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Mutational analysis of cell development in Paramecium tetraurelia Jones, Donald 1977

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MUTATIONAL ANALYSIS of CELL DEVELOPMENT i n PARAMECIUM TETRAURELIA by DONALD JONES B . S c , Un ive rs i t y of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s thes i s as conforming to the required standard THE WlVERSITY OF BRITISH VoLUMBIA October, 1977 © Donald Jones, 1977 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e g u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Zoology The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 Date Deo. 6. 1977 i i ABSTRACT -Temperature-sensitive mutants have been used i n this study to examine development i n Paramecium tetraurelia. Ten mutations af-fecting development were described. Since two of the mutants were a l l e l i c , the effects of nine genes on Paramecium development were studied. Of these, two affect formation of the fission-zone (called dfz or defective fission-zone mutants), five affect constriction of the fission-furrow (called dc or defective constriction mutants), and two produce a reduction i n c e l l size (called sm or small mutants). Morphometric measurements were made on inter-fission and dividing wild-type and mutant c e l l s to examine two aspects of Paramecium devel-opment: changes i n c e l l shape and size which preceed and accompany c e l l division and positioning of new structures on the c e l l surface during c e l l division. This analysis suggested the following hypo-theses: 1. The shape and size changes which preceed and accompany c e l l d i v i -sion i n Paramecium are causally related to c e l l division. Defec-tive constriction mutants undergo exaggerated contraction prior to fission-furrow formation. This contraction appears to in t e r -fere with the decrease i n c e l l width which ordinarily occurs during division. Although the mutant c e l l s are able to make a normal amount of furrow surface, the abnormal c e l l width prevents furrow comple-tion. Premature contractions seen i n dfz mutants similarly interfere with furrow formation. 2. Surface growth i n Paramecium i s dependent on prior basal body i i i proliferation. Basal bodies appear to act as organizing centres for surface growth. Reduced basal body proliferation in mutant cells was always associated with reduced surface growth: There was a consistent relationship between the number of new basal bodies produced proceeding cell division and the amount of sur-face growth which occurred. The order of the causal relationship was suggested by the observation that basal body proliferation was completed prior to the beginning of surface growth. 3. The positioning of new structures during cell division in Paramecium can be affected by the pre-existing cell shape, size, or structure. This model, called mechanical guidance, was based on observations of the movement and positioning of the vestibule (the opening leading to the mouth) in wild-type and mutant cells. This model was discussed in relation to other developmental mechanisms proposed to account for protozoan morphogenesis. i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i i i LIST OF FIGURES x LIST OF PLATES x v i i INTRODUCTION 1 MATERIALS AND METHODS 10 RESULTS Section A: Genetics 21 Section B: Morphometric Analysis of Ce l l Division 28 1. INTRODUCTION .28 2. THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS 32 i . Growth Phase 32 i i . Contraction Phase 34 i i i . Fission-Furrow Phase 36 i v . C e l l Width 37 v. Basal Body Proliferation 38 v i . Contour Drawings 39 v i i . Growth of the Dorsal Surface 39 v i i i . Growth of the Ventral Surface 40 3. THE MORPHOGENETIC PERIOD IN MUTANT CELLS 42 (a) Mutant sm2 42 i . Growth Phase 42 i i . Contraction Phase 44 i i i . Fission-Furrow Phase 44 i v . Basal Body Proliferation 46 V v. Width .47 v i . Stomatogenesis 48 v i i . Juvenile Cells 49 v i i i . Contour Drawings 50 ix. Growth of the Dorsal Surface 50 x. Growth of the Ventral Surface 50 (b) Mutant dc4 51 i . Growth Phase 53 i i . Contraction Phase 53 i i i . Fission-Furrow Phase 54 iv. Basal Body Proliferation 55 v. Width 56 v i . Stomatogenesis 57 v i i . Contour Drawings 57 v i i i . Growth of the Dorsal Surface 57 ix. Growth of the Ventral Surface 58 (c) Mutant dfz2 58 i . Growth Phase 60 i i . Contraction Phase 60 i i i . Fission-Furrow Phase 61 iv. Stomatogenesis 63 v. Basal Body Proliferation 63 v i . Width 63 v i v i i . Contour Drawings and Surface Growth ... 64 (d) Mutant dc2 a 64 i . Growth Phase 64 i i . Contraction Phase 66 i i i . Fission-Furrow Phase 66 iv. Stomatogenesis 67 v. Basal Body Proliferation 67 v i . Width 67 v i i . Contour Drawings 68 v i i i . Growth of the Dorsal Surface 68 ix. Growth of the Ventral Surface 68 Section C: The Pattern of Temperature-Sensitivity During the Cell Cycle 70 1. INTRODUCTION 70 2. EXPERIMENTAL DESIGN 71 3.. TEMPERATORE-SENSITIVITY OF WILD-TYPE AND MUTANT CELLS 72 Section D: Cortical Patterning 76 1. INTRODUCTION 76 2. EXPERIMENTAL DESIGN 77 3. MEASUREMENTS 77 4. PROBIT ANALYSIS ..77 5. TEMPERATURE EFFECT ON sm2 CELL LENGTH 80 6. THE LENGTHS OF PARTS OF THE-CELL AS A FUNCTION OF CELL LENGTH 82 7. CORRELATION ANALYSIS 86 v i i 8 . BIYARIATE SCATTERGRAMS 94 Section E: Gullet Defects and Feeding Discussion 100 A. Genetics and Fhenotypes of the Mutants 105 B. Ce l l Division I l l (a) Shape and Size Changes During the C e l l Cycle 112 (b) Surface Growth 118 C. Cortical Pattern 128 (a) Cytotaxis i n Paramecium 131 (b) Positional Control i n Paramecium 139 SUMMARY 150 v i i i LIST OF TABLES TABLE: PAGE: I. a. C o r t i c a l Abnormal i t ies Found i n Morpho-l o g i c a l Var iants 14-16 b. C o r t i c a l Abnormal i t ies i n Selected Variant L ines 17-18 c. ts Var iant Stocks used i n the Study . . . 19-20 II. Exautogamous Segregation of t s Heterorygotes 23 III. _ Complimentation Matr ix 26 IV. Age-Dependent Changes i n Wild-Type C e l l S ize 33 V. Contour Lengths of Ventra l Regions 4-1 VI. Age-Dependent Changes i n sm2 C e l l S ize . . . . 43 VII. Age-Dependent Changes i n dc4 C e l l S ize . . . . 52 VIII. Age-Dependent Changes i n d f z 2 C e l l S ize . . . 59 IX. Age-Dependent Changes i n dc2 C e l l S ize . . . 65 X. Size-Dependent Changes i n sm2 C e l l Dimensions 78 XI. D i s t r i bu t i ons Obtained from Prob i t Analys is • 81 XII. Morphometric Measurements as a Frac t ion of C e l l Length 84 XIII. L inear Regressions of C e l l Dimensions Against Time at the Res t r i c t i v e Temperature 85 XIV. Matrix of Cor re l a t ion Coe f f i c i en t s I 8 8 - 8 9 XV. Matrix of Cor re l a t i on Coe f f i c i en t s II 9 0 - 9 1 LIST OF TABLES - (Cont 'd . ) TABLE: PAGE XVI. Parametric Cor re l a t ion Coe f f i c i en t s for Sets of Homogeneous Cor re la t ions 92 XVII. P r i n c i p a l Axes of B i va r i a te Scattergrams . . 96 XVIII. Morphometric Parameters - Food Vacuole Experiment 101 XIX. Matrix of Cor re la t ion Coe f f i c i en t s fo r Data from the Food Vacuole Experiment . . . . 103 XX. Predicted Growth of the Furrow Surface on Wild-Type and Mutant Ce l l s 123 LIST OF FIGURES NUMBER: PAGE: 1. A Schematic Diagram of the K ine t i es on the Dorsal and Ventra l Surfaces of an I n t e r f i s s i o n Paramecium 160 2. A Schematic Diagram of a Por t ion of the Paramecium Cortex 162 3 . Structure of the Paramecium Gu l l e t 164 • 4. A Schematic Diagram of Oral Anlage Development 166 5 . Camera Lucida Drawings of Con t rac t i l e Vacuole Pore Abnormal i t ies i  tx-0 C e l l s . 168 6 . Measurements Made on C e l l s of Known Age . . . 170 7-12. Sample S t a t i s t i c s fo r Wild-Type and sm2 C e l l s of Known Age a f t e r a S h i f t t o the Restrictive Temperature 7a. Length 172 7b. Length of the Anterior Suture and Vestibule Length. 172 8a. Length of the Posterior Suture 174 8b. Width 174 9a. CVA 176 9b. CVP 176 10a. CVA' 178 10b. CVP' 178 11a. Length-to-Width Ratio 180 l i b . CVINT * 180 xi LIST OF FIGURES - (Cont 'd. ) NUMBER: PAGE: 12a. Number of Basal Bodies 182 12b. Spacing Between Basal Bodies 182 1 3 . Out l ine Drawings of Wild-Type and sm2 D i v id ing Ce l l s 184 14-. Growth of Various Regions on the Dorsa l .Surface of D i v i d i ng C e l l s 186 15. Basal Body P r o l i f e r a t i o n i n sm2 C e l l s During the F i r s t C e l l Cycle at the Res t r i c t i v e Temperature 188 16. Abnormal i t ies i n Gu l l e t Anlage Development i n sm2 Ce l l s 1 9 1 17-22. Sample S t a t i s t i c s of Wild-Type and dc4 Ce l l s of Known Ages A f te r a Sh i f t to the Res t r i c t i v e Temperature 17a. Length 193 17b. Length of the Anter io r Suture and Vest ibu le Length 193 18a. Length of the Pos te r io r Suture 195 18b. Width 195 19a. CVA 197 19b. CVP 197 20a. CVA' 199 20b. CVP' 199 21a. Length-to-Width Rat io 201 21b. CVINT 201 x i i LIST OF FIGURES - (Cont 'd. ) NUMBER: PAGE: 22a. Number of Basal Bodies 203 22b. Spacing Between Basal Bodies 203 2 3 . Camera Lucida Drawing of a dc4 C e l l A f t e r 0 . 9 3 C e l l Cycles at the Res t r i c t i v e Temperature 205 24. Outl ine Drawings of Wild-Type and dc4 D i v id ing C e l l s . . . . 207 2 5 . Growth of Regions of the Dorsa l Surface of D i v id ing C e l l s . 209 2 6 - 3 1 . Samples S t a t i s t i c s of Wild-Type and dfz2 Ce l l s of Known Age A f te r a Sh i f t to the Res t r i c t i v e Temperature 26a. Length * 211 26b. Length of the Ante r io r Suture and the V e s t i b u l e . . . 211 27a. Length of the Pos te r io r S u t u r e . . . . 213 27b. Width 213 28a. CVA 215 28b. CVP 215 29a. CVA' 217 29b. CVP' • 217 3 0 a . Length-to-Width Rat io 219 3 0 b . CVINT 219 3 1 a . Number of Basal Bodies 221 3 1 b . Spacing Between Basal Bodies 221 xLii LIST OF FIGURES - (Cont 'd . ) NUMBER; PAGE: 3 2 . Camera Lucida Drawings of d f z 2 Ce l l s Which Were Arrested During D i v i s i on at the Res t r i c t i v e Temperature 223 3 3 . Outl ine Drawings of d f z 2 and d c 2 a Div id ing Ce l l s 225 3 4 - 3 9 . Sample S t a t i s t i c s fo r Wild-Type and d c 2 a Ce l l s A f t e r a S h i f t to the Res t r i c t i v e Temperature 3 4 a . Length 227 3 4 b . Length of the Ante r io r Suture and the Vest ibu le 227 3 5 a . Length of the Pos te r io r Suture 229 3 5 b . Width 229 3 6 a . CVA 231 3 6 b . c v p . . 2 3 1 3 7 a . CVA' 233 3 7 b . CVP' 233 3 8 a . Length-to-Width Rat io 235 3 8 b . CVINT 235 3 9 a . Number of Basal Bodies 237 3 9 b . Spacing Between Basal Bodies 237 40. An Example of the P lots Used to Obtain the Mean C e l l Cycle Length of a Synchronous Populat ion of C e l l s 239 41. The Pattern of Temperature-Sensit iv i ty During the Paramecium C e l l Cycle 2 4 l xlv LIST OF FIGURES - (Cont ' d . ) NUMBER: PAGE: 4 2 . The Pattern of Temperature-Sensit iv i ty During the Paramecium C e l l Cycle 243 4 3 . The Pattern of Temperature-Sensit iv i ty During the Paramecium C e l l Cyc le . 245 4 4 . A Schematic Diagram Showing the Measurements made on sm2 C e l l s of Various S i z e s . . . 247 -4 5 . Probi t P lo ts of sm2 C e l l Length f o r Asynchronous Samples Taken at Various Times A f t e r a S h i f t to the Res t r i c t i v e Temperature 249 4 6 . Probi t P lo ts of C e l l Width f o r Asychronous Samples of sm2 Ce l l s Taken at Various Times A f t e r a S h i f t to the Res t r i c t i v e Temperature 251 4 7 . The Length and Width of sm2 Ce l l s P lo t ted Against Time at the Res t r i c t i v e Temperature 253 4 8 . Sample S t a t i s t i c s of Asynchronous sm2 Ce l l s at Various Times A f t e r a Sh i f t to the Res t r i c t i v e Temperature 4 8 a . Ante r io r and Pos te r io r Suture Lengths 255 48b. Anter io r and Pos te r io r Suture Lengths as a F rac t ion of C e l l Length 255 4 8 c . CVINT, CVA, and CVP. . . 255 4 8 d . CVINT, CVA, and CVP as a F rac t i on of C e l l Length 255 4 9 . Sample S t a t i s t i c s of Asychronous sm2 Ce l l s at Various Times A f t e r a Sh i f t to the Res t r i c t i v e Temperature. . . XV LIST OF FIGURES - (Cont 'd . ) NUMBER; PAGE: 4 9 a . Port ions of C e l l L e n g t h . . . . 257 4 9 b . Port ions of C e l l Length as a F rac t ion of C e l l Length 257 4 9 c . Cytoproct Length 2 5 7 4 9 d . Cytoproct Length as a F rac t ion of C e l l Length . 257 5 0 - 5 5 . B i va r ia te Scatter P lo ts of Sample S t a t i s t i c s of sm2 Ce l l s 5 0 a . The Length of the Anter io r Suture P lo t ted against C e l l Length 259 5 0 b . The Length of the Pos te r io r Suture P lo t ted against C e l l Length 259 5 1 a . The Cytoproct Length P lo t ted against C e l l Length 2 6 l 5 1 b . The Length of the Vest ibu le P lo t ted against C e l l Length 261 5 2 a . The Distance Between the Ante r io r CVP and the Anter ior of the C e l l (CVA) P lo t ted against C e l l Length 263 5 2 b . The Distance Between the Pos te r io r CVP and the Pos te r io r of the C e l l (CVP) P lo t ted against C e l l Length 263 5 3 a . The Distance Between the CVPs (CVINT) P lo t ted against C e l l Length 265 5 3 b . The Number of Basal Bodies Between the CVPs P lo t ted against C e l l Length. 265 5 4 a . The Spacing Between Basal Bodies P lo t ted against C e l l Length 267 5 4 b . The To ta l Number of K ine t i es P lo t ted Against C e l l Width 267 LIST OF FIGURES - (Cont 'd. ) NUMBER: PAGE 5 5 a . The To ta l Number of K inet ies Per C e l l P lo t ted against the Number of K ine t i es Between the Le f t Ves t ibu la r Wall and the F i r s t CVP to the C e l l ' s Le f t (KTCV) 269 5 5 b . The Value KTCV as a Percentage of the Tota l Number of K ine t ies P lo t t ed against C e l l Width 269 5 6 . Probi t P lo ts of C e l l Length Measurements from the Feeding Experiment 271 5 7 . B i va r i a te Scatter P lo ts of Sample S t a t i s t i c s of sm2 Ce l l s 273 5 8 . A Schematic Diagram I l l u s t r a t i n g Poss ib le Mechanisms for the Gain and Loss of K ine t ies 275 5 9 . The Fate of CVPs i n a C e l l Lineage Diagram 277 6 0 . The Re la t i ve Pos i t ions of CVPs i n D i v id ing Ce l l s 279 xvii LIST OF PLATES NUMBER; PAGE; 1. Gu l l e t Structure 281 2. Somatic Cortex-Normal and Abnormal Ce l l s . . 283 3. Gu l l e t Abnormal i t ies 285 4-. Cytoproct Structure 28? 5. C e l l Shapes 289 6. C e l l Shapes 291 ?. C e l l Shapes 293 8. C e l l D i v i s i o n Stages 295 9. Basal Body P r o l i f e r a t i o n i n Wild-Type C e l l s 297 10. D i v i s i on Stages of sm2 Ce l l s 299 x v l i i ACKNOWLEDGEMENT I thank Dr. James D. Berger fo r h i s thoughtful advice and encouragement during the course of t h i s t h e s i s . For h e l p f u l conversations and c r i t i c i s m s I am indebted to P h y l l i d a Riseborough, Caro l Po l l ock , Glenn Morton, and Connie Boogaard. I am espec i a l l y g r a t e fu l to E r i c Peterson and Adele Hunter fo r p rov id ing me with the mutant Paramecium stocks used i n t h i s study. F i nanc i a l support fo r the thes i s work was provided by the Nat ional Research Counc i l of Canada through grants to Dr. J . D . Berger. 1 INTRODUCTION Mutat ional ana lys is i s a p o t e n t i a l l y powerful approach to the study of developmental b io logy . This approach en ta i l s the use of chemical or phys i ca l agents to produce mutations which a l t e r some aspect of development. I dea l l y , the mutant phenotype i s produced by a s ing le mutated gene. The ac t ion of i nd i v i dua l genes i n development can therefore be examined. The i d e n t i f i c a t i o n of these genes a lso lays open the p o s s i b i l i t y that t h e i r gene products can be i d e n t i -f i ed and the ro l e of these products i n development analysed. Th is approach has been used success fu l l y i n the ana lys is of numerous aspects of development. These inc lude f l a g e l l a r development i n Chlamydomonas (McV i t t i e , 1972), t r i chocys t development i n Paramecium (Pol lock, 1974), and development of the feeding apparatus i n Tetrahymena (Orias and Po l lock , 1975). C e l l d i v i s i o n has been examined by mutational ana lys is i n bac te r i a (reviewed by S l a t e r and Schechter, 1975)» yeast (Hartwel l , 1974), Tetrahymena (Frankel et a l . , 1976), and Chinese Hamster c e l l s (Hatzfe ld and Bu t t i n , 1975). Mutants such as these, where c e l l d i v i s i o n or other essent i a l c e l l funct ions are impaired, can only be maintained i f t h e i r expression i s cond i t i ona l . These are most commonly temperature-sensit ive (ts) cond i -t i ona l mutants. 2 The two holotr ichous c i l i a t e s Paramecium and Tetrahymena are wel l su i ted f o r studies invo l v ing mutational ana l ys i s . In Paramecium the genetic process of autogamy r esu l t s i n homozygosis of a l l gene l o c i i n a s ing l e genera-t i on (Sonneborn, 1974). Recessive mutations may therefore be brought to expression r ap id l y . Although autogamy does not occur i n Tetrahymena, a l ternate methods are ava i l ab le to br ing recess ive a l l e l e s in to expression (A l l en , 196?)• Frankel (Frankel et a l . , 1976, a,b) has i s o l a t e d s ix gene l o c i involved i n c e l l d i v i s i o n i n Tetrahymena  py r i fo rmis . Both heat and co ld-sens i t i ve mutant a l l e l e s of these l o c i have been found. They produce d i v i s i o n a r res t at various c e l l ages ranging from the i n i t i a t i o n of furrowing to completion of cy tok ines i s . Non-condit ional c e l l d i v i s i o n mutants of Paramecium  t e t r a u r e l i a have been reported by Maly (1958), Sonneborn (1974b), and Whitt le and Chen-Shan (1972). Whitt le and Chen-Shan (1972) a l so i so l a t ed a ts c e l l d i v i s i o n mutant. The purpose of the present study i s to use severa l new cond i t iona l c e l l d i v i s i o n and c e l l growth mutants to analyse the mechanisms under ly ing c y t o d i f f e r e n t i a t i o n and c e l l d i v i s i o n i n Paramecium. The study w i l l focus on a morpho-metric ana lys is of growth, s t ruc tu re , and morphogenesis of the Paramecium c e l l and w i l l compare the changes i n c e l l dimensions proceeding and accompanying c e l l d i v i s i o n i n 3 wild-type c e l l s with those i n mutant c e l l s . P. t e t r a u r e l i a i s wel l su i ted to morphometric ana lys is . of c y tod i f f e r en t i a t i on due to i t s c e l l shape and complex c e l l a rch i tec ture . The i n t e r f i s s i o n c e l l i s 100 pm long and 43 pm wide (Kaneda and Hanson, 1974). These dimensions vary with temperature (Whitson, 1964) and n u t r i t i o n a l s ta te . The c e l l has an enigmatic shape; names such as s l ipper-shaped, pear-shaped, or pro la te spheroid are only roughly app l i cab le . The c e l l i s wider i n the pos te r io r than the anter io r region and the c e l l i s d i s to r ted by a one-sided groove which extends from the anter io r t i p to a depression (the ves t ibu le ) which leads i n to the feeding apparatus. Two components of the c i l i a t e c e l l are t y p i c a l l y analysed i n developmental s tud ies . These are the somatic cortex and the feeding apparatus (the g u l l e t ) . Development of the f i r s t i s r e fe r red to as c o r t i c a l development and that of the l a t t e r as stomatogenesis. Both components can be revealed by s i lver- impregnat ion techniques. S i l v e r-impregnation of paramecia by the Chatton-Lwoff technique (Chatton and Lwoff, 1930) r e s u l t s i n impregnation of only the cortex and g u l l e t . The i n t e r i o r of the c e l l remains transparent. As a l l s ides of the c e l l are impregnated i t i s poss ib le to examine a l l of the c e l l ' s ex te r io r . D i p p e l l (1962) has shown that the s i t e s of s i l v e r depos i t ion are at the juncture of each c i l i um with i t s basal 4 body, at the juncture of each t r i chocys t with the c e l l sur face , i n the parasomal sacs , i n the con t r a c t i l e . vacuole pores, and i n the cytoproct . The l o ca t i on of a l l of these st ructures can be found i n f igures 1 and 2 and t h e i r u l t r as t ruc tu re w i l l be considered i n more d e t a i l l a t e r . C i l i a r y basal bodies are arranged on the c e l l surface i n l ong i tud ina l rows ca l l ed k i ne t i e s . The arrangement of k ine t i es Is shown i n f igure 1. On the dorsa l surface the k ine t i es are continuous from pole to po le . On the vent ra l surface the kinety pattern i s in ter rupted by the ves t i bu l e . K inet ies to the l e f t ( c e l l ' s l e f t ) of the ves t ibu le bend sharply around i t and meet k ine t i e s on the r i gh t of the ves t ibu le along two "suture l i n e s " . The pos te r io r suture contains within i t the cytoproct which appears as a wavy black l i n e i n s i l v e r ed preparat ions ( f i g . l ) . The cytoproct i s the s i t e of egest ion of food vacuoles. K ine t ies are composed of an a l t e rna t ing array of c i l i a r y basa l bodies , parasomal sacs, and t r i chocys t s ( f i g . l ) . The space between t r i chocys t s cons t i tu tes a c o r t i c a l un i t . In add i t ion to e i ther one or two basal bodies and a parasomal sac each c o r t i c a l un i t contains a complex of membranes and a va r i e t y of microtubules and microf ibers (Ehret and Powers, 1959; Hufnagel , 1969). The anatomy of a c o r t i c a l un i t i s shown i n f igure 2. 5 Of the complex array of s t ructures shown i n th i s f igure only ce r ta in ones are evident i n s i l v e r ed preparat ions (see above). As t h i s study w i l l r e l y heav i l y on s i l v e r ed mater ia l a c loser examination of these a rgentoph i l i c organel les i s warranted. Paramecium c i l i a r y basal bodies and the i r development have been extens ive ly studied by D i p p e l l ( 1 9 6 8 , 1 9 7 6 ) . The basal body i s composed of nine groups of t r i p l e t microtubules arranged to form a cy l i nder ( f i g . 2 ) . The two inner microtubules of each t r i p l e t are continuous with the outer doublet microtubules of the c i l i a . The outer micro-tubule of each t r i p l e t ends at a basal p la te which separates the basa l body from the c i l i um above i t . Development of new basal bodies i s r e s t r i c t e d to the l a t e r stages of the c e l l cyc le (D ippe l l , 1 9 6 8 ; Kaneda and Hanson, 1 9 7 4 ) . New basal bodies appear at r i gh t angles and anter io r to old ones (D ippe l l , 1 9 6 8 ) . During t h e i r deve l -opment they t i l t and move upwards to the c e l l sur face . De ta i l s of t he i r attachment to surface membranes and the subsequent growth of c i l i a from the basa l bodies have not yet been determined. Tr ichocysts are large p r o j e c t i l e organel les with a carrot-shaped body and a pointed t i p ( f i g . 2 ) . The body of the t r i chocys t with i t s attached t i p can be expel led as a f ine thread. Tr ichocysts a r i s e deep wi th in the cytoplasm 6 and undergo a complex ser ies of movements which br ing the mature organel les to the c e l l surface (Jurand and Selman, 1 9 7 0 ) . The t r i chocys t attachment s i t e on the c e l l membrane cons is ts of a " r ose t t e " or c i r c u l a r arrangement of granules. Mutants l ack ing the roset tes have t r i chocys t s incapable of at taching to the c e l l membrane (Beisson et a l . , 1 9 7 & a ) • The parasomal sac i s an invag inat ion of the outer c e l l membrane (Hufnagel, 1 9 & 9 ; f i g - 2 ) . These sacs are located to the . r i gh t and an te r io r to the basa l body i n each c o r t i c a l un i t . I f two basal bodies occur within a uni t the parasomal sac i s associated with the pos te r io r one (Hufnagel, 1 9 & 9 ) . The o r i g i n of new parasomal sacs during development has not been determined. On the dorsa l surface of the c e l l s i l v e r impregnation reveals the pores of the con t r a c t i l e vacuoles. The con-t r a c t i l e vacuoles are the f a m i l i a r water-expulsion organel les of paramecia. The vacuoles themselves are not revealed by s i l v e r impregnation. The pores of the vacuoles are 1 . 2 pm long and 1 pm wide invaginat ions of the outer membrane and are surrounded by a h e l i c a l microtubule complex (McKanna, 1 9 7 3 ) . The gu l l e t can a lso be seen i n s i l v e r e d preparat ions . Basal bodies i n the gu l l e t are arranged i n twelve k i ne t i e s . The four k ine t ies on the dorsa l gu l l e t wal l are c a l l ed the quadrulus. The remaining eight k ine t i e s comprise the 7 peniculus which i s a r b i t r a r i l y separated in to the dorsa l and ven t ra l p e n i c u l i , each conta in ing four k ine t i e s ( f ig .3)« Basal bodies i n the peniculus are spaced more c l o se l y than those i n the quadrulus ( f i g .3). An add i t i ona l gu l l e t k inety occurs at the juncture between the r i gh t ves t i bu l a r wal l and the g u l l e t . This short k inety i s ca l l ed the endoral membrane. Stomatogenesis i n I__ t e t r a u r e l i a has been descr ibed by Roque (1956), Kaneda and Hanson (197*0, and Jones (1976). Figure 4 i s an out l ine of stomatogenesis as presented by these authors. The fo l lowing account of stomatogenesis i s given by Jones (1976). In si lver-impregnated mater ia l the ora l anlage appears at age 0.75 c e l l cyc les above the endoral k inety on the r i gh t ves t ibu l a r wa l l ( f i g . 4 ) . At th i s time the anlage cons is ts of only a few i r r e g u l a r l y spaced argentoph i l i c l o c i . Up to age 0.84 c e l l cyc les add i t i ona l l o c i appear i n the anlage and the endoral k inety i s resorbed. The anlage eventual ly becomes organized in to three, and l a t e r s i x , k ine t i e s ( f i g . 4 ) . By age O.85 c e l l cyc les the anlage has invaginated and taken a pos i t i on to the r i gh t of the ves t i bu l e . The anlage now looks l i k e an inver ted "J" when viewed from above (plate 1) . The number of k ine t i e s i n the anlage doubles a f t e r age O.85 c e l l cyc les and the anlage separates from the anter ior end of the ves t ibu le (plate 1 ) . 8 I t then moves pos t e r i o r l y to occupy a pos i t i on under the op i s the ' s ves t ibu le (plate l ) . Add i t iona l d e t a i l s of stomatogenesis are given by Jones (1976). The endoral k ine ty , which i s resorbed during stomatogenesis, reappears on the r i gh t ves t ibu l a r wal l of both proter and opisthe p r i o r to separat ion of the daughter c e l l s . In add i t i on , the anlage has been shown to be present throughout the c e l l cyc le (Jones, 1976). P rotargo l impreg-nat ion of i n t e r f i s s i o n c e l l s reveals basa l bodies located beneath the c e l l surface i n the region of the r i gh t v e s t i -bular wa l l . At age 0.75 c e l l cyc les these l o c i move to the c e l l surface where they are suscept ib le to Chatton-Lwoff s i lver- impregnat ion. The new gu l l e t anlage ac tua l l y forms around age O.87 c e l l cyc les at which time two new anlagen form, one destined fo r the opisthe and the other fo r the proter ( f i g .4). The complexity of the Paramecium c e l l and i t s morpho-genesis should be evident from the foregoing desc r ip t ions . I t i s hoped, however, that i n sp i te of the spec i a l i z a t i ons which al low Paramecium to ex i s t as a f r e e - l i v i n g s ing le-ce l l ed organism there are fundamental developmental processes which paramecia share with other eukaryotic c e l l s . These processes may inc lude c e l l growth, formation and cons t r i c t i on of the f i ss ion-fur row, and deployment of c e l l organel les such as basal bodies. In add i t i on , i t i s poss ib le that at 9 the molecular l e v e l the processes governing development i n Paramecium w i l l prove to be common to c y t o d i f f e r e n t i a t i o n i n many c e l l types. 10 MATERIALS AND METHODS: 1. CULTURE TECHNIQUE Paramecium t e t r a u r e l i a stock 51 was grown i n grass medium (pH 6 . 8 ) innoculated with K l e b s i e l l a  aerogenes as described by Sonneborn ( 1 9 7 0 ) . 2. SYNCHRONIZATION OF CULTURES Populations of synchronous c e l l s were obtained by manual s e l e c t i o n of d i v i d i n g c e l l s from log-phase cultures. 3- TEMPERATURE - SHOCK EXPERIMENTS The procedure f o r temperature-shock experiments was modified from Natchway and Cameron ( 1 9 6 7 ) . Individual c e l l s from a synchronous population were i s o l a t e d i n small drops of culture f l u i d i n a Falcon p l a s t i c p e t r i - d i s h ( # 3 0 0 1 ) . The drops containing the c e l l s were then covered with a layer of mineral o i l . To heat-shock the c e l l s , the p e t r i - d i s h was submerged fo r 30 minutes i n a water bath set at 3 4 . 5°C. The median c e l l cycle length was determined by 11 3. TEMPERATURE - SHOCK EXPERIMENTS (Cont'd.) tabulating (every 10 minutes) the number of c e l l s i n the population that had completed d i v i s i o n . The cumulative percentage of divided c e l l s was plotted against time on p r o b a b i l i t y paper. The i n t e r s e c t of a str a i g h t l i n e drawn through the points at the 5°^ l e v e l gave the median c e l l cycle length. Natchway and Cameron (1967) present a de s c r i p t i o n of t h i s technique and a discussion of i t s v a l i d i t y . 4. CHATT0N-LW0FF SILVER-IMPREGNATION. Silver-impregnation was performed as outlined by Frankel and Heckmann (1968). 5. IDENTIFICATION OF CELL CYCLE STAGES The i d e n t i f i c a t i o n of c e l l cycle stages i n s i l v e r -impregnated material was based on photographs and figures presented by Kaneda and Hanson (1974) and Jones (1976). The age of c e l l s was l i s t e d as a decimal f r a c t i o n of the c e l l cycle length. (An age O.75 c e l l was three-quarters of the way through the c e l l cycle.) 12 6. MUTAGENESIS AND MUTANT STOCKS The temperature-sensitive (ts) morphologically abnormal variants used i n t h i s study were kindly supplied by Adele Hunter and E r i c Peterson. These variants were part of a group of sixty morphologically abnormal variants found among 12,000 separate clones i s o l a t e d a f t e r mutagenesis. The mutagenesis procedure i s given i n Peterson and Berger (1976) . A l l 60 variants were temperature-sensitive. The r e s t r i c t i v e and permissive temperatures were 34.5°C and 17°C respectively. 7. ANALYSIS OF MUTANT PHENOTYPES To examine the morphological abnormalities of variants, a log-phase p e t r i - d i s h culture of variant c e l l s was held at 34 .5°C f o r 20 hours. The c e l l s were then fi x e d and silver-impregnated. C l a s s i f i c a t i o n of the abnormalities i n the silver-impregnated c e l l s was achieved by separating the Paramecium cortex i n seven components. A number of d i s t i n c t abnormalities were found f o r each of these components. Table l a l i s t s 13 7. ANALYSIS OF MUTANT PHENOTYPES - (Cont'd.) the seven components of the cortex and the abnormal-i t i e s noted i n each. Samples of variant c e l l s were examined with respect to these seven c o r t i c a l components and the number of variant c e l l s with each of the abnormalities l i s t e d i n table l a were noted. 8. SELECTION OF VARIANTS FOR STUDY Thirty-eight variants were examined by the procedure given above. Ten variants were selected f o r further study. These included s i x variants with a defect i n fission-furrow c o n s t r i c t i o n (variants 2, k, 8, 23, 62 and 63), two variants with a defect fission-zone formation (variants 29 and 30), and two variants which produce small c e l l s (variants 21 and 26). The c o r t i c a l abnormalities present i n these ten variants are l i s t e d i n table l b and a general description of t h e i r phenotypes i s given i n table l c . 14 TABLE Ia C o r t i c a l Abnormalities Found i n Morphological Variants C o r t i c a l Component Type Description Somatic Cortex G u l l e t Normal Abnormal, type 1 Abnormal, type 2 Abnormal, type 3 Abnormal, extra Normal Abnormal, type 1 Abnormal type 2 Abnormal type 3 Abnormal, everted Kineties and basal bodies are evenly spaced (plate 2). An occasional basal body or tri c h o c y s t i s not aligned within a kinety (plate 2). Many basal bodies and tric h o c y s t s are not aligned within k i n e t i e s , parts of the cortex may have no kin e t i e s or k i n e t i e s may be wavy or bent at odd angles (plate 2). Kineties are highly disorganized, basal bodies are scattered i n a more or l e s s random manner (plate 2). More than the usual number of argentophilie bases are found on the cortex (plate 2). See figure 3. Loss or disorganization of the innermost part of the g u l l e t , the disorganized region i s not more extensive than one-quarter of the g u l l e t length (plate 3). Loss or disorganization of the innermost one-quarter to one-third of the g u l l e t (plate 3). Loss or disorganization of more than one-third of the g u l l e t (plate 3) . Gullet k i n e t i e s are on the c e l l surface i n t e r c a l a t e d between somatic k i n e t i e s (plate 1). 15 TABLE Ia (con't.) C o r t i c a l Component Type Description Gullet C o r t i c a l Pattern Abnormal, astomatous Normal Abnormal, patchy Cytoproct Normal Abnormal C e l l Shape Normal Abnormal, large Abnormal, small Abnormal, swollen Abnormal, round Abnormal, truncated Abnormal, d i v i s i o n a r r e s t . No g u l l e t present (plate 5 ) ' The entire c e l l surface i s covered by c o r t i c a l units. Patches on the cortex are devoid of c o r t i c a l organelles (plates 5 and 6 ) . The cytoproct silver-impregnates as a th i n , wavy black l i n e about 20 um long (plate 4). Abnormalities include short, very wide (distended), and disrupted cytoprocts (plate 4). See figure 1 . C e l l s are abnormally long and wide (plates 5 and 6 ) . C e l l s are unusually short and narrow (plate 5)• C e l l s are wide with c o r t i c a l b l i s t e r s or blebs (plate 6 ) . C e l l s are equally wide as long (plate 6 ) . C e l l s lack either an anterior or posterior c e l l region (plate 5 ) • C e l l s cannot complete c o n s t r i c t i o n of the fission-furrow (plate 7 ) « 16 TABLE l a (con't.) C o r t i c a l Component Type Description C e l l Abnormal, shape monster Abnormal, i r r e g u l a r Contractile Normal vacuole pores Abnormal, extra Abnormal, p l a t e - l i k e a large, irregularly-shaped c e l l which i s the r e s u l t of continued c e l l growth i n the absence of c e l l d i v i s i o n (plate 7) • C e l l has an i r r e g u l a r outline (plates 5 and 6). C e l l s have two ( r a r e l y three or more) pores which are about 1 um i n diameter (figure 1). C e l l s have three or more c o n t r a c t i l e vacuole pores (plates 6 and 7)• The pores are very broad, with an i r r e g u l a r outline (plate 6 and figure 5) 1 7 TABLE l b C o r t i c a l Abnormalities i n Selected Variant Lines C o r t i c a l 3 -Character Variant Number CELL SHAPE Normal Normal divider D i v i s i o n a r r e s t Monster Irregular Truncation Large Swollen CELL SIZE 0 - 80pm 80-lOOpm 100-120;am >120pm CORTEX Normal Type 1 Type 2 Type 3 Extra mtr'l. CORTICAL PATTERN Normal Patchy CYTOPROCT Normal Abnormal Absent GULLET Normal Type 1 Type 2 Type 3 On surface Astomatous % of sampled c e l l s with given character 8 2 1 23 2 6 2 9 30 62 63 8 0 92 4 6 30 50 4 4 7 7 1 4 - 1 4 - 2 2 2 2 6 - - 3 2 0 6 1 5 4 - - - -• 4 2 8 1 8 1 0 6 2 4 6 2 2 4 1 8 4 8 4 1 - 4 -. - 3 1 4 - - 1 8 - . - 2 1 — • — — • 8 1 - - 1 2 -1 1 1 1 9 _ 1 * 4 - - 63 3 _ 4 8 4 7 1 4 4 1 6 8 7 5 5 8 9 1 1 29 5 6 2 1 0 4 5 6 3 1 8 1 4 mm 9 1 9 4 2 50 1 4 1 3 8 27 51 3 7 2 6 1 8 4 6 1 8 1 8 8 1 8 6 2 4 1 1 0 3 2 — - 50 1 2 3 4 ^ 3 2 0 3 5 1 4 4 8 15 30 . 9 7 5 6 5 8 6 52 8 5 70 9 1 25 7 6 90 7 4 52 5 6 7 9 6 1 2 2 1 0 2 6 4 8 4 1 2 1 3 7 2 mm — — 3 - 2 1 6 2 8 36 8 15 3 3 23 * 1 9 4 0 1 6 2 0 25 3 3 3 3 1 8 1 6 1 4 1 6 13 9 9 32 1 0 32 4 7 39 1 2 27 1 3 - 4 — 9 1 1 4 3 2 4 8 4 7 18 TABLE l b - (Cont'd.) C o r t i c a l Abnormalities i n Selected Variant Lines C o r t i c a l 3 " v. Character % of sampled Variant Number 8 21 2; CONTRACTILE VACUOLE PORES Normal number 85 88 62 Supernumerary Ik 12 3^ P l a t e - l i k e 1 - 2 a. A f u l l d escription of thes table l a . *.. No values available. b. Sample si z e s : variants 8 , . variants 2 6 - 8 0 c e l l s ; remaining variants - 50 c e l l s . c e l l s with given character 26 29 30 62 63 90 7k 61 66 * 10 26 39 33 1 characters may be found i n , and 62 - 100 c e l l s ; 19 TABLE l c ts Variant Stocks Used i n the Study Stock Fission-Zone Fission-Furrow  Number Formation Formation Normal Normal 8 Normal 23 Normal 62 Normal 63 Normal P a r t i a l , arrested P a r t i a l , arrested P a r t i a l , arrested P a r t i a l , arrested P a r t i a l , arrested P a r t i a l , arrested Notes Forms very short d i v i d -ing c e l l s (about 13Cvum lo n g ) - d i v i s i o n arrest occurs during the f i r s t c e l l cycle at 34.5 c-c e l l behaviour i s abnormal at 34.5 C-the c e l l s frequently swim backwards f o r short periods. C e l l swelling and death occurs at 34-5 C-gullet defects and astomatous c e l l s were common-g u l l e t s occasionally have supernumerary k i n e t i e s . C e l l swelling and death occurs at 34.5 C - c e l l s only r a r e l y are arrested i n fission-supernumerary g u l l e t k i n e t i e s are common. Gulle t abnormality i s severe with astomatous c e l l s common-has super-numerary c o n t r a c t i l e vacuole pores. Penetrance of the mis-di v i d e r t r a i t ranged from 50 to 95$ i n d i f -ferent experiments. Chains of 3 and 4 c e l l s 20 TABLE l c - (Cont'd.) ts Variant Stocks Used i n the Study Stock Fission-Zone Fission-Furrow  Number Formation Formation 63 29 30 21 26 Normal Does not occur or p a r t i a l Does not occur or p a r t i a l Normal Normal P a r t i a l , arrested Does not occur or p a r t i a l Does not occur or p a r t i a l Normal Normal Cont'd. as well as heteropolar doublets are common-g u l l e t and c o r t i c a l abnormalities are severe. C e l l s arrested p r i o r to fission-zone forma-t i o n are rounded, and are astomatous i n either the proter or opisthe. Same or s i m i l a r to stock 2 9 . C e l l s are small with severe c o r t i c a l abnormality. C e l l s are small with severe c o r t i c a l and g u l l e t abnormality-c e l l s have a transverse gap across g u l l e t k i n e t i e s (plate l ) . 21 RESULTS; SECTION A - GENETICS From a series of 60 morphological variants obtained following nitrosoguanidine mutagenesis, ten variants were selected f o r genetic analysis. These included six variants with a defect i n fission-furrow c o n s t r i c t i o n (variants 2, 4, 8, 23, 62, and 63), two variants with a defect i n fission-zone formation (variants 29 and 3°)> and two variants that produce small c e l l s (variants 21 and 26). Two serie s of genetic crosses were made i n the study. The f i r s t was designed to determine whether the phenotype of each variant was due to a single gene and the second was designed to determine the a l l e l i c r e l a t i o n s h i p of selected mutant l i n e s . In the f i r s t series of genetic crosses, c e l l s of each variant were mated with wild-type c e l l s carrying the behavioral marker gene, pawn (pw). The heterozygous progeny of t h i s cross were induced to undergo autogamy, which r e s u l t s i n homozygosis at a l l genetic l o c i (Sonneborn, 1950). The exautogamous c e l l l i n e s constituted the F^ generation. These l i n e s were Scored for the morphological and pawn phenotypes. The scored r e s u l t s were compared with those that would have been expected on the basis of the following hypotheses: (a) a single recessive gene caused the abnormality, (b) two recessive genes acted i n concert 22 to cause the abnormality, and (c) two recessive genes were present i n the variant and each caused a morphological abnormality. The fr a c t i o n s of F 2 c e l l s i n the phenotypic classes morphological (m~/m~; pw +/pw +), morphological pawn (m~/m~; pw~/pw~), and pawn (m+/m+; pw~/pw~) which were expected on the basis of these hypotheses were 1 to 1 to 1, 3 to 3 to 1, and 1 to 1 to 3 respectively. The number of wild-type c e l l s was ignored as t h i s class may contain heterozygous c e l l s (those which had not gone through autogamy). The observed numbers of c e l l s i n the other phenotypic classes were compared by means of the G-test (Sokal and Rohlf, 1969) with the numbers expected. • In most cases the r e s u l t s indicated that a single recessive gene caused the morphological abnormality (table 2). In several cases (variants 26, 3°» and 63) the mating of variant c e l l s with pawn-marked wild-type c e l l s was done twice. In two of these cases (variants 26 and 30) the experimental re s u l t s from one of the genetic crosses did not s a t i s f y the hypothesis that a single gene was respons-i b l e f o r the morphological abnormality. When the matings with these two variants were repeated, however, the re s u l t s indicated that a single gene was responsible for the abnormality (table 2). The aberrant r e s u l t s i n the f i r s t crosses made using these l i n e s apparently resulted from inconsistencies i n scoring the mutant phenotypes or from TABLE 2 EXAUTOGAMOUS SEGREGATION OF ts HETEROZYGOTES F 2 PHENOTYPE  Variant Mutant Pawn- Pawn+ Dead ** Number t t m- m+ m- m+ pw- pw+ ?+ 2 dc5 1 4 12 4 6 47 1 8 73 4 dc4 23 20 27 4 6 — — 8 8 dc3 54 49 38 49 4 3 — 21 sm3 - - 69 88 — 10 _ 23 dc6 4 0 4 2 4 0 73 2 2 10 2 6 ( 1 ) sm2(l) 37 27 80 4 0 — — 23 26(2) sm2(2) 6 1 62 79 74 2 4 2 9 d f z l 38 57 44 47 . - - 2 4 30(1) dfz2(l) 31 36 29 83 . 2 3 34 30(2) dfz2(2) 30 66 26 4 8 — 2 62 dc2 u 38 58 60 52 3 1 3 63(1) dc2 a(l) 30 32 35 70 — 7 63(2) dc2 a(2) 2 8 30 27 50 2 1 2 VALUE O F "G" Ratio Tested  l i l i l 3: 1: 3 1: 3: 1 2 8 . 1 3 * 1.06 2 . 9 6 1 4 . 3 8 * . 0 2 3 1 - 3 9 * 2 . 9 6 4.04 ' . 7 9 2 2 . 3 9 * 6.00* • 3 9 . 1 9 1 8 . 4 1 9 - 7 8 3 9 . 5 5 83.85 3 0 . 9 9 1 8 . 4 4 3 6 . 7 0 5 9 . 3 6 3 1 . 7 2 1 0 6 . 2 0 5 5 - 0 4 2 1 . 9 3 2 3 . 4 1 7 5 . 3 2 2 8 . 6 3 3 9 - 3 7 3 7 . 3 4 3 2 . 4 2 1 1 8 . 9 8 ' 7 3 . 2 3 2 1 . 5 9 1 9 . 8 2 2.09 3 7 . 9 6 2 9 . 1 8 2 1 . 1 3 * adjusted g using Yate's correction. G values are to be compared to X 2 (.05) 2= 5 - 9 9 1 and X 2 (.05) 1 = 3 . 8 4 1 + - l i n e s which died before being tested f o r phenotype **- the pw~ and pw+ l i n e s were tested for t h i s phenotype before they died. ++- the bracketed numbers r e f e r to experiment numbers where any one mutant was crossed to pawn c e l l s i n more than one experiment. 24 incomplete penetrance. The r e s u l t s from crosses of several of the variants with pawn-marked c e l l s did not conform to what was expected i f a single recessive gene.caused the abnormality. These l i n e s were: (a) Variant 2 - The re s u l t s d i d not agree with any of the hypotheses tested. The number of m~/m~ and m /m c e l l s were equal, however, i n both the pw~/pw~ and pw+/pw+ classes. This suggested that a single gene was responsible for the abnormality but that many of the pw~/pw~ li n e s died before they were scored. (b) Variant 21 - The number of mutant c e l l s may have been underestimated due to d i f f i c u l t y i n scoring the phenotype of t h i s l i n e . Although the c e l l s were small, they were of normal shape and therefore abnormal clones may have been overlooked. (c) Variant 62 - The r e s u l t s d i f f e r only s l i g h t l y from what was expected i f a single gene caused the abnormality. The nine mutant genes were given names rela t e d to t h e i r phenotypes. These names are l i s t e d i n table 2. Mutants with defects i n furrow c o n s t r i c t i o n were c a l l e d defective c o n s t r i c -t i o n (dc) mutants, those with defective f i s s i o n zone formation were c a l l e d f i s s i o n zone (dfz) mutants, and those producing small c e l l size were c a l l e d small (sm) mutants. Each gene 25 was numbered (sml, sm2, etc.) and each a l l e l e was given a superscript (dc2 a, dc2 b, e t c . ) . The second series of genetic crosses were made to determine i f a l l e l i s m occurred among any of the ten genes examined. To do t h i s , c e l l s of a mutant l i n e , which had been marked with the pawn gene, were crossed with c e l l s of every other mutant l i n e . The heterozygous progeny were tested f o r the presence of morphological abnormality. (Permanent disappearance of the pawn t r a i t ensured that r e c i p r o c a l f e r t i l i z a t i o n had taken place.) I f the hetero-zygous progeny were morphologically abnormal then the two parent l i n e s were assumed to be a l l e l i c . The complimentation assay (table 3) revealed only one a l l e l i c pair: dc2 a and dc2 b (variants 63 and 62). Four mutants were selected for further study. These included three genes (dc2 a, dc4, and dfz2) which a f f e c t c e l l d i v i s i o n and one (sm2) which a f f e c t s c e l l growth. These mutants provide the basic material f o r an analysis of c e l l growth and d i v i s i o n i n Paramecium. In addition, the reduction i n c e l l size produced by the sm2 gene makes possible an analysis of the e f f e c t s of a change i n c e l l s ize on morphogenesis. Morphometric analysis was the main technique used i n examining the phenotypes of the mutant c e l l s . Two 26 TABLE 3  COMPLIMENTATION MATRIX dc2 a dc2 b dc3 dc4 dc5 dc6 d f z l dfz2 sm2 sm3 dc2 a + + + + + + + + dc2 b * + + + + a + a dc3 * * * + + + a dc4 + a ' + + + a dc5 + + + + a dc6 + + + a d f z l + + + dfz2 + + sm2 + sm3 a - repeatedly refused to mate * - mating that was not tested 27 types of morphometric data were colle c t e d : data on c e l l s of known age and data on populations of c e l l s of unknown age. The f i r s t type of morphometric data w i l l be used to examine the age-dependent changes i n s i z e and morphology of wild-type and mutant c e l l s . The second type of data w i l l be used to examine size-dependent aspects of c e l l morphogenesis and surface structure. 28 SECTION B; MORPHOMETRIC ANALYSIS OF CELL DIVISION INTRODUCTION: ' ' Kaneda and Hanson (197*0 separated the Paramecium c e l l cycle into three periods. The f i r s t period (post-f i s s i o n period) spans ages 0 to 0.1 c e l l cycles and i s a time of rapid c e l l growth. The second, or morpho-s t a t i c , period extends up to age 0-75 c e l l cycles. During t h i s time the c e l l does not change i n length. The t h i r d , or morphogenetic, period begins with the appearance of the or a l anlage at age 0.75 and culminates i n the c o n s t r i c t i o n of the f i s s i o n furrow. During t h i s period the c e l l undergoes a series of siz e and shape changes whose origi n s and functions are poorly under-stood. The i s o l a t i o n of mutants defective i n c e l l d i v i s i o n and c e l l growth, however, provides a means of assessing the importance of these s i z e and shape changes i n normal c e l l morphogenesis. In order to use the mutants for t h i s type of analysis, i t was necessary to compare the size and shape changes during the morpho-genetic period i n wild-type c e l l s with the corresponding changes i n mutant c e l l s . Four mutants were used, one with defective f i s s i o n zone formation (dfz2), two with defective c o n s t r i c t i o n of the f i s s i o n furrow (dc2 a and dc*0, and one which produced small c e l l s (sm2). 29 EXPERIMENTAL DESIGN: The purpose of the morphometric study of wild type and mutant c e l l s was to correlate differences i n the temporal pattern of s i z e and shape changes between these two types of c e l l s with the defects i n c e l l d i v i s i o n or development i n the mutants. Since the mutant c e l l s express t h e i r defects only at the r e s t r i c -t i v e temperature, a l l experiments were done at 34.5°C. To provide a suitable c o n t r o l , wild-type c e l l s were also cultured at 34.'5°C. (Kaneda and Hanson's study was done on c e l l s grown at room temperature and there-fore does not constitute a suitable control.) Two types of experiments were done: (a) EXPERIMENTS WITH SYNCHRONOUS CULTURES Synchronous c e l l samples were placed i n 35 x 100 m.m. p l a s t i c p e t r i dishes containing about 2 ml. of culture f l u i d with bacteria. The dishes were immediately immersed i n a 34.5°C water bath. The culture was p e r i o d i c a l l y examined u n t i l d i v i d i n g c e l l s were noted, and the culture was then fixed and silver-impregnated. (b) EXPERIMENTS WITH ASYNCHRONOUS CULTURES Asynchronous log-phase c e l l samples were placed i n culture f l a s k s containing culture f l u i d with bacteria such that the depth of f l u i d i n the f l a s k 30 EXPERIMENTAL DESIGN; - (Cont'd.) (b) EXPERIMENTS WITH ASYNCHRONOUS CULTURES - (Cont'd.) was no. greater than 1 .5 cm. A synchronous sample of c e l l s was removed from the asynchronous culture and prepared as indicated above. Both the asynchronous and synchronous cultures were then heated to 34.5°C i n a water bath. When div i d i n g c e l l s were seen i n the synchronous sample, the asynchronous culture was fixe d and s i l v e r -• impregnated. The r e s u l t s reported below f o r sm2 and dc2 a c e l l s were based e n t i r e l y , and f o r dfz2 and dc4 c e l l s p a r t i a l l y , on synchronous material, while the res u l t s reported f o r wild-type c e l l s were based e n t i r e l y on asychronous material. MORPHOMETRIC ANALYSIS OF SILVER-IMPREGNATED CELLS; The age"*" of silver-impregnated c e l l s was determined by comparing the morphology of the c e l l with data presented by Kaneda and Hanson (1974) and Jones (1976), (see plate 8). A v a r i e t y of measurements, indicated i n figure 6, were then made on each c e l l of known age. These measurements were made with an ocular micrometer and represent the l i n e a r length of the c e l l or parts of the c e l l . As the c e l l s are not f l a t , however, l i n e a r length measurements do not represent the actual length ages are indicated as a decimal f r a c t i o n of the length of the c e l l cycle. 31 EXPERIMENTAL DESIGN: - (Cont'd.) MORPHOMETRIC ANALYSIS OF SILVER-IMPREGNATED CELLS: - (Cont'd.) of the c e l l surface. The a c t u a l , or contour, surface lengths were obtained by constructing outline drawings of d i v i d i n g c e l l s using the l i n e a r length measurements. Separate outline drawings were made f o r c e l l s of ages 0.94, 0.95. 0.96 - 0.97. and 0.98 - 0.99 c e l l cycles. To obtain contour lengths from the c e l l o u tlines, a piece of t h i n wire was shaped to conform to the contour and then was straightened and measured. In constructing and using the outline drawings, two assumptions were -made: that the reference points used .in the measure-ments (for example the CVPs) did not migrate or move during c e l l d i v i s i o n and that the f i s s i o n furrow c o n s t r i c t s at a constant rate that i s equal i n both mutant and wild-type c e l l s . The l a t t e r assumption i s at least q u a l i t a t i v e l y true based on observations of l i v e and f i x e d material. Observations on contour length changes w i l l be presented separately from l i n e a r length changes. Unless stated otherwise, a l l measurements r e f e r to l i n e a r length. 32 2. THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; The data c o l l e c t e d on wild-type c e l l s are l i s t e d i n table 4. Morphometric parameters have been plotted against c e l l age i n figures 7 through 12. Figure 7a shows the changes i n length of wild-type c e l l s that occurred during one c e l l cycle at 34.5°C. Based on t h i s f i g u r e , the morphogenetic period was separated i n t o three phases: (a) a growth phase from age 0.75 to O.83 c e l l cycles; (b) a contraction phase from age O.83 to 0.95 c e l l cycles; (c) . a fission-furrow phase from age 0.95 to completion of cytokinesis. i . Growth Phase: Between ages 0.75 and O.83 c e l l cycles the c e l l length increased by 7 um (from 123 to 130 /am). Growth occurred p r i m a r i l y i n the posterior part of the c e l l , with both the p o s t e r i o r suture (GP) and region CVP (fig.9b) increasing. Other parts of the c e l l did not change i n length with the exception of the middle of the anterior h a l f of the c e l l (CVA") which decreased i n length. During the growth phase two new CVP's appeared (plate 8), one anterior to the old TABLE 4 VARIABLE AGE-DEPENDENT CHANGE IN WILD-TYPE CELL SIZE  DIMENSION ( i n urn) WITH STANDARD ERRORS •75 123+3.0 41.1.6 3.0^.15 43-0.6 CELL AGE Length Width L/Wc CVA CVA* CVINT CVP' -----CVP 24J1.7 GA 54jl.6 G 17+0.7 GP d 52.1.3 #b.b.d o 365O.6 CVINT/b.b.l . 6 - .04 PROTER LENGTH -OPISTHE LENGTH PG OG PGP GAO N f 5 .8-.84 .87-.89 .9-.91 .92-.93 130+2.5 41+0.5 3-1+.04 24,1.0 23*1.2 38?1.2 18-0.9 27+1.5 56+2.1 17+0.7 57+1.4 26^2.4 1.5-.05 124x2.2 44.1.0 2.9Z.06 24+1.1 19+0.5 39+1.4 19+1.0 25+1.4 54x1.0 17+0.4 53+1.9 34^2.4 1.2-.08 124-1.1 43,0.4 2.9+.05 25+0.7 18.1.0 40-1.1 19+1.0 23+1.3 • 53+1.0 19+0.4 51+0.4 43J1.4 I.O-.06 121,2.4 44T0.7 2.8-.06 24,1.1 16,1.0 41.1.3 17+1.8 24-0.9 50+1.5 22T1.6 49*1.4 47+2.3 O.9-.O3 8 a-see figure 6 f o r explanation of abbreviations b-in f r a c t i o n s of a c e l l cycle c-length to width r a t i o ( a r b i t r a r y units) d-the number of basal bodies i n region CVINT 7 used .94 123+3-5 47J0.8 2.6-.10 24T2.2 19x1.1 37+2.2 20-1.1 23+1.6 49+2.7 27+1.6 48J2.3 49^2.2 O . 8 - . O 3 •95 .96-.97 .98-.99 126.2.9 42x0.5 3.0-.05 20T1.1 22rl . 7 43- 1.2 20,0.9 21+0.5 44- 1.1 43Jo.7 47^2.4 0.9-.06 65-1.9 59.1.0 16^0.4 13+0.4 7-1.5 1 3 8 J 3 . 5 42Jo.6 3-5+.08 24Jo.8 25+1.1 44,1.7 25+0.5 20- 1.6 35-1-1 43+0.7 46.1.2 I.O-.03 71-2.2 67+1.9. 15+0.5 14-0.4 21- 1.8 8 153+3.3 40- 1.2 3.9+.18 25.O.8 32,2.2 45x2.9 27^2.3 25+4.5 35-2.1 45+1.0 41- 3-3 1.1-.04 79-1.9 75+1.7 15+0.3 15+0.3 29-1.4 e-the spacing between basal bodies f-sample size 3* 2. THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; - (Cont'd.) i . Growth Phase: - (Cont'd.) anterior CVP and one anterior to the old posterior CVP. Kaneda and Hanson ( 1 9 7 ^ ) described a s i m i l a r growth phase and also found that growth was r e s t r i c t e d to the ends of the c e l l . They stated that the maximum length attained during t h i s phase (ages 0.75 to 0.89) was 132 pm at age 0.89-i i . Contraction Phase: Between ages O.83 and 0.95 the c e l l length decreased by 10 pm. This contraction was accompanied by a change i n c e l l shape, which became flattened on the sides (plate 7). The contraction occurred i n two regions of the c e l l , the poster i o r end (CVP) and the mid-region of the anterior h a l f of the c e l l (CVA'). Region CVA' was therefore continuing a contraction which began i n the growth phase. During the contraction phase region CVINT (fig.6) began an expansion that would continue into the f i n a l phase of morphogenesis. Near age 0 . 9 the f i s s i o n l i n e (or zone), a clear space devoid of c o r t i c a l organelles, 35 2. THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; - (Cont'd.) i i . Contraction Phase; - (Cont'd.) appeared on'either side of the vestibule (plate 8). This l i n e spread around the c e l l and was complete by age 0.93' Between ages 0.93 and 0.95 there was an apparent t w i s t i n g of the posterior h a l f of the c e l l i n r e l a t i o n to the anterior h a l f . Note i n Plate 9 ( f i g s . . c and d) that the k i n e t i e s bend at the f i s s i o n l i n e as i f the posterior region has twisted clockwise i n r e l a t i o n to the anterior region. The twisting was also seen on the vent r a l surface as the l e f t wall of the opisthe's vestibule was aligned with the r i g h t wall of the proter's vestibule (plate 8, f i g . e ) . The two separate vestibules i n the daughter c e l l s are produced by the b i s e c t i o n of the o r i g i n a l vestibule by the f i s s i o n l i n e and subsequently the f i s s i o n furrow. The anterior vestibule remains attached to the e x i s t i n g g u l l e t . The posterior vestibule subsequently attaches to the developing g u l l e t anlage. Extensive development of the anlage occurs at t h i s time, as w i l l be described below. 36 2. THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS: - (Cont'd.) i i . Contraction Phase: - (Cont'd.) P r o l i f e r a t i o n of basal bodies also began during t h i s phase, the d e t a i l s of which w i l l be presented l a t e r . The end of the contraction phase was marked by the appearance of a con-s t r i c t i o n i n the region of the f i s s i o n l i n e , i i i . Fission-Furrow Phase: With the c o n s t r i c t i o n of the f i s s i o n furrow the c e l l underwent a rapid increase i n length from 123 um (age 0.95) to 154 pm (age 0.98). This growth occurred p r i m a r i l y i n the mid-regions of the proter and opisthe (CVA', fig.10a; CVP', fig.10b). Minor growth occurred i n the posterior region (CVP) and the c e l l mid-region (CVINT). ( I t should be noted here that once c o n s t r i c t i o n begins the c e l l becomes highly curved i n the furrow region and there-fore l i n e a r measurements do not accurately gauge growth i n t h i s area.) A dramatic shortening of the anterior and posterior sutures (GA and GP, f i g s . 7b and 8a) accompanies the increase i n c e l l length. This resulted from migration of the vestibules to central locations on the proter and opisthe. 37 2. THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS: - (Cont'd.) i i i . Fission-Furrow Phase: - (Cont'd.) The forward movement of the proter's vestibule (beginning at age 0.94) l e f t i n i t s wake a proter posterior suture. The increase i n length of t h i s suture exceeded by 5 pm the decrease i n length of the anterior suture, which indicated that the poster i o r region of the proter was growing, a change also seen on the dorsal surface (region CVINT was increasing). Migration of the opisthe's vestibule (also beginning at age 0.94) was less dramatic. As a r e s u l t , at f i s s i o n the opisthe had i t s vestibule located i n the anterior t h i r d of the c e l l (plate 8, f i g . h ) . The opisthe was therefore shaped rather d i f -f e r e n t l y from the proter. The former was pointed at the anterior end and blunt at the posterior while the l a t t e r was e l l i p s o i d a l (plate 7 ) . i v . C e l l Width: Width was f a i r l y constant during the growth and contraction phases. There was a s l i g h t increase i n width just before formation of the f i s s i o n furrow (fig.8b). During the 38 THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS; - (Cont'd.) i v . C e l l Width: - (Cont'd.) fission-furrow phase the c e l l width decreased s t e a d i l y from 48 pm at age 0.95 to 36 pm at age 0.98. v. Basal Body P r o l i f e r a t i o n : The number of basal bodies between the innermost CVPs on the dorsal surface began to increase at age 0.82 (fig.12a). Between ages 0.82 and 0.95 the number of basal bodies i n t h i s region doubled (from 25 to 50). A f t e r age 0.95 the mean number of basal bodies i n CVINT decreased by nine. Although t h i s decrease was not s t a t i s t i c a l l y s i g n i f i c a n t i t constituted a consistent trend for decrease i n basal body numbers and was seen i n some mutant l i n e s as well as i n wild-type c e l l s . Basal body p r o l i f e r a t i o n during c e l l d i v i s i o n i s i l l u s t r a t e d i n Plate 9. The spacing ( i n um) between basal bodies i s shown i n figure 12b. This spacing accounts f o r both the number of basal bodies i n the region CVINT and the length of CVINT. At age 0.75 basal bodies were 1.6 pm apart (fig.12b). The spacing decreased to a minimum of 0.8 jura 39 THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS: - (Cont'd.) v. Basal Body P r o l i f e r a t i o n : by age 0.94 at which time basal body p r o l i f -eration was at a maximum (Kaneda and Hanson, 1974). By age 0.98 the spacing increased to 1.1 Lun. A f t e r f i s s i o n , the basal body spacing was s t i l l l e s s than that i n i n t e r f i s s i o n c e l l s (plate 9» f i g . e ) . v i . Contour Drawings: Contour drawings of wild-type c e l l s ( f i g . 13) indicate a l l of the length and width changes discussed above. Note that the positions of the CVPs and vestibules changed i n a very regular manner with increasing c e l l age. There i s also a marked difference i n the extent of vestibule migration i n the proter and opisthe. v i i : Growth of the Dorsal Surface: Between ages 0.93 and 0.98 there was a l i n e a r increase i n the contour length of the mid-regions of the proter and opisthe (CVA' and CVP *; fig.14). The c e l l mid-region (CVINT) i n i t i a l l y contracted and then began to increase i n contour length ( f i g . l 4 ) . The polar regions (CVA and CVP) showed no net growth during t h i s part of the c e l l cycle. 40 THE MORPHOGENETIC PERIOD IN WILD-TYPE CELLS: - (Cont'd.) v i i i . Growth of the Ventral Surface: Contour lengths of regions on the ve n t r a l surface are l i s t e d i n table 5- Between ages 0.94 and 0.95 the anter i o r suture decreased i n contour length by 7 tun while the proter's posterior suture increased by 7 pm> In the opisthe, the decrease i n length of the poster i o r suture was s i m i l a r l y equal to the increase i n length of the opisthe's anterior suture. The 3 p.m increase i n the t o t a l contour length of the ventral surface occurring at t h i s time appeared as an increase i n the length of the proter's vestibule, (from 13 to 16 jum). Between ages 0.95 and O.97 the growth of the posterior suture i n the proter exceeded the decrease i n the length of the anter i o r suture. Surface growth behind the vestibule was there-fore accounting f o r some of the apparent vestibule movement. In the opisthe, the length of the anterior suture continues to increase a f t e r age 0.95 without any further change i n the posterior suture length. TABLE 5 CONTOUR LENGTHS OF VENTRAL REGIONS MORPHOMETRIC PARAMETER* Sample Age GAp Gp GPp GAo Go GPo Wild-Type .94 59 13 0 0 13 60 .95 52 16 7 9 13 52 .96-.97 43 15 23 14 14 52 .98-.99 42 15 35 22 15 53 sm2 .91-.94 56 11 0 0 11 53 •95-.96 38 16 11 5 12 47 .97-.98 38 15 20 10 13 43 • .99 32 15 31 22 14 41 dc4 .91-.93 52 11 0 0 11 52 •94-.95 40 14 6 5 12 42 .96-.97 28 15 25 10 14 45 .98-.99 31 15 35 12 14 45 dc2 a .92-.94 55 13 0 0 13 57 • 95 47 15 5 2 12 55 .96 37 15 21 10 14 44 •97-.99 35 15 33 20 15 50 dfz2 •93 63 14 0 0 14 55 •94-.95 44 17 10 5 14 50 .96-.97 35 15 21 14 14 46 .98-.99 36 15 35 21 15 44 * i n pm. a b b r e v i a t i o n s : GAp, a n t e r i o r s u t u r e o f p r o t e r ; Gp, p r o t e r v e s t i b u l e ; GPp, p r o t e r p o s t e r i o r s u t u r e ; GAo, o p i s t h e a n t e r i o r s u t u r e ; Go, o p i s t h e v e s t i b u l e ; GPo, o p i s t h e p o s t e r i o r s u t u r e . 42 THE MORPHOGENETIC PERIOD IN MUTANT LINES: a. Mutant sm2 At the r e s t r i c t i v e temperature sm2 c e l l s were small, with severe c o r t i c a l and g u l l e t abnormalities (plate 5)' C e l l s could complete two to four c e l l cycles at 34-5°C before d i v i s i o n arrest occurred. D i v i s i o n arrest was not accompanied by misdivision. Data c o l l e c t e d on sm2 c e l l s are given i n table 6. The parameters are plot t e d against c e l l age i n figures 7 to 12. D i v i s i o n stages of sm2 c e l l s are i l l u s t r a t e d i n plate 10. With the exception of width, the standard errors of measurement parameters were s i m i l a r to those recorded f o r wild-type c e l l s . Width i s more variable i n sm2 than i n wild type, i . Growth Phase: Between ages 0.75 and 0.83 c e l l cycles there was no s i g n i f i c a n t increase i n length (fig.7a). The only part of the c e l l which changed i n length was the mid-region (CVINT) which contracted ( f i g . l i b ) . Region CVA' did not change i n length (fig.10a) whereas the same region contracted i n wild-type c e l l s during t h i s phase. I n t e r f i s s i o n and growth phase sm2 c e l l s had an unusual c e l l outline with a conspicuous TABLE 6 AGE-DEPENDENT CHANGE IN sm2 CELL SIZE DIMENSION ( i n um) WITH STANDARD ERRORS VARIABLE3" CELL AGE b 0.75 0.8 0.82 O.85-.86 0.91-.94 0.95-.96 0.97--98 .99 1.05 Length 123+1.2 120Jl.7 117+5 .1 122^ 2.2 lllj2.4 108,2.9 114x2.9 128^3-7 81^4.5 Width L/Wc CVA 55-1.1 52-1.0 53-3 .2 52-1.5 48-1.3 46-1.5 44-1.4 42-1.3 50-2.9 41-1.0 39-0.9 22±2 .1 25+1.3 22^ 1.0 19^1.5 19+0.9 25Jo.9 28-2.5 CVA' + 20-1 .6 18-1.8 17+0.8 19+1.0 19J0.6 . 24-1.3 CVINT 57-1.4 54-1.2 . 40±2 -7 34^ 2.6 32^ 0.8 31+1.1 36T2.0 34x2.2 35-3-8 CVP' _ — — — 4. 20-1 .4 20^ 1.1 18-1.0 22jl.O 18-2.3 - 25+1-2 CVP 25+0.8 26-1.0 18^0 .6 25+L3 22-1.0 17+0.5 23+L7 20x1.2 18^1.8 GA 52-0.9 51+1.2 49^4 .0 51+1.9 47+1.7 31-L5 29-2.1 25-1.1 31+2.5 G 18-0.3 18^0.4 17+0 .6 18-1.0 23x0.6 *4* 17+0.4 2 P d 53+0.9 51+0.9 34jl.4 53+2 • 7 52-1.8 5 35+0.8 33+L3 29x1-8 33+1.5 32-3.1 #b.b.a 35+0.7 23x1 .8 20-1.3 25+0.9 27+1.4 30+3.2 22x2.7 CVINT/b.b. I.6-.03 1.6-.05 1.7-.07 1.7-.05 1-3-.04 1.2-.07 1.3-.05 1.2-.12 1.6-.12 PROTER LENGTH 56-2.4 61-1.2 68-1.8 — — OPISTHE LENGTH 50,1.0 51+-.2 60^2.3 ' PG 16-1.0 15+1.0 15+1.0 OG 12x1.0 13+1.0 I4jl.0 - — . — PGP 10-2.4 18X2.0 27+1.3 GAO 3-1.0 6-1.0 13-1.0 n f 23 16 4 6 16 8 7 7 6 a-see f i g u r e 6 f o r explanation of abbreviations used e-the spacing between basal bodies b-in f r a c t i o n s of a c e l l cycle f-sample si z e c-length to width r a t i o ( a r b i t r a r y units) _p_ d-the number of basal "bodies i n region CVINT u> 44 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i . Growth Phase: - (Cont'd.) bulge on the c e l l ' s l e f t side (plate 10). The mean c e l l width at age 0.75 was 55 pm (fig.8b) which i s s i g n i f i c a n t l y greater than the width of wild-type c e l l s of the same age (42 pm). i i . Contraction Phase: The contraction phase i n sm2 c e l l s began at age 0.85 and continued to age 0.95- C e l l s decreased i n mean length by 14 pm during t h i s phase compared to a 9 pm contraction i n wild-type c e l l s . At the end of the contraction phase sm2 c e l l s were s i g n i f i c a n t l y shorter than wild type (fig.7a). Most of the contraction i n sm2 c e l l s was l o c a l i z e d i n the c e l l extremities (CVA and CVP) and the mid-region (CVINT). Region CVA' did not show the contraction i t did i n wild-type c e l l s (fig.10a). i i i . Fission-Furrow Phase: This phase begins i n sm2 c e l l s at the same age as i n wild-type c e l l s (0.95). Increase i n length during t h i s phase occurred at a s i m i l a r rate i n both wild-type and sm2 c e l l s but sm2 c e l l s were shorter than wild type when they 45 THE MORPHOGENETIC PERIOD IN MUTANT LINES; - (Cont'd.) i i i . Fission-Furrow Phase; - (Cont'd.) entered t h i s phase. By age 0.98 the mean length of sm2 c e l l s reached 128 pm, 6 pm longer than i n t e r f i s s i o n c e l l s (fig.7a). During the f i r s t c e l l cycle at the r e s t r i c t i v e temperature furrowing was normal i n most sm2 c e l l s , but a few cases of misdivision (arrested at about age 0.99) were encountered. Increase i n length during t h i s phase occurred p r i m a r i l y i n the mid-regions of the proter and opisthe (CVA' and CVP', figs.10a and b). The extremities of the c e l l (CVA and CVP) also increased i n length during t h i s phase. The s t a r t of the fission-furrow phase i n wild-type c e l l s was marked by a dramatic shortening of the anter i o r (GA) and poster i o r (GP) sutures (figs.7b and 8a). In sm2 c e l l s the anterior suture r a p i d l y contracted between ages 0.94 and 0.96 (fig.7b). The posterior suture showed no dramatic shortening but de-creased s t e a d i l y i n length from age O.85 to age 0.97 (fig.8a). The poster i o r suture i n sm2 c e l l s at age 0.95 was s i g n i f i c a n t l y shorter (35 pm) than i n wild-type c e l l s (43 jum). This 46 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i i i . Fission-Furrow Phase: - (Cont'd.) difference arose from the difference i n the o v e r a l l c e l l length and not a difference i n vestibule movement, i v . Basal Body P r o l i f e r a t i o n : Mutant sm2 c e l l s had a d i f f e r e n t pattern of basal body p r o l i f e r a t i o n from that seen i n wild-type c e l l s (fig.12a). The number of basal bodies i n CVINT increased s t e a d i l y from age 0.85 to age 0.99. Unlike the case i n wild-type c e l l s , there was no doubling of basal body numbers. At age O.85 there was a mean of 20 basal bodies i n CVINT and t h i s increased to 30 by age 0.99- There was no reduction i n basal body number during the fission-furrow phase and p r o l i f e r a t i o n continued up to the time of f i s s i o n . Despite the differences i n the pattern of basal body p r o l i f e r a t i o n between wild type and sm2 c e l l s there was no s i g n i f i c a n t d i f f e r -ence i n the number of basal bodies i n CVINT of these two c e l l l i n e s at age 0.99 (fig.12a). The length of the region CVINT increased s t e a d i l y during the morphogenetic period of wild-type c e l l s but decreased s t e a d i l y i n sm2 4? THE MORPHOGENETIC PERIOD IN MUTANT LINES; - (Cont'd.) i v . Basal Body P r o l i f e r a t i o n ; - (Cont'd.) c e l l s ( f i g . lib)'. This may have been re l a t e d to the differences noted i n basal body pro-l i f e r a t i o n . Examination and comparison of basal body spacing eliminates the ef f e c t of differences i n the length of the region i n which the basal bodies are counted. Decrease i n basal body spacing i n sm2 c e l l s was not as obvious as i n wild-type c e l l s (fig.12b) i n d i c a t i n g a reduction i n p r o l i f e r a t i o n i n sm2 c e l l s . At age 0.98 the basal body spacing on wild-type and sm2 c e l l s was i d e n t i c a l . Although basal body p r o l i f e r a t i o n was reduced i n sm2 c e l l s t h i s was of f s e t by a reduction i n c e l l s i z e . C e l l s which had been at 34.5°C f o r 1.05 c e l l cycles had the same basal body spacing as i n t e r f i s s i o n c e l l s (table 3). Basal body p r o l i f e r a t i o n i n sm2 c e l l s of various ages i s shown i n figure 15• v. Width; Age 0.75 sm2 c e l l s were much wider than wild-type c e l l s of the same age (fig.20b). In sm2 c e l l s width decreased s t e a d i l y during the morphogenetic period and reached 40 LUTI by age 48 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) v. Width: - (Cont'd.) 0.99. Wild-type c e l l s of age 0.99 were also 40 pm wide. The length to width r a t i o i n sm2 c e l l s was 2.3 + 0.1 at age 0.75* This remained constant u n t i l the fission-furrow phase. By age 0.99 i t increased to 3.1 +0.2 (fig.11a). At the s t a r t of the fission-furrow phase the length to width r a t i o was about 2.5 ± 0.1 i n both wild-type and sm2 c e l l s , v i . Stomatogenesis: The o r a l anlage i s f i r s t seen i n sm2 c e l l s of age 0.75- I t s morphology at that age was s i m i l a r to anlagenin age 0.75 wild-type c e l l s . Invagination of the anlage occurred and i t developed to the three kinety stage ( f i g . l 6 ; a, b, c ) . Abnormalities i n the anlage were appar-ent i n c e l l s age 0.9 or older. Most c e l l s of this.age had anlagenwith i r r e g u l a r l y spaced argentophilic l o c i but had no kin e t i e s (fig.16). In some cases the anlagen were abnormally shaped or reduced i n size ( f i g . l 6 ; d, g). Migration of the sm2 anlage began at age 0.92 or 0.93 and proceeded i n a manner s i m i l a r 49 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) v i . Stomatogenesis - (Cont'd.) to that seen i n wild-type c e l l s . In many cases granular argentophilic material was located between the posterior of the anlage and i t s o r i g i n a l point of contact with the vestibule (fig.16; g to j ) . The endoral kinety, which i s resorbed during the early stages of stomatogenesis ( f i g . 4 ) , reappeared i n sm2 c e l l s around age 0.93- In some c e l l s the new ehdoral kinety was normal (fig.16; f) while i n others i t consisted of only a few irregularly-spaced argentophilic bases ( f i g . 16; e, h). The newanlagen, which should have formed at age 0.88, were found i n some c e l l s but not i n others ( f i g . l 6 ) . v i i . Juvenile C e l l s : C e l l s aged 1.05 c e l l cycles constitute the proters and opisthes r e s u l t i n g from the f i r s t d i v i s i o n at the r e s t r i c t i v e temperature. In mutant sm2 these c e l l s had a mean length of 81 pm (table 6) and were 20 pm shorter than age 1.05 wild-type c e l l s (these are about 100 jum long). The juvenile c e l l s were rounded i n out-l i n e and had a-twisted body (plate 10). The 50 THE MORPHOGENETIC PERIOD IN MUTANT LINES; - (Cont'd.) v i i . Juvenile C e l l s ; - (Cont'd.) kinety pattern,- g u l l e t , and cytoproct were often abnormal on these c e l l s , v i i i . Contour Drawings; The contour drawings of sm2 d i v i d i n g c e l l s i l l u s t r a t e the size difference between these and wild type d i v i d i n g c e l l s ( f i g . 13) • Not-e that the contraction i n sm2 c e l l s was more pro-longed (to age 0.95) a n d that the displacement of CVPs and vestibules did not change with c e l l age i n as regular a manner as i n wild-type c e l l s . i x . Growth of the Dorsal Surface; The greatest r e l a t i v e increase i n surface length occurred i n the portion of the furrow surface anterior to the f i s s i o n - l i n e ( f i g . l 4 b ) . Posterior to the f i s s i o n l i n e , the furrow surface showed no net growth. Growth of the c e l l mid-regions was reduced i n sm2 c e l l s ( i n comparison to wild type) and there was no net growth i n the polar regions, x. Growth of Ventral Surface; Between ages 0.94 and 0.95 the proter's anterior suture decreased by 18 p.m i n length 51 THE MORPHOGENETIC PERIOD I N MUTANT LINES;. - ( C o n t ' d . ) x. G r o w t h o f V e n t r a l S u r f a c e ; - ( C o n t ' d . ) ( t a b l e 5) w h i l e ' i t s p o s t e r i o r s u t u r e i n c r e a s e d by 11 pm i n l e n g t h . D u r i n g t h e same p e r i o d t h e p r o t e r ' s v e s t i b u l e i n c r e a s e d i n l e n g t h b y 5 pm. A f t e r a ge 0.95 t h e r e , was no f u r t h e r c h a n g e i n t h e l e n g t h o f t h e p r o t e r ' s a n t e r i o r s u t u r e . B e t w e e n a g e s 0.94 a n d 0.95 i n t h e o p i s t h e , t h e p o s t e r i o r s u t u r e d e c r e a s e d b y 6 pm i n l e n g t h w h i l e t h e a n t e r i o r s u t u r e i n c r e a s e d b y 5 pm i n l e n g t h . A f t e r age 0.95 t h e p o s t e r i o r s u t u r e c o n t i n u e d a s l o w d e c r e a s e i n l e n g t h , b. M u t a n t dc4 I n a s y n c h r o n o u s c u l t u r e a t 35°C t h e dc4 p h e n o -t y p e was c h a r a c t e r i z e d b y d i v i s i o n a r r e s t a n d r e d u c e d c e l l s i z e . S e v e r e c o r t i c a l and g u l l e t a b n o r m a l i t i e s w e re n o t e d a f t e r 20 h o u r s a t t h e r e -s t r i c t i v e t e m p e r a t u r e . D a t a c o l l e c t e d on dc4 c e l l s a r e g i v e n i n t a b l e ?. P l o t s o f t h e m o r p h o m e t r i c p a r a m e t e r s a g a i n s t c e l l age a r e g i v e n i n f i g u r e s 17 t o 22. The s t a n d a r d e r r o r s a n d c o e f f i c i e n t s o f v a r i a t i o n f o r m e a s u r e m e n t s o f dc4 c e l l s w e r e s i m i l a r t o o r l e s s t h a n t h o s e f o r w i l d - t y p e c e l l s . TABLE 7 AGE-DEPENDENT CHANGE IN dc4 CELL SIZE  DIMENSION ( i n mn) WITH STANDARD ERROR VARIABLE CELL AGE b •75 .8- .81 .83-.84 .85-. 87 .88-.9 1 •91-.93 Length 109J2. 1 111 J l . 6 108jl. 5 112T"2 .1 113x3. 1 106Jl. 6 Width 42^0. 6 45 J l . 0 46x1. 2 4 4 j l .2 4741. 6 48T1. 7 L/Wc 2.6X.05 2.5 -.06 2.4-T.09 2.6J. 08 2.4-.12 2-3x-10 CVA 37-1. 0 20 9 19+0. 5 21x1 •3 22x1. 3 20 x0. 3 CVA ' - — 19 8 17 Jo. 8 18x0 •5 19+0. 9 16 Jo. 5 CVINT 52-1. 7 32 I1- 5 33+1. 0 33+0 .9 32-1. 0 33+0. 7 CVP • — — 20 I1- 2 L S j l . 6 19+1 .1 21.0. 7 18 J l . 0 CVP GA ' 20-1. 45^1. 3 8 19 43 h-1 5 21x1. 43 J l . 8 2 22x1 45^1 •5 .1 20x0. 45 J l . 4 ! 1 19+1. 4 2 j l . 0 6 G 16-0. 2 17 +°- 5 16 Jo. 3 18x0 .4 18 Jo. 4 23x1- 0 SP d 49x0. 6 51 J i - 4 4 9 j l . 5 49^1 • 7 51+2. 0 4 l j l . 7 #b.b.a 37x1- 8 25 l l . 0 23x1- 3 2 6 j l .8 33T1. 3 40T2. 7 CVINT/b.b. 1.4-.04 1-3 -.04 1.5-.05 1.3-. 09 1.0-.04 0.9-.06 PROTER LENGTH OPISTHE LENGTH PG OG PGP GAO n f 8 10 8 8 a-see figure 6 for explanation of abbreviations used b-in f r a c t i o n s of a c e l l cycle c-length to width r a t i o ( a r b i t r a r y units) d-the number of basal bodies i n region CVINT 10 .94-.95 -96-.97 -98-.99 98-3-2 45J1.6 2.2T.13 18Jo.6 17+1.4 30+1.4 18Jo.6 15+1.1 30-1.5 32J2.4 39+1.3 O.8-.03 50-1.6 47+1.7 l4 +0.3 12+0.5 6 j l . l 3-1-0 10 120-1.9 38|l.4 3.2-.13 20x0.8 25+1.1 36Jo.8 25-0.8 15+0.4 23-1.0 35+1.2 36jl.6 1.0-.05 62-1.5 58jl.3 15+0.1 14x0.2 24jl.8 8-I.3 10 128x1.2 38J1.1 3-^+.09 24Jo.7 26jl.2 37+1.2 26-0.9 15+1.2 23-0.9 35+1.3 39+2.0 I.O-.05 63-1.0 6 4 j l . l 15+0.2 14x0.2 26jl.O 15-1.4 10 e-the spacing between basal bodies f-sample size ^ 53 THE MORPHOGENETIC PERIOD IN MUTANT LINES;. - (Cont'd.) i . Growth Phase; Age 0.75 dc4 c e l l s had a mean le n g t h of 109 pm ( t a b l e 7). The d i f f e r e n c e i n leng t h between wild-type and dc4 c e l l s at t h i s age (109 pm as compared to 120 pm f o r w i l d - t y p e c e l l s ) was l a r g e l y due to d i f f e r e n c e i n the len g t h of the a n t e r i o r h a l f of the c e l l . An apparent defect i n c e l l growth occurred du r i n g the i n t e r f i s s i o n p e r i o d of dc^ c e l l s . From age 0.75 to age 0.9 there was no inc r e a s e i n dc4 c e l l l e n g t h ( f i g . 1 7 a ) . The inc r e a s e i n p o s t e r i o r suture (GP) l e n g t h noted i n w i l d - t y p e c e l l s .did not occur i n dc4 c e l l s ( f i g . 1 8 a ) . i i . C o n t r a c t i o n Phase; C o n t r a c t i o n of dc4 c e l l s occurred between ages 0.9 and 0.95 with a l e n g t h decrease of 16 pm. At age 0.95 the mean c e l l l e n g t h was 98 jam as compared to 126 um i n age 0.95 wild - t y p e c e l l s ( f i g . 1 7 a ) . The c o n t r a c t i o n of dc4 c e l l s was l o c a l i z e d i n the c e l l e x t r e m i t i e s (CVA and CVP, f i g s . 1 9 a and b) . I n the r e g i o n CVA* , none of the c o n t r a c t i o n seen i n wild - t y p e c e l l s was found i n dc4 c e l l s ( f i g . 2 0 a ) . 54 THE MORPHOGENETIC PERIOD IN MUTANT LINES; - (Cont'd.) i i i . Fission-Furrow Phase; Between ages 0.95 and 0.98 the mean length of dc4 c e l l s increased from 95 to 128 pm ( f i g . 17a). The length at age 0.98 was s i g n i f i c a n t l y less than that of wild-type c e l l s (153 .um) • Growth was l o c a l i z e d i n the mid-regions of the proter and opisthe (CVA 1 and CVP1; figs.20a and b). Some growth also occurred i n the anterior end of the c e l l (CVA; fig.19a) and i n CVINT (fig.21b). This pattern of growth was iden-t i c a l to that of wild-type c e l l s . I t was d i f f i c u l t to score dc4 c e l l s of age 0.98 or 0.99 since many of these c e l l s were misdividers. Such c e l l s were not used i n the analysis as they could not be accurately aged. Thus, f o r dc4 c e l l s which appeared as i f they would not be arrested i n the f i r s t c e l l cycle at 3^-5°C there was a siz e reduction s i m i l a r to that found i n sm2 c e l l s . Decrease i n length of the anterior suture (GA; fig.17b) i n dc4 c e l l s began i n the contrac-t i o n phase at about age 0.92. The suture reached a minimum length of 23 pm at age O.96. The length of t h i s suture i n wild-type c e l l s 55 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i i i . Fission-Furrow Phase: - (Cont'd.) never f e l l below 35 pm (fig.17b). In dc4 c e l l s the vestibule moved forward i n the proter a distance of 26 pm (table 4; PGP). This i s s i m i l a r to the extent of vestibule movement i n wild-type c e l l s (29 pm) and i n sm2 c e l l s (27 pm). The p o s t e r i o r suture i n dc4 c e l l s began to decrease i n length around age 0.9 (GP; f i g . 18a). I t reached a mean length of 35 pm at age 0.95 and maintained t h i s length u n t i l f i s s i o n . The pattern of decrease i n length of t h i s suture was s i m i l a r to that found i n wild-type c e l l s . The minimum suture length i n wild-type c e l l s however, exceeded by 10 pm that i n dc4 c e l l s (fig.18a). Patches appeared on the cortex of dc4 c e l l s during the f i r s t c e l l cycle at 34.5°C. These brown patches had a grainy appearance i n silver-impregnated material and were i n t e r -calated between kineties or were found i n the anterior or p o s t e r i o r suture (fig.23). i v . Basal Body P r o l i f e r a t i o n The number of basal bodies i n region CVINT of dc4 c e l l s increased from 22 to 40 between 56 THE MORPHOGENETIC PERIOD IN MUTANT LINES;. - (Cont'd.) i v . Basal Body P r o l i f e r a t i o n - (Cont'd.) ages O.83 and 0.92 (fig.22a). The number of basal bodies did not change aft e r age 0.92. This pattern of basal body p r o l i f e r a t i o n was s i m i l a r to that i n wild-type c e l l s . There i s however, a small difference i n the number of basal bodies present i n the mid-regions of wild-type and m4 c e l l s at age 0.94 with ten fewer i n the dc4 c e l l s (fig.22a). The basal body spacing i n dc4 c e l l s changed during the c e l l cycle i n a manner s i m i l a r to that i n wild-type c e l l s (fig.22b). The s l i g h t reduction i n basal .body p r o l i f e r a t i o n i n dc4 c e l l s was therefore associated with a reduction i n c e l l length, v. Width; At age O.75 the mean width of dc4 c e l l s was 42 _um. This increased to 48 pm by age 0.92. A steady decrease i n width occurred during the fission-furrow phase. The pattern of change i n width (and length to width r a t i o ) was s i m i l a r to that i n wild-type c e l l s ( f i g s . l 8 b and 21a) except that no decrease i n width was noted i n dc4 c e l l s a f t e r age O.96. 57 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) v i . Stomatogenesis: Stomatogenesis i n dc4 c e l l s was normal dur-ing the f i r s t c e l l cycle at 34.5°C. v i i . Contour Drawings: Contour drawings (fig.24) indicate the reduced size of dc4 d i v i d i n g c e l l s as well as the marked and prolonged contraction of the c e l l . This contraction was p a r t i c u l a r l y prom-inent i n the poster i o r part of the c e l l , v i i i . Growth of the Dorsal Surface: Surface growth i n a l l c e l l regions was delayed u n t i l a f t e r age 0.94 (fig.25a). From age 0.94 to age 0.97 growth of the c e l l mid-regions (CVA' and CVP') increased the length of these regions by 50% of t h e i r o r i g i n a l length. After age 0.97 however, growth of the c e l l mid-regions was attenuated. Furrow surface growth began at age 0.95 and continued to age 0.98. There was i n i t i a l l y a s l i g h t contraction i n the furrow surface posterior to the f i s s i o n l i n e . No net growth occurred i n the polar regions, but the posterior region (CVP) contracted to 80$ of i t s o r i g i n a l length and remained contracted to age 0.99. 58 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i x . Growth of the Ventral Surface: Between ages 0.93 and 0.95 the decrease i n length of the anterior suture equalled the increase the length of the proter's posterior suture (table 5)• Surface growth i n the post e r i o r suture began a f t e r age 0.95 and con-tinued u n t i l f i s s i o n . Between ages 0.93 and 0.95> contraction of the posterior suture i n the opisthe exceeded the increase i n length of the opisthe*s anterior suture. After age 0.95 there was l i t t l e further change i n the length of ei t h e r the anterior or posteri o r suture i n the opisthe. The opisthe's anterior suture at age 0.98 was considerably-shorter (12 pm long) than that found i n either wild-type (22 ;um long) or sm2 (22 jm long) c e l l s of the same age. c. Mutant dfz2 Af t e r 20 hours at the r e s t r i c t i v e temperature the dfz2 phenotype was characterized by i n a b i l i t y to form a f i s s i o n - l i n e or furrow. Data col l e c t e d on dfz2 c e l l s are given i n table 8. Plots of the morphometric parameters against c e l l age are given i n figures.26 to 31. TABLE 8 AGE-DEPENDENT CHANGE IN dfz2 CELL SIZE  DIMENSION ( i n pm) WITH STANDARD ERROR VARIABLE CELL AGE b .75 120T2.2 44JO.8 Length Width L/Wc CVA CVA' CVINT CVP' CVP GA G #b.b.d CVINT^ b. b. PROTER LENGTH OPISTHE LENGTH PG OG PGP GAO n f .81-.83 .84-.85 .86-.87 .88-.89 12ljl.9 I30J4.I 123J2 49x3-1 47x1-4 45x1 2.7+.07 2.5x-20 2.8x-10 2.8T.12 2.6T.10 2.5T.17 2.5 43-2.3 56-2.7 21x1.5 53x1-0 17.0.4 50-1.8 56J2 36-1.6 22-1 21^0 23x1 37x1 22^2 18 Jo 48 J l 17x0 2 4 j l 23jl 37x2 25jl 20x0 53x1 20J0 57x2 25-1 24^2 20jl 36j3 19j0 25x2 51+2 18 Jo 54jl 26-1 .91 2 13lj4.0 128J2.6 125 3 50x0.8 52x2.7 49 26-0 22±2 0 26x1-9 1 20jl.8 2 4lj2.2 5 22jl.4 4 23x1-3 1 56J2.9 55Jl 6 20-0.6 21-1 5 55x2.4 52Jl 2 3O-O.7 40-3 36-;i 20J1 23x1 22 21 39 21 22 48 22 56 37 .92 -I3-3 Jl.4 X-12 Jo. 7 Jl.8 +1.9 Jl.O Jl.8 J2.1 Jo. 6 Jl.6 -2.3 •93 126J3.O 50jl.2 2.5J.O8 27J2.5 20jl.9 38J2.6 19J2.0 23+1.7 53J3.0 28-2.1 44Jl.4 41-1.4 .94-.95 118J3.4 45+2.3 2.7+.13 L S j l . l 21Jo.8 39+3.0 22J0.8 17jl.5 36jl.8 .96-.97 123J2.2 40x2.3 3.1J.17 21J1.1 25J1.2 37+1.8 25jl.5 I6J1.O 28-1.8 39jl.6 37+1.5 42-2.5 40-2.5 .98-.99 142J3.5 37+1.3 3.9J.17 24jl.9 33J2.6 40x2.1 29 Jl.4 16+1.3 29-1.6 38+1.1 38-1.8 1.1-.08 1.6-.10 1.5-.07 1.4-.13 1.4-.09 0.9-.07 1.1-.04 0.9-.06 0.9-.04 O.9-.05 1.1-.04 60-1.4 6I-5.I 74-2.6 5 12 8 8 f o r explanation of abbreviations used of a c e l l cycle c-length to width r a t i o ( a r b i t r a r y units) d-the number of basal bodies i n region CVINT a-see figur e 6 b-i n f r a c t i o n s 57J2.2 6 l j l . 4 19Jo.4 15Jo.3 14Jo.6 14J0.6 8J1.9 19+1.1 4-1.6 10-0.8 9 7 11 9 e-the spacing between basal f-sample size 67jl.5 15Jo.4 15Jo.3 3 l j l . l 15-1.6 8 bodies VA 60 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i . Growth Phase: In dfz2 c e l l s t h i s phase extended from age 0.75 to age O.89. During t h i s time the c e l l increased i n length (from 120 jam to 131 LIUI; fig.26a) i n a manner s i m i l a r to that of wild-type c e l l s , i i . Contraction Phase: The contraction phase of dfz2 c e l l s ex-tended from age 0.89 to age 0.95 with a minimum length of 118 _um attained at age 0.95' Wild-type c e l l s of the same age had a mean length of 126 jum ( f i g . 26a). The majority of the dfz2 c e l l contraction was l o c a l i z e d i n the c e l l extrem-i t i e s (CVA and CVP; figs.28a and b). The standard error of the mean dfz2 c e l l length at age 0.95 was greater than that found i n wild-type c e l l s (table 4) despite greater, sample size i n the dfz2 experiment. The range of lengths of age 0.95 dfz2 c e l l s was from 101 to 141 jum. The smaller c e l l s were very abnormal. They were wide (up to 62 ;um), lacked a f i s s i o n -l i n e , and showed no sign of furrowing. This group of c e l l s appeared to be arrested i n the c e l l cycle p r i o r to the formation of the 61 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i i . Contraction Phase: - (Cont'd.) f i s s i o n - l i n e . ' i i i . Fission-Furrow Phase: Those c e l l s which did not have a f i s s i o n -l i n e or furrow were not included i n the morpho-metric analysis as they had a grossly abnormal body form (cases of p a r t i a l furrowing were also excluded). Figure 32 shows the serie s of changes which occurred i n those c e l l s which underwent d i v i s i o n a rrest. The c e l l s became twisted such that the k i n e t i e s of the opisthe eventually l a y at r i g h t angles to those of the proter. Monsters.produced by t h i s type of div-i s i o n arrest were apparently unable to continue into a second d i v i s i o n at the r e s t r i c t i v e temp-erature. Rounded forms with more than two g u l l e t s were not seen even i n samples l e f t at 35°C f o r 20 hours. The vestibules and g u l l e t s of d i v i s i o n -arrested dfz c e l l s were usually exposed on the c e l l surface (fig.32d). Abnormalities i n the structure of the g u l l e t k ineties were however, rare. For dfz2 c e l l s which were not arrested 62 THE MORPHOGENETIC PERIOD I N MUTANT LINES:. - ( C o n t ' d . ) i i i . F i s s i o n - F u r r o w P h a s e : - ( C o n t ' d . ) d u r i n g t h e f i r s t c e l l c y c l e a t t h e r e s t r i c t i v e t e m p e r a t u r e t h e f i s s i o n - f u r r o w p h a s e e x t e n d e d f r o m age 0.95 t o f i s s i o n . D u r i n g t h i s t i m e t h e c e l l l e n g t h i n c r e a s e d f r o m 118 t o 142 pm. A t age 0.98 t h e dfz2 c e l l s w e r e a b o u t 10 ^ jm s h o r t e r t h a n t h e i r w i l d t y p e c o u n t e r p a r t s ( f i g . 2 6 a ) . The i n c r e a s e i n l e n g t h was l o c a l -i z e d i n t h e m i d - r e g i o n s o f t h e p r o t e r a n d o p i s t h e , a s was t h e c a s e i n w i l d - t y p e c e l l s ( f i g s . 2 9 a a n d b ) . The a n t e r i o r a n d p o s t e r i o r s u t u r e s b e g a n a r a p i d s h o r t e n i n g .at age 0.92 ( f i g s . 2 6 b a n d 27a). The c o n t r a c t i o n o f t h e a n t e r i o r s u t u r e was v e r y r a p i d a n d c o n t i n u e d u n t i l age 0.98 ( f i g . 2 6 b ) . A minimum l e n g t h o f 29 jum was r e a c h e d a t t h i s t i m e , 6 pm s h o r t e r t h a n t h e l e n g t h i n w i l d - t y p e c e l l s . The p o s t e r i o r s u t u r e r e a c h e d a l e n g t h o f 35 um a t age 0.95> a b o u t 10 p m s h o r t e r t h a n i t s l e n g t h i n age 0.95 w i l d - t y p e c e l l s ( f i g . 2 7 a ) . The r a t e o f f o r w a r d movement o f t h e p r o t e r ' s v e s t i b u l e was i d e n t i c a l i n w i l d - t y p e and dfz2 c e l l s (GF; t a b l e s 2 and 5). T h i s 63 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) i i i . Fission-Furrow Phase: - (Cont'd.) indicated that 'the differences i n length of the anterior suture between dfz2 and wild-type c e l l s was not due to differences i n vestibule movement but to differences i n growth of the proter. i v . Stomatogenesis: Stomatogenesis i n dfz2 c e l l s during the f i r s t c e l l cycle at 34.5°C was normal, v. Basal Body P r o l i f e r a t i o n : The increase i n basal body numbers i n dfz2 c e l l s followed the same pattern as seen i n wild-type c e l l s (fig.31a). The numbers were s l i g h t l y lower i n dfz2 c e l l s but so was the length of the region CVINT. .The spacing between basal bodies at a l l c e l l ages was i d e n t i c a l i n wild-type and dfz2 c e l l s (fig.31b). v i . Width: Throughout the i n i t i a l growth phase the width of dfz2 c e l l s was s i m i l a r to that of wild-type c e l l s (fig.27b). During the contraction phase a s i g n i f i c a n t difference between the widths of wild-type and dfz2 c e l l s appeared. This may have been associated with the defect i n furrow formation i n dfz2 c e l l s . In the 64 THE MORPHOGENETIC PERIOD IN MUTANT LINES: - (Cont'd.) v i . Width: - (Cont'd.) f i s s i o n furrow phase the widths of dfz2 c e l l s were i d e n t i c a l to t h e i r w i l d type counterparts, v i i . Contour Drawings and Surface Growth: The i n t e r e s t i n g d e f e c t i n dfz2 c e l l s was a l a c k of f i s s i o n - z o n e and furrow formation. A d e t a i l e d a n a l y s i s of surface growth was not c a r r i e d out on c e l l s e xpressing t h i s d e f e c t . Contour drawings of dfz c e l l s which formed a p a r t i a l or complete furrow i n d i c a t e d t h a t there was a s l i g h t r e d u c t i o n i n surface growth. The phenotype of these dfz c e l l s was s i m i l a r t o th a t of dc2 a c e l l s (compare the o u t l i n e drawings of dfz2 and dc2 a c e l l s , fig.33). d. Mutant dc2 a At the r e s t r i c t i v e temperature dc2 a c e l l s have the m i s d i v i d e r phenotype. Data c o l l e c t e d on dc2 a c e l l s i s given i n t a b l e 9. P l o t s of the morphometric parameters aga i n s t c e l l age are given i n f i g u r e s 34 to 39. i . Growth Phase: In the dc2 c e l l c y c l e t h i s phase extended from age O.75 to age 0.9. No inc r e a s e i n mean c e l l l e n g t h occurred d u r i n g t h i s time (fig.34a). TABLE 9 a AGE-DEPENDENT CHANGE IN dc2~ CELL SIZE  DIMENSION ( i n pi) WITH STANDARD ERROR •75 121T2.6 122-T2 46Jo.9 2.7-.10 2.3+.10 2.6Z.08 2-5+.07 43-1-5 57-2.8 24^1 18 Jo 34j2 - X " 19+1 20-1.7 27x3 ^7x1.5 51+2 17x0.5 17x0 56-1.2 54x1 32x2.2 23 +l 119x2 46jl 23+1 23x1 4lj7 I6j2 I6j5 46^2 17j0 56jl 29-> 4 122x2 50-0 VARIABLE CELL AGE b Length Width L/W c CVA . CVA' CVINT CVP' CVP GA G GP . -#b.b.d CVINT/b.b. PROTER LENGTH OPISTHE LENGTH PG PGP GAO n ** 9 5 4 8 a-see figure 6 f o r explanation of abbreviations b-in f r a c t i o n s of a c e l l cycle c-length to width r a t i o ( a r b i t r a r y units) d-the number of basal bodies i n region CVINT .8-.84 .85-.86 .87 .88-.89 .9-..91 -92-.94 .95 .96 .97-.99 23x1 19x0 38j2 21x1 21 J L 52JL 18J0 52x1 32x3 120x2.8 48 J L . 2.5X.05 — + ~ 5 22x0 23.1 35x2 I8j2 23.3 47j4 I8J0 56j3 34j4 120Jr.3 115J2.2 l l l j l . l 114^3.4 138J3.4 50^0.9 49^0.8 48?!,' '•-+— '•'+- -1.8-.07 1.5-.07 1.4-.17 1.2-.15 1.1-.10 23x0 22j2 35jl 18 J l 22^2 47^2 19J1 54jl 38J2 21x0 20jo 35jl 19jl 19j2 44jl 26jl 44J1 4l±l . _ . 6 43J1.1 41Jl.3 2.4J.07 2.4J.07 2.3J.IO 2.7J.IO 3.4J.18 r, ~,+- ~ , 20jl.O 23jl.8 - + - . _ 23jl.6 - + ' -34^1 - ~ 20^0 17+0 38-1 4 l j l 43j2 0.9-.09 0.9-.03 0.8J.04 0.8J.05 56J1 ~ '~ +~ -54jo 15x1 12x1 4 j l 1-0 32.1.7 24jl.7 l6jl.2 29-3.2 35jl.7 39J2.6 used 60x2.0 54J2.1 15x0.4 14-0.7 17x1-7 5-2.5 5 29.1.5 4lj3.1 28jl.5 17jl.6 30-1.2 38-2.2 72Jo.9 66Jo.3 15x0-5 15.0.5 27x0.1 12-0.2 8 e-the spacing between basal bodies f-sample size *-value not available due to poor • qua l i t y of silver-impregnation. ON 66 THE MORPHOGENETIC PERIOD I N MUTANT LI N E S : - ( C o n t ' d . ) i . G r o w t h P h a s e : - ( C o n t ' d . ) A t t h e e n d o f t h i s p h a s e dc2 c e l l s w e r e 120 pm l o n g , i i . C o n t r a c t i o n P h a s e : B e t w e e n a g e s 0.9 a n d 0.95 t h e mean c e l l l e n g t h d e c r e a s e d b y 10 pm ( f i g . 3 4 a ) . The minimum l e n g t h a t t a i n e d was 110 pm ( a t age 0.95) w h i c h was 10 pm s h o r t e r t h a n w i l d - t y p e c e l l s o f t h e same age ( f i g . 3 4 a ) . M o s t o f t h e c o n t r a c t i o n o c c u r r e d i n t h e p o s t e r i o r e n d o f t h e c e l l (CVP; fig.36b) w i t h no c o n t r a c t i o n s e e n i n t h e a n t e r i o r e n d (CVA; f i g . 3 6 a ) . C o n t r a c t i o n o f . t h e a n t e r i o r a n d p o s t e r i o r s u t u r e s b e g a n a t age 0.9 ( f i g s . 3 4 b a n d 35a). The p a t t e r n o f c h a n g e i n t h e l e n g t h o f t h e s u t u r e s was s i m i l a r t o t h a t f o u n d i n w i l d - t y p e c e l l s . i i i . F i s s i o n - F u r r o w P h a s e : T h i s p h a s e b e g a n a t age 0.95 i n t h e dc2 a c e l l c y c l e . A mean c e l l l e n g t h o f 138 pm was r e a c h e d b y age 0.98. M o s t o f t h e g r o w t h o c c u r r e d i n r e g i o n s CVA' a n d CVP' ( f i g s . 3 ? a a n d b) w i t h some g r o w t h i n r e g i o n CVINT ( f i g . 3 8 b ) . T h i s p a t t e r n o f g r o w t h was i d e n t i c a l t o t h a t 6 7 THE MORPHOGENETIC PERIOD I N MUTANT L I N E S : - ( C o n t ' d . ) i i i . F i s s i o n - F u r r o w P h a s e : - ( C o n t ' d . ) f o u n d i n w i l d - t y p e c e l l s . A t a g e O.98 dc2 a c e l l s w e r e a b o u t 12 pm s h o r t e r t h a n t h e i r w i l d t y p e c o u n t e r p a r t s ( f i g . 3 4 a ) . P a t c h e s s i m i l a r t o t h o s e s e e n on dc4 c e l l s w e r e a l s o f o u n d on t h e c o r t e x o f dc2 c e l l s ( f i g . 2 3 ) . i v . S t o m a t o g e n e s i s : S t o m a t o g e n e s i s i n dc2 c e l l s was n o r m a l d u r i n g t h e f i r s t c e l l c y c l e a t t h e r e s t r i c t i v e t e m p e r a t u r e . v. B a s a l Body P r o l i f e r a t i o n : The c h a n g e i n n u m b e r s a n d s p a c i n g o f b a s a l b o d i e s t h r o u g h o u t t h e m o r p h o g e n e t i c p e r i o d was s i m i l a r t o t h a t i n w i l d - t y p e c e l l s ( f i g s . 3 9 a a n d b ) . v i . W i d t h : T h r o u g h o u t t h e i n i t i a l g r o w t h a n d c o n t r a c -t i o n p h a s e s dc2 c e l l s w e r e c o n s i s t e n t l y w i d e r t h a n w i l d - t y p e c e l l s ( f i g . 3 5 b ) . A t age O.95 h o w e v e r , t h e w i d t h s w e r e i d e n t i c a l (47 p.m). A r a p i d d e c r e a s e i n w i d t h o c c u r r e d d u r i n g t h e f i s s i o n - f u r r o w p h a s e . 68 THE MORPHOGENETIC PERIOD I N MUTANT LINES;. - ( C o n t ' d . ) v i i . C o n t o u r D r a w i n g s : C o n t o u r ' d r a w i n g s ( f i g . 3 3 ) o f dc2 c e l l s i n d i c a t e t h a t t h e r e i s a s l i g h t r e d u c t i o n i n s u r f a c e g r o w t h a n d t h a t t h e c o n t r a c t i o n o f t h e p o l a r r e g i o n s o f t h e c e l l i s p r o l o n g e d , v i i i . G r o w t h o f t h e D o r s a l S u r f a c e : S u r f a c e g r o w t h i n dc2 a c e l l s f o l l o w s t h e same g e n e r a l t r e n d a s i n w i l d - t y p e c e l l s ( f i g . 25b) b u t g r o w t h i n t h e c e l l m i d - r e g i o n s (CVA' a n d CVP') i s n o t i c e a b l y r e d u c e d . T h e s e r e g i o n s i n c r e a s e b y k0% o f t h e i r o r i g i n a l l e n g t h i n dc2 c e l l s w h i l e t h e y i n c r e a s e b y ?0fo o f t h e i r o r i g i n a l l e n g t h i n . w i l d - t y p e c e l l s . The g r o w t h o f t h e f u r r o w s u r f a c e i n dc2 a c e l l s i s n o r m a l , a n d t h e r e i s no n e t g r o w t h o f t h e p o l a r r e g i o n s o f t h e c e l l , i x . G r o w t h o f t h e V e n t r a l S u r f a c e : B e t w e e n a g e s 0.93 a n d 0.95 v e s t i b u l e m i g r a t i o n was e v i d e n t i n b o t h t h e p r o t e r a n d o p i s t h e . G r o w t h o f t h e p o s t e r i o r s u t u r e i n t h e p r o t e r a n d t h e a n t e r i o r s u t u r e o f t h e o p i s t h e b e g a n a f t e r age O.95 ( t a b l e 9). 69 Continuous heat treatment throughout the c e l l cycle of mutant and wild-type c e l l s was necessary f o r consistency of experimental design when morphometric studies of the mutant phenotypes were carried out. I t was possible however, that the temperature-sensitivity of the mutants varied temporally during the c e l l cycle. I f a temporal v a r i a t i o n i n temp-e r a t u r e - s e n s i t i v i t y was found, i t might have proven valuable i n determining the o r i g i n of the mutant phenotypes. Exper-iments were therefore c a r r i e d out to determine the pattern of temperature-sensitivity throughout the c e l l cycle of wild-type and mutant c e l l s . 70 SECTION C: THE PATTERN OF TEMPERATURE-SENSITIVITY DURING  THE CELL CYCLE: 1. INTRODUCTION: This section w i l l examine the e f f e c t of heat-shocks on synchronous polulations of wild-type and mutant c e l l s . This part of the study i s designed to answer two questions: (a) i s there a temporal pattern of temp-erature s e n s i t i v i t y during the c e l l cycle, and i f so, does t h i s d i f f e r i n wild type and mutant l i n e s , and (b) does the pattern of temperature s e n s i t i v i t y i n the mutants correlate with the v i s i b l e morphological defects i n the mutants? Similar experiments with Tetrahymena have suggested that there i s a h e a t - l a b i l e d i v i s i o n protein that i s e s s e n t i a l for c e l l d i v i s i o n (see Mitchison, 1971 or Zeuthen and Rasmussen, 1971 for a review). Heat-shocks applied during the c e l l cycle presumably destroy t h i s protein and r e s u l t i n a delay i n c e l l d i v i s i o n . The c e l l must replace the protein destroyed by the heat shock before i t can divide. At some time i n the c e l l cycle the Tetrahymena d i v i s i o n protein i s s t a b i l i z e d and i s no longer heat-sensitive. Temperature-shocks applied a f t e r t h i s time produce no d i v i s i o n delay. The time at which the protein i s s t a b i l i z e d i s referred to by Mitchison (1971) as the 71 INTRODUCTION: - ( C o n t ' d . ) t r a n s i t i o n p o i n t . EXPERIMENTAL DESIGN: • • S y n c h r o n o u s s a m p l e s o f l o g - p h a s e c e l l s were i s o l a t e d a s d e s c r i b e d a b o v e ( s e e m a t e r i a l s a n d m e t h o d s ) . The s a m p l e s w ere h e a t - s h o c k e d f o r 30 m i n u t e s a t known t i m e s d u r i n g t h e c e l l c y c l e . (The c o n t r o l c e l l c y c l e l e n g t h was d e t e r m i n e d f r o m a s a m p l e t h a t was n o t h e a t - s h o c k e d . ) The d i v i s i o n d e l a y p r o d u c e d by a h e a t - s h o c k was d e t e r -m i n e d by c o m p a r i n g t h e c e l l c y c l e l e n g t h o f h e a t - s h o c k e d s a m p l e s w i t h t h a t o f t h e c o n t r o l . The c e l l c y c l e l e n g t h was d e t e r m i n e d by t h e " s h o r t c u t m e t h o d " ( N a t c h w a y a n d C a m eron, 1968). T h i s i n v o l v e d c o u n t i n g t h e number o f c e l l s p r e s e n t i n e a c h s y n c h r o n o u s s a m p l e a t t e n m i n u t e • i n t e r v a l s o n c e c e l l d i v i s i o n h a d b e g u n i n t h a t s a m p l e . The d a t a w ere t h e n t r a n s f o r m e d i n t o t h e p e r c e n t a g e o f c e l l s w h i c h h a d d i v i d e d a t e a c h t e n m i n u t e i n t e r v a l a n d p l o t t e d on p r o b a b i l i t y p a p e r as a c u m u l a t i v e p e r c e n t a g e o f d i v i d e d c e l l s a g a i n s t t i m e . The m e d i a n c e l l c y c l e l e n g t h a nd i t s s t a n d a r d d e v i a t i o n w e r e r e a d d i r e c t l y f r o m t h e g r a p h . An e x a m p l e o f a p r o b a b i l i t y p l o t i s g i v e n i n f i g u r e 40. The c e l l c y c l e l e n g t h o f e x p e r i m e n t a l g r o u p s was c a l c u l a t e d a s a p e r c e n t a g e o f t h e c o n t r o l c e l l c y c l e l e n g t h . T h u s , a 120$ l o n g c e l l c y c l e i s 20$ l o n g e r t h a n 72 2. EXPERIMENTAL DESIGN: - ( C o n t ' d . ) t h e c o n t r o l . I n a l l c a s e s t h e c o n t r o l c e l l c y c l e l e n g t h was d e t e r m i n e d a t 27°C. The t e m p e r a t u r e o f t h e h e a t - s h o c k v a r i e d among t h e e x p e r i m e n t s . 3. TEMPERATURE-SENSITIVITY OF WILD-TYPE AND MUTANT CELLS: W i l d - t y p e c e l l s w e r e t e s t e d w i t h h e a t - s h o c k s o f 34.5°C and 35°C. The 34.5°C h e a t - s h o c k s p r o d u c e d v e r y l i t t l e d e l a y r e g a r d l e s s o f t h e c e l l age a t t h e t i m e o f t h e h e a t - s h o c k ( f i g . 4 l a ) . The maximum d e l a y was f i v e p e r c e n t a t age 0.6. A t r a n s i t i o n p o i n t o c c u r r e d some-w h e r e b e t w e e n a g e s 0.6 a n d O.65 a n d c e l l s h e a t - s h o c k e d a f t e r age 0.7 showed no d e l a y . H e a t - s h o c k s a t 35°C r e v e a l e d a d e f i n i t e p a t t e r n o f t e m p e r a t u r e s e n s i t i v i t y i n t h e P a r a m e c i u m c e l l c y c l e ( f i g . 4 l a ) . V e r y l i t t l e d e l a y o c c u r r e d up t o age 0.2 b u t a f t e r t h a t t i m e t h e r e was a r o u g h l y l i n e a r i n c r e a s e i n d e l a y up t o age 0.5. The d e l a y a t t h i s age was 20$ a n d i t r e m a i n e d a t t h i s v a l u e u n t i l age O.65. A f t e r age O.65 t h e d e l a y d e c r e a s e d r a p i d l y t o a b o u t 5$-The e f f e c t o f 30 m i n u t e , 36°C h e a t - s h o c k s h a s b e e n e x a m i n e d b y W h i t s o n (1964). The maximum d e l a y was 23$ a t age 0.9. The t r a n s i t i o n p o i n t was f o u n d a t age 0.92, c o n s i d e r a b l y l a t e r t h a n t h a t f o u n d f o r 35°C h e a t -s h o c k s ( a g e O.65). The p a t t e r n o f t e m p e r a t u r e s e n s i t i v i t y o f m u t a n t 73 TEMPERATURE-SENSITIVITY OF WILD-TYPE AND MUTANT CELLS: ( Cont'd.) dc5 to 34.5°C heat-shocks was s i m i l a r to that found fo r wild-type c e l l s at 35°C ( f i g . 4 l b ) . Both the maximum delay (120%) and the t r a n s i t i o n point (age O.65) were i d e n t i c a l . In dc5 c e l l s however, heat-shocks applied a f t e r the t r a n s i t i o n point decreased the c e l l cycle length by as much as 10$. This was not the case i n wild-type c e l l s . Variant 48 c e l l s showed a constant delay of seven percent up to age 0.3 (fig.42a). Between ages 0.3 and 0.45 the delay produced by 34.5°C heat-shocks increased up to a maximum of 16%. The t r a n s i t i o n point occurred at age 0.7 with c e l l s heat-shocked a f t e r age 0.82 showing c e l l cycle acceleration. Mutant dc4 was the only l i n e examined that had a clear bimodal pattern of temperature-sensitivity ( f i g . 42b). Two t r a n s i t i o n points occurred i n the c e l l cycle. The f i r s t was at age 0.2 and the second at age 0.7. Between ages 0.3 and 0.45 and a f t e r age 0.7 heat-shocks did not produce s i g n i f i c a n t delay. Two experiments were done with sm2 c e l l s and both indicated a temperature-sensitive period extending from age O.35 to age O.65 (fig.43). There also appeared to be a second heat-sensitive period beginning at age O.85. 74 3. TEMPERATURE-SENSITIVITY OF WILD-TYPE AND MUTANT CE L L S : ( C o n t ' d . ) As no d a t a a r e a v a i l a b l e f o r c e l l s o l d e r t h a n age O.85 t h e e x t e n t o f t h i s t e m p e r a t u r e - s e n s i t i v e p e r i o d c a n n o t be d e t e r m i n e d . SECTION D: CORTICAL PATTERNING 76 SECTION D; CORTICAL PATTERNING: 1. INTRODUCTION: The Paramecium c e l l r e p l i c a t e s the highly-ordered array of organelles on i t s cortex at each c e l l d i v i s i o n . The mechanisms which determine the l o c a t i o n and orien-t a t i o n of newly-forming structures could operate i n two ways: by p o s i t i o n i n g a new structure at a f i x e d distance from a reference point or by assessing the distance between two reference points and then p o s i t i o n i n g the new structure at a l o c a t i o n that i s a fixed f r a c t i o n of that length from one of the reference points. These two models of c o r t i c a l patterning can be c a l l e d f i x e d d i s -tance assessment and proportional distance assessment respectively. The f i r s t model operates without regard to the c e l l length, while the second model i s length-dependent. In order to d i s t i n g u i s h between these two models, therefore, i t i s necessary to examine the positions of c o r t i c a l structures i n paramecia of d i f -ferent length. This study w i l l attempt to d i s t i n g u i s h between these models by examining the l o c a t i o n of c o r t i c a l structures i n small c e l l s produced by the sm2 gene. The r e s u l t s of t h i s study w i l l be discussed i n r e l a t i o n to recent models f o r p o s i t i o n a l determination i n c i l i a t e s (Frankel, 197k; Sonneborn, 1974a, 1975). 77 2. EXPERIMENTAL DESIGN; A s y n c h r o n o u s l o g - p h a s e p e t r i - d i s h ( 1 5 x 1 0 0 m.m.) c u l t u r e s o f sm2 c e l l s were h e a t e d t o 3 5 ° C i n a n a i r i n c u b a t o r . F o u r c u l t u r e s w e r e u s e d and w e r e f i x e d a f t e r 6 , 8, 1 1 . 5 a n d 24 h o u r s . An a d d i t i o n a l a s y n -c h r o n o u s s a m p l e o f sm2 c e l l s f r o m a c u l t u r e k e p t a t t h e p e r m i s s i v e t e m p e r a t u r e was f i x e d and u s e d a s a c o n t r o l . The f i x e d c e l l s w e r e s i l v e r - i m p r e g n a t e d . 3 . MEASUREMENTS; The p o s i t i o n s o f t h e c o n t r a c t i l e v a c u o l e p o r e s ( C V P s ) a n d t h e v e s t i b u l e were m e a s u r e d i n r e l a t i o n t o t h e e n d s o f t h e c e l l ( f i g . 4 4 ) . ( O n l y i n t e r f i s s i o n c e l l s w ere u s e d i n t h e a n a l y s i s . ) The l e n g t h o f t h e c y t o p r o c t was m e a s u r e d b u t i t s p o s i t i o n i n r e l a t i o n t o t h e e n d s o f t h e c e l l was n o t . T h e s e m e a s u r e m e n t s were made on one e a c h o f a c o n t r o l , 6 , 8, 1 1 . 5 and 24 h o u r s a m p l e o f c e l l s . F o r a s e p a r a t e c o n t r o l a n d 24 h o u r c e l l s a m p l e a more e x t e n s i v e s e r i e s o f m e a s u r e m e n t s w e r e made, i n c l u d i n g t a l l i e s o f t h e n u m b e r s o f b a s a l b o d i e s a n d k i n e t i e s on t h e c e l l s ( f i g . 4 4 ) . The m e a s u r e m e n t d a t a a r e l i s t e d i n t a b l e 1 0 . 4 . PROBIT ANALYSIS: A p r o b i t a n a l y s i s was p e r f o r m e d on a l l v a r i a b l e s t o d e t e r m i n e i f t h e y w e re n o r m a l l y d i s t r i b u t e d . P r o b i t p l o t s o f l e n g t h and w i d t h a r e shown i n f i g u r e s 4 5 a n d TABLE 10 SIZE-DEPENDENT CHANGE IN sm2 CELL DIMENSIONS VARIABLE b DIMENSION (i n um) WITH STANDARD ERROR TIME at 34.5°C 0 hrs. 6 hrs. 8 hrs. 11.5 hrs. 24 h r s . a 24 hrs ; LENGTH 123+1.2 111+1.6 112+1.3 101+1.5 92+1.1 80+1.6 WIDTH 50+0.6 39+0.6 45+O.6 34+0.4 31+0.6 40+0.6 L/W RATIO C 2.5+.05 2.9+.07 2.5+.05 3.0+.07 3.1+.10 2.0+.11 CVA 41+0.7 38+O.7 38+O.8 35+0.9 32+0.6 29+0.8 CVINT 58+0.8 48+0.9 44+0.8 40+0.8 36+O.9 28+1.1 CVP 23+0.6 2510.5 28+0.5 26+0.5 23+0.5 24+0.8 GA 58+0.8 51+0.9 52+0.7 50+0.8 46+0.6 ' 38+1.1 G 15+0.6 17+0.4 17+0.3 17+0.5 14+0.3 . 14+0.4 GP ' 50+O.7 43+0.9 43+O.8 34+0.8 32+0.7 28+1.2 CYTL 19+0.4 18+0.4 18+0.4 15+0.4 12+0.4 * n d e 44 ' 54 46 44 53 47 #b.b. £ 35+2.2 •8- # •H- •* 23+6.0 um/b.b. _ 1.5+.04 * # 1.2+.25 KINETIES g 72+1.6 •a- # * 72+0.4 KTCV 39+2.1 41+0.5 KTCL 3311.6 * # •* 30+0.4 KTCVV . 1+0.1 •a- •H- # 1+0.3 KTCV/TOTAL 5312.6 * •H- 58+1.0 CO TABLE 10 - (Cont'd.) SIZE-DEPENDENT CHANGE IN sm2 CELL DIMENSIONS * not measured i n t h i s sample a- two separate 2k hour samples (from separate experiments) are reported b- see fi g u r e kk f o r explanation of the abbreviations used c- r a t i o of length to width i n a r b i t r a r y units d- sample siz e e- number of basal bodies i n region CVINT f- spacing between basal bodies g- the t o t a l number of kin e t i e s on the c e l l h- KTCV expressed as a $ of the t o t a l number of kineties. VO 80 PROBIT ANALYSIS: - (Cont'd.). 46. Other probit p l o t s are not shown but i n t h e i r place descriptions of the d i s t r i b u t i o n s obtained through probit analysis are given i n table 11. Length was normally d i s t r i b u t e d i n the control sample and one of the 24 hour samples. A l l others showed a mixture of two normally d i s t r i b u t e d populations. In cases where length was bimodally d i s t r i b u t e d other variables for that sample were not necessarily b i -modally d i s t r i b u t e d . The following variables tended to be normally d i s t r i b u t e d i n those samples with a b i -modal length d i s t r i b u t i o n : GA, G, GP, CYTL, CVP, and WIDTH (abbreviations are l i s t e d i n figure 44). Most of the bimodality i n length arose from bimodality i n the length of regions CVA and CVINT. As w i l l be shown l a t e r , i t was i n these regions where the majority of the loss i n c e l l length occurred during the heat shock. TEMPERATURE EFFECT ON sm2 CELL LENGTH: The mean length of sm2 c e l l s decreased l i n e a r l y during culture at 35°C (fig.47a) . The c e l l length was reduced by about 35$ during the 24 hour heat period. Most of the length decrease occurred i n the posterior region of the c e l l (GP; fig.48a), or more s p e c i f i c a l l y , i n the p o s t e r i o r part of the c e l l mid-region (fig.49a) . Region CVP (fig.48d) and the vestibule (fig.6 ) did 81 TABLE 11 THE TYPES OF DISTRIBUTIONS OBTAINED FROM A PROBIT ANALYSIS OF MEASUREMENT VARIABLES TIME AT 34.5 °C (HOURS) MORPHOMETRIC a PARAMETER 0 6 8 11.5 24 24 b LENGTH N BM BM BM BM N GA N N N N BM N G S L N N N N N GP N BM N N N N CYTL N N LK LK N n a CVA N BM N BM BM BM CVINT N N SR BM BM BM CVP N N SR BM SR N WIDTH N SR N LK N N KTCV n a n a n a n a n a N (ABBREVIATIONS: N = n o r m a l l y d i s t r i b u t e d , BM = b i m o d a l l y d i s t r i b u t e d , S L = s k e w e d l e f t , SR = ske w e d r i g h t , L K = l e p t o k u r t i c , n a = n o t a p p l i c a b l e ) a -b -T h e s e m e a s u r e m e n t v a r i a b l e s a r e d e f i n e d i n f i g u r e 44. Two i n d e p e n d e n t 24 h o u r s a m p l e s were e x a m i n e d . 82 TEMPERATURE EFFECT ON sm2 C E L L LENGTH:- ( C o n t ' d . ) n o t c h a n g e i n l e n g t h d u r i n g t h e h e a t p e r i o d w h i l e a l l o t h e r r e g i o n s d e c r e a s e d i n l e n g t h ( f i g s . 4 8 a n d 4-9). D u r i n g t h e f i r s t 12 h o u r s o f t h e h e a t p e r i o d t h e mean c e l l w i d t h d e c r e a s e d f r o m 50 t o 34 jum a f t e r w h i c h no f u r t h e r c h a n g e o c c u r r e d ( f i g . 4 7 a ) . The l e n g t h t o w i d t h r a t i o i n c r e a s e d f r o m 2.5 t o 3*1 d u r i n g t h e h e a t p e r i o d ( f i g . 4 7 b ) . • The c y t o p r o c t l e n g t h was m e a s u r e d w i t h o u t r e g a r d t o i t s p o s i t i o n on t h e c e l l . T h i s s t r u c t u r e showed a r e g u l a r d e c r e a s e i n l e n g t h t h r o u g h o u t t h e h e a t p e r i o d ( f i g . 4 9 c ) . The mean number o f k i n e t i e s p e r c e l l i n t h e 17°C c o n t r o l was t h e same a s t h e n umber f o u n d a f t e r 24 h o u r s a t 35°C ( t a b l e 10). The number o f b a s a l b o d i e s b e t w e e n t h e CVPs d e c r e a s e d f r o m 35 t o 23 d u r i n g t h e h e a t p e r i o d ( t h e d i s t a n c e b e t w e e n t h e CVPs a l s o d e c r e a s e d ) . B a s a l b o d y s p a c i n g d e c r e a s e d s l i g h t l y d u r i n g t h e h e a t p e r i o d ; f r o m 1.5 /im t o 1.2 pm ( t a b l e 10). THE LENGTHS OF PARTS OF THE C E L L AS A FUNCTION OF C E L L LENGTH: One o f t h e r e a s o n s f o r e x a m i n i n g t h e s i z e r e d u c t i o n i n sm2 c e l l s was t o d e t e r m i n e w h e t h e r c o r t i c a l s t r u c t u r e s f o r m e d a t f i x e d d i s t a n c e s f r o m r e f e r e n c e p o i n t s on t h e c e l l ( t h e C V P s , v e s t i b u l e , o r e n d s o f t h e c e l l c o u l d be 83 THE LENGTHS OF PARTS OF THE CELL AS A FUNCTION OF CELL LENGTH: - (Cont'd.) used as reference points) or whether the location of these structures was determined with reference to the length of the c e l l . To d i s t i n g u i s h "between these two p o s s i b i l i t i e s , the length of each c e l l region was calculated as a f r a c t i o n of the c e l l length (table 12) and was plot t e d against time at 35°C (figs.48 and 49). The regression of each variable against time was determined (table 13)» and s i g n i f i c a n t regression l i n e s were added to the appropriate plots. The anterior suture i n i t i a l l y made up 4?$ of the c e l l length, the vestibule, 12$; and the posterior suture, 41$ (fig.48b). The f r a c t i o n s of the c e l l length occupied by these regions changed s l i g h t l y during the f i r s t 12 hours of the heat period, with the anterior suture increasing to 50$ of the c e l l length, the v e s t i -bule increasing to 16$ of the length, and the posterior suture decreasing to 34$ of the c e l l length. L i t t l e further change occurred during the remainder of the heat period (fig.48b). There was no s i g n i f i c a n t regression of any region of the ventral surface with time at the r e s t r i c t i v e temperature (table 13). The anterior and posterior sutures therefore occupied constant f r a c t i o n s of the c e l l length. 84 TABLE 12 MORPHOMETRIC MEASUREMENTS EXPRESSED AS A FRACTION OF THE C E L L LENGTH - MEANS AND STANDARD ERRORS TIME AT 35°C (HOURS) 2 L O 6 8 11.5 24 24 G/L .12 GP/L . 41 S x * MEAN Sx MEAN Sx MEAN Sx MEAN Sx MEAN Sx .01 •34 .01 • 34 .01 • 35 .02 • 35 .01 .36 .02 .01 .44 .01 .40 .01 .40 .02 • 39 .02 •35 .02 .01 .23 .01 .25 .01 .26 .01 • 25 .01 • 30 .02 .01 .46 .02 .4? .01 .50 .02 .50 .02 .47 .02 .01 .16 .01 .15 .01 .17 .01 .16 .02 .18 .01 .01 •39 .01 •39 .01 • 34 .02 •34 .01 • 35 .02 .01 .16 .01 .16 .01 •15 .02 .13 .02 n / a n/a .02 .41 .02 .41 .02 .44 .03 •39 .03 n / a n/a * s t a n d a r d e r r o r o f p r o p o r t i o n s + r a t i o s o f m o r p h o m e t r i c p a r a m e t e r s ( s e e f i g u r e 44 f o r a b b r e v i a t i o n s ) . 85 TABLE 13 LINEAR REGRESSIONS OF CELL DIMENSIONS AGAINST TIME AT THE RESTRICTIVE TEMPERATURE _ x REGRESSION EQUATION ts CVA/L TIME y=33.4+0.97x 0.10 CVINT/L TIME y-45.2-0.88x -O.38 CVP/L TIME y=20.3+0.90x O.36 GA/L TIME y=46.5+0.56x 0.11 * G/L TIME y=13.6-0.80x 0.16 * GP/L TIME y=40.0-0.?9x -0.27* CYTL/L TIME y=l6.1-0.45x -0.11 LENGTH TIME y=83.5-0.92x -1.05 CYTL/GP TIME y=40.3+0.04x 0.01 * * ts < t .05 (3) and the slope does not d i f f e r s i g n i f i c a n t l y from zero. 86 6. THE LENGTHS OF PARTS OF THE C E L L AS A FUNCTION OF C E L L LENGTH: - ( C o n t ' d . ) On the. d o r s a l s u r f a c e r e g i o n CVA i n i t i a l l y o c c u p i e d 34$ o f t h e c e l l l e n g t h , r e g i o n CVINT 47$, and r e g i o n CVP 19$. M o s t o f t h e c h a n g e i n r e l a t i v e CVP p o s i t i o n s o c c u r r e d w i t h i n t h e f i r s t e i g h t h o u r s a t t h e r e s t r i c t i v e t e m p e r a t u r e ( f i g . 4 8 d ) . R e g i o n s CVA a n d CVP i n c r e a s e d t o o c c u p y 38 a n d 25$ o f t h e c e l l l e n g t h r e s p e c t i v e l y . R e g i o n CVINT d e c r e a s e d t o 39$ o f t h e c e l l l e n g t h . Of t h e t h r e e r e g i o n s , o n l y CVA a p p r o a c h e d b e i n g a c o n s t a n t f r a c t i o n o f t h e c e l l l e n g t h . T h e r e was h o w e v e r , a s m a l l b u t s i g n i f i c a n t r e g r e s s i o n o f CVA/L w i t h t i m e ( t a b l e 13)' I t t h e r e f o r e a p p e a r s t h a t t h e p o s i t i o n o f t h e a n t e r i o r CVP i s r e g u l a t e d when c e l l s i z e i s r e d u c e d w h i l e t h a t o f t h e p o s t e r i o r CVP i s n o t . The c y t o p r o c t l e n g t h was c a l c u l a t e d a s a f r a c t i o n o f t h e c e l l l e n g t h a n d t h e p o s t e r i o r s u t u r e l e n g t h . The c y t o p r o c t o c c u p i e d a c o n s t a n t f r a c t i o n o f t h e p o s t e r i o r s u t u r e l e n g t h b u t n o t o f t h e c e l l l e n g t h ( t a b l e 13 and fig.49d). 7. CORRELATION ANALYSIS: A c o r r e l a t i o n a n a l y s i s was p e r f o r m e d f o r a l l m e a s u r e m e n t v a r i a b l e s t o d e t e r m i n e w h i c h p a r a m e t e r s v a r i e d t o g e t h e r . T h i s a n a l y s i s w o u l d t h e r e f o r e a l l o w one t o d e t e r m i n e , f o r e x a m p l e , i f a d e c r e a s e i n c e l l 87 7. CORRELATION ANALYSIS: - ( C o n t ' d . ) l e n g t h was a c c o m p a n i e d b y a d e c r e a s e i n w i d t h o r a c h a n g e i n t h e number o f k i n e t i e s on t h e c e l l . M a t r i c e s o f p r o d u c t moment c o r r e l a t i o n c o e f f i c i e n t s ( S o k a l a n d R o h l f , 1969) f o r a l l p a i r - w i s e c o m p a r i s o n s o f m orpho-m e t r i c p a r a m e t e r s a r e g i v e n i n t a b l e s lk and 15. I n t h e t a b l e s , c o r r e l a t i o n c o e f f i c i e n t s ( r ^ ' s ) a r e g i v e n f o r a l l s a m p l e s o f b o t h e x p e r i m e n t s and t h e manner i n w h i c h t h e r ^ ' s c h a n g e d u r i n g t h e h e a t s h o c k may t h e r e -f o r e be e x a m i n e d . Some o f t h e g r o u p s o f c o r r e l a t i o n c o e f f i c i e n t s w e r e t e s t e d f o r h o m o g e n e i t y ( S o k a l and R o h l f , 1969). C e r t a i n c o r r e l a t i o n s d i d n o t c h a n g e s i g n i f i c a n t l y i n m a g n i t u d e d u r i n g t h e h e a t p e r i o d . T h ese g r o u p s and t h e i r p a r a m e t r i c c o r r e l a t i o n c o e f f i c i e n t s ( p ) a r e l i s t e d i n t a b l e 16. I n t e r p r e t a t i o n o f c o r r e l a t i o n s w h i c h w e r e n o t homogeneous t h r o u g h o u t t h e h e a t p e r i o d was d i f f i c u l t . T h o s e c o r r e l a t i o n s w h i c h showed a d e c l i n i n g v a l u e o f r ^ ( t h e c o r r e l a t i o n c o e f f i c i e n t ) d u r i n g t h e h e a t p e r i o d may h a v e b e e n i n d i c a t i n g a n e f f e c t o f t h e h e a t t r e a t -ment b u t t h o s e w h i c h showed an i n c r e a s i n g v a l u e o f r ^ w e r e l i k e l y s p u r i o u s . I n t h e l a t t e r c a s e s i t a p p e a r e d t h a t t h e c o r r e l a t i o n was b e c o m i n g a more s i g n i f i c a n t p a r t - w h o l e c o r r e l a t i o n , t h e m a g n i t u d e o f w h i c h w o u l d 88 TABLE_14 WIDTH __ LENGTH GA CYTL 0 * 6 . 4 4 2 8 .598 H-5 .322 24 CVP CVINT CVA GP 0 6 8 11.5 24 0 6 8 11.5 24 0 6 8 11.5 -24 0 6 8 11.5 24 0 6 8 11.5 24 • 574 • 544 • 542 • 386 •538* •517 •375 • 579 •353 • 316 •309 • 504 • 605 .606 .684** • 920 • 742 •730 • 784 • 758* . 8 3 1 •734 . 8 0 8 .648 • 784* • 774 .812 • 722 . 6 8 8 GA 0 6 8 11.5 24 0 6 8 11.5 24 # . 4 8 0 •359 . 4 2 9 •554 . 8 2 9 * .881 • 787 . 8 5 5 •719 . 6 8 5 • 768 • 560 . 4 5 3 . 4 5 7 • 509 •758 • 719 .652 •529 •557 • 436 •339 •375 - . 4 2 4 . 4 6 7 G GP CVA CVINT CVP CYTL .461 .333 .413 -374 .342 . 4 5 6 . 5 0 8 . 3 8 I . 3 6 4 .300 . 3 2 7 . 3 7 4 - . 3 6 1 . 6 9 0 * . 3 1 6 . 6 3 2 . 4 8 7 . 5 4 9 . 6 9 4 . 5 6 9 .717 . 4 6 2 .344 . 4 6 0 . 5 8 0 . 3 0 7 . 3 5 2 - - - -. 3 5 2 .684 . 4 7 8 . 2 9 2 . 3 2 6 . 3 0 7 . 2 9 9 . 3 2 8 . 5 4 3 . 5 6 8 . 3 2 9 .802 . 6 6 5 _^ s w \J . 6 2 5 -753 .472 .626 — — .498 .524 . 5 6 5 . 3 4 3 .440 .420 . 5 7 8 . 4 0 3 -.436 • 310 • 420 • 328 .286 89 TABLE 14 - ( C o n t ' d . ) MATRIX OF CORRELATION COEFFICIENTS * * ( e x c l u s i v e o f t h e two s a m p l e s w h e r e k i n e t y a n d b a s a l b o d y numbers w e r e t a l l i e d . ) LEGEND: + = TIME AT 35 ° C ; * = t h e c o r r e l a t i o n s shown i n t h e b o x a r e homogeneous; ** = t h e c o r r e l a t i o n s shown i n t h e b o x a r e n o t homogeneous. B l a n k s i n d i c a t e n o n - s i g n i f i c a n t c o r r e l a t i o n s . TABLE 15 MATRIX OF CORRELATION COEFFICIENTS T LENGTH GA G GP CVA CVINT CVP WIDTH TOTAL KINETIES KTCV BB 0 2 4 • 5 8 7 • 3 9 3 . 7 2 8 . 8 8 9 • 5 5 8 . 8 4 8 — KTCVV 0 2 4 •532 .434 . 4 2 0 - . 6 1 8 KTCL 0 2 4 .303 . 4 8 6 . 4 9 6 . 3 6 4 . 2 8 2 . 7 6 3 - . 3 7 7 KTCV 0 2 4 . 3 4 3 • 5 8 7 .405 . 3 3 4 _ _ _ _ _ _ _ _ _ _ _ _ . 4 2 2 . 4 1 8 . 6 8 4 . 7 1 1 TOTAL KINETIES 0 2 4 .529 .372 • 6 7 5 .540 . 4 9 1 • 577 .361 . 3 2 8 . 4 6 1 .403 . 6 2 2 • WIDTH 0 2 4 • 7 1 5 • 5 7 6 . 4 8 5 • 2 7 9 .452 . 4 9 2 CVP 0 2 4 •333 . 5 7 6 — . 3 2 6 .852 CVINT 0 2 4 . 7 8 2 . 7 3 6 .452 • 5 1 3 . 4 8 1 _ _ _ _ CVA 0 2 4 .530 • 592 • 570 . 4 7 7 . 6 2 9 GP 0 2 4 . 7 6 3 . 5 5 4 G 0 2 4 . 6 1 9 .650 GA 0 2 4 . 6 2 1 KTCL KTCW VO o TABLE 15 - (Cont'd.) MATRIX OF CORRELATION COEFFICIENTS LEGEND: Blanks indicate non-significant c o r r e l a t i o n s . * In addition to the correlations l i s t e d , LENGTH and basal body spacing are p o s i t i v e l y correlated (r- = .325) f o r the 2k hour sample and WIDTH and KTCV/TOTAL KINETIES are not s i g n i f i c a n t l y correlated f o r t h i s same sample. TABLE 16 PARAMETRIC CORRELATION COEFFICIENTS  FOR THE HOMOGENEOUS SETS OF CORRELATION COEFFICIENTS FROM THE MATRIX IN TABLE 14. CORRELATED PARAMETERS p* L - CVA . ? 3 8 L - CVP . L - GA .821 L - G .ixlili L - GP L - WIDTH .730 . 341 GP - CYTL ,iei * parametric c o r r e l a t i o n c o e f f i c i e n t s . 93 CORRELATION ANALYSIS: - ( C o n t ' d . ) n a t u r a l l y be h i g h . F o r t h e p u r p o s e s o f a n a l y s i s o n l y t h o s e c o r r e l a t i o n s w h i o h w e r e i n i t i a l l y h i g h ( a n d may o r may n o t h a v e s u b s e q u e n t l y d e t e r i o r a t e d ) w e re c o n s i d e r e d . B o t h t h e a n t e r i o r a n d p o s t e r i o r s u t u r e s h a d a s i g n i f i c a n t a n d homogeneous c o r r e l a t i o n w i t h c e l l l e n g t h ( t a b l e 1 4 ) . The v e s t i b u l e a l s o showed a homo-g e n e o u s , b u t l o w , c o r r e l a t i o n w i t h l e n g t h . The l e n g t h o f t h e a n t e r i o r s u t u r e was p o s i t i v e l y c o r r e l a t e d w i t h t h a t o f t h e p o s t e r i o r s u t u r e , b u t t h e m a g n i t u d e o f r ^ d e c r e a s e d d u r i n g t h e h e a t p e r i o d a n d was n o t s i g n i f i c a n t a f t e r 24 h o u r s . The l e n g t h o f t h e a n t e r i o r s u t u r e was a l s o c o r r e l a t e d w i t h t h e l e n g t h s o f r e g i o n s CVA and CVINT ( b u t n o t C V P ) . The l e n g t h o f t h e p o s t e r i o r s u t u r e was s i m i l a r l y c o r r e l a t e d w i t h CVA a n d CVINT, b u t n o t w i t h CVP. The l e n g t h o f r e g i o n CVINT was w e a k l y c o r r e l a t e d w i t h CVA b u t was n o t c o r r e l a t e d w i t h CVP. The c o r r e l a t i o n a n a l y s i s s h o w e d t h a t , a s was s e e n when v a r i o u s p a r a m e t e r s w ere e x a m i n e d a s a f r a c t i o n o f c e l l l e n g t h , p a r a m e t e r s GA, GP a n d CVA h a d a c o n s i s t e n t r e l a t i o n s h i p w i t h c e l l l e n g t h . P a r a m e t e r s CVP and G w e r e n o t c o r r e l a t e d w i t h l e n g t h , n o r d i d t h e y o c c u p y c o n s t a n t f r a c t i o n s o f t h e c e l l l e n g t h . C e l l w i d t h was w e a k l y , b u t h o m o g e n e o u s l y , c o r r e l a t e d 9k 7. CORRELATION ANALYSIS; - ( C o n t ' d . ) w i t h c e l l l e n g t h . The w i d t h was more s t r o n g l y c o r r e -l a t e d w i t h o t h e r m o r p h o m e t r i c p a r a m e t e r s , n o t a b l y CVINT. The c y t o p r o c t l e n g t h was w e a k l y c o r r e l a t e d w i t h c e l l l e n g t h ( h e t e r o g e n e o u s ) a n d s t r o n g l y c o r r e l a t e d w i t h t h e p o s t e r i o r s u t u r e l e n g t h ( h o m o g e n e o u s ) . T h e s e f i n d i n g s a g r e e d w i t h t h o s e o b t a i n e d when t h e same p a r a -m e t e r s were e x a m i n e d a s f r a c t i o n s o f t h e c e l l l e n g t h . The t o t a l number o f k i n e t i e s p e r c e l l was c o r r e -l a t e d w i t h c e l l l e n g t h , c e l l w i d t h , a n d t h e a n t e r i o r s u t u r e l e n g t h ( t a b l e 15). ^he number o f k i n e t i e s c o u n t e d c o u n t e r c l o c k w i s e f r o m t h e v e s t i b u l e t o t h e f i r s t CVP (KTCV) was s i m i l a r l y c o r r e l a t e d w i t h t h e s e t h r e e v a r i a b l e s . KTCV was a l s o s t r o n g l y c o r r e l a t e d w i t h t h e t o t a l number o f k i n e t i e s , s u g g e s t i n g t h a t when t h e number o f k i n e t i e s on t h e c e l l i n c r e a s e d t h e CVPs s h i f t e d c o u n t e r c l o c k w i s e t o m a i n t a i n a f i x e d p o s i t i o n on t h e c e l l c i r c u m f e r e n c e . The number o f k i n e t i e s s e p a r a t i n g t h e two CVPs (KTCVV) was n e g a t i v e l y c o r r e -l a t e d w i t h KTCV. When KTCV was s m a l l t h e two CVPs t e n d e d n o t t o be i n t h e same m e r i d i a n . 8. B I V A R I A T E SCATTERGRAMS; To c l a r i f y t h e c o r r e l a t i o n a n a l y s i s , b i v a r i a t e s c a t t e r g r a m s w e r e c o n s t r u c t e d f o r s e l e c t e d p a i r s o f v a r i a b l e s . The p r i n c i p a l a x e s o f t h e s c a t t e r g r a m s w e r e 95 B I V A R I A T E SCATTERGRAMS; - ( C o n t ' d . ) c a l c u l a t e d a n d a d d e d t o t h e a p p r o p r i a t e p l o t s . E q u a t i o n s f o r t h e p r i n c i p a l a x e s a r e g i v e n i n t a b l e 17. I n c a s e s w here t h e r e was no s i g n i f i c a n t c o r r e l a t i o n b e t w e e n two p l o t t e d v a r i a b l e s a l i n e was d r a w n t h r o u g h t h e mean o f one o f t h e v a r i a b l e s . The a n t e r i o r s u t u r e was p r e v i o u s l y shown t o o c c u p y a b o u t 47$ o f t h e v e n t r a l s u r f a c e . A l i n e d r a w n a t t h e 47$ l e v e l on a b i v a r i a t e s c a t t e r g r a m o f GA a n d c e l l l e n g t h (fig.50a) p a s s e d t h r o u g h t h e c l u s t e r o f p l o t t e d p o i n t s a n d h a d s i m i l a r s l o p e a n d i n t e r c e p t t o t h e p r i n c i p a l a x e s o f t h e s c a t t e r g r a m s . The s l o p e o f t h e . p r i n c i p a l a x e s f o r a s c a t t e r g r a m o f GP a n d c e l l l e n g t h was s i m i l a r t o t h a t f o r GA and l e n g t h , b u t t h e r e was a d i f f e r e n t i n t e r c e p t ( f i g . 5 0 b ) . S c a t t e r p l o t s o f c e l l l e n g t h a n d v e s t i b u l e l e n g t h i n d i c a t e d a l a c k o f c o r r e l a t i o n b e t w e e n t h e s e v a r i a b l e s ( f i g . 5 1 b ) . C e l l s s m a l l e r t h a n 65 pm, h o w e v e r , h a d v e s t i b u l e s l e s s t h a n 15 pm l o n g . T h e s e v e s t i b u l e s w e r e o f t e n a b n o r m a l i n s h a p e . The c y t o p r o c t o c c u p i e d a b o u t 15$ o f t h e c e l l l e n g t h ( f i g . 4 9 d ) . P r i n c i p a l a x e s f o r s c a t t e r p l o t s o f CYTL a n d c e l l l e n g t h r a n p a r a l l e l a n d c l o s e t o t h e 15$ l e v e l ( f i g . 5 1 a ) . On t h e d o r s a l s u r f a c e , r e g i o n CVA o c c u p i e d a b o u t 96 TABLE 17 PRINCIPAL AXES OF BIVARIATE SCATTERGRAMS CONFIDENCE INTERVALS _1 Length G* Length GA** Length GP* Length CVA* Length CVINT** Length CVP* T 0 6 8 11-5 24M1 24M2 0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2 0 6 8 11.5 24M1 24M2 EQUATION OF PRINCIPAL AXIS ns Y n = - 4 6 . 3 + 9 . 1 Y , Y t = - 9 8 . 1 + 1 2 .7Y' Y i = YI= " Y l = " Y l = Y i =  Y i = Y t = -12 3 6.8 : Y±= -9-7+5-7 4.6+6.0 32.3+1.6 11.5+2.0 2.9+2.1 0.6+2.0 2.2+2.0 61.2+3.5 7.8+2.3 16.2+2.2 32.1+1.8 26.1+2.2 27.4+2.0 31.0+1.7 38.6+2.1 10.9+2.6 31.9+2.1 31.7+2.0 9.1+2.6 5.0+2.9 11 .8+1 .9 Y0 17.2+1.9 Yp 27 .4+1 .9 Y% 9.1+2.3 Y% 43.4+1.3 Y% 33 .6+1 .6 Y^ 48.0+3.3 Y „ Y^= - 4 4 . 9 + 6.3 Y 2 Y, = ns Yt= 48.3+2.0 Y9 Y|=-l49.2+10 .7Y^ Y^= ns * Y i = Y l = Y l = Y l = Y , = FOR SLOPES  OF PRINCIPAL  AXES  Lower Upper 6.1 17.7 7.2 53.7 4.2 17.7 4.0 9.5 4.4 9.3 1-3 1.9 1.7 2.3 1.7 2.7 1.7 2.4 1.6 2.7 2.7 4.9 1.8 3-1 1.8 2.8 1.5 2.3 1.7 3.0 1.6 2.8 1.1 2.8 1.6 2.7 2.2 3.2 1.6 2.8 1.6 2.5 1.9 3-7 2.1 4.7 1.4 2.7 1.7 2.2 1.5 2.5 1.8 3.1 1.1 1.6 1.3 2.0 2.2 5.9 4.3 11.2 1.8 2.2 4.2 20.3 97 TABLE 17 - (Cont'd.) PRINCIPAL AXES OF BlVARIATE SCATTERGRAMS Length Length Length Width tl CYTL** BB SPACING BETWEEN BASAL BODIES KTCV TOTAL KINETIES T 0 6 8 11.5 24M1 24M2 24M2 EQUATION OF PRINCIPAL AXIS Y, - ns Y l =  Y l =  Y l =  Y l =  Y l = I7.2+7.3 1.8+6.5 2.6+6.6 4.1+7.2 n/a Y 1= - 3.1+2.5 Y 2 CONFIDENCE INTERVALS FOR SLOPES OF PRINCIPAL AXES Lower Upper 5.5 4.5 4.5 4.4 1.8 11.1 11.7 12.1 20.0 4.0 24M2 Y±= 44.9+11.8Y2 9-7 15-0 24M2 Y,=. -46.3+1.0 Y, * the slopes of the p r i n c i p a l axes of these two variables are homogeneous throughout a l l of the samples (0-24 hr s . ) . ** the slopes of the p r i n c i p a l axes are not homogeneous. n/a not applicable ns not s i g n i f i c a n t (there i s no c o r r e l a t i o n between the variables, see tables 14 and 15)• 98 B I V A R I A T E SCATTERGRAMS: - ( C o n t ' d . ) 35$ o f t h e c e l l l e n g t h ( f i g . 4 8 d ) . A l i n e a t t h e 35$ l e v e l on a s c a t t e r p l o t o f CVA.and c e l l l e n g t h was i n -d i s t i n g u i s h a b l e f r o m t h e p r i n c i p a l a x e s ( f i g . 5 2 a ) . R e g i o n CVP a p p e a r e d t o be a c o n s t a n t l e n g t h (22 pm) d e s p i t e c h a n g e s i n c e l l l e n g t h ( f i g . 4 8 C ) . A s c a t t e r -p l o t o f CVP a n d c e l l l e n g t h i n d i c a t e d a l a c k o f c o r r e l a t i o n b e t w e e n t h e s e p a r a m e t e r s ( f i g . 5 2 b ) . The o t h e r d o r s a l r e g i o n ( C V I N T ) o c c u p i e d t h e r e m a i n d e r o f t h e c e l l l e n g t h . T h i s c a n be d e f i n e d by t h e f o r m u l a ; CVINT = L - (0.35L + 22 pm). A l i n e d e s c r i b i n g t h i s e q u a t i o n was s i m i l a r t o t h e p r i n c i p a l a x e s o f a s c a t t e r -p l o t o f CVINT a n d c e l l l e n g t h ( f i g . 5 3 a ) . A s c a t t e r p l o t o f w i d t h , a nd k i n e t y number i s g i v e n i n f i g u r e 54b. The s l o p e o f t h e p r i n c i p a l a x e s showed t h a t a n i n c r e a s e i n 1.4 pm i n c e l l w i d t h was a c c o m p a n i e d b y t h e a d d i t i o n o f one k i n e t y . A s c a t t e r p l o t o f KTCV ( a s a p e r c e n t a g e o f t h e t o t a l n umber o f k i n e t i e s ) a n d c e l l w i d t h i n d i c a t e d n o c o r r e -l a t i o n ( r ^ = 0.05). The f r a c t i o n o f t h e t o t a l number o f k i n e t i e s o c c u p i e d b y KTCV was t h e r e f o r e c o n s t a n t a n d e q u a l l e d a b o u t 58$ ( t a b l e 1 0 ) . When KTCV was p l o t t e d a g a i n s t t h e t o t a l number o f k i n e t i e s (fig.55a) t h e r e was s i g n i f i c a n t c o r r e l a t i o n ( r ^ = 0.71) a n d t h e p r i n c i p a l a x e s a p p r o a c h e d t h e 58$ l e v e l . 99 8. B I V A R I A T E SCATTERGRAMS: - ( C o n t ' d . ) A s c a t t e r g r a m o f c e l l l e n g t h a n d t h e number o f b a s a l b o d i e s b e t w e e n ' t h e CVPs i n d i c a t e d a c o r r e l a t i o n b e t w e e n t h e s e p a r a m e t e r s ( f i g . 5 3 b ) . T h e r e was a l s o a c o r r e l a t i o n b e t w e e n b a s a l b o d y s p a c i n g a n d c e l l l e n g t h ( r ^ = O.38). The p r i n c i p a l a x e s f o r t h e b i v a r i a t e s c a t t e r g r a m o f t h e s e two v a r i a b l e s i n d i c a t e d t h a t s m a l l e r c e l l s t e n d e d t o h a v e t h e i r b a s a l b o d i e s s p a c e d more c l o s e l y t o g e t h e r ( f i g . 5 4 a ) . 100 SECTION E: GULLET DEFECTS AND FEEDING; M u t a n t sm2 c e l l s a n d c e l l s o f many o t h e r v a r i a n t l i n e s h a d s e v e r e g u l l e t d e f e c t s w h i c h m i g h t h a v e a f f e c t e d t h e i r f e e d i n g a b i l i t y . The a p p e a r a n c e o f p h e n o t y p e s s u c h a s r e d u c e d c e l l s i z e m i g h t t h e r e f o r e be r e l a t e d t o s u c h a d e f e c t i n f e e d i n g a b i l i t y . To e x a m i n e t h i s p r o b l e m t h e f o l l o w i n g e x p e r i m e n t was done w i t h w i l d - t y p e a n d sm2 c e l l s . L o g - p h a s e c u l t u r e s o f b o t h l i n e s w e r e grown o v e r n i g h t a t 34.5°C ( f l a s k c u l t u r e s i n a w a t e r b a t h ) . The n e x t m o r n i n g t h e c e l l s were c o n -c e n t r a t e d b y c e n t r i f u g a t i o n a n d r e s u s p e n d e d i n a d i l u t e s o l u t i o n o f r e d a c r y l i c p a i n t i n c u l t u r e medium. A f t e r s e v e n m i n u t e s t h e c e l l s were f i x e d i n Champy's s o l u t i o n and t h e s a m p l e s w ere s u b s e q u e n t l y s i l v e r i m p r e g n a t e d . The s i l v e r e d m a t e r i a l was s c o r e d f o r t h e number o f p a i n t - f i l l e d f o o d v a c u o l e s , t h e c e l l l e n g t h , t h e l e n g t h o f t h e g u l l e t p a r t s ( q u a d r u l u s a n d p e n i c u l u s ) , a n d t h e e x t e n t o f t h e g u l l e t d e f e c t ( t y p e 1, 2, o r 3; p l a t e 4). The mean l e n g t h o f sm2 c e l l s a f t e r t h e h e a t p e r i o d was 73 pm a s c o m p a r e d t o 111 pm f o r w i l d - t y p e c e l l s ( t a b l e 1 8 ) . The m u t a n t c e l l s c o m p r i s e d a n o r m a l l y d i s t r i b u t e d p o p u l a t i o n on t h e b a s i s o f l e n g t h w h i l e t h e w i l d - t y p e c e l l s h a d a b i m o d a l l e n g t h d i s t r i b u t i o n ( f i g . 5 6 ) . The sm2 c e l l s f o r m e d f e w e r f o o d v a c u o l e s t h a n d i d t h e 101 TABLE 18  MORPHOMETRIC PARAMETERS - FOOD VACUOLE EXPERIMENT LENGTH (pm-s. e. ) Wild-Type C e l l s sm2 Ce l l s C e l l Length Quadrulus Peniculus Number of Food Vacuoles Number of Food Vacuoles i n C e l l s with a: (a) normal g u l l e t (b) type 1 g u l l e t (c) type 2 g u l l e t (d) type 3 g u l l e t 111.0-1.6 21.1-0.2 20.0-0.2 7.6-0.2 7.6-0.2 n/a n/a n/a 73-3-1.6 18.8-0.4 15.7-0.4 1.8-0.2 3.6-O.3 2.0-0.4 O.6-O.3 0.1-0.1 (na = not applicable; sample sizes - f o r wild-type c e l l s 32, f o r . sm2 c e l l s 100). 102 SECTION E; GULLET DEFECTS AND FEEDING: - ( C o n t ' d . ) w i l d - t y p e c e l l s ( t a b l e 1 8 ) . I n a d d i t i o n , t h e number f o r m e d was r e l a t e d t o t h e d i s o r g a n i z a t i o n o f t h e g u l l e t ( t a b l e 1 8 ) . O n l y one o f t h e sm2 c e l l s w i t h a t y p e 3 g u l l e t f o r m e d an y f o o d v a c u o l e s d u r i n g t h e s e v e n m i n u t e p a i n t p u l s e . C e l l s w i t h t y p e s 1 and 2 g u l l e t s a l s o h a d r e d u c e d numbers o f f o o d v a c u o l e s . E v e n sm2 c e l l s w i t h n o r m a l g u l l e t s f o r m e d f e w e r f o o d v a c u o l e s t h a n w i l d - t y p e c e l l s . The c e l l l e n g t h was p o s i t i v e l y c o r r e l a t e d w i t h t h e l e n g t h s o f t h e q u a d r u l u s and p e n i c u l u s i n sm2 c e l l s , b u t n o t i n w i l d - t y p e c e l l s ( t a b l e 1 9 ) . W i l d - t y p e g u l l e t s w e r e c o n s t a n t i n l e n g t h w h i l e sm2 c e l l s l e s s t h a n 80 pm l o n g t e n d e d t o h a v e s h o r t e r g u l l e t s ( f i g . 5 1 a ) . The number o f f o o d v a c u o l e s was c o r r e l a t e d w i t h t h e l e n g t h o f b o t h sm2 a n d w i l d - t y p e c e l l s . L a r g e r c e l l s t e n d e d t o c o n t a i n more f o o d v a c u o l e s t h a n s m a l l e r o n e s . The l e n g t h o f t h e g u l l e t p a r t s was h o w e v e r , o n l y w e a k l y c o r r e l a t e d w i t h v a c u o l e n u m b e r s . The r e d u c t i o n i n f o o d v a c u o l e n u m b e r s i n sm2 c e l l s was n o t e n t i r e l y due t o r e d u c t i o n i n l e n g t h o f t h e g u l l e t s . E v e n sm2 c e l l s w i t h n o r m a l g u l l e t l e n g t h s had f e w e r f o o d v a c u o l e s t h a n w i l d - t y p e c e l l s ( f i g . 5 7 b ) . The number o f f o o d v a c u o l e s c o n t a i n e d i n sm2 c e l l s was t h e r e f o r e r e l a t e d , n o t j u s t t o t h e d i s o r g a n i z a t i o n o f t h e g u l l e t , b u t t o some o t h e r f a c t o r n o t e v i d e n t f r o m t h e s i z e o r a n a tomy o f t h e g u l l e t . The r e d u c e d s i z e o f sm2 c e l l s may a l s o h a v e TABLE 19 MATRIX OF CORRELATION COEFFICIENTS . f o r DATA FROM THE FOOD VACUOLE EXPERIMENT Number of C e l l Quadrulus S Food Vacuoles Length Length PENICULUS LENGTH . 9 .300 .238 .357 24 .496 .831 QUADRULUS LENGTH .9 .264 24 .387 .623 CELL LENGTH © .682 24 .429 S = sample, + = wild-type c e l l s , 24 = sm2 c e l l s l e f t at 35°C for 24 hours. Blanks indicate i n s i g n i f i c a n t c o r r e l a t i o n s . 104 SECTION Et GULLET DEFECTS AND FEEDING: - ( C o n t ' d . ) c o n t r i b u t e d t o t h e r e d u c t i o n i n t h e number o f f o o d v a c u o l e s p e r c e l l a s s m a l l e r c e l l s w o u l d h a v e a n i n c r e a s e d p r o b a -b i l i t y o f l o s i n g f o o d v a c u o l e s a t t h e c y t o p r o c t . 105 DISCUSSION I n t h i s s t u d y s e v e r a l n e w l y - d e s c r i b e d P a r a m e c i u m m u t a n t s h a v e b e e n u s e d t o e x a m i n e m o r p h o g e n e s i s i n t h i s o r g a n i s m . The s t u d y i s b a s e d on a m o r p h o m e t r i c a n a l y s i s o f t h e s h a p e a nd s i z e o f m u t a n t a nd w i l d - t y p e c e l l s . M o r p h o m e t r i c a n a l y s i s h a s b e e n a p p r o a c h e d f r o m t w o v i e w -p o i n t s : a n e x a m i n a t i o n o f a g e - d e p e n d e n t c h a n g e s i n c e l l s i z e a n d a n e x a m i n a t i o n o f s i z e - d e p e n d e n t c e l l p a t t e r n ( o r how t h e p o s i t i o n s o f c o r t i c a l s t r u c t u r e s a r e r e l a t e d t o c e l l s i z e ) . The r e s u l t s o f t h e m o r p h o m e t r i c s t u d y w i l l be d i s c u s s e d i n r e l a t i o n t o c e l l d i v i s i o n , c o r t i c a l g r o w t h d u r i n g d i v i s i o n , a n d c o r t i c a l p a t t e r n i n g . P r i o r t o exam-i n i n g t h e s e a s p e c t s o f P a r a m e c i u m m o r p h o g e n e s i s , h o w e v e r , t h e g e n e t i c s and p h e n o t y p e s o f t h e t e n m u t a n t s u s e d i n t h e s t u d y w a r r a n t a f e w comments. A) GENETICS AND PHENOTYPES OF THE MUTANTS: Ten new m o r p h o l o g i c a l m u t a n t s o f P a r a m e c i u m h a v e b e e n d e s c r i b e d i n t h i s s t u d y . Two o f t h e s e m u t a n t s a r e a l l e l e s a t t h e same l o c u s . The m u t a n t s i n c l u d e e x a m p l e s o f t h r e e t y p e s o f m o r p h o l o g i c a l d e f e c t s ; d e f e c t s i n f i s s i o n - z o n e f o r m a t i o n ( d f z m u t a n t s , i n c l u d i n g d f z l a n d dfz2), d e f e c t s i n f i s s i o n - f u r r o w c o n s t r i c t i o n ( d c m u t a n t s , i n c l u d i n g dc2 a, dc2 b, dc3, dc4, dc5, and dc6), a n d d e f e c t s i n c e l l g r o w t h r e s u l t i n g i n s m a l l c e l l s 106 GENETICS AND PHENOTYPES OF THE MUTANTS: - ( C o n t ' d . ) (sm m u t a n t s , i n c l u d i n g sm2 a n d sm3). B o t h sm a n d dc m u t a n t s h a v e b e e n p r e v i o u s l y d e s c r i b e d i n P a r a m e c i u m ( S o n n e b o r n , 1974b). The d f z m u t a n t s , h o w e v e r , c o n s t i -t u t e a new t y p e o f P a r a m e c i u m m u t a n t a l t h o u g h m u t a n t s w i t h a s i m i l a r p h e n o t y p e h a v e b e e n r e p o r t e d i n T e t r a h y m e n a ( F r a n k e l , e t a l , 1976a). The i s o l a t i o n o f o n l y one p a i r o f a l l e l e s among t e n d e v e l o p m e n t a l m u t a n t s s u g g e s t s t h a t a l a r g e number o f g e n e s a f f e c t d e v e l o p m e n t i n P a r a m e c i u m . I s o l a t i o n a b o f t h e a l l e l e s dc2 and dc2 s h o u l d n o t be i n t e r p r e t e d a s i n d i c a t i n g t h a t o n l y a f e w g e n e s p r o d u c e p r o d u c t s e s s e n t i a l f o r c e l l d i v i s i o n , a s t h e v a r i a n t s t h a t w e r e u s e d i n t h e g e n e t i c s t u d y were c a r e f u l l y s e l e c t e d f r o m a g r o u p o f 60 v a r i a n t s s u c h t h a t v a r i a n t s w i t h s i m i l a r p h e n o t y p e s , a s w e l l a s v a r i a n t s w i t h w i d e l y d i f f e r e n t p h e n o t y p e s , w e r e i n c l u d e d . T h i s s e l e c t i o n w o u l d e n -h a n c e t h e p r o b a b i l i t y o f i s o l a t i n g a l l e l i c m u t a n t s . O t h e r s t u d i e s on d e v e l o p m e n t a l m u t a n t s i n P a r a m e c i u m h a v e s i m i l a r l y r e v e a l e d f e w c a s e s o f a l l e l i s m ( W h i t t l e a n d C h e n - S h a n , 1969). T h i s c o n t r a s t s s h a r p l y w i t h t h e c a s e i n T e t r a h y m e n a , w h e r e a s u r p r i s i n g number o f t h e d e v e l o p m e n t a l m u t a n t s a r e a l l e l i c ( F r a n k e l , e t a l , 1976a). A d e t e r m i n a t i o n o f t h e a c t u a l n u m b e r s o f g e n e s whose p r o d u c t s a r e e s s e n t i a l f o r c e l l d i v i s i o n o r o t h e r 107 GENETICS AND PHENOTYPES OF THE MUTANTS; - ( C o n t ' d . ) d e v e l o p m e n t a l e v e n t s i n b o t h P a r a m e c i u m a nd T e t r a h y m e n a a w a i t s f u r t h e r m u t a g e n e s i s a n d g e n e t i c e x p e r i m e n t s . The t e n m u t a t i o n s e x a m i n e d h a v e p l e i o t r o p i c e f f e c t s on c e l l m o r p h o l o g y . They a l l p r o d u c e a b n o r m a l a r r a n g e m e n t s o f o r g a n e l l e s on t h e c e l l c o r t e x a n d i n t h e g u l l e t , a f f e c t t h e s t r u c t u r e o f t h e c y t o p r o c t , and i n f l u e n c e t h e number o f c o n t r a c t i l e v a c u o l e p o r e s on t h e c e l l . T h e s e t y p e s o f a b n o r m a l i t i e s w e r e s u r p r i s -i n g l y s i m i l a r i n a l l o f t h e m u t a n t s . T h e s e d e f e c t s a r e n o t , h o w e v e r , t h e p r i m a r y ( o r f i r s t t o a p p e a r ) m orpho-l o g i c a l d e f e c t s i n t h e m u t a n t s b u t r a t h e r r e s u l t f r o m a g r a d u a l d e t e r i o r a t i o n o f t h e n o r m a l c o r t i c a l s t r u c t u r e d u r i n g p r o l o n g e d h e a t - t r e a t m e n t . L o e t a l (1976) h a v e p r o d u c e d a b n o r m a l a r r a n g e m e n t s o f c o r t i c a l o r g a n e l l e s i n T e t r a h y m e n a b y g r o w i n g t h e c e l l s a t e l e v a t e d t e m p e r -a t u r e i n a medium c o n t a i n i n g s a t u r a t e d p h o s p h o l i p i d s . T h i s r e s u l t s i n a l o s s i n c e l l s h a p e ( p r e s u m a b l y b y a f f e c t i n g t h e s t r u c t u r e o f c o r t i c a l membranes) a n d s e c o n d a r i l y r e s u l t s i n a b n o r m a l c o r t i c a l p a t t e r n . The a b n o r m a l c e l l s h a p e p r o b a b l y a f f e c t s t h e a t t a c h m e n t o f o r g a n e l l e s t o t h e c e l l s u r f a c e a n d t h e r e f o r e i s t h e p r i m a r y c a u s e o f t h e a l t e r e d c o r t i c a l m o r p h o l o g y . C e l l s h a p e i s a b n o r m a l i n a l l o f t h e P a r a m e c i u m m u t a n t s e x a m i n e d i n t h e p r e s e n t s t u d y a nd one m i g h t t h e r e f o r e 108 GENETICS AND PHENOTYPES OF THE MUTANTS: - ( C o n t ' d . ) assume t h a t t h i s a f f e c t s t h e p o s i t i o n i n g o f new o r g a n e l l e s on t h e c o r t e x . I n a d d i t i o n , b o t h c e l l s h a p e a n d c o r t i c a l p a t t e r n become i n c r e a s i n g l y a b n o r m a l d u r i n g c o n t i n u e d g r o w t h a t t h e r e s t r i c t i v e t e m p e r a t u r e , a n d a c a u s a l c o n n e c t i o n b e t w e e n t h e s e two d e f e c t s i s s t r o n g l y s u g g e s t e d . The p r i m a r y m o r p h o l o g i c a l d e f e c t s w e re d e t e r m i n e d f o r t h o s e m u t a n t s t h a t were e x a m i n e d m o r p h o m e t r i c a l l y . T hese m u t a n t s and t h e i r p r i m a r y d e f e c t s were: sm2 -d e f e c t i v e b a s a l b o d y p r o l i f e r a t i o n , d c 2 - p r o l o n g e d c e l l c o n t r a c t i o n p r i o r t o f o r m a t i o n o f a f i s s i o n - f u r r o w , d c 4 - f a i l u r e o f c e l l g r o w t h i n t h e e a r l y p a r t o f t h e c e l l c y c l e , a n d d f z 2 - a p r e m a t u r e c e l l c o n t r a c t i o n p r i o r t o f u r r o w f o r m a t i o n . A t t h e p r e s e n t t i m e i t i s n o t p o s s i b l e t o c o r r e l a t e a n y o f t h e s e m o r p h o l o g i c a l d e f e c t s w i t h s p e c i f i c b i o c h e m i c a l l e s i o n s . The p r i m a r y d e f e c t s i n sm2 and d c 4 c e l l s , h o w e v e r , o c c u r a t t i m e s d u r i n g t h e c e l l c y c l e when t h e s e c e l l s a r e h e a t -s e n s i t i v e . I n d c 4 c e l l s , h e a t - s e n s i t i v i t y o c c u r s d u r i n g t h a t p a r t o f t h e c e l l c y c l e when c e l l g r o v / t h s h o u l d o c c u r t o b r i n g t h e c e l l t o i t s i n t e r f i s s i o n l e n g t h . T h i s g r o w t h i s s u b n o r m a l i n d c 4 c e l l s . I n sm2 c e l l s , h e a t - s e n s i t i v i t y o c c u r s d u r i n g t h a t p a r t o f t h e c e l l c y c l e when b a s a l b o d y p r o l i f e r a t i o n n o r m a l l y o c c u r s . 109 A) GENETICS AND PHENOTYPES OF THE MUTANTS: - ( C o n t ' d . ) B a s a l "body p r o l i f e r a t i o n i s d e f e c t i v e i n sm2 c e l l s . T h i s c o r r e l a t i o n b e t w e e n h e a t - s e n s i t i v i t y a n d v i s i b l e m o r p h o l o g i c a l d e f e c t s s u g g e s t s t h a t t h e h e a t t r e a t m e n t d e s t r o y s some p r o d u c t e s s e n t i a l f o r t h e d e v e l o p m e n t a l e v e n t c o n c e r n e d . W h i l e t h i s d o e s n o t a l l o w t h e n a t u r e o f t h e b i o c h e m i c a l l e s i o n i n t h e s e m u t a n t s t o be d e t e r m i n e d , i t d o e s r e l a t e t h e h y p o t h e t i c a l b i o c h e m i c a l l e s i o n t o a s p e c i f i c d e v e l o p m e n t a l e v e n t . . I t i s n o t c l e a r w h e t h e r h e a t - s e n s i t i v i t y d u r i n g t h e P a r a m e c i u m c e l l c y c l e i s a l w a y s r e l a t e d t o s p e c i f i c m o r p h o g e n e t i c e v e n t s . A l l c e l l l i n e s e x a m i n e d ( w i l d t y p e , dc4, dc5i sm2, a n d v a r i a n t 48) h a v e a p e a k i n h e a t - s e n s i t i v i t y a t o r n e a r age O.65. H e a t s h o c k s a p p l i e d a f t e r t h i s age p r o d u c e l i t t l e o r no d e l a y i n c e l l d i v i s i o n ( e x c e p t i n m u t a n t sm2). The u n i f o r m o c c u r r e n c e o f t h i s p e a k i n h e a t - s e n s i t i v i t y s u g g e s t s t h a t i t c o r r e s p o n d s t o some p r o c e s s t h a t i s h e a t -s e n s i t i v e i n w i l d - t y p e c e l l s , o f i n c r e a s e d h e a t -s e n s i t i v i t y i n m u t a n t c e l l s , a n d e s s e n t i a l f o r c e l l d i v i s i o n . A p e a k i n s e n s i t i v i t y t o i n h i b i t o r s o f RNA an d p r o t e i n s y n t h e s i s a l s o o c c u r s n e a r age O.65 ( G i l l a n d H a n s o n , 1968; Suhama a n d H a n s o n , 1971; R a s m u s s e n , 1967) a n d i t i s p o s s i b l e t h a t b o t h h e a t - s h o c k s and t h e s e m e t a b o l i c i n h i b i t o r s a f f e c t t h e same d e v e l o p m e n t a l 110 A) GENETICS AND PHENOTYPES OF THE MUTANTS; - ( C o n t ' d . ) e v e n t . The o n l y m o r p h o g e n e t i c e v e n t o c c u r r i n g a t age O.65 i s t h e p r o l i f e r a t i o n o f new b a s a l b o d i e s i n t h e e n d o r a l k i n e t y ( J o n e s , 1976). T h i s e v e n t s i g n a l s t h e s t a r t o f o r a l d e v e l o p m e n t . I t i s t e m p t i n g t o s u g g e s t t h a t o r a l d e v e l o p m e n t and c e l l d i v i s i o n a r e i n t i m a t e l y r e l a t e d i n P a r a m e c i u m and t h a t i n t e r f e r e n c e w i t h o r a l d e v e l o p m e n t by h e a t - s h o c k s o r m e t a b o l i c i n h i b i t o r s d e l a y s t h e o n s e t o f c e l l d i v i s i o n . T h i s t y p e o f r e l a -t i o n s h i p b e t w e e n o r a l d e v e l o p m e n t and c e l l d i v i s i o n i s w e l l d o c u m e n t e d i n a n o t h e r c i l i a t e , T e t r a h y m e n a , a nd i t s o c c u r r e n c e i n P a r a m e c i u m a l s o seems p r o b a b l e . The p o s s i b i l i t y t h a t t h e h e a t - s e n s i t i v e p e r i o d e n d i n g a t age O.65 i s r e l a t e d t o some p r o c e s s o t h e r t h a n o r a l d e v e l o p m e n t c a n n o t be e x c l u d e d , h o w e v e r . I n m u t a n t s , h e a t - s e n s i t i v i t y f o r o r a l d e v e l o p m e n t may mask o t h e r h e a t - s e n s i t i v e p r o c e s s e s , s u c h a s p r e p a r a t i o n f o r f u r r o w f o r m a t i o n . O n l y t h o s e m u t a n t s w h i c h h a v e a p r i m a r y d e f e c t e i t h e r v e r y e a r l y o r v e r y l a t e i n .the c e l l c y c l e ( a s i n dc4 and sm2 c e l l s ) w o u l d be e x p e c t e d t o show m u t a n t - s p e c i f i c p e r i o d s o f h e a t - s e n s i t i v i t y . D e t e c t i o n o f s e p a r a t e h e a t - s e n s i t i v e p e r i o d s i n t h e c e n t r a l p o r t i o n o f t h e c e l l c y c l e may be b e y o n d t h e r e s o l u t i o n o f t h e e x p e r i m e n t a l t e c h n i q u e . I l l CELL D I V I S I O N : The r e s u l t s o b t a i n e d f r o m a m o r p h o m e t r i c a n a l y s i s o f c e l l d i v i s i o n i n w i l d - t y p e a n d m u t a n t c e l l s r a i s e a number o f i s s u e s c o n c e r n i n g b i n a r y f i s s i o n i n P a r a m e c i u m . T h e r e a r e a w e l l d e f i n e d s e r i e s o f c e l l s h a p e a n d s i z e c h a n g e s w h i c h p r e c e d e and a c c o m p any c e l l d i v i s i o n i n P a r a m e c i u m . T h e s e d e s e r v e a f e w comments, p a r t i c u l a r l y i n t e r m s o f t h e i r r e l a t i o n t o c e l l d i v i s i o n . T h e s e s i z e a n d s h a p e c h a n g e s s h o u l d a l s o be c o m p a r e d w i t h t h o s e f o u n d i n o t h e r c e l l s t o d e t e r m i n e w h e t h e r s i m i l a r m e c h a n i s m s may c o n t r o l c e l l d i v i s i o n i n d i v e r s e c e l l t y p e s . The d a t a o b t a i n e d f r o m m u t a n t s w i t h d e f e c t s i n t h e c e l l d i v i s i o n p r o c e s s ( d c a n d d f z m u t a n t s ) t h e n a l l o w a n e x a m i n a t i o n o f some o f t h e p o s t u l a t e d m e c h a n i s m s f o r c e l l d i v i s i o n i n P a r a m e c i u m . The r e s u l t s o f t h i s p a r t o f t h e s t u d y i n d i c a t e t h a t p r e m a t u r e o r a b n o r m a l l y p r o l o n g e d c o n t r a -c t i o n o f t h e p o l a r c e l l r e g i o n s i s a s s o c i a t e d w i t h t h e dc and d f z p h e n o t y p e s . The f i n a l i s s u e e x a m i n e d i s t h e r e l a t i o n b e t w e e n b a s a l b ody p r o l i f e r a t i o n a nd s u r f a c e g r o w t h i n m u t a n t a n d w i l d - t y p e c e l l s . T h i s p a r t o f t h e s t u d y l e a d s t o t h e c o n c l u s i o n t h a t b a s a l b o d y p r o l i f -e r a t i o n p r e c e d e s a n d i s a c a u s a l a g e n t o f s u r f a c e g r o w t h . T h i s r e s u l t i s i n d i r e c t c o n t r a d i c t i o n t o a p r e s e n t m o d e l o f s u r f a c e g r o w t h i n P a r a m e c i u m . 112 C E L L D I V I S I O N : - ( C o n t ' d . ) a) Shape a n d S i z e C h a nges D u r i n g t h e C e l l C y c l e : The s h a p e a n d s i z e c h a n g e s d u r i n g t h e l a s t q u a r t e r o f t h e c e l l c y c l e n o t e d i n t h i s s t u d y a r e v e r y s i m i l a r t o t h o s e r e p o r t e d by K a n e d a a n d H a n s o n ( 1 9 7 4 ) . P r e c e d i n g f i s s i o n - f u r r o w f o r m a t i o n , t h e c e l l f i r s t i n c r e a s e s i n l e n g t h and t h e n c o n t r a c t s . B o t h o f t h e s e e v e n t s i n v o l v e o n l y t h e p o l a r c e l l . r e g i o n s and a r e n o t a s s o c i a t e d w i t h t h e a p p e a r a n c e o f new c o r t i c a l u n i t s o r o r g a n e l l e s . T hese s i z e c h a n g e s t h e r e f o r e r e p r e s e n t f i r s t a s t r e t c h i n g a n d t h e n a c o m p a c t i n g o f t h e a v a i l a b l e c o r t i c a l u n i t s . Once t h e c e l l c o n t r a c t s , i t assumes a s h a p e w h i c h c a n be l o o s e l y d e s c r i b e d a s a c y l i n d e r w i t h e l l i p - -s o i d a l e n d s . D o e r n e r ( 1 9 7 6 ) h a s s u g g e s t e d t h a t t h e a t t a i n m e n t o f c y l i n d r i c a l s h a p e f a c i l i t a t e s c e l l d i v i s i o n . O t h e r t y p e s o f c e l l s , i n c l u d i n g s e a -u r c h i n ( H i r a m o t o , 1 9 5 8 ) a n d newt ( S a w a i , 1 9 7 6 ) e g g s , a l s o a t t a i n a c y l i n d r i c a l s h a p e p r i o r t o c e l l d i v i s i o n , a l t h o u g h t h i s s h a p e c h a n g e i s m e d i a t e d by c o n t r a c t i o n i n t h e c e n t r e o f t h e c e l l r a t h e r t h a n i n t h e p o l a r r e g i o n s a s i n P a r a m e c i u m . T h e r e i s a l s o e v i d e n c e t o s u g g e s t t h a t c o n t r a c t i o n i n T e t r a h y m e n a r e s u l t s i n a s h a p e c h a n g e p r e c e d i n g c e l l d i v i s i o n ( D o e r d e r , e t a l . , 1 9 7 5 ) . A l t h o u g h t h e means 113 C E L L D I V I S I O N : - ( C o n t ' d . ) a) S h a p e a n d S i z e C h a nges D u r i n g t h e C e l l C y c l e : - ( C o n t ' d . ) b y w h i c h t h e c y l i n d r i c a l s h a p e i s o b t a i n e d i n t h e s e d i f f e r e n t c e l l t y p e s may be d i v e r s e , t h e p u r p o s e f o r a t t a i n i n g t h i s s h a p e may be t h e same. The f u n c t i o n a l r e l a t i o n s h i p b e t w e e n t h i s s h a p e c h a n g e and c e l l d i v i s i o n h o w e v e r , r e m a i n s o b s c u r e . W h i l e i t i s d i f f i c u l t t o p r o v e t h a t a f u n c -t i o n a l r e l a t i o n s h i p e x i s t s b e t w e e n c e l l c o n t r a c t i o n a n d c e l l d i v i s i o n , i t i s e v i d e n t t h a t r e l a x a t i o n o r r e c o v e r y f r o m t h e c o n t r a c t i o n i s n e c e s s a r y f o r s u c c e s s f u l c e l l d i v i s i o n . I n two m u t a n t s d e f e c t i v e i n f u r r o w c o n s t r i c t i o n ( d c 2 a and d c 4 ) t h e amount o f s u r f a c e g r o w t h o c c u r r i n g d u r i n g t h e c e l l c y c l e was s u f f i c i e n t t o c o m p l e t e c o n s t r i c t i o n o f t h e f i s s i o n f u r r o w . The f a c t t h a t t h e s e c e l l s w e r e n o t a b l e t o c o m p l e t e f u r r o w c o n s t r i c t i o n d e s p i t e n o r m a l s u r f a c e g r o w t h l e d t o a s e a r c h f o r o t h e r p o s s i b l e d e f e c t s common t o b o t h m u t a n t s . Two s u c h d e f e c t s w e r e f o u n d : c o r t i c a l l e s i o n s a n d e n h a n c e d and p r o l o n g e d c o n t r a c t i o n p r i o r t o f u r r o w f o r m a t i o n . No r e c o v e r y f r o m t h e c o n t r a c t i o n i n t h e p o s t e r i o r p o l a r r e g i o n o f d c 4 c e l l s was n o t e d a n d o n l y l i m i t e d r e c o v e r y o c c u r r e d i n d c 2 c e l l s . F o r c e s p r o d u c e d by t h e 114 C E L L D I V I S I O N ; - ( C o n t ' d . ) a) S h a p e a n d S i z e C h a n g e s D u r i n g ; t h e C e l l C y c l e ; - ( C o n t ' d . ) ~ ~ s u s t a i n e d c o n t r a c t i o n o f t h e p o l a r r e g i o n s o f t h e c e l l m u s t i n t e r f e r e w i t h o r e q u a l i z e t h e f o r c e s a s s o c i a t e d w i t h c o n s t r i c t i o n o f t h e f i s s i o n f u r r o w . G r o w t h o f t h e f u r r o w s u r f a c e i s n o r m a l i n dc c e l l s and t h e f u r r o w i n g d e f e c t t h e r e f o r e r e s u l t s f r o m an i n a b i l i t y o f t h e f u r r o w t o move i n w a r d s r a t h e r t h a n a d e f e c t i n s u r f a c e g r o w t h . The m e c h a n i s m s w h i c h c a u s e t h e f u r r o w t o move i n w a r d a r e p o o r l y u n d e r -s t o o d , b u t i t i s o b v i o u s t h a t d u r i n g c l e a v a g e t h e P a r a m e c i u m c e l l b o t h i n c r e a s e s i n l e n g t h a n d d e c r e a s e s i n w i d t h . The d e c r e a s e I n w i d t h i s p a r t i c u l a r l y i m p o r t a n t a s i t a i d s f u r r o w c o n s t r i c t i o n b y e f f e c t i v e l y r e d u c i n g t h e c e l l d i a m e t e r a n d t h e r e -f o r e t h e amount o f s u r f a c e n e e d e d t o c o m p l e t e c o n s t r i c t i o n . A n y t h i n g i m p e d i n g t h e w i d t h r e d u c t i o n m i g h t t h e r e f o r e be e x p e c t e d t o i n d i r e c t l y i n t e r f e r e w i t h f u r r o w c o m p l e t i o n . I n d c 4 c e l l s t h e r e d u c t i o n i n c e l l w i d t h i s s h a r p l y a t t e n u a t e d i n t h e l a t t e r s t a g e s o f c e l l d i v i s i o n . T h i s may be due t o t h e s u s t a i n e d c o n t r a c t i o n i n t h e p o s t e r i o r p o l e o f t h e c e l l . D u r i n g f u r r o w c o n s t r i c t i o n , c y t o p l a s m f r o m t h e f u r r o w r e g i o n w o u l d be f o r c e d o u t w a r d s , t o w a r d s 115 C E L L D I V I S I O N ; - ( C o n t ' d . ) a) S h a p e a nd S i z e C h a nges D u r i n g t h e C e l l C y c l e ; - ( C o n t ' d . ) t h e c e l l p o l e s . C o n t i n u e d c o n t r a c t i o n o f t h e p o l a r r e g i o n s , h o w e v e r , w o u l d p r e v e n t c y t o p l a s m f r o m t h e f u r r o w r e g i o n f r o m m o v i n g i n t o t h e p o l a r a r e a s , t h e r e b y i n d i r e c t l y b l o c k i n g f u r t h e r c o n s t r i c t i o n o f t h e f u r r o w . I n dc2 c e l l s t h e e x t e n t o f w i d t h r e d u c t i o n d u r i n g c o n s t r i c t i o n o f t h e f u r r o w i s n o r m a l , b u t dc2 c e l l s a r e w i d e r t h a n w i l d - t y p e c e l l s t h r o u g h o u t t h e c e l l c y c l e . I n a d d i t i o n , t h e w i d t h d e c r e a s e i n dc2 c e l l s i s d e l a y e d i n r e l a t i o n t o t h a t i n w i l d - t y p e c e l l s . I n b o t h dc m u t a n t s c o r t i c a l l e s i o n s may p r o m o t e t h e i n c r e a s e d a n d s u s t a i n e d c o n t r a c t i o n , p r o b a b l y b y a l l o w i n g a n i n f l u x o f c a l c i u m i o n s , w h i c h a r e known t o s t i m u l a t e c o n t r a c t i o n o f p a r a m e c i a (Kamada a n d K i n o s i t a , 19^5)• C o r t i c a l l e s i o n s p r o d u c e d b y t r e a t i n g p a r a m e c i a w i t h d e t e r g e n t s i m i l a r l y r e s u l t i n c e l l c o n t r a c t i o n , ( D r y l a n d M e h r , 1976). The a b n o r m a l b e h a v i o u r o f dc4 c e l l s a t t h e r e s t r i c t i v e t e m p e r a t u r e , w i t h f r e q u e n t r e v e r s a l s o f t h e d i r e c t i o n o f s w i m m i n g , may a l s o be r e l a t e d t o t h e c o r t i c a l l e s i o n s . K u n g a n d E c k e r t (1972) h a v e shown t h a t P a r a m e c i u m b e h a v i o u r a l m u t a n t s h a v e 116 C E L L D I V I S I O N ; - ( C o n t ' d . ) a) S h a p e a n d S i z e C h a n g e s D u r i n g t h e C e l l C y c l e ; - ( C o n t ' d . ) d e f e c t s i n t h e a b i l i t y o f t h e s u r f a c e membranes t o c o n d u c t s p e c i f i c i o n s e i t h e r i n t o o r o u t o f t h e c e l l The c o r t i c a l l e s i o n s may p o s s i b l y a l s o i n t e r f e r e w i t h n o r m a l i o n f l u x a c r o s s t h e s u r f a c e membranes. D e f e c t s i n f i s s i o n - z o n e f o r m a t i o n a n d i n i t i a t i o n o f f u r r o w i n g a r e a l s o r e l a t e d t o a b n o r m a l c e l l c o n t r a c -t i o n . A t t h e r e s t r i c t i v e t e m p e r a t u r e , d f z 2 c e l l s do n o t f o r m e i t h e r a f i s s i o n - z o n e o r f u r r o w . T h e s e c e l l s u n d e r g o a c o n t r a c t i o n o f t h e p o l a r r e g i o n s a t age 0.8 c e l l c y c l e s , a p p r o x i m a t e l y 0 .15 c e l l c y c l e s ( o r a b o u t k-S m i n u t e s ) p r i o r t o t h e t i m e when c o n t r a c t i o n n o r m a l l y o c c u r s . T h i s p r e m a t u r e c o n t r a c t i o n i s a c c o m p a n i e d b y a m a r k e d i n c r e a s e i n c e l l w i d t h . T h e s e h i g h l y - c o n t r a c t e d c e l l s do n o t f o r m a f i s s i o n -z o ne o r f u r r o w . T h e i r r o u n d e d o u t l i n e i s q u i t e d i f f e r e n t f r o m t h e c y l i n d r i c a l f o r m o f n o r m a l d i v i d i n g c e l l s a n d i t i s p o s s i b l e t h a t t h e r o u n d e d s h a p e p o s e s a s t r u c t u r a l o b s t a c l e t o f u r r o w f o r m a t i o n . I n d f z 2 c e l l s , a p o l a r c o n t r a c t i o n a t age 0.8 i s a s s o c i a t e d w i t h r o u n d i n g o f t h e c e l l o u t l i n e , w h i l e a p o l a r c o n t r a c t i o n a t age 0 .95 i n w i l d - t y p e 117 B) C E L L D I V I S I O N : - ( C o n t ' d . ) a ) S h a p e - a n d S i z e C h a nges D u r i n g t h e C e l l C y c l e : - ( C o n t ' d . ) c e l l s i s a s s o c i a t e d w i t h t h e a t t a i n m e n t . o f a c y l i n d r i c a l c e l l s h a p e . T h i s s u g g e s t s t h a t s t r u c t u r a l r e i n f o r c e m e n t o f t h e c o r t e x i n t h e f u r r o w r e g i o n o c c u r s "between a g e s 0.8 and 0.95 and t h a t t h i s p r e v e n t s c e l l r o u n d i n g d u r i n g c o n t r a c t i o n a t a g e 0.95- B a s a l b o d y p r o l i f e r a t i o n i s c o m p l e t e by a ge 0.95» b u t i t i s d i f f i c u l t t o i m a g i n e how a d d i t i o n a l b a s a l b o d i e s , l y i n g p e r p e n d i c u l a r t o t h e c e l l s u r f a c e , w o u l d p r o v i d e r e s i s t a n c e t o a n o u t w a r d b u l g i n g o f t h e c e l l i n t h e f u r r o w r e g i o n . L o g i c a l l y , a r e i n f o r c e m e n t p a r a l l e l t o t h e l o n g a x i s o f t h e c e l l i s r e q u i r e d . S u n d a r a r a m a n a n d H a n s o n (1976) h a v e r e c e n t l y d e m o n s t r a t e d t h e p r e s e n c e o f l o n g i t u d i n a l m i c r o t u b u l e s i n t h e P a r a m e c i u m c o r t e x d u r i n g c e l l d i v i s i o n . T h e s e m i c r o t u b u l e s a p p e a r a t age 0.9 and p e r s i s t u n t i l c e l l d i v i s i o n i s c o m p l e t e and may p r o v i d e t h e c o r t i c a l r e i n f o r c e m e n t n e c e s s a r y d u r i n g c e l l c o n -t r a c t i o n . T h i s c o n c l u s i o n i s s u p p o r t e d b y t h e f i n d i n g t h a t d i s r u p t i o n o f t h e l o n g i t u d i n a l m i c r o -t u b u l e s b y l o w t e m p e r a t u r e o r c o l c h i c i n e r e s u l t s i n c e l l r o u n d i n g ( S u n d a r a r a m a n a nd H a n s o n , 1976). 118 C E L L D I V I S I O N : - ( C o n t ' d . ) a) Shape and S i z e Changes D u r i n g t h e C e l l C y c l e : - ( C o n t ' d . ) S u n d a r a r a m a n a n d H a n s o n d i d n o t e x a m i n e t h e e f f e c t s o f m i c r o t u b u l e d i s r u p t i o n d u r i n g s p e c i f i c s t a g e s o f c e l l d i v i s i o n , h o w e v e r , and i t w o u l d be i n t e r e s t i n g t o c o mpare t h e e f f e c t s o f t h i s t r e a t m e n t on c o n t r a c -t i n g c e l l s w i t h i t s e f f e c t s on c e l l s l a t e r d u r i n g c e l l d i v i s i o n , a f t e r c o n t r a c t i o n i s c o m p l e t e d . The p r e f i s s i o n c o n t r a c t i o n s a n d e x p a n s i o n s o f t h e p o l a r c e l l r e g i o n s a r e t h e r e f o r e c r i t i c a l f o r b o t h i n i t i a t i o n o f f u r r o w i n g a n d c o m p l e t i o n o f c y t o k i n e s i s . A l t h o u g h t h i s s t u d y h a s n o t r e v e a l e d t h e n a t u r e o f t h e m e c h a n i s m s w h i c h t r i g g e r o r c a u s e t h e c o n t r a c t i o n s , i t d o e s i n d i c a t e t h a t a c a r e f u l s t u d y o f t h e s e p r o c e s s e s i n P a r a m e c i u m i s w a r r a n t e d i f c e l l d i v i s i o n i n t h i s o r g a n i s m i s t o be p r o p e r l y u n d e r s t o o d . I t a l s o i n d i c a t e s t h a t , s i n c e s h a p e c h a n g e s s i m i l a r t o t h o s e f o u n d i n P a r a m e c i u m o c c u r i n o t h e r t y p e s o f c e l l s , P a r a m e c i u m p r o v i d e s a m o d e l o r g a n i s m f o r a n a l y z i n g t h e f u n c t i o n o f p r e f u r r o w c e l l s h a p e c h a n g e s . b ) S u r f a c e G r o w t h : P r o l i f e r a t i o n o f b a s a l b o d i e s i n t h e c e n t r a l p o r t i o n o f t h e P a r a m e c i u m c e l l i s c o m p l e t e d p r i o r 119 C E L L D I V I S I O N ; - ( C o n t ' d . ) b) S u r f a c e G r o w t h ; - ( C o n t ' d . ) t o t h e s t a r t o f s u r f a c e g r o w t h a n d f u r r o w f o r m a t i o n . T h i s s u g g e s t s t h a t b a s a l b o d y p r o l i f e r a t i o n i s a p r e r e q u i s i t e f o r s u r f a c e g r o w t h . T h i s c o n c l u s i o n i s s t r o n g l y s u p p o r t e d b y t h e o b s e r v a t i o n t h a t m u t a n t c e l l s w i t h r e d u c e d b a s a l b o d y p r o l i f e r a t i o n (sm2 a n d dc4) a l s o h a v e r e d u c e d s u r f a c e g r o w t h . " I n f a c t , t h e r e i s a c o n s i s t e n t r e l a t i o n s h i p b e t w e e n t h e number o f new b a s a l b o d i e s f o r m e d p r i o r t o s u r f a c e g r o w t h a n d t h e amount o f s u r f a c e g r o w t h w h i c h s u b -s e q u e n t l y o c c u r s . I t a p p e a r s t h a t , when new b a s a l b o d i e s f o r m t h e y a c t a s o r g a n i z i n g c e n t r e s w h i c h s t i m u l a t e t h e f o r m a t i o n , o f o t h e r s t r u c t u r e s a r o u n d them r e s u l t i n g i n a d d i t i o n o f new c o r t i c a l u n i t s t o t h e s o m a t i c k i n e t i e s , e l o n g a t i o n o f t h e s e k i n e t i e s , a nd s u r f a c e g r o w t h . A c c o r d i n g t o t h i s m o d e l t h e s t i m u l u s f o r s u r f a c e g r o w t h r e s i d e s i n t h e c o r t e x and n o t , a s K a n e d a and H a n s o n (1974) h a v e s u g g e s t e d , i n t h e f i b r i l l a r l a y e r u n d e r n e a t h t h e c o r t e x . S u p p o r t f o r t h e m o d e l p r e s e n t e d h e r e comes n o t o n l y f r o m t h e e x a m i n a t i o n o f m u t a n t c e l l s , b u t a l s o f r o m t h e o b s e r v a t i o n s o f H u f n a g e l (1969) a n d S o n n e b o r n (1970, 1974a) t h a t c o r t i c a l u n i t s a r e o r g a n i z e d w i t h t h e b a s a l b o d y a s t h e c e n t r a l c o m p o n e n t . The 120 C E L L D I V I S I O N ; - ( C o n t ' d . ) b) S u r f a c e G r o w t h : - ( C o n t ' d . ) o r g a n i z i n g i n f l u e n c e o f t h e b a s a l b o d i e s on c o r t i c a l d e v e l o p m e n t h a s a l s o p r e v i o u s l y b e e n s u g g e s t e d by L w o f f (1950) a n d S o n n e b o r n (1974a). K a n e d a a n d H a n s o n ' s m o d e l f o r s u r f a c e g r o w t h was b a s e d on m o r p h o m e t r i c d a t a on d i v i d i n g w i l d -t y p e c e l l s , j u s t a s t h e m o d e l i n t h i s p a p e r was b a s e d on a s i m i l a r , b u t i n d e p e n d e n t , s e t o f meas-u r e m e n t s . T h e i r m o d e l , h o w e v e r , was b a s e d on t h e c o n c l u s i o n t h a t t h e r e was no t e m p o r a l c o r r e l a t i o n b e t w e e n s u r f a c e g r o w t h a n d c o r t i c a l u n i t p r o l i f -e r a t i o n . T h i s i s o b v i o u s l y t r u e i n t h e p o l a r r e g i o n s o f t h e c e l l w h e r e no p r o l i f e r a t i o n o f b a s a l b o d i e s a c c o m p a n i e s s u r f a c e g r o w t h . T h i s g r o w t h i s o n l y t e m p o r a r y , h o w e v e r , a n d r e f l e c t s a s t r e t c h i n g o f t h e a v a i l a b l e c o r t i c a l u n i t s r a t h e r t h a n a c t u a l s u r f a c e g r o w t h . P a r t s o f t h e c e l l w h i c h show s t a b l e g r o w t h d u r i n g c e l l d i v i s i o n a l w a y s h a v e b a s a l b ody p r o l i f e r a t i o n p r e c e d i n g s u r f a c e g r o w t h . W h i l e i t i s t r u e t h a t t h e t e m p o r a r y c h a n g e s i n t h e s u r f a c e a r e a o f t h e p o l a r r e g i o n s p r o b a b l y r e s u l t f r o m c o n t r a c t i o n s i n t h e s u b - c o r t i c a l f i b r e l a t t i c e , s u r f a c e g r o w t h a p p e a r s t o be i n i t i a t e d b y t h e a d d i t i o n o f new b a s a l b o d i e s t o k i n e t i e s on t h e 121 C E L L D I V I S I O N ; - ( C o n t ' d . ) b) S u r f a c e G r o w t h : - ( C o n t ' d . ) c o r t e x . B a s e d on t h e m o d e l t h a t b a s a l b o d i e s s t i m u l a t e s u r f a c e g r o w t h by o r g a n i z i n g new c o r t i c a l u n i t s , i t i s p o s s i b l e t o p r e d i c t t h e amount o f s u r f a c e a c e l l c o u l d p r o d u c e f r o m a g i v e n number o f b a s a l b o d i e s . F o r e x a m p l e , t h e number o f b a s a l b o d i e s f o u n d i n t h e f u r r o w r e g i o n o f p r e f i s s i o n w i l d -t y p e and m u t a n t c e l l s c a n be u s e d t o p r e d i c t t h e amount o f f u r r o w s u r f a c e t h a t t h e s e c e l l s c o u l d p r o d u c e . T h i s p r e d i c t e d amount c a n t h e n be c o m p a r e d w i t h t h e o b s e r v e d amount o f s u r f a c e g r o w t h i n t h e s e c e l l s , t h e c o m p a r i s o n s e r v i n g a s a s i m p l e t e s t o f t h e m o d e l o f s u r f a c e g r o w t h p r e s e n t e d . The r e s u l t s o f s u c h an a n a l y s i s ( t a b l e 20) n o t o n l y i n d i c a t e t h a t t h e p r e d i c t e d and o b s e r v e d a m ounts o f s u r f a c e g r o w t h a r e v e r y s i m i l a r , b u t t h a t i n some c a s e s t h e c e l l s h a v e t h e c a p a c i t y t o make more s u r f a c e t h a n r e q u i r e d t o c o m p l e t e t h e f i s s i o n f u r r o w . S u r p r i s -i n g l y , i n e a c h s u c h c a s e ( w i l d - t y p e , dc2 , a n d d f z c e l l s ) t h e number o f b a s a l b o d i e s i n t h e f u r r o w r e g i o n was r e d u c e d d u r i n g f u r r o w s u r f a c e g r o w t h . The number o f b a s a l b o d i e s l o s t a t t h i s t i m e c o r r e s p o n d s v e r y c l o s e l y t o t h e number t h a t w e r e 122 C E L L D I V I S I O N ; - ( C o n t ' d . ) b) S u r f a c e G r o w t h : - ( C o n t ' d . ) p r e d i c t e d t o be i n e x c e s s o f t h e number r e q u i r e d t o f o r m a c o m p l e t e f u r r o w ( t a b l e 20). The m o d e l f o r s u r f a c e g r o w t h t h e r e f o r e seems t o be s u p p o r t e d by t h e r e s u l t s o b t a i n e d f r o m m u t a n t a n d w i l d - t y p e c e l l s . The r e s u l t s , h o w e v e r , r a i s e two u n e x p e c t e d s e t s o f p r o b l e m s r e l a t e d t o s u r f a c e g r o w t h a n d c h a l l e n g e t h e v a l i d i t y o f d e t e r m i n i n g s u r f a c e g r o w t h by m e a s u r i n g d i s t a n c e s b e t w e e n CVPs. A l t h o u g h t h e amount o f s u r f a c e g r o w t h a p p e a r s t o be c o n t r o l l e d b y t h e e x t e n t o f b a s a l b o d y p r o l i f e r a t i o n i t i s n o t e v i d e n t what c o n t r o l s t h e e x t e n t o f t h e p r o l i f e r a t i o n i t s e l f . The e x t e n t o f b a s a l b ody p r o l i f e r a t i o n can. a t l e a s t be i n f l u e n c e d b y g e n e s , a s shown by t h e e f f e c t s o f t h e sm2 gene. The n a t u r e o f t h e mech-a n i s m w h i c h c o n t r o l s t h e e x t e n t o f p r o l i f e r a t i o n c a n n o t , h o w e v e r , be d e t e r m i n e d s o l e l y f r o m morpho-m e t r i c d a t a . R e l a t e d t o t h i s p r o b l e m o f t h e c o n t r o l o f b a s a l b o d y p r o l i f e r a t i o n i s t h e q u e s t i o n o f t h e f a t e o f t h e b a s a l b o d i e s w h i c h d i s a p p e a r f r o m t h e f u r r o w r e g i o n d u r i n g f u r r o w s u r f a c e g r o w t h . T h e s e b a s a l b o d i e s c o u l d be r e s o r b e d , a s c l a i m e d by K a n e d a a n d H a n s o n (1974), o r t h e y c o u l d move o u t -s i d e o f t h e r e g i o n b o u n d e d b y t h e two CVPs w h i c h TABLE 20 PREDICTED GROWTH OF THE FURROW SURFACE ON WILD-TYPE AND MUTANT C E L L S #•* + ATTAINABLE LENGTH CONTOUR ' *»» LENGTH OF ++ NO. BASAL +++ NUMBER OF BASAL USING NO. FURROW MAX. BODIES WHICH ACTUAL CHANGE BODIES/CVINT BASAL BODIES AT SURFACE AT D I F F . CORRESPOND I N NUMBER OF AGE 0.95 MAX.* AGE 0.98 AGE.96 MAX. AGE.98 FISSION(um) (um) TO DIFFERENCE BASAL BODIES Wild-Type 49 49 41 73-5 73.5 61.5 63 -10.5 -7 -8 dc4 39 40 39 58.5 60 58.5 58 - 2.0 -1 -1 sm2 25 30 30 37-5 45 45 53 8.0 5 • 0 dc2 a 41 43 • 39 61.5 64.5 58.5 60 " M -3 -4 dfz2 42 42 38 63 63 57 60 - 3-0 -2 -4 The maximum mean number of basal bodies/CVlNT at any c e l l age Attainable lengths were calculated assuming that the available basal bodies would move apart to assume 1.6 um spacing; the values l i s t e d correspond to the number of basal bodies at the indicated ages. + Taken from contour drawings of d i v i d i n g c e l l s (see text) ++ The difference between the maximum attainable length and the mean contour length at f i s s i o n *** Assuming a 1.6 pm spacing +++ The maximum number of basal bodies minus the number at age 0.98-0.99. 124 CELL DIVISION; - (Cont'd.) b) Surface Growth; - (Cont'd.) were used as reference points i n measuring the furrow surface. I f the l a t t e r a l t e r n a t i v e i s true then either the basal bodies migrate past the CVPs or the CVPs migrate towards the furrow region. I f the CVPs do migrate towards the furrow (a conclusion reached by King (1954)) then the use of these structures as reference points i n measuring surface growth becomes unacceptable. It would therefore be preferable i f the other a l t e r n a t i v e , that the basal bodies move past the CVPs, were true. Evidence to support t h i s a l t e r n a t i v e i s tenuous, but the available r e s u l t s give some i n d i r e c t support for t h i s model. I f the basal bodies move past the CVPs into the c e l l mid-regions they would s t i l l contribute to surface growth. The amount of basal body p r o l i f e r a t i o n should therefore control the t o t a l amount of surface growth during c e l l d i v i s i o n and the size of the r e s u l t i n g daughter c e l l s . A di r e c t demonstration of the control of c e l l length by basal body p r o l i f e r a t i o n i s provided by the data on sm2 c e l l s where reduced basal body p r o l i f e r a t i o n r e s u l t s i n reduced c e l l length. A more i n d i r e c t demonstration of thi s r e l a t i o n s h i p i s provided by 125 C E L L D I V I S I O N : - ( C o n t ' d . ) b) S u r f a c e G r o w t h : - ( C o n t ' d . ) t h e o b s e r v a t i o n t h a t t h e l e n g t h o f p a r a m e c i a i s d i r e c t l y r e l a t e d t o t h e c u l t i v a t i o n t e m p e r a t u r e , w i t h h i g h e r t e m p e r a t u r e s p r o d u c i n g l o n g e r c e l l s ( W h i t s o n , 1 9 6 4 ) . I f c e l l l e n g t h i s r e l a t e d t o t e m p e r a t u r e , t h e n b a s a l b o d y p r o l i f e r a t i o n m u s t a l s o be r e l a t e d t o t e m p e r a t u r e . A l t h o u g h no d i r e c t • t e s t o f t h i s r e l a t i o n s h i p was made, i t a p p e a r s t h a t a t 3 4.5°C ( t h e t e m p e r a t u r e u s e d i n t h e c o n t r o l e x p e r i m e n t i n t h i s s t u d y ) w i l d - t y p e c e l l s make a b o u t 1 0 more b a s a l b o d i e s p e r k i n e t y t h a n K a n e d a and H a n s o n ( 1 9 ? 4 ) f o u n d i n w i l d - t y p e c e l l s g r o w n a t room t e m p e r a t u r e . A t e m p e r a t u r e e f f e c t on b a s a l b o d y p r o l i f e r a t i o n t h e r e f o r e seems p l a u s i b l e . I f t h i s w e r e c o n c l u s i v e l y d e m o n s t r a t e d , i t w o u l d c o n s t i t u t e t h e o n l y known e x a m p l e o f t h i s e f f e c t i n a n y o r g a n i s m . The p r o p o s a l t h a t e x c e s s b a s a l b o d i e s m i g r a t e o u t o f t h e f u r r o w r e g i o n t h e r e f o r e h a s c o n s i d e r a b l e i n d i r e c t s u p p o r t a n d i t a p p e a r s t h a t t h e u s e o f t h e CVPs a s r e f e r e n c e p o i n t s f o r c e l l m e a s u r e m e n t s i s v a l i d . R e d u c t i o n i n b a s a l b o d y p r o l i f e r a t i o n a n d s u r f a c e g r o w t h i n sm2 c e l l s d o e s n o t a f f e c t t h e 126 B) C E L L D I V I S I O N : - ( C o n t ' d . ) b ) S u r f a c e G r o w t h : - ( C o n t ' d . ) a b i l i t y o f t h e s e c e l l s t o c o m p l e t e c o n s t r i c t i o n . P r e d i c t i o n s b a s e d on t h e m o d e l t h a t s u r f a c e g r o w t h i s m e d i a t e d by b a s a l b o d y p r o l i f e r a t i o n i n d i c a t e t h a t sm2 c e l l s h a v e 5 f e w e r b a s a l b o d i e s p e r k i n e t y t h a n a r e r e q u i r e d t o p r o d u c e a c o m p l e t e f i s s i o n f u r r o w ( t a b l e 2 0 ) . The a b i l i t y o f sm2 c e l l s t o ' d i v i d e s u g g e s t s t h a t t h e s e c e l l s h a v e a m e c h a n i s m w h i c h c o m p e n s a t e s f o r t h e r e d u c e d s u r f a c e g r o w t h i n t h e f u r r o w r e g i o n . F u r r o w s u r f a c e g r o w t h i n sm2 c e l l s o c c u r s o n l y a n t e r i o r t o t h e f i s s i o n - z o n e w i t h t o t a l f a i l u r e o f s u r f a c e g r o w t h p o s t e r i o r t o t h e f i s s i o n - z o n e . A l t h o u g h f u r r o w , s u r f a c e g r o w t h i s l i m i t e d , i t i s r e d i s t r i b u t e d s u c h t h a t f u r r o w g r o w t h a n t e r i o r t o t h e f i s s i o n - z o n e i s n o r m a l . The f u r r o w s u r f a c e p o s t e r i o r t o t h e f i s s i o n - z o n e i s a p p a r e n t l y s u p p l i e d by g r o w t h o f t h e m i d - r e g i o n o f t h e o p i s t h e t o w a r d s t h e f u r r o w . On c o n t o u r d r a w i n g s o f sm2 c e l l s t h i s g r o w t h i s i n d i c a t e d by movement o f t h e a n t e r i o r CVP o f t h e o p i s t h e t o w a r d s t h e f u r r o w . S u c h a CVP movement was s e e n o n l y i n sm2 c e l l s . The n e c e s s a r y f u r r o w s u r f a c e t o c o m p l e t e f u r r o w c o n s t r i c t i o n i n sm2 c e l l s i s t h e r e f o r e p r o v i d e d b y a r e d i s t r i b u t i o n o f s u r f a c e g r o w t h a n d 127 CELL DIVISION; - (Cont'd.) b) Surface Growth: - (Cont'd.) a contribution to furrow surface growth of c e l l regions d i s t a l to the furrow. R e s t r i c t i o n s on furrow completion are not produced by a lack of basal body p r o l i f e r a t i o n i n the furrow region, suggesting that the inward directed growth of the furrow i s not due to surface growth alone. A l l of the dfz and dc mutants examined have s u f f i c i e n t c o r t i c a l organelles to complete growth of the furrow surface. The defect i n these mutants i s therefore not due to a lack of s u f f i c i e n t organelles to construct the furrow. This reinforces the con-clusions previously made about the origins of the dfz and dc phenotypes. 128 C) CORTICAL PATTERN; The i s o l a t i o n o f a m u t a t i o n (sm2) r e s u l t i n g i n a s i z e r e d u c t i o n i n P a r a m e c i u m h a s a l l o w e d e x a m i n a t i o n o f t h e e f f e c t s o f a s i z e c h a n g e on t h e p o s i t i o n i n g o f c o r t i c a l s t r u c t u r e s i n t h i s o r g a n i s m . I n o t h e r c i l i a t e s t h i s p r o b l e m h a s b e e n f r e q u e n t l y e x a m i n e d b y a l t e r i n g c e l l s i z e b y c u t t i n g away p a r t s o f t h e c e l l . S u c h o p e r a t i o n s on P a r a m e c i u m a r e d i f f i c u l t a s c u t t i n g t h e c e l l s o f t e n p r o v e s f a t a l a n d s i n c e t h e c e l l s h a v e p o o r r e g e n e r a t i v e c a p a b i l i t i e s e v e n i f t h e c u t i s s u c c e s s f u l ( T a r t a r , 195^; C h e n - S h a n , 1969» 1970). The s m a l l m u t a n t s , t h e r e f o r e , p r o v i d e a n i m p r o v e d means f o r r e -d u c i n g t h e s i z e o f p a r a m e c i a a n d h a v e a l l o w e d f o r a c o m p a r i s o n o f t h e r e s u l t s o f s u c h a s i z e c hange w i t h t h e o b s e r v a t i o n s made on o t h e r c i l i a t e s w h ere s i z e h a s b e e n s u r g i c a l l y a l t e r e d . A s t u d y o f sm2 c e l l m o r p h o l o g y h a s shown t h a t t h e p o s i t i o n s o f c e r t a i n c o r t i c a l s t r u c t u r e s c h a n g e w i t h c h a n g e s i n c e l l s i z e i n a m a n n e r a d e q u a t e l y p r e d i c t e d b y t h e a v a i l a b l e m o d e l s f o r c i l i a t e d e v e l o p m e n t . The v e s t i b u l e , h o w e v e r , a p p e a r s t o be p o s i t i o n e d b y mech-a n i s m s t h a t a r e n o t p r e d i c t e d b y t h e a v a i l a b l e m o d e l s . A new m o d e l , m e c h a n i c a l p o s i t i o n i n g , i s t h e r e f o r e p r o -p o s e d t o a c c o u n t f o r v e s t i b u l e p o s i t i o n i n g . T h i s m o d e l c a n be e x t e n d e d t o a c c o u n t f o r o t h e r d e v e l o p m e n t a l 129 CORTICAL PATTERN; - (Cont'd.) processes i n Paramecium. A number of models' have been proposed to account f o r morphogenesis i n the c i l i a t e s . Many of these have been adapted from models o r i g i n a l l y designed to explain development i n m u l t i c e l l u l a r organisms. Frankel (1974) has proposed, for example, that Wolpert's hypothesis of p o s i t i o n a l information (Wolpert, 1969) can be ap-p l i e d to c i l i a t e s . The need to introduce t h i s hypo-thesis arose out of a lack of a suitable framework f o r thinking about a large number of accumulated obser-vations on c i l i a t e development that could not adequately be explained by the previous models of development, notably Sonneborn's cytotaxis'theory (Sonneborn, 1964). Cytotaxis was defined by Sonneborn (1964) as the "ordering and arranging of new c e l l structure under the influence of p r e - e x i s t i n g c e l l structure". While t h i s model can account f o r the growth of k i n e t i e s where developing organelles are i n close proximity to s i m i l a r p r e - e x i s t i n g structures, i t was d i f f i c u l t to apply t h i s model to situations where a new structure formed at a large distance from any s i m i l a r p r e - e x i s t i n g structure. Frankel (1974) has therefore proposed a retention of cytotaxis theory i n a more r e s t r i c t e d sense, renaming i t s t r u c t u r a l guidance, and suggesting 130 C) CORTICAL PATTERN; - (Cont'd.) that s t r u c t u r a l guidance involves only short-range control over the orientation and p o s i t i o n of newly a r i s i n g structures. He suggests that long range control over development of new structures i s more adequately explained by a model based on Wolpert's hypothesis of p o s i t i o n a l information. This model, c a l l e d p o s i t i o n a l c o n t r o l , i s proposed to function through gradient f i e l d s which operate i n accordance with the r u l e s defined i n Wolpert's hypothesis. When applied to c i l i a t e s , the main points of Wolpert's hypothesis are; (a) there are mechanisms involved i n surface patterning that u t i l i z e reference points either i n or on the c e l l , (b) the main type of surface patterning mechanism operating from or between these points i s a graded d i s t r i b u t i o n of some substance or q u a l i t y of the surface structure, (c) the reference points and gradients are regulated or re-established i f they are l o s t or disturbed, (d) regions of the c e l l surface along the gradients can i n t e r p r e t the quan-ry^- t i t a t i v e v a r i a t i o n i n the hypothetrical factors which constitute the gradient as a source of information r e l a t e d to how that part of the c e l l w i l l develop, and (e) t h i s information i s translated l o c a l l y into morpho-genetic events. The novel aspect of t h i s model i s the 131 CORTICAL PATTERN; - (Cont'd.) separation of the information ( p o s i t i o n a l information) for morphogenesis from the v i s i b l e morphogenetic events normally associated with patterning. Unfor-tunately, as Sonneborn (1974a and 1975) has indicated, there i s no evidence from any study of c i l i a t e morpho-genesis to support the existence of t h i s novel aspect of the model. The r e s u l t s on sm2 c e l l s s i m i l a r l y do not provide support f o r the existence of p o s i t i o n a l information i n c i l i a t e s . Deprived of i t s p o s i t i o n a l information aspect, Frankel's p o s i t i o n a l control becomes a model, based on gradient f i e l d s , which i s s t i l l a p p l i -cable and useful i n thinking about c i l i a t e development. A study of sm2 c e l l s indicates that both t h i s model and cytotaxis can be applied to Paramecium development, a) Cytotaxis i n Paramecium; In order to apply the cytotaxis model to some of the observations made on sm2 c e l l s i t i s nec-essary to restate the d e f i n i t i o n of cytotaxis as: the organizing and arranging of new c e l l structures under the influence of the p r e - e x i s t i n g c e l l shape, s i z e , or components. This does not s i g n i f i c a n t l y a l t e r the o r i g i n a l d e f i n i t i o n of the model and i s s i m i l a r to other l i b e r a l i nterpretations of cyto-t a x i s (Grimes, 1976). I t i s also possible to 132 C) CORTICAL PATTERN: - (Cont'd.) a) Cytotaxis i n Paramecium: - (Cont'd.) di s t i n g u i s h two types of c y t o t a c t i c events: short range e f f e c t s and long range e f f e c t s . Cytotactic events operating over a short range, such as the addition of new basal bodies to a kinety, can be referred to by Frankel's term, s t r u c t u r a l guidance. Cytotactic events operating over a long range can be c a l l e d mechanical guidance. Examples of t h i s l a t t e r type of cytotaxis i n Paramecium include the po s i t i o n i n g of the vestibule, the determination of the number of k i n e t i e s on the c e l l , and control of the length of k i n e t i e s . The Paramecium vestibule does not develop i n s i t u at each binary f i s s i o n but i s derived from bi s e c t i o n of the parental vestibule and migration of the r e s u l t i n g parts to a ce n t r a l l o c a t i o n on the ventral surface of the proter and opisthe. The study on wild-type c e l l morphogenesis has shown that two factors contribute to vestibule movement: vestibule migration and surface growth posterior to the d i r e c t i o n of migration. I n i t i a l l y , migration accounts f o r a l l of the movement of the vestibule, but l a t e r , surface growth po s t e r i o r to the migration adds to the apparent vestibule movement. Surface 133 CORTICAL PATTERN; - (Cont'd.) a) Cytotaxis i n Paramecium; - (Cont'd.) growth poste r i o r to the d i r e c t i o n of vest i b u l e movement does not a c t u a l l y contribute to the move-ment, but contributes to vestibule p o s i t i o n i n g by creating new c e l l surface behind or ante r i o r to the v e s t i b u l e . Both t h i s surface growth and the i n i t i a l vestibule migration are therefore necessary fo r proper p o s i t i o n i n g of the vestibule. Vestibule migration begins when the c e l l width decreases during f i s s i o n furrow formation. This r a p i d decrease i n width r e s u l t s i n a narrowing of the c e l l circumference and a decrease i n the spacing of kineties around the c e l l circumference. A decrease i n kinety spacing would be expected to produce tensions i n the cortex that would p u l l the vestibule i n the proter forward and the vestibule i n the opisthe backward. These tensions a r i s e since the kin e t i e s meet the l e f t sides of the a n t e r i o r and posterior sutures at a sharp angle ( f i g . l ) . A decrease i n kinety spacing w i l l therefore shorten the length of the sutures and create vestibule migration i n the required d i r e c t i o n . The extent of vestibule migration i s therefore c o n t r o l l e d by the number of k i n e t i e s on the c e l l (which i s 134 C) CORTICAL PATTERN; - (Cont'd.) a) Cytotaxis i n Paramecium: - (Cont'd.) p o s i t i v e l y correlated with c e l l width) and the amount of width decrease which occurs during c e l l d i v i s i o n . This model fo r v e s t i b u l e migration adequately explains Porter's observation that the number of k i n e t i e s i n the a n t e r i o r suture does not change during vestibule movement i n the proter (Porter, 1962). A change i n the number of k i n e t i e s would not be expected i f the vestibule movement were due to an i n t e r - k i n e t y contraction. The model also predicts and explains the following observations made on mutant and wild-type c e l l s : (a) In mutant c e l l s where a normal width decrease occurs during c e l l d i v i s i o n at the end of the f i r s t c e l l cycle at the r e s t r i c t i v e tempera-ture the vestibule moves forward a constant distance without regard to the t o t a l c e l l length. (The change i n kinety spacing i s dependent on the width of the c e l l but not the length.) (b) The length of the anterior suture i s highly correlated with the number of k i n e t i e s on the c e l l . The suture i s longer (and contains more kineties) on c e l l s with a large number 135 C) CORTICAL PATTERN; - (Cont'd.) a) Cytotaxis i n Paramecium; - (Cont'd.) (b) - (Cont'd.) of k i n e t i e s . Since the number of k i n e t i e s on the c e l l i s correlated with both c e l l length and width, t h i s implies that i n long c e l l s there w i l l be more k i n e t i e s i n the anterior suture, and vest i b u l e movement w i l l be more extensive than i n short c e l l s where there are fewer k i n e t i e s i n the suture. Regulation of vestibule p o s i t i o n i n g when a change i n c e l l length occurs i s therefore mediated through changes i n the number of kin e t i e s on the c e l l . (c) The extent of vestibule migration i s greater i n the proter than i n the opisthe. The anteri o r suture has more ki n e t i e s abutting i t than does the poster i o r suture, and the kin e t i e s meeting the ante r i o r suture are at an angle approaching 90° while those meeting the p o s t e r i o r suture are at a less acute angle. Changes i n kinety spacing w i l l there-fore a f f e c t the length of the anterior, more than the length of the posterior, suture. 136 CORTICAL PATTERN; - (Cont'd.) a) Cytotaxis i n Paramecium: - (Cont'd.) (c) - (Cont'd.)• . Vestibule p o s i t i o n i n g i s a c y t o t a c t i c phenom-enon as the extent of migration i s determined by the c e l l width and the number of kineties on the c e l l (pre-existing form and s t r u c t u r e s ) . Vestibule p o s i t i o n i n g i s an example of mechan-i c a l guidance since the mechanism responsible for vestibule migration operates over a large distance on the cortex. Two other examples of mechanical p o s i t i o n i n g are the control of kinety number and control of the number of basal bodies within each kinety. Kinety number i s d i r e c t l y r e l a t e d to c e l l width i n sm2 c e l l s . During a temperature-induced width reduction, sm2 c e l l s lose k i n e t i e s such that the number of k i n e t i e s per unit of c e l l width (or circumference) remains constant. These kineties are probably l o s t by the mechan-ism proposed by Heckmann and Frankel (1968) to explain loss of kineties i n Euplotes. In t h i s model, kineties which do not reach the poles of the c e l l , but which terminate somewhere i n 137 CORTICAL PATTERN; - (Cont'd.) a) Cytotaxis i n Paramecium; - (Cont'd.) (c) - (Cont'd.) the c e n t r a l portion of the cortex, are even-t u a l l y l o s t during c e l l d i v i s i o n when either the proter or opisthe receives no c o r t i c a l organelles from t h i s incomplete kinety ( f i g . 5 8 ) . On the Paramecium cortex i t i s not possible for a l l of the somatic k i n e t i e s to terminate at the c e l l poles or along the two suture l i n e s . Kineties are frequently seen that terminate at other points on the cortex presumably due to overcrowding of k i n e t i e s at the c e l l poles. One might therefore expect there to be a tendency f o r paramecia to lose k i n e t i e s during c e l l d i v i s i o n . To counteract t h i s e f f e c t the c e l l s must have a mechanism for increasing the number of k i n e t i e s . The c o r r e l a t i o n between kinety numbers and c e l l width i n sm2 c e l l s suggests that when the inte r k i n e t y spacing exceeds a c e r t a i n value, a new kinety w i l l form on the c e l l surface by i n s e r t i o n between e x i s t i n g kineties ( f i g . 5 8 ) . The width of the c e l l would therefore deter-mine whether new kineties would form, q u a l i f y i n g 138 C) CORTICAL PATTERN: - (Cont'd.) a) Cytotaxis i n Paramecium: - (Cont'd.) (c) - (Cont'd.) control over kinety numbers as a c y t o t a c t i c phenomenon. The precise mechanism by which new ki n e t i e s are added to the cortex, however, has not been determined. The amount of basal body p r o l i f e r a t i o n during c e l l d i v i s i o n i n sm2 c e l l s appears to influence the c e l l length, vestibule length, and g u l l e t length by inf l u e n c i n g the length of the cor- -t i c a l k i n e t i e s . This suggests that paramecia can control t h e i r size by c o n t r o l l i n g the amount of basal body p r o l i f e r a t i o n occurring at c e l l d i v i s i o n . Regulation of c e l l s i z e must be associated with regulation of basal body p r o l i f e r a t i o n . When abnormally large c e l l s return to normal s i z e , they must do so by l i m i t i n g the amount of basal body pro-l i f e r a t i o n . When abnormally small c e l l s return to normal s i z e , they must increase the amount of p r o l i f e r a t i o n . The extent of pro-l i f e r a t i o n must be r e l a t e d to c e l l s i z e ( e x i s t i n g size) and would constitute a 139 CORTICAL PATTERN: - (Cont'd.) a) Cytotaxis i n Paramecium: - (Cont'd.) (c) - (Cont'd.) c y t o t a c t i c phenomenon. The three examples of mechanical guidance that have been presented constitute a new class of c y t o t a c t i c events. They either operate over large distances or i n ways not predicted by Frankel's model of s t r u c t u r a l guidance. P o s i t i o n a l mech-anisms operating by mechanical guidance are also not d i r e c t l y under genie control. Genes may i n f l u -ence these developmental mechanisms only by l i m i t i n g the a v a i l a b i l i t y of necessary proteins or p o s s i b l y by a l t e r i n g the rate constants of the biochemical reactions involved i n the mechanisms. Ph y s i o l o g i c a l or environmental factors may s i m i l a r l y a f f e c t these mechanisms by a f f e c t i n g the a v a i l a b i l i t y of materials necessary f o r the normal functioning of the mechanisms. The basic processes by which mechanical p o s i t i o n i n g occurs, however, are i n -dependent of d i r e c t gene control, b) P o s i t i o n a l Control i n Paramecium: The mechanisms for the p o s i t i o n i n g of c e r t a i n structures i n Paramecium (the CVPs and the cytoproct) 140 CORTICAL PATTERN: - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium: - (Cont'd.) remain unknown. The study of these mechanisms i s therefore l i m i t e d to a desc r i p t i o n of the topo-graphical rules by which these mechanisms operate. These rules are discussed as examples of p o s i t i o n a l control only because t h i s i s presently the model which best explains the observations made on the pos i t i o n i n g of ce r t a i n structures. These obser-vations should not be interpreted, however, as proof of the existence of p o s i t i o n a l information i n c i l i a t e s . What the observations do indicate i s that the size or s i t e of formation of c o r t i c a l structures can be interpreted as being due to a morphogenetic f i e l d or gradient as defined by Wolpert (1969). This i n t e r p r e t a t i o n of the r e s u l t s r e l i e s on the demonstration that the s i t e s of organellogenesis are determined with reference to the size of the c e l l . In Paramecium the an t e r i o r CVP forms at a distance from the anter i o r end of the c e l l that i s a fixed f r a c t i o n of the c e l l length. The poster i o r CVP, however, i s located at a f i x e d distance from the posterior end of the c e l l , regardless of c e l l length. The anter i o r CVP, but not the posterior CVP, i s therefore positioned by 141 CORTICAL PATTERN; - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium; - (Cont'd.) the r u l e s governing Wolpert's hypothetical morpho-genetic f i e l d s . Another structure which conforms to these rules i s the cytoproct, the length of which i s a constant f r a c t i o n of the length of the p o s t e r i o r suture i n which i t i s located. The anterior CVP i n paramecia not only main-tains a r e l a t i v e p o s i t i o n along the polar axis of the c e l l , but also maintains a r e l a t i v e p o s i t i o n on the c e l l circumference, being located approximately opposite the mouth regardless of the number of k i n e t i e s on the c e l l . There are therefore at l e a s t two reference points f o r CVP positioning; the anterior end of the c e l l and the mouth. Whether gradients or f i e l d s operate from or between these reference points cannot be determined from the morphometric data. The data indicate only the pos-s i b i l i t y that such gradients may occur. S i m i l a r gradients, with the o r a l apparatus as one of the gradient reference points, have been proposed f o r the control of CVP p o s i t i o n i n g i n Tetrahymena (Nanny, 1972) and C h i l o d i n e l l a (Kaczanowska, 1974; 1975). The p o s i t i o n of the posterior CVP on the 142 CORTICAL PATTERN: - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium: - (Cont'd.) Paramecium c e l l i s not regulated according to the p o s i t i o n a l control model since t h i s CVP does not form de novo during c e l l d i v i s i o n but i s i n h e r i t e d i n s i t u from the parent c e l l . Any CVP which forms on a c e l l w i l l , within two c e l l s generations, become the p o s t e r i o r CVP of an opisthe l i n e of • descent (fig.59). In sm2 c e l l s the distance of t h i s CVP from the p o s t e r i o r of the c e l l i s constant, regardless of c e l l s i z e . This means that once a CVP forms on the cortex (by p o s i t i o n a l control) i t s p o s i t i o n i n the cortex i s f i x e d and i s no longer subject to regulation. This i s possibly due to the lack of basal body p r o l i f e r a t i o n and surface growth poste r i o r to t h i s CVP during c e l l d i v i s i o n . Small c e l l s w i l l i n h e r i t t h i s c o r t i c a l region i n -tact and could only reduce the distance from the p o s t e r i o r CVP to the p o s t e r i o r of the c e l l by resorbing c o r t i c a l units between these two points. Evidently, the c e l l s are not capable of doing t h i s . During a s i z e reduction i n sm2 c e l l s the length of the cytoproct remains a constant f r a c t i o n of the length of the posterior suture. I t i s d i f f i c u l t , however, to define the reference points f o r any 143 CORTICAL PATTERN: - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium: - (Cont'd.) gradient that would determine the cytoproct length. The cytoproct forms during c e l l d i v i s i o n p r i o r to the separation of the daughter c e l l s . Growth of the posterior suture i s not yet complete when the cytoproct forms. The hypothetical gradient con-t r o l l i n g cytoproct length therefore cannot use the length of t h i s suture as a reference f o r determining the cytoproct length. Although a gradient-based mechanism f o r control of cytoproct length cannot be ruled out, i t i s d i f f i c u l t to imagine how such a gradient would operate. During c e l l d i v i s i o n the opisthe retains the parental cytoproct. During a siz e reduction i n sm2 c e l l s one would therefore expect to see two kinds of cytoprocts; the long parental cytoprocts and shorter newly-formed cytoprocts. No such bimodality i n cytoproct length was found, suggesting that there i s a mechanism f o r reducing the length of i n h e r i t e d cytoprocts when c e l l s i z e i s decreased. Ng (1976) has reported a s i m i l a r observation on paramecia and also found that the cytoproct i n the opisthe occa-s i o n a l l y disappears and reappears during c e l l d i v i -sion although he does not indicate how frequently 144 CORTICAL PATTERN; - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium; - (Cont'd.) t h i s happens. I f the parental cytoproct i s re-structured during c e l l d i v i s i o n the c e l l could presumably control the length of the newly forming cytoproct. The preceding account has demonstrated that the s i t e s of formation of new CVPs and the length of the cytoproct are determined with reference to the length of the c e l l . The topographical r e l a t i o n -ship between c e l l length and s i t e s of CVP formation described above are therefore s i m i l a r to those reported for Tetrahymena (Nanney, 1972) and C h i l o d i n e l l a (Kaczanowska, 1974). Whether or not these topographical r e l a t i o n s h i p s are determined by a gradient system, however, i s not a question that can be answered by morphometric data alone. Re-gardless of t h i s , the p r e v a i l i n g opinion expressed i n the l i t e r a t u r e i s that these types of topo-graphical relationships are based on gradient sys-tems (Frankel, 1974; Jerka-Dziadosz, 1974; Kaczanowska, 1974; Lynn and Tucker, 1976; Sonneborn, 1975). Alternately, structures which form on the cortex at locations that are independent of c e l l s i z e are thought to be positioned by mechanisms 145 CORTICAL PATTERN; - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium: - (Cont'd.) that are not based on gradients. These mechanisms include cytotaxis (Sonneborn, 1 9 6 4 ) , p o s i t i o n a l control (Frankel, 1 9 7 4 ) , and s t r u c t u r a l p o s i t i o n i n g (Lynn and Tucker, 1 9 7 6 ) . There i s t h e o r e t i c a l l y no reason, however, why c y t o t a c t i c mechanisms can-not account f o r size-dependent p o s i t i o n i n g of new c o r t i c a l structures. The vestibule i n Paramecium appears to be positioned by a c y t o t a c t i c mechanism, and yet i t i s not located at a f i x e d distance from any reference point on or i n the c e l l . I t s p o s i t i o n remains i n the approximate center of the v e n t r a l surface regardless of the c e l l length. I t i s there-fore not e s s e n t i a l that a l l size-dependent mechan-isms fo r p o s i t i o n i n g c o r t i c a l structures be based on gradient systems. The main objection to a gradient-based mechan-ism f o r p o s i t i o n i n g the CVPs or determining the length of the cytoproct i n paramecia i s that these structures form before the s i z e of the daughter c e l l s i s determined. The CVPs, i n f a c t , form p r i o r to any surface growth i n the d i v i d i n g c e l l . This objection to the gradient model would not be a serious one i f surface growth were uniform throughout 146 CORTICAL PATTERN: - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium: - (Cont'd.) the length of the c e l l . As shown "by the morpho-metric study, however, surface growth i s decidedly non-uniform. I f gradients account f o r the s i z e -dependent p o s i t i o n i n g of c o r t i c a l structures, they must therefore be able to "predict" how much and where surface growth w i l l occur during c e l l d i v i s i o n . To i l l u s t r a t e t h i s point, consider the l o c a t i o n of the CVPs on the dorsal cortex i n terms of t h e i r r e l a t i v e p o s i t i o n i n regard to the anterior end of the c e l l . In the i n t e r f i s s i o n c e l l the two CVPs are located at positions which are 35 and 80$ of the c e l l length from the a n t e r i o r end of the c e l l ( f i g . 60). Two new CVPs form during c e l l d i v i s i o n which w i l l become the anterior CVPs of the proter and opisthe and they should therefore be located at 35$ of the c e l l length from the anterior of the proter and opisthe. They do not form at t h i s s i t e , how-ever, and only reach i t just p r i o r to the completion of cytokinesis (fig .60). Although a gradient-based mechanism f o r CVP formation cannot be excluded, i t i s also possible that the c e l l uses c y t o t a c t i c mechanisms to deter-mine the s i t e s of CVP formation. As previously 147 CORTICAL PATTERN; - (Cont'd.) b) Positional Control i n Paramecium; - (Cont'd.) shown, any CVP which forms on a c e l l w i l l become the posterior CVP of an opisthe within two c e l l cycles. Once i t has reached t h i s p o s i t i o n i t re-mains as the p o s t e r i o r CVP of an opisthe throughout a l l future generations of the clone. The p o s t e r i o r CVP of the opisthe i s located i n a d i s t i n c t part of the cortex which has the following features; (a) during c e l l d i v i s i o n , there i s no basal body pro-l i f e r a t i o n p o s t e r i o r to the CVP i n t h i s region but .there i s extensive p r o l i f e r a t i o n anterior to i t and (b) the c e l l surface p o s t e r i o r to the CVP i n t h i s region contracts p r i o r to c e l l d i v i s i o n while the surface anterior to i t does not. The p o s t e r i o r CVP of the opisthe i s therefore located near to or i n a boundary zone between regions of the cortex that have s i g n i f i c a n t l y d i f f e r e n t properties. I t i s possible that these properties of the cortex, as well as the p o s i t i o n of the CVP, are correlated with some yet unknown s t r u c t u r a l features of the cortex. Regional s p e c i a l i z a t i o n s of the Paramecium cortex have been described by Naitoh and Eckert (1969)» and i t i s possible that the CVPs can form only i n c e r t a i n parts of the cortex. The regions 148 CORTICAL PATTERN: - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium: - (Cont'd.) of the cortex i n which the CVPs form may be deter-mined long before the CVPs appear i n them. The o r i g i n a l positions of these regions would therefore be d i f f i c u l t to predict by examining the locations where CVPs form. Although the discussion of these s p e c i a l c o r t i c a l regions i s highly-speculative, i t • has been introduced to show that an apparently simple topographical r e l a t i o n s h i p between the loca-t i o n of a structure on the c e l l surface and the length of the c e l l may turn out to be a complex re l a t i o n s h i p not r e a d i l y explainable by any simple model of c i l i a t e morphogenesis. Regardless of whether mechanical guidance (or other c y t o t a c t i c mechanisms) proves to account for CVP p o s i t i o n i n g or the control of cytoproct length, t h i s model does provide an a l t e r n a t i v e to gradient-based models of c i l i a t e morphogenesis and therefore removes the bias imposed by having only one type of model to apply to size-dependent p o s i t i o n i n g of c o r t i c a l structures. The adherence, i n the present study, to mechanical guidance and other c y t o t a c t i c models should not be interpreted as i n d i c a t i n g that gradient systems i n c i l i a t e s are not possible. 149 C) CORTICAL PATTERN: - (Cont'd.) b) P o s i t i o n a l Control i n Paramecium: - (Cont'd.) V i s i b l e gradients of c e l l structure are common i n c i l i a t e s : f o r example, the spacing of k i n e t i e s around the Paramecium c e l l circumference shows a gradation from narrow to wide (Sonneborn, 1975). Chemical gradients, although they have never been demonstrated i n c i l i a t e s , could e x i s t i f they were r e s t r i c t e d to the c o r t i c a l layer where they would not be disturbed by cytoplasmic flow. A l t e r -n a t i v e l y , the gradients could be " b u i l t i n " to the cortex by gradations i n molecular structure as proposed by Sonneborn ( 1 9 7 5 ) and Roth et a l . ( 1 9 7 7 ) . 150 SUMMARY Ten mutants a f f e c t i n g morphogenesis of Paramecium  t e t r a u r e l i a have been described i n t h i s study. The pheno-type of each of the mutants was shown to be due to a mutated single gene. Since two of the mutants were a l l e l i c , nine genes a f f e c t i n g morphogenesis i n Paramecium have been studied. Of these, two a f f e c t formation of the f i s s i o n zone ( c a l l e d defective f i s s i o n zone or dfz mutants), f i v e a f f e c t c o n s t r i c t i o n of the f i s s i o n furrow ( c a l l e d defective c o n s t r i c t i o n or dc mutants), and two produce a reduction i n c e l l s i z e ( c a l l e d small or sm mutants). To analyse the origins of the d i v i s i o n - a r r e s t phenotypes of the mutants, wild-type c e l l s were examined to determine how they changed, i n length and width p r i o r to and during c e l l d i v i s i o n . I t was found that the c e l l s f i r s t increased i n length and then contracted p r i o r to forming a f i s s i o n furrow. Once the c e l l s contracted, they assumed a shape roughly equivalent to a cylinder with e l l i p s o i d a l ends. Furrowing, which began when the c e l l s reached t h i s c y l i n d r i c a l shape, was accom-plished by a rapid increase i n the c e l l length and a rapid decrease i n the c e l l width. In the dfz mutant examined (dfz2) there was a premature c e l l contraction which produced a rounding of the c e l l outline. These rounded c e l l s did not form eith e r a f i s s i o n zone or furrow. I t was suggested that 1 5 1 the abnormal shape of these' c e l l s prevents f i s s i o n furrow formation and that the attainment of the normal c y l i n d r i c a l p r e - d i v i s i o n shape i s e s s e n t i a l for furrowing. The two defective c o n s t r i c t i o n mutants examined (dc2 a and dc4) contract at the usual time i n the c e l l cycle, but t h i s contraction i s excessive and prolonged. The enhancement of the contraction was possibly due to the presence of c o r t i c a l lesions on the dc c e l l s that would permit the entry of calcium ions which stimulate contraction i n Paramecium. The prolonged contraction of the dc c e l l s probably i n t e r -feres with cytokinesis by preventing the width of the c e l l from decreasing during c e l l d i v i s i o n . The width decrease i s necessary to reduce the diameter of the c e l l and hence, the amount of surface needed f o r furrow completion. Although the dc mutants produce a normal amount of furrow surface, without the normal width reduction the furrow cannot complete c o n s t r i c t i o n of the c e l l . In the small mutant examined (sm2), there was a s i g n i f i c a n t reduction i n surface growth during c e l l d i v i s i o n . The extent of c i l i a r y basal body r e p l i c a t i o n was also reduced and i t was suggested that a causal r e l a t i o n existed between basal body r e p l i c a t i o n and surface growth. This r e l a t i o n s h i p was tested i n other mutants (dc4, dc2 a, and dfz2) and was found 152 to hold true i n a l l of them. C o r t i c a l pattern was examined by creating small c e l l s using the sm2 mutation (or mutant s t r a i n ) . This allowed fo r an analysis of the positions of c o r t i c a l structures i n r e l a t i o n to the s i z e of the c e l l on which they were found. This study revealed that the p o s i t i o n i n g or number of certai n c o r t i c a l structures were determined by the pre-ex i s t i n g shape, s i z e , or structure of the c e l l . A morpho-genetical model c a l l e d mechanical guidance was proposed to account f o r the po s i t i o n i n g or r e p l i c a t i o n of these structures (including the vestibules, k i n e t i e s , and basal bodies). The study also described the topographical r e l a t i o n s h i p between c o n t r a c t i l e vacuole positions and c e l l s ize and the r e l a t i o n between cytoproct length and c e l l s i z e . I t seemed u n l i k e l y that these topographical r e l a t i o n s were due to gradient-based developmental mechanisms, a l -though such mechanisms could not be t o t a l l y excluded. The study also showed that the locations of c e r t a i n structures, such as the CVPs and the vestibules were determined by the c e l l with reference to the c e l l length. 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Berger (1976) Mutational blockage o f DNA synthesis i n Paramecium t e t r a u r e l i a . Canadian Journal of Zoology j>4: 2089-2097. Pollock, S.L. ( 1 9 7 4 ) Mutations a f f e c t i n g the trichocysts i n Paramecium a u r e l i a . I. Morphology and description of the mutants. Journal of Protozoology 21: 352-362. Porter, E.D. (1962) A theory of morphogenetic migration i n Paramecium a u r e l i a . Journal of Protozoology 2 : 27a. Rasmussen, L. (1967) E f f e c t s of metabolic i n h i b i t o r s on Paramecium a u r e l i a during the c e l l cycle. Experimental C e l l Research 4 8 : 132-139. Roque, M.' (1956) L'evolution de l a c i l i a t u r e buccale pendant l'autogamie et l a conjugaison chez Paramecium a u r e l i a . Comptes Rendus des Seances de 1'Academic des Sciences 242: 2592-2595. Roth, L.E. and D.J. P i h l a j a (1976) Gradionation: hypothesis f o r p o s i t i o n i n g and patterning. Journal of Protozoology 2 4 : 2-9. Sawai, T. (1976) Movement of the c e l l surface and change i n surface area during cleavage i n the newt's egg. Journal of C e l l Science 21: 537-551. Sl a t e r , M. and M. Schaechter ( 1 9 7 4 ) Control of c e l l d i v i s i o n i n bacteria. B a c t e r i o l o g i c a l Reviews _3_8: 199-221. Sokal, R.R. and F.J. Rohlf (1969) Biometry. W.H. Freeman and Co. San Francisco. Sonneborn, T.M. (1950) Methods i n the general biology and genetics of Paramecium a u r e l i a . Journal of Experimental Zoology 113: 87 - 1 4 8 . Sonneborn, T.M. (1964) The d i f f e r e n t i a t i o n of c e l l s . Proceedings of the National Academy of Science £ L : 915-929. • Sonneborn, T.M. (1970) Methods i n Paramecium research. In: Methods i n C e l l Physiology; v o l . 4. ed. D.M. Prescott pp. 182-203. 158 Sonneborn, T.M. (1974a) C i l i a t e morphogenesis and i t s bearing on general c e l l u l a r morphogenesis. A c t u a l i t e s Protozoologiques (proceedings of the fourth Inter-national Conference on Protozoology, Clermont-Ferrand) 1: 327 -355-Sonneborn, T.M. (1974b) Genetics of the 14 species of Paramecium a u r e l i a . In: Handbook of Genetics, v o l . II,. ed. R.C. King. Plenum Press, New York, N.Y. Sonneborn, T.M. (1975) P o s i t i o n a l information and nearest neighbour in t e r a c t i o n s i n r e l a t i o n to s p a t i a l patterns i n c i l i a t e s . L'Anne Biologique 14: 5 6 5 - 5 8 4 . Suhama, M. and E.D. Hanson (1971) The r o l e of protein synthesis i n p r e f i s s i o n morphogenesis of Paramecium  a u r e l i a . Journal of Experimental Zoology 177: 4 6 3 -478: Sundararaman, V. and E.D. Hanson (1976) Longitudinal micro-tubules and t h e i r functions during asexual reproduction i n Paramecium t e t r a u r e l i a . Genetical Research 27_: 205-211. Tartar, V. (1954) Anomalies i n the regeneration of Paramecium  a u r e l i a . Journal of Protozoology 1: 11-17. Whitson, G.L. (1964) Temperature s e n s i t i v i t y and i t s r e l a t i o n to changes i n growth, control of c e l l d i v i s i o n , and s t a b i l i t y of morphogenesis i n Paramecium a u r e l i a , syngen 4 , stock 51« Journal of c e l l u l a r and Comparative Physiology 64: 4 5 5 - 4 6 5 . Whittle, J.R.S. and L. Chen-Shan (1972) C o r t i c a l morphogenesis i n Paramecium aurel i a : mutants a f f e c t i n g c e l l shape. Genetical Research 1 2 : 271 -279-Wolpert, L. (1969) P o s i t i o n a l information and the s p a t i a l pattern of c e l l u l a r d i f f e r e n t i a t i o n . Journal of Theoretical Biology 2$: 1 - 4 7 . Zeuthen, E. and L. Rasmussen (1971) Synchronized c e l l d i v i s i o n i n Protozoa. In: Research i n Protozoology; v o l . 4. ed. T.T. Chen. Oxford, Pergamon Press. Figure 1. A schematic diagram of the k i n e t i e s on the dorsal and v e n t r a l surfaces of an i n t e r f i s s i o n Paramecium c e l l . The kineties are represented by s o l i d l i n e s . The pattern of c o r t i c a l organ-e l l e s within the kineties i s shown i n the lower figure. anterior right field posterior right 'field anterior left field vestibule posterior left field cytoproct VENTRAL contractile vacuole pores DORSAL para soma I sac basal body trichocyst Figure 2. Schematic diagram of a portion of the Paramecium cortex showing the arrangement of various c o r t i c a l structures (based on published observations of other authors). Abbreviations used ares amr, anterior microtubular ribbon; axp, a x i a l plate; bb, basal body; bp, basal plate; c, cilium; e l , epiplasmic layer; f i b , f i b r i l s ; iam, inner alveolar membrane; kf, kinetodesmal f i b e r ; oam, outer alveolar mem-brane; ps, parasomal sac; pso, parasomal sac opening; pmr, posterior microtubular ribbon; tmr, transverse microtubular ribbon; t r , t r i c h o -cyst. Figure Structure of the Paramecium g u l l e t . The s o l i d and dashed l i n e s indicate the paths of the g u l l e t kineties. 164 Figure 4 . A schematic diagram of o r a l anlage develop-ment during binary f i s s i o n based on the published observations of Kaneda and Hanson ( 1974) and Jones ( 1 9 7 6 ) . C e l l ages are given as fr a c t i o n s of the c e l l cycle length. Between ages 0 . 2 and 0 . 6 there i s a p r o l i f e r a t i o n of basal bodies i n the endoral membrane (the row of large dots on the r i g h t v e s t i b u l a r wall). At age 0 . 7 5 the anlage appears just above the endoral membrane and by age O .85 the anlage organizes into three ki n e t i e s while the endoral membrane vanishes. By age O .87 the anlage has invaginated and consists of s i x kin e t i e s . At t h i s time, two add i t i o n a l anlagen appear (the i r r e g u l a r l y spaced dots i n the f i g u r e ) , one adjacent to the large, develop-ing anlage, and one on the r i g h t v e s t i b u l a r wall. These two anlagen develop to the three kinety stage and then sink below the c e l l surface (age 0 - 9 8 ) and w i l l l a t e r reappear during the sub-sequent c e l l cycle at age 0 . 7 5 * At age 0 . 9 the large anlage has developed to the 12 kinety stage and moved p o s t e r i o r l y to a p o s i t i o n beneath the opisthe's vestibule. The c i r c l e d numerals indicate the stages of gu l l e t development given by Kaneda and Hanson ( 1 9 7 4 ) . Figure 5« Camera l u c i d a drawings of c o n t r a c t i l e vacuole pore abnormalities i n ts-0 c e l l s . The shaded areas are presumeably abnormal c o n t r a c t i l e vacuole pores. The kineties ( s o l i d l i n e s ) follow abnormal paths around these structures. The po s i t i o n of the g u l l e t i s indicated by s o l i d l i n e s . The v e r t i c a l bar indicates 15 pm. Figure 6 . Measurements made on c e l l s of known age. A- Measurements made on c e l l s with two contrac-t i l e vacuole pores (CVPs). B- Measurements made on c e l l s with four CVPs and no fission-furrow. C- Measurements made on c e l l s which have a fission-furrow. ^ O G P > O G I O G A I P G P G P -Figure 7 . Sample s t a t i s t i c s of c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars indicate 95% confidence i n t e r -vals . A- Sample s t a t i s t i c s : length of wild type (o) and sm2 c e l l s (©). B- Sample s t a t i s t i c s : length of the anterior suture (GA) of wild type (o) and sm2 (•) c e l l s ; vestibule length (G) of wild type (o) and sm2 (©) c e l l s . 172 —i i 1 1 r— •8 -85 .9 .95 t o AGE (cell cycles) — i 1 1 1 1 1 — •75 - 8 -85 -9 -95 1 0 A G E (cell c yc l es ) F i g u r e 8 . Sample s t a t i s t i c s of c e l l s o f known age a f t e r a s h i f t t o the r e s t r i c t i v e temperature.. The v e r t i c a l bars are 95f° c o n f i d e n c e i n t e r v a l s of the mean. A- The l e n g t h o f the p o s t e r i o r suture (GP) of w i l d type (o) and sm2 (©) c e l l s . B- The width o f w i l d type (o) and sm2 (©) c e l l s . I 1 1 1 1 1— •75 -8 -85 • 9 -95 1-0 A G E ( eel I cycles) ^75 ^ li5 ^ 35 V'o AGE (cell cycles) Figure 9« Sample s t a t i s t i c s of c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95% confidence i n t e r v a l s the mean. A- CVA (see f i g . 6 ) of wild-type (o) and sm2 (o) c e l l s . B- CVP (see f i g . 6 ) of wild-type (o) and sm2 (©) c e l l s . 176 —i 1 1 1 1 1 — •75 -8 -85 -9 -95 1-0 A G E ( eel I eye les) Figure 1 0 . Sample s t a t i s t i c s of c e l l s of known age aft e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 9 5 $ confidence i n t e r v a l s the mean. A- CVA' (see f i g . 6 ) of wild-type (o) and sm2 (•) c e l l s . B- CVP' (see f i g . 6) of wild-type (o) and sm2 (©) c e l l s . Figure 1 1 . Sample s t a t i s t i c s of c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95% confidence i n t e r v a l s the mean. A- The length-to-width r a t i o of wild-type (o) and sm2 (e) c e l l s . B- CVINT of wild-type (o) and sm2 (•) c e l l s . 180 Figure 12. Sample s t a t i s t i c s of c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A- The number of basal bodies between the two innermost CVPs (see f i g . 6) of wild-type (o) and sm2 (©) c e l l s . B- The spacing between basal bodies on wild-type (o) and sm2 (e) c e l l s . Figure 13. Outline drawings of wild-type ( l e f t ) and sm2 (right) d i v i d i n g c e l l s of ages 0.94 (a), 0.95 (b), 0.96-0.97 (c), and 0.98-0.99 (d). The open c i r c l e s indicate the positions of the CVP's The scallops on the l e f t of each diagram i n d i c a t the p o s i t i o n and siz e of the vestibule. Figure 14. Growth of various regions on the dorsal surface of d i v i d i n g c e l l s of ages 0.93 to 0.99-To produce these p l o t s , the contour lengths of c e l l regions at the indicated ages were compared to the contour length of that region at age 0.93 and the " r e l a t i v e growth" r a t i o was calculated. The graphs show the amount of increase or decrease i n contour length as compared to the length at age 0.93' The contour lengths indicated are: (©) - CVA (+) - CVA' (o) - The distance from the fission-furrow to the p o s t e r i o r CVP of the proter. (o) - The distance from the fission-furrow to the anterior CVP of the opisthe. (A) - CVP' (v) - CVP figure A -figure B -wild-type c e l l s . sm2 c e l l s . 201 I 1 1 T-•93 - 9 5 - 9 7 - 9 9 A G E (cell eye les ) Figure 1 5 « Basal body p r o l i f e r a t i o n i n sm2 c e l l s during the f i r s t c e l l cycle at the r e s t r i c t i v e temperature. The figures show the positions of basal bodies and parasomal sacs i n k i n e t i e s i n the midregion (CVINT) of the dorsal surface. The ages of the c e l l s ( i n c e l l cycles) are given below each figure. Large dark spots are CVPs. The extent of basal body p r o l i f e r a t i o n can be compared with that of the wild-type c e l l s of s i m i l a r ages shown i n plate 9« Abnormalities i n sm2 c e l l s include reduced p r o l i f e r a t i o n , c o r t i c a l units lacking e i t h e r a basal body or parasomal sac, c o r t i c a l units with two parasomal sacs, and disorganization of the k i n e t i e s i n the furrow region. The v e r t i c a l bar represents 20 ;um. 188 Figure 16. Abnormalities i n g u l l e t anlage development i n sm2 c e l l s during the f i r s t c e l l cycle at the r e s t r i c t i v e temperature. a - A c e l l of about age 0.85 with a normal anlage containing three k i n e t i e s (the s o l i d l i n e s ) . b - A c e l l of about age 0.87 with an anlage con-t a i n i n g three k i n e t i e s . The anterior end of the anlage has moved p o s t e r i o r l y while the posterior end has not, r e s u l t i n g i n an i n -verted "C"-shaped anlage. c - An anlage from a c e l l of about age 0.9. There are three wide s t r i a t i o n s i n the anlage which may comprise s i x k i n e t i e s . Dark granular material l i e s between the anterior of the anlage and the vestibule wall. d, e, f - Anlagen from c e l l s of age 0.92. In each case no kineties are v i s i b l e i n the anlage. In figure f, an anlage and endoral kinety have appeared on the r i g h t v e s t i b u l a r wall. In figure d, only the endoral kinety can be seen and i n figure e the endoral kinety i s very i r r e g u l a r . g - An anlage from a c e l l of age 0.93« The anlage i s exceedingly small and contains no k i n e t i e s . Figure 16 (con't) h, i , j - Anlagen from c e l l s of about age 0 .95* In figures h and i there are no k i n e t i e s i n the anlagen. In figure j there are f i v e k i n e t i e s . In each case the endoral kinety can be found along the r i g h t v e s t i b u l a r wall and there i s i r r e g u l a r , grainy material between the poste r i o r of the anlage and the vestibule. 1 9 1 Figure 1 7 - Sample s t a t i s t i c s of c e l l s of known ages af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A - The length of wild-type (o) and dc4 (©) c e l l s . B - The length of the anterior suture (GA) and vestibule (G) of wild-type (o) and dck (©) c e l l s . ~—T- I 1 1 p-•75 .8 .85 .9 .95 A G E ( ce l l cyc les) — i 1 — 1 1 — r -•75 8 -85 -9 .95 A G E (cell cyc les ) > Figure 18. Sample s t a t i s t i c s of c e l l s of known age a f t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 9 5 $ confidence i n t e r v a l s of the mean. A - The length of the posterior suture (GP) of wild-type (o) and dc4 (©) c e l l s . B - The width of wild-type (o) and dc4 (©) c e l l s . Figure 1 9 « Sample s t a t i s t i c s of c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 9 5 $ confidence i n t e r v a l s of the mean. A - CVA (see f i g . 6) of wild-type (o) and dc4 (©) c e l l s . B - CVP (see f i g . 6) of wild-type (o) and dc4 (•) c e l l s . 10 A 0 J , 1 1 1— 1 r— . 7 5 - 8 - 8 5 -9 - 9 5 1 0 A G E ( ce 11 eye les ) Figure 20. Sample s t a t i s t i c s of c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A - CVA' (see f i g . 6) of wild-type (o) and dc4 (•) c e l l s . B - CVP' (see f i g . 6) of wild-type (o) and dc4 («) c e l l s . Figure 21. Sample s t a t i s t i c s of c e l l s of known age a f t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95% confidence i n t e r v a l s of the mean. A - The length-to-width r a t i o of wild-type (o) and dc4 (•) c e l l s . B - CVINT of wild-type (o) and dck («) c e l l s . 6 C H 204 I 1 1 I 1 1 -• 75 -8 -85 -9 -95 1-0 A G E ( c e l l c y c l e s ) Figure 22. Sample s t a t i s t i c s f o r c e l l s of known age a f t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95% confidence i n t e r v a l s of the mean. A - The number of basal bodies between the two innermost CVPs of wild-type (o) and dc4 (•) c e l l s . B - The spacing between basal bodies on wild-type (o) and dc4 (©) c e l l s . Figure 23. Camera l u c i d a drawing of a dc4 c e l l a f t e r 0.93 c e l l cycles at the r e s t r i c t i v e temperature. Large, i r r e g u l a r patches have, appeared i n the central portion of the dorsal surface. The patche interrupt the paths of kin e t i e s around them. The entire c e l l shown i s about 110 pm long. 205 Figure 24. Outline drawings of wild-type ( l e f t ) and dc4 (right) d i v i d i n g c e l l s of ages 0.94 (a), 0.95 (b), 0.96-0.97 (c), and' 0.98-0.99 (d). The open c i r c l e s represent the positions of the CVPs. The scallops on the l e f t of the diagrams indicate the siz e and po s i t i o n of the vestibules. 2QZ Figure 2 5 . Growth of regions of the dorsal surface of di v i d i n g c e l l s . The p l o t s were made by comparing the contour lengths of each dorsal region at various c e l l ages with the length of that region at age 0 . 9 2 - 0 . 9 3 . The r a t i o of lengths was then plotted against c e l l age. The graphs show the r e l a t i v e amount of increase or decrease i n contour length at various c e l l ages. The regions on the dorsal surface are: (•) - CVA (+) - CVA' (o) - The distance from the fission-furrow to the posteri o r CVP of the proter. (0.) - The distance from the fission-furrow to the anterior CVP of the opisthe. (A) - CVP' (v) - CVP figure A - d c 4 c e l l s , figure B - d c 2 a c e l l s . 209 Figure 26. Sample s t a t i s t i c s f o r c e l l s of known age a f t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A - The length of wild-type (o) and dfz2 («) c e l l s . B - The length of the anterior suture (GA) and the vestibule (G) of wild-type (o) and dfz2 (©) c e l l s . 211 —i 1 1 1 1 1— •75 . 8 - 8 5 9 9 5 1 -0 A G E ( ee l I eye les ) Figure 27. Sample s t a t i s t i c s f o r c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A - The length of the posterior suture (GP) of wild-type (o) and dfz2 (©) c e l l s . B - The width of wild-type (o) and dfz2 ( a ) c e l l s . 213 Figure 28. Sample s t a t i s t i c s f o r c e l l s of known age a f t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 9 5 $ confidence i n t e r v a l s of the mean. A - CVA of wild-type (o) and dfz2 (•) c e l l s . B - CVP of wild-type (o) and dfz2 (o) c e l l s . ure 29. Sample s t a t i s t i c s f o r c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. Th v e r t i c a l bars are 95$ confidence i n t e r v a l s of th mean. A - CVA' of wild-type (o) and dfz2 (9) c e l l s . B - CVP* of wild-type (o) and dfz2 (®) c e l l s . 217 0 1 1 1 1 r— 1— .75 -8 -85 -9 -95 1-0 A G E (cell cycles) Figure 30. Sample s t a t i s t i c s f o r c e l l s of known age aft e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 9 5 $ confidence i n t e r v a l s of the mean. A - The length-to-width r a t i o of wild-type (o) and dfz2 (o) c e l l s . B - CVINT of wild-type (o) and dfz2 (©) c e l l s . a 5 —i 1 1 1 i i 7 5 -8 - 8 5 -9 - 9 5 1 0 A G E (ce l l c y c l e s Figure yi. Sample s t a t i s t i c s f o r c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95% confidence i n t e r v a l s of the mean. A - The number of basal bodies between the inner-most CVPs on wild-type (o) and d f z 2 (©) c e l l s . B - The spacing between basal bodies on wild-type (o) and d f z 2 (•) c e l l s . Figure 32. Camera l u c i d a drawings of dfz2 c e l l s which were arrested during d i v i s i o n at the r e s t r i c t i v e temperature. a and e - Both sides of a c e l l which had been at the r e s t r i c t i v e temperature f o r about 0.9 c e l l cycles. There are 4 CVPs but no f i s s i o n l i n e or furrow. The k i n e t i e s i n the central portion of the c e l l are bent, b and f - Both sides of a c e l l which had been at the r e s t r i c t i v e temperature for about 0.95 c e l l cycles. There are 4 CVPs and 2 vestibules but only a very short f i s s i o n - l i n e and no fission-furrow, c and g - Both sides of a c e l l which had been at the r e s t r i c t i v e temperature for about 0.97 c e l l cycles. The opisthe portion of the c e l l has twisted i n r e l a t i o n to the proter. d and h - Both sides of a c e l l which had been at the r e s t r i c t i v e temperature f o r about 1.0 c e l l cycles. The g u l l e t s of the proter and opisthe are exposed on the c e l l surface. The opisthe's g u l l e t l i e s to the r i g h t of the proter's. On the dorsal surface ( f i g . h) the kineties of the opisthe are at r i g h t angles to those of the proter. 223 3 Figure 33. Outline drawings of dfz2 ( l e f t ) and dc2 a (right) d i v i d i n g c e l l s of ages 0.94 (a), 0.95 (b), 0.96-0.97 ( c ) , and 0.98-0.99 (d). The open c i r c l e s indicate the positions of the CVP's. The scallops on the l e f t of the diagrams indicate the size and positions of the vestib u l e s . 225 Figure 34. Sample s t a t i s t i c s f o r c e l l s of known age aft e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A - The length of wild-type (o) and dc2 a (o) c e l l s . B - The length of the anterior suture (GA) and vestibule (G) of wild-type (o) and d c 2 a ( © ) c e l l s . 227 AGE (cell cycles) Figure 35- Sample s t a t i s t i c s f o r c e l l s of known age aft e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A - The length of the posterior suture (GP) of wild-type (o) and d c 2 a (©) c e l l s . B - The width of wild-type (o) and d c 2 a (•) c e l l s . Figure 36. Sample s t a t i s t i c s f o r c e l l s of known age aft e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95f° confidence i n t e r v a l s of the mean. A - CVA of wild-type (o) and d c 2 a ( 0 ) c e l l s . B - CVP of wild-type (o) and d c 2 a (•) c e l l s . Figure 37- Sample s t a t i s t i c s f o r c e l l s of known age aft e r a s h i f t to the r e s t r i c t i v e temperature. Th v e r t i c a l bars are 95$ confidence i n t e r v a l s of th mean. A - CVA' of wild-type (o) and dc2 a (•) c e l l s . B - CVP' of wild-type (o) and dc2 a (©) c e l l s . 40 1 30-U 20-10 0 • r • 75 -] I 1 1— • 8 - 8 5 -9 -95 A G E ( ce II cycles ) 1-0 Figure 38 • Sample s t a t i s t i c s f o r c e l l s of known age aft e r a s h i f t to the r e s t r i c t i v e temperature. Th v e r t i c a l bars are 95$ confidence i n t e r v a l s of th mean. A - The length-to-width r a t i o of wild-type (o) and d c 2 a ( e ) c e l l s . B - CVINT of wild-type (o) and d c 2 a (•) c e l l s . ro Figure 39- Sample s t a t i s t i c s f o r c e l l s of known age af t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95$ confidence i n t e r v a l s of the mean. A - The number of basal bodies between the two innermost CVPs of wild-type (o) and d c 2 a (•) c e l l s . B - The spacing between basal bodies on wild-type (o) and d c 2 a (o) c e l l s . 237 Figure 40. An example of the p l o t s used to obtain the mean c e l l cycle length of a synchronous population of c e l l s . The cumulative percentage of divided c e l l s i s plotted on a semi-log scale against the time elapsed since the c e l l popul-ation was i s o l a t e d . The intercept of a straight l i n e drawn through the points with the 50$ l e v e l on the X-axis gives the median c e l l cycle length. A further discussion of th i s technique may be found i n Natchway and Cameron (1972). The two plots shown are f o r populations of dc5 c e l l s heat-shocked at age 0.55 (*) and at age 0.7 (o). CUMULATIVE PERCENTAGE DIVIDED CELLS ro -» O i O U J O O O O O - W O i .W I i i i i i I i l I | i 1 L _ L _ I |_ ro VJ Figure 4 i . The pattern of temperature-sensitivity during the Paramecium c e l l cycle. T h i r t y minute heat-shocks were applied to synchronous popul-ations of c e l l s of known age. The c e l l cycle length of the treated samples was compared to that of a non-heat-shocked control sample and the r e l a t i v e c e l l cycle length was calculated. The r e l a t i v e c e l l cycle lengths were plot t e d against the time during the c e l l cycle at which the c e l l s were heat-shocked. The v e r t i c a l bars are 95$ confidence i n t e r v a l s f o r proportions. A - Wild-type c e l l s heat-shocked at 34.5°C (•) and at 35°C (o). B - dc5 c e l l s heat-shocked at 34.5°C. C E L L C Y C L E L E N G T H (°/ 0 of cont ro l ) C E L L C Y C L E L E N G T H (•/. of c o n t r o l ) t\3 Figure 4 2 . The pattern of temperature-sensitivity during the Paramecium c e l l cycle. T h i r t y minute heat-shocks were applied to synchronous popul-ations of c e l l s of known age. The c e l l cycle length of the treated samples was compared to that of a non-heat-shocked control and the r e l a t i v e c e l l cycle length was calculated. The r e l a t i v e c e l l cycle lengths were plo t t e d against the time during the c e l l cycle at which the c e l l s were heat-shocked. The v e r t i c a l bars are 95% confidence i n t e r v a l s f o r proportions. A - Variant 48 c e l l s heat-shocked at 3 4 . . 5 ° C . B - d c 4 c e l l s heat-shocked at 34.5°C.. - H c O o 115 r 110-i r -O 105-UJ _i ui 100-U 9 5 H U 90 T" -i 1 1 1 1 r 0 20 40 60 80 100 TIME OF HEAT SHOCK ( %> of cell c y c l e ) o 1 2 0 t_ c O « 115 110 O 105 z UJ o O 95 LU O 90 — i 1 1 1 1 1 20 40 60 TIME OF HEAT SHOCK (% T 1— 1 1 80 100 of cel l cyc le ) Figure 43. The pattern of temperature-sensitivity during the Paramecium c e l l cycle. T h i r t y minute heat-shocks were applied to synchronous popul-ations of c e l l s of known age. The c e l l cycle length of the treated samples was compared to that of non-heat-shocked controls and the r e l a t i v e c e l l cycle length was calculated. The r e l a t i v e c e l l cycle lengths were pl o t t e d against the time during the c e l l cycle at which the c e l l s were heat-shocked. The v e r t i c a l bars are 9 5 $ confid-ence i n t e r v a l s of proportions. The graph shown i s f o r sm2 c e l l s heat-shocked at 34.5°C. The r e s u l t s of two separate experiments are shown. C E L L C Y C L E L E N G T H <•/. of c o n t r o l ) § Figure kk. A schematic diagram showing the measurements made on sm2 c e l l s of various s i z e s . The measure-ments shown i n B were made on only two c e l l samples. Abbreviations include: CYTL - cytoproct length. KTCV - the number of kin e t i e s to the f i r s t con-t r a c t i l e vacuole pore (CVP) encountered counting counterclockwise from the vest i b -ule. KTCL - the number of kineties from the f i r s t CVP encountered to the r i g h t edge of the vestibule. KTCVV - the number of kin e t i e s between the CVPs. Figure 45. Probit p l o t s of sm2 c e l l length f o r asynchronous samples taken at various times afte r a s h i f t to the r e s t r i c t i v e temperature. A - sm2 samples: (x) - non-heat-shocked control (.) - sample taken 6 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature. (•) - sample taken 11 .5 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature, (o) - sample taken 24 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature. B - sm2 samples: (•) - sample taken 8 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature, (o) - sample taken 24 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature. 8 7 6 \— CO 4 O CL 3 2 5 0 ® ,.' J x •8 70 90~ 110 LENGTH (pm) 130 8 7 5 h-CQ O Cd CL 3 B 68 7 0 >o6 9 0 110 130 £ LENGTH (pm) Figure 46. Probit p l o t s of c e l l width f o r asynchronous samples of sm2 c e l l s taken at various times a f t e r a s h i f t to the r e s t r i c t i v e temperature. A - sm2 samples: (x) - non-heat-shocked control, (o) and (o) - two independant samples taken af t e r 24 hours at the r e s t r i c t i v e temperature. B - sm2 samples: (x) - sample taken a f t e r 8 hours at the r e s t r i c t i v e temperature. (••) - sample taken a f t e r 6 hours at the r e s t r i c t i v e temperature, (o) - sample taken a f t e r 11.5 hours at the r e s t r i c t i v e temperature. 8 7 ® 5 CD O 4 or 0_ o 3 O -5 5 30 4 0 50 WIDTH (pm) 6 0 8-7-6 5 CD O A cc 0_ 3 1 0 B o o o o o \ X 30 4 0 WIDTH (jjm) Figure 4?. UPPER - The length (o) and width (•) of sm2 c e l l s p lo t t ed against time at the r e s t r i c t i v e temperature. LOWER - The length-to-width r a t i o of sm2 c e l l s p lo t t ed against time at the r e s t r i c t i v e temperature. 140 0-J , , , , r 0 6 12 18 24 TIME(hours) Figure 4 8 . Sample s t a t i s t i c s of asynchronous sm2 c e l l s at various times a f t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95% confidence i n t e r v a l s of the mean. A - Sample s t a t i s t i c s : (o) - anterior suture length. (©) - posterior suture length. (o, lower) - vestibule length. B - Sample s t a t i s t i c s shown i n figure A as a f r a c t i o n of the c e l l length: (o) - GA/length (•) - GP/length (o, lower) - G/length C - Sample s t a t i s t i c s : (o) - CVINT (see f i g . 4 4 ) (©) - CVA (see f i g . 4 4 ) (x) - CVP (see f i g . 44 ) D - Sample s t a t i s t i c s shown i n figure C as a f r a c t i o n of the c e l l length; (o) - CVINT/length (•) - CVA/length (x) - CVP/length L E N G T H (p m) L E N G T H ( p m ) Figure 49 • Sample s t a t i s t i c s of asynchronous sm2 c e l l s at various times a f t e r a s h i f t to the r e s t r i c t i v e temperature. The v e r t i c a l bars are 95% confidence i n t e r v a l s of the mean. A - Sample s t a t i s t i c s : (o) - the distance from the poster i o r CVP to the anterior of the vesti b u l e . (•) - the distance from the anterior CVP to the pos t e r i o r of the vestibule, (x) - the distance from the po s t e r i o r of the vestibule to the poster i o r CVP. (+) - the distance from the anterior CVP to the an t e r i o r of the vestibule. B - Sample s t a t i s t i c s shown i n figure A expressed as a f r a c t i o n of the c e l l length. The symbols used are the same as i n figure A. C - Cytoproct length. D - The cytoproct length as a f r a c t i o n of the c e l l length (e) and as a f r a c t i o n of the length of the posterior suture (o). Figure 50- Bivariate scatter p l o t s of sample st a t -i s t i c s of sm2 c e l l s . A - The length of the anterior suture (GA) p l o t t e d against the length of sm2 c e l l s which had been at the r e s t r i c t i v e temperature f o r 24 hours. The dotted l i n e s are: ( ) - The p r i n c i p a l axis of the s c a t t e r p l o t of GA and length f o r a sample taken a f t e r 8 hours at the r e s t r i c t i v e temperature. (-.-) - The p r i n c i p a l axis of the s c a t t e r p l o t of GA and length f o r a non-heat-shocked sample. The s o l i d l i n e indicates values of GA equal to 47.5$ of the c e l l length (see t e x t ) . The slopes of a l l p r i n c i p a l axes are given i n table 17. B - The length of the p o s t e r i o r suture (GP) pl o t t e d against the length of sm2 c e l l s which had been at the r e s t r i c t i v e temperature for 24 hours. The dotted l i n e i s the p r i n c i p a l axis. Figure 51 • Bivariate scatter p l o t s of sample s t a t i s -t i c s of asynchronous sm2 c e l l s . A - The cytproct length (CYTL) pl o t t e d against c e l l length f o r a non-heat-shocked sample (o) and a sample taken a f t e r 24 hours at the r e s t r i c t i v e temperature (•). The p r i n c i p a l axes of the non-heat-shocked sample ( ) and the 24 hour sample (-.-) are shown. The • s o l i d l i n e represents cytoproct lengths equal to 15% of the c e l l length. B - The length of the vestibule (G) p l o t t e d against c e l l length: (o) - non-heat-shocked sample (©) - sample taken 24 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature. The p r i n c i p a l axis of the 24 hour sample i s shown (-.-). The length of the vestibule i s not correlated with c e l l length i n the non-heat-shocked sample ( ). 261 F i g u r e 52. B i v a r i a t e s c a t t e r p l o t s o f s a m p l e s t a t -i s t i c s o f sm2 c e l l s . A - The d i s t a n c e "between t h e . a n t e r i o r CVP and t h e a n t e r i o r o f t h e c e l l (CVA) p l o t t e d a g a i n s t c e l l l e n g t h : ( o ) - n o n - h e a t - s h o c k e d s a m p l e . (©) - s a m p l e t a k e n 24 h o u r s a f t e r a s h i f t t o . t h e r e s t r i c t i v e t e m p e r a t u r e . B - The d i s t a n c e b e t w e e n t h e p o s t e r i o r CVP a n d t h e p o s t e r i o r o f t h e c e l l (CVP) p l o t t e d a g a i n s t c e l l l e n g t h : ( o ) - n o n - h e a t - s h o c k e d s a m p l e . (•) - s a m p l e t a k e n a f t e r 24 h o u r s , a t t h e r e s t r i c t i v e t e m p e r a t u r e . On b o t h p l o t s , t h e p r i n c i p a l a x e s a r e i n d i c a t e d a s f o l l o w s : ( - — ) - n o n - h e a t - s h o c k e d s a m p l e . (-.-) - 24 h o u r s a m p l e . 263 Figure 5 3 - Bivariate scatter p l o t s of sample st a t -i s t i c s of sm2 c e l l s . A - The distance between the CVPs (CVINT) p l o t t e d against the c e l l length: (o) - non-heat-shocked sample. (•) - sample taken 24 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature. The p r i n c i p a l axes of the of the non-heat-. shocked sample ( ) and the 24 hour sample (-.-) are indicated. The s o l i d l i n e describes the equation CVINT = LENGTH - (-35LENGTH + 22 um) (see te x t ) . B - The number of basal bodies between the CVPs plotted against c e l l length for a sample of c e l l s taken 24 hours a f t e r a s h i f t to the r e s t r i c t i v e temperature. 265 F i g u r e 5 ^ . B i v a r i a t e s c a t t e r p l o t s of sample s t a t -i s t i c s o f asynchronous sm2 c e l l s . A - The s p a c i n g between b a s a l bodies p l o t t e d a g a i n s t c e l l l e n g t h f o r a sample a f c e l l s t aken 2k hours a f t e r a s h i f t t o the r e s t r i c -t i v e temperature. The dashed l i n e i s the p r i n c i p a l a x i s . B - The t o t a l number of k i n e t i e s p e r c e l l p l o t t e d a g a i n s t c e l l width f o r a sample taken 2k hours a f t e r a s h i f t to the r e s t r i c -t i v e temperature. The dashed l i n e i s the p r i n c i p a l a x i s . 104 •8H E V 6 ' o CD < 3 •4J / O / O / oo qfo O O Q / 0 Q o o o of o 0 • / ° . - o / 0 / O O o o o/' 40 60 80 100 CELL LENGTH (pm) 120 e 8 0 E 2 76 * 68-I _ i < 0 64-j 604 B o o / o o o / o O O O/Q CD o o o CD O A D O / O o is o <t>/ / s o o /O o o o 34 38 42 46 50 54 WIDTH (pm) Figure 5 5 - Bivariate scatter p l o t s of sample stat-i s t i c s of asynchronous sm2 c e l l s . A - The t o t a l number of ki n e t i e s per c e l l p lotted against the number of kin e t i e s between the l e f t v e s t i b u l a r wall and the f i r s t CVP to the c e l l ' s l e f t (KTCV) f o r a sample of c e l l s taken 24 hours a f t e r a s h i f t to the r e s t r i c -t i v e temperature. The dashed l i n e i s the p r i n c i p a l axis. The s o l i d l i n e describes the equation: KTCV = .58TOTAL KINETIES (see t e x t ) . B - The value KTCV expressed as a percentage of the t o t a l number of ki n e t i e s p l o t t e d against c e l l width. There i s no c o r r e l a t i o n . KTCV C/o of k ine t ies ) TOTAL KINETIES (number ) i ro O N NO Figure 56. Probit plots of c e l l length measurements from the feeding experiment. (•) - non-heat-shocked'sample. (o) - sample taken aft e r 24 hours at the r e s t r i c -t i v e temperature. 8 4 0 6 0 8 0 ' 100 C E L L LENGTH (pm) 120 Figure 57 • Bivariate scatter p l o t s of sample s t a t -i s t i c s of sm2 c e l l s . A - The length of the quadrulus plotted against c e l l length: (o) - non-heat-shocked sample. (•) - sample taken a f t e r 24 hours at the r e s t r i c t i v e temperature. B - .The number of p a i n t - f i l l e d food vacuoles plotted against the length of the quad-rulus: (o) - non-heat-shocked sample. (•) - sample taken a f t e r 24 hours at the r e s t r i c t i v e temperature. E X 20-r -o z LU _1 i/) 16-Z> j GC Q vn 12-0 © e» o o o 9 S3 oa o oo o » 0 • O OO O 9 0 0 OO g C O O O o eas eo o «D <D <ID O&> es o o o o o • o e o o e d o a o e ©e> e e e». o e 0 0 o 0 1 1 1 1 — 35 55 75 95 115 CELL LENGTH (um) 135 273 o o o o fe 8 0 0 9 0 8 8 S S 8 o o 8 S o . • 8 • S I • % • • • • • - i 1 1 1 — — — ' 1 77 0 4 8 12 16 20 24 OUADRULUS LENGTH (pm) Figure 58. A schematic diagram i l l u s t r a t i n g possible mechanisms for the gain and loss of kineties (from Heckmann and Frankel, 1968). The zone of basal body p r o l i f e r a t i o n on d i v i d i n g c e l l s i s represented by the dotted l i n e s . Short kineties which are outside of t h i s zone are eventually l o s t (a). Short kineties which extend into t h i s zone (b) eventually increase i n length to extend pole-to-pole. GAIN AND LOSS OF KINETIES F i g u r e 5 9 . The f a t e o f CVPs i n a c e l l l i n e a g e d i a g r a m . The 4 CVPs o f d i v i d i n g c e l l s a r e shown. E a c h new g e n e r a t i o n o f CVPs i s i n d i c a t e d b y a s e p a r a t e s y m b o l . See t h e t e x t f o r f u r t h e r e x p l a n a t i o n . F i g u r e 60. The r e l a t i v e p o s i t i o n s o f CVPs i n d i v i d i n g c e l l s . The u p p e r f i g u r e shows t h e r e l a t i v e p o s i t i o n s o f t h e k CVPs on a d i v i d i n g c e l l o f age 0.8. The CVP p o s i t i o n s a r e g i v e n a s a p r o p o r t i o n o f t h e c e l l l e n g t h , m e a s u r e d f r o m t h e a n t e r i o r end o f t h e c e l l . The r e l a t i v e p o s i t i o n s t h a t t h e CVPs w o u l d assume a f t e r c e l l d i v i s i o n i f g r o w t h were u n i f o r m t h r o u g h o u t t h e c o r t e x a r e shown t o t h e r i g h t . The l o w e r f i g u r e shows t h e r e l a t i v e p o s i -t i o n s o f t h e 4 CVPs i n d i v i d i n g c e l l s o f t h e i n d i c a t e d a g e s . I n t h i s f i g u r e , d i v i d i n g c e l l s w e r e t r e a t e d a s i f t h e y were c o m p r i s e d o f two s e p a r a t e c e l l s ( t h e p r o t e r a n d o p i s t h e ) e v e n t h o u g h a f i s s i o n - f u r r o w may n o t h a v e b e e n p r e s e n t . The p o s i t i o n o f t h e CVPs i s g i v e n i n t e r m s o f b o t h l i n e a r l e n g t h (©) a n d c o n t o u r l e n g t h ( o ) . The h a t c h e d l i n e s a t 35$ and 80$ i n d i c a t e t h e p o s i -t i o n s o f t h e 2 CVPs a l o n g t h e l i n e a r l e n g t h o f i n t e r f i s s i o n c e l l s . The h a t c h e d l i n e s a t 35$ a n d 75$ i n d i c a t e t h e p o s i t i o n s o f t h e 2 CVPs a l o n g t h e c o n t o u r l e n g t h o f i n t e r f i s s i o n c e l l s . 279 100--85 .9 - 9 5 A G E ( celJ c y c l e s ) . 8 5 -9 -95 1 -0 A G E ( cel l cyc les) Plate 1. Gul l e t structure. a - Inverted " J " anlage from a c e l l at age 0.9-b - I n t e r f i s s i o n age 0.92. The anterior end of the anlage i s pulled away from the anterior of the vestibule, c - I n t e r f i s s i o n age 0.95* The anlage l i e s p o sterior to the f i s s i o n l i n e , d - A g u l l e t everted on the surface of a dfz2 c e l l . e and f - Transverse gap across a l l g u l l e t k i n e t i e s i n sm2 c e l l s . 231 Plate 2. Somatic cortex - normal and abnormal c e l l s . a - A normal cortex i n a wild-type c e l l . The l a r g e r black spots' ( s i l v e r deposits) indicate the positions of c i l i a r y basal bodies and parasomal sacs, the smaller spots indicate the positions of t r i c h o c y s t s . b - Supernumerary tri c h o c y s t s i n a t s O - l a c e l l . (This mutant, along with several others, although not considered i n d e t a i l i n the present study, w i l l be used i n t h i s and other plates f o r i l l u s t r a t i v e purposes.) c - Supernumerary components on a t s O - l a c e l l . d, e, f - C o r t i c a l abnormalities of types 1, 2, and 3 respectively, ( f i g . d - variant 46, f i g s , e and f - sm2) g, h - C o r t i c a l patches (p) apparently containing no c o r t i c a l organelles, (variants 42 and 46) 283 »••••.»«. * ' * • • > • • * * » . 4 AM********' 0 '** .»>r * A CT) • 11 Plate 3' Gullet abnormalities. a, b - Type 1 abnormality. The quadrular ki n e t i e s are i r r e g u l a r , (variant 53 c e l l s ) c, d - Type 2 abnormality. The innermost one-third of the g u l l e t i s missing, the k i n e t i e s are very i r r e g u l a r . (sm2 c e l l s ) e, f - Type 3 abnormality. The innermost one-half of the g u l l e t i s missing. (sm2 c e l l s ) 285 Plate 4. Cytoproct structure, a - normal cytoproct. b - disrupted cytoproct - d f z l c e l l , c - disrupted cytoproct - dc2 b c e l l , d - p l a t e - l i k e cytoproct - t s O - l a c e l l , e - distended cytoproct - t s O - l a c e l l . 287 Plate 5 ' C e l l shapes. a - normal shape - wild-type c e l l . b - large, pear-shaped' c e l l - variant 27-c - large c e l l - tsO-1 c e l l . d - irregularly-shaped c e l l - variant 18. e - t h i n , astomatous, c e l l - sml c e l l . f - anterior truncation - the specimen has no anterior suture or vestibule - sm2 c e l l , g p o s t e r i o r truncation - sm2 c e l l , h - small c e l l - sm2 c e l l . i , j - twisted c e l l s - note that the ki n e t i e s s p i r a l around the c e l l surface - dc6 c e l l s . Plate 6 . C e l l shapes. a - large tsO-1 c e l l . b - large, round, and deeply-argentophilic tsO-3 c e l l . c - swollen, irregularly-shaped dc6 c e l l . d - large tsO-1 c e l l with abnormal c o n t r a c t i l e vacuole pores, e - i r r e g u l a r l y shaped dfz2 c e l l with patchy cortex. f - dc6 c e l l with supernumerary c o n t r a c t i l e vacuole pores (most are s l i g h t l y out of focus). 29/ Plate 7. C e l l shapes. a - normal d i v i d i n g c e l l . b and d - blockage of c e l l d i v i s i o n l a t e i n the c e l l cycle - mutants dc4 ( f i g . b) and d f z l . c and e - blockage of c e l l d i v i s i o n during the early stages of furrowing - mutant dfz2 c e l l s , f - monster c e l l (mutant dc2^) r e s u l t i n g from continued c e l l growth i n the absence of normal c e l l d i v i s i o n . 273 P l a t e 8. C e l l d i v i s i o n s t a g e s . a - i n t e r f i s s i o n c e l l w i t h no o b v i o u s s i g n s o f m o r p h o g e n e s i s , b - a p p e a r a n c e o f t h e f i s s i o n - l i n e ( f l ) o n a c e l l o f a ge 0.92. c - age 0.94 c e l l w i t h a f i s s i o n - l i n e c o m p l e t e a r o u n d t h e c e l l a n d w i t h t h e s i d e s o f t h e c e l l f l a t t e n e d . d - f o u r CVPs on t h e d o r s a l s u r f a c e o f a n age 0.94 c e l l ( t h e new CVPs f o r m a r o u n d age 0 . 8 ) . e - a c e l l a g e d O.95 w i t h a s l i g h t c o n s t r i c t i o n a n d a l a r g e c l e a r s p a c e p o s t e r i o r t o t h e v e s t -i b u l e i n t h e p r o t e r . f - a n age 0.99 c e l l w i t h a n e a r l y c o m p l e t e f i s s i o n - f u r r o w . g - p r o t e r f r o m a r e c e n t l y d i v i d e d c e l l - t h e a n t e r i o r o f t h e c e l l i s p o i n t e d w h i l e t h e p o s t e r i o r i s b l u n t . h - an o p i s t h e f r o m a r e c e n t l y d i v i d e d c e l l - t h e a n t e r i o r a n d p o s t e r i o r e n d s o f t h e c e l l a r e b l u n t a n d t h e v e s t i b u l e i s n o t c e n t r a l l y l o c a t e d . 2 9 5 " Plate 9- Basal body p r o l i f e r a t i o n i n wild-type c e l l s . a - c e l l age 0.75 - no basal body p r o l i f e r a t i o n has occurred on the dorsal surface. b - c e l l age 0.9 - basal body p r o l i f e r a t i o n extends a few c o r t i c a l units p o s t e r i o r to the f i s s i o n -l i n e . c - c e l l age 0.93 - basal body p r o l i f e r a t i o n extends p o s t e r i o r l y beyond the anterior CVP of the opisthe and a n t e r i o r l y to the posterior CVP of the proter. d - c e l l age 0.96 - basal body p r o l i f e r a t i o n extends . a n t e r i o r l y past the posterior CVP of the proter and the kineties appear bent i n the furrow region. e - c e l l age 0.99 - surface growth i s increasing the spacing of previously c l o s e l y packed (compare with f i g u r e d) basal bodies. 297 - * * T 2 • * % 3 r*»r% • > * • * • • * • t « • .• « • • 3 j) ••2-5! ftps W W W • ill * • * i l 1 lit P l a t e 10. C e l l d i v i s i o n s t a g e s o f sm2 c e l l s a t t h e r e s t r i c t i v e t e m p e r a t u r e . C e l l s w e r e g r o w n f o r one c e l l c y c l e a t t h e r e s t r i c t i v e t e m p e r a t u r e , a - age 0.75 b - age 0.8 - The c e l l i s a b n o r m a l l y w i d e . c - age 0.93 - F i s s i o n - l i n e f o r m a t i o n i s n o r m a l . d - age 0.94 - The c e l l i s c o n t r a c t e d a t t h e a n t e r i o r a n d p o s t e r i o r e n d s . C o n t r a c t i o n i n t h e o r a l g r o o v e r e g i o n i s p a r t i c u l a r l y n o t i c e a b l e . e - age O.96 - The c e l l i s a b n o r m a l l y s h o r t b u t f u r r o w f o r m a t i o n i s n o r m a l . The a n t e r i o r e n d o f t h e c e l l i s a b n o r m a l l y p o i n t e d , p o s s i b l y due t o c o n t i n u e d c o n t r a c t i o n o f t h e a n t e r i o r p o l e o f t h e c e l l . f - age 0.99 - An a b n o r m a l l y s h o r t d i v i d e r w i t h a v e r y d i s o r g a n i z e d c o r t e x . g - p r o t e r a n d o p i s t h e ( a g e 1.05) r e s u l t i n g f r o m c e l l d i v i s i o n a t t h e r e s t r i c t i v e t e m p e r -a t u r e . h - w i l d - t y p e c e l l a g e 0.99 - Compare w i t h t h e s i z e o f sm2 d i v i d e r s shown i n f i g u r e f . The v e r t i c a l b a r i n d i c a t e s 20 pm. A l l o t h e r f i g u r e s a r e t o t h e same s c a l e . 299 

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