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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 L I P I D  COMPOSITION  IN WILD-TYPE AND MUTANT PARAMECIUM TETRAURELIA by CAROL  POLLOCK  B. Sc. ( H o n s ) , The U n i v . o f M a n i t o b a , W i n n i p e g , M a n i t o b a , 1972 M.Sc., The U n i v . o f M a n i t o b a , W i n n i p e g , M a n i t o b a , 1974 A THESIS SUBMITTED I N P A R T I A L FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in \ THE FACULTY OF GRADUATE STUDIES'*, (Department o f Zoology) i We a c c e p t t h e t h e s i s a s c o n f o r m i n g t o t h e required standard \ .  THE UNIVERSITY OF B R I T I S H COLUMBIA O c t o b e r 1980 (6) CAROL POLLOCK, 1980  In presenting t h i s thesis in p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by his representatives.  It i s understood that copying or p u b l i c a t i o n  of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department n f  '^  C > 0  * ® ] C  The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  • E-6  BP  75-51  1 E  ii  ABSTRACT  Eighty-twc  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 o f e n d o c y t o s i s o r v a c u o l a r processing Two  of  were i s o l a t e d  these,  clumping)  the  and  following  recessive  dmj_ ( d e f e c t i v e  nitrosoguanidine treatment.  mutations  fvc  (food  vacuole  membrane) , were e x a m i n e d i n some  detail. . 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 , of  fvc  cells  clumped  p o s t e r i o r . Fewer food  in  one  34.5°C, t h e f o o d  area of t h e c e l l ,  vacuoles accumulated  usually the  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 m i n u t e s i n a f o o d v a c u o l e as blue  watercolor a  ( B P ) . , The  recovered  as  double  (standard  deviation)  fvc  mutation  map  units  to  s e n s i t i v e , trichocyst. non-discharge) The which,  mutant,  dmj,  i n the light  had  nutation  and  was  the  tnd  integrity  of the food  i n which  missing,  presumably  vacuoles  been  enzymes t h a t were r e l e a s e d f r o m  cells  palmitic linolenic  indicated  (16:0), s t e a r i c  that  (temperature-  vacuoles or  revealed that the  was  lost.  Abnormal  of  the  digested  cytoplasm  as  were  by t h e h y d r o l y t i c  the fcod vacuole.  A g a s - l i g u i d chromatographic mutant  by 34 ± 3  s t r u c t u r e s were a l s o o b s e r v e d ,  large portions having  originally  a p p e a r e d a s a mass o f f u s e d  structural  w e l l as c e l l s  such  lccus. .  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  vesicular  was linked  disrupted  and  marker,  m o r p h o l o g i c a l l y abnormal  microscope,  mitochondria  vacuoles  analysis  the  major  (18:0), o l e i c  of  wild-type  fatty  acids  (18:1),  linoleic  (18:3), a r a c h i d o n i c (20:4), d o c o s a t e t r a e n o i c  and were:  (18:2), (22:4),  te t r a c o s a d i e n o i c an  increase  i n wild-type  t h a t o f 24:4  occurred, 24:4  and  the  only  in  percent  the.  slight  (PC)  changes  PE  and  and  decreased  i n PE,  U.L.in  increased A  dm_1  dibucaine,  wild-type food  vacuole  of  The  was  the  of  fatty  The  100  acids  unsaturation acyl  fatty  and  agents and  and  gullet  groups) acids  and  However,  phospholipids  indicated  colchicine sensitive  phenocopies of  A f t e r treatment fused  acids  similar  temperature.  more  separation was  the  «ith 6-12% of  inhibited.,  inhibited  intracellular  transport  .Dimethyl  nascent  was  which  in  sulfoxide  dmj. c e l l s  mutant, f v c  sodium  mutant  dimethyl the  that  to  dmj  s u l f o x i d e t h a n were w i l d - t y p e  r e s u l t s i n d i c a t e d that the  aspect  the  c o l c h i c i n e than wild-type c e l i s .  produced p a r t i a l  vacuoles  2-  temperature.  mutant, f vc,  cells.  phosphonolipid,  per  fatty  sulfate  s e n s i t i v e to dimethyl The  cell  dodecyl  and  with  cells.  in  chemical  sulfate  from  increase  in  (PE)  i n whcle c e l l  several  sodium  endocytosis.  sulfoxide  whole  w i t h an i n c r e a s e  survey  dodecyl  cells,  w i t h an  phospholipids  cells,  bonds  and  reflected  t e m p e r a t u r e i n a manner  double  wild-type  opposite  decreased  the  the  percent  the  c h a n g e s were  ( A E P L ) . . I n dm1  AEPL v a r i e d w i t h  in  particularly  some  in  With  increased  phosphatidylethanolamine  occurring  ( 0 . 1 . = number o f  cells,  of  the p a t t e r n o b s e r v e d w i t h w h o l e dm_1  index  the  acids  the  o f 16:0  o f 16:0  these  (24:4).  34.5°C  lipids,  composition  fatty  aminoethylphosfhonolipid o f PC,  to  whole c e l l  i n c r e a s e d . In w i l d - t y p e c e l l s  phosphatidylcholine  the  27°C  d e c r e a s e d . . H o w e v e r , i n dm1_  i.e.  mainly  to  tetracosatetraenoic  i n temperature from  composition, and  (24^2) ,  food  were more  cells.. abnormal i n may  involve  iv  microtubules  or  microfilaments.  undergo t h e same c h a n g e s i n f a t t y cells  i n response  membranes normally  in  The acid  mutant,  composition  t o an i n c r e a s e i n t e m p e r a t u r e .  this  seguestered  mutant  degenerated  i n the vacuoles  c e l l u l a r destruction..,  dmj[,  d i d not  as w i l d - t y p e The  vacuolar  and m a t e r i a l which  was  *as r e l e a s e d , r e s u l t i n g i n  V  TABLE OF CONTENTS ABSTRACT  i  L I S T OF TABLES  ....  ,  i xi  L I S T OF FIGURES  xiv  L I S T OF PLATES ,  xVi  ACKNOWLEDGEMENTS.  .........................................xvii  CHAPTER I ENDOCYTOSIS IN PARAMECIUM A..  Description  of  eadocytosis  TETRAU RELIA  1  and i n t r a c e l l u l a r  digestion B.  . 1  Kinetics  of  accumulation  and  loss  of  food  vacuoles  6  C. . Membrane r e c y c l i n g D.  ...  Purpose of t h i s t h e s i s  8 11  CHAPTER I I STANDARDIZATION OF CONDITIONS . . . . . . . . . . . . . . . 1 5 A. . I n t r o d u c t i o n B.  Materials  15  a n d Methods  1. . G r o w t h and p r e p a r a t i o n 2.. S e l e c t i o n 3.  of food  .17 o f Paramecium t e t r a u r e l i a  v a c u o l e markers  E f f e c t o f t e m p e r a t u r e ..  4.. E f f e c t o f t i m e i n m a r k e r 5.. E f f e c t o f c u l t u r e Results  1..  G r o w t h and p r e p a r a t i o n  Effect  •.  , 18  ........................18 .18 19  2.. S e l e c t i o n 3.  .17  medium  C.  17  of food  o f P. t e t r a u r e l i a  vacuole markers  of temperature  ...19 ..19  ...  19  4.. E f f e c t o f t i m e i n m a r k e r ........................ 5.. E f f e c t o f c u l t u r e D. . D i s c u s s i o n  medium  2C  ...20 21  CHAPTER I I I MUTAGENESIS  AND  DESCRIPTION  OF  PUTATIVE  MUTANTS  32  A. . I n t r o d u c t i o n  32  B. . M a t e r i a l s and Methods  .........32  1.. Mutagenesis  32  2.. S e l e c t i o n procedure ..............................33 a.  individual line selection  33  b. . mass s e l e c t i o n ............................... 34 3.. D e s c r i p t i o n of p u t a t i v e mutants ................. 35 4.  Genetic a n a l y s i s  36  C. . R e s u l t s  , 36  1.. Mutagenesis ..................................... 36 2.. Y i e l d of p u t a t i v e mutants  37  3.. D e s c r i p t i o n of p u t a t i v e mutants .................. 37 4.  Genetic a n a l y s i s  38  a.  A10 x pawn A  38  b.  A5 x pawn A .............................••.••38  c.  A5 x spot  ...39  D. . Discussion CHAPTER  40  IV FURTHER CHAR ACT ERIZATICN  AND f v c  56  A. . I n t r o d u c t i o n B.  OF THE MUTANTS dm_1  .. 56  M a t e r i a l s and Methods  56  1. . E f f e c t of time i n BP  56  2.. L o s s c f c o l o r e d vacuoles a f t e r removal from BP ...57 3.. E f f e c t of c o n c e n t r a t i o n 4.. E f f e c t of time at 34.5°C a.,  asynchronous c e l l s  of BE  58 .58 58  b. 5..  synchronous c e l l s  58  Down-shift t o p e r m i s s i v e temperature  6.. E f f e c t o f i n c r e a s i n g  59  t e m p e r a t u r e ................ 59  C. _ R e s u l t s . 1.. E f f e c t  • 59  o f t i m e i n BP  2. L o s s o f c o l o r e d  59  v a c u o l e s a f t e r removal from  3. . E f f e c t  o f c o n c e n t r a t i o n o f BP  4..Effect  o f t i m e a t 34. 5°C  a.  asynchronous c e l l s  b.  synchronous c e l l s  BP ... 60 .61 61 61  ............................ 62  5. . D o w n - s h i f t t o p e r m i s s i v e t e m p e r a t u r e .............. 63 6. . E f f e c t o f 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 B.  .................................... 94  M a t e r i a l s a n d Methods  1.  Procedure  98  f o r determining effects  of c h e m i c a l  agents  98  a. . p r e p a r a t i o n o f 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  d.  analysis of results  and chemical agents  .....99 ........100  2. , DMSO a. , e f f e c t  10 1 of concentration  ......................101  b.  observations of l i v e c e l l s  c.  effect of treatment duration  d. . a d d i t i o n o f RP f o l l o w e d  by DMSO a n d a  101 .....102 second  viii  water c o l o r  ..  .  102  3.. Dibucaine 4.  .........103  SDS  103  5.. C o l c h i c i n e  103  6.. C y t o c h a l a s i n B  104  C. . R e s u l t s ,..  • 104  1. . DMSO  104  a.  e f f e c t of c o n c e n t r a t i o n  104  b.  observations  105  c.  e f f e c t of treatment d u r a t i o n  d.  of l i v e  cells  ..107  a d d i t i o n of RP f o l l o w e d by DMSO and a second  watercolor  ....107  2.. Dibucaine 3.  ...........110  SDS  4. . C o l c h i c i n e  ...  11C  ......................................110  5.  Cytochalsin B  D.  Discussion  .....111 111  CHAPTER VI 0LTRA52? RU CT U R E OF WILD-TYPE AND dm_1 CELLS ...137 A. . I n t r o d u c t i o n B.  ....137  M a t e r i a l s and Methods  ..137  C. . R e s u l t s D.  Discussion  1.. S l i g h t a b n o r m a l i t i e s 2.. Degeneration of s t r u c t u r e ..  ...138 •  .142 143 ..........143  3.. Large vacuoles  143  4. . T o t a l degeneration  143  CHAPTER VII FATTY ACID COMPOSITION OF WILD-TYPE AND dml. CELLS  158  ix  A.  Introduction  158  B. . M a t e r i a l s and Methods ...........................160 1.. E x t r a c t i o n of l i p i d s  .........16C  2.. T h i n l a y e r chromatography  16C  3. . P r e p a r a t i o n of g l y c e r y l ethers 4.  ..161  Methylation  ...162  a. , f a t t y a c i d s b.  .162  g l y c e r y l ethers  162  5.. Gas l i g u i d chromatography ........................ 163 C.  Results  164  1. . F a t t y acid composition  of whcle c e l l s  2.  of p h o s p h o l i p i d s  F a t t y acid composition  D. . Discussion CHAPTER  ........... 164 .........167  ...........168  VIII CONCLUDING REMARKS ,  ..190  LITERATURE CITED  192  APPENDIX I CULTURE TECHNIQUES .......................... 215 A. . Axenic medium  .................215  B. . D r y l s o l u t i o n  217  APPENDIX I I EHENOTx PES OF PUTATIVE MUTANTS A.  .224  Individual l i n e selection  224  B. . Mass s e l e c t i o n  ....225  APPENDIX I I I ELECTRON MICROS COPY APPENDIX IV DETERMINATION  226  OF FATTY ACID COMPOSITION  A. . L i p i d e x t r a c t i o n  ....228  •  .......228  B.  Thin l a y e r chromatography  229  C.  Removal of p h o s p h o l i p i d s from s i l i c a  D. . Preparation of g l y c e r y l E.  g e l ........229  ethers .................. 230  M e t h y l a t i o n of f a t t y e s t e r s  ....231  Trimethylsilation Gas  liquid  of g l y c e r y l  chromatography  ethers  LIST OF TABLES 2-1. E f f e c t  of  temperature  vacuoles •,.  on  the  accumulation  of  ,  23  3-1. Phenotypes of p u t a t i v e mutants  . .<  44  3-1, cont. Phenotypes of p u t a t i v e mutants ............... 45 3- 2. . A1 0 x  joawn ,  3-3. A5 x  pawn  3-4. A5 x  sjcot  3- 5.  tnd x spot  4- 1. E f f e c t  46 47 -  ... 48  ; f v c x spot  49  of blue watercolor  at  34.5°C  on  vacuolar  morphology 4-2. E f f e c t  68 of removal from blue watercolor on vacuolar  morphology 4-3. E f f e c t  69 of  concentration  cf  blue  watercolor  on  vacuolar morphology  70  4-4. E f f e c t  of time at 34.5°C on vacuolar morphology ... 71  4-5. E f f e c t  c f time a t 34.5°C on vacuolar clumping ..... 72  4-6. Vacuolar clumping i n A5 and f v c 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 a f t e r  75  4-9. Vacuolar clumping a f t e r  s h i f t t o 27°C  shift  t c 27°C  4-10. Vacuolar clumping i n f v c and A5 c e l l s to 27°C . ,  76 after  ,  77  4-11..Effect of temperature on vacuolar morphology 4- 12. E f f e c t  ......78  of temperature on vacuolar clumping ....... 79  5- 1 . . E f f e c t o f dimethyl s u l f o x i d e 5-2. E f f e c t  shift  at 27°C .............. 116  of dimethyl s u l f o x i d e a t 34.5°C  5-3. .Length of c o n t r a c t i l e  vacuole c y c l e  ............117 ..118  xii  5-4.  Effect  o f 12% d i m e t h y l s u l f o x i d e a t 34.5°C  5-5.  Effect  cf  dimethyl  sulfoxide  and  red  ...119 and  blue  w a t e r c o l o r a t 34.5°C 5-6..Effect  of  120  dimethyl  sulfoxide  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 a t 27°C  122  5-8. . E f f e c t  o f d i b u c a i n e a t 34. 5°C  5-9.  o f s o d i u m d o d e c y l s u l f a t e a t 27°C  Effect  5 - 1 0 . . E f f e c t o f sodium d o d e c y l s u l f a t e  .123 124  a t 34.5°C  125  5 - 1 1 . . E f f e c t o f c o l c h i c i n e a t 34.5°C  126  7-1.  174  Fatty  a c i d composition of Khole c e l l s  7-2..Changes i n w h o l e c e l l 7-3. . D i s t r i b u t i o n 7-4*  Fatty  fatty  o f whale c e l l  acid composition  ......175  fatty acids  ....176  acids of phosphatidylcholine  .177  7-5..Fatty  acids of phosphatidylethanolamine  ........... 178  7-6.  Fatty  acids of 2-aminoethylphosphonolipid  7-7.  C h a n g e s 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  179  7 - 8 . . 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 c h o l i n e f a t t y a c i d s ...181 7-9..Distribution  of  phosphatidylethanolamine  fatty  acids  182  7-10..Distribution  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  fatty  acids  183  Appendix 1-1. .Glass t u b i n g r e g u i r e d  f o r adaptation  to  a x e n i c medium Appendix IV-1. F a t t y experiment  219 a c i d c o m p o s i t i o n of whole c e l l s -  1..  Appendix IV-2. F a t t y a c i d c o m p o s i t i o n o f experiment 2  233 whole  cells 234  Appendix IV-3. F a t t y experiment 3.  a c i d composition of whole c e l l s •  235  xiv  L I S T OF FIGURES 1- 1. . M o r p h o l o g i c a l  changes  in  feed  vacuoles  during  digestion.  13  2- 1.. A c c u m u l a t i o n red  of vacuoles  i n carmine  p a r t i c l e s and  watercolor.  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.  Accumulation  of vacuoles  2- 4.  Accumulation  of  i n blue watercolor  vacuoles  in  buffer  28  and  axenic  medi urn  30  3- 1. A c c u m u l a t i o n 3- 2. . S c h e m a t i c  o f v a c u o l e s i n 4y-21  illustration  4-1..Accumulation  of  .50  o f t h e dmt p h e n o t y p e .  vacuoles  in  .52  blue watercolor a t  27°C. 4- 2.  80  Accumulation  of  vacuoles  in  blue  watercolor  at  34.5°C 4-3.  82  Loss  of c o l o r e d  v a c u o l e s a t 27°C.................. 84  4-4. L o s s  of c o l o r e d  v a c u o l e s a t 34.5°C  4-5. . A c c u m u l a t i o n at  of vacuoles  86  i n w i l d - t y p e a n d dm_1  cells  34.5°C  88  4- 6. . A c c u B u l a t i o n o f v a c u o l e s  i n w i l d - t y p e and A5  cells  a t 34.5°C.  90  4- 7. . A c c u m u l a t i o n  of vacuoles i n synchronized  cells  at  34. 5°C 5- 1. S c h e m a t i c  92 illustration  of morphological e f f e c t s of  dimethyl sulfoxide. 5- 2. F r a m e - b y - f r a m e  127 movement c f a f o o d 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 o f 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  biosynthesis.  fatty  acid .184  7-2. F a t t y acids of wild-type  cells  .186  7-3. F a t t y acids of dm1 c e l l s . Appendix  1-1. Glass  c e l l s t o axenic  tubing  188 reguired  medium..  Appendix 1-2. T r a n s f e r o f c e l l s t c axenic  f o r t r a n s f e r of 220 medium........ 222  xvi  I I I - 1 . Wild-type  and  LIST OF  PLATES  mutant  cells  in  watercolor  34.5°C  at 54  V- 1..Morphological e f f e c t s o f d i m e t h y l s u l f o x i d e . ....... 135 VI- 1..Food vacuoles of a w i l d - t y p e c e l l . VI-2. . Wild-type c e l l t a n g e n t i a l VI-3. . Cortex  t c the s u r f ace. .......... 15C  and g u l l e t r e g i o n s cf djtl c e l l s .  VI-4..Food vacuoles of dml  148  cells.  VI-5. . Extreme cases of the dmjt phenotype.  ...152 ........154 156  xvii  ACKNOWLEDGEMENTS  I  would l i k e t o thank my s u p e r v i s o r , Dr. J.D. Berger, f o r  h i s support, thesis.  I  encouragement wculd  also  and  like  guidance  to  thank  i n preparing several  c o n t r i b u t e d g r a c i o u s l y of t h e i r time and a d v i s e :  this  others Adrian  who Smith  f o r h i s help with the s e c t i o n i n g , Dr. C.E. .Vance and t h e people in  his laboratory,  p a r t i c u l a r l y Harry Paddon, f o r t h e i r help  with the f a t t y acid i s o l a t i o n and a n a l y s i s , P h y l l i d a Morton f o r t h e b e a u t i f u l f i g u r e s , the s t u d e n t s Phyllida in  Morton  i n the  l a b , Don  Jones,  and C o l i n Rasmussen, the f a c u l t y and students  the G e n e t i c s and C e l l B i o l o g y Programme a t the U n i v e r s i t y of  B r i t i s h Columbia, my f a m i l y and f r i e n d s who have encouraged and supported me over the years, and f i n a l l y ,  my wonderful husband,  J i m , f o r h i s constant love and understanding.. Research funds were p r o v i d e d by N a t i o n a l Research  Council  Grant 67-6300 t o Dr. . J. D.. Berger. T h i s t h e s i s i s d e d i c a t e d t o the memory of Glenn  Morton.  1  CHAPTER  I  ENDOCxTOSIS IN PARAMECIUM  A.  Description  of e n d o c y t o s i s  and  intracellular  E n d o c y t o s i s i s t h e p r o c e s s by is  internalized  occurs  as  properties, membrane the  In  a  conseguence  surface  membrane  represent  ciliates, is  rows  of  that  s u c h as Paramecium The groove  cytopharynx) cilia  cytopharynx them al.,  form t h e o r a l  1965,  and  K o r n and  1 9 7 5 ) . . Food  cytopharynx  tube  (Figure  a  food  is  and the  i n hymenostomes  i s a shallow  the tody c a v i t y  ciliated gullet  (Mast,  such as b a c t e r i a ,  coat  the p a r t i c l e s ,  concentrating  Weisoaan,  1967,  and  entrapment  .  particles,  vacuoles  complex  specialization  thereby  1 — 1 A)  sites  1977).  extremely  (the b u c c a l c a v i t y ,  extends i n t o  suspended  or  areas of  receptor  membranelles  where mucus s e c r e t i o n s  together  Rappitt,  for  of s u c h  to a c i l i a t e d  force  1977)  (Anderson e t a l . , an  specialized  and T e t r a h y m e n a  which  membrane  o r g a n e l l e s adapted f o r the b i n d i n g  f e e d i n g a p p a r a t u s i n Paramecium  leading  in  Fera,  contain  apparatus,  An example  and  material  endocytosis  1979). S p e c i a l i z e d  macrophages  oral  changes  (Berlin  of macromolecules  highly  cilia  Generally,  local  et a l . ,  specific  the  internalization.  The  of  internalization  structure,  of  s u c h as m i c r o v i s c o s i t y (Reis  dijgestion  which e x t r a c e l l u l a r  vesicularization.  potential  which may and  via  TETRAURELIA  them  Nilsson,  form  at  circulate  1970,  the  1947).  into  the  sticking  (Jahn Ricketts  base  or  of  et and the  i n t h e c y t o p l a s m where  2  digestion  occurs (Jurand,  The  changes  throughout  the  extensively Allen,  in  1961).  food  vacuole  digestive  described  1974,1976).  cycle  (Jurand,  Young  morphology in  Paramecium  1961, J u r a n d and  vacuoles  cr  membrane  occur  have  Selman,  phagosomes  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 vacuolar  that  with  proximity Dunihue, These  to  young  small  granules  vacuoles  (Hall  contain  Eosenbaum forming  were l a t e r  hydrolytic  and  Ricketts,  enzymes  De  vacuoles Duve  and  Dunihue,  as l y s o s o m e s , and  secondary  lysosomes  (De  Esteve,  1970,  acid  p h o s p h a t a s e , w h i c h i s an enzyme commonly u s e d  for  lysosomes  and  i n the small v e s i c l e s surrounding,  t h e young  vacuoles (Esteve,  In  stage  II  unchanged. inside around  By  this  the t a c t e r i a  1.4  but acid around  (Esteve,  drops gradually at f i r s t around  1-1C) ,  staje  the f o o d vacuoles,  but  not  marker  1962), i s within,  t h e v a c u o l e s w e l l s and t h e  tacterial  the  1 9 7 0 ) . The  Mast,  morphology  phosphatase a c t i v i t y  and f i n a l l y  ( S h a p i r o , 1927,  as a  1970).  (Figure  membrane becomes i r r e g u l a r  phagosome,  Anderson,  localized  1962,  phagosomes  197C) . P r i o r t o t h e f u s i o n o f l y s o s o m e a n d  Barka  1930,  organelles Toro,  W a t t i a u x , 1966,  ( G o m o r i , 1 952,  close  W h i t t n e r , 1962).  (Muller  or  vitally  in  W h i t t n e r , 1962). lysosomes f u s e w i t h  digestive  Duve, 1963,  identified  and  The  vesicles are  appear  and  1931, V o l k o n s k y , 1934, Eosenfcaum  granules  that  red,  I)  ( F i g u r e 1-1B).  i s v e r y r e g u l a r and many s m a l l  neutral  1969),  (stage  o b s e r v e d a r o u n d t h e g r o w i n g v a c u o l e . When p a r a m e c i a a r e stained  been  may  1947)..  membranes  as  remains appears well  as  pH of t h e f o o d v a c u o l e reach  a  minimum  of  3  In  older  are undergoing membrane  vacuoles (stage I I I , F i g u r e 1-1D), the d i g e s t i o n and condense away  which  i s very i r r e g u l a r .  from  to  that  of food vacuoles and  phosphatase vesicles The  nature  of  the  freeze-fracture  1972)  food  the  and  of the p a r t i c l e compared  vacuole  al.,  has  also  changes  as  the  cytoplasm.  Changes  in  pH  1962,  McKanna  distribution  been  in  (1973a,b)  tha  1947). by  vacuoles result  distribution  food  vacuole  in  (Pinto  and  in  detail  : a r a s s o , 1964,  suggests  that  membranes  as  1976) .  ultrastiucture  described  Favard  (Mast,  the  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  peritrichs,  vesicles  similar  membrane, as determined  with the plasma membrane ( A l l e n ,  In  density  and the pH l e g i n s t o r i s e  techniques,  through  Silva,  (pinocytotic  pinch c f f from the vacuole. Acid  a l t e r a t i o n s i n the intramembrane p a r t i c l e da  vacuolar  a c t i v i t y i s very high i n both the vacuoles and  (Esteve, 1970)  proceed  the  Small v e s i c l e s  or cup-shaped v e s i c l e s ) c o n t a i n m a t e r i a l of a  bacteria  of  the  cup-shaped  (Faure-Fremiet  et  Carasso et a l . , 1964).  these  cup-shaped  coated  v e s i c l e s , which have a modified membrane c o n s i s t i n g of a h i g h l y ordered  coat  similar  ( S l a u t t e r b a c x , 1967), may absorption  of  to  that  observed  be important i n the  macromolecules.  As  shaped  digestion  extended  form  In the extended (eg. f e r r i t i n ) .  Hydra  recognition  of  5 nm  x 20  l i k e pegs with a g l o b u l e near the d i s t a l  proceeds,  the  and  In Hydra, empty v e s i c l e s i n the  cytoplasm e x h i b i t a condensed coat c o n s i s t i n g subunits  in  coat  is  transformed  into  nm  end. the  with the s u b u n i t s resembling extended f i l a m e n t s . form  the  coat  can  bind  specific  molecules  The membrane with bound macromolecules pinches  4  o f f i n t o the cytoplasm  at  condensed conformation  and r e l e a s e s the macromolecules i n t o the  lumen  of  which  time  the  membrane  from  and migrate  the  surface  membrane  where they fuse with lysosomes ultrastructure in  of the coated  Hydra,  which  extended  coat  vacuolar  contents  Coated  (Pearse, and and  and  have  transfer  Fine,  et  a l . , 1977).  The  a  binds  similar  function  macromolecules  i».e. the from  the  (McKanna, 1973a,b, see s e c t i o n  also of  been  implicated  membranes  in  in  mammalian  the cells  (Cachon (Rothman  1980) .  with  (stage IV, F i g u r e 1 - I E )  t h e c y t o p r o c t and the undigested  v i a exocytosis,. By t h i s stage a c i d apparent  in  the  vacuoles  old  r  with  Microtubules  the  plasma  a r e important  in  vacuoles  debris i s expelled  phosphatase a c t i v i t y  i s no  (Esteve, 1970). The a c t u a l  process o f e x o c y t o s i s i n v o l v e s the f u s i o n of t h e membrane  off  through the cytoplasm  and v i r u s i n f e c t e d mammalian c e l l s  In the f i n a l stage  longer  pinch  1976, Mollenhauer et a l . , 1977), r a d i o l a r i a n s  Cachon, 1977)  fuse  similarly  and the condensed coat r e p r e s e n t s a " c l e a n "  vesicles  intracellular  the  v e s i c l e s i n p e r i t r i c h s i s the same  suggests  selects  invaginate,  (Anderson  membrane a v a i l a b l e f o r r e c y c l i n g 3) .  resumes  the v e s i c l e . .Ia c u l t u r e d human f i b r o b l a s t s  coated r e g i o n s of the plasma  as  coat  membrane guiding  food  at  food  the  vacuole  cytoproct.  vacuoles  t o the  c y t o p r o c t as well as p o s s i b l y s u p p l y i n g the f o r c e s r e g u i r e d f o r fusion  ( A l l e n and Wolf, 1974)..  The  morphological  changes which occur during the vacuolar  c y c l e o f Tetrahymena , a c i l i a t e are  very  similar  closely related to  Paramecium  ( E l l i o t t and Clemmcns, 1966, R i c k e t t s , 1972,  5  Nilsson,  1976, 1977a), as are  Greenside, also  1976,  been  the  exccytic  (Blum  described  and  Muller  and  localized  R a p p i t t , 1974a),  and  in  Toro, 1962,  Klamer and F e n n e l l , 1963, E l l i o t t  Allen  Tetrah ymena e t a 1. , 1963,  and Clemmons, 1966,  Ricketts  the d i s t r i b u t i o n i s very s i m i l a r t o  t h a t i n Paramecium except t h a t i n stage I I a c i d phosphatase not  initially  limited  to  the vacuolar membrane  throughout the food vacuole. .In p e r i t r i c h s , not  localized  i n membrane-bound  concentration food al.,  vacuoles  and  A l l e n and Wolf, 1979). Acid phosphatases have  (Seaman, 1961a,  and  events  within  vesicles  endoplasmic  but i s found  a c i d phosphatase i s  but  reticulum  (Faure-Fremiet e t  is  occurs  in  high  around the young  a l . , 1962,  Goldfisher  et  1963, Carasso et a l . , 1964).. E n d o c y t o s i s has been d e s c r i b e d i n many organisms i n c l u d i n g  other  ciliates:  (Rudzinska et a l . , (Bradbury, 1973), (Tucker, 1972), Tokophyra  Blepharisma 1966), E u p l o t e s Paraciaita  Terebrpspira  AiS2§_!£|  Blum, 1965), P l a n a r i a (Essner, 1960,  (Bradbury as  1960).  1974), Hyalophysa  as Euglena  (Rosenbaum and Eclon, and  Colpoda  Phascolodon  and Goyal, 1 976), and  well  (Mercer, 1959),  Cohn, 1971),  (Conn and H i r s c h ,  (Klcetzel,  (Hauser, 1970),  (Rudzinska, 19 70^,  (Roth, 1960),  (Dembitzer, 1968) ,  1960),  in  Pelomyxa  (Sommer  and  macrophages  polymorphonuclear l e u k o c y t e s  6  B.  K i n e t i c s of accumulation and C i l i a t e s can  l e s s cf food  be fed substances which allow  labeled  vacuoles which accumulate or are l o s t  period  to  be  easily  be c a l c u l a t e d . In  time  a  nature of the 0.44  to  form  ingested  minutes  (Lea,  s i n g l e food  minutes  Ricketts,  1942a)  (Muller  1971a,  decreases  to  6.0  and  and  minutes  of  feeding  s t a t e between accumulation and and  Pollock, In  Pollock,  Pollock,  ,  the  no  exocytosis  before  Orias, personal  age  a  Pollock,  this  1980), but  not  due  to  eguilibrium  vacuoles  changes  t h a t d i g e s t i o n has not from  deoxyribonucleic  of  of the  vacuoles  (Berger  is  begun  (DNA)  vacuole  can  ingested  (Gebauer, 1977). where  and  period,  i s g e n e r a l l y around  period during  i n the  Paramecium acid  but  non-  vacuole (Berger  be  communication). T h i s  50 minute p r e - d i g e s t i o n  ultrastructural  different  1980).  However, i n Tetrahymena , a maturation  60 minutes, i s reguired  with the  1971,  vacuoles,  l e s s of l a b e l e d  independent of the  1580).  and  and  Berger,  attainment of an  which v a r i e s with the m a t e r i a l i n g e s t e d  (McBeath  1912)  1980) . .  Paramecium  s e l e c t i v e and  from  (Metalnikov,  and  Berger and  but r a t h e r  the  Tetrahymena between 1.5  Berger  ( N i l s s o n , 1972,  the  varies  Tore, 1962,  N i l s s o n , 1972,  ,  vacuole depends on  double l a b e l i n g experiments i n d i c a t e that t h i s i s cessation  of  specific  Paramecium  A f t e r an i n i t i a l r a p i d r a t e of accumulation of rate  number  during a  p a r t i c l e s (Mast, 1947)  with most values f o r Paramecium and 4.0  the  determined. In t h i s manner the r a t e s of  accumulation and l o s s can required  vacuoles  exocytosed may  coincide  which t h e r e bacteria This  are  implying is  quite  radioactivity  from  of t r i t i u m - l a b e l e d b a c t e r i a appears  7  i n macronuclear DN& 4-5 bacteria elapsed,  (Berger,  minutes  1971}..  Once  and  Blum, 1974, some  vacuoles  in  Eappitt,  observed  cells  r e s u l t s obtained starved  studies starved  undergo  ultrastructural  many  of  cells  cells  well  personal loss  of  ( E i c k e t t s and  ( E i c k e t t s , 1 979).  the t r u e  1976) the  situation  since  changes, such a s b i o c h e m i c a l and  modifications  i n amounts  sequential  has  as  Orias,  ( E i c k e t t s and E a p p i t t ,  may not r e p r e s e n t  cells  differences  are used  and  o f the  period  i n Tetrahymena  1976) as well as i n w e l l - f e d  starved  addition  maturation  McEeath  communication) although i n was  the  the  e g e s t i o n i s probably random  (Eothstein  When  after  (Levy  acid  and  Elliott,  phosphatase  1968),  released after  exposure t o d i g e s t i b l e p a r t i c l e s ( E i c k e t t s , 1971a), d i f f e r e n c e s in  the  rates  of  N i l s s o n , 1976) ,  uptake  and  of  changes  particles  in cell  volume  1974b).  When  spheres  followed  by carmine p a r t i c l e s , s e q u e n t i a l e x c r e t i o n of  vacuoles  results  of  also  occurred  the r e c i p r o c a l  Tetrahymena  ( E i c k e t t s and  Eappitt,  the  well-fed  ( E i c k e t t s , 1971b,  were  ( E i c k e t t s , 1979).. However, the experiment  (carmine  l a t e x ) a r e l e s s c l e a r - c u t and suggest t h e observed elimination  may  have  been  minutes  f o l l o w e d by pattern  i n f l u e n c e d by experimental  s i n c e the time between f e e d i n g 75  fed latex  of  design  the f i r s t and second markers i s  which i s c l o s e to t h e maturation time r e g u i r e d f o r  food vacuoles  i n Tetrahymena  (McBeath  and  Orias,  personal  the formation  of a food  communication) . The  time  that  elapses  vacuole and i t s subseguent guite  between  egestion  (i.e..turnover  v a r i a b l e and depends t o a c e r t a i n extent  time) i s  on the m a t e r i a l  8  ingested  (Metalnikov,  1912,  around  45  Gebauer, 1977,  Pollock,  1980) .  vacuoles  to t u r n over i n c e l l s t h a t have been  Berger  and  minutes i s r e q u i r e d f o r 50% of the fed  labeled  unlabeled  b a c t e r i a (Berger,  vacuoles,  and thus the e f f i c i e n c y c f f e e d i n g , may be r e l a t e d t o  the  availability  of  Pollock,  1580) . In  vacuoles  increases  cells  amoebae,  of turnover  is  of food  plentiful  vacuole  but  i f food  Pollock,  of  (Berger  and  and the c e l l s  the  feeding In  of  (Berger  of  food  with  starved  Chapman-Andresen  and  P o l l o c k , 1980) . . Thus, i f w e l l - f e d , the h a l f - l i f e of a  half-life  would  of  feeding  vacuoles  increase  and the  (Berger  s p i t e of d i f f e r e n c e s i n the turnover  accumulation  and  low  and rates  non-nutritive  particles,  l o s s of these  vacuoles i s  and P o l l o c k , 1980).  Membrane r e c y c l i n g Actively  phagocytizing  greater  than  minutes  (Kloetzel,  al.,  turnover  and  i n c r e a s e s when n o n - n u t r i t i v e  c o n t a i n i n g n u t r i t i v e and  kinetics  similar  vacuoles  i s scarce  1S80) .  of vacuoles  of  (Berger  would be s h o r t and the e f f i c i e n c y of  efficiency  C.  rate  of food  1S70, Chapman-Andresen, 1977). In Paramecium , the  food  the  the  materials  i n w e l l - f e d c e l l s as compared  p a r t i c l e s are ingested food  digestible  (Chapman-Andresen, 1968,  Christensen, rate  1971). The r a t e of turnover  or  1972,  Eappitt,  their  cells  use  amounts  of  membrane  t o t a l s u r f a c e i n p e r i o d s of l e s s than t e n 1970,1974,  McKanna, 1973a,  1974b). The source  of  E i c k e t t s , 1971a, Allen, this  1974,  membrane  Goodall  et  Eicketts  and  is  a  pool  of  9  cytoplasmic al.,  membranes  1972,  (Chlapowski  W e i d e n b a c h and of v e s i c l e s  base  buccal  the  (Roth,  1957,  Randall  Stone,  1963)  and  could  s i n c e t h e y have b e e n o b s e r v e d a t t h e  cavity and  are  B a n d , 197 1, G o o d a l l e t  Thompson, 1 9 7 4 ) . T h i s membrane  be i n t h e f o r m of  and  and  around  growing food vacuoles  Fitton-Jackson,  freguently  1958,  seen f u s i n g  Miller  with the  and plasma  membrane a t t h e base o f 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  food  vacuoles  1966,  (Kennedy,  1965,  McKanna, 1969,1973a, Bardele, Paulin,  1972,  Bradbury,  plasma  areas  1973,  of the G o l g i  memlrane  Band, 1971).  and  R u d z i n s k a , 1970,  (Hicks,  Ey l a b e l i n g  1966,  Howell  and  observed  arising  from  Falk,  1969,  p o s s i b l e to f o l l o w the i n t e r n a l i z a t i o n  of  the plasma  plasma  membrane  al.,  apparatus 1977,  transfer  the  Golgi In  where  reticulum  (Cachon a n d Paramecium  similar  Chlapowski  and  labeled  occur  in  in  diameter,  structure  to  1 9 7 4 ) . They a r i s e by  pinocytcsis  during  digestion  the  fuses with the cytoproct  from  also  (Mollenhauer 1980)  et  or they via  1977).  (Allen,  and  vesicles  without proceeding  c a a da turn , t h e s e v e s i c l e s (um)  at the  then fuse with the  1979, Rothman and F i n e ,  Cachon,  portions  reappearance  and may  membrane p r o d u c t s d i r e c t l y  0.2-0.5 m i c r o m e t e r s thick,  of  subsequent  modifications  Morre e t a l . ,  may  with  ( T h i l o and V o g e l , 1 9 8 0 ) . C o a t e d  a r i s e from t h e endoplasmic Golgi  then f u s i n g  s u r f a c e of P i e t y o s t e l i u m , i t  is  their  1970,1974,  1974,  a p p a r a t u s and  the c e l l  membrane and  Clemmons,  Kloetzel,  Allen,  1 976). V e s i c l e s have a l s o been  specialized the  Elliott  ( A l l e n and  are disk-shaped,  with  membranes  the  plasma  membrane  food  vacuoles  from  9  nm  former food vacuole a f t e r i t W c l f , 1 9 7 4 ) . The  disks  l i e  10  along microtubules, i n s i n g l e f i l e from  them.  cytostomal the  The  disks  to the l e f t  l i p which i s a r e g i o n s p e c i a l i z e d  for  seguestering  As  new  direct  membrane i s r e q u i r e d f o r e n d o c y t o s i s , the  d i s k s f u s e with th=- growing v a c u o l a r membrane No c r o s s - b r i d g e s between have  been  process result the  observed  in  (Allen,  microtubules  1974).  and  i n the micrctubule-mediated  vesicles  disk recycling  between  microtubules  the d i r e c t e d movement of c e l l u l a r components, as i s with  the  (Eobison, 1966) , (Green,  the  ( A l l e n , 1975).. C r o s s - b r i d g e s  case  40 nm away  the  disks.  microtubules  and approximately  mobility  the  cf  movement  of  non-flagellated granules  1968), chromosome movement during  in  mitosis  melanocytes (Hepler  al.,  1970), and s y n a p t i c v e s i c l e s i n nerve axons (Smith,  as  well  Porter,  as  changes  1967) ,  contraction  tentacles  (Giimestone  Bannister  and  propulsion ciliate,  and  Tatchell,  of  food  ghascoloion  microtubules  in  into  nuclear  and  shape  expansion  of  (Tucker,  cytopharyngeal 1972).  In  1971), and  axostyles  and  E a r d e l e , 1974),  the  et  (Mcintosh  C l e v e l a n d , 1965, Eudzinska  1968,  sperm  and  , 1967, in  the  basket  o f the  P._ caudatum  , the  are f i r m l y bound, a t a d i s t a n c e of 30-40 nm, i n a  f i l a m e n t o u s m a t e r i a l . . The v e s i c l e s c r i e n t with the microtubules but the r e s u l t i n g movement c o u l d be due to breakage  of  linkages  within  p o l y m e r i z a t i o n at one end and  the  The  study  of  formation  filamentous  concurrent  the other end cf the microtubules  the  m a t e r i a l or a  depolymerization  distribution  of  freeze-fracture  8. 5  nm  at  {Allen, 1975). sections  membranes provides a d d i t i o n a l evidence f o r membrane The  and  of  vacuolar recycling.  p a r t i c l e s on t h e two f a c e s of a  11  fractured  membrane  (Branton, 1971, temperature (Pinto  are  Dempsey  can r e s u l t  da  Silva,  caudatum  ,  characteristic et  a l . , 1974).  Changes  i n r e v e r s i b l e clumping  1972,  the  f o r membrane  Speth  and  cytopharyngeal  face  ( A l l e n , 1976),  particulate particles  A  face.  per  Circulating  u n i t - a r e a than  attached  the way  particulate  food  vacuoles  have  fewer  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  than  their  B  face. . This  reversed  When  food vacuole fuses with t h e c y t o p r c c t , the r e s u l t i n g as t h o s e a t t h e  t h e same v e s i c l e s g e n e r a t e d vesicles back  vacuole  t h a t p i n c h o f f from  to  the  (Allen,  Dm . P u r p o s e  formation, the  both  gullet  through e x o c y t o s i s as w e l l as the food  where  indicating  vacuole  make  their  t h e y a r e added t o a new f o o d  thesis  known  food  vacuole  t h e d i g e s t i v e a n d membrane r e c y c l i n g  aspects,  implications  Endocytosis  cytopharynx  disks  1976)..  of this  Much i s new  and  food  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.  h a v e t h e same p o l a r i t y that  1973).. I n  c o u l d be due t o t h e l o w pH o f t h e v a c u o l e o r i t m i g h t  indicate the  or  w h e r e a s t h e p l a s m a membrane h a s a more  f a c e i s more p a r t i c u l a t e polarity  pH  particles  Wunderlich,  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 B  in  of t h e  membrane,  types  about  of  the  this  and i n t r a c e l l u l a r  process  knowledge  cf  are  far-reaching.  d i g e s t i o n are widespread,  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 o f  eukaryotes.  In  protozoa  and  i s responsible  f o r the  lower  ingestion  metazoa,  this  process  and d i g e s t i o n o f n u t r i e n t s *  In vertebrates, the  same  12  type  of  process  degradation phagocytic  cf  foreign  cells  of  e x c e l l e n t organism easily  i s responsible  been  organisms  of  studied  endocytosis  way  organism.  of  in  to understand  and  the  since  i tis  many  aspects  time  of i t s  i n organisms i s an  t h e nature of a given process and  mutants  in  particular  facilitate  the  events t h a t a r e c r u c i a l t o t h e s u r v i v a l of the  The g e n e t i c s of P. t e t r a u r e l i a  understood  by  (Sonneborn, 197C).  temperature-sensitive examination  and  has a s h o r t g e n e r a t i o n  i n d u c t i o n and a n a l y s i s of mutations  excellent  many  (Sonneborn, 1S74)..The examine  study  extsnsively  and  ingestion  immune system. P__ t e t r a u r e l i a i s an  and maintained,  b i o l o g i c a l processes The  the  fora  cultivated  and has  substances  f o r the  endocytosis  mutants purpose and  have of  i s relatively  well-  been i s o l a t e d and s t u d i e d this  thesis  will  be  to  the vacuolar c y c l e i n P. . t e t r a u r e l i a  u s i n g t h e i n d u c t i o n of t e m p e r a t u r e - s e n s i t i v e mutants t o f u r t h e r e l u c i d a t e some of the events o f t h i s  process.  13  Figure  1 - 1 . Morphological  changes  in  food  vacuoles  during  digestion. A. accumulation  and  concentration  of b a c t e r i a a t the base of  the g u l l e t (g) B. .accumulation food  of small v e s i c l e s  (1) around the  newly  formed  becomes  highly  vacuole  C D. as r  digestion  proceeds,  i r r e g u l a r and p i n o c y t o t i c  the  vesicles  membrane  (p) pinch o f f from the  food  vacuole E. the  old  food  vacuole  c y t o p r o c t and the vacuolar  fuses  vith  the  membrane  at the  membrane fragments i n t o v e s i c l e s (v)  15  CHAPTER I I  STANDARDIZATION OF CONDITIONS  A..  Introduction Many f a c t o r s i n f l u e n c e the f o r m a t i c n of food  ciliates: 1942b,  temperature  Nilsson,  (Nilsson,  1972)  (Lee,.  1976), and  (Chapman-Andresen and  1942a,  N i l s s o n , 1968,  m a t e r i a l s (Mast, 1947,  M u l l e r e t a l . , 1965, of  p a r t i c l e s are  Cellular  age  is  Eicketts,  not  fed  also  accompanied by a decrease Andresen  and  Nilsson,  and  nutritional  1972). Starved c e l l s can d i s t i n g u i s h nutritive  N i l s s o n , 1972), pH  centrifugaticn the  media  state  Eicketts,  of  the  1971b,  cells  Nilsson,  Bcsenbaum and  1962,  1971a), as long as the two  types  important;  1968,  (Eicketts,  increased  phagocytic  well  Aufderheide,  non-  Whittner,  1971a).  clonal  competence  Smith-Sonneborn  c e l l s do not form food  N i l s s o n , 1968,  (Lee,  changes  and  age  vacuoles  Eodermel, because  (Chapman-Andresen  1976)..The r a t e of  endocytosis  as the number of vacuoles per c e l l changes throughout  c e l l cycle  (Berger,  1971,  Eicketts,  1971b,  is  (Chapman-  1976). The stage i n the c e l l c y c l e i s a l s o s i g n i f i c a n t dividing  in  between n u t r i t i v e and  simultaneously  in  vacoules  Nilsson,  and as the 1976,  Smith-Sonneborn and Eodermel, 1976) . Tetrahymena such  as  require  peptone-yeast  (Seaman, 1961a),  the broth  proteins,  (RNA) , p o l y s a c c h a r i d e s , and  presence or  the  of i n d u c e r dye,  polypeptides,  glucose  (Eicketts,  substances  trypan  ribonucleic 1972). These  blue acid may  16  bind  at  specific  sites  at  (Seaman, 1961b, R i c k e t t s , The food  concentration  vacuoles  according  the  of  p a r t i c l e s may  their  cells  Pollock,  1980)  diameter of 4 um, vacuoles  result  markers  nutritive  is  nutritive  an  and  in  the  particles  the  medium  Rodermel, 1976, such as  formation  Berger  yeast  of  with a  larger  and  non-nutritive materials vacuoles.  indication  of  The  food  (Mast, 1947),  phagocytic  (Seaman, 196 1b), 1971)  killed  tritium-labeled and  radioactively  ( R i c k e t t s and  Rappitt,  non-nutritive  markers  (Lee, 1 942a,b),  heat  these  r a t e . . Some of stained  Serratia  that  have  Interofcacter  blue  ferritin  utilized:  albumin  India  (Seaman, 1961a,b) ,  and  colloidal  (Favard  and  carmine  p a r t i c l e s (Chapman-Andresen and N i l s s o n , 1968), a l c i a n  Pollock,  1975),  acrylic  communication) , and Therefore, endocytosis, standardized  tantalum colors  watercolors  considering  procedures i n order  to  the  and  and  ink  1964) , c o l l o i d a l  ( N i l s s o n , 1972) ,  (Elliott  beads  of  Carasso,  blue  gold  latex  with  aeroqenes  l a b e l e d sucrose and  been  the  marcescens  1975).,There are a l s o a wide v a r i e t y  trypan  oxide,  are commonly  r a t e of uptake of  markers t h a t have been used a r e : yeast  Red  thorium  in  number of  phagocytize  and l a r g e r p a r t i c l e s ,  used as markers for food  (Berger,  gullet  (Mast, 1947)..  Both  Congo  the  a f f e c t the  concentration  ( R i c k e t t s , 197 1a, Smith-Sonneborn and  cf  1972).  formed... W e l l - f e d to  base  Clemmons, 1966) ,  particles (McEeath  (Berger and  and  (Orias Orias,  personal  P o l l o c k , 1 980).  myriad f a c t o r s  that  influence  c o n d i t i o n s were i n v e s t i g a t e d  reduce,  as  much  and  as  possible,  and 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 p r e p a r a t i o n of Paramecium P..tetraurelia  aurelia  , syngen  inoculated  (Sonneborn , 1975), 4,  with  was  grown  Enterobacter  and  resuspended  fresh culture f l u i d . After  aerogenes  minutes  the  cells  in  an  were  formerly  i n autoclaved  E x p o n e n t i a l l y growing c e l l s approximately centrifuged  tetraurelia Paramecium  grass  medium  (Sonneborn, 1970).  20 f i s s i o n s o l d were  twice the o r i g i n a l volume o f  eguilibration  incubated  period  for 6  of  hours  5-15  a t the  experimental temperature. _ The« c e l l s were then exposed t o a food vacuole marker at the e x p e r i m e n t a l drops  i n a g i v e n time p e r i o d was counted  S e l e c t ion of food vacuole The  (Gunter  in a  few  a b i l i t y of P.. t e t r a u r e l i a Wagner  designer's  0.8 mg/ml carmine p a r t i c l e s carmine  particles  were  to  colors)  phagocytize  watercolors  was compared  with carmine  (EP, P e l i k a n , carmine #34),  (Fisher S c i e n t i f i c )  suspended  i n culture  volumes of marker were mixed with c e l l s i n samples  were removed and f i x e d a f t e r  minutes.  i n 30-50 c e l l s . .  markers  p a r t i c l e s . . 1.0 mg/ml r e d w a t e r c o l o r  40  fixed  of 37% formaldehyde and t h e number of food vacuoles t h a t  accumulated  2«  temperature,  and  1.0  mg/ml  f l u i d . _• Egual  culture  fluid  and  1, 5, 10, 15, 20, 30, and  18  3..  E f f e c t of temperature C e l l s were incubated a t 17°C,  and  then  f e d RP  (1  27°C and 34.5°C f o r 6  mg/ml) or blue watercolor  Ultramarine  #120, 1.5 mg/ml) f o r 20  fixed  the  and  number  of  (BP, P e l i k a n ,  minutes. . The  c o l o r e d food vacoules  hours  cells  were  per c e l l was  counted.  4.  E f f e c t o f time i n marker C e l l s were incubated  RP or BP prepared and  fixed  after  f o r 6 hours a t 27°C or 34.5°C and f e d  as p r e v i o u s l y i n d i c a t e d . Samples were removed 1, 3, 5, 10 ,  15, 20, 30, 40, 50,  and  60  minutes i n the watercolor.  5.  E f f e c t of c u l t u r e medium Cells  by  were adapted t o axenic medium (Keenen e t a l . , 1978)  a m o d i f i c a t i o n of the method  (Appendix  grown  i n bacterized  in  and  medium.. In  Nerad  (1978)  ( P e l i k a n , Mixing addition,  cells  medium, adapted to D r y l ^ s s o l u t i o n  ( D r y l , 1959) (Appendix I) and were  Allen  I ) . They were fed black watercolor  Black #012, 1.0 mg/ml) i n a x e n i c were  of  f e d black  watercolor.  Samples  removed a f t e r 1 , 3, 5, 10, 15, 20, 30, 45, and 60 minutes  watercolor, f i x e d and examined.  19  C.  1  «  Eg s a l t s  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 The o u t l i n e d procedure was r o u t i n e l y f o l l o w e d . In s p i t e o f  these p r e c a u t i o n s v a r i a t i o n s  between  T h e r e f o r e , wild-type c o n t r o l c e l l s  experiments  d i d occur.  were i n c l u d e d f o r comparison  i n a l l experiments. .  2..  S e l e c t i o n of food vacuole markers Bed watercolor (EP) was r e a d i l y phagocytized by Paramecium  cells  and the food vacuoles formed  easy t o count formed in  after  the  (Plate III-1a)..By comparison, t h e food  number of c o l o r e d  A  significantly  vacuoles accumulated a f t e r 20 minutes  watercolor (Figure 2-1) and t h e r e was no d i f f e r e n c e number  vacuoles  i n g s s t i o n of carmine p a r t i c l e s were l e s s r e g u l a r  shape and they were not as c l e a r l y defined;  higher in  were c l e a r l y d e l i n e a t e d and  that  accumulated  in  0.4  or  0.5  between  mg/ml carmine  particles..  3..  E f f e c t of temperature There was an  vacuoles  increase  no  the  mean  number  of  colored  that accumulated a f t e r 20 minutes i n EP o r BP with an  i n c r e a s e i n temperature from was  in  further  increase  17°C t o 27°C at  34.5°C,  (Table  2-1).. There  indicating  a  wide  temperature range f o r maximum e n d o c y t i c a c t i v i t y . There was  no  s i g n i f i c a n t d i f f e r e n c e between the mean number of vacuoles that  20  accumulated  4.  a f t e r 20  Effect  2-3) .  EP.  of time i n marker  There colored  minutes i n EP or  was  an  initial  rapid  vacuoles t h a t accumulated This  increase  was  cases a p l a t e a u at around  increase  i n EP or BP  i n the number of (Figures 2-2  sharper a t 34.5°C than  and  27°C. In a l l  14 v a c u o l e s per c e l l was  reached  which p o i n t an e g u i l i b r i u m s t a t e between the formation and of  colored  vacuoles  was  apparent.  There were no  at loss  significant  d i f f e r e n c e s between c e l l s i n c u b a t e d at 27°C or 34.5°C once  the  steady s t a t e was reached . _  5»  Effect The  of c u l t u r e medium number  of  food  vacuoles that accumulated  medium as well as t h e i r s i z e and  shape were  present  (Figure  in  tacterized  medium  i n axenic  similar  2-4) .  to  those  However, those  c o l o r e d vacuoles formed i n D r y l ' s s o l u t i o n were very small the  mean  interval  number  increased  greatly  ( c . i . ) = 20. 8-24.5,  after  22.6,  60  95%  minutes  confidence in  black  watercolor  (compared with  14.1,  = 12.9-15.2, i a b a c t e r i z e d medium). In both cases an  c.i.  eguilibrium  state  12.9,  to  and  between  c . i . = 12.0-13.7, i n a x e n i c  formation  and  loss  was  and  reached  although i t took longer to reach i n D r y l ' s s o l u t i o n . The  reason  for  Dryl's  the  unusual  results  with  black  watercolor  s o l u t i o n i s unknown. I t i s known t h a t the s i z e of the influences  the  size  of  the  food  vacuoles  in  particles  (Mast,  1947),  21  therefore i t i s possible that black small  p a r t i c l e s . . When  cells  watercolor  in  contains  very  D r y l ' s s o l u t i o n were f e d EP  i n s t e a d of b l a c k w a t e r c o l o r , the mean number of vacuoles formed a f t e r 60  D.  minutes was  10.3  ( c . i . .= 9.1-11.6)..  Discussion Watercolors  are  very  useful  because  they are non-toxic and  clearly  v i s i b l e i n the cytoplasm  of  exposure  formation  to  watercolors  and  loss  of  w a t e r c o l o r s are a r t i f i c i a l and  loss  are  similar  b a c t e r i a are used  the c c l c r e d  a  steady  colored  phagocytosis  vacuoles formed are  o f the c e l l .  Within 20  state  the  between  rate  of  appears  of 27°C and  20°C and  formation  34.5°C are both e x c e l l e n t f o r with  Lee  (1942a)  35°C t h e r e i s l i t t l e  decreases.. N i l s s o n (1972)  or  above  When number  watercolor  between 27°C and  has  endocytosis  who  14  vacuoles  increase i n or  above  a  decrease  In Paramecium i t  per  cell  can  vacuoles  used.  This  be  34.5°C.  c e l l s are f e d w a t e r c o l o r i n D r y l ' s s o l u t i o n the of  has  found t h a t i n  with  t h a t temperature.  t h a t a mean of around  maintained  and  below  these  t o those obtained when t r i t i u m - l a b e l e d  Tetrahymena 28°C i s optimum f o r occurring  between  vacuoles.. Although  t h e r a t e of e n d o c y t o s i s i n P . . a u r e l i a but below 20°C 35°C  minutes  exists  markers, the k i n e t i c s  e n d o c y t o s i s . This i s i n agreement that  for  (Berger and P o l l o c k , 1980). .  The temperatures  found  markers  that  accumulate  variability  can  varies  with  size the  be decreased  i f dead  b a c t e r i a are added to the w a t e r c o l o r i n D r y l ' s s o l u t i o n  (Berger  22  and  Pollock,  1980).  particles  frcm  accumulate  in  In  the  presence  watercolor  and  particularly  advantageous, t h e r e  cells.  of  hours  a t 25°C  mitochondria,  have  lower  are  of  are  medium  would  seem  several  drawbacks,  techniques, the  compared  the these  generation  of the e f f e c t  associated  to  cells  when and grown  condensed  l e v e l s of  ATPase,  a l lcharacteristic  o f a g i n g on  Eodermel,  ten  Prince  have  lower  cytochromes,  cn E n t e r o b a c t e r  endocytosis  1976) , a s  with cultivating  and  w e l l as  of in the  maintaining  c a r r i e d out u s i n g c e l l s  grown  aerogenes.  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 h a v e been  adopted  as s t a n d a r d  cells  approximately  procedure, 20  temperatures  u n l e s s o t h e r w i s e s t a t e d : use  fissions of  27°C  suspended i n c u l t u r e  m i n u t e s as f o o d  shape  axenically  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  watercolors  and  compared w i t h s i x h o u r s  that  ( S m i t h - S o n n e b o r n and  Therefore,  vacuoles that  ( S o n n e b o r n , 1970).-  grown  amounts  problems  incubation  axenic  oxygen c o n s u m p t i o n ,  I n view  monoxenically  size  or  a x e n i c medium i s g r e a t e r t h a n  shown  those  decreased  routine  in  monoxenically  monoxenically,  Paramecium  of  ( T h i e l e e t a l . , 1980)  (1978)  aging c e l l s .  use  mass c u l t u r i n g  Pj_ t e t r a u r e l i a  c e l l s a r e grown Gibson  bacteria  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  E v e n w i t h new  time  and  the  their  Whereas  important  live  a x e n i c medium t h e number o f f o o d  consistant..  most  of  vacuole  markers.  eld and  grown 34.5°C  f l u i d and  of  monoxenically, and  use  of  f e d t o c e l l s f o r 20  23  Table  2-1..Effect  of  temperature  en  the  accumulation  of  vacuoles  BP (2)  Temp. (°C) EP (1)  17. 0  5. 9 (5. 3-6. 4)  6. 1 (5.7-6.5)  27. 0  14.0(12.7-15.3)  15. 1 (14.0-16.2)  34. 5  13. 9(12.9-1 5.0)  14.5(13.0-15.9)  (1) means and 95% c o n f i d e n c e i n t e r v a l s  of the number of c o l o r e d  vacuoles t h a t accumulated a f t e r 20 minutes i n r e d watercolor (2) means and 95% c o n f i d e n c e i n t e r v a l s cf the number o f c o l o r e d vacuoles t h a t accumulated  a f t e r 20 minutes i n blue  watercolor  24  Figure red  2-1. Accumulation  of vacuoles i n carmine  p a r t i c l e s and  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 i n c u b a t i o n time  Vertical  bars  watercolor, mg/ml carmine  at  27°C.  represent 95% c o n f i d e n c e i n t e r v a l s . . (• — • = red =0.4 mg/ml particles)  carmine  particles,  +—+  =  0.5  IS  26  Figure of  2-2. A c c u m u l a t i o n 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 . E f f e c t  i n c r e a s i n g time  and  34.5°C.  V e r t i c a l bars represent 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)  in  red  watercolor  at  27°C  28  F i g u r e 2-3. of  Accumulation  increasing  time  in  of vac uoles i n blue watercolor. blue  V e r t i c a l bars represent 95% •  = 34.5°C)  watercclor  at 27°C and  con f i d e n c e i n t e r v a l s .  (•—•  Effect 34.5°C. = 27°C,  NO. OF COLORED_VACUOL.ES/CELL cn o cn  30  Figure 2-4. Accumulation medium.. E f f e c t V e r t i c a l bars  of  vacuoles  of i n c r e a s i n g time represent  95%  in  buffer  and  axenic  i n black w a t e r c o l o r a t 27°C.  confidence  D r y l ' s b u f f e r , • — • = axenic medium)  i n t e r v a l s . . (•--•  =  31  32  CHAPTER I I I  MUTAGENESIS AND DESCRIPTION CF PUTATIVE MUTANTS  A. . I n t r o duct ion Phagocytosis i n v o l v e s the  is  activities  components.  Each  potentially  mutable  sensitive  an  (ts)  elaborate and  aspect  biological  interactions  process  of  many  event.  The  mutations  selection  makes  of  have  t o feeding  been  temperature  i t possible because  can be maintained at the p e r m i s s i v e temperature. endocytosis  cellular  o f t h i s complex process r e p r e s e n t s a  indispensible functions related  affect  that  isolated  to  examine  the  cells  Mutations that in  Tetrahymena  th ermophila  (Orias and P o l l o c k , 1975, S i l b e r s t e i n e t a l . , 1975,  Suhr-Jessen  and O r i a s , 1979a,b) and have been found  with  other  mutations  Mutagenesis was undertaken  in  P. t e t r a u r e l i a  to r e c o v e r mutations  associated  (Jones,  1977).  involving  some  aspect of endocytosis or food vacuole p r o c e s s i n g .  B. . M a t e r i a l s and Methods  1.  Mutagenesis Mutagenesis was performed  Cells  approximately  centrifugation Dryl's  50  a c c o r d i n g t o Kung et a l . (1971).  fissions  o l d were  (100xg, 3 minutes) and  solution  containing  75  concentrated  resuspended  ug/ml  in  100  N-methyl-N'nitro-  by ml N-  33  nitrosoguanidine (determined fluid)  by  were  (MNNG, serial  treated  temperature.  After  Sigma).  A  total  of  3 x 10  cells  5  d i l u t i o n of 1.0 ml of c e l l s i n c u l t u r e with  three  MNNG  f o r 60  minutes  at  washes i n D r y l ' s s o l u t i o n  the c e l l s  were suspended i n s u f f i c i e n t c u l t u r e  f l u i d to  four  and the onset of autogamy,  divisions  which r e s u l t s of  cell  death  before  starvation  i n homozygosis o f any induced  growth  and  was  autogamous  cell  prior  to  determined lines.  isolated  three  mutations.  to  Failure  autogamy was c o n s i d e r e d v e g e t a t i v e by  examining  Failure  84  isolated  pre-  o f c e l l growth subseguent t o  autogamy was considered ex-autogamous recessive  allow  room  death,  attributable  to  l e t h a l mutations, and was determined by examining 84 ex-autogamous c e l l  2.- S e l e c t i o n  lines.  procedure  Ex-autogamous l i n e s were t r e a t e d i n one of  the  following  ways:  a..  individual 1,26C  line  cell  selection  l i a e s were i s o l a t e d  a f t e r approximately 20 c e l l d i v i s i o n s is  the  maximum  drops  of  blue  (12  cell  slides  divisions  observed phencmic l a g , Berger, 1 976) . The  c e l l s were r e p l i c a p l a t e d set of i s o l a t e s  i n depression  (Sonneborn, 1 970)  was s h i f t e d watercolor  and the second  t o 34.5°C f o r 24 hours. .A few (EP)  l i n e . . T h e c e l l s were f i x e d a f t e r  were added to each  cell  20 minutes and each clone  was scored i n d i v i d u a l l y f o r a b n o r m a l i t i e s i n  size,  shape  34  a n d number o f f o o d v a c u o l e s . C e l l s t h a t were a b n o r m a l retested  w i t h BP and r e d w a t e r c o l o r ( H P ) . .  b.. mass  selection  This  procedure  synchronization modified  were  method  by  Aufderheide  was  based developed  ten-fold  the density-labeling by  Silberstein  (1976) . C e l l s  diluted  on  Wolfe  et  (1973)  were  minutes)  and  20  old  approximately i n culture fluid  fissions  and i n c u b a t e d a t  containing Bell, and  by  centrifugation  resuspended  in  5  then  The  cells  layered consisting  on  a  at  300xg.  endocytosis  to  resuspended  grow a t 27°C f o r 3-4  repeated.. Five which  ( M a t h e s o n , Coleman and  was  f o r 10  minutes  centrifuged  (Sigma) Ficoll for  5  t h a t had i n g e s t e d t a n t a l u m  i n the  pellet  and  those  with  were e x p e c t e d t o be l o c a t e d a t t h e  10%/ 2 0 % i n t e r f a c e . _ C e l l s collected,  3  r  solution  discontinuous F i c o l l  Those c e l l s  time  (100xg  o f 4 ml o f 20% and 4 ml o f 10%  were e x p e c t e d t o s e d i m e n t decreased  which  Dryl's  were g e n t l y s h a k e n  Dryl's solution..The gradient  minutes  ml  15 mg t a n t a l u m p a r t i c l e s  Co.).  gradient in  concentrated  and  and  a l . . (1975)  34.5°C f o r 24 h o u r s i n an a i r i n c u b a t o r a f t e r they  were  found  at  the  interface  i n fresh culture fluid days  enrichment  before cycles  2 10 p o t e n t i a l m u t a n t l i n e s  the were  and a l l o w e d  procedure performed  were i s o l a 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 ) . .  were  and  was after  tested  35  3.  D e s c r i p t i o n of p u t a t i y e mutants A  total  of  61  ts  putative  (a)..Of t h e s e ,  mutants 11,  were  denoted  i s o l a t e d by  selection  procedure  A1-A11,  were  selected  f o r f u r t h e r study..The f o l l o w i n g c h a r a c t e r i s t i c s were  ex am i n e d : a. . generation time at 34. 5°C (Nachtwey and Cameron, 1972) b. _ number of food vacuoles t h a t accumulated i n EP in  morphology  determined  ( F r a n k e l and Heckmann,  by  s i l v e r impregnation  1968)  d. t r i c h o c y s t d i s c h a r g e determined by adding a a  saturated p i c r i c a c i d  (Pollack,  few  drops  s o l u t i o n t o a sample o f c e l l s  1974)  e. .endocytosis i n synchronous samples - synchronous of  the  synthesis cells  ts  mutant  4Y-2 1,  (Peterson, 1974),  which were  is  deficient  obtained  by  in  DNA  collecting  for  20, 40, 60, and  100  minutes  2, 2.5, 3 , 3.5, 4, 4.5, and 5 hours. BP was added minutes,  the  cells  gelatin  or  albumin and photographed using Nomarski  optics..  and for  20  were f i x e d and the number of c o l o r e d  vacuoles per c e l l was counted. F i x e d c e l l s saline  cells  w i t h i n 10 minutes o f d i v i s i o n . These were s h i f t e d t o  34.5°C  in  BP  20 minutes  c. g u l l e t  of  and  air-dried  were  embedded  on g l a s s s l i d e s coated with  on a L e i t z Orthomat-W  photomicroscope  36  4.. G e n e t i c a n a l y s i s Two  putative  temperature  resistant  behavioral  marker  generation  (F2)  inducing  mutants,  A5  line  segregation  for  patterns  cross. The l i n e  manner  temperature  were  i n . the  the  discharge  (A10)  Results  1.  Mutagenesis  line  of  the  death  toxicity  frequency of ex-autogamous death freguency  experiment  of  lethal  v e g e t a t i v e death was  28%. These experiments rate  freguencies  were  (Morton, 1977)  varied  accumulation  food  (A5), and  of  vacuole  trichocyst  an  indication  mutations. In t h i s  of  low  in  although  comparison the  experiments  autogamous death r a t e s y i e l d e d  more p u t a t i v e  the  mutagenesis  23% ard ex-autogamous death  general,  In  mutagenesis  of the mutagen and the  experiments  1977).  greatly  gave  following  between  Morton,  same  homozygous f o r the an  or p o s i t i o n i n the c e l l  indication  relative  i n the  (Morton, 1977)..The progeny of the  The freguency of v e g e t a t i v e an  by  (A5) .  C.  gave  second  determined  c r o s s e s were scored f o r the pawn or spot markers, morphology  recessive  1971). The  A5 was a l s o crossed  resistant  cytoplasm  crossed t o a  g e n e r a t i o n (F1) c l o n e s produced  r e c e s s i v e spot Jspi_ mutation which caused crystals  were  pawn A lpw]_ (Rung et a l . ,  by the i n i t i a l a  A10,  homozygous  autogamy i n the f i r s t  to  and  to  was  similar  ex-autogamous death (Peterson, 1974, with  higher  mutants.  ex-  37  2.  Y i e l d of p u t a t i v e mutants Through  putative  the  individual  mutants were i s o l a t e d  s e l e c t i o n procedure 21/2 10  =  105?),  than expected or  3.  61  ( r a t e = 61/1260 = 5%)..The mass  r e s u l t e d i n 21  a  procedure  putative  mutants  (rate  two-fold i n c r e a s e . T h i s i n c r e a s e was  because the procedure  also selected f o r  =  lower  dividing  (Aufderheide,  1976).  D e s c r i p t i o n of p u t a t i v e mutants  listed the  characteristics  of  i n Table 3-1.„Of these,  most  study  dramatic  and  (the l i n e , A7,  was  A5  a ts c e l l  (Peterson, EP was  11  putative  A5,  A7 and A10  and  lost).  mutant l i n e s have been l i s t e d  in  selection  c o n j u g a t i n g c e l l s and c e l l s i n autogamy  The  was  line  A1C  mutants were  have been considered  were s e l e c t e d f o r f u r t h e r  B r i e f d e s c r i p t i o n s of the  other  i n Appendix I I . The mutant  c y c l e mutant with e r r a t i c DNA  4Y-21  s y n t h e s i s at 34.5°C  1S74) . The mean number of c o l o r e d vacuoles per 4.14  significantly  {95% lower  confidence than  interval,  wild-type  cell  c. i . = 3.81-4.47),  cells  (Table  3-1).  In  synchronous p o p u l a t i o n s s h i f t e d t o 34.5°C a gradual decrease i n t h e number of food vacuoles t h a t accumulated o c c u r r e d a f t e r  the  temperature s h i f t  2A2  and  2A5  at  34.5°C  (Figure 3-1).  ether t s DNA-  mutants,  (Peterson, 1974), a l s o formed fewer food v a c u o l e s i n EP (means  and  3. 42, 2.8-4. 04 for 2A5) after  Two  division  vacuoles to 6.S6  also  95% c . i . =  5. 24,  Synchronous samples of 2A2 showed  a  decrease  (c.i.= 6. ,33-7.59) a f t e r  r e s t r i c t i v e temperature  3. 4- 7.08  [Z.Easmussen,  in three  f o r 2A2 shifted  and up  numbers of food hours  at  the  personal communication).  38  4. . Genetic a n a l y s i s  a. . A10 x pawn A The  first  generation  (F1) i n d i v i d u a l s o f t h i s c r o s s  were a l l wild-type. The s e g r e g a t i o n p a t t e r n o f the ( F 2 ) was c o n s i s t e n t with a r e c e s s i v e  generation at a s i n g l e  locus  designated this  (Table  defective  mutant  have  3-2). . T h i s  membrane  been  mutant  second mutation  has  been  or dml. The phenotypes o f  schematically  illustrated  in  F i g u r e 3-2.  b.  A5 x pawn A Two  crosses  were performed and i n both  progeny were a l l wild-type..The (Table  3-3)  were  r e c e s s i v e mutation vacuoles  and  segregated  in  probabilities  not  consistent  affecting  trichocyst a  1:1  F2  both  =  significant  homogeneity,  segregate  a  single  clumping  The  Chi  of  gene food  Each  locus  sguare  (X ) 2  .99,.5,  probability  (P)  trichocyst  non-  vacuole clumping (fvc) <.05  was  considered  f o r r e j e c t i o n of the o r i g i n a l h y p o t h e s i s ; The  data from both  presented  the  Jtnd)_ = . 1 5 , . 33 and food  .15,.28.. A  with  patterns  t o as P) f o r each l o c u s f o r each  of t h e two c r o s s e s were: pawn = discharge  segregation  non-discharge..  fashion.  (referred  cases the F1  crosses P  =.71)  were and  homogeneous the  pooled  (X  2  data  i n Table 3-3. The c r o s s o v e r phenotypes i n a 1:1:1:1 f a s h i o n  of pawn c e l l s with clumped  test have  for been  d i d not  (P = .02) due t o an excess  vacuoles. In order to determine  39  if  t h e .pawn p a e n o t y p e was  cells  c.  w i t h clamped  A5  x  i n t e r f e r i n g w i t h the  vacuoles,  A5  was  crossed to  F l p r o g e n y were a l l w i l d - t y p e . The  patterns  have  been  presented  recombinants segregated indicating  t h a t the  in  in  a  increase  morpholcgical  in  the  with the  two  A5  and  the  34±3 ( s t a n d a r d  resulting fvc  and  from t h e  A5  fashion  abnormalities  sometimes  wild-type  and  consistant  t h e F2  with  x  spot  cross  abnormal The fvc  cells  as having  is  and  map  units.  t o s j r o t . The  and  The  F1  patterns  (Table  c h e c k o f 60 F2 no  cells  c h e c k o f 60 F2  morphological  but  abnormalities. .  be  3-5)  and  as  were single  clumped  l i n e s from  may  tnd  l i n e s from  with  l i n e s e a c h w i t h two  known  lines  p r o g e n y were a l l  fj/c each s e g r e g a t i n g  yielded  two  were d e s i g n a t e d  t h a t d i d not d i s c h a r g e  not  clumped  homogeneous  the  the food  fvc  x  morphologically  their  trichocysts.  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 cells  an  combined l i n k a g e between t n d  segregation  c r o s s y i e l d e d two  caused  .56  x spo_t c r o s s  tnd  vacuoles..A s i m i l a r spot  34.5°C  for parentals,  gene r e c e s s i v e m u t a t i o n s . . A tnd  at  (F = .82  deviation)  were e a c h c r o s s e d  F2  =.6)  o f t h i s c r o s s were  x pawn c r o s s e s  for recombinants)  segregation  3-4. . The  (P  number o f l i n e s s c o r e d  vacuoles..The r e s u l t s  was  spot.  F2  Table  1:1:1:1  a s s o c i a t e d w i t h t h e pawn p h e n o t y p e  fvc  of  spot  The  food  scoring  related  in to  these their  40  D.  Discussion The  mutants o b t a i n e d  f o u r main  i n t h i s study  can  be  divided  into  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  in  this  a b n o r m a l i t i e s which  2-  Decreased  interfere  any  interfere  obvious  fewer  food  food  normal  cf food  vacuoles  but t h o s e  It  interesting  A9,  f v c , A8  A4,  without  that  would  4 Y - 2 1 , 2A2,  2A5  not  be  atypically accompanied  vacuoles o r may are  not  form  n o r m a l number o f  abnormal  in  size  and/or  A1 1  t h a t no l i n e s h a v e been i s o l a t e d  are d e f e c t i v e i n e x o c y t o s i s . I f  exccytosis  is  H o w e v e r , i f t h e r e i s a l i m i t e d s u p p l y of  vacuoles,  which  might accumulate rate  of  is  recycled  and  turnover  of  an  might  membrane  (Allen,1974) ,  i n a g i v e n p e r i o d o f t i m e due  exocytosis  which  inhibited,  i n t h e numbers o f f o o d v a c u o l e s t h a t a c c u m u l a t e  be e x p e c t e d .  decreased  A6,  eg.  formed  djn_1, A2,  vacuoles  A3,  abnormalities  c r may  i n endocytosis.  s h a p e . . ecj. is  vacoules than  T h i s may  M u t a n t s i n t h i s g r o u p may  food  A7  vacuoles  4. A b n o r m a l m o r p h o l o g y o f f o o d  for  gullet A1,  v a c o u l e s of mutants i n t h i s group are  within the c e l l .  by a d e c r e a s e  increase  and/or ecj.  w i t h e n d o c y t o s i s . , ecj.  distributed  food  cellular  with endocytcsis.  morphological  3. A b n o r m a l p o s i t i o n i n g The  have  endocytosis  These mutants form having  group  fewer to  the pool  the of  41  membrane a v a i l a b l e f o r e n d o c y t o s i s . mutants  i n group  Due  by  observing  the  loss  is  Group  time-saving  (1) could be and  gullet  procedure  P_ t e t r a u r e l i a  initially, range  the mass  selection  individual selection i s  of mutants.  subdivided  to  differentiate  (1977) has d e s c r i b e d a t s  with  an  abnormal  e n d o c y t o s i s which i s due t o both the  between  gullet  decreased  g u l l e t . . Using a mass s e l e c t i o n system  small  mutant  and  decreased  size  and the  s i m i l a r t o the  one p r e v i o u s l y described, Suhr-Jesscn and O r i a s (1 979a,b) recovered  fall  a b n o r m a l i t i e s although i n some cases the  two are r e l a t e d . .Jones  abnormal  as t h e  (1) or (2) whereas those s e l e c t e d i n d i v i d u a l l y a r e  r e g u i r e d t o obtain a complete  of  well  the m a j o r i t y of  system  d i s t r i b u t e d among the f o u r groups..Although  cellular  as  to the nature of the s e l e c t i o n system  group  procedure  of the  of c o l o r e d v a c u o l e s .  mutants i s o l a t e d by the mass s e l e c t i o n into  some  (2) could i n f a c t be e x o c y t o s i s mutants. T h i s  c o u l d be determined accumulation  Therefore,  13 t s mutants i n T._  phagocytosis apparatus.  due  to  defects  have  thermophila which have decreased in  the development of the o r a l  Most of these mutants have a m o r p h o l o g i c a l l y  normal  but n o n - f u n c t i c n a l o r a l apparatus and a l l but one belong t o the same complementation Three  group,  vac A.  DNA- mutants ( P e t e r s o n , 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 a l s o e x h i b i t a decrease i n DNA s y n t h e s i s (Smith-Sonneborn (Smith-Sonnebcin  and and  K l a s s , 1974)  Rodermel, 1976)  between the two i s not known. The which  does  not  fall  and  mutant  endocytic but of  capacity  the  relationship  T._  thermophila  i n t o the vac A complementation  group has  42  characteristics similar division and  ceases  to  the  DNA-  mutants;  f o r example,  a f t e r t r a n s f e r to the r e s t r i c t i v e  e n d o c y t i c capacity i s g r a d u a l l y l e s t . T h i s mutant  been  tested  f o r DNA s y n t h e s i s at the r e s t r i c t i v e  Mutants i n group (2) may prove important  in  The  mutant  recessive  fvc in  mutation  vacuoles  group  that  (3)  results  in  ncn-discharge. . There  cases of l i n k a g e reported i n there  identified  the  clumping  are  170  vacuoles  which  been  tetraurelia  ( D i p p e l l , 1954),.  in  researchers  mutation  have  presumptively  the  to  There  micronucleus  l i n k e d genes or there may  The  be  undertake  is  ts for  only e i g h t  other  different  genie  loci  may  be  some  features  t h a t preclude the d e t e c t i o n of a  reluctance  such  on  projects  the  part  of  (T.M. Sonneborn,  communication).. mutant that  dm!  i  results  f  l  group  in  (4)  the  carries  abnormal  a  ts  recessive  morphology  g i v i n g the appearance of a mass of d i s r u p t e d  i n t e r a c t i o n s during  valuable  food  which i s s u r p r i s i n g  T h i s mutant should be u s e f u l i n the study of  The  of  (T.M. Sonneborn, p e r s o n a l communication) and 43 ± 2  chromosomes  personal  and aging.  i n ere area o f the c e l l , u s u a l l y the posterior*. I t i s  trichocyst  inherent  temperature.  c a r r i e s a t s s i n g l e gene  l i n k e d by 34 ± 3 map .units to t h e t n d locus  since  has not  i n v e s t i g a t i n g the  r e l a t i o n s h i p s between DNA s y n t h e s i s , phagocytosis  of food vacuoles.  membrane-membrane  phagocytosis.  mutants i n groups f o r examining  (3) and (4) are p o t e n t i a l l y the most the  during p h a g o c y t o s i s . . T h e r e f o r e , will  temperature  intracellular the  remainder  events t h a t of  this  cccur study  be based on f u r t h e r i n v e s t i g a t i o n and a n a l y s i s of the two  43  mutants f v c and dmj_.  44  Table  3 - 1 . _ P h e n o t y p e s of p u t a t i v e  mutants  L i n e G T (1)BP (2)  DE(3)  BP(4)  Remarks  wild5. 3 type  13. 9 (12.9-15.0)  0.00  5.2 (4.4-6.0)  0.27  5.8 (5.1-6.5)  chains, misdividers gullet-abnormal cells-large vacuoles-abnormal gullet-normal  11.6 Elate (10.5-12.8)  III-1a,b  A1  6.8  A2  5. 6  10. 1 (9.2-11.1)  0.09  6.2 (5.3-7.1)  A3  5.6  1 1.7 (10.6-12.8)  0.00  6.6 (5.7-7.4)  A4  5.5  9. 4 (8.7-10.1)  0.00  6.3 (5.2-7.3)  cells-large vacuoles-large  A5  6.6  9.7 (8.6-10.8)  0.00  7.3 (6.5-8.1)  Elatelll-1d;cells-large vacuoles-clumped gullet-normal trichocysts-nondischarge  A6  5.6  6. 7 (6.2-7.3)  0.00  5.9 (4.9-69)  A7* >20  2.4 (1.9-2.9)  0.62  2.8 (2.0-3.6)  cells-large,monsters  .  ...  continued  45  Table 3-1, cont. Phenotypes of p u t a t i v e mutants LineGT (1)fiP (2)  DE(3) BP (4)  Remarks  A8  nd  7.7 (7.C-8.4)  0.00  6. 1 (5.2-7.0)  vacuo l e s - c l u mped  A9  5.8  8. 1 (7.2-8.9)  0.00  6. 1 (5.6-6.7)  A1 0 5.5  12.0 (10.5-13.5)  0.30  7.8 (6.9-8.7)  Plate III-1c vacuoles-disrupted gullet-norma 1 t r i c h o c ysts-normal  A11 5.3  8.2 (7.C-9.4)  0.00  8.0 (7.0-8.9)  vacuoles-disrupted  (1) GT = generation time ( i n hours) a t 34.5°C (2) RP = means and 95% c o n f i d e n c e i n t e r v a l s of the number colored vacuoles that accumulated after 20 minutes i n w a t e r c o l o r at 34.5°C: (3) DE = the f r a c t i o n of c e l l s t h a t had no c o l o r e d vacuoles 34.5 °C (4) BP = means and 95% c o n f i d e n c e i n t e r v a l s of the number c o l o r e d vacuoles t h a t accumulated i n blue w a t e r c o l o r after minutes at 34.5°C * t h i s l i n e has been l o s t nd = not determined  of red at of 20  Table 3-2. A10 x  pawn .F2.  Phenctype  Number  £W+;  dmj+  16  £ W  dmj  23  +  ;  £w  ; drnj*  17  £w  ; dm 1  8  Total X 2  (1)  P (2)  64 7.12 0.07  pw = pawn phenotype at 27°C; p_w+ = wild-type dmj = d i s r u p t e d vacuoles at 34.5°C; dml* = w i l d - t y p e (1) C h i Sguare Test f o r goodness of f i t (2) p r o b a b i l i t y geaerated  by C h i Sguare Test  47  T a b l e 3-3. A5 x  pawn .F2.  Phenotype  * 2** ; tnd + ; _pw,I tnd+ pw+ ; t n d ; * £2. ;tnd + ; * pw ; tnd ; pw ; tnd*, pw ; tnd * -E" ;'tnd +  +  fvc+ fvc f vc+ fyc+ fvc f vc fvc  To t a l X (1) P (2) X Parentals P X Recombinants P No. p a r e n t a l s No. recombinants Linkage 2  2  2  cross#1  crcss#2  pooled data  32 18 10 25 26 20 9 31  33 13 14 29 33 20 9 26  65 31 24 54 59 40 18 57  171 25. 05 0. 007 1.30 0. 73 6. 51 0. 09 114 57 33. 3fc  177 28.25 <.001 1. 15 0.77 4.41 0.22 121 56 32.0%  348 50.45 <.00 1 1.09 0. 78 9.56 0.02 235 113 32.5%  pw = pawn phenctype a t 27°C; pw = wild-type tnd = t r i c h o c y s t non-discharge a t 34.5°C; tnd+ = wild-type f v c = clumped vacuoles at 34.5°C; J v c = wild-type * p a r e n t a l phenotypes f o r tnd and f_yc a l l e l e s (1) C h i Square Test f o r goodness of f i t (2) p r o b a b i l i t y generated by C h i Sguare Test +  +  Table 3-4..A5 x  spot . F2.  Phenotype  Number  * sp*,, tnd + S£  +  s£ ; +  * *  34 15 21 30 31 20 15 26  fvc* tnd*. f vc tnd , fvc*  1 tnd*-, f y_c + ; tnd ; f v c fvc ; tnd* sp ; tnd ; f y c * * sp ; tnd ; f v c S£ S£  +  Total X (1) P (2) X Parentals P X Recombinants P No. p a r e n t a l s No. recombinants Linkage  192 15.69 0.03 1.14 0. 77 1.89 0.60 121 71 37%  2  2  2  phenctype a t 27°C; s p = wild-type tnd = t r i c h o c y s t non-discharge at 34.5°C; tnd+ = wild-type f v c = clumped vacuoles a t 3 4 . 5 ° C ; f_yc = wild-type * p a r e n t a l phenotypes f o r tnd and f v c a l l e l e s (1) C h i Sguare Test f o r goodness of f i t (2) p r o b a b i l i t y generated by the C h i Sguare Test +  +  49  Table 3-5. ., tnd x spot  tnd  ; f v c x spot . F2.  x spot  Phenotype  sp *•; tnd  fvc Number  x spot  Phenotype  Number  56  sp*-; f v c  sp*; t n d  38  sp ; fvc  48  sp ; t n d *  55  sp ; fvc*-  48  sp ; t n d  51  sp ;  43  +  Total X2  (1)  P (2)  200  +  200  4.12  X2  3.56  0.25  P  0.31  = t r i c h o c y s t non-discharge  f v c = clumped  fvc  Total  sp = spot phenotype at 27° C; s p tnd  61  +  +  = wild-type  at 34.5°C; tnd*- = w i l d - t y p e  vacuoles a t 34.5°C; f_vc  +  = wild-type  (1) C h i Sguare Test f o r goodness of f i t (2) p r o b a b i l i t y generated  by C h i Sguare Test  50  Figure  3-1.  Accumulation  of vacuoles i n 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  CJi  V A C U O L E S / C E L L  O  52  Figure  3-2. Schematic  illustration  represent blue watercolor  a. wild-type c e l l  b, c..some  o f t h e dml  Dots  (BP) .  (m = mouth, f v = food  vacuoles  phenctype.  appear  normal,  vacuole)  others  are d i s r u p t e d and  appear to fuse  d,e.  no normal vacuoles are present and some  are v i s i b l e  i n the cytoplasm  particles  of  BP  53  54  III-1.  Plate  Wild-type  34.5°C. A l l c e l l s slides  and  were f i x e d  wild-type cells  i n red  b.  wild-type  i n blue  dnM  visible  cells  cells  in  blue  near the p o s t e r i o r  d. . A5 c e l l s  i n blue  i n the p o s t e r i o r  cells  in  i n f o r m a l d e h y d e and  (a-c) c r embedded i n g e l a t i n  a.  Cm  mutant  at  air-dried  on  vacuoles  are  (d) .  watercolor  watercclcr  watercolor.  Disrupted  of the c e l l .  w a t e r c o l o r . Clumped v a c u o l e s  of the  watercolor  cell..  are  visible  56  CHAPTEE IV  FUBTHEB CHAEACTEEIZATION OF TEE MUTANTS dmj. AND f v c  A.  Introduction In  order  to  further  investigate  mutants dm_1 and fvc (or A5 mutations),  the  were compared watercolor at on  food  which  vacuoles  the phenotypes  carries  the  tnd  o f the  and f v c  of wild-type and mutant  cells  under a v a r i e t y of c o n d i t i o n s ; The e f f e c t of blue  (BE), the accumulation and l o s s of c o l o r e d  vacuoles  27°C and 34.5°C and the e f f e c t s of i n c r e a s i n g time at 34.5°C synchronous  cells.  cells  were  determined  in  wild-type and dmj  The e f f e c t s of i n c r e a s i n g time at 34.5°C on asynchronous  c e l l s , the r e t u r n t o 27°C a f t e r the the  were determined i n w i l d - t y p e , dml and f v c c e l l s . .  E f f e c t o f time i n BP  to  Wild-type and dml. c e l l s were incubated at 27°C for  a  mutant  temperatures  1.  cells  the  and  M a t e r i a l s and Methods  of  of  phenotypes  B.  responses  induction  range  or  of  34.5°C  6 hours and f e d BP f o r 1 , 3, 5, 10, 15, 20, 30, 40, 50, o r  60 minutes. The c e l l s  were f i x e d  vacuoles that accumulated disrupted  vacuoles  were  and  the  number  of  colored  per c e l l and the number o f c e l l s determined.  In  with  t h i s and subseguent  experiments the mean number of vacuoles i n 30 c e l l s and the 95%  57  confidence i n t e r v a l s cells  (c.i.)  were c a l c u l a t e d . Between 30 and  were examined f o r the presence  clumped vacuoles when f v c or A5 c e l l s Sguare  (X )  with wild-type and mutant c e l l s was  homogeneity  considered  (when  only  c o r r e c t i o n f a c t o r was disrupted  c f d i s r u p t e d vacuoles (or were examined).  The  Chi  t e s t f o r homogeneity between the r e s u l t s obtained  2  (P) < 0.05  50  vacuoles  was  performed  significant  two  and  for  rejection  of  s e t s of data were compared Yates'  employed). Ihe frequency was  a probability  calculated  of  cells  with  by d i v i d i n g the number of  c e l l s with d i s r u p t e d vacuoles by the t o t a l number of c e l l s t h a t formed c o l o r e d food vacuoles i e . . these c e l l s  that  t  forming  2..  were  not  food vacuoles were excluded. .  Loss of c o l o r e d vacuoles a f t e r removal frpm Wild-type  for  6 hours,  was  removed  and  dm1  BP was and  cells  at  were incubated at 27°C or 34.5°C  added f o r 20 minutes and a sample f i x e d . . The  v o l u m e t r i c f l a s k t h a t was maintained  BP  27°C  c e l l s were decanted  then f i l l e d  with  i n t o a 50  culture  or 34.5°C. A f t e r 5 and  (time 0)  fluid  ml 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 t r a n s f e r e d to a 25 ml volumetric f l a s k t h a t was  also  filled  with c u l t u r e 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 separated  from  Additional minutes  the  samples  geotactic,  w a t e r c o l o r , which remained a t the were  taken  a f t e r removal from BP,  vacuoles per c e l l  negatively  and  the  15, 20, 30,  40, 50,  were  bottonu and  60  f i x e d and the number of c o l o r e d  number  cf  cells  with  disrupted  58  vacuoles were determined. .  3.  E f f e c t of c o n c e n t r a t i o n of BP Wild-type  hours  and  and  were  dm1  cells  f e d BP  at  were incubated at 34.5°C f o r 6 a  final  concentration  0. 75, 1. 0, 2.5, 5.0, 7.5, 10.0, 25. 0, 50.0, optical  density  Spectronic culture fixed  20  fluid after  of  2.5  mg/ml  EP  was  spectrophotometer  or 75.0 mg/ml. The  determined  with  (wavelength = 660)  without w a t e r c o l o r as a  of  blank.  The  a  using  cells  were  20 minutes and t h e number of c e l l s with d i s r u p t e d  vacuoles was determined. ..  4.  E f f e c t o f time at 34_,5£C  a. . asynchronous  cells  Wild-type, dmj, f v c and A5, 34.5°C  and  after  cells  were  shifted  1, 2, 3,, 4', 5 and 6 hours samples  removed, fed BP f o r 20 minutes, number  cells  cf colored  vacuoles  fixed,  and  to were  examined.. The  per c e l l and the number o f  with d i s r u p t e d or clumped vacuoles were determined.  b. . synchronous  cells  Twenty to 50 d i v i d i n g collected  wild-type  dm_1  pairs  sample  were  shifted  t c 34.5,°C f o r 1, 2, 2.5, 3, 3.5, 4, 4.5,  hours. . D i v i d i n g  within  and  dm1 c e l l s  per  10 minutes of d i v i s i o n and  were a l s c  collected,  5, or 6 incubated  59  at or  27°C f o r 3.5 hours and then s h i f t e d 2 hours. The c e l l s number  were f e d BP f o r  and  the  cell  and the number of c e l l s  to 34.5°C f o r 0 20  minutes,  f  1  fixed  of c o l o r e d vacuoles that accumulated per with d i s r u p t e d vacuoles  were  determined.  5.  Down-shift t o p e r m i s s i y e temperature Wild-type,  dmj., f v c and A5 c e l l s  f o r 6 hours a f t e r which time the c e l l s Samples  were  were incubated at 34.5°C were  shifted  with d i s r u p t e d or clumped  6. „ E f f e c t o f i n c r e a s i n g Wild-type,  dm1_,  and the  20  number  vacuoles was determined.  temperature  f v c and A5 c e l l s were incubated a t 33.5,  34. 1 , 34. 3, 34.5, 34. 7, 34. 9, and 35. 1°C f o r 6' hours, for  27°C.  removed a f t e r 0 , . 3 3 , .67, 1, 2, 3, 4, 6, 8, and  16 hours at 27°C, f e d BP f o r 20 minutes, f i x e d , of c e l l s  to  minutes, f i x e d , and the number of c e l l s with  fed  BP  disrupted  or clumped vacuoles was determined.  C. . Be s u i t s  1. E f f e c t  of time i n BP  There was an i n i t i a l colored 27°C  rapid  vacuoles t h a t accumulated  increase  in  the  number  of  with i n c r e a s i n g time i n BP at  and 34.5°C i n w i l d - t y p e and dml c e l l s  (Figures 4-1 and 4-  60  2).  A plateau  was  establishment of  vacuoles..  formed  34.5°C. A f t e r  in  Taere  minutes  indicating  The  o f dmj. c e l l s  freguency  differed with  individual  wild-type  (Table  i n EP g r e a t e r indicated  4-1).  was between  than 5  minutes.  t h a t e x p o s u r e s o f 24 wild-type  dml. c e l l s . .  2..Loss of c o l o r e d vacuoles At  after  27°C t h e l o s s o f c o l o r e d  c e l l s was c o m p l e t e by one hour minutes  (Figure 4-4).  vacuoles  The  was i n i t i a l l y  removal frcm vacuoles  exocytosis  increased  ( F i g u r e 4-3) and a t 34.5°C by 20  rate  greater  of  of  colored  a t 34.5°C t h a n a t 27°C  (Figures  at the higher  f r o m BP t h e number o f dml 34.5°C was s i g n i f i c a n t l y minutes  disrupted exocytosis freguency  (Table vacuoles cf  BP  i n w i l d - t y p e a n d dml  accumulation  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 c f b o t h  15  food  a t 27°C and  h o u r s a t 27°C c r 2 h o u r s a t 34.5°C were n o t t o x i c t o or  and l o s s  fewer  disrupted vacuoles  cells  the  i n BE t h e number o f dml. c e l l s from  and .71 f o r i n c u b a t i o n t i m e s with  significantly  i n wild-type c e l l s  3 minutes or l o n g e r vacuoles  Studies  20  were  dml. t h a n  with disrupted  .57  after  o f an e q u i l i b r i u m b e t w e e n t h e f o r m a t i o n  colored  vacuoles  reached  cells  endocytosis  temperature.  with  disrupted  After  and  removal  vacuoles  at  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  4-2) . Howe v e r , t h e f r e q u e n c y decreased  disrupted  steadily  vacuoles  a s e x o c y t o s i s of normal  occurred  vacuoles.  of c e l l s  indicating with  the  with that same  61  3 . . E f f e c t of c o n c e n t r a t i o n of BP At a l l c o n c e n t r a t i o n s of BP t e s t e d t h e number of dmj c e l l s with d i s r u p t e d 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  type c e l l s  At c o n c e n t r a t i o n s higher than  (optical  (Table 4-3). density  disrupted  vacuoles  concentrations  freguency between  than  and  vacuoles..  the  varied  greater  the number of dmj colored  .5)  =  dmj  .45  2.5 mg/ml  cells  and  with  .55.  At  25 mg/ml there was an i n c r e a s e i n  wild-type  Therefore,  an i n h i b i t o r y e f f e c t  of  wild-  cells  which  d i d not  form  a t higher c o n c e n t r a t i o n s , BP had  on e n d o c y t o s i s  i n wild-type and dmj  cells  but the e f f e c t on dmj c e l l s was g r e a t e r .  4- E f f e c t o f time a t 3__5_C  a.. asynchronous c e l l s There  was  no  significant  number of c o l o r e d vacuoles or  dm 1  cells  after  6  reduced cells  that  hours  accumulated  3  hours  these c e l l s by 6 phenotypic  at  34.5°C  the  wild-type  (Figure 4-5) .  number  of  colored  vacuoles appeared  i n dmj  34.5°C and were evident i n 71% o f  hours  expression  at  the  i n A5 c e l l s was s i g n i f i c a n t l y  (Figure 4-6)..Disrupted by  between  which accumulated i n  However, a f t e r 2 hours a t 34.5°C vacuoles  difference  (Table of  4-4). A  wide  abnormal vacuoles  range  was  of  observed  (Figure 3-2) and i n c u b a t i o n periods l o n g e r than 6 hours a t 34.5°C r e s u l t e d Significant  in  numbers  increased of  lethality  of  dm 1  cells.  A5 c e l l s had clumped vacuoles by  62  the  end c f 2 hours a t 3 4 . 5 ° C  (Table 4 - 5 ) .  There  were  s i g n i f i c a n t d i f f e r e n c e s between A 5 and f v c c e l l s  no  (Table 4 -  6) .  b.. synchronous When  cells  synchronized  cells  d o u b l i n g cf the number of hours  after  division  sere  shifted  to 3 4 . 5 ° C a  occurred  within 3 - 4  i n dml c e l l s  only a small  vacuoles  whereas  i n c r e a s e i n the number of v a c u o l e s was observed (Figure 4 7) . .Disrupted at 3 4 . 5 ° C  vacuoles appeared i n dmj c e l l s a f t e r  and t h i s i n c r e a s e d  to 70% of c e l l s  by  2 hours 4  hours  (Table 4 - 7 ) . The same p a t t e r n was observed i n asynchronous cells  although  gradual  the  increase  (Table 4 - 4 ) . . T h e  accumulated  i n dml c e l l s  mean  t o 71% penetrance was more number  11.Q-12.4)..  (c.i.=  different  from dml. c e l l s  following  division  vacuoles  at 27°C 3 . 5 hours a f t e r  was 10.1 (c.i.= 9.1-11.1) and a f t e r 11.7  of  This  shifted  (Figure  division  2 hours a t 3 4 . 5 ° C was  not  to  34.5°C  4 - 7 ) . There  that  was  significantly immediately was  a  sharp  i n c r e a s e i n the freguency o f c e l l s with d i s r u p t e d vacuoles to  .72 by the end of 2 hours a t 3 4 . 5 ° C i n those c e l l s  were incubated at 27°C f o r 3 . 5 hours Thus, the stage i n the c e l l time  at 3 4 . 5 ° C  vacuoles  but  temperature  following  was  length  of  less  division.  c y c l e as w e l l as the l e n g t h of  c o n t r i b u t e d to the appearance the  that  of  time  importance  number of food vacuoles formed.  at  the  of d i s r u p t e d restrictive  i n d e t e r m i n i n g the  63  5. . Down-shift dmj at  27°C  to permissive  temperature  c e l l s r e q u i r e d between 8 and to  return  to  normal  r e q u i r e d l e s s than 2 hours  16 hours  (Table 4-8).  (Tables 4-9 fox  vacuoles  27°C to 34.5°C  from  6) . There were no s i g n i f i c a n t cells of  (Table  vacuoles  separate  low  in  penetrance this  6. . E f f e c t of i n c r e a s i n g At temperatures  was  were  normal  (52%)  with  a  temperatures cells  were very few fvc  more  .35  endocytosis  in  dmj  cells  but s i m i l a r r e s u l t s were was  higher. .  at  there  of c e l l s with d i s r u p t e d  34.5°C.  In  wild-type  occurring  at  34.9°C.  decreased  in  dmj  was  dmj  cells  above 34.5°C  and  At  higher  wild-type  i n h i b i t e d and at 35.1°C there  c e l l s capable of forming c o l o r e d vacuoles. In  c e l l s the e x p r e s s i o n of the clumped vacuole  vacuole  A5  vacuoles  (Table 4-11)..Above t h i s temperature  of  4-  as having normal  when the penetrance  v a r i e d d i f f e r e n t l y with i n c r e a s i n g disrupted  and  l e s s than 34.3°C the food vacuoles of  t h i s freguency  because  and  fvc  vacuoles began to appear a t temperatures maximum  clumped  temperature  vacuoles t o a maximum of .53  and  with 2 or  a g r a d u a l i n c r e a s e i n the freguency  disrupted  of  the  a gradual moving apart  of d i s r u p t e d vacuoles  experiment  fvc c e l l s  (Tables 4-5  the clump they were c l a s s i f i e d  o b t a i n e d i n ether experiments  cells  appearance  d i f f e r e n c e s between  (when c e l l s were observed  vacuoles) . The was  the  4-10). .In both, t h e r e was  from  A5 and  cycles)  and 4-10), which was  same l e n g t h of time r e q u i r e d after shifting  (2 c e l l  temperature  phenotype. At temperatures  than  A5  phenotype did  the  above 34.1°C the  64  freguency of c e l l s  with  remained  .50  at around  clumped  vacuoles  f o r temperatures  12). Temperatures above t h i s caused poor  D.  rose  rapidly  up to 34.9°C  decreased  and  (Table 4-  endocytosis  and  viability.  Discussion Within  two  hours a t temperatures  above 34.1°C t h e r e i s a  decrease i n the number of food vacuoles that accumulate and  fvc  cells  after  a  20  minute  exposure  vacuoles clump i n one area of the c e l l , This  phenotype  i s reversible;  to the p e r m i s s i v e temperature asynchronous are  exposure  to  significant  t o BP and  u s u a l l y the  the vacuoles appear normal.  restrictive  differences  these  posterior.  .50 of A 5  or  fvc  T h i s freguency does net i n c r e a s e with the  A5  w i t h i n two hours a f t e r a r e t u r n  p o p u l a t i o n approximately  affected.  in  temperature..  between  AS  and  cells  prolonged  There fvc  In an  are  cells  no which  i n d i c a t e s that although the genes f o r 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  if  has  little,  any, e f f e c t on the e x p r e s s i o n of the f v c phenotype. Because the mutant  phenotype  is  fully  expressed a f t e r a r e l a t i v e l y  time at the r e s t r i c t i v e temperature,  gene  product  The decrease i n number of food vacuoles and t h e i r  clumping  probably t u r n s over  could  both  the a l t e r e d  short  guickly.  be explained by a d e f e c t i n e i t h e r the  or m i c r o f i l a m e n t vacuoles  would  recycled  as  system  in  the  cytoplasm.. The  microtubule numbers  of  decrease i f the membrane components, which are  disk-shaped  vesicles  ;(Allen, 1 974),  cannot  be  65  transported  by  associated  the  (Allen,  impaired.  microtubules 1975) . . V a c u o l a r  Alternatively,  movement o f v e s i c l e s and In  dm 1  cells  food vacuoles although  there  is  and  five  also  in  i s a l s o a decrease  of  turn  after  the  only the  i n t h e number o f  restrictive  temperature  when s y n c h r o n i z e d  number  division  o b t a i n e d a t room t e m p e r a t u r e and  Eodermel,  of  food  c e l l s are  vacuoles  i s consistent  (Berger,  1971)  and  with  (Smith-  1 9 7 6 ) . H o w e v e r , i n dmj c e l l s  there i s  to  division.  The d i f f e r e n c e b e t w e e n t h e number o f  the  number  g r e a t e r than number  of  since  i s not apparent  a v e r a g e number o f  feeding  ceases  with  for  (Aufderheide,  1976)  Conseguently,  t h e 95% c o n f i d e n c e  type than periods all  their  and  dmj c e l l s of  BP  fed  time,  with  much  by  the  balanced number o f  minutes  vacuoles  during for  division  20  minutes.  intervals are larger f o r wild-  ( f i g u r e 4-5)..When BP  i s  fed  for  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  longer  time f o r  v a c u o l e s t o become c o l o r e d and under t h e s e c o n d i t i o n s  dm 1 c e l l s do h a v e f e w e r v a c u o l e s cells  i s  a decreased  was  in  i n asynchronous samples  vacuoles  20-25  prior  vacuoles  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  post-dividers  three  27°C  m a j o r i n c r e a s e i n t h e number o f f o c d v a c u o l e s p e r c e l l  because  in  results  no  w i l d - t y p e and dml c e l l s  be  inhibit  s a m p l e s o f w i l d - t y p e c e l l s a t 34.5°C b e t w e e n  hours  Sonneborn  would  vacuoles.  apparent  examined..The d o u b l i n g synchronized  that could  that accumulate a t  this  movement  are closely  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 the  with which they  per  cell  than  do  wild-type  ( F i g u r e 4-2). , Asynchronous  dmj c e l l s r e g u i r e a r o u n d  s i x h o u r s a t 34.5°C  66  f o r t h e maximum p e n e t r a n c e  o f the disrupted vacuole  Above a minimum t i m e 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  cells  mg/ml)  with  these f a c t o r s  disrupted  phenotype.  do n e t i n f l u e n c e t h e number o f  v a c u o l e s . . The  inhibitory  effect  on with  endocytosis  of  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 c o m p a r a b l e  the  cf  other  effect  detergents,  which  depending  on  agents, can  their  such  stimulate  as  calcium  or  ions  inhibit  concentraticn  and  phagocytosis  (Nilsson,  1971,1976,  B r u t k o w s k a and Mehr, 19 76)... In.  synchronized  immediately  samples  following  of  i f the  cells  per  cell  T h i s c a n be  are s h i f t e d  hours a f t e r d i v i s i o n . vacuoles  s h i f t e d t o 34.5°C  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  appearance o f d i s r u p t e d vacuoles. hours  dm.1 c e l l s  begins  reduced  t o 34.5°C t h r e e  In wild-type c e l l s  the  to  of  t o r i s e d r a m a t i c a l l y by t h i s  o f membrane c o m p o n e n t s ; a d e c r e a s e  a d e f e c t i n t h e membrane c o m p o s i t i o n of  time,  i n increasing  i n t h e amount  o f membrane p r o d u c e d would r e d u c e t h e number o f v a c u o l e s  loss  food  The m u t a n t , d m j , 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  the production  and  two  and o n e - h a l f  number  t h e r e f o r e , membrane c o m p o n e n t s must be r e g u i r e d amounts._  f o r the  the  structural  integrity  could  result  formed i n the  o f t h e membranes l e a d i n g t o  their disrupticn. . Perhaps, (Allen,  because  1974) ,  membrane  a period of time  components  at the r e s t r i c t i v e  (egual t o s i x hours i n asynchronous c e l l s , cells)  is  reguired  incorporated vacuoles;  into  for sufficient food  Similarly,  vacuoles  after  a  are  to  less i n  recycled temperature  synchronous  d e f e c t i v e membrane t o be result  down-shift  to  in  disrupted  the permissive  67  temperature, normal  between one and two c e l l c y c l e s are  membrane  components  reguired f o r  t o r e p l a c e the d e f e c t i v e ones and  r e s u l t i n normal vacuoles. _ Ihe d i f f e r e n c e i n time the  two  temperatures  accumulation possibly  i s consistent  and l o s s of c o l o r e d  indicative  of  reguired at  with the i n c r e a s e d r a t e of  vacuoles  at  34.5°C  and i s  a higher metabolic r a t e a t the higher  temperature. Since d i s r u p t e d vacuoles cells,  they  response  are  to  an  composition  cf  temperature and  probably increase  membrane  a  Tetrahymena  (Conner  Aspergillus  in  trout  in  wild-type  some  cellular  The  fatty  acid  p h o s p h o l i p i d s changes i n response t o  and  as E s c h e r i c h i a c o l i Ckuyama  et  S t r e t t o n , 1976),  (Johnston and Roots,  (Hazel, 1979),  et  1976),  Neurpspora  1964)  planktcnic  (Sato  (Marr  a l . , 1977),  1976, Fukushima et a l . ,  (Farkas, 1979) and blue-green algae  mackerel  crustaceans  a l . , 1 S79) .  In  the rate of p h o s p h o l i p i d s y n t h e s i s i n E. c o l i may be  r e l a t e d to the stage i n the c e l l above  of  temperature..  and Stewart,  1977), g o l d f i s h  appear  consequence  Cronan, 1975,  (Dart  (Ueda, 1976),  addition,  alsc  i n such d i v e r s e organisms  Ingraham, 1962,  (Friedman,  can  cycle  (Pierucci,  1979) .  The  observations suggest t h a t the d e f e c t of dm1 c e l l s may be  a conseguence of a b n o r m a l i t i e s i n the f a t t y a c i d composition of phospholipids  which  thermotolerance disrupted  be  responsible  for  a  decreased  of membranes as evidenced by the appearance of  vacuoles a t a lower  c e l l s * . This VII.  may  aspect w i l l  temperature  than  in  wild-type  be i n v e s t i g a t e d more f u l l y i n Chapter  68  Table 4 - 1 . . E f f e c t of blue morphology  T i me in BP(1)  Cell Type  watercolor  DV (2)  N (3)  at  X2  1  wt dJBi  0 0  30 30  0.00  3  wt dmj  1 29  29 46  5  wt dml  0 30  10  wt dmj  15  34.5°C  P (5)  on  vacuolar  Freque DV (6)  1. 00  O.CO 0.00  11.43  <. 001  0.03 0. 45  30 23  24.2  <. 001  O.CO 0.57  1 30  29 20  23. 14  <.001  0.03 C.60  wt dml  1 30  29 19  23.78  <.001  0.03 0.61  20  wt dml  1 30  29 13  26.07  <. 001  0.07 0. 70  25  wt dml  0 30  30 23  24.20  <. 001  0.00 0.57  30  wt drnj  0 30  30 12  33. £5  <. 001  0.00 0.7 1  40  wt dml  0 30  30 20  26.30  <. 110  O.CO 0.60  60  wt dml  0 30  30 22  24.86  <. 001  0.00 0. 58  wt = wild-type dml = mutant with d i s r u p t e d vacuoles a t 34.5°C ( D minutes i n blue watercolor (2) number of c e l l s with d i s r u p t e d vacuoles (3) number of c e l l s with normal vac ucles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) fre guency of c e l l s with d i s r u p t e d vacuoles  69  Table 4-2. E f f e c t morpholog y  of removal frcm blue watercolor on vacuolar  Cell Type  DV (2)  N (3)  wt dmj  3 24  wt dmi 10  Time  Freguency (6)  (4)  (5)  46 25  20.45  <.001  0.06 0.49  5 24  45 26  15.74  <.001  0. 10 0.48  wt dmj  0 18  50 30  20.54  <.001  0.00 0. 36  15  wt dmj  1 16  46 28  15.35  <. 001  0.02 0.38  20  wt dmi  0 3  19 18  0. 27  0.00 0. 14  1.24  wt = wild-type dmj = mutant with d i s r u p t e d vacuoles a t 34.5°C (1) time (minutes) a f t e r removal frcm blue watercolor (2) number of c e l l s with d i s r u p t e d vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency of c e l l s with d i s r u p t e d vacuoles  70  T a b l e 4--3. E f f e c t of vacuolar morphology  Cone. . Cell mg/ml( 1) Type  concentration  DV (2)  N (3)  of  X2  (*»)  blue  watercolor  P (5)  Freguency (6)  0.75  wt dm!  2 12  44 33  7.07  . 008  0.04 0. 27  0. 10  wt dml  4 .14  44 36  5.07  . 02  0.C8 0.28  4 21  44 26  14.36  <. 001  0.08 0.45  wt  Ml  3 21  42 26  15.31  <. 001  0.07 0.45  7.5  wt dmj  1 24  45 23  25.84  <. 001  0.02 0. 51  10.9  wt dml  5 24  41 26  13.96  <.001  0. 1 1 0.48  25.0  wt dm!  7 23  33 19  10.71  . 001  50.0  wt dml  3 19  29 16  13.32  <. 001  0.09 0.54  75.0  wt dm 1  1 6  21 9  5. 18  . 02  0.05 0. 40  2.5 5.0  M t  dm!  wt = wild-type dm! mutant with d i s r u p t e d vacuoles a t 34.5°C (1) c o n c e n t r a t i o n of blue watercolor (2) number o f c e l l s with d i s r u p t e d vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency of c e l l s with d i s r u p t e d vacuoles =  on  0. 18 0.55  71  T a b l e 4- 4. E f f e c t of time a t 34.5°C cc vacuolar morphology  T i me (hr) (1)  _  Cell Type  __  DV (2)  -  N (3)  X* (4)  P (5)  .__  Frequency (6)  o7oc  dmi  0  "~30 30  1  wt dmj  0 0  30 30  0  1  0.00 0.00  2  wt dmj  0 0  30 30  0  1  o.co  3  wt dmj  1 30  29 50  10.95  . 001  0.03 0.38  4  wt dmj  0 30  30 19  27.07  <. 001  0.00 0.61  5  wt dmj  5 30  25 17  14.58  <.Q01  0.17 0. 64  6  wt dmj  1 30  29 12  30.38  <.001  0.C3 0.71  0.00  wt = wild-type dmj = mutant with d i s r u p t e d v a c u o l e s at 34.5°C (1) number of hours a t 34. 5°C (2) number o f c e l l s with d i s r u p t e d vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Square Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) frequency of c e l l s with d i s r u p t e d vacuoles  0.00  72  Table 4 - 5 . . E f f e c t  time at 34.5°C on v a c u o l a r  of  wt A5  1 0  27 30  0.0012 0. 97  0.04 O.CO  1  wt A5  0 0  29 30  0.00  1. 00  0.00 0.00  2  wt A5  1 11  29 19  8.44  0. 004  0.03 0.37  3  Wt A5  1 14  28 16  12.34  <.001  0.03 0. 47  4  wt A5  1 17  27 13  16.68  <. 001  0.04 0. 57  5  wt A5  2 19  27 11  18. 10  <. 001  0.07 0.63  6  wt A5  2 16  28 14  13.41  <. 001  0.07 0.53  Cell Type  0  X  2  (<»)  P (5)  Freguency (6)  12)  N (3)  T i me (hr) (1)  CV  clumping  wt = w i l d - t y p e A5 = double nutant with t r i c h o c y s t non-discharge vacuoles a t 34. 5°C (1) number of hours a t 34.5°C (2) number o f c e l l s with clumped vacuoles (3) number o f c e l l s with normal vacuoles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency c f c e l l s with clumped vacuoles  and clumped  73  Table 4-6. Vacuolar clumping  i n A5 and J v c c e l l s  (4)  P (5)  Freguency (6)  33 27  2.6 3  0. 10  0.27 0. 45  18 28  25 20  1.85  0. 17  0.42 0.58  25 20  21 17  0.00  1. 00  0.54 0-. 54  Time (hr) (1)  Cell Type  CV (2)  N (3)  X2  2  A5 fyc  12 22  4  A5 fyc  6  A5 fvc  A5 = double mutant with t r i c h o c y s t non-discharge vacuoles at 34.5°C f y c = mutant with clumped vacuoles at 34.5°C (1) number of hours a t 34. 5°C (2) number of c e l l s with clumped vacuoles (3) number o f c e l l s with normal vacuoles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency of c e l l s with clumped vacuoles  and  clumped  74  T a b l e 4-7. Vacuolar morphology i n synchronous c e l l s at 34.5°C  DV (2)  N (3)  X (4)  P (5)  Freguency (6)  wt dml  0 0  30 17  0.00  1. 00  0.00 0.00  1.0  wt dm!  0 0  30 30  0.00  1. 00  0.00 O.CO  2. 0  wt dml  0 0  39 30  0.00  1.00  0.00 0.00  3.0  wt dm!  0 7  30 23  5. 82  0. 02  0.00 0.23  3. 5  wt djl  0 7  30 23  5.82  0.02  0.00 0. 23  4. 0  wt dm!  0 21  30 9  29.30  <.001  0.00 0. 70  5. 0  wt dml  0 20  30 10  27.C8  <.001  O.CO 0.67  T i me (hr) (1)  Cell Type  0. 0  2  wt = wild-type dm! = mutant with d i s r u p t e d v a c u o l e s at 34.5°C (1) number of hours at 34. 5°C ( c e l l s s h i f t e d up after divisicc) (2) number of c e l l s with d i s r u p t e d vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency of c e l l s with d i s r u p t e d vacuoles  immediately  75  T a b l e 4-8. Vacuolar morphology a f t e r s h i f t t o 27°C Time (hr) (1)  Cell Type  DV (2)  N (3)  X (4)  P (5)  Fregi (6)  o7oo  wt dmj  2 23  31 21  16.32  <. 001  0.06 0. 52  0. 33  wt dmj  6 22  43 28  10.79  0. 001  0. 12 0.44  0. 67  wt dmj  7 18  42 28  e.33  0. 01  0. 14 0. 39  1. 00  wt dmj  7 18  42 32  5.09  0.02  0. 14 0.36  1. 50  wt dmj  8 18  40 28  4.85  0. 03  0.17 0. 39  2. 00  wt dmj  7 19  43 28  7.33  0. 007  0. 14 0.40  4. 00  wt dmj  7 21  41 28  6. 11  0. 004  0. 15 0.43  6. 00  wt dmj  6 18  44 27  8.41  0. 004  0. 12 0.40  8. 00  wt dmj  7 14  41 25  4.24  0. 04  0.15 0. 36  16. 0  wt dm1  3 2  42 47  0.01  0. 92  0.07 0.04  2  wt = wild-type dmj = mutant with d i s r u p t e d v a c u o l e s at 34.5°C (1) number of hours a f t e r s h i f t down t o 27°C ( a f t e r 6 hours 34. 5°C) (2) number o f c e l l s with d i s r u p t e d vacuoles (3) number of c e l l s with normal vacuoles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency of c e l l s with d i s r u p t e d vacuoles  at  76  Table 4- 9. Vacuolar clumping  a f t e r s h i f t t c 27 °C  Cell Type  CV (2)  N (3)  X (4)  0. 00  wt fvc  3 26  31 24  ""14783  0.33  wt fvc  3 19  40 24  13.74  <. 00 1  0.07 0.44  0.67  wt . fvc  1 19  41 26  17. 29  <. 001  0.02 0.42  1.00  wt fvc  1 14  41 29  11.32  <. 001  0.02 0.42  1. 50  wt fvc  0 4  40 42  1.95  0. 16  0.00 0.09  2. 00  wt fvc  4 5  39 42  0.00  1.00  0. 09 0.11  4.00  wt fvc  1 2  40 40  0. 00  1. oo  0.02 0. 05  6. CO  wt fvc  1 6  • 43 36  2. 70  0. 10  0.02 0. 14  2  P (5)  Fregi (6)  Time (hr) (1)  ~<7oo"'  oToT 0.52  wt = w i l d - t y p e f v c = mutant with clumped vacuoles at 34.5°C (1) number of hours a f t e r shift t o 27°C 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) C h i Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency of c e l l s with clumped vacuoles  ( a f t e r 6 hours at  77  Table 4-10. Vacuolar clumping to 27°C  i n f y c and A5 c e l l s  after  shift  Time (hr) (1)  Cell Type  CV (2)  N (3)  X2  (4)  P (5)  Freguency (6)  0.00  fvc A5  26 17  24 17  0*00  1.00  0752 0. 50  0.33  fvc A5  19 10  24 20  0. 48  0. 49  0.44 0. 33  0. 67  fvc A5  19 10  26 20  0. 28  0.60  0. 42 0.33  1. 00  fvc A5  14 7  29 23  0.35  0. 55  0.42 0.23  1.50  fvc A5  4 5  42 25  0.47  0. 49  0.09 0. 17  2. 00  fvc A5  5 1  42 29  0. 53  0. 47  0. 1 1 0.03  6. 00  fvc A5  6 2  36 28  0. 40  0. 53  0. 14 0.07  f y c = mutant with clumped 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) number of hours a f t e r 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) C h i Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency of c e l l s with clumped vacuoles  78  Table 4- 11.  E f f e c t of temperature DV (2)  N (3)  wt Is!  3 2  47 48  34. 1  wt dm!  0 5  34.3  wt dm!  34. 5  en vacuolar morphology P (5)  Freguency (6)  0.00  1. 00  0.06 0.C4  28 25  3.21  0.07  0.00 0. 17  0 7  29 19  6.69  0. 01  0.00 0. 27  wt dm!  0 19  33 17  <. 001  0.00 0.53  34 .7  wt dm!  8 21  40 28  6.74  0. 009  0. 17 0.43  34.9  wt dm!  11 12  19 18  0.00  1. 00  0. 37 0. 40  35. 1  wt dm!  6 5  24 15  0.005  0. 94  0.20 0. 25  Te mp. l°C) (1)  Cell Type  33 .5  X  2  CO  21.46  wt = wild-type dm! mutant with d i s r u p t e d vacuoles a t 34.5°C (1) incubated f o r 6 hours at the temperature i n d i c a t e d (2) number of c e l l s with d i s r u p t e d vacuoles (3) number o f c e l l s with normal vacuoles (4) Chi Sguare Test f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare Test (6) freguency o f c e l l s with d i s r u p t e d vacuoles =  79  T a b l e 4-12. E f f e c t of temperature on vacuolar Temp. (°C) (1)  Cell Type  3375™  clumping  (2)  N (3)  wt fyc  0 5  47 44  3. 20 " o 7 o 7  34. 1  wt A5  0 7  28 30  4.13  34.3  wt A5  4 17  25 12  34.5  wt A5  0 13  34 .7  wt fyc  34.9 35. 1  CV  X2  (4)  P (5)  Freguency (6)  o7oo 0. 10  0.04  0.00 0.19  10.75  0. 001  0. 14 0.59  29 17  13.69  <.001  0.00 0. 43  3 23  37 19  19.01  <. 001  0.08 0.55  wt A5  0 18  19 11  16.31  <. 001  0.00 0.62  wt A5  1 0  22 3  0.00  1. 00  0.04 0.00  wt = wild-type f v c = mutant with clumped vacuoles at 34.5 ° c A5 = double mutant with t r i c h c c y s t ncn-d i s c h a r g e and clumped vacuoles at 34.5°C (1) incubated f o r 6 hours at t h e temperature i n d i c a t e d (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 f o r homogeneity (5) p r o b a b i l i t y generated by C h i Sguare T e s t (6) freguency c f c e l l s with clumped vacuoles :  80  Figure 4-1..Accumulation 27°C.  The  of  vacuoles  in  blue  number of c o l o r e d vacuoles per c e l l  time i n blue watercolor. V e r t i c a l i n t e r v a l s . . (•--•  = wild-type,  watercolor  with i n c r e a s i n g  bars r e p r e s e n t 95% = dm 1)  at  confidence  81  82  Figure 4-2..Accumulation of 34.5°C. The  vacuoles  in  blue  number of c o l o r e d vacuoles per c e l l  watercolor  with i n c r e a s i n g  time i n blue watercolor. V e r t i c a l bars r e p r e s e n t 95% i n t e r v a l s . . (•--•  = wild-type,  •—•  =  dml)  at  confidence  N O .  O F  C O L O R E D  oi  \ A C U O L E S / C E L L  O  cn  84  Figure  4-3.  loss  of c o l o r e d vacuoles at 27°C. Loss o f c o l o r e d  vacuoles with time a f t e r removal frcm blue watercolor. 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 . =  dml)  (•--•  = wild-type, A — A  b 2  20 40 60 TIME AFTER REMOVAL FROM B.P (min)  86  F i g u r e 4-4.  Less of c o l o r e d vacuoles at 34. 5°C. Loss of c o l o r e d  vacuoles with time a f t e r removal frcm blue watercolor. bars 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 . =  dmj.)  (•--•  Vertical  = w i l d - t y p e , *>—*  87  _l _l  LU O \  88  Figure at  4-5..Accumulation  34.5°C..Blue  incubation confidence  of vacuoles  watercolor  periods  was  added  indicated..  i n t e r v a l s . . (•--•  i n wild-type f o r 20  Vertical  and  minutes bars  dml  cells  after  the  represent  95%  = w i l d - t y p e , • — • =dm 1)  NO. OF COLORED VACUOLES /CELL cn o  oo  90  F i g u r e 4-6. at  34.5°C.  incubation  accumulation of vacuoles i n wild-type and A5 Blue watercolor was times  added f o r 20 minutes a f t e r  indicated.. Vertical  confidence i n t e r v a l s .  (•--•  cells  bars  represent  = w i l d - t y p e , m — • = A5)  the 95%  92  Figure  4-7.  Accumulation  34.5°C. Synchronized after  of vacuoles i n synchronized  c e l l s were s h i f t e d t o  34.5°C  c e l l s at  immediately  d i v i s i o n and fed blue watercolor f o r 20 minutes a f t e r  incubation  period  indicated.  confidence i n t e r v a l s . . (•--•  Vertical  bars  = wild-type, • — •  =  represent dmi)  the 95%  94  CHAPTEB V  EFFECTS OF CHEMICAL AGENTS ON WI1D-TYEE AND MUTANT CELLS  A.  Introduction Many chemical agents  mutant,  dmj.#  is  p e r t u r b membrane  defective  in  seme  s t r u c t u r e . . I f the aspect  composition,  i t i s p o s s i b l e t h a t these agents  phenocopies  of  t h i s mutant, i . e .  cf  could  d i s r u p t e d food  membrane  result  in  vacuoles, i n  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  different  s e n s i t i v i t y than wild-type c e l l s t c these chemicals. S i m i l a r l y , if  the  mutant,  microtubules c r these  f y c , i s abnormal due t o the m a l f u n c t i o n i n g of microfilaments,  structures  may  result  i..e. food vacuole clumping, cells  (which  have a l t e r e d agents  were  in  sensitivity selected  to on  of t h i s mutant* f v c or  A5  these  the  chemicals.  The  following  b a s i s of t h e i r known a c t i o n on  (DMSO) i s a p c l a r s o l v e n t which  has a  (Jacob, 1971). DMSO induces or  f u s i o n of hen e r y t h r o c y t e s (eg. Ahkong e t a l . , 1975,  1976)  Kalinina,  with  organisms.  sulfoxide  Wilairat et a l . , al.,  phenocopies  interfere  i n wild-type c e l l s .  wide range o f b i o l o g i c a l e f f e c t s enhances  that  contain the r e c e s s i v e genes f y c and tnd) may a l s o  c i l i a t e s or other Dimethyl  agents  1978),  and  the  1 979) .  It  human  pseudopodia has  differentiation  in  Friend  a l . , 1976),  a  radio-  as  epithelial of  also  amoeba  been  leukemic and  cells  cells  (Norwood (Grebecka  used  to  (eg. -  cryoprotectant  et and  induce Lyman  et  (Ashwood-  95  Smith,  1971),  solvent  a drug c a r r i e r  f o r water i n s o l u b l e  Nilsson,  1976).  Bucholtz  (1977)  accounts  of  Paramecium  Reports and  (eg.  compounds by  Sibley  et  c o n t r a c t i l e vacuoles aggregates,  .. In  (eg.  Nilsscn  the a f f e c t s of DM SO  tetraurelia  Weed and Wood, 1975), and a cytochalasin  (1977b),  a l . (1977)  Reisner  provide  B, and  detailed  on Tetrahymena p y r i f ormis and  addition  to  causing  swollen  (CVs) , a n u c l e a t e daughter c e l l s , n u c l e o l a r  abnormal  chromatin  bodies,  mitochondria,  peroxisomes, and ribosomal  aggregates  affecting  polysome s t r u c t u r e and c e l l s i z e i n  fission  rates,  Paramecium , lower c o n c e n t r a t i o n s decreased  endocytosis  concentrations in  (10&) cause food  Paramecium  considered  in  .  In  view  i n Tetrahymena  of  DMSO  (7.5%)  Tetrahymena vacuole  of  , and  result i n  whereas  membrane  higher  abnormalities  the l a s t two e f f e c t s ,  to be a drug that might have i n t e r e s t i n g  DMSO was  effects  on  the mutant, dmj.. L o c a l a n a s t h e t i c s , such a s d i b u c a i n e , r e a c t with the p o l a r groups of p h o s p h o l i p i d s a t the membrane surface as well as with the  hydrophobic  fluidity  of  organization  interior  membranes  (Ohki,  (Hubbell  of c y t o s k e l e t a l  and may d i s p l a c e  et  et  In  a n a s t h e t i c s cause c i l i a r y r e v e r s a l and of  Nelson,  1976) but t h e r e are no  endocytosis. in  calcium  and e f f l u x  i n c r e a s e the  a l . , 1970),  calcium  a l . , 1975)..  influx  They  affect  components (Poste e t a l . ,  membrane-bound  (Papahadjopoulos  1970).  and  magnesium  P. . a u r e l i a  stimulate  of potassium  ions  reports  their  of  the  ,  the 1975) ions local  passive  (Browning and effects  on  Therefore, the e f f e c t o f d i b u c a i n e on e n d o c y t o s i s  P. t e t r a u r e l i a was examined. .  96  Detergents dodecylsulfate  are l i p i d s (SDS)  concentrations,  i s an  binds  anionic  of the membrane  stimuli  morphological (Dryl  and  g/ml)  sensitive  to  in  Mehr, 1976),  SDS  and  intracellar  than  of  (8x10 with  and  biopterin, also a  g/ml)  in  many  (Allen,  being  binds  to  1974,  i n a c t i v a t i o n of  sugars  (Cheng  and  cells. of  the  1975,  Allen causes  microtubules  Katsoyannis,  transport  (Mizel  and  19 75)  and  Langentach, 1978) . P o d o p h y l l o t o x i n ,  inhibitor, algae  more  tubulin,  eg. n u c l e o s i d e s  green  inhibitory  facets  cells,  and  6  i n Paramecium , i n c l u d i n g the  subseguent  (Eembold  4x10~  Paramecium  are  into  mitotic  symbiotic  and  B o r i s y , 1973) . C o l c h i c i n e a l s o i n h i b i t s  1972),  ectoplasm  . Concentrations i n t h i s  role  process  -6  SDS  causes  wild-type and mutant  movement of food v a c u l e s  materials  Wilson,  and  Paramecium  Tetrah ymena  exocytic  depolymerization and  1975)  Tetrahymena  Wolf, 1974) . C o l c h i c i n e , which  (Olmsted  Dryl,  and  of P..caudatum t o  c o n c e n t r a t i o n s ( l e s s than  M i c r o t u b u l e s have a major  and  and  have d i f f e r e n t e f f e c t s on  endocytic  Simons, 1 S75) .  motor response  concentrations  and  low  membrane c a u s i n g changes i n  (Helenius and  (Bujwid-Cwik  phagocytosis  higher  range may  and  Mehr, 1976). , Low  (Brutkowska  in  changes owing to the c o n t r a c t i o n of the  stimulate  whereas  which,  e g u i l i b r i u m t h a t can lead t o l y s i s  i n c r e a s e s the e x c i t a b i l i t y external  amphipiles;.Sodium  detergent  to the c e l l  p e r m e a b i l i t y and osmotic disintegration  which are s o l u b l e  delays in  the  _fiY . .a d  r  normal yiridis  migration  of  (Cooper  and  M a r g u l i s , 1977). C o l c h i c i n e does net have an e f f e c t on the r a t e of p h a g o c y t o s i s leukocytes  in  (fKLs)  isolated but  membranes  of  polymorphonuclear  does change the membrane topography of  97  these  cells  colchicine  (Berlin similar  and  F e r a , 1977)..  Concentrations  t o those used on PMLs (1-5 mg/ml)  of  decrease  t h e e n d o c y t i c rate i n Paramecium without causing any changes i n the  ul t r a s t r ucture  effects  of  this  of  microtubules  agent  on  ( T o l l o c z k o , 1977).  The  the mutant, f v c , as compared  wild-type c e l l s may p r o v i d e more i n f o r m a t i o n on the  nature  with of  the a b n o r m a l i t i e s i n t h i s mutant. . Cytochalasins  (eg. . c y t o c h a l a s i n  B) are products of f u n g i  with a wide range of b i o l o g i c a l e f f e c t s into  two  major  molecules  categories:  i n t o c e l l s and  morphogenesis  (1)  (2)  ( L i n , 1978).  that  can  membrane  changes  and  effects They  which  resulting  inhibit  microfilaments  from  in  on  cell  cause  motility  rapid by  and  and  plasma  with  the  division  novement  Miranda, 1978). macrophages  ultimately  membrane  (Davies  N i l s s o n , 1974,  (Schroeder, cells  Cytochalasin  ( T o l l o c z k o , 1977)  of  of  and  and  B  release  (Spooner, 1 978).  in  1978)  culture  inhibits Allison,  T.. p y r i f ormis  and  1978),  (Nilsson  the  cell  interferes (Godman  and in  P..caudatum et  a l . , 1973,  1976)..There i s no change i n the u l t r a s t r u c t u r e  the m i c r o f i l a m e n t s i n t r e a t e d P. caudatum  unclear  without  phagocytosis  ( T o l l o c z k o , 1977).  Since the r o l e of m i c r o f i l a m e n t s i n endocytosis and is  severe  penetrating  C y t o c h a l s i n B prevents the s e p a r a t i o n cf daughter c e l l s impairing nuclear  and  conformational o r a l l o s t e r i c  enzymes the  divided  e f f e c t s on the t r a n s p o r t of  i n h i b i t i o n of membrane-bound t r a n s p o r t systems the  be  exocytosis  (Korn e t a l . , 1974) , c y t o c h a l a s i n B may be a c t i n g on membrane  i n i t s i n h i b i t i o n of phagocytosis  may have i n t e r e s t i n g e f f e c t s on both  and thus  the d i l and f v c mutants..  98  The  agents,  cytochalasin  B,  DMSO,  dibucaine,  a l l have  SDS,  demonstrated  colchicine,  e f f e c t s on membranes  and/or phagocytosis and a study of these e f f e c t s and  mutant c e l l s has been c a r r i e d  c h a r a c t e r i z e the mutants,  B.  Mat e r i a l s  dmi  and  on  wild-type  cut i n an attempt t o f u r t h e r  and f y c . .  and Methods  1. . Procedure f o r d e t e r m i n i n g e f f e c t s cf chemical agents  a.  p r e p a r a t i o n of c e l l s E x p o n e n t i a l l y growing 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 5-6 cells/ml cells  as  in  hours  (cell  determined  culture  d e n s i t y was by  approximately  serial dilution  fluid).  The  cells  10*  of 1.0 ml of  were  centrifuged  (100xg, 3 minutes) and adapted t o a b u f f e r c o n s i s t i n g of 4 mM  potassium c h l o r i d e , 1 mM  with  a  pH  of  calcium c h l o r i d e and  7.0-7.2 (see Appendix  c e l l s to t u f f e r ) . . 2 ml of c e l l s buffer  containing  0.25  bacteria  JEnterobacter  buffer.  The  cells  ml  were  of  The  fyc,  and A5  buffer  same  with  could a r i s e  procedure was  c e l l s . . The dead  also  then  from  the  3  in  added  ml  of  the to  (see  same various  section  1-  followed f o r wild-type, dmi.,  experiments  bacteria  to  concentrated, autoclaved  c o n c e n t r a t i o n s of the agent t o be t e s t e d b) .  Tris  I f o r a d a p t a t i o n of added  aerpgenes), were  1 mM  to  metabolism  were  performed  minimize v a r i a b i i t y of  bacteria  and  in that the  99  undefined  nature of t h e c u l t u r e  b. . preparation The using  of chemical  concentration  published  concentrations  organisms. . The  times  their  one for  range to be t e s t e d was determined (where  to  similar  described  agents  reports known  final  fluid.  be  available)  effective  chemicals  were  concentration  diluted  in  the  determined using a Corning  same  of  four  buffer  duplicate;  solution  was  pH meter.  c. _ mixing of c e l l s and chemical ml  to  at 27°C and the other s e t a t 34.5°C  30 minutes p r i o r t o use. The pH of each  0.25  the  on Paramecium or  a b o v e . . S e r i a l d i l u t i o n s were made i n  s e t was incubated  on  chemical  agents  was  to  glass  followed  0 . 5 ml of b u f f e r c o n t a i n i n g e i t h e r 1.5 mg/ml  blue watercolor mg/ml  black  watercolor,  0.25 ml of the  mixture  (section  that  /autoclaved  was  1.0  particles. bacteria  were  fixed  with  formaldehyde*  prepared i n e x a c t l y the same manner except  0.25 ml of b u f f e r  chemical  (EP) ,  or 1.0 mg/ml carmine Paramecium  This  1-a) were added and a f t e r 20 minutes a t  27°C or 34.5°c: t h e c e l l s were  tube.  (BP), 1.0 mg/ml red watercolour  Finally,  Controls  culture  a 13 x 100 mm  borosilicate by  disposable  added  i n buffer.  alone  replaced  the  0.25  ml of  100  d..  a n a l y s i s of r e s u l t s Thirty  agent  to 50 c e l l s  at each c o n c e n t r a t i o n of chemical  were examined i n a Z e i s s l i g h t  phenotypes were d e s c r i b e d as i.  iii.  the  (N)  with swollen con-tractile vacuoles  cells  and  fcllcws:  c e l l s with normal vacuoles  i i . . cells  microscope  with clumped  vacuoles  (SCV)  (CV) u e . . vacuoles  l o c a t e d i n one area of th i v. . c e l l s with decrease d than  f i v e c o l o r ed  wild  type c e l l s = 11.3,  v. c e l l s  cells  membrane Sibley  c  with d i s r u p t e d  morphologi c a l l y vi.  vacuole  abnormal cortical  with and  cortical  and Hans on  (1974)  Freguently a s i n g l e c e l l abnormality and having  the  had  more than  type  of  when t h i s occurred c e l l s were d e s c r i b e d as  most  severe  phenotype  ( i - v i with i n c r e a s i n g  s e v e r i t y ) . For example, i f a c e l l had disrupted  one  vacuoles  it  was  a  swollen  placed i n group  CV  and  (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 c o n c e n t r a t i o n * the  Chi  Sguare  (X )  correction performed  2  test  factor i n two  for with  homogeneity one  different  s p e c i f i c c o n c e n t r a t i o n was  degree  (employing of  Yates'  freedom)  ways. F i r s t , t o determine  was if a  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) of  a t that c o n c e n t r a t i o n was compared with the number  cells  same was  with each phenotype i n the c o n t r o l sample o f the  cell found  type..Second, a f t e r (P < .05), each mutant  wild-type  cells  wild-type  was  concentration  compared  with t h e  at the same c o n c e n t r a t i o n . T h i s i n d i c a t e d  whether the mutants were being  2.  an e f f e c t i v e  affected  differently  than  cells.  DMSO  a. . e f f e c t The  cf c o n c e n t r a t i o n c o n c e n t r a t i o n s o f DMSO (Sigma, Molecular  weight,  M. W..= 78.13) t e s t e d at 27°C and 34.5°C ranged from 5-15%. The  pH of 10% DMSO a t both  added  to  the c e l l s embedded coated  f o r 20 minutes and  were  Fixed  fixed  and  examined.  i n saline-gelatin  or a i r - d r i e d  cells  were  on g l a s s s l i d e s  with albumin and photographed on a L e i t z Orthomat-W  b. . o b s e r v a t i o n s of l i v e Wild-type, 6%  optics.  cells  dmj. and f y c c e l l s  20  were adapted  to buffer  c r 12% DMSO i n b u f f e r and BP was added a t 27°C or  34.5°C. Samples of 10 c e l l s for  6.6. . BP was  wild-type, dmi and f y c c e l l s  microscope using Nomarski  and  temperatures was  to  3%  methyl  minutes on a  microscope s l i d e and examined i n a Z e i s s microscope  using  optics.,  added  5  cellulose  Nomarski  minutes,  were removed every  a l t e r n a t i v e l y , s i n g l e c e l l s were placed  102  i n a rcto-compressor o p t i c s . . The r a t e  and  also  examined  using  of c o n t r a c t i o n of the CVs was noted a s  w e l l as t h e formation and movement of food cells  were  Nomarski  recorded  vacuoles.  The  on videotape using an RCA TC1C00 TV  camera and a Sanyo videotape  recorder  (VTR 2000).. Frame-  by- frame t r a c i n g s were made from an 11-inch Electrohome TV monitor. . The  osmolality  with and without  of b u f f e r and 6% DMSO i n b u f f e r  added BP were determined  on  an  Advanced  Wide-Range Osmometer. .  c.  e f f e c t of treatment BP  i n buffer  c e l l s , also minutes,  duration  was  added  t o w i l d - t y p e , dmj. and f v c  i n buffer.  After  0, 5, 10, 15, 17, and 19  samples  were  removed and DMSO i n b u f f e r and BP  (to maintain a f i n a l c o n c e n t r a t i o n of 12% DMSO mg/ml of  BP)  and 0.75  were added. The c e l l s were f i x e d a f t e r a t o t a l  20 minutes from the i n i t i a l a d d i t i o n of BP had  i.e.  cells  were  i n BP f o r a t o t a l  elapsed  o f 20 minutes and i n  DMSO f o r varying p o r t i o n s of t h a t 20 minutes. .  d.  addition  of  RP  followed  t_y DMSO  and a  second  watercolor Wild-type, for  6  hours  dm1_ and f v c c e l l s  were incubated a t 34.5°C  and then f e d EP i n c u l t u r e f l u i d . The c e l l s  were c e n t r i f u g e d (100xg, 5 minutes) and adapted t o b u f f e r . 6% DMSO i n BP minutes. for  or  black  Alternativsly,  watercolor  was  added  BP or black watercolor  10 minutes f o l l o w e d by an a d d i t i o n a l  10  f o r 20 was added  minutes  with  103  DMSO  in  final  c o n c e n t r a t i o n s o f 6% o r 12? DMSO a n d 0.75  or  BP o r b l a c k w a t e r c o l o r  w a t e r c o l o r ) . The c e l l s  o f 20 m i n u t e s i n t h e s e c c n d  (to m a i n t a i n mg/ml  were f i x e d  BP  after  watercolor..  Dib u c a i n e Wild-type,  and  and  0.5 mg/ml b l a c k  a total  3«  buffer  dml  a f l  d  A5 c e l l s  3.8 x 1 0 ~ g/ml o f d i b u c a i n e 5  were t e s t e d w i t h 3.8 x 1 0 ~ (ICN P h a r m a c e u t i c a l s ,  6  M.W.=  379. 92) i n b u f f e r a n d BP f o r 20 m i n u t e s a t 27°C a n d 34.5°C. The pH  of  cells  both  concentrations  a t both  t e m p e r a t u r e s was 6.7. The  were f i x e d a n d e x a m i n e d . .  4. . SDS Wild-type, 10-7,  10-6, 5  d m l a n d A5 c e l l s were t r e a t e d w i t h x  10-6, n d a  10  - 7  , 5  x  10-s g/ml o f SDS ( S i g m a , M.W. =  288.4) i n b u f f e r a n d BP f o r 20 m i n u t e s a t 27°C a n d 34.5°C.„ The pH  of  cells  10-s g/ml a t  27°C was 6.9 a n d a t 34.5°C was 7.0. The  were f i x e d and e x a m i n e d .  5«- C o l c h i c i n e Wild-type, and  2.5  mg/nl  dml  a f i  d  A5 c e l l s  of c o l c h i c i n e  were t r e a t e d w i t h  0 . 2 5 , 0.5,  ( S i g m a , 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 a n d 34.5°C. The c e l l s  and  examined.  were  fixed  104  6.  Cytochalasin B Cytochalasin  B  i s g e n e r a l l y d i s s o l v e d i n DMSO but s i n c e  t h i s was one of the agents t e s t e d , agueous suspensions 17, in  18,  19, and 20 ug/ml c y t o c h a l a s i n B (Sigma,M. W. .= 479.62)  b u f f e r were mixed with  carmine  C.  1.  wild-type,  dm]  and A5  cells  p a r t i c l e s . . The suspensions were shaken during  minute i n c u b a t i o n at 34. 5°C  of 16,  and  the 20  p e r i o d a t 27°C o r 34.5°C..The pH of 20  ug/ml  was 6.7. The c e l l s were f i x e d and examined*  Results  DMSO  a.  e f f e c t of c o n c e n t r a t i o n After  and dmj  treatment  34.5°C, the d i s t r i b u t i o n of phenotypes and  fvc cells  controls  swollen  was  (Tables  concentration  were  with 5% DMSO f o r 20 minutes a t 27°C  of  5-1  different and  in  wild-type,  (P < .00 1)  5-2).  With  than i n increasing  DMSO from 8% t c 15%, the CVs were o f t e n  (Figure 5-1a and P l a t e  occasionally  normal  V-1a).  The food  vacuoles  t u t were most o f t e n  disrupted  (Figure 5-1b-e and P l a t e V-1b). Abnormal vacuoles were of f o u r major  i.  types:  cells  with  vacuoles present larger  several i n t h e same  and m o r p h o l o g i c a l l y  normal cell  watercolor-filled as  one o r  abnormal vacuoles  more  (Figure  105  5-lb) ii.  cells  with  vacuole (s) iii.  or  mere  large  and  irregular  (Figure 5-1c and P l a t e V-1b)  cells  particles  one  with  no  apparent  of w a t e r c o l o r  vacuoles  throughout  but many  the  cytoplasm  (Figure 5-1d) iv.  cells  CVs)  with very l a r g e c o l o r l e s s vacuoles  and p a r t i c l e s  cytoplasm All  of  watercolor  (swollen  throughout  the  (Figure 5-1e)  four types of d i s r u p t e d vacuoles  were  found  in  w i l d - t y p e , dm1 and f v c c e l l s . . At  high  increase  in  concentrations the  number  of DMSO (15%) , there was an of  cells  with  cortical  a b n o r m a l i t i e s where blebs o f v a r i o u s s i z e s appeared cell  surface  dmj c e l l s  (Figure 5-1f,g and P l a t e s V-1c and V-1d).  were more s e n s i t i v e t o  evidenced  by  a  greater  number  5%  DMSO  a t 34.5°C  were more dm1 c e l l s with c o r t i c a l  b._  as  of c e l l s with d i s r u p t e d  vacuoles than i n w i l d - t y p e and i n 12% DMSO at  type  on the  27°C  there  a b n o r m a l i t i e s than w i l d -  cells.  o b s e r v a t i o n s of l i v e One  of  cells  the f i r s t  abnormalities  a d d i t i o n c f DMSO a t 27°C and 34.5°C was  apparent  the decrease  the r a t e cf c o n t r a c t i o n o f the CVs. In untreated cells seconds  at  27°C  (Cii..=  after in  wild-type  the mean length of the CV c y c l e was 7.59 7.11 - 8.C7) ( l a b l e 5-3). The CV c y c l e was  l o n g e r i n dmj c e l l s  (8.44 seconds,  c . i . .= 7.65 - 9.23) and  106  fvc  cells  the  (9.3 s e c o n d s ,  length  of  c. i .  = 8.49 - 1 0 . 1 1 ) . . A t  34.5°C  t h e CV c y c l e i n c r e a s e d i n a l l c e l l  types.  T h e r e 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 thec y c l e lengths  o f w i l d - t y p e and f y c c e l l s when  BP  was  when  was  5 m i n u t e s i n 6% DMSO a t  34.5°C, t h e CV c y c l e s o f a l l c e l l  reguiring  at least  began s w e l l i n g for  gullet  and a f t e r  was  into the gullet and  within  (Figure base  reached  t h e presence  fused with o t h e r breakdown  other  (Figure  became formed. DMSO  p a r t i c l e s were swept  vacuoles present  of  t h e food  areas  rapidly  of  vacuoles  extended..  (Figure 5-3). membrane directly  and  34.5°C,  o f t e n f u s e d w i t h each abnormalities  cytoplasm  also  c l e a r e d and bulges sensitive  appeared  as  minutes  t o 6%  early  a s 10  i n  wild-  1 2 % DMSO t h e e f f e c t s o c c u r r e d  and by 20 m i n u t e s many c e l l s  their c i l i a  at the  vacuole  15 m i n u t e s a t 27°C  m i n u t e s a f t e r a d d i t i o n ( c o m p a r e d t o 15 t y p e and f v c c e l l s ) . . I n  of the c e l l  were r e l e a s e d  i n the cytoplasm  fused  pinched o f f  o f DMSO t h e v a c u o l e  dml c e l l s a t 34.5°C were more  i n that  vacuoles  (15 m i n u t e s  the posterior  5-4). Morphological  apparent;  long,  f o r m a t i o n a t t h e base o f  and t h e v a c u o l a r c o n t e n t s  present  27°C  d i d n e t pinch o f f but e n l a r g e d and  i n t o t h e cytoplasm. . A f t e r vacuoles  vacuole  abnormal. Normally,  seconds  Alternatively, occurred  10 m i n u t e s i n 6% DMSO  food  the g u l l e t  eventually  The f o o d  and a membrane-bound v a c u o l e  5-2)..In  of  t y p e s were v e r y  10 s e c o n d s p e r c y c l e .  wild-type cells)  the  a d d e d . . However,  added t o dml. c e l l s , t h e l e n g t h o f 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 or  BP  were m o t i o n l e s s  very with  107  The mOs had  o s m o l a l i t y o f 6% DMSO i n b u f f e r i n c r e a s e d t o 627  (frcm the 45 mOs of b u f f e r a l o n e ) . The a d d i t i o n of BP no e f f e c t  on the o s m o l a l i t y c f b u f f e r alone  or DMSO i n  buffer.  c.. e f f e c t  of treatment  duration  A f t e r one minute i n phenotypes  in  significantly The  CVs  12%  wild-type  different  were swollen  abnormalities  dmj  the d i s t r i b u t i o n and  fyc  from that o f c o n t r o l s  dmj and f y c c e l l s  became evident  minutes and i n wild-type  disrupted  d-  vacuoles  addition  of  RP  vacuoles  (Table 5-4).  the f i r s t  different  from  (P < .01)..By 15 minutes i n 12% DMSO  the c e l l s i n a l l three c e l l types had  (P < .001).  and f y c c e l l s a f t e r 10 minutes,  dmi c e l l s were s i g n i f i c a n t l y  type c e l l s  was  i n dmj c e l l s a f t e r 5  dmj c e l l s were, more s e n s i t i v e t o DMSO; f o r minutes  of  cells  and by 3 minutes d i s r u p t e d  were observed i n wild-type, Cortical  DMSO  (wild-type,  and/or c o r t i c a l  followed  by  10  wild-  most o f  dmi and fyc)  abnormalities.  DMSO  and  a  second  watercolor RP was added to w i l d - t y p e , minutes  followed  minutes of which  f o r 20  by BP f o r 20 minutes during the l a s t 10 12% DMSO i n b u f f e r and BP were added. The  distribution  of phenotypes  manner  significantly  was  dmj and f y c c e l l s  i n samples different  treated  this  than i n c o n t r o l c e l l s  (P < .00 1). T h i s d i f f e r e n c e was mainly due to of c e l l s with d i s r u p t e d vacuoles  in  the number  (Table 5-5). Both dmj and  1C8  fvc  were  significantly  different  from  wild-type  (P = .006), t h e d i f f e r e n c e again t e i n g mainly number  t o the  of c e l l s with d i s r u p t e d vacuoles. When 6% DMSO was  similarly abnormal the  due  cells  added, the d i s t r i b u t i o n o f phenotypes  was  also  (P < .001) with the g r e a t e s t changes o c c u r r i n g i n  number  vacuoles.  The  d i s t r i b u t i o n of phenotypes i n wild-type and f y c c e l l s  was  similar  of  cells  (P = .21)  with  while  disrupted  that c f dm 1 c e l l s was d i f f e r e n t  (P < .001) because o f a g r e a t e r number of dml c e l l s disrupted  with  vacuoles..  The  greatest  incidence  cf c e l l s  with  disrupted  vacuoles occurred when 6% DMSO was added f o r 20 minutes i n b u f f e r and BP. There was no d i f f e r e n c e and  dm 1 c e l l s  (P = .54),  between  wild-type  and although t h e r e appears t o be  a d i f f e r e n c e between wild-type and fyc c e l l s , t h i s may not be v a l i d sample  due t o the s m a l l (15)...The  u s u a l l y a mixture vacuoles  number  of f v c c e l l s  watercolor i n the d i s r u p t e d vacuoles was of  formed  BP  and  i n DMSO  EE  indicating  were  t h e ones  abnormal. O c c a s i o n a l l y vacuoles with disrupted When  i n the  only  that that  EP  were  those became also  u s u a l l y , however, these vacuoles were i n t a c t . EP  was  added f o r 20 minutes followed by black  watercolor for 20 minutes with  12% DMSO added f o r t h e l a s t  10 minutes of i n c u b a t i o n , t h e r e s u l t s were very s i m i l a r t o those obtained with BP (Table 5-6). watercolor  and b u f f e r was added f o r 10 o r 20 minutes, the  r e s u l t s obtained with dnQ c e l l s obtained  When 6% DMSO i n black  with  BP  were  similar  to  those  but the r e s u l t s f o r wild-type and f v c  109  c e l l s were d i f f e r e n t * in  T h i s can be e x p l a i n e d by d i f f e r e n c e s  the watercolors themselves. .Red and  consist  of very  t i g h t l y held  small  black  watercolors  p a r t i c l e s evenly d i s t r i b u t e d and  i n the watercolor  suspension  whereas  BP  c o n s i s t s c f l a r g e r c r y s t a l s of pigment not h e l d t i g h t l y i n suspension.  Therefore,  i f miner  membrane  disruptions  o c c u r r e d , the vacuoles with red or black watercolor retain  their  pigment  vacuole  would r e a d i l y  in a  lead  w e l l as  but BP i n a  seep out. Major d i s r u p t i o n s  vacuolar membrane caused would  cohesive sphere  would  of the  by a higher c o n c e n t r a t i o n of DMSO  t o a d i s r u p t i o n c f red and black vacuoles as  blue  vacuoles.  This  was  observed  when  black  watercolor was added i n 12% DMSO f o r 10 minutes; d i s r u p t e d vacuoles  were present i n a l l three c e l l  were more s e n s i t i v e than f o r 10 minute treatment  types. . dm 1 c e l l s  w i l d - t y r e o r f vc c e l l s t o 6% DMSO (P = .001) o r 20 minute  treatment  (P = .002) i n d i c a t i n g t h a t the lower c o n c e n t r a t i o n of DMSO induces  more  e x t e n s i v e membrane damage i n dm_1 c e l l s  i n wild-type or f y c c e l l s . dmj  cells  The vacuolar  at 34.5°C without  i n the d i s r u p t i o n  vacuoles.  In  BP i t s e l f  abnormalities i n  DMSO added were  to r e s u l t  insufficient  of red or b l a c k - c o l o r e d  a d d i t i o n , t h e r e i s the p o s s i b i l i t y  c o n t r i b u t e s to vacuolar d i s r u p t i o n s .  b a : c l a r i f i e d i n Chapter VI.  than  t h a t the  This  will  110  2..  Dibucaine At  a l l c o n c e n t r a t i o n s t e s t e d at 27°C and 34.5°C i n w i l d -  tyP #  dmj and f y c c e l l s ,  that  of untreated c o n t r o l s (Tables 5-7 and 5-8). At 3,.8 x 1 C  e  e n d o c y t o s i s was decreased  mg/ml a t 34.5°C morphological  vacuoles were  i n v a c u o l a r morphology  observed.  e n d o c y t o s i s with no  resulted -5  in  decreased  endocytosis  ; 5 x 10  A5  cells  at  10  - 6  i n A5 c e l l s a t 27°C  g/ml were reguired f o r e g u i v a l e n t decrease of  wild-type and d i j c e l l s . . A t  in  change  (Tables 5-9 and 5-10) . .However, A5 c e l l s  were more s e n s i t i v e than w i l d - t y p e or dm 1 c e l l s  in  - 5  SDS SDS a l s o r e s u l t e d i a decreased  10  to  a b n o r m a l i t i e s were a l s o apparent.  No changes i n morphology of food  3.  relative  g/ml while  endocytosis  34.5°C e n d o c y t o s i s was i n h i b i t e d  g/ml, i n d j j c e l l s a t 10~ g/ml and i n  - 7  6  w i l d - t y p e c e l l s at 10~ g/ml ( t h i s c o n c e n t r a t i o n was l e t h a l f o r 5  A5 c e l l s ) .  4.  Colchicine Colchicine  changes  in  also  inhibited  the food  vacuoles  endocytosis (Table  without  5-11). . At  causing 27°C  c o n c e n t r a t i o n s t e s t e d had no e f f e c t on any o f the c e l l However, 0.25  at  mg/ml  reguired  to  34.5°C  endocytosis  colchicine decrease  whereas the  wild-type and dmj c e l l s . . .  the  types.  was i n h i b i t e d i n A5 c e l l s a t t e n times  number  that  amount  was  c f food vacuoles formed i n  111  5.  Cytochalsin B Aqueous  variable  suspensions  results  due  of  to  cytochalasin  B  gave  the impossibility  of  extremely obtaining a  uniform s o l u t i o n . T h i s agent appeared to decrease but  no  conclusive  statement  cculd  be  observed e f f e c t s of low c o n c e n t r a t i o n s considered  endocytosis  made. I n view of the  of  DMSO,  i t was not  wise t o d i s s o l v e c y t o c h a l a s i n B i n DMSO i n order t o  obtain a uniform s o l u t i o n . .  D.  Discussion The  observed  endocytosis reports al.,  effects  i n wild-type  of cells  ( S k r i v e r and N i l s s o n ,  1977) and i n c l u d e  DMSO  on  CV  function  are consistent  with  1974, N i l s s o n , 1977b,  and other  Sibley  et  the.following:  1. s w e l l i n g and decreased r a t e of c o n t r a c t i o n of CVs 2.  decreased r a t e of food  3..fusion  of  exposures  abnormalities the e x t e n s i v e  (up to 24  Paramecium  formation  and d i s r u p t i o n o f vacuoles  4. c o r t i c a l Many  vacuole  hours)  changes to  DMSO  by  long-term  i n Tetrahymena  and  a r e s i m i l a r t o the e f f e c t s of s t a r v a t i o n and,can be  a t t r i b u t e d t o the c e s s a t i o n of endocytosis the preceding minutes)  caused  ( N i l s s o n , 1977b). In  experiments exposure times were short  Therefore,  (maximum 20  the observed e f f e c t s a r e u n l i k e l y  due t o  starvation. The  CV  i s an o r g a n e l l e  used by f r e s h water p r o t i s t s f o r  112  osmoregulation  t o prevent  s w e l l i n g and t o maintain  concentrations  of o s m o t i c a l l y  active  ( P a t t e r s o n , 1980)., The r a t e  of  intracellular  contraction  i n v e r s e l y p r o p o r t i o n a l to the o s m o l a l i t y environment  (Prusch,  f o r P i caudatum  of  t h e CV i s  the  surrounding  DMSO were s h o r t e r than  (Patterson,  f o r P. m u l t i j i c r o n u c l e a t u m  particles  of  1977). The values obtained  of the CV c y c l e without  and r e g u l a t e  f o r the l e n g t h those  obtained  1977) but s i m i l a r to those  obtained  (Organ e t a l . , 1968). The reason f o r  the i n c r e a s e d length of the c y c l e i n the mutants i s unclear but may  be r e l a t e d to a p o s s i b l e impairment i n some a s p e c t  of t h i s  complex osmoregulatory mechanism. The increase, i n o s m o l a l i t y of buffer  with  isomolar probably  added DMSO i s f a r above 50 - 60 mOs  concentration  f o r Paramecium  accounts f o r the i n c r e a s e d  which  (Prusch,  length  i s the  1977) and t h i s  of  the  vacuolar  c y c l e when DMSO i s added. The difficult  decrease  i n endocytosis  t o e x p l a i n because N i l s s o n  caused  by  DMSO  (1977b) found  i s more  no  changes  i n the u l t r a s t r u c t u r e of the c y t o s t c m a l  region o f T. p y r i f o r m i s  after  hour  treatment  endocytosis vesicles  DMSO  f o r one  the pharyngeal  membrane  mitochondrial consumption energy  structure  to  those  the i n c o r p o r a t i o n  vacuoles..DMSO  also  affects  ( N i l s s o n , 1977b) and decreases oxygen  (franz and Van Bruggen, 1S67) thereby production  cells  t h e r e were s m a l l  similar  i n starved cells..DMSO may prevent  of v e s i c l e s i n t o the growing food  with  ( i n these  ceased a f t e r t e n minutes) although  near  occurring  with  which  (Chapman-Andresen and N i l s s o n ,  i s essential  interfering  f o r endocytosis  1968, Yamada, 1974).  DMSO a l s o changes the d i s t r i b u t i o n  of  particles  i n the  113  membranes of mouse lymphocytes may  also  occur  i n food  r e f l e c t i o n of modified interactions  (Mclntyxe e t a l . , 1974)  vacuole  enzyme  since  membranes. T h i s c o u l d be a  function  DMSO  and t h i s  alters  and/or  protein-lipid  protein  conformation  (Rammler, 1971) and f a t t y  acid  M a r t i n - E s t e v e , 1965).. T h i s  i s c o n s i s t e n t with o b s e r v a t i o n s o f  disrupted al.,  stereoisometry  vacuoles i n these and other  experiments  (Muset  and  (Sibley  et  1977) . " T o l l o c s k o (1977)  found  t h a t i n E_ ca udatum c e l l s  treated  with c y t o c h a l a s i n B d i s s o l v e d i n DMSO, d e f e c t i v e s e p a r a t i o n of food  vacuoles from the g u l l e t  o c c u r r e d . T h i s was a t t r i b u t e d t o  the c y t o c h a l a s i n B but may have been caused by the the  same  lack  of  separation  was  observed  (Figure 5-3)..Fusion of vacuoles a f t e r exposure been p r e v i o u s l y reported but t h i s difficulty  i n observing  such  t h i s study t h i s d i f f i c u l t y videotape  where  the discernment  was  night  be  DMSO  since  with DMSO alone t o DMSO has not  explained  by t h e  r a p i d events i n l i v e c e l l s . I n circumvented  by  the use of  t h e a v a i l a b i l i t y of " i n s t a n t r e p l a y s " i n s u r e d of a l l events.. The  (Rammler, 1971) and l i p i d s  e f f e c t s of DMSO cn p r o t e i n s  (Muset  and Martin-Esteve, 1 965),  which are based, i n p a r t , on the i n t e r a c t i o n of DMSO with water (Cowie and Toporowski, water  196 1) and i t s  (MacGregor, 1967),  may  possible  contribute  to  replacement  of  the f u s i o n  of  vacuoles. Paramecia separations) and  tend  to  i n response  chemicals  form  tlebs  (membrane  and  cortical  t o many agents, such as p r e s s u r e , heat  (Sibley and Hanson, 1974) and i n s e n s i t i v e  i n response t o kappa symbionts  cells  (Jurand e t a l . , . 1978). Since the  114  shape  of  Paramecium  infracilliary  based  lattice,  abnormalities (Sibley  is  with  i n d i r e c t l y as  is  decrease  contraction  the  case  with  can  kappa of  and blebs rate  pressure  cortical  cytoskeleton  occur  directly  endosymbionts  the  i n c r e a s e d r e t e n t i o n of water c a u s i n g an hydrostatic  cause  subcortical  This  rate  microfilamentous  which  this  Hanson, 1 974). the  the  agents  interfere  and  on  CV..  which  This results i n  increase  in  internal  that l e a d s t o the formation of s w e l l i n g s  (Jurand et a l . , 1978). Since DMSO a l s o decreased  of  or  c o n t r a c t i o n of the CVs,  the observed  the  blebs could also  be due t o an i n c r e a s e i n i n t e r n a l h y d r o s t a t i c pressure. Dibucaine, SDS wild-type and anasthetics and Nelson, normal with  1S76,  Helenius and  be due  Simons, 1975)  functioning. agents  abnormalities)  could  bilayer  inhibited  endocytosis  inserting (Browning  (eg.. be into  and  due  than dmi,  to  opposite  sensitive  wild-type  cells cells,  mutants have i r r e g u l a r i t i e s i n  which  (Browning  may  different effects  dibucaine  Nelson,  s e n s i t i v e t o SDS than  The  caused  of  impede observed  morphological  anasthetics faces  in  to the i n s e r t i o n of  and detergents i n t o the membrane b i l a y e r  two  detergents  colchicine  mutant c e l l s . T h i s may  membrane the  and  and the  anionic membrane  1976). . A5  cells  were  more  which,  turn,  were  more  in  thereby i n d i c a t i n g t h a t both  their  membranes  (probably  at  d i f f e r e n t s i t e s ) which make them mere s u s c e p t i b l e t o the a c t i o n of the detergent. . Colchicine causing  changes  (Tolloczko,  1977)  inhibits in and  the  endocytosis  in  ultrastructure  t h i s i n h i b i t i o n may  P._ caudatum of  without  microtubules  r e s u l t from  changes  115  i n membrane topography of  PMLs  (Berlin  and  as occurs i n i s o l a t e d F e r a , 1977).  plasma  Normally,  membranes  phagocytosis i n  these c e l l s i s accompanied by a decrease i n the m i c r o v i s c o s i t y of  the  membrane.  B e r l i n and Fera mediated  T h i s change i s e l i m i n a t e d by c o l c h i c i n e and  (1977)  that  extensive  microtubule-  membrane m o d i f i c a t i o n s are r e g u i r e d f o r phagocytosis.  The t e n - f o l d i n c r e a s e c o l c h i c i n e suggests system i n t h i s The  suggest  vast  in  sensitivity  of  a possible lesion i n a  the  A5  mutant  to  microtubule-mediated  mutant. range  of b i o l o g i c a l e f f e c t s o f DMSO l i m i t s i t s  u s e f u l l n e s s i n f u r t h e r c h a r a c t e r i z i n g the mutant, dmj..However, s i n c e i t does a f f e c t membranes, the cells  are  consistent  with  mutant..Its  g r e a t e s t value  selection  system  for  observed  membrane  effects  abnormalities  may  l i e in  more  membrane  i t s use mutants.  in  on in  this  a  mass  Similarly,  c o l c h i c i n e may a i d i n the s e l e c t i o n of other mutants which d e f e c t i v e i n the i n t e r n a l movement of vacuoles. .  dmj  are  116  Table 5 - 1 . . E f f e c t of dimethyl s u l f o x i d e at 2 7°C Cone. DMSO (1)  Cell N Type (2)  c o n t r o l ~Wt dmj fyc  SCV (3)  42~ 43 39  o~~ 0 0  wt dm! fyc  11 10 5  1 0 0  wt dmj fyc  12 3 4  10%  wt dml fyc  12%  15%  5%  8%  DV  CO 0 0 0  DE (5)  X2  (7)  P (8)  7 9  C C 0  0.25 1. 24  0.62 0. 26  30 30 30  8 10 5  0 0 0  1. 27 2.87  0.74 0. 41  9 11 15  20 30 30  9 1 0 5  0 c 0  8.20 5.51  0.04 0.04  4 1 2  24 18 13  16 26 20  6 5 5  0 0 0  5. 13 3. 4  0. 16 0.33  wt dm! fyc  0 0 1  10 5 7  30 23 28  6 4 5  3 18 9  17.70 4.68  0.001 0. 32  wt ami fyc  3 0 0  10 9 12  14 16 15  3. 27 3.22  0 23 0 . 25 0 23  ~5  CA (6)  0.35 0.36  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 vacuoles at 34.5°C (1) c o n c e n t r a t i o n of d i m e t h y l s u l f o x i d e added f o r 20 minutes with b l u e watercolor (2) number o f 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 d i s r u p t d vacuoles (5) number o f c e l l s with decreased e n d o c y t o s i s (6) number of c e l l s with c o r t i c a l a b n o r m a l i t i e s (7) Chi Sguare Test f o r homogeneity ( 8 ) p r o b a b i l i t y generated by C h i Sguare Test =  117  T a b l e 5-2. E f f e c t of dimethyl s u l f o x i d e a t 34.5°C Cone.. DM SO (1)  Cell N Type (2)  SCV (3)  CA (6)  X (7)  P (8)  control  wt dmj fvc  40 19 18  0 0 0  0 10 19 12 0(9) 1 0  0 12 C  26.66 30.34  <.001 <.00 1  5%  wt dmj fyc  4 2 4  0 0 0  23 38 21  23 10 16  0 0 0  9. 48 4.50  0.004 0.21  8%  wt dlJ fyc  4 5 4  6 11 6  28 31 30  6 2 7  0 1 2  4.37 1.88  0.36 0.76  10%  wt dmj fvc  5 4 5  8 7 9  33 32 29  3 2 6  1 1  3.06 1. 32  0.55 0.86  12%  wt dmj fyc  1 0 0  9 1 2  30 38 37  4 4 5  6 7 6  8.42 6.3  0.08 0. 18  15%  wt dmj fyc  1 0 0  0 0 0  29 26 27  5 9 6  15 15 17  2. 31 1.29  0.53 0. 73  DV  (4)  DE (5)  c  2  wt = wild-type dmj = mutant with d i s r u p t e d vacuoles at 34.5°C f y c = mutant with clumped vacuoles at 34.5°C (1) c o n c e n t r a t i o n of dimethyl s u l f o x i d e 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 d i s r u p t e d vacuoles (5) number o f 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 a b n o r m a l i t i e s (7) Chi Sguare Test f o r homogeneity (8) p r o b a b i l i t y generated by C h i Sguare Test (9) number of f y c c e l l s with clumped vacuoles = 22, number o f wild-type c e l l s with clumped v a c u o l e s = 0  118  Table 5-3. Length  of c o n t r a c t i l e vacuole  cycle  Temp. °C(1)  Cell Type  BP added (2)  Length of c y c l e (sec) (3)  27  wt dmjl dmj fyc  -  7.59(7. 11-8.07) 8.44 (7. 65-9.23) 6. 21 (5. 44-6. 98) 9.30 (8. 49-10. 1 1)  wt wt dm! dm! fyc fyc  + " «• *  6. 38 (5. 78-6.98) 7.55(5.91-9.19) 5.63 (4. 72-6.54) 8. 78 (7. 36-10. 20) 6. 13 (5. 69-6.57) 7.42 (6. 38-8. 46)  34.5  +  wt = wild-type dm! mutant with d i s r u p t e d vacuoles a t 34.5°C f y c = 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 i n d i c a t e d («•) blue watercolor was added f o r 20 minutes (3) means and 95% confidence i n t e r v a l s of the length o f the c o n t r a c t i l e vacuole c y c l e =  119  T a b l e 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 Time Cell (min) (1) Type  N  (2)  scv  DV  DE  (3)  (4)  (5)  "HI 18 14  0 0 0  wt dmj fyc  30 4 9  wt dmj fyc  5  10  c o n t r o l ~wt djl fyc 1  3  15  20  ___  CA (6)  X2  P  (7)  (8)  __  24 8 1 (*) 8  0 0  27. 26 39.45  <.001 <.001  11 15 1 1  7 0 28 2 9 (+) 4  1 1 0  35.09 13.97  <.00 1 0.C07  0 0 1  30 19 18  16 23 14  0 5 5  4 3 4  12.87 8. 5  0.01 0.07  wt dmj fyc  1 0 0  28 13 22  15 14 20  3 1 1 2  3 12 4  16.49 2.74  0.002 0. 60  wt dmj fyc  1 0 . 0  18 13 15  23 17 28  2 6 3  4 14 4  wt dmj fyc  0 0  c  1 1 8 8  26 7 21  2 3 0  11 32 16  wt dmi fvc  0 0 0  2 1 0  4 3 1  1 1 7 1 1  33 40 38  9. 18 1.92 21.87 3.68 2.03 4. 15  0.04 0.75 <. 001 0.30 0. 57 0.25  wt = wild-type dmi = mutant with d i s r u p t e d v a c u o l e s at 34. 5°C f y c = mutant with clumped vacuoles at 34.5°C (*) number f y c c e l l s with clumped vacuoles = 27, wild-type (+•) number f y c c e l l s with clumped vacuoles = 1 7 , wild-type (1) number o f minutes i n 12% d i m e t h y l s u l f o x i d e (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 v a c u o l e s (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 endocytosis (6) number of c e l l s with c o r t i c a l a b n o r m a l i t i e s (7) Chi Sguare Test f o r homogeneity (8) p r o b a b i l i t y generated by C h i Sguare Test  =0 = 1  120  Table 5-5. E f f e c t of w a t e r c o l o r a t 34.5°C  P (7)  0 0  2 19 1 (8)  7 10 6  21.51 18. 38  <.001 <.001  14 7 1  7 0 0  26 40 42  3 2 4  12*49 22. 10  0.006 <. 001  14 1 5  25 33 23  C 4 1  16. 36 4.47  0.001 0.21  46 45 9  0 1 1  2. 15 17.9  0. 54 <.001  control  wt  41 23 24  Wt dml fyc  r e d and b l u e  X (6)  N (2)  12% DM SO 10 min  and  DE (5)  Cell Type  6% DMSO 1 0 min  wt djl fyc  3 3 1  6% DMSO 20 min  wt dm! fyc  3 4 0  SCV  sulfoxide  DV  Co nc., Time (1)  Ml fVG  dimethyl  (3)  1 . 0 5  2  wt = w i l d - t y p e dm! mutant 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 f y c = mutant with clumped v a c u o l e s a t 3 4 . 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 u e watercolor f o r 20 minutes w i t h t h e c o n c e n t r a t i o n and t i m e o f e x p o s u r e t o DMSO a s indicated (2) number o f normal c e l l s (3) number o f 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 c a 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 o f c e l l s w i t h d e c r e a s e d e n d c c y t o s i s (6) C h i Sguare T e s t f o r h o m o g e n e i t y (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 S g u a r e T e s t (8) number o f f y c c e l l s with clumped vacuoles =14, wildtype = 0 =  121  Table 5-6. E f f e c t of w a t e r c o l o r a t 34.5°C  dimethyl  Cone., Time (1)  Cell Type  N (2)  SCV (3)  control  wt fyc  50 32 30  0 0 0  12% DM SO 10 min  wt dmj fyc  6 1 1  13 8 8  6% DMSO 10 min  wt dmj fyc  17 7 14  6% DMSO 20 min  wt dmj fyc  13 8 2  DV (4)  sulfoxide  r e d and b l a c k  X (6)  P (7)  0~ 2 3  5.83 27.94  0. 05 <.001  31 40 32  1 0 0  5.89 3.43  0.05 0. 14  17 4 7  13 19 7  1 6 6  15.50 7.66  0.001 0.05  1 8 2  10 22 4  23 12 4  14.51 5.52  0.002 0. 14  0 2 0(8)  DE (5)  and  2  wt = w i l d - t y p e dmi = mutant 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 f y c = m u t a n t w i t h c l u m p e d 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 watercolor f o r 20 minutes w i t h t h e c o n c e n t r a t i o n a n d t i m e o f e x p o s u r e t o DMSO a s indicated (2) number o f n o r m a l c e l l s (3) number o f 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 o f c e l l s w i t h d e c r e a s e d e n d o c y t o s i s (6) C h i S g u a r e T e s t f o r h o m o g e n e i t y (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 S g u a r e (8) number f y c c e l l s w i t h c l u m p e d v a c u o l e s = 2 0 , w i l d - t y p e = 0  1 22  Table 5-7. E f f e c t of dibucaine at 27°C  Cone. (D  Cell Type  N [2)  DE (3)  control  wt ami A5~  30 30 30  0 0 0  0.00 0. 00  1.00, 1.00  3.8x10-6 wt dml A5~  17 13 22  13 24 13  16.60 30.30 13.93  <.001 <.001 <.001  3.8x10-5 wt dml A5~  0 0 0  39 31 16  69.00 61.00 46.00  <.001 <.001 <.001  X2  P (5)  wt = wild-type dm_1 = mutant with d 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 non-discharge and clumped vacuoles at 34.5°C (1) c o n c e n t r a t i o n of d i b u c a i n e (grams per ml) added f o r 20 minutes (2) number of normal of c e l l s (3) number of c e l l s with decreased e n d o c y t o s i s (4) C h i Sguare Test f o r homogeneity i . i n c o n t r o l s between w i l d - t y p e and each mutant i i . i n d i b u c a i n e between each c e l l type and i t s c o n t r o l distribution (5) p r o b a b i l i t y generated by C h i Sguare Test  123  T a b l e 5- 8. E f f e c t of  Conc. (2) control  Cell Type  dibucaine a t 34.5°C  N (2)  ___ dml A5  30 (7) 30(8)  DE (3) 5 1 5  X*  m  P (5)  ~ 1.44 0.02  0. 09 0. 88  3.8x1C-6 wt dml A5  15 7 2  21 27 28  13.10 38.38 40. 38  <.001 <. 001 <. 001  3.8x10-5 wt dml A5  0 0 0  30 30 30*  44.84 57.13 47.76  <.00 1 <. 001 <. 001  wt = w i l d - t y p e dmj = mutant with d i s r u p t e d vacuoles a t 34.5°C A5 = double mutant with t r i c h o c y s t non-discharge and clumped vacuoles a t 34.5°C (1) c o n c e n t r a t i o n of dibucaine (grams per ml) added f o r 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) C h i Square Test f o r homogeneity (see T a b l e 5-7) (5) p r o b a b i l i t y generated by C h i Sguare Test (7) number drnj c e l l s with d i s r u p t e d vacuoles = 25, wildtype = 0 (8) number A5 c e l l s with clumped vacuoles = 2 1 , wild-type = C * a l l c e l l s m o r p h o l o g i c a l l y abnormal  124  T a b l e 5-9. E f f e c t of sodium dodecyl s u l f a t e a t 27°C Cone. (1)  Cell Type  N (2)  control  wt dm! A5  30 30 31  0 0 1  0.00 0.95  1.00 0. 33  t dm! A5  30 30 30  0 0 0  0.00 0.00 0.95  1. 00 1.00 0.33  dm! A5  29 30 30  1 1 0  1.02 C.98 0.95  0.31 0. 34 0.33  10-6  wt dm! A5  29 29 30  1 1 0  0.98 1.02 0.95  0.32 0.31 0.33  5x10-6  wt dm! A5  30 32 19  1 1 11  0.98 0.92 11.16  0. 32 0.34 <.001  4 0 3  26 30 27  45.88 57. 13 47.19  <.001 <.001 <.001  10-7  5x10-7  10-s  K  w  w  t  t  dm! A5  DE (3)  X (4) 2  P (5)  * t = wild-type dm! = mutant with d 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 ncn-discharge and clumped vacuoles at 34.5°C (1) c o n c e n t r a t i o n (g/ml) sodium dodecyl s u l f a t e added f o r 20 minutes (2) number of normal c e l l s (3) number o f c e l l s with decreased endocytosis (4) Chi Sguare Test f o r homogeneity (see Table 5-7) (5) p r o b a b i l i t y generated by C h i Sguare Test  125  Table 5- 10. . E f f e c t of sodium d o d e c y l s u l f a t e a t 34. 5°C  Conc. d)  Cell Type  N (2)  control  wt dmi A5  29 29(6) 22 (7)  10-7  wt dm1 A5  30 24 3 .  5x10-7  wt ' dm1 A5  10-6  wt dmi A5  5x10-6  wt  DE (3) 1 1 8  X2  (4)  P (5)  0.00 6.40  1. 00 0.01  1 6 27  0.0006 4.0 4 24.75  0.98 0. 04 <.001  30 . 27 4  0 3 26  1.02 1.07 22.99  0.31 0. 30 <.001  26 16 4  4 20 20  1.96 20. 57 35.90  0. 16 <. 001 <. 001  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) c o n c e n t r a t i o n sodium dodecyl s u l f a t e (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 e n d o c y t o s i s (4) Chi Sguare Test f o r homogeneity (see Table 5-7) (5) p r o b a b i l i t y generated by C h i Sguare Test (6) number dnQ c e l l s with disrupted vacuoles = 21, w i l d type = 0 (7) number A5 c e l l s with clumped vacuoles = 12, wild-type = 2  126  T a b l e 5- 11. E f f e c t of  c o l c h i c i n e at 34.5°C  Conc. (1)  Cell Type  N (2)  control  wt dml A5  30 (6) 27 (7)  0. 25  wt dml A5  0. 50  2. 50  DE (3)  X (*») 2  P (5)  0 3  0.00 3. 16  1. 00 0. 08  30 24 8  0 6 22  0.00 6.67 24.75  1.00 0.01 <. 001  wt dml A5  27 22 8  3 8 26  3. 16 9.23 28.42  0. 08 0. 002 <. 001  wt dml A5  17 9 3  13 21 27  16.60 32.30 38.40  <.001 < .001 <. 001  wt = wild-type dm_1 = mutant with d i s r u p t e d vacuoles a t 34.5°C A5 = double mutant with t r i c h o c y s t non-discharge and clumped vacuoles a t 34.5°C (1) c o n c e n t r a t i o n c o l c h i c i n e (mg/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 f o r homogeneity (see Table 5-7) (5) p r o b a b i l i t y generated by C h i Sguare T e s t (6) number dml. c e l l s with disrupted vacuoles = 19, w i l d type = 1 (7) number A5 c e l l s with clumped vacuoles = 2 1 , wild-type = 0  127  Figure  5-1Schematic  dimethyl s u l f o x i d e .  i l l u s t r a t i o n cf morphological e f f e c t s of  Dots r e p r e s e n t p a r t i c l e s of blue watercolor  (BP) .  a. normal vacuoles and swollen  b. s e v e r a l  c o n t r a c t i l e vacuoles (CV)  normal vacuoles and one  or  more  large,  irregular  vacuoles  c. one or more l a r g e ,  d. no c l e a r  e. very  irregular  delineation  large,  of vacuoles  colorless  p a r t i c l e s of BE throughout  f. a cortical  vacuoles  (formerly  the  CVs)  present throughout  the c o r t e x .  Particles  of  appears BP  are  the cytoplasm  blebs where the c o r t e x appears  from t h e cytoplasm  and  the cytoplasm  s e p a r a t i o n where t h e u n d e r l y i n g cytoplasm  to have condensed away from  g. c o r t i c a l  vacuoles  t o have l i f t e d away  which has many p a r t i c l e s of BP  throughout  129  F i g u r e 5-2. Frame-by-frame newly  formed  movement  vacuole moves frcm  the p o s t e r i o r c f a w i l d - t y p e c e l l . second..  of  a  food  vacuole.  the base o f the g u l l e t Each number  The  (g) t o  represents  1/6  130  131  Figure  5-3..Endocytosis  frame t r a c i n g the  gullet  (g)  2.  5 second  a.  a food  b.  instead  vacuole  c.  the  vacuole  d.  c f abnormal  in  6%  food  sulfoxide..Frame-by-  formation a t the  Tracings are  base o f  separated  by  intervals..  of  i s forming  pinching  a t the base  off  remains attached t o the  nascent  fusion  vacuole  of a w i l d - t y p e c e l l .  vacuole  and  dimethyl  vacuole  t h e two  begin  the g u l l e t  (g)  moving t o t h e p o s t e r i o r ,  gullet  comes v e r y  i s c o m p l e t e and  which i s s t i l l  and  of  close  and  increases in  to a p r e v i o u s l y  the  size  formed  to fuse  a large,  attached to the  irregular  gullet  vacuole  i s formed  13Z  i  1  IOJJM  133  Figure  5-4.  Fusion of vacuoles  by-frame t r a c i n g of  a dml c e l l .  a. one l a r g e  of t h e f u s i o n  i n 6SS d i m e t h y l o f two v a c u o l e s  Tracings are separated  and one s m a l l  sulfoxide.  vacuole  by 2 . 5  approach  i n the second  Frame-  cytoplasm intervals  each o t h e r  in  the  cytoplasm  b. .the v a c u o l e s  c.  fusion  d. . f u s i o n vacuole  meet  proceeds  is  and b e g i n  t o fuse  and the c o n t e n t s  complete  resulting  o f t h e two v a c u o l e s  in  one  larger,  mix  irregular  I 3 H  135  P l a t e V-1. Morphological e f f e c t s  of  dimethyl  sulfoxide. 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  a. Wild-type  cell  (DMSO) f o r 10 vacuoles  minutes. . Note  filled  c o n t r a c t i l e vacuole  b. Wild-type  a f t e r treatment  with  blue  watercolor  of  sulfoxide  normal  (BP) and  swollen  with 6% DMSO f o r 10  vacuoles f i l l e d  i n r e d watercolor  minutes.  with BP.  (EP) f o r 20  minutes  f o l l o w e d by BP f o r 20 minutes and 12% EMSO f o r 10 minutes. normal vacuoles are present  (these were f i l l e d  as swollen CVs (small arrows) and a bleb  c e l l i s a mass of watercolor p a r t i c l e s (arrows)  on the c e l l  surface.  Some  with EP) as w e l l  (large  d. Wild-type c e l l i n 12% DMSO f o r 10 minutes.  blebs  food  (CV) (arrow).  Note t h e l a r g e , disrupted  cell  with 12% dimethyl  the presence  c e l l a f t e r treatment  c. Wild-type  on s l i d e s .  arrow).  The c e n t r e o f t h e  (BP) and t h e r e are s m a l l  (36  137  CHAPTER VI  ULTRESTRUCTURE OF WILD-TYPE AND  A.  dml  CELLS  Introduction In the l i g h t microscope,  watercolor  (BE)  dm!  c e l l s that have been f e d blue  appear to have very l a r g e d i s r u p t e d vacuoles  compared with the h i g h l y r e g u l a r , s p h e r i c a l wild-type  cells  f i x e d and  vacuoles  (Plate I I I - 1 b , c ) . However, when dml  examined  previously  food  been  in  the  fed  BP,  light  microscope  of  c e l l s are  without  t h e r e i s l i t t l e evidence  as  having  of vacuolar  a b n o r m a l i t i e s . In order to c l a r i f y the s t r u c t u r a l nature of the vacuolar  abnormalities,  wild-type  and  mutant  cells  were  examined by t r a n s m i s s i o n e l e c t r o n microscopy. .  B.  Materials  and  Methods  (see  Appendix I I I f o r d e t a i l s of  procedure) Wild-type hours..  Half  minutes,  the  c e l l s were incubated  cells  centrifuged  phosphate i n 6 mM  and dm!  (100xg,  phosphate buffer..The  f i x e d c e l l s were then one  The c e l l s  each 5  sample  34.5°C  were  minutes) ,  except  remaining for  the  washed t w i c e i n 6mM  in  6  omission  6  mM  glutaraldehyde  h a l f o f each sample of  BP..  was The  b u f f e r and p o s t - f i x e d  hour i n 1S osmium t e t r o x i d e i n 25 mM were wasied and dehydrated  for  f e d BP f o r 20  washed  b u f f e r , and f i x e d f o r 2 hours i n 0.5%  t r e a t e d i n the same way  for  in  at  step-wise  phosphate b u f f e r . through  ethanol  138  and  propylene  stained Epon  oxide.. While  70$  e t h a n o l , the c e l l s were  with uranyl a c e t a t e . The c e l l s were  and  sectioned on a S o r v a l l MT-1  Thin s e c t i o n s (60-90 nm) 30  in  30 dmi. c e l l s  embedded  in  U l t r a Microtome. acetate  (20 -  (5 - 10 minutes) and examined on  10 transmission e l e c t r o n  w i l d - t y p e and  or MT-2  were s t a i n e d i n u r a n y l  minutes) and l e a d c i t r a t e  a Z e i s s EM  then  microscope.  A minimum of 30  were examined.  C. . Re s u It s The and  i d e n t i f i c a t i o n of the s t r u c t u r e s of  the  terminology  used  in  describing  Allen  (1974)..The i d e n t i f i c a t i o n  food  vacuoles  Selman  based  on  McArdle  was  based  (1961)  on Jurand and Selman  and  and  age  Jurand  at the r e s t r i c t i v e  temperature  on of and  cytoplasmic  (1969) and E h r e t and  (1974). Unless otherwise i n d i c a t e d , a l l r e s u l t s  cells  region  them were based  of the s t r u c t u r e Jurand  oral  (1969) and t h a t of other c o r t i c a l u n i t s and  structures  to  was  the  t h a t had  refer  not been fed  BP p r i o r t o t h e i r f i x a t i o n . .. P l a t e VI-1 cell  near  r e p r e s e n t s a t r a n s v e r s e s e c t i o n of a  the  posterior  part  of the g u l l e t  wild-type  (g). Three food  vacuoles are present, each at a d i f f e r e n t stage. Food vacuole a (fva)  is a  membrane and  newly  formed  vacuole  a  relatively  smooth  and the b a c t e r i a i n s i d e are r e l a t i v e l y w e l l - p r e s e r v e d  undigested. Food vacuole b  stage;  with  the  at  a  slightly  later  membrane i s more i r r e g u l a r and the b a c t e r i a are i n  various stages of beginning  (fvb) i s  digestion  with  the  bacterial  cell  walls  to separate from the membranes. Food vacuole c (fvc)  139  is  an o l d e r vacuole with many outwardly  directed projections i n  the membrane. I t c o n t a i n s mainly  ghosts and s m a l l fragments  digested  (approximately 0.2  bacteria.  The  small  um) , dense,  membrane-bound v e s i c l e s c l o s e to the feed vacuoles are in  s i z e and d i s t r i b u t i o n t o n e u t r a l red granules  which  were  which were Whittner,  shown  to be the s i t e ,  later  associated  with  lysosomes  similar  (Jurand,  i n | j _ ca udatum  of  1961)  , o f enzymes  (Rosenbaum  and  1962).  Other  components  of the o r a l apparatus  are a l s o e v i d e n t .  Bordering the g u l l e t i s the p o s t e r i o r end of the guadrulus  (g),  which c o n s i s t s cf f o u r rows of c i l i a that begin at the a n t e r i o r end of the b u c c a l o v e r t u r e , pass half  of  the  buccal  cavity  dorsally  and  over  the  anterior  then angle over to the  (animal's l e f t ) s i d e and t r a n s v e r s e the ' p o s t e r i o r  end  left  of  the  b u c c a l c a v i t y . The l e f t c y t o s t o m a l l i p i s a s p e c i a l i z e d area of the  gullet  vesicles (Allen,  which (d)  1974).  is  for The  10 - 12 microtubules regular bundle  important recycling  in  seguestering  disk-shaped  cf  membrane  components  cytopharyngeal arranged  in  ribbons one  (cr) are bands of  plane  and  placed  i n t e r v a l s along the l i p . The cytostomal cord of  microfibrils  which  lies  over  the  at  (ce) i s a  ends  of  the  cytopharyngeal ribbons and extends along the f u l l l e n g t h o f the left  edge  of the cytostome  (Allen,  (po) are bundles of hexagonally beyond the b u c c a l c a v i t y and end  1S74). .The  postoral fibers  packed microtubules t h a t extend near  the  posterior  of  the  cell. . A mature t r i c h o c y s t near  the  upper  right  (t) i s v i s i b l e i n l o n g i t u d i n a l of  P l a t e VI-1.  section  I t s body appears  clear  140  because of a l a c k of a f f i n i t y f o r osmium t e t r o x i d e (Jurand Selman, 1969)  and  while  the t i p and  surrounding sheath are d a r k l y  also  represents  a  stained._ P l a t e VI-2 section  has  been  cut  tangential  c o r t i c a l u n i t s are c l e a r l y (bb)  surrounded  parasomal sac the  by  an  of  to  the  alveolus  (a)  (p).,The kinetodesmal  on  fibers  s u r f a c e . The  its  basal  either (k)  this  body  side,  are  and  joined  to  trichocyst  tips  (tt)  are  i n c r o s s s e c t i o n and are s i t u a t e d between the b a s a l bodies adjacent  fibrils  cortical  (Hufnagel,  The  units  infracilliary  (m)  inf r a c i l l i a r y  kinetodesmal  fibers  within  lattice  1969),  s e v e r a l mitochondria  is  appearance  to  of  a  wild-type  structure  (arrows). The proximity  and  presanse  to  drnj  have  basal cell  appears  smaller  fine are  (bb)  and  in  a  section  cut  similar  in  However, the mitochondria  (m)  areas abnormal  that  have  portions  is  a l s o unusual  lost of  their  membrane  (v) i n  close  (Plate VI-3b).  (t) of a dmj  In  cell.  mature t r i c h o c y s t , i s o s m o p h i l l i c and  normal i n comparison with ether u l t r a s t r u c t u r a l s t u d i e s  wild-type  McArdle,  a  of as  bodies  of membrane-bound v e s i c l e s  mitochondria  than  of b a s a l  visible  (Plate VI-3a) are  P l a t e VI-3c there i s an immature t r i c h o c y s t is  network  clearly  (if),  cells.  are very a t y p i c a l with i n t e r n a l ordered  a  (a row  with t h e i r t u b u l a r c r i s t a e . lattice  (k)  a kinety  (if) ,  also  t a n g e n t i a l l y t o the c e l l s u r f a c e  of  cell  but  b a s a l bodies and run a n t e r i o r l y from the b a s a l body to the  bodies) . The  It  cell  v i s i b l e , each with  same s i d e as the parasomal sac. The seen  wild-type  1974).  cells The  (Jurand gullet  and  Selman, 1969,  area  of a dmj c e l l  Ehret  and  ( P l a t e VI-3d)  141  also  compares  vesicles chord  well  (d) ,  cytopharyngeal  food vacuoles of dmj  those of wild-type VI-4a  i s a relatively  vacuole  ribbons  As  surrounded  by  lysosomes.  The  bacterial  cell  The  (cr)  disk-shaped  and  cytostomal  appear normal.  cells  are  very  different  in  where  wild-type  membrane-bound  the  membrane  cells,  the  vesicles  is  separating  no c l e a r d e l i n e a t i o n  have  seems food  pulled  away  membranous and  from  have  vacuole  slightly  is  probably  older  (the  the membrane). The  between v a c u o l a r contents and  from  the  (large  memtrane,  arrow).  There  which is  v e s i c u l a r m a t e r i a l throughout  arrows) ... The o l d food vacuole  the  to  of  there  cytoplasm  is as  wild-type c e l l s . The c o n t e n t s of the vacuole i n P l a t e VI-4c  discontinuities  that  area  (1) which are  vacuole i a P l a t e VI-4b i s wall  an  membrane of the food vacuole can b a r e l y be detected;  in  from  young vacuole as evidenced by the  undigested b a c t e r i a . There i s  (arrow)  degenerated..  cells.  (Plates VI-4a to VI-4d). The food vacuole i n  smooth membrane and the  wild-type  (cc) are a l s o v i s i b l e and The  Plate  with  a  also  has  profusion  of  the vacuole  (small  ( P l a t e VI-4d) i s most unusual  in  the vacuolar c o n t e n t s have completely condensed away from membrane  extensions  which  normally  does  not  show  the  highly  convoluted  seen i n o l d e r vacuoles of wild-type  cells  (Plate VI- 1, f vc). The  dmj  cells  in  Plates  Vl-5a  and  VI-5b  demonstrate  extreme cases of the mutant phenotype. In the c e l l i n P l a t e VI5a,  pieces  bacteria represents  cf  are a  membrane scattered  are  still  throughout  intact the  (arrows)  area. . Plate  and  the  Vl-5b  cross s e c t i o n 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 It  should be noted  that of  the  (arrows)  34  dmj  a r e apparent.  cells  that  had  v a c u o l e s , 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 t h a t had food vacuoles,  t h r e e c e l l s had vacuoles with s l i g h t  i n the v a c u o l a r membranes, one c e l l contents  slightly  condensed  bacteria  were undigested)  discontinuities  had a food vacuole with i t s  away  from  the  membrane  (the  and two out of 30 c e l l s examined had  a few abnormal mitochondria. There were no w i l d - t y p e c e l l s  with  p o o r l y d e f i n e d vacuolar membranes as i n dmj  VI-  4b),  huge vacuoles  cells  (Plate  (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 d e p i c t e d i n P l a t e s  VI-5c  and  VI-5d r e s p e c t i v e l y , were f e d BP before being f i x e d . These c e l l s were  much  more  difficult  t o s e c t i o n than c e l l s which had not  been f e d BP. The food vacuoles and mitochondria that  had  of  dmj  cells  been fed BP were s i m i l a r t o those dml c e l l s t h a t had  not been f e d BE whereas the w i l d - t y p e c e l l s  appeared  normal.  D. . D i s c u s s i o n In within  wild-type c e l l s the  a food vacuole corresponds  vacuolar vacuoles,  membrane the  membrane  of  the  vacuole  of  is  In  smooth, a s food  is  digestion  of  to the p h y s i c a l  1961).  (Jurand,  ( P l a t e V I - 1 , f va).. Normally, swelling  state  newly  the  bacteria  s t a t e o f the formed  bacteria  food  undigested  vacuoles age, a p e r i o d  of  f o l l o w e d by e v a g i n a t i o n s of the  v a c u o l a r membrane t h a t are accompanied  by  the  appearance  of  143  pinocytotic Finally,  vesicles  the  exocytosis  (Jurand,  old v a c u o l e s  of  the  1961, Favard and Carasso, fuse  undigested  with  the  1964).  cytoproct  m a t e r i a l occurs  and  (Jurand, 1961,  A l l e n and Wolf, 1974)..In dmj c e l l s t h e r e a r e no stages of t h e digestive  cycle  in  which  the p h y s i c a l s t a t e of the vacuolar  membrane c o r r e l a t e s c l e a r l y  with the c o n d i t i o n of the b a c t e r i a .  Instead, the food vacuoles can  be  grouped  according  t o the  s e v e r i t y of t h e i r d e f e c t s , i n the f o l l o w i n g manner:  1.  Slight abnormalities These  vacuoles  appear  normal  except  for  slight  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 u l t r a s t r u c t u r e o f the membrane  2.  ( P l a t e s Vl-4a and VI-4c) .  Degeneration  of s t r u c t u r e  The membrane surrounding these food vacuoles changes t h a t i t i s no longer c l e a r l y d i s t i n g u i s h a b l e  3..  Large  such  (Plate VI-4b) .  vacuoles  In o l d e r vacuoles a h i g h l y convoluted membrane i s expected but  i n s t e a d the vacuole s w e l l s and the membrane remains smooth  and extended  4.. T o t a l The  (Plate  VI-4d)..  degeneration vacuolar membranes degenerate  s m a l l patches  are d i s c e r n i b l e  of the cytoplasm i s d e s t r o y e d Mitochondrial  t o t h e extent t h a t  only  (Plate VI-5a) and u l t i m a t e l y most (Plate  abnormalities  VI-5b).  are a s s o c i a t e d with a l l f o u r  144  groups  ( P l a t e s VI-3a and VI-3b).  If cell  any of these four types of anomalies a r e present  which  has  been  f e d BP  and then examined i n the l i g h t  microscope, the BP would leak  into  appearance  fused  of  a  mass  of  the  cytoplasm  vacuoles  S i m i l a r l y , i f c e l l s are viewed i n the l i g h t previously  having  been  fed  BP,  d i s c e r n i b l e s i n c e these d e f e c t s not  be r e s o l v e d  detection  results  four  structure  could  be  membrane  responsible membranes  susceptible  phagosomes  impairment vacuolar structure food  enabling  causing of  could the  BP does not cause the facilitates i t s  to  (Muller  the  in  enzymes  f o r the  or the the  the  fusion  of  tc  leak  destroy  primary  into  portions  could  be  the  state  vacuoles between  of  the cytoplasm  mutant  relationship  could  powerful d i g e s t i v e  (Plate  the  observed  change  widespread d e s t r u c t i o n food  in  and Toro, 1962, E l l i o t t and  Clemmons, 1966)..The a c i d h y d r o l a s e s may  swelling  would be  i n membrane s t r u c t u r e . T h i s change  directly  membrane  and  eventually  without  abnormalities  that  enzymes which are a c t i v a t e d f o l l o w i n g  the  microscope  i n t h e membrane s t r u c t u r e  indicate  degeneration of the vacuolar  lysosomes  III-1c) .  degrees D f s e v e r i t y of the mutant phenotype a r e  with a d e f e c t  the  the  microscope.  consistent  render  (Plate  of the mutant phenotype, i t merely i n the l i g h t  All  no  giving  at the l i g h t microscope l e v e l . . Therefore,  ultrastructural expression  in a  VI-5b).  The  due t o an o f the  contents and the s t r u c t u r e of t h e membrane i t s e l f . The o f the vacuolar  vacuole and t h i s  is  membrane changes with the age o f the reflected  i n 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 f r e e z e - f r a c t u r e images of vacuoles of  145  H i caudataj  (Allan,  Wunderlich,  and  .1. P i r i f o r m i s  1S76). In wiLd-type c e l l s the  of the vacuoles particle  1976)  (eg. _ pH) may themselves  density  and  membrane  (Batz  internal  maintaining  v a c u o l e s , throughout The  conditions  mediate t h e changes i n  topography  (Allen,  However, i n dmj c e l l s the membranes may be unable thereby  and  to  1976).  respond,  the smooth c o n f i g u r a t i o n , common i n young the v a c u o l a r  cycle.  m i t o c h o n d r i a l a b n o r m a l i t i e s are a l s o c o n s i s t e n t with a  d e f e c t i n membrane composition vacuolar  membranes  components  are  (reviewed  Thompson, 1974).  s i n c e m i t o c h o n d r i a l as  synthesized in  from  Schatz,  Weidenbach  Mitochondrial abnormalities are also  The  mitochondria  are  (mainly  treated  and  (Jurand  a l . , 1978)... The  et  osmoregulatory  spaces  between  with  i n the c o r t i c a l  properties  p r o p e r t i e s might a l s o  be  cristae  symbionts  (Jurand  et  disturbed  by  appear may  kappa region)  electron  much  be  wider  affecting  a l . , 1978) changes  and  observed  appear s w o l l e n , the m i t o c h o n d r i a l matrix becomes more translucent  as  a c e l l u l a r pool o f  1970,  a f t e r s e n s i t i v e stocks of P. a u r e l i a symbionts.  well  and  in  these  membrane  composition. The  outer membrane o f the c e l l  t h e e f f e c t s of the dmj mutation membrane b l e b s which  Hanson  (1974) but not V-1c  although  immune t o  occasionally  double  (Plate VI-5b) o r r a r e l y , c y t o p l a s m i c s e p a r a t i o n s  occur  (Plates  seems r e l a t i v e l y  are s i m i l a r as  to  those  severe  as  and V-1d). The c i l i a r y  T . . p y r i f o r m i s are Bore r e s i s i t a n t t o changes i n the l i p i d  described those  by S i b l e y and  induced  by  DMSO  membranes and p e l l i c l e of  than the microsomal membranes  composition  that are induced by changes  146  i n temperature al.,  or  diet  (Martin  et  Fukushima  1976). In Paramecium , t h e p h o s p h o l i p i d composition  ciliary  membranes  (Andrews and  Nelson,  1979,  Rhoads and Kaneshiro,  the f l u i d i t y of the c i l i a r y  different (Nozawa  from and  the  Thompson, 1978). . The in  and  and  linkage  between  the nitrogenous base, acids  and  in  1979)  phospholipid  Paramecium Nelson,  1979,  properties  differences  ciliary  fiosenberg,  Tetrahymena  are  membranes, of linkage  1973),  ,  composition  the  of  and  the  high  phosphonolipids rather  than  polyunsaturated sterol-like  the  Ehoals and Kaneshiro, is  instrumental  (Ferguson  other  et  an  outer  of  fatty  pentacyclic tetrahymenol,  membranes  of  s t e r o l s , such  p r o t e i n s (Borochov  Although  no  the p e l l i c u l a r lipids  sterols  1S79). I t i s p o s s i b l e  Ccnner  and  Landry,  patterns  et a l . ,  1979,  of  extrinsic  and  Davis e t a l . , 1980).  or s t e r o l - l i k e substances a r e present i n  stable  structures  s p h i n g o l i p i d s , Rhoads and Kaneshiro, membranes  1976)  as c h o l e s t e r o l , change the membrane  membranes of Paramecium , there i s an with  that  i n maintaining optimal membrane  a l . , 1975,  intrinsic  these  Martin  the phosphorous atom and the carbon  f l u i d i t y and the s u r f a c e l a b e l i n g  of  in  i s very s i m i l a r t o t h a t of Tetrahymena. (Andrews and  tetrahymenol  since  and  membranes  et a l . , 1976,  t r i t e r p e n o i d , tetrahymenol. . With the exception of the  cell  membranes are very  mitochondrial  Martin  major  the  pellicle  ( p h o s p h o l i p i d s that c o n t a i n an e t h e r ester  of the  f a t t y a c i d composition as well  microsomal  Thompson, 1971,  concentrations,  et  i s very d i f f e r e n t from the r e s t of the  Tetrahymena the p h o s p h o l i p i d and as  a l . , 1976,  somewhat  from  the  1979)  abundance  (phosphonolipids which  effects  of  may  and  protect  changes  in  147  membrane c o m p o s i t i o n  induced  Thus, i t a p p e a r s t h a t mutation  are  mitochondrial cytoplasm,  manifested  by t h e dm 1 m u t a t i o n . .  the phenctypic e f f e c t s primarily  in  the  membranes. ..tfhen t h e h y d r c l y t i c  systematic destruction  of t h e c e l l  food  of  the  dm 1  v a c u o l e and  enzymes e n t e r occurs..  the  148  VI-1.  Plate section The the the  cilia left  the  lining  near  the  (animal's  postoral  and  longitudinal visible.  Three  surrounded  the  a wild-type  posterior  part  belong  the  to  cytostomal  vesicles  fibers  of  gullet  left)  cytopharyngeal  microtubules. age  vacuoles  cell  disk-shaped  as t h e The  of  Food  (d) ,  ribbons (po)  food  lip  the  (cr) are  vacuoles  bands  of  a  of  the  bundles (fva,  mature  A transverse gullet  guadrulus the of  of  fvb,  (g) .  (g).  gullet  Along  are  microtubules  and c y t o s t o m a l  by m e m b r a n e - b o u n d  section  of  cell.  the known  chord  (cc).  hexagonally  packed  fvc)  vesicles trichocyst  as  of  increasing well  (t)  are  as  a  also  150  Plate VI-2..Wild-type c e l l cortical  unit  is  centred  tangential around  to  the  surface.  a b a s a l body  (bb)  with i t s  kinetodesmal f i b e r  (k) running a n t e r i o r l y , an a l v e o l u s  either  parasomal sac  side  and  (p). The  are  seen  Mitochondria  (m)  in  cross  section  with t h e i r t u b u l a r  (a)  on  infracilliary lattice  ( i f ) c o n s i s t s cf a network of f i n e f i b r i l s . (tt)  Each  The  between  trichocyst tips basal  c r i s t a e are a l s o  bodies. visible.  152  P l a t e VI-3. Cortex and g u l l e t r e g i o n s c f dmj c e l l s .  a. .A (m)  section are  tangential  abnormal  kinetodesmal  t o the c e l l s u r f a c e . The mitochondra  (arrows)  fibers  but  the  basal  (k) and i n f r a c i l l i a r y  bodies  lattice  (bb) ,  ( i f ) appear  normal. .  b. Higher m a g n i f i c a t i o n  of an abnormal mitochondrion (m) and  a  membrane-bound v e s i c l e (v) . .  c. L o n g i t u d i n a l sheath  s e c t i o n through an immature t r i c h o c y s t  (ts) surrounds the t i p  d. .Transverse  section  (tt).  through  the  gullet  cytopharyngeal ribbons (cr) , c y t o s t c m a l chord shaped cells. .  vesicles  (d)  are  ( t ) . The  similar  in  (g).  (cc)  structure  and  The disk-  t o wild-type  153  154  P l a t e VI-4. Food vacuoles of dmj c e l l s .  a. A s e c t i o n degeneration  c f a young food vacuole with an area (arrow) and surrounded  ( 1 ) . The b a c t e r i a  of  membrane  ty membrane-bound v e s i c l e s  (b) a r e well-preserved and are i n  the  early  stages of d i g e s t i o n .  b. A  section  of  an  older  s e p a r a t i n g from the cytoplasm  vacuole  (the b a c t e r i a l  (arrows). There  is  no  walls are distinct  membrane surrounding the vacuole..  c. A  section  degeneration moving  away  of (large from  an  older  arrows) the  v e s i c l e s and fragments  and  the  membrane.  vacuolar  There  are  contents  are  many membranous  (small a r r c w s ) .  d. A s e c t i o n c f an o l d food contents  vacuole. The membrane has areas o f  have completely  vacuole  in  which  condensed away from  t h a t remains c f most of the b a c t e r i a  the  vacuolar  the membrane. A l l  (b) are membrane ghosts.  155  156  P l a t e VI-5. Extreme cases of t h e dmj phenotype.  a. A s e c t i o n c f degenerated  a  dmj  membrane.  cell  with  Parts  a  large  vacuole  with  o f the membrane are s t i l l  a  intact  (arrows). .  b. A c r o s s s e c t i o n of a dmj c e l l disintegrated.  There  are  and s m a l l c o r t i c a l blebs  c. A s e c t i o n  of  surface. . This fixation.  fixation..The  interior  (cb) along the c u t e r c e l l  cell  tangential  (m) ,  trichocyst  has  (arrows)  surface.  to  c e l l had been f e d blue watercolor  of  a  dmi  cell  mitochondria  membrane  (a rrows) . .  the  the  cell  (BP) p r i o r t o  tips  (tt)  and  (t) are v i s i b l e .  section  vacuole  which  many abnormal mitochondria  wild-type  Mitochondria  trichocysts  d..A  a  in  has  (m)  that are  degenerated  had been f e d BP p r i o r t o abnormal except  in  and a  the few  food areas  158  CHAPTER VII  FATTY ACID COMPOSITION Of WILD-TYPE AND dmi. CELLS  A.  Introduction The  phospholipids  l e s s e r extent, Fukushima Nelson,  et  Paramecium  have  Kaneshiro  also  et  contain  aminoethylphosphonolipid replaced,  is  position  (Mangnall  a l . , 1979,  Rhoads  (AEPL),  i n which the  such  replaced  also  by  and have  phosphoryl  Getz,  1973,  an Setz,  fatty  base  acids  and  the  glycerol  ether  linkage  1973,  Rosenberg, 1973).. These  at the carbon 1  sphingophospholipids  (Mangnall  and p r o t e c t i o n t o the membrane  Rosenberg, 1973,  Rhoads and Kaneshiro,  and  diet  (Wisnieski  Andrews  and  cells  (Mangnall  Nelson, 1979,  et  can be modified by many f a c t o r s , such a l . , 1973,  Ferguson  et  Dewailly e t a l . , 1977, C h r i s t i ansson and Weislander, the  provide  1979).,  Membrane composition  of  2-  1973, Sugita et a l . , 1979). Since ether l i n k a g e s are l e s s  a degree o f s t a b i l i t y  as  and  as  s u s c e p t i b l e t c h y d r o l y t i c and p h o s p h o l y t i c a t t a c k , they  and  (eg.  f o r example, by 2-amincethylphosphonic a c i d , and  backbone  Getz,  characterized  phosphonclipids,  the ester l i n k a g e between the  organisms  heen  1979). In a d d i t i o n t o p h o s p h o l i p i d s , Tetrah ymena and  Paramecium  is  ,  a l . , 1976, Conner and Stewart, 1976, Andrews and  1979,  Kaneshiro,  and f a t t y a c i d s of Tetrahymena and, to a  (Kaneshiro  et  a l . , 1975, 1980),. age  a l . , 1979) and c o n c e n t r a t i o n o f  oxygen and carbon d i o x i d e . (Erwin,  1973). Changes i n temperature  159  a l s o have many e f f e c t s on membranes and and  these  membrane  changes  are  fluidity  (Chapman, 1973). results  in  Fukushima  an et  necessary In  increase a l . , 1976),  in  the  Kaneshiro is  are  1979,  to  synthesized  of  acids  a l . , 1976)  particles  (eg.  and  from  acid  ,  (18:1) and linoleic  (18:2)  polyunsaturated 1973, (16:0)  via, p a l m i t o l e i c a c i d fatty  acids  a c i d v i a chain e l o n g a t i o n and  carboxyl  the  by  1973).  polyunsaturated  introduced  toward  an  as determined  Wunderlich,  Tetrahymena  all  subseguent  double  end  of  bonds  are  the  molecule  temperature-sensitive  mutation  1973).  The  dmi  resulting react  fatty  (Martin et  (Speth and as  i n temperature  i n c r e a s e i n the a c t i v i t y o f the  f u r t h e r d e s a t u r a t i o n where  (Erwin,  a decrease  functions  see F i g u r e 7-1) . P a l m i t i c a c i d  linoleic  (16:1) and o l e i c a c i d are  ,  cellular  s y n t h e s i z e d v i a the w6 pathway (Erwin,  et. a l . ,  converted  an  aggregation  In Paramecium as w e l l acids  normal  unsaturated  desaturase  f r e e z e - f r a c t u r e technigues  fatty  for  in  constituents  f o r maintaining the c o r r e c t  Tetrahymena  enzyme p a l m i t c y l CoA increase  reguired  membrane  a  i n abnormal membranes at 34. 5°C. S i n c e c e l l s  to  temperature  composition restrictive type and  mutant c a r r i e s  (Chapman, 1973), a  by study  altering of  the  their fatty effect  temperature on the f a t t y a c i d composition  dm 1 c e l l s ,  permissive  changes  as compared with  temperature,  may  r e g a r d i n g the d i s r u p t i o n of dmj  the  composition  provide membranes..  normally  further  of  acid the  i n wildat  the  information  160  B. . M a t e r i a l s and Methods  1.  Extraction  of  lipids  (see  Appendix  IV  for  complete  procedure) Wild-type for  6 hours.  by  serial  Approximately  10  of  1  filter  were  (Folch  paper  reagent  in  grade)  et  (#1). The l i p i d  and  reduced  according  to  B l i g l i and  twice  f i l t e r e d through  with  Whatman  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 acid  composition  w i t h t h a t o f P. . t e t r a u r e l i a  grown t o s t a t i o n a r y phase  were  chloroform/methanol/water  phase was washed  a l . , 1957)  -5°C..To c o m p a r e - t h e f a t t y  a e r o gen es  determined  ml o f c e l l s i n c u l t u r e f l u i d )  ( 1 9 5 9 ) . The f i n a l c h l o r o f o r m  methanol  (as  ( 1 0 0 x g , 5 m i n u t e s ) a n d t h e f i n a l volume was  solvents  Dyer  a t 27°C o r 34.5°C  c e l l s per sample  6  2.4 m l . L i p i d s were e x t r a c t e d  (all  at  dm 1 c e l l s w e r e i n c u b a t e d  dilution  centrifuged to  and  (10  cells  9  of  Enterobacter  , 1. 5 1 o f b a c t e r i a were per  ml)  and  half  the  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 h o u r s a n d t h e o t h e r h a l f a t 34.5°  C.  The  c e l l s were c e n t r i f u g e d a n d t h e r e s u l t i n g  pellet  was t r e a t e d as d e s c r i b e d a b o v e . .  2» . T h i n This remainder the  fatty  l a y e r c h r o m a t o g r a p h y (TIC) procedure of acid  the  separated l i p i d s which  composition  phospholipids  facilated  acid  from  the  the determination of  of t h e i n d i v i d u a l  the d e t e r m i n a t i o n of the f a t t y this  the  phospholipids. For  composition  o f whole c e l l s ,  s t e p was o m i t t e d . . The  lipid  extracts  were  evaporated  under  nitrogen  and  161  redissolved  i n 200  ul  chloroform.  Half  of each sample was  a p p l i e d to s i l i c a g e l G TLC p l a t e s (the remaining to  determine  were  chloroform/acetic and  separated  using  the  a c i d / methanol/water  Thompson, 1971)..  phosphatidylethanolamine  The  (PE,  10  solvent  of  were k i n d l y  system  (75:25:5:2.2 by volume, standards, mg/ml  pig  liver  chloroform)  p h o s p h a t i d y l c h o l i n e . ( P C , 20 mg/ml chloroform) Laboratory)  was used  the f a t t y a c i d composition of whole c e l l s ) and the  phospholipids  Nozawa  half  (Serdary  s u p p l i e d by Dr. D.E. Vance,  Research  Department  Biochemistry, U n i v e r s i t y o f B r i t i s h Columbia. The separated  p h o s p h o l i p i d s were i d e n t i f i e d  by s t a i n i n g with i o d i n e and areas  corresponding t o the i n d i v i d u a l p h o s p h o l i p i d s were scraped the p l a t e . P h o s p h o l i p i d s were removed from  the  silica  washing twice with c h l o r o f o r m / acetcne/methanol/water Andrews  and  Nelson,  1979).  The  chloroform  c o n t a i n e d t h e p h o s p h o l i p i d s , was evaporated the  samples  silica  were  stored  at  (6:8:2:2,  layer,  which  under n i t r o g e n and  -5°C. As a c o n t r o l , an area o f and  procedure.  P r e p a r a t i o n of g l y c e r y l e t h e r s Because  paramecia  contain  necessary t o convert the before adding  they  could  be  phosphonolipids,  phosphonolipids methylated.  to  This  hours..  glyceryl  i t was ethers  was accomplished by  2 ml of a c e t i c a c i d / a c i d anhydride  Kapoulos, 4  from  g e l by  g e l with no p h o s p h o l i p i d was scraped from t h e p l a t e  c a r r i e d through the e n t i r e  3.  and  (3:2, Thompson  and  1969) and heating i n t i g h t l y capped tubes at 90°C f o r The  tubes  were  cooled  and  1  ml of 6N potassium  162  hydroxide i n 95% e t h a n o l was s l o w l y tightly  capped.  The  samples  added and  were  the  tubes  then heated a t 90°C f o r 2  hours, cooled and e x t r a c t e d with ether twice. The e t h e r were  combined,  washed  twice  with  were  water,  layers  evaporated  under  n i t r o g e n , and stored a t room temperature..  4.  Met h y l a t i c n  a.  fatty  acids  The methyl e s t e r s of f a t t y adding  1  ml  of  acids  were  90°C  by  methanol i n 1N hydrogen c h l o r i d e gas t o  each sample. The tubes were t i g h t l y capped at  prepared  overnight.^  After  coding,  the  and  incubated  methanol  evaporated under n i t r o g e n and the samples were  stored  was at  room temperature.  b.  g l y c e r y l ethers A f t e r the methyl e s t e r s of f a t t y a c i d s were prepared,  the  same  samples were t r e a t e d i n the f o l l o w i n g manner to  prepare the t r i m e t h y l s i l a n e e s t e r s of g l y c e r y l e t h e r s . .150 ul  of  pyridine/hexamethyldisili zane/trichlorotrimethylsilane (10:4:2, Vance and Sweeley, 1967) was added t o each sample for  15  minutes  at  room  temperature.. The samples were  evaporated under n i t r o g e n and r e d i s s o l v e d i n 20-100 u l of heptane and s t o r e d at room temperature.  1.63  5.  Gas  liquid  chromatography  Quantitative Hewlett 6-foot 15%  Packard  a n a l y s i s of m e t h y l  761GA H i g h  U-shaped  r  ethylene  column  glycol  from  was  155°C t o  minutes  of  were  used t h r o u g h o u t  injected  sample  the percent  cutting  the  p e a k s and  1%  of  16:1  acids  identified  were and  Vance and  (20:1),  arachidonic  first  in  palmitoleic  (22:1),  (20:4) were p u r c h a s e d e s t e r s was  also  the  right  notation  hydrocarbons  of  bonds i n t h a t a c y l  from  provided  number o f t h e s h o r t h a n d  the f a t t y weighing  by  (16:1),  docosenoic  methyl  composition  w h i c h was  (24:0)  the  colon  chain.  h e p t a n e p l u s 2 u l of h e p t a n e were  the t o t a l  e x c e p t i o n of  standards  ( 2 2 : 0 ) , and t e t r a c o s a n o i c  t h e number t o  Xeroxing  than  and  (18:0),  by  less  2  6  (16:0), s t e a r i c  determined out  4 or 8 x 10  (eg. .16:0) i n d i c a t e s t h e number o f  of  and  beginning  acids, palmitic  number o f d o u b l e  ul  programmer,  methyl  of r a t l i v e r  t h e a c y l c h a i n and  0.5  minute  column  standards*  Vance. The  i n d i c a t e s the  the  The  (20:2) and  D.E.  with  Laboratory,  200°C and  per  a  inches/second.  aicosenoic  Sigma..The m i x t u r e  in  0.5  fatty  (18:1),  Dr.  packed  Science  was  2°C  s u p l i e d by Dr. .D.E.  docosadienoic  by  = 2 mm)  (Applied  temperature  (20:0), docosanoic  kindly  oleic  flame  diameter  i n j e c t i o n . . T h e a t t e n u a t i o n was  the  eicosanoic  p e r f o r m e d on a  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  t h e c h a r t s p e e d was esters  (inside  185°C a t a r a t e o f  after  e s t e r s was  E f f i c i e n c y gas c h r o m a t o g r a p h w i t h  succinate  I n c . , H 1 - E F F 2 B ) . The temperature  (GLC)  cf  each  fatty  acid  a c i d p r o f i l e of each then*  weight  sample,  Fatty acids that  were d i s r e g a r d e d  i n some.cases s t a n d a r d s  the  were  (with the  always i n c l u d e d ) . I n d i v i d u a l comparing  was  fatty  e l u t i o n times  were a d d e d t o t h e  with  samples  164  to a c t as i n t e r n a l standards. A l l but the l a s t 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  were  tentatively  identified  in this  analyzed  1970).  those  separate  these  stationary  phase  was  and t h e f a t t y  determined  was acid  f o r two  of  experiments.  Results  1.  Fatty acid The.  c o m p o s i t i o n o f whole  GLC  cells  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 s a m p l e s o f m e t h y l  e s t e r s of f a t t y  a c i d s have b e e n i l l u s t r a t e d  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  temperatures lower  temperatures  assymetric  ( i e . . L e s s than  the  peaks  the peaks.  increase did  not  in  155°C)..The b a s e - l i n e s l o p e d column  present  acid  of  experiments  wild-type  (18:0),  both  oleic  arachidonic  problems  but t h e  i n the a n a l y s i s  by t h e r e l a t i v e  weights  acids  temperatures (18:1),  (20:4),  and  dml  cells  from  three  a t 27°C a n d 34.5°C h a v e 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 at  temperature  The means and s t a n d a r d d e v i a t i o n s o f t h e p e r c e n t  of each f a t t y  cells  and  were n o t r e s o l v e d a t t h e s e  b e c a u s e t h e c o m p o s i t i o n was d e t e r m i n e d  separate  i n F i g u r e s 7-2  s i n c e t h e y were more v o l a t i l e a n d were r e s o l v e d a t  upward due t o  of  same  experiments  of the p h o s p h o l i p i d s  C.  7-3.  and  The f a t t y a c i d c o m p o s i t i o n o f w h o l e c e l l s  f o r three  composition  manner  eluting)  on t h e b a s i s o f e x t r a p o l a t i o n a s  well as the l i t e r a t u r e values f o r the (Jamieson,  (latest  in  were:  linoleic  both  palmitic  (18:2),  docosotetraenoic  wild-type (16:0),  linolenic  and  dml  stearic (18:3),  (22:4), t e t r a c o s a d i e n o i c  165  (24:2), and t e t r a c o s a t e t r a e n o i c (24:4). The acyl  chains  positively  that  were l e s s than  identified  because  methyl  16 carbons  they  esters  of  long c o u l d not be  eluted  very  closely  together. T h e r e f o r e , they were considered as a unit and omitted from  most  calculations..  The percent composition  a c i d s o f t h e b a c t e r i a was not determined differences  i n the  polyunsaturated carbons  bacteria  fatty  acids  but there were obvious  particularly and  of the f a t t y  the  lack  of  a c y l c h a i n s g r e a t e r than 20  l o n g . The c o n t r o l sample with s i l i c a g e l G alone showed  no d i s c e r n i b l e  peaks. .  The standard d e v i a t i o n s were g u i t e l a r g e , p a r t i c u l a r l y the  mutant  (ec[.  Ohxi  samples, et  experiments  similar  a l . , 1979) .  were  became apparent  but  examined  t h i s range o f v a r i a b i l i t y i s common When  the  results  s e p a r a t e l y (see  that experiments  data and experiment  in  one  and  of  the  Appendix three  three  IV),  yielded  i t  very  two was g u i t e d i f f e r e n t : t h e r e were  e l e v a t e d amounts of 22:4 and 24:4 and decreased amounts of 18:1 i n experiment The  two. .  change  in  composition  whole c e l l s was c a l c u l a t e d composition (Table  by  of  the major f a t t y a c i d s i n  subtracting  the  mean  at 27°C from the mean percent composition  7-2). The  a t 34.5°C  major d i f f e r e n c e s between wild-type and dm_1  c e l l s occurred i n 16:0 and 24:4. The l a r g e p o s i t i v e 16:0  percent  value f o r  i n wild-type c e l l s i n d i c a t e d that the percent of p a l m i t i c  a c i d i n c r e a s e d with i n c r e a s e d temperature  whereas i t  decreased  i n mutant c e l l s . The l a r g e n e g a t i v e value f o r 24:4 i n wild-type cells  indicated  a  decrease  in this  unsaturated f a t t y a c i d i n wild-type  cells  long-chain, and  an  highly  eguivalent  166  increase  in  mutant  ceils..  This  t h r e e experiments (Appendix IV) in  composition  r e g a r d l e s s of  differences  m a j o r i t y of f a t t y a c i d s were g r e a t e r than 20 carbons (Table  7-  i n wild-type  c e l l s the trend was  l e n g t h a f t e r an i n c r e a s e i n part,  among  the  The  and  acid  consistent i n a l l  the t h r e e experiments.  3)  fatty  t r e n d was  temperature  to decrease the (contributed  by the i n c r e a s e i n 16:0). T h i s trend  c e l l s where the carbons content  percent  increased  of  with  acyl  chains  was  chain  to,  in  reversed i n dm 1  with  more  than  an i n c r e a s e i n temperature. The  of saturated f a t t y a c i d s i n wild-type c e l l s was  20  total  similar  f o r each temperature i n a l l three  experiments i n d i c a t i n g t h a t a  constant  fatty acid r a t i o for a  s a t u r a t e d to unsaturated  temperature  may  of unsaturated 34.5°C index  but  the  U.I.  decreased  in  wild-»type  cells.  The  percent  cells  at  unsaturation  the number cf double bonds per  100  acyl  cf each unsaturated  fatty  i s c a l c u l a t e d as f o l l o w s : sum  of:  U(%  composition  (number of double bonds)/100  decreased  temperature  in  wild-type  t u t i n c r e a s e d i n dmj  at 27°C was  wild-type  i n wild-type c e l l s . .The  underwent no change i n dmj  acid) x  two  fatty acids  (U.I.) expresses  groups and  The  be maintained  given  cells  higher there  occur i n dm 1 c e l l s .  cells  an  c e l l s . . T h e U.I.  i n both wild-type was  with  and  dmj  increase  in  i n experiment cells  but  in  a decrease at 34.5°C which d i d not  167  2.  Fatty, a c i d The  composition of p h o s p h o l i p i d s  means of the percent composition of the f a t t y a c i d s of  the p h o s p h o l i p i d s , PC,  PE and  AEPL, of w i l d - t y p e and dmj  have been presented i n T a b l e s 7-4 of  all  three  phospholipids  were  a c i d s of whole c e l l s i._e. .. 16:0, 22:4,  24:2,  and between  34.5°C  previously  described  and  percent  in  greatest  difference  27°C  of  16:0  18:2,  18:3,  in  were  the  20:4, percent  determined  as  increased  substantially  p a r t i c u l a r l y i n PE, but i n  very s l i g h t .  The  p r o p o r t i o n of 18:0  AEPL i n wild-type but not i n dmj  compositions  increase  18:1,  have been presented i n T a b l e 7-7. .In  with an i n c r e a s e i n temperature,  i n PC and  major f a t t y acids  differences  and  w i l d - t y p e c e l l s the percent  c e l l s the i n c r e a s e was  The  s i m i l a r to the major f a t t y  18:0  24:4.. The  composition  increased  t c 7-6.  cells  of  18:1  temperature  and  20:4  cells.  The  with  an  decreased  wild-type  and  dm 1  cells.  between  wild-type  and  mutant  cells in  i n c r e a s e occurred i n 24:4;  i n wild-  type c e l l s the percent composition i n EC and PE decreased temperature  substantially composition composition the  In  similar  PE and  in  dmj  cells  i t  of AEPL remained more constant.. In of  AEPL  as  with the temperature  well  as  PC  ir  the  long i n PC, to  the  with  in  PE  cells, varied  change. resulted  p r o p o r t i o n of a c y l c h a i n s g r e a t e r than PE and  changes  AEPL  the  PE while the  mutant and  The  increased  AEPL. .The g r e a t e s t changes  wild-type c e l l s an i n c r e a s e i n temperature  decrease  carbons  but  of wild-type c e l l s occurred i n PC and  composition  considerably  a  i n PC,  also  in  response t o the temperature  increased  dmj  (Tables 7-8  observed  t o 7-10)..This  with whole c e l l  in 20 was  f a t t y acids  168  (Table 7-3)» increase  In dmi  cells  i n temperature,  there  was  an  increase,  with  i n the percent of f a t t y a c i d s i n t h i s  category. .In wild-type c e l l s t h e r e was  an i n c r e a s e i n the  level  of s a t u r a t e d f a t t y a c i d s a t 34.5°C, p a r t i c u l a r l y i n PC and and  to  a  saturated  lesser fatty  temperature..  extent acids  The  in  AEE1.  decreased  U.I..deereased  in  In dmi  cells  with  an  dmi.  cells  PE,  the l e v e l of increase  wild-type  i n c r e a s e i n tenperature; the g r e a t e s t change PE. . In  an  in  c e l l s with an  was  observed  the U. I. . i n c r e a s e d at 34.5°C i n PC,  PE  in and  AEPL. .  D.  Discussion The  27°C  observed f a t t y a c i d composition of wild-type c e l l s  agrees,  a l . ( 1979)  -for  difference amounts  in  is  of  exception  with r e s u l t s obtained by  a u r e l i a grown a x e n i c a l l y at the  fatty of  part,  presence, acids  24:2  in  this  containing  and 24:4,  24  25°C.  study, of  The  major  substantial the  the major f a t t y a c i d s p e c i e s are  i n e l e v a t e d amounts i n the second  the  Kaneshiro et  carbons.. With  the same i n both s t u d i e s . . T h e f a t t y a c i d , 24:4,  may  at  experiment  i s a l s o present  (see R e s u l t s )  and  be a r e f l e c t i o n of v a r i a b l e growth c o n d i t i o n s or the age o f paramecia  since the degree of u n s a t u r a t i o n and the l e n g t h  of the f a t t y a c y l c h a i n s i n c r e a s e s with c l o n a l  age  (Kaneshiro  e t a l . , 1979). The c o n t r i b u t i o n of the b a c t e r i a l f a t t y a c i d s t o the  total  composition  b a c t e r i a l composition Paramecium  was  not c a l c u l a t e d and changes i n the  would be r e f l e c t e d i n the composition  membranes. . T h i s may  a l s o c o n t r i b u t e t o the  of  results  169  o b t a i n e d i n experiment and  two. S i n c e i n each experiment  dml. c e l l s at both temperatures  same batch, v a r i a t i o n s due fatty  a c i d composition The  changes  in  f o l l o w i n g an i n c r e a s e  to  were f e d b a c t e r i a from the  differences  w i t h i n an experiment fatty in  wild-type  acid  in were  composition  temperature  can  the  be  bacterial  minimized. that  occurred  summarized  as  follows: 1.  wild-type c e l l s a. i n c r e a s e i a s a t u r a t e d f a t t y  acids, particularly  16:0 i n  whole c e l l s and i n PE b. i n c r e a s e i n f a t t y in fatty  a c i d s with  16-20 carbons  acids with more than 20  c. .decrease particularly  in  highly  carbons  unsaturated  fatty  acids,  20:4 and 24:4 i n whcle c e l l s and i n PC and PE  d. .decrease i n U.I.,, p a r t i c u l a r l y 2.  and decrease  i n PE  dml c e l l s a. s l i g h t i n c r e a s e or decrease b. _decrease i n f a t t y in fatty  a c i d s with  i n saturated f a t t y acids 16-20 carbons  and i n c r e a s e  acids with more than 20 carbons  c. . i n c r e a s e i a the h i g h l y unsaturated f a t t y a c i d , 24:4 d. i n c r e a s e i n U.I. . A l l changes occurred i n PC, PE and AEPL.. A l l f o u r changes can be a t t r i b u t e d t o occur at  the  changes  i n 16:0 and 24:4. An i n c r e a s e i n 16:0 i n wild-type c e l l s  34.5°C i n c r e a s e s the percent  cf saturated fatty  a c i d s and a t  the same time the percent of a c y l chains 16-20 carbons decrease and  which  i n 24:4 decreases  long.  A  the l e v e l c f unsaturated f a t t y a c i d s  thereby the U.I._as w e l l as the percent of a c y l chains more  170  than  20 c a r b o n s l o n g . .In dmj c e l l s  decrease, in  or s l i g h t  i n c r e a s e , i n 16:0 and a  24:4 w h i c h c o m p l e t e l y In  Tetrahymena  there  i s  an  a c i d s when  from  (Conner  (Martin al.,  to  et  1976,  observed  15°C  a l . , 1 976) Watanabe  the  of  39.5°C  wild-type  cells  reported  f o r Tetrahymena  (primarily  enzyme Kasai  palmitoleic  acid  may have o c c u r r e d present  and  activation  (Nozawa  of  (Martin  et  (Fukushima  on  this  20  the study  carbons  et  fatty  acid  although the  has  which c o n v e r t s  not  fatty  been  acids  Although  Fukushima  et  the  a l . , 1976,  chaages  about Kasai  study in  (Martin e t a l . , fluidity  acid  desaturase  a l . , 1979)  enzyme i n r e s p o n s e  brought  in this  palmitic  (16:1). T h i s enhanced  of  the 1976  i n response  a g r e a t e r e x t e n t by f a t t y  no  decreased  et  a l . , 1976,  the  temperature Martin  and  1S79)..  attempt amounts have  or  t o a change i n t h e  by  Thompson, 1 9 7 8 , S k r i v e r and Thompson,  membrane  15°C  a n d K a s a i , 1 9 7 8 , Nozawa e t a l . , 1979,  existing  fluidity  themselves  1 9 7 6 ) , 39°C t o 15°C  a s a r e s u l t o f i n c r e a s e d amounts  Nozawa, 1980,  membrane  guantify  lowered  18:2 and 18:3) i s due t o t h e i n c r e a s e d a c t i o n o f t h e  to  activity  i s  , the increase i n unsaturated  enzyme p a l m i t c y l CoA d e s a t u r a s e , (16:0)  to  in  of f a t t y a c i d s longer than  Tetrahymena  increase  a 1. , 1 9 7 9 ) . T h i s i s c o m p a r a b l e t o t h e  presence  In  a  i n t h e amount o f  temperature  and S t e w a r t ,  or  et  increase  e f f e c t s of i n c r e a s e d temperature  composition  definite  i^e.  reverse the results..  desaturation of fatty 35°C  the opposite occurs  has of  shown  t o temperature  acid composition  been  made  to  the phospholipids that  changes  in  are influenced t o  than  by  head  group  17 1  composition),  it  appears  t h a t the f a t t y a c i d s of PC and PE i n  w i l d - t y p e c e l l s undergo g r e a t e r changes than do the f a t t y of  AEPL i n response  suggest  that  PE  to  temperature.  plays  chains d u r i n g temperature al. the  ( 1 979) most  increases  the  have  a l . (1979)  a c c l i m a t i o n i n Tetrahymena and Nozawa  treatment  fluidity  et  p r i n c i p a l r o l e as a c c e p t o r of a c y l  have found that PC and PE of after  a l . . (1979)  a  Watanabe  with  of  found  phenethyl  membranes. that  Tetrahymena  However,  the  fluidity  of  after  whereas the f l u i d i t y  PC decreases g r a d u a l l y f o r the f i r s t  first  24 hours  ten  than does t h a t of AEPL..The decreases  the  most  with  0.1. an  suggesting  in  temperature  and  and that  results  that  the  PE undergoes a g r e a t e r change of  the  fatty  acids  of  PE  i n c r e a s e i n temperature. In dml  c e l l s the f a t t y acids of a l l the increase  in  hours  of PE  o b t a i n e d with wild-type c e l l s i n t h i s study i n d i c a t e a c i d composition of PC and  et  AEPL  i s important i n the i n i t i a l thermal response. The  fatty  which  Ohki  during  a decrease i n temperature  the  change  alcohol,  T . p y r i f o r m i s increases r a p i d l y  AEPL  acids  phospholipids the  vary  U.I. i n c r e a s e s  with  an  i n a l l the  phospholipids. Mutants o f other eukaryotes have been i s o l a t e d d e f i c i e n t i n seme aspect of l i p i d et  a l . , 1973),  (Hill, been in  1980).  Neurospora  metabolism, eg,.  (Friedman,  1977)  which yeast  and  the c u l t u r e medium. The pawn  mutants  of  P  L  (Keith  Tetrahymena  These are mainly f a t t y a c i d auxotrophs t h a t  used t o i n v e s t i g a t e the e f f e c t s of f a t t y a c i d  are  have  supplements  tetraurelia , ,  which have d e f e c t s i n the.gated calcium channel of the membrane (Kung, 1975),  show  no d i f f e r e n c e s i n t h e i r f a t t y  a c i d content  172  as compared although  with  by  minor p h o s p h o l i p i d s  et  Kung  (1971),  (2%) i n  fast  deciliated  a 1> , 1979)  of pawn c e l l s i s  ether  behavioral  (fa97) , has two extra bodies  but  c e l l s show no d i f f e r e n c e s i n p h o s p h o l i p i d  when compared Another  (Kaneshiro  i n axenic medium. One of the  isolated  paranoic  cells  i t i s i n t e r e s t i n g t h a t the v i a b i l i t y  s e v e r l y reduced mutants  wild-type  with wild-type c e l l s  mutant  of  (Andrews and  P. . t e t r a u r e l i a  ,  nd9,  pawn  composition  Nelson,  isa  at  27°C  1979).  temperature-  s e n s i t i v e mutant with normal t r i c h o c y s t discharge a t non-discharge  18°C but  (Beisson e t a l . , 1976). At 18°C t h e nd9  gene product  i s abnormal but  temperature  enables  the  the  membrane  protein  fluidity  at  d i s c h a r g e of t r i c h o c y s t s . _At i f fatty  in  fatty  place  acid s y n t h e s i s i s i n h i b i t e d d u r i n g t h e s h i f t down  restored  composition  normal  27°C no i n t e r a c t i o n can take  to the p e r m i s s i v e temperature normal not  that  t c i n t e r a c t with e i t h e r the  t r i c h o c y s t membrane or plasma membrane r e s u l t i n g i n the  and  and  (Beisson  trichocyst  e t a l . , 1980). Thus, although  i s not r e s p o n s i b l e f o r the nd9 acid  discharge  composition  is  membrane  phenotype,  changes  at the p e r m i s s i v e temperature can  compensate f o r an abnormal p r o t e i n . 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 i n t h e s y n t h e t i c pathway t h a t would r e s u l t i n the However,  accumulation  i t does  appear  of that  membrane f a t t y acid composition I f the c o n t r o l l i n g fatty  step i n  one the  species  of  fatty  mechanism f o r c o n t r o l o f  i s impaired. the  pathway  for  unsaturated  a c i d s y n t h e s i s i s the same as i n Tetrah ymena , i . e.  palmitoyl  Cofl  desaturase  acid.  conversion  of  palmitic  acid  the to  173  palmitoleic  acid  (Martin  a c i d would increase the  i f the enzyme was i n h i b i t e d and decrease i f  enzyme was a c t i v a t e d .  palmitic  acid  consistent  In wild-type  increases  with  an  cells  the  increase  in  percent  change  in  i n temperature i t s e l f  fluidity  resulting  from  increasing  palmitic  to  temperature paLmitoleic  Tetracosatetraenoic pathway  and  acid  indicating acid  has  the  the not  (24:4) may be an end  i t s depletion  either  or i n d i r e c t l y by increase  temperature. I n dml c e l l s the amount of p a l m i t i c a c i d with  of  temperature,  with the i n h i b i t i o n of the desaturase enzyme  d i r e c t l y by the increase the  et a l . , 1576), the l e v e l of p a l m i t i c  decreases  conversion been  in  of  inhibited.  product  of  the  i n wild-type c e l l s and accumulation  i n dml c e l l s f u r t h e r a t t e s t 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,  involving ba  dml,  may  represent  a  genetic  lesion  the c o n t r o l of desaturase a c t i v i t y and as such would  an extremely valuable t o o l f o r i l l u c i d a t i n g the f a c t o r s  influence  the mechanism of a c t i o n  of t h i s enzyme..  that  174  T a b l e 7-1. F a t t y a c i d composition  Fatty Ac i d  <TeT 0  wild-type 27°C  cf whole  cells  Percent Composition (1) dm1 wild-type 27°C 34.5°C  *16: 0 10. 54 (3.43) 2.40 (0.72) 16: 1 17: 1 (3) 1.24 *1 8:0 2. 98 (1.21) *1 8: 1 15. 98 (7.11) * 18:2 4. 33 (1.37) 6. 96 (1.99) *18: 3(4) 20: 0 3. 17 (1.50) 20: 2 3.08 (1.32) 5. 13 (3. 18) *20: 4 22: 1 3.93 22: 2 1.86 1 1. 13 (6.61) *22: 4 22.6 1.32 24: 0 2.96 24: 1 1. 82 *24: 2 4.03 (1.97) *24: 4 18.56 (10.02)  t. 25 16.92 (6. 54) 4. 31 (2. 55) 1. 54 2. 53 (1 .66) 14. 22 (6.3 4) 3.69 (1.27) 6. 01 (2.41) 3. 96 (0.54) 2. 86 2.99 (1. 67) 2. 28 2. 35 12.40 (6. 50) 2. 27 2. 07 1.96(0. 58) 4.11(1. 50) 14.99(11 .69)  dm1 34. 5°C  7.45 1.48 15.29 (3. 06 ) 14.80 (7. 76) 3.89(2. 05) 3. 48 (2.86) 1.01 2. 63 3.45 (2.33) 3. 47 (2. 78) 14.99(2.98) 13. 85 (6.40) 4.19 (0. 71) 3. 81 (1. 36) 8. 6 9 (1. 95 ) 4.29 (2. 51) 2. 16(0. 59) 3. 20 (1.59) 2.58 1 .78 (0.50) 3.66 (0. 53) 3. 27 3.43 (2. 67) 1.81 1.82 1.50 9.65 (3. 36) 10. 67 (6.38) 2.27(0.97) 3. 41 1.88(1. 15) 3. 29 (1.51) 2.01 1.94 4. 67 (2.99) 4.69 12.31 (7.31) 21. 46 (15. 20)  (1) means and standard d e v i a t i o n s f c r values from three experiments, means only where data a v a i l a b l e from o n l y two experime nts. (2) t r = t r a c e (3) may a l s o i n c l u d e 17:0 (4) a l s o c o n t a i n s 20:1 * major f a t t y acids  Table 7-2. Changes i n whole c e l l f a t t y a c i d  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  (1) c a l c u l a t e d by s u b t r a c t i n g  composition  6. 47  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  acyl  wild-type 27°C  chain  <16  o f whole  cell  draj  a c i d s (1)  wild-type  27°C  0.00  fatty  34.5°C  1. 25  dml 34.5°C  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)  0. I. (5)  (1)  4.03  2. 68  2.96  2.74  2. 15 (0. 51)  1. 87(0. 34)  1.88 (0.47)  2.03 (0. 68)  means  and  standard  deviations  for  values  from  three.,  experiments (2)  S = sum  (3) U = sum of  mixed  (4) U/S  of saturated f a t t y of unsaturated  composition = ratio  (5) U.I..=  acids  fatty  or l e s s  than  of unsaturated  u n s a t u r a t i o n index  acids  (U*S<100% b e c a u s e  peaks  16 c a r b o n s were n o t i n c l u d e d )  to saturated f a t t y  acids  (see E e s u l t s f o r c a l c u l a t i o n )  177  T a b l e 7- 4. F a t t y  Fa t t y Ac i d  wild-type 27°C  <16: 0 5.00 *1 6: 0 6.66 16: 1 0.42 17:1(2) 3. 54 *1 8: 0 5. 93 *18: 1 1 1. 24 1. 78 *1 8:2 * 18: 3 1.01 20: 0 2. 04 20: 1 3.83 17. 48 *20: 4 22: 1 1. 59 22: 2 2.65 *22:4 12.63 1.07 22: 6 24:0 2. 74 24: 1 7.98 *24:2 4. 31 *24: 4 24.45  a c i d s of  phosphatidylcholine  Percent Compo s i t i o n (1) dml wild-type 27°C 34.5°C 3. 29 12. 68 0.90 3.45 8. 11 11. 69 3. 86 1. 39 2. 12 1. 69 13. 69 tr 1,. 76 13. 39 3.60 2. 70 1. 1 9 6. 49 15. 53  (1) means o f two experiments (2) may a l s o i n c l u d e 17:0 (3) t r = t r a c e * major f a t t y acids  5.35 11.60 1,28 1.65 7.55 8.65 5.84 4.85 2.0 3 7.19 4.66 1.97 tr 16.53 3. 34 2.78 1.31 5.31 18.80  dml 34.5°( 2.20 13.74 2.08 1 .50 5. 97 8.64 2.90 1.23 t r (3) 6.00 5.54 1.55 1.37 18.22 3.86 3. 84 2. 45 4.18 20.03  Table 7-5. . F a t t y a c i d s of phosphatidylethanolamine  Fatty Acid  wild-type 27°C  ~67oc <"l6 *1 6: 0 11.83 16: 1 1.65 2.00 17:1 (3) *18:0 7. 21 *1 8: 1 12. 58 *18: 2 4. 86 3.49 *18:3 20:0 2. 34 20: 1 1. 87 *20:4 7.04 22:1 tr 1. 01 22: 2 *22: 4 12. 80 22:6 4.28 24: 0 tr 24: 1 tr *24:2 6.41 18. 49 *24: 4  Percent Composition (1) dm1 wild-type 27°C 34.5°C 4. 98 15. 13 2.25 2. 6 3 6.78 13. 27 2. 93 tr 2. 94 1.07 12. 69 tr 1. 49 13. 37 4. 67 1. 96 1. 09 4. 93 14.25  (1) means of two experiments (2) t r = t r a c e (3) may a l s o include 17:0 * major f a t t y acids  1.71 24.42 3.73 1.33 5.29 10.90 3.87 2.09 3.52 1.50 4.89 3.37 tr 12.95 2.33 2.60 2.34 3.49 14.02  dm1 34. 5°( t r (2) 16.65 1.97 1 .97 4.81 7.90 2.61 1. 36 2.48 2.79 4. 39 1.43 1.38 15.61 1.97 2.04 1.98 4.94 25.00  Table 7-6..Fatty a c i d s of 2-aminoethylphosphonolipid  Fatty Acid  ___ *1 6: 0 16: 1 17:1 (2) *18: 0 *18: 1 *18: 2 *18:3 20: 0 20 : 1 *20: 4 22:1 22. 2 *22: 4 24:0 24: 1 *24: 2 *24:4  wild-type 27°C 4707 8. 33 0. 81 1.42 8. 1 1 12. 34 3. 16 12.19 1. 35 1. 41 7.94 1.04 2. 33 18. 45 1.57 1.78 3. 18 18.78  Percent Composition (1) wild-type dml 34.5°C 27°C 7. 17 7. 80 0. 76 2. 43 9. 35 17.10 4. 41 tr 1. 43 tr 14. 38 1. 42 3. 32 17. 26 1. 91 tr 4. 71 15. 45  (1) means of two experiments (2) may a l s o c o n t a i n 17: 0 (3) t r = t r a c e * major f a t t y acids  10.26 9. 16 1.19 tr(3) 11.79 15.02 3.58 11.12 1.46 3.22 2.94 4.93 1.45 16.10 2.01 tr 5. 27 19.54  dml 34. 5°( 1 .99 9.68 1.59 1.10 8.29 1 1.05 2.69 1.20 tr 3. 75 2.90 tr 2. 11 23.00 2.28 1.66 3.64 26.61  180  T a b l e 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 composition (1)  PC  Fatty Acid  PE  (2)  wild-type dmi  w i l d - t y p € dmi  16:0  4. 94  1.06  12.59  18:0  1.62  -2. 14  18^ 1  - 2.59  AEPI• (4)  (3)  w i l d - t y pe dmi  1.52  0.83  1. 88  - 1.92  - 1.97  3.68  - 1.06  -3.05  - 1 .68  - 5.37  2.68  - 6.05  0.42  - 1.72  18:2  4.06  -0. 96  - 0.99  - 0.32  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  - 5.65  4.50  - 4.47  0.76  11. 16  24: 4  10.75  (1) c a l c u l a t e d by s u b t r a c t i n g t h e mean composition at 27°C from t h e mean composition a t 34.5°C (2) PC = p h o s p h a t i d y l c h o l i n e (3) PE =  phosphatidylethanolamine  (4) AEPL = 2-aminoethylphosphonolipid  181  Table 7-8. D i s t r i b u t i o n  acyl chain  <16  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 (1)  wild-type  dmj[  27°C  27°C  wild-type 34.5°C  dm 1 34.5°C  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  5. 10  3. 13  3.06  3. 15  2.44  2. 23  2.27  2. 34  U/S  (4)  U.I. (5)  (1) means of two experiments (2) S = sum of s a t u r a t e d f a t t y a c i d s (3) U = sum of unsaturated f a t t y a c i d s of mixed composition  or l e s s than  (U+S<100% because  peaks  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. = u n s a t u r a t i c n index  (see R e s u l t s f o r c a l c u l a t i o n )  182  Table  7-9. D i s t r i b u t i o n  of phosphatidylethanolamine  fatty  acids  (D  acyl  wild-type  chain  dmi  27°C  <16  wild-type  27°C  dmi  34.5°C  34.5°C  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  (4)  3.05  2. 76  1.76  2.72  U.I. (5)  2.43  1. 89  1. 81  2. 15  U/S  (1) mean o f two  experiments  (2) S = sum c f s a t u r a t e d (3)  0 = sum  of unsaturated  of mixed c o m p o s i t i o n (4) U/S (5)  = ratio  U.I..=  fatty  fatty  or l e s s  than  of unsaturated  unsaturation  acids  index  acids  (0*S<100% b e c a u s e  peaks  16 c a r b o n s were n o t i n c l u d e d )  to saturated fatty {see R e s u l t s f o r  acids  calculation)  183  Table 7-10.„Distribution  of  2-aminoethylphcsphonolipid  fatty  a c i d s (1)  ac y l  wild-type  chain  27°C  4.04  <16  dml  wild-type  27°C  dmj  34.5°C  7. .17  34.5°C  10.16  1.99  16<20  39. 54  40.63  42.33  34.44  >20  56.44  55. 75  52.59  64.56  S (2)  18.68  18. 82  22.68  19. 10  U (3)  77. 30  77.60  72.24  79.90  U/S (4)  4. 14  4. 12  3. 19  4.18  U. S. (5)  2. 36  2. 33  2.09  2.59  (1) means of two  experiments  (2) S = sum of s a t u r a t e d f a t t y (3) U = sum of  acids  of unsaturated f a t t y  mixed compcsition o r l e s s than  c a i d s (S+U<100% because peaks 16 carbons  were not included)  (4) U/S = r a t i o of unsaturated t o s a t u r a t e d f a t t y (5) U.I. .,= unsatur a t i o n index  acids  (see E e s u l t s f o r c a l c u l a t i o n )  184  Figure  7-1.  w6  pathway  biosynthesis. .Linoleic acid desaturated  (Erwin,  1973)..  of (18:2)  polunsaturated i s sucessively  fatty elongated  acid and  LO  O +  o  CVJ Cvj  cvj cvj  O  o 00  U +  cvj  186  Figure  7-2. .Fatty  acids  of w i l d - t y p e c e l l s . T r a c i n g of a  l i g u i d chromatographic a n a l y s i s of whole c e l l 34.5°C  {reduced  time and fatty  by  50%).  v e r t i c a l axis  acids.  fatty  Horizontal axis represents  represents  relative  acids  gas at  retention  concentration  of  l 8 \  188  Figure  7-3.  Fatty  chromatographic  5G%).  (reduced  by  vertical  axis  acids  analysis  of of  Horizontal  represents  dmj. c e l l s . whole axis  relative  cell  Tracing fatty  represents  of  a gas  liquid  acids  at  34.5°C  retention  concentration  of  time  fatty  and  acids.  190 I  CHAPTER  VIII  CONCLUDING REMARKS A t o t a l c f 82 t e m p e r a t u r e - s e n s i t i v e mutants t h a t i n t e r f e r e with  some  aspect  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 have  been i s o l a t e d . Two of these have been d e s c r i b e d i n each i s d e f i c i e n t i n a d i f f e r e n t The  aspect of t h i s  mutant, f y c , r e s u l t s i n the clumping  near t h e p o s t e r i o r movement  is  of  the  hampered,  c e l l . . It  normal f u n c t i o n i n g of c y t o p l a s m i c time,  the  time decreases, which suggests  vacuolar  i n t e r f e r e n c e with the At  the  same  which accumulate i n a given  and  vacuolar  movement.. The  of vacuolar movement may i n t e r r u p t the r e c y c l i n g of  membranes, thereby reducing the amount for  that  vacuoles  an i n t e r r e l a t i o n s h i p between the  process of food vacuole f o r m a t i o n inhibition  of food  microtubules.  number of food vacuoles  and  process.  appears  p o s s i b l y through  detail  endocytosis. . A l t e r n a t i v e l y ,  of  membrane  both processes  available  may r e g u i r e the  normal f u n c t i o n i n g of the same f a c t o r s . The f v c mutant i s more sensitive  to  the  detergent,  wild-type c e l l s and provide  a  mass  the  use  selection  SES, and to c o l c h i c i n e than a r e of  these  system  chemical  agents  may  f o r the g e n e r a t i o n of more  mutants with s i m i l a r phenotypes. . The  mutant, dmj, r e s u l t s i n  membranes  at  the  restrictive  the  disruption  temperature.  of  The  vacuolar membranes  degenerate and the changes i n the f a t t y a c i d composition, occur  in  wild-type  temperature, could be  due  cells  as  a  function  of  do not occur i n t h i s mutant. The e f f e c t s to  the  malfuncticning  of  the  which  increased observed  palmitoyl  CoA  191  desaturase  enzyme  composition  of membranes i n Tetrahymena  This and  which  is  known to r e g u l a t e t h e f a t t y (Martin et  acid  a l . , 1976).  mutant has an i n c r e a s e d s e n s i t i v i t y t o the s o l v e n t , DMSO, t h i s agent  may prove u s e f u l i n s e l e c t i n g other mutants with  phenotypes s i m i l a r t o dm!. Although i._e..ts  the o b j e c t i v e of t h i s p r o j e c t has  mutants  vacuolar cycle  in  various  aspects  have been i s o l a t e d  characterized,  of  been  attained  e n d o c y t o s i s and the  and two of  these  have  been  these r e s u l t s r e p r e s e n t only a b a s i s f o r asking  more g u e s t i o n s about the events i n v o l v e d i n these processes. study  of  mutants  intracellular also  in  fvc  may  a i d i n the understanding o f  movement of m a t e r i a l s net only i n e n d o c y t o s i s but  embryology  understanding  and  differentiation  in  general.  of the c o n t r o l of membrane composition,  of prime importance be  like  A  facilitated  by  An  which i s  i n a l l a s p e c t s of c e l l u l a r metabolism,  may  a f u r t h e r i n v e s t i g a t i o n of dm 1 and s i m i l a r  mutants..Thus, the study of e n d o c y t o s i s i n  Paramecium  i s not  limited  i n a p p l i c a t i o n to t h i s organism  but can be expanded t o  include  membrane  i n t e r c o n v e r s i o n s , and  intracellular  specializations  and  movement of m a t e r i a l s i n other  eukaryotes..  192  LITERATURE  CITED  Ahkong, Q. 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The i n v i v o e f f e c t of d i m e t h y l s u l f o x i d e on p r o t e i n s y n t h e s i s and the polyribosome p r o f i l e i n Paramecium . J . C e l l . P h y s i o l . . 90J.169- 178. Rembold, H. and T. Langenbach. 1978. E f f e c t of c o l c h i c i n e on c e l l membranes and on b i o p t e r i n t r a n s p o r t i n C r i t h i d i a f a s c i c u l a t a . J . . P r o t o z o o l . . 2 _ i 404-408. Rhoads, D.E. and E. S. .. Kaneshiro. . 1979. C h a r a c t e r i z a t i o n s p h o s p h o l i p i d s from Paramecium t e t r a u r e l i a c e l l s and c i l i a . J . Protozool.,26:329-338.  of  209  E i c k e t t s , T.E. . 197,1 a. . E n d o c y t o s i s i n Tetrahymena p i r i f o r m i s . . The s e l e c t i v e uptake of p a r t i c l e s and the adaptive i n c r e a s e i n c e l l u l a r a c i d phosphatase a c t i v i t y . E x p t l . C e l l E e s . 66__49-58. . 1971b. 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S c i . H i r o s h . Univ. Ser. B. Div. 1..25:243-2577  215  APPENDIX I  CULTURE TECHNIQUES  A.  Axenic  medium  Composition  (Keenan e t a l . , 1 978)  1. L i v e r #2 ( N u t r i t i o n a l Biochemical Company)  0.1%  2. Brewer's yeast ( N u t r i t i o n a l Biochemical Company) 0.1% 3« Micrococcus  lysodeikticus dried c e l l s  0.1%  Preparation 1. suspend components i n d i s t i l l e d water 2. heat g e n t l y with s t i r r i n g 3. dispense 4. a u t o c l a v e  f o r 10 minutes  i n t o screw-cap t e s t - t u b e s , s t i r r i n g v i g o r o u s l y 15 minutes,  120°C  5. when c o o l , add p e n i c i l l i n - s t r e p t o m y c i n 100  ( f i n a l concentrations  units/ml and 100 ug/ml r e s p e c t i v e l y )  6. tape caps to prevent  evaporation  7. shake tubes immediately  Transfer  of  cells  from  a f t e r i n o c u l a t i o n with Paramecium  monoxenic to axenic medium  ( A l l e n and  Nerad, 1978) Glass t u b i n g r e g u i r e d (Table Appendix 1-1, F i g u r e  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  t u b i n g and covered  with aluminum  foil  216  2. . the other end i s covered with the i n v e r t e d t e s t - t u b e , E 3. tube C i s f i t t e d  with 4 cm  and covered with aluminum  of t u b i n g with a clamp around  it  foil  4. tube E i s placed on the o t h e r end 5..all  tubes are a u t o c l a v e d  T r a n s f e r of c e l l s 1. . c e n t r i f u g e  {100xg,  5 minutes)  100 ml of a dense c u l t u r e of  Paramecium 2. suck concentrated c e l l s  into a sterile  pasteur  pipette  (A)  with a cotton plug i n the end 3..heat  s e a l open end of p i p e t t e  4. remove c o t t o n plug, flame l i p of p i p e t t e 5.  remove f o i l  and i n s e r t p i p e t t e i n t o t u b i n g of tube B  6..add 5 ml axenic medium through top, remove a i r bubbles*  fill  column with axenic medium 7. remove insert  tube  E  from  tube  B  the top of tube B i n t o the  Appendix  I-2b)  8. f i l l  tube  C  and the f o i l tubing  of  from tube C and tube  C  (Figure  to the top with axenic medium, r e p l a c e tube E  and open clamp 9. allow c e l i s t o migrate o v e r n i g h t 10..close clamp and remove tube C frcm tube B 11. remove the f o i l insert  tube C i n t o the tubing of tube D (Figure Appendix  12. f i l l for  from tube D, remove tube E from tube C  tube  and  I-2c)  D with axenic medium and a l l o w c e l l s t o migrate  8 hours  13. remove 1 ml of c e l l s from the top with  a  sterile  pasteur  217  pipette,  transfer  to  a sterile  test-tube  and add 5 ml axenic  medium 14. a f t e r s e v e r a l days t h i s c u l t u r e can be subdivided  by adding  s e v e r a l drops of c e l l s t o f r e s h axenic medium  B.  Dryl solution  ( D r y l , 19 59)  Composi t i o n S o l u t i o n A: calcium  chloride  distilled  1.47 g  water  100.00 ml  S o l u t i o n B: sodium c i t r a t e  2.67 g  sodium phosphate  (dibasic)  5.63 g  sodium phosphate  (monobasic)  5.63 g  distilled  water  800.00 ml  a u t o c l a v e s o l u t i o n s A and B  separately  Preparation 1. 945 ml d i s t i l l e d  water  2..40 ml s o l u t i o n A 3..15 ml s o l u t i o n B  A d a p t a t i o n of c e l l s t o D r y l ' s 1. . c e n t r i f u g e c e l l s 2. place c e l l s  in  as  solution  (100xg, 5 minutes) small  a  volume  of  culture  fluid  as  218  possible  in  3,.  f i l l  flask  to  the  bottom with  of  a 25  Dryl's  ml v o l u m e t r i c  solution  flask  and a l l o w  cells  to  migrate  top  4. r e m o v e tube  the  cells  and  place  in  Dryl's  solution  in  a sterile  test-  219  Table Appendix  1-1.. Glass t u b i n g  reguired  for  adaptation  a x e n i c medium  Tube  Inner  Outer  Eiam(mm)  Diam (mm)  Length (mm)  B,D  5  7  60 0  C  8  7  230  E  8  10  75  to  220  Figure  Appendix  c e l l s t o axenic  1-1..Glass  tubing  medium.  a. the component p i e c e s of glassware  reguired  for transfer  221  a.  TUBE A  TUBE B, D  cotton plug  —pasteur pipette  TUBE C inverted "culture tube -glass tubealuminum foil naigene tubing clamp  -E  222  F i g u r e Appendix 1 - 2 . _ T r a n s f e r  of c e l l s t o axenic medium.  b..assembly  f o r the f i r s t  migration  c. assembly  f o r the second m i g r a t i o n . See t e x t f o r 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 I I  PHENOTYPES OF PUTATIVE MUTANTS  A.  Indiyidual  line  selection  Group 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  B6, B8, B9, B11, B12, B16, B23, E40, B43, C11, C28, D2, D9  Group 2. Decreased  • B1, B25,  + B2, + B27,  endocytosis  B3, *B4, + B5, *B10, B29,  + E14,  B30, B31, B37, B38  C10, C18, C21, C26, D7,  +B19, B20 , B21, r  + B22,  B4 1, + B42, B44, C1,  *D16  Group 3. Abnormal p o s i t i o n i n g  of food  vacuoles  B28, B32, B33, C19  Group 4. Abnormal morphology o f food  B7, + B15, +B17,  B24, B26,  vacuoles  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 s e r i e s but i t s true i s unknown  identity  225  B.  Mass  Group  selection  1. M o r p h o l o g i c a l  MB19, MB3 1, MD1,  G r o u p 2. D e c r e a s e d  abnormalities  MD21  endocytosis  + MA7, MA33, MB10, MB14, MB16, MD11, MD16, MD18,MD24,  Group 3 . _ P o s i t i c n i n g  of food  ME19, ME23  vacuoles  none  Group 4. M o r p h o l o g y o f f o o d  +MA26, MD20  ••these  lines  have been  lost  ME17,  vacuoles  +MB39,  MC29,  MC33,  226  APPENDIX I I I  ELECTRON  MICROSCOPY  1. c e n t r i f u g e mass c u l t u r e (100xg, 5 minutes) 2. wash i n 6 mM  phosphate b u f f e r  (pb), pH = 7  3.. f i x f o r 2 hcurs i n 0.5i& g l u t a r a l d e h y d e i n 6 mM pb 4. c e n t r i f u g e 50 seconds 5. wash twice i n 6 mM pb 6. p o s t - f i x  f o r 1 hour i n 1% osmium t e t r o x i d e i n 25 mM pb  7..centrifuge  50 seconds  8. wash twice i n 25 mM pb 9. c e n t r i f u g e  30  seconds,  remove  a l l washing  fluid  with a  pi pe tte 10. add 1 volume of 30% e t h a n o l t o p e l l e t , mix, l e t stand f o r 5 minutes 11. add 1 volume of 95% e t h a n o l to above, mix, l e t stand f o r 5 minutes 12. repeat step  (11), e t h a n o l now = 70%  13. c e n t r i f u g e , remove  fluid  14. dehydrate 5-10 minutes i n 70% ethanol with u r a n y l a c e t a t e 15. c e n t r i f u g e , remove  fluid  16. add 95% ethanol f o r 5 minutes 17. c e n t r i f u g e , remove f l u i d 18..two changes, 20 minutes each, of 100% e t h a n o l , f i n a l change in  1 volume  227  19. add (total  0.5  volume  propylene  oxide every 5 minutes*  4 times  = 3 volumes)  20. c e n t r i f u g e , remove f l u i d 21. two changes, in  20 minutes  each,  propylene oxide, f i n a l change  1 volume  22. t r a n s f e r t c v i a l 23..add 0.5  ml  Epon  every  5  minutes,  4  times  volumes) 24. l e a v e overnight uncapped 25. f i l l  BEEM capsules 1/3  full  with f r e s h Epon  26..add sample to each capsule 27. leave at rccm temperature  f o r 4 hcurs  28. incubate a t 60°C f o r 24-36 hours  (total  = 3  228  APPENDIX IV  DETERMINATION OF FATTY ACID COMPOSITION  A  - L i p i d extraction  I. . f i n a l  (Bligh and Dyer,  volume of c e l l s  = 2.4  1959, F o l c h e t a l . ,1957)  ml  2. add 9 ml chloroform/methanol (1:2), homogenize 3..add 3 ml chloroform, homogenize 4. .add 3 ml d i s t i l l e d  2 minutes  30 sec  water, homogenize  30 seconds  5. t r a n s f e r t c 50 ml round-bottom c e n t r i f u g e tubes 6..rince  hcmogenizer  with -4.5  ml  chloroform  and  add  to  amount  of  c e n t r i f u g e tube 7..vortex 8. c e n t r i f u g e  (200xg,  10minutes)  9. remove upper l a y e r 10..add  a volume of methanol/water  (1:0.8) egual t o  f l u i d removed I I . repeat  7-10  12. c e n t r i f u g e , remove upper l a y e r 13..add  1.0 ml 95% e t h a n o l , v o r t e x  14. f i l t e r 15. f i l l  through Whatman f i l t e r  (should go c l e a r ) paper (#1) i n t o  test-tube  t e s t - t u b e with c h l o r o f o r m , cover with T e f l o n - l i n e d cap  and s t o r e i n f r e e z e r  229  B  «  Thin l a y e r chromatography  1. evaporate 2i  (Nozawa and Thompson, 19 71)  under n i t r o g e n  r e d i s s o l v e i n 200  3..spot s i l i c a  u l chloroform  gel G p l a t e with 4 a p p l i c a t i o n s each of 25 u l of  sample 4..apply 10 u l i 5. p l a c e  standards  solvent  c h l o r o f o r m / a c e t i c acid/methanol/water/  (75: 25: 5: 2. 2) i n tank with l a r g e f i l t e r 6 . . a f t e r s o l v e n t has migrated p l a t e and 7..place  i n i o d i n e chamber u n t i l spots darken  Removal  Nelson,  L.add  (around  3  hours)  remove  dry  8 . . c i r c l e spots i n p e n c i l and  C._  to top  paper  of  (10 minutes)  scrape frcm p l a t e  phospholipids  from  silica  gel  (Andrews and  1979)  4  ml  c h l o r o f 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, c e n t r i f u g e (200xg, 5 minutes) 4. remove upper l a y e r , t r a n s f e r bottom l a y e r to c l e a n t e s t - t u b e 5. repeat 6.  combine  1-4  (not necessary  chloroform  t o l e a v e overnight  (bottom)  layers  and  again) evaporate  under  n i trogen 7. i f a s m a l l amount of water i s present add a few e t h a n o l to speed evaporation  drops o f  90%  230  8. s t o r e i n f r e e z e r  D  « -  Preparation  Kapoulos,  1969)  1. add  ml  2  of  acetic  glyceryl  ethers  acid/acetic  anhydride  (Thompson  and  (3:2), cap  tubes  tightly 2. heat to 90°C i n water bath f o r 4 hcurs, c o o l 3..slowly add  1.0 ml 6N  ( f r e s h l y prepared) , cap  potassium  hydroxide  in  95%  ethanol  tightly  4. heat t o 90°C i n water bath f o r 2 hours, c o o l 5. ether e x t r a c t i o n a. add  3 ml water  b. .add  1.5 ml e t h y l e t h e r , shake v i g o r o u s l y  c. remove and save ether phase d. .repeat  (b) and  5. removal of potassium a. .add  a  few  (c) combining  (top l a y e r ) ether l a y e r s  hydroxide  drops  of  water to combined e t h e r phases,  vortex b. remove water c. .repeat  (bottom  (a) and  layer)  (b)  7. evaporate under n i t r o g e n and  s t o r e a t room  temperature  231  E. . M e t h y l a t i c c  of f a t t y  esters  1. p r e p a r e f r e s h m e t h a n o l / h y d r o g e n a. _ w e i g h  c h l o r i d e g a s (HClg)  90 ml m e t h a n o l i n b e a k e r  b. b u b b l e HClg t h r o u g h m e t h a n o l f o r a r o u n d 5 m i n u t e s c . w e i g h and add more H C l g o r m e t h a n o l t o g i v e d. .CAUTICN  HClg v e r y  1N H C l g  dangerous  2. a d d 1 ml m e t h a n o l / H C l g , c a p t i g h t l y 3. _heat t o 90°C i n w a t e r b a t h  overnight  4. c o o l , e v a p o r a t e u n d e r n i t r o g e n  F.  Trimeth y l s i l a t i o n  of  a n d s t o r e a t room t e m p e r a t u r e  glyceryl  ethers  (Vance  and  S w e e l e y , 1967)  1. a d d  150  ul  pyridine/hexamethyldisilazane/trimethylsilane  (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 m i n u t e s 3. e v a p o r a t e under  nitrogen  4. r e d i s s o l v e i n 23-100 u l h e p t a n e i n s m a l l v i a l o r in  1  ml  nitrogen  heptane,  transfer  to  small  vial,  a n d r e d i s s o l v e i n 20-100 u l h e p t a n e  5. s t o r e a t room t e m p e r a t u r e  redissolve  e v a p o r a t e under  232  G.  Gas l i q u i d  1. machine  chromatography  settings  a. .flame temperature = 200°C b. range =  10  2  c. .attenuation d. „ chart  = 8 or 4  speed = 0.5 inches/minute  2. m u l t i l e v e l programmer a. .post i n j e c t i o n time = 6 minutes b. r a t e  1 = 2°/minute;  c. .time  1=6  rate  minutes; time  2,3 = 0 2=2  minutes;  time  3 = 10  minutes d. .to of  initiate  press advance  immediately a f t e r i n j e c t i o n  sample  3..injection  of sample  a. take up 2 u l heptane b. .take up 5 u l sample  and move gauge on s y r i n g e  to  9  ul  mark c. . i n j e c t injecting 4. . a n a l y s i s  into  portal  A  holding  end  of  syringe  and  whole sample a t once  of r e s u l t s  a. Xerox  peaks  b. . c u t out and weigh each peak c. f o r each f a t t y a c i d c a l c u l a t e the percent of the weight  total  233  Table Appendix IV-1. F a t t y experiment 1. .  Fatty Ac id  wild-type 27°C  acid  composition  Percent Composition dm1 wild-type 27°C 34.5°C  <16: 0 tr *16:0 14.33 16: 1 3. 16 17: 1 (1) 1. 0 1 *18: 0 3.62 *18:1 17.53 *18: 2 5. 58 •18:3 (2) 9. 03 20: 0 2. 25 2.36 20:2 *20: 4 5. 66 22: 1 1.82 22: 2 2. 26 22:4 7.57 22: 6 1.39 24:0 1. 71 24: 1 1.60 1.77 *24:2 *24: 4 14. 35  (1) may 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 acids  1. 08 20. 86 3.64 1. 96 2.24 17. 02 4. 58 8. 65 4. 2 7 3. 83 4. 91 3. 38 1. 84 5. 31 2. 73 tr 1.38 2. 52 8.04  3.54 18.39 2.37 1. 10 6.14 16.19 4.74 10.94 2.02 2.27 4.13 6.50 tr 6.47 2.92 1. 42 2.01 tr 7.49  of whole c e l l s -  drnj 34.5° 1. 47 19.40 2.45 3.57 6.59 16.95 3.72 1 .66 3.49 tr tr 2.21 tr 6.72 5.32 3.20 1.78 3.22 14. 13  234  T a b l e Appendix I V - 2 . F a t t y e x p e r i m e n t 2. .  Fatty Acid  wild-type 27°C  <16:0 *1 6: 0 16: 1 17:1 (1) *18:0 *18: 1 *1 8:2 • 1 8 : 3(2) 20:0 20: 2 *20:4 22: 1 22:2 *22: 4 22: 6 24:0 24. 1 *24:2 *24: 4  tr 7.65 1.74 tr 1. 59 8 . 23 2.86 6 . 7S 2. 36 2. 28 5.86 tr 1. 45 18.76 1. 24 4. 21 2. 04 5.38 30.00  acid composition  Percent Composition dml nild-type 27°C 34.5°C 1.41 9. 37 2. 16 tr 1.04 7. 18 2. 23 3.91 4 . 27 1. 89 1. 9 1 tr 2. 86 18. 07 tr 5. 96 1. 98 5. 51 28.49  (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 2 0 : 1 * major f a t t y a c i d s  tr 10.97 3.08 tr 2.06 11.60 4.444 7.66 1.65 2.03 3.08 1.59 1.8 2 15.84 2.74 3. 19 tr 4.70 20.62  of  whole  dml 34. 5°'  ___ 5.85 1. 28 tr 1.27 6.49 2.50 4.55 1 .49 tr 1.62 tr 1.50 18.03 tr 4 . 84 2. 10 8 . 11 38.93  cells -  235  T a b l e Appendix IV-3. F a t t y experiment 3. .  Fatty Acid  <16To  wild-type 27°C __  *16: 0 9. 75 1 6: 1 2. 29 17:1 (1) 1.46 *18: 0 3. 72 *18: 1 22. 19 4.55 *18: 2 5.06 *1 8:3 (2) 20: 0 4. 89 20:2 4. 61 *20: 4 7.88 6. 04 22:1 22:2 1. 84 7. 07 *22: 4 22:6 tr 24: 0 tr 24: 1 tr *24: 2 4.93 *24: 4 1 1. 34  acid  composition of whole c e l l s -  Percent Composition dml wild-type 27°C 34.5°C tr 20. 52 7. 13 1. 11 4.32 18. 46 4. 26 5. 48 3. 33 tr 2. 14 1. J7 tr 13. 83 1. 80 1.17 2. 53 4. 30 8.45  (1) may a l s o contain 17:0 (2) a l s o c o n t a i n s 20:1 * major f a t t y acids  11.28 16.52 6.22 tr 2. 15 17. 19 3.39 7. 48 2.80 3. 43 3.78 2.21 tr 6.64 1.16 1.02 tr 4.68 8.82  dml 34. 5°( 1 .48 19.17 6.72 1.69 2.54 18.11 5. 22 6.67 4.62 1 .43 4.92 1 .40 tr 7. 27 1.50 1.82 tr 2.69 1 1.31  

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