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UBC Theses and Dissertations

A comparative study of the cell surface and the mechanism of macromolecule internalization in three phytoflagellates de Andrade Alves de Sá Klut, Maria Emilia 1985

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A COMPARATIVE STUDY OF THE CELL SURFACE AND THE MECHANISM OF MACROMOLECULE INTERNALIZATION IN THREE PHYTOFLAGELLATES by MARIA EMILIA DE ANDRADE ALVES DE SA^KLUT Master of Science, The U n i v e r s i t y of B r i t i s h Columbia 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY FACULTY OF SCIENCE UNIVERSITY OF BRITISH COLUMBIA we accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA November 1985 © Maria E m i l i a K l u t , 1985 32 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of */ ~ </5/0£>Q(by The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) i i ABSTRACT A cytocheraical i n v e s t i g a t i o n on the c e l l s u r f a c e of three p h y t o f l a g e l l a t e s , one chlorophyte, D u n a l i e l l a t e r t i o l e c t a and two d i n o f l a g e l l a t e s , Amphidinium c a r t e r a e and Prorocentrum roicans showed that they a l l possess an a n i o n i c and heterogenous o l i g o s a c c h a r i d e coat on the e x t e r n a l s i d e of the plasma membrane. Studies with l e c t i n s gave c l u e s to the nature of the sugar residues of t h e i r g l y c o c o n j u g a t e s . In p a r t i c u l a r , they may co n t a i n N-acetylglucosamine and/ or s i a l i c a c i d , N-acetylgalactosamine and D-mannose or gl u c o s e . Among other p r o p e r t i e s , the carbohydrate m o i e t i e s of the c e l l s u r f a c e components were found to play an important r o l e i n c e l l r e c o g n i t i o n , c e l l to c e l l adhesion and i n e n d o c y t o s i s . A l l three p h y t o f l a g e l l a t e s were able to i n t e r n a l i z e macromolecules such as h o r s e r a d i s h peroxidase, c a t i o n i z e d f e r r i t i n , l e c t i n s and l e c t i n - c o l l o i d a l gold conjugates. In D u n a l i e l l a p i n o c y t o s i s was observed for the f i r s t time, and was found to occur i n the v i c i n i t y of the f l a g e l l a , away from the cup-shaped c h l o r o p l a s t . During f l u i d - p h a s e and a d s o r p t i v e e n d o c y t o s i s , macromolecules were trapped w i t h i n uncoated or coated p i t s and then d e l i v e r e d , v i a endosomes, to the lysosomal system. The reverse process of metabolic s e c r e t i o n as part of membrane r e c y c l i n g i s a l s o d e s c r i b e d . i i i In Prorocentrum f exogenous macromolecules were i n t e r n a l i z e d v i a s u r f a c e pores, while i n Amphidinium the l i g a n d s were taken i n by plasma membrane-derived v e s i c l e s with subsequent i n c o r p o r a t i o n i n t o the p e r i p h e r a l c y t o p l a s m i c vacuole. In both d i n o f l a g e l l a t e s , the major s i t e of macromolecule i n t e r n a l i z a t i o n was the f l a g e l l a r c a n a l-pusule system. Morphological and morphometric s t u d i e s on Prorocentrum showed pusule s i z e changes i n response to d i f f e r e n t l e v e l s of s a l i n i t y and temperature. A three-dimensional model of Amphidinium pusule i s proposed based on s e q u e n t i a l s e c t i o n s . I t i s suggested that a f i b r i l l a r c o l l a r system i n c o n j u n c t i o n with the f l a g e l l a r - b e a t i n g p l a y s an important r o l e i n r e g u l a t i n g the flow of m a t e r i a l s i n and out of the pusule. I t appears that the pusule i s a complex s t r u c t u r e i n v o l v e d i n osmoregulation, c e l l u l a r uptake and p o s s i b l y s e c r e t i o n . With regard to the r e l a t i o n s h i p between the u l t r a s t r u c t u r e of the c e l l c o v e r i n g and the mechanism of e n d o c y t o s i s , two h y p o t h e t i c a l e v o l u t i o n a r y l i n e s are suggested, one i n c l u d e s those organisms such as D u n a l i e l l a , amoeba and some other protozoa that lack a r i g i d c e l l w all and a s p e c i a l i z e d compartment for macromolecule i n t e r n a l i z a t i o n ; the other comprises those forms such as d i n o f l a g e l l a t e s , cryptomonads, euglenoids and some k i n e t i d a l protozoa that possess a complex c e l l c o v e r i n g and a s p e c i f i c compartment for macromolecule uptake. i v TABLE OF CONTENTS page ABSTRACT i i TABLE OF CONTENTS . iv LIST OF FIGURES x LIST OF TABLES x i i LIST OF ABBREVIATIONS x i i i ACKNOWLEDGEMENTS xiv DEDICATION X V INTRODUCTION 1 A l g a l s t r a i n s 1 General s t r u c t u r e and nature of the c e l l s u r f a c e of the chlorophytes and d i n o f l a g e l l a t e s 2 Gly c o c a l y x and i t s p o s s i b l e f u n c t i o n s 4 Endocytosis , endosomes and lysosomes : d e f i n i t i o n and terminology 5 Types of e n d o c y t o s i s : phagocytosis and p i n o c y t o s i s 7 Phagocytosis of l a r g e p a r t i c l e s 7 P i n o c y t o s i s of f l u i d and s o l u t e s 8 P i n o c y t o s i s i n p l a n t c e l l s 10 Markers used to t e s t p i n o c y t o s i s i n p l a n t c e l l s 12 Mechanisms of p i n o c y t o s i s : f l u i d - p h a s e and a d s o r p t i v e p i n o c y t o s i s . Methods of study 14 Fluid-phase p i n o c y t o s i s 14 V page F a c t o r s r e g u l a t i n g p i n o c y t o s i s 15 Markers f o r s t u d y i n g f l u i d - p h a s e p i n o c y t o s i s 15 A d s o r p t i v e p i n o c y t o s i s 16 Markers f o r s t u d y i n g a d s o r p t i v e p i n o c y t o s i s 19 P u s u l e 19 O b j e c t i v e and methology used i n t h i s study 22 T a b l e 1 24 T a b l e 2 25 MATERIAL AND METHODS 26 C u l t u r e c o n d i t i o n s 26 Reagents 26 L i g h t m i c r o s c o p y 27 A. C e l l a g g l u t i n a t i o n e x periments 27 B. M o r p h o l o g i c a l a n a l y s i s of P. micans p u s u l e 27 F l u o r e s c e n c e m i c r o s c o p y 28 A. C e l l s u r f a c e b i n d i n g and macromolecule i n t e r n a l i z a t i o n .. 28 B. I d e n t i f i c a t i o n and l o c a l i z a t i o n of l i p i d b o d i e s 29 F r eeze e t c h i n g p r o c e d u r e 29 Scanning e l e c t r o n m i c r o s c o p y 30 T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y 30 A. C e l l s u r f a c e b i n d i n g 30 1. C a t i o n i c dye s t a i n i n g 30 2. L e c t i n - p e r o x i d a s e l a b e l i n g 31 B. A c i d phosphatase l o c a l i z a t i o n 32 v i page C. C y t o c h e m i c a l l o c a l i z a t i o n of v a r i o u s m o l e c u l a r markers . 33 1. H o r s e r a d i s h p e r o x i d a s e 33 2. C a t i o n i z e d f e r r i t i n 33 3. Wheat g e r m - c o l l o i d a l g o l d . P r e p a r a t i o n of CG p a r t i c l e s and CG-WGA c o n j u g a t e 34 Procedure 34 Tab l e 3 36 Tab l e 4 37 Tab l e 5 38 RESULTS . . 39 I . D u n a l i e l l a t e r t i o l e c t a 39 A. C e l l s t r u c t u r e 39 B. Nature of the c e l l s u r f a c e . C e l l s u r f a c e r e l a t e d a c t i v i t i e s 39 L i g h t m i c r o s c o p y 39 L e c t i n - i n d u c e d a g g l u t i n a t i o n 39 F l u o r e s c e n c e m i c r o s c o p y 40 a. C e l l s u r f a c e b i n d i n g and l e c t i n i n t e r n a l i z a t i o n 40 b. HRP i n t e r n a l i z a t i o n 41 T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y 41 a. C e l l s u r f a c e b i n d i n g . Evidence f o r l e c t i n i n t e r n a l i z a t i o n 41 b. Macromolecule i n t e r n a l i z t i o n u s i n g two m o l e c u l a r markers 42 v i i page HFP 42 CF 43 C. A c i d phosphatase l o c a l i z a t i o n 45 I I . Arophidiniuni c a r t e r a e 46 A. C e l l s t r u c t u r e 46 B. C e l l c o v e r i n g morphology and morphometry 46 C. C e l l s u r f a c e r e l a t e d s t r u c t u r e s . L i p i d bodies and t r i c h o c y s t s 48 D. Nature of the c e l l s u r f a c e . C e l l s u r f a c e - r e l a t e d a c t i v i t i e s 49 Fluorescence microscopy 49 C e l l s u r f a c e b i n d i n g using v a r i o u s fluorochromes 49 Transmission e l e c t r o n microscopy 50 C e l l s u r f a c e b i n d i n g using SBA or c a t i o n i c dyes .... 50 E. Macromolecule i n t e r n a l i z a t i o n using v a r i o u s molecular markers 52 SEM and Fluorescence microscopy 52 Transmission e l e c t r o n microscopy 53 1. I n t e r n a l i z a t i o n of HRP 53 2. C e l l s u r f a c e b i n d i n g and i n t e r n a l i z a t i o n of WGA-CG 54 I I I . Prorocentrum micans 55 A. C e l l s t r u c t u r e 55 B. Nature of the c e l l s u r f a c e components 55 v i i i page F l u o r e s c e n c e m i c r o s c o p y 55 a. C e l l s u r f a c e b i n d i n g and l e c t i n i n t e r n a l i z a t i o n . 55 b. T h e c a l p l a t e s 56 T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y 57 a. Nature of the c e l l s u r f a c e 57 b. T r i c h o c y s t pores and macromolecule i n t e r n a l i z a t i o n 58 C. P u s u l e 60 a. Morphology and morphometry of P. micans p u s u l e .. 60 b. S t r u c t u r e of P. micans p u s u l e . Macromolecule 61 i n t e r n a l i z a t i o n 62 c. S t r u c t u r e of _. c a r t e r a e p u s u l e . Macromolecule i n t e r n a l i z a t i o n 62 T a b l e 6 64 T a b l e 7 65 T a b l e 8 66 DISCUSSION 67 A. S t r u c t u r e of the c e l l s u r f a c e 67 B. Nature of the c e l l s u r f a c e coat 71 C. P o s s i b l e r o l e of the g l y c o c a l y x 74 D. C e l l s u r f a c e - r e l a t e d s t r u c t u r e s 75 1 . L i p i d b o d i e s 75 2. S p i n d l e t r i c h o c y s t s 77 E. C e l l w a l l morphology and s y n t h e s i s 79 F. Nature of the c e l l w a l l 81 i x page G. T h e c a l o r n a m e n t a t i o n s 81 H. R o l e s of the c y t o s k e l e t o n i n c e l l - s u r f a c e a c t i v i t i e s . 82 J . A d s o r p t i v e p i n o c y t o s i s 83 K. A c i d phosphatase l o c a l i z a t i o n 86 L. F l u i d - p h a s e p i n o c y t o s i s 87 M. Macromolecule i n t e r n a l i z a t i o n i n Arophidinium and Proro c e n t r um 88 1. Macromolecule i n t e r n a l i z a t i o n v i a plasma-membrane i n v a g i n a t i o n and d e r i v e d endosomes 88 2. Macromolecules i n t e r n a l i z a t i o n v i a s u r f a c e pores . 90 3. Macromolecule i n t e r n a l i z a t i o n v i a the f l a g e l l a r c a n a l - p u s u l e system 91 N. F a c t o r s r e g u l a t i n g p i n o c y t o s i s 98 0. Evi d e n c e f o r p i n o c y t o s i s i n oth e r p l a n t c e l l s 98 P. Membrane r e c y c l i n g 100 Q. Some comments on the e v o l u t i o n a r y mode of macromolecule i n t e r n a l i z a t i o n i n p r o t i s t s 101 KEY FOR FIGURES 105 FIGURES 106 LITERATURE CITED 142 X LIST OF FIGURES FIGURE page 1 Diagram of D u n a l i e l l a c e l l i l l u s t r a t i n g p i n o c y t o s i s 106 2-5 L e c t i n a g g l u t i n a t i o n t e s t s on D u n a l i e l l a 107 6-11 C e l l s u r f a c e b i n d i n g and i n t e r n a l i z a t i o n of FITC-WGA 108 12-18 D u n a l i e l l a c e l l s t r e a t e d w i t h l e c t i n s . C o n t r o l 109 19-26 HRP i n t e r n a l i z a t i o n . C o n t r o l 110 27-28 The s t r u c t u r a l arrangement of s u b c e l l u l a r o r g a n e l l e s 111 29-32 C e l l s u r f a c e coat l a b e l e d w i t h CF 111 33-38 CF i n t e r n a l i z a t i o n 112 39-40 CF w i t h i n secondary lysosomes 113 41-43 G o l g i - d e r i v e d v e s i c l e s and membrane r e c y c l i n g 113 44-47 A c i d - p h o s p h a t a s e l o c a l i z a t i o n . C o n t r o l 114 48-49 SEM of _. c a r t e r a e c e l l s 115 50-51 A p i c a l and d o r s a l view of Amphidiniuro c e l l s 116 52-53 The p o l y g o n a l appearance of the amphiesmal u n i t s 116 54-55 F r e e z e - e t c h i n g r e p l i c a of the amphiesma v e s i c l e s 117 56 Histogram r e p r e s e n t i n g the s u r f a c e area of the amphiesmal u n i t s 118 57-58 L o c a l i z a t i o n of l i p i d b o d i e s u s i n g Rhodamine B 119 59-60 L o c a l i z a t i o n of l i p i d b o d i e s u s i n g F l u o r o B o r a P 119 61-62 TEM of l i p i d - l i k e s t r u c t u r e s 120 63-64 T r i c h o c y s t s 120 x i 65-68 C e l l coat of Amphidinium c e l l s 121 69-72 C e l l coat l a b e l e d w i t h SBA 122 73-77 C e l l coat s t a i n e d w i t h AlcB-RR. C o n t r o l 123 78-82 C e l l s u r f a c e and - r e l a t e d s t r u c t u r e s 124 83 Diagram of the c e l l c o v e r i n g r e g i o n 125 84-88 I n t e r n a l i z a t i o n of FITC-HRP/FITC 126 89-93 V a c u o l e s and the p u s u l e c o n t a i n i n g HRP 127 94-99 C e l l s u r f a c e b i n d i n g and i n t e r n a l i z a t i o n of WGA-CG 128 100-101 2. micans c e l l s t r e a t e d w i t h AO or ConA 129 102-103 C e l l w a l l s t a i n e d w i t h C a l c o f l u o r White 129 104-109 S t r u c t u r a l d e t a i l s on Prorocentrum c e l l s u r f a c e 130 110-115 T r i c h o c y s t s b e f o r e and d u r i n g e x t r u s i o n . 131 116-119 M o r p h o l o g i c a l appearance of s u r f a c e pores 132 120 Diagram i l l u s t r a t i n g t h r e e t y p e s s of pores 133 121-126 CF and HRP i n t e r n a l i z a t i o n v i a pores or s u t u r e s 134 127-129 P u s u l e s i z e changes w i t h s a l i n i t y 135 130-132 M o r p h o l o g i c a l appearance of Prorocentrum p u s u l e 135 133 Coated v e s i c l e s c l o s e t o the p u s u l e 135 134-137 HRP i n t e r n a l i z a t i o n v i a the p u s u l e 136 138-141 CF and WGA-CG i n t e r n a l i z a t i o n v i a the p u s u l e 137 142 Diagram of the f l a g e l l a r r e g i o n of Amghidinium 138 143-148 S t r u c t u r e of the f l a g e l l a r c a n a l - p u s u l e system 139 149-151 I n t e r n a l i z a t i o n of WGA_CG and HRP v i a the p u s u l e 140 151-152 V a c u o l e s , c o a t e d v e s i c l e s c l o s e t o the p u s u l e 141 x i i LIST OF TABLES page Table 1. A l g a l s t r a i n s 24 Table 2. Markers used to t e s t p i n o c y t o s i s i n p l a n t c e l l s . . . . 25 Table 3. Composition of the c u l t u r e medium used i n t h i s study 36 Table 4. Fluorochromes used and t h e i r known s p e c i f i c i t i e s . . . 37 Table 5. Vari o u s TEM schedules used 38 Table 6. D u n a l i e l l a a g g l u t i n a t i o n induced with v a r i o u s l e c t i n s 64 Table 7. Morphometric a n a l y s i s f o r the s u r f a c e area of the amphiesmal v e s i c l e s of A. c a r t e r a e 65 Table 8. S a l i n i t y e f f e c t on the s i z e of the pusule and c e l l of £. micans 66 LIST OF ABBREVIATIONS AcPase a c i d phosphatase AlcB-RR a l c i a n blue-ruthenium red AO a c r i d i n e orange CF c a t i o n i z e d f e r r i t i n CG c o l l o i d a l gold p a r t i c l e s Con A concanavalin A DAB 3 , 3 ' - d i a m i n o b e n z i d i n e FITC f l u o r e s c e i n i s o t h i o c y a n a t e HRP h o r s e r a d i s h peroxidase LPA Limulus polyphemus a g g l u t i n i n OSO4 osmium t e t r o x i d e PBS phosphate b u f f e r s a l i n e SBA soybean a g g l u t i n i n SEM scanning e l e c t r o n microscopy TEM t r a n s m i s s i o n e l e c t r o n microscopy WGA wheat germ a g g l u t i n i n D-GlcNAc N-acetyl-D-glucosamine D-GalNAc N-acetyl-D-galactosamine °(-D-ManMe or-methyl-D-mannoside xiv ACKNOWLEDGEMENTS The author g r a t e f u l l y acknowledges the f i n a n c i a l support and the i n s i g h t f u l guidance of Dr. T. B i s a l p u t r a , her t h e s i s s u p e r v i s o r . She wishes to thank Dr. N. J. A n t i a for h i s encouragement, help and a d v i c e . Sincere g r a t i t u d e i s a l s o extended to the other committee members, Dr. A. Acton, Dr. A. G. Lewis, and Dr. P. J . H a r r i s o n f o r t h e i r help i n c l a r i f y i n g key ideas expressed i n t h i s t h e s i s . She thanks Dr. L. O l i v e i r a for reading part of the f i n a l manuscript. She a l s o expresses h e a r t f e l t g r a t i t u d e for her f e l l o w students and t e c h n i c i a n s whose c o n t r i b u t i o n s helped r e a l i z e t h i s t h e s i s . L a s t , but not l e a s t , she thanks her parents whose unceasing encouragement and love kept her going. X V T h i s t h e s i s i s d e d i c a t e d t o Dr. Naval J. A n t i a on the o c c a s i o n of h i s r e t i r e m e n t . A w i l l i n g n e s s t o share h i s r i c h r e s e a r c h e x p e r i e n c e has p r o v i d e d c o n t i n u i n g i n s p i r a t i o n . INTRODUCTION 1 A l g a l s t r a i n s The growing i n t e r e s t i n u n i c e l l u l a r a l g a l c u l t u r e s , t o g e t h e r w i t h the a b i l i t y t o c o n t r o l t h e i r growth, under s p e c i f i e d c o n d i t i o n s , has l e d t o new p e r s p e c t i v e s i n the development of e x p e r i m e n t a l c e l l b i o l o g y and p h y s i o l o g y . The a l g a l s t r a i n s used i n t h i s study were the c h l o r o p h y t e D u n a l i e l l a t e r t i o l e c t a Butcher and two d i n o f l a g e l l a t e s , Amphidinium c a r t e r a e H u l b u r t and Prorocentrum micans Ehrenberg (Table 1 ) . These p h y t o f l a g e l l a t e s were chosen because of the f o l l o w i n g f e a t u r e s : the n a t u r e and d i v e r s i t y of t h e i r c e l l s u r f a c e , i . e . the presence or absence of a complex c e l l c o v e r i n g w i t h or w i t h o u t t h e c a l p l a t e s , t h e i r d i f f e r e n t c a p a c i t y t o w i t h s t a n d h o s t i l e e n v i r o n m e n t s , t h e i r p o t e n t i a l a b i l i t y t o i n t e r a c t w i t h exogenous macromolecules, and the s i m p l i c i t y of m o n i t o r i n g t h e i r growth under w e l l - d e f i n e d n u t r i t i o n a l c o n d i t i o n s . On the b a s i s of t h e o r e t i c a l grounds e s t a b l i s h e d f o r ot h e r e u k a r y o t i c c e l l s , i t i s h y p o t h e s i z e d t h a t these p h y t o f l a g e l l a t e s do have a g l y c o c a l y x - l i k e s u r f a c e l a y e r and t h a t they a re a b l e t o i n t e r n a l i z e and, c o n c e i v a b l y , u t i l i z e l a r g e m o l e c u l e s such as p r o t e i n s . 2 G e n e r a l s _ r _ c j _ _ e ajid n a t u r e of the ceJLl §j_r___e. o f The c h l o r o p h y t e s a,nd d j n o f ; U g e 3 , 3,3tes V e g e t a t i v e c e l l s of most c h l o r o p h y t e s a re e n c l o s e d i n a c e l l w a l l made up of two components: the f i b r i l l a r framework and the amorphous m a t r i x . The former i s composed of c e l l u l o s e , mannan or x y l a n , w h i l e the l a t t e r forms the n o n f i b r i l l a r embedding m a t e r i a l made up of p r o t e i n , and h e m i c e l l u l o s e or o t h e r p o l y s a c c h a r i d e r e s i d u e s such as D - g a l a c t o s e , L - a r a b i n o s e or D - x y l o s e . Some u n i c e l l u l a r c h l o r o p h y t e s have a t r i l a m i n a r w a l l l a y e r composed of s p o r o p o l l e n i n (Mackie and P r e s t o n 1974). In c o n t r a s t , members of the f a m i l y P o l y b l e p h a r i d a c e a e ( V o l v o c a l e s ) a re c h a r a c t e r i z e d by t h e i r l a c k of c e l l w a l l , as e x e m p l i f i e d by D u n a l i e l l a Teodoresco (Bold and Wynne 1985). D i n o f l a g e l l a t e s a re known t o be e n c l o s e d i n a unique complex s t r u c t u r e v a r i o u s l y r e f e r r e d t o as the "amphiesma" (Schu'tt 1895, L o e b l i c h 1970, M o r r i l l and L o e b l i c h 1983), " t h e c a " (Dodge and Craw f o r d 1968, 1970; K a l l e y and B i s a l p u t r a 1970, 1971; Dodge 1973, 1974; S t e i d i n g e r and Cox 1980) or " c e l l c o r t e x " (Spector 1984, N e t z e l and Durr 1984). The c e l l c o v e r i n g of a v e g e t a t i v e m o t i l e c e l l c o n s i s t s of a l i m i t i n g membrane (plasma membrane) and a s i n g l e u n d e r l y i n g l a y e r of f l a t t e n e d v e s i c l e s w i t h or w i t h o u t t h e c a l p l a t e s ( r e f e r r e d t o 3 as t h e c a t e or armored and a t h e c a t e or unarmored r e s p e c t i v e l y ) . Amphidiniuro C l a p a r e d e and Lachmann (from Gr. amphi, around + d i n e i n , t o whorl) i s a genus r e p r e s e n t i n g a t h e c a t e d i n o f l a g e l l a t e s which p o s s e s s numerous amphiesmal v e s i c l e s , w h i l e P r o r o c e n t r u m Ehrenberg (from L. p r o r a , prow + L. centrum, c e n t e r ) r e p r e s e n t s t h e c a t e d i n o f l a g e l l a t e s w i t h o n l y two v a l v e s . I t has been r e p o r t e d t h a t the t h e c a l p l a t e s of d i n o f l a g e l l a t e s a re p o l y g o n a l and made up of c e l l u l l o s e (Dodge 1971) or c e l l u l o s e - l i k e g l u c a n (Nevo and Sharon 1969). O s m i o p h i l i c m a t e r i a l w i t h i n the amphiesmal v e s i c l e s of some a t h e c a t e d i n o f l a g e l l a t e s ( t r a n s i t i o n a l type) has been d e s c r i b e d (Dodge and Craw f o r d 1969, Dodge 1974 , W i l c o x e_t a l . 1982) and i s c o n s i d e r e d t o be homologous t o the p l a t e s of t h e c a t e d i n o f l a g e l l a t e s . A p e l l i c u l a r l a y e r made of s p o r o p o l l e n i n - l i k e m a t e r i a l l i e s between the amphiesmal v e s i c l e s and the c y t o p l a s m i c membrane i n s p e c i e s t h a t a re known t o encyst ( L o e b l i c h 1970, M o r r i l l and L o e b l i c h 1983). S e v e r a l t y p e s of c e l l s u r f a c e o r n a m e n t a t i o n s i n c l u d i n g p o r e s , s p i n e s , r i d g e s and o r g a n i c s c a l e s have been r e p o r t e d i n these f l a g e l l a t e s ( A n d r e i s et a l . 1982). The s t r u c t u r e and o r g a n i z a t i o n of the d i n o f l a g e l l a t e c e l l c o v e r i n g are c o n s i d e r e d t o be of p h y l o g e n e t i c s i g n i f i c a n c e . These key f a c t o r s , among o t h e r s , have enabled 4 t a x o n o m i s t s t o e s t a b l i s h h y p o t h e t i c a l l i n e s i n the e v o l u t i o n of the P y r r o p h y t a ( T a y l o r 1980, L o e b l i c h 1984). Glycocglyx, and i t s p o s s i b l e f u n c t i o n s An e a r l y b i o c h e m i c a l i n v e s t i g a t i o n ( J o k e l a 1969) i n d i c a t e d t h a t the plasma membrane of D u n a l i e l l a t e r t i o l e c t a i s composed p r e d o m i n a n t l y of p r o t e i n s and p h o s p h o l i p i d s . The p resence of a g l y c o c o n j u g a t e coat was suggested o n l y r e c e n t l y from c a t i o n i c dye s t a i n i n g and p r o t e o l y t i c enzyme d i g e s t i o n ( O l i v e i r a et a l . 1980). However, s i n c e these dyes are known t o s t a i n a c i d i c and s u l f a t e d p o l y s a c c h a r i d e s ( P a r k e r and D i b o l l 1966, Ramus 1977), the i n f e r e n c e on the presence of g l y c o p r o t e i n s i n the c e l l c oat of D. t e r t i o l e c t a r e q u i r e d f u r t h e r s u p p o r t i v e e v i d e n c e . Such e v i d e n c e has been p r o v i d e d from s t u d i e s on l e c t i n - i n d u c e d a g g l u t i n a t i o n ( K l u f e i a l . 1983). S p e c i f i c i n t e r a c t i o n s of g l y c o p r o t e i n - c o n t a i n i n g plasma membrane w i t h " c e l l - b i n d i n g or c e l l a g g l u t i n a t i n g " p r o t e i n s termed l e c t i n s have been p r e v i o u s l y shown w i t h o t h e r e u k a r y o t i c c e l l s (Hughes 1976, Warton and Honiberg 1980). A l t h o u g h a g l y c o c o n j u g a t e coat has l o n g been r e c o g n i z e d i n a wide range of a n i m a l (Hughes 1976, Tumanova 1978) and a l g a l c e l l s ( O l i v e i r a e£ a l . 1980, S h a r a b i and G i l b o a - G a r b e r 1980, Surek and Sengbusch 1981, V a n n i n i _± a_l. 1981, Sengbusch and 5 M u l l e r 1 9 83), the presence of such a coat i n d i n o f l a g e l l a t e s has not been r e p o r t e d . Among o t h e r f u n c t i o n s , c e l l s u r f a c e g l y c o c o n j u g a t e s p l a y an i m p o r t a n t r o l e i n r e g u l a t i n g e s s e n t i a l a c t i v i t i e s such as c e l l r e c o g n i t i o n , a d h e s i o n and e n d o c y t o s i s ( M a r s h a l l and Nachmias 1965, Hughes 1976). E n d o c y t o s i s . endosomes, a n d lysosoroes: d e f i n i t i o n ajjd t e r m i n o l o g y E n d o c y t o s i s i s the p r o c e s s of i n g e s t i o n or uptake of e x t r a c e l l u l a r m a t e r i a l s v i a plasma membrane i n v a g i n a t i o n l e a d i n g t o subsequent f o r m a t i o n of endosomes. Endosomes are e n d o c y t i c compartments (0.2-1 jum i n dia m e t e r ) of heterogeneous morphology and c e l l u l a r d i s t r i b u t i o n . They are c h a r a c t e r i z e d by an a c i d i c pH, g e n e r a l l y pH 5, but d i f f e r from lysosomes by h a v i n g much lower e l e c t r o n d e n s i t y and by l a c k i n g a c i d h y d r o l a s e s . The low pH c o n d i t i o n of these compartments has been p a r t i a l l y a t t r i b u t e d t o the a c t i v i t y of a H+-ATPase and i s thought t o be r e s p o n s i b l e f o r the u n c o u p l i n g of r e c e p t o r s from l i g a n d s (Galloway e£ a_l. 1983). These l i g a n d s are a p p a r e n t l y r o u t e d i n t o lysosomes w h i l e r e c e p t o r s are e i t h e r r e c y c l e d back t o the c e l l s u r f a c e ( s h o r t - c i r c u i t pathway) or degraded 6 ( H e l e n i u s _£ a l . 1983). Endosomes not o n l y a c t as p r e - l y s o s o m a l compartments but are i n v o l v e d i n m a i n t a i n i n g the i n t r a c e l l u l a r t r a f f i c from the plasma membrane to the G o l g i endoplasmic r e t i c u l u m - l y s o s o m e system (GEFL) or a c r o s s the c e l l s ( i . e . the t r a n s c e l l u l a r pathway, Fothman and Lenard 1984). Endosomes have been v a r i o u s l y r e f e r r e d to as phagosomes, pinosomes, receptosomes, smooth or " C U R L " v e s i c l e s , and e n d o c y t i c - , r e s o r p t i v e - , i n t e r m e d i a t e - , p r i m a r y f o o d - , p r e - l y s o s o m a l v a c u o l e s . The term endosome appears t o be more g e n e r a l and adequate, s i n c e i t does not evoke e q u i v o c a l i n t e r p r e t a t i o n s (as phagosomes), nor does i t always imply the presence of s u r f a c e r e c e p t o r s (as receptosomes) or f u s i o n w i t h lysosomes (as p r e - l y s o s o m a l v a c u o l e s , H e l e n i u s _t a l . 1983). Lysosomes are s u b c e l l u l a r o r g a n e l l e s c h a r a c t e r i z e d by an a c i d i c pH t h a t may be a t t r i b u t e d to an a c t i v e H+-flTPase pump. They c o n t a i n s e v e r a l h y d r o l y t i c enzymes (e.g. p h o s p h a t a s e s , n u c l e a s e s , g l y c o s i d a s e s , p r o t e a s e s , l i p a s e s , s u l f a t a s e s , p h o s p h o l i p a s e s ) . A p p a r e n t l y , many of these h y d r o l a s e s s y n t h e s i z e d i n the endoplasmic r e t i c u l u m have a p h o s p h o r y l a t e d mannose r e s i d u e which i s r e c o g n i z e d by the G o l g i and then packed i n t o p r i m a r y lysosomes v i a c l a t h r i n - c o a t e d v e s i c l e s . In c o n t r a s t t o p r i m a r y lysosomes, 7 secondary lysosomes ( f r e q u e n t l y termed d i g e s t i v e v a c u o l e s , m u l t i v e s i c u l a r b o d i e s , a u t o p h a g i c v a c u o l e s , secondary v a c u o l e s , heterophagosomes) d i s p l a y a heterogenous morphology and a c q u i r e s u b s t r a t e s by e n d o c y t o s i s or autophagy ( S i l v e r s t e i n e_£ a l . 1977). In p l a n t c e l l s , plasma membrane-derived v e s i c l e s or v a c u o l e s (endosomes) are e i t h e r i n c o r p o r a t e d i n t o a p r e - e x i s t i n g v a c u o l e t o form the secondary v a c u o l e (Mahlberg 1972, Mahlberg e_t_ a l . 1970 , 1974, N i s h i z a w a and M o r i 1977) or are s urrounded by endoplasmic r e t i c u l u m (ER) or i t s d e r i v e d v e s i c l e s f o r m i n g heterophagosomes ( N i s h i z a w a and M o r i 1978). In e i t h e r c a s e , the v a c u o l e or the ER may c o n t a i n the l y t i c enzymes f o r d i g e s t i o n of endocytosed m a t e r i a l . Types of e n d o c y t o s i s : p h a g o c y t o s i s and p i n o c y t o s i s . P h a g o c y t o s i s of l a r g e p a r t i c l e s P h a g o c y t o s i s ("phago" (G) , eat) or» " c e l l e a t i n g " i s an e n d o c y t i c p r o c e s s i n v o l v e d i n the i n g e s t i o n of l a r g e p a r t i c l e s such as m i c r o o r g a n i s m s , l a t e x beads, e t c . I t i s r e g u l a t e d by the p h y s i c o - c h e m i c a l p r o p e r t i e s of the s u r f a c e of both the p a r t i c l e to be i n g e s t e d and the phagocyte. I t may r e q u i r e Ca2+ f o r p a r t i c l e a d h e s i o n and energy f o r i t s 8 subsequent d i g e s t i o n . A f t e r the c a p t u r e of the p a r t i c l e i n a " p h a g o c y t i c cup", the phagosome (endosome) forms and f u s e s w i t h lysosomes. These c e l l u l a r compartments c o n t a i n the a c i d h y d r o l a s e s which d i g e s t the i n t e r n a l i z e d m a t e r i a l . P h a g o c y t o s i s i s a temperature-dependent p r o c e s s mediated by m i c r o f i l a m e n t s and may be i n v o l v e d i n host defense (e.g. i n l e u c o c y t e s such as macrophages and polymorphonuclear l e u c o c y t e s ) or used as a f e e d i n g mechanism (e.g. i n P r o t o z o a such as Amoeba, C h l o r o h y d r a f Tetrahymena. Acantamoeba. as reviewed by Chambers and Thompson 1976, Chapman-Andresen 1977, S i l v e r s t e i n et a l . 1977, N i l s s o n 1979, M c N e i l 1981). S e v e r a l a l g a e , p a r t i c u l a r l y h e t e r o t r o p h i c (e.g. c o l o r l e s s c h l o r o p h y c e a n , e u g l e n o i d s , d i n o f l a g e l l a t e s ) or m i x o t r o p h i c (e.g. c h r y s o p h y t e s , d i n o f l a g e l l a t e s ) s p e c i e s , are a l s o known to phagocytose l a r g e p a r t i c l e s or b a c t e r i a , p o s s i b l y as a food s o u r c e . I n t r a c e l l u l a r b a c t e r i a have been r e p o r t e d i n many c e l l - w a l l e d p r o t i s t s , but i n most c a s e s , the mechanism of t h e i r e n t r y i n t o the host remains t o be e l u c i d a t e d ( T a y l o r 1982). P i n o c y t o s i s of f l u i d and s o l u t e s P i n o c y t o s i s , (G, P i v e l v ) was f i r s t d e s c r i b e d i n macrophages (Lewis 1931) and l a t e r i n amoebae (Mast and Doyle 1934) as an e n d o c y t i c p r o c e s s of a c t i v e " c e l l " d r i n k i n g " , v i z 9 uptake of f l u i d and s o l u t e s . Despite the s i g n i f i c a n c e of such a d i s c o v e r y , i t r e c e i v e d very l i t t l e a t t e n t i o n during the f o l l o w i n g twenty years. According to Lewis (1931, 1937) and H o l t z e r (1959) p i n o c y t o s i s i s an intense but dis c o n t i n u o u s process of f l u i d uptake through plasma membrane i n v a g i n a t i o n and v e s i c u l a t i o n . The reasons f o r i t s i n i t i a t i o n and c e s s a t i o n are not we l l understood, although c e r t a i n p r o t e i n s and i n o r g a n i c s a l t s are known to induce p i n o c y t o s i s (Holter 1959, MacRobbi 1969, Nassery and Jones 1976). Plasma membrane i n v a g i n a t i o n s or i n f o l d i n g s i n p l a n t and animal c e l l s are f r e q u e n t l y d e s c r i b e d i n the l i t e r a t u r e . G e n e r a l l y they are con s i d e r e d as a means to inc r e a s e s u r f a c e area and enhance the e f f i c i e n c y of a d s o r p t i o n and s e l e c t i v e p e r m e a b i l i t y (Palade 1956, Buvat and Lance 1957, Benedetti and B e r t o l i n 1963). Based on Palade's s t u d i e s , Bennett (1956) proposed the "membrane flow and v e s i c u l a t i o n " theory i n which he p o s t u l a t e d that p a r t i c l e s , molecules and ions bound to the c e l l s u r f a c e flow l a t e r a l l y i n t o membrane i n v a g i n a t i o n s , with subsequent formation of endosomes that become i n c o r p o r a t e d i n t o the cytoplasm. P i n o c y t o s i s i n animal c e l l s i s a well-known process and has been e x t e n s i v e l y d e s c r i b e d i n d i f f e r e n t types of c e l l s : l e u c o c y t e s (Chapman-Andresen 1957, Mesrobeanu .ei aj,. 1965 , B i b e r f e l d 1971), human p l a t e l e t s ( Z u c k e r - F r a n k l i n 1981), 10 macrophages (Cohn 1966, Steinman and Cohn 1972 , Steinman e_t a l . 1976, P i a s e k and Thyberg 1980, S t a h l and S c h l e s i n g e r 1980, Thyberg and S t e n s e t h 1981), f i b r o b l a s t s (Steinman _ t a l . 1974, Van Deurs and N i l a u s e n 1982), L c e l l s (Steinman e_ a l . 1976), v a r i o u s normal and m a l i g n a n t c e l l t y pes (Cormack and Ambrose 1962), and s e v e r a l P r o t o z o a (Schumaker 1958, F a v a r d and Carasso 1964, N o i r o t - T i m o t h e e 1966, Bowers and O l s z e w s k i 1972, Chapman-Andresen 1977, Prush 1980). P i n o c y t o s i s i n p l a n t c e l l s remains a s u b j e c t of c o n t r o v e r s y among p l a n t p h y s i o l o g i s t s . A c c o r d i n g t o Cram (1980), t h e r e i s i n s u f f i c i e n t e v i d e n c e i n f a v o u r of p i n o c y t o s i s i n p l a n t s and, from a p h y s i o l o g i c a l p o i n t of v i e w , he b e l i e v e s t h a t p l a n t systems do not s a t i s f y the b a s i c r e q u i r e m e n t s f o r t h i s p r o c e s s t o occur as i s known i n a n i m a l systems. He p o i n t e d out the l i m i t a t i o n s of s t a t i c morphology and s t r e s s e d the l a c k of time sequenced s t u d i e s and q u a n t i t a t i v e a n a l y s i s . He a l s o assumed t h a t p l a n t c e l l s do not have c e l l s u r f a c e c o a t , so t h a t the i n t a k e of e x t r a c e l l u l a r m a t e r i a l s can not be s e l e c t i v e . A d d i t i o n a l l y , he s t r e s s e d t h a t p i n o c y t o s i s i n p l a n t s would r e q u i r e a l a r g e amount of energy t o overcome the e l e v a t e d t u r g o r c r e a t e d by the water f l o w from such uptake. 11 Other s t u d i e s f a i l e d t o observe p r o t e i n i n t e r n a l i z a t i o n i n i n t a c t l i v i n g p l a n t c e l l s ( B r a d f u t e and McLaren 1964), or a t t r i b u t e d the e n t r y of f o r e i g n p a r t i c l e s t o plasma membrane i n j u r y (Burgess et a l . 1973 a , b ) . On the o t h e r hand, the o c c u r r e n c e of p i n o c y t o s i s has been suggested by the presence of p e r i p h e r a l or secondary v a c u o l e s and m u l t i v e s i c u l a r b o d i e s l o c a t e d c l o s e t o the plasma membrane i n r o o t s of h i g h e r p l a n t s such as A l l i u m cepa L. and Lup i n u s a l b u s L. (Mesquita 1970), T r q d e s c g n t j a v j y g i n i a n a (Mahlberg e_fc a_l. 1970), S c o r z o n e r a h i s p a n i c a (Coulomb 1973) and i n c u l t u r e d c e l l s of g l y c i n e ma_x Merr, H e l i a n t h u s anuus L. and P i o u s e l l i o t t i Engelm (Mahlberg et a l . 1974). F u r t h e r m o r e , i t has been shown t h a t these v a c u o l e s , whether empty or f i l l e d w i t h membranous i n c l u s i o n s , can be t r a n s l o c a t e d t o the v i c i n i t y of the c e n t r a l or p r i m a r y v a c u o l e by c y t o p l a s m i c s t r e a m i n g (Mahlberg 1972). S t u d i e s on r i c e r o o t s suggested t h a t p i n o c y t o t i c v e s i c l e s c o u l d be surrounded e i t h e r by endoplasmic r e t i c u l u m , or become i n c o r p o r a t e d i n t o p r e - e x i s t i n g v a c u o l e s ( N i s h i z a w a and M o r i 1978). In t h i s c o n n e c t i o n , i t i s i n t e r e s t i n g t o note t h a t the p r o c e s s of d i g e s t i o n w i t h i n heterophagosomes has been c o r r e l a t e d w i t h slow uptake r a t e s (Schumaker 1958) and h i g h l y s o s o m a l a c t i v i t y (Coulomb 1973) . 12 Markers used to test pinocytpsjq i n plant c e l l s P i n o c y t i c uptake of u r a n y l a c e t a t e (MW 388.15) i n r o o t cap c e l l s of o a t s , b a r l e y and c o r n (Wheeler and Hanchey 1971, Wheeler et _±. 1972, Robards and Robb 1972, 1974, Wheeler e i a l . 1972 , Wheeler and Baker 1973) and of lanthanum n i t r a t e and c o l l o i d a l lanthanum h y d r o x i d e i n endodermal c e l l s and the s t e e l of b a r l e y has been observed (Robards and Robb 1974). A d d i t i o n a l l y , a wide range of enzymes or o t h e r p r o t e i n s (MW 13,000-747,000), have been a p p l i e d t o the r o o t s of s e v e r a l h i g h e r p l a n t s and a l g a l c e l l s t o study the mechanism of uptake. These i n c l u d e : lysozyme (MW 15,000) f o r l i v i n g r o o t s of b a r l e y , tomato, o n i o n , wheat, c o r n , v e t c h and maize (Jensen and McLaren 1960, McLaren e_t a l . 1960 , B r a d f u t e and McLaren 1964, B r a d f u t e _t _1, 1965, U l r i c h £i _1. 1964, U l r i c h and McLaren 1965 , McLaren and B r a d f u t e 1966, Seear _±. a l . 1968, Drew and McLaren 1970, Drew £i aJL. 1970, Sung and McLaren 1974); c a l f thymus h i s t o n e (MW 20,000) f o r r o o t s of b a r l e y (Drew and McLaren 1970, Drew _t a l . 1970). r i b o n u c l e a s e (MW 13,000) f o r r o o t s of b a r l e y , pea and o n i o n (Brachet 1954, 1955, H o l z b a u e r and R i g l e r 1954, Bhide and B r a c h e t 1960, Jensen and McLaren 1960, McLaren e_t a l . 1960, Yeoman 1962, B r a d f u t e and McLaren 1964); hemoglobin (MW 65,000) f o r b a r l e y , tomato and r i c e r o o t s (Jensen and McLaren 1960, McLaren e_fc a l . 1960, U l r i c h and McLaren 1965 , N i s h i z a w a 13 and M o r i 1977, 1978); l a c t o g l o b u l i n (MW 41,500) and ovalbumin (MW 45,000) f o r b a r l e y , tomato and bean p l a n t s ( U l r i c h et a l . 1964, U l r i c h and McLaren 1965); f e r r i t i n f o r tomato, pea and bean (Barton 1964, Seear et a l . 1968, Mayo and C o c k i n g 1969a, Joachim and Robinson 1984); p o l y - L - l y s i n e (MW 74,000) f o r b a r l e y r o o t s (Drew and McLaren 1970, Drew et a l . 1970); and h o r s e r a d i s h p e r o x i d a s e (MW 40,000) f o r Euglena g r a c i l i s c e l l s ( K i v i c and Vesk 1974), (Table 2 ) . Among the p r o t e i n s l i s t e d above, hemoglobin was the o n l y one found t o be s u c c e s s f u l l y used as an o r g a n i c n i t r o g e n s ource f o r the growth of r i c e r o o t s ( N i s h i z a w a and M o r i 1977) . The p h y s i o l o g i c a l s i g n i f i c a n c e of the e n t r y of the ot h e r s u b s t a n c e s remained obscure because of t h e i r t o x i c e f f e c t s . Ion t r a n s p o r t through p i n o c y t o s i s was suggested to occur i n both p l a n t and a n i m a l c e l l s (Bennett 1956). I t was proposed t h a t i o n s are t r a n s p o r t e d by p i n o c y t i c v e s i c l e s and d i s c h a r g e d d i r e c t l y e i t h e r i n t o v a c u o l e s or i n t o the endoplasmic r e c t i c u l u m from which new v e s i c l e s may form and d i s i n t e g r a t e i n the c y t o p l a s m ( H a l l 1970, Baker and H a l l 1973) . Of p a r t i c u l a r i n t e r e s t i s the study of p i n o c y t o s i s i n p l a n t p r o t o p l a s t s . I t has been shown t h a t tomato f r u i t p r o t o p l a s t s c o n s t i t u t e a good system f o r i n v e s t i g a t i n g the 14 e n t r y of tobac c o mosaic v i r u s (Cocking and P o j n a r 1969), the uptake of p h o s p h o t u n g s t i c a c i d (Mayo and C o c k i n g 1969 b) and p o l y s t y r e n e l a t e x p a r t i c l e s , 0.1-0.3 /im i n d i a m e t e r , (Mayo and C o c k i n g 1969 a, W i l l i s o n e_t a_. 1971). Mechanisms of p i n Q c y t Q S J E j ; f j , q l d phase and a d s o r p t i v e p i n o c y t o s i s . Methods of s t u d y , F l u i d - p h a s e p i n o c y t o s i s F l u i d - p h a s e or b u l k - t r a n s p o r t i s a n o n - s p e c i f i c p r o c e s s of uptake i n which s m a l l a l i q u o t s of the e x t e r n a l f l u i d and s o l u t e s are i n t e r n a l i z e d by, plasma membrane i n v a g i n a t i o n , i n t o endosomes. F o l l o w i n g r e p e a t e d f u s i o n these v e s i c l e s become l a r g e r and are e v e n t u a l l y i n c o r p o r a t e d i n t o lysosomes as p a r t of the h e t e r o p h a g i c p r o c e s s (Steinman and Cohn 1972). High r a t e s of f l u i d - p h a s e p i n o c y t o s i s ( S i l v e r s t e i n e_t a l . 1977) have been r e p o r t e d i n macrophages, f i b r o b l a s t s , L c e l l s or c e l l s p r i m a r i l y i n v o l v e d i n s e c r e t i o n (e.g. e x o c r i n e or e n d o c r i n e p a n c r e a t i c c e l l s ) . Such f i n d i n g s s u pport P a l a d e ' s p r e d i c t i o n (1956) of membrane r e c y c l i n g t o account f o r the i n t e n s e membrane t r a f f i c between the plasma membrane and the endosomes. Q u a n t i t a t i v e s t u d i e s showed t h a t the t o t a l s u r f a c e a r e a of c e l l s w i t h h i g h r a t e s of p i n o c y t o s i s remains c o n s t a n t ( S i l v e r s t e i n e_ a_. 1977, Besterman and Low 1983). 15 However, d_g novo s y n t h e s i s of membrane components i s too slow to ensure t h i s e q u i l i b r i u m ( S i l v e r s t e i n e i a l . 1977). A l l these c o n s i d e r a t i o n s l e d to the assumption that e n d o c y t o s i s and e x o c y t o s i s ( e x o c y t o s i s , i . e mode of r e l e a s i n g s e c r e t o r y or degradative products) are concomittant processes that must occur c o n t i n u o u s l y . Fa,ctopg reajalqting P±Docyio_gis P i n o c y t o s i s depends not only on the c e l l type and i t s p h y s i o l o g i c a l s t a t e but a l s o on other f a c t o r s such as temperature, and the nature as we l l as e l e c t r i c a l charge of the marker used. The i n t e r a c t i o n of calcium with s u r f a c e a s s o c i a t e d cytoplasmic c o n t r a c t i l e f i l a m e n t s has a l s o been suggested to r e g u l a t e p i n o c y t o s i s i n some protozoan c e l l s (Chapman-Andresen 1977, Herzog 1981, Besterman and Low 1983). Native f e r r i t i n , r a d i o l a b e l e d sucrose, i n u l i n , and p o l y v i n y l p y r r o l i d o n e have been used as markers for f l u i d - p h a s e p i n o c y t o s i s (Besterman and Low 1983). Although n a t i v e peroxidase (HRP) has been widely used as a f l u i d - p h a s e marker (Gonatas e i a l . 1980, Thyberg 1980, Thyberg and Stenseth 1981), according to Straus (1981, 1983) t h i s 16 g l y c o p r o t e i n has s p e c i f i c a f f i n i t i e s f o r mannosyl r e s i d u e s . The i n t e r n a l i z a t i o n of HEP p r i m a r i l y by f l u i d - p h a s e and t o a l e s s e r degree by a d s o r p t i v e p i n o c y t o s i s has been p r e v i o u s l y suggested (Goud _t a l . 1981, O l i v e r 1982). The use of HRP as a marker has s e v e r a l advantages: i t i s n o n - t o x i c , i n e x p e n s i v e , easy t o assay and f o l l o w i n p u l s e - c h a s e s t u d i e s . However, i t s h o u l d be p o i n t e d out t h a t HRP becomes i n a c t i v a t e d when the pH f a l l s below 4, which may c o n s t i t u t e a l i m i t a t i o n f o r l o n g p e r i o d s of i n c u b a t i o n . A d s o r p t i v e p i n o c y t o s i s i s the uptake of exogenous m o l e c u l e s bound t o the e x t e r n a l s u r f a c e of the plasma membrane by e i t h e r i ) n o n - s p e c i f i c i o n i c i n t e r a c t i o n or i i ) by s p e c i f i c r e c e p t o r s , v i z . " r e c e p t o r - m e d i a t e d p i n o c y t o s i s " . In n o n s p e c i f i c a d s o r p t i v e p i n o c y t o s i s , b i n d i n g o c c u r s between the l i g a n d and the n e g a t i v e s u r f a c e charges due t o s u r f a c e components such as s i a l i c a c i d , PO^, OH~,S04 g r o u p s , p h o s p h a t i d i c a c i d , s u l f u r and a s p a r a g i n e . These l i g a n d s then e n t e r the c e l l s by e i t h e r c o a t e d or uncoated v e s i c l e s and become accumulated i n the lysosomes and/or i n the t r a n s - c i s t e r n a e of the G o l g i (Thyberg 1980, Thyberg _± a j . 1980 , Van Deurs _t aJL. 1981, Takata _t _1. 1982). 17 In receptor-mediated p i n o c y t o s i s , the l i g a n d s (such as macr o g l o b u l i n , l e c t i n s , hormones, growth f a c t o r s ) bind s e l e c t i v e l y to s p e c i f i c r e c e p t o r s on the plasma membrane to form l i g a n d - r e c e p t o r (L-R) complexes. A f t e r l a t e r a l movement these complexes are c l u s t e r e d and then trapped i n coated p i t s (Willingham et a i . 1981, Steinman e_ a_. 1983). Coated p i t s are d i f f e r e n t i a t e d microdomains of the plasma membrane with a b r i s t l e - l i k e coat on the cy t o p l a s m i c s u r f a c e . T h i s coat d i s p l a y s a b a s k e t - l i k e s t r u c t u r e and i s composed of a hexagonal (and pentagonal) l a t t i c e . The b a s i c u n i t of t h i s l a t t i c e i s the " t r i s k e l i o n " (Ungewickwell and Branton 1982) that c o n s i s t s of c l a t h r i n (MW 180,000, Pearse 1980) and accessory p r o t e i n s (MW 100,000/ 50,000). C o n t r o v e r s i a l views have been p o s t u l a t e d as to whether the l i g a n d - r e c e p t o r complexes, trapped i n coated p i t s , are t r a n s f e r r e d i n t o smooth-surfaced v e s i c l e s or endosomes v i a c r y p t i c p i t s (Willingham and Pastan 1984, 1985, Pastan and Willingham 1981 a,b) or v i a coated v e s i c l e s (Pearse 1980, Wild 1980, Herzog 1981, Petersen and van Deurs 1983). In the "receptosome" model, the smooth s u r f a c e d endosomes, s o - c a l l e d "receptosomes" (Pastan and Willingham 1981a,b) are thought to form from t r a n s i e n t " c r y p t i c p i t s " 18 ( W i l l i n g h a m _£ a_l. 1981). I t has been suggested t h a t the c l a t h r i n l a t t i c e of these s u r f a c e domains has a s k e l e t a l f u n c t i o n and remains a s s o c i a t e d w i t h the plasma membrane, thus c o a t e d v e s i c l e s would not e x i s t ( W i l l i n g h a m and Pastan 1984). A f t e r the u n c o u p l i n g of l i g a n d - r e c e p t o r complexes, the r e c e p t o r s a re r e c y c l e d back t o the c e l l s u r f a c e (Steinman et a l . 1983) w h i l e the receptosomes move, by s a l t a t o r y m o t i o n , a l o n g m i c r o t u b u l e s (MT) and d e l i v e r the l i g a n d s i n t o the G o l g i - e n d o p l a s m i c r e t i c u l u m l y s o s o m a l system (GERL) and/or lysosomes (Goud £± a_l. 1981). A l t e r n a t i v e l y , i n the "coated v e s i c l e " model, the endosomes a r i s e from c o a t e d v e s i c l e s . Coated v e s i c l e s a l s o termed pinosomes are c h a r a c t e r i z e d by t h e i r " b r i s t l e " c o a t which can be q u i c k l y (15 - 60 sec) shed and r e t u r n e d t o the c e l l s u r f a c e . I t has been suggested t h a t the d i s s o c i a t i o n of l i g a n d - r e c e p t o r complexes i s due t o a pH s h i f t w i t h i n endosomes (Farquhar 1983, H e l e n i u s £± _1. 1983) or "CURL" v e s i c l e s ( i . e . compartments of u n c o u p l i n g of r e c e p t o r s and l i g a n d s , D a u t r y - V a r s a t and L o d i s h 1984). "CURL" v e s i c l e s a c t as p r e - l y s o s o m a l compartments, from which a t u b u l a r p o r t i o n detaches and c a r r i e s the r e c e p t o r s t o the c e l l s u r f a c e w h i l e the r e m a i n i n g v e s i c u l a r p a r t f u s e s w i t h lysosomes. In comparison w i t h f l u i d - p h a s e p i n o c y t o s i s , i t has been 19 suggested t h a t a d s o r p t i v e p i n o c y t o s i s i s more e f f i c i e n t i n removing s u b s t a n c e s from the e x t e r n a l medium. Indeed, h i g h e r uptake r a t e s have been r e p o r t e d f o r su r f a c e - b o u n d m o l e c u l e s (reviewed by S i l v e r s t e i n e_t a_. 1977, Steinman e_t a l . 1983). D e s p i t e these h i g h p i n o c y t i c r a t e s , s t e r o l o g i c a l a n a l y s e s have y i e l d e d s u b s t a n t i a l i n f o r m a t i o n on the " h o m e o s t a t i c s t a t e " of the c e l l . Markers f o r s t u d y i n g a d s o r p t i v e p i n o c y t o s i s C a t i o n i z e d f e r r i t i n (CF) has been used t o l a b e l n e g a t i v e l y charged c e l l s u r f a c e s and t o t r a c e membrane f l o w (Danon e_ a l . 1972). L e c t i n s c o n j u g a t e d t o FITC, Fhodamine, or c o l l o i d a l g o l d p a r t i c l e s have been employed to determine the s p e c i f i c i t y , d i s t r i b u t i o n , and v a r i a b i l i t y of c e l l s u r f a c e r e c e p t o r s and t o f o l l o w the mechanism of l e c t i n uptake . L e c t i n - c o l l o i d a l g o l d p a r t i c l e c o n j u g a t e s are easy t o assay and t o i d e n t i f y by TEM or X-ray m i c r o a n a l y s i s . However, i t s h o u l d be p o i n t e d out t h a t c o l l o i d a l g o l d p a r t i c l e s do not p r o v i d e a v e r y s t a b l e s o l u t i o n as they tend t o f l o c c u l a t e . Pu.SU. l e The d i n o f l a g e l l a t e p u s u l e i s a unique c e l l u l a r s t r u c t u r e o c c u r r i n g i n many marine and f r e s h - w a t e r s p e c i e s , whether 20 f r e e - l i v i n g or p a r a s i t i c . The u l t r a s t r u c t u r e of t h i s o r g a n e l l e was f i r s t d e s c r i b e d i n W o l o s z y n s k i a m i c r a by Leadbeater and Dodge (1966) and i t i s known t o be a s s o c i a t e d w i t h the base of each f l a g e l l u m . Based on morphology Dodge (1972) i d e n t i f i e d seven d i s t i n c t types of p u s u l e s , which he grouped i n two main c a t e g o r i e s : i ) v e s i c u l a r p u s u l e , such as t h a t i n flmphidinium c a r t e r a e and i i ) t u b u l a r or sack p u s u l e , as i n P r o r o c e n t r u m micans. D e s p i t e these m o r p h o l o g i c a l v a r i a t i o n s , the b a s i c s t r u c t u r e of the p u s u l e i s remarkably u n i f o r m . In a l l c a s e s , the p u s u l e i s formed by the i n v a g i n a t i o n of the plasma membrane and i s e n c l o s e d by one or two a d d i t i o n a l membrane(s). Whether v e s i c u l a r or t u b u l a r , each p u s u l e c o n s i s t s of a main chamber l e a d i n g t o the f l a g e l l a r r e g i o n by a c o n s t r i c t i o n or a s l e n d e r c a n a l . C o n t r o v e r s i a l hypotheses have been p o s t u l a t e d c o n c e r n i n g the b i o l o g i c a l r o l e of the p u s u l e system. The e a r l i e s t and most commonly a c c e p t e d n o t i o n of the p u s u l e ' s f u n c t i o n , p a r t i c u l a r l y i n marine d i n o f l a g e l l a t e s , i s t h a t of a r o l e i n m e t a b o l i t e e x c r e t i o n (Schu'tt 1895 , Cachon e_t a_l. 1970 , 1983, Dodge 1972). More s p e c i f i c a l l y , the e x c r e t i o n of m u c i l a g e was suggested f o r some s p e c i e s of P r o r o c e n t r u m ( L o e b l i c h __ a l . 1979). I n t e r e s t i n g l y , a s e c r e t o r y ( r a t h e r than e x c r e t o r y ) r o l e was suggested i n the case of Heterocapsa n i e i ( M o r r i l l and L o e b l i c h 1984), where the p u s u l e seemed t o be the source of d e v e l o p i n g amphiesmal membranes d u r i n g c e l l 21 d i v i s i o n . Other c y t o l o g i c a l s t u d i e s have suggested t h a t t h i s s t r u c t u r e might be analogous t o the c o n t r a c t i l e v a c u o l e of s e v e r a l p r o t i s t s , i n c l u d i n g chrysomonads (Aaronson and Behrens 1974), chloromonads (Heywood 1978), cryptomonads ( P a t t e r s o n 1980, P a t t e r s o n and Hausmann 1981), c i l i a t e s ( B a n n i s t e r and T a t c h e l l 1972) and amoebae (Prusch and Dunham 1970). The p u s u l e has a l s o been regarded as an o s m o r e g u l a t o r y system r e q u i r e d t o m a i n t a i n the i n t r a c e l l u l a r i o n i c b a l a n c e p a r t i c u l a r l y i n f r e s h water s p e c i e s (Dodge 1971, 1972, 1973, Cachon et a l . 1970, 1983, S a r j e a n t 1974, Tappan 1980). F u r t h e r m o r e , N o r r i s (1966) suggested t h a t i t may f u n c t i o n as a f l o t a t i o n a p p a r a t u s i n c o n j u n c t i o n w i t h the v a c u o l a r system. I n t e r e s t i n g l y , one of the e a r l i e s t h y p o t h e s i s suggested p u s u l a r involvement i n the uptake of exogenous food p a r t i c l e s ( K o f o i d 1909; K o f o i d and Swezy 1921). S i n c e a l l these p r o p o s a l s were p r i m a r i l y based on the m o r p h o l o g i c a l appearance of the p u s u l e w i t h l i t t l e e x p e r i m e n t a l e v i d e n c e , a s e t of e x p e r i m e n t s , u s i n g v a r i o u s p h y s i o l o g i c a l parameters and c y t o l o g i c a l markers, were conducted t o i n v e s t i g a t e the p o s s i b l e f u n c t i o n (s) of the p u s u l e i n the b i o l o g y of these p r o t i s t s . 22 O b j e c t i v e and methodology used i n t h i s s tudy In view of the g r e a t p o t e n t i a l s i g n i f i c a n c e of the c e l l s u r f a c e and p i n o c y t o s i s i n the p h y s i o l o g y of p l a n t a l g a l c e l l s , i t i s proposed i n t h i s s t u d y : 1. to examine i n d e t a i l the morphology and c h e m i c a l n a t u r e of the c e l l s u r f a c e of t h r e e marine p h y t o f l a g e l l a t e s , a c h l o r o p h y t e ( D u n g l i e l l a , t e r t i o l e c t a ) and two d i n o f l a g e l l a t e s (Amphidiniuro c a r t e r a e and P r o r o c e n t r u m micans) 2. to i n v e s t i g a t e the mechanism of macromolecule i n t e r n a l i z a t i o n i n these a l g a l c e l l s u s i n g m o l e c u l a r markers, such as f l u o r o c h r o m e s , a l c i a n b l u e -ruthenium red ( A l c B - R R ) , h o r s e r a d i s h p e r o x i d a s e (HRP), c a t i o n i z e d f e r r i t i n ( C F ) , l e c t i n s and l e c t i n - c o l l o i d a l g o l d c o n j u g a t e s . A l c i a n B l u e i s a c a t i o n i c copper p h t h a l o c y a n i n e dye used t o l o c a l i z e s u l f a t e d and n o n - s u l f a t e d a c i d p o l y s a c c h a r i d e s of the c e l l s u r f a c e (Parker and D i b o l l 1966, Ramus 1977). Ruthenium Red i s an ammoniated ruthenium o x y c h l o r i d e ( c a . 1.13 nm d i a m e t e r ) used t o l o c a l i z e a c i d m u c o p o l y s a c c h a r i d e s and t o examine p i n o c y t i c uptake ( S z u b i n s k a and L u f t 1971, Benedeezky and Smith 1972, Blanquet 1976 a,b, D a v i e s and Kuczera 1981) . HRP i s a heme-containing g l y c o p r o t e i n enzyme, a p p r o x i m a t e l y 5 nm i n d i a m e t e r , ( K e i l i n and H a r t r e e 1951, Ugarova and Lebedeva 1979) f r e q u e n t l y used as a t o o l t o 23 i n v e s t i g a t e e n d ocytosis i n p l a n t and animal c e l l s (Graham and Karnovsky 1966, Straus 1969, Steinman and Cohn 1972, K i v i c and Vesk 1974 , Steinman e_t a l . 1976, Gonatas e_ aJL. 1980 , Harper e_ __. 1980 , Piasek and Thyberg 1980 , Goud e_t a l . 1981, Thyberg and Stenseth 1981, Kadota and Kadota 1982). CF i s a p o l y c a t i o n i c l i g a n d used to detect negative charges of the c e l l s u r f a c e and to study the mechanism of uptake i n d i f f e r e n t types of c e l l s (Danon e_fc aJL. 1972 , Souza et a l . 1977, Joachim and Pobinson 1984). L e c t i n s (L. l e g e r e = to choose) or phytohemaglutinins, are p r o t e i n s used to determine the s p e c i f i c i t y , d i s t r i b u t i o n and v a r i a b i l i t y of c e l l s u r f a c e r e c e p t o r s and to promote c e l l a g g l u t i n a t i o n (Bernhard and Avrameas 1971, Sharon and L i s 1972, L i s and Sharon 1973, Hughes 1976, Kahane and T u l l y 1976, Wang __ _ L . 1976, Sharon 1977, C a l v e r t e_ a_l. 1978, Rabat 1978, L a r k i n 1978, Araujo e_ a_t. 1980 , Bourguignon and Pozek 1980, Carbonetto and Argon 1980, De F e l i c i and S i r a c u s a 1980, Warton and Honiberg 1980, L i s and Sharon 1981, S p i c e r e_t a__. 1981, Watanabe e_t a_L. 1981, Vannini e_t _L 1981). 2 4 T a b l e 1. A l g a l s t r a i n s A l g a C u l t u r e S t r a i n / c l o n e I s o l a t o r L o c a l o f c o l l e c t i o n * d e s i g n a t i o n i s o l a t i o n CHLOPOPHYTA D u n a l i e l l a t e r t i o l e c t a Woods H o l e Dun ? ? B u t c h e r PYFRHOPHYTA A m p h i d i n i u m c a r t e r a e Woods H o l e Amphi 1 P. G u i l l a r d F a l m o u t h H u l b u r t G r e a t Pound, Mass P r o r o c e n t r u m m i c a n s N.E.P.C.C. D33 P. J o w e t t Mayne Bay, E h r e n b e r g B.C. * O b t a i n e d f r o m : Woods H o l e . O c e a n o g r a p h i c I n s t i t u t i o n a t Woods H o l e ; U.£.1.C.C., N o r t h e a s t P a c i f i c C u l t u r e C o l l e c t i o n B r i t i s h C o l u m b i a , B.C., c o u r t e s y o f D r . F. J . P. T a y l o r . T a b l e 2. M a r k e r s used t o t e s t p i n o c y t o s i s i n p l a n t c e l l s 25 Marker M o l . w e i g h t P l a n t D i a m e t e r (pm) T e s t e d R e f e r e n c e s F i b o n u c l e a s e 13,000 B a r l e y , O n i o n Pea B r a c h e t 1954, 1955; H o l z b a u e r and R i g l e r 1954; B h i d e and B r a c h e t 1960; J e n s e n and McLaren 1960;McLaren a t a l . 1960; Yeoman 1962; B r a d f u t e and McLaren 1964 Lysozyme 15 ,000 B a r l e y , Corn M a i z e , O n i o n V e t c h Tomato, Wheat J e n s e n and McLaren 1960; McLaren a t a l . 1960; B r a d f u t e and McLaren 1964; B r a d f u t e a t a l . 1965 ; u l r i c h a t a l . 1 9 6 4 ; u l r i c h and McLaren 1965; McLaren and B r a d f u t e 1966; Seear a t a l . 1968; Drew and McLaren 1970; Drew a t a l . 1970; Sung and McLaren 1974 C a l f thymus h i s t o n e H o r s e r a d i s h P e r o x i d a s e 20,000 40,000 B a r l e y E u g l e n o i d Drew and McLaren 1970; Drew a t a l . 1970 K i v i c and Vesk 1974 fi-Lactoglobulin 41,500 B a r l e y , Bean U l r i c h a t a l . 1964; u l r i c h and McLaren Tomato 1965 Ov a l b u m i n 45 ,000 Tomato U l r i c h and McLaren 1965 He m o g l o b i n 65,000 B a r l e y , P i c e Tomato J e n s e n and McLaren 1960; McLaren a t a l . 1960; U l r i c h and McLaren 1 9 6 5 ; N i s h i z a w a and M o r i 1977, 1978 P o l y - L - L y s i n e 74,000 B a r l e y Drew and McLaren 1970; Drew a t a l . 1970 F e r r i t i n 545,000 Bean, Pea Tomato B a r t o n 1964; Seear a t a l . 1968; Mayo and C o c k i n g 1969a; J o a c h i m and R o b i n s o n 1984 U r a n y l A c e t a t e 388.15 B a r l e y , Corn Oat Wheeler and Hanchey 1971;Wheeler a t a l . 1972; Robards and Rob 1972, 1974; Wheeler and Baker 1973 1973 Lanthanum N i t r a t e or C o l . L. H y d r o x i d e B a r l e y Robards and Robb 1974 P o l y s t y r e n e 0.1-0.3 L a t e x P a r t i c l e s Tomato f r u i t p r o t o p l a s t Mayo and C o c k i n g 1969a; W i l l i s o n a t a l . 1971 Tobacco M o s a i c 40,000,000 V i r u s Tomato f r u i t p r o t o p l a s t C o c k i n g and P o j n a r 1969 P h o s p h o t u n g s t i c A c i d Tomato f r u i t p r o t o p l a s t Mayo and C o c k i n g 1969b 26 MATERIALS AND METHODS Cu l t u r e c o n d i t i o n s Three marine p h y t o p l a n k t e r s : D u n a l i e l l a t e r t i o l e c t a , Amphidinium c a r t e r a e and Prorocentrum micans were chosen from two a l g a l d i v i s i o n s (Table 1 ) . These s p e c i e s were maintained p h o t o a u t o t r o p h i c a l l y , as u n i a l g a l or axenic batch c u l t u r e s , i n seawater medium of composition shown i n Table 3. C e l l growth was monitored at 18°C under continuous c o o l - w h i t e l i g h t of photon f l u x d e n s i t y of cj_. 25 jjmol m-2 s ~ l . Reagents The fluorochromes c a l c o f l u o r white ST (4,4'-bis [ 4 - a n i l i n o - 6 - b i s ( 2 - h y d r o x y e t h y l ) a m i n o - s - t r i a z i n - 2 - y l a m i n o ] - 2 , 2 ' - s t i l b e n e d i s u l f o n i c acid) and f l u o r o B o r a P were purchased from P o l y s c i e n c e s , Inc. (Warrington, PA, USA). The other fluorochromes (Table 4), or reagents (Table 4), the l e c t i n s (or a g g l u t i n i n s , wheat-germ, WGA , soybean, SBA , Concanavalin A, Con A) and a l l l e c t i n - s p e c i f i c sugars were obtained from Sigma Chemical Co. (St. L o u i s , MO, USA). Limulus polyphemus (Horseshoe crab) a g g l u t i n i n (LPA) was purchased from E.Y. Labs Inc. (San Mateo, CA, USA). 27 A. C e l l a g g l u t i n a t i o n e x p e r i m e n t s £>. t e r t i o l e c t a c e l l s were c o l l e c t e d by g e n t l e c e n t r i f u g a t i o n , washed w i t h 2% NaCl s o l u t i o n and suspended i n the same s a l t s o l u t i o n . C e l l s were then i n c u b a t e d w i t h each l e c t i n at c o n c e n t r a t i o n s of 100, 200 and 400 jug.ml-1. In each c a s e , c o n t r o l experiments were performed by i n c o r p o r a t i n g a l o n g w i t h the l e c t i n an e x c e s s of the s p e c i f i c sugar known to b i n d the l e c t i n and t h e r e b y i n h i b i t c e l l - a g g l u t i n a t i o n ( G o l d s t e i n 1981, Hughes 1976, L i s and Sharon 1981). A l l the a g g l u t i n a t i o n t e s t s were c a r r i e d out at room temperature (20-25°C) and r e p e a t e d a t l e a s t t h r e e times w i t h d i f f e r e n t c u l t u r e s of the a l g a . O b s e r v a t i o n s were made u s i n g phase c o n t r a s t o p t i c s i n a Z e i s s P h o t o m i c r o s c o p e . B. M o r p h o l o g i c a l a n a l y s i s of P. micans p u s u l e The e f f e c t s of some p h y s i c o c h e m i c a l f a c t o r s such as s a l i n i t y (17-30%<J , t e m p e r a t u r e (4-30OC), pH (6-8) and r e a g e n t s such as c o l c h i c i n e , (10 -4M) and c y t o c h a l a s i n B, (10~^M) on the c e l l s t r u c t u r e and p a r t i c u l a r l y on the morphology of P.. micans p u s u l e s were examined u s i n g phase 28 c o n t r a s t o p t i c s on a Z e i s s P h o t o n i i c r o s c o p e . Morphometric a n a l y s e s were c a r r i e d out u s i n g the computer morphometric program e l a b o r a t e d by Danai B i s a l p u t r a . F l u o r e s c e n c e m i c r o s c o p y A. C e l l s u r f a c e b i n d i n g and macromolecule i n t e r n a l i z a t i o n L i v i n g c e l l s , at d i f f e r e n t phases of growth, were i n c u b a t e d , at 20 - 25°C , w i t h v a r i o u s f l u o r o c h r o m e s (Table 4) d i s s o l v e d i n f r e s h c u l t u r e medium. For each s p e c i e s , the f o l l o w i n g f l u o r o c h r o m e s and i n c u b a t i o n p e r i o d s of time were used: P.. t e r t i o l e c t a ; f l u o r e s c e i n wheat germ a g g l u t i n i n (FITC-WGA) , f o r 5, 10, 30, 60 min. _. c a r t e r a e : a c r i d i n e orange (AO) f o r 5 min, f l u o r e s c e i n - l e c t i n s , such as c o n c a n a v a l i n A, wheat germ and soybean a g g l u t i n i n (FITC-Con A, WGA, SBA) f o r 30 min or h o r s e r a d i s h p e r o x i d a s e (HRP) f o r 10, 30, 60, min. £. micans: c a l c o f l u o r w h i t e or congo r e d f o r 2 min, a c r i d i n e orange (AO) f o r 5 min or f l u o r e s c e i n - C o n A (FITC-CON A) f o r l h . In the case of l e c t i n s , c o n t r o l experiments were c a r r i e d out i n c o r p o r a t i n g the s p e c i f i c sugar a l o n g w i t h the l e c t i n . A f t e r i n c u b a t i o n , c e l l s were r i n s e d t w i c e i n f r e s h c u l t u r e medium or i n b u f f e r TAPSO ( 3 - ( N - t r i s ( h y d r o x y m e t h y l ) 29 roe t h y l amino) -2-hydroxy propane s u l fori i c a c i d ) . Photographs were o b t a i n e d w i t h a L e i t z D i a l u x 20 ED m i c r o s c o p e equipped w i t h the Ploemopak 2.4 a r e f l e c t e d - l i g h t f l u o r e s c e n c e system. B. I d e n t i f i c a t i o n and l o c a l i z a t i o n of l i p i d b o d i e s To i l l u s t r a t e l i p i d b o d i e s the f l u o r o c h r o m e f l u o r o B o r a P ( 3 - P y r e n e s u l f a m i d o ) - p h e n y l b o r o n i c a c i d ) , 1 mg, was d i s s o l v e d i n 100 u l o f N,N-dimethylacetamide and d i l u t e d w i t h 10 ml of p h y s i o l o g i c a l l y i s o t o n i c c a r r i e r b u f f e r TAPSO. The s o l u t i o n was f i l t e r e d w i t h a M i l l i p o r e f i l t e r ( G a l l o p _t _1. 1982). L i v i n g c e l l s of c a r t e r a e at s t a t i o n a r y phase of growth were i n c u b a t e d a t room temperature f o r 5 min w i t h e i t h e r rhodamine B d i s s o l v e d i n f r e s h c u l t u r e medium or f l u o r o B o r a P d i s s o l v e d i n TAPSO b u f f e r (Table 4 ) . A f t e r i n c u b a t i o n , c e l l s were r i n s e d , c o l l e c t e d by c e n t r i f u g a t i o n and observed w i t h a L e i t z D i a l u x 20 ED m i c r o s c o p e , equipped w i t h the Ploemopak 2.4 r e f l e c t e d - l i g h t f l u o r e s c e n c e system. pr o c e d u r e C e l l samples ( D u n a l i e l l a or Amphidinium), c o n c e n t r a t e d on a support g o l d d i s c , were f r o z e n i n l i q u i d n i t r o g e n a f t e r a q u i c k immersion i n l i q u i d Freon 22. Frozen samples were 30 f r a c t u r e d at -100°C and e t c h e d f o r 30 to 60 seconds u s i n g a B a l z e r s BA 360 d e v i c e . A f t e r c l e a n i n g ( i n 70% s u l f u r i c a c i d , d i s t i l l e d w a t e r , sodium h y p o c h l o r i t e , d i s t i l l e d w a t e r ) , the r e p l i c a was mounted on a Formvar-coated g r i d and examined w i t h a Z e i s s EM 9S e l e c t r o n m i c r o s c o p e . Scanning e l e c t r o n m i c r o s c o p y A l g a l c e l l s (Amphidinium or Prorocentrum) were c o n c e n t r a t e d by the f i l t r a t i o n t e c h n i q u e of B i s a l p u t r a _ t a l . (1973) and p r e - f i x e d (5 min) w i t h g l u t a r a l d e h y d e - o s m i u m m i x t u r e . Samples were then f i x e d (30 min) w i t h g l u t a r a l d e h y d e (2% v/v) i n a phosphate s a l i n e (PBS, 0.17M, pH 7.4) and p o s t - f i x e d (30 min) w i t h 1% OsO^ b u f f e r e d w i t h PBS. Samples were d e h y d r a t e d i n a graded e t h a n o l s e r i e s , f o l l o w e d by c r i t i c a l p o i n t d r y i n g u s i n g l i q u i d C 0 2 and g o l d c o a t i n g (Hayat 1978). Specimens were then examined w i t h a Cambridge s t e r e o s c a n 250T s c a n n i n g e l e c t r o n m i c r o s c o p e . T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . A. C e l l S u r f a c e b i n d i n g 1. C a t i o n i c dye s t a i n i n g To p r e s e r v e the amphiesmal complex, A., c a r t e r a e or _. 31 micans c e l l s were c o n c e n t r a t e d by f i l t r a t i o n t e c h n i q u e ( B i s a l p u t r a e_£ a_l. 1973) and p r e - f i x e d at room temperature , f o r 5 min, w i t h a m i x t u r e of 2% g l u t a r a l d e h y d e and 1% 0 s 0 4 i n 0.1M sodium c a c o d y l a t e b u f f e r , pH 7.4. C e l l s were then f i x e d f o r 2 h at room temperature i n 2.5% g l u t a r a l d e h y d e ( i n Na c a c o d y l a t e b u f f e r , pH 7.4) c o n t a i n i n g a l c i a n b l u e (0.5% w/v). A f t e r r i n s i n g t w i c e i n the same b u f f e r , samples were p o s t - f i x e d f o r 2 h, i n 1% Os0 4 ( i n Na c a c o d y l a t e b u f f e r ) c o n t a i n i n g ruthenium r e d (0.05% w/v) ; (Table 5 ) . A f t e r r i n s i n g i n the same b u f f e r , c e l l s were dehydrated i n a graded e t h a n o l s e r i e s and embedded i n Epon 812 ( L u f t 1961). S e c t i o n s were cut w i t h a R e i c h e r t OMU-3 U l t r a m i c r o t o m e and p o s t - s t a i n e d (or not) w i t h s a t u r a t e d aqueous u r a n y l a c e t a t e and l e a d c i t r a t e ( Reynolds, 1963). O b s e r v a t i o n s were c a r r i e d out w i t h a Z e i s s EM 9S e l e c t r o n m i c r o s c o p e . 2. L e c t i n - p e r o x i d a s e l a b e l i n g 12. t e r t i o l e c t a or e a s t e r n s c e l l s were c o n c e n t r a t e d on a M i l l i p o r e f i l t e r (pore s i z e 0.45 /im) . Only A. c a r t e r a e c e l l s were p r e - f i x e d , f o r 5 min, w i t h 2% g l u t a r a l d e h y d e - 1 % ° s 04 m i x t u r e . Both samples were f i x e d , f o r 2 h at room te m p e r a t u r e , i n 2.5% v/v g l u t a r a l d e h y d e w i t h 0.1 M s a l i n e phosphate b u f f e r (PBS) pH 7.4. The m a t e r i a l was then t r e a t e d w i t h the f o l l o w i n g r e a g e n t s d i s s o l v e d i n PBS: ( i ) 0.5 mg.ml-1 l e c t i n (30 m i n ) ; ( i i ) 0.5 mg.ml-1 HRP (30 m i n ) ; ( i i i ) 32 DAB-H 202 s o l u t i o n (30 m i n ) , (T a b l e 5 ) . T r e a t e d specimens were p o s t - f i x e d i n 1% Os0 4 b u f f e r e d w i t h PBS and deh y d r a t e d i n a graded e t h a n o l s e r i e s . A f t e r embedding i n Epon 812, t h i n s e c t i o n s were examined w i t h a Z e i s s EM 9S e l e c t r o n m i c r o s c o p e . C o n t r o l samples were p r e p a r e d by i n c o r p o r a t i n g the s p e c i f i c sugar (0.5 M) a l o n g w i t h the l e c t i n . B. A c i d Phosphatase l o c a l i z a t i o n C y t o c h e m i c a l l o c a l i z a t i o n of a c i d phosphatase (AcPase) was c a r r i e d out as d e s c r i b e d by Poux (1967,1974), (Table 5 ) . £• t e r t i o l e c t a c o n c e n t r a t e d c e l l s were f i x e d i n 2.5% (v/v) g l u t a r a l d e h y d e b u f f e r e d w i t h 0.1M sodium c a c o d y l a t e , pH 7.4 at room t e m p e r a t u r e . C e l l s were i n c u b a t e d a t 32°C f o r 1 h i n a medium composed of Na- (3 - g l y c e r o p h o s p h a t e (10 mM), a c e t a t e b u f f e r (50 mM, pH'5.0), and l e a d n i t r a t e (3.5 mM) . A f t e r i n c u b a t i o n , c e l l s were r i n s e d i n 0.1 M Na c a c o d y l a t e b u f f e r (pH 7.4) c o n t a i n i n g 5% (w/v) s u c r o s e , and post f i x e d i n 1% OsO^ u s i n g the same b u f f e r . P a r a l l e l c o n t r o l e x periments were performed w i t h the f i x e d c e l l s where the s u b s t r a t e was o m i t t e d from the i n c u b a t i o n medium. 33 C. C y t o c h e m i c a l l o c a l i z a t i o n of v a r i o u s m o l e c u l a r markers 1 . H o r s e r a d i s h p e r o x i d a s e £. t e r t i o l e c t a , A., c a r t e r a e or £. micans l i v i n g c e l l s were i n c u b a t e d w i t h h o r s e r a d i s h p e r o x i d a s e , HRP (0.5 mg.ml-1 f o r 5, 10, 30 or 60 min) at 25oc, c o n c e n t r a t e d by f i l t r a t i o n and f i x e d f o r 2 h w i t h 2.5% (v/v) g l u t a r a l d e h y d e i n s a l i n e phosphate (0.1M) b u f f e r , (PBS) pH 7.4. C e l l s were then t r e a t e d f o r 30 min w i t h 3 , 3 ' - d i a m i n o b e n z i d i n e (DAB, 0.05% w/v) s o l u t i o n c o n t a i n i n g H 2 o 2 (0.01% v / v ) . The m a t e r i a l was p o s t - f i x e d f o r 2 h i n 1% Os0 4 b u f f e r w i t h PBS and de h y d r a t e d i n a graded e t h a n o l s e r i e s (Table 5 ) . A f t e r embedding i n Epon 812, u l t r a - t h i n s e c t i o n s were examined w i t h a Z e i s s EM 9S m i c r o s c o p e . C o n t r o l experiments were c a r r i e d out by o m i t t i n g e i t h e r the HRP i n c u b a t i o n or the DAB-H 202 tre a t m e n t (based on Graham and Karnovsky method, 1966). 2. C a t i o n i z e d f e r r i t i n A f t e r i n c u b a t i o n w i t h c a t i o n i z e d f e r r i t i n , CF (1 mg.ml-1 f o r 10, 30, 60 min a t 250c, £. t e r t i o l e c t a or £. micans l i v i n g c e l l s were c o n c e n t r a t e d by f i l t r a t i o n and p r e - f i x e d f o r 5 min, i n a m i x t u r e of 2% g l u t a r a l d e h y d e and 1% Os0 4 w i t h 0.1 M s a l i n e phosphate b u f f e r (PBS), pH 7.4 (Table 5 ) . The specimens were then f i x e d i n 2.5% (v/v) g l u t a r a l d e h y d e (PBS), f o r 2 h at room temperature and r i n s e d t w i c e i n the same 34 b u f f e r . Post f i x a t i o n was c a r r i e d out f o r 2 h i n 1% OSO4 b u f f e r e d w i t h PBS, pH 7.4 and dehy d r a t e d through a graded e t h a n o l s e r i e s . A f t e r embedding i n Epon 812, t h i n s e c t i o n s were cut w i t h a R e i c h e r t OMD-3 u l t r a m i c r o t o m e . O b s e r v a t i o n s were c a r r i e d out w i t h a Z e i s s EM 9S e l e c t r o n m i c r o s c o p e . C o n t r o l samples were p r e p a r e d by o m i t t i n g t he i n c u b a t i o n w i t h CF. 3. W G A - C o l l o i d a l Gold P r e p a r a t i o n of c o l l o i d a l g o l d p a r t i c l e s and CG-WGA c o n j u g a t e C o l l o i d a l g o l d p a r t i c l e s (CG) of a p p r o x i m a t e l y 20 nm were p r e p a r e d by the r e d u c t i o n of g o l d c h l o r i d e ( c h l o r o a u r i c a c i d , HAu 3 C I 4 ) w i t h sodium c i t r a t e (Frens 1973, R a i k h e l e_£ a l . 1984). T h i s s o l u t i o n was a d j u s t e d t o pH 7 w i t h 0.2 M K 2 c 0 3 f mixed w i t h wheat germ a g g l u t i n i n (WGA), (0.1 mg.rnl -!) as d e s c r i b e d by Roth and B i n d e r (1978) and s t a b i l i z e d w i t h 1% p o l y e t h y l e n e g l y c o l (mol. wt. 20,000 d a l t o n s ) . To pre p a r e the working s o l u t i o n the g o l d - c o n t a i n i n g sediment was re c o v e r e d by u l t r a c e n t r i f u g a t i o n and resuspended i n 5 ml of c u l t u r e medium c o n t a i n i n g p o l y e t h y l e n e g l y c o l . P r o c e d u r e A. c a r t e r a e or P.. micans l i v i n g c e l l s were i n c u b a t e d w i t h WGA-CG, f o r 30 min, 1, 2 h at 250C. C e l l s were c o n c e n t r a t e d by f i l t r a t i o n , p r e - f i x e d f o r 5 min i n 35 g l u t a r a l d e h y d e - O s 0 4 , and then f i x e d f o r 2 h i n 2.5% (v/v) g l u t a r a l d e h y d e w i t h 0.1M sodium c a c o d y l a t e b u f f e r , pH 7.4. A f t e r r i n s i n g , samples were p o s t - f i x e d f o r 2 h i n 1% Os0 4 u s i n g the same b u f f e r (Table 5 ) . A f t e r d e h y d r a t i o n i n a graded e t h a n o l s e r i e s , specimens were embedded i n Epon 812. C o n t r o l e x p e r i m e n t s were c a r r i e d out by i n c u b a t i n g w i t h w h e a t - g e r m - c o l l o i d a l g o l d a l o n g w i t h the s p e c i f i c sugar, N - a c e t y l g l u c o s a m i n e (0.5M)'. 36 T a b l e 3. C o m p o s i t i o n of the c u l t u r e medium used i n t h i s study KNO-: NaH 2P0 4 *H 20 Na 2 s i 0 3 «9H 2 o 250 mg (2500 jumole) 3 34.5 mg (250 jumole) 3 84.0 mg (300 jumole) 3 V i t a m i n s : * Tniamine-HCl B i o t i n B 12 500 ;jg (1 .48 umole) 1 jjg (0.0041 jumole) 2 ug (0.00134 jumole) T r a c e - m e t a l i o n s ( c h e l a t e d ) 3 Na 2.EDTA.2H 2 o F e C l 3 . 6 H 2 o MnS0 4-4H 2 o ZnS0 4.7H 20 Na 2Mo04 '2B 2 0 CUS0 4.5H 2 o CoS0 4-7H 2 o 8 .1 mg (21 .8 jumole) 2 .7 mg (10.0 jjmole) 1 .125 mg (5.0 jumole) 0 .575 mg (2.0 jumole) 0 .243 mg (1.0 umole) 0 .025 mg (0.1 jumole) U .014 mg (0.05 jumole) B u f f e r : T r i s . H C l 41.3 mM, pH 6.8-6.9b b e f o r e a u t o c l a v i n g 200 ml T r i s ) (1 g or 8.3 mmol Sea water, P a c i f i c Ocean, s a l i n i t y 33%o To 1 l i t e r *Omitted f o r D. t e r t i o l e c t a . apH of s t o c k s o l u t i o n s was a d j u s t e d t o i n c o r p o r a t i o n w i t h the r e s t of the medium. DThe pH of the medium i s 7.6-8.0 a f t e r a u t o c l a v i n g . (From" A n t i a and Cheng, 1 9 / 5 ) . 7.6-7.8 b e f o r e 37 T a b l e 4. F l u o r o c h r o m e s u s e d a n d t h e i r k n o w n s p e c i f i c i t i e s N a m e C o n c e n t r a t i o n ( m g «L ~ 1 ) A f f i n i t y R e f e r e n c e s A c r i d i n e o r a n g e 50 C a l c o f l u o r w h i t e S T 10 C o n g o r e d 50 R h o d a m i n e B 0.5 F l u o r o B o r a P 100 F I T C 50 F I T C - H R P 100 F I T C - W G A 100 F I T C - S B A 100 F I T C - C o n A 100 a c i d m u c o p o l y s a c c h a r i d e s S a u n d e r s 1964 T i m a r e_fc a _ l . 1979 | J-1,3 a n d (3-1,4 g l u c a n s H a i g l e r e_ t al. 1980 ft-1,3 g l u c a n s W o o d 1980 l i p o , h y d r o p h y l i c a r e a s G a l l o p si. a l . 1982 D - m a n n o s e D - G l c N A c s i a l i c a c i d D - G a l N A c D - g a l a c t o s e D - m a n n o s e D - g l u c o s e S t r a u s 1983 G o l d s t e i n 1981 L i s a n d S h a r o n 1981 Table 5. Various TEM schedules used Marker*** Incubation P r e - f i x a t i o n F i x a t i o n L e c t i n DAB/H 20 2 AcPase P o s t - f i x a t i o n time GA/Os 2.5% GA HRP t e s t medium** 1* ° s°4 (min) (h) (min) (h) (min) (min) (h) (h) CF 10,30, 1 5 2 - _ - 2 WGA-CG 30, 1,2 5 2 - - - 2 HRP 5,10,30,1 - 2 - 30 - 2 L e c t i n - 5 2 30 30 - 2 AB-RR - 5 2* - - - 2* AcPase - - 2 - - 1 2 * A l c i a n blue (0.5% w/v) or ruthenium red (0.05% w/v) added to the above f i x a t i v e s o l u t i o n . ** Composed of Na- Ii-glycerophosphate (10 mM), acetate b u f f e r (50 mM, pH 5.0) and l e a d n i t r a t e (3.5 mM), according to Poux 1974. ***££. c a t i o n i z e d f e r r i t i n ; WGA-CG. wheat germ a g g l u t i n i n - c o l l o i d a l g o l d ; HJLE. Horseradish peroxidase; AB-RR. A l c i a n blue-ruthenium red; AcPase. a c i d phosphatase. 39 RESULTS I . D u n a l i e l l a t e r t i o l e c t a : A. C e l l s t r u c t u r e The u l t r a s t r u c t u r e of D. t e r t i o l e c t a i s , i n g e n e r a l , s i m i l a r t o t h a t p r e v i o u s l y d e s c r i b e d by Hoshaw and Maluf (1981). The l a r g e cup-shaped c h l o r o p l a s t c l o s e l y apposed to the plasma membrane, o c c u p i e s most of the b a s a l p a r t of the• c e l l ( F i g . 1 ) . The a p i c a l c y t o p l a s m i c r e g i o n , below the f l a g e l l a , c o n t a i n s G o l g i a p p a r a t u s , endoplasmic r e t i c u l u m , m i t o c h o n d r i a and the n u c l e u s surrounded by numerous v a c u o l e s (or secondary l y s o s o m e s ) . A d d i t i o n a l l y , a g l y c o c o n j u g a t e coat i s a s s o c i a t e d w i t h the plasma membrane of t h i s f l a g e l l a t e ( O l i v e i r a et a l . 1980 , K l u t et aJL. 1983). B. Nature of the c e l l s u r f a c e . C e l l s u r f a c e r e l a t e d a c t i v i t i e s L i s M E i c l O s c o E y . L e c t i n - i n d u c e d a g g l u t i n a t i o n The t e s t - r e s u l t s i n d i c a t e d t h a t L i m u l u s polyphemus a g g l u t i n i n (LPA.) and wheat germ a g g l u t i n i n (WGA) were the most e f f e c t i v e a g g l u t i n a t i n g agents (Table 6 ) , and, i n both 40 c a s e s , a g g l u t i n a t i o n was d e c r e a s e d by s i m u l t a n e o u s i n t e r a c t i o n w i t h the s p e c i f i c s a c c h a r i d e ( F i g s . 2-5). Soybean a g g l u t i n i n (SBA) caused weak a g g l u t i n a t i o n a t the lowest c o n c e n t r a t i o n , which was enhanced by d o u b l i n g the c o n c e n t r a t i o n and i n h i b i t e d by the use of the s p e c i f i c sugar (Table 6 ) . C o n c a n a v a l i n A (Con A) caused r e l a t i v e l y weaker a g g l u t i n a t i o n at the lower c o n c e n t r a t i o n s , and t h i s was i n h i b i t e d by i t s s p e c i f i c s u g a r . In terms of the molar c o n c e n t r a t i o n s used, the observed ord e r of l e c t i n e f f e c t i v e n e s s i n c a u s i n g D u n a l i e l l a a g g l u t i n a t i o n was: LPA >y WGA > SBA > Con A. Z i u o r ^ s c e j i c ^ micxoscorr^ a. C e l l s u r f a c e b i n d i n g and l e c t i n i n t e r n a l i z a t i o n E xperiments w i t h f l u o r e s c e i n l a b e l e d wheat-germ a g g l u t i n i n (FITC-WGA), a p p l i e d to l i v i n g D u n a l i e l l a c e l l s , at 25°C, showed c e l l s u r f a c e random b i n d i n g ( F i g . 6 ) , f o l l o w e d by c a p p i n g ( F i g . 7) and p r o t e i n i n t e r n a l i z a t i o n through e n d o c y t i c v e s i c l e s ( F i g s . 8, 1 0 ) . A f t e r 15 min of i n c u b a t i o n , FITC-WGA.-loaded v e s i c l e s were observed i n the a n t e r i o r r e g i o n of the c y t o p l a s m ( F i g . 8 ) . For i n c u b a t i o n p e r i o d s e x t e n d i n g t o 1 h, v a c u o l e s b e a r i n g the l i g a n d were mai n l y c o n c e n t r a t e d i n the p e r i n u c l e a r r e g i o n ( F i g s . 9-11). I t was observed t h a t such uptake was temperature-dependent, thereby s u g g e s t i n g a 41 s p e c i f i c mechanism of i n t e r n a l i z a t i o n . Some v a r i a t i o n s i n the r a t e of l e c t i n uptake were observed among c e l l s of the same or d i f f e r e n t c u l t u r e s . D u r i n g these o b s e r v a t i o n s , attempts were made to i n s u r e t h a t the t r e a t e d c e l l s were i n t a c t and m o t i l e w i t h no s i g n s of c e l l damage. As a c o n t r o l , the e f f e c t of FITC-WGA was i n h i b i t e d w i t h the s p e c i f i c sugar N - a c e t y l g l u c o s a m i n e . b. HRP i n t e r n a l i z a t i o n L i v i n g D u n a l i e l l a c e l l s i n c u b a t e d w i t h f l u o r e s c e i n l a b e l e d h o r s e r a d i s h p e r o x i d a s e (FITC-HRP), at 25°C f o r 30 min, showed a c c u m u l a t i o n of t h i s p r o t e i n w i t h i n v a c u o l e s of the p e r i n u c l e a r r e g i o n ( F i g . 1 9 ) . T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y a. C e l l s u r f a c e b i n d i n g . E v i d e n c e f o r l e c t i n i n t e r n a l i z a t i o n C e l l s u r f a c e b i n d i n g r e s u l t s from t h r e e l e c t i n s WGA, LPA and Con A i n d i c a t e d the h e t e r o g e n e i t y of the sugar r e s i d u e s f o r m i n g the g l y c o c a l y x . E l e c t r o n m i c r o g r a p h s of u n s t a i n e d s e c t i o n s ( w i t h o u t U & Pb p o s t - s t a i n i n g ) showed t h a t WGA produced an i n t e n s e b i n d i n g p a t t e r n a l o n g D u n a l i e l l a c e l l s u r f a c e i n c l u d i n g the s u r f a c e of the f l a g e l l a ( F i g s . 12-14). The b i n d i n g was d i m i n i s h e d by the l e c t i n - s p e c i f i c sugar N - a c e t y l g l u c o s a m i n e ( F i g . 1 8 ) . For LPA, the b i n d i n g p a t t e r n 42 on the c e l l s u r f a c e of t h i s f l a g e l l a t e was almost e q u a l l y i n t e n s e ( F i g . 1 5 ) . As f o r c o n t r o l e x p e r i m e n t s , t h i s b i n d i n g d e c r e a s e d u s i n g s i a l i c a c i d as a c o m p e t i t i v e i n h i b i t o r . Plasma membrane i n v a g i n a t i o n s , i n the a p i c a l c e l l u l a r r e g i o n , were f r e q u e n t l y seen t o c o n t a i n c l u s t e r s of su r f a c e - b o u n d LPA s u g g e s t i n g i n t e r n a l i z a t i o n ( F i g . 1 6 ) . In comparison t o WGA and LPA, Con A produced a moderate s u r f a c e l a b e l i n g ( F i g . 1 7 ) . P r o f i l e s of s u r f a c e - c o n n e c t e d s t r u c t u r e s c o n t a i n i n g e l e c t r o n dense m a t e r i a l , p o s s i b l y Con A, were a l s o seen. ( F i g . 1 7 ) . The s p e c i f i c i t y of these Con A e f f e c t s was c o n f i r m e d by t h e i r i n h i b i t i o n when the specimens were s u b j e c t e d t o a p r e v i o u s treatment w i t h c< - m e t h y l - D - g l u c o p y r a n o s i d e . b. Macromolecule i n t e r n a l i z a t i o n u s i n g two m o l e c u l a r markers In view of s u g g e s t i o n s of l e c t i n i n t e r n a l i z a t i o n d e s c r i b e d above, f u r t h e r u l t r a s t r u c t u r a l s t u d i e s were undertaken t o examine t o what e x t e n t , and i n which mode macrom o l e c u l e s , such as h o r s e r a d i s h p e r o x i d a s e (HPP) and c a t i o n i z e d f e r r i t i n ( C F ) , e n t e r D u n a l i e l l a c e l l s . HRP Sh o r t - t e r m exposure t o h o r s e r a d i s h p e r o x i d a s e (5 min) r e v e a l e d a weak and patchy s u r f a c e l a b e l i n g w i t h c l e a r / 43 e v i d e n c e of plasma membrane i n v a g i n a t i o n ( F i g . 2 0 ) . In c e l l s exposed to HHP f o r 10 min, two types of p i n o c y t i c v e s i c l e s were observed underneath the plasma membrane: one c o n t a i n e d a p e r i p h e r a l zone of p e r o x i d a s e r e a c t i o n p r o d u c t whereas the oth e r was c o m p l e t e l y f i l l e d w i t h t h i s p r o d u c t ( F i g . 2 1 ) . Longer i n c u b a t i o n time (30-60 min) showed a p r o g r e s s i v e i n c r e a s e of HRP l a b e l e d s t r u c t u r e s . These i n c l u d e the l a s t two (or one) c i s t e r n a e of the t r a n s G o l g i compartment, as w e l l as the p r o x i m a l t u b u l e s and v e s i c l e s ( F i g s . 22,23). When the i n c u b a t i o n p e r i o d was extended t o 60 min, HFP-packed • s t r u c t u r e s o c c u p i e d an e x t e n s i v e area of the a p i c a l c e l l u l a r r e g i o n . As i l l u s t r a t e d i n F i g . 24, s m a l l v e s i c l e s appeared to undergo f u s i o n w i t h l a r g e p e r i n u c l e a r v a c u o l e s . S i m i l a r r e a c t i o n was not observed i n specimens p r o c e s s e d t o r u l e out the p o s s i b i l i t y of endogenous p e r o x i d a s e a c t i v i t y ( F i g . 25) or i n specimens exposed t o exogenous HRP i n the absence of DAB/H2C>2 treatment ( F i g . 2 6 ) . CF L i g h t m i c r oscopy and f r e e z e - e t c h i n g s t u d i e s on D u n a l i e l l a c e l l s r e v e a l e d the s t r u c t u r a l arrangement of the s u b c e l l u l a r o r g a n e l l e s ( F i g s . 27,28). TEM i n v e s t i g a t i o n s w i t h the n o n - s p e c i f i c marker, c a t i o n i z e d f e r r i t i n , unmasked the a n i o n i c p o l y s a c c h a r i d e coat 44 of t h i s f l a g e l l a t e . Such s u r f a c e coat i s c h a r a c t e r i z e d by e l e c t r o n - d e n s e patches a l t e r n a t i n g w i t h areas of l e s s dense b i n d i n g ( F i g s . 29,30). M i c r o t u b u l e s , presumably i n v o l v e d i n s t a b i l i z i n g s u r f a c e - r e c e p t o r m o b i l i t y , d i s p l a y an i r r e g u l a r arrangement underneath the plasma membrane. In a d d i t i o n , a c l o s e a s s o c i a t i o n between c y t o p l a s m i c MT and s m a l l v e s i c l e s was c l e a r l y v i s u a l i z e d ( F i g . 3 1 ) . As i l l u s t r a t e d i n F i g . 32, c e l l - t o c e l l - a d h e s i o n appears t o occur randomly a l o n g the c e l l s u r f a c e . In the e a r l y s t a g e s of i n c u b a t i o n w i t h CF (10-30 m i n ) , numerous plasma membrane i n v a g i n a t i o n s were obse r v e d i n the v i c i n i t y of the f l a g e l l a . Of p a r t i c u l a r i n t e r e s t was the presence of adsorbed f e r r i t i n w i t h i n s t r u c t u r e s known as "coated p i t s " ( F i g s . 33-38). These c o a t e d p i t s were c h a r a c t e r i z e d by t h e i r p e c u l i a r b r i s t l e - l i k e c l a t h r i n c o a t (arrow) on the c y t o p l a s m i c s i d e of the plasma membrane. S i m u l t a n e o u s l y w i t h these p i t s , numerous smooth v e s i c l e s or endosomes c o n s t i t u t e d a prominent f e a t u r e of the p e r i p h e r a l c y t o p l a s m ( F i g . 3 5 ) . A f t e r 1 h of i n c u b a t i o n i t appeared t h a t CF was f u r t h e r i n t e r n a l i z e d i n t o secondary lysosomes ( F i g s . 39,40). I t s h o u l d be noted t h a t l i t t l e marker remained bound to the i n n e r s u r f a c e of the v a c u o l a r membrane. T h i s may have r e s u l t e d from the u n c o u p l i n g of the r e c e p t o r s on account of the h y d r o l y t i c enzyme a c t i v i t y . F i g . 41 i l l u s t r a t e s the 45 h y p e r a c t i v i t y of the G o l g i a p p a r a t u s where dense c o a t e d v e s i c l e s can be v i s u a l i z e d budding o f f from the c i s t e r n a e , or s c a t t e r e d i n the c y t o p l a s m . A d d i t i o n a l l y , smooth s u r f a c e d v e s i c l e s w i t h or w i t h o u t e l e c t r o n dense c o n t e n t s appeared to o r i g i n a t e i n the t r a n s G o l g i r e g i o n ( F i g s . 42,43). Such v e s i c l e s , a r r a n g e d i n rows towards the c e l l s u r f a c e , are p o s s i b l y i n v o l v e d i n m e t a b o l i c s e c r e t i o n and/or membrane r e c y c l i n g . C. A,cjd PhospMfca_g.e. aocglisatjori A c i d phosphatase l o c a l i z a t i o n i n d i c a t e d the s i t e s of enzyme a c t i v i t y w i t h i n c y t o p l a s m i c v e s i c l e s c l o s e t o the c i s f a c e of the G o l g i , as w e l l as i n the t r a n s G o l g i compartment, w i t h i n p r i m a r y lysosomes ( F i g . 44) and w i t h i n l a r g e v a c u o l e s (secondary lysosomes) around the n u c l e u s ( F i g s . 45,46). C o n t r o l experiments showed t h a t such r e a c t i o n p r o d u c t was not observed i n specimens p r o c e s s e d w i t h o u t the a c i d phosphatase s u b s t r a t e ( F i g . 4 7 ) . 46 I I . ^ m r j h i ^ l i i i u j i i c a r t e r a e A. . C e l l s t r u c t u r e The marine d i n o f l a g e l l a t e A., c a r t e r a e ( G y m n o d i n i a l e s ) , i s c h a r a c t e r i z e d by a s m a l l epicone s e p a r a t e d from the hypocone by the g i r d l e , ( F i g s . 48-51). The r i b b o n - l i k e t r a n s v e r s e f l a g e l l u m d i s p l a y s a s t r i a t e d s t r a n d (arrow F i g . 50) f i t t i n g t h i s g i r d l e , w h i l e the l o n g i t u d i n a l f l a g e l l u m p r o t r u d e s from the s u l c u s which i s o r i e n t a t e d p e r p e n d i c u l a r l y t o the g i r d l e . Among o t h e r f e a t u r e s of t h i s f l a g e l l a t e , a t t e n t i o n i s drawn t o the s i n g l e p e r i p h e r a l c h l o r o p l a s t w i t h a p y r e n o i d t r a v e r s i n g the c e l l and one " v e s i c u l a r " p u s u l e at the base of each f l a g e l l u m . The s t r u c t u r e of the n u c l e u s , m i t o c h o n d r i a , G o l g i complex, and t r i c h o c y s t s i s g e n e r a l l y s i m i l a r t o t h a t d e s c r i b e d f o r o t h e r d i n o f l a g e l l a t e s (Dodge and C r a w f o r d 1968). B. C e l l c o v e r i n g morphology and morphometry M o r p h o l o g i c a l s t u d i e s on the amphiesmal v e s i c l e s of A.. c a r t e r a e c e l l s , at s t a t i o n a r y phase of growth, were c a r r i e d out u s i n g s c a n n i n g e l e c t r o n m i c r o s c o p y (SEM), t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y (TEM) and f r e e z e f r a c t u r e t e c h n i q u e s . R e s u l t s from SEM p r o v i d e d a t h r e e - d i m e n s i o n a l image of the whole c e l l and y i e l d e d v a l u a b l e i n f o r m a t i o n on the s p a t i a l 47 arrangement and d i s t r i b u t i o n of the amphiesmal v e s i c l e s ( F i g s . 48,51). TEM r e s o l v e d the f i n e s t r u c t u r e of the plasma membrane and the membrane of the amphiesmal v e s i c l e s (up t o 0.2 nm), ( F i g s . 52,53). The f r e e z e f r a c t u r e t e c h n i q u e p r o v i d e d v a l u a b l e i n f o r m a t i o n on the morphology of the amphiesmal v e s i c l e s . These u n i t s have a p o l y g o n a l appearance w i t h 4, 5, 6, or 7 s i d e s ( F i g s . 54,55). Hexagons were predominant w h i l e q u a d r a n g l e s were r a r e . I t was a l s o noted t h a t these amphiesmal v e s i c l e s were f r e q u e n t l y e q u i l a t e r a l and t r a v e r s e d by one t o t h r e e t r i c h o c y s t p o r e s . Morphometric a n a l y s e s were conducted t o determine the s u r f a c e area of the p o l y g o n a l amphiesmal u n i t s (Table 7) . R e l a t i v e l y u n i f o r m r e s u l t s were o b t a i n e d from m a t e r i a l p r e p a r e d by the t h r e e t e c h n i q u e s mentioned above. In SEM m a t e r i a l , the s t a n d a r d d e v i a t i o n was s i g n i f i c a n t l y l a r g e r than t h a t of TEM or f r e e z e - e t c h e d samples. The h i s t o g r a m ( F i g . 56) f o r the s u r f a c e area of these v e s i c l e s showed a normal d i s t r i b u t i o n w i t h a predominant s u r f a c e area r a n g i n g from 1.1 to 1.7 pm2. The unduly low e s t i m a t e s o b t a i n e d between 1.2-1.3 c o u l d be due t o a sampling e r r o r . Apart from the medium s i z e v e s i c l e s , two o t h e r s i z e s were a p p a r e n t , i . e . , the s m a l l (<1.1 jum2) and the l a r g e (>1.7 Jjm2) . 48 C. C e l l s u r f a c e r e l a t e d s t r u c t u r e s . L i p i d b o d i e s and t r i c h o c y s t s F l u o r e s c e n c e and TEM s t u d i e s were c a r r i e d out to i d e n t i f y and c h a r a c t e r i z e dense rounded b o d i e s l o c a t e d beneath the amphiesma. The f l u o r o g e n i c r e a c t i o n from rhodamine B and f l u o r o B o r a P a p p l i e d t o l i v i n g Amphidinium c e l l s , s uggested the l i p o i d n a t u r e of these b o d i e s ( F i g s . 57-60). L i p i d d i s t r i b u t i o n and abundance appear t o v a r y from c e l l t o c e l l , p o s s i b l y r e f l e c t i n g d i f f e r e n t p h y s i o l o g i c a l s t a t e s . Both s a t u r a t e d and u n s a t u r a t e d l i p i d s became a prominent f e a t u r e of aged Amphidinium c e l l s ( F i g s . 61,62). As i l l u s t r a t e d i n these f i g u r e s , the presumably s a t u r a t e d l i p i d s a re l a r g e r and d i s p l a y a more i r r e g u l a r appearance. F u r t h e r TEM s t u d i e s showed t h a t t r i c h o c y s t p r i m o r d i a a re l o c a t e d i n the G o l g i - e n d o p l a s m i c r e t i c u l u m r e g i o n . They a r e c h a r a c t e r i z e d by a s i n g l e l i m i t i n g membrane and a f i n e g r a n u l a r c o n t e n t w i t h or w i t h o u t a c r y s t a l - l i k e i n c l u s i o n ( F i g . 6 3 ) . When mature, these o r g a n e l l e s e x h i b i t two c o m p l e t e l y d i f f e r e n t i a t e d p a r t s : the "neck" and the " c r y s t a l l i n e body". C r y s t a l - l i k e rods were f r e q u e n t l y found w i t h i n s i n g l e - membrane bound v a c u o l e s surrounded by EF (autophagosomes), ( F i g . 6 4 ) . 49 D. Nature of the c e l l s u r f a c e . C e l l s u r f a c e - r e l a t e d a c t i v i t i e s F l u o r e s c e n c e m i c r o s c o p y C e l l s u r f a c e b i n d i n g u s i n g v a r i o u s f l u o r o c h r o m e s Amphidinium c e l l s ( c a . 18 u^m l e n g t h ) s t a i n e d w i t h a c r i d i n e orange r e v e a l e d not o n l y the condensed chromosomes but a l s o the presence of an a c i d i c m u c o p o l y s a c c h a r i d e l a y e r on the o u t e r s u r f a c e of the plasma membrane ( F i g . 6 5 ) . The s p e c i f i c i t y of t h i s f l u o r o c h r o m e f o r a c i d i c m u c o p o l y s a c c h a r i d e or g l y c o s a m i n o g l y c a n s has been p r e v i o u s l y demonstrated on the s u r f a c e of animal c e l l s (Saunders 1964, Timar e_£ a l . 1979). L e c t i n b i n d i n g r e s u l t s , o b t a i n e d from the i n c u b a t i o n of l i v i n g c e l l s w i t h v a r i o u s F I T C - l e c t i n c o n j u g a t e s (WGA, SBA, Con A ) , suggested a r a t h e r heterogeneous and random d i s t r i b u t i o n of the r e c e p t o r sugar r e s i d u e s on the c e l l s u r f a c e of t h i s f l a g e l l a t e ( F i g s . 66-68). In f a c t , a f t e r a s h o r t p e r i o d of i n c u b a t i o n , f l u o r e s c e n t a r e a s were f r e q u e n t l y observed i n s i d e the c e l l s and, u n l i k e the s u r f a c e - b o u n d l e c t i n - , these i n t r a c e l l u l a r b r i g h t a r e a s p e r s i s t e d a f t e r subsequent t r e a t m e n t w i t h the l e c t i n - s p e c i f i c s u g a r . These o b s e r v a t i o n s suggested l e c t i n i n t e r n a l i z a t i o n presumably v i a s p e c i f i c r e c e p t o r s . The b i n d i n g observed w i t h F I T C - l e c t i n c o n j u g a t e s was found t o be 50 temperature i n s e n s i t i v e i n the range of 4 - 2 5 o c . The s p e c i f i c i t y of t h i s b i n d i n g was c o n f i r m e d by u s i n g the a p p r o p r i a t e sugar s i m u l t a n e o u s l y w i t h each l e c t i n (D-mannose, N - a c e t y l g l u c o s a m i n e and N - a c e t y l g a l a c t o s a m i n e f o r Con A, WGA, and SBA r e s p e c t i v e l y ) . In these c o n t r o l e x p e r i m e n t s , the f l u o r e s c e n c e i n t e n s i t y was reduced or v i r t u a l l y d i s a p p e a r e d . F u r t h e r m o r e , i n c u b a t i o n w i t h FITC alone d i d not produce s u r f a c e l a b e l i n g . T r a n s m i s s i o n e l e c t r o n m i c r o s c o p y C e l l s u r f a c e b i n d i n g u s i n g SBA or c a t i o n i c dyes The r e s u l t s from u l t r a s t r u c t u r a l o b s e r v a t i o n s i n d i c a t e d t h a t the treatment of the d i n o f l a g e l l a t e c e l l s w i t h the l e c t i n SBA produced an i n t e n s e b i n d i n g p a t t e r n a l o n g the c e l l s u r f a c e ( F i g s . 6 9 - 7 2 ) . T h i s l e c t i n b i n d i n g was i n h i b i t e d i n the presence of N - a c e t y l g a l a c t o s a m i n e . C y t o c h e m i c a l s t a i n i n g w i t h the c a t i o n i c dyes, a l c i a n b l u e and ruthenium red (Alc B - R R ) , c o n f i r m e d the presence of an a n i o n i c p o l y s a c c h a r i d e s u r f a c e coat c o n s i s t i n g of a dense l a y e r o v e r l y i n g the plasma membrane and a f i n e o u t e r f i l a m e n t o u s l a y e r (c_a. 15-70 nm t h i c k ) , ( F i g s . 73-76 ; 78-80) . The dense l a y e r appeared to be of un i f o r m t h i c k n e s s (40-60 nm) and was c h a r a c t e r i z e d by an a l t e r n a t i n g p a t t e r n of dense 51 and l i g h t s t a i n i n g a r e a s (see arrows i n F i g . 74) somewhat s i m i l a r t o the b i n d i n g p a t t e r n observed w i t h F I T C - l e c t i n c o n j u g a t e s . I n t e r e s t i n g l y , the t h i c k n e s s of the s u r f a c e coat seemed t o i n c r e a s e w i t h c e l l age, e_.cj. i n c e l l s of 4 months o l d c u l t u r e (presumably n u t r i e n t d e p l e t e d ) t h i s c e l l c o a t was found t o be 70-300 nm t h i c k ( F i g s . 81,82). Large dense g l o b u l a r b o d i e s ( a p p a r e n t l y mucocysts) connected to the c e l l s u r f a c e by a channel were f r e q u e n t l y observed underneath the amphiesmal v e s i c l e s . The c o n t e n t of these s t r u c t u r e s appeared t o be s i m i l a r i n n a t u r e t o t h a t found i n the m a t r i x of the c e l l s u r f a c e coat of t h i s f l a g e l l a t e ( F i g . 8 1 ) . The i n n e r s u r f a c e of the presumably "empty" amphiesma v e s i c l e showed d i s t i n c t p o s i t i v e r e a c t i o n f o r a c i d m u c o p o l y s a c c h a r i d e s ( F i g . 7 4 ) . Of p a r t i c u l a r i n t e r e s t was the presence of e n d o c y t i c v e s i c l e s , c o n t a i n i n g e l e c t r o n - d e n s e m a t e r i a l , which e v e n t u a l l y became i n c o r p o r a t e d i n t o the p e r i p h e r a l c y t o p l a s m i c v a c u o l e ( F i g . 76) . C e l l u l a r a d h e s i o n through the s u r f a c e coat was f r e q u e n t l y observed and appeared t o be g e n e r a l l y random a l o n g the c e l l s u r f a c e ( F i g . 7 7 ) . In c o n t r a s t t o the above r e s u l t s , the c o n t r o l samples r e v e a l e d the u n s t a i n e d s u r f a c e c o a t , where the t h r e e membranes c o n s t i t u t i n g the c e l l c o v e r i n g were c l e a r l y v i s u a l i z e d ( F i g . 7 7 ) . In a d d i t i o n , a l a y e r of i r r e g u l a r l y spaced m i c r o t u b u l e s was observed beneath the s o - c a l l e d amorphous l a y e r u n d e r l y i n g the amphiesmal v e s i c l e s ( F i g . 7 7 ) . T r a n s v e r s e s e c t i o n s 52 through the amphiesmal v e s i c l e s showed t h e i r p o l y g o n a l appearance and the o u t l i n e of v a r i o u s pores g e n e r a l l y about 60 nm i n diameter ( F i g . 7 8 ) . Mature s p i n d l e t r i c h o c y s t s , ready t o emerge from these p o r e s , were found t o be o r i e n t a t e d almost p e r p e n d i c u l a r l y t o the c e l l s u r f a c e ( F i g . 7 9 ) . Summing up a l l these o b s e r v a t i o n s , a diagrammatic s k e t c h i s i n c l u d e d t o i l l u s t r a t e the new i n f o r m a t i o n added to the then e x i s t i n g knowledge of A. c a r t e r a e c e l l s u r f a c e ( F i g . 83) . E. Macromolecule i n t e r n a l i z a t i o n u s i n g v a r i o u s m o l e c u l a r markers A f t e r examining the s t r u c t u r e and n a t u r e of Amphidinium c e l l s u r f a c e and i t s r e l a t e d s t r u c t u r e s , f u r t h e r s t u d i e s were conducted to i n v e s t i g a t e the mode and f a t e of macromolecule i n t e r n a l i z a t i o n . SEM and f l u o r e s c e n c e m i c r o s c o p y As r e v e a l e d i n the SEM m i c r o g r a p h , A. ca_£ie_r§a_e c e l l s have two f l a g e l l a r pores l i n k e d by the amphiesmal r i d g e . These pores l e a d t o the c a n a l of the t r a n s v e r s e or l o n g i t u d i n a l f l a g e l l u m ( F i g . 8 4 ) . FITC or FITC-HFP when a p p l i e d t o l i v i n g c e l l s were obser v e d t o e n t e r the f l a g e l l a r p o r e s . A f t e r a s h o r t p e r i o d 53 of i n c u b a t i o n (15 m i n ) , the f l u o r o c h r o m e s would appear i n patch e s a l o n g the c e l l s u r f a c e and at the base of the f l a g e l l a r c a n a l ( F i g s . 85-87) . When the i n c u b a t i o n p e r i o d was extended to 60 min, most c e l l s e x h i b i t e d a p e c u l i a r p a t t e r n from the endocytosed f l u o r o c h r o m e . As i l l u s t r a t e d i n F i g . 88, these f l u o r e s c e n t v e s i c l e s were a r r a n g e d i n an a r c between the two f l a g e l l a r p o r e s . S i n c e the enzyme HRP e x h i b i t s a n a t u r a l brownish c o l o u r , i t i s p o s s i b l e , w i t h b r i g h t f i e l d i l l u m i n a t i o n , t o i d e n t i f y s t r u c t u r e s c o n t a i n i n g the enzyme and r e l a t e them w i t h p r o x i m a l o r g a n e l l e s . HRP uptake i n Amphidinium i s a temperature dependent p r o c e s s , so i t was d i m i n i s h e d a t 4°C and g r e a t l y i n c r e a s e d a t tempe r a t u r e s r a n g i n g from 20-25°C. T r g n s m i s s j o n e 1 e c t ron mjcroseopy 1. I n t e r n a l i z a t i o n of HEP TEM s t u d i e s , u s i n g the enzyme h o r s e r a d i s h p e r o x i d a s e , c o n f i r m e d , i n g e n e r a l , the r e s u l t s from f l u o r e s c e n c e m i c r o s c o p y ; the HRP-DAB r e a c t i o n product was i n i t i a l l y o b s erved w i t h i n concave are a s a l o n g the c e l l s u r f a c e and l a t e r on w i t h i n v a c u o l e s of the p e r i p h e r a l c y t o p l a s m . Numerous v e s i c l e s c o n t a i n i n g HRP were found between the two f l a g e l l a r c a n a l s and around the p y r e n o i d ( F i g s . 89-91). Some of these H R P - l a b e l e d v e s i c l e s appear to have been d e r i v e d 54 d i r e c t l y from the f l a g e l l a r c a n a l by membrane i n v a g i n a t i o n . A d d i t i o n a l l y , a c o n s i d e r a b l e amount of HRP r e a c t i o n p r o d u c t was found d e p o s i t e d i n the f l a g e l l a r c a n a l and chamber w i t h subsequent appearance w i t h i n the p u s u l e ( F i g . 9 2 ) . Secondary lysosomes or heterophagosomes c o n t a i n i n g s i m i l a r e l e c t r o n - d e n s e m a t e r i a l and p o r t i o n s of degraded o r g a n e l l e s were f r e q u e n t l y v i s u a l i z e d throughout the c y t o p l a s m ( F i g . 9 3 ) . A c l o s e s p a t i a l r e l a t i o n s h i p between these s t r u c t u r e s and the endoplasmic r e t i c u l u m was f r e q u e n t l y o b s e r v e d . 2. C e l l s u r f a c e b i n d i n g and I n t e r n a l i z a t i o n of WGA-CG F u r t h e r c y t o l o g i c a l s t u d i e s w i t h wheat-germ a g g l u t i n i n - c o l l o i d a l g o l d c o n j u g a t e c o n f i r m e d p r e v i o u s o b s e r v a t i o n s on the s p a t i a l d i s t r i b u t i o n of WGA r e c e p t o r s on Amphidinium c e l l s u r f a c e ( F i g s . 94-96) . I n p a r t i c u l a r , the s u r f a c e of the amphiesmal r i d g e was h e a v i l y s t a i n e d by t h i s type of c o n j u g a t e ( F i g . 9 4 ) . C l u s t e r s of l e c t i n - r e c e p t o r complexes were o f t e n v i s u a l i z e d c l o s e t o areas of a c t i v e plasma membrane i n v a g i n a t i o n . I t was a l s o observed t h a t l i g a n d i n t e r n a l i z a t i o n o c c u r r e d v i a membrane i n f o l d i n g s through the amphiesmal v e s i c l e s ( F i g s . 96-98). These i n v a g i n a t i o n s o c c u r r e d randomly a l o n g the c e l l s u r f a c e and were c l o s e l y a s s o c i a t e d w i t h m i c r o f i l a m e n t s (arrow F i g . 9 6 ) . A d d i t i o n a l l y , s m a l l v e s i c l e s b e a r i n g the l i g a n d (WGA-CG) were f r e q u e n t l y seen i n the lumen of the l a r g e p e r i p h e r a l v a c u o l e ( F i g s . 96,98). 55 I I I . P r o rocentrum micans A. C e l l s t Q c t g y e The s t r u c t u r e of P. micans ( P r o r o c e n t r a l e s ) has been p r e v i o u s l y d e s c r i b e d by Bursa (1959), Dodge and Bibby (1973), and Dodge (1975). T h i s marine d i n o f l a g e l l a t e (c_§. 40 urn l e n g t h ) has a wide, rounded, a p i c a l r e g i o n w i t h a narrow or p o i n t e d p o s t e r i o r end ( F i g s . 100-103). The c e l l w a l l c o n s i s t s of two l a r g e t h e c a l ( v a l v e s ) p l a t e s and v a r i o u s p l a t e l e t s . The "two" f l a g e l l a are d i s s i m i l a r i n s t r u c t u r e and i n s e r t e d a p i c a l l y w h i l e the "sack" p u s u l e s a r e l o c a l i z e d at t h e i r base. The m u l t i - l o b e d p e r i p h e r a l c h l o r o p l a s t (one under each v a l v e ) c o n t a i n s one t o two p y r e n o i d s . The n u c l e u s , G o l g i a p p a r a t u s , and m i t o c h o n d r i a are b a s i c a l l y s i m i l a r to those d e s c r i b e d f o r o t h e r d i n o f l a g e l l a t e s . B. N a t u r e _ o f the,, c e l l ,SJJ.r.face, comrjongiats F l u o r e s c e n c e m i c r o s c o p y a. C e l l s u r f a c e b i n d i n g and l e c t i n i n t e r n a l i z a t i o n The i n t e r a c t i o n of a c r i d i n e orange w i t h P. micans showed the condensed chromosomes of the U-shaped n u c l e u s which o c c u p i e s a l a r g e p o r t i o n of the c e l l . T h i s f l u o r o c h r o m e a l s o r e v e a l e d the presence of a c i d i c m u c o p o l y s a c c h a r i d e s i n the 56 s u r f a c e coat ( F i g . 1 0 0 ) . A d d i t i o n a l e v i d e n c e f o r the presence of a g l y c o c a l y x was o b t a i n e d from FITC-Con A s u r f a c e b i n d i n g . T h i s s u r f a c e coat extends to the a p i c a l s p i n e and i n t o the s u r f a c e p o r e s . S u r f a c e pores are numerous ( c a . 70- 100 per c e l l ) and d i s p l a y a r e g u l a r arrangement i n r a d i a l rows. Uptake of FITC-Con A o c c u r r e d m a i n l y through the s l e n d e r f l a g e l l a r c a n a l w i t h subsequent a c c u m u l a t i o n i n the c o r r e s p o n d i n g "sack " p u s u l e ( F i g . 101). T h i s 1 e c t i n - f i l l e d f l a g e l l a r c a n a l - p u s u l e system i s r e m i n i s c e n t of a "bag-pipe" i n which e x p a n s i o n and c o n t r a c t i o n may depend on the p a r t i c u l a t e s t r e n g t h of the s u r r o u n d i n g medium. C o l d t e m p e r a t u r e (ca.. 4°C) suppressed l e c t i n i n t e r n a l i z a t i o n and i n d u c e d Brownian motion i n l o c a l i z e d c y t o p l a s m i c a r e a s . b. T h e c a l p l a t e s Two main p o l y s a c c h a r i d e t h e c a l p l a t e s were v i s u a l i z e d by c a l c o f l u o r w h i t e or congo red s t a i n i n g . The a f f i n i t y of these f l u o r o c h r o m e s f o r (1-1,4 p o l y g l u c a n s such as c e l l u l o s e and c h i t i n has been p r e v i o u s l y demonstrated (Wood 1980, H a i g l e r et a l . 1980, Herth and Schnepf 1980 ) . P r o r o c e n t r u m t h e c a l p l a t e s v a r i e d i n t h i c k n e s s w i t h i n i n d i v i d u a l c e l l s or among c e l l s from c u l t u r e s at d i f f e r e n t s t a g e s of growth ( F i g s . 102,103). P a r t i c u l a r l y t h i n p l a t e s (ca.. 150 nm wide) were observed i n c e l l s at e x p o n e n t i a l growth phase ( F i g . 102) w h i l e markedly t h i c k p l a t e s (ca.,830 nm) were o f t e n observed 57 i n c e l l s at s t a t i o n a r y phase of growth ( F i g . 103). A set of r i d g e d s t r i a t i o n s was v i s u a l i z e d at the edges of the opposing v a l v e s ( l e f t and r i g h t ) while p l a t e l e t s were found i n the a p i c a l r e gion c l o s e to the f l a g e l l a r pore complex. T h i s complex i s l o c a t e d on the l e f t s i d e of the a p i c a l s p i n e . It was a l s o observed that c e l l d i v i s i o n occurs by " o b l i q u e " f i s s i o n i n which the cleavage furrow i s i n i t i a t e d at the p o s t e r i o r end. Transmission e l e c t r o n microscopy a. Nature of the c e l l s u r f a c e Cytochemical s t u d i e s , using a combination of a l c i a n blue and ruthenium red r e v e a l e d the inner l a y e r of the c a r b o h y d r a t e - r i c h coat a s s o c i a t e d with the plasma membrane ( F i g . 104). T h i s coat i s continuous with the f l a g e l l a r s u r f a c e and the spine and extends to the inner s u r f a c e of the "pores" ( F i g s . 105,107). S e c t i o n s through the a n t e r i o r region of the c e l l showed 6-8 p l a t e l e t s (some of which are i l l u s t r a t e d i n F i g . 109). Among these p l a t e l e t s , one has two w a l l p r o j e c t i o n s (double a p i c a l spine, F i g . 107) and another appears to possess at l e a s t one s p i n e . As shown i n these u l t r a s t r u c t u r a l s t u d i e s , the t h e c a l p l a t e s of P. micans have an amorphous appearance. 58 S t u d i e s w i t h c a t i o n i z e d f e r r i t i n c o n f i r m e d the presence of a w e l l developed a n i o n i c c e l l - s u r f a c e coat ( F i g . 1 0 6 ) . T h i s coat c o n s i s t e d of a c o n t i n u o u s l a y e r a d j a c e n t t o the plasma membrane and an e x t e r n a l p a t c h y , f r i n g e - l i k e l a y e r . B a c t e r i a - l i k e s t r u c t u r e s were o f t e n seen c l o s e l y a s s o c i a t e d w i t h the c e l l s u r f a c e of Prorocentrum or i n t e g r a t e d in. the p l a t e l e t s ( F i g s . 108,109). b. T r i c h o c y s t pores and macromolecule i n t e r n a l i z a t i o n The s p i n d l e t r i c h o c y s t s of Pro r o c e n t r um a p p a r e n t l y have the same b a s i c s t r u c t u r e as those of oth e r d i n o f l a g e l l a t e s i n c l u d i n g Amphidinium. They c o n s i s t of a "neck" r e g i o n and a " c r y s t a l l i n e body" both surrounded by numerous f i b r o u s s t r a n d s . R e s u l t s from t h i s study i n d i c a t e d t h a t f u l l y d e v eloped t r i c h o c y s t s l o c a t e d i n the n u c l e a r r e g i o n d i s p l a y a f r o n t membranous cap ( F i g s . 110,111). I t was a l s o observed t h a t some of these o r g a n e l l e s have m i g r a t e d towards the c e l l s u r f a c e and came t o a r e s t i n g s t a t e beneath t r i c h o c y s t " p o r e s " a r r a n g e d i n rows a l o n g the c e l l s u r f a c e . As r e v e a l e d by AlcB-RR or c a t i o n i z e d f e r r i t i n - t r e a t m e n t , some of these pores were cov e r e d by a mucous a c i d i c l a y e r ( F i g s . 112-115). B e s i d e the t y p i c a l p o r e s , d e f i n e d here as deep plasma membrane i n v a g i n a t i o n s u s u a l l y u n d e r l a i n by inward t h e c a l p r o j e c t i o n s ( F i g . 1 1 3 ) , SEM and f u r t h e r TEM s t u d i e s r e v e a l e d two a d d i t i o n a l types of pore c o n f i g u r a t i o n s ( F i g s . 116-119). 59 As summarized i n F i g . 120, s u r f a c e pores can be s i m p l e or e l e v a t e d i n a cone-shaped s t r u c t u r e or d i s p l a y a c r a t e r - l i k e appearance. I t s h o u l d be p o i n t e d out t h a t t h i s t h i r d type of pore c o n f i g u r a t i o n was f r e q u e n t l y observed when t r i c h o c y s t d i s c h a r g e was induced by s l i g h t o s m o t i c changes p r i o r t o g l u t a r a l d e h y d e f i x a t i o n . As i l l u s t r a t e d i n F i g . 116, some t r i c h o c y s t s remained a t t a c h e d t o the c r a t e r - l i k e p o r e s . S t u d i e s w i t h CF suggested t h a t t h i s macromolecule e n t e r e d P r o r o c e n t r u m c e l l s through presumably opened s u r f a c e p o r e s . T h i s marker was e i t h e r found i n the lumen of the cone-shaped s t r u c t u r e s ( F i g s . 121,123) or bound t o the n e g a t i v e l y charged groups of plasma membrane c o n s t i t u e n t s ( F i g . 1 2 2 ) . A comparison between F i g s . 118 and 122 c l e a r l y shows the b i n d i n g of CF t o the plasma membrane w i t h i n ( i ) or o u t s i d e (o) the pore. In c r o s s s e c t i o n , these CF c o n t a i n i n g s t r u c t u r e s were u s u a l l y e n c i r c l e d by the t h e c a l c e l l w a l l , were o u t s i d e the c y t o p l a s m i c membrane and o c c a s i o n a l l y found, out of the plane of c o n n e c t i o n w i t h the t h e c a . CF was a l s o observed t o e n t e r the c e l l s through the t h e c a l s u t u r e s (arrow, F i g . 124) w i t h subsequent a c c u m u l a t i o n w i t h i n v a c u o l e s of the p e r i p h e r a l c y t o p l a s m . Moreover, v a c u o l e s c o n t a i n i n g CF or HFP were found s c a t t e r e d i n the cy t o p l a s m ( F i g s . 125, 1 2 6 ) . 60 C. P u s u l e a. Morphology and morphometry of £. micans p u s u l e The e f f e c t of s e v e r a l p h y s i c o - c h e m i c a l f a c t o r s on P. micans showed t h a t the p u s u l e s i z e v a r i e s w i t h d i f f e r e n t l e v e l s of s a l i n i t y and t e m p e r a t u r e . As compared t o c o n t r o l ( F i g . 128) c o n d i t i o n s , low s a l i n i t y (17-18 ppt) or low temperature (4°C f o r 45 min) caused s i z e r e d u c t i o n ( F i g . 1 2 7 ) , w h i l e h i g h s a l i n i t y (28-34 p p t , u s i n g N a C l , C a C l 2 or M g C l 2 ) , or h i g h temperature (25-30°C) produced p u s u l e enlargement ( F i g . 129). I t was a l s o noted t h a t these s a l i n i t y - i n d u c e d s i z e changes o c c u r r e d a f t e r t r e a t i n g the c e l l s , f o r two hou r s , w i t h e i t h e r c o l c h i c i n e or c y t o c h a l a s i n B . A l t h o u g h no s i g n i f i c a n t s i z e r e d u c t i o n appeared t o occur w i t h any of these r e a g e n t s per se, the shape of the p u s u l e was s l i g h t l y deformed. On the o t h e r hand, pH and presence/absence of l i g h t had v i r t u a l l y no e f f e c t on the morphology of the p u s u l e . Morphometric a n a l y s i s of £. micans c e l l s and p u s u l e s were undertaken t o q u a n t i f y m o r p h o l o g i c a l changes i n d u c e d by h i g h s a l i n i t y l e v e l s . As shown i n Ta b l e 8, when the s a l i n i t y was r a i s e d from 24 to 30 p p t , the area of the p u s u l e was i n c r e a s e d by 100%. An e x t e n s i o n of the p u s u l e l e n g t h by 47% and w i d t h by 33% was c l e a r l y o b s e r v e d . In s p i t e of such 61 l a r g e changes of the pusule, the o v e r a l l s i z e of the c e l l i n c r e a s e d only s l i g h t l y . These data a l s o y i e l d e d i n f o r m a t i o n on the pusule s i z e as a f r a c t i o n (10-17%) of the c e l l s i z e . b. S t r u c t u r e of £. micans pusule. Macromolecule i n t e r n a l i z a t i o n The f i n e s t r u c t u r e of JP. micans pusular region r e v e a l e d an a p i c a l "pocket" l e a d i n g to the slender canal of the "sack" pusule ( F i g . 1 3 0 ) . A s e r i e s of v e s i c l e s p r o t r u d i n g from t h i s pocket appeared to c o n t a i n membranous m a t e r i a l (arrows F i g . 1 3 0 ) . The lumen of the pusule could vary from being e l e c t r o n t r ansparent to a f a i r l y packed c o n d i t i o n with g r a n u l a r , f i b r i l l a r , v e s i c u l a r , or b a c t e r i a - l i k e s t r u c t u r e s ( F i g s . 130-132). Vacuoles, mitochondria, endoplasmic r e t i c u l u m , G o l g i apparatus, coated v e s i c l e s were often observed i n the v i c i n i t y of the pusule ( F i g s . 131,133). A f t e r a short exposure of P. micans c e l l s to HEP (jc§. 10 min), the s t a i n i n g of the pusular membrane became prominent. With longer p e r i o d s of i n c u b a t i o n (ca.. 30 min), some peroxidase r e a c t i o n product was observed i n the lumen of the pusule ( F i g . 1 3 4 ) . Subsequently, HEP-labeled s t r u c t u r e s were observed w i t h i n e x t e n s i v e vacuolar c a n a l i c u l i (arrows, F i g s . 134,137). These c a n a l i c u l i protrude i n a l l d i r e c t i o n s and can be seen adjacent to the p e r i p h e r a l c y t o p l a s m i c v a c u o l e s . A d d i t i o n a l l y , HEP-containing v e s i c l e s were observed around 62 i the "sack" p u s u l e ( F i g . 1 3 6 ). As i l l u s t r a t e d i n F i g . 135, m i c r o t u b u l e s are a r r a n g e d i n a r e g u l a r p a t t e r n on the border of the the p u s u l a r c a n a l i c u l i . F u r t h e r c y t o l o g i c a l s t u d i e s w i t h e i t h e r f e r r i t i n or WGA-CG c o n j u g a t e showed t h a t these l i g a n d s were l o c a l i z e d w i t h i n the f l a g e l l a r c a n a l and the lumen of the p u s u l e ( F i g s . 138-141). F u r t h e r m o r e , these markers were v i s u a l i z e d i n e x t e n s i v e p u s u l a r r a m i f i c a t i o n s t r a v e r s i n g the c y t o p l a s m ( F i g . 140) and i n s m a l l v e s i c l e s around the p u s u l e (arrow F i g . 1 4 1 ) . c. S t r u c t u r e of A., c a r t e r a e p u s u l e . Macromolecule i n t e r n a l i z a t i o n . The u l t r a s t r u c t u r e of h. c a r t e r a e f l a g e l l a r c a n a l - p u s u l e system, i s p r e s e n t e d d i a g r a m m a t i c a l l y i n a model e l a b o r a t e d from s e q u e n t i a l s e c t i o n s ( F i g . 1 4 2 ) . The amphiesma (or c e l l c o v e r i n g ) extends i n t o the f l a g e l l a r c a n a l and appears to t e r m i n a t e i n the r e g i o n of a f i b r i l l a r " p r i m a r y c o l l a r " ( F i g s . 143-145) . Below t h i s c o l l a r the plasma membrane c o n t i n u e s and forms a l a r g e f l a g e l l a r chamber ( F i g s . 146-148) . T h i s chamber opens i n t o the p u s u l e which i s composed of numerous v e s i c u l a r u n i t s (40 a c c o r d i n g to Dodge 1968) . Each of these u n i t s has a c o n s t r i c t e d neck r i n g e d by a f i b r i l l a r "secondary c o l l a r " ( F i g . 1 4 6 ) . Double 63 c a t i o n i c - d y e (AlcB-FF) s t a i n i n g showed t h a t the f l a g e l l a r c a n a l i s c o a t e d by a n i o n i c p o l y s a c c h a r i d e m a t e r i a l s i m i l a r to t h a t of the c e l l s u r f a c e . A l t h o u g h m o s t l y c o n t r a c t e d ( p o s s i b l y due t o the f i x a t i o n p r o c e d u r e ) , p u s u l e s can d i s p l a y a h i g l y d i s t e n d e d form. T h e i r c o n t e n t s appeared t o be v a r i a b l e and c o n s i s t e d of g r a n u l a r , t u b u l a r or v e s i c u l a r m a t e r i a l . I n c u b a t i o n of l i v i n g c e l l s of A., c a r t e r a e w i t h HFP showed t h a t , w i t h i n 30 min of i n c u b a t i o n , t h i s p r o t e i n would e n t e r i n t o the f l a g e l l a r c a n a l , f l a g e l l a r chamber and s u b s e q u e n t l y i n t o the v e s i c u l a r u n i t s of the p u s u l e s of both the t r a n s v e r s e and l o n g i t u d i n a l f l a g e l l a ( F i g . 1 5 0 ) . S i m i l a r r e s u l t s were not observed i n the c o n t r o l experiments w i t h o u t exogenous HFP. S t u d i e s u s i n g W G A - c o l l o i d a l g o l d c o n j u g a t e showed t h a t , a f t e r 2h of i n c u b a t i o n , " g o l d " p a r t i c l e s were found i n the f l a g e l l a r c a n a l , the f l a g e l l a r chamber and w i t h i n the v e s i c u l a r u n i t s of both p u s u l e s (arrowheads, F i g s . 149,151). A s e r i e s of (8 to 11) m i c r o t u b u l e s were observed b o r d e r i n g the f l a g e l l a r chamber ( F i g . 1 5 1 ) . A c l o s e a s s o c i a t i o n between the p u s u l e s and m i t o c h o n d i a , endoplasmic r e t i c u l u m , G o l g i a p p a r a t u s , numerous c o a t e d v e s i c l e s can be c l e a r l y v i s u a l i z e d ( F i g s . 151-153) . 6 4 T a b l e 6. D u n a l i e l l a a g g l u t i n a t i o n 3 i n d u c e d w i t h v a r i o u s l e c t i n s L e c t i n s ^ L e c t i n 0 c o n c e n t r a t i o n s 0 100 200 (pg/raL) 400 L e c t i n (100 yug/mL) p l u s s u g a r d poo pg/mL) WGA ( 36,000) - ++ (2.8) +++ +++ - (D-GlcNAc) LPA (400,000) - ++ (0.25) +++ +++ ( s i a l i c a c i d ) SBA (120,000) - + (0.83) ++ ++ - (D-GalNAc) Con A (102, OOOj - + (0.98) + ++ - (4-D-ManMe) a A g g l u t i n a t i o n was a r b i t r a r i l y s c o r e d as s t r o n g ( + + + ) , medium (++), weak (+), or n e g a t i v e ( - ) . D T h e v a l u e s i n p a r e n t h e s e s d e n o t e l e c t i n known m o l e c u l a r w e i g h t s ( L i s and Snaron 1981) . c I n t h e c a s e o f 100 /jg/mL, each l e c t i n c o n c e n t r a t i o n i s a l s o shown i n te r m s o t m o l a r i t y (JJM) i n p a r e n t h e s e s . ^Tne l e c t i n - s p e c l f i c s u g a r s a r e d e n o t e d i n p a r e n t h e s e s by t h e f o l l o w i n g a D b r e v i a t i o n s : D-GlcNAc = N - a c e t y l - D - g l u c o s a m i n e ; D-GalNAc = N - a c e t y l - D - g a l a c t o s a m i n e ; o(-D-ManMe =«-methyl-D-mannoside. T a b l e 7. Morphometric a n a l y s i s f o r the s u r f a c e area of the amphiesmal v e s i c l e s of A. c a r t e r a e S t a t i s t i c a l parameters Number of Technique mean s t a n d a r d v a r i a n c e samples d e v i a t i o n 21 SEM 1 .50 0.68 0.46 31 f r e e z e - e t c h 1 .44 0.37 0.13 58 f r e e z e - e t c h 1 .46 0.30 0.09 4 TEM 1 .44 0.28 0.01 2 TEM 1 .44 _ — 66 T a D l e 8. S a l i n i t y e f f e c t on t h e s i z e o f t h e p u s u l e and c e l l o t £. m i c a n s S a l i n i t y ( P P t ) w i d t h {pm) P u s u l e l e n g t h (pm) a r e a ( p i 2 ) w i d t h (pm) C e l l P u s u l e / c e l l a r e a l e n g t h a r e a (pm2) 24 30* 12 (100) 15 (100) 121 (100) 16 (133) 22 (147) 242 (200) 33 (100) 52 (100) 1188 (100) 10 39 (100) 59 (113) 1391 (117) 17 Tne v a l u e o t each p a r a m e t e r shown w i t h o u t p a r e n t h e s e s r e p r e s e n t s t h e mean fro m measurements made on 30 random s a m p l e s . * V a l u e s i n p a r e n t h e s e s d e n o t e t h e p e r c e n t a g e o f i n c r e a s e d e t e r m i n e d i n r e l a t i o n t o t h a t o r 24 p a r t s p e r t h o u s a n d ( p p t ) . DISCUSSION 67 A. S t r u c t u r e of the c e l l s u r f a c e I t i s w e l l known t h a t the c h l o r o p h y t e D u n a l i e l l a c e l l l a c k s a r i g i d c e l l w a l l and i s surrounded by o n l y the plasma membrane, w h i l e the d i n o f l a g e l l a t e c e l l s (e.g. Amphidinium or Prorocentrum) a r e e n c l o s e d i n a complex c o v e r i n g c o n s i s t i n g of the plasma membrane and a l a y e r of f l a t t e n e d v e s i c l e s . S e v e r a l terms such as t h e c a , c e l l c o r t e x and amphiesma have been used t o denote t h i s d i n o f l a g e l l a t e c e l l c o v e r i n g . From t h e s e , I have chosen the term "amphiesma" which was o r i g i n a l l y c o i n e d by S c h u t t (1895) and r e c e n t l y r e i n t r o d u c e d by L o e b l i c h (1970). T h i s term appears t o be more p r e c i s e i n r e p r e s e n t i n g the o v e r a l l c e l l c o v e r i n g , i r r e s p e c t i v e of the presence (e.g. Pro r o c e n t r u m micans) or absence of t h e c a l p l a t e s (e.g. A m p h i d i n i u m c a y t e r a s ) . In some d i n o f l a g e l l a t e s (e.g. S c r i p s i e l l a t r o c h o i d e a . G o n y a u l d X p p l y e d r a ) a d d i t i o n a l f e a t u r e s such as the p e r i p h e r a l c y t o p l a s m i c membrane and the p e l l i c l e have a l s o been c o n s i d e r e d as p a r t of the amphiesmal complex ( L o e b l i c h 1970, M o r r i l l and L o e b l i c h , 1981,1983). Based on r e p o r t s t h a t the outermost l i m i t i n g membrane and the p e r i p h e r a l c y t o p l a s m i c membrane are both c o n t i n u o u s around the d i n o f l a g e l l a t e c e l l and t h a t the former can be l o s t as a r e s u l t of e c d y s i s , c o n t r o v e r s i a l views have been 68 e x p r e s s e d as t o which of these two membranes r e p r e s e n t s the plasma membrane ( K a l l e y and B i s a l p u t r a 1975, S t e i d i n g e r e_£ a l . 1978, Durr 1979 a,b, Spector and Triemer 1979, S t e i d i n g e r and Cox 1980, M o r r i l l and L o e b l i c h 1983, M o r r i l l 1984). Based on the n a t u r e of the s u r f a c e s t a i n i n g , the outermost l i m i t i n g membrane o v e r l y i n g the amphiesma v e s i c l e s of amphidinium and Pror o c e n t r u m appears t o r e p r e s e n t the plasma membrane. N e v e r t h e l e s s , i t s h o u l d be p o i n t e d out t h a t t h i s membrane i s v e r y v u l n e r a b l e t o the c o n v e n t i o n a l double f i x a t i o n f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y , and i s n o r m a l l y d i s r u p t e d by g l u t a r a l d e h y d e . T h i s membrane can, however, be p r e s e r v e d by a m i x t u r e of Os0 4 and g l u t a r a l d e h y d e . W i l c o x e i s i . (1982) made s i m i l a r o b s e r v a t i o n s w i t h another s p e c i e s of Amphidinium, w h i l e Dodge and Crawford (1968) used o n l y Os0 4 to p r e s e r v e t h i s membrane i n c a r t e r a e . Pi comparison between the c e l l c o v e r i n g of d i n o f l a g e l l a t e s w i t h t h a t of e u g l e n o i d s or cryptomonads showed t h a t i n a l l t h r e e groups, the plasma membrane i s o u t s i d e the s t r u c t u r a l components of the " c e l l w a l l " (when p r e s e n t ) . I n e u g l e n o i d s the plasma membrane i s u n d e r l a i n by the p e l l i c l e made of h e l i c a l l y a r r a n g e d s t r i p s ( L e e d a l e 1982), w h i l e i n cryptomonads i t i s s u p p o r t e d by p o l y g o n a l p e r i p l a s t p l a t e s ( S a n t o r e 1985). I n t e r e s t i n g l y , the c e l l c o v e r i n g of a l l t h r e e groups extends i n t o the f l a g e l l a r c a n a l . 69 Morphometric a n a l y s i s of m i c r o g r a p h s of A. c a r t e r a e c e l l s p r o c e s s e d by t h r e e d i f f e r e n t t e c h n i q u e s : SEM, TEM and f r e e z e f r a c t u r e , showed a r e l a t i v e l y u n i f o r m s t r u c t u r e and s u r f a c e a rea of the amphiesmal v e s i c l e s . In comparison t o TEM and f r e e z e - f r a c t u r e , SEM showed h i g h e r s t a n d a r d d e v i a t i o n . T h i s e l e v a t e d v a l u e c o u l d have a r i s e n from the degree of m o r p h o l o g i c a l v a r i a b i l i t y i n d u ced by specimen p r e p a r a t i o n . S i z e v a r i a b i l i t y was a l s o e x p e c t e d f o r t w o - d i m e n s i o n a l image produced from d i f f e r e n t s p a t i a l l e v e l s . As i l l u s t r a t e d i n the h i s t o g r a m ( F i g . 56) the i n t e r m e d i a t e s i z e v e s i c l e s (1.1 - 1 . 7 /jm2) were predominant w h i l e the l a r g e and p a r t i c u l a r l y the s m a l l v e s i c l e s showed lower f r e q u e n c y d i s t r i b u t i o n . I t i s p o s s i b l e t h a t s i z e v a r i a b i l i t y of these amphiesmal u n i t s may r e f l e c t d i f f e r e n t s t a g e s of development as r e l a t e d t o c e l l d i v i s i o n . L i t t l e i s known about the mechanism by which new amphiesmal v e s i c l e s a re formed d u r i n g c y t o k i n e s i s of a t h e c a t e d i n o f l a g e l l a t e s . The p r e s e n t study showed t h a t when c y t o k i n e s i s i s almost complete, each Amphidinium daughter c e l l has a s i m i l a r number of amphiesmal v e s i c l e s as the n o n - d i v i d i n g mature c e l l . Whether these new amphiesmal v e s i c l e s a r e formed by p a r t i t i o n of the p a r e n t a l u n i t s or are newly s y n t h e s i z e d i s not y e t c l e a r . However, i t i s i n t e r e s t i n g t o note t h a t , i n c e l l s of p o s t - e x p o n e n t i a l phase of growth the m a j o r i t y of the amphiesmal v e s i c l e s , appeared 70 to be hexagonal and o c c a s i o n a l l y h e p t a g o n a l or q u a d r a n g u l a r . Based on these o b s e r v a t i o n s , i t i s tempting t o suggest t h a t the p a r e n t a l hexagonal v e s i c l e s c o u l d e n l a r g e i n t o s e v e n - s i d e d p o l y g o n s which would d i v i d e g i v i n g r i s e t o s m a l l e r f o u r or f i v e - s i d e d u n i t s . A l t e r n a t i v e l y , based on the g e n e r a l concept of c e l l d i v i s i o n i n d i n o f l a g e l l a t e s , each daughter c e l l c o u l d r e t a i n h a l f of the p a r e n t a l amphiesmal v e s i c l e s ( w i t h 6 or 7 s i d e s ) and r e s y n t h e s i z e an eq u a l number of new v e s i c l e s ( w i t h 4 or 5 s i d e s ) . I t i s known t h a t d u r i n g c y t o k i n e s i s , t h e c a t e d i n o f l a g e l l a t e s can e i t h e r shed t h e i r p a r e n t a l t h e c a l p l a t e s and the out e r membranes (e.g. Z o o x a n t h e l l a m i c r o a d r i a t i c a , L o e b l i c h and S h e r l e y 1979) or r e t a i n h a l f of the p a r e n t a l amphiesma (e.g. Prorocentrum, i n i c a n s , S i l v a 1959, C e r a t i u m , t r i p o s , Wetherbee 1975 a,b and He t e r o c a p s a n i e i , M o r r i l l and L o e b l i c h 1984). In £. t r i p o s , which l a c k s a p e l l i c l e , h a l f of the c e l l c o v e r i n g , i n p a r t i c u l a r the plasma membrane and the t h e c a , i s r e g e n e r a t e d by i n t e r c a l a r y growth t h a t o c c u r s beneath the s u t u r e s . Such growth was shown t o occur by c o a l e s c e n c e of c y t o p l a s m i c v e s i c l e s (Wetherbee 1975 a , b ) . In the p e l l i c u l a t e fl. n i e i , the d e v e l o p i n g amphiesmal membranes were suggested t o be formed by p o l y v e s i c u l a r b o d i e s , g r a n u l a r v e s i c l e s or membranous m a t e r i a l r e l e a s e d from the p u s u l e ( M o r r i l and L o e b l i c h 1984). P r e v i o u s s t u d i e s 71 on Gonyauxlax p o l y e d r a or P e r i d i n i u m c i n c t u m c e l l s , have suggested t h a t g r a n u l a r v e s i c l e s s e r v e d t o t r a n s p o r t the t h e c a l p r e c u r s o r m a t e r i a l (Du'rr 1979 a , b ) . On the o t h e r hand, s i n c e the c l e a v a g e f u r r o w of fl. n i e i o c c u r s a l o n g the f l a g e l l a - p u s u l e r e g i o n and s i n c e p u s u l e s e x h i b i t membranous f e a t u r e s s i m i l a r t o those of the new amphiesma of ecdysed c e l l s , i t was suggested t h a t p u s u l e s c o u l d be a source of membranous m a t e r i a l f o r the new amphiesma ( M o r r i l l and L o e b l i c h 1984) . B. Nature of the c e l l s u r f a c e c o a t I t i s w e l l known t h a t the c e l l - s u r f a c e c o a t , a l s o r e f e r r e d to as " f u z z " or g l y c o c a l y x , i s a dynamic and complex s u r f a c e m a t r i x made up o l i g o s a c c h a r i d e s c o v a l e n t l y bound t o p r o t e i n s and l i p i d s of the plasma membrane of most e u k a r y o t i c c e l l s (such as f u n g i , h i g h e r p l a n t s , a l g a l and a n i m a l c e l l s ; Fawcett 1965, Hughes 1976, Sengbusch and M u l l e r 1983). By u s i n g a c r i d i n e orange, c a t i o n i c dyes and l e c t i n s , i t was demonstrated t h a t the e x t e r i o r s u r f a c e of D u n a l i e l l a . Amphidinium, or P r o r o c e n t r u m plasma membrane d i s p l a y s c h a r a c t e r i s t i c s t y p i c a l of a c i d i c o l i g o s a c c h a r i d e s . A c r i d i n e orange has been shown t o i n t e r a c t w i t h a c i d i c m u c o p o l y s a c c h a r i d e s (Saunders 1964 , Timar e_t a_l. 1979) w h i l e c a t i o n i c dyes (AlcB-RR) have been r e c o g n i z e d t o b i n d t o a c i d i c and s u l f a t e d p o l y s a c c h a r i d e s ( L u f t 1971a,b, Ramus 1977, D y k s t r a and A l d r i c h 1978). 72 The l a b e l i n g p a t t e r n produced by c a t i o n i z e d f e r r i t i n (CF) on D u n a l i e l l a c e l l s u r f a c e s u g gests t h a t the s u r f a c e a n i o n i c s i t e s a r e d i s t r i b u t e d i n random pat c h e s or l a r g e r a g g r e g a t e s . These o b s e r v a t i o n s c o n f i r m p r e v i o u s r e s u l t s of D u n a l i e l l a s u r f a c e s t a i n i n g w i t h a l c i a n b l u e and ruthenium red ( O l i v e i r a et a l . 1980). S i m i l a r s t u d i e s w i t h CF on P r o r o c e n t r u m added c o n s i d e r a b l e s t r u c t u r a l d e t a i l on the u n i f o r m n a t u r e of the b i n d i n g p a t t e r n o b t a i n e d from f l u o r o c h r o m e or c a t i o n i c dye s t a i n i n g . T h i s c o a t i s composed of two l a y e r s , one a p p a r e n t l y u n i f o r m and o v e r l y i n g the plasma membrane w h i l e the o t h e r i s patchy and p r o j e c t s i n t o the e x t e r n a l aqueous environment. In a d d i t i o n t o c a t i o n i c s t a i n i n g , the d i f f e r e n t degrees of s u r f a c e b i n d i n g and a g g l u t i n a t i o n response t o the v a r i o u s l e c t i n t r e a t m e n t s enabled f u r t h e r i n f e r e n c e s on the s p e c i f i c c a r b o h y d r a t e n a t u r e of the l e c t i n r e c e p t o r s i n the manner r e p o r t e d f o r p r o t o z o a (Warton and Honigberg 1980, Benchimol et a l . 1981). Such r e s u l t s suggest t h a t N - a c e t y l g l u c o s a m i n e or s i a l i c a c i d , N - a c e t y g a l a c t o s a m i n e and g l u c o s e or mannose might f u n c t i o n as l e c t i n r e c e p t o r s on the c e l l s u r f a c e of D u n a l i e l l a and Amphidinium. A d d i t i o n a l l y , r e s u l t s from FITC-Con A b i n d i n g suggested D-mannose as a p o s s i b l e l e c t i n - r e c e p t o r on the plasma membrane of P r o r o c e n t r u m . These 73 l e c t i n - s p e c i f i c sugars i n f e r r e d f o r the s u r f a c e of the t h r e e p h y t o f l a g e l l a t e s have been p r e v i o u s l y r e p o r t e d t o occur i n ot h e r e u k a r y o t i c m i c r o a l g a e but not i n c o n n e c t i o n w i t h l e c t i n - r e c e p t o r s . S i a l i c a c i d was i d e n t i f i e d as a component of u n d e f i n e d g l y c o p r o t e i n s from C h l o r e l l a ( C o r r e l l 1964), N - a c e t y l g l u c o s a m i n e as the b u i l d i n g b l o c k of c h i t i n a c e o u s f i b r i l s produced by c e r t a i n diatoms (McLachlan e_t a l . 1965 , Herth and Zugenmaier 1977) , N - a c e t y l g a l a c t o s a n i i n e as a mating-type substance s e c r e t e d by Chlamydomonas eugametos ( W i l l i a m s 1981) and D-mannose was found as p a r t of the c e l l s u r f a c e of many a l g a l c e l l s (Sengbusch and M u l l e r 1983). The b i n d i n g p a t t e r n produced by the c e l l - s u r f a c e markers d i s p l a y s c o n s i d e r a b l e v a r i a t i o n i n i n d i v i d u a l c e l l s as w e l l as between c e l l s of the same ( a l b e i t u n s y n c h r o n i z e d ) c u l t u r e . Such v a r i a t i o n may r e f l e c t q u a l i t a t i v e and/or q u a n t i t a t i v e d i f f e r e n c e s i n the t e r m i n a l sugar r e s i d u e s of the g l y c o c a l y x . V a r i a t i o n s i n c e l l s u r f a c e c a r b o h y d r a t e s have been w e l l documented f o r both u n i c e l l u l a r and m u l t i c e l l u l a r organisms at d i f f e r e n t s t a g e s of l i f e - c y c l e ( A r a u j o e_t a l . 1980 , S p i c e r e_t a l . 1981) . The c a t i o n i c - d y e s t a i n i n g a l s o r e v e a l e d a t h i n but d i s t i n c t l a y e r of a c i d m u c o p o l y s a c c h a r i d e m a t e r i a l a l o n g the i n n e r s u r f a c e of each amphiesmal v e s i c l e of Amphidinium. A l t h o u g h the l o c a t i o n of t h i s l a y e r c o r r e s p o n d s a p p r o x i m a t e l y t o t h a t of the p l a t e s i n t h e c a t e d i n o f l a g e l l a t e s , i t s 74 s t a i n i n g r e a c t i o n c o n t r a d i c t s the a l l e g e d " c e l l u l o s i c " n a t u r e of the t h e c a l p l a t e s . A p p a r e n t l y , t h i s l a y e r r e p r e s e n t s the o l i g o s a c c h a r i d e components of the amphiesmal v e s i c l e membrane, r e m i n i s c e n t of the g l y c o p r o t e i n sugar r e s i d u e s l o c a t e d on the i n n e r s u r f a c e of G o l g i v e s i c l e s . A l t h o u g h t h i s study p r e s e n t s the f i r s t c y t o c h e m i c a l d e s c r i p t i o n of a g l y c o c a l y x i n d i n o f l a g e l l a t e s , a l i t e r a t u r e s e a r c h has r e v e a l e d photographs of s e v e r a l o t h e r d i n o f l a g e l l a t e s p e c i e s (e.g. Ce r a t i u m h i r u n d e l l a , Gymnodinium s i m p l e x , H e t e rocapsa t r i q u e t r a l w i t h r e c o g n i z a b l e s u r f a c e coat p r o f i l e s (Dodge and Crawford 1970, Dodge 1974). I t ap p e a r s , t h e r e f o r e t h a t t h i s s u r f a c e s t r u c t u r e i s a common o c c u r r e n c e i n t h i s group of p h y t o f l a g e l l a t e s . In l i n e w i t h the f i n d i n g of an a c i d m u c o p o l y s a c c h a r i d e coat on the s u r f a c e of Amphidinium c e l l s , e v i d e n c e i s p r o v i d e d t o support Dragesco's (1965) o b s e r v a t i o n of at l e a s t two types of extrusomes, the s p i n d l e t r i c h o c y s t s and the mucocysts. The appearance and the c a t i o n i c - d y e s t a i n i n g p r o p e r t i e s of the l a t t e r suggest t h a t mucocyst s e c r e t i o n may p l a y a s i g n i f i c a n t r o l e i n the d e p o s i t i o n of the g l y c o c a l y x l a y e r . C. P o s s i b l e r o l e of the g l y c o c a l y x Among ot h e r f u n c t i o n s , c e l l - s u r f a c e coat or g l y c o c a l y x may p l a y an i m p o r t a n t r o l e i n c e l l - t o - c e l l a d h e s i o n . 75 I n t e r c e l l u l a r a d h e s i o n i s known t o occur i n p r o c e s s e s of f e r t i l i z a t i o n , c e l l d i f f e r e n t i a t i o n and host p a r a s i t e i n t e r a c t i o n ( r e v i e w e d by Rauvala 1983, Sengbusch and M u l l e r 1983, Sharon 1984). In c o n t r a s t t o the " l o n g - t e r m " c e l l - t o - c e l l a d h e s i o n u s u a l l y e f f e c t i v e among c e l l s of a t i s s u e ( L a c k i e 1980), temporary or " s h o r t - l i v e d " a d h e s i o n o c c u r r e d i n D u n a l i e l l a c e l l s exposed to CF. Based on p r e v i o u s models proposed f o r c e l l - t o - c e l l a d h e s i o n ( L a c k i e 1980), i t can be suggested t h a t CF when bound t o s u r f a c e a n i o n i c s i t e s may c o u n t e r the e l e c t r o s t a t i c r e p u l s i o n between s i m i l a r l y charged c e l l s . Then the b a l a n c e e s t a b l i s h e d between the r e p u l s i v e and the a t t r a c t i o n f o r c e s may b r i n g c e l l s t o g e t h e r a l l o w i n g temporary a d h e s i o n . However, the c e l l s u r f a c e i s not an autonomous system and a c c o r d i n g t o L a c k i e (1980), the s t r u c t u r a l o r g a n i z a t i o n of i t s components may depend upon a combined a c t i o n of e x t e r n a l f a c t o r s , c y t o p l a s m i c c o n t r a c t i l e systems and c y t o s k e l e t o n . D. C e l l s u r f a c e - r e l a t e d s t r u c t u r e s 1. L i p i d b o d i e s Two f l u o r o c h r o m e s (rhodamine B and f l u o r o B o r a P) were used t o i n v e s t i g a t e the n a t u r e of dense g l o b u l a r b o d i e s f r e q u e n t l y observed underneath the c e l l s u r f a c e of 76 Amphidinium. The b a s i c dye rhodamine B has been v a r i a b l y used as v i t a l s t a i n t o l o c a l i z e s u b c e l l u l a r s t r u c t u r e s such as G o l g i a p p a r a t u s and l i p i d s . F l u o r o B o r a P i s a f l u o r e s c e n t b o r o n i c a c i d d e r i v a t i v e , used t o l o c a l i z e l i p o p h i l i c and h y d r o p h i l i c a r e a s of l i v i n g c e l l s ( G a l l o p et a l . 1982). T h i s w a t e r - i n s o l u b l e f l u o r o p h o r e was s o l u b i l i z e d w i t h a c a r r i e r b u f f e r (TAPSO) c o n t a i n i n g a r e c e p t o r group f o r r e c e p t o r - b o r o n i c a c i d complex f o r m a t i o n . In c o n t a c t w i t h the plasma membrane, t h i s complex i s c l e a v e d so t h a t the f r e e d f l u o r o p h o r e e n t e r s the c e l l and s t a i n s the l i p o p h i l i c p o c k e t s w i t h a v i o l e t - f l u o r e s c e n c e w h i l e the h y d r o p h i l i c r e g i o n s s t a i n w h i t i s h - y e l l o w f l u o r e s c e n t . In c a r t e r a e l i p o p h i l i c m a t e r i a l o c c u r s as s m a l l areas randomly d i s t r i b u t e d underneath the c e l l s u r f a c e . These rounded t o a n g u l a r c y t o p l a s m i c i n c l u s i o n s v a r y i n the degree of o s m i o p h i l y and were found i n c e l l s at d i f f e r e n t s t a g e s of development. Such l i p o i d i n c l u s i o n s were g e n e r a l l y l o c a t e d between the amphiesmal v e s i c l e s and the c h l o r o p l a s t . T h e i r s i z e appeared t o be g r e a t l y i n c r e a s e d i n c e l l s of o l d c u l t u r e s . B i o c h e m i c a l s t u d i e s have shown t h a t the n a t u r e of d i n o f l a g e l l a t e l i p i d s i s v e r y s i m i l a r t o t h a t of o t h e r e u k a r y o t i c "chromophy t e s " ( H a r r i n g t o n £± aJL. 1970). A c c o r d i n g t o Wood (1974), they c o n t a i n h i g h amounts of u n s a t u r a t e d f a t t y a c i d s . The m a j o r i t y of these f a t t y a c i d s 77 are even-numbered but they d i f f e r i n the l e n g t h of t h e i r carbon c h a i n and i n the number and p o s i t i o n of carbon t o carbon double bond. &• c a r t e r a e has h i g h c o n t e n t s of 22:6, 20:5, 18:4 and 16:0 and r e l a t i v e l y s m a l l amounts of o t h e r a c i d s (Ackman e_£ a l . 1968, Wood 1974). In c o n t r a s t , P.. micans i s c h a r a c t e r i z e d by s i g n i f i c a n t amounts of 16:1 and a l s o 16:2 and 16:3. T h i s s p e c i e s has more 18:3 than A., c a r t e r a e (Chuecas and R i l e y 1969). A p p a r e n t l y , the l i p i d and f a t t y a c i d c o m p o s i t i o n are p h y l o g e n e t i c a l l y s i g n i f i c a n t . In f a c t , they have been used as a c r i t e r i o n t o e s t a b l i s h e v o l u t i o n a r y r e l a t i o n s h i p s among d i f f e r e n t a l g a l groups (as r e viewed by Wood 1974). 2. S p i n d l e t r i c h o c y s t s The s t r u c t u r e of s p i n d l e t r i c h o c y s t s i n A,, c a r t e r a e or £. micans was i n g e n e r a l s i m i l a r t o t h a t p r e v i o u s l y d e s c r i b e d f o r o t h e r d i n o f l a g e l l a t e s (Hausmann 1978). Of p a r t i c u l a r i n t e r e s t was the presence of a membranous cap on the a n t e r i o r r e g i o n of t r i c h o c y s t s l y i n g w i t h i n P r o r o c e n t r um c e l l . I t i s p o s s i b l e t h a t t h i s membranous s t r u c t u r e may p r o v i d e the t r i c h o c y s t w i t h s p e c i f i c r e c o g n i t i o n s i t e s r e q u i r e d f o r i t s m i g r a t i o n towards the c e l l s u r f a c e . A d d i t i o n a l l y , t h i s membranous cap may s e r v e t o r e c o g n i z e the p e r i p h e r a l c y t o p l a s m i c membrane and t r i g g e r membrane f u s i o n b e f o r e t r i c h o c y s t d i s c h a r g e . In o r d e r to e x p l a i n the mechanism of 78 t r i c h o c y s t d i s c h a r g e i n c i l i a t e p r o t o z o a (e.g. Paramecium), i t has been proposed t h a t upon e x t e r n a l s t i m u l i , transmembrane p r o t e i n s of the plasma membrane undergo c o n f o r m a t i o n changes which a l l o w the i n f l u x of Ca2+ through h y d r o p h i l i c c h a n n e l s . S u b s e q u e n t l y , the i n c r e a s e of Ca2+ between the plasma membrane and the t r i c h o c y s t membrane w i l l i n duce f u s i o n of these membranes f o l l o w e d by t r i c h o c y s t d i s c h a r g e ( e x o c y t o s i s , S a t i r 1978) . The s p i n d l e t r i c h o c y s t s of the two d i n o f l a g e l l a t e s examined here appeared t o o r i g i n a t e i n the v i c i n i t y of the E F - G o l g i a p p a r a t u s and were found, i n v a r i a b l e numbers a t v a r i o u s s t a g e s of development, throughout the c y t o p l a s m . S p i n d l e t r i c h o c y s t s have been suggested t o o r i g i n a t e i n the m i t o c h o n d r i a (Soyer 1969), endoplasmic r e t i c u l u m (Dragesco and H o l l a n d e 1965), or i n the G o l g i a p p a r a t u s (Bouck and Sweeney 1966, Messer and Ben-Shaul 1971). The a s s o c i a t i o n of G o l g i - d e r i v e d c o a t e d v e s i c l e s w i t h the t r i c h o c y s t p r i m o r d i a i n Amphidinium, suggests t h a t c o a t e d v e s i c l e s may p l a y an i m p o r t a n t r o l e i n the t r a n s p o r t of enzymes or o t h e r p r o t e i n s f o r t r i c h o c y s t f o r m a t i o n . S e v e r a l f u n c t i o n s have been suggested f o r the d i n o f l a g e l l a t e extrusomes i n c l u d i n g o s m o r e g u l a t i o n and s e c r e t i o n of t o x i c or m u c i f e r o u s s u b s t a n c e s (Dragesco 1965, Bouck and Sweeney 1966). A l t h o u g h the r o l e of s p i n d l e t r i c h o c y s t s remains open to c o n j e c t u r e , i t appears t h a t , at l e a s t i n P r o r o c e n t r u m , p o s s i b l y as a response t o e x t e r n a l s t i m u l i , they can e x c r e t e 79 a c i d i c s u b s t a n c e s which accumulate i n patches j u s t o u t s i d e the t r i c h o c y s t p o r e . I t i s p o s s i b l e t h a t these p a t c h e s may c o r r e s p o n d t o B a l e c h ' s (1980) d e s c r i p t i o n s of " i r r e g u l a r l i n e s " t h a t extend from s u r f a c e pores and v a r y w i t h c e l l age. I t was a l s o observed t h a t autophagosomes c o n t a i n remains of u n d i s c h a r g e d t r i c h o c y s t s which appear t o be used as p a r t of the normal t u r n o v e r of c e l l s . E. C e l l w a l l morphology and s y n t h e s i s The c e l l w a l l (theca) of P. micans i s comprised of two major p l a t e s (or v a l v e s ) , l i n k e d by r i d g e d m a r g i n s , and s e v e r a l p l a t e l e t s . T h i s c e l l w a l l was observed t o vary i n t h i c k n e s s a c c o r d i n g t o the growth phase of the c u l t u r e . I t appears t h a t t h i n w a l l s a re c h a r a c t e r i s t i c of a c t i v e l y d i v i d i n g c e l l s or they may r e p r e s e n t the i n i t i a l s t a t e of new w a l l s y n t h e s i s a f t e r e c d y s i s w h i l e t h i c k w a l l s appear t o be c h a r a c t e r i s t i c of agi n g c e l l s . I t i s known t h a t P_. micans d i v i d e s by " o b l i q u e " f i s s i o n , i n which each daughter c e l l r e t a i n s one o l d v a l v e and s y n t h e s i z e s the o t h e r v a l v e . However, s i n c e e c d y s i s i s a f r e q u e n t phenomenon) i n P. micans c u l t u r e s and s i n c e two t h i n p l a t e s were observed i n some c e l l s , i t i s suggested t h a t s i m i l a r l y t o o t h e r d i n o f l a g e l l a t e s p e c i e s , P. micans i s a b l e t o r e g e n e r a t e a complete new w a l l a f t e r e c d y s i s . B e s i d e the two l a r g e t h e c a l p l a t e s , s e v e r a l p l a t e l e t s 80 (6-8 i n s e c t i o n ) are l o c a t e d i n a depression on the l e f t s i d e of the a p i c a l r e g i o n . These p e r i f l a g e l l a r p l a t e l e t s d i s p l a y v a r i a b l e morphology and are j o i n e d together by s u t u r e s . There i s no agreement i n the l i t e r a t u r e as to the number of small p l a t e s i n P r o r o c e n t r a l e s . In P. micans, f o r i n s t a n c e , 1,2,8 or 10-14 p l a t e l e t s have been repo r t e d ( L o e b l i c h 1970, Dodge 1975, Taylor 1980). Eight p l a t e l e t s have a l s o been i d e n t i f i e d i n other members of P r o r o c e n t r a l e s (Faust 1974, Balech 1980). Further o b s e r v a t i o n s on the per i f l a g e l l a r region of P. micans r e v e a l e d the double s t r u c t u r e of the a p i c a l s p i n e . T h i s f e a t u r e has been p r e v i o u s l y d e s c r i b e d as a winged spine (Dodge 1965) or a s i n g l e a p i c a l spine (Dodge and Bibby 1973). Pi second spine seems to protrude from the a p i c a l area. T h i s f l a g e l l a r complex i s m o r p h o l o g i c a l l y s i m i l a r to that p r e v i o u s l y d e s c r i b e d i n Prorocentrum m a r i a e - l e b o u r i a e (Dodge and Bibby 1973, Faust 1974). L i g h t microscopy s t u d i e s confirmed the " wing" aspect of the a p i c a l spine d e s c r i b e d by Dodge (1965). However, TEM o b s e r v a t i o n s suggested that such a winged appearance could be provided by the outer amphiesmal membranes o v e r l y i n g the double a p i c a l s p i n e . The arrangement of these a n t e r i o r spines i n r e l a t i o n to the f l a g e l l a r pores appears to suggest that they may act as " f r o n t " s t r u c t u r e s to concentrate and d i r e c t the water cu r r e n t flow towards the f l a g e l l a r canal and the sutures of adjacent p l a t e l e t s . 81 F. Nature of the c e l l w a l l F l u o r e s c e n c e s t u d i e s u s i n g c a l c o f l u o r w h i t e and congo red suggested the p o l y g l u c a n ( p o s s i b l y c e l l u l o s e ) n a t u r e of P. micans t h e c a . P r e v i o u s s t u d i e s u s i n g X-ray d i f f r a c t i o n p a t t e r n s , i o d i n e KI-H 2S04, z i n c - c h l o r - i o d i d e s t a i n i n g , c e l l u l a s e r e a c t i o n or c e l l u l o s e s o l v e n t s ( S c h w e i t z e r ' s reagent or Cadoxen) i n c o m b i n a t i o n w i t h a c i d p r e - t r e a t m e n t , suggested t h a t the t h e c a l p l a t e s were composed of i ) c e l l u l o s e - l i k e g l u c a n w i t h both (3-1,3 and (S-1,4 l i n k a g e s (e.g. i n P e r i d i n i u m w e s t i i , Nevo and Sharon 1969), or i i ) c e l l u l o s e e i t h e r made of f i n e m i c r o f i b r i l s (Dodge 1971) or w i t h low degree of c r y s t a l l i n i t y ( L o e b l i c h 1970, M o r r i l l and L o e b l i c h 1983) . A l t h o u g h amorphous i n u l t r a s t r u c t u r a l appearance, the c e l l w a l l of P. micans, a f t e r t r eatment w i t h hot a c e t i c a c i d and h y p o c h l o r i t e s o l u t i o n , was shown to be made of g r a n u l a r or f i b r i l a r m a t e r i a l (Dodge 1965). G. T h e c a l o r n a m e n t a t i o n s The c e l l s u r f a c e of Prorocentrum showed numerous d e p r e s s i o n s and "po r e s " of v a r i a b l e s i z e and morphology. The term d e p r e s s i o n was . used t o c h a r a c t e r i z e s h a l l o w concave t h e c a l i n d e n t a t i o n s w h i l e the term " p o r e s " was employed t o d i f f e r e n t i a t e deeper s u r f a c e i n v a g i n a t i o n s o f t e n l o d g e d i n cone-shaped s t r u c t u r e s . The number of pores i d e n t i f i e d by l i g h t m i c r o s c o p y and SEM i n P_. micans ( c a . 100 per c e l l ) was 82 w i t h i n the range p r e v i o u s l y r e p o r t e d by Dodge (1965) and Dodge and Crawford (1970). ( D i f f e r e n t types of o r n a m e n t a t i o n s ( d e p r e s s i o n s , p o r e s , and r e l i e f s ) have been d e s c r i b e d f o r the t h e c a l p l a t e s of v a r i o u s d i n o f l a g e l l a t e s ( A n d r e i s _± _1. 1982). Based on the m o r p h o l o g i c a l c h a r a c t e r i s t i c s and a s s o c i a t i o n p a t t e r n s of these o r n a m e n t a t i o n s , s e v e r a l s t r u c t u r a l models ( s i m p l e t o complex) have been proposed. A c c o r d i n g t o A n d r e i s and c o l l a b o r a t o r s , the s o - c a l l e d " p r i m i t i v e " s p e c i e s (such as those of Prorocentrum) o n l y p o s s e s s s i m p l e pores (as i l l u s t r a t e d i n t h e i r F i g . 13 a-j). S u r f a c e pores of P.. micans e x h i b i t e d v a r i o u s s i z e s (6-100 nm, Dodge 1975) and c o n f o r m a t i o n a l t y p e s ( d i a g r a m m a t i c a l l y i l l u s t r a t e d i n F i g . 1 2 0 ) . SEM s t u d i e s suggested t h a t the cone-shaped prominent s t r u c t u r e s l o c a t e d i n concave t h e c a l d e p r e s s i o n s might have r e s u l t e d from the outward movement of t r i c h o c y s t s d u r i n g e x t r u s i o n . H. P o l e s of the c y t o s k e l e t o n i n c e l l - s u r f a c e a c t i v i t i e s A s i g n i f i c a n t advance i n the u n d e r s t a n d i n g of the o r g a n i z a t i o n of the c e l l ' s c y ' t o s k e l e t a l elements ( m i c r o t u b u l e s , m i c r o f i l a m e n t s and i n t e r m e d i a t e f i l a m e n t s ) and m i c r o t r a b e c u l a r l a t t i c e has been made w i t h h i g h v o l t a g e e l e c t r o n m i c r o s c o p y ( P o r t e r and Tucker 1981, Wardrop 1983). 83 Elements of such complex and dynamic network are known t o p a r t i c i p a t e i n many b i o l o g i c a l a c t i v i t i e s . Among thes e a r e : maintenance of c e l l - s u r f a c e topography, transmembrane s i g n a l l i n g , c y t o p l a s m i c s t r e a m i n g , o r g a n e l l e t r a n s p o r t , c e l l w a l l d e p o s i t i o n , d i s t r i b u t i o n of c e l l - s u r f a c e r e c e p t o r s , f o r m a t i o n of plasma membrane-derived v e s i c l e s , t r a n s p o r t of e n d o c y t i c or e x o c y t i c v e s i c l e s and c o u p l i n g of e x o - e n d o c y t o s i s (Fobards 1968, Sloboda 1980, Thyberg 1980, Thyberg e£ s i . 1980, Couchman and Fees 1982, Nakamura and M i k i - H i r o s i g e 1982 , B i r c h m e i e r 1984, Traas 1984, Koonce and S c h l i w a 1985). In D u n a l i e l l a the randomly d i s t r i b u t e d or o r d e r l y a r r a n g e d m i c r o t u b u l e s (MT) observed underneath the plasma membrane may p l a y an a c t i v e r o l e i n a n c h o r i n g and s t a b i l i z i n g the l a t e r a l m o b i l i t y of c e l l s u r f a c e r e c e p t o r s d u r i n g p a t c h i n g or c a p p i n g of l i g a n d - r e c e p t o r complexes . A c l o s e a s s o c i a t i o n between c y t o p l a s m i c MT and s m a l l v e s i c l e s may a l s o suggest t h a t MT c o u l d p l a y a key r o l e i n c o n t r o l l i n g the d i r e c t i o n and f l o w of both s e c r e t o r y v e s i c l e s and endosomes. J . A d s o r p t i v e p i n o c y t o s i s P i n o c y t o s i s i n B. t e r t i o l e c t a was e v i d e n t from both f l u o r e s c e n c e m i c r o s c o p y and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y u s i n g v a r i o u s m o l e c u l a r markers. F I T C - l e c t i n s have been p r e v i o u s l y used t o l o c a l i z e o l i g o s a c c h a r i d e s u r f a c e r e c e p t o r s 84 i n v a r i o u s c e l l t y pes (Cormak and Ambrose 1962) i n c l u d i n g a l g a l c e l l s (Surek and Sengbush 1982, Sengbusch and M u l l e r 1981) and t o f o l l o w the e n d o c y t i c uptake i n animal c e l l s (Maher and Molday 1979). The i n t e r a c t i o n of the p o l y v a l e n t l i g a n d , FITC-WGA, w i t h l i v i n g D u n a l i e l l a c e l l s , at 4°C, produced i n t e n s e l a b e l i n g p a t t e r n s u g g e s t i n g random d i s t r i b u t i o n of the l e c t i n b i n d i n g s i t e s i . e . GlcNAc r e s i d u e s of s u r f a c e g l y c o c o n j u g a t e s . A c c o r d i n g t o the f l u i d mosaic membrane model (as reviewed by P a l a d e 1982), these g l y c o c o n j u g a t e s are a b l e t o move l a t e r a l l y i n the plane of the membrane by d i f f u s i o n . At a p p r o x i m a t e l y 25°C, the p a t c h i n g and c a p p i n g of the 1 i g a n d - r e c e p t o r complexes o c c u r r e d a t the a n t e r i o r end of D u n a l i e l l a c e l l s . S i m i l a r to animal c e l l s , f o l l o w i n g c a p p i n g and plasma membrane i n v a g i n a t i o n , the l i g a n d was t r a n s p o r t e d , by endosomes, i n t o the l y s o s o m a l system and became accumulated w i t h i n v a c u o l e s of the p e r i n u c l e a r r e g i o n . As e x p e c t e d , the b i n d i n g and i n t e r n a l i z a t i o n of FITC-WGA was s i g n i f i c a n t l y reduced i n the presence of the c o r r e s p o n d i n g s p e c i f i c s u g a r , N - a c e t y l g l u c o s a m i n e . The uptake of l e c t i n s i n D u n a l i e l l a c e l l s i s t h e r e f o r e t y p i c a l of r e c e p t o r - m e d i a t e d p i n o c y t o s i s p r e v i o u s l y r e p o r t e d i n animal c e l l s . F u r t h e r e v i d e n c e f o r a d s o r p t i v e p i n o c y t o s i s was p r o v i d e d , by the o b s e r v a t i o n of c l u s t e r s of l i g a n d - r e c e p t o r 85 complexes t r a p p e d i n d i s t i n c t " c oated p i t s " of c e l l s t r e a t e d w i t h c a t i o n i z e d f e r r i t i n . In animal c e l l s , c o a t e d p i t s a r e known t o be i n v o l v e d i n a d s o r p t i v e e n d o c y t o s i s ( S i l v e r s t e i n et a l . 1977 P e t t e r s e n and van Deurs 1983, B r e t s c h e r 1984, Rothman and Lenard 1984). However, e n d o c y t o s i s i n p l a n t c e l l s remains a s u b j e c t of c o n t r o v e r s y , and c o a t e d p i t s , when r e p o r t e d , have been c o n s i d e r e d as s t r u c t u r e s r e l a t e d t o e x o c y t o s i s , membrane r e c y c l i n g ( O ' N e i l and La C l a i r e 1984, Traas 1984) and r a r e l y i n t e r p r e t e d as an i n d i c a t o r of e n d o c y t o s i s ( K i v i c and Vesk 1974, Joachim and Robinson 1984). Coated p i t s a r e d i f f e r e n t i a t e d s u r f a c e microdomains known t o p l a y a major r o l e i n the t r a p p i n g of l i g a n d - r e c e p t o r (L-R) complexes p r i o r t o the i n t e r n a l i z a t i o n of l i g a n d s ( W i l l i n g h a m et a l . 1981). As has been reviewed e a r l i e r , the q u e s t i o n remains as t o whether these L-R complexes are t r a n s f e r r e d t o the endosomes termed "receptosomes" v i a " c r y p t i c p i t s " (receptosoroe model, Pastan and W i l l i n g h a m 1981a,b; W i l l i n g h a m and P a s t a n , 1984) or v i a c o a t e d v e s i c l e s , termed "pinosomes" ( c o a t e d v e s i c l e model, P e t e r s e n and van Deurs 1983). In the f i r s t model, the smooth-surfaced receptosomes are formed from c r y p t i c p i t s . "Coated v e s i c l e s " a r e not f r e e s t r u c t u r e s but are p o r t i o n s of c o n v o l u t e d l o n g and narrow c r y p t i c p i t s . In D u n a l i e l l a , the i n t e r m e d i a t e s t a g e s between c r y p t i c p i t and receptosomes were not 86 ob s e r v e d , and a c c o r d i n g t o Kolb-Bachofen (1985) the e x i s t e n c e of e i t h e r receptosomes or f r e e c o a t e d v e s i c l e s may depend on the c e l l t y p e . I t can however be c o n c l u d e d t h a t CF bound t o the a n i o n i c s i t e s of D u n a l i e l l a plasma membrane and c o a t e d p i t s , was i n t e r n a l i z e d and then t r a n s p o r t e d t o the lysosomes. S u b s e q u e n t l y , i t appears t h a t the l i g a n d became degraded w i t h i n these compartments. Other s t u d i e s have shown t h a t i n t e r n a l i z e d CF was t r a n s p o r t e d t o the G o l g i (Thyberg 1980, Takata et a l . 1982) or i t was c a r r i e d a c r o s s the c e l l by t r a n s c e l l u l a r r o u t e ( P e t e r s e n and van Deurs 1983, Farquhar 1983). In the p r e s e n t study the o c c u r r e n c e of CF w i t h i n the G o l g i was not c l a r i f i e d . K. A c i d phosphatase l o c a l i z a t i o n In D u n a l i e l l a , a c i d phosphatase was d e t e c t e d i n v e s i c l e s c l o s e t o the EE, i n the t r a n s G o l g i compartment, w i t h i n v e s i c l e s of both c i s and t r a n s f a c e , as w e l l as w i t h i n l a r g e secondary lysosomes i n the p e r i n u c l e a r r e g i o n . P r e v i o u s s t u d i e s have shown t h a t h y d r o l a s e s are s y n t h e s i z e d i n the rough endoplasmic r e t i c u l u m and t r a n s f e r r e d t o the G o l g i ( r e v i e w e d by Steinman £t a l . 1983). Based on these f i n d i n g s , i t i s suggested t h a t , i n D u n a l i e l l a , the enzyme a c i d phosphatase was s y n t h e s i z e d i n the EE, t r a n s f e r r e d by t r a n s i t i o n a l v e s i c l e s t o the G o l g i , and then s o r t e d out and t r a n s p o r t e d t o the p r i m a r y lysosomes. S i n c e i n t e r n a l i z e d 87 markers were found w i t h i n v a c u o l e s t h a t c o n t a i n a c i d p hosphatase, i t i s suggested t h a t endocytosed l i g a n d s are p o s s i b l y degraded or accumulated i n the l y s o s o m a l system. L. F l u i d - p h a s e p i n o c y t o s i s E v idence f o r f l u i d - p h a s e p i n o c y t o s i s , i n D u n a l i e l l a , was o b t a i n e d from TEM s t u d i e s of c e l l s exposed t o HRP. D e s p i t e the o c c a s i o n a l r e p o r t s on c e l l s u r f a c e b i n d i n g (Goud e_t_ a l . 1981, Thyberg and S t e n s e t h 1981), HRP has been w i d e l y used as a marker f o r f l u i d phase e n d o c y t o s i s (Steinman and Cohn 1972, Steinman e± a l . 1974, Thyberg 1980, Adams £t i l . 1982). A c c o r d i n g t o S t r a u s (1981, 1983), HRP has s p e c i f i c a f f i n i t y f o r mannosyl r e s i d u e s . However, s i n c e these sugar r e s i d u e s u s u a l l y do not occur i n a t e r m i n a l p o s i t i o n , HRP r e a c t i o n has been m a i n l y suggested t o be i n d i c a t i v e of f l u i d - p h a s e e n d o c y t o s i s . In D u n a l i e l l a , the i n t e r n a l i z a t i o n of HRP o c c u r r e d i n both c o a t e d and uncoated p i t s . The r e s u l t a n t endosomes were e i t h e r c o m p l e t e l y f i l l e d w i t h HRP r e a c t i o n p r o d uct or c o n t a i n e d a t h i n l i n i n g of t h i s p r o d u c t . S i m i l a r r e s u l t s have been r e p o r t e d i n macrophages and f i b r o b l a s t s and thought t o i n d i c a t e d i f f e r e n t degrees of HRP i n t e r n a l i z a t i o n (Steinman Zt s i . 1974) . I t i s l i k e l y t h a t i n D u n a l i e l l a , HRP e n t e r s the c e l l s by 88 both f l u i d - p h a s e and a d s o r p t i v e p i n o c y t o s i s as r e c e n t l y proposed by Goud et a l . (1981) and O l i v e r (1982) f o r lymphoid and e x o c r i n e a c i n a r c e l l s . A f t e r i n t e r n a l i z a t i o n , HRP was then r o u t e d t o the G o l g i c i s t e r n a e and lysosomes. These r e s u l t s a r e i n agreement w i t h p r e v i o u s r e p o r t s of HRP i n the G o l g i c i s t e r n a e ( S i l v e r s t e i n _± a l . 1977) and lysosomes (Steinman and Cohn 1972 , Steinman e_t a l . 1976, Adams £i a l . 1982, Farquhar 1983, O l i v e r 1982). M. Macromolecule i n t e r n a l i z a t i o n i n Amphidinium and In c o n t r a s t t o the s i m p l e s t r u c t u r a l o r g a n i z a t i o n of P u n a l i e l l a c e l l s u r f a c e , M f f i M d j j i i u n i and Ixoroceutruffi. are surrounded by the amphiesmal complex which p r o v i d e s the c e l l s w i t h s p e c i f i c r o u t e s f o r macromolecule i n t e r n a l i z a t i o n . F l u o r e s e n c e and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y s t u d i e s r e v e a l e d t h a t macromolecules e n t e r e d the c e l l s v i a (1) random plasma-membrane i n v a g i n a t i o n s and d e r i v e d endosomes; (2) s u r f a c e p o r e s ; or (3) the f l a g e l l a r c a n a l - p u s u l e system. 1. Macromolecule i n t e r n a l i z a t i o n v i a plasma-membrane i n v a g i n a t i o n and d e r i v e d endosomes The i n t e r a c t i o n of WGA-CG w i t h A,, c a r t e r a e c e l l s r e s u l t e d i n the " p a t c h i n g " of l i g a n d - r e c e p t o r complexes 89 i r r e s p e c t i v e of the t e m p e r a t u r e . M i c r o f i l a m e n t s ( a c t i n -myosin) are known t o be i n v o l v e d i n the p r o c e s s of p a t c h i n g and f o r m a t i o n of l a r g e aggregates or c l u s t e r s t h a t move i n t o membrane i n f o l d i n g s ( L a c k i e 1980). Such s u r f a c e events have been suggested t o occur by "transmembrane l i n k a g e s " i n which c r o s s - l i n k e d a ggregated p r o t e i n s b i n d t o an i n t e g r a l membrane p r o t e i n , which i n t u r n i n t e r a c t s w i t h a c t i n i n the c y t o p l a s m . ( L a c K i e 1980). In c a r t e r a e , a meshwork of m i c r o f i l a m e n t s was observed l y i n g the plasma membrane i n v a g i n a t i o n s . S m a l l e n d o c y t i c v e s i c l e s detached from the plasma membrane were then i n c o r p o r a t e d i n t o the p e r i p h e r a l c y t o p l a s m i c v a c u o l e . S i m i l a r o b s e r v a t i o n s were o b t a i n e d i n i n t a c t r o o t s of Oryza  s a t i v a ( N i s h i z a w a and M o r i 1977). In s p i t e of membrane i n c o m p a t i b i l i t y between the plasma membrane and the t o n o p l a s t , such an event had been suggested t o r e p r e s e n t the f l o w of exogenous m a t e r i a l i n t o the v a c u o l a r system. Large v a c u o l e s ( p o s s i b l y secondary lysosomes) or a c c u m u l a t i o n b o d i e s c o n t a i n i n g heterogeneous m a t e r i a l were o f t e n a s s o c i a t e d w i t h endoplasmic r e t i c u l u m (ER). I t i s p o s s i b l e t h a t ER may c a r r y the l y t i c enzymes f o r d i g e s t i o n of endogenous (autophagy) or exogenous (heterophagy) m a t e r i a l s . The presence of these l a r g e v a c u o l e s i n Amphidinium and Proro c e n t r u m i s r e m i n i s c e n t of s i m i l a r s t r u c t u r e s found i n ot h e r d i n o f l a g e l l a t e s and v a r i o u s l y r e f e r r e d t o as food b o d i e s or food v a c u o l e s ( K o f o i d and Swezy 1921, Dodge and 90 C r a w f o r d 1970), r e s i d u a l b o d i e s ( S t e i d i n g e r e_t s i . 1978), or a c c u m u l a t i o n b o d i e s (Dodge 1971, Lee 1977, W i l c o x e_t a l . 1982) . The presence of these o r g a n e l l e s has been i n t e r p r e t e d as i n d i c a t i v e of phagotrophy or autophagy. P h a g o c y t o s i s has been p r e v i o u s l y d e s c r i b e d i n d i n o f l a g e l l a t e s (Cachon and Cachon 1971, Dodge and C r a w f o r d 1974). There are s e v e r a l r e p o r t s of b a c t e r i a found ( i ) i n s i d e v a c u o l e s of d i n o f l a g e l l a t e s i n c l u d i n g P r o r o c e n t r u m micans ( S i l v a 1959), C e r a t i u m h i r u n d e l l a (Dodge and Crawford 1970), ( i i ) underneath the amphiesma of C r y p t h e c o d i n i u m (Gyrodinium) c o h n i i ( I s h i o e_fc s i . 1977), ( i i i ) i n the c y t o p l a s m of G y r o d i n i u m l e b o g r i s e (Lee 1977), p r o t o g o n y a u l a x ( G o n y a u l a x ) t a m a r e n s i s . Gyrodinium i n s t r i a t u m ( S i l v a and Sousa 1981), ( i v ) i n the n u c l e u s of Gymnodinium s p l e n d e n s , G l e n o d i n i u m j o l i g c e u m ( s i l v a 1978), G y r o d i n i u m i n s t r i a t u r o ( s i l v a and Sousa 1981) or (v) i n s i d e the p u s u l e s of P r o t o p e r i d i n i u m ma j u s (Abe 1981) or o t h e r d i n o f l a g e l l a t e s (Cachon e_t a l . 1983) . However, l i t t l e i s known about the p r o c e s s of b a c t e r i a e n t r y and t h e i r p o t e n t i a l m e t a b o l i c i n t e r a c t i o n s w i t h the d i n o f l a g e l l a t e c e l l . 2. Macromolecule i n t e r n a l i z a t i o n v i a s u r f a c e pores M o r p h o l o g i c a l s t u d i e s on P r o r o c e n t r u m i n d i c a t e d t h a t upon t r i c h o c y s t d i s c h a r g e the s u r f a c e area around the 91 t r i c h o c y s t was m o d i f i e d t o a p r o m i n e n t l y cone-shaped p r o f i l e . I t i s p o s s i b l e t h a t a f t e r t r i c h o c y s t e x t r u s i o n , the m o d i f i e d s t r u c t u r e s s e r v e as a s i t e f o r macromolecule i n t e r n a l i z a t i o n as p r e v i o u s l y d e s c r i b e d i n Paramecium caudatum ( A l l e n and Fok 1984). Moreover, these f i n d i n g s r e c a l l p r e v i o u s d e s c r i p t i o n s of " p r o l i f i c v e s i c u l a t i o n " a d j a c e n t t o the sac of a d i s c h a r g e d t r i c h o c y s t i n Gonyaulax p o l y e d r a ( f i g . 15, Bouck and Sweeney 1966) . Such v e s i c l e s were a s c r i b e d t o the c o l l a p s e ( c o n t r a c t i o n ) of the t r i c h o c y s t c o n t a i n e r . The p r e s e n t i n v e s t i g a t i o n s u p p o r t s B a l e c h 's view t h a t s u r f a c e " p o r e s " , g e n e r a l l y c a l l e d t r i c h o c y s t p o r e s , a r e not e x c l u s i v e l y t o e x t r u d e t r i c h o c y s t s but they may p l a y an i m p o r t a n t r o l e i n f a c i l i t a t i n g c e l l - e n v i r o n m e n t exchanges. To r e i n f o r c e h i s p o i n t of view, B a l e c h p o i n t e d out t h a t pores of the cingulum are . i n f r o n t of the " a x i a l p l a n e of the t r a n s v e r s e f l a g e l l u m " thus r e n d e r i n g t r i c h o c y s t d i s c h a r g e d i f f i c u l t . A l t e r n a t i v e l y , B a l e c h suggested t h a t pores of t h i s r e g i o n may s e r v e t o e s t a b l i s h the c o n n e c t i o n between the p r o t o p l a s m and the f l a g e l l u m ( v i a f i n e f i l a m e n t s ) . 3. Macromolecule i n t e r n a l i z a t i o n v i a the f l a g e l l a r c a n a l - p u s u l e system The major s i t e f o r uptake of macromolecules i n Amphidinium or P r o r o c e n t r u m was the f l a g e l l a r - c a n a l p u s u l e system. Among the hypotheses p o s t u l a t e d f o r the b i o l o g i c a l 92 r o l e of the d i n o f l a g e l l a t e p u s u l e , the f o l l o w i n g w i l l be c o n s i d e r e d : e x c r e t i o n , o s m o r e g u l a t i o n , f l o t a t i o n , and n u t r i e n t uptake. As f o r the e x c r e t i o n r o l e , p u s u l e s have been c o n s i d e r e d analogous t o c o n t r a c t i l e v a c u o l e s ( S c h u t t 1895, Cachon _± a l . 1983, Dodge 1972). A l t h o u g h the c o n t r a c t i l e aspect of the p u s u l e remains c o n t r o v e r s i a l ( S t e i d i n g e r and W i l l i a m s 1970), f l u c t u a t i o n s i n the p u s u l a r volume have been r e p o r t e d ( K o f o i d 1909, B i e c h e l e r 1952, Abe 1981) and c o r r e l a t e d w i t h the d i s c h a r g e of r e f r i n g e n t p a r t i c l e s from the f l a g e l l a r c a n a l (Cachon _± a l . 1983). These p a r t i c l e s were suggested t o be d e r i v e d from the multimembranous s t r u c t u r e s f r e q u e n t l y found w i t h i n the p u s u l e . F u r t h e r m o r e , Ca2+-dependent n o n - a c t i n f i l a m e n t s of the s t r i a t e d f l a g e l l a r r o o t l e t s were suggested t o p l a y a r o l e i n the c o n t r a c t i o n of the p u s u l e (Cachon £± a l . 1 983). However, the invo l v e m e n t of such f i l a m e n t s i n the c o n t r a c t i o n of v a c u o l e s i s c o n s i d e r e d u n l i k e l y . In c o n t r a s t t o the c o n t r a c t i l e v a c u o l e ( P a t t e r s o n 1980) , no r e g u l a r s y s t o l e - d i a s t o l e p r o c e s s e s have been observed f o r the p u s u l e (Dodge 1972, Abe' 1981, Cachon _±. al. 1983). A d d i t i o n a l l y the c o n t r a c t i l e v a c u o l e s are bounded by a s i n g l e membrane, whereas p u s u l e s are f r e q u e n t l y e n c l o s e d by a double or t r i p l e l a y e r of membranes. I t s h o u l d a l s o be p o i n t e d out t h a t the c o n t r a c t i l e v a c u o l e s are known t o p o s s e s s a c h a r a c t e r i s t i c mechanism f o r t h e i r o s m o r e g u l a t i o n , and such a mechanism i s 93 not suggested from the s t r u c t u r a l o b s e r v a t i o n s of the p u s u l e . The events of o s m o r e g u l a t i o n i n the c o n t r a c t i l e v a c u o l e i n v o l v e the a c t i v e pumping and c o l l e c t i o n of i n t r a c e l l u l a r f l u i d s i n t o s m a l l v e s i c l e s w i t h subsequent emptying i n t o the l a r g e r c o l l e c t i n g chamber, f o l l o w e d by an e x t r a c e l l u l a r d i s c h a r g e . M i c r o t u b u l e s a s s o c i a t e d w i t h the margins of the c o l l e c t i n g chamber p l a y a v e r y i m p o r t a n t r o l e i n the emptying of t h i s chamber ( P a t t e r s o n 1980). I t i s known t h a t the p u s u l e i s permanently connected t o the f l a g e l l a r c a n a l and o c c u r s i n both marine and f r e s h water s p e c i e s . From a s t r u c t u r a l p o i n t of view, i f o s m o r e g u l a t i o n i s a f u n c t i o n of the d i n o f l a g e l l a t e p u s u l e (Dodge 1971, 1972, 1973, S a r j e a n t 1974, Tappan 1980, Cachon _± a l . 1983, S t e i d i n g e r _i a l . 1978, Chapman _t a l . 1981), the mechanism may be d i f f e r e n t from t h a t of the c o n t r a c t i l e v a c u o l e . The f l o t a t i o n f u n c t i o n suggested f o r the d i n o f l a g e l l a t e p u s u l e ( N o r r i s 1966) remains c o n j e c t u r a l , w i t h o u t any s u p p o r t i n g e v i d e n c e . In the p r e s e n t s t u d y , p u s u l e volume v a r i e d i n response t o d i f f e r e n t s a l i n i t y l e v e l s and these changes were not a f f e c t e d e i t h e r by c o l c h i c i n e or c y t o c h a l a s i n t r e a t m e n t . Such o b s e r v a t i o n i n d i c a t e d t h a t m i c r o t u b u l e s and m i c r o f i l a m e n t s found i n the v i c i n i t y of the p u s u l e s may not be i n v o l v e d i n the a l l e g e d c o n t r a c t i l e f u n c t i o n . The m o r p h o l o g i c a l changes caused by s a l i n i t y and temperature may r e g u l a t e f l o t a t i o n . S i n c e c o n s i d e r a b l e 94 f l u c t u a t i o n of p h y s i c a l parameters i s expected i n the n a t u r a l environment (Kamykowski and Z e n t a r a 1977), i t i s p o s s i b l e t h a t d i n o f l a g e l l a t e s use a f l o t a t i o n mechanism ( r e g u l a t e d by the p u s u l e s i z e ) d u r i n g t h e i r d i u r n a l v e r t i c a l m i g r a t i o n . However, r i g o r o u s e x p e r i m e n t a l p r o o f i s r e q u i r e d t o i n v e s t i g a t e the involvement of the p u s u l e i n the e x e r c i s e of t h i s mechanism. The r o l e of the p u s u l e i n the uptake of food p a r t i c l e s was proposed by K o f o i d (1909) and K o f o i d and Swezy (1921), who compared the d i n o f l a g e l l a t e p u s u l e t o the " c y t o p h a r y n x " of o t h e r p r o t i s t s . I t was then suggested t h a t the water f l o w , g e n e r a t e d by the b e a t i n g of the f l a g e l l a might s e r v e t o d i r e c t f o o d - p a r t i c l e s i n t o the p u s u l e . These p a r t i c l e s would be then t r a n s p o r t e d i n t o the c y t o p l a s m , p o s s i b l y d i g e s t e d and s u b s e q u e n t l y e x c r e t e d . Lebour (1925) as w e l l as Gaines and T a y l o r (1984) suggested f u r t h e r t h a t the p u s u l e might have a n u t r i t i o n a l r o l e i n c o l o r l e s s d i n o f l a g e l l a t e s . A l t h o u g h such a r o l e was not d i s r e g a r d e d by N o r r i s (1966), i t has not been g e n e r a l l y a c c e p t e d . I t may be worth n o t i n g t h a t the f l a g e l l a r c a n a l - p u s u l e system of d i n o f l a g e l l a t e s shows some s t r u c t u r a l and f u n c t i o n a l s i m i l a r i t y w i t h the t u b u l a r c a n a l - r e s e r v o i r of e u g l e n o i d s ( L e e d a l e 1982) and the f u r r o w - g u l l e t system of cryptomonads (Sa n t o r e 1985). In £. c a r t e r a e , the f l a g e l l a r c a n a l i s l i n e d by the amphiesmal v e s i c l e s and opens i n t o the f l a g e l l a r chamber which l e a d s 95 i n t o the p u s u l e . In e u g l e n o i d s , the f l a g e l l a r ( t u b u l a r ) c a n a l i s u n d e r l a i n by the p e l l i c l e , and opens i n t o the r e s e r v o i r . In cryptomonads, the f u r r o w - g u l l e t system c o n s i s t of the f u r r o w ( a l s o termed v e s t i b u l e ) l i n e d by p e r i p l a s t p l a t e s , and opens i n t o the g u l l e t (or p h a r y n x ) . I n t e r e s t i n g l y , the r e s e r v o i r of Euglena has been suggested t o be i n v o l v e d i n the uptake of HRP ( K i v i c and Vesk 1974) w h i l e the g u l l e t of Cyathomonas has been suggested t o p l a y a r o l e i n the uptake of food m a t e r i a l ( S c h u s t e r 1968) . R e s u l t s from the p r e s e n t i n v e s t i g a t i o n i n d i c a t e the movement of m o l e c u l a r markers i n t o the f l a g e l l a r c a n a l - p u s u l e system. I t appears t h a t t h i s i n f l u x of m o l e c u l e s may be r e g u l a t e d by the f i b r i l l a r c o l l a r s . In the case of amphidinium, macromolecules were observed p r i m a r i l y i n the c o n s t r i c t e d r e g i o n of both p u s u l e s w i t h a tendency t o move f u r t h e r towards the lumen of the v e s i c u l a r chamber. T h i s o b s e r v a t i o n does not support Abe's hypotheses (1981) t h a t the two p u s u l e s i n the d i n o f l a g e l l a t e c e l l a r e m o r p h o l o g i c a l l y and f u n c t i o n a l l y d i f f e r e n t ( i . e . one f o r uptake and the o t h e r f o r e x c r e t i o n ) . I t was a l s o observed t h a t exogenous HRP became l o c a l i z e d i n both the c y t o p l a s m i c v e s i c l e s and the e x t e n s i v e c a n a l i c u l i of P r o r o c e n t r u m . S i n c e the p u s u l e s r e p r e s e n t a l a r g e p o r t i o n of the c e l l s u r f a c e a r e a , i t i s l i k e l y t h a t they p o s s e s s h i g h t r a n s p o r t c a p a b i l i t i e s . The c l o s e a s s o c i a t i o n of s m a l l 96 c o a t e d v e s i c l e s , EE and G o l g i a p p a r a t u s w i t h the p u s u l e suggested t h a t i n t r a c e l l u l a r components may a l s o p l a y an i m p o r t a n t r o l e i n r e g u l a t i n g the topography of the p u s u l a r s u r f a c e . Coated v e s i c l e s have been r e p o r t e d t o be a s s o c i a t e d w i t h G o l g i a p p a r a t u s (e.g. Gonyaulax p o l y e d r a , Gaudsmith and Dawes 1972; Amphidinium c r y o p h i l u m , W i l c o x _± a l . 1982) and the p u s u l e (e.g. K o f o i d i n i g m p a v i l l a r d i f Cachon _t _1. 1983). F u r t h e r m o r e , the o c c u r r e n c e of many m i t o c h o n d r i a i n the v i c i n i t y of the p u s u l e s u g g e s t s a h i g h energy r e q u i r e m e n t . An exposure t o low t e m p e r a t u r e would then be expected t o i n h i b i t the energy r e q u i r i n g p r o c e s s e s of the p u s u l e . The presence of p e c u l i a r H R P - l a b e l e d s t r u c t u r e s w i t h i n c a n a l i c u l i t r a v e r s i n g the c y t o p l a s m and w i t h i n v a c u o l e s beneath the amphiesma v e s i c l e s of £. micans r e c a l l s analogous c r y s t a l - l i k e i n c l u s i o n s p r e v i o u s l y r e p o r t e d i n Gonyaulax  p o l y e d r a , P y r o d i n i u m bahamense (Gaudsmith and Dawes 1972), C r y p t h e c o d i n i u m c o h h i i , gymnodinlmi SPlendejjS (Gold and Pokorny 1973). I n t e r e s t i n g l y , some of these i n c l u s i o n s were shown t o be i n t r a c e l l u l a r l y produced i n response t o c e r t a i n modes of n u t r i t i o n (Gold and Pokorny 1973, Pokorny and G o l d 1973) . In t h i s c o n n e c t i o n , i t i s i n t e r e s t i n g t o l o o k at the c o r r e l a t i o n between d i n o f l a g e l l a t e morphology and n u t r i t i o n . The known modes of d i n o f l a g e l l a t e n u t r i t i o n i n c l u d e a u t o t r o p h i c w i t h (e.g. P y r o d i n i u m limbatum) or w i t h o u t 97 v i t a m i n r e q u i r e m e n t s (e.g. P e r i d i n i u m w i l l e i ) , p h a g o t r o p h i c (e.g. Gymnodinium h e l v e t i c u m ) ; m i x o t r o p h i c (e.g. C e r a t i u m h i r u n d e l l a ) : p a r a s i t i c (e.g. Haplozoon a x i o t h e l l a e ) and s y m b i o t i c (e.g. P e r i d i n i u m f o l i a c e u m ) , ( K o f o i d and Swezy 1921, Lebour 1925, T a y l o r 1973, S i e b e r t and West 1974, Lee 1977, I r i s h 1979, M o r r i l l and L o e b l i c h 1979, Tappan 1980, H o l t and P f i e s t e r 1981, Spero and Moree 1981, Spero 1982). In r e l a t i o n t o these n u t r i t i o n a l modes, the t h e c a t e p h o t o s y n t h e t i c s p e c i e s are b e l i e v e d t o show a p r e d o m i n a n t l y h o l o p h y t i c type of n u t r i t i o n , w h i l e the a t h e c a t e n o n - p h o t o s y n t h e t i c s p e c i e s a r e m o s t l y h o l o z o i c . I n t e r e s t i n g l y the t h e c a t e c o l o r l e s s s p e c i e s , thought t o be s a p r o p h y t e s , e x h i b i t a w e l l - d e v e l o p e d p u s u l e ( s ) (e.g. p y o t o p e r i d i n i u m (Peridin,ium,) £e_llncidum» ( K o f o i d and Swezy 1921, Lebour 1925). N e v e r t h e l e s s , i t s h o u l d be noted t h a t the t o t a l absence or the presence of p o o r l y developed p u s u l e has been r e p o r t e d i n some osm o t r o p h i c p a r a s i t i c or s y m b i o n t i c forms as w e l l as i n the c y s t i c phase of o t h e r d i n o f l a g e l l a t e s (Bibby and Dodge 1972). The p r e s e n t i n v e s t i g a t i o n w i t h two p h o t o s y n t h e t i c d i n o f l a g e l l a t e s showed t h a t the t h e c a t e £. micans has w e l l developed p u s u l e s w i t h g r e a t e r c a p a c i t y f o r the uptake of m o l e c u l a r markers used i n t h i s study than the n o n - t h e c a t e A., c a r t e r a e . At p r e s e n t , the p h y s i o l o g i c a l s i g n i f i c a n c e of t h i s uptake i s not f u l l y u n d e r s t o o d . 98 N. F a c t o r s r e g u l a t i n g p i n o c y t o s i s In t h i s s t u d y , c e l l s of the same or from d i f f e r e n t c u l t u r e s showed some degree of v a r i a b i l i t y on the r a t e of l i g a n d uptake. T h i s c o u l d be due t o the e f f e c t of f a c t o r s such a s , temperature and the p h y s i o l o g i c a l s t a t e of the c e l l i . e . s t a t e of n u t r i t i o n and stage of the l i f e c y c l e . P i n o c y t o s i s , s p e c i a l l y r e c e p t o r - m e d i a t e d p i n o c y t o s i s , i s known t o be a temperature-dependent p r o c e s s ( S i l v e r s t e i n et a l . 1977). As e x p e c t e d , the i n c r e a s e of l i g a n d uptake i n the upper temperature range (ca.. 25°C) was observed i n a l l t h r e e p h y t o f l a g e l l a t e s . I t s h o u l d be p o i n t e d out t h a t these p h y t o f l a g e l l a t e s were p h o t o a u t o t r o p h i c a l l y m a i n t a i n e d i n a n u t r i e n t complete medium, t h e r e f o r e t h e r e was no apparent n u t r i t i o n a l requirement f o r p i n o c y t o s i s . However, D u n a l i e l l a i s known t o take i n c e r t a i n d i s s o l v e d o r g a n i c s u b s t a n c e s , such as v i t a m i n B 1 2 , which i t does not r e q u i r e as a n u t r i e n t f o r growth ( S c o t t 1981). The uptake of v i t a m i n s has been suggested t o occur e i t h e r by means of osmosis (Ginzburg 1969) or by p i n o c y t o s i s (Steinman _ £ s i . 1983). 0. Evidence f o r p i n o c y t o s i s i n o t h e r p l a n t c e l l s P i n o c y t o s i s has been r a r e l y r e p o r t e d f o r the o s m o t r o p h i c p h y t o f l a g e l l a t e s . Without d i r e c t e v i d e n c e , p i n o c y t o s i s was p r e v i o u s l y s u r mised f o r D u n a l i e l l a p r i m o l e c t a based on the abundance of v e s i c l e s beneath the plasma membrane, and the 99 presence of l y s o s o m e - l i k e b o d i e s . The p i n o c y t i c p r o c e s s was b e l i e v e d to p l a y a r o l e i n the r e c o v e r y of n u t r i e n t s from the l y s i s of moribund c e l l s (Eyden 1975). In the case of Euglena  g r a c i l i s , the i n t e r n a l i z a t i o n of HFP through the t u b u l a r c a n a l - r e s e r v o i r r e g i o n was thought to r e p r e s e n t a mechanism f o r r e c o v e r i n g l a r g e m o l e c u l e s , o t h e r w i s e l o s t d u r i n g c o n t r a c t i l e v a c u o l e d i s c h a r g e . M u l t i v e s i c u l a r or membranous s t r u c t u r e s a l s o termed "lomasomes" (Esau .§_£ a l . 1966, Walker and B i s a l p u t r a 1967, Heyn 1971) have been commonly c o n s i d e r e d as a r t i f a c t u a l or i f n o t , as a means f o r the d i s c h a r g e of c e l l w a l l p r e c u r s o r s and s e c r e t o r y p r o d u c t s . S i m i l a r p e r i p h e r a l s t r u c t u r e s have been d e s c r i b e d as "plasmalemasomes" (Mesquita 1970) and s uggested t o p l a y a r o l e i n p i n o c y t o s i s . A l t h o u g h s i m i l a r i n appearance lomasomes and plasmalemasomes d i f f e r i n t h e i r o r i g i n and f u n c t i o n . Based on p r e v i o u s s t u d i e s , i t i s suggested t h a t some of the ambiguous r e s u l t s o b t a i n e d f o r p i n o c y t o s i s i n h i g h e r p l a n t s c o u l d be a t t r i b u t e d t o the n a t u r e of the markers used, v i z . r i b o n u c l e a s e , lysozyme, p o l y - L - l y s i n e , or u r a n y l a c e t a t e . A p p a r e n t l y these markers induced m o r p h o l o g i c a l and m e t a b o l i c d e l e t e r i o u s e f f e c t s , such as l e s i o n s and a l t e r a t i o n s i n the p e r m e a b i l i t y of the plasma membrane, changes i n the FNA m e t a b o l i s m , p r o t e i n s y n t h e s i s and c e l l 100 growth (Brachet 1954, 1955, Yeoman 1962, McLaren and B r a d f u t e 1966, Drew and McLaren 1970 , Drew .§_t a_l. 1970). On the o t h e r hand, s t u d i e s on p l a n t p r o t o p l a s t s were more s u c c e s s f u l i n d e m o n s t r a t i n g the uptake of p o l y s t y r e n e l a t e x p a r t i c l e s (Mayo and C o c k i n g 1969a), l a t e x spheres ( W i l l i s o n jet a l . 1971), to b a c c o mosaic v i r u s (Cocking and P o j n a r 1969) or c a t i o n i z e d f e r r i t i n (Joachim and Robinson 1984). P. Membrane r e c y c l i n g In s p i t e of i n t e n s i v e p i n o c y t o s i s observed i n macrophages and f i b r o b l a s t s , the t o t a l s u r f a c e a r e a of the plasma membrane and the v a c u o l a r system remains c o n s t a n t (Steinman and Cohn 1972, Cohn and Steinman 1982 ) . These f i n d i n g s s u p port the i d e a t h a t membrane c o n s t i t u e n t s are r e c y c l e d back t o the c e l l s u r f a c e p r i o r t o d e g r a d a t i o n or r e s y n t h e s i s . L i t t l e i s known about the mechanism of membrane r e c y c l i n g . However, t i n y v e s i c l e s c l o s e l y r e l a t e d t o endosomes, lysosomes or G o l g i a p p a r a t u s have been suggested t o p l a y a r o l e i n such event ( S i l v e r s t e i n .£_£ a l . 1977). I t i s p o s s i b l e t h a t i n D u n a l i e l l a the abundant G o l g i - d e r i v e d v e s i c l e s c o u l d be i n v o l v e d i n the e x o c y t o s i s of s e c r e t o r y g r a n u l e s , thus r e t u r n i n g to the c e l l s u r f a c e p o r t i o n s of the plasma membrane r e t r i e v e d d u r i n g p i n o c y t o s i s . The i nvolvement of smooth and c o a t e d v e s i c l e s i n s e c r e t i o n and membrane r e c y c l i n g have been s t u d i e d i n o t h e r p l a n t systems (Mollenhauer and Whaley 101 1963, Bonnett and Newcomb 1966, N o r t h c o t e and P i c k e t t - H e a p s 1966, Ryser 1979, Swanson et a l . 1981, Van Der V a l k and Fowke 1981, Singh 1984). The s i m u l t a n e o u s o c c u r r e n c e of membrane r e c y c l i n g and i n t e n s i v e f l u i d - p h a s e e n d o c y t o s i s has a l s o been r e p o r t e d i n c e l l s p r i m a r i l y i n v o l v e d i n s e c r e t i o n (e.g. i n n e u r o h y p o p h y s i s , a d r e n a l m e d u l l a and i s l e t c e l l s ; Steinman £i a l . 1976). There i s a l r e a d y c o n s i d e r a b l e i n f o r m a t i o n , i n the l i t e r a t u r e , t o support the view t h a t e x c r e t i o n of o r g a n i c matter i s normal f o r D u n a l i e l l a . Depending upon the growth c o n d i t i o n s used, D u n a l i e l l a spp. have been r e p o r t e d t o show c o n s i d e r a b l e e x c r e t i o n of o r g a n i c carbon ( C r a i g i e .§_£. a l . 1966, Hunstsman 1972) o r g a n i c n i t r o g e n (Newell e± a l . 1 9 72), and o r g a n i c phosphorus ( K u e n z l e r 1970). Wh i l e a s m a l l p r o p o r t i o n of the e x c r e t e d o r g a n i c carbon may c o n s i s t of the g l y c e r o l p h o t o s y n t h e s i z e d by D u n a l i e l l a as an osmoticum i n i t s s e l f - d e f e n s e a g a i n s t s a l i n i t y changes (Enhuber and Gimmler 1980, Jones and Galloway 1979) the g r e a t e r p r o p o r t i o n i s e x p e c t e d t o be composed of c a r b o h y d r a t e r e p o r t e d as c o n t i n u o u s l y r e l e a s e d by p_. t e r t i o l e c t a r e g a r d l e s s of p h y s i o l o g i c a l s t a t e (Hunstsman 1972). Q. Some comments on the e v o l u t i o n a r y mode of macromolecule i n t e r n a l i z a t i o n i n p r o t i s t s The p r o c e s s e s of e n d o c y t o s i s and e x o c y t o s i s w i t h membrane r e c y c l i n g have been t r a c e d back t o p o s t u l a t e d 102 p r o k a r y o t i c forms which had the a b i l i t y t o r e p l a c e t h e i r c e l l w a l l by an a c t i n - m y o s i n - l i k e m i c r o f i l a m e n t system ( C a v a l i e r - S m i t h 1975). S i n c e most a v a i l a b l e data on t h i s s u b j e c t have d e a l t w i t h n o n - p h o t o s y n t h e t i c organisms, c o n s i d e r a b l e work on p h o t o s y n t h e t i c forms must be done b e f o r e making any g e n e r a l statement on the f l e x i b i l i t y of t h e i r e v o l u t i o n a r y b e h a v i o r . However, i t i s noteworthy t h a t p r e s e n t l i v i n g forms, whether p h o t o s y n t h e t i c or n o n - p h o t o s y n t h e t i c , have e v o l v e d from s i m p l y o r g a n i z e d a n c e s t o r s and are s u c c e s s f u l s u r v i v o r s of a l o n g e v o l u t i o n a r y p r o c e s s . The t h r e e p h y t o f l a g e l l a t e s used i n t h i s study may r e p r e s e n t two h y p o t h e t i c a l e v o l u t i o n a r y l i n e s . The f i r s t i n c l u d e s those organisms (e.g. D u n a l i e l l a ) t h a t may l a c k a r i g i d c e l l w a l l as w e l l as a s p e c i a l i z e d compartment f o r macromolecule i n t e r n a l i z a t i o n . The second comprises those forms (e.g. P r o r o c e n t r u m and Amphidinium) t h a t are e n c l o s e d i n a complex c o v e r i n g and p o s s e s s a s p e c i a l i z e d d e v i c e f o r f o o d uptake. 1. As p r e v i o u s l y d e s c r i b e d i n t h i s work, D u n a l i e l l a i n t e r n a l i z e s exogenous m o l e c u l e s t h a t b i n d t o s p e c i f i c r e c e p t o r s of i t s plasma membrane. These l i g a n d - r e c e p t o r complexes, i n i t i a l l y randomly d i s t r i b u t e d , move al o n g the c e l l s u r f a c e and form pa t c h e s f o l l o w e d by c a p p i n g b e f o r e 103 macromolecule i n t e r n a l i z a t i o n . I t i s thus v e r i f i e d t h a t , i n D u n a l i e l l a , p i n o c y t o s i s o c c u r s i n the r e g i o n of f l a g e l l a r emergence and away from the cup-shaped c h l o r o p l a s t . I t proceeds through a d e g r a d a t i v e l y s o s o m a l system and i s c o u n t e r b a l a n c e d by G o l g i - d e r i v e d v e s i c l e s g u i d e d by m i c r o t u b u l e s . In c o n t r a s t the p h o t o s y n t h e t i c chrysomonad Ochromonas d a n i c a changes from the f l a g e l l a t e s t a t e t o an amoeboid form t o e n g u l f l a r g e food m a t e r i a l such as b a c t e r i a or c o c c o i d c y a n o b a c t e r i a (Daley e i a l . 1973). Such b e h a v i o r t r i g g e r e d the s p e c u l a t i o n t h a t amoebae arose from a c h r y s o m o n a d - l i k e p r e c u r s o r . A l t h o u g h p r e s e n t l y regarded as h i g h l y s p e c i a l i z e d (as m i c r o c o n s u m e r s ) , amoebae depend on pseudopodia f o r f e e d i n g and l o c o m o t i o n . A c c o r d i n g t o Hanson (1976), t h i s c h a r a c t e r i s t i c has " d e n i e d them the e v o l u t i o n a r y p o t e n t i a l " g e n e r a l l y r e c o g n i z e d i n h i g h e r p r o t o z o a . A l s o i n c l u d e d i n the f i r s t g r o u p i n g of organisms are those p r o t o z o a (such as r a d i o l a r i a n s a c a n t h a r i a n s and h e l i o z o a n s ) t h a t r e l y on pseudopodia f o r c a p t u r i n g p a s s i n g p r e y . 2. The p r o c e s s . o f macromolecule i n t e r n a l i z a t i o n through the f l a g e l l a r c a n a l - p u s u l e system i n the d i n o f l a g e l l a t e s s t u d i e d here shows s t r o n g s i m i l a r i t y t o t h a t known t o occur v i a the f u r r o w - g u l l e t system of cryptomonads or the t u b u l a r c a n a l - r e s e r v o i r system of e u g l e n o i d s . Such s i m i l a r i t y , a l o n g w i t h t h a t of the c e l l c o v e r i n g morphology, l e n d s some weight 104 to c u r r e n t hypotheses ( T a y l o r 1980, L o e b l i c h 1984) on e v o l u t i o n a r y p a r a l e l l i s m between these t h r e e groups of p r o t i s t s . S u p p o r t i n g e v i d e n c e f o r t h i s second e v o l u t i o n a r y l i n e i s a l s o p r o v i d e d by the cytostonie - c y t o p h a r y n x system of k i n e t i d a l p r o t o z o a (e.g. z o o f l a g e l l a t e s and c i l i a t e s ) . In p r i m i t i v e c i l i a t e s ( i . e . gymnostomes), such c o r t i c a l s p e c i a l i z a t i o n , l o c a t e d a t the c e l l s u r f a c e or at the bottom of the v e s t i b u l u m , i s m a i n l y i n v o l v e d i n the uptake of l a r g e f o o d m a t e r i a l (Jones 1974, F e n c h e l 1980). As c i l i a t e e v o l u t i o n proceeded (e.g. i n o l i g o and polyhymenophorans), the uptake of s m a l l s i z e p a r t i c l e s became e f f e c t e d through a m i c r o t u b u l e - r i c h c y t o s t o m a l system (Hutner and C o r l i s s 1976). As a r e s u l t of t h i s e n d o c y t i c development i n Paramecium  caudatum f food e g e s t i o n and membrane r e c y c l i n g i s a s s u r e d by a complex m i c r o t u b u l a r system which mediates the t r a n s p o r t of food v a c u o l e s t o the c y t o p r o c t ( A l l e n 1974, A l l e n and Wolf 1974) . In r e l a t i n g e n d o c y t o s i s s u c c e s s t o the s t r u c t u r a l o r g a n i z a t i o n of the c e l l c o v e r i n g , i t i s remarkable t h a t D u n a l i e l l a has a c h i e v e d g r e a t s u c c e s s w i t h the s i m p l e s t c o v e r i n g of a plasmalemma w h i l e d i n o f l a g e l l a t e s have deve l o p e d a l t e r n a t i v e modes t o surmount the s t r u c t u r a l c o m p l e x i t y of an amphiesma. 105 KEY FOR FIGURES a amorphous l a y e r ar amphiesmal r i d g e ap a p i c a l s p i n e apo a p i c a l p o c k e t bb b a s a l body c s l e n d e r c a n a l c l p r i m a r y c o l l a r c 2 se c o n d a r y c o l l a r C c h l o r o p l a s t e e n d o c y t i c v e s i c l e (endosome) E e p i c o n e EP e n d o p l a s m i c r e t i c u l u m f f i b r i l l a r m a t e r i a l F f l a g e l l u m Fc f l a g e l l a r c a n a l Fch f l a g e l l a r chamber G G o l g i a p p a r a t u s gr g i r d l e Gy g l y c o c a l y x H hypocone i i n n e r membrane i l i n n e r c o a t l a y e r L l i p i d i n c l u s i o n LF l o n g i t u d i n a l f l a g e l l u m Ly l y sosome m mucocyst M m i t o c h o n d r i a MT m i c r o t u b u l e s N n u c l e u s o o u t e r membrane o l o u t e r l a y e r p s u r f a c e pore p p u s u l e p i p i a t e l e t PM plasma membrane s s u l c u s t t h e c a l p l a t e T t r i c h o c y s t TF t r a n s v e r s e f l a g e l l u m V v e s i c l e V v a c u o l e 106 F i g . 1. Diagrammatic r e p r e s e n t a t i o n of D u n a l i e l l a t e r t i o l e c t a c e l l i l l u s t r a t i n g the p i n o c y t i c uptake of exogenous m o l e c u l e s bound t o the c e l l s u r f a c e ( r e c e p t o r - m e d i a t e d e n d o c y t o s i s ) or i n the d i s s o l v e d form ( f l u i d - p h a s e e n d o c y t o s i s ) . The broken arrow i n d i c a t e s a c o a t e d p i t i n the a p i c a l r e g i o n of the c e l l . V a c u o l e s (V) c o n t a i n i n g the l i g a n d are d i s t r i b u t e d i n the c y t o p l a s m i c area s u r r o u n d i n g the n u c l e u s (N). Note the s p a t i a l arrangement of o t h e r o r g a n e l l e s such as the G o l g i a p p a r a t u s (G) m i t o c h o n d r i a (M) and the cup-shaped c h l o r o p l a s t (C) w i t h the p y r e n o i d ( P y ) . 106 .1 107 F i g s . 2-5. F i g . 2. F i g . 3. F i g . 4. F i g . 5. L i g h t m i c r o g r a p h s of £. t e r t i o l e c t a c e l l s t r e a t e d w i t h l e c t i n s (100 pq/mL) and t h e i r s p e c i f i c s u g a rs (100 jjq/ mL) . Wheat germ a g g l u t i n i n (WGA) o n l y . x350. WGA p l u s N - a c e t y l - D - g l u c o s a m i n e . x250. Limvilus polyphemus a g g l u t i n i n (LPA) o n l y . x350. LPA p l u s s i a l i c a c i d , x 300. 108 F i g s . 6-11 . C e l l s u r f a c e b i n d i n g and i n t e r n a l i z a t i o n of FITC-WGA a p p l i e d t o l i v i n g D u n a l i e l l a c e l l s , at 250C. F i g . 6. C e l l s u r f a c e b i n d i n g (arrows) a f t e r 5 rain of i n c u b a t i o n . x l , 8 0 0 . F i g . 7. Capping of WGA-receptor complexes (arrow) i n the a n t e r i o r r e g i o n of the c e l l (10 min of i n c u b a t i o n ) . x2 ,400. F i g . 8. E n d o c y t i c v e s i c l e s a f t e r 15 min of i n c u b a t i o n , x 2,000. F i g . 9. The arrow i n d i c a t e s c a p p i n g d u r i n g the p r o c e s s of e n d o c y t o s i s (20 min of i n c u b a t i o n ) . x2,400. F i g . 10. V a c u o l e s c o n t a i n i n g the l e c t i n are c o n c e n t r a t e d i n the p e r i n u c l e a r r e g i o n a f t e r 30 min of i n c u b a t i o n . x2,500. F i g . 11. Capping of l e c t i n - r e c e p t o r complexes (arrow) and l e c t i n a c c u m u l a t i o n w i t h i n v a c u o l e s a f t e r 30 min of i n c u b a t i o n . x2,400. 109 E l e c t r o n m i c r o g r a p h s of £. t e r t i o l e c t a c e l l s t r e a t e d w i t h t h r e e d i f e r e n t l e c t i n s (WGA, LPA, ConA), and WGA i n presence of i t s s p e c i f i c s u g a r . I n t e n s e b i n d i n g p a t t e r n a l o n g the c e l l s u r f a c e , i n c l u d i n g the s u r f a c e of the f l a g e l l a ( F ) . x45,000 and x36,000 r e s p e c t i v e l y . C l u s t e r i n g of WGA-receptor complexes ( a r r o w ) . x l 8 , 0 0 0 . C e l l s u r f a c e l a b e l i n g produced by LPA. Some ev i d e n c e f o r l i g a n d i n t e r n a l i z a t i o n through plasma membrane i n v a g i n a t i o n ( a r r o w ) . Nucleus (N). x25,000 and x21,400 r e s p e c t i v e l y . S u r f a c e b i n d i n g produced by Con A. L i g a n d i n t e r n a l i z a t i o n (arrow) p r o c e e d i n g v i a v a c u o l e - l i k e s t r u c t u r e s (V) . Nucleus (N). x25,500. C o n t r o l . WGA p l u s N - a c e t y l g l u c o s a m i n e . Note the absence of e l e c t r o n dense m a t e r i a l w i t h i n v a c u o l e s (V) of the a p i c a l r e g i o n of the c e l l . C h l o r o p l a s t ( C ) , n u c l e u s (N), and G o l g i a p p a r a t u s (G). x l 0 , 2 0 0 . 10'J.l 110 F i g . 19. F l u o r e s c e n c e m i c r o g r a p h of J), t e r t i o l e c t a c e l l i n c u b a t e d w i t h (FITC-HFP) f o r 30 min. x3,000. F i g s . 20-24. E l e c t r o n m i c r o g r a p h s of D u n a l i e l l a c e l l s i n c u b a t e d w i t h HFP. F i g . 20. S e c t i o n showing p i n o c y t i c uptake of HFP v i a c o a t e d p i t s (arrow) a f t e r 5 min of i n c u b a t i o n . x66,500. F i g 21. Two types of p i n o c y t i c v e s i c l e s (arrows) i n c e l l s exposed t o HFP, f o r 10 min. x59,700. F i g s . 22,23. HFP r e a c t i o n p r o d uct i n the G o l g i c i s t e r n a e (G) and p r o x i m a l v e s i c l e s (arrows) of c e l l s t r e a t e d w i t h HFP f o r 20 min. x74,000 and x73,300 r e s p e c t i v e l y . F i g . 24. S m a l l l a b e l e d v e s i c l e s (arrows) f u s e d w i t h l a r g e v a c u o l e s (V) a f t e r 60 min of HEP i n c u b a t i o n , x 20,600. F i g . 25. S e c t i o n of a c o n t r o l c e l l t r e a t e d w i t h DAB/H2O2 w i t h o u t p r e v i o u s i n c u b a t i o n w i t h HFP. x l 6 , 6 0 0 . F i g . 26. C o n t r o l c e l l exposed t o HFP w i t h o u t DAB/H2O2 t r e a t m e n t . The arrow i n d i c a t e s plasma membrane i n f o l d i n g s l a c k i n g HFP r e a c t i o n p r o d u c t . x30,280. 110.1 I l l F i g s . 27,28. D u n a l i e l l a c e l l s showing the s t r u c t u r a l arrangement of s u b c e l l u l a r o r g a n e l l e s . F i g s . 29-32. S e c t i o n of c e l l s t r e a t e d w i t h c a t i o n i z e d f e r r i t i n ( CF). F i g . 27. L i g h t m i c r o g r a p h of c e l l s showing the h i g h d e n s i t y of v a c u o l e s (arrow) i n the a p i c a l r e g i o n of the c e l l . x2,350. F i g . 28. F r e e z e - e t c h i n g of a c e l l showing the v a c u o l e s (arrows) i n the p e r i n u c l e a r r e g i o n j u s t above the cup-shaped c h l o r o p l a s t ( C ) . Nucleus (N). x7,500. F i g s . 29,30. C e l l s u r f a c e c o a t c h a r a c t e r i z e d by e l e c t r o n - dense pat c h e s a l t e r n a t i n g w i t h l e s s dense areas ( a r r o w s ) . Plasma membrane (PM). x23,000 and x24,500 r e s p e c t i v e l y . F i g . 31. V e s i c l e s (arrows) c l o s e l y a s s o c i a t e d w i t h m i c r o t u b u l e s (MT). x26,250. F i g . 32. C e l l - c e l l a d h e s i o n through the s u r f a c e c o a t . x21 ,050. 112 F i g s . 33-38. S e c t i o n s of £. t e r t i o l e c t a c e l l s showing c o a t e d p i t s a f t e r t r e a t m e n t w i t h CF f o r 10 min. F i g s . 33,34. The arrows i n d i c a t e some c o a t e d a r e a s of the i n n e r s u r f a c e of the plasma membrane (PM). x65,000 and x70 ,680 r e s p e c t i v e l y . F i g s . 35,37. Note the presence of c o a t e d p i t s (arrow) and uncoated v e s i c l e s (v) i n the v i c i n i t y of the f l a g e l l a ( F ) . x31,570 and x73,300 r e s p e c t i v e l y . F i g s . 36,38. High m a g n i f i c a t i o n of c o a t e d p i t s showing the b r i s t l e - l i k e c o a t ( a r r o w ) . x23,000 and x87,430 r e s p e c t i v e l y . 1J 2. J 113 F i g s . 39-43. D i f f e r e n t s e c t i o n s of £. w i t h CF f o r 1 h. c e l l s t r e a t e d F i g . 39. F i n e s t r u c t u r e of one c e l l d e p i c t i n g s m o o t h - s u r f a c e d v e s i c l e s (e) beneath the plasma membrane (PM), c o a t e d p i t s (arrows) and v a c u o l e s (V) c o n t a i n i n g CF. Nucleus (N) , C h l o r o p l a s t ( C ) , G o l g i a p p a r a t u s (G). x24 ,100. F i g . 40. V a c u o l e (V) w i t h some CF detached from i t s i n n e r s u r f a c e . x35,700. F i g . 41. Note the a c c u m u l a t i o n of G o l g i - d e r i v e d c o a t e d v e s i c l e s (arrows) i n the a p i c a l r e g i o n of the c e l l . x37,000. F i g . 42. The arrows i n d i c a t e s m o o t h - s u r f a c e d v e s i c l e s w i t h or w i t h o u t e l e c t r o n dense c o n t e n t s i n the G o l g i (G) r e g i o n . x40,000. F i g . 43. Tin y v e s i c l e s (arrows) a r r a n g e d i n rows, almost p e r p e n d i c u l a r t o the c e l l s u r f a c e . x62,500. 113.1 114 F i g s . 44-47. A c i d phosphatase a c t i v i t y i n D. t e r t i o l e c t a c e l l s and c o r r e s p o n d i n g c o n t r o l . F i g . 44. S e c t i o n showing some s i t e s of a c i d phosphatase a c t i v i t y : i n c y t o p l a s m i c v e s i c l e s c l o s e t o the c i s f a c e of the G o l g i ( a r r o w h e a d s ) , i n the G o l g i compartment (G) and w i t h i n p r i m a r y lysosomes ( L y ) . x49,450. F i g s . 45,46. Secondary lysosomes (V) c o n t a i n i g a c i d phosphatase r e a c t i o n p r o d uct are l o c a t e d around the n u c l e u s (N). x!7,230 and x20,000 r e s p e c t i v e l y . F i g 47. C o n t r o l . Note the absence of a c i d phosphatase r e a c t i o n p r o d uct w i t h i n v a c u o l e s (V) of c e l l s p r o cessed. w i t h o u t the s u b s t r a t e , sodium g l y c e r o p h o s p h a t e . x!8,000. 115 F i g s . 48,49. Scanning e l e c t r o n m i c r o g r a p h s (SEM) of h. c a r t e r a e c e l l s ( v e n t r a l s i d e ) i l l u s t r a t i n g the p o l y g o n a l amphiesmal v e s i c l e s bounded m a i n l y by f i v e or s i x s i d e s . Each p o l y g o n a l u n i t i s t r a v e r s e d by one t o t h r e e pores (arrowheads). Epicone ( E ) , hypocone (H) , t r a n s v e r s e f l a g e l l u m ( T F ) , l o n g i t u d i n a l f l a g e l l u m ( L F ) , g i r d l e (gr) and s u l c u s ( s ) . x7,140 and xl6,700 r e s p e c t i v e l y . 116 F i g s . 50 ,51 . F i g . 50. F i g . 51 . F i g s . 52,53. SEM of A. c a r t e r a e c e l l s . A p i c a l view of a c e l l showing the epicone ( E ) , the t r a n s v e r s e f l a g e l l u m ( T F ) , the s t r i a t e d s t r a n d ( a r r o w ) , and the g i r d l e ( g r ) . x5,900. D o r s a l view . Epicone (E) and hypocone (H). x5,170. Transmission e l e c t r o n micrographs (TEM) i l l u s t r a t i n g some amphiesmal v e s i c l e s with s i x s i d e s . The arrow i n f i g . 53 i n d i c a t e s a newly formed septum between two daughter amphiesmal v e s i c l e s . Note the presence of s u r f a c e pores (arrowheads), and g l y c o c a l y x (Gy). x40,000 and x45,830 r e s p e c t i v e l y . 116.1 117 F i g s . 54, 55. F r e e z e - e t c h i n g r e p l i c a of the amphiesmal v e s i c l e s of h. c a r t e r a e c e l l s d i s p l a y i n g f o u r t o seven s i d e s . One t o t h r e e pores ( l a r g e arrowheads) a r e r e c o g n i z e d as w e l l as f i n e p u n t u a t i o n s ( s m a l l a r r o w h e a d s ) . xl4,170 and x l l , 0 0 0 r e s p e c t i v e l y . 117.1 118 Histogram r e p r e s e n t i n g the s u r f a c e area of the amphiesmal v e s i c l e s of A., c a r t e r a e c e l l s p r e p a r e d by SEM, TEM, or the f r e e z e f r a c t u r e t e c h n i q u e . Note t h a t the i n t e r m e d i a t e - s i z e v e s i c l e s r a n g i n g from 1.1 to 1.7 pv\2 are predominant, w h i l e the l a r g e r ( g r e a t e r than 1.7 jum2) or the s m a l l e r s i z e v e s i c l e s ( s m a l l e r than 1.1 ;jm2) show low freq u e n c y d i s t r i b u t i o n . 118.1 119 F i g s . 57-60. F l u o r e s c e n c e m i c r o g r a p h s of l i v e c a r t e r a e c e l l s t r e a t e d w i t h rhodamine B or f l u o r o B o r a P showing d i s t r i b u t i o n of l i p i d b o d i e s . F i g s . 57, 58. Rhodamine B. x3,580 and x3,500 r e s p e c t i v e l y . F i g s . 59,60. F l u o B o r a P. x3,500 and x3,400 r e s p e c t i v e l y . 119.1 120 TEM s e c t i o n s showing u n s a t u r a t e d and s a t u r a t e d (?) l i p i d s (L) of aged A. c a r t e r a e c e l l s . x32,140 and x25,000 r e s p e c t i v e l y . T r i c h o c y s t s of Amphidinium. P r i m o r d i a of t r i c h o c y s t s (T) a r e observed i n the v i c i n i t y of the endoplasmic r e t i c u l u m - G o l g i (G) r e g i o n . Note the presence of numerous c o a t e d v e s i c l e s ( a r rowheads). x42,000. T r i c h o c y s t s rods (T) w i t h i n v a c u o l e s ( V ) , presumably f u n c t i o n i n g as autophagosomes, surrounded by endoplasmic r e t i c u l u m ( E F ) . x54 ,000. 120.1 121 F i g s . 65-68. F l u o r e s c e n c e m i c r o g r a p h s showing the g l y c o c a l y x of l i v e A. c a r t e r a e c e l l s a f t e r the f o l l o w i n g t r e a t m e n t : F i g . 65. A c r i d i n e orange, x 2,900. F i g . 66 FITC-ConA. Note the heterogenous s u r f a c e b i n d i n g p a t t e r n , x 3,000. F i g . 67. WGA. x 3,500. F i g . 68. SBA. x3,600. 122 F i g s . 69-72. M i c r o g r a p h s of u n s t a i n e d ( l a c k i n g p o s t s t a i n i n g ) ft. c a r t e r a e c e l l s , t r e a t e d w i t h the l e c t i n SBA to show the i n t e n s e l a b e l i n g of the c e l l s u r f a c e . F i g . 69. x l l , 2 0 0 . F i g . 70. x l 3 , 4 0 0 . F i g . 71. x l 4 , 0 0 0 . F i g . 72. x!2,000. 122.1 123 F i g s . 73-76. S e c t i o n s of A., c a r t e r a e c e l l s d o u b l e - s t a i n e d w i t h a l c i a n b l u e and ruthenium red (AlcB-RF) . x l 7 , 5 0 0 . F i g . 73. A n i o n i c p o l y s a c c h a r i d e coat ( g l y c o c a l y x ) a l o n g the c e l l s u r f a c e . x!7,500. F i g . 74. 2 d i s t i n c t l a y e r s ( i l , o l ) of the c e l l c o a t on the o u t s i d e of the plasma membrane (PM). Note the p a t t e r n of a l t e r n a t i n g l i g h t and dense s t a i n i n g a r e a s (arrows) and the " f u z z " m a t e r i a l on the i n n e r s u r f a c e ( i ) of the amphiesmal v e s i c l e s . x65,000. F i g . 75. Amphiesmal area u n d e r l a i n by f i b r i l l a r m a t e r i a l ( f ) and an a r r a y of m i c r o t u b u l e s (MT). x37,100. F i g . 76. C e l l - t o - c e l l a d h e s i o n mediated by the g l y c o c a l y x (Gy). Note a c e l l s u r f a c e pore (p) formed by plasma membrane (PM) i n v a g i n a t i o n . An endosome (e) i s p r o j e c t e d i n t o the p e r i p h e r a l c y t o p l a s m i c v a c u o l e ( V ) . x47,850. F i g . 77. C o n t r o l c e l l s adhered by t h e i r u n s t a i n e d g l y c o c a l y x (Gy). Note each s e t of the apposing membranes: the plasma membrane (PM), the o u t e r and the i n n e r membrane ( o , i ) of the amphiesmma and the amorphous l a y e r ( a ) . x67,700. 124 F i g s . 78-82. TEM s e c t i o n s of A., c a r t e r a e c e l l s s t a i n e d w i t h c a t i o n i c dyes. F i g . 78. T a n g e n t i a l s e c t i o n t h rough the p o l y g o n a l amphiesmal v e s i c l e s showing the g l y c o c a l y x (Gy) and the o u t l i n e of s u r f a c e pores ( p ) . x37,500. F i g . 79. Mature u n d i s c h a r g e d " r e s t i n g " t r i c h o c y s t l y i n g almost p e r p e n d i c u l a r l y t o the c e l l s u r f a c e . Note both the c o r r u g a t e d membrane of the t r i c h o c y s t (tm) and the i n n e r membrane ( i ) of the amphiesmal v e s i c l e . x58,500. F i g . 80. G l y c o c a l y x of a f l a g e l l u m ( F ) . x29,300. F i g . 81. M i c r o g r a p h of an aged c e l l i l l u s t r a t i n g the t h i c k e r (76 nm) i n n e r coat l a y e r ( i l ) and a p o s i t i v e l y s t a i n e d g l o b u l a r body (mucocyst, m) l o c a t e d beneath a c e l l s u r f a c e p ore. x72,000. F i g . 82. S e c t i o n of an aged c e l l showing the t h i c k g l y c o c a l y x (Gy) and the p o l y g o n a l u n i t s u n d e r l a i n by m i c r o t u b u l e s (MT). Note the o u t l i n e of a s u r f a c e pore i n which the t r i c h o c y s t membrane (tm) i s surrounded by the i n n e r membrane ( i ) of the amphiesmal v e s i c l e . x48,000. 124.1 125 Diagrammatic r e p r e s e n t a t i o n of the c e l l c o v e r i n g r e g i o n of A., c a r t e r a e (1) as v i s u a l i z e d by Dodge and Crawf o r d (1968), (2) as c o n s t r u c t e d from the r e s u l t s of the p r e s e n t i n v e s t i g a t i o n . Note the a d d i t i o n a l f e a t u r e s of g l y c o c a l y x (Gy) o v e r l y i n g the plasma membrane (PM), the " f u z z " l a y e r on the i n n e r s u r f a c e ( i ) of the amphiesmal v e s i c l e , the s u r f a c e pore ( p ) , the amorphous l a y e r ( a ) , and the endosome (e) p r o j e c t e d i n t o the c y t o p l a s m i c v a c u o l e ( V ) . M i c r o t u b u l e s (MT) , o u t e r membrane ( o ) . c_a x52 ,500. 225 ,J 126 F i g . 84. SEM m i c r o g r a p h of A., c a r t e r a e c e l l s showing the f l a g e l l a r pores (arrows) l i n k e d by the amphiesmal r i d g e ( a ) . T r a n s v e r s e f l a g e l l u m ( T F ) , l o n g i t u d i n a l f l a g e l l u m ( L F ) . x6,760. F i g . 85. Patches of FITC-HFP a l o n g the c e l l s u r f a c e . x2,680. F i g s . 8 6 - 8 8 . FITC obse r v e d i n the area of the f l a g e l l a r p o r e s . F i g . 86. x3,400. F i g . 87. x3,750. F i g . 88. F I T C - l o a d e d v e s i c l e s a r r a n g e d i n the shape of an a r c between the 2 f l a g e l l a r p o r e s . x3,430. 126.1 127 F i g s . 89-93. E l e c t r o n m i c r o g r a p h of A., c a r t e r a e c e l l s t r e a t e d w i t h HRP f o r 30 min. F i g s . 89-91. H R P - l a b e l e d v a c u o l e s (V) l o c a t e d between the two f l a g e l l a r c a n a l s and around the p y r e n o i d ( P y ) . x!3,000, x35,200 and x48,100 r e s p e c t i v e l y . F i g . 92. HRP r e a c t i o n p r o d uct observed w i t h i n the f l a g e l l a r c a n a l (Fc) and the p u s u l e ( P ) . x50,650. F i g . 93. V a c u o l e s (presumably heterophagosomes) c o n t a i n i n g heterogenous m a t e r i a l are c l o s e l y a s s o c i a t e d w i t h the endoplasmic r e t i c u l u m (ER). x34,550. 127.1 128 E l e c t r o n m i c r o g r a p h s of A., c a r t e r a e c e l l s t r e a t e d w i t h W G A - c o l l o i d a l g o l d c o n j u g a t e (WGA-CG). S u r f a c e l a b e l i n g of the amphiesmal r i d g e ( a r ) . x20,330. C l u s t e r s (arrows) of WGA-CG r e c e p t o r complexes a l o n g the c e l l s u r f a c e . x35,200. I n t e r n a l i z a t i o n of l e c t i n - C G v i a plasma membrane (PM) i n v a g i n a t i o n . V e s i c l e s (arrowheads) c o n t a i n i n g the l i g a n d a r e observed w i t h i n the p e r i p h e r a l v a c u o l e ( V ) . The arrow i n d i c a t e s m i c r o f i l a m e n t s a l i g n i n g the plasma membrane (PM) i n f o l d i n g s . x74,000. x45,000. x76,000. x70 ,000. 128.1 129 F i g . 100. F l u o r e s c e n c e m i c r o g r a p h of P. micans c e l l t r e a t e d w i t h a c r i d i n e orange (5 m i n ) , showing the p o l y s a c c h a r i d e l a y e r of the s u r f a c e . Nucleus (N). x l , 6 0 0 . F i g . 101. Pror o c e n t r u m c e l l t r e a t e d w i t h FITC-Con A f o r 1 h. Note t h a t the membrane (arrow) f o r m i n g the sack p u s u l e (P) i s l a b e l e d w i t h the l e c t i n . x l , 8 0 0 . F i g s . 102, 103. Pr o r o c e n t r u m c e l l s t r e a t e d w i t h c a l c o f l u o r - w h i t e f o r 2 min. F i g . 102. Note the t h i n t h e c a l p l a t e s (c_a 150nm) of c e l l s i n e x p o n e n t i a l phase of growth. x2,100. F i g . 103. Th i c k t h e c a l p l a t e s (CJI 830 nm) t y p i c a l of c e l l s i n s t a t i o n a r y phase of growth. x l , 9 0 0 . 129.1 130 G l y c o c a l y x (Gy) of Pror o c e n t r um c e l l d o u b l e - s t a i n e d w i t h A l c B - E F . x80,000. S u r f a c e pore formed by i n v a g i n a t i o n of both the plasma membrane (PM) and the o u t e r amphiesmal membrane ( o ) . Note the g l y c o c a l y x (Gy) e x t e n d i n g i n t o t h i s pore ( p ) . x48,000. P o r t i o n of a c e l l t r e a t e d w i t h c a t i o n i z e d f e r r i t i n (CF) showing the a n i o n i c c e l l s u r f a c e coat (Gy) w i t h the c o n t i n u o u s i n n e r l a y e r ( i l ) o v e r l a i n by the patchy o u t e r l a y e r ( o l ) . x l 5 , 3 0 0 . Double a p i c a l s p i n e ( a p ) . x l 4 , 2 0 0 . A b a c t e r i a - l i k e s t r u c t u r e c l o s e l y a s s o c i a t e d w i t h the c e l l - s u r f a c e coat of Pr o r o c e n t r u m . x49,000. S e c t i o n of Pro r o c e n t r u m p e r i f l a g e l l a r r e g i o n . The arrowhead i n d i c a t e s the presence of a b a c t e r i a - l i k e s t r u c t u r e i n t e g r a t e d i n one of the p l a t e l e t s ( p i ) . x8,920. 130.1 131 F i g s . 110,111. S p i n d l e t r i c h o c y s t ( T ) , d i s p l a y i n g a f r o n t membranous cap (arrowhead) . Nucleus (N). x21,930 and x9,600 r e s p e c t i v e l y . F i g s . 112,113. Mucous a c i d i c l a y e r c o v e r i n g the t r i c h o c y s t "pore". T h i s pore appears t o have been formed by i n v a g i n a t i o n of the two o u t e r membranes (arrowhead). Theca ( t ) . x45,230 and x50,000 r e s p e c t i v e l y . F i g s . 114,115. T r i c h o c y s t (T) d u r i n g the p r o c e s s of d i s c h a r g e . x29,250 and x40,000. 131.1 132 F i g s . 116-117. SEM m i c r o g r a p h s of Pr o r o c e n t r u m c e l l i l l u s t r a t i n g the c r a t e r - l i k e s u r f a c e pores (arrowhead). Some t r i c h o c y s t s remain l i n k e d t o these pores ( f i g . 1 1 6 ) . x3,100 and x7,400 r e s p e c t i v e l y . F i g . 118. Higher m a g n i f i c a t i o n of a c r a t e r - l i k e pore ( p ) . x38,000. . F i g . 119. E l e c t r o n m i c r o g r a p h showing t h r e e p o s s i b l e c o n f i g u r a t i o n s (A,B,C) of the s u r f a c e p o r e s . x21 ,600. 132.1 133 F i g . 120. Diagram i l l u s t r a t i n g t h r e e m o r p h o l o g i c a l a s p e c t s of the s u r f a c e pores of £. micans c e l l s : s i m p l e ( A ) , e l e v a t e d i n a cone-shaped s t r u c t u r e (B) or w i t h a c r a t e r - l i k e appearance ( C ) . CO 134 F i g . 121. P o r t i o n of a EiQLQSenttm c e l l showing the w e l l - developed g l y c o c a l y x (Gy) and one s u r f a c e pore c o n t a i n i g CF (arrowhead). T h e c a l p l a t e ( t ) . x20,200. F i g s . 122,123. Cr o s s s e c t i o n s of c r a t e r - l i k e pores l a b e l e d w i t h CF. T h i s marker was observed i n the lumen of the pore ( F i g . 123) or bound t o the plasma membrane w i t h i n ( i ) and o u t s i d e (o) the p o r e . T h e c a l p l a t e ( t ) . x95,750 and x84,000 r e s p e c t i v e l y . F i g . 124 I n t e r n a l i z a t i o n of CF through the p l a t e s u t u r e s (arrowhead). T h e c a l p l a t e ( t ) , v a c u o l e ( V ) . x48,000. F i g . 125. Va c u o l e s (V) c o n t a i n i n g CF a r e s c a t t e r e d i n the c y t o p l a s m . x20,000. F i g . 126. V a c u o l e s c o n t a i n i n g e l e c t r o n - d e n s e m a t e r i a l were found i n c e l l s t r e a t e d w i t h HFP f o r 1 h. x l 6 , 7 0 0 . 134.1 135 F i g s . 127-129. E f f e c t of d i f f e r e n t s a l i n i t y l e v e l s on the s i z e of the p u s u l e s of P. micans. F i g . 127. P u s u l e s a re not v i s i b l e , by l i g h t m i c r o s c o p y , when the c e l l s a r e s u b m i t t e d t o low s a l i n i t y (S=17 p p t ) . x l , 6 5 0 . F i g . 128. Normal p u s u l e (P) ar e a (.ca. 121 urn2) at S=24 p p t . x l , 7 1 0 . F i g . 129. E n l a r g e d p u s u l e ( P ) , ( c a . 242 um 2) at S=30 p p t . x l ,750. F i g s . 130-133. U l t r a s t r u c t u r e of Prorocentrum p u s u l a r r e g i o n . F i g . 130. L o n g i t u d i n a l s e c t i o n through the sack p u s u l e (P) showing the s l e n d e r c a n a l ( c ) , the a p i c a l pocket (apo) and some p r o t r u d i n g v e s i c l e s c o n t a i n i n g membranous m a t e r i a l ( a r r o w s ) . x l l , 1 8 0 . F i g s . 131,132. P u s u l e c o n t a i n i n g g r a n u l a r , v e s i c u l a r or b a c t e r i a - l i k e s t r u c t u r e s . V a c u o l e ( V ) . x8,700 and xl8,000 r e s p e c t i v e l y . F i g . 133. G o l g i ( G ) - d e r i v e d c o a t e d v e s i c l e s (arrows) and m i t o c h o n d r i a (M) i n the v i c i n i t y of the p u s u l e . x!4 ,000. 135.1 136 S e c t i o n s through the p u s u l e of P. micans c e l l s t r e a t e d w i t h HFP f o r 30 min. L a b e l e d s t r u c t u r e s (arrows) w i t h i n v a c u o l a r c a n a l i c u l i and the p e r i p h e r a l c y t o p l a s m i c v a c u o l e ( V ) . x l 8 , 9 0 0 and x20,000 r e s p e c t i v e l y . M i c r o t u b u l e s (MT) b o r d e r i n g the p u s u l a r c a n a l i c u l i . x42,300. V e s i c l e s (v) c o n t a i n i n g HFP r e a c t i o n p r o d uct a r e l o c a t e d around the sack p u s u l e ( P ) . x22,500. 137 S e c t i o n s t h rough the p u s u l e (P) of JE. micans c e l l s t r e a t e d w i t h CF. F i g . 140. Note the presence of m i t o c h o n d r i a (M) i n the v i c i n i t y of t h i s o r g a n e l l e . x21 ,820. x81 ,000. x l 6 ,000. P u s u l e (P) of P r o r o c e n t r um c e l l s t r e a t e d w i t h WGA-gold c o n j u g a t e . The arrows i n d i c a t e s m a l l v e s i c l e s c o n t a i n i n g t h i s l i g a n d . x l l , 7 0 0 . 3 37 .1 138 F i g . 142. Diagrammatic r e p r e s e n t a t i o n of the f l a g e l l a r c a n a l - p u s u l e system of c a r t e r a e c e l l s . Note the amphiesmal v e s i c l e s e x t e n d i n g through the f l a g e l l a r c a n a l (Fc) and r e a c h i n g the p r i m a r y c o l l a r r e g i o n ( c 2 ) . The f l a g e l l a r chamber (Fch) l e a d s t o the p u s u l e (P) through a c o n s t r i c t e d r e g i o n surrounded by the secondary c o l l a r ( C 2 ) . Plasma membrane (PM), g l y c o c a l y x (Gy), and m i c t o t u b u l e s (MT) . ca, x54 ,500. 138.1 139 F i g s . 143-148. S e q u e n t i a l s e c t i o n s of the f l a g e l l a r c a n a l - p u s u l e system of A., c a r t e r a e c e l l s . F i g s . 143,144. T a n g e n t i a l s e c t i o n s i l l u s t r a t i n g the plasma membrane (PM) of the amphiesma e x t e n d i n g i n t o the f l a g e l l a r c a n a l ( F c ) , f l a g e l l a r chamber ( f c h ) , and the p u s u l e ( P ) . Note the presence of the p r i m a r y c o l l a r (cj) and the secondary c o l l a r ( c 2 ) . G l y c o c a l y x (Gy), v a c u o l e (V) and c o a t e d v e s i c l e s ( a r r o w s ) . x29,420 and x50,000 r e s p e c t i v e l y . F i g . 145. Cr o s s s e c t i o n i l l u s t r a t i n g the p r i m a r y c o l l a r ( c j ) j u s t above the " v e s i c u l a r p u s u l e ( P ) . V a c u o l e ( V ) . x63 ,460 . F i g s . 146,147. S e c t i o n s showing the secondary c o l l a r ( c 2 ) around the c o n s t r i c t e d r e g i o n of each u n i t of the p u s u l e ( P ) . F l a g e l l a r chamber ( F c h ) , m i c r o t u b u l e s (MT). x52,500 and x48,900 r e s p e c t i v e l y . F i g . 148. S e c t i o n through the b a s a l body (bb) of the f l a g e l l u m ( F ) . The p r i m a r y c o l l a r (c^) and the f l a g e l l a r chamber (Fch) can be r e c o g n i z e d . x50 ,200. 139.1 140 F i g s . 149-151. 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