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Identification and organization of the cytoskeleton in the alga Vaucheria longicaulis var. macounii Peat, Lucinda Jane 1992

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IDENTIFICATION AND ORGANIZATION OF THE CYTOSKELETON IN THE ALGA Vaucheria l o n q i c a u l i s var. macounii by LUCINDA JANE PEAT B.Sc, The University of Leeds, U.K., 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1992 (c)Lucinda Jane Peat, 1992 MASTER OF SCIENCE in In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my wri t ten permission. Department The University of British Columbia Vancouver, Canada -6 (2/88) i i Abstract The presence of the cytoskeletal proteins a c t i n and t u b u l i n i n the alga Vaucheria l o n g i c a u l i s Hoppaugh var. macounii Blum i s studied by SDS-PAGE and immunoblotting techniques. These techniques also indicate the presence of the mechanomotor protein myosin. The o v e r a l l organization of the cytoskeleton i n the cytoplasm of i n t a c t filaments, t h e i r i d e n t i f i c a t i o n and d i s t r i b u t i o n , are investigated by immunofluorescence and epifluorescence microscopy using monoclonal anti-B-tubulin and a n t i - a c t i n antibodies and FITC-labelled p h a l l o i d i n . ' Anti-myosin antibodies were also u t i l i z e d , but proved to be inadequate f o r my work. P h a l l o i d i n l a b e l l i n g of F-actin proved to be more su i t a b l e f o r v i s u a l i z i n g microfilaments than a n t i - a c t i n antibodies. P h a l l o i d i n l a b e l l i n g of F-actin reveals a dense array of microfilament cables i n the c o r t i c a l cytoplasm of vegetative filaments, which appear to be sub-divided into two morphologically d i s t i n c t sets. One set consists of s t r a i g h t e r elements, p r e f e r e n t i a l l y occupying the cytoplasm adjacent to the plasma membrane and possibly providing tracks f o r organelle m o t i l i t y . A second set i s made up of wavy elements and extends deeper into the cytoplasm where i t may be part of the force generating system responsible for organelle m o t i l i t y . Immunofluorescence for tubulin reveals that the microtubule array i s much les s dense than that made up of microfilaments. Microtubule bundles appear shorter and s t r a i g h t e r and are located i i i throughout the width of the cytoplasm. They show no p a r t i c u l a r r e l a t i o n s h i p to organelles, except n u c l e i . With respect to n u c l e i , they seem to be involved i n t h e i r arrangement within the filament, p a r t i c u l a r l y i n the a p i c a l region, and t h i s may have implications f o r the organization of the tip-growth processes. High r e s o l u t i o n scanning electron microscopy i s also u t i l i z e d i n the study of the organization and d i s t r i b u t i o n of the cytoskeleton. D i f f e r e n t i a l interference contrast microscopy reveals cytoplasmic tracks o r i g i n a t i n g from f o c a l regions i n l i v i n g vegetative filaments of the coenocytic alga Vaucheria l o n q i c a u l i s var. macounii. Fluorescein-labelled p h a l l o i d i n s t a i n i n g also reveals regions (foci) of s i m i l a r structure and dimensions among the F-actin array. Immunofluorescence using monoclonal antibodies to B-tubulin shows punctate fluorescence i n a s s o c i a t i o n with the microtubule array. Cytochalasins are used to breakdown the F-actin array, an e f f e c t that i s concentration dependent. Cytochalasin D causes a gradual breakdown of the F-a c t i n array, revealing the close association between f o c i and F-a c t i n fluorescence. Recovery from treatment with t h i s i n h i b i t o r confirms t h i s association and suggests that f o c i act as organizing centers for the F-actin array. Cold temperature, O r y z a l i n and taxol are used to disrupt the microtubule bundle array. Depolymerization i s evident i n the appearance of many fluorescent spots ( f o c i ) , which may be c o - l o c a l i z e d with nuclei or associated with the ends of microtubule bundles. Recovery i v from these treatments suggests that s p o t - l i k e f o c i act as nucleation centers f o r microtubule bundles. The existence of microtubule-associated f o c i i s supported by the r e s u l t s of t a x o l treatment. The d i f f e r e n t roles played by microfilaments and microtubules i n the organization of the polarized structure of the c e l l are discussed with respect to the function of t h e i r respective organizing centers. V TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS V LIST OF FIGURES v i ACKNOWLEDGEMENTS x i INTRODUCTION 1 MATERIALS AND METHODS 6 RESULTS - PART 1 12 PROTEIN EXTRACTION AND ANALYSIS 12 DIFFERENTIAL INTERFERENCE MICROSCOPY OBSERVATIONS 13 MICROFILAMENT ARRAY AS VISUALIZED BY FLUORESCENCE 13 MICROTUBULE ARRAY AS VISUALIZED BY IMMUNOFLUORESCENCE..16 HIGH RESOLUTION ELECTRON MICROSCOPY OBSERVATIONS 18 DISCUSSION - PART 1 19 RESULTS - PART 2 28 FOCAL REGIONS IN UNTREATED CELLS 28 F-ACTIN FOCI 29 MICROTUBULE ASSOCIATED FOCI 32 DISCUSSION - PART 2 35 CONCLUSIONS 41 FIGURES 43 LITERATURE CITED 59 v i LIST OF FIGURES FIGURE 1 SDS-PAGE gels and immunoblots of Vaucheria homogenate proteins 44 2 DIC image of a x i a l l y aligned array of fibrous structures d i s t r i b u t e d throughout the cytoplasm 45 3 DIC image showing r e l a t i o n s h i p between fibrous structures,chloroplasts, and nuclei 45 4 F-actin microfilament array v i s u a l i z e d using f l u o r e s c e i n - l a b e l l e d p h a l l o i d i n . inset: d i s t r i b u t i o n of F-actin i n c o r t i c a l region of the filament 45 5 Microfilament array showing branches between long i t u d i n a l cables and around organelles 45 6 Branching of microfilament cables i n close proximity to organelles 46 7 C r y s t a l l i n e - l i k e microfilament arrays i n Vaucheria filament. 46 8 Association of microfilament cables and chloroplasts...47 9 Microfilament cables appear to radiate from a f o c a l area i n the a p i c a l region of a vegetative filament 47 10 Microfilament cable organization into two sub-sets: a s t r a i g h t e r sub-set, and a more r e t i c u l a t e one 47 11 Focal point of microfilament cables ......47 v i i 12 Confocal scans showing the p a r t i t i o n i n g of the microfilament array 48 13 Confocal scans showing a l o n g i t u d i n a l l y aligned microfilament set and a r e t i c u l a t e set beneath i t 48 14 Confocal cross-sectional scan of microfilament d i s t r i b u t i o n within the cytoplasm 48 15 Confocal graphical representation of microfilament d i s t r i b u t i o n along a l a b e l l e d l o n g i t u d i n a l axis of the c e l l 48 16 Microtubule bundle array of a Vaucheria vegetative filament 49 17 Vaucheria c e l l showing microtubule bundles curving and crossing over into a branch 49 18 Confocal composite image of microtubule bundles 49 19 Microfilament bundle array i n the t i p region of the filament 49 20 Cross-sectional confocal scan and graphical representation of microtubule bundle d i s t r i b u t i o n throughout the cytoplasm 50 21 S e r i a l confocal scans at d i f f e r e n t o p t i c a l depths showing microtubule bundles throughout the cytoplasm...50 22 Cross-section of a Vaucheria filament showing punctate microtubule fluorescence throughout the cytoplasm 50 2 3 Extruded chloroplast with c l o s e l y associated microtubule bundles ......50 24 HRSEM of a fractured end of a Vaucheria c e l l . . . 51 v i i i 25 Higher magnification HRSEM of fibrous material 51 26 HRSEM of a fractured filament showing st r a i g h t e r cables within the cytoplasm 51 27 Higher magnification of Fig . 26 51 2.1 DIC image showing cytoplasmic track f o c i 53 2.2 Microfilament array showing f o c i adjacent to a cross -wall 53 2.3 Microtubule array of an untreated (control) c e l l 53 2.4 Oblique end view of a vegetative filament revealing filamentous and punctate B-tubulin fluorescence 53 2.5 F-actin fluorescence af t e r treatment with Cytochalasin D 54 2.6 F-actin f o c i a f t e r treatment with Cytochalasin B 54 2.7 Early stage of F-actin breakdown by Cytochalasin D 54 2.8 F-actin array of an untreated vegetative filament 54 2.9 F-actin f o c i i n the v i c i n i t y of a cross-wall a f t e r Cytochalasin D treatment 54 2.10 F-actin f o c i amidst F-actin cables a f t e r treatment with Cytochalasin D 54 2.11 Web-like array of F-actin with f o c i a f t e r treatment with Cytochalasin D 55 2.12 Web-like array of F-actin with f o c i a f t e r treatment with Cytochalasin D - t i p region of vegetative filament 55 2.13 Advanced stage of F-actin depolymerization a f t e r treatment with Cytochalasin D 55 2.14 Extensive depolymerization of F-actin a f t e r treatment with Cytochalasin D 55 2.15 Almost complete F-actin depolymerization a f t e r treatment with Cytochalasin D 55 2.16 F-actin array a f t e r cold treatment - appearance of f o c i 55 2.17 Foci during recovery from Cytochalasin D treatment 56 2.18 Microtubule array a f t e r cold treatment 56 2.19 Depolymerization of the microtubule array a f t e r cold treatment 56 2.20 Depolymerization of the microtubule array a f t e r cold treatment - t i p region 56 2.21 D i s t r i b u t i o n of B-tubulin fluorescence a f t e r cold treatment 56 2.22 DAPI l o c a l i z a t i o n of nuclei as a reference to Figure 2.21 56 2.23 Fragmentation of microtubule bundles during depolymerization 57 2.24 Bright spots at ends of depolymerizing microtubule bundles 57 2.25 Bright spot at end of depolymerizing microtubule bundle 57 2.26 Microtubule bundle array a f t e r treatment with taxol....57 2.27 Fluorescent spots v i s i b l e during repolymerization of the microtubule array ......58 X 2.28 Microtubule bundles during the process of repolymerization 58 2.29 Fluorescent spot terminated microtubule bundles during repolymerization 58 2.30 Repolymerization of the microtubule array 58 x i ACKNOWLEDGEMENTS It has been an enriching experience to study and l i v e abroad. I would l i k e to express thanks to those who made t h i s possible; my supervisor Dr. Luis O l i v e i r a , UBC, Canada Immigration, and my family and friends on both sides of the world for t h e i r unceasing support of my endeavours. Special thanks are i n order f o r technical advice and assistance to Dr. Brian Oates and Dr. Edith Camm and her laboratory s t a f f at the Department of Botany, UBC, and Dr. John W. La C l a i r e II at the University of Texas, Austin, f o r performing the anti-Dictyostelium myosin immunoblot. 1 Introduction The cytoskeleton of higher plant and a l g a l c e l l s i s , f o r the most part, considered to be made up of microfilaments and microtubules (Seagull 1989, Derksen et a l . 1990), although recently intermediate filament-like structures have been described (Goodbody et a l . 1989). Multiple functions have been assigned to these cytoskeletal elements. They include i n t r a c e l l u l a r m o t i l i t y (Williamson 1986), r o l e s i n mitosis and cytokinesis (Forer 1982, Gunning 1982), s p a t i a l organization (Van Lammeren 1988), maintenance of c e l l shape (Marchant 1982) and alignment of c e l l wall m i c r o f i b r i l s (Robinson and Quader 1982). The involvement of the cytoskeleton i n i n t r a c e l l u l a r m o t i l i t y and the creation of motive force have been studied i n depth using giant a l g a l c e l l s as models, e.g. Chara (Williamson 1975, G r o l i g et a l . 1988). In such c e l l s i t has been determined that the major mechanism underlying these processes u t i l i z e s an actomyosin system. In many higher plant and a l g a l c e l l s e x h i b i t i n g cytoplasmic streaming, extensive a x i a l l y aligned arrays of a c t i n microfilaments can be seen using immunofluorescence and fluorochrome l a b e l l e d phallotoxins (Menzel 1987, Heslop-Harrison and Heslop-Harrison 1989). They are often presumed to function i n a si m i l a r fashion to the a l g a l c e l l models. In contrast, microtubule based motive systems which have been found to be largely responsible f o r i n t r a c e l l u l a r m o t i l i t y within animal c e l l s (Adams and Po l l a r d 1986), where they seem to 2 pa r t i c i p a t e i n t h i s function by acting i n conjunction with mechanoenzymes such as dynein, kinesin and other microtubule associated proteins (Hollenbeck 1988). In streaming higher plant and a l g a l c e l l s immunofluorescence staining of tu b u l i n has revealed microtubule arrays which are sometimes a x i a l l y co-aligned with F-actin arrays. As a r e s u l t of i n h i b i t o r studies and molecular analysis, they have also been implicated to function i n the movement of organelles within c e l l s (Menzel and Schliwa 1986b, Schnepf 1986, T i e z z i et a l . 1992, Tornbom and O l i v e i r a , unpublished). Tip growing c e l l s , examples of which include p o l l e n tubes (Steer and Steer 1989), moss protonemata (Sievers and Schnepf 1981), a l g a l r h i z o i d s (Tewinkel et a l . 1989) and fungal hyphae (Temperli et a l . 1990), demonstrate a unique property of l o c a l i z e d growth and zonation of organelles as a manifestation of p o l a r i z a t i o n (Sievers and Schnepf 1981, Schnepf 1986). Cytoskeleton has also been found to be i n t e g r a l i n the establishment and maintenance of t i p growth. In such c e l l s microtubules and microfilaments are also usually a x i a l l y aligned (Lloyd 1987) and extend into the ap i c a l dome of the growing t i p where they are implicated to function i n the maintenance of s t r u c t u r a l i n t e g r i t y as well as i n the processes of exocytosis and streaming (Doonan et a l . 1988, Steer and Steer 1989). Vaucheria l o n g i c a u l i s Hoppaugh var. macounii Blum i s a coenocytic filamentous alga of the D i v i s i o n Chrysophyta, which exhibits t i p growth i n the ap i c a l dome of each filament (Kataoka, 3 1975). Rapid cytoplasmic streaming of organelles, including chloroplasts, n u c l e i , mitochondria and small v e s i c l e s (Ott 1992, Tornbom and O l i v e i r a , unpublished), i s also observed throughout the body of the filament into the t i p region. Previous studies have revealed the presence of cy t o s k e l e t a l elements i n Vaucheria (Ott and Brown 1972, B l a t t et a l . 1980, O l i v e i r a and F i t c h 1988, Ott 1992, Tornbom and O l i v e i r a , unpublished) but these d i d not include the use of fluorescence microscopy and protein analysis. These techniques are used i n t h i s study i n order to elucidate the organization of, and r o l e , the cytoskeleton plays i n t h i s unusual c e l l type with complex i n t r a - c e l l u l a r m o t i l i t y and polarized growth (Tornbom and O l i v e i r a , unpublished). Since the recognition of the cytoskeleton as an ubiquitous component of eukaryotic c e l l s , the subject of how the i n d i v i d u a l proteinaceous elements are organized and assembled has been of great i n t e r e s t . Observations of how i n h i b i t o r s disrupt (depolymerize) c y t o s k e l e t a l elements can reveal information such as the existence of microtubule organizing centers (MTOCs) (Brown et a l . 1982) or centers of a c t i n assembly (polymerization) (Stossel 1989). The organization of filamentous a c t i n (F-actin) i n plant c e l l s has been the subject of numerous studies (Staiger and Schliwa 1987, Seagull et a l . 1987). Despite t h i s fact, our knowledge of the subject i s meager compared to that of F-actin i n animal c e l l s . In animal c e l l s i t has been shown that a c t i n , a c t i n binding proteins and the plasma membrane are c l o s e l y 4 associated with one another (Luna 1991, Isenberg 1991). Depolymerization experiments with the fungal metabolites Cytochalasins, which s p e c i f i c a l l y bind to F-actin (Brenner and Korn 1980, Goddette and Frieden 1986), show varied e f f e c t s often including fragmentation and the formation of f o c i amidst a c t i n microfilaments. As a r e s u l t of t h e i r structure and behaviour, f o c i have been described as centers of F-actin organization (Schliwa 1982, Yahara et a l . 1982). Repolymerizing arrays of F-a c t i n a r i s i n g from these f o c a l regions give supporting evidence for t h e i r p a r t i c i p a t i o n i n F-actin assembly and provide information on how the organization of the microfilament network and i t s association with organelles i s established (Parthasarathy 1985). Polymerization of tubulin i n plant c e l l s i s observed i n conjunction with nucleation s i t e s (Hogetsu 1986). However, the occurrence of highly structured microtubule organizing centers (MTOCs) i s less common in higher plant than animal c e l l s (Lloyd 1987). Possible s i t e s for nucleation of microtubules include the nuclear envelope (Wick and Duniec 1983, Clayton et a l . 1985, Lloyd 1987, Falconer et a l . 1988, Marc and Palevitz 1990, Astrom et a l . 1991), regions of the c o r t i c a l cytoplasm (Hogetsu 1986, Cleary and Hardham 1990, Marc and Palevitz 1990) and the plasma membrane (Falconer et a l . 1988, Marc and Palevitz 1990, Astrom et a l . 1991). Microtubule i n h i b i t o r s and low temperatures can be used to disrupt the organization of the microtubular system and often to depolymerize microtubules completely. Repolymerization 5 experiments can then give insight into the establishment of microtubule growth, the pattern of o r i e n t a t i o n and i n t e r a c t i o n with d i f f e r e n t c e l l u l a r compartments, p a r t i c u l a r l y nucleation s i t e s (Mitchison and Kirschner 1984, Murray 1984, Bre et a l . 1987) . In t h i s study Cytochalasins are used to investigate the involvement of microfilament f o c i i n the assembly and organization of the F-actin array. The microtubule depolymerizing herbicide Oryzalin (Strachan and Hess 1983, Cleary and Hardham 1988 & 1990, Wasteneys and Williamson 1989), the s t a b i l i z i n g drug taxol (Horwitz 1992), and cold temperature which depolymerizes some microtubule systems (Troutt et a l . 1990, Akashi and Shibaoka 1991, Astrom et a l . 1991), are also used to investigate the assembly and organization of the microtubule bundle array and to determine whether or not the B-tubulin spots act as nucleation centers. 6 Materials and Methods Culture procedures Vaucheria l o n q i c a u l i s var. macounii was c o l l e c t e d from an i n t e r t i d a l region of North Vancouver, B.C., and grown on i t s natural substrate, supplemented with half strength Instant Ocean (Aquarium Systems, Inc., Eastlake, Ohio) medium with added minor elements (Lewin 1966) and s o i l extract. Cultures were kept at 10 °C, with v i t a - l i t e fluorescent l i g h t i n g providing 250 /xE m - 2 s - 1 under a 16-8h light-dark photoperiod. D i f f e r e n t i a l interference contrast and fluorescence microscopy Filaments of Vj. l o n q i c a u l i s var. macounii selected f o r microscopical studies were removed from a c t i v e l y growing cultures. Detailed observations and fluorescence micrographs were obtained using a L e i t z Dialux 20EB compound l i g h t microscope equipped with epifluorescence optics, and a Zeiss Axiophot microscope equipped with d i f f e r e n t i a l contrast optics. Confocal laser scanning microscopy images were obtained from a BioRad apparatus with attached Zeiss Axiophot microscope. Fluorescein l a b e l l e d p h a l l o i d i n (Sigma Chemical Co, USA) stock s o l u t i o n was made up i n 100% methanol at 2 x 10~ 5 M and stored at -20 °C. The s t a i n was reconstituted i n 0.2 M phosphate buffered s a l i n e (PBS) (osmolarity equal to that of the growth medium) containing 10 mM EGTA (ethylene g l y c o l - b i s (B-aminoethyl ether) N,N,N,N'-tetraacetic acid) and 1 mM MgS04 (and 2%'ethanol or 1% dimethylsulphoxide (DMSO) for permeabilization purposes 7 (Pierson 1988, Heslop-Harrison and Heslop-Harrison 1991). Incubations were c a r r i e d out for 15 minutes at room temperature. For controls, competition staining was c a r r i e d out by pre-incubation with unlabelled p h a l l o i d i n at 1.2 x 10~ 3 M before incubation with f l u o r e s c e i n - l a b e l l e d p h a l l o i d i n , as previously described. C e l l s were rinsed i n PBS before examination.. For i n d i r e c t immunofluorescence, c e l l s were f i x e d using f r e s h l y made 3% formaldehyde, 0.1% glutaraldehyde i n PBS or cytoskeleton s t a b i l i z i n g buffer (5 mM MgSC^, 100 mM PIPES (piperazine-N,N'-bis [2-ethanesulphonic a c i d ] ) , 10 mM EGTA pH 7.3) for 1 hour at room temperature or 4 °C. Use of sodium borohydride a f t e r f i x a t i o n did not v i s i b l y improve the fluorescence image. Fixed c e l l s were placed on a poly-L-lysine (Sigma Co., USA, > 300,000 MW) coated s l i d e , i n a drop of PBS and cut into small fragments (1 mm or shorter). The PBS was then removed and replaced with 10% skim milk as a blocking agent against non-specific antibody binding. Primary antibodies were monoclonal mouse anti-B-tubulin at d i l u t i o n 1:300 - 1:500, monoclonal mouse a n t i - a c t i n at d i l u t i o n 1:1000. Secondary antibody was goat anti-mouse at d i l u t i o n 1:30. A l l antibodies were from Amersham Corp., USA. A polyclonal r a b b i t a n t i - t u b u l i n (Sigma Co, USA) at d i l u t i o n 1:10 was used as c o n t r o l . Dilutions were made using PBS containing 0.1% NaN3 and 1 mg/ml BSA (bovine serum albumin) pH 7.3. Staining was improved with the use of 1% DMSO i n PBS as a r i n s i n g solution between antibody incubations (Schroeder et a l . 1985, Traas et a l . 1987). F i n a l mounting 8 medium was a 1:1 s o l u t i o n of g l y c e r o l and PBS, containing 0.1% phenylenediamine as an anti-quenching agent. When required, sections of f i x e d c e l l s were obtained using a freeze stage microtome. High r e s o l u t i o n scanning electron microscopy Specimen preparation followed the protocol of Blackmore et a l . (1984), modified for the preservation of c y t o s k e l e t a l elements. This involved rapid freezing i n l i q u i d propane before f r a c t u r i n g the specimens i n l i q u i d nitrogen. Specimens were examined using a H i t a c h i S-4100 high resolution f i e l d emission scanning electron microscope, at an accelerating voltage of 5.0 kV. Electrophoresis and immunoblotting Crude protein extracts of Vaucheria were made by freezing approximately 0.4 g of t i s s u e i n l i q u i d nitrogen, and grinding i t i n extraction buffer on i c e (La C l a i r e 1991 and references t h e r e i n ) . The remaining suspension was centrifuged, the supernatant d i l u t e d 1:4 with electrophoresis sample buffer and incubated i n b o i l i n g water for 5 minutes. Approximately 5 nq of Vaucheria protein per well was loaded (calculated with the BioRad Protein Assay using the Bradford method) before electrophoresis was performed. The bovine brain tubulin was a generous g i f t from Dr. W. Vogl, Dept. Anatomy, UBC, and the chicken gizzard myosin was from Sigma Co, USA. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) was c a r r i e d out according to Laemmli (1970) using a BioRad Protean II apparatus and 16 cm x 9 0.75 mm thi c k 12% polyacrylamide gels, except f o r the myosin immunoblot when a 7.5% gel was used. Semi-dry transfers were performed onto n i t r o c e l l u l o s e (Towbin et a l . 1979) which was then blocked using 3% f i s h skin g e l a t i n or 0.5% skim milk (Johnson et a l . 1984). A n t i - a c t i n and anti-B t u b u l i n monoclonal primary antibodies were as used for immunofluorescence but at a higher d i l u t i o n (1:1000). Monoclonal anti-pan myosin (Amersham Corp., USA) was used at a d i l u t i o n of 1:10. The secondary antibody was goat anti-mouse a l k a l i n e phosphatase (Gibco BRL, Canada) used at a d i l u t i o n of 1:1500, and the colour reaction s o l u t i o n contained Fast Red s a l t and Naphthol AS-MX phosphoric ac i d (3-hydroxy-2-naphthoic acid-2,4-dimethylanilide phosphate) (Sigma Co, USA) i n 50 mM T r i s HC1 pH 8.0. A f f i n i t y p u r i f i e d r a b b i t polyclonal a n t i -Dictyostelium myosin (Dr. J. W. La C l a i r e , U n i v e r s i t y of Austin, Texas) was d i l u t e d 1:250 i n T r i s buffered s a l i n e with 5% milk and 0.5 mg/ml NaN3, and the secondary antibody was goat anti - r a b b i t a l k a l i n e phosphatase (Sigma Co, USA) d i l u t e d at 1:1000. Depolymerization experiments Stock solutions of Cytochalasin B and D were made at 20 mg/ml and 1 mg/ml i n 100% DMSO, and d i l u t e d with growth medium to working concentrations of 200 jxg/ml and 10 /ig/ml respectively. Stock s o l u t i o n of Oryzalin was made at 7.5 mM i n 100% acetone and di l u t e d with growth medium to a working concentration of 10 jiM. Stock s o l u t i o n of taxol was made at 2.3 mM i n 100% DMSO and di l u t e d with growth medium to 30 - 50 /iM. A l l stock solutions were stored at -20° C. DAPI (4,6-Diamidino-2-phenylindole) was 10 made up d i r e c t l y i n growth medium at a concentration of 5 /xg/ml. A l l chemicals were purchased from Sigma Chemical Co., except for Oryzalin [4(Dipropylamino)-3,5-dinitrobenzenesulfonamide, synonym Surflan] which was a generous g i f t from Dow Elanco Co., Greenfield, Indiana, and taxol, which was a generous g i f t from the National Cancer I n s t i t u t e , Bethesda, Maryland. When appropriate controls using 1% DMSO or 0.1% acetone i n growth medium were also c a r r i e d out, and these solvents revealed no changes i n the respective cytoskeletal arrays. C e l l s were incubated at room temperature for approximately 10 minutes with Cytochalasins and 15 mins with Oryzalin. Cold temperature treatments were performed by cooling v i a l s of growth medium to freezing temperature with a thermoelectric cold p l a t e (Thermoelectrics Unlimited, Inc), the average temperature was -2° C and the cooling period approximately 30 minutes. Temperatures were measured using a c a l i b r a t e d d i g i t a l thermometer probe. Repolymerization experiments A f t e r the depolymerization treatments, c e l l s were transferred to i n h i b i t o r - f r e e medium, or medium at 10 °C, as appropriate. They were then p e r i o d i c a l l y examined under a microscope f o r the restoration of cytoplasmic streaming, and stained to assess the degree and sequence of re c o n s t i t u t i o n of both the microfilament and microtubule arrays. 11 S t a t i s t i c a l analysis T h i r t y microfilament cables and microtubule bundles were measured from micrographs i n order to ca l c u l a t e the mean width, and t e s t whether microtubule bundles are s i g n i f i c a n t l y wider than microfilament cables. Means i n the text are provided with standard error f i g u r e s . The difference between microfilament cable and microtubule bundle mean width was found to be s i g n i f i c a n t (p = .002), using the Student t t e s t (Parker 1980) where population variances are not assumed to be equal. However, these measurements are considered to be approximate due to the exaggerated e f f e c t of the emitted l i g h t from the fluorochrome. 12 Results - Part 1 Protein extraction and analysis Bands of the appropriate molecular weight f o r a c t i n (~42 kDa) and t u b u l i n (-55 kDa) are consistently present i n the t o t a l protein extracts of Vaucheria l o n q i c a u l i s var. macounii. In contrast a putative myosin band (-180 Mr) which co-migrates with the myosin molecular weight marker, i s only present upon c a r e f u l extraction using protease i n h i b i t o r s and conditions designed to reduce protease a c t i v i t y (Fig. 1). Immunoblots using commercial monoclonal primary antibodies raised to bovine brain t u b u l i n (B sub-unit) and chicken gizzard a c t i n revealed p o s i t i v e cross-reactions with Vaucheria proteins. The a n t i - a c t i n cross-reacted with only a sing l e band of approximately 47 Mr, whereas the a n t i -B t u b u l i n cross-reacted with two bands of approximately 55 Mr and 46.5 Mr (Figs. 1A & B). Immunoblots using Amersham's monoclonal anti-pan myosin gave a p o s i t i v e cross-reaction with bands of approximately 47 Mr, i n d i c a t i n g the presence of probable myosin breakdown products (Fig. 1C). Immunoblots were also performed using a polyclonal anti-Pietyostelium myosin antibody, which showed only a very f a i n t i n d i c a t i o n of cross-reaction at approximately 116 Mr (Fig. ID). Controls f o r the immunoblotting procedure included omission of the primary antibody (negative control) and use of p u r i f i e d protein standards (positive c o n t r o l ) . The negative controls showed no cross-reaction, i n d i c a t i n g absence of endogenous phosphatase a c t i v i t y , while the 13 pos i t i v e controls showed cross-reactions with the tub u l i n and myosin bands, confirming the effectiveness of these antibodies for t h e i r i d e n t i f i c a t i o n . D i f f e r e n t i a l interference contrast microscopy observations D i f f e r e n t i a l interference contrast microscopy shows the cytoplasm of vegetative filaments of Vaucheria l o n g i c a u l i s var. macounii to contain an array of c a b l e - l i k e filamentous structures which resemble tracks (Figs. 2 & 3). These are observed throughout the length of the vegetative filaments. The elements of t h i s array appear as long, mostly unbranched cables l y i n g p a r a l l e l to the l o n g i t u d i n a l axis of the c e l l . Their density varies along the length of the c e l l . The organization of the elements of t h i s array i s constantly changing with time. This occurs concurrently with the transport of the organelles they are clo s e l y associated with, such as n u c l e i , small p a r t i c l e s and chloroplasts. Microfilament organization: fluorescein conjugated p h a l l o i d i n studies Figure 4 shows l a b e l l i n g of F-actin using f l u o r e s c e i n -conjugated p h a l l o i d i n i n the mid-filament region of a Vaucheria l o n g i c a u l i s var. macounii c e l l . Controls for t h i s procedure involved examination of the c e l l s a f t e r incubation i n p h a l l o i d i n -free s o l u t i o n , or a f t e r incubation with high concentration unconjugated p h a l l o i d i n (in order to saturate the a c t i n binding 14 sites) followed by incubation with fluorescein-conjugated p h a l l o i d i n . The controls showed l i t t l e or no p o s i t i v e s t a i n i n g (not shown). A c t i n microfilaments are not e a s i l y v i s u a l i z e d by i n d i r e c t immunofluorescence using the Amersham a n t i - a c t i n antibody, or by phalloidin-FITC staining a f t e r conventional aldehyde f i x a t i o n . However, good preservation of these elements can be achieved by incubating the c e l l s with p h a l l o i d i n (which binds to a c t i n and s t a b l i z e s the microfilaments) before aldehyde f i x a t i o n (Fig. 5). F-actin i s seen to be organized into c a b l e - l i k e strands, which extend throughout the length of the vegetative filaments (Figs. 4 & 5) . Each c a b l e - l i k e strand averages 0.37 ± 0.03 /xm i n diameter, and approximately 16 strands seem to occur across the c e l l per f i e l d of view. Taking into consideration that the average thickness of a microfilament i s 5-7 nm (Staiger and Schliwa 1987, Derksen et a l . 1990), each c a b l e - l i k e strand consists of about 60 i n d i v i d u a l microfilaments. The preponderant ori e n t a t i o n of these strands i s p a r a l l e l to the lo n g i t u d i n a l axis of the vegetative filaments. Local branching of microfilament cables occurs i n close proximity to organelles (Fig. 6). Occasionally microfilaments were v i s u a l i s e d as shorter and more angular c r y s t a l l i n e arrays (Fig. 7), consistent with a l t e r a t i o n s due to osmotic e f f e c t s on the molecular structure of a c t i n (Janmey 1991). Microfilament strands are often seen to be c l o s e l y associated with chloroplasts (Fig. 8), forming channel-l i k e regions through which these organelles may t r a v e l during 15 cytoplasmic streaming. Towards the t i p region of each vegetative filament, the c a b l e - l i k e strands extend i n t o the a p i c a l dome, become more r e t i c u l a t e , and form areas of very intense fluorescence (Fig. 9). When observed from a d i f f e r e n t perspective a f i n e network of cables can be seen to radiate from these bright fluorescent areas (Fig. 9, i n s e t ) . Through m u l t i - l e v e l focussing (Fig. 4, inset) and confocal laser scanning microscopy (Figs. 12 & 13), i t i s possible to demonstrate that the whole microfilament array i s almost exclusively located i n the f i r s t few microns of the ( c o r t i c a l ) cytoplasm (actual depth depending on the i n d i v i d u a l s i z e of the c e l l ) . Figures 14 and 15 quantify F-actin d i s t r i b u t i o n along the transverse and longitudinal axes of the vegetative filament res p e c t i v e l y . The microfilament population i s d i s t r i b u t e d uniformly both circumferentially (Fig. 14) and l o n g i t u d i n a l l y (Fig. 15) i n the c e l l . Microfilament cables closer to the plasma membrane seem st r a i g h t e r and a x i a l l y aligned, whereas those placed at a greater depth are more r e t i c u l a t e and i n closer contact with chloroplasts (Fig. 10). This i s v e r i f i e d by the confocal s e r i a l scans (Fig. 13) which demonstrate r e t i c u l a t e a c t i n cables (3rd and 4th scan) at a greater depth than the a x i a l l y aligned ones (1st and 2nd scan). A variable number (up to 20) of f o c a l points from which 4 to 10 microfilament cables are seen to radiate occurs i n each c e l l . These are found both i n the a p i c a l region (Fig. 9) as well as throughout the vegetative filament (Fig. 11 & i n s e t ) . Figure 16 13 also indicates the existence of f o c i amidst the r e t i c u l a t e population of microfilaments seen i n the 3rd and 4th scans. Microtubule organization: i n d i r e c t immunofluorescence studies Optimum p o s i t i v e staining of the microtubular compartment was obtained by subjecting fixed material, cut into small fragments, to permeabilization with low concentration DMSO (1%) and subsequent s t a i n i n g with the appropriate antibody sol u t i o n . Incubation periods of up to 72 hours were necessary to allow d i f f u s i o n of the antibodies into the cut ends and throughout the body of the c e l l fragment. Controls for t h i s procedure consisted of incubation with only the secondary antibody, or incubation with a primary antibody raised i n a d i f f e r e n t animal (rabbit) to that of the experimental primary antibody (mouse). Both types of control methods resulted i n c e l l s with no s p e c i f i c l a b e l l i n g and only dim chloroplast autofluorescence was v i s i b l e (not shown). Figure 16 shows l a b e l l i n g of the B-tubulin i n the mid-filament region of a vegetative c e l l . B-tubulin can be seen to be organized into c a b l e - l i k e structures measuring approximately 0.57 + 0.05 jum i n diameter, s i g n i f i c a n t l y wider than the a c t i n microfilament cables. Assuming that the average microtubule measures 25 nm i n diameter (Bershadsky and V a s i l i e v 1988, Derksen et a l . 1990), these cables consist of approximately 23 individual microtubules. The mean number of microtubule bundles per f i e l d of view i s 7, s i g n i f i c a n t l y less than the density of microfilament cables. Microtubule bundles are always straight 17 and shorter (tens of microns i n length) than microfilament cables (hundreds of microns). This i s determined through confocal scanning lase r microscopy observations where successive o p t i c a l scans show that microtubule bundles do not span the e n t i r e length of the vegetative filament (Fig. 18). The preponderant orient a t i o n i s p a r a l l e l to the l o n g i t u d i n a l axis of the c e l l . Microtubule bundles follow c l o s e l y the curvature of the c e l l ' s surface. This i s noticeable at points where the vegetative filament branches (Fig. 17), and i n the a p i c a l dome of the c e l l where they form a loose network (Fig. 19). Branching or r e t i c u l a t i o n of the microtubule network i s not seen i n other areas of the c e l l , and the bundles have no obvious physical association with the majority of the organelles . However, an i n t e r a c t i o n between microtubule bundles and chloroplasts seems to exi s t , as indicated by t h e i r close association with extruded cytoplasm preparations (Fig. 23). There are no obvious f o c a l points, s i m i l a r to those observed f o r microfilaments, from which the microtubule bundles seem to, o r i g i n a t e . Successive o p t i c a l sections taken with the confocal laser scanning microscope show that microtubule bundles occur throughout the f u l l width of the cytoplasm i n vegetative filaments (Fig. 21). This i s confirmed by Figure 20 that shows a cross-sectional scan of microtubule bundle fluorescence. Figure 22 shows a transverse section with punctate B-tubulin fluorescence (presumably microtubule bundle ends) occupying the 18 f u l l width of the cytoplasm of the vegetative filament to a depth (of up to about 15 um) close to the vacuole. High r e s o l u t i o n scanning electron microscopy studies Figure 24 shows a low magnification high r e s o l u t i o n scanning electron micrograph of a transverse fractured face of a filament of Vaucheria l o n g i c a u l i s var. macounii. Chloroplasts occupy most of the vegetative filament cytoplasm. These organelles and t h e i r i n t e r n a l membranes seem to be well preserved. Two types of fibrous structures have been observed i n such preparations. In Figure 24 (arrowhead) a f i b r i l l a r network can be discerned. At higher magnification, t h i s f i b r i l l a r network appears as an i r r e g u l a r web of fibrous elements connecting d i f f e r e n t organelles (Fig. 25). These fibrous elements measure approximately 50 nm i n diameter. Spherical bodies, measuring 50-100 nm i n diameter, are frequently found i n c l u s t e r s i n association with the fibrous elements of t h i s network. Figure 26 shows another type of c a b l e - l i k e fibrous strand i n the cytoplasm of a vegetative filament of Vaucheria. In t h i s case, the i n d i v i d u a l elements are unbranched, s t r a i g h t and of determinate length. Figure 27 shows a higher magnification of these structures. The thinner elements measure approximately 50 nm i n diameter and the wider ones 100 nm. Their i n t e r a c t i o n with organelles seems to be better defined than i n the case of the web-like elements. 19 Discussion - Part 1 The cytoskeleton of Vaucheria species has previously been studied by both d i f f e r e n t i a l interference contrast (DIC) optics and electron microscope studies (Ott and Brown 1972, B l a t t and Briggs 1980, O l i v e i r a and F i t c h 1988, Ott 1992, Tornbom and O l i v e i r a , unpublished). Images of arrays of p a r a l l e l fibrous cables observed i n close association with organelles by d i f f e r e n t i a l interference contrast microscopy are s i m i l a r to those seen i n t h i s study. Ott and Brown (1972) and O l i v e i r a and Fi t c h (1988) showed using transmission electron microscopy, that microtubules are organized i n bundles of 10-20 units, either i n close contact with n u c l e i or the surface membrane of the c e l l . Microfilaments are not e a s i l y v i s u a l i z e d by transmission electron microscopy, apparently due to the disruptive e f f e c t s of aldehyde f i x a t i v e s (Ott and Brown 1974, B l a t t et a l . 1980, Doonan et a l . 1988, and our own observations). Kengen and de Graaf (1990) suggested that the s t a b i l i t y of a c t i n during f i x a t i o n may be dependent upon i t s i n t e r a c t i o n with other cytoskeleton proteins (e.g. microtubules or a c t i n binding proteins), and t h i s i s worth bearing i n mind when considering the organization and d i s t r i b u t i o n of Vaucheria a c t i n . Overall, the studies have been rather fragmented and no cle a r understanding of the dynamics of the organization of the cytoskeleton exists i n t h i s genus. In t h i s study SDS-PAGE and immunoblots confirm that two of the major cytoskeletal proteins, a c t i n and tubul i n , are present 20 i n t h i s alga. Commercially obtained monoclonal antibodies, raised against mammalian forms of the proteins cross-react with them demonstrating conserved epitopes. The a c t i n molecule i s a highly conserved one (La C l a i r e 1991), and i n Vaucheria i t appears to be of s i m i l a r molecular weight to actins from animal sources (43 kDa) and plant c e l l s such as Nitella (46 kDa, A l l e n and A l l e n 1978) and tomato (42 - 45 kDa, Seagull 1989). The upper band cross-reacting with the anti-B t u b u l i n i s of a s i m i l a r molecular weight to bovine brain tubulin, while the lower band of approximately 46.5 Mr i s probably a proteolysis breakdown product of B-tubulin. The same antibodies have also been used to demonstrate the presence of a c t i n i n Chara (Williamson et a l . 1987), Pisum (Abe and Davies 1991), pollen tubes (Tang et a l . 1989) and the fungus Phytophthera (Temperli et a l . 1990), and tubulin i n Allium (Gubler 1989) and Phytophthera (Temperli et a l . 1990) . These antibodies are therefore suitable f o r i d e n t i f i c a t i o n and l o c a l i z a t i o n of a c t i n and tubulin containing structures by immunofluorescence i n Vaucheria filaments. There i s now a substantial body of evidence for the existence of an actomyosin system i n plant and a l g a l c e l l s , e s p e c i a l l y i n association with cytoplasmic movement and wound healing (Williamson 1986, Williamson et a l . 1987, Kohno and Shimmen 1988, La C l a i r e 1991). Our r e s u l t s indicate that myosin i s also present i n Vaucheria. However, t h i s i s known to be a very l a b i l e protein and not as highly conserved as a c t i n (Parke et a l . 1986, La C l a i r e 1991). Only a very f a i n t band of 21 approximate molecular weight 116 Mr showed a cross-reaction with anti-Dictyostelium myosin heavy chain polyclonal antibody. This antibody was shown to cross react with proteins, probably including myosin, i n the alga Ernodesmis (La C l a i r e 1991). The 55 Mr band, which cross-reacts with the commercial monoclonal anti- xpan' myosin, i s l i k e l y a proteolysis breakdown product. This anti-myosin has been shown to cross-react with higher plant myosin (Parke et a l . 1986). At present, the absence of a recognizable myosin cross-reaction renders these antibodies unsuitable f o r myosin i d e n t i f i c a t i o n and l o c a l i z a t i o n by immunofluorescence i n Vaucheria filaments. P h a l l o i d i n and immunofluorescence-labelling give c l e a r images of a c t i n and tubulin d i s t r i b u t i o n i n vegetative filaments of Vaucheria. res p e c t i v e l y . Actin immunofluorescence did not give s a t i s f a c t o r y r e s u l t s , perhaps due to d i f f i c u l t y of penetration of the large Amersham IgM monoclonal a n t i - a c t i n antibody (La C l a i r e 1989). C e l l s f i x e d with aldehyde a f t e r p h a l l o i d i n s t a b i l i z a t i o n show the d i s t r i b u t i o n of the F-actin arrays to be s i m i l a r to that of c e l l s permeabilized with low concentration DMSO, known to improve fine s t a i n i n g of F-actin arrays (see materials and methods for references). I t i s also important to note that i n the present study, the d i s t r i b u t i o n and organization of fluorescent microfilament bundles i s s i m i l a r to that of fibrous structures seen by d i f f e r e n t i a l interference contrast microscopy of l i v i n g specimens. Ott (1992) describes the existence of a membranous ER-like compartment ( m o t i l i t y 22 associated reticulum or MAR), which he proposes to be equivalent to the DIC cytoplasmic tracks and to p a r t i c i p a t e i n cytoplasm and organelle m o t i l i t y . The XMAR' elements were shown to .be associated with a d i f f u s e a c t i n - l i k e network. Our observations tend to support Ott's (1992) conclusions that the AMAR' i s co-lo c a l i z e d with microfilaments i n vegetative filaments of Vaucheria. However, bearing i n mind that B l a t t et a l . (1980) presented good evidence for the presence of F-actin bundles of sim i l a r dimensions to those seen i n t h i s study, and that Ott (1992) himself proposes that microfilaments may form bundles during m o t i l i t y , our r e s u l t s showing the occurrence of microfilament bundles i n Vaucheria more l i k e l y represents the true configuration (Sonobe and Shibaoka 1989). The system of p a r a l l e l a c t i n cables running l o n g i t u d i n a l l y to the c y l i n d r i c a l body of the vegetative filament i n Vaucheria i s s i m i l a r i n organization to that found i n other elongated plant and a l g a l c e l l s e x h i b i t i n g cytoplasmic streaming (Palevitz and Hepler 1975, Parthasarathy et a l . 1985, Menzel and Schliwa 1986a, Tewinkel et a l . 1989, Jackson and Heath 1990). The microfilament cables of Vaucheria are approximately 0.37 jum i n diameter, s i m i l a r to estimates obtained from other plant and a l g a l c e l l s (Kersey and Wessels 1976, B l a t t et a l . 1980, Parthasarathy and Pesacreta 1980). Estimates of approximately 60 i n d i v i d u a l microfilaments per cable i n Vaucheria are also s i m i l a r to those of Chara a c t i n cables (Staiger and Schliwa 1987). High resolution scanning electron micrographs (HRSEMs) show an 23 i r r e g u l a r web-like fibrous array s i m i l a r to the f i n e , r e t i c u l a t e fluorescence of F-actin, and with dimensions that could correspond to bundles of several microfilaments. However, estimates of numbers of microfilaments per bundle c a r r i e d out with fluorescence observations may be d i f f e r e n t due to the 'cone' e f f e c t of fluorescent l i g h t emissions that increases the o v e r a l l diameter of the cabl e - l i k e structures. In addition, metal coating i n HRSEM can add several nanometers to the dimensions of structures v i s u a l i z e d by t h i s technique (Ip and Fischman 1979). The microfilament array of Vaucheria seems to be composed of two sub-sets of cables: one made up of f a i r l y s t r a i g h t elements, oriented p a r a l l e l to the longitudinal axis of the filament and adjacent to the plasma membrane, and beneath i t a r e t i c u l a t e set in close association with organelles, p a r t i c u l a r l y chloroplasts. This observation i s supported by confocal scan images which reveal a more r e t i c u l a t e F-actin array, at a greater depth i n the cytoplasm than the a x i a l l y aligned microfilament cables. Two si m i l a r populations of a c t i n microfilaments were observed i n fern protonemata (Kadota and Wada 1989). Wasteneys and Williamson (1991) also observed d i s t i n c t a c t i n microfilaments i n N i t e l l a . with s u b - c o r t i c a l cables associated with chloroplasts and e n c i r c l i n g n u c l e i which rotate i n t h i s species. One possible i n t e r p r e t a t i o n i s that the straighter microfilament bundles provide guiding tracks, and the r e t i c u l a t e ones, i n association with organelles, enable them to move along those tracks. This could be s p e c i a l l y true for chloroplasts, that show a very close 24 association with the r e t i c u l a t e microfilament bundles located beneath the straighter set. In Vaucheria chloroplasts frequently reverse t h e i r d i r e c t i o n of t r a v e l , implying the existence of microfilaments of d i f f e r e n t p o l a r i t i e s within the array (Tornbom and O l i v e i r a , unpublished). The existence of two sets of microfilaments could then provide a system f o r the regulation of complex organelle movement. Large coenocytic algae such as Vaucheria may require the a b i l i t y to accomplish sudden d i r e c t i o n a l changes, as i n response to wounding (Tornbom and O l i v e i r a , unpublished). Large c r y s t a l l i n e - l i k e arrays of microfilament bundles occasionally seen i n Vaucheria filaments seem to a r i s e from, the aggregation of a c t i n as a r e s u l t of osmotic stress (Suzuki et a l . 1989). Similar structures, v i s u a l i z e d using p h a l l o i d i n staining, have been documented by Heslop-Harrison et a l . (1986), who postulated they were the r e s u l t of F-actin storage i n l i n e a r deposits. Pierson et a l . (1986) reported a c t i n f o c i i n pollen tubes which are *dense and star shaped', and suggested a possible r o l e i n a c t i n organization although acknowledging that they could be stress f i b r e s . The c r y s t a l l i n e - l i k e a c t i n aggregates i n Vaucheria are d i s t i n c t from the microfilament f o c i seen throughout the microfilament array. There are a few other reports of a c t i n f o c i i n the l i t e r a t u r e , but these show l i t t l e s i m i l a r i t y to those observed i n Vaucheria (Menzel 1987, Quader and Schnepf 1989). The r o l e of f o c i i n F-actin organization w i l l be considered i n Part 2 of t h i s study. 25 The organization of F-actin i n the a p i c a l dome of the t i p region of Vaucheria i s characterized by dense r e t i c u l a t i o n associated with intense pools of fluorescence, po s s i b l y f o c i . A si m i l a r organization observed at the apex i n other tip-growing c e l l s i s consistent with a r o l e i n exocytosis of v e s i c l e s carrying materials needed f o r the expansion of the c e l l wall (Schnepf 1986, Segawa and Yamashina 1989, Steer and Steer 1989). Microtubule bundles are l o n g i t u d i n a l l y oriented to the c y l i n d r i c a l body of Vaucheria filaments. The short, s t r a i g h t microtubule bundles v i s u a l i z e d i n t h i s study are consistent with Ott's (1992) de s c r i p t i o n of a 'microtubular probe', attached to the anterior end of each nucleus and apparently involved i n i t s m o t i l i t y . The close association of nuclei and microtubule bundles i n Vaucheria (Ott and Brown 1972) and t h e i r response to the microtubule depolymerizing agent Oryzalin (Tornbom and O l i v e i r a , unpublished) give strong evidence supporting a r o l e for microtubules i n nuclear m o t i l i t y . Vaucheria i s a tip-growing c e l l (Kataoka 1975) and simi l a r organizations of microtubule arrays are seen i n other c e l l s e x h i b i t i n g t i p growth (Derksen et a l . 1985, Doonan et a l . 1988, Temperli et a l . 1990). Therefore, the common features of microtubule arrays of tip-growing c e l l s imply i n t e g r a l functions related to the formation and maintenance of a pol a r i z e d c e l l structure (Lloyd 1984, Schnepf 1986). O l i v e i r a and F i t c h (1988) observed the constant presence of a group of up to 20 nuclei behind the api c a l dome of the t i p of the Vaucheria filament, while hundreds of other n u c l e i are 26 d i s t r i b u t e d randomly throughout the remaining cytoplasm. These nuclei are associated with a loose network of microtubules present i n the t i p region. In t h i s manner they may also p a r t i c i p a t e i n the establishment of conditions preserving the i n t e g r i t y of the a p i c a l region (Doonan et a l . 1988). Indeed, nuclei have been shown to influence the s i t e of t i p i n i t i a t i o n and to be transported equidistant to growing t i p s i n a number of tip-growing systems (see Schnepf 1986 for review), although a d i r e c t r o l e of n u c l e i i n the establishment of p o l a r i t y could not be demonstrated so f a r . There are no obvious f o c a l regions f o r microtubule organization i n Vaucheria. However, bright B-tubulin fluorescence spots observed i n association with nuclei are consistent with the p o s s i b i l i t y that they may represent nucleation or polymerization s i t e s (Part 2). Observations of depolymerization of tubulin a f t e r cold treatment, revealing a pool of fluorescence at the end of each shortened microtubule bundle are also consistent with t h i s i n t e r p r e t a t i o n . Further support comes from studies showing s i m i l a r pools of fluorescence i n repolymerizing microtubule bundles experiments (Part 2). L i t t l e c o - d i s t r i b u t i o n of microtubule and microfilament arrays i s seen i n Vaucheria. probably due to the marked differ e n c e i n numbers of elements of the two arrays. However, the e f f e c t of both Cytochalasins and Oryzalin on organelle behaviour and cytoplasmic streaming suggests that both are involved i n c e l l m o t i l i t y and i n t r a c e l l u l a r organization (Tornbom 27 and O l i v e i r a , unpublished). Cross-bridging molecules such as MAP-2 could l i n k the two systems as suggested i n Acetabularia (Menzel and Elsner-Menzel 1989) and microtubule bundles could also serve as a template for orien t a t i o n and a s t a b i l i z e r of microfilament cables and the acto-myosin motor system (Hepler and Palevitz 1974, Menzel and Schliwa 1986a, La C l a i r e 1987). 28 Results - Part 2 Untreated c e l l s Figures 2.1a and b are d i f f e r e n t i a l interference contrast (DIC) micrographs, taken at an i n t e r v a l of approximately one minute apart, of a portion of a vegetative filament of Vaucheria l o n q i c a u l i s var. macounii. The cytoplasm i s organized into a network of c a b l e - l i k e strands (tracks) which seem to radiate from f o c a l regions (arrows). Cytoplasmic tracks are not s t a t i c within the c e l l ; hence, t h e i r reorganization from f o c a l regions i s i n a constant state of f l u x . Observed with f l u o r e s c e i n - p h a l l o i d i n (Ph-FITC) s t a i n i n g , these regions appear as bright fluorescent areas from which F-actin cables seem to radiate (Fig. 2.2). The si z e of these f o c i i s quite variable and they can measure from 1 to 6 microns i n diameter. In comparison, f o c i - l i k e structures v i s i b l e i n DIC microscopy measure approximately 2 to 5 p . No clear r e l a t i o n s h i p seems to exis t between organelles and the f o c i , although the association of organelles with cytoplasmic tracks i s well defined when observed by DIC microscopy (Part l ) . No f o c i s i m i l a r to those observed f o r microfilaments have been i d e n t i f i e d i n association with the microtubule bundle array of the vegetative Vaucheria filament (Fig. 2.3). However, fix e d c e l l s , sectioned with a freeze-stage microtome, often produce oblique cut ends which reveal an image of the cytoplasm viewed from e i t h e r the plasma membrane or the tonoplast sides. These reveal punctate fluorescence when viewed from the tonoplast side (Fig. 2.4a) and filamentous fluorescence when viewed from the 29 plasma membrane side (Fig. 2.4b). Given the f a c t that the predominant orien t a t i o n of the microtubule bundles i s p a r a l l e l to the c e l l surface and lo n g i t u d i n a l axis of the vegetative filament (Fig. 2.3), these punctate structures possibly represent aggregates of B-tubulin sub-units which could function as microtubule-associated f o c a l regions. F-actin f o c i In order to determine whether f o c i are r e l a t e d to the assembly and organization of the microfilament network, the c e l l s were treated with Cytochalasins (F-actin i n h i b i t o r s ) . Most cytoplasmic streaming ceases a f t e r 5 to 10 minutes of incubation with 10 /ig/ml Cytochalasin D. Tornbom and O l i v e i r a (unpublished) found that a much higher concentration (200 /zg/ml) of Cytochalasin B was required to stop cytoplasmic streaming almost immediately. Streaming i s restored 15 to 20 minutes a f t e r transfer to Cytochalasin-free growth medium. No change of c e l l shape i s observed upon treatment with Cytochalasins. Fluorescence microscopy reveals that treatment of the c e l l s with Cytochalasins B and D r e s u l t s i n the breakdown of the microfilament bundle array. The extent of the breakdown depends on the concentration and duration of the treatments. Cytochalasin D has a stronger e f f e c t , with very l i t t l e F-actin fluorescence observed when applied at 100 /ug/ml (Fig. 2.5). Cytochalasin B requires a much higher concentration (200 /xg/ml) to achieve a v i s i b l e breakdown of the F-actin array (Fig. 2.6). 30 When applied at 10 /xg/ml fo r approximately 10 minutes, Cytochalasin D was observed to cause breakdown of the microfilament array and hence i t was r o u t i n e l y used at t h i s concentration i n a l l the subsequent studies. The pattern of fluorescence observed a f t e r treatment with Cytochalasin D takes several forms, which may represent d i f f e r e n t stages of the breakdown of the F-actin array. These may r e s u l t from the f a c t that i n d i v i d u a l c e l l s may react to drugs d i f f e r e n t l y so as to produce d i f f e r e n t rates of depolymerization (Cleary and Hardham 1988) . The f i r s t i n d i c a t i o n of a disruption of the microfilament array i s the appearance of specks of brighter fluorescence amidst the a x i a l l y aligned cables (Fig. 2.7, arrows). The F-actin array i s now more r e t i c u l a t e and disorganized when compared to controls (Fig. 2.8). Intense, r e t i c u l a t e F-actin fluorescence becomes p a r t i c u l a r l y apparent adjacent to cross walls formed during the sealing o f f of o l d wound s i t e s (Fig. 2.9). Foci are v i s i b l e amidst the remaining F-actin cables (arrowheads). These f o c a l regions are s i m i l a r to those observed i n c o n t r o l images (Fig. 2.2) and compare well with s i m i l a r DIC structures (Fig. 2.1). Figure 2.10 shows another aspect of F-actin f o c i situated i n t e r m i t t e n t l y along microfilament cables (arrowheads). This pattern of F-actin depolymerization i n close association with f o c a l regions, i s seen to extend throughout most of the length of Cytochalasin-treated vegetative filament (Fig. 2.11, arrow), and into the t i p region (Fig. 2.12). Figure 2.13 shows a region of a 31 c e l l with l i t t l e r e t i c u l a t e fluorescence, but with several F-a c t i n cables that appear to be merging into a larger extremely bright structure. These and other s i m i l a r structures are seen to radiate from a bright fluorescent spot (arrow). Extensive depolymerization and disruption of F-actin i s seen i n Figure 2.14, where there are large areas of the c e l l without any F-actin, except f o r those areas associated with f o c i . Figure 2.15 shows a more advanced stage of F-actin depolymerization with only very compact and b r i g h t l y fluorescent f o c i remaining. The microfilament array becomes highly disorganized a f t e r cold temperature treatment (< 0 °C), with the bundles of microfilaments l o s i n g t h e i r uniform orientation p a r a l l e l to the longitudinal axis of the c e l l and displaying a more i r r e g u l a r configuration. The disorganized remnants of F-actin filaments are seen to be c l o s e l y associated with numerous f o c i - l i k e regions (Fig. 2.16, arrows). The microtubule depolymerizing herbicide Oryzalin has no v i s i b l e e f f e c t on the microfilament array of the vegetative filament, as seen by Ph-FITC l a b e l l i n g (not shown). Recovery of Vaucheria c e l l s a f t e r exposure to i n h i b i t o r s was measured by the r e s t o r a t i o n of cytoplasmic streaming. Fluorescence images reveal microfilament arrays i n the process of reassembly. The elements of t h i s array are better defined and c l e a r l y associated with f o c a l regions (Fig. 2.17, arrows). Similar F-actin arrays containing f o c i are observed a f t e r re-warming of cold-treated c e l l s , again suggesting that the new arrays are i n the process of reassembly (not shown). 32 Microtubule-associated f o c i C e l l s were treated with cold temperature, the herbicide Oryzalin, and the s t a b i l i z i n g agent taxol, to investigate the organization of the microtubule array and i t s possible r e l a t i o n s h i p to the bright fluorescent B-tubulin spots observed amongst the microtubule bundle array (Fig. 2.4). The e f f e c t s observed a f t e r cold and Oryzalin treatments are s i m i l a r . The r e s u l t s reveal that Vaucheria c e l l s have cold-tolerant microtubules. Depolymerization was never observed unless c e l l s were treated with temperatures below 0 °C, and an apparently normal microtubule array was sometimes s t i l l present a f t e r exposure to temperatures as low as -3 °C (Fig. 2.18). Cytoplasmic streaming ceased at temperatures below 0 °C, and a f t e r treatment with 10 /nM Oryzalin. I t was restored upon rewarming of the medium or upon placement of vegetative filaments i n Oryzalin-free growth medium. Recovery occurred within a period of 30 minutes to one hour. Incomplete depolymerization of the microtubule bundles by these treatments reveal intermediate stages of breakdown. The f i r s t i n d i c a t i o n of depolymerization i s marked by the appearance of fragmented microtubule bundles (Fig. 2.23). In t h i s process, bright spots of fluorescence become v i s i b l e at one end of the microtubule bundle (Fig. 2.25, arrow). Other e f f e c t s of incomplete depolymerization include d i s o r i e n t a t i o n of short microtubule bundles so that they l i e at an angle to the 33 longitudinal axis of the c e l l . Some bright spots of fluorescence are observed amidst the increasingly disorganized array (Fig. 2.24 arrows). Complete depolymerization of microtubules e i t h e r by cold temperature or Oryzalin treatments r e s u l t i n the breakdown of microtubule cables into a s e r i e s of l o c a l i z e d bright spots of fluorescence. These sometimes occur i n rows, as though the whole cable has been fragmented into multiple pools of B-tubulin ( f i g . 2.19). Figure 2.20 shows the t i p region of a vegetative filament a f t e r complete depolymerization of microtubules by cold treatment. Once more, only punctate fluorescence i s observed. In some cases, the l o c a t i o n of these pools of tubulin correlates well with the po s i t i o n s of n u c l e i (as v i s u a l i z e d using DAPI staining) (Figs. 2.21 and 2.22). Upon closer inspection, the tubulin staining sometimes appears to be bipolar i n r e l a t i o n to each tear-shaped nucleus. Nuclear autofluorescence was never observed. The microtubule array remains v i s i b l e a f t e r incubation i n the s t a b i l i z i n g agent taxol, but some disorganization i s apparent. This appears i n the form of wavy and b r i g h t l y fluorescent spot-terminated cables (Fig. 2.26). Taxol did not s t a b i l i z e the microtubules against cold depolymerization (data not shown). C e l l s e x h i b i t i n g cytoplasmic movement a f t e r re-warming revealed microtubule arrays i n d i f f e r e n t stages of apparent repolymerization and reorganization. The f i r s t i n d i c a t i o n of repolymerization i s evident i n the formation of large areas of bright B-tubulin fluorescence i n association with bright f o c i -34 l i k e spots (Fig. 2.27, arrowhead). Curved, short microtubules are also observed i n contact with bright spots of fluorescence at a more advanced stage of tubulin polymerization (Fig. 2.28). Figures 2.29 and 2.30 show an apparent progressive formation of microtubule bundles oriented p a r a l l e l to the lo n g i t u d i n a l axis of the vegetative filament. Cytochalasins show no e f f e c t on the structure or d i s t r i b u t i o n of the microtubule array (not shown). 35 Discussion - Part 2 Part 1 reported the existence of extensive arrays of a x i a l l y aligned microfilament cables and microtubule bundles i n Vaucheria l o n q i c a u l i s var. macounii. Part 2 investigates how the organization of t h i s large and complex cytoskeleton system might be accomplished, and the possible role(s) of f o c a l regions as organizing centers. Cytochalasin D causes a s t e p - l i k e breakdown of the microfilament array of Vaucheria. This i s dependent upon the concentration and the duration of the treatments (Williamson 1978, Schliwa 1982, Yahara et a l . 1982, Tang et a l . 1989, Murali Krishna Rao et a l . 1992) and i t provides a u s e f u l t o o l to analyse the organization of the microfilament array. A consistent feature of the disorganization of the microfilament array i s that i t always occurs i n close association with regions of the F-actin network c a l l e d f o c i . These observations are consistent with those of B l a t t et a l . (1980) who observed r e t i c u l a t i o n and branching of c o r t i c a l f i b e r s by d i f f e r e n t i a l interference contrast microscopy a f t e r a p p l i c a t i o n of Cytochalasin B. Yumura and Kitanishi-Yumura (1990) observed s i m i l a r structures i n Dictyostelium and proposed them to be organizing centers of F-a c t i n . Formation of F-actin aggregates i n plant c e l l s i s common under the influence of Cytochalasin D (Parthasarathy 1985, Menzel and Schliwa 1986b, Palevitz 1988). D i f f e r e n t i a l interference contrast observations of l i v i n g vegetative filaments show that 36 the cytoplasmic track network seems to originate from highly dynamic f o c a l regions which appear to have a s i m i l a r d i s t r i b u t i o n to the F-actin f o c i (Part 1). Therefore the F-actin f o c a l regions are not l i k e l y to be an a r t i f a c t of the methods of preparation. Foci may be formed by contraction of F-actin. This i s proposed to be mediated by a c t i n binding proteins located within the f o c a l regions (Schliwa 1982, Yahara et a l . 1982, Yumura and Kitanishi-Yumura 1990). I t i s i n t e r e s t i n g to notice i n r e l a t i o n to t h i s i n t e r p r e t a t i o n that images show that as depolymerization progresses, f o c i seem to increase i n s i z e concurrent with the a l t e r a t i o n and eventual disappearance of the F-actin cables. The probable presence of myosin, i n association with the microfilament array (Part 1) may allow for a s i m i l a r phenomenon to occur i n Vaucheria. Repolymerization studies further support the existence of F-actin f o c i i n association with F-actin cables and suggest the p o s s i b i l i t y that they act as polymerization centers. Yumura and Kitanishi-Yumura (1990) showed that i n Dictvostelium f o c i are located on the plasma membrane. In Vaucheria vegetative filaments, the location of the whole F-actin array, including f o c i , i n the c o r t i c a l cytoplasm suggest that the plasma membrane may have an organizational function (Traas 1990). A possible model for t h i s type of organizational structure could include a c t i n binding i n t e g r a l membrane proteins s i m i l a r to those known to occur i n animal c e l l s , such as p o n t i c u l i n (Wuestehube 37 and Luna 1987) and v i n c u l i n (Jockusch and Isenberg 1981). These molecular interactions could act to transmit signals v i a the plasma membrane to the c l o s e l y associated cytoskeleton (Isenberg 1991). This would enable the c e l l to co-ordinate the d i s t r i b u t i o n of microfilament bundles i n the coenocytic vegetative filament of Vaucheria. The existence of multiple f o c i d i s t r i b u t e d throughout the microfilament array i n untreated c e l l s of Vaucheria i s consistent with t h i s i n t e r p r e t a t i o n . These signals could p a r t i c i p a t e i n the organization of F-actin into two d i s t i n c t , f u n c t i o n a l l y d i f f e r e n t sub-sets of microfilament cables (Part 1). However, no p r e f e r e n t i a l l o c a l i z a t i o n of f o c i between the two sub-sets of F-actin has been observed. I t i s possible then that polymerization at the f o c i may not d i s t i n g u i s h between d i f f e r e n t sub-sets of microfilaments. Foci and extensive arrays of F-actin are observed close to cross-walls formed i n response to wounding. Under these circumstances, no d i f f e r e n t i a t i o n between the two sub-sets of F-actin cables can be seen, suggesting that t h i s may be occurring a f t e r polymerization. The e f f e c t s of Oryzalin and cold temperature on cytoplasmic movement i n Vaucheria are r e v e r s i b l e . These i n h i b i t o r s depolymerize microtubule bundles into a s e r i e s of l o c a l i z e d spots of t u b u l i n , s i m i l a r i n appearance to the punctate fluorescence observed i n untreated c e l l s . This e f f e c t i s also observed i n other plant c e l l s where i t i s interpreted as i n d i c a t i v e of microtubule depolymerization (Menzel and Schliwa 1986b, Cleary and Hardham 1988, Wacker et a l . 1988, Wasteneys and Williamson 38 1989, Akashi et a l . 1990, Schwuchow et a l . 1990, Astrom et a l . 1991) . Microtubule depolymerization i n response to cold treatment i s p a r t i c u l a r l y pertinent for t h i s i n v e s t i g a t i o n because of i t s relevance to the natural habitat of Vaucheria. where low temperatures within the range used i n t h i s study are frequently experienced during the winter. Transmission electron microscopy of a v a r i e t y of species of Vaucheria suggests a close r e l a t i o n s h i p between microtubule bundles and the anterior end of each nucleus, including a pair of c e n t r i o l e s proposed to act as a microtubule organizing centre (MTOC) (Ott and Brown 1972, Ott 1992) . Some of the b r i g h t l y fluorescent spots (foci) seen a f t e r depolymerization appear to be c o - l o c a l i z e d with n u c l e i . These are also observed i n repolymerization studies, which indicates nucleation of tubulin at these s i t e s . Nuclear-related, MTOC anchored, and xcapped' microtubules are cold-stable (Doonan et a l . 1988, McBeath and Fujiwara 1990), and t h i s i s consistent with our observations of the extreme tolerance of Vaucheria microtubules to cold temperatures. Taxol causes the appearance of microtubule bundles i n association with bright spots ( f o c i ) . Similar e f f e c t s of taxol were suggested to be due to reorganization of microtubules through increased bundling and aggregation of tubulin (Kuss-Wymer and Cyr 1992), and establishment of microtubule cross-links (Melan 1990). These observations further supports the i n t e r p r e t a t i o n that they are involved with the polymerization and organization of tubulin. Polymerization may be occurring at the d i s t a l (plus) end (McBeath 39 and Fujiwara 1990), i f the microtubule bundle i s anchored at the cen t r i o l e s (MTOC). In Vaucheria. nuclei do not divide synchronously (Ott and Brown 1972). Therefore the nucleus associated tubulin fluorescence i s not always observed. Microtubule-associated fluorescent spots i n Vaucheria which do not c o - l o c a l i z e with nu c l e i may represent other nucleation s i t e s , such as those found in regions adjacent to the plasma membrane (Falconer et a l . 1988). This i s supported by Ott's (1992) observations that not a l l microtubule bundles i n Vaucheria seem to be attached to nu c l e i . In t h i s case i t i s possible that microtubule bundles are organized by a nucleus-associated MTOC that detaches from i t at a ce r t a i n point during t h e i r growth (McBeath and Fujiwara 1990). The existence of xmicrotubule probes' recently described by Ott (1992) i n Vaucheria l o n q i c a u l i s may be the r e s u l t of such a mechanism. Our repolymerization studies did not reveal r a d i a l growth of microtubules from f o c a l points (Lloyd 1987) . Instead, l o n g i t u d i n a l l y oriented microtubule bundles with u n i d i r e c t i o n a l growth are seen to be associated with s p o t - l i k e f o c i . This organizational pattern may be the r e s u l t of the presence of MAPs which cause increased bundling of polymerizing microtubules (Wasteneys and Williamson 1989), and perhaps a *pre-programmed' pattern of p a r a l l e l growth at the MTOC (Brown et a l . 1982, Kalnins 1992). This pattern of growth of microtubule bundles i s consistent with the r o l e they and associated n u c l e i are thought 40 to play i n the polarized growth of Vaucheria vegetative filaments (Kataoka 1975, Part 1). The lack of e f f e c t of Cytochalasins, on the organization of the microtubule array, and Oryzalin, on the microfilament array, i s i n t e r e s t i n g when considering the proposed i n t e r a c t i o n between the two cytoskeleton systems i n organelle movement (Ott 1992, Tornbom and O l i v e i r a , unpublished, Part 1). Cold treatment d i d , however, depolymerize the microfilament cables to reveal f o c i . These observations suggest that i n t e r a c t i o n between the two arrays may be mediated by proteins such as MAPs (Pollard et a l . 1984, Menzel and Elsner-Menzel 1989) which allow the two systems to function i n tandem, whilst enabling them to maintain s p e c i a l i z e d r o l e s (Ott 1992, Tornbom and O l i v e i r a , unpublished). These s p e c i a l i z a t i o n s are r e f l e c t e d i n the differences i n t h e i r organization from d i s t i n c t nucleating centers. A nucleus and i t s associated MTOC seem to organize the microtubule bundle as an independent unit with growth from the d i s t a l end of the microtubule, whereas F-actin cables seem to be organized at the proximal end from centers d i s t r i b u t e d throughout the cytoplasm (Dustin 1984). 41 Conclusion This study has i d e n t i f i e d two cytoskeleton components, microtubules and microfilaments, and described t h e i r o v e r a l l organization i n vegetative filaments of Vaucheria l o n g i c a u l i s var. macounii. The extensive array of l o n g i t u d i n a l l y oriented microfilaments, and the less dense array of microtubules undoubtedly play important r o l e s i n the polarize d organization of t h i s coenocytic alga which exhibits vigourous cytoplasmic streaming. There i s also evidence for the existence of the mechanomotor myosin, although not as conclusive as for the other two components. Experimental manipulation of these cytoskeleton elements, through disruption (depolymerization) and recovery (repolymerization), gives some insight into how control over the arrays of cyt o s k e l e t a l elements may be accomplished. This seems to occur through organizing centers with d i f f e r e n t properties and d i s t r i b u t i o n s for microfilaments and microtubules. Microfilaments may be organized by f o c i l o c a l i z e d at s i t e s adjacent to the plasma membrane and d i s t r i b u t e d throughout the filament, whereas microtubule bundles are thought to be organized at centers associated with the cen t r i o l e s and n u c l e i . Nuclei-free organization centers are also observed and thought to r e s u l t from detachment of microtubules from t h e i r o r i g i n a l organizing center. An obvious d i r e c t i o n i n which to continue t h i s study i s to unequivocally i d e n t i f y myosin, and to probe f o r other proteins 42 such as MAPs and intermediate filament related proteins using SDS-PAGE and immunoblotting. L o c a l i z a t i o n of these proteins using immunofluorescence confocal microscopy w i l l add further depth of knowledge to the organization and functioning of the cytoskeleton. Treatment of vegetative filaments with N-ethylmaleimide, a dynein and myosin poison, w i l l provide further evidence as to the presence of molecular motors and the rol e ( s ) they play i n t h i s organism. Immunofluorescence studies of c e l l u l a r extracts w i l l add to a better i d e n t i f i c a t i o n of the relat i o n s h i p of d i f f e r e n t organelles with both F-actin and microtubule bundles. In addition, c e l l u l a r extracts, as used for other a l g a l systems (Kohno et a l . 1990), may provide a useful t o o l for the study of the r o l e d i f f e r e n t cytoskeleton molecules play i n organelle transport and c e l l u l a r organization. Microscopical work involving l a b e l l i n g of F-actin with heavy meromyosin f o r TEM w i l l provide information on the p o l a r i t y of the microfilaments, which i s important when considering the m o t i l i t y mechanism. Immunoelectron microscopy (TEM and HRSEM) can also be used to locate some of the proteins discussed i n t h i s study and t h e i r r e l a t i o n s h i p to the putative organizing centers ( f o c i ) , while providing at the same time knowledge about t h e i r u l t r a s t r u c t u r a l organization. FIGURES - PART 44 Fig. 1 SDS-PAGE gels and immunoblots of Vaucheria homogenate proteins A. 12% gel of Vaucheria proteins (a), a n t i - a c t i n immunoblot of lane a (b), negative control ( n i t r o c e l l u l o s e treated only with secondary antibody) (c) B. 12% gel of Vaucheria proteins (d), bovine brain t u b u l i n standard (e), a n t i - t u b u l i n immunoblot of lane d ( f ) , a n t i -t u b u l i n immunoblot of lane e (g) C. 12% gel of Vaucheria proteins - note f a i n t band at approximately 200 kDa (arrowhead) (h), anti-myosin (Amersham) immunoblot of lane h (i) D. 7.5% gel of Vaucheria proteins - note band at approximately 200 kDa (arrowhead) ( j ) , anti-Dictyostelium myosin immunoblot of lane j - note f a i n t skewed band at approximately 110 kDa (arrowhead) (k), anti-Dictyostelium myosin immunoblot of myosin standard (1), negative control ( n i t r o c e l l u l o s e treated only with secondary antibody) (m) 45 Figs. 2 to 7 Bars = 10 jum Figs. 2 & 3 D i f f e r e n t i a l interference contrast microscopy of a Vaucheria filament. Figure 2 shows an a x i a l l y aligned array of fibrous structures d i s t r i b u t e d throughout the cytoplasm, i n close association with c e l l organelles such as chloroplasts (arrow) and v e s i c l e s (arrowheads). Figure 3 shows a d i f f e r e n t region of a Vaucheria filament, demonstrating the r e l a t i o n s h i p between the fibrous structures and chloroplasts as well as nuclei (arrowheads) Fi g . 4 F-actin microfilament array v i s u a l i z e d using f l u o r e s c e i n - l a b e l l e d p h a l l o i d i n (unfixed material). A dense network of p a r a l l e l , a x i a l l y aligned cables can be seen. Inset: D i s t r i b u t i o n of F-actin by focussing on the c o r t i c a l region of the filament, showing that microfilament cables are present within approximately 2 jum of the c e l l ' s surface Fi g . 5 Microfilament array of an aldehyde-fixed c e l l showing branching of longitudinal cables i n the cytoplasm (arrowhead) and around a chloroplast (c) (arrow) 46 Fi g . 6 Branching of microfilament cables i n close proximity to organelles (arrowheads) i n an unfixed filament stained with f l u o r e s c e i n - l a b e l l e d p h a l l o i d i n F i g . 7 C r y s t a l l i n e - l i k e microfilament arrays i n an osmotically stressed filament of Vaucheria 47 Fi g . 8 Fluorescein-labelled p h a l l o i d i n s t a i n i n g shows the association of microfilament cables and chloroplasts (c) . Bar = 10 /itm Fi g . 9 Microfilament cables appear to radiate from a f o c a l area (arrow) i n the ap i c a l region of a vegetative filament stained with f l u o r e s c e i n - l a b e l l e d p h a l l o i d i n . Bar = 10 /xm. Inset: intense, f i n e F-actin fluorescence i n the t i p region of a filament. Bar = 5 /xm Fi g . 10 Microfilament cables appear to occur i n two forms: s t r a i g h t e r ones, p a r a l l e l to the l o n g i t u d i n a l axis of the c e l l and adjacent to the c e l l ' s surface (arrows), and more r e t i c u l a t e ones i n apparent association with organelles and located deeper i n the cytoplasm (arrowheads). Bar = 10 p Fi g . 11 Focal point of microfilament cables with many ra d i a t i n g from and becoming p a r a l l e l to the l o n g i t u d i n a l axis of the c e l l . Bar = 10 ftm. Inset: f o c a l point with fewer microfilament cables. Bar = 5 fim 48 Fig. 12 Confocal scans (sequence l e f t to right) at i n t e r v a l s of 0.8 /nm, showing the p a r t i t i o n i n g of the microfilament array throughout the cytoplasm of a Vaucheria filament l a b e l l e d with f l u o r e s c e i n - l a b e l l e d p h a l l o i d i n . Bar = 50 /xm Fig. 13 Confocal scans (sequence l e f t to right) at i n t e r v a l s of 1 /urn (upper se r i e s , and upper to lower series) and 2 /xm (lower s e r i e s ) . Fluorescein-labelled p h a l l o i d i n s t a i n i n g of a vegetative filament shows l o n g i t u d i n a l l y aligned microfilaments within the f i r s t 2 lim of the c e l l ' s surface, and r e t i c u l a t e d regions of a c t i n microfilaments at a depth of approximately 4 /xm into the cytoplasm. Focal regions are also observed i n a l l l e v e l s of o p t i c a l sectioning. Bar = 50 jum Fig. 14 Confocal cross-sectional scan of a f l u o r e s c e i n - l a b e l l e d p h a l l o i d i n stained vegetative filament, with graphical representation of fluorescence d i s t r i b u t i o n within the cytoplasm. The highest density of fluorescence occurs i n the c o r t i c a l region. Bar = 10 jxm Fig . 15 Confocal graphical representation of fluorescence d i s t r i b u t i o n along a l a b e l l e d l o n g i t u d i n a l axis of a vegetative filament. A s i m i l a r pattern of fluorescence d i s t r i b u t i o n seems to occur throughout the filament. Bar = 10 jim 49 Figs. 16 to 19 Bars = 10 jura F i g . 16 Microtubule bundles are v i s u a l i z e d using i n d i r e c t immunofluorescence for B-tubulin. They show a x i a l o r i e n t a t i o n and determinate length, and sparse d i s t r i b u t i o n throughout the cytoplasm F i g . 17 Vaucheria c e l l showing microtubule bundles curving and crossing over into a branch (arrowheads) F i g . 18 Confocal composite image of 30 scans showing the o v e r a l l d i s t r i b u t i o n of microtubule bundles i n the mid-section of a vegetative filament. F i g . 19 Microtubule bundles form a loose network i n the a p i c a l dome of the t i p region of a filament 50 Figs. 20 to 23 Bars = 10 jm, except F i g . 21 Bar = 50 nm F i g . 20 Cross-section confocal scan and graphical representation of B-tubulin fluorescence d i s t r i b u t i o n . High fluorescence i n t e n s i t y i s not equally d i s t r i b u t e d throughout the cytoplasm. F i g . 21 S e r i a l confocal scans at depth i n t e r v a l s of 3 jum ( f i r s t scan at a depth of 10 /xm into the cytoplasm) . Microtubule bundles show that they are present throughout most of the cytoplasm width. F i g . 22 Cross-section of a Vaucheria filament showing punctate microtubule fluorescence (arrowheads) deeper into the cytoplasm. F i g . 23 Extruded chloroplast (c) with c l o s e l y associated microtubule bundles (arrowheads). 5< 51 Fig. 24 Fractured end of a Vaucheria c e l l as seen by HRSEM. Chloroplasts (c), c e l l wall (cw) and fibrous material (arrowhead) are v i s i b l e . Bar = 5 /urn Fig . 25 Higher magnification of fibrous material i n Figure 24 showing i t s web-like appearance, attachment to organelles (arrowheads) and small v e s i c l e s (arrow). Bar = 5 /zm Fig. 26 Fractured filament of Vaucheria showing c a b l e - l i k e structures within the cytoplasm. These structures are la r g e l y unbranched and run s t r a i g h t between d i f f e r e n t areas of the cytoplasm. Bar = 2.5 /xm Fig. 27 Higher magnification of Fi g . 26 showing the association between an organelle (arrowhead) and thinner cables. Larger cables (arrow) appear to be made up of thinner cables. Bar = 150 nm FIGURES - PART 53 Figs. 2.1 to 2.4 Bars = 10 jum Figs. 2.1a & b a. D i f f e r e n t i a l interference microscopy micrograph showing a region of a vegetative filament adjacent to a cross-wall formed i n response to wounding (arrow). A network of cytoplasmic tracks i s seen throughout the cytoplasm and these seem to radiate from f o c a l areas (arrowheads). b. Same area of the c e l l one minute l a t e r , demonstrating that these are dynamic structures with anastamosing elements. F i g . 2.2 Reticulate F-actin array, v i s u a l i z e d by fl u o r e s c e i n - l a b e l l e d p h a l l o i d i n , i s seen to be associated with numerous f o c i (arrowheads). These are observed adjacent to a cross-wall of a vegetative filament formed i n response to wounding (arrow). F i g . 2.3 Microtubule array of a vegetative filament v i s u a l i z e d using B-tubulin immunofluorescence. Figs. 2.4a & b a. Oblique cut end of a vegetative filament reveals filamentous and punctate B-tubulin fluorescence when focussing on the tonoplast side. b. Same filament shows only filamentous fluorescence when focussed from the plasma membrane side. 54 Figs. 2.5 to 2.10 Bar = 10 jxm, except F i g . 2.6 Bar = 5 /xm Fig. 2.5 Absence of F-actin fluorescence i n a vegetative filament of Vaucheria a f t e r treatment with 100 /zg/ml Cytochalasin D. Some autofluorescence of chloroplasts, and small specks of fluorescein are v i s i b l e (arrowheads). Fi g . 2.6 F-actin fluorescence of a vegetative filament a f t e r treatment with 200 fig/ml Cytochalasin B. The F-actin array i s la r g e l y disorganized, with i t s remnants associated with f o c i - l i k e structures (arrowheads). Fig. 2.7 The f i r s t i n d i c a t i o n of breakdown of the F-actin array upon treatment with 10 /xg/ml Cytochalasin D i s represented by bright specks of fluorescence (arrowheads) amidst the more i r r e g u l a r and r e t i c u l a t e F-actin cables. F i g . 2.8 F-actin array of an untreated vegetative filament. Note the o v e r a l l a x i a l alignment of the F-actin cables. F i g . 2.9 Region adjacent to a cross-wall (formed i n response to wounding) a f t e r treatment with 10 ng/ml Cytochalasin D. Large (arrow) and small (arrowhead) f o c i of F-actin are v i s i b l e amongst the remaining F-actin cables. F i g . 2.10 Foci of F-actin (arrowheads) are v i s i b l e along the length of F-actin cables a f t e r treatment with 10 Mg/ml Cytochalasin D. 55 Figs. 2.11 to 2.16 Bar = 10 ym, except F i g . 2.12 Bar = 5 /xm Fig. 2.11 Web-like array of F-actin with interspersed f o c i (arrow) i s seen throughout the mid-filament region of a vegetative filament a f t e r treatment with 10 iig/ml Cytochalasin D. Fig. 2.12 Similar web-like array with interspersed f o c i i s seen to extend into the t i p region of a vegetative filament. Fig . 2.13 Advanced stage of depolymerization of the F-actin array by Cytochalasin D. A x i a l l y aligned F-actin cables appear to merge into one, large b r i g h t l y fluorescent structures that are seen converging into a central f o c a l region (arrow). Fig . 2.14 Extensive depolymerization of F-actin. Large areas of the c e l l show no fluorescence, and only a few f o c i with short, disorganized F-actin cables remain. Fig. 2.15 F-actin depolymerization i s almost complete, except for a few intensely fluorescent compact f o c i . Fig. 2.16 Disorganization of the F-actin array of a vegetative filament a f t e r cold (< -2 °C) treatment. Foci appear amidst the disrupted F-actin cables (arrows). 56 Figs. 2.17 to 2.22 Bar = 10 urn, except F i g . 2.18 Bar = 5 /zm Fig. 2.17 Recovery from Cytochalasin D treatment. Bright fluorescent f o c i are v i s i b l e (arrows), amidst p a r t i a l l y reorganized F-actin cables. Fig. 2.18 Microtubule array present i n a vegetative filament of Vaucheria a f t e r exposure to sub-zero temperatures (-2 °C). Fi g . 2.19 Complete depolymerization of the microtubule array i n a vegetative filament, a f t e r exposure to cold temperature (-2 °C). Fluorescence i s v i s i b l e as a series of bright spots (arrowheads), often i n l i n e a r alignment. Fig . 2.20 Complete depolymerizatin of the microtubule array i n the t i p region of a filament a f t e r exposure to cold temperature (-2 °C). Only bright spots of fluorescence are v i s i b l e . Figs. 2.21 & 2.22 Demonstration of the c o - l o c a l i z a t i o n of B-tubulin spots and n u c l e i (compare Figs. 2.21 and 2.22). Figure 2.21 shows punctate B-tubulin fluorescence a f t e r cold treatment (-2 °C). Figure 2.22 shows nuclei v i s u a l i z e d by DAPI staining. The B-tubulin fluorescence displays a bipolar d i s t r i b u t i o n with respect to the nuclei (arrowheads). 57 Figs. 2.23 to 2.26 Bars = 10 /m F i g . 2.23 F i r s t i n d i c a t i o n of depolymerization i s evident by the fragmentation of microtubule bundles. Fig . 2.24 Disorganization of microtubule bundles i s accompanied by the appearance of bright fluorescent spots at t h e i r ends (arrows). F i g . 2.25 Bright spot of fluorescence at the end of a microtubule bundle (arrow). 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