h e r e a r e s e v e r a l drawbacks, t h e most i m p o r t a n t being the g u e s t i o n a t l e m e t a b o l i c s t a t e of these c e l l s . Even w i t h new mass c u l t u r i n g t e c h n i q u e s , t h e g e n e r a t i o n time of Pj_ t e t r a u r e l i a i n a x e n i c medium i s g r e a t e r t h a n t e n h o u r s a t 25°C ( T h i e l e e t a l . , 1980) compared w i t h s i x h o u r s when c e l l s a r e grown m o n o x e n i c a l l y (Sonneborn, 1970).- P r i n c e and Gibson (1978) have shown t h a t compared t o c e l l s grown m o n o x e n i c a l l y , those grown a x e n i c a l l y have condensed m i t o c h o n d r i a , lower oxygen c o n s u m p t i o n , l o w e r l e v e l s of ATPase, and d e c r e a s e d amounts of c y t o c h r o m e s , a l l c h a r a c t e r i s t i c o f a g i n g c e l l s . I n view of t h e e f f e c t o f a g i n g on e n d o c y t o s i s i n Paramecium (Smith-Sonneborn and Eodermel, 1976) , as w e l l as the r o u t i n e problems a s s o c i a t e d w i t h c u l t i v a t i n g and m a i n t a i n i n g c e l l s a x e n i c a l l y , t h i s s t u d y was c a r r i e d out u s i n g c e l l s grown m o n o x e n i c a l l y cn E n t e r o b a c t e r aerogenes. T h e r e f o r e , i u summary, t h e f o l l o w i n g c o n d i t i o n s have been adopted as s t a n d a r d p r o c e d u r e , u n l e s s o t h e r w i s e s t a t e d : use o f c e l l s a p p r o x i m a t e l y 20 f i s s i o n s e l d grown m o n o x e n i c a l l y , i n c u b a t i o n temperatures of 27°C and 34.5°C and use o f w a t e r c o l o r s suspended i n c u l t u r e f l u i d and f e d t o c e l l s f o r 20 minutes as f o o d v a c u o l e markers. 23 Table 2-1..Effect of temperature en the accumulation of vacuoles Temp. (°C) EP (1) BP (2) 17. 0 27. 0 34. 5 5. 9 (5. 3-6. 4) 14.0(12.7-15.3) 13. 9(12.9-1 5.0) 6. 1 (5.7-6.5) 15. 1 (14.0-16.2) 14.5(13.0-15.9) (1) means and 95% confidence i n t e r v a l s of the number of colored vacuoles that accumulated a f t e r 20 minutes i n red watercolor (2) means and 95% confidence i n t e r v a l s cf the number of colored vacuoles that accumulated after 20 minutes i n blue watercolor 24 Figure 2-1. Accumulation of vacuoles in carmine p a r t i c l e s and red watercolor..Effect of increasing incubation time at 27°C. V e r t i c a l bars represent 95% confidence i n t e r v a l s . . (• — • = red watercolor, =0.4 mg/ml carmine p a r t i c l e s , +—+ = 0.5 mg/ml carmine particles) IS 26 F i g u r e 2-2. Acc u m u l a t i o n of v a c u o l e s i n red w a t e r c o l o r . E f f e c t of i n c r e a s i n g time i n r e d w a t e r c o l o r a t 27°C and 34.5°C. V e r t i c a l b a r s r e p r e s e n t 95% c o n f i d e n c e i n t e r v a l s . . ( • — • = 27°C, +-- + = 34.5°C) 28 Figure 2-3. Accumulation of vac of increasing time i n blue V e r t i c a l bars represent 95% con • = 34.5°C) uoles in blue watercolor. Effect watercclor at 27°C and 34.5°C. fidence i n t e r v a l s . (•—• = 27°C, NO. OF COLORED_VACUOL.ES/CELL cn o cn 30 Figure 2-4. Accumulation of vacuoles i n buffer and axenic medium.. Ef f e c t of increasing time in black watercolor at 27°C. V e r t i c a l bars represent 95% confidence i n t e r v a l s . . (•--• = Dryl's buffer, • — • = axenic medium) 31 32 CHAPTER III MUTAGENESIS AND DESCRIPTION CF PUTATIVE MUTANTS A. . Intro duct ion Phagocytosis i s an elaborate b i o l o g i c a l process that involves the a c t i v i t i e s and interactions of many c e l l u l a r components. Each aspect of t h i s complex process represents a po t e n t i a l l y mutable event. The selection of temperature sensitive (ts) mutations makes i t possible to examine indispensible functions related to feeding because the c e l l s can be maintained at the permissive temperature. Mutations that affect endocytosis have been isolated in Tetrahymena th ermophila (Orias and Pollock, 1975, S i l b e r s t e i n et a l . , 1975, Suhr-Jessen and Orias, 1979a,b) and have been found associated with other mutations in P. te t r a u r e l i a (Jones, 1977). Mutagenesis was undertaken to recover mutations involving some aspect of endocytosis or food vacuole processing. B. . Materials and Methods 1. Mutagenesis Mutagenesis was performed according to Kung et a l . (1971). C e l l s approximately 50 f i s s i o n s old were concentrated by centrifugation (100xg, 3 minutes) and resuspended in 100 ml Dryl's solution containing 75 ug/ml N-methyl-N'nitro- N-33 nitrosoguanidine (MNNG, Sigma). A t o t a l of 3 x 10 5 c e l l s (determined by s e r i a l d i l u t i o n of 1.0 ml of c e l l s i n culture fluid) were treated with MNNG for 60 minutes at room temperature. After three washes i n Dryl's solution the c e l l s were suspended in s u f f i c i e n t culture f l u i d to allow three to four d i v i s i o n s before starvation and the onset of autogamy, which r e s u l t s i n homozygosis of any induced mutations. Failure of c e l l growth prior to autogamy was considered vegetative death and was determined by examining 84 i s o l a t e d pre-autogamous c e l l l i n e s . F a i l u r e of c e l l growth subseguent to autogamy was considered ex-autogamous death, attributable to recessive l e t h a l mutations, and was determined by examining 84 iso l a t e d ex-autogamous c e l l l i n e s . 2.- Selection procedure Ex-autogamous l i n e s were treated in one of the following ways: a.. individual l i n e s e l e c t i o n 1,26C c e l l l i a e s were isolated i n depression s l i d e s a f t e r approximately 20 c e l l d i v i s i o n s (12 c e l l d i v i s i o n s is the maximum observed phencmic lag, Berger, 1 976) . The c e l l s were r e p l i c a plated (Sonneborn, 1 970) and the second set of is o l a t e s was s h i f t e d to 34.5°C for 24 hours. .A few drops of blue watercolor (EP) were added to each c e l l line..The c e l l s were f i x e d after 20 minutes and each clone was scored indi v i d u a l l y f o r abnormalities in size, shape 34 and number of f o o d v a c u o l e s . C e l l s t h a t were abnormal were r e t e s t e d with BP and r e d w a t e r c o l o r (HP).. b.. mass s e l e c t i o n T h i s procedure was based on t h e d e n s i t y - l a b e l i n g s y n c h r o n i z a t i o n method developed by Wolfe (1973) and m o d i f i e d by S i l b e r s t e i n et a l . . (1975) and A u f d e r h e i d e (1976) . C e l l s a p p r o x i m a t e l y 20 f i s s i o n s o l d were d i l u t e d t e n - f o l d i n c u l t u r e f l u i d and i n c u b a t e d a t 34.5°C f o r 24 hours i n an a i r i n c u b a t o r a f t e r which t i m e t h e y were c o n c e n t r a t e d by c e n t r i f u g a t i o n (100xg r 3 minutes) and resuspended i n 5 ml D r y l ' s s o l u t i o n c o n t a i n i n g 15 mg t a n t a l u m p a r t i c l e s (Matheson, Coleman and B e l l , Co.). The c e l l s were g e n t l y shaken f o r 10 minutes and t h e n l a y e r e d on a d i s c o n t i n u o u s F i c o l l (Sigma) g r a d i e n t c o n s i s t i n g o f 4 ml of 20% and 4 ml of 10% F i c o l l i n D r y l ' s s o l u t i o n . . T h e g r a d i e n t was c e n t r i f u g e d f o r 5 minutes a t 300xg. Those c e l l s t h a t had i n g e s t e d t a n t a l u m were expected t o sediment i n t h e p e l l e t and t h o s e w i t h d e c r e a s e d e n d o c y t o s i s were expected t o be l o c a t e d a t t h e 10%/ 20% i n t e r f a c e . _ C e l l s found at t h e i n t e r f a c e were c o l l e c t e d , resuspended i n f r e s h c u l t u r e f l u i d and a l l o w e d t o grow a t 27°C f o r 3-4 days b e f o r e t h e procedure was r e p e a t e d . . F i v e e n r i c h m e n t c y c l e s were performed a f t e r which 2 10 p o t e n t i a l mutant l i n e s were i s o l a t e d and t e s t e d i n d i v i d u a l l y w i t h BP as i n p r o c e d u r e ( a ) . . 35 3. Description of putatiye mutants A t o t a l of 61 ts putative mutants were isola t e d by sele c t i o n procedure (a)..Of these, 11, denoted A1-A11, were selected f o r further study..The following c h a r a c t e r i s t i c s were ex am ined: a. . generation time at 34. 5°C (Nachtwey and Cameron, 1972) b. _ number of food vacuoles that accumulated in EP and BP in 20 minutes c. g u l l e t morphology determined by s i l v e r impregnation (Frankel and Heckmann, 1968) d. trichocyst discharge determined by adding a few drops of a saturated p i c r i c acid solution to a sample of c e l l s (Pollack, 1974) e. .endocytosis i n synchronous samples - synchronous c e l l s of the ts mutant 4Y-2 1, which i s def i c i e n t i n DNA synthesis (Peterson, 1974), were obtained by c o l l e c t i n g c e l l s within 10 minutes of d i v i s i o n . These were shi f t e d to 34.5°C for 20, 40, 60, and 100 minutes and 2, 2.5, 3 , 3.5, 4, 4.5, and 5 hours. BP was added for 20 minutes, the c e l l s were fixed and the number of colored vacuoles per c e l l was counted. Fixed c e l l s were embedded i n saline gelatin or a i r - d r i e d on glass s l i d e s coated with albumin and photographed on a L e i t z Orthomat-W photomicroscope using Nomarski optics.. 36 4.. Genetic analysis Two putative mutants, A5 and A10, were crossed to a temperature resistant l i n e homozygous f o r the recessive behavioral marker pawn A lpw]_ (Rung et a l . , 1971). The second generation (F2) segregation patterns were determined by inducing autogamy in the f i r s t generation (F1) clones produced by the i n i t i a l cross. The lin e A5 was also crossed in the same manner to a temperature r e s i s t a n t l i n e homozygous for the recessive spot Jspi_ mutation which caused an accumulation of c r y s t a l s in . the cytoplasm (Morton, 1977)..The progeny of the crosses were scored for the pawn or spot markers, food vacuole morphology (A10) or position i n the c e l l (A5), and trichocyst discharge (A5) . C. Results 1. Mutagenesis The freguency of vegetative death following mutagenesis gave an indication of the t o x i c i t y of the mutagen and the frequency of ex-autogamous death gave an indication of the r e l a t i v e freguency of l e t h a l mutations. In t h i s mutagenesis experiment vegetative death was 23% ard ex-autogamous death was 28%. These freguencies were low i n comparison to s i m i l a r experiments (Morton, 1977) although the ex-autogamous death rate varied greatly between experiments (Peterson, 1974, Morton, 1977). In general, experiments with higher ex-autogamous death rates yielded more putative mutants. 37 2. Yield of putative mutants Through the i n d i v i d u a l l i n e selection procedure 61 putative mutants were i s o l a t e d (rate = 61/1260 = 5%)..The mass sel e c t i o n procedure resulted i n 21 putative mutants (rate = 21/2 10 = 105?), a two-fold increase. This increase was lower than expected because the procedure also selected for dividing or conjugating c e l l s and c e l l s i n autogamy (Aufderheide, 1976). 3. Description of putative mutants The c h a r a c t e r i s t i c s of 11 putative mutants have been l i s t e d i n Table 3-1.„Of these, A5, A7 and A10 were considered the most dramatic and A5 and A1C were selected for further study (the l i n e , A7, was l o s t ) . Brief descriptions of the other mutant l i n e s have been l i s t e d in Appendix I I . The mutant 4Y-21 was a ts c e l l cycle mutant with e r r a t i c DNA synthesis at 34.5°C (Peterson, 1S74) . The mean number of colored vacuoles per c e l l in EP was 4.14 {95% confidence i n t e r v a l , c. i . = 3.81-4.47), s i g n i f i c a n t l y lower than wild-type c e l l s (Table 3-1). In synchronous populations s h i f t e d to 34.5°C a gradual decrease i n the number of food vacuoles that accumulated occurred a f t e r the temperature s h i f t (Figure 3-1). Two ether ts DNA- mutants, 2A2 and 2A5 (Peterson, 1974), also formed fewer food vacuoles i n EP at 34.5°C (means and 95% c.i.= 5. 24, 3. 4- 7.08 f o r 2A2 and 3. 42, 2.8-4. 04 for 2A5) Synchronous samples of 2A2 s h i f t e d up afte r d i v i s i o n also showed a decrease in numbers of food vacuoles to 6.S6 (c.i.= 6. ,33-7.59) after three hours at the r e s t r i c t i v e temperature [Z.Easmussen, personal communication). 38 4. . Genetic analysis a. . A10 x pawn A The f i r s t generation (F1) individuals of t h i s cross were a l l wild-type. The segregation pattern of the second generation ( F 2 ) was consistent with a recessive mutation at a single locus (Table 3-2). . This mutant has been designated defective membrane or dml. The phenotypes of t h i s mutant have been schematically i l l u s t r a t e d i n Figure 3-2. b. A5 x pawn A Two crosses were performed and i n both cases the F1 progeny were a l l wild-type..The F2 segregation patterns (Table 3-3) were not consistent with a single gene recessive mutation a f f e c t i n g both the clumping of food vacuoles and trichocyst non-discharge.. Each locus segregated in a 1:1 fashion. The Chi sguare (X2) p r o b a b i l i t i e s (referred to as P) for each locus for each of the two crosses were: pawn = . 9 9 , . 5 , trichocyst non-discharge Jtnd)_ = . 1 5 , . 33 and food vacuole clumping (fvc) = . 15 , .28.. A pr o b a b i l i t y (P) <.05 was considered s i g n i f i c a n t f o r re j e c t i o n of the o r i g i n a l hypothesis; The data from both crosses were homogeneous (X 2 test for homogeneity, P =.71) and the pooled data have been presented i n Table 3-3. The crossover phenotypes did not segregate in a 1:1:1:1 fashion (P = .02) due to an excess of pawn c e l l s with clumped vacuoles. In order to determine 39 i f t h e .pawn paenotype was i n t e r f e r i n g w i t h the s c o r i n g of c e l l s w i t h clamped v a c u o l e s , A5 was c r o s s e d t o s p o t . c. A5 x spot The F l progeny were a l l w i l d - t y p e . The F2 s e g r e g a t i o n p a t t e r n s have been p r e s e n t e d i n T a b l e 3-4. . The F2 r e c o m b i n a n t s s e g r e g a t e d i n a 1:1:1:1 (P =.6) f a s h i o n i n d i c a t i n g t h a t the m o r p h o l c g i c a l a b n o r m a l i t i e s sometimes a s s o c i a t e d w i t h t h e pawn phenotype a t 34.5°C ca u s e d an i n c r e a s e i n the number o f l i n e s s c o r e d as h a v i n g clumped f o o d vacuoles..The r e s u l t s of t h i s c r o s s were homogeneous w i t h t h e two A5 x pawn c r o s s e s (F = .82 f o r p a r e n t a l s , .56 f o r recombinants) and the combined l i n k a g e between t n d and f v c was 34±3 ( s t a n d a r d d e v i a t i o n ) map u n i t s . The two l i n e s r e s u l t i n g from th e A5 x spo_t c r o s s were d e s i g n a t e d t n d and f v c and were each c r o s s e d t o s j r o t . The F1 progeny were a l l w i l d - t y p e and t h e F2 s e g r e g a t i o n p a t t e r n s (Table 3-5) were c o n s i s t a n t w i t h tnd and fj/c each s e g r e g a t i n g as s i n g l e gene r e c e s s i v e m u t a t i o n s . . A check o f 60 F2 l i n e s from the t n d x spot c r o s s y i e l d e d no c e l l s w i t h clumped food v a c u o l e s . . A s i m i l a r check of 60 F2 l i n e s from the f v c x s p o t c r o s s y i e l d e d two l i n e s each w i t h two m o r p h o l o g i c a l l y abnormal c e l l s t h a t d i d not d i s c h a r g e t h e i r t r i c h o c y s t s . The r e a s o n f o r the n o n - d i s c h a r g e c f t r i c h o c y s t s i n t h e s e f v c c e l l s i s not known but may be r e l a t e d t o t h e i r m o r p h o l o g i c a l a b n o r m a l i t i e s . . 40 D. D i s c u s s i o n The mutants o b t a i n e d i n t h i s s t u d y can be d i v i d e d i n t o f o u r main groups: 1. M o r p h o l o g i c a l a b n o r m a l i t i e s Mutants i n t h i s group have c e l l u l a r and/or g u l l e t a b n o r m a l i t i e s which i n t e r f e r e w i t h e n d o c y t c s i s . ecj. A1, A7 2- Decreased e n d o c y t o s i s These mutants form fewer food v a c o u l e s t h a n normal w i t h o u t h a v i n g any o b v i o u s m o r p h o l o g i c a l a b n o r m a l i t i e s t h a t would i n t e r f e r e w i t h e n d o c y t o s i s . , ecj. A3, A6, A9, 4Y-21, 2A2, 2A5 3. Abnormal p o s i t i o n i n g c f f o o d v a c u o l e s The f o o d v a c o u l e s of mutants i n t h i s group are a t y p i c a l l y d i s t r i b u t e d w i t h i n t h e c e l l . T h i s may c r may not be accompanied by a d e c r e a s e i n e n d o c y t o s i s . eg. f v c , A8 4. Abnormal morphology of f o o d v a c u o l e s Mutants i n t h i s group may or may not form normal number of f o o d v a c u o l e s but t h o s e formed a r e abnormal i n s i z e and/or shape. . ecj. djn_1, A2, A4, A1 1 I t i s i n t e r e s t i n g t h a t no l i n e s have been i s o l a t e d which are d e f e c t i v e i n e x o c y t o s i s . I f e x c c y t o s i s i s i n h i b i t e d , an i n c r e a s e i n the numbers of f o o d v a c u o l e s t h a t accumulate might be e x p e c t e d . However, i f t h e r e i s a l i m i t e d s u p p l y of membrane f o r f o o d v a c u o l e s , which i s r e c y c l e d (Allen,1974) , fewer v a c u o l e s might accumulate i n a g i v e n p e r i o d o f t i m e due t o t h e decreased r a t e of e x o c y t o s i s and t u r n o v e r o f t h e p o o l of 41 membrane available for endocytosis. Therefore, some of the mutants i n group (2) could i n fact be exocytosis mutants. This could be determined by observing the loss as well as the accumulation of colored vacuoles. Due to the nature of the sel e c t i o n system the majority of mutants is o l a t e d by the mass se l e c t i o n system procedure f a l l i n t o group (1) or (2) whereas those selected i n d i v i d u a l l y are dis t r i b u t e d among the four groups..Although the mass selection procedure i s time-saving i n i t i a l l y , i n d i v i d u a l selection i s reguired to obtain a complete range of mutants. Group (1) could be subdivided to d i f f e r e n t i a t e between c e l l u l a r and gullet abnormalities although i n some cases the two are related. .Jones (1977) has described a ts small mutant of P_ t e t r a u r e l i a with an abnormal g u l l e t and decreased endocytosis which i s due to both the decreased size and the abnormal g u l l e t . . Using a mass selection system s i m i l a r to the one previously described, Suhr-Jesscn and Orias (1 979a,b) have recovered 13 ts mutants in T._ thermophila which have decreased phagocytosis due to defects i n the development of the oral apparatus. Most of these mutants have a morphologically normal but non-functicnal oral apparatus and a l l but one belong to the same complementation group, vac A. Three DNA- mutants (Peterson, 1974) are c h a r a c t e r i s t i c of group (2). Aging c e l l s also exhibit a decrease i n DNA synthesis (Smith-Sonneborn and Klass, 1974) and endocytic capacity (Smith-Sonnebcin and Rodermel, 1976) but the relationship between the two i s not known. The mutant of T._ thermophila which does not f a l l into the vac A complementation group has 42 c h a r a c t e r i s t i c s similar to the DNA- mutants; for example, d i v i s i o n ceases after transfer to the r e s t r i c t i v e temperature and endocytic capacity i s gradually l e s t . This mutant has not been tested for DNA synthesis at the r e s t r i c t i v e temperature. Mutants in group (2) may prove important i n investigating the relationships between DNA synthesis, phagocytosis and aging. The mutant fvc in group (3) ca r r i e s a ts single gene recessive mutation that r e s u l t s in the clumping of food vacuoles in ere area of the c e l l , usually the posterior*. I t i s linked by 34 ± 3 map .units to the tnd locus which i s ts for tric h o c y s t ncn-discharge. . There have been only eight other cases of linkage reported i n t e t r a u r e l i a which i s surprising since there are 170 presumptively di f f e r e n t genie l o c i i d e n t i f i e d (T.M. Sonneborn, personal communication) and 43 ± 2 chromosomes (Dippell, 1954),. There may be some features inherent i n the micronucleus that preclude the detection of linked genes or there may be a reluctance on the part of researchers to undertake such projects (T.M. Sonneborn, personal communication).. The mutant dm! i f l group (4) c a r r i e s a t s recessive mutation that results in the abnormal morphology of food vacuoles giving the appearance of a mass of disrupted vacuoles. This mutant should be useful i n the study of membrane-membrane interactions during phagocytosis. The mutants in groups (3) and (4) are po t e n t i a l l y the most valuable for examining the i n t r a c e l l u l a r events that cccur during phagocytosis..Therefore, the remainder of t h i s study w i l l be based on further investigation and analysis of the two 43 mutants fvc and dmj_. 44 Tabl e 3-1._Phenotypes of p u t a t i v e mutants LineGT (1)BP (2) DE(3) BP(4) Remarks wild5. 3 13. 9 0.00 11.6 E l a t e III-1a,b type (12.9-15.0) (10.5-12.8) A1 6.8 5.2 0.27 5.8 c h a i n s , m i s d i v i d e r s (4.4-6.0) (5.1-6.5) gullet-abnormal A2 5. 6 10. 1 0.09 6.2 c e l l s - l a r g e (9.2-11.1) (5.3-7.1) vacuoles-abnormal g u l l e t - n o r m a l A3 5.6 1 1.7 0.00 6.6 (10.6-12.8) (5.7-7.4) A4 5 .5 9. 4 0.00 6.3 c e l l s - l a r g e (8.7-10.1) (5.2-7.3) v a c u o l e s - l a r g e A5 6.6 9.7 0.00 7 . 3 E l a t e l l l - 1 d ; c e l l s - l a r g e (8.6-10.8) (6.5-8.1) vacuoles-clumped g u l l e t - n o r m a l t r i c h o c y s t s - n o n d i s c h a r g e A6 5.6 6. 7 0.00 5.9 (6.2-7.3) (4.9-69) A7* >20 2.4 0.62 2.8 c e l l s - l a r g e , m o n s t e r s (1.9-2.9) (2.0-3.6) . ... continued 45 Table 3-1, cont. Phenotypes of putative mutants LineGT (1)fiP (2) DE(3) BP (4) Remarks A8 nd 7.7 0.00 6. 1 vacuo l e s - c l u mped (7.C-8.4) (5.2-7.0) A9 5.8 8. 1 0.00 6. 1 (7.2-8.9) (5.6-6.7) A1 0 5.5 12.0 0.30 7.8 Plate III-1c (10.5-13.5) (6.9-8.7) vacuoles-disrupted gullet-norma 1 trichoc ysts-normal A11 5.3 8.2 0.00 8.0 vacuoles-disrupted (7.C-9.4) (7.0-8.9) (1) GT = generation time (in hours) at 34.5°C (2) RP = means and 95% confidence intervals of the number of colored vacuoles that accumulated after 20 minutes i n red watercolor at 34.5°C: (3) DE = the fraction of c e l l s that had no colored vacuoles at 34.5 °C (4) BP = means and 95% confidence i n t e r v a l s of the number of colored vacuoles that accumulated in blue watercolor after 20 minutes at 34.5°C * th i s l i n e has been lost nd = not determined Table 3-2. A10 x pawn .F2. Phenctype Number £ W + ; dmj+ 16 £ W + ; dmj 23 £w ; drnj* 17 £w ; dm 1 8 Total 64 X 2 (1) 7.12 P (2) 0.07 pw = pawn phenotype at 27°C; p_w+ = wild-type dmj = disrupted vacuoles at 34.5°C; dml* = wild-type (1) Chi Sguare Test for goodness of f i t (2) probability geaerated by Chi Sguare Test 47 Table 3-3. A5 x pawn .F2. Phenotype cross#1 crcss#2 pooled data * 2** ; tnd + ; fvc+ 32 33 65 _pw+, I tnd+ fvc 18 13 31 pw+ ; tnd ; f vc+ 10 14 24 * £2. ; tnd + ; fyc+ 25 29 54 * pw + ; tnd ; fvc 26 33 59 pw ; tnd*, f vc 20 20 40 pw ; tnd 9 9 18 * -E" ' ; tnd fvc 31 26 57 To t a l 171 177 348 X 2 (1) 25. 05 28.25 50.45 P (2) 0. 007 <.001 <.00 1 X 2 Parentals 1.30 1. 15 1.09 P 0. 73 0.77 0. 78 X 2 Recombinants 6. 51 4.41 9.56 P 0. 09 0.22 0.02 No. parentals 114 121 235 No. recombinants 57 56 113 Linkage 33. 3fc 32.0% 32.5% pw = pawn phenctype at 27°C; pw+ = wild-type tnd = t r i c h o c y s t non-discharge at 34.5°C; tnd+ = wild-type fvc = clumped vacuoles at 34.5°C; J v c + = wild-type * parental phenotypes f o r tnd and f_yc a l l e l e s (1) Chi Square Test for goodness of f i t (2) probability generated by Chi Sguare Test Table 3-4..A5 x spot . F2. Phenotype Number * sp*, , tnd + fvc* 34 S £ + tnd*. f vc 15 s£ +; tnd , fvc* 21 * S £ 1 tnd*-, f y_c + 30 * S £ + ; tnd ; fvc 31 ; tnd* fvc 20 sp ; tnd ; fyc* 15 * sp ; tnd ; fvc 26 Total 192 X 2 (1) 15.69 P (2) 0.03 X 2 Parentals 1.14 P 0. 77 X 2 Recombinants 1.89 P 0.60 No. parentals 121 No. recombinants 71 Linkage 37% phenctype at 27°C; sp + = wild-type tnd = t r i c h o c y s t non-discharge at 34 .5°C; tnd+ = wild-type fvc = clumped vacuoles at 34 .5°C; f_yc+ = wild-type * parental phenotypes for tnd and fvc a l l e l e s (1) Chi Sguare Test for goodness of f i t (2) probability generated by the Chi Sguare Test 49 Table 3-5. ., tnd x spot ; fvc x spot . F2. tnd x spot fvc x spot Phenotype Number Phenotype Number sp *•; tnd + 56 sp*-; fvc + 61 sp*; tnd 38 sp +; fvc 48 sp ; tnd* 55 sp ; fvc*- 48 sp ; tnd 51 sp ; fvc 43 Total 200 Total 200 X 2 (1) 4.12 X2 3.56 P (2) 0.25 P 0.31 sp = spot phenotype at 27° C; s p + = wild-type tnd = tr i c h o c y s t non-discharge at 34.5°C; tnd*- = wild-type fvc = clumped vacuoles at 34.5°C; f_vc+ = wild-type (1) Chi Sguare Test for goodness of f i t (2) pr o b a b i l i t y generated by Chi Sguare Test 50 Figure 3-1. Accumulation of vacuoles in 4y-21. E f f e c t of time at 34.5°C on synchronized samples of c e l l s . . N O . O F C O L O R E D V A C U O L E S / C E L L CJi O 52 Figure 3-2. Schematic i l l u s t r a t i o n of the dml phenctype. Dots represent blue watercolor (BP) . a. wild-type c e l l (m = mouth, fv = food vacuole) b, c..some vacuoles appear normal, others are disrupted and appear to fuse d,e. no normal vacuoles are present and some p a r t i c l e s of BP are v i s i b l e in the cytoplasm 53 54 P l a t e I I I - 1 . Wild-type and mutant c e l l s i n w a t e r c o l o r a t 34.5°C. A l l c e l l s were f i x e d i n formaldehyde and a i r - d r i e d on s l i d e s (a-c) c r embedded i n g e l a t i n (d) . a. w i l d - t y p e c e l l s i n red w a t e r c o l o r b. w i l d - t y p e c e l l s i n b l u e w a t e r c c l c r Cm dnM c e l l s i n b l u e w a t e r c o l o r . D i s r u p t e d v a c u o l e s a r e v i s i b l e near the p o s t e r i o r o f t h e c e l l . d. . A5 c e l l s i n blue w a t e r c o l o r . Clumped v a c u o l e s are v i s i b l e i n the p o s t e r i o r of t h e c e l l . . 56 CHAPTEE IV FUBTHEB CHAEACTEEIZATION OF TEE MUTANTS dmj. AND fvc A. Introduction In order to further investigate the phenotypes of the mutants dm_1 and fvc (or A5 which carries the tnd and fvc mutations), the food vacuoles of wild-type and mutant c e l l s were compared under a variety of conditions; The e f f e c t of blue watercolor (BE), the accumulation and loss of colored vacuoles at 27°C and 34.5°C and the e f f e c t s of increasing time at 34.5°C on synchronous c e l l s were determined i n wild-type and dmj c e l l s . The effects of increasing time at 34.5°C on asynchronous c e l l s , the return to 27°C a f t e r the induction of the mutant phenotypes and the responses of c e l l s to a range of temperatures were determined i n wild-type, dml and fvc c e l l s . . B. Materials and Methods 1. Effect of time i n BP Wild-type and dml. c e l l s were incubated at 27°C or 34.5°C for 6 hours and fed BP for 1 , 3, 5, 10, 15, 20, 30, 40, 50, or 60 minutes. The c e l l s were fixed and the number of colored vacuoles that accumulated per c e l l and the number of c e l l s with disrupted vacuoles were determined. In t h i s and subseguent experiments the mean number of vacuoles in 30 c e l l s and the 95% 57 confidence inte r v a l s (c.i.) were calculated. Between 30 and 50 c e l l s were examined for the presence cf disrupted vacuoles (or clumped vacuoles when fvc or A5 c e l l s were examined). The Chi Sguare (X2) test for homogeneity between the re s u l t s obtained with wild-type and mutant c e l l s was performed and a probability (P) < 0.05 was considered s i g n i f i c a n t for r e j e c t i o n of homogeneity (when only two sets of data were compared Yates' correction factor was employed). Ihe frequency of c e l l s with disrupted vacuoles was calculated by dividing the number of c e l l s with disrupted vacuoles by the t o t a l number of c e l l s that formed colored food vacuoles i t e . . these c e l l s that were not forming food vacuoles were excluded. . 2.. Loss of colored vacuoles a f t e r removal frpm BP Wild-type and dm1 c e l l s were incubated at 27°C or 34.5°C fo r 6 hours, BP was added for 20 minutes and a sample (time 0) was removed and f i x e d . . The c e l l s were decanted into a 50 ml volumetric f l a s k that was then f i l l e d with culture f l u i d and maintained at 27°C or 34.5°C. After 5 and 10 minutes samples were removed and fixed..The c e l l s that swam to the top of the f l a s k were transfered to a 25 ml volumetric flask that was also f i l l e d with culture f l u i d and maintained at 27°C or 34.5°C. In t h i s manner the c e l l s , which were negatively geotactic, were separated from the watercolor, which remained at the bottonu Additional samples were taken 15, 20, 30, 40, 50, and 60 minutes after removal from BP, fi x e d and the number of colored vacuoles per c e l l and the number cf c e l l s with disrupted 58 vacuoles were determined. . 3. Effect of concentration of BP Wild-type and dm1 c e l l s were incubated at 34.5°C for 6 hours and were fed BP at a f i n a l concentration of 0. 75, 1. 0, 2.5, 5.0, 7.5, 10.0, 25. 0, 50.0, or 75.0 mg/ml. The op t i c a l density of 2 .5 mg/ml EP was determined with a Spectronic 20 spectrophotometer (wavelength = 660) using culture f l u i d without watercolor as a blank. The c e l l s were fixe d after 20 minutes and the number of c e l l s with disrupted vacuoles was determined. .. 4. Effect of time at 34_,5£C a. . asynchronous c e l l s Wild-type, dmj, fvc and A5, c e l l s were shi f t e d to 34.5°C and after 1, 2, 3,, 4', 5 and 6 hours samples were removed, fed BP for 20 minutes, fixed, and examined.. The number cf colored vacuoles per c e l l and the number of c e l l s with disrupted or clumped vacuoles were determined. b. . synchronous c e l l s Twenty to 50 div i d i n g wild-type and dm_1 pairs per sample were collected within 10 minutes of d i v i s i o n and shifted tc 34.5,°C for 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, or 6 hours. . Dividing dm1 c e l l s were alsc c o l l e c t e d , incubated 59 at 27°C for 3.5 hours and then shifted to 34.5°C for 0 f 1 or 2 hours. The c e l l s were fed BP for 20 minutes, fixed and the number of colored vacuoles that accumulated per c e l l and the number of c e l l s with disrupted vacuoles were determined. 5. Down-shift to permissiye temperature Wild-type, dmj., fvc and A5 c e l l s were incubated at 34.5°C for 6 hours after which time the c e l l s were shifted to 27°C. Samples were removed afte r 0,.33, .67, 1, 2, 3, 4, 6, 8, and 16 hours at 27°C, fed BP for 20 minutes, fixed, and the number of c e l l s with disrupted or clumped vacuoles was determined. 6. „ Effect of increasing temperature Wild-type, dm1_, fvc and A5 c e l l s were incubated at 33.5, 34. 1 , 34. 3, 34.5, 34. 7, 34. 9, and 35. 1°C for 6' hours, fed BP for 20 minutes, fixed, and the number of c e l l s with disrupted or clumped vacuoles was determined. C. . Be s u i t s 1. Effect of time i n BP There was an i n i t i a l rapid increase i n the number of colored vacuoles that accumulated with increasing time i n BP at 27°C and 34.5°C in wild-type and dml c e l l s (Figures 4-1 and 4-6 0 2 ) . A p l a t e a u was reached a f t e r 20 minutes i n d i c a t i n g t h e e s t a b l i s h m e n t of an e q u i l i b r i u m between the f o r m a t i o n and l o s s of c o l o r e d v a c u o l e s . . Taere were s i g n i f i c a n t l y fewer food v a c u o l e s formed i n dml. th a n i n w i l d - t y p e c e l l s at 27°C and 34.5°C. A f t e r 3 minutes or l o n g e r i n BE the number of dml. c e l l s w i t h d i s r u p t e d v a c u o l e s d i f f e r e d from w i l d - t y p e (Table 4-1). The f r e g u e n c y of dmj. c e l l s w i t h d i s r u p t e d v a c u o l e s was between .57 and .71 f o r i n c u b a t i o n t i m e s i n EP g r e a t e r than 5 minutes. S t u d i e s w i t h i n d i v i d u a l c e l l s i n d i c a t e d t h a t e x p o s u r e s o f 24 hours a t 27°C c r 2 hours a t 34.5°C were not t o x i c t o w i l d - t y p e or dml. c e l l s . . 2..Loss of c o l o r e d v a c u o l e s a f t e r removal f r c m BP At 27°C t h e l o s s of c o l o r e d vacuoles i n w i l d - t y p e and dml c e l l s was complete by one hour ( F i g u r e 4-3) and a t 34.5°C by 20 minutes ( F i g u r e 4-4). The r a t e of a c c u m u l a t i o n of c o l o r e d v a c u o l e s was i n i t i a l l y g r e a t e r a t 34.5°C th a n a t 27°C ( F i g u r e s 4-1 and 4-2) i n d i c a t i n g t h a t t h e r a t e cf both e n d o c y t o s i s and e x o c y t o s i s i n c r e a s e d at t h e h i g h e r t e m p e r a t u r e . A f t e r removal from BP t h e number of dml c e l l s w i t h d i s r u p t e d v a c u o l e s a t 34.5°C was s i g n i f i c a n t l y d i f f e r e n t from w i l d - t y p e f o r t h e f i r s t 15 minutes (Table 4-2) . Howe ver , the f r e q u e n c y o f c e l l s w i t h d i s r u p t e d v a c u o l e s d e c r e a s e d s t e a d i l y i n d i c a t i n g t h a t e x o c y t o s i s c f d i s r u p t e d v a c u o l e s o c c u r r e d w i t h t h e same f r e g u e n c y as e x o c y t o s i s of normal v a c u o l e s . 61 3..Effect of concentration of BP At a l l concentrations of BP tested the number of dmj c e l l s with disrupted vacuoles was s i g n i f i c a n t l y d i f f e r e n t from wild-type c e l l s (Table 4-3). At concentrations higher than 2.5 mg/ml (optical density = . 5 ) the freguency of dmj c e l l s with disrupted vacuoles varied between .45 and .55. At concentrations greater than 25 mg/ml there was an increase i n the number of dmj and wild-type c e l l s which did not form colored vacuoles.. Therefore, at higher concentrations, BP had an i n h i b i t o r y effect on endocytosis in wild-type and dmj c e l l s but the eff e c t on dmj c e l l s was greater. 4- Effect of time at 3__5_C a.. asynchronous c e l l s There was no s i g n i f i c a n t difference between the number of colored vacuoles which accumulated i n wild-type or dm 1 c e l l s after 6 hours at 34.5°C (Figure 4-5) . However, after 2 hours at 34.5°C the number of colored vacuoles that accumulated i n A5 c e l l s was s i g n i f i c a n t l y reduced (Figure 4-6)..Disrupted vacuoles appeared in dmj c e l l s by 3 hours at 34.5°C and were evident i n 71% of these c e l l s by 6 hours (Table 4-4). A wide range of phenotypic expression of abnormal vacuoles was observed (Figure 3-2) and incubation periods longer than 6 hours at 34.5°C resulted i n increased l e t h a l i t y of dm 1 c e l l s . S i g n i f i c a n t numbers of A5 c e l l s had clumped vacuoles by 62 the end cf 2 hours at 3 4 . 5 ° C (Table 4 - 5 ) . There were no s i g n i f i c a n t differences between A 5 and fvc c e l l s (Table 4 -6) . b.. synchronous c e l l s When synchronized c e l l s sere s h i f t e d to 3 4 . 5 ° C a doubling cf the number of vacuoles occurred within 3 - 4 hours after d i v i s i o n whereas in dml c e l l s only a small increase i n the number of vacuoles was observed (Figure 4 -7) . .Disrupted vacuoles appeared in dmj c e l l s a f t e r 2 hours at 3 4 . 5 ° C and t h i s increased to 70% of c e l l s by 4 hours (Table 4-7). The same pattern was observed in asynchronous c e l l s although the increase to 71% penetrance was more gradual (Table 4 - 4 ) . . T h e mean number of vacuoles that accumulated i n dml c e l l s at 27°C 3 . 5 hours a f t e r d i v i s i o n was 10.1 (c.i.= 9.1-11.1) and after 2 hours at 3 4 . 5 ° C was 11.7 (c.i.= 11.Q-12 . 4 ) . . This was not s i g n i f i c a n t l y d i f f e r e n t from dml. c e l l s shifted to 3 4 . 5 ° C immediately following d i v i s i o n (Figure 4-7). There was a sharp increase in the freguency of c e l l s with disrupted vacuoles to .72 by the end of 2 hours at 3 4 . 5 ° C i n those c e l l s that were incubated at 27°C for 3 . 5 hours following d i v i s i o n . Thus, the stage i n the c e l l cycle as well as the length of time at 3 4 . 5 ° C contributed to the appearance of disrupted vacuoles but the length of time at the r e s t r i c t i v e temperature was of l e s s importance in determining the number of food vacuoles formed. 63 5. . Down-shift to permissive temperature dmj c e l l s required between 8 and 16 hours (2 c e l l cycles) at 27°C to return to normal (Table 4-8). A5 and fvc c e l l s required l e s s than 2 hours (Tables 4-9 and 4-10), which was the same length of time required fox the appearance of clumped vacuoles after s h i f t i n g from 27°C to 34.5°C (Tables 4-5 and 4-6) . There were no s i g n i f i c a n t differences between fvc and A5 c e l l s (Table 4-10). .In both, there was a gradual moving apart of vacuoles (when c e l l s were observed with 2 or more vacuoles separate from the clump they were c l a s s i f i e d as having normal vacuoles) . The penetrance of disrupted vacuoles i n dmj c e l l s was low i n this experiment (52%) but si m i l a r results were obtained in ether experiments when the penetrance was higher. . 6. . Effect of increasing temperature At temperatures less than 34.3°C the food vacuoles of dmj c e l l s were normal (Table 4-11)..Above t h i s temperature there was a gradual increase in the freguency of c e l l s with disrupted vacuoles to a maximum of .53 at 34.5°C. In wild-type c e l l s disrupted vacuoles began to appear at temperatures above 34.5°C with a maximum of .35 occurring at 34.9°C. At higher temperatures t h i s freguency decreased i n dmj and wild-type c e l l s because endocytosis was inhibited and at 35.1°C there were very few c e l l s capable of forming colored vacuoles. In A5 and fvc c e l l s the expression of the clumped vacuole phenotype varied d i f f e r e n t l y with increasing temperature than did the disrupted vacuole phenotype. At temperatures above 34.1°C the 64 freguency of c e l l s with clumped vacuoles rose rapidly and remained at around .50 for temperatures up to 34.9°C (Table 4-12). Temperatures above t h i s caused decreased endocytosis and poor v i a b i l i t y . D. Discussion Within two hours at temperatures above 34.1°C there i s a decrease in the number of food vacuoles that accumulate i n A5 and fvc c e l l s after a 20 minute exposure to BP and these vacuoles clump in one area of the c e l l , usually the posterior. This phenotype i s re v e r s i b l e ; within two hours after a return to the permissive temperature the vacuoles appear normal. In an asynchronous population approximately .50 of A 5 or fvc c e l l s are affected. This freguency does net increase with prolonged exposure to the r e s t r i c t i v e temperature.. There are no s i g n i f i c a n t differences between AS and fvc c e l l s which indicates that although the genes for food vacuole clumping and t r i c h o c y s t discharge are l i n k e d , the tnd gene has l i t t l e , i f any, e f f e c t on the expression of the fvc phenotype. Because the mutant phenotype i s f u l l y expressed after a r e l a t i v e l y short time at the r e s t r i c t i v e temperature, the altered gene product probably turns over guickly. The decrease i n number of food vacuoles and t h e i r clumping could both be explained by a defect in either the microtubule or microfilament system in the cytoplasm.. The numbers of vacuoles would decrease i f the membrane components, which are recycled as disk-shaped v e s i c l e s ;(Allen, 1 974), cannot be 65 t r a n s p o r t e d by the m i c r o t u b u l e s w i t h which they a re c l o s e l y a s s o c i a t e d ( A l l e n , 1975) . . V a c u o l a r movement would a l s o be i m p a i r e d . A l t e r n a t i v e l y , an a b n o r m a l i t y i n t h e m i c r o f i l a m e n t s would i m p a i r c y t o p l a s m i c m o t i l i t y t h a t c o u l d i n t u r n i n h i b i t t h e movement o f v e s i c l e s and v a c u o l e s . In dm 1 c e l l s t h e r e i s a l s o a decrease i n t h e number of f o o d v a c u o l e s t h a t accumulate a t t h e r e s t r i c t i v e t e mperature a l t h o u g h t h i s i s a p p a r e n t o n l y when s y n c h r o n i z e d c e l l s a r e examined..The d o u b l i n g of t h e number of food v a c u o l e s i n s y n c h r o n i z e d samples of w i l d - t y p e c e l l s a t 34.5°C between t h r e e and f i v e h o u r s a f t e r d i v i s i o n i s c o n s i s t e n t w i t h r e s u l t s o b t a i n e d a t room t e m p e r a t u r e ( B e r g e r , 1971) and 27°C (Smith-Sonneborn and Eodermel, 1976). However, i n dmj c e l l s t h e r e i s no major i n c r e a s e i n t h e number of f o c d v a c u o l e s p e r c e l l p r i o r t o d i v i s i o n . The d i f f e r e n c e between t h e number of v a c u o l e s i n w i l d - t y p e and dml c e l l s i s not a p p a r e n t i n asynchronous samples because the number of p r e - d i v i s i o n w i l d - t y p e c e l l s w i t h much g r e a t e r than average number of v a c u o l e s i s b a l a n c e d by the number of p o s t - d i v i d e r s w i t h a decreased number of v a c u o l e s s i n c e f e e d i n g ceases f o r 20-25 minutes d u r i n g d i v i s i o n ( A u f d e r h e i d e , 1976) and BP was f e d f o r 20 minutes. C o n s e g u e n t l y , the 95% c o n f i d e n c e i n t e r v a l s a r e l a r g e r f o r w i l d -t y pe t h a n dmj c e l l s ( f i g u r e 4-5)..When BP i s f e d f o r l o n g e r p e r i o d s o f tim e , p o s t - d i v i d e r s a l s o have s u f f i c i e n t t i m e f o r a l l t h e i r v a c u o l e s t o become c o l o r e d and under th e s e c o n d i t i o n s dm 1 c e l l s do have fewer v a c u o l e s per c e l l t h a n do w i l d - t y p e c e l l s ( F i g u r e 4-2). , Asynchronous dmj c e l l s r e g u i r e around s i x hour s a t 34.5°C 66 f o r the maximum penetrance o f t h e d i s r u p t e d v a c u o l e phenotype. Above a minimum time i n BP ( t h r e e minutes) and c o n c e n t r a t i o n o f BP (0.75 mg/ml) thes e f a c t o r s do net i n f l u e n c e t h e number o f c e l l s w i t h d i s r u p t e d v a c u o l e s . . The i n h i b i t o r y e f f e c t on e n d o c y t o s i s o f BP a t h i g h e r c o n c e n t r a t i o n s i s comparable w i t h t h e e f f e c t c f o t h e r a g e n t s , such as c a l c i u m i o n s and d e t e r g e n t s , which can s t i m u l a t e or i n h i b i t p h a g o c y t o s i s depending on t h e i r c o n c e n t r a t i c n ( N i l s s o n , 1971,1976, Brutkowska and Mehr, 19 76)... I n . s y n c h r o n i z e d samples of dm.1 c e l l s s h i f t e d t o 34.5°C i m m e d i a t e l y f o l l o w i n g d i v i s i o n f o u r hours i s s u f f i c i e n t f o r the appearance o f d i s r u p t e d v a c u o l e s . T h i s can be reduced t o two hours i f t h e c e l l s are s h i f t e d t o 34.5°C t h r e e and o n e - h a l f h o u r s a f t e r d i v i s i o n . I n w i l d - t y p e c e l l s t h e number of food v a c u o l e s per c e l l b e g i n s t o r i s e d r a m a t i c a l l y by t h i s t i m e , t h e r e f o r e , membrane components must be r e g u i r e d i n i n c r e a s i n g amounts._ The mutant, dmj, c o u l d be d e f e c t i v e i n some a s p e c t o f t h e p r o d u c t i o n of membrane components; a decrease i n t h e amount of membrane produced would reduce the number o f v a c u o l e s formed and a d e f e c t i n the membrane c o m p o s i t i o n c o u l d r e s u l t i n the l o s s o f the s t r u c t u r a l i n t e g r i t y of t h e membranes l e a d i n g t o t h e i r d i s r u p t i c n . . Perhap s , because membrane components a r e r e c y c l e d ( A l l e n , 1974) , a p e r i o d of t i m e a t t h e r e s t r i c t i v e temperature ( e g u a l t o s i x hours i n asynchronous c e l l s , l e s s i n synchronous c e l l s ) i s r e g u i r e d f o r s u f f i c i e n t d e f e c t i v e membrane t o be i n c o r p o r a t e d i n t o f o o d v a c u o l e s t o r e s u l t i n d i s r u p t e d v a c u o l e s ; S i m i l a r l y , a f t e r a d o w n - s h i f t t o t h e p e r m i s s i v e 67 temperature, between one and two c e l l cycles are reguired for normal membrane components to replace the defective ones and resul t i n normal vacuoles. _ Ihe difference i n time reguired at the two temperatures i s consistent with the increased rate of accumulation and loss of colored vacuoles at 34.5°C and i s possibly i n d i c a t i v e of a higher metabolic rate at the higher temperature. Since disrupted vacuoles can alsc appear i n wild-type c e l l s , they are probably a consequence of some c e l l u l a r response to an increase in temperature.. The fat t y acid composition cf membrane phospholipids changes i n response to temperature in such diverse organisms as Escherichia c o l i (Marr and Ingraham, 1962, Cronan, 1975, Ckuyama et a l . , 1977), Tetrahymena (Conner and Stewart, 1976, Fukushima et a l . , 1976), Aspergillus (Dart and Stretton, 1976), Neurpspora (Friedman, 1977), goldfish (Johnston and Roots, 1964) mackerel (Ueda, 1976), trout (Hazel, 1979), planktcnic crustaceans (Farkas, 1979) and blue-green algae (Sato et a l . , 1 S79) . In addition, the rate of phospholipid synthesis i n E. c o l i may be related to the stage i n the c e l l cycle (Pierucci, 1979) . The above observations suggest that the defect of dm1 c e l l s may be a conseguence of abnormalities i n the fatty acid composition of phospholipids which may be responsible for a decreased thermotolerance of membranes as evidenced by the appearance of disrupted vacuoles at a lower temperature than i n wild-type c e l l s * . This aspect w i l l be investigated more f u l l y i n Chapter VII. 68 Table 4-1..Effect of blue watercolor at 34.5°C on vacuolar morphology Ti me in C e l l DV N X2 P Freque BP(1) Type (2) (3) (5) DV (6) 1 wt 0 30 0.00 1. 00 O.CO dJBi 0 30 0.00 3 wt 1 29 11.43 <. 001 0.03 dmj 29 46 0. 45 5 wt 0 30 24.2 <. 001 O.CO dml 30 23 0.57 10 wt 1 29 23. 14 <.001 0.03 dmj 30 20 C.60 15 wt 1 29 23.78 <.001 0.03 dml 30 19 0.61 20 wt 1 29 26.07 <. 001 0.07 dml 30 13 0. 70 25 wt 0 30 24.20 <. 001 0.00 dml 30 23 0.57 30 wt 0 30 33. £5 <. 001 0.00 drnj 30 12 0.7 1 40 wt 0 30 26.30 <. 110 O.CO dml 30 20 0.60 60 wt 0 30 24.86 <. 001 0.00 dml 30 22 0. 58 wt = wild-type dml = mutant with disrupted vacuoles at 34.5°C ( D minutes in blue watercolor (2) number of c e l l s with disrupted vacuoles (3) number of c e l l s with normal vac ucles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) fre guency of c e l l s with disrupted vacuoles 69 Table 4-2. Effect of removal frcm blue watercolor on vacuolar morpholog y Time C e l l Type DV (2) N (3) (4) (5) Freguency (6) 10 15 20 wt dmj wt dmi wt dmj wt dmj wt dmi 3 24 5 24 0 18 1 16 0 3 46 25 45 26 50 30 46 28 19 18 20.45 <.001 15.74 <.001 20.54 <.001 15.35 <. 001 1.24 0. 27 0.06 0.49 0. 10 0.48 0.00 0. 36 0.02 0.38 0.00 0. 14 wt = wild-type dmj = mutant with disrupted vacuoles at 34.5°C (1) time (minutes) after removal frcm blue watercolor (2) number of c e l l s with disrupted vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency of c e l l s with disrupted vacuoles 70 Table 4--3. Effect of concentration of blue watercolor on vacuolar morphology Cone. . C e l l DV N X2 P Freguency mg/ml( 1) Type (2) (3) (*») (5) (6) 0.75 wt 2 44 7.07 . 008 0.04 dm! 12 33 0. 27 0. 10 wt 4 44 5.07 . 02 0.C8 dml .14 36 0.28 2.5 M t 4 44 14.36 <. 001 0.08 dm! 21 26 0.45 5.0 wt 3 42 15.31 <. 001 0.07 Ml 21 26 0.45 7.5 wt 1 45 25.84 <. 001 0.02 dmj 24 23 0. 51 10.9 wt 5 41 13.96 <.001 0. 1 1 dml 24 26 0.48 25.0 wt 7 33 10.71 . 001 0. 18 dm! 23 19 0.55 50.0 wt 3 29 13.32 <. 001 0.09 dml 19 16 0.54 75.0 wt 1 21 5. 18 . 02 0.05 dm 1 6 9 0. 40 wt = wild-type dm! = mutant with disrupted vacuoles at 34.5°C (1) concentration of blue watercolor (2) number of c e l l s with disrupted vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency of c e l l s with disrupted vacuoles 71 Table 4-4. Effect of time at 34.5°C cc vacuolar morphology Ti me C e l l DV N X* P Frequency (hr) (1) Type (2) (3) (4) (5) (6) _ _ _ - "~30 .__ o7oc dmi 0 30 0.00 1 wt 0 30 0 1 0.00 dmj 0 30 0.00 2 wt 0 30 0 1 o.co dmj 0 30 0.00 3 wt 1 29 10.95 . 001 0.03 dmj 30 50 0.38 4 wt 0 30 27.07 <. 001 0.00 dmj 30 19 0.61 5 wt 5 25 14.58 <.Q01 0.17 dmj 30 17 0. 64 6 wt 1 29 30.38 <.001 0.C3 dmj 30 12 0.71 wt = wild-type dmj = mutant with disrupted vacuoles at 34.5°C (1) number of hours at 34. 5°C (2) number of c e l l s with disrupted vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Square Test for homogeneity (5) probability generated by Chi Sguare Test (6) frequency of c e l l s with disrupted vacuoles 72 Table 4 -5..Effect of time at 34.5°C on vacuolar clumping Ti me C e l l CV N X 2 P Freguency (hr) (1) Type 12) (3) (<») (5) (6) 0 wt 1 27 0.0012 0. 97 0.04 A5 0 30 O.CO 1 wt 0 29 0.00 1. 00 0.00 A5 0 30 0.00 2 wt 1 29 8.44 0. 004 0.03 A5 11 19 0.37 3 Wt 1 28 12.34 <.001 0.03 A5 14 16 0. 47 4 wt 1 27 16.68 <. 001 0.04 A5 17 13 0. 57 5 wt 2 27 18. 10 <. 001 0.07 A5 19 11 0.63 6 wt 2 28 13.41 <. 001 0.07 A5 16 14 0.53 wt = wild-type A5 = double nutant with t r i c h o c y s t non-discharge and clumped vacuoles at 34. 5°C (1) number of hours at 34.5°C (2) number of c e l l s with clumped vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency cf c e l l s with clumped vacuoles 73 Table 4-6. Vacuolar clumping i n A5 and Jvc c e l l s Time C e l l CV N X2 P Freguency (hr) (1) Type (2) (3) (4) (5) (6) 2 A5 12 33 2.6 3 0. 10 0.27 fyc 22 27 0. 45 4 A5 18 25 1.85 0. 17 0.42 fyc 28 20 0.58 6 A5 25 21 0.00 1. 00 0.54 fvc 20 17 0-. 54 A5 = double mutant with t r i c h o c y s t vacuoles at 34.5°C fyc = mutant with clumped vacuoles at 34.5°C (1) number of hours at 34. 5°C (2) number of c e l l s with clumped vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency of c e l l s with clumped vacuoles non-discharge and clumped 74 Table 4-7. Vacuolar morphology i n synchronous c e l l s at 34.5°C T i me C e l l DV N (hr) (1) Type (2) (3) 0. 0 wt 0 30 dml 0 17 1.0 wt 0 30 dm! 0 30 2. 0 wt 0 39 dml 0 30 3.0 wt 0 30 dm! 7 23 3. 5 wt 0 30 d j l 7 23 4. 0 wt 0 30 dm! 21 9 5. 0 wt 0 30 dml 20 10 X 2 P Freguency (4) (5) (6) 0.00 1. 00 0.00 0.00 0.00 1. 00 0.00 O.CO 0.00 1.00 0.00 0.00 5. 82 0. 02 0.00 0.23 5.82 0.02 0.00 0. 23 29.30 <.001 0.00 0. 70 27.C8 <.001 O.CO 0.67 wt = wild-type dm! = mutant with disrupted vacuoles at 34.5°C (1) number of hours at 34. 5°C (c e l l s s hifted up immediately after d i v i s i c c ) (2) number of c e l l s with disrupted vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency of c e l l s with disrupted vacuoles 75 Table 4-8. Vacuolar morphology a f t e r s h i f t to 27°C Time C e l l DV N X 2 P Fregi (hr) (1) Type (2) (3) (4) (5) (6) o7oo wt 2 31 16.32 <. 001 0.06 dmj 23 21 0. 52 0. 33 wt 6 43 10.79 0. 001 0. 12 dmj 22 28 0.44 0. 67 wt 7 42 e.33 0. 01 0. 14 dmj 18 28 0. 39 1. 00 wt 7 42 5.09 0.02 0. 14 dmj 18 32 0.36 1. 50 wt 8 40 4.85 0. 03 0.17 dmj 18 28 0. 39 2. 00 wt 7 43 7.33 0. 007 0. 14 dmj 19 28 0.40 4. 00 wt 7 41 6. 11 0. 004 0. 15 dmj 21 28 0.43 6. 00 wt 6 44 8.41 0. 004 0. 12 dmj 18 27 0.40 8. 00 wt 7 41 4.24 0. 04 0.15 dmj 14 25 0. 36 16. 0 wt 3 42 0.01 0. 92 0.07 dm1 2 47 0.04 wt = wild-type dmj = mutant with disrupted vacuoles at 34.5°C (1) number of hours afte r s h i f t down to 27°C (after 6 hours at 34. 5°C) (2) number of c e l l s with disrupted vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency of c e l l s with disrupted vacuoles 76 Table 4-9. Vacuolar clumping after s h i f t tc 27 °C Time C e l l CV N X 2 P Fregi (hr) (1) Type (2) (3) (4) (5) (6) 0. 00 wt 3 31 ""14783 ~<7oo"' oToT fvc 26 24 0.52 0.33 wt 3 40 13.74 <. 00 1 0.07 fvc 19 24 0.44 0.67 wt . 1 41 17. 29 <. 001 0.02 fvc 19 26 0.42 1.00 wt 1 41 11.32 <. 001 0.02 fvc 14 29 0.42 1. 50 wt 0 40 1.95 0. 16 0.00 fvc 4 42 0.09 2. 00 wt 4 39 0.00 1.00 0. 09 fvc 5 42 0.11 4.00 wt 1 40 0. 00 1. oo 0.02 fvc 2 40 0. 05 6. CO wt 1 • 43 2. 70 0. 10 0.02 fvc 6 36 0. 14 wt = wild-type fvc = mutant with clumped vacuoles at 34.5°C (1) number of hours after s h i f t to 27°C (after 6 hours at 34.5° C) (2) number of c e l l s with clumped vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) pr o b a b i l i t y generated by Chi Sguare Test (6) freguency of c e l l s with clumped vacuoles 77 Table 4-10. Vacuolar clumping in fyc and A5 c e l l s after s h i f t to 27°C Time C e l l CV N X 2 P Freguency (hr) (1) Type (2) (3) (4) (5) (6) 0.00 fvc 26 24 0*00 1.00 0752 A5 17 17 0. 50 0.33 fvc 19 24 0. 48 0. 49 0.44 A5 10 20 0. 33 0. 67 fvc 19 26 0. 28 0.60 0. 42 A5 10 20 0.33 1. 00 fvc 14 29 0.35 0. 55 0.42 A5 7 23 0.23 1.50 fvc 4 42 0.47 0. 49 0.09 A5 5 25 0. 17 2. 00 fvc 5 42 0. 53 0. 47 0. 1 1 A5 1 29 0.03 6. 00 fvc 6 36 0. 40 0. 53 0. 14 A5 2 28 0.07 fyc = mutant with clumped vacuoles at 34.5°C A5 = double mutant with trichocyst non-discharge and clumped vacuoles at 34.5°C (1) number of hours after s h i f t t c 27°C (after 6 hours at 34.5°C) i . (2) number of c e l l s with clumped vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency of c e l l s with clumped vacuoles 78 Table 4- 11. Effect of temperature en vacuolar morphology Te mp. C e l l DV N X 2 P Freguency l°C) (1) Type (2) (3) CO (5) (6) 33 .5 wt 3 47 0.00 1. 00 0.06 I s ! 2 48 0.C4 34. 1 wt 0 28 3.21 0.07 0.00 dm! 5 25 0. 17 34.3 wt 0 29 6.69 0. 01 0.00 dm! 7 19 0. 27 34. 5 wt 0 33 21.46 <. 001 0.00 dm! 19 17 0.53 34 .7 wt 8 40 6.74 0. 009 0. 17 dm! 21 28 0.43 34.9 wt 11 1 9 0.00 1. 00 0. 37 dm! 12 18 0. 40 35. 1 wt 6 24 0.005 0. 94 0.20 dm! 5 15 0. 25 wt = wild-type dm! = mutant with disrupted vacuoles at 34.5°C (1) incubated for 6 hours at the temperature indicated (2) number of c e l l s with disrupted vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency of c e l l s with disrupted vacuoles 79 Table 4-12. Effect of temperature on vacuolar clumping Temp. C e l l CV N X2 P Freguency (°C) (1) Type (2) (3) (4) (5) (6) 3375™ wt 0 47 3. 20 "o7o7 o7oo fyc 5 44 0. 10 34. 1 wt 0 28 4.13 0.04 0.00 A5 7 30 0.19 34.3 wt 4 25 10.75 0. 001 0. 14 A5 17 12 0.59 34 .5 wt 0 29 13.69 <.001 0.00 A5 13 17 0. 43 34 .7 wt 3 37 19.01 <. 001 0.08 fyc 23 19 0.55 34.9 wt 0 19 16.31 <. 001 0.00 A5 18 11 0.62 35. 1 wt 1 22 0.00 1. 00 0.04 A5 0 3 0.00 wt = wild-type fvc = : mutant with clumped vacuoles at 34.5 ° c A5 = double mutant with tri c h c c y st ncn-discharge and clumped vacuoles at 34.5°C (1) incubated for 6 hours at the temperature indicated (2) number of c e l l s with clumped vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test for homogeneity (5) probability generated by Chi Sguare Test (6) freguency cf c e l l s with clumped vacuoles 80 Figure 4-1..Accumulation of vacuoles i n blue watercolor at 27°C. The number of colored vacuoles per c e l l with increasing time i n blue watercolor. V e r t i c a l bars represent 95% confidence i n t e r v a l s . . (•--• = wild-type, = dm 1) 81 82 Figure 4-2..Accumulation of vacuoles i n blue watercolor at 34.5°C. The number of colored vacuoles per c e l l with increasing time i n blue watercolor. V e r t i c a l bars represent 95% confidence in t e r v a l s . . (•--• = wild-type, • — • = dml) N O . O F C O L O R E D \ A C U O L E S / C E L L oi O cn 84 Figure 4-3. loss of colored vacuoles at 27°C. Loss of colored vacuoles with time after removal frcm blue watercolor. V e r t i c a l bars represent 95% confidence i n t e r v a l s . (•--• = wild-type, A — A = dml) 20 40 60 b TIME AFTER REMOVAL 2 FROM B.P (min) 86 Figure 4-4. Less of colored vacuoles at 34. 5°C. Loss of colored vacuoles with time after removal frcm blue watercolor. V e r t i c a l bars represent 95% confidence i n t e r v a l s . (•--• = wild-type, *>—* = dmj.) 87 _ l _ l LU O \ 88 F i g u r e 4-5..Accumulation of vacuoles i n wild-type and dml c e l l s a t 34.5°C..Blue watercolor was added f o r 20 minutes a f t e r the i n c u b a t i o n periods i n d i c a t e d . . V e r t i c a l bars r e p r e s e n t 95% confidence i n t e r v a l s . . (•--• = w i l d - t y p e , • — • =dm 1) NO. OF COLORED VACUOLES /CELL cn o oo 90 Figure 4-6. accumulation of vacuoles in wild-type and A5 c e l l s at 34.5°C. Blue watercolor was added for 20 minutes after the incubation times indicated.. V e r t i c a l bars represent 95% confidence i n t e r v a l s . (•--• = wild-type, m — • = A5) 92 Figure 4 - 7 . Accumulation of vacuoles in synchronized c e l l s at 34.5°C. Synchronized c e l l s were shifted to 34.5°C immediately after d i v i s i o n and fed blue watercolor for 20 minutes after the incubation period indicated. V e r t i c a l bars represent 95% confidence i n t e r v a l s . . (•--• = wild-type, • — • = dmi) 94 CHAPTEB V EFFECTS OF CHEMICAL AGENTS ON WI1D-TYEE AND MUTANT CELLS A. Introduction Many chemical agents perturb membrane structure. . I f the mutant, dmj.# i s defective i n seme aspect cf membrane composition, i t i s possible that these agents could r e s u l t i n phenocopies of t h i s mutant, i . e . disrupted food vacuoles, in wild-type c e l l s . A l t e r n a t i v e l y , dmj c e l l s may have a di f f e r e n t s e n s i t i v i t y than wild-type c e l l s tc these chemicals. S i m i l a r l y , i f the mutant, fy c , i s abnormal due to the malfunctioning of microtubules cr microfilaments, agents that i n t e r f e r e with these structures may r e s u l t i n phenocopies of thi s mutant* i..e. food vacuole clumping, i n wild-type c e l l s . fvc or A5 c e l l s (which contain the recessive genes fyc and tnd) may also have altered s e n s i t i v i t y to these chemicals. The following agents were selected on the basis of t h e i r known action on c i l i a t e s or other organisms. Dimethyl sulfoxide (DMSO) i s a pclar solvent which has a wide range of b i o l o g i c a l e f f e c t s (Jacob, 1971). DMSO induces or enhances fusion of hen erythrocytes (eg. Ahkong et a l . , 1975, Wilairat et a l . , 1978), human e p i t h e l i a l c e l l s (Norwood et a l . , 1976) and the pseudopodia of amoeba (Grebecka and Kalinina, 1 979) . I t has also been used to induce d i f f e r e n t i a t i o n in Friend leukemic c e l l s (eg. - Lyman et a l . , 1976), as a radio- and cryoprotectant (Ashwood-95 Smith, 1971), a drug c a r r i e r (eg. Weed and Wood, 1975), and a solvent for water insoluble compounds (eg. cytochalasin B, Nilsson, 1976). Reports by Nilsscn (1977b), Reisner and Bucholtz (1977) and Sibley et a l . (1977) provide detailed accounts of the affects of DM SO on Tetrahymena p y r i f ormis and Paramecium t e t r a u r e l i a .. In addition to causing swollen c o n t r a c t i l e vacuoles (CVs) , anucleate daughter c e l l s , nucleolar aggregates, abnormal chromatin bodies, mitochondria, peroxisomes, and ribosomal aggregates i n Tetrahymena , and affe c t i n g f i s s i o n rates, polysome structure and c e l l size in Paramecium , lower concentrations of DMSO (7.5%) r e s u l t i n decreased endocytosis i n Tetrahymena whereas higher concentrations (10&) cause food vacuole membrane abnormalities i n Paramecium . In view of the l a s t two e f f e c t s , DMSO was considered to be a drug that might have interesting e f f e c t s on the mutant, dmj.. Local anasthetics, such as dibucaine, react with the polar groups of phospholipids at the membrane surface as well as with the hydrophobic i n t e r i o r (Ohki, 1970). They increase the f l u i d i t y of membranes (Hubbell et a l . , 1970), a f f e c t the organization of cytoskeletal components (Poste et a l . , 1975) and may displace membrane-bound calcium and magnesium ions (Papahadjopoulos et a l . , 1975).. In P. . aurelia , l o c a l anasthetics cause c i l i a r y r eversal and stimulate the passive i n f l u x of calcium and efflux of potassium ions (Browning and Nelson, 1976) but there are no reports of their e f f e c t s on endocytosis. Therefore, the ef f e c t of dibucaine on endocytosis in P. t e t r a u r e l i a was examined. . 96 Detergents are l i p i d s which are soluble amphipiles;.Sodium dodecylsulfate (SDS) i s an anionic detergent which, i n low concentrations, binds to the c e l l membrane causing changes in permeability and osmotic eguilibrium that can lead to l y s i s and disi n t e g r a t i o n of the membrane (Helenius and Simons, 1 S75) . SDS increases the e x c i t a b i l i t y and motor response of P..caudatum to external stimuli (Bujwid-Cwik and Dryl, 1975) and causes morphological changes owing to the contraction of the ectoplasm (Dryl and Mehr, 1976). , Low concentrations (less than 4x10~ 6 g/ml) stimulate phagocytosis i n Tetrahymena and Paramecium whereas higher concentrations (8x10 - 6 g/ml) are i n h i b i t o r y (Brutkowska and Mehr, 1976), with Paramecium being more se n s i t i v e to SDS than Tetrah ymena . Concentrations i n t h i s range may have different e f f e c t s on wild-type and mutant c e l l s . Microtubules have a major role in many facets of the endocytic and exocytic process in Paramecium , including the i n t r a c e l l a r movement of food vacules (Allen, 1974, 1975, Allen and Wolf, 1974) . Colchicine, which binds to tubulin, causes depolymerization and subseguent in a c t i v a t i o n of microtubules (Olmsted and Borisy, 1973) . Colchicine also i n h i b i t s transport of materials into c e l l s , eg. nucleosides (Mizel and Wilson, 1972), sugars (Cheng and Katsoyannis, 19 75) and bi o p t e r i n , (Eembold and Langentach, 1978) . Podophyllotoxin, also a mitotic i n h i b i t o r , delays the normal migration of symbiotic green algae in _fiYd.r.a y i r i d i s (Cooper and Margulis, 1977). Colchicine does net have an effect on the rate of phagocytosis in i s o l a t e d membranes of polymorphonuclear leukocytes (fKLs) but does change the membrane topography of 97 these c e l l s (Berlin and Fera, 1977).. Concentrations of col c h i c i n e similar to those used on PMLs (1-5 mg/ml) decrease the endocytic rate i n Paramecium without causing any changes i n the ul t r a s t r ucture of microtubules (Tolloczko, 1977). The eff e c t s of t h i s agent on the mutant, f v c , as compared with wild-type c e l l s may provide more information on the nature of the abnormalities in this mutant. . Cytochalasins (eg. .cytochalasin B) are products of fungi with a wide range of b i o l o g i c a l e f f e c t s that can be divided into two major categories: (1) effects on the transport of molecules into c e l l s and (2) ef f e c t s on c e l l m o t i l i t y and morphogenesis (Lin, 1978). They cause rapid and severe i n h i b i t i o n of membrane-bound transport systems by penetrating the membrane and re s u l t i n g i n conformational or a l l o s t e r i c changes which i n h i b i t enzymes and ultimately release microfilaments from the plasma membrane (Spooner, 1 978). Cytochalsin B prevents the separation cf daughter c e l l s without impairing nuclear d i v i s i o n (Schroeder, 1978) and i n t e r f e r e s with the novement of c e l l s i n culture (Godman and Miranda, 1978). Cytochalasin B i n h i b i t s phagocytosis i n macrophages (Davies and A l l i s o n , 1978), P..caudatum (Tolloczko, 1977) and T.. p y r i f ormis (Nilsson et a l . , 1973, Nilsson, 1974, 1976)..There i s no change i n the ultrastructure of the microfilaments i n treated P. caudatum (Tolloczko, 1977). Since the role of microfilaments i n endocytosis and exocytosis i s unclear (Korn et a l . , 1974) , cytochalasin B may be acting on the c e l l membrane i n i t s i n h i b i t i o n of phagocytosis and thus may have interesting e f f e c t s on both the d i l and fvc mutants.. 98 The agents, DMSO, dibucaine, SDS, colchicine, and cytochalasin B, a l l have demonstrated e f f e c t s on membranes and/or phagocytosis and a study of these ef f e c t s on wild-type and mutant c e l l s has been car r i e d cut in an attempt to further characterize the mutants, dmi and fyc.. B. Mat e r i a l s and Methods 1. . Procedure for determining e f f e c t s cf chemical agents a. preparation of c e l l s Exponentially growing c e l l s were incubated at 27°C or 34.5°C f o r 5-6 hours ( c e l l density was approximately 10* cells/ml as determined by s e r i a l d i l u t i o n of 1.0 ml of c e l l s i n culture f l u i d ) . The c e l l s were centrifuged (100xg, 3 minutes) and adapted to a buffer consisting of 4 mM potassium chloride, 1 mM calcium chloride and 1 mM T r i s with a pH of 7.0-7.2 (see Appendix I for adaptation of c e l l s to tuffer)..2 ml of c e l l s were added to 3 ml of buffer containing 0.25 ml of concentrated, autoclaved bacteria JEnterobacter aerpgenes), also i n the same buffer. The c e l l s were then added to various concentrations of the agent to be tested (see section 1-b) . The same procedure was followed f o r wild-type, dmi., fyc, and A5 c e l l s . . The experiments were performed i n buffer with dead bacteria to minimize v a r i a b i i t y that could a r i s e from the metabolism of bacteria and the 99 undefined nature of the culture f l u i d . b. . preparation of chemical agents The concentration range to be tested was determined using published reports (where available) on the concentrations known to be e f f e c t i v e on Paramecium or si m i l a r organisms. . The chemicals were diluted to four times their f i n a l concentration i n the same buffer described above..Serial d i l u t i o n s were made i n duplicate; one set was incubated at 27°C and the other set at 34.5°C f o r 30 minutes prior to use. The pH of each solution was determined using a Corning pH meter. c. _ mixing of c e l l s and chemical agents 0.25 ml of chemical was added to a 13 x 100 mm bo r o s i l i c a t e glass disposable culture tube. This was followed by 0 .5 ml of buffer containing either 1.5 mg/ml blue watercolor (BP), 1.0 mg/ml red watercolour (EP) , 1.0 mg/ml black watercolor, or 1.0 mg/ml carmine p a r t i c l e s . F i n a l l y , 0.25 ml of the Paramecium /autoclaved bacteria mixture (section 1-a) were added and after 20 minutes at 27°C or 34.5°c: the c e l l s were fixed with formaldehyde* Controls were prepared i n exactly the same manner except that 0.25 ml of buffer alone replaced the 0.25 ml of chemical i n buffer. 100 d.. analysis of r e s u l t s Thirty to 50 c e l l s at each concentration of chemical agent were examined i n a Zeiss l i g h t microscope and the phenotypes were described as fcllcws: i . c e l l s with normal vacuoles (N) i i . . c e l l s with swollen con-tractile vacuoles (SCV) i i i . c e l l s with clumped vacuoles (CV) u e . . vacuoles located i n one area of th i v. . c e l l s with decrease d than five color ed vacuole wild type c e l l s = 11.3, c v. c e l l s with disrupted morphologi c a l l y abnormal v i . c e l l s with c o r t i c a l membrane and c o r t i c a l Sibley and Hans on (1974) Freguently a single c e l l had more than one type of abnormality and when thi s occurred c e l l s were described as having the most severe phenotype ( i - v i with increasing s e v e r i t y ) . For example, i f a c e l l had a swollen CV and disrupted vacuoles i t was placed in group (v) , DV. Once the number of c e l l s with each phenotype was determined for wild-type and mutant c e l l s at each concentration* the Chi Sguare (X2) test for homogeneity (employing Yates' correction factor with one degree of freedom) was performed in two d i f f e r e n t ways. F i r s t , to determine i f a s p e c i f i c concentration was having an e f f e c t , the number of c e l l s with each phenotype of one c e l l type ( eg. wild-101 type) at that concentration was compared with the number of c e l l s with each phenotype in the control sample of the same c e l l type..Second, a f t e r an e f f e c t i v e concentration was found (P < .05), each mutant was compared with the wild-type c e l l s at the same concentration. This indicated whether the mutants were being affected d i f f e r e n t l y than wild-type c e l l s . 2. DMSO a. . e f f e c t cf concentration The concentrations of DMSO (Sigma, Molecular weight, M. W..= 78.13) tested at 27°C and 34.5°C ranged from 5-15%. The pH of 10% DMSO at both temperatures was 6.6. . BP was added to wild-type, dmi and fyc c e l l s for 20 minutes and the c e l l s were fixed and examined. Fixed c e l l s were embedded i n s a l i n e - g e l a t i n or air - d r i e d on glass s l i d e s coated with albumin and photographed on a L e i t z Orthomat-W microscope using Nomarski optics. b. . observations of l i v e c e l l s Wild-type, dmj. and fyc c e l l s were adapted to buffer and 6% cr 12% DMSO in buffer and BP was added at 27°C or 34.5°C. Samples of 10 c e l l s were removed every 5 minutes f o r 20 minutes, added to 3% methyl c e l l u l o s e on a microscope s l i d e and examined in a Zeiss microscope using Nomarski optics., a l t e r n a t i v e l y , single c e l l s were placed 102 in a rcto-compressor and also examined using Nomarski optics.. The rate of contraction of the CVs was noted as well as the formation and movement of food vacuoles. The c e l l s were recorded on videotape using an RCA TC1C00 TV camera and a Sanyo videotape recorder (VTR 2000).. Frame-by- frame tracings were made from an 11-inch Electrohome TV monitor. . The osmolality of buffer and 6% DMSO in buffer with and without added BP were determined on an Advanced Wide-Range Osmometer. . c. e f f e c t of treatment duration BP in buffer was added to wild-type, dmj. and fvc c e l l s , also in buffer. After 0, 5, 10, 15, 17, and 19 minutes, samples were removed and DMSO i n buffer and BP (to maintain a f i n a l concentration of 12% DMSO and 0.75 mg/ml BP) were added. The c e l l s were fixed a f t e r a t o t a l of 20 minutes from the i n i t i a l addition of BP had elapsed i . e . c e l l s were i n BP for a to t a l of 20 minutes and in DMSO for varying portions of that 20 minutes. . d. addition of RP followed t_y DMSO and a second watercolor Wild-type, dm1_ and fvc c e l l s were incubated at 34.5°C for 6 hours and then fed EP in culture f l u i d . The c e l l s were centrifuged (100xg, 5 minutes) and adapted to buffer. 6% DMSO i n BP or black watercolor was added f o r 20 minutes. Alternativsly, BP or black watercolor was added for 10 minutes followed by an additional 10 minutes with 103 DMSO i n b u f f e r and BP or b l a c k w a t e r c o l o r (to m a i n t a i n f i n a l c o n c e n t r a t i o n s of 6% or 12? DMSO and 0.75 mg/ml BP or 0.5 mg/ml b l a c k w a t e r c o l o r ) . The c e l l s were f i x e d a f t e r a t o t a l of 20 minutes i n t h e seccnd w a t e r c o l o r . . 3« Dib u c a i n e W i l d - t y p e , dml a f l d A5 c e l l s were t e s t e d w i t h 3.8 x 1 0 ~ 6 and 3.8 x 10~ 5 g/ml of d i b u c a i n e (ICN P h a r m a c e u t i c a l s , M.W.= 379. 92) i n b u f f e r and BP f o r 20 minutes a t 27°C and 34.5°C. The pH of both c o n c e n t r a t i o n s a t both t e m p e r a t u r e s was 6.7. The c e l l s were f i x e d and examined.. 4. . SDS W i l d - t y p e , dml and A5 c e l l s were t r e a t e d with 1 0 - 7 , 5 x 10-7, 10-6, 5 x 10-6, a n d 10-s g/ml of SDS (Sigma, M.W. = 288.4) i n b u f f e r and BP f o r 20 minutes at 27°C and 34.5°C.„ The pH of 10-s g/ml a t 27°C was 6.9 and a t 34.5°C was 7.0. The c e l l s were f i x e d and examined. 5«- C o l c h i c i n e W i l d - t y p e , dml a f i d A5 c e l l s were t r e a t e d w i t h 0.25, 0.5, and 2 . 5 mg/nl of c o l c h i c i n e (Sigma, M.W. ,= 399.43) i n b u f f e r and BP f o r 20 n i n u t e s a t 27°C and 34.5°C. The c e l l s were f i x e d and examined. 104 6. Cytochalasin B Cytochalasin B i s generally dissolved i n DMSO but since t h i s was one of the agents tested, agueous suspensions of 16, 17, 18, 19, and 20 ug/ml cytochalasin B (Sigma,M. W. .= 479.62) in buffer were mixed with wild-type, dm] and A5 c e l l s and carmine p a r t i c l e s . . The suspensions were shaken during the 20 minute incubation period at 27°C or 34.5°C..The pH of 20 ug/ml at 34. 5°C was 6.7. The c e l l s were fixed and examined* C. Results 1. DMSO a. e f f e c t of concentration After treatment with 5% DMSO for 20 minutes at 27°C and 34.5°C, the d i s t r i b u t i o n of phenotypes i n wild-type, dmj and fvc c e l l s was d i f f e r e n t (P < .00 1) than i n controls (Tables 5-1 and 5-2). With increasing concentration of DMSO from 8% tc 15%, the CVs were often swollen (Figure 5-1a and Plate V-1a). The food vacuoles were occasionally normal tut were most often disrupted (Figure 5-1b-e and Plate V-1b). Abnormal vacuoles were of four major types: i . c e l l s with several normal wa t e r c o l o r - f i l l e d vacuoles present i n the same c e l l as one or more larger and morphologically abnormal vacuoles (Figure 105 5-lb) i i . c e l l s with one or mere large and ir r e g u l a r vacuole (s) (Figure 5-1c and Plate V-1b) i i i . c e l l s with no apparent vacuoles but many part i c l e s of watercolor throughout the cytoplasm (Figure 5-1d) iv. c e l l s with very large co l o r l e s s vacuoles (swollen CVs) and p a r t i c l e s of watercolor throughout the cytoplasm (Figure 5-1e) A l l four types of disrupted vacuoles were found i n wild-type, dm1 and fvc c e l l s . . At high concentrations of DMSO (15%) , there was an increase in the number of c e l l s with c o r t i c a l abnormalities where blebs of various sizes appeared on the c e l l surface (Figure 5-1f,g and Plates V-1c and V-1d). dmj c e l l s were more se n s i t i v e to 5% DMSO at 34.5°C as evidenced by a greater number of c e l l s with disrupted vacuoles than i n wild-type and in 12% DMSO at 27°C there were more dm1 c e l l s with c o r t i c a l abnormalities than wild-type c e l l s . b._ observations of l i v e c e l l s One of the f i r s t abnormalities apparent after addition cf DMSO at 27°C and 34.5°C was the decrease i n the rate cf contraction of the CVs. In untreated wild-type c e l l s at 27°C the mean length of the CV cycle was 7.59 seconds ( C i i . . = 7.11 - 8.C7) (lable 5-3). The CV cycle was longer in dmj c e l l s (8.44 seconds, c . i . .= 7.65 - 9.23) and 106 f v c c e l l s (9.3 seconds, c. i . = 8.49 - 10.11).. A t 34.5°C t h e l e n g t h of the CV c y c l e i n c r e a s e d i n a l l c e l l t y p e s . There were no s i g n i f i c a n t d i f f e r e n c e s i n the c y c l e l e n g t h s of w i l d - t y p e and f y c c e l l s when BP was added.. However, when BP was added t o dml. c e l l s , the l e n g t h of t h e c y c l e changed s i g n i f i c a n t l y . . A f t e r 5 minutes i n 6% DMSO a t 27°C or 34.5°C, the CV c y c l e s of a l l c e l l t y p e s were v e r y l o n g , r e g u i r i n g at l e a s t 10 seconds per c y c l e . The food v a c u o l e s began s w e l l i n g and a f t e r 10 minutes i n 6% DMSO (15 minutes f o r w i l d - t y p e c e l l s ) food v a c u o l e f o r m a t i o n a t t h e base of the g u l l e t was abnormal. N o r m a l l y , p a r t i c l e s were swept i n t o t h e g u l l e t and a membrane-bound v a c u o l e pinched o f f and w i t h i n seconds r e a c h e d the p o s t e r i o r of the c e l l ( F i g u r e 5-2)..In t h e pre s e n c e o f DMSO t h e v a c u o l e a t the base o f the g u l l e t d i d net pinch o f f but e n l a r g e d and e v e n t u a l l y fused w i t h o t h e r v a c u o l e s p r e s e n t ( F i g u r e 5-3). A l t e r n a t i v e l y , breakdown of t h e food v a c u o l e membrane o c c u r r e d and t h e v a c u o l a r c o n t e n t s were r e l e a s e d d i r e c t l y i n t o t h e cytoplasm. . A f t e r 15 minutes a t 27°C and 34.5°C, v a c u o l e s present i n the c y t o p l a s m o f t e n f u s e d w i t h each o t h e r ( F i g u r e 5-4). M o r p h o l o g i c a l a b n o r m a l i t i e s a l s o became apparent; a r e a s o f cy t o p l a s m c l e a r e d and bu l g e s formed. dml c e l l s a t 34.5°C were more s e n s i t i v e t o 6% DMSO i n t h a t f u s e d v a c u o l e s appeared as e a r l y as 10 minutes a f t e r a d d i t i o n (compared t o 15 minutes i n w i l d -t y p e and f v c c e l l s ) . . I n 12% DMSO the e f f e c t s o c c u r r e d v e r y r a p i d l y and by 20 minutes many c e l l s were m o t i o n l e s s w i t h t h e i r c i l i a extended.. 107 The osmolality of 6% DMSO i n buffer increased to 627 mOs (frcm the 45 mOs of buffer alone). The addition of BP had no effect on the osmolality cf buffer alone or DMSO i n buffer. c.. e f f e c t of treatment duration After one minute i n 12% DMSO the d i s t r i b u t i o n of phenotypes i n wild-type dmj and fyc c e l l s was s i g n i f i c a n t l y d i f f e r e n t from that of controls (P < .001). The CVs were swollen and by 3 minutes disrupted vacuoles were observed in wild-type, dmj and fyc c e l l s (Table 5-4). C o r t i c a l abnormalities became evident i n dmj c e l l s after 5 minutes and in wild-type and fyc c e l l s after 10 minutes, dmj c e l l s were, more s e n s i t i v e to DMSO; for the f i r s t 10 minutes dmi c e l l s were s i g n i f i c a n t l y d i f f e r e n t from wild-type c e l l s (P < .01)..By 15 minutes i n 12% DMSO most of the c e l l s in a l l three c e l l types (wild-type, dmi and fyc) had disrupted vacuoles and/or c o r t i c a l abnormalities. d- addition of RP followed by DMSO and a second watercolor RP was added to wild-type, dmj and fyc c e l l s f o r 20 minutes followed by BP for 20 minutes during the l a s t 10 minutes of which 12% DMSO in buffer and BP were added. The di s t r i b u t i o n of phenotypes i n samples treated in t h i s manner was s i g n i f i c a n t l y d i f f e r e n t than i n control c e l l s (P < .00 1). This difference was mainly due to the number of c e l l s with disrupted vacuoles (Table 5-5). Both dmj and 1 C 8 fvc were s i g n i f i c a n t l y d i f f e r e n t from wild-type c e l l s (P = .006), the difference again teing mainly due to the number of c e l l s with disrupted vacuoles. When 6% DMSO was s i m i l a r l y added, the d i s t r i b u t i o n of phenotypes was also abnormal (P < .001) with the greatest changes occurring i n the number of c e l l s with disrupted vacuoles. The d i s t r i b u t i o n of phenotypes i n wild-type and fyc c e l l s was si m i l a r (P = .21) while that cf dm 1 c e l l s was diff e r e n t (P < .001) because of a greater number of dml c e l l s with disrupted vacuoles.. The greatest incidence cf c e l l s with disrupted vacuoles occurred when 6% DMSO was added for 20 minutes i n buffer and BP. There was no difference between wild-type and dm 1 c e l l s (P = .54), and although there appears to be a difference between wild-type and fyc c e l l s , t h i s may not be v a l i d due to the small number of fvc c e l l s in the sample (15)...The watercolor in the disrupted vacuoles was usually a mixture of BP and EE indicating that those vacuoles formed in DMSO were the ones that became abnormal. Occasionally vacuoles with only EP were also disrupted usually, however, these vacuoles were i n t a c t . When EP was added for 20 minutes followed by black watercolor for 20 minutes with 12% DMSO added for the l a s t 10 minutes of incubation, the results were very s i m i l a r to those obtained with BP (Table 5-6). When 6% DMSO in black watercolor and buffer was added for 10 or 20 minutes, the res u l t s obtained with dnQ c e l l s were s i m i l a r to those obtained with BP but the results f o r wild-type and fvc 109 c e l l s were different* This can be explained by differences i n the watercolors themselves. .Red and black watercolors consist of very small p a r t i c l e s evenly d i s t r i b u t e d and t i g h t l y held in the watercolor suspension whereas BP consists cf larger c r y s t a l s of pigment not held t i g h t l y i n suspension. Therefore, i f miner membrane disruptions occurred, the vacuoles with red or black watercolor would re t a i n their pigment i n a cohesive sphere but BP i n a vacuole would readily seep out. Major disruptions of the vacuolar membrane caused by a higher concentration of DMSO would lead to a disruption cf red and black vacuoles as well as blue vacuoles. This was observed when black watercolor was added in 12% DMSO for 10 minutes; disrupted vacuoles were present in a l l three c e l l types. . dm 1 c e l l s were more sensitive than wild-tyre or f vc c e l l s to 6% DMSO for 10 minute treatment (P = .001) or 20 minute treatment (P = .002) indicating that the lower concentration of DMSO induces more extensive membrane damage i n dm_1 c e l l s than i n wild-type or fyc c e l l s . The vacuolar abnormalities i n dmj c e l l s at 34.5°C without DMSO added were i n s u f f i c i e n t to r e s u l t in the disruption of red or black-colored vacuoles. In addition, there i s the p o s s i b i l i t y that the BP i t s e l f contributes to vacuolar disruptions. This w i l l b a : c l a r i f i e d i n Chapter VI. 110 2.. Dibucaine At a l l concentrations tested at 27°C and 34.5°C i n wild-tyP e# dmj and fyc c e l l s , endocytosis was decreased r e l a t i v e to that of untreated controls (Tables 5-7 and 5-8). At 3,.8 x 1C - 5 mg/ml at 34.5°C morphological abnormalities were also apparent. No changes i n morphology of food vacuoles were observed. 3. SDS SDS also resulted i a decreased endocytosis with no change in vacuolar morphology (Tables 5-9 and 5-10) . .However, A5 c e l l s were more sensitive than wild-type or dm 1 c e l l s ; 5 x 10 - 6 g/ml resulted in decreased endocytosis i n A5 c e l l s at 27°C while 10 - 5 g/ml were reguired f o r eguivalent decrease of endocytosis i n wild-type and d i j c e l l s . . A t 34.5°C endocytosis was inhibited in A5 c e l l s at 10 - 7 g/ml, i n d j j c e l l s at 10~6 g/ml and i n wild-type c e l l s at 10~ 5 g/ml ( t h i s concentration was l e t h a l for A5 ce l l s ) . 4. Colchicine Colchicine also i n h i b i t e d endocytosis without causing changes i n the food vacuoles (Table 5-11). . At 27°C the concentrations tested had no e f f e c t on any of the c e l l types. However, at 34.5°C endocytosis was i n h i b i t e d i n A5 c e l l s at 0.25 mg/ml colchicine whereas ten times that amount was reguired to decrease the number cf food vacuoles formed i n wild-type and dmj cells... 111 5. Cytochalsin B Aqueous suspensions of cytochalasin B gave extremely variable results due to the imposs i b i l i t y of obtaining a uniform sol u t i o n . This agent appeared to decrease endocytosis but no conclusive statement cculd be made. In view of the observed e f f e c t s of low concentrations of DMSO, i t was not considered wise to dissolve cytochalasin B in DMSO in order to obtain a uniform solution.. D. Discussion The observed e f f e c t s of DMSO on CV function and endocytosis in wild-type c e l l s are consistent with other reports (Skriver and Nilsson, 1974, Nilsson, 1977b, Sibley et a l . , 1977) and include the.following: 1. swelling and decreased rate of contraction of CVs 2. decreased rate of food vacuole formation 3..fusion and disruption of vacuoles 4. c o r t i c a l abnormalities Many of the extensive changes caused by long-term exposures (up to 24 hours) to DMSO i n Tetrahymena and Paramecium are similar to the e f f e c t s of starvation and,can be attributed to the cessation of endocytosis (Nilsson, 1977b). In the preceding experiments exposure times were short (maximum 20 minutes) Therefore, the observed effects are unlikely due to starvation. The CV i s an organelle used by fresh water p r o t i s t s for 112 osmoregulation to prevent swelling and to maintain and regulate concentrations of osmotically active i n t r a c e l l u l a r p a r t i c l e s (Patterson, 1980)., The rate of contraction of the CV i s inversely proportional to the osmolality of the surrounding environment (Prusch, 1977). The values obtained for the length of the CV cycle without DMSO were shorter than those obtained for P i caudatum (Patterson, 1977) but similar to those obtained for P. multijicronucleatum (Organ et a l . , 1968). The reason for the increased length of the cycle in the mutants i s unclear but may be related to a possible impairment in some aspect of t h i s complex osmoregulatory mechanism. The increase, in osmolality of buffer with added DMSO i s far above 50 - 60 mOs which i s the isomolar concentration for Paramecium (Prusch, 1977) and t h i s probably accounts for the increased length of the vacuolar cycle when DMSO i s added. The decrease i n endocytosis caused by DMSO i s more d i f f i c u l t to explain because Nilsson (1977b) found no changes i n the ultrastructure of the cytostcmal region of T. pyriformis a f t e r treatment with DMSO for one hour (in these c e l l s endocytosis ceased a f t e r ten minutes) although there were small vesic l e s near the pharyngeal membrane sim i l a r to those occurring i n starved cells..DMSO may prevent the incorporation of vesicles into the growing food vacuoles..DMSO also affects mitochondrial structure (Nilsson, 1977b) and decreases oxygen consumption (franz and Van Bruggen, 1S67) thereby i n t e r f e r i n g with energy production which i s essential for endocytosis (Chapman-Andresen and Nilsson, 1968, Yamada, 1974). DMSO also changes the d i s t r i b u t i o n of p a r t i c l e s i n the 113 membranes of mouse lymphocytes (Mclntyxe et a l . , 1974) and this may also occur in food vacuole membranes. This could be a r e f l e c t i o n of modified enzyme function and/or p r o t e i n - l i p i d interactions since DMSO a l t e r s protein conformation (Rammler, 1971) and f a t t y acid stereoisometry (Muset and Martin-Esteve, 1965).. This i s consistent with observations of disrupted vacuoles i n these and other experiments (Sibley et a l . , 1977) . " Tollocsko (1977) found that i n E_ ca udatum c e l l s treated with cytochalasin B dissolved i n DMSO, defective separation of food vacuoles from the gul l e t occurred. This was attributed to the cytochalasin B but may have been caused by the DMSO since the same lack of separation was observed with DMSO alone (Figure 5-3)..Fusion of vacuoles after exposure to DMSO has not been previously reported but t h i s night be explained by the d i f f i c u l t y i n observing such rapid events in l i v e c e l l s . In th i s study t h i s d i f f i c u l t y was circumvented by the use of videotape where the a v a i l a b i l i t y of "instant replays" insured the discernment of a l l events.. The effects of DMSO cn proteins (Rammler, 1971) and l i p i d s (Muset and Martin-Esteve, 1 965), which are based, i n part, on the interaction of DMSO with water (Cowie and Toporowski, 196 1) and i t s possible replacement of water (MacGregor, 1967), may contribute to the fusion of vacuoles. Paramecia tend to form tlebs (membrane and c o r t i c a l separations) in response to many agents, such as pressure, heat and chemicals (Sibley and Hanson, 1974) and i n sensitive c e l l s in response to kappa symbionts (Jurand et al.,. 1978). Since the 114 shape of Paramecium i s based on the microfilamentous i n f r a c i l l i a r y l a t t i c e , agents which cause c o r t i c a l abnormalities interfere with t h i s subcortical cytoskeleton (Sibley and Hanson, 1 974). This can occur d i r e c t l y or i n d i r e c t l y as i s the case with kappa endosymbionts which decrease the contraction rate of the CV.. This r e s u l t s i n increased retention of water causing an increase in i n t e r n a l hydrostatic pressure that leads to the formation of swellings and blebs (Jurand et a l . , 1978). Since DMSO also decreased the rate of contraction of the CVs, the observed blebs could also be due to an increase i n i n t e r n a l hydrostatic pressure. Dibucaine, SDS and colc h i c i n e i n h i b i t e d endocytosis i n wild-type and mutant c e l l s . This may be due to the in s e r t i o n of anasthetics and detergents into the membrane bilayer (Browning and Nelson, 1S76, Helenius and Simons, 1975) which may impede normal membrane functioning. The different e f f e c t s observed with the two agents (eg.. dibucaine caused morphological abnormalities) could be due to anasthetics and anionic detergents inserting into opposite faces of the membrane bilayer (Browning and Nelson, 1976). . A5 c e l l s were more sens i t i v e to SDS than dmi, c e l l s which, in turn, were more sensit i v e than wild-type c e l l s , thereby i n d i c a t i n g that both mutants have i r r e g u l a r i t i e s in t h e i r membranes (probably at d i f f e r e n t sites) which make them mere susceptible to the action of the detergent. . Colchicine i n h i b i t s endocytosis in P._ caudatum without causing changes in the u l t r a s t r u c t u r e of microtubules (Tolloczko, 1977) and t h i s i n h i b i t i o n may result from changes 115 i n membrane topography as occurs i n isolated plasma membranes of PMLs (Berlin and Fera, 1977). Normally, phagocytosis in these c e l l s i s accompanied by a decrease i n the microviscosity of the membrane. This change i s eliminated by col c h i c i n e and Berlin and Fera (1977) suggest that extensive microtubule-mediated membrane modifications are reguired for phagocytosis. The ten-fold increase i n s e n s i t i v i t y of the A 5 mutant to colchicine suggests a possible l e s i o n in a microtubule-mediated system i n t h i s mutant. The vast range of b i o l o g i c a l effects of DMSO l i m i t s i t s usefullness i n further characterizing the mutant, dmj..However, since i t does affect membranes, the observed e f f e c t s on dmj c e l l s are consistent with membrane abnormalities i n t h i s mutant..Its greatest value may l i e in i t s use i n a mass selection system for more membrane mutants. S i m i l a r l y , c o l c h i c i n e may aid i n the se l e c t i o n of other mutants which are defective i n the in t e r n a l movement of vacuoles. . 116 Table 5-1..Effect of dimethyl sulfoxide at 2 7°C Cone. C e l l N SCV DV DE CA X 2 P DMSO (1) Type (2) (3) CO (5) (6) (7) (8) control ~Wt 4 2 ~ o~~ 0 ~5 C dmj 43 0 0 7 C 0.25 0.62 fyc 39 0 0 9 0 1. 24 0. 26 5% wt 11 1 30 8 0 dm! 10 0 30 10 0 1. 27 0.74 fyc 5 0 30 5 0 2.87 0. 41 8% wt 12 9 20 9 0 dmj 3 11 30 1 0 c 8.20 0.04 fyc 4 15 30 5 0 5.51 0.04 10% wt 4 24 16 6 0 dml 1 18 26 5 0 5. 13 0. 16 fyc 2 13 20 5 0 3. 4 0.33 12% wt 0 10 30 6 3 dm! 0 5 23 4 18 17.70 0.001 fyc 1 7 28 5 9 4.68 0. 32 15% wt 3 0 23 10 14 ami 0 0 . 25 9 16 3. 27 0.35 fyc 0 0 23 1 2 15 3.22 0.36 wt = wild-type dm! = mutant with disrupted vacuoles at 34.5°C fyc = mutant with clumped vacuoles at 34.5°C (1) concentration of dimethyl sulfoxide added f o r 20 minutes with blue watercolor (2) number of normal c e l l s (3) number of c e l l s with swollen c o n t r a c t i l e vacuoles (4) number of c e l l s with disruptd vacuoles (5) number of c e l l s with decreased endocytosis (6) number of c e l l s with c o r t i c a l abnormalities (7) Chi Sguare Test for homogeneity (8) probability generated by Chi Sguare Test 117 Table 5-2. Effect of dimethyl sulfoxide at 34.5°C Cone.. C e l l N SCV DV DE C A X 2 P DM SO (1) Type (2) (3) (4) (5) (6) (7) (8) control wt 40 0 0 10 0 dmj 19 0 19 12 12 26.66 <.001 fvc 18 0 0(9) 1 0 C 30.34 <.00 1 5% wt 4 0 23 23 0 dmj 2 0 38 10 0 9. 48 0.004 fyc 4 0 21 16 0 4.50 0.21 8% wt 4 6 28 6 0 dl J 5 11 31 2 1 4.37 0.36 fyc 4 6 30 7 2 1.88 0.76 10% wt 5 8 33 3 1 dmj 4 7 32 2 c 3.06 0.55 fvc 5 9 29 6 1 1. 32 0.86 12% wt 1 9 30 4 6 dmj 0 1 38 4 7 8.42 0.08 fyc 0 2 37 5 6 6.3 0. 18 15% wt 1 0 29 5 15 dmj 0 0 26 9 15 2. 31 0.53 fyc 0 0 27 6 17 1.29 0. 73 wt = wild-type dmj = mutant with disrupted vacuoles at 34.5°C fyc = mutant with clumped vacuoles at 34.5°C (1) concentration of dimethyl sulfoxide added for 20 minutes with blue watercolor (2) number of normal c e l l s (3) number of c e l l s with swollen c o n t r a c t i l e vacuoles (4) number of c e l l s with disrupted vacuoles (5) number of c e l l s with decreased endocytosis (6) number of c e l l s with c o r t i c a l abnormalities (7) Chi Sguare Test for homogeneity (8) probability generated by Chi Sguare Test (9) number of fyc c e l l s with clumped vacuoles = 22, number of wild-type c e l l s with clumped vacuoles = 0 118 Table 5-3. Length of c o n t r a c t i l e vacuole cycle Temp. C e l l BP added Length of °C(1) Type (2) cycle (sec) (3) 27 wt - 7.59(7. 11-8.07) dmjl - 8.44 (7. 65-9.23) dmj + 6. 21 (5. 44-6. 98) fyc - 9.30 (8. 49-10. 1 1) 34.5 wt - 6. 38 (5. 78-6.98) wt + 7.55(5.91-9.19) dm! " 5.63 (4. 72-6.54) dm! «• 8. 78 (7. 36-10. 20) fyc - 6. 13 (5. 69-6.57) fyc * 7.42 (6. 38-8. 46) wt = wild-type dm! = mutant with disrupted vacuoles at 34.5°C fyc = mutant with clumped vacuoles at 34.5°C (1) c e l l s were incubated at the temperature indicated for 6 hours (2) where indicated («•) blue watercolor was added for 20 minutes (3) means and 95% confidence i n t e r v a l s of the length of the co n t r a c t i l e vacuole cycle 119 Table 5-4. Effect of 12% dimethyl sulfoxide at 34.5°C Time C e l l N s c v DV DE CA X 2 P (min) (1) Type (2) (3) (4) (5) (6) (7) (8) control ~wt "HI 0 ___ __ d j l 18 0 24 8 0 27. 26 <.001 fyc 14 0 1 (*) 8 0 39.45 <.001 1 wt 30 11 7 0 1 dmj 4 15 28 2 1 35.09 <.00 1 fyc 9 1 1 9 (+) 4 0 13.97 0.C07 3 wt 0 30 16 0 4 dmj 0 19 23 5 3 12.87 0.01 fyc 1 18 14 5 4 8. 5 0.07 5 wt 1 28 15 3 3 dmj 0 13 14 1 1 12 16.49 0.002 fyc 0 22 20 2 4 2.74 0. 60 10 wt 1 18 23 2 4 dmj 0 . 13 17 6 14 9. 18 0.04 fyc 0 15 28 3 4 1.92 0.75 15 wt 0 1 1 26 2 11 dmj 0 8 7 3 32 21.87 <. 001 fyc c 8 21 0 16 3.68 0.30 20 wt 0 2 4 1 1 33 dmi 0 1 3 7 40 2.03 0. 57 fvc 0 0 1 1 1 38 4. 15 0.25 wt = wild-type dmi = mutant with disrupted vacuoles at 34. 5°C fyc = mutant with clumped vacuoles at 34.5°C (*) number fyc c e l l s with clumped vacuoles = 27, wild-type (+•) number fyc c e l l s with clumped vacuoles =17, wild-type (1) number of minutes i n 12% dimethyl sulfoxide (2) number of normal c e l l s (3) number of c e l l s with swollen c o n t r a c t i l e vacuoles (4) number of c e l l s with disrupted vacuoles (5) number of c e l l s with decreased endocytosis (6) number of c e l l s with c o r t i c a l abnormalities (7) Chi Sguare Test for homogeneity (8) probability generated by Chi Sguare Test = 0 = 1 120 Table 5-5. E f f e c t of dimethyl s u l f o x i d e and red and blue watercolor at 34.5°C Co nc., C e l l N SCV DV DE X 2 P Time (1) Type (2) (3) (5) (6) (7) c o n t r o l wt 41 2 7 Ml 23 0 19 10 21.51 <.001 fVG 24 0 1 (8) 6 18. 38 <.001 12% Wt 14 7 26 3 DM SO dml 7 0 40 2 12*49 0.006 10 min f y c 1 0 42 4 22. 10 <. 001 6% wt 3 14 25 C DMSO d j l 3 1 33 4 16. 36 0.001 1 0 min f y c 1 5 23 1 4.47 0.21 6% wt 3 1 . 46 0 DMSO dm! 4 0 45 1 2. 15 0. 54 20 min fy c 0 5 9 1 17.9 <.001 wt = wild-type dm! = mutant with d i s r u p t e d vacuoles at 34.5°C f y c = mutant with clumped vac u o l e s at 34.5°C (1) c e l l s were fed red f o l l o w e d by blue watercolor f o r 20 minutes with the c o n c e n t r a t i o n and time of exposure t o DMSO as i n d i c a t e d (2) number of normal c e l l s (3) number of c e l l s with swollen c o n t r a c t i l e vcauoles (4) number of c e l l s with d i s r u p t e d vacuoles (5) number of c e l l s with decreased e n d c c y t o s i s (6) Chi Sguare Test f o r homogeneity (7) p r o b a b i l i t y generated by C h i Sguare Test (8) number of f y c c e l l s with clumped vacuoles =14, w i l d -type = 0 121 Table 5-6. E f f e c t o f d i m e t h y l s u l f o x i d e and r e d and b l a c k w a t e r c o l o r at 34.5°C Cone., C e l l N SCV DV DE X 2 P Time (1) Type (2) (3) (4) (5) (6) (7) c o n t r o l wt 50 0 0 0~ 32 0 2 2 5.83 0. 05 f y c 30 0 0(8) 3 27.94 <.001 12% wt 6 13 31 1 DM SO dmj 1 8 40 0 5.89 0.05 10 min f y c 1 8 32 0 3.43 0. 14 6% wt 17 17 13 1 DMSO dmj 7 4 19 6 15.50 0.001 10 min f y c 14 7 7 6 7.66 0.05 6% wt 13 1 10 23 DMSO dmj 8 8 22 12 14.51 0.002 20 min f y c 2 2 4 4 5.52 0. 14 wt = w i l d - t y p e dmi = mutant with d i s r u p t e d v a c u o l e s a t 34.5°C f y c = mutant w i t h clumped v a c u o l e s a t 34.5°C (1) c e l l s were f e d r e d f o l l o w e d by b l a c k w a t e r c o l o r f o r 20 minutes w i t h t he c o n c e n t r a t i o n and time of exposure t o DMSO as i n d i c a t e d (2) number of normal c e l l s (3) number of c e l l s w i t h s w o l l e n c o n t r a c t i l e v a c u o l e s (4) number o f c e l l s w i t h d i s r u p t e d v a c u o l e s (5) number of c e l l s with decreased e n d o c y t o s i s (6) C h i Sguare Test f o r homogeneity (7) p r o b a b i l i t y g e n e r a t e d by C h i Sguare (8) number f y c c e l l s w i t h clumped v a c u o l e s = 20, w i l d - t y p e = 0 1 22 Table 5-7. Effect of dibucaine at 27°C Cone. C e l l N DE X2 P (D Type [2) (3) (5) control wt 30 0 ami 30 0 0.00 1.00, A5~ 30 0 0. 00 1.00 3.8x10-6 wt 17 13 16.60 <.001 dml 13 24 30.30 <.001 A5~ 22 13 13.93 <.001 3.8x10-5 wt 0 39 69.00 <.001 dml 0 31 61.00 <.001 A5~ 0 16 46.00 <.001 wt = wild-type dm_1 = mutant with disrupted vacuoles at 34.5°C A5 = double mutant with trichocyst non-discharge and clumped vacuoles at 34.5°C (1) concentration of dibucaine (grams per ml) added for 20 minutes (2) number of normal of c e l l s (3) number of c e l l s with decreased endocytosis (4) Chi Sguare Test for homogeneity i . i n controls between wild-type and each mutant i i . in dibucaine between each c e l l type and i t s control d i s t r i b u t i o n (5) p r o b a b i l i t y generated by Chi Sguare Test 123 Table 5- 8. Effect of dibucaine at 34.5°C Conc. C e l l N DE X* P (2) Type (2) (3) m (5) control ___ 5 ~ dml 30 (7) 1 1.44 0. 09 A5 30(8) 5 0.02 0. 88 3.8x1C-6 wt 15 2 1 13.10 <.001 dml 7 27 38.38 <. 001 A5 2 28 40. 38 <. 001 3.8x10-5 wt 0 30 44.84 <.00 1 dml 0 30 57.13 <. 001 A5 0 30* 47.76 <. 001 wt = wild-type dmj = mutant with disrupted vacuoles at 34.5°C A5 = double mutant with t r i c h o c y s t non-discharge and clumped vacuoles at 34.5°C (1) concentration of dibucaine (grams per ml) added for 20 mi n u te s (2) number of normal c e l l s (3) number of c e l l s with decreased endocytosis (4) Chi Square Test for homogeneity (see Table 5-7) (5) probability generated by Chi Sguare Test (7) number drnj c e l l s with disrupted vacuoles = 25, wild-type = 0 (8) number A5 c e l l s with clumped vacuoles =21, wild-type = C * a l l c e l l s morphologically abnormal 124 Table 5-9. Eff e c t of sodium dodecyl sulfate at 27°C Cone. C e l l N DE X 2 P (1) Type (2) (3) (4) (5) control wt 30 0 dm! 30 0 0.00 1.00 A5 31 1 0.95 0. 33 10-7 K t 30 0 0.00 1. 00 dm! 30 0 0.00 1.00 A5 30 0 0.95 0.33 5x10-7 w t 29 1 1.02 0.31 dm! 30 1 C.98 0. 34 A5 30 0 0.95 0.33 10-6 wt 29 1 0.98 0.32 dm! 29 1 1.02 0.31 A5 30 0 0.95 0.33 5x10-6 wt 30 1 0.98 0. 32 dm! 32 1 0.92 0.34 A5 19 11 11.16 <.001 10-s w t 4 26 45.88 <.001 dm! 0 30 57. 13 <.001 A5 3 27 47.19 <.001 * t = wild-type dm! = mutant with disrupted vacuoles at 34.5°C A5 = double mutant with t r i c h o c y s t ncn-discharge and clumped vacuoles at 34.5°C (1) concentration (g/ml) sodium dodecyl sulfate added for 20 minutes (2) number of normal c e l l s (3) number of c e l l s with decreased endocytosis (4) Chi Sguare Test for homogeneity (see Table 5-7) (5) probability generated by Chi Sguare Test 125 Table 5- 10. .Effect of sodium dodecyl sulfate at 34. 5°C Conc. C e l l N DE X2 P d) Type (2) (3) (4) (5) control wt 29 1 dmi 29(6) 1 0.00 1. 00 A5 22 (7) 8 6.40 0.01 10-7 wt 30 1 0.0006 0.98 dm1 24 6 4.0 4 0. 04 A5 3 . 27 24.75 <.001 5x10-7 wt ' 30 . 0 1.02 0.31 dm1 27 3 1.07 0. 30 A5 4 26 22.99 <.001 10-6 wt 26 4 1.96 0. 16 dmi 16 20 20. 57 <. 001 A5 4 20 35.90 <. 001 5x10-6 wt 2 28 48.65 <.001 wt = wild-type dmi = mutant with i i s r u p t e d vacuoles at 34.5°C A5 = double mutant with t r i c h o c y s t hen-discharge and clumped vacuoles at 34.5°C (1) concentration sodium dodecyl sulfate (g/ml) added f o r 20 minutes (2) number of normal c e l l s (3) number of c e l l s with decreased endocytosis (4) Chi Sguare Test for homogeneity (see Table 5-7) (5) pr o b a b i l i t y generated by Chi Sguare Test (6) number dnQ c e l l s with disrupted vacuoles = 21, wild-type = 0 (7) number A5 c e l l s with clumped vacuoles = 12, wild-type = 2 126 Table 5- 11. Effect of c o l c h i c i n e at 34.5°C Conc. C e l l N DE X 2 P (1) Type (2) (3) (*») (5) control wt dml 30 (6) 0 0.00 1. 00 A5 27 (7) 3 3. 16 0. 08 0. 25 wt 30 0 0.00 1.00 dml 24 6 6.67 0.01 A5 8 22 24.75 <. 001 0. 50 wt 27 3 3. 16 0. 08 dml 22 8 9.23 0. 002 A5 8 26 28.42 <. 001 2. 50 wt 17 13 16.60 <.001 dml 9 2 1 32.30 < .001 A5 3 27 38.40 <. 001 wt = wild-type dm_1 = mutant with disrupted vacuoles at 34.5°C A5 = double mutant with t r i c h o c y s t non-discharge and clumped vacuoles at 34.5°C (1) concentration colchicine (mg/ml) added for 20 minutes (2) number of normal c e l l s (3) number of c e l l s with decreased endocytosis (4) Chi Sguare Test for homogeneity (see Table 5-7) (5) probability generated by Chi Sguare Test (6) number dml. c e l l s with disrupted vacuoles = 19, wild-type = 1 (7) number A5 c e l l s with clumped vacuoles =21, wild-type = 0 127 Figure 5 - 1 S c h e m a t i c i l l u s t r a t i o n cf morphological effects of dimethyl sulfoxide. Dots represent p a r t i c l e s of blue watercolor (BP) . a. normal vacuoles and swollen c o n t r a c t i l e vacuoles (CV) b. several normal vacuoles and one or more large, i r r e g u l a r vacuoles c. one or more large, i r r e g u l a r vacuoles d. no clear delineation of vacuoles e. very large, colorless vacuoles (formerly the CVs) and p a r t i c l e s of BE throughout the cytoplasm f . a c o r t i c a l separation where the underlying cytoplasm appears to have condensed away from the cortex. P a r t i c l e s of BP are present throughout the cytoplasm g. c o r t i c a l blebs where the cortex appears to have l i f t e d away from the cytoplasm which has many p a r t i c l e s of BP throughout 129 Figure 5-2. Frame-by-frame movement of a food vacuole. The newly formed vacuole moves frcm the base of the g u l l e t (g) to the posterior cf a wild-type c e l l . Each number represents 1/6 second.. 1 3 0 131 Figure 5-3..Endocytosis i n 6% dimethyl sulfoxide..Frame-by-frame t r a c i n g cf abnormal food vacuole formation a t the base of the g u l l e t (g) of a wild-type c e l l . T r a c i n g s are separated by 2. 5 second i n t e r v a l s . . a. a food vacuole i s forming a t the base of the g u l l e t (g) b. i n s t e a d of p i n c h i n g o f f and moving to the p o s t e r i o r , the vacuole remains attached t o the g u l l e t and i n c r e a s e s i n s i z e c. the nascent vacuole comes very c l o s e to a p r e v i o u s l y formed vacuole and the two begin to f u s e d. f u s i o n i s complete and a l a r g e , i r r e g u l a r vacuole i s formed which i s s t i l l attached to the g u l l e t 13Z i 1 IOJJM 133 F i g u r e 5 - 4 . Fusion of vacuoles i n 6SS dimethyl s u l f o x i d e . Frame-by-frame t r a c i n g of the f u s i o n of two vacuoles i n the cytoplasm of a dml c e l l . T r a c i n g s are separated by 2 . 5 second i n t e r v a l s a. one l a r g e and one small vacuole approach each other i n the cytoplasm b. .the vacuoles meet and begin t o fuse c. f u s i o n proceeds and the contents of the two vacuoles mix d. . f u s i o n i s complete r e s u l t i n g i n one l a r g e r , i r r e g u l a r vacuole I 3 H 135 Plate V-1. Morphological e f f e c t s of dimethyl sulfoxide. A l l c e l l s were fixed in formaldehyde and air-dried on s l i d e s . a. Wild-type c e l l after treatment with 12% dimethyl sulfoxide (DMSO) for 10 minutes. . Note the presence of normal food vacuoles f i l l e d with blue watercolor (BP) and swollen c o n t r a c t i l e vacuole (CV) (arrow). b. Wild-type c e l l after treatment with 6% DMSO for 10 minutes. Note the large, disrupted vacuoles f i l l e d with BP. c. Wild-type c e l l i n red watercolor (EP) for 20 minutes followed by BP for 20 minutes and 12% EMSO for 10 minutes. Some normal vacuoles are present (these were f i l l e d with EP) as well as swollen CVs (small arrows) and a bleb (large arrow). d. Wild-type c e l l i n 12% DMSO for 10 minutes. The centre of the c e l l i s a mass of watercolor p a r t i c l e s (BP) and there are small blebs (arrows) on the c e l l surface. ( 3 6 137 CHAPTER VI ULTRESTRUCTURE OF WILD-TYPE AND dml CELLS A. Introduction In the l i g h t microscope, dm! c e l l s that have been fed blue watercolor (BE) appear to have very large disrupted vacuoles as compared with the highly regular, spherical food vacuoles of wild-type c e l l s (Plate III-1b,c). However, when dml c e l l s are fixed and examined i n the l i g h t microscope without having previously been fed BP, there i s l i t t l e evidence of vacuolar abnormalities. In order to c l a r i f y the structural nature of the vacuolar abnormalities, wild-type and mutant c e l l s were examined by transmission electron microscopy. . B. Materials and Methods (see Appendix III f o r d e t a i l s of procedure) Wild-type and dm! c e l l s were incubated at 34.5°C for 6 hours.. Half the c e l l s i n each sample were fed BP for 20 minutes, centrifuged (100xg, 5 minutes) , washed i n 6 mM phosphate buffer, and fi x e d f o r 2 hours in 0.5% glutaraldehyde in 6 mM phosphate buffer..The remaining half of each sample was treated in the same way except for the omission of BP.. The fixed c e l l s were then washed twice in 6mM buffer and post-fixed for one hour i n 1S osmium tetroxide in 25 mM phosphate buffer. The c e l l s were wasied and dehydrated step-wise through ethanol 138 and propylene oxide.. While i n 70$ ethanol, the c e l l s were stained with uranyl acetate. The c e l l s were then embedded in Epon and sectioned on a S o r v a l l MT-1 or MT-2 Ultra Microtome. Thin sections (60-90 nm) were stained in uranyl acetate (20 -30 minutes) and lead c i t r a t e (5 - 10 minutes) and examined on a Zeiss EM 10 transmission electron microscope. A minimum of 30 wild-type and 30 dmi. c e l l s were examined. C. . Re s u It s The i d e n t i f i c a t i o n of the structures of the o r a l region and the terminology used i n describing them were based on A l l e n (1974)..The i d e n t i f i c a t i o n of the structure and age of food vacuoles was based on Jurand (1961) and Jurand and Selman (1969) and that of other c o r t i c a l units and cytoplasmic structures was based on Jurand and Selman (1969) and Ehret and McArdle (1974). Unless otherwise indicated, a l l r e s u l t s refer to c e l l s at the r e s t r i c t i v e temperature that had not been fed BP prior to t h e i r f i x a t i o n . .. Plate VI-1 represents a transverse section of a wild-type c e l l near the posterior part of the g u l l e t (g). Three food vacuoles are present, each at a d i f f e r e n t stage. Food vacuole a (fva) i s a newly formed vacuole with a r e l a t i v e l y smooth membrane and the bacteria i n s i d e are r e l a t i v e l y well-preserved and undigested. Food vacuole b (fvb) i s at a s l i g h t l y l a t e r stage; the membrane i s more i r r e g u l a r and the bacteria are i n various stages of digestion with the b a c t e r i a l c e l l walls beginning to separate from the membranes. Food vacuole c (fvc) 139 i s an older vacuole with many outwardly directed projections i n the membrane. I t contains mainly ghosts and small fragments of digested bacteria. The small (approximately 0.2 um) , dense, membrane-bound vesicles close to the feed vacuoles are si m i l a r i n size and d i s t r i b u t i o n to neutral red granules (Jurand, 1961) which were shown to be the s i t e , in | j _ ca udatum , of enzymes which were l a t e r associated with lysosomes (Rosenbaum and Whittner, 1962). Other components of the oral apparatus are also evident. Bordering the gullet i s the posterior end of the guadrulus (g), which consists cf four rows of c i l i a that begin at the anterior end of the buccal overture, pass dorsally over the anterior half of the buccal cavity and then angle over to the l e f t (animal's l e f t ) side and transverse the ' posterior end of the buccal cavity. The l e f t cytostomal l i p i s a specialized area of the g u l l e t which i s important i n seguestering disk-shaped vesicles (d) for recycling cf membrane components (Allen, 1974). The cytopharyngeal ribbons (cr) are bands of 10 - 12 microtubules arranged i n one plane and placed at regular intervals along the l i p . The cytostomal cord (ce) i s a bundle of m i c r o f i b r i l s which l i e s over the ends of the cytopharyngeal ribbons and extends along the f u l l length of the l e f t edge of the cytostome (Allen, 1S74). .The postoral f i b e r s (po) are bundles of hexagonally packed microtubules that extend beyond the buccal cavity and end near the posterior of the c e l l . . A mature trichocyst (t) i s v i s i b l e i n lo n g i t u d i n a l section near the upper right of Plate VI-1. Its body appears clear 140 because of a lack of a f f i n i t y f o r osmium tetroxide (Jurand and Selman, 1969) while the t i p and surrounding sheath are darkly stained._ Plate VI-2 also represents a wild-type c e l l but t h i s section has been cut tangential to the c e l l surface. The c o r t i c a l units are c l e a r l y v i s i b l e , each with i t s basal body (bb) surrounded by an alveolus (a) on either side, and parasomal sac (p).,The kinetodesmal f i b e r s (k) are joined to the basal bodies and run a n t e r i o r l y from the basal body to the same side as the parasomal sac. The trichocyst t i p s (tt) are seen in cross section and are situated between the basal bodies of adjacent c o r t i c a l units within a kinety (a row of basal bodies) . The i n f r a c i l l i a r y l a t t i c e (if) , a network of fine f i b r i l s (Hufnagel, 1969), i s also c l e a r l y v i s i b l e as are several mitochondria (m) with t h e i r tubular c r i s t a e . The inf r a c i l l i a r y l a t t i c e ( i f ) , basal bodies (bb) and kinetodesmal fibers (k) of a drnj c e l l in a section cut tangentially to the c e l l surface (Plate VI-3a) are s i m i l a r i n appearance to wild-type c e l l s . However, the mitochondria (m) are very a t y p i c a l with i n t e r n a l areas that have l o s t t h e i r ordered structure and have abnormal portions of membrane (arrows). The presanse of membrane-bound vesicles (v) in close proximity to mitochondria i s also unusual (Plate VI-3b). In Plate VI-3c there i s an immature trichocyst (t) of a dmj c e l l . It is smaller than a mature trichocyst, i s osmophillic and appears normal i n comparison with ether u l t r a s t r u c t u r a l studies of wild-type c e l l s (Jurand and Selman, 1969, Ehret and McArdle, 1974). The g u l l e t area of a dmj c e l l (Plate VI-3d) 141 also compares well with wild-type c e l l s . The disk-shaped vesic l e s (d) , cytopharyngeal ribbons (cr) and cytostomal chord (cc) are also v i s i b l e and appear normal. The food vacuoles of dmj c e l l s are very d i f f e r e n t from those of wild-type (Plates VI-4a to VI-4d). The food vacuole in Plate VI-4a i s a r e l a t i v e l y young vacuole as evidenced by the smooth membrane and undigested bacteria. There i s an area of the vacuole (arrow) where the membrane seems to have degenerated.. As i n wild-type c e l l s , the food vacuole i s surrounded by membrane-bound vesicl e s (1) which are probably lysosomes. The vacuole i a Plate VI-4b i s s l i g h t l y older (the bac t e r i a l c e l l wall i s separating from the membrane). The membrane of the food vacuole can barely be detected; there i s no c l e a r delineation between vacuolar contents and cytoplasm as in wild-type c e l l s . The contents of the vacuole i n Plate VI-4c have pulled away from the memtrane, which also has d i s c o n t i n u i t i e s (large arrow). There i s a profusion of membranous and vesicular material throughout the vacuole (small arrows) ... The old food vacuole (Plate VI-4d) i s most unusual i n that the vacuolar contents have completely condensed away from the membrane which does not show the highly convoluted extensions normally seen i n older vacuoles of wild-type c e l l s (Plate VI- 1, f vc). The dmj c e l l s i n Plates Vl-5a and VI-5b demonstrate extreme cases of the mutant phenotype. In the c e l l in Plate VI-5a, pieces cf membrane are s t i l l i n t a c t (arrows) and the bacteria are scattered throughout the area. . Plate Vl-5b represents a cross section of a c e l l i n which the i n t e r i o r i s 142 gone and numerous abnormal mitochondria (arrows) are apparent. It should be noted that of the 34 dmj c e l l s that had vacuoles, only four c e l l s had normal vacuoles and a l l c e l l s had some abnormal mitochondria. In 25 wild-type c e l l s that had food vacuoles, three c e l l s had vacuoles with s l i g h t d i s c o n t i n u i t i e s in the vacuolar membranes, one c e l l had a food vacuole with i t s contents s l i g h t l y condensed away from the membrane (the bacteria were undigested) and two out of 30 c e l l s examined had a few abnormal mitochondria. There were no wild-type c e l l s with poorly defined vacuolar membranes as in dmj c e l l s (Plate VI-4b), huge vacuoles (Plate VI-4d) or holes i n the c e l l s (Plates VI-5a and VI-5b). The wild-type and dml c e l l s depicted i n Plates VI-5c and VI-5d respectively, were fed BP before being fixed. These c e l l s were much more d i f f i c u l t to section than c e l l s which had not been fed BP. The food vacuoles and mitochondria of dmj c e l l s that had been fed BP were s i m i l a r to those dml c e l l s that had not been fed BE whereas the wild-type c e l l s appeared normal. D. . Discussion In wild-type c e l l s the state of digestion of bacteria within a food vacuole corresponds to the physical state of the vacuolar membrane (Jurand, 1 9 6 1 ) . In newly formed food vacuoles, the membrane i s smooth, the bacteria undigested (Plate VI-1 , f va).. Normally, as food vacuoles age, a period of swelling of the vacuole i s followed by evaginations of the vacuolar membrane that are accompanied by the appearance of 143 pinocytotic vesicles (Jurand, 1961, Favard and Carasso, 1964). F i n a l l y , the old vacuoles fuse with the cytoproct and exocytosis of the undigested material occurs (Jurand, 1961, Allen and Wolf, 1974)..In dmj c e l l s there are no stages of the digestive cycle i n which the physical state of the vacuolar membrane correlates c l e a r l y with the condition of the bacteria. Instead, the food vacuoles can be grouped according to the severity of t h e i r defects, i n the following manner: 1. Slight abnormalities These vacuoles appear normal except for s l i g h t i r r e g u l a r i t i e s and d i s c o n t i n u i t i e s i n the ultrastructure of the membrane (Plates Vl-4a and VI-4c) . 2. Degeneration of structure The membrane surrounding these food vacuoles changes such that i t i s no longer c l e a r l y distinguishable (Plate VI-4b) . 3.. Large vacuoles In older vacuoles a highly convoluted membrane i s expected but instead the vacuole swells and the membrane remains smooth and extended (Plate VI-4d).. 4.. Total degeneration The vacuolar membranes degenerate to the extent that only small patches are discernible (Plate VI-5a) and ultimately most of the cytoplasm i s destroyed (Plate VI-5b). Mitochondrial abnormalities are associated with a l l four 144 groups (Plates VI-3a and VI-3b). If any of these four types of anomalies are present i n a c e l l which has been fed BP and then examined in the l i g h t microscope, the BP would leak into the cytoplasm giving the appearance of a mass of fused vacuoles (Plate III-1c) . S i m i l a r l y , i f c e l l s are viewed in the lig h t microscope without previously having been fed BP, no abnormalities would be discernible since these defects i n the membrane structure could not be resolved at the l i g h t microscope l e v e l . . Therefore, the u l t r a s t r u c t u r a l r e s u l t s indicate that BP does not cause the expression of the mutant phenotype, i t merely f a c i l i t a t e s i t s detection in the l i g h t microscope. A l l four degrees Df severity of the mutant phenotype are consistent with a defect i n membrane structure. This change in structure could be d i r e c t l y responsible for the observed degeneration of the vacuolar membranes or the change could render the membrane susceptible to the powerful digestive enzymes which are activated following the fusion of primary lysosomes and phagosomes (Muller and Toro, 1962, E l l i o t t and Clemmons, 1966)..The acid hydrolases may destroy portions of the membrane enabling enzymes tc leak into the cytoplasm eventually causing widespread destruction (Plate VI-5b). The swelling of the mutant food vacuoles could be due to an impairment in the rel a t i o n s h i p between the state of the vacuolar contents and the structure of the membrane i t s e l f . The structure of the vacuolar membrane changes with the age of the food vacuole and t h i s i s ref l e c t e d in the d i s t r i b u t i o n of p a r t i c l e s obtained i n the freeze-fracture images of vacuoles of 145 H i caudataj (Allan, 1976) and .1. P i r i f o r m i s (Batz and Wunderlich, 1S76). In wiLd-type c e l l s the in t e r n a l conditions of the vacuoles (eg. _ pH) may themselves mediate the changes i n p a r t i c l e density and membrane topography (Allen, 1976). However, in dmj c e l l s the membranes may be unable to respond, thereby maintaining the smooth configuration, common in young vacuoles, throughout the vacuolar cycle. The mitochondrial abnormalities are also consistent with a defect in membrane composition since mitochondrial as well as vacuolar membranes are synthesized from a c e l l u l a r pool of components (reviewed i n Schatz, 1970, Weidenbach and Thompson, 1974). Mitochondrial abnormalities are also observed afte r s e n s i t i v e stocks of P. a u r e l i a are treated with kappa symbionts. The mitochondria (mainly in the c o r t i c a l region) appear swollen, the mitochondrial matrix becomes more electron translucent and spaces between cr i s t a e appear much wider (Jurand et a l . , 1978)... The symbionts may be affecting osmoregulatory properties (Jurand et a l . , 1978) and these properties might also be disturbed by changes in membrane composition. The outer membrane of the c e l l seems r e l a t i v e l y immune to the effects of the dmj mutation although occasionally double membrane blebs (Plate VI-5b) or r a r e l y , cytoplasmic separations occur which are similar to those described by Sibley and Hanson (1974) but not as severe as those induced by DMSO (Plates V-1c and V-1d). The c i l i a r y membranes and p e l l i c l e of T..pyriformis are Bore r e s i s i t a n t than the microsomal membranes to changes in the l i p i d composition that are induced by changes 146 in temperature or diet (Martin et a l . , 1976, Fukushima et a l . , 1976). In Paramecium , the phospholipid composition of the c i l i a r y membranes i s very d i f f e r e n t from the rest of the c e l l (Andrews and Nelson, 1979, Rhoads and Kaneshiro, 1979) and i n Tetrahymena the phospholipid and f a t t y acid composition as well as the f l u i d i t y of the c i l i a r y and p e l l i c l e membranes are very d i f f e r e n t from the microsomal and mitochondrial membranes (Nozawa and Thompson, 1971, Martin et a l . , 1976, Martin and Thompson, 1978). . The major differences are the high concentrations, in the c i l i a r y membranes, of phosphonolipids (phospholipids that contain an ether linkage rather than an ester linkage between the phosphorous atom and the carbon of the nitrogenous base, fiosenberg, 1973), polyunsaturated fa t t y acids and in Tetrahymena , the s t e r o l - l i k e pentacyclic t r i t e r p e n o i d , tetrahymenol. . With the exception of tetrahymenol, the phospholipid composition of the outer membranes of Paramecium i s very s i m i l a r to that of Tetrahymena. (Andrews and Nelson, 1979, Ehoals and Kaneshiro, 1S79). I t i s possible that tetrahymenol i s instrumental i n maintaining optimal membrane properties (Ferguson et a l . , 1975, Ccnner and Landry, 1976) since other sterols, such as cholesterol, change the membrane f l u i d i t y and the surface l a b e l i n g patterns of e x t r i n s i c and i n t r i n s i c proteins (Borochov et a l . , 1979, Davis et a l . , 1980). Although no sterols or s t e r o l - l i k e substances are present i n the p e l l i c u l a r membranes of Paramecium , there i s an abundance of l i p i d s with stable structures (phosphonolipids and sphingolipids, Rhoads and Kaneshiro, 1979) which may protect these membranes somewhat from the e f f e c t s of changes in 147 membrane composition induced by the dm 1 mutation.. Thus, i t appears that the phenctypic e f f e c t s of the dm 1 mutation are manifested p r i m a r i l y i n the food vacuole and m i t o c h o n d r i a l membranes. .. tf hen the h y d r c l y t i c enzymes enter the cytoplasm, systematic d e s t r u c t i o n of the c e l l o ccurs.. 148 P l a t e V I - 1 . Food v a c u o l e s o f a w i l d - t y p e c e l l . A t r a n s v e r s e s e c t i o n o f t h e c e l l n e a r t h e p o s t e r i o r p a r t o f t h e g u l l e t (g) . T h e c i l i a l i n i n g t h e g u l l e t b e l o n g t o t h e g u a d r u l u s ( g ) . A l o n g t h e l e f t ( a n i m a l ' s l e f t ) c y t o s t o m a l l i p o f t h e g u l l e t a r e t h e t h e d i s k - s h a p e d v e s i c l e s (d) , t h e b a n d s o f m i c r o t u b u l e s known as t h e c y t o p h a r y n g e a l r i b b o n s ( c r ) a n d c y t o s t o m a l c h o r d ( c c ) . The p o s t o r a l f i b e r s (po) a r e b u n d l e s o f h e x a g o n a l l y p a c k e d m i c r o t u b u l e s . T h r e e f o o d v a c u o l e s ( f v a , f v b , f v c ) o f i n c r e a s i n g a g e and s u r r o u n d e d by m e m b r a n e - b o u n d v e s i c l e s as w e l l as a l o n g i t u d i n a l s e c t i o n o f a m a t u r e t r i c h o c y s t ( t ) a r e a l s o v i s i b l e . 150 Plate VI -2 ..Wild-type c e l l tangential to the surface. Each c o r t i c a l unit i s centred around a basal body (bb) with i t s kinetodesmal fiber (k) running a n t e r i o r l y , an alveolus (a) on either side and parasomal sac (p). The i n f r a c i l l i a r y l a t t i c e (if) consists cf a network of fine f i b r i l s . The trichocyst t i p s (tt) are seen in cross section between basal bodies. Mitochondria (m) with their tubular c r i s t a e are also v i s i b l e . 152 Plate VI-3. Cortex and gul l e t regions cf dmj c e l l s . a. .A section tangential to the c e l l surface. The mitochondra (m) are abnormal (arrows) but the basal bodies (bb) , kinetodesmal fibers (k) and i n f r a c i l l i a r y l a t t i c e (if) appear normal. . b. Higher magnification of an abnormal mitochondrion (m) and a membrane-bound vesicle (v) . . c. Longitudinal section through an immature trichocyst ( t ) . The sheath (ts) surrounds the t i p ( t t ) . d. .Transverse section through the gul l e t (g). The cytopharyngeal ribbons (cr) , cytostcmal chord (cc) and disk-shaped vesi c l e s (d) are s i m i l a r in structure to wild-type c e l l s . . 153 154 Plate VI-4. Food vacuoles of dmj c e l l s . a. A section cf a young food vacuole with an area of membrane degeneration (arrow) and surrounded ty membrane-bound vesicles ( 1 ) . The bacteria (b) are well-preserved and are i n the early stages of digestion. b. A section of an older vacuole (the bac t e r i a l walls are separating from the cytoplasm (arrows). There i s no d i s t i n c t membrane surrounding the vacuole.. c. A section of an older vacuole. The membrane has areas of degeneration (large arrows) and the vacuolar contents are moving away from the membrane. There are many membranous vesicles and fragments (small arrcws). d. A section cf an old food vacuole in which the vacuolar contents have completely condensed away from the membrane. A l l that remains cf most of the bacteria (b) are membrane ghosts. 155 156 Plate VI-5. Extreme cases of the dmj phenotype. a. A section cf a dmj c e l l with a large vacuole with a degenerated membrane. Parts of the membrane are s t i l l i ntact (arrows). . b. A cross section of a dmj c e l l in which the i n t e r i o r has disintegrated. There are many abnormal mitochondria (arrows) and small c o r t i c a l blebs (cb) along the cuter c e l l surface. c. A section of a wild-type c e l l tangential to the c e l l surface. . This c e l l had been fed blue watercolor (BP) prior to f i x a t i o n . Mitochondria (m) , trichocyst t i p s (tt) and trichocysts (t) are v i s i b l e . d..A section of a dmi c e l l that had been fed BP prior to fixation..The mitochondria (m) are abnormal and the food vacuole membrane has degenerated except i n a few areas (a rrows) . . 158 CHAPTER VII FATTY ACID COMPOSITION Of WILD-TYPE AND dmi. CELLS A. Introduction The phospholipids and fatty acids of Tetrahymena and, to a lesser extent, Paramecium , have heen characterized (eg. Fukushima et a l . , 1976, Conner and Stewart, 1976, Andrews and Nelson, 1979, Kaneshiro et a l . , 1979, Rhoads and Kaneshiro, 1979). In addition to phospholipids, Tetrah ymena and Paramecium also contain phosphonclipids, such as 2-aminoethylphosphonolipid (AEPL), in which the phosphoryl base i s replaced, for example, by 2-amincethylphosphonic acid, and the ester linkage between the f a t t y acids and the glycerol backbone i s replaced by an ether linkage at the carbon 1 posi t i o n (Mangnall and Setz, 1973, Rosenberg, 1973).. These organisms also have sphingophospholipids (Mangnall and Getz, 1973, Sugita et a l . , 1979). Since ether linkages are less susceptible tc hydrolytic and phospholytic attack, they provide a degree of s t a b i l i t y and protection to the membrane (Mangnall and Getz, 1973, Rosenberg, 1973, Andrews and Nelson, 1979, Rhoads and Kaneshiro, 1979)., Membrane composition can be modified by many factors, such as diet (Wisnieski et a l . , 1973, Ferguson et a l . , 1975, Dewailly et a l . , 1977, C h r i s t i ansson and Weislander, 1980),. age of the c e l l s (Kaneshiro et a l . , 1979) and concentration of oxygen and carbon dioxide . (Erwin, 1973). Changes i n temperature 159 also have many effects on membranes and membrane constituents and these changes are reguired for maintaining the correct membrane f l u i d i t y necessary for normal c e l l u l a r functions (Chapman, 1973). In Tetrahymena , a decrease i n temperature results in an increase i n unsaturated f a t t y acids (eg. Fukushima et a l . , 1976), an increase i n the a c t i v i t y of the enzyme palmitcyl CoA desaturase (Martin et a l . , 1976) and an increase in the aggregation of p a r t i c l e s as determined by freeze-fracture technigues (Speth and Wunderlich, 1973). In Paramecium as well as Tetrahymena , polyunsaturated f a t t y acids are synthesized via the w6 pathway (Erwin, 1973, Kaneshiro et. a l . , 1979, see Figure 7-1) . Palmitic acid (16:0) i s converted to l i n o l e i c acid (18:2) via, palmitoleic acid (16:1) and o l e i c acid (18:1) and polyunsaturated f a t t y acids are synthesized from l i n o l e i c acid via chain elongation and further desaturation where a l l subseguent double bonds are introduced toward the carboxyl end of the molecule (Erwin, 1973). The dmi mutant carr i e s a temperature-sensitive mutation r e s u l t i n g in abnormal membranes at 34. 5°C. Since c e l l s normally react to temperature changes by a l t e r i n g t h e i r f a t t y acid composition (Chapman, 1973), a study of the e f f e c t of the r e s t r i c t i v e temperature on the f a t t y acid composition i n wild-type and dm 1 c e l l s , as compared with the composition at the permissive temperature, may provide further information regarding the disruption of dmj membranes.. 160 B. . M a t e r i a l s and Methods 1. E x t r a c t i o n of l i p i d s (see Appendix IV f o r complete procedure) W i l d - t y p e and dm 1 c e l l s were i n c u b a t e d a t 27°C or 34.5°C f o r 6 h o u r s . A p p r o x i m a t e l y 10 6 c e l l s per sample (as de t e r m i n e d by s e r i a l d i l u t i o n o f 1 ml of c e l l s i n c u l t u r e f l u i d ) were c e n t r i f u g e d (100xg, 5 minutes) and the f i n a l volume was reduced t o 2.4 ml. L i p i d s were e x t r a c t e d i n c h l o r o f o r m / m e t h a n o l / w a t e r ( a l l s o l v e n t s were reagent grade) a c c o r d i n g t o B l i g l i and Dyer ( 1959). The f i n a l c h l o r o f o r m phase was washed t w i c e w i t h methanol ( F o l c h e t a l . , 1957) and f i l t e r e d t h r o u g h Whatman f i l t e r paper (#1). The l i p i d e x t r a c t s were s t o r e d i n c h l o r o f o r m at -5°C..To compare-the f a t t y a c i d c o m p o s i t i o n of E n t e r o b a c t e r aero gen es w i t h t h a t of P. . t e t r a u r e l i a , 1. 5 1 of b a c t e r i a were grown t o s t a t i o n a r y phase (10 9 c e l l s per ml) and h a l f t h e c u l t u r e was i n c u b a t e d a t 27°C f o r 6 hours and the o t h e r h a l f a t 3 4 . 5 ° C. The c e l l s were c e n t r i f u g e d and t h e r e s u l t i n g p e l l e t was t r e a t e d as d e s c r i b e d above.. 2» . T h i n l a y e r chromatography (TIC) T h i s procedure s e p a r a t e d t h e p h o s p h o l i p i d s from t h e remainder of the l i p i d s which f a c i l a t e d t h e d e t e r m i n a t i o n o f t h e f a t t y a c i d c o m p o s i t i o n of the i n d i v i d u a l p h o s p h o l i p i d s . F o r the d e t e r m i n a t i o n of the f a t t y a c i d c o m p o s i t i o n of whole c e l l s , t h i s s t e p was o m i t t e d . . The l i p i d e x t r a c t s were e v a p o r a t e d under n i t r o g e n and 161 redissolved i n 200 ul chloroform. Half of each sample was applied to s i l i c a gel G TLC plates (the remaining half was used to determine the fatty acid composition of whole c e l l s ) and the phospholipids were separated using the solvent system chloroform/acetic acid/ methanol/water (75:25:5:2.2 by volume, Nozawa and Thompson, 1971).. The standards, pig l i v e r phosphatidylethanolamine (PE, 10 mg/ml chloroform) and phosphatidylcholine.(PC, 20 mg/ml chloroform) (Serdary Research Laboratory) were kindly supplied by Dr. D.E. Vance, Department of Biochemistry, University of B r i t i s h Columbia. The separated phospholipids were i d e n t i f i e d by staining with iodine and areas corresponding to the i n d i v i d u a l phospholipids were scraped from the plate. Phospholipids were removed from the s i l i c a gel by washing twice with chloroform/ acetcne/methanol/water (6:8:2:2, Andrews and Nelson, 1979). The chloroform layer, which contained the phospholipids, was evaporated under nitrogen and the samples were stored at -5°C. As a c o n t r o l , an area of s i l i c a gel with no phospholipid was scraped from the plate and carried through the entire procedure. 3. Preparation of gl y c e r y l ethers Because paramecia contain phosphonolipids, i t was necessary to convert the phosphonolipids to gl y c e r y l ethers before they could be methylated. This was accomplished by adding 2 ml of acetic acid/acid anhydride (3:2, Thompson and Kapoulos, 1969) and heating in t i g h t l y capped tubes at 90°C for 4 hours.. The tubes were cooled and 1 ml of 6N potassium 162 hydroxide i n 95% ethanol was slowly added and the tubes were t i g h t l y capped. The samples were then heated at 90°C for 2 hours, cooled and extracted with ether twice. The ether layers were combined, washed twice with water, evaporated under nitrogen, and stored at room temperature.. 4. Met hylaticn a. fatty acids The methyl esters of f a t t y acids were prepared by adding 1 ml of methanol i n 1N hydrogen chloride gas to each sample. The tubes were t i g h t l y capped and incubated at 90°C overnight.^ After c o d i n g , the methanol was evaporated under nitrogen and the samples were stored at room temperature. b. g l y c e r y l ethers After the methyl esters of fatty acids were prepared, the same samples were treated i n the following manner to prepare the trimethylsilane esters of g l y c e r y l ethers. .150 ul of pyridine/hexamethyldisili zane/trichlorotrimethylsilane (10:4:2, Vance and Sweeley, 1967) was added to each sample f o r 15 minutes at room temperature.. The samples were evaporated under nitrogen and redissolved i n 20-100 ul of heptane and stored at room temperature. 1.63 5. Gas l i q u i d chromatography (GLC) Q u a n t i t a t i v e a n a l y s i s of m e t h y l e s t e r s was performed on a H e w l e t t P a c k a r d 761GA High E f f i c i e n c y gas chromatograph w i t h a 6 - f o o t r U-shaped column ( i n s i d e diameter = 2 mm) packed w i t h 15% e t h y l e n e g l y c o l s u c c i n a t e ( A p p l i e d S c i e n c e L a b o r a t o r y , I n c . , H1-EFF2B). The flame t e m p e r a t u r e was 200°C and t h e column temp e r a t u r e was i n c r e a s e d , u s i n g a 7600 m u l t i l e v e l programmer, from 155°C t o 185°C a t a r a t e o f 2°C per minute b e g i n n i n g 6 minutes a f t e r i n j e c t i o n . . T h e a t t e n u a t i o n was 4 o r 8 x 10 2 and t h e c h a r t speed was 0 . 5 i n c h e s / s e c o n d . The s t a n d a r d s * methyl e s t e r s o f t h e f a t t y a c i d s , p a l m i t i c ( 1 6:0), s t e a r i c (18:0), e i c o s a n o i c ( 2 0 : 0 ) , d o c o s a n o i c ( 2 2 : 0 ) , and t e t r a c o s a n o i c (24:0) were k i n d l y s u p l i e d by Dr. .D.E. Vance and p a l m i t o l e i c ( 1 6:1), o l e i c (18:1), a i c o s e n o i c ( 2 0 : 1 ) , d o c o s e n o i c (22:1), d o c o s a d i e n o i c (20:2) and a r a c h i d o n i c (20:4) were purc h a s e d from Sigma..The mi x t u r e of r a t l i v e r m e t h y l e s t e r s was a l s o p r o v i d e d by Dr. D.E. Vance. The f i r s t number of the s h o r t h a n d n o t a t i o n used t h r o u g h o u t (eg. .16:0) i n d i c a t e s t h e number of hydrocarbons i n the a c y l c h a i n and the number t o the r i g h t of the c o l o n i n d i c a t e s the number of double bonds i n t h a t a c y l c h a i n . 0.5 u l of sample i n heptane p l u s 2 u l of heptane were i n j e c t e d and the p e r c e n t c o m p o s i t i o n c f each f a t t y a c i d was d e t e r m i n e d by X e r o x i n g t h e f a t t y a c i d p r o f i l e of each sample, c u t t i n g out the peaks and weighing then* F a t t y a c i d s t h a t were l e s s t h a n 1% of the t o t a l weight were d i s r e g a r d e d ( w i t h t h e e x c e p t i o n of 16:1 which was always i n c l u d e d ) . I n d i v i d u a l f a t t y a c i d s were i d e n t i f i e d by comparing the e l u t i o n t i m e s w i t h s t a n d a r d s and i n some.cases s t a n d a r d s were added t o t h e samples 164 t o a c t as i n t e r n a l s t a n d a r d s . A l l but the l a s t ( l a t e s t e l u t i n g ) two f a t t y a c i d s c o u l d be i d e n t i f i e d i n t h i s manner and the s e were t e n t a t i v e l y i d e n t i f i e d on the b a s i s of e x t r a p o l a t i o n as w e l l as the l i t e r a t u r e v a l u e s f o r the same s t a t i o n a r y phase (Jamieson, 1970). The f a t t y a c i d c o m p o s i t i o n of whole c e l l s was a n a l y z e d f o r thr e e s e p a r a t e e x p e r i m e n t s and t h e f a t t y a c i d c o m p o s i t i o n of the p h o s p h o l i p i d s was determined f o r two of t h o s e e x p e r i m e n t s . C. R e s u l t s 1. F a t t y a c i d c o m p o s i t i o n of whole c e l l s The. GLC r e s u l t s f o r two r e p r e s e n t i t i v e samples of methyl e s t e r s of f a t t y a c i d s have been i l l u s t r a t e d i n F i g u r e s 7-2 and 7-3. The t r i m e t h y l s i l a n e d e r i v a t i v e s were not r e s o l v e d a t t h e s e t e m p e r a t u r e s s i n c e t h e y were more v o l a t i l e and were r e s o l v e d a t l o w e r t e m p e r a t u r e s ( i e . . L e s s than 155°C)..The b a s e - l i n e s l o p e d upward due t o the i n c r e a s e i n column t e m p e r a t u r e but t h e a s s y m e t r i c peaks d i d not p r e s e n t problems i n the a n a l y s i s because the c o m p o s i t i o n was de t e r m i n e d by t h e r e l a t i v e w e i g h t s of the peaks. The means and s t a n d a r d d e v i a t i o n s of t h e p e r c e n t of each f a t t y a c i d of w i l d - t y p e and dml c e l l s from t h r e e s e p a r a t e experiments at 27°C and 34.5°C have been p r e s e n t e d i n T a b l e 7-1..The major f a t t y a c i d s i n both w i l d - t y p e and dml c e l l s at both t e m p e r a t u r e s were: p a l m i t i c (16:0), s t e a r i c (18:0), o l e i c (18:1), l i n o l e i c (18:2), l i n o l e n i c (18:3), a r a c h i d o n i c (20:4), d o c o s o t e t r a e n o i c (22:4), t e t r a c o s a d i e n o i c 165 (24:2), and tetracosatetraenoic (24:4). The methyl esters of acy l chains that were l e s s than 16 carbons long could not be p o s i t i v e l y i d e n t i f i e d because they eluted very cl o s e l y together. Therefore, they were considered as a unit and omitted from most calculations.. The percent composition of the f a t t y acids of the bacteria was not determined but there were obvious differences i n the bacteria p a r t i c u l a r l y the lack of polyunsaturated fatty acids and acyl chains greater than 20 carbons long. The control sample with s i l i c a gel G alone showed no discernible peaks. . The standard deviations were guite large, p a r t i c u l a r l y in the mutant samples, but t h i s range of v a r i a b i l i t y i s common (ec[. Ohxi et a l . , 1979) . When the re s u l t s of the three experiments were examined separately (see Appendix IV), i t became apparent that experiments one and three yielded very similar data and experiment two was guite d i f f e r e n t : there were elevated amounts of 22:4 and 24:4 and decreased amounts of 18:1 in experiment two. . The change i n composition of the major f a t t y acids i n whole c e l l s was calculated by subtracting the mean percent composition at 27°C from the mean percent composition at 34.5°C (Table 7-2). The major differences between wild-type and dm_1 c e l l s occurred i n 16:0 and 24:4. The large p o s i t i v e value f o r 16:0 i n wild-type c e l l s indicated that the percent of palmitic acid increased with increased temperature whereas i t decreased i n mutant c e l l s . The large negative value f o r 24:4 in wild-type c e l l s indicated a decrease in t h i s long-chain, highly unsaturated f a t t y acid in wild-type c e l l s and an eguivalent 166 increase i n mutant c e i l s . . This trend was consistent i n a l l three experiments (Appendix IV) regardless of the differences i n f a t t y acid composition among the three experiments. The majority of fatty acids were greater than 20 carbons (Table 7-3) and in wild-type c e l l s the trend was to decrease the chain length af t e r an increase in temperature (contributed to, i n part, by the increase in 16:0). This trend was reversed i n dm 1 c e l l s where the percent of acyl chains with more than 20 carbons increased with an increase in temperature. The t o t a l content of saturated f a t t y acids in wild-type c e l l s was similar for each temperature i n a l l three experiments indicating that a constant saturated to unsaturated fatty acid r a t i o for a given temperature may be maintained i n wild-type c e l l s . .The percent of unsaturated fatty acids decreased in wild-»type c e l l s at 34.5°C but underwent no change in dmj c e l l s . The unsaturation index (U.I.) expresses the number cf double bonds per 100 acyl groups and i s calculated as follows: the sum of: U(% composition cf each unsaturated f a t t y acid) x (number of double bonds)/100 The U.I. decreased i n wild-type c e l l s with an increase i n temperature tut increased in dmj cells..The U.I. i n experiment two at 27°C was higher in both wild-type and dmj c e l l s but i n wild-type c e l l s there was a decrease at 34.5°C which did not occur i n dm 1 c e l l s . 167 2. Fatty, acid composition of phospholipids The means of the percent composition of the f a t t y acids of the phospholipids, PC, PE and AEPL, of wild-type and dmj c e l l s have been presented i n Tables 7-4 tc 7-6. The major f a t t y acids of a l l three phospholipids were similar to the major fatty acids of whole c e l l s i._e. .. 16:0, 18:0 18:1, 18:2, 18:3, 20:4, 22:4, 24:2, and 24:4.. The differences i n the percent composition between 34 .5°C and 27°C were determined as previously described and have been presented in Table 7-7. .In wild-type c e l l s the percent of 16:0 increased substantially with an increase in temperature, p a r t i c u l a r l y i n PE, but i n dmj c e l l s the increase was very s l i g h t . The proportion of 18:0 also increased i n PC and AEPL i n wild-type but not i n dmj c e l l s . The percent compositions of 18:1 and 20:4 decreased with an increase i n temperature i n wild-type and dm 1 c e l l s . The greatest difference between wild-type and mutant c e l l s i n response to the temperature increase occurred in 24:4; i n wild-type c e l l s the percent composition i n EC and PE decreased with increased temperature but i n dmj c e l l s i t increased su b s t a n t i a l l y in PC, PE and AEPL. .The greatest changes i n the composition of wild-type c e l l s occurred i n PC and PE while the composition of AEPL remained more constant.. In mutant c e l l s , the composition of AEPL as well as PC and PE varied considerably with the temperature change. In wild-type c e l l s an increase in temperature resulted i n a decrease i r the proportion of acyl chains greater than 20 carbons long i n PC, PE and AEPL (Tables 7-8 to 7-10)..This was s i m i l a r to the changes observed with whole c e l l f a t t y acids 168 (Table 7-3)» In dmi c e l l s there was an increase, with an increase in temperature, i n the percent of fatty acids i n t h i s category. .In wild-type c e l l s there was an increase in the l e v e l of saturated fatty acids at 34.5°C, p a r t i c u l a r l y i n PC and PE, and to a lesser extent in AEE1. In dmi c e l l s the l e v e l of saturated f a t t y acids decreased with an increase i n temperature.. The U.I..deereased i n wild-type c e l l s with an increase in tenperature; the greatest change was observed i n PE. . In dmi. c e l l s the U. I. . increased at 34.5°C i n PC, PE and AEPL. . D. Discussion The observed f a t t y acid composition of wild-type c e l l s at 27°C agrees, in part, with r e s u l t s obtained by Kaneshiro et a l . ( 1979) -for aurelia grown axenically at 25°C. The major difference i s the presence, i n t h i s study, of substantial amounts of fatty acids containing 24 carbons.. With the exception of 24:2 and 24:4, the major f a t t y acid species are the same in both studies..The fatty acid, 24:4, i s also present i n elevated amounts i n the second experiment (see Results) and may be a r e f l e c t i o n of variable growth conditions or the age of the paramecia since the degree of unsaturation and the length of the fatty acyl chains increases with clonal age (Kaneshiro et a l . , 1979). The contribution of the b a c t e r i a l fatty acids to the t o t a l composition was not calculated and changes i n the b a c t e r i a l composition would be reflected i n the composition of Paramecium membranes. . This may also contribute to the re s u l t s 169 obtained in experiment two. Since i n each experiment wild-type and dml. c e l l s at both temperatures were fed bacteria from the same batch, variations due to differences i n the b a c t e r i a l f a t t y acid composition within an experiment were minimized. The changes i n f a t t y acid composition that occurred following an increase in temperature can be summarized as follows: 1. wild-type c e l l s a. increase ia saturated fatty acids, p a r t i c u l a r l y 16:0 in whole c e l l s and i n PE b. increase i n fatty acids with 16-20 carbons and decrease i n f a t t y acids with more than 20 carbons c. .decrease i n highly unsaturated fatty acids, p a r t i c u l a r l y 20:4 and 24:4 in whcle c e l l s and i n PC and PE d. .decrease in U.I.,, p a r t i c u l a r l y i n PE 2. dml c e l l s a. s l i g h t increase or decrease in saturated f a t t y acids b. _decrease in fatty acids with 16-20 carbons and increase in f a t t y acids with more than 20 carbons c. .increase ia the highly unsaturated fatty acid, 24:4 d. increase in U.I. . A l l changes occurred in PC, PE and AEPL.. A l l four changes can be attributed to the changes which occur in 16:0 and 24:4. An increase in 16:0 i n wild-type c e l l s at 34.5°C increases the percent cf saturated fatty acids and at the same time the percent of acyl chains 16-20 carbons long. A decrease in 24:4 decreases the l e v e l cf unsaturated f a t t y acids and thereby the U.I._as well as the percent of acyl chains more 170 t h a n 20 ca r b o n s l o n g . .In dmj c e l l s the o p p o s i t e o c c u r s i ^ e . a d e c r e a s e , or s l i g h t i n c r e a s e , i n 16:0 and a d e f i n i t e i n c r e a s e i n 24:4 which c o m p l e t e l y r e v e r s e t h e r e s u l t s . . In Tetrahymena t h e r e i s an i n c r e a s e i n t h e amount o f d e s a t u r a t i o n o f f a t t y a c i d s when t h e temperature i s l o w e r e d from 35°C t o 15°C (Conner and S t e w a r t , 1976), 39°C t o 15°C ( M a r t i n et a l . , 1 976) o r 39.5°C to 15°C (Fukushima e t a l . , 1976, Watanabe e t a 1. , 1979). T h i s i s comparable t o t h e ob s e r v e d e f f e c t s of i n c r e a s e d t e m p e r a t u r e on the f a t t y a c i d c o m p o s i t i o n of w i l d - t y p e c e l l s i n t h i s s t u d y a l t h o u g h t h e presence o f f a t t y a c i d s l o n g e r t h a n 20 carbons has not been r e p o r t e d f o r Tetrahymena In Tetrahymena , the i n c r e a s e i n u n s a t u r a t e d f a t t y a c i d s ( p r i m a r i l y 18:2 and 18:3) i s due t o the i n c r e a s e d a c t i o n of the enzyme p a l m i t c y l CoA d e s a t u r a s e , which c o n v e r t s p a l m i t i c a c i d (16:0) t o p a l m i t o l e i c a c i d (16:1). T h i s enhanced d e s a t u r a s e a c t i v i t y may have o c c u r r e d as a r e s u l t of i n c r e a s e d amounts of enzyme p r e s e n t (Nozawa and K a s a i , 1978, Nozawa e t a l . , 1979, K a s a i and Nozawa, 1980, Fukushima e t a l . , 1979) o r t h e a c t i v a t i o n of e x i s t i n g enzyme i n response t o a change i n the membrane f l u i d i t y brought about by decreased t e m p e r a t u r e ( M a r t i n e t a l . , 1976, K a s a i e t a l . , 1976, M a r t i n and Thompson, 1978, S k r i v e r and Thompson, 1S79).. A l t h o u g h i n t h i s s t u d y no attempt has been made t o g u a n t i f y t h e chaages i n t h e amounts of t h e p h o s p h o l i p i d s t h e m s e l v e s ( M a r t i n e t a l . , 1976 have shown t h a t changes i n membrane f l u i d i t y i n response t o temperature a re i n f l u e n c e d t o a g r e a t e r e x t e n t by f a t t y a c i d c o m p o s i t i o n than by head group 17 1 composition), i t appears that the fatty acids of PC and PE i n wild-type c e l l s undergo greater changes than do the f a t t y acids of AEPL i n response to temperature. Watanabe et a l . (1979) suggest that PE plays a p r i n c i p a l r o l e as acceptor of a c y l chains during temperature acclimation in Tetrahymena and Nozawa a l . ( 1 979) have found that PC and PE of Tetrahymena change the most after treatment with phenethyl alcohol, which increases the f l u i d i t y of membranes. However, Ohki et a l . . (1979) have found that the f l u i d i t y of AEPL i n T . p y r i f o r m i s increases rapidly during the f i r s t ten hours after a decrease i n temperature whereas the f l u i d i t y of PE and PC decreases gradually for the f i r s t 24 hours suggesting that AEPL i s important in the i n i t i a l thermal response. The results obtained with wild-type c e l l s i n t h i s study indicate that the f a t t y acid composition of PC and PE undergoes a greater change than does that of AEPL..The 0.1. of the fatty acids of PE decreases the most with an increase in temperature. In dml c e l l s the f a t t y acids of a l l the phospholipids vary with an increase i n temperature and the U.I. increases i n a l l the phospholipids. Mutants of other eukaryotes have been i s o l a t e d which are def i c i e n t i n seme aspect of l i p i d metabolism, eg,. yeast (Keith et a l . , 1973), Neurospora (Friedman, 1977) and Tetrahymena ( H i l l , 1980). These are mainly fa t t y acid auxotrophs that have been used to investigate the e f f e c t s of f a t t y acid supplements i n the culture medium. The pawn mutants of P L t e t r a u r e l i a , , which have defects in the.gated calcium channel of the membrane (Kung, 1975), show no differences i n their fatty acid content 172 as compared with wild-type c e l l s (Kaneshiro et a 1> , 1979) although i t i s intere s t i n g that the v i a b i l i t y of pawn c e l l s i s severly reduced in axenic medium. One of the ether behavioral mutants isolated by Kung (1971), fast (fa97) , has two extra minor phospholipids (2%) in d e c i l i a t e d bodies but pawn and paranoic c e l l s show no differences in phospholipid composition when compared with wild-type c e l l s (Andrews and Nelson, 1979). Another mutant of P. . t e t r a u r e l i a , nd9, i s a temperature-se n s i t i v e mutant with normal trichocyst discharge at 18°C but non-discharge at 27°C (Beisson et a l . , 1976). At 18°C the nd9 gene product i s abnormal but the membrane f l u i d i t y at that temperature enables the protein tc i n t e r a c t with either the tr i c h o c y s t membrane or plasma membrane resulting i n the normal discharge of trichocysts. _At 27°C no interaction can take place and i f fatty acid synthesis i s inhibited during the s h i f t down to the permissive temperature normal trichocyst discharge i s not restored (Beisson et a l . , 1980). Thus, although membrane composition i s not responsible for the nd9 phenotype, changes i n f a t t y acid composition at the permissive temperature can compensate for an abnormal protein. The mutant, dmi., does not appear to be an auxotroph; there i s no i n d i c a t i o n of a block in the synthetic pathway that would result in the accumulation of one species of fatty acid. However, i t does appear that the mechanism f o r control of membrane f a t t y acid composition i s impaired. I f the cont r o l l i n g step i n the pathway for unsaturated f a t t y acid synthesis i s the same as in Tetrah ymena , i . e. the palmitoyl Cofl desaturase conversion of palmitic acid to 173 palmitoleic acid (Martin et a l . , 1576), the l e v e l of palmitic acid would increase i f the enzyme was inhi b i t e d and decrease i f the enzyme was activated. In wild-type c e l l s the percent of palmitic acid increases with an increase i n temperature, consistent with the i n h i b i t i o n of the desaturase enzyme either d i r e c t l y by the increase i n temperature i t s e l f or i n d i r e c t l y by the change i n f l u i d i t y r e s u l t i n g from the increase i n temperature. In dml c e l l s the amount of palmitic acid decreases with increasing temperature in d i c a t i n g the conversion of palmitic to paLmitoleic acid has not been i n h i b i t e d . Tetracosatetraenoic acid (24:4) may be an end product of the pathway and i t s depletion i n wild-type c e l l s and accumulation in dml c e l l s further attest to decreased a c t i v i t y of the enzyme in wild-type but not dmj c e l l s . Thus the mutaat, dml, may represent a genetic lesion involving the control of desaturase a c t i v i t y and as such would ba an extremely valuable t o o l for i l l u c i d a t i n g the factors that influence the mechanism of action of t h i s enzyme.. 174 Table 7-1. Fatty acid composition cf whole c e l l s Percent Composition (1) Fatty wild-type dm1 wild-type dm1 Ac id 27°C 27°C 34.5°C 34. 5°C <TeT 0 10. 54 (3.43) t. 25 7.45 1. 48 *16: 0 16.92 (6. 54) 15.29 (3. 06 ) 14 .80 (7. 76) 16: 1 2.40 (0.72) 4. 31 (2. 55) 3.89(2. 05) 3. 48 (2.86) 17: 1 (3) 1.24 1. 54 1.01 2. 63 *1 8: 0 2. 98 (1.21) 2. 53 (1 . 66) 3.45 (2.33) 3. 47 (2. 78) *1 8: 1 15. 98 (7.11) 14. 22 (6. 3 4) 14.99(2.98) 13. 85 (6.40) * 18: 2 4. 33 (1.37) 3.69 (1. 27) 4.19 (0. 71) 3. 81 (1. 36) *18: 3(4) 6. 96 (1.99) 6. 01 (2. 41) 8. 6 9 (1. 95 ) 4. 29 (2. 51) 20: 0 3. 17 (1.50) 3. 96 (0. 54) 2. 16(0. 59) 3. 20 (1.59) 20: 2 3.08 (1.32) 2. 86 2.58 1 . 78 (0.50) *20: 4 5. 13 (3. 18) 2.99 (1. 67) 3.66 (0. 53) 3. 27 22: 1 3.93 2. 28 3.43 (2. 67) 1. 81 22: 2 1.86 2. 35 1.82 1. 50 *22: 4 1 1. 13 (6.61) 12.40 (6. 50) 9.65 (3. 36) 10. 67 (6.38) 22.6 1.32 2. 27 2.27(0.97) 3. 41 24: 0 2.96 2. 07 1.88(1. 15) 3. 29 (1.51) 24: 1 1. 82 1.96(0. 58) 2.01 1. 94 *24: 2 4.03 (1.97) 4.11(1. 50) 4.69 4. 67 (2.99) *24: 4 18.56 (10.02) 14.99(11 .69) 12.31 (7.31) 21. 46 (15. 20) (1) means and standard deviations f c r values from three experiments, means only where data available from only two experime nts. (2) tr = trace (3) may also include 17:0 (4) also contains 20:1 * major fatty acids Table 7-2. Changes i n whole c e l l f a t t y acid composition Fatty Acid wild-type dmj 16:0 4. 75 -2. 12 18:0 0.47 0.94 18:1 -0. 99 -0. 37 18:2 -0. 14 0. 12 18:3 1. 73 -1.72 20:4 - 1. 47 0. 28 22:2 0. 66 0. 56 22:4 -1. 48 -1.73 24:4 -6. 26 6. 47 (1) calculated by subtracting the m#an composition at 27°C from the mean composition at 34.5°C 176 Table 7-3. D i s t r i b u t i o n of whole c e l l f a t t y a c i d s (1) a c y l wild-type draj wild-type dml chain 27°C 27°C 34.5°C 34.5°C <16 0.00 1. 25 7.41 1.48 16<20 44.53(13.98) 48. 71 (19. 79) 50.85 (10. 15) 45.47 (20 .58) >20 55.47 (13.98) 50.48 (19.25) 44. 21 (13.8 1) 53.71 (21 .71) S (2) 19.89(2.17) 26. 93 (3. 99) 23.89 (4.97) 26. 49 (10 .40) U (3) 80. 12 (2. 17) 72.21 (3. 47) 70.76 (8.27) 72.66 (11 .07) U/S (4) 4.03 2. 68 2.96 2.74 0. I. (5) 2. 15 (0. 51) 1. 87(0. 34) 1.88 (0.47) 2.03 (0. 68) (1) means and standard d e v i a t i o n s f o r values from three., experiments (2) S = sum of s a t u r a t e d f a t t y acids (3) U = sum of unsaturated f a t t y a c i d s (U*S<100% because peaks of mixed composition or l e s s than 16 carbons were not included) (4) U/S = r a t i o of unsaturated to s a t u r a t e d f a t t y a c i d s (5) U.I..= un s a t u r a t i o n index (see E e s u l t s f o r c a l c u l a t i o n ) 177 Table 7-4. Fatty acids of phosphatidylcholine Percent Compo s i t i o n (1) Fa tty wild-type dml wild-type dml Ac id 27°C 27°C 34.5°C 34.5°( <16: 0 5.00 3. 29 5.35 2.20 *1 6: 0 6.66 12. 68 11.60 13.74 16: 1 0.42 0.90 1,28 2.08 17:1(2) 3. 54 3.45 1.65 1 .50 *1 8: 0 5. 93 8. 11 7.55 5. 97 *18: 1 1 1. 24 11. 69 8.65 8.64 *1 8:2 1. 78 3. 86 5.84 2.90 * 18: 3 1.01 1. 39 4.85 1.23 20: 0 2. 04 2. 12 2.0 3 t r (3) 20: 1 3.83 1. 69 7.19 6.00 *20: 4 17. 48 13. 69 4.66 5.54 22: 1 1. 59 tr 1.97 1.55 22: 2 2.65 1,. 76 t r 1.37 *22:4 12.63 13. 39 16.53 18.22 22: 6 1.07 3.60 3. 34 3.86 24:0 2. 74 2. 70 2.78 3. 84 24: 1 7.98 1. 1 9 1.31 2. 45 *24:2 4. 31 6. 49 5.31 4.18 *24: 4 24.45 15. 53 18.80 20.03 (1) means of two experiments (2) may also include 17:0 (3) t r = trace * major f a t t y acids Table 7-5. .Fatty acids of phosphatidylethanolamine Percent Composition (1) Fatty wild-type dm1 wild-type dm1 Acid 27°C 27°C 34.5°C 34. 5°( <"l6 ~67oc 4. 98 1.71 t r (2) *1 6: 0 11.83 15. 13 24.42 16.65 16: 1 1.65 2.25 3.73 1.97 17:1 (3) 2.00 2. 6 3 1.33 1 .97 *18:0 7. 21 6.78 5.29 4.81 *1 8: 1 12. 58 13. 27 10.90 7.90 *18: 2 4. 86 2. 93 3.87 2.61 *18:3 3.49 tr 2.09 1. 36 20:0 2. 34 2. 94 3.52 2.48 20: 1 1. 87 1.07 1.50 2.79 *20:4 7.04 12. 69 4.89 4. 39 22:1 t r tr 3.37 1.43 22: 2 1. 01 1. 49 t r 1.38 *22: 4 12. 80 13. 37 12.95 15.61 22:6 4.28 4. 67 2.33 1.97 24: 0 t r 1. 96 2.60 2.04 24: 1 t r 1. 09 2.34 1.98 *24:2 6.41 4. 93 3.49 4.94 *24: 4 18. 49 14.25 14.02 25.00 (1) means of two experiments (2) t r = trace (3) may also include 17:0 * major f a t t y acids Table 7-6..Fatty acids of 2-aminoethylphosphonolipid Percent Composition (1) Fatty wild-type dml wild-type dml Acid 27°C 27°C 34.5°C 34. 5°( ___ 4707 7. 17 10.26 1 .99 *1 6: 0 8. 33 7. 80 9. 16 9.68 16: 1 0. 81 0. 76 1.19 1.59 17:1 (2) 1.42 2. 43 tr(3) 1.10 *18: 0 8. 1 1 9. 35 11.79 8.29 *18: 1 12. 34 17.10 15.02 1 1.05 *18: 2 3. 16 4. 41 3.58 2.69 *18:3 12.19 tr 11.12 1.20 20: 0 1. 35 1. 43 1.46 t r 20 : 1 1. 41 tr 3.22 3. 75 *20: 4 7.94 14. 38 2.94 2.90 22:1 1.04 1. 42 4.93 t r 22. 2 2. 33 3. 32 1.45 2. 11 *22: 4 18. 45 17. 26 16.10 23.00 24:0 1.57 1. 91 2.01 2.28 24: 1 1.78 tr t r 1.66 *24: 2 3. 18 4. 71 5. 27 3.64 *24:4 18.78 15. 45 19.54 26.61 (1) means of two experiments (2) may also contain 17: 0 (3) t r = trace * major fatty acids 180 Table 7-7. Changes i n phospholipid fatty acid composition (1) Fatty PC (2) PE (3) AEPI • (4) Acid wild-type dmi wild-typ € dmi wild-ty pe dmi 16:0 4. 94 1.06 12.59 1.52 0.83 1. 88 18:0 1.62 -2. 14 - 1.92 - 1.97 3.68 - 1. 06 18^ 1 - 2.59 -3.05 - 1 .68 - 5.37 2.68 - 6. 05 18:2 4.06 -0. 96 - 0.99 - 0.32 0.42 - 1. 72 18:3 3.7£ -0.16 - 1.40 1.36 -1.17 1. 20 20 :4 -12.26 -8. 15 -2.15 - 8.30 -5.00 -11. 48 22:4 3.90 4. 83 0.13 2.24 -2.35 5. 74 24:2 1.00 -2. 31 - 2.92 0. 01 2.09 - 1. 63 24: 4 - 5.65 4.50 - 4.47 10.75 0.76 11. 16 (1) calculated by subtracting the mean composition at 27°C from the mean composition at 34.5°C (2) PC = phosphatidylcholine (3) PE = phosphatidylethanolamine (4) AEPL = 2-aminoethylphosphonolipid 181 Table 7-8. Distribution of phosphatidylcholine f a t t y acids (1) acyl wild-type dmj[ wild-type dm 1 chain 27°C 27°C 34.5°C 34.5°C <16 5.00 3.29 5.35 2.20 16<20 28.34 39.65 37.57 35.30 >20 69. 16 57. 10 59.74 65. 52 S (2) 15.99 23. 20 23.95 23.55 0 (3) 81.51 72.65 73. 36 74.27 U/S (4) 5. 10 3. 13 3.06 3. 15 U.I. (5) 2.44 2. 23 2.27 2. 34 (1) means of two experiments (2) S = sum of saturated f a t t y acids (3) U = sum of unsaturated fatty acids (U+S<100% because peaks of mixed composition or less than 16 carbons were not included) (4) U/S = r a t i o of unsaturated to saturated f a t t y acids (5) U.I. = unsaturaticn index (see Results for calculation) 182 T a b l e 7-9. D i s t r i b u t i o n o f p h o s p h a t i d y l e t h a n o l a m i n e f a t t y a c i d s ( D a c y l w i l d - t y p e dmi w i l d - t y p e dmi c h a i n 27°C 27°C 34.5°C 34.5°C <16 6.00 5.98 1.71 3.32 16<20 42.62 42.97 50.94 35.02 >20 51.39 54.05 47.35 63.13 S (2) 22. 1 1 25. 82 35„57 26.47 0 (3) 67.49 71. 20 65.53' 71.87 U/S (4) 3.05 2. 76 1.76 2.72 U.I. (5) 2.43 1. 89 1. 81 2. 15 (1) mean of two ex p e r i m e n t s (2) S = sum c f s a t u r a t e d f a t t y a c i d s (3) 0 = sum of u n s a t u r a t e d f a t t y a c i d s (0*S<100% because peaks of mixed c o m p o s i t i o n or l e s s than 16 carbons were not i n c l u d e d ) (4) U/S = r a t i o of u n s a t u r a t e d t o s a t u r a t e d f a t t y a c i d s (5) U.I..= u n s a t u r a t i o n index {see R e s u l t s f o r c a l c u l a t i o n ) 183 Table 7-10.„Distribution of 2-aminoethylphcsphonolipid fatty acids (1) ac yl chain wild-type 27°C dml 27°C wild-type dmj 34.5°C 34.5°C <16 16<20 >20 S (2) U (3) U/S (4) U. S. (5) 4.04 39. 54 56.44 18.68 77. 30 4. 14 2. 36 7. .17 40.63 55. 75 18. 82 77.60 4. 12 2. 33 10.16 42.33 52.59 22.68 72.24 3. 19 2.09 1.99 34.44 64.56 19. 10 79.90 4.18 2.59 (1) means of two experiments (2) S = sum of saturated fatty acids (3) U = sum of unsaturated f a t t y caids (S+U<100% because peaks of mixed compcsition or l e s s than 16 carbons were not included) (4) U/S = r a t i o of unsaturated to saturated fatty acids (5) U.I. .,= unsatur ation index (see Eesults f o r calculation) 184 F i g u r e 7-1. w6 pathway of polunsaturated f a t t y a c i d b i o s y n t h e s i s . . L i n o l e i c a c i d (18:2) i s s u c e s s i v e l y elongated and desaturated (Erwin, 1973).. cvj O O + o cvj U + LO CVJ Cvj cvj o 00 186 Figure 7-2. .Fatty acids of wild-type c e l l s . Tracing of a gas l i g u i d chromatographic analysis of whole c e l l f a t t y acids at 34.5°C {reduced by 50%). Horizontal axis represents retention time and v e r t i c a l axis represents r e l a t i v e concentration of f a t t y acids. l 8 \ 1 8 8 F i g u r e 7 - 3 . F a t t y a c i d s o f dmj. c e l l s . T r a c i n g o f a g a s l i q u i d c h r o m a t o g r a p h i c a n a l y s i s o f w h o l e c e l l f a t t y a c i d s a t 3 4 . 5 ° C ( r e d u c e d by 5G%). H o r i z o n t a l a x i s r e p r e s e n t s r e t e n t i o n t i m e a n d v e r t i c a l a x i s r e p r e s e n t s r e l a t i v e c o n c e n t r a t i o n o f f a t t y a c i d s . 190 I CHAPTER VIII CONCLUDING REMARKS A t o t a l cf 82 temperature-sensitive mutants that i n t e r f e r e with some aspect of endocytosis or vacuolar processing have been i s o l a t e d . Two of these have been described in d e t a i l and each i s d e f i c i e n t i n a di f f e r e n t aspect of t h i s process. The mutant, fyc, r e s u l t s in the clumping of food vacuoles near the posterior of the c e l l . . It appears that vacuolar movement i s hampered, possibly through interference with the normal functioning of cytoplasmic microtubules. At the same time, the number of food vacuoles which accumulate in a given time decreases, which suggests an in t e r r e l a t i o n s h i p between the process of food vacuole formation and vacuolar movement.. The i n h i b i t i o n of vacuolar movement may interrupt the recycling of membranes, thereby reducing the amount of membrane available f o r endocytosis. .Alternatively, both processes may reguire the normal functioning of the same fac t o r s . The fvc mutant i s more sensitive to the detergent, SES, and to colchicine than are wild-type c e l l s and the use of these chemical agents may provide a mass selection system for the generation of more mutants with similar phenotypes. . The mutant, dmj, results in the disruption of vacuolar membranes at the r e s t r i c t i v e temperature. The membranes degenerate and the changes in the fatty acid composition, which occur in wild-type c e l l s as a function of increased temperature, do not occur i n t h i s mutant. The ef f e c t s observed could be due to the malfuncticning of the palmitoyl CoA 191 desaturase enzyme which i s known to regulate the f a t t y acid composition of membranes i n Tetrahymena (Martin et a l . , 1976). This mutant has an increased s e n s i t i v i t y to the solvent, DMSO, and t h i s agent may prove useful i n selecting other mutants with phenotypes similar to dm!. Although the objective of t h i s project has been attained i._e..ts mutants in various aspects of endocytosis and the vacuolar cycle have been i s o l a t e d and two of these have been characterized, these r e s u l t s represent only a basis f o r asking more guestions about the events involved i n these processes. 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Isolation and genetic characterization., Genet. .9 2:1061-1077. . and E. .Orias.. 1979b. .Mutants of Tetrahymena thermophila with temperature-sensitive feed vacuole formation. I I . Physiological and morphological studies. Exptl. C e l l Ees. 124^317-3 27. 213 Thiele, J. , 0. Honer-Schmid, J . Wahl, G. Kleefeld, and J. Schultz. 1980. .A new method for axenic mass culture of Paramecium t e t r a u r e l i a . J . Protozool..27:118-121. Thilo, L. ..and G. Vogel. . 19 80. . Kinetics of membrane i n t e r n a l i z a t i o n and recycling during pinocytosis i n Dictyostelium discoideum.. Proc. Natl. Acad. S c i . U.S. 77: 1015-1019. Thompson, G.A. . and V.M._Kapoulos.. 1969. Preparation and assay of g l y c e r y l ethers, i n Methods i n Enzymoloqy. . XIV. L i p i d s . J.M. Lowenstein (ed.). Acad. Press. New York, pp 668-678. Tolloczko, B. . 1977.. Endocytosis i n Paramecium . I I I . E f f e c t of cytochalasin B and c o l c h i c i n e . Acta. Protozool. 16:185-193. Tucker, J.B. 1972..Microtubular-arms and the propulsion of food p a r t i c l e s inside a large feeding organelle i n the c i l i a t e Phasacolodon y o r t i c e l l a . J. . C e l l S c i . .,10: 883- 903. Ueda, T. 1976. Changes i n the fatty acid composition of mackerel l i p i d and probably related factors. I..Influence of the season, body length and l i p i d content*.Bull. Jap. Soc. . S c i . . Fisheries. . 42^479-484. Vance, D.E and C.C. ^ Sweelay.. 1967. .Quantitive determination of the neutral glycosyl ceramides in human blood. J. L i p i d Res..8 :621-630. . Volkonsky, M..1934. _L'aspect cytologigue de la digestion i n t r a c e l l u l a i r e . . A r c h * E x p t l . Zellforsch..15:355-372. Watanabe, T., H. Fukushima and Y. Nczawa. 1979. Studies on temperature adaptation i n Tetrahymena . . P o s i t i o n a l d i s t r i b u t i o n of f a t t y acids and species analysis of phosphatidylathanolamine from Tetrahymena p i r i f o r m i s grown at different temperatures. .Biochim. Biophys. Acta. . 5751365-374. Weidenbach, A..L.S. and G.A. .Thompson. 1974. Studies of membrane formation in Tetrahymena pyriformis ..VIII. on the o r i g i n of membranes surrounding food vacuoles. J . Protozool. 21: 745-751. 214 Wilairat, P., Y. Yuthavong and E. Khungvanlert. . 1978. .Effect of membrane modifications on c e l l fusion of hen erythrocytes induced by dimethyl sulfoxide . L i f e S c i . 22J.1993-1998. Wisnieski, B.J., E. E. . Williams and C F . Fox. 1973. Manipulation of fatty acid composition i n animal c e l l s grown i n culture. Proc. . Natl... Acad. S c i . U.S. 10: 3669-3673. Wolfe, J. 1973. D i f f e r e n t i a l density labeling and gradient centrifugation of Tetrahymena . Exptl. C e l l Ees. 77^232-238. Wood, D.C. and J. Wood. 1975. Eharmacolcgical and biochemical considerations of dimethyl sulfoxide . Ann. N.Y. Acad. Sc i . 24J4.1-19. Yamada, K. 1974. Effects of 2,4-dinitrophenol on cy c l o s i s and output of water i n Paramecium caudatum . J. Sci. Hirosh. Univ. Ser. B. Div. 1..25:243-2577 215 APPENDIX I CULTURE TECHNIQUES A. Axenic medium Composition (Keenan et a l . , 1 978) 1. Liver #2 (Nutritional Biochemical Company) 0.1% 2. Brewer's yeast (Nutritional Biochemical Company) 0.1% 3« Micrococcus lysodeikticus dried c e l l s 0.1% Preparation 1. suspend components in d i s t i l l e d water 2. heat gently with s t i r r i n g for 10 minutes 3. dispense into screw-cap test-tubes, s t i r r i n g vigorously 4. autoclave 15 minutes, 120°C 5. when cool, add penicillin-streptomycin ( f i n a l concentrations 100 units/ml and 100 ug/ml respectively) 6. tape caps to prevent evaporation 7. shake tubes immediately after inoculation with Paramecium Transfer of c e l l s from monoxenic to axenic medium (Allen and Nerad, 1978) Glass tubing reguired (Table Appendix 1-1, Figure Appendix I-1a) 1. one end of tubes E and D i s f i t t e d with a 2 cm length of nalgene tubing and covered with aluminum f o i l 216 2. . the other end i s covered with the inverted test-tube, E 3. tube C i s f i t t e d with 4 cm of tubing with a clamp around i t and covered with aluminum f o i l 4. tube E i s placed on the other end 5 . . a l l tubes are autoclaved Transfer of c e l l s 1. . centrifuge {100xg, 5 minutes) 100 ml of a dense culture of Paramecium 2. suck concentrated c e l l s i n t o a s t e r i l e pasteur pipette (A) with a cotton plug i n the end 3..heat seal open end of pipette 4. remove cotton plug, flame l i p of pipette 5 . remove f o i l and ins e r t pipette into tubing of tube B 6..add 5 ml axenic medium through top, remove ai r bubbles* f i l l column with axenic medium 7. remove tube E from tube B and the f o i l from tube C and inse r t the top of tube B into the tubing of tube C (Figure Appendix I-2b) 8. f i l l tube C to the top with axenic medium, replace tube E and open clamp 9. allow c e l i s to migrate overnight 10..close clamp and remove tube C frcm tube B 11. remove the f o i l from tube D, remove tube E from tube C and inse r t tube C into the tubing of tube D (Figure Appendix I-2c) 12. f i l l tube D with axenic medium and allow c e l l s to migrate f o r 8 hours 13. remove 1 ml of c e l l s from the top with a s t e r i l e pasteur 217 pipette, transfer to a s t e r i l e test-tube and add 5 ml axenic medium 14. after several days this culture can be subdivided by adding several drops of c e l l s to fresh axenic medium B. Dryl solution (Dryl, 19 59) Composi tion Solution A: calcium chloride 1.47 g d i s t i l l e d water 100.00 ml Solution B: sodium c i t r a t e 2.67 g sodium phosphate (dibasic) 5.63 g sodium phosphate (monobasic) 5.63 g d i s t i l l e d water 800.00 ml autoclave solutions A and B separately Preparation 1. 945 ml d i s t i l l e d water 2..40 ml solut i o n A 3..15 ml solution B Adaptation of c e l l s to Dryl's solution 1. . centrifuge c e l l s (100xg, 5 minutes) 2. place c e l l s in as small a volume of culture f l u i d as 218 p o s s i b l e i n t h e b o t t o m o f a 25 m l v o l u m e t r i c f l a s k 3,. f i l l f l a s k w i t h D r y l ' s s o l u t i o n and a l l o w c e l l s t o m i g r a t e t o t h e t o p 4. r e m o v e c e l l s and p l a c e i n D r y l ' s s o l u t i o n i n a s t e r i l e t e s t -t u b e 219 Table Appendix 1-1.. Glass tubing reguired for adaptation to axenic medium Tube Inner Outer Length Eiam(mm) Diam (mm) (mm) B,D 5 7 60 0 C 8 7 230 E 8 10 75 220 Figure Appendix 1 - 1 . .Glass tubing reguired for transfer c e l l s to axenic medium. a. the component pieces of glassware 221 a. TUBE A cotton plug —pasteur pipette TUBE B, D -E TUBE C inverted "culture tube -glass tube-aluminum foil naigene tubing clamp 222 Figure Appendix 1 - 2 ._Transfer of c e l l s to axenic medium. b..assembly for the f i r s t migration c. assembly for the second migration. See text for d e t a i l s . inverted culture t u b e — glass tube clamp nalgene tubing D glass tube nalgene tubing. pasteur pipette clamp-" heat sealed 224 APPENDIX II PHENOTYPES OF PUTATIVE MUTANTS A. Indiyidual l i n e selection Group 1. Morphological abnormalities B6, B8, B9, B11, B12, B16, B23, E40, B43, C11, C28, D2, D9 Group 2. Decreased endocytosis • B1, + B2, B3, *B4, + B5, *B10, + E14, +B19, B20 , B21, + B22, B25, + B27, B29, B30, B31, B37, B38r B4 1, + B42, B44, C1, C10, C18, C21, C26, D7, *D16 Group 3. Abnormal positioning of food vacuoles B28, B32, B33, C19 Group 4. Abnormal morphology of food vacuoles B7, + B15, +B17, B24, B26, B35 +• these l i n e s have been l o s t * t h i s was o r i g i n a l l y one of the B series but i t s true i d e n t i t y i s unknown 225 B. Mass s e l e c t i o n Group 1. Mo r p h o l o g i c a l a b n o r m a l i t i e s MB19, MB3 1, MD1, MD21 Group 2. Decreased e n d o c y t o s i s + MA7, MA33, MB10, MB14, MB16, ME17, +MB39, MC29, MC33, MD11, MD16, MD18,MD24, ME19, ME23 Group 3 . _ P o s i t i c n i n g of food vacuoles none Group 4. Morphology of food vacuoles +MA26, MD20 ••these l i n e s have been l o s t 226 APPENDIX III ELECTRON MICROSCOPY 1. centrifuge mass culture (100xg, 5 minutes) 2. wash i n 6 mM phosphate buffer (pb), pH = 7 3.. f i x for 2 hcurs i n 0.5i& glutaraldehyde in 6 mM pb 4. centrifuge 50 seconds 5. wash twice i n 6 mM pb 6. post-fix for 1 hour i n 1% osmium tetroxide i n 25 mM pb 7..centrifuge 50 seconds 8. wash twice i n 25 mM pb 9. centrifuge 30 seconds, remove a l l washing f l u i d with a pi pe tte 10. add 1 volume of 30% ethanol to p e l l e t , mix, l e t stand for 5 minutes 11. add 1 volume of 95% ethanol to above, mix, l e t stand for 5 minutes 12. repeat step (11), ethanol now = 70% 13. centrifuge, remove f l u i d 14. dehydrate 5-10 minutes in 70% ethanol with uranyl acetate 15. centrifuge, remove f l u i d 16. add 95% ethanol f o r 5 minutes 17. centrifuge, remove f l u i d 18..two changes, 20 minutes each, of 100% ethanol, f i n a l change in 1 volume 227 19. add 0.5 volume propylene oxide every 5 minutes* 4 times (total = 3 volumes) 20. centrifuge, remove f l u i d 21. two changes, 20 minutes each, propylene oxide, f i n a l change in 1 volume 22. transfer tc v i a l 23..add 0.5 ml Epon every 5 minutes, 4 times (t o t a l = 3 volumes) 24. leave overnight uncapped 25. f i l l BEEM capsules 1/3 f u l l with fresh Epon 26..add sample to each capsule 27. leave at rccm temperature f o r 4 hcurs 28. incubate at 60°C for 24-36 hours 228 APPENDIX IV DETERMINATION OF FATTY ACID COMPOSITION A- L i p i d extraction (Bligh and Dyer, 1959, Folch et a l . ,1957) I. . f i n a l volume of c e l l s = 2.4 ml 2. add 9 ml chloroform/methanol (1:2), homogenize 2 minutes 3..add 3 ml chloroform, homogenize 30 sec 4. .add 3 ml d i s t i l l e d water, homogenize 30 seconds 5. transfer tc 50 ml round-bottom centrifuge tubes 6..rince hcmogenizer with -4.5 ml chloroform and add to centrifuge tube 7..vortex 8. centrifuge (200xg, 10minutes) 9. remove upper layer 10..add a volume of methanol/water (1:0.8) egual to amount of f l u i d removed I I . repeat 7-10 12. centrifuge, remove upper layer 13..add 1.0 ml 95% ethanol, vortex (should go clear) 14. f i l t e r through Whatman f i l t e r paper (#1) into test-tube 15. f i l l test-tube with chloroform, cover with Teflon-lined cap and store in freezer 229 B« Thin layer chromatography (Nozawa and Thompson, 19 71) 1. evaporate under nitrogen 2i redissolve in 200 ul chloroform 3..spot s i l i c a gel G plate with 4 applications each of 25 u l of sample 4..apply 10 ul standards i 5. place solvent chloroform/acetic acid/methanol/water/ (75: 25: 5: 2. 2) i n tank with large f i l t e r paper 6..after solvent has migrated to top (around 3 hours) remove plate and dry 7..place i n iodine chamber u n t i l spots darken (10 minutes) 8 . . c i r c l e spots in pencil and scrape frcm plate C._ Removal of phospholipids from s i l i c a gel (Andrews and Nelson, 1979) L.add 4 ml chlorof orm/acetone/methanol/water (6:8:2:2) to sa mple 2..vortex, store in r e f r i g e r a t o r overnight 3. vortex, centrifuge (200xg, 5 minutes) 4. remove upper layer, transfer bottom layer to clean test-tube 5. repeat 1-4 (not necessary to leave overnight again) 6. combine chloroform (bottom) layers and evaporate under ni trogen 7. i f a small amount of water i s present add a few drops of 90% ethanol to speed evaporation 230 8. store in freezer D« - Preparation of g l y c e r y l ethers (Thompson and Kapoulos, 1969) 1. add 2 ml acetic acid/acetic anhydride (3:2), cap tubes t i g h t l y 2. heat to 90°C i n water bath for 4 hcurs, cool 3..slowly add 1.0 ml 6N potassium hydroxide i n 95% ethanol (freshly prepared) , cap t i g h t l y 4. heat to 90°C in water bath for 2 hours, cool 5. ether extraction a. add 3 ml water b. .add 1.5 ml ethyl ether, shake vigorously c. remove and save ether phase (top layer) d. .repeat (b) and (c) combining ether layers 5. removal of potassium hydroxide a. .add a few drops of water to combined ether phases, vortex b. remove water (bottom layer) c. .repeat (a) and (b) 7. evaporate under nitrogen and store at room temperature 231 E. . M e t h y l a t i c c of f a t t y e s t e r s 1. p r e p a r e f r e s h methanol/hydrogen c h l o r i d e gas (HClg) a. _ weigh 90 ml methanol i n beaker b. bubble HClg through methanol f o r around 5 minutes c. weigh and add more HClg or methanol t o g i v e 1N HClg d. .CAUTICN HClg very dangerous 2. add 1 ml methanol/HClg, cap t i g h t l y 3. _heat t o 90°C i n water bath o v e r n i g h t 4. c o o l , e vaporate under n i t r o g e n and s t o r e a t room temperature F. T r i m e t h y l s i l a t i o n of g l y c e r y l e t h e r s (Vance and Sweeley, 1967) 1. add 150 u l p y r i d i n e / h e x a m e t h y l d i s i l a z a n e / t r i m e t h y l s i l a n e (10: 4: 2) 2.. i n c u b a t e a t room t e m p e r a t u r e f o r 15 minutes 3. e v a p o r a t e under n i t r o g e n 4. r e d i s s o l v e i n 23-100 u l heptane i n s m a l l v i a l o r r e d i s s o l v e i n 1 ml heptane, t r a n s f e r t o s m a l l v i a l , e v a p o r a t e under n i t r o g e n and r e d i s s o l v e i n 20-100 u l heptane 5. s t o r e a t room temperature 232 G. Gas l i q u i d chromatography 1. machine settings a. .flame temperature = 200°C b. range = 102 c. .attenuation = 8 or 4 d. „ chart speed = 0 .5 inches/minute 2. multilevel programmer a. .post i n j e c t i o n time = 6 minutes b. rate 1 = 2°/minute; rate 2,3 = 0 c. .time 1 = 6 minutes; time 2 = 2 minutes; time 3 = 10 minutes d. .to i n i t i a t e press advance immediately aft e r i n j e c t i o n of sample 3..injection of sample a. take up 2 ul heptane b. .take up 5 u l sample and move gauge on syringe to 9 ul mark c. . i n j e c t into portal A holding end of syringe and i n j e c t i n g whole sample at once 4. .analysis of results a. Xerox peaks b. .cut out and weigh each peak c. for each fatty acid c a l c u l a t e the percent of the t o t a l weight 233 Table Appendix IV-1. Fatty acid composition of whole c e l l s -experiment 1. . Percent Composition Fatty wild-type dm1 wild-type drnj Ac id 27°C 27°C 34.5°C 34.5° <16: 0 t r 1. 08 3.54 1. 47 *16:0 14.33 20. 86 18.39 19.40 16: 1 3. 16 3.64 2.37 2.45 17: 1 (1) 1. 0 1 1. 96 1. 10 3.57 *18: 0 3.62 2.24 6.14 6.59 *18:1 17.53 17. 02 16.19 16.95 *18: 2 5. 58 4. 58 4.74 3.72 •18:3 (2) 9. 03 8. 65 10.94 1 .66 20: 0 2. 25 4. 2 7 2.02 3.49 20:2 2.36 3. 83 2.27 t r *20: 4 5. 66 4. 91 4.13 t r 22: 1 1.82 3. 38 6.50 2.21 22: 2 2. 26 1. 84 tr t r 22:4 7.57 5. 31 6.47 6.72 22: 6 1.39 2. 73 2.92 5.32 24:0 1. 71 tr 1. 42 3.20 24: 1 1.60 1.38 2.01 1.78 *24:2 1.77 2. 52 t r 3.22 *24: 4 14. 35 8.04 7.49 14. 13 (1) may contain 17:0 (2) also contains 20:1 * major fatty acids 234 T a b l e Appendix I V - 2 . F a t t y a c i d c o m p o s i t i o n o f whole c e l l s -e xperiment 2. . P e r c e n t C o m p o s i t i o n F a t t y w i l d - t y p e dml n i l d - t y p e dml A c i d 27°C 27°C 34.5°C 34. 5°' <16:0 t r 1.41 t r ___ *1 6: 0 7.65 9. 37 10.97 5.85 16: 1 1.74 2. 16 3.08 1. 28 17:1 (1) t r t r t r t r *18 :0 1. 59 1.04 2.06 1.27 *18 : 1 8. 23 7. 18 11.60 6.49 *1 8:2 2.86 2. 23 4.444 2.50 • 1 8 : 3(2) 6. 7S 3.91 7.66 4 .55 20 :0 2. 36 4. 27 1.65 1 .49 20: 2 2. 28 1. 89 2.03 t r *20:4 5.86 1. 9 1 3 .08 1.62 22 : 1 t r t r 1.59 t r 22 :2 1. 45 2. 86 1.8 2 1.50 *22 : 4 18.76 18. 07 15.84 18.03 22: 6 1. 24 t r 2.74 t r 24:0 4. 21 5. 96 3. 19 4. 84 24 . 1 2. 04 1. 98 t r 2. 10 *24 :2 5.38 5. 51 4 .70 8 . 11 *24: 4 30 .00 28 .49 20.62 38 .93 (1) may a l s o c o n t a i n 17:0 (2) a l s o c o n t a i n s 20:1 * major f a t t y a c i d s 235 Table Appendix IV-3. Fatty acid composition of whole c e l l s -experiment 3. . Percent Composition Fatty wild-type dml wild-type dml Acid 27°C 27°C 34.5°C 34. 5°( <16To __ tr 11.28 1 .48 *16: 0 9. 75 20. 52 16.52 19.17 1 6: 1 2. 29 7. 13 6.22 6.72 17:1 (1) 1.46 1. 11 t r 1.69 *18: 0 3. 72 4.32 2. 15 2.54 *18: 1 22. 19 18. 46 17. 19 18.11 *18: 2 4.55 4. 26 3.39 5. 22 *1 8:3 (2) 5.06 5. 48 7. 48 6.67 20: 0 4. 89 3. 33 2.80 4.62 20:2 4. 61 tr 3. 43 1 .43 *20: 4 7.88 2. 14 3.78 4.92 22:1 6. 04 1. J7 2.21 1 .40 22:2 1. 84 tr t r t r *22: 4 7. 07 13. 83 6.64 7. 27 22:6 t r 1. 80 1.16 1.50 24: 0 t r 1.17 1.02 1.82 24: 1 tr 2. 53 tr t r *24: 2 4.93 4. 30 4.68 2.69 *24: 4 1 1. 34 8.45 8.82 1 1.31 (1) may also contain 17:0 (2) also contains 20:1 * major fatty acids 

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