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The effects of aluminum on cytoplasmic organization, the F-actin array and calcium homeostasis in Vaucheria… Alessa, Lilian 1998

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T H E E F F E C T S O F A L U M I N U M O N C Y T O P L A S M I C O R G A N I Z A T I O N , T H E F-A C T I N A R R A Y A N D C A L C I U M H O M E O S T A S I S I N VAUCHERIA LONGICAULIS V A R . MACOUNIi by L I L I A N A L E S S A Bachelor of Science (B.Sc.) University of British Columbia, 1989 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Botany) We accept tikis' thesis as conforming to the required standard T H E 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 October 1997 Copyright, Li l ian Alessa, 1997 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 written permission. I Department of fy) The University of British Columbia Vancouver, Canada Date )Gr£mhjrii i<flf~ . DE-6 (2/88) 11 Abstract The single-celled alga, Vaucheria longicaulis var.macounii B l u m was used as a model organism to determine the effects of aluminum on cytoplasmic organization, the F -actin array and calcium homeostasis. The translocation of chloroplasts and mitochondria is a microfilament-dependent process whereas that of nuclei translocation, is microtubule-dependent. The addition of aluminum (80 p M ) resulted in the inhibition of microfilament-based organelle translocation (within 60 seconds). Aluminum also caused the disorganization of cortical, cytoplasmic strands, which were visible using differential interference contrast (DIC) microscopy. These strands were shown to represent the endoplasmic reticulum in this species. Since the endoplasmic reticulum is supported by an intact F-actin array in many types of plant cells, this suggested that A l caused changes in the F-actin array in cells of V. longicaulis. Consistent with this, fluorescein labeled (FITC) phalloidin was used to show that exposure to A l resulted in the fragmentation of the F-actin array and the formation of F-actin aggregates which appeared to self-assemble in vivo. The hypothesis that aluminum causes cytoplasmic disorganization and fragmentation of the F -actin array via the perturbation of calcium homeostasis was tested. The calcium-sensitive tetracarboxylate dye, fluo-3, was used to image free cytosolic calcium in this system. Treatment with aluminum caused an increase in cytosolic calcium levels within two minutes of exposure. It was noted that the response of cytosolic calcium to aluminum treatment varied between the youngest, apical region and the older, vacuolated region of the cell. Consistent with this, aluminum toxicity was particulary acute in the cell tip. The aluminum-induced rise in cytosolic calcium correlates with aluminum-induced effects on cytoplasmic streaming and the F-actih array using calcium modulators in an aluminum-free system. The use of ionomycin resulted in changes in the F-actin array which were similar, but not exactly the same as those observed during aluminum treatment. Thapsigargin and T M B - 8 , used to stimulate and inhibit release of calcium from intracellular stores, Ul respectively, were used to determine whether the source of the aluminum-induced rise in cytosolic calcium was internal. Data obtained using these latter calcium modulators were inconclusive. Table of Contents; Page Abstract i i Table of Contents iii List of Tables v List of Figures v i List of Abbreviations xi Acknowledgement xi i Dedication xi i i 1.0 Chapter One: General Introduction 1 The Plant Cytoskeleton 4 Microfilaments and Cytoplasmic Streaming 7 Calcium Homeostasis in Plant Cells 9 Aluminum Effects on Tip Growth 13 The Experimental Organism: Vaucheria longicaulis 14 Functional Zonation 17 1.12. Statement of Aims of Thesis and Hypotheses to be Tested 18 2.0. Chapter Two: Materials and Methods 20 3.0. Chapter Three: Effects of Aluminum on Organelle Streaming and Cytoplasmic Organization. 33 3.1. Introduction 33 3.2. Results 36 3.3. Discussion 54 V 4.0. Chapter Four: Effects of Aluminum on the F-actin Array 60 4.1. Introduction 60 4.2. Results 63 4.3. Discussion 82 5.0. Chapter Five: Calcium Studies 90 5.1. Introduction 90 5.2. Results 92 5.3. Discussion 116 6.0. Chapter Six: Calcium Modulator Studies : 124 6.1. Introduction 124 6.2. Results 127 6.3. Discussion 151 7.0. Chapter Seven: Conclusions and Future Research 161 Literature Cited 164 vi List of Tables: Table 1 42 Effect of aluminum on growth rates Table 2 67 Effect of aluminum on G-actin levels in the cell Table 3 96 Effects of time of addition after treatment with aluminum of 8.0 m M calcium on cytoplasmic streaming of chloroplasts and mitochondria. Table 4 Effects of concentration of calcium added 5 seconds after treatment with aluminum on cytoplasmic streaming of chloroplasts and mitochondria. 97 List of Figures Page F i g u r e 1 43 Region of control filament, 100 um distal from apical tip, observed using DIC microscopy F i g u r e 2 43 Region of control filament, 100 um distal from apical tip, observed using D I C microscopy F i g u r e 3a-c 43 Reticulate cytoplasmic strands located in sub-cortical region of the cell F i g u r e 4 45 DiOC6(3)-positive structures observed in cortical cytoplasm F i g u r e 5a 45 DiOC6(3)-positive structure observed in sub-cortical cytoplasm F i g u r e 5b 45 Apical portion of cell showing intense DiOC6(3) staining F i g u r e 6 45 Fluo-3 fluorescence demonstrating compartmentalization into a cortical, tubular compartment F i g u r e 7a,b 45 Fluo-3 fluorescence demonstrating compartmentalization into a sub-cortical reticulate compartment F i g u r e 8 47 Graph demonstrating dependence of aluminum-induced cessation of cytoplasmic streaming of chloroplasts and mitochondria F i g u r e 9 48 D I C micrograph series showing preferential inhibition by aluminum of cytoplasmic streaming of chloroplasts and mitochondria but not nuclei F i g u r e 10a 50 Normal (control) morphology of vegetative filaments F i g u r e 10b Morphology of vegetative filaments after treatment with aluminum 50 F i g u r e 11 50 Brightfield image showing swollen apical tip and retraction of plasma membrane from cell wall F i g u r e 12a,b 50 Aluminum treated cells. Patterns of disorganization in cytoplasmic strands. Linear strands become associated with each other F i g u r e 12c 50 Aluminum treated cells. Observation of cytoplasmic strands which form "rings" F i g u r e 12d 50 Aluminum treated cells. Change from linear to transverse cytoplasmic strands F i g u r e 13a-c 52 DiOC6(3) staining of endoplasmic reticulum and demonstration of UV-induced toxicity F i g u r e 14 52 DiOC6(3) staining of endoplasmic reticulum after 18 hours of continuous exposure to aluminum F i g u r e 15 52 DIC image of chloroplast/mitochondria aggregate and larged vesicles in an aluminum-treated cell F i g u r e 16a, b 66 FITC-phalloidin staining showing F-actin array in fixed cells F i g u r e 17 66 Control cell vital stained with FITC-phalloidin using gelatin embedding protocol F i g u r e 18 66 FITC-phalloidin staining showing reticulate F-actin array located in sub-cortical region of the cell ix F i g u r e 19 66 FITC-phalloidin staining showing F-actin array in apical tip F i g u r e 20 , 70 FTTC-phalloidin staining in 30 minute aluminum treated apical tip F i g u r e 21 70 FITC-phalloidin staining of a region 100 um distal from the apical tip in 30 minute aluminum-treated cell F i g u r e 22 70 FITC-phalloidin staining of a region 100 um distal from the apical tip in 2 hour aluminum-treated cell F i g u r e 23 70 FITC-phalloidin staining of a region 100 um distal from the apical tip showing heterogeneity of disorganization in the F-actin array F i g u r e 24 73 Alteration in chloroplast shape noted in 8 hour aluminum treated cell F i g u r e 25 73 FITC-phalloidin staining of an isolated region of the cell, 100 um distal from the apical tip, showing localized disorganization of F-actin array F i g u r e 26 73 FTTC-phalloidin staining showing formation of focal mass in close proximity to a chloroplast F i g u r e 27 73 FTTC-phalloidin staining of F-actin array in 8 hour aluminum treated cell F i g u r e 28 75 FITC-phalloidin staining of F-actin array in >8 hour aluminum treated cell showing focal mass and aggregates of F-actin F i g u r e 29 75 FITC-phalloidin staining of F-actin array in >8 hour aluminum treated cell showing various F-actin aggregates X F i g u r e 30 75 FITC-phalloidin staining of F-actin array in >8 hour aluminum treated cell showing stellate and amorphous F-actin aggregates F i g u r e 31 75 FTTC-phalloidin staining of F-actin array in >8 hour aluminum treated cell showing F-actin aggregates of thick, curved bundles F i g u r e 32 77 FITC-phalloidin staining of F-actin array in >8 hour aluminum treated cell showing curled and ring-form F-actin aggregates F i g u r e 33 77 FITC-phalloidin staining showing persistence of F-actin ring structures and focal masses after long term aluminum treatment F i g u r e 34 77 FTTC-phalloidin staining showing various morphologies of ring aggregates of F-actin F i g u r e 35 77 FITC-phalloidin staining of F-actin in >12 hour aluminum treated cell showing various F-actin ring-form aggregates which remain in the cytoplasm F i g u r e 36 , 80 FITC-phalloidin staining of F-actin showing variations in ring aggregates F i g u r e 37 80 FITC-phalloidin staining showing formation of large, coiled F-actin aggregate F i g u r e 38 80 FITC-phalloidin staining showing collapse of sub-cortical, reticulate F-actin array into focal masses F i g u r e 39 80 FITC-phalloidin staining showing persistence of F-actin ring aggregates in proximity to large, amorphous focal masses F i g u r e 40 98 C T C localization of Ca2+ ions in proximity to hydrophobic surfaces. F i g u r e 41 100 Fluo-3 imaging of free cytosolic calcium in the apical zone (AZ) F i g u r e 42 102 Fluo-3 imaging of free cytosolic calcium in the zone of vacuolation (ZV) F i g u r e 43 104 Fluo-3 imaging of free cytosolic calcium. Localized contact with A l causes a localized rise in cytosolic calcium F i g u r e 44 106 Graph showing typical response in the zone of vacuolation (ZV) of a population of cells loaded with fluo-3 to A l treatment in low external calcium F i g u r e 45 108 Graph showing typical response in the zone of vacuolation (ZV) of a population of cells loaded with fluo-3 to A l treatment in high external calcium F i g u r e 46 110 Graph showing typical response in the apical zone (AZ) of a population of cells loaded with fluo-3 to A l in 2.5 m M external calcium F i g u r e 47 112 Graph showing typical response in the apical zone (AZ) of a population of cells loaded with fluo-3 to A l in high external calcium F i g u r e 48 114 Graph showing typical response in the apical zone (AZ) of a population of cells loaded with fluo-3 to A l in low external calcium F i g u r e 49 131 Graph showing typical response in the apical zone (AZ) of a population of cells loaded with fluo-3 and treated with 1 u M thapsigargin F i g u r e 50 133 FTTC-phalloidin and C T C staining in control cell tips and cell tips treated with A l F i g u r e 51 135 Graph showing typical response in the apical zone (AZ) of a population of cells loaded with fluo-3, pre-treated with thapsigargin and exposed to A l F i g u r e 52 , 137 Graph showing typical response in the apical zone (AZ) of a population of cells loaded with fluo-3 and treated with T M B - 8 in varying concentrations of external calcium F i g u r e 53 139 Graph showing typical response in the apical zone (AZ) of a population of cells loaded with fluo-3, pre-treated with T M B - 8 and exposed to A l F i g u r e 54 141 Graph showing typical response in the zone of vacuolation (ZV) of a population of cells loaded with fluo-3 and treated with thapsigargin F i g u r e 55 143 Graph showing typical response in the zone of vacuolation (ZV) of a population of cells loaded with fluo-3, pre-treated with thapsigargin and exposed to A l F i g u r e 56 145 Graph showing typical response in the zone of vacuolation (ZV) of a population of cells loaded with fluo-3 and treated with T M B - 8 F i g u r e 57 147 Graph showing typical response in the zone of vacuolation (ZV) of a population of cells loaded with fluo-3, pre-treated with T M B - 8 and exposed to A l F i g u r e 58 149 FITC-phalloidin staining showing F-actin array after treatment with T M B - 8 and exposure to A l F i g u r e 59 149 FITC-phalloidin staining showing F-actin array after treatment with thapsigargin and exposure to A l xii i F i g u r e 60 149 FITC-phalloidin staining showing showing F-actin array after treatment with ionomycin F i g u r e 61 149 FITC-phalloidin staining showing F-actin array in a cell which represents the Al-only control for Figures 68-60. List of Abbreviations: A l Aluminum A Z Apical Zone Ca 2 +(C yt) Cytosolic calcium [ Ca 2 + ] e x External calcium concentration C T C Chlorotetracycline DIC Differential Interference Contrast DiOC 6 (3 ) 3,3'-Dioctadecyloxacarbocyanine perchlorate E R Endoplasmic reticulum F I T C Fluorescein isothiocyanate IP 3 Inositol trisphosphate p\m micrometres m M millimolar p M micromolar O T C Oxytetracycline R F I Relative fluorescence Intensity ROI Region of interest Z V Zone of vacuolation XV Acknowledgements: M y deepest thanks go to my best friend, David Pfeiffer. Without his rational thinking, humour and love, I would have thrown a few microscopes across the room. Many thanks go to Dr. T i m O'Connor who provided friendship, knowledge and equipment when any were needed. Thanks also to Drs. Anthony Glass and Herbert Kronzucker who often helped me see things from different perspectives, and who provided me an umbrella when it rained. Thanks to Dr. Wayne Vog l who, early on, allowed me to have access to his laboratory and equipment and whose inspired curiousity gave me a great appreciation for the scientific method. Thanks to all those, who via email, helped me design experiments and who supported and discussed my findings. Finally, I am in debt to the following people: Dr. Lacey Samuels for her on-going discussions and faith, to Dr. John Church for allowing me the use of his Attofluor system, to Dr. Elaine Humphrey for not kicking me out of the E . M . lab at closing time and to Dr. Mark Rand for helping me with the localized aluminum exposure experiment. Last but not least, thanks to Judy, Lebby and Fiona who are outstanding individuals. xvi This thesis is dedicated to my parents, who never understood but always believed. 1 1.0. General Introduction Aluminum (Al) is a common constituent in acidic soils and the most abundant metal in the earth's crust. It is toxic to plants primarily by restricting root growth (Foy, 1988). Tip-growing cells such as the root hair (which is suspected to be particulary sensitive to A l ) exhibit polarized, apical growth which is affected by a number of factors, including A l . The F-actin cytoskeleton is central to the vitality of tip-growing cells, as is the maintenance of a tip-focused calcium gradient (Cai et al. 1997). Calcium affects such cells in various ways, for example, by maintaining cytoplasmic streaming of organelles and the integrity of the F-actin cytoskeleton. The latter is organized to facilitate organelle translocation throughout the older, vacuolated portions of the cell. In the non-vacuolated, growing tip, F-actin is maintained as a loose meshwork which appears to facilitate vesicle secretion and fusion with the plasma membrane (as well as deposition of cell wall constituents) and hence cell tip expansion (Cai et al. 1997). Thus, factors which perturb calcium homeostasis also affect the F-actin cytoskeleton and hence, cell viability. Aluminum is considered an environmental stress to the roots of plants which grow on acidic soils (Foy, 1988). The mechanism by which A l affects plant cells is unresolved (Rengel, 1996) and the information concerning its effect on the F-actin cytoskeleton is scarce. This study examines the effects of A l on the F-actin cytoskeleton and calcium homeostasis and represents the first cytological study of the effects of this environmental stressor on these inter-related parameters. Although most soil A l is bound in inert form as aluminosilicates, small quantities may appear in forms which are capable of affecting biological systems (Driscoll and Schecher, 1988; M a y and Nordstrom, 1991). This solubilization of reactive A l species is enhanced in acidic soils (Bolan et al. 1991). Factors such as acid precipitation have increased soil acidity and enhanced A l solubility in large areas particularly in North American and northern European forest stands (Cronan, 1991). 2 The speciation of A l in aqueous solutions at p H values less than 5.0 is complex. It may exist in solution as monomelic or polymeric complexes. With respect to solutions bathing the root these classes include the mononuclear form of A l ^ + , which designates the octahedral hexahydrate ( A 1 ( H 2 0 ) 6 ^ + ) , considered to be the toxic species for wheat roots. This is the form that wi l l be considered in this study since it may be the most relevant to biological systems (Kochian, 1995). It is designated as " A l " throughout the text. The contribution of various aluminum ionic species to toxicity in plant cells is compounded by its complex dynamic chemistry. The chemical speciation of aluminum in dilute solutions undergoes rapid changes upon small changes in pHs (Kinraide, 1991). Thus, variations in the formation of toxic aluminum polynuclear complexes may arise due to small local differences in aluminum concentrations and/or localized p H (McDonald-Stephens and Taylor, 1995). Aluminum may interact with the plant cell at the level of the plasma membrane (apoplasmically) and/or by direct interaction with cytosolic constituents via penetration into the cytosol (symplasmically). There are insufficient data to fully support one or the other hypothesis. However, it is agreed that toxicity may be divided into short and long term effects. Short term effects are measurable within seconds to minutes of exposure to A l whereas long term, effects manifest themselves only over prolonged periods of continuous exposure (hours to days) (Taylor, 1988). Information on the events which occur within seconds or minutes of exposure is scarce. Recently, A l was shown to cause an increase in cytosolic calcium in protoplasts of Triticum aestivum and Vaucheria longicaulis within one minute after treatment (Lindberg and Strid, 1997, Alessa and Oliveira, manuscript submitted). A common, long term feature of A l toxicity is the inhibition of root growth. This occurs from 1 to 6 hours after exposure and is detected by a decrease in indices of root cell mitosis as well as rates of elongation (Ownby and Popham, 1989; Horst et al . 1991; Tepperetal . 1989). 3 Much evidence suggests that at least one major site of the A l - induced toxic lesion may exist at the level of the plasma-membrane. Numerous studies indicate that A l causes a reduction in rates of calcium uptake suggesting its potential action as a calcium channel blocker (Huang et al. 1992; Rengel and Elliot, 1992a & b). These effects seem to be similar to the action of some known calcium channel blockers such as lanthanum ( L a ^ + ) . This polyvalent cation binds in the apoplasm and inhibits root growth (Clarkson, 1965). It has been reported to compete for calcium binding to membranes as well as inhibit calcium fluxes across the plasma membrane in a manner similar to A l (Tester, 1990). Verapamil, another calcium channel blocker, has also been shown to inhibit root growth (Drakeford and Trevawas, 1986). Overall, these data seem to indicate that A l toxicity may be mediated, at least in part, via its interference with normal calcium regulation (e.g. Baskin and Cande, 1990). For example, A l ions have been shown to inhibit adsorption of calcium to the negative charges in Donnan free space (Cronan, 1991; Jentschke et al. 1991), to reduce net calcium uptake rates (Rengel and Robinson, 1989; Jan, 1991) and to 45 2+ competitively reduce short-term net Ca uptake into intact roots (Asp and Berggren, 1990; Huang et al. 1992). Whether or not A l enters the cell and manifests toxicity via symplasmic interactions is still under debate. A n internal primary toxic lesion would require uptake of A l across the root-cell plasma membrane. Data obtained from Chora 26 using sensitive detection techniques for A l indicate that A l may enter the cytosol but not within 30 minutes after exposure (Dr. Gregory Taylor, personal communication). It is generally agreed that no one approach wi l l be sufficient to elucidate the location or locations and nature of the lesions caused by exposure to A l . It seems likely that A l toxicity arises as the result of a hierarchical series of events rather than a single internal or external toxic lesion. This study provides information on the short-term effects of A l on cytomorphology and the inter-related parameters of the F-actin cytoskeleton and calcium homeostasis. 4 The Plant Cytoskeleton Plant cells may grow via uniform as well as locally restricted expansion of the cell wall resulting in an unlimited variety of cell morphologies. Growth within a single cell may be localized to specific regions such as is the case in tip-growing systems (Derksen et al. 1995). The cytoskeleton of these plant cells, which include types such as root hairs, pollen tubes, moss protonemata and some algal cells, is a dynamic three dimensional array consisting of microtubules, microfilaments and possibly intermediate filaments plus various cytoskeleton-associated proteins. Microfilaments (F-actin) Polymerized or filamentous actin (F-actin) in the plant cell exists in equilibrium with its monomeric subunit, globular actin (G-actin). Contrary to animal cells, most of the actin in mature plant cells is F-actin which is usually cross-linked by (putative) actin-binding proteins to form bundled microfilaments. Individual microfilaments in tip-growing plant cells have a similar diameter to those found in animal cells, approximately 6-7 nm. The arrays are usually oriented parallel to the long axis of the cell, and their position is consistent with a role in cytoplasmic streaming (Pierson and Cresti, 1992). In this study, the term "F-actin" is used since individual microfilaments are not visible using the techniques employed in this study. Actin which is visualized by staining FITC-phalloidin and using light microscopy is assumed to be filamentous and bundled. A s in animal systems, plant microfilaments are sensitive to cytochalasins and other F-actin destabilizing drugs. They are also particularly sensitive to aldehyde fixatives which has necessitated the development of staining protocols that allow vital staining of F-actin (Andersland and Parasarathy, 1993). The organization of F-actin in plant cells has been the subject of numerous studies (Staiger and L loyd , 1991 and references cited therein). However, our knowledge of the biochemistry and molecular biology of actin and possible isoforms remains meager compared that in animals cells. The identification and localization of F-actin in plant cells has been facilitated by the introduction of fluorescent probes such 5 as R h (rhodarnine) and FITC (fluorescein isothiocyanate) labeled phallotoxins which were first developed for widespread use in animal cells (Wulf, et al; 1979; Barak, et al. 1980). The sensitivity of plant actins to aldehyde fixatives and a low sensitivity to stabilization by phallotoxins has led to the development of a wide range of protocols for vital staining using phalloidin (e.g. Collings et al. 1995; Ott, 1992). These protocols ensure that the concentration of phalloidin used be below that considered to cause stabilization or promotion of polymerization of actin (Collings et al. 1995). Bioassays, such as rates of cytoplasmic streaming, are used to assess the cell's viability during the procedure (Perdue and Parasarathy 1985; Wasteneys et al. 1996). Such techniques have been used to visualize the dynamics of F-actin arrays in living cells. Actin Associated/Binding Proteins There is little doubt that a full complement of proteins comparable to the actin-binding proteins found in animals, slime moulds and yeast w i l l be reliably identified in plants. Such actin-binding proteins would modify the behaviour of microfilaments by cross-linking, bundling or severing in response to changes in calcium homeostasis as is the case in other eukaryotic cells (Pollard and Cooper, 1986; Drubin et al. 1990). Actin-binding proteins belonging to the monomer-binding class have recently been identified in plants (Staiger, et al. 1994). These proteins closely resemble profilin, found in animal cells and bind to G-actin with relatively high affinity and are known to have interactions with three types of ligand: linear tracts of proline residues, actin and polyphosphoinositide lipids. Genes encoding profilin-type proteins have been characterized from maize (Staiger, et. al. 1994), timothy grass (Valenta, et al. 1993) and tobacco (Mittermann, et al. 1995). The full significance for plant cell actin behaviour of these proteins has yet to be determined, however, plant profilins can bind to muscle-derived actin demonstrating that the plant molecule is a functional homologue with conserved properties (Valenta et al. 1993). Plant profilin can also act as a sequestering protein in living cells since when microinjected into Tradescantia stamen hair cells it has a 6 rapid and dose-dependent effect on cytoplasmic streaming and on the organization of cytoplasmic strands; these structures, which mirror the F-actin array, fragment and disorganize within an hour after exposure to the actin-binding protein. Profilin-type protein interactions in plants are also apparently sensitive to calcium (Staiger, et al. 1994). A family of actin-binding proteins, known as actin-depolymerizing factors (ADF) have also been recently identified in Lilium longiflorum (Trewavas and Knight, 1994) and Zea mays (Rozycka, et al. 1995). The isolated polypeptides share 40 to 60 percent homology with A D F members from lower eukaryotes and vertebrates. They share the ability to cause actin depolymerization at the steady state through the combined ability to bind monomeric actin and to sever actin filaments, thereby creating new ends for disassembly. Another molecule with the potential to regulate the actin cytoskeleton in plants is the calcium dependent protein kinase (CDPK) (McCurdy and Williamson, 1991 and references cited therein). These proteins are encoded by a small family of genes and each contains a catalytic kinase domain and a calmodulin-like calcium binding domain (Harper, et al. 1991). At least one isoform of C D P K interacts with the actin cytoskeleton and has been found to co-localize with F-actin arrays in onion root cells, Tradescantia pollen tubes (Putnam-Evans, et al. 1989) and Chara internodal cells (McCurdy and Harmon, 1992). Experimental evidence suggests that it is more likely that C D P K interacts with an actin-associated protein rather than with actin itself since the purified components do not interact in vitro. Streaming in plants is inhibited by increased levels of cellular calcium, and may involve phosphorylation (Tominaga, et al. 1987). The C D P K can phosphorylate a putative myosin light chain homologue from Chara in a calcium-dependent manner (McCurdy and Harmon, 1992), which suggests that it may play a role in the inhibition of cytoplasmic streaming in plant cells by calcium (Williamson, 1993). Aluminum may interfere with the functions of these proteins by direct interaction (if it enters the cytosol) or indirect interaction via the perturbation of calcium homeostasis. In either case, changes due 7 to A l toxicity could, in turn, affect the F-actin array and hence have an impact on the overall function of the cell. Microfilaments and Cytoplasmic Streaming Early work on the giant cells of the algae Chara and Nitella produced descriptions of protoplasmic streaming, crucial for cell viability, but did not relate the process to cytoskeletal elements (Kamiya, 1960; Allen and Kamiya, 1964). Subsequent studies utilizing new and improved electron microscopy techniques revealed a variable number of filamentous structures at the interface between the stationary ectoplasm and the moving endoplasm. These structures were also found to be associated with organelles which streamed (Negai and Rebhun, 1966). Clues to the chemical identity of these fibres were first suggested by Wessels et al. (1971) when they showed that cytoplasmic streaming could be inhibited by cytochalasins. Furthermore, the requirement of cytoplasmic streaming to maintain cell function was assessed as a primary functional process and hence most likely controlled by some basic factor of the cell's structure (Willamson, 1972). Conclusive evidence that bundled F-actin composed the filamentous structures, observed by electron microscopy, came from work using decoration with heavy meromyosin. Several researchers showed that the microfilaments within the bundles had the same polarity, thus explaining the unidirectional movement of organelles along the actin "cables" (Palevitz and Hepler, 1975; Kersey et al. 1976). Kato and Tonomura (1977) first isolated 2+ myosin-like, calcium sensitive molecules from Nitella. These proteins possessed M g -ATPase activity and were activated by muscle actin. Nagai and Hayama (1979), as well as Al len , et al., (1980) also showed that small filaments, which turned out to be myosin-like molecules with a long "tail" and a globular "head" , associated directly with streaming vesicles and organelles. In the absence of A T P or at elevated calcium levels, such organelles would cease to move but would remain anchored to the F-actin cables by the small filamentous structures. 8 Our understanding of the cellular regulation of streaming was rapidly advanced using the perfusion model for characean cells. This system was used to demonstrate the participation of A T P in organelle movement and association with microfilaments. It also confirmed the previously suspected role which calcium played in the regulation of cytoplasmic streaming (Nagai and Hayama, 1979; Williamson and Ashley, 1982 ). It was eventually determined that free calcium concentrations above 10 7 M rapidly inhibited microfilament-dependent organelle streaming. Furthermore, organelle translocation rates responded to elevated calcium levels in a dose-dependent manner with increasing calcium levels causing a progressive decrease in streaming. These observations showed that regulation of the algal actomyosin system differed from the that of skeletal muscle where low levels of calcium were inhibitory (Spudich, 1990). Similarly, in pollen tubes, ionomycin inhibits in situ organelle movement and fragments actin cables when external calcium concentrations are raised from 1.5 m M to 5 m M (Kohno and Shimmen, 1987). Cytoplasmic streaming in tip-growing plant cells most likely involves myosin as the driving force for organelle movement. Myosin II-like proteins have been immunolocalized in a variety of plant species including V. longicaulis (Peat and Oliveira, 1994) but it is actually myosins belonging to the myosin I family which are postulated to translocate organelles (Sinard and Pollard, 1989) and which would be inhibited at elevated levels of cytosolic calcium. Small homologous proteins which fall within the size range of myosin Is have been isolated from plants and may be involved in microfilament-based cytoplasmic streaming of organelles (Ma and Yen, 1989). The correlation between cytoplasmic streaming and calcium wi l l prove to be a key tool in assessing some of the rapid effects of Al-induced toxicity as well as monitoring the system for viability during the course of this study. Vegetative filaments of V. longicaulis exhibit vigorous basal-apical-basal cytoplasmic streaming of chloroplasts, mitochondria, nuclei and various other inclusions (Tornbom and Oliveira, 1993b). Chloroplasts and mitochondria translocate along the cortical cytoplasmic strands which are visible using 9 differential interference microscopy. These structures are located peripherally and oriented longitudinally throughout the cell. Nuclei also translocate along the long axis of the cell, endoplasmically, and do not appear to retain a specific association with the cortical, DIC-visible cytoplasmic strands (Ott, 1992). Evidence that both processes are cytoskeleton-dependent comes from studies using cytoskeleton-disrupting drugs. Cytochalasins B and D both inhibit streaming chloroplasts and mitochondria at the same rate but do not affect the translocation of nuclei. Conversely, oryzalin, a microtubule de-stabilizing herbicide, effectively stops the streaming of nuclei without affecting chloroplasts and mitochondria (Tornbom and Oliveira, 1993b). This distinct division between organelle translocation systems in the cell allows the further resolution of the rapid effects of Al-induced toxicity. Since a large amount of data suggest that a putative mechanism of Al-induced toxic effects may be mediated by the interference of A l with cytosolic calcium, it is necessary to provide a brief background on the plant cell calcium homeostat. Calcium Homeostasis in Plant Cells-Background Calcium is the most dynamic and ubiquitous of signaling molecules. It can elicit such a seemingly endless variety of responses to an equally endless number of stimuli. Brief interactions or low levels of compounds which block calcium channels have been shown to have effects on cytosolic calcium homeostasis. The recent development of technologies in the form of dyes (Tsien and Tsien, 1990) and equipment capable of imaging the distribution and dynamics of calcium in single cells has revealed the extensive variability of responses to numerous stimuli (Bush et al. 1993; Bush, 1995; also, See Appendix). Some aspects of calcium homeostasis in plants are similar to animals. However the temporal resolution of calcium dynamics in plant cells is typically slower, on the order of seconds to minutes, compared to milliseconds to seconds in animal cells (Knight et al. 1993). Cytosolic calcium concentrations in plant cells at rest are generally within the range of 100 to 200 n M , typical of those found so far in all eukaryotes (Tester, 1990). In 10 vacuolated cells such as V". longicaulis, the cytoplasm is a thin layer, bounded on the outside by a plasma membrane which is tightly appressed to the cell wal l . The cell wal l , especially in young or actively growing tissue, is porous, allowing for the diffusion of water and nutrients (Carpita, 1982). Within the matrix of the wal l , which is generally composed of polysaccharides and pectins chelated calcium concentrations can be high (approximately 0.1-10 mM) . These calcium ions are strongly chelated by pectins and generally considered non-exchangable under normal growth conditions (Evans, et al. 1991). Furthermore, for most plant cells, the external environment contains calcium concentrations in the range of 1 to 10 m M (Runge and Rode, 1991). The membrane potential for the plant cell plasma membrane is usually around -110 m V (Clarkson, 1984). Hence, a steep gradient for calcium normally exists across the plasma membrane (Kochian, 1995). The cytoplasm surrounds a vacuole which can occupy up to 96% of the cell's volume. This compartment represents the largest accessible store of calcium within the plant cell with the average concentration of vacuolar calcium estimated to be on the order of up to 10 m M and the membrane potential usually around -120 m V (Bush, 1995). Consequently, another steep gradient for calcium exists across the tonoplast membrane (Canut et al. 1993). Recent studies have strongly supported the vacuole's role in maintaining cytosolic levels of calcium and providing a source of messenger calcium during IP3-mediated signal transduction (Canut et al. 1993). In plants, as in animal cells and yeast, the IP 3 pathway appears to be an important second messenger pathway leading to changes in the cell's calcium homeostasis (Berridge, 1993). A n IP3 releasable pool of calcium has been identified in isolated oat tonoplasts vesicles (Schumaker and Sze, 1986). Other intracellular compartments such as the endoplasmic reticulum (ER) may function as calcium stores in plant cells (Quader, 1990). Measurement of calcium efflux from E R vesicles indicate that a rapid exchange of calcium across the E R membrane is possible (Bush et al. 1993). The efflux of calcium from the E R of animals cells may be stimulated by agents such as IP 3 (Kraus-Friedman, 1994), cyclic ADP-Ribose (Leung et 11 al. 1994) or intracellular mediators involved in signalling pathways (Tsien and Tsien, 1990). Similar stimulation of efflux by IP 3 has yet to be confidently reported in plant cells (Bush, 1995) and the degree of importance of the E R as a stimulus-releasable source of calcium has yet to be determined. Since not all plant cells contain large vacuoles, it may be that the role the E R plays in cytosolic calcium homeostasis varies between species, cell type and cell age. Finally, chloroplasts may contain millimolar levels of calcium but whether they play a significant role in the maintenance of resting cytosolic calcium levels or in the stimulus-mediated calcium response is currently unknown. It has been shown that U V light can cause large calcium fluxes across the chloroplast envelope (Miller and Sanders, 1987). To date, a consistent pattern has not emerged for describing changes of calcium levels in plant cells. They can increase, decrease, stay the same or demonstrate a combination of all three traits anywhere from 10 seconds to 10 minutes after a primary stimulus (Bush, 1995). It is generally agreed that cellular calcium homeostasis is maintained by a group of calcium transport proteins similar to those found in animal and other eukaryotic cells (Evans, 1994). Functionally, these proteins fall into two classes: efflux transporters, those which mediate efflux from the cytoplasm and influx transporters, those which mediate influx from intracellular compartments or the external environment (i.e. calcium channels). Plant cells contain a group of primary ion pumps known collectively as the C a 2 + -ATPases which mediate efflux from the cytosol. In plants, calcium transport in isolated membrane vesicles has been attributed to the P-type Ca2+ -ATPase. In animal cells P-type C a 2 + -ATPases, to which the ER-type Ca2+ -ATPase belongs, have molecular masses of 100-120 kDa, are not stimulated by calmodulin, transport two calcium ions for each A T P hydrolyzed and are prevented from forming a phosphorylated intermediate by L a ^ + (Schatzmann, 1989). Calcium transport activities with these characteristics have been found in virtually all membrane preparations from plant cells (Evans, 1994; DuPont et al. 12 1990; Olbe and Sommarin, 1991). In addition to possessing some of the qualities of animal cell P-type Ca2+ -ATPases, those found in plant cells have a slightly higher affinity for calcium (Evans et al. 1991), are inhibited by decreases in resting cytosolic calcium levels and erythrosin B (Wang et al. 1991) and have low specificity for A T P as the substrate for formation of phosphoenzyme and hence transport (Chen et al. 1991). The importance of Ca2+ -ATPases in regulating calcium in plant cells has been inferred from their abundance, intracellular locations and affinities for calcium. However, few studies have been able to assess the function of these pumps in vivo. These difficulties arise from variations in the effects caused by the use of inhibitors such as L a ^ + , vanadate and calmodulin inhibitors. For example, L a ^ + causes an increase in cytosolic calcium levels in guard cells but a decrease in root hairs (Gilroy et al. 1990). During signal transduction, Ca2+ -ATPases are presumed to function primarily to restore cytosolic calcium levels to prestimulus values but, due to difficulties in measuring and standardizing Ca2+ -ATPase activities in living plant cells, validations of this assumption are lacking. The principle routes of calcium entry into the cell from the external environment are mostly likely to be through calcium channels (Bush, 1995). The speed of calcium flux through these channels together with sensors in the channel that determine the open or closed state, permit fine control over the kinetic and spatial properties of calcium influx. Calcium channels in animal cells are extraordinarily diverse in intracellular location and in stimuli that lead to the opening or closing of the pore (Tsien and Tsien, 1990). Calcium channels may be classified by their intracellular localization, those found on the plasma membrane are termed "influx" channels and those found on an intracellular membrane are called "release" channels. Direct evidence has been obtained for two types of selective, voltage operated release channels on the tonoplast (Bush, 1995). The plant vacuole is unique, it serves as both a primary source of stimulus-releasable calcium as well as a primary sink for calcium in unstimulated cells. 13 Aluminum Effects on Tip Growth Calcium ions are a requirement for the growth of tip polarized plant cells such as pollen tubes (Hepler, 1997) and algal cells such as V. longicaulis (Oliveira, 1992). Pollen tubes possess an intracellular calcium gradient which is limited to the tip (Cai et al. 1997). This gradient may be maintained by both the influx of calcium through the localization of selective channels in the tip plasma membrane (See Figure 1.1) as well as the sequestering activity of the endoplasmic reticulum (Pierson et al. 1994). The tip-focused calcium gradient is postulated to mark the precise area of the tip membrane where secretory vesicles fuse. A calcium-dependent actin-binding protein belonging to the annexin family has been shown to mediate vesicle fusion. Furthermore, this process, and hence that of growth, is reliant on the presence of a mesh-like F-actin array at the tube tip. Aluminum has been shown to affect the growth of Riccia fluitans rhizoids (Alfano et al. 1993a). The elevated (resting) cytosolic calcium within the first 20 pm of the tip of apically elongating cells (up to 1.39 p M ) appears necessary for polar growth. It is most likely the consequence of localized calcium influx through calcium channels which are localized to the apical plasma membrane (Hepler, 1997). Aluminum ions could reduce such an influx, possibly by blocking calcium channels (Rengel and Elliot, 1992b) and rapidly compromise growth. Hence, in cells, such as those of V. longicaulis, one predicts that the tip would exhibit particular sensitivity to A l exposure. 14 C a z + « Activated Ca 2 + -channel £ Annexin-like protein 4> Calmodulin jj^jt Calcium V Myosin XXDGCODGCrjCnXD XXXXDGOODCCCCCXX) * MT-based motor ( ( t ) Microtubule C a 2 + C C C O Actin filament (^^) Secretory vesicle • r r r F r r Clathrin Plasma membrane Fig. 5. Diagram showing the principal components of a pollen tube and a hypothetical interpre-tation of the involvement of these components in the growth process. The Ca2' gradient may well play a central role in determining the polarized growth of pollen tubes. It is likely that this is maintained by the opposing activities of ion channels, which allow influx of Ca2' to the tip. and of Ca2*-sequestering organelles, such as the endoplasmic reticulum (ER). High Ca2* levels in the tip may facilitate the docking and fusion of secretory vesicles through the activity of annexin-like pro-teins. Clathrin molecules are likely to take part in membrane recycling. Also, Ca2' and Ca**-acti-vated proteins, such as calmodulin, may exert regulatory activity on both cytoskeletal fibrils and motor proteins (open arrows). At the present time, however, this hypothesis is only speculative (indicated by question marks). The several distinct myosin molecules that have been identified in pollen tubes are indicated as a single generic motor involved in the translocation of organelles (Org) and vesicles. Microtubule (MT) based motors are also shown interacting with generic organelles (X), although this role remains to be clearly determined. For clarity, the cell wall of the pollen tube is not included. • Figure 1.1. (Taken from: Cai, G . , Moscatelli, A . and Cresti, M . (1997). Cytoskeletal organization and pollen tube growth. Trends in Plant Science 2(3): 86-91) Elucidating the rapid effects of aluminum in a model plant cell: the experimental organism. Vaucheria longicaulis Many complex processes in biological systems have been elucidated using "model organisms". There are some fundamental requirements which must be met before an organism is adopted as an experimental system: 1) Does it share a feature common to other plant cells? (e.g.tip growth). 2) Is it easily obtained, maintained and handled with a minimum of perturbation to the system during the course of the study? (e.g. tolerant of low pH, easily cultured) 15 3) Can a cellular process be unequivocally identified such that it may be used as a bioassay to assess the viability of the system during the course of the study? (e.g. cytoplasmic streaming) 4) Does the organism possess a level of simplicity such that elicited effects may be confidently attributed to the primary stimulus? (e.g. a single, optically transparent cell) The unicellular, filamentous alga, V. longicaulis, fulfills all of these criteria and offers an opportunity to resolve some of the early cytological events which contribute to A l toxicity in tip-growing plant cells. Specific Advantages of V. longicaulis as a Model System In terms of elucidating the possible mechanism(s) by which A l causes detrimental effects in plant cells, information on those events which occur during the first half hour of exposure to A l are valuable but scarce (Rengel, 1996). Vaucheria. longicaulis represents a simple, tip-growing system which is ideal for the documentation of cytological events which occur shortly following exposure to A l . Cells of this alga exhibit an inhibition of rates of cytoplasmic streaming for chloroplasts and mitochondria within one minute of exposure. Owing to its relatively rapid growth rates, the impact of A l upon this process is measurable within one hour. Distinct cellular processes (i.e. cytoplasmic streaming) coupled with the rapid onset of measurable toxic effects upon them makes this alga an ideal model system for documenting the early events (within half an hour to one hour of exposure) of A l toxicity. Furthermore, one may work with an identifiable tissue type (i.e. a single cell) and a population which exhibits low morphological variability. The alga thrives in low p H environments (i.e. p H 4.5) and is therefore amenable to ion-trapping (acid loading) protocols, for loading calcium-sensitive tetracarboxylate dyes such as fluo-3. L o w p H conditions are often not feasible in tissues or cells of many species of higher plants. This alga is also periodically subject to wave action which results in weighting from above and hence is not affected by embedding in a colloidal gel. This procedure is advantageous since it allows the specific isolation of the functionally distinct apical zone 16 (AZ) and zone of vacuolation (ZV) . The ability to work with each of these zones independently yields a system by which calcium dynamics, changes in the cytoskeleton and the contribution of the vacuole, can be observed. History and Habitat In 1801, Vaucher described a group of algae which we now know as the genus "Vaucheria". Vaucheria is a member of the Xanthophyceae and is widely distributed in brackish-water and marine environments. Although it has been a generally overlooked member of the marine algal flora of B . C . and northern Washington, it is an abundant organism in salt marshes and estuaries. Morphology and Cytological Structure Vegetative filaments of V. longicaulis are nonsepatate, coenocytic filamentous cells possessing apical growth. Growth occurs at a rate of 172 + 34.2 p m hour"1. Branching and convolution are rare. The cells are cylindrical (up to approximately 40 p m in diameter) and of variable lengths (up to 3 centrimetres in culture). The vacuole occupies a central position within the cell and may consist of up to 93% of the cell's volume (Ott, 1992). Chloroplasts and Mitochondria Chloroplasts in this system are conspicuous organelles due to their large size (13.9 3 + 0.5 pm), density (approximately 5 chloroplasts per 900 mm of cytoplasm), pigmentation and shape. In V. longicaulis, each plastid appears to function as an independent unit and mass movement of the organelles is not often observed. Chloroplasts display dorsoventral symmetry with the ventral side of the plastid facing the plasma membrane and the dorsal side exhibiting a bulge due to the pyrenoid embedded in the plastid. Streaming chloroplasts have a flattened edge on their ventral side which is not observed in the isolated organelles (Ott, 1992). Chloroplasts stream at an average rate of 36.0 + 8.2 pm min" 1. Mitochondria are present within the cytoplasm in large numbers, 17 thousands of these organelles translocate bidirectionally throughout the cell at an average rate of 63.4 + 5.2 pm min" 1. Both mitochondria and chloroplasts may be seen to be associated with a system of "cytoplasmic strands" which are proposed to represent the E R (Ott, 1992). Nuclei The third type of noticeable organelle is that of the pyriform nuclei, approximately 5.2 pm in length, which are numerous throughout the cell and translocate bidirectionaly throughout the cell. It is estimated that a vegetative cell may have on the order of several thousand nuclei for reasons that are currently unknown. These organelles generally stream in the endoplasm. Each nucleus moves as an independent unit with a fairly stiff rod-like structure projecting anteriorly from the narrow end of each nucleus. This structure has been shown, using electron microscopy to be a bundle of microtubules, 10-200 p m long, which Ott has termed a microtubular probe (MP) (Ott, 1992). The method of translocation of the microtubule/nuclei complex is unknown. Nuclei may be seen to translocate over "open areas" where no cytoplasmic strands are visible but may also associate with cortical cytoplasmic strands, as well . Functional zonation in Vaucheria longicaulis: The Apical Zone and Zone of Vacuolation. The cytoplasmic zonation observed in V. longicaulis is similar to other tip-growing cells which possess a distinct polarity (Menzel, 1994a). The central vacuole does not extend into the youngest tip region of the vegetative filament. This area, termed the apical zone, is the youngest region and site of active growth. Organelle density in this region is high and movements are complex, three-dimensional and difficult to interpret. Cytoplasmic streaming is absent from this portion of the cell. The region of the cell found behind the tip is older, contains the central vacuole and is termed the "zone of vacuolation". 18 Cytoplasmic streaming occurs throughout the cortical cytoplasm which may be as thin as 15 p,m. Vaucheria longicaulis vegetative filaments have well developed wound healing capabilities to counteract damage inflicted through grazing, fragmentation by debris in winter storms and sand abrasion. In order to accomplish this, the alga must possess the ability to rapidly direct and control specific cellular processes on a localized scale. Statement of Aims of Thesis and Hypotheses to be Tested Therein This is the first study to document the cytological events which occur in a single-celled, tip-growing algal model system during exposure to A l . I focus on changes in the inter-related parameters of cytoplasmic organization, the F-actin array and cytosolic calcium. I suggest a mechanism whereby A l may affect key cytological structures and functions, at least in part, by perturbing calcium homeostasis. Support for this argument comes from the observation that modulation of calcium mimicks the toxic effects of A l in an Al-free system. Specifically, the hypotheses to be tested in this thesis are as follows: a) . Aluminum affects key cellular processes and structures such as cytoplasmic streaming, stability of the E R and the cytoplasmic strands in vegetative filaments of Vaucheria longicaulis. This wi l l be tested by treating cells with A l and utilizing light microscopical techniques and fluorescent, lipophilic dyes to visualize changes in cytomorphology. b) . The effects of A l on these processes and structures arise, in part, due to the disorganization of the F-actin cytoskeleton. This hypothesis w i l l be tested by treating cells with A l and visualizing the F-actin array in living cells. c) . Short term A l toxicity is the result of changes in calcium homeostasis. If this is true, then exposure to A l should cause changes in calcium homeostasis within the cell, in a time-frame consistent with the disruption of the aforementioned processes and structures. This prediction wi l l be verified by utilizing calcium specific dyes, fluorescent phallotoxins and lipophilic dyes. d) . Calcium modulators should mirnick the effects of A l in an Al-free system. 19 e). The effects of A l should be blocked by inhibiting the putative cytosolic calcium increase, using inhibitors. The purpose in exploring the effects of A l on these parameters stems from observations that the viability of tip-growing cells relies on F-actin (Cai et al. 1997; Piersonetal . 1994; Hepler, 1997; Pierson and Cresti, 1992). This arises from the fact that many processes (such as cytoplasmic streaming) which occur in tip-growing cells require an intact F-actin array. Furthermore, cellular events which occur in the tip are mediated primarily by cytoskeletal dynamics a few of which include vesicle fusion mediated by F-actin and annexins (Cai et al. 1997) cell wall deposition ( L i , 1996), and the maintenance of high concentrations of calcium channels in the plasma membrane located at the cell's tip (Pierson et al. 1994; Malcolm et al. 1996). This study provides the first data on the effects of A l on the inter-related parameters of cytoplasmic organization, organelle streaming, the F-actin array and calcium dynamics. It attempts to provide insight into some putative mechanisms by which Al-toxicity may arise. However, it is acknowledged that these data do not provide unequivocal proof of either an external or internal toxic lesion. Chapter 3 provides data which suggest that A l affects cytoplasmic streaming and organization via the disruption of the F-actin array. Chapter 4 justifies this suggestion by providing data which demonstrate that A l has pronounced effects on F-actin. Chapters 5 and 6 provide data which show that calcium homeostasis is perturbed upon exposure to A l . These are discussed in a context which links Al-induced changes in calcium homeostasis to the Al-induced effects documented in the previous two chapters. Furthermore, data are presented which support the hypothesis that the apical portion of theV. longicaulis vegetative cell exhibits particular sensitivity to A l . 20 2.0. Materials and Methods Culture conditions V. longicaulis var. macounii was collected from intertidal (brackish) regions in North Vancouver, B . C . Sampling was conducted every week from a different part of the site. This was done in order to mimmize culture-induced artefacts (Ott, personal communication) as well as to obtain individual cells from a broad range of populations of the same species. Ten stock cultures were grown on their natural substrate in 500 ml clear plastic containers, supplemented with Instant Ocean medium (Aquarium systems, Eastlake, OH) diluted 2.5 times with distilled, de-ionized water (since the alga lives in brackish environments). This is referred to as artificial sea water (ASW) throughout the text. The composition of Instant Ocean is given in the Apprendix. Thirty experimental cultures were established (three from each of the ten cultures) in p H 7.5 and p H 4.5 (for low p H controls). For the latter, the p H of the A S W was adjusted by drop-wise addition of 1 M and 0.1 M HC1 at room temperature. Cultures were kept at 12 degrees C with vita-lite -2 -1 fluorescent lighting providing 250 pmol m s under a 16-8 hour light/dark photoperiod. Experimental cultures were established by removing vegetative filaments from stock cultures and maintaining them in plastic chambers filled with A S W with or without the addition of 80 | i M A l prepared as the chloride salt in both p H 4.5 and p H 7.5. Potassium chloride solutions (80 u M ) made up in A S W , p H 4.5 were added to some cultures as a control. A n 80 p:M solution of A I C I 3 was prepared by dissolving the solid in A S W at p H 4.5. Aluminum stock solutions were prepared by adding solid AICI3 to A S W to make a 1 m M solution according to to Lindberg (1990). The p H of the stock was adjusted to p H 4.5 over the course of two days prior to use. The solid reagent was visibly dissolved in the media at this p H . However, G E O C H E M predictions indicate that only 21 26% of A l C l j is present as the A l 3 + form, primarily due to complexes formed with sulphate present in the A S W medium (See Appendix). G E O C H E M was run on an I B M pc computer. The algae did not tolerate Instant Ocean solutions which were diluted more than 2.5 times. Temperature and lighting parameters for experimental cultures remained the same as those for stock cultures. L o w p H (4.5) controls in the absence of A l were carried out. A l l experiments were carried out at room temperature unless otherwise stated. Controls for varied temperature were also carried out at 17, 20 and 23 (i.e. room temperature) degrees C. The duration of the A l treatments and other pertinent information is given in the text. The volume of A S W in each stock culture chamber was 500 ml and 300 ml in each experimental culture. The p H of each chamber was monitored daily for deviations from p H 4.5. Sampling At least three independent trials were carried out (n=3) for each experiment conducted (unless otherwise stated). For each trial, three experimental cultures were established from each of ten independent stock cultures. The number of cells which remained viable per trial (i.e. from which data were used) varied considerably but was >25 unless otherwise indicated. Where necessary, standard deviation was calculated by a formula application to raw data in CricketGraph III. Data presented as micrographs or Relative Fluorescence Intensity (RFI) calcium profiles are respresentative examples from at least three individual replicates unless otherwise stated. Controls for each experimental procedure are given under each separate heading. Gelatin Embedding Protocol for Immobilization of Cells Solutions of 1% (w/v) P h y t a g e f ^ (Sigma Chemical Corp.) were made using A S W and cooled to 20 degrees C (this alga is periodically uncovered by tidal action and tolerates temperatures up to 26 degrees C for 6 hours at a time with no changes in rates of cytoplasmic streaming). A loop measuring approximately 4 cm in diameter which had been constructed from stainless steel wire attached to a dissection probe was dipped into the 22 gelatin solution and raised carefully such that an intact film occupied the centre of the loop. Since the thin layer cooled rapidly, the loop required quick and smooth transfer onto a glass coverslip such that a homogenous layer of gelatin was laid upon it. Vegetative filaments were then quickly transferred onto this layer, separated using a small stream of medium dispersed through a Pasteur pipette and blotted using filter paper without touching the cells. Cells were gently impressed into the gelatin using air blown through a Pasteur pipette attached by rubber tubing to a compressed air outlet. Cells were examined after resting for 2 hours at room temperature. Immobilizing cells in this manner minimizes artefacts which may arise as a result of direct mechanical handling. DiOCf iG) Staining for the Endoplasmic Reticulum and Mitochondria A stock solution of DiOC6(3) (Molecular Probes) was prepared by dissolving 25 mg of the compound in 0.25 ml of absolute D M S O and adding 4.75 ml A S W to make a final volume of 5 ml (5mg/ml). A working solution of 0.5 mg/ml was prepared from this A S W stock and added directly to gelatin embedded cells, using a Pasteur pipette. Cells were exposed to the dye for 5 minutes. Cells were then washed in A S W , p H 4.5 and examined under the microscope immediately. DiOCg(3) fluorescence was observed on an epifluorescence microscope (Axiophot, Carl Zeiss Co. , Ltd. F R G ) equipped with a Zeiss integrated camera system. Images were recorded on Kodak Tmax 400 film (pushed to 1600 A S A ) and processed accordingly. For each type of optics used, the aperture setting, f-stop and exposure time were kept constant. Photographs were obtained within 1 minute of U V irradiation to minimize dye-induced cytotoxicity. Microscopic Observations for Growth Rates. Cytoplasmic Streaming and Cytoplasmic Strands Growth measurements were recorded using a Wi ld M P S 11 dissecting microscope equipped with a calibrated eyepiece micrometer. Cells were placed on a petri dish in a minimal amount of A S W such that they were completely covered by media. The position of their cell tips was marked on the bottom of the dish using a permanent marker. Dishes 23 were left on the microscope stage so as not to disturb the position of the filaments by moving the petri dish. Measurements were done every hour for 6 hours and every 8 hours for up to 48 hours. Each trial consisted of measuring 75 cells. Three separate trials were conducted (n=3). Cytoplasmic streaming was analyzed from videotaped material. Data for dose-dependent inhibition by A l of cytoplasmic streaming (Figure 8, Chapter 3) utilized various concentrations of A l . Each was prepared by diluting a 1.2 m M stock solution prepared in A S W , p H 4.5 at room temperature. Cells were incubated in each concentration of A l in low (0.025 mM) , medium (2.5 mM) and high (8.0 mM) C a C l 2 solutions for 60 minutes each. Calcium chloride solutions were prepared by dissolving the solid reagent in A S W , p H 4.5. This was the case in each experiment were these solutions were used. For amelioration studies (Tables 3 and 4), solutions of C a C l 2 were made up at concentrations of 1.0 m M , 2.0 m M , 3.0 m M , 4.0 m M , 6.0 m M and 8.0 m M in A S W at p H 4.5. Videotaping was conducted with a Sony D X C 101 camera attached to a Zeiss Axiophot microscope (Carl Zeiss Co., Ltd. F R G ) and observed on a J V C T M - 9 0 C A high resolution video monitor. Thirty filaments were videotaped for each experimental set (n=3). The distance travelled by various organelles (chloroplasts, mitochondria and nuclei) was measured over a period of 3 minutes. Values are expressed as the mean speed + SD expressed in p m m i n 1 for each organelle or as a percentage of the values obtained in control sets. The organization of the cytoplasmic strands and organelle distribution was studied with a Zeiss Axiophot microscope equipped with differential interference contrast (DIC) optics. Images were recorded on Tmax 100 (shot at A S A 400) using a Zeiss integrated camera system. For each type of optics used, the aperture setting, f-stop and exposure time were kept constant. 24 Fixation Protocol for Visualization of F-actin Prior to aldehyde fixation three drops of methylcyanoacrylate (Crazy G l u e ™ ' ) were placed on a glass slide and slowly heated with a small flame from an alcohol burner until fuming began. Filaments were placed on a glass coverslip and inverted over the fumes of the methylcyanoacrylate for 45 minutes. The filaments were then processed by placing the coverslip into a glass container containing a freshly prepared solution of 3 % formaldehyde and 0.1 % glutaraldehyde in phosphate buffered saline (PBS) for 1 hour at room temperature. A solution of 0.1 %(w/v) sodium borohydride was used after fixation to reduce aldehyde induced fluorescence. Vital Staining for F-actin Living cells placed on a gelatin overlaid coverslip were pre-incubated sequentially in solutions containing 0.001% (w/v) saponin + 1 % D M S O (v/v), 0.01% saponin + 1% D M S O (v/v) and 0.1% saponin + 1% D M S O (v/v). Both reagents (Sigma Chemical Corp.) were prepared in A S W , p H 4.5. Incubations were done at 17 degrees C for 10 minutes per step. N o changes in viability were observed in cells which were incubated in this fashion. Cells were washed for 30 minutes in A S W , p H 4.5 and transferred to a solution containing 0.025 m M FITC-phalloidin which was prepared from a stock solution of 2.5 m M . The stock solution was prepared in 100 % methanol and stored at -20 degrees C . Prior to staining the cells, the FITC-phalloidin was desiccated and reconstituted in A S W , p H 4.5 containing 10 m M E G T A , 1% D M S O (v/v) and 0.1 M M g S 0 4 to produce a working solution of 0.025 m M . Cells were incubated in this solution for up to 2 hours at 17 degrees C in total darkness. Controls for the specificity of FITC-phalloidin binding were conducted by incubating saponin-permeabilized living and fixed cells with unlabeled phallacidin (300 mg/ml in A S W , p H 4.5) for 2 hours prior to staining with F I T C -phalloidin. Separate controls using 1% D M S O and 0.001%-0.1% saponin in A S W , p H 4.5 were carried out to determine i f either compound affected cell viability. Each trial contained at least 75 cells and seven independent trials (n=7) were carried out. Control 25 cells which demonstrated a significant reduction or complete cessation in cytoplasmic streaming during staining with FITC-phalloidin were not considered (<5% per trial, n=7). Aluminum-treated cells, in which cytoplasmic streaming could not be monitored, were assessed for recovery by removal to Al-free media after staining with FITC-phalloidin. Only those cells which exhibited recovery in rates of cytoplasmic streaming were considered as data in this study. FITC-phalloidin fluorescence was observed on an epifluorescence microscope (Axiophot, Carl Zeiss Co . , Ltd . F R G ) equipped with a Zeiss integrated camera system. Images were recorded on Kodak Tmax 400 film (pushed to 1600 A S A ) and processed accordingly. For each type of optics used, the aperture setting, f-stop and exposure time were kept constant. Assay for quantification of G actin G-actin was measured by the DNase I inhibition assay of Heacock and Bamburg (1975). Approximately 28 g (wet weight) of cells were lysed in a petri dish at -10 degrees C in a solution containing 500 ml of 10 m M Tris HC1 containing 2 m M M g C ^ , 0.025 m M dithioerythritol, 1% (v/v) Triton X-100 and 15% (v/v) glycerol. Twenty microlitres of DNase I (0.1 mg/ml) (Sigma Chemical Corp.) and 1.0 ml calf thymus D N A (Sigma Chemical Corp.) (0.1 mg/ml) were added and mixed with the sample in succession. Absorbance was recorded over 5 minutes using a Perkin-Elmer Lambda spectrophotometer. The percentage decrease in DNase activity was converted to mg actin with a standard curve constructed using freshly prepared monomelic rabbit muscle actin (Sigma Chemical Corp.). Two independent trials were carried out (n=2). Cytochalasin D and Oryzalin Studies Cytochalasin D is a drug used to disrupt microfilament integrity and inhibit microfilament-dependent processes such as translocation of chloroplasts and mitochondria. Oryzalin is an herbicide used to disrupt microtubules and inhibit microtubule-dependent processes such as nuclei translocation. Cytochalasin D (Sigma Chemical Corp.) was 26 prepared as a 5 ml stock solution containing 1 mg/ml in absolute D M S O and was diluted with A S W , p H 4.5 to 20 ml of a 10 mg/ml working solution. A stock solution (7.5 m M in 100% acetone) of Oryzalin (Dow Elanco Co. , Greenfield, I N , U . S . A ) was diluted to a working concentration of 10 m M . Controls using 1% (v/v) D M S O in A S W , and 1% (v/v) acetone in A S W , p H 4.5 (as maximum concentrations) were also carried out. C T C (chlortetracycline) Staining for Membrane Associated Calcium Gelatin embedded cells were incubated in total darkness and at room temperature for 5 minutes with 100 p M C T C . The working solution of 100 u M C T C was prepared in A S W from a stock solution of 1 m M in A S W , p H 4.5. The duration of the treatment did 2+ not exceed 5 minutes to minimize the artefactual binding of Ca to membranes by C T C (Reiss et al. 1983). After staining, cells were washed three times using A S W , p H 4.5. 2+ 2+ Since C T C may also fluoresce in the presence of M g as well as C a , Oxytetracycline (OTC) staining was carried out as a control. Oxytetracycline is an analog of C T C which is 2+ specific for M g (Wolniak et al. 1980). Parameters for the preparation of O T C and the experimental procedures were identical to those for C T C . Chlortetracycline fluorescence was observed on an epifluorescence microscope (Axiophot, Carl Zeiss Co. , Ltd . F R G ) equipped with a Zeiss integrated camera system. Excitation wavelengths were achieved by a filter system which passed 340-380 nm from a U V light source. Emission fluorescence was directed through a filter which passed frequencies >430 nm. Images were recorded on Kodak Tmax 400 fi lm (pushed to 1600 A S A ) and processed accordingly. For each type of optics used, the aperture setting, f-stop and exposure time were kept constant. Use of Fluo-3 for the Visualization of Free Cytosolic Calcium: Properties of Fluo-3 The fluorochrome component of fluo-3 is based on fluorescein and, as a result, it is 2+ excited in the visible light range. Fluo-3 does not undergo a wavelength shift on C a binding and is used in single wavelength mode. Fluorescence intensity increases 27 approximately 40 fold upon binding of the dye to Ca with the unbound form of the dye 2+ being almost non-fluorescent. The K d of fluo-3 for Ca (400 nM) is higher than for any other indicator available for this purpose. Although fluo-3 cannot be used in the ratio mode 2+ it has some advantages over other Ca sensitive dyes such as the ratiometric stilbene fura-2: (a) It is less demanding on optics and excitation light sources for effective illumination (b) the excitation wavelength does not overlap with pyridine nucleotide autofluorescence and (c) it can be used with low power visible light sources, and (d) a standard fluorescein 2+ filter set is suitable. The disadvantage of fluo-3 is that is that small changes in C a (Cyt) may be undetectable i f cytoplasmic streaming is present or i f the cell does not lie flat, as is the case in this system. However, the major problem, that of compartmentalization, was less severe with fluo-3 than with fura-2. Ac id Loading Procedure for Fluo-3 Working solutions of fluo-3 were made up in A S W at p H 4.5 from a stock solution of 8 m M in A S W , p H 4.5 and 1% D M S O (v/v). These were pipetted into petri dishes and consisted of sequentially increasing concentrations of the dye which ranged from 0.04 m M to 4 m M in A S W , p H 4.5. containing 0.001% (w/v) saponin. Cells were incubated in fluo-3 in a sequence of 3 incubation steps. Prior to dye loading, cells were treated with 0.01% digitonin (w/v) for 1 hour before incubation in the fluo-3 solutions. Cells were incubated in each concentration of fluo-3 for 45 minutes at room temperature. The number of viable cells which became loaded with the dye was higher using the stepwise dye gradient than when a single, high concentration of the dye was used. Prior to observation, cells were washed three times in A S W , p H 4.5 for 10 minutes each time. Approximately 15% of cells demonstrated fluo-3 in their cytoplasm as monitored by brief observation under a fluorescence microscope (Excitation, 490 nm; Emission, 525 nm). On average, this corresponded to 8 cells for each trial. Four independent trials were carried out (n=4). 28 The method of "acid loading" cells with C a -sensitive dyes was developed specifically for plant cells because of technical diffculties associated with using other loading strategies (Read et al. 1992). At acidic pH's (eg p H 4.5), cells may become 2+ permeable to the Ca -dyes because these compounds are in their uncharged, undissociated (i.e. protonated) form. Once inside the cell, the dye should become trapped in the cytosol due to the loss of its protons upon encountering physiological pH's. Picospritzing for localized exposure of A l Picospritzing for localized exposure to A l in a single cell was conducted by Dr . Mark N . Rand from the University of Washington according to the procedure of Rand and Breedlove (1992). Briefly, a double-barrelled pipette was constructed by pulling two pipettes from 1 mm glass tubing and taping them together. One acted as an outflow (for A1C1 3 solution) while the other acted as suction. Each pipette was connected to a Beckman picospritzer (pump system) via 0.75 mm rubber tubing. Approximately 50 picolitres of A1C1 3 (in A S W ) was expelled from one pipette and contacted the cell for approximately 1 second before it was suctioned into the other pipette. This allowed A l to be introduced to a specific portion of the cell for a very short period of time. Determination of Relative Cytosolic Calcium Levels from Fluo-3 This method serves to provide the researcher with confirmation that fluo-3 fluorescence can be modulated by the use of ionophores and hence can report changes in 2+ C a (cyt)- However, it does not give any indication of quantitative (absolute) values of 2+ C a (cyt)- The contribution of the extracellular indicator was assessed by the addition of 0.1 m M M n C l 2 and the subsequent addition of 0.3 m M D T P A approximately 30 seconds later. Ionomycin was added at a concentration of 40 m M such that a maximal signal increase occured. N o serum albumins were present in the A S W , p H 4.5 (serum albumins bind ionomycin). A solution of 3 m M M n C l 2 was added to the medium and the entire procedure was repeated for cells not loaded with fluo-3. Due to the difficulties in 29 calibrating single wavelength dyes no quantitative values are given for cytosolic calcium. Rather, changes are expressed as relative levels of calcium within the cell. Calcium imaging instrumentation Cytosolic calcium levels were observed with the use of the single-excitation dye, fluo-3 and an Attofluor digital fluorescence microscopy system (Atto Instruments Inc.; Carl Zeiss Canada, Ltd.). Using an excitation wavelength of 490 nm, fluorescence intensities (525 nm) were obtained for single cells (to obtain spatial distribution) or cell 2+ populations (to observe Ca (Cyt) dynamics in response to A l ) . Populations of cells were used preferentially over single cell images because of problems with signal to noise ratios. 2+ However, single cells were used to determine spatial patterns of C a (cyt)-For cell populations, cells embedded on gelatin overlaid coverslips were placed in an experimental chamber (plastic petri dish) and covered with 100 ml of A S W . Solutions containing various concentrations of calcium, A l or calcium modulators could be introduced via a plastic pipette connected to a pump by rubber tubing and fixed on the edge of the petri dish using plasticene. Similarly, solutions could be removed via a separate tube such that the cells were not touched. Controls were conducted to ensure that movement of the solution did not cause any changes in calcium dynamics. Regions of Interest (ROI) were defined for each individual cell during each trial using the Attofluor software. Regions of interest were indicated for either the (apical zone) A Z at one per cell or for the zone of vacuolation (ZV) at approximately three per cell. Images were sampled every 2 or 5 seconds in order to minimize photobleaching, U V mediated cytotoxicity and U V induced compartmentalization of the dye. Each experiment was performed on at least two independent cultures (n=2) and the sample number of cells imaged per culture was, on average, 15 cells (unless otherwise stated). Data were obtained for each R O I and recorded. Changes in Relative Fluorescent Intensity (RFI) were recorded immediately after the addition of 80 p M A I C I 3 to fluo-3 loaded cells and after re-focusing. Representative 30 graphs were obtained from cell populations containing approximately 15-40 viable cells. Data from dead cells, which tend to accumulate the dye, were discarded. Raw data were imported into CricketGraph III and expressed with the x-axis representing "time" and the y-axis representing each RFI value. Each graph is a representative graph and each point on the graph represents a mean response for a single trial. Digital Image Processing of Cytosolic Calcium Images Images obtained from the Attofluor system were imported as T IFF files into N I H Image. For all data processing, imaging conditions were kept constant. Brightness/contrast enhancement and pixel level filtering were performed for final pseudocolour images. The final fluo-3 images included a background substraction step as well as a threshold setting in which regions of low signal were set to black. A l l data processing was conducted on a Power Macintosh 9500/132. Calcium modulators T M B - 8 is used as an inhibitor of calcium release from internal stores (Brumell and Machlachlan, 1989). This reagent was purchased from Calbiochem and a working solution of 2 p M was prepared from a 20 m M stock solution in methanol. Since T M B - 8 was not entirely soluble in methanol, the concentrations reported are the maximums possible. Ionomycin was purchased from Sigma Chemical Corp. and dissolved in D M S O to make a stock solution at a concentration of 4 m M . Working solutions of ionomycin contained a final concentration of 40 p M . Ionomycin was used instead of Br-A23187 since the former compound can lose two acidic hydrogens and form an uncharged 1:1 complex with a divalent ion. Br-A23187 forms a 2:1 complex making ionomycin a more 2+ 2+ efficient C a transporter. Furthermore, ionomycin has better selectivity for C a over 2+ 2+ 2+ M g , favouring C a approximately twice as much as M g . The ionophore Br -A23187 shows no preference for one cation over the other in Chara (Reid and Smith, 1993). Perhaps most importantly, however, is the fact that Br-A23187 is an efficient 31 proton exchanger and rapidly acidifies the cytoplasm (Reid and Smith, 1993) whereas ionomycin does not. Hence, major effects associated with lowering cytoplasmic p H are avoided by using ionomycin. When used, E G T A was made up in A S W , p H 4.5 and 2+ added at concentrations approximately 5 times that of [Ca ] e x used in the experiment. It should be noted that E G T A is p H sensitive and the pCa at p H 4.5 is at least 10 fold less than at p H 7.4 (Bers, 1994). Speciation predictions obtained from the G E O C H E M program state that 30 % of E G T A is present as C a - E G T A at p H 4.5. Thapsigargin was used to stimulate the release of calcium from IP3-sensitive intracellular stores. Thapsigargin was purchased from Sigma Chemical Corp. and a 1 m M stock solution was made up in 10 ml of absolute D M S O . Working solutions were made by pipetting a small amount of the stock solution into A S W , p H 4.5 to make concentrations of 500 p M and 1 u M . Thapsigargin was used at 500 u M on isolated cell tips and 1 u M on whole cells and isolated vacuolated regions. For unknown reasons, isolated cell tips did not tolerate the higher concentration of the drug. Due to the distinct differences observed in calcium dynamics between the A Z and Z V , both regions were treated separately, using the gelatin embedding method. External calcium concentrations and internal stores of calcium were manipulated in the presence or absence of A l in order to see i f various treatments would lead to changes in calcium dynamics and a rearrangement of the F-actin cytoskeleton. Only cells which exhibited chloroplast/mitochondrial streaming and growth rates comparable to those of controls were initially selected for experimentation. 2+ Furthermore, experiments were done in low [Ca ] e x as opposed to "zero calcium" concentrations since cells displayed abnormal cytoplasmic streaming rates or rapid tip lysis under the latter condition. Cytotoxicity Criteria Many fluorescent probes are potentially cytotoxic. Cytotoxicity wi l l depend on the concentration of the probe employed, the duration of its application and its interaction with 32 other cellular constituents. Therefore, it is important to consider the level of cytotoxicity of a probe in a living system before describing changes which may be attributed to the variable(s) in question. Some factors that were considered throughout the course of this study are as follows, (a) is the cell viable?, (b) does the cell grow normally following application of the probe?, (c) are cytoplasmic streaming rates and/or organelle motion and/or morphology unaffected by the probe and (d) do the cells continue to function normally? In this system, viability is defined primarily by cytoplasmic streaming and growth rates. Only those cells which fulfilled all the criteria of possessing patterns and rates of cytoplasmic streaming and growth, as well as patterns of cytoplasmic strands comparable to those in controls were selected for A l studies. Such cells were considered viable. Furthermore, experimental cultures were placed in Al-free A S W , p H 4.5 following each trial in order to determine whether or not cells could recover. Those cultures which did not recover were not considered in the evaluation of data. Thus, the effects of 80 | i M A l could be considered those of toxicity and not the responses of dying cells. 33 3.0. Effects of Aluminum on Organelle Streaming and Cytoplasmic Organization 3.1. Introduction The role of A l as a factor in yield decline of wheat and rye grown in acid soils was documented as early as 1918 (Hartwell and Pember, 1918). Prior to this, toxic effects of A l in plants were already suspected (Rothert, 1906; Flur i , 1909). Data from whole plant studies indicate that higher plant root hair cells undergo severe morphological changes which result in a loss of viability when exposed to A l . These include effects such as the inhibition of cell division (Horst, et al. 1983) and cell elongation (Matsomuto, et al. 1977). Although the tip-growing, root hair cell is suspected to be particularly sensitive to A l (Jones et al. 1995), few studies have utilized a cytological approach to describe its toxic effects on key cellular processes and structures such as cytoplasmic streaming and the E R . Studies dealing with the inhibition of cation transport across the plasma membrane of cells are numerous (Rengel, 1996 and references cited therein). Studies which look at the effects of A l on cell wall biosynthesis (Le Van et al. 1994) and the homeostasis of calcium ions (Rengel, 1992; Kochian, 1995; Huang, et al. 1992, 1993; Nichol and Oliveira, 1995; Ryan and Kochian, 1993) are also well represented in the literature. This study provides data which document the effects of A l on cytoplasmic streaming, cytoplasmic organization and the E R , functions and structures known to be dependent on the cytoskeleton as well as calcium (Doree and Picard, 1980; Williamson and Ashley, 1982; Tominaga, et al. 1983; Quader, 1990). Due to the myriad toxic effects documented after exposure to A l , it is important to discern those which arise from the primary stimulus (Al) versus those which are caused by a degeneration of the cell's viability due to experimental treatment. Part of the problem, limiting the use of in vivo conditions in A l toxicity studies, is the difficulty in finding suitable organisms amenable to easy use in reproducible and rigorous experimental testing. 34 Isolated tissues or cells of higher plants are generally not tolerant of the low p H environments required to conduct A l toxicity studies, and hence p H effects may complicate the interpretation of A l induced effects. Vaucheria longicaulis not only thrives in lower p H environments but is also sensitive to environmentally meaningful levels of A l (80 (J,M). Bioassay parameters are easily established due to the large size of the cell and vigorous cytoplasmic streaming which occurs throughout zone of vacuolation. Furthermore, Vaucheria longicaulis demonstrates two distinct motility systems. One is a microfilament-based system which transports chloroplasts, mitochondria and other inclusions within this size range and the other is a microtubule-based system which transports nuclei. Cytoplasmic streaming proceeds as a continuous flow of organelles and other cytoplasmic inclusions. The direction of movement is parallel to the length of the filament and all organelles move from the basal to apical to basal region in continuous motion (Tornbom and Oliveira, 1993b). This allows the resolution of preferential effects of A l on one or both of these cytoskeletal-based motility systems. Monitoring the dynamics of the cytoplasmic strands (Tornbom and Oliveira, 1993a & b) and demonstrating their identity as E R is valuable in obtaining a more accurate picture of the effects of A l . This follows the fact that the E R shows an association with microfilaments in many plant species (Lichtscheidl et al. 1990; Fuhrman, et al. 1990). In V. longicaulis, a close relationship has been described between the cytoplasmic strands and organelles such as the mitochondria and chloroplasts which exhibit microfilament dependent streaming (Tornbom and Oliveira, 1993a). In this work, the versatile features offered by V. longicaulis are utilized to conduct the first study of the effects of A l in vivo upon the basic cellular processes of cytoplasmic streaming and cytoplasmic organization using both DIC and DiOC6(3) epifluorescence microscopy. Addition of A l (80 uM) results in rapid inhibition of cytoplasmic streaming as well as the dissipation of the cortical longitudinal E R . Subsequently, the DIC visible cortical 35 cytoplasmic strands exhibit progressive degrees of disorganization. However, chloroplasts and mitochondria maintain a close association with these structures suggesting that microfilaments may represent another component of the DIC visualized cortical cytoplasmic strands. 36 3.2. Results Control Cultures Vegetative filaments grown in A S W at p H 7.5 exhibited vigorous cytoplasmic streaming. Chloroplasts and mitochondria streamed at 34.3 + 6.8 p m /min and 65.7 + 2.4 pm /min, respectively. Nuclei streamed at 176.4 + 5.9 pm min" 1. Growth rates were 110 + 5.5 p m hour"1 and cells grew as straight filaments with no branching or convolution. The vacuole occupied a central position within the cylinderical cells but did not extend into the youngest, most actively growing region, termed the apical zone (AZ) . Furthermore, cytoplasmic streaming was restricted to the region of the cell which contained the vacuole, termed the zone of vacuolation (ZV) and was not observed to occur in the A Z . In the Z V , chloroplasts and mitochondria were seen to be associated with a network of DIC visible, longitudinally oriented structures termed "cytoplasmic strands". Chloroplasts possessed an oval shape with narrowed ends and both chloroplasts and mitochondria were evenly spaced throughout the Z V (not shown). Images were recorded for low p H cultures (see below) and these represent controls for the A l studies. Low p H Control Cultures Vegetative filaments of V. longicaulis grown in A S W , p H 4.5 for two weeks exhibited apical growth. Growth rates averaged 110 + 14.2 p m hour"1 and filaments remained straight with little branching or curling. The vacuole retained its position in the central cytoplasm and did not extend into the apical zone. Nuclei and mitochondria were distributed evenly throughout the cytoplasm and chloroplasts remained evenly spaced and oriented parallel to the long axis of the filament (Figure 1, taken 100 pm from the cell tip and Figure 2, taken 250 pm from the cell tip). Cytoplasmic streaming remained restricted to the zone of vacuolation and was not observed in the apical zone. 37 The chloroplasts retained their oval shape with conspicuously narrowed ends and flattened edges on the ventral side (Figure 2, "chp"). They exhibited rates of streaming comparable to those in control cultures of 35 + 3.8 u,m min" 1. Similarly, nuclei retained their teardrop shape (Figure 2, short arrow) and possessed streaming rates comparable to those in control cultures (189 + 5.8 | i m min"1). Mitochondria remained evenly spaced throughout the filament and streamed at a rate comparable to control cultures (62 + 5.1 u,m min"1). The DIC-visible strands, referred to throughout the text as "cytoplasmic strands", remained present in the cytoplasm and occasionally exhibited activity such as tubule merging or branching (data not shown). Both morphological configurations were observed in low p H grown cells: the thin, linear strands observed when focusing toward the plasma membrane (Figures 1 and 2) and the reticulate pattern, which was found when focusing toward the vacuole (Figure 2, inset and Figure 3). Although the high density of organelles in the cell makes visualization of this latter component difficult, it was observed to possess the anastomosing qualities observed in control cultures. In the apical zone, neither reticulate nor longitudinal strands were detected due to the density of organelles in this region where it was difficult to interpret data obtained using DIC microscopy. In the zone of vacuolation, it was difficult to discern whether the longitudinally arranged cytoplasmic strands may develop into the polygonal-type network and vice versa. Filaments treated with DiOC6(3) using the procedure of Terasaki, et al. (1984), revealed the presence of two morphological types of DiOC6(3)-positive structures: one appeared as long, tubular elements oriented parallel to the longitudinal axis of the cell and occupied a cortical position close to the plasma membrane (hereafter referred to as the longitudinal component). B y modifying the concentration of DiOC6(3) used it was possible to achieve simultaneous visualization of mitochondria which were seen to be specifically associated with individual tubular elements (Figure 4, arrows). For comparision, a portion of the cell stained for mitochondria only is shown in Figure 4, 38 inset. A second morphological configuration of DiOC6(3)-positive structure existed closer to the tonoplast and appeared as a polygonal network connected by tubular structures, (hereafter referred to as the lamellar component) (Figure 5a and inset). It was interesting to note that the longitudinal component existed only in the zone of vacuolation and did not extend into the apical regions of the cell. The lamellar component, however, did exist in the tip region where intense staining was observed (Figure 5b). As with observations of the DIC-visible cytoplasmic strands, it was difficult to determine whether or not one morphological type of DiOC5(3)-positive structure could transform into the other or whether the two existed as distinct populations independent of each other. The endoplasmic lamellar component exhibited activity much as was observed in the reticulate cytoplasmic strands such as splitting and merging, and emptying/filling of lamellae which are connected by tubules. The DiOCg(3)-positive, cortical, longitudinal and lamellar components closely resembled the cortical DIC-visible longitudinal and sub-cortical reticulate cytoplasmic strands, although, the former appeared smaller in diameter. Support for the DiOC6(3)-positive structures visualized as being E R came from loading cells for long periods of time (2 hours) with the calcium indicator fluo-3 and allowing the dye to accumulate into various intracellular compartments. Photobleaching occurred rapidly during the process of taking photographs, which made obtaining images difficult. In addition, in most cells, compartmentalization into the vacuole predominated (not shown). However, cells which were observed prior to fluo-3 accumulation in the vacuole demonstrated that both morphologies of the DiOCg(3)-positive structures accumulated the dye. This suggested that its identity was that of a calcium-containing intracellular compartment and most likely represented the E R in this species (Figure 6 and 7 a and b). Aluminum-Treated Cultures Compared to controls, cells treated with A l possessed growth rates which decreased by more than 20% during the first hour of exposure. These rates continued to 39 drop slowly over the next two hours of continuous exposure to A l until they were only 40% of those in controls (See Table 1). Aluminum did not affect cytoplasmic streaming within the cell at exactly the same time. Inhibition of streaming due to A l exposure was characterized by spatially discrete regions where rates of organelle translocation were affected first. Calculation of streaming rates in exposed cells was conducted by measuring the speeds of chloroplast and mitochondria translocation throughout the entire length of the zone of vacuolation of the filament and averaging. Cytoplasmic streaming of chloroplasts and mitochondria decreased by 55% within 60 seconds of exposure to 80 u M A l and ceased entirely within approximately 3 minutes after treatment. The speed of inhibition of cytoplasmic streaming by A l was dose-dependent and the temporal onset of effects varied depending on the concentration of external calcium supplied in the medium (Figure 8). Initially, the cessation of streaming was not accompanied by changes in the distribution of the chloroplasts and mitochondria which remained evenly spaced throughout the filaments. This is documented in Figures 9a through d. These figures represent 4 successive exposures taken at 30 second intervals of the same portion of a vegetative filament. Three chloroplasts are identified by the numbers "1" , "2" and "3". Their position, relative to that in previous frames, remained unchanged throughout the entire exposure period. This demonstrates not only the complete cessation of streaming for these organelles but also their continued association with a cytoplasmic strand. Mitochondria are identified by arrowheads and were also observed to remain stationary throughout the series of micrographs. In contrast, nuclei continued to stream at rates unaffected by the presence of A l in the system. This is clearly seen in this series of micrographs by observing the relative position of the nucleus, identified by an arrow. This nucleus is aligned with a chloroplast (Figure 9a) and as time passes becomes progressively distanced from it. The rates of streaming of nuclei remained similar to those observed in controls, at 192 + 8.1 p m min" 1. Nuclei were seen to translocate over "open areas" but were also observed to 40 temporarily associate with the DIC-visible, cortical cytoplasmic strands. Both morphological types of cytoplasmic strands remained visible after chloroplast and mitochondrial streaming ceased. However, little or no activity (such as tubular splitting or merging) was observed in longitudinal strands in the majority of cells when observed using D I C microscopy. Within the first hour of A l treatment, both chloroplasts and mitochondria remained closely associated with the disorganized, DIC-visible, longitudinal, cytoplasmic strands. Within 2 to 4 hours, the majority of cells displayed a convoluted appearance rather than the linear shape characteristic of control filaments over the same time period (Figure 10a, b). The treated cells continued to grow although growth rates were reduced by 4 hours after continuous exposure (i .e. treated cells possessed 30% of growth rates measured in control cells). Aberrations in tip morphology, such as an initial swelling and subsequent pulling away of the protoplast from the cell wall in the cell tip was noted in approximately 85% (n=3) of the cells observed (Figure 11). After 4 to 8 hours of continuous A l exposure, chloroplasts and mitochondria retained their association with fragments of cytoplasmic strands. However, these displayed various patterns of disorganization. The predominant forms were those in which strands became associated with each other (Figure 12a, b) or in which the strands became transverse (Figure 12d). Less frequently, rings of cytoplasmic strands were observed (Figure 12c). Once again, these changes did not always occur at the same time throughout the cell. Rather, discrete regions exhibited different degrees of disorganization at any one time of observation. The reticulate strands remained unchanged in terms of their morphology throughout the filament. After 60 minutes of exposure to A l , few continuous longitudinal cytoplasmic strands were observed in the cytoplasm whereas the reticulate network remained intact and continued to exhibit dynamic motion such as splitting, merging and filling in of lamellae comparable to that observed in controls. Staining with DiOCg(3) revealed the presence of the lamellar morphology of the E R but not that of the 41 cortical longtitudinal type (Figure 13a). Tubular connections between polygons remained intact and straight (Figure 13a) but their definition as well as that of the polygons became more diffuse after 5 minutes of exposure to U V light in the process of obtaining photographs (Figure 13b and c). After prolonged periods of exposure to A l (approximately 8 to 24 hours) motionless aggregates of chloroplasts and mitochondria formed within the cytoplasm. Chloroplasts displayed an altered morphology relative to controls over the same time period by ultimately assuming a spherical shape (Figure 15). Nuclei associated with these chloroplast/mitochondria aggregates but this may have been the result of encountering a physical barrier rather than their participation in aggregrate formation. This hypothesis was supported by the fact that, in regions free of major aggregates of chloroplasts and mitochondria, many nuclei continued to stream at reduced rates (80 + 14.6 pm min"1) and were restricted only upon encountering large aggregates in their path. Growth rates of convoluted filaments eventually ceased and the cells became chlorotic and fragile, making their removal from culture difficult. Streaming of nuclei ceased at approximately the same time that the lamellar E R began to disintegrate and formed patches of polygons and large vesicles which were intensely stained by DiOC6(3) (Figure 14). Large vesicles and aggregates of organelles predominated in the cytoplasm when cells were observed using DIC microscopy (Figure 15). This was noted in almost all cells observed. 42 Table 1. Effect of 80 pM Al on growth rates of vegetative filaments of V. longicaulis. Seventy five filaments were measured per trial (n=3). Rates are reported + S.D. Time (hours) Growth Rate + S.D. (pm hour 1 ) 0 (Control, p H 4.5) 162.7 + 8.1 1 120.3 + 3.7 2 65.8 + 7.3 3 30.2 + 10.1 4 9.7 + 4.0 10 neg. 16 neg. 24 neg. 4 8 neg. 44 Figure 1 A region of the vegetative filament, 100 pm distal from the cell tip, observed using DIC microscopy. A longitudinal cytoplasmic strand is labelled as "tub.cyt.str." (white arrow). Mitochondria ("mitoch", black arrow) and chloroplasts ("chp") are seen to be associated with these structures. Bar=10 pm. Figure 2 A region of the vegetative filament, 250 pm distal from the cell tip, observed using DIC microscopy. Note basoventral shape of chloroplast with tapered ends ("chp"). A s in Figure 1, mitochondria ("mitoch", black arrowhead) and chloroplasts are associated with the longitudinal cytoplasmic strands. Nuclei ("nuc", short, black arrow) are visible throughout the cell. Bar=8.5 pm. Inset: reticulate cytoplasmic strands located toward the tonoplast. Bar=5 pm. Figure 3a-c Reticulate cytoplasmic strands, located in the subcortical cytoplasm in regions of temporarily free of organelles display anastomosing qualities. Bar=5 pm 46 Figure 4 DiOC6(3)-positive structures seen in the cortical cytoplasm of a region of the vegetative filament. Mitochondria (arrows) are seen to associate specifically with thin tubules which run parallel to the long axis. Bar=10 pm. Inset: DiOC6(3) staining showing mitochondria which are distributed evenly throughout the cell. Bar=15 pm. Figure 5 (a) DiOC6(3)-positive structure located toward the tonoplast in a region of the vegetative filament consisting of a polygonal network connected by tubules. Bar=10 pm. Inset: enlarged area of 5a showing detail of polygons and tubular connections. Bar=2 pm. (b). Apical portion of cell showing intense DiOC6(3) fluorescence. Bar=10 pm. Figure 6 Fluo-3 fluorescence compartmentalizes into a tubular component which runs parallel to the long axis of the cell and with which mitochondria are seen to associate. Bar=30 pm. Figure 7a and b Fluo-3 fluorescence compartmentalizes into a reticulate network located toward the tonoplast. (a), Bar=15 pm; (b), Bar=18 p m . 47 Figure 8 Graph showing the dependence of Al-induced cessation of chloroplast and mitochondria translocation on the external calcium concentration. Low=0.025 m M , Medium=2.5 m M , High=8mM. Rates are determined by sampling three separate regions of the cell, 2 minutes after exposure to 80 ( i M A l , and averaging. Approximately 80 cells were used for each trial (n=5). 120 Low external calcium Medium external calcium High external calcium 40 60 80 100 120 140 160 Aluminum Concentration (micromolar)pH 4.5 180 200 49 Figure 9a-d Micrograph series demonstrating the preferential effects of Al on cytoplasmic streaming of chloroplasts ("1", "2" and "3") and mitochondria (arrowheads). Nuclei labelled with short arrow continues to stream away from chloroplast "2" whereas chloroplasts and mitochondria have ceased to stream but remain associated with the longitudinal cytoplasmic strands. Images were taken 20 seconds apart. Bar=10 p:m 5 0 51 Figure 10 (a) Control morphology of vegetative filaments and (b) 4 hour treated cells displaying convoluted appearance. Bar=250 | im. Figure 11 Brightfield image of cell tip showing cell wall morphology indicative of swelling and retraction of protoplast from apical wall. Bar=50 urn. Inset: enlarged image of the cell tip, approximately 8 hours after A l treatment. Note spherical chloroplast morphology. Bar=12 | i m Figure 12a-d Patterns of disorganization in cytoplasmic strands, (a, b) Linear strands with which associate with each other are observed. Elongated, immobile chloroplast ("chp") is associated with the strands, Bar=4 pm. (c) Cytoplasmic strands which form rings are also seen (arrow). Note chloroplast ("chp") association Bar=8 fim. (d) Transverse arrangement of linear strands. Note aggregates of chloroplasts and mitochondria associated with strands. Bar=12 pm. 53 Figure 13 DiOC6(3) staining of lamellar E R in cell exposed to A l for 8 hours, (a) Tubules connecting polygons remain straight and well defined Bar=5 pm. Exposure to U V light under the light microscope results in the DiOC6(3) fluorescence becoming more diffuse with increasing time of exposure, (b) 3 minutes and (c) 5 minutes. Bars=10 pm. Figure 14 DiOC6(3) staining of lamellar E R after approximately 18 hours of continuous exposure to A l . Intensely stained polygons and vesicles are observed within the cell. Bar=5 pm. Figure 15 DIC image of chloroplast/mitochondria aggregate and large vesicles in a cell which has been treated with A l for approximately 18 hours. Note spherical shape of chloroplasts ("chp"). Cells which possess large vesicles in their cytoplasm do not generally recover when returned to A l free media. Bar=5 pm. 54 3.3. Discussion This study suggests that, in this system, A l preferentially effects the process of microfilament-based mitochondria and chloroplast translocation but not microtubule-dependent nuclear translocation. It also suggests that exposure to A l results in the disorganization of the cortical longitudinal cytoplasmic strands. Cel l distortions such as tip swelling and overall convolution are features common to other tip-growing cells which have been treated with cytoskeleton-disrupting agents (Heslop-Harrsion et al. 1991; Steer 1990; Heslop-Harrison and Heslop-Harrsion 1991) as well as A l (Alfano et al. 1993a). Perturbation of calcium homeostasis has been shown to cause tip swelling and abnormal growth in Lilium pollen tubes (Pierson, et al. 1994). These features are similar to those noted in vegetative filaments of V. longicaulis after exposure to A l (e.g. Figures 10a and b). Since the morphology of a plant cell is ultimately designated by deposition of the cell wall , in this case, at the cell tip, and deposition of cell wall materials is generally controlled by cytoskeletal elements (Simmonds, 1992), one possible interpretation of the data is that A l could be altering the orientation of the cytoskeletal elements which control cell wall deposition, however, the data do not allow any further speculation. The cytoskeleton is central to tip growth in cells such as pollen tubes and fungal hyphae (Cai et al. 1997; Hepler, 1997). Perturbation of the F-actin array in tip-focused cells such as pollen tubes results in the inhibition of tube growth (Pierson and Cresti 1992). This may also be the case for V. longicaulis since A l treatment reduces growth rates even though streaming of nuclei continues at rates comparable to those in controls. This supports data which show that an intact F-actin array is necessary for the maintenance of cell viability. Microfilament-based cytoplasmic streaming is affected by exposure to A l within 60 seconds whereas microtubule-based processes (e.g. translocation of nuclei) are not affected. The fact that chloroplasts and mitochondria retain their close association with the 55 longitudinal cytoplasmic strands even after cytoplasmic streaming has stopped may suggest that A l could be interfering with a putative myosin motor. It has been shown that the F -actin array plays a role in organelle anchorage via myosin (Kadota and Wada 1992; Alfano et al. 1993a, b). This is interesting in the context that treatment with A l causes a change in chloroplast shape from the normal baso-ventral to a spherical morphology (Figure 15). In V. longicaulis, inhibitor studies have shown that chloroplasts are translocated via microfilament bundles (Tornbom and Oliveira, 1993b). Thus, it is possible that A l not only affects the streaming of these organelles but also, after longer periods of exposure to A l , their association with and possibly anchorage to F-actin. Tip swelling may be caused by the inhibition of exocytosis by A l at the cell apex, a process shown to be mediated by the F-actin array (Cai et al. 1997). Preferential effects of A l on the actin based versus microtubule based processes in this system is supported by the observation that nuclei continue to stream even after chloroplasts and mitochondria form immobile aggregates in the cytoplasm. One factor which may account for the observations obtained in this part of the study is calcium. It has been shown that dissipation of the calcium gradient in pollen tubes results in the loss of cytoplasmic organization (Pierson et al. 1994). Dynamic processes such as organelle translocation by myosin motor proteins appear to be regulated primarily by calcium, either directly or via calcium regulated proteins (Cai et al. 1997). The dependence of cytoplasmic streaming on calcium has been demonstrated in perfusion studies (Williamson and Ashley, 1982), permeabilized whole cells (Tominaga, et al. 1983) and in vivo (Doree and Picard, 1980; Menzel and Elsner-Menzel, 1989). In all cases, even small increases in the level of cytoplasmic calcium were sufficient to affect organelle streaming. Cytoplasmic streaming of mitochondria and chloroplasts in vegetative filaments of V. longicaulis may occur as a result of a number of cellular structures and their putative functions. Both mitochondria and chloroplasts are always observed to move in fairly 56 straight trajectories and always in association with the longitudinal cytoplasmic strands (Figures 1 and 2). These strands have been described in other species of Vaucheria (Ott and Brown, 1972; Blatt and Briggs, 1980; Blatt, Wessels and Briggs, 1980) as well as in other plant systems such as Bryopsis (Menzel and Schliwa, 1986) and onion epidermal cells (Knebel et al. 1990). Such strands are generally found only in the region of the cell which displays vigorous cytoplasmic streaming, (i.e. the Z V ) , and are absent from the apical portion suggesting a specialized role for these structures in facilitating organelle motility (Palevitz and Hepler, 1975). Aluminum appears to interfere with calcium channels in the plasma membrane (Rengel, 1992; Kochian, 1995; Huang, et al. 1992, 1993; Nichol and Oliveira, 1995; Ryan and Kochian, 1993). Thus, it may be speculated that perturbation of calcium homeostasis may be part of the mechanism by which A l rapidly affects organelle streaming. For the early symptoms of A l toxicity on cytoplasmic streaming this hypothesis is favoured since A l does not appear to be present in the cytosol until 30 minutes after exposure (Dr. Gregory Taylor, personal communication). Thus, the fact that A l causes these initial visible effects so quickly suggests that its interaction at the plasma membrane may occur before A l enters the cell. Identification of the Cytoplasmic Strands as Endoplasmic Reticulum Generally, the E R of plant cells consists of a dense, extensive network of interconnected tubules and small cisternae or polygons (Menzel, 1994b). Some cells, such as adaxial epidermal cells of onion bulb, possess two forms which have been shown to be continuous with each other. The first type is arranged in a complex network of flat cisternae or polygons connected by thin tubules and the other form consists of bundles of long, thin tubular elements (Quader and Schnepf, 1986; Knebel, et al. 1990). Although a number of techniques exist with which to visualize the E R in plant and animal cells, the lipophilic dye, DiOC6(3) is the most prevalent protocol used today (Terasaki et al. 1989). The basis for the E R staining by DiOC6(3) and other dyes in its 57 class is still under debate. However, specificity to mitochondria and E R is maintained by the fact that these dyes carry a delocalized positive charge at physiological pHs which interacts with a strong net negative internal membrane potential in both these structures (Terasaki, 1989). Several lines of evidence support the observations that the longitudinal and reticulate cytoplasmic strands observed using DIC microscopy represent the E R in V . longicaulis. Staining with DiOC6(3) reveals structures which closely resemble the cytoplasmic strands in both their relative positions and morphologies. Electron microscopy of vegetative filaments of V. longicaulis reveals the presence of longitudinal tubular strands whose measurements range from 75-250 nm, running parallel to the long axis of the cell, as well as lamellar ER, located toward the tonoplast (Ott, 1992). Another line of evidence that supports the identity of a component of the longitudinal and reticulate cytoplasmic strands as being E R comes from images obtained after compartmentalization of the calcium sensitive dye, fluo-3 (Figures 6 and 7). After different times of exposure to the dye, compartmentalization occurs in various intracellular compartments, most notably the vacuole (data not shown). Fluo-3 also accumulates into an intracellular compartment which closely resembles the morphology and location of the cytoplasmic strands and E R . Intense staining in the most apical portion of the cell is consistent with observations reporting the participation of the E R as a source of stored calcium in plant cells (McCauley and Hepler, 1990). Hence, the identity of a component which associates with the cortical longitudinal cytoplasmic strands, may be a specialized form of E R . The other component may consist of bundles of microfilaments which would be visible using DIC microscopy. This would rectify the difference in relative sizes between the cytoplasmic strands and the cortical longitudinal tubular E R observed using E M while taking into account optical aberrations caused by light microscopy (Kachar and Reese, 1988). The disorganized, cortical cytoplasmic strands do not fluoresce when stained with DiOC6(3) but are clearly visible using DIC-microscopy after the longitudinal E R is no longer located (using 58 DiOC6(3) staining). This supports the hypothesis that the other component of the cortical, DIC-visible, longitudinal cytoplasmic strands may consist of bundles of microfilaments. The longitudinal E R appears to disintegrate rapidly upon A l treatment whereas the lamellar component does not. This may be related to the function of the lamellar component in protein synthesis required to support the apical growth of the vegetative filament as evidenced by its density in the tip. Furthermore, staining for R N A co-localizes with the lamellar, but not the tubular component of the E R and subsequent treatment with cycloheximide, an inhibitor of protein synthesis, results in the cessation of apical growth and disorganization of the lamellar component (Chu and Oliveira, unpublished results). In addition, Lichtsheidl and Weiss (1989) used light microscopy techniques to observe both the E R and to report that vesicular organelles at the periphery of the cell were moving along strands of E R (which were supported by individual F-actin bundles). In Nitella, cortical actin cables, which support a myosin driven rapidly streaming internal cytoplasm, have been identified and tubular membranes were found to move along these actin cables at a rate which corresponded to the rate of cytoplasmic streaming of mitochondria in intact cells (Kachar and Reese, 1988). Conclusions The effects of A l on microfilament based cytoplasmic streaming in V. longicaulis are rapid, supporting data which indicate that A l may exert its toxic effects at the level of the plasma membrane (Rengel, 1992a; Kochian, 1995; Huang, et al. 1992, 1993; Nichol and Oliveira, 1995; Ryan and Kochian, 1993). The DIC-visible, cytoplasmic strands, which represent the F-actin/ER network of this alga, disorganize within 30 minutes of exposure to A l . Chloroplasts and mitochondria retain their association with these disorganized structures even though cytoplasmic streaming is inhibited. It has been observed that manipulation of cytosolic calcium levels can produce tip irregularities, inhibition of cytoplasmic streaming and changes in the distribution of F-actin array in fungal hyphae (Jackson and Heath 1989) and pollen tubes (Kohno and Shimmen 1987). 59 Thus, the effects noted in vegetative cells of V. longicaulis upon treatment with Al appear to be similar to those noted in cells upon perturbation of calcium homeostasis. If Al toxicity is initiated at the level of the plasma membrane, this could explain the speed of inhibition of organelle streaming. I propose that Al causes changes in cytoplasmic organization, in this system, primarily via the disorganization of the F-actin cytoskeleton (which is calcium sensitive). This hypothesis is tested in Chapters Four and Five. 60 4.0. Effects of Aluminum on the F-actin Array 4.1. Introduction Aluminum toxicity in plants is recognized as the foremost factor hmiting crop production on acid soils. The development of Al-tolerant crops could save billions of dollars in lime which is currently purchased to raise soil p H . Aluminum has been shown to have an impact on the structure and function of tip-growing root hair cells in higher plants (Rengel, 1992a; Horst et al. 1991). It has also been shown to impair numerous processes such as the net uptake of calcium (Godbold, 1991; Huang et al. 1992), cell division (Horst et al. 1983) and cell elongation (Matsumoto et al. 1977). However, the exact mechanism of Al-induced toxicity remains unclear. Despite the importance of identifying the nature of the primary toxic lesions in plant cells, studies which relate the effects of A l to the cytoskeleton are lacking. Works which have examined the effects of A l on the cytoskeleton consist of an examination of changes in the tension of F-actin in protoplasts of soybean (Grabski and Schindler, 1995) and an investigation of the effects of A l on the organization of the actin cytoskeleton in rhizoids of Riccia fluitans (Alfano et al . 1993a). The lack of cytoskeletal studies is surprising since it is well known that plant cells possess dynamic cortical F-actin arrays which are crucial to diverse cellular processes. The actin cytoskeleton of plants has proven to be sensitive to disturbance and may play a key role in both rapid and long-term cellular re-organization in response to environmental stimuli. Actin filaments have been shown to be disorganized in response to a number of stimuli, both chemical and physical (Quader, 1990; Alfano et al. 1993a, b; Kaminskyj and Heath 1994; Liebe and Quader 1994). Cytoplasmic streaming, crucial to the viability of tip-growing algal cells (reviewed by L a Claire, II., 1989), involves the movement of putative organelle-associated myosins along bundles of F-actin filaments (Palevitz and Hepler, 1975). Also , the E R has been shown to be supported by various structural 61 patterns of bundled microfilaments (Quader et al. 1987; Fuhrman et al. 1990; Allen and Kurbinski , 1995). Hence, compounds which perturb microfilament structure also impact primary cellular functions and result in a loss of viability of the cell as a whole (Faure, 1996). Tip-growing cells such as pollen tubes, root hairs and fungal hyphae demonstrate a unique property of apically localized growth and distal zonation of organelle streaming as a manifestation of polarity which is reflected in the cytoskeleton (Lloyd et al. 1987; Jackson and Heath 1993; Tiwari and Polito, 1988). Microfilaments have been found to be crucial in the establishment and maintenance of tip polarized growth (Pierson and Cresti, 1992; Kropf et al. 1992). In cells such as pollen tubes, bundled F-actin filaments are axially aligned and extend almost to the apical dome of the growing tip. In the apical zone, however, the aligned F-actin bundles may end abruptly, fan out and tend to appear more diffuse and randomly distributed (Steer, 1990; Pierson and Cresti, 1992). The lack of distinct bundles in the cell tip also occurs in germinating Fucus embryos and may indicate that F-actin is present as a fine mesh (Kropf et al. 1992). In this region actin is postulated to facilitate the processes of exocytosis (Steer and Steer, 1989) and tip extensibility (Jackson and Heath 1993). Vegetative filaments of V. longicaulis represent a model system in which to study the impact of A l on the actin cytoskeleton in a tip-growing plant cell. Here, by utilizing vital staining protocols for F-actin and establishing stringent cell viability criteria, I document the changes which occur in the microfilament array in this alga following exposure to A l . I provide evidence that A l affects the cortical F-actin cytoskeleton, thus accounting for the effects noted in the previous chapter on cytoplasmic streaming of chloroplasts and mitochondria as well as the cortical cytoplasmic strands visualized using D I C microscopy. Evidence is also presented that F-actin fragments can assemble to form unusual but stable structures which persist in the cytoplasm after long term A l treatment. These data are discussed in a context that supports the hypothesis that Al-induced changes 62 in the F-actin cytoskeleton may be mediated by changes in calcium homeostasis. The potential role of calcium-sensitive actin-binding proteins in the modification of the F-actin array is also discussed in this respect. 63 4.2. Results The use of aldehyde fixatives in vegetative filaments of V. longicaulis resulted in the visualization of an array where only thick bundles of F-actin were seen (Figure 16 a and b). Chloroplast morphology was altered, resulting in a deviation from their normal baso-ventral shape (Figure 16 b). Vital staining for F-actin in unfixed cells produced reproducible results and cells remained viable with no changes in the rates of cytoplasmic streaming or growth observed compared to controls. Therefore, unfixed cells were used in this study to minimize artefacts due to fixation, particularly while attempting to describe cytoskeletal changes which occurred during A l treatment. Control Cells When stained with FITC-phalloidin, the region of the zone of vacuolation revealed long, straight bundles of F-actin located in the cortical cytoplasm. These often branched and merged with each other as well as exhibited a close interaction with both chloroplasts and mitochondria (Figure 17 , arrows). Identification of these bundles as F-actin was affirmed by the fact that pre-treatment of cells with phallacidin (which shares the same binding site) sharply reduced the fluorescence upon subsequent staining with F I T C phalloidin and that treatment with cytochalasin D caused the disappearance of the bundles whereas oryzalin, known to depolymerize microtubules in this system (Tornbom and Oliveira, 1993b), did not. Control cells were embedded and/or overlaid with gelatin for the equal amount of time as those undergoing Al-treatment. Cells which were covered by a thin layer of gelatin did not label with FITC-phalloidin (Figure 17). Embedding cells in this manner did not affect their viability, or the appearance of the F-actin array and filaments continued to grow in a moist environment until they over-ran the glass slide. This method was modified for use in vital staining of F-actin in this alga after it was noted that normal experimental handling of control cells was sufficient to cause changes in the actin array. These included the appearance of brightly stained phalloidin-positive material 64 which were identified as focal masses in the zone of vacuolation where they do not normally occur. When focusing toward the tonoplast, a reticulate pattern of actin was observed (Figure 18). In the cortical cytoplasm of the apical zone, linear actin bundles gave way to bright, diffuse staining (Figure 19b), whereas discrete, dimly labeled focal masses were observed slightly deeper in the cell tip (Figure 19a). Aluminum-treated Cells In the apical zone, exposure to 80 p M A l over 20 minutes resulted in a loss of the normally diffuse phalloidin-positive staining. This array was replaced with distinctly bundled and disorganized F-actin which extended toward the very tip of the cell (Figure 20). Cel l tips became fragile upon treatment with A l and often exhibited swelling and/or rupture (Figure 20, inset). In the zone of vacuolation, Al-induced changes involved the loss of the axial alignment of cortical F-actin bundles which appeared as an overall undulating pattern (Figure 21), seen in approximately 80% of cells observed (n=7). Cells displaying this pattern possessed thicker bundles which appeared to be made up of large numbers of individual F-actin filaments which converged at specific points (Figure 21 , compare with Figure 17). Chloroplasts remained in contact with F-actin at distinct points (Figure 22, arrow). After approximately 4 hours of A l treatment, discrete areas of focal masses were observed to form (Figure 23 and 23 b). Double labeling with D i O C g (3) to visualize mitochondria and FITC-phalloidin (to visualize F-actin) indicated that mitochondria remained attached to the disintegrating F-actin array, even in the areas where small focal masses were forming (Figure 23, arrowheads). Focal masses tended to form in regions of the cell which were occupied by chloroplast/mitochondria aggregates (also see Figures 28, 30 and 33). The extent of F-actin disorganization within a cell at any given time was not homogenous. Some regions still possessed longitudinal bundles of F-actin and mitochondria were still observed to be regularly spaced and associated with a 65 microfilament (Figure 23 a). It is noted that chloroplast shape was altered when the association with a microfilament was lost and the plastids took on a more spherical shape (Figure 24). In contrast to the rapidly disorganizing cortical F-actin array, the sub-cortical array did not undergo changes even after 6 hours of exposure to A l and retained an appearance similar to those of control cells. Assays for unpolymerized actin conducted on cells (n=2) after >4 hours of exposure to A l indicated that the amount of G-actin begins to increase (Table 2). In order to test whether or not Al-induced changes in the cortical F-actin array could occur, at the same time, distal to an area of A l exposure while not being directly exposed to A l , the gelatin embedding method was used to expose specific areas of the cell (areas "a" and "c", Figure 25) to A l . Another portion was protected from exposure to A l (area "b", Figure 25). After approximately 120 minutes of localized exposure, the protected section revealed F-actin which displayed close associations with mitochondria (Figure 25, arrows). Conversely, the adjacent areas, which were exposed to A l , exhibited changes in the F-actin array and subsequent loss of spacing of mitochondria. In another cell, a small area of the cell which was exposed to A l showed an amorphous F-actin focal region forming in contact with a chloroplast (Figure 26, arrow). B y approximately 8 hours after exposure to A l , a disorganized microfilament array was observed in the cortical cytoplasm (Figure 27). Thick F-actin bundles lost their co-axial alignment and often crossed each other at various angles (Figure 28). Between 8 and 12 hours in culture, four distinct patterns of disorganized cortical F-actin were observed in the cytoplasm. These included short curved or linear rods (Figure 29 a, b and c), star-shaped or amorphous structures (Figure 30 a, b and c), thickened aggregates of bundled F -actin (Figure 31) and circular F-actin structures (Figure 32 a, b and c). These latter structures were notable in that after long term A l exposure, ring-form actin structures were observed to form, mature (i.e. increase in fluorescent intensity) and persist in the cortical cytoplasm often in association with the large amorphous focal masses (Figure 33 and inset, 66 arrows). Numerous thick bundles of actin were also associated with a single ring structure (Figure 34 a) which could possess multiple linear'emanations or "tails" of varied lengths. At later stages of exposure to A l , any individual ring structure could possess two, one or no tails. These emanating tails appeared to be continuous with the central core of the bundles (Figure 34 b). In some cases, ring form aggregates of F-actin were open and teardrop shaped but still possessed one or two emanations (Figure 34 c). Rings exhibited variation in their sizes, the presence or absence of tails and their proximity to amorphous focal masses (Figure 35 and insets). In some cases a ring became twisted such that it resembled a "figure eight" (Figure 36 a and b). In addition, large cyclone shaped formations were noted infrequently in the cytoplasm (Figure 36 c). The formation of such structures was often associated with the presence of an undulating aggregate of thick F -actin bundles (Figure 37 a) that gradually transformed into a distinctly coiled ring with emanating bundles of F-actin (Figure 37 b). Attempts to videotape such processes using conventional microscopy resulted in photo-bleaching of the F I T C and in some cases, cytoplasmic dissolution. The subcortical reticulate array persisted after the disorganization of the cortical array had progressed to an advanced stage (i.e. only large amorphous focal masses and F -actin ring aggregates remain in the cytoplasm). However, eventually (>20 hours of continuous exposure to Al) the reticulate F-actin array collapsed and appeared to be incorporated into the focal masses as well (Figure 38 and inset). N o ring form actin or other unusual F-actin structures were noted in the subcortical region of the cell. In the cortical region, these aggregates persisted, usually proximal to the large focal masses associated with chloroplast/mitochondria aggregates and F-actin tangles (Figure 39) or independently, with no apparent association with a plastid or focal mass (Figures 33, inset and 35). 67 Table 2 Effects of exposure to Al on G-actin content as assessed using the method of Heacock and Bamburg (1975) (See Materials and Methods). Approximate weight (wet) of cells used in each trial is 25 g (n=2). One gram wet weight equals approximately 1500 cells. Effect of Aluminum Exposure on G-actin Time of Exposure to G-actin Aluminum fhounrt Cug/mT) 0 12+6 4 21±5 8 49+9 12 53±9 16 57±8 69 Figure 16 FrTC-phalloidin staining for F-actin in fixed cells. Only thick bundles of F-actin are visible (a). Chloroplast (chp) shape is altered and intense F-actin staining is noted around the periphery of these organelles (arrow) (b). Bars (a)=15 pm; (b)=20 pm. Figure 17 Liv ing , control cell double stained with FITC-phalloidin for F-actin showing association of F-actin with chloroplasts (chp) and DiOC6(3) to visualize mitochondria (mitoch.) (a). Unstained, living control cell which has been covered by a thin layer of gelatin showing chloroplasts appressed to the plasma membrane and the relative optical transparency of the cel l(b) . Bar=40um. Figure 18 FITC-phalloidin staining of a living control cell showing reticulate F-actin array located when focusing toward the vacuole. Bar=30 urn. Figure 19 (a) FITC-phalloidin staining showing dimly fluorescent, amorphous F-actin focal masses located deeper in the cytoplasm in the tip (apical) region of a control, living cell. Bar=35 p,m. (b) Cortical F-actin array showing longitudinal bundles which give way to diffuse staining in the apical 10 um of the cell. Bar=15 pm. 71 Figure 20 FITC-phalloidin staining in 30 minute Al-treated tip showing loss of diffuse F-actin staining and disorganization of F-actin array. Bar=10 pm. Inset: lysis of the cell tip and association of F-actin with chloroplast (chp) (arrow). Bar=12 um. Figure 21 FTTC-phalloidin staining in 30 minute Al-treated cell, 100 p.m distal from the cell tip showing undulating bundles of F-actin which merge and diverge at points (arrows). Bar=20 pm. Figure 22 FTTC-phalloidin staining in a 2 hour Al-treated cell, 100 um distal from the cell tip, showing F-actin surrounding a chloroplast (arrow). The chloroplast is identified by its shape as well as its inherent autofluorescence. Bar=20 u m Figure 23 FITC-phalloidin in a 4 hour Al-treated cell, 100 p m distal from the cell tip, showing heterogeneity of disorganization in the F-actin array. Mitochondria (arrowheads), visualized using DiOC6(3) remain associated with F-actin in regions of disorganization as well as in regions which retain a longitudinal orientation. Bar=10 u.m. Area showing close association of DiOC6(3)-stained mitochondrion with F-actin (arrow) (a). Bar=2 u.m. Discrete areas of stellate phalloidin-positive material are seen to form adjacent to the plasma membrane (b). Bar=15 pm 73 Figure 24 ( Alteration in chloroplast (chp) shape resulting in a spherical plastid is noted in an approximately 8 hour Al-treated cell. Note also a loss of F-actin staining around the periphery of the organelle. Bar=8 pm. Figure 25 Isolation of a region of the cell ("b"), 100 pm distal from the cell tip, using the gelatin embedding method and stained with FITC-phalloidin. Areas "a" and "c" exposed to Al for 120 minutes. Region "b" retains longitudinal F-actin bundles with associated mitochondria (m, arrow) whereas regions "a" and "c" exhibit disorganization of the F-actin array. Bar=10 pm. 74 Figure 26 Area of cell exposed to A l and stained with FTTC-phalloidin showing formation of f-actin focal mass in association with a chloroplast (arrow). Bar=20 pm. Figure 27 Representative cell treated with A l for approximately 8 hours. FTTC-phalloidin staining observed 100 p m distal from the cell tip, showing disorganized F-actin array. Bar=40 pm. 75 76 Figure 28 Representative cell treated with Al for more than 8 hours. FTTC-staining shows a focal mass which is present in the centre of the image and thick bundles of intensely stained F-actin cross each other at various angles. Bar=20 pm. Figure 29 Representative cell treated with Al for more than 8 hours. Various types of short F-actin rods visualized with FITC-phalloidin. These include curved rods which may represent intermediate forms of F-actin in the process of F-actin ring structure formation (a, c) as well as short, linear rods of consistent lengths (b). Bars=12pm. Figure 30 Representative cell treated with Al for more than 8 hours. Various types of stellate and amorphous F-actin structures visualized with FITC-phalloidin. Region containing stellate, amorphous and ring structures of F-actin (a). Bar=10 pm. Stellate structures forming adjacent to the plasma membrane (b). Bar=35 pm. Stellate and amorphous F-actin structures which are interconnected by linear F-actin (c). Bar=15 pm. Figure 31 Representative cell treated with Al for more than 8 hours. Intensely stained F-actin visualized with FITC-phalloidin showing aggregates of thick curved bundles. Bar=5 pm. 77 78 Figure 32 Representative cell treated with Al for more than 8 hours. Various types of circular and curved F-actin visualized with FITC-phalloidin. Large, curling structures (a). Bar=8 pm. Numerous ring and twisted ring structures (b). Bar=10 pm. Amorphous region of F-actin showing intensely staining short rods as well as ring structures (c). Bar=15 pm. Figure 33 Representative cell treated with Al for more than 12 hours. FITC-phalloidin staining shows persistence of ring structures after collapse of thick F-actin bundles into amorphous focal masses. Note radially emanations from ring structure (arrow). Bar=8 pm. Inset: same cell, same focal plane approximately 25 minutes later showing loss of emanations from ring structure and an increase in fluorescent intensity of the ring (arrow). Bar=20 pm. Figure 34 Representative cell treated with A l for more than 8 hours. FITC-phalloidin staining shows radial emanation of linear F-actin from a single ring structure (a). Bar=8 pm. Later morphologies of ring structures showing two linear emanations which are continuous with the ring (b). Bar=5 pm. Teardrop shaped F-actin ring structures (c). Bar=10 pm. Figure 35 Representative cell treated with Al for more than 12 hours. FITC-phalloidin staining shows variations in ring structures which remain in the cytoplasm. Bar=5 pm. Top inset: F-actin ring structure (arrow) which is continuous with long F-actin bundles. Bar=10 pm. Bottom inset: Larger, intensely staining ring structures. Bar=15 pm. 80 Figure 36 Representative cell treated with A l for more than 12 hours. FITC-phalloidin staining shows twisted ring structures of various forms, (a, b). Bars=5 pm. Also , large, a stable cyclone structure which persists in the cytoplasm after long term A l treatment is observed (c). Bar=10 pm. Figure 37 Representative cell treated with A l for approximately 8 hours. FITC-phalloidin staining shows the formation of large, coiled F-actin structure from aggregate of thick, curved bundles. F-actin bundles appear to associate at a specific point (a). Structure observed approximately 25 minutes later showing closed F-actin ring structure and associated, emanating bundles (b). Bar=15 pm. 81 Figure 38 Representative cell treated with A l for approximately 20 hours. FITC-phalloidin staining showing collapse of reticulate array into a focal mass. Bar=8 pm. Inset: same cell, same focal plane, approximately 35 minutes later showing increase in fluorescent intensity of F -actin focal mass. Bar=8 pm. Figure 39 Representative cell treated with A l for more than 24 hours. FITC-phalloidin showing persistence of ring structures in the cytoplasm proximal to large, amorphous focal masses. Bar=15 pm. 82 4.3. Discussion Assessment of the Vital Staining Methodology Since the morphology of the F-actin array in plants is affected by fixation, a discussion of the advantages of vital staining is neccessary. Fixation procedures have major disadvantages in plants which possess fine cortical F-actin arrays since these are destabilized by aldehyde fixatives (Parathasarthy et al. 1985 and references therein). Conversely, vital staining has traditionally been criticized for the fact that, at certain concentrations, phalloidin may introduce artefacts through the stabilization of microfilaments (Guiliano and Taylor, 1995). Permeabilization of living cells presents further problems since perturbation of the cell wall and/or the plasma membrane to allow phallotoxins, fluorochromes and antibodies to penetrate the cytosol may produce changes in the cell. These changes can have an impact upon the organization of the cytoskeleton (Pont-Lezica et al. 1993). In V. longicaulis , the incubation of cells in a gradient of low concentrations of saponin (from 0.001% to 0.01%) in media containing 1 % D M S O at 15-17 degrees C prior to staining (See Materials and Methods) produces consistent staining of the F-actin array without affecting cell viability. It has been shown that the use of up to 0.08 nmol/ml of phalloidin does not affect the polymerization state of actin (Tewinkel et al. 1989; Traas et al. 1987). A number of researchers have successfully carried out vital staining (Wasteneys et al. 1996; Grolig et al. 1988) mostly in actively streaming characean internodal cells using slightly higher concentrations of phalloidin with reportedly no effect on rates of cytoplasmic streaming (Wasteneys et al. 1996). In contrast, fixation of vegetative filaments results in cytoplasmic disorganization and the loss of fine arrays of F -actin as well as changes in chloroplast morphology. Hence, vital staining is used in order to obtain a more accurate picture of cytoskeletal changes during A l exposure and rrmiimize artefacts which may arise from fixation. 83 The gelatin overlay protocol used in this study reduces artefacts which may arise due to experimental handling. Thus, any changes observed in the F-actin array may be attributed to exposure to A l rather than factors such as mechanical trauma which can cause extensive fragmentation and aggregation of the F-actin cytoskeleton (Frost and Roberts, 1996). The reticulate-type F-actin array, seen in Figure 18, is located in the sub-cortical cytoplasm and has been reported in a number of plant cells (Parthasarathy, 1985; Collings etal . 1995; Wasteneys et al. 1996). Al-Induced Changes in the F-actin Array The debate regarding the site of the initial toxic lesion of A l remains unresolved. A large body of evidence suggests that A l may interfere with calcium homeostasis since the effect of A l on calcium transport is rapid, reversible and kinetically consistent with competitive inhibition (Lindberg, 1990; Huang et al. 1992; Rengel and Elliot, 1992a; Rengel, 1996). Aluminum has been shown to disrupt calcium homeostasis in a variety of other systems (Huang et al. 1992; Jones and Kochian, 1995; Nichol and Oliveira, 1995) including V. longicaulis where A l treatment of vegetative filaments causes an increase in intracellular calcium levels (Alessa and Oliveira, manuscript submitted). This is consistent with the fact that cessation of cytoplasmic streaming is noted within the first minute of exposure to A l and visible changes in the F-actin array are noted as early as 30 minutes after treatment. The initial changes from the linear microfilament array to an undulating and fragmented one (Figures 21 and 22) is reproducible. The progressive disorganization results in visibly thickened structures of F-actin which exhibit a variety of morphologies often within a single cell; these include rods of various dimensions (Figure 29a, b and c) as well as long bundles of F-actin which occur at angles to each other (Figure 28), fragmented F-actin (Figure 27), stellate or amorphous F-actin (focal mass) structures (Figure 30a, b and c) and circular or curved F-actin (rings, twisted rings and cyclones) (Figures 34, 35, 36 and 37). It is interesting to note that almost identical structures have 84 been reported in Zinnia mesophyll cells in response to wounding (Frost and Roberts, 1996). Fragmentation of the F-actin array and formation of F-actin aggregates Fragmentation and disorganization of actin filaments has been previously observed with various forms of chemical and physical treatment other than A l (Menzel and Schliwa, 1986; Hasezawa et al. 1988; Tiwari and Polito, 1988; Quader and Schnepf, 1989; Tewinkel et al. 1989; Wasteneys and Williamson, 1991; Hasek and Steirblova, 1994; Collings et al. 1995; Alfano et al. 1993a and 1993b; Peat and Oliveira, 1994 and Frost and Roberts, 1996). Structures such as F-actin rings not associated with chloroplasts and rings associated with chloroplasts as well as cyclone-like actin aggregates have been reported (Masuda et al. 1992; Kadota and Wada, 1992; Alfano, et al. 1993b; Dong et al. 1996; Frost and Roberts, 1996) as well as stellate actin aggregates (Pierson et al. 1994; Menzel, 1994a; Tewinkel, et al. 1989; Peat and Oliveira, 1994). The ring structures in V.longcaulis are most often observed to be located independently in the cytoplasm rather than associated with chloroplasts or chloroplast/mitochondria aggregates. However, they may exist in close proximity to the growing focal masses. A unique feature of these structures is the presence of associated bundles of F-actin filaments or "tails" which emanate either directionally (e.g. Figures 34 c and 37 a and b) or radially (e.g. Figures 33 and 34a). Observations of the formation of such structural features (Figures 37 a and b) suggests that these forms of F-actin are an intermediate structure in the formation of the large, stable, closed ring and cyclone structures (e.g. Figure 33, inset and 36 c, respectively). Furthermore, since only the focal masses and ring-like structures but not linear bundles are seen after long term A l treatment, these may represent a stable form of F -actin organization which persist after the overall array has been disassembled (e.g. Figure 35). This is supported by the fact that relative amounts of G-actin do not increase after 85 approximately 8 hours of continuous exposure to A l (Table 1). Ring form F-actin appears consistently in V. longicaulis cells during A l treatment but not upon treatment with cytochalasins (Peat and Oliveira, 1994). However, they are noted in Al-treated rhizoids of the liverwort Ricciafluitans (Alfano et al. 1993a). In V. longicaulis , the progressive disorganization of the array which results in curved F-actin structures consisting of fragmented, bundled actin filaments is consistent with the observations of Grabski and Schindler (1995) which suggest that the microfilament array is normally constrained under tension. The self-assembly of F-actin aggregates from F-actin filament fragments is supported by the following observations: a) the former replace the latter during exposure to A l and, b) the large ring and cyclone F-actin structures consistently occur after long term (>8 hours) A l treatment and persist in the cytoplasm. Formation of F-actin aggregates: Speculation on the involvement of actin-binding proteins A similarity in the response of F-actin to diverse forms of cell disturbance both physical and chemical has been demonstrated in a variety of cells types (e.g. Yahara et al. 1982; Alfano et al. 1993a, b). Similarly, artificial elevation of the calcium-activated barbed-end actin capping protein, gelsolin, in animal cells (Huckriede et al. 1990; Borovikov et al. 1995) also yields F-actin distribution patterns typical of stressed or disturbed cells. Data show that diverse forms of stress, known to affect calcium homeostasis, also cause the fragmentation of the F-actin array (Bush, 1995). This latter event is postulated to be facilitated by calcium-activated, actin associated proteins. It has been suggested that one putative mechanism which could result in the fragmentation of F-actin may involve the activation of barbed end capping protein analogs (Frost and Roberts, 1996) such as gelsolin in animal cells, (Cooper, 1987; Huckriede et al. 1990 and Borovikov, 1995). In plants, an actin-binding protein resembling gelsolin has been recently isolated from Medicago sativa (Allen and Kubinski , 1995). Al so , actin-86 binding proteins belonging to the monomer-binding class have recently been identified (Staiger et al. 1994; Valenta etal . 1993). These proteins closely resemble profilin, found in animal cells and bind to G-actin with relatively high affinity and are known to have interactions with actin. Profilin-type proteins encoding genes have been characterized from maize (Staiger et al. 1994), timothy grass (Valenta et al. 1993) and tobacco (Mittermann et al. 1995). The full significance for plant cell actin behaviour of these proteins has yet to be determined, however, plant profilins can also act as a sequestering protein in living cells since when microinjected into Tradescantia stamen hair cells they have a rapid and dose-dependent effect on cytoplasmic streaming and on the organization of cytoplasmic strands. These structures, which mirror the F-actin array, fragment and disorganize within an hour after exposure to the actin-binding protein. Furthermore, these profilin-type proteins appear to be sensitive to calcium (Staiger et al. 1994). Another molecule with the potential to regulate the actin cytoskeleton in plants is the calcium dependent protein kinase ( C D P K ) (McCurdy and Harmon, 1992). C D P K s are encoded by a small family of genes and each contains a catalytic kinase domain and a calmodulin-like calcium binding domain (Harper et al. 1991). At least one isoform of C D P K interacts with the actin cytoskeleton and has been found to co-localize with F-actin arrays in onion root cells, Tradescantia pollen tubes (Putnam-Evans et al. 1989) and Chora internodal cells (McCurdy and Harmon, 1992). Lines of evidence suggest that one of the putative substrates is myosin since C D P K can phosphorylate gizzard myosin light chain (Harper et al. 1991). Also , C D P K can phosphorylate a putative myosin light chain homologue from Chora in a calcium dependent manner (McCurdy and Harmon, 1992) and this strongly suggests that C D P K may play a key role in the calcium dependent inhibition of cytoplasmic streaming in plant cells (reviewed by Williamson, 1993). The fact that curved aggregates of F-actin bundles are observed to precede the appearance of stable ring or coiled structures (Figures 37 a and b) may suggest that a type of F-actin bundling protein or an F-actin associated mechanical protein (i.e. a myosin) may 87 be involved. Since A l has been shown to cause an increase in intracellular calcium (Lindberg and Strid, 1997; Alessa and Olveira, manuscript submitted) such a myosin would have to be activated rather than inhibited at elevated internal calcium levels. Plant myosin genes have not been cloned and their gene products have been difficult to study biochemically or to localize in situ. B y the criterion of heavy chain M r , most putative plant myosins fall within the myosin II range. Several are small and he within the size range of the myosin I family (Ma and Yen, 1989). These molecules would be favoured to move organelles (Sinard and Pollard, 1989) whereas myosin l i s are expected to generate tension by cross-linking parallel actin filaments as well as facilitate the sliding of one bundle of microfilaments against another. Myosin l i s have been shown to be active when intracellular calcium levels are elevated (Spudich, 1990) and their action could conceivably account for the contraction of F-actin filaments into some of the structures observed inV. longicaulis. The association of the thick, curled bundles at a specific point or points is further suggestive of the initiation of directional contraction by a process of myosin facilitated F-actin bundle sliding (Figures 37 a and b). In V. longicaulis a protein band has been immunolocalized from a total protein extract and identified as being comparable to the myosin II protein found in Dictyostelium but no myosin I band was identified unequivocally (Peat and Oliveira, 1994). When mitochondria are visualized in the early stages of A l exposure, they are seen to remain attached to the disorganizing and depolymerizing F-actin array, therefore, although cytoplasmic streaming ceases rapidly upon treatment with A l , the organelles retain their attachment to the F-actin fragments. This supports the hypothesis that it is, initially, loss of the function of the putative myosin motor which generates organelle movement, most likely via a rise in intracellular calcium, rather than an upcoupling of its association with the microfilament motility tracks. The large focal masses observed may also be formed by the contraction of F-actin. In other systems, the process of focal mass formation is believed to be mediated by actin-binding proteins as well as myosin (Yumura 88 and Kitanishi-Yumura, 1992). Focal masses and stellar aggregates are seen to form close to the plasma membrane; however, due to the thin depth of the cytoplasm and density of the organelles in the cortical cytoplasm, it is difficult to assess whether these foci may, in fact, be forming on or near a plastid or the plasma membrane. The fact that the sub-cortical reticulate array is not disorganized suggests that it may be regulated by a different class or set of actin-binding proteins which are either calcium independent or less sensitive to changes in cytosolic calcium. Spatial localization of Al-induced changes in the F-actin array A s with Al-induced effects noted on cytoplasmic streaming and the organization of cytoplasmic strands in cells of V. longicaulis (See Chapter 3), spatially distinct regions which differ in the severity of disorganization of the F-actin array are noted (e.g. Figure 23). Generally, changes in the F-actin array which fall within any of the descriptive categories occur within a single vegetative filament but rarely is the temporal or spatial occurrence of disorganization homogenous. With the use of the gelatin overlay protocol, the isolation of small portions of the cell from the bathing solution and exposure of others is possible. In this manner, observations may be made, upon the addition of A l to the media, of spatially heterogenous changes in the F-actin array. This is demonstrated by observing an overlaid portion of the cell which retains the longitudinal orientation of bundled microfilaments as well as specific interaction with mitochondria (Figure 23). Conversely, star-shaped focal masses which appear to be in direct contact with a chloroplast may form in portions of the cell which are locally exposed to A l (Figure 26). These findings suggest that the toxic effects of A l may be restricted to localized regions of the cell. There may be a few potential reasons for this observed phenomenon. For instance, is the spatial distribution of A l homogenous throughout the media bathing the cell? If it is not, this could affect the points (spatially) where A l comes into contact with the plasma membrane. If A l affects calcium homeostasis, then one of the potential reasons for 89 the temporally heterogeneous changes which occur on the F-actin array may be the poor mobility of calcium in the cytoplasm (Hille, 1984). This does not exclude the possibility that rates of entry of A l into the cytoplasm (and hence interaction with the F-actin array) may also vary depending on the spatial distribution of A l in the media. Conclusions In this study, it has been shown that the A l affects the F-actin array within minutes of exposure. It is of note that similar, and in some cases, identical morphological changes occur in the F-actin cytoskeletons of Riccia fluitans and Zinnia in response to A l treatment and wounding, respectively. Such comparisions suggest that the formation of F-actin aggregates in V. longicaulis could involve the action of specific, as of yet unidentified, actin-binding proteins which sever, bundle and contract F-actin (Pollard and Cooper, 1986; Vandekerchkhove, 1990). Many stimuli which produce physiological stress in plant cells also cause a rise in Ca^+^y^ (See Appendix). The similarities in the response of the F -actin array to such diverse treatments suggests that the pattern of F-actin fragmentation observed in this study could be a generalized response of the array to stress. This hypothesis would be consistent i f the mechanism of Al-induced effects on the F-actin array in V.longicaulis occurs, at least in part, via a perturbation of calcium homeostasis (Lindberg and Strid 1997; Alessa and Oliveira, manuscript submitted). It should be noted that these data do not exclude the possibility that A l could also enter the cytosol and effect the F-actin array by direct interaction (e.g. Grabski and Schindler, 1995). 90 5.0. Calcium Studies 5.1. Introduction In the previous three chapters, this study provides evidence that the initial effects of A l are rapid and may be observed to have an impact upon cellular functions such as cytoplasmic streaming (within seconds), and the cytoskeleton (within minutes), processes and structures known to be dependent on calcium (Pierson et al. 1994). Beyond the consensus that A l ions are toxic to plants and that A l toxicity produces a myriad different symptoms in a wide variety of plant cells little agreement exists on the putative site of the primary Al-induced toxic lesion (Rengel, 1996). A number of authors have speculated on the internal (symplasmic) (Jones and Kochian, 1995) or external (apoplasmic) sites of the primary lesion (Rengel, Pineros and Tester, 1995) but unequivocal experimental proof for either is lacking. The fact that exposure to A l results in the rapid, dose-dependent inhibition of cytoplasmic streaming suggests that A l may bind to the apoplasmic side of the plasma membrane. Consistent with this, preliminary data show that A l does not enter the cytosol within 30 minutes of exposure (Taylor, personal communication). Immediately following exposure to A l , one scenario is that A l interacts with the plasma membrane calcium channels and initiates calcium mediated signal transduction (Bennet and Breen, 1991b; Rengel et al. 1995). Numerous data indicate that the effects of A l on calcium transport are rapid, reversible and kinetically consistent with competitive inhibition (Lindberg, 1990). The putative involvement of calcium as a mediator of Al ' s toxic effects is suggested when noting some of the major changes in cellular parameters which occur rapidly after exposure to A l . Cytoplasmic streaming is a calcium dependent process which is sensitive to small increases in cytosolic calcium concentrations and is inhibited in a dose-dependent manner by exposure to 80 p M A l (See Chapter 3). Furthermore, the F-actin cytoskeleton and the longitudinal tubular E R are disrupted during exposure to A l . The former structure is calcium sensitive in most plant cells, possibly via modification by 91 calcium sensitive actin-binding proteins (Staiger and L loyd , 1991) and the latter being dependent on the integrity of the linear F-actin array (See Chapter 4). Also , new data that A l causes rapid (within seconds) changes in calcium homeostasis have been obtained from protoplasts of Triticum aestivum (Lindberg and Strid, 1997). In this chapter, the calcium sensitive tetracarboxylate dye, fluo-3, is used to test the hypothesis that A l causes a rise in cytosolic calcium (Ca^ + ^- C y t p , within 1 minute of exposure, in vegetative filaments ofV. longicaulis. 92 5.2. Results Visualization of membrane associated calcium in low p H grown cells A s previously discussed, culturing vegetative filaments in low p H A S W did not affect parameters such as cytoplasmic streaming and apical growth (See Chapter 3). Staining with C T C (chlorotetracycline) revealed a sharply defined region of fluorescence which was observed to be restricted to the most apical portion of the cell's tip (Figure 40 a). In contrast, material treated with oxytetracychne (OTC), a calcium insensitive analog of C T C exhibited no fluorescence (Figure 40 c). No C T C staining was observed in other regions of the cell distal to the cell tip (Figure 40 d). Dye loading for C T C was readily achieved at all experimental pHs attempted (pH 4.5 to 7.5) and required no permeabilization of the cell to give a strong signal with short loading times. Long term _4 exposure to 10 M C T C did not result in any differences in rates of cytoplasmic streaming, growth or changes in overall morphology from those observed in control cells. Visualization of distribution of free cytosolic calcium in low p H grown cells Fluo-3 was chosen preferentially over fura-2 as the calcium reporter dye in this system since fura-2 compartmentalized into the chloroplasts and vacuole more rapidly than did fluo-3 upon illumination with U V light. Visualization of resting levels of calcium in the tip region of the cell, which is not vacuolated, was achieved more easily than in the distal, vacuolated portions (Figures 41 a and 42 c, respectively). Images from the Z V and the A Z were obtained separately since gain settings could not be set at the same level for both regions simultaneously. Contrary to C T C loading, permeabilization was necessary in order to obtain a measurable fluorescence signal using fluo-3. Fluo-3 fluorescence was not uniform in either region of the cell. High loading concentrations of the dye were necessary to detect resting levels of cytosolic calcium in the Z V . Pre-incubation in growth media containing 0.02 mg/ml of saponin gave the most consistent fluo-3 signal. A l l data were 93 obtained within the first 180 seconds after illumination with U V light after which time observations became unreliable due to dye compartmentalization. Cel l viability was not affected at the concentration of dye used. The dye was excluded from the vacuole as indicated by the decreased fluorescence in this region. At 2+ present, it is difficult to determine quantitative Ca (Cyt) directly using fluo-3. However, the addition of ionomycin resulted in an increase in fluo-3 fluorescence intensity demonstrating that the reporter was able to be modulated and hence reflected relative 2+ C a (cyt) levels (Figures 42 a and b). This distinction is important to establish. Aluminum-induced changes in membrane associated and free cytosolic calcium Zone of Vacuolation (ZV) Observations in single cells Images obtained using the Zeiss Attofluor system show that A l induced a rise in 2+ Ca (cyt) (Figures 42 d and 42 e). Difficulties were encountered finding cells which were lying perfectly flat since even small changes in the focal plane resulted in problems detecting the signal. In a cell (n=l) which occupied an ideal position on a gelatin overlaid coverslip, exposure to A l at a specific point, caused an increase in the fluo-3 signal, which was observed to spread to other regions within approximately 2 minutes (Figures 43 a-d). Observations in cell populations A typical response to A l exposure in the Z V for a population of viable cells is shown in Figure 44. Addition of A l did not result in immediate changes in fluo-3 fluorescence intensity but rather showed an initial lag period during which fluo-3 fluorescence remained on par with pre-Al exposure baselines (Figure 44, Section "a"). A subsequent large rise in fluo-3 fluorescence occurred (Figure 44, Section "c"). Fol lowing this, a gradual decrease in RFI values was noted (Figure 44, Section "d"). 94 Compartmentalization of the dye after approximately 180 seconds made data obtained after this time unreliable. In media containing low external concentrations of calcium (0.025 m M ) , the initial decrease in fluo-3 fluorescence occurred slightly sooner in 60% of cells observed (n=2). However, the overall RFI profile was similar (Figure 45). Apical Zone (AZ) Observations in single cells C T C Studies N o differences in C T C staining (compared to controls) were visualized in cell tips treated in low external calcium (Figure 40 e). Observations of cells loaded with C T C revealed that A l exposure resulted in more diffuse C T C staining (Figure 40 f). Treatment in high external calcium resulted in staining similar to that of cells treated in low external calcium (Figure 40 g). The majority of cell tips did not tolerate A l treatment in media containing low calcium (0.025 mM) and lysis occurred in approximately 90% of cells observed (n=3). Fluo-3 Studies Resting levels of calcium in the A Z , as indicated by the fluorescence intensity of fluo-3, were relatively higher than in the Z V (Figure 41a). Treatment with A l resulted in a rise in fluo-3 fluorescence intensity which then decreased and remained slightly higher than in the control (Figures 41 b and 41 c). The distribution of calcium did not appear homogenous in the apical portion of the cell. Observations in cell populations A typical example of time courses for fluo-3 fluorescence in the A Z in response to A l is shown in Figure 46. Addition of A l resulted in a short lag period during which no changes in fluo-3 fluorescence were noted. Following this, a drop in R F I values occurred concommitantly with the lysis of approximately 20% of cell tips. Remaining cells exhibited a sustained increase to reach a maximum fluorescence intensity on the order of a 95 2 fold elevation from baseline values. This was then followed by a steep decline in R F I values to levels below those of pre Al-exposure. Compartmentalization of fluo-3 after approximately 180 s made data obtained after this time unreliable. On average, the R F I m a x value in the A Z was achieved sooner than those in the Z V for all three concentrations of external calcium tested (70 s < R F I m a x < 85 s for A Z vs 90 s < R F I m a x < 100 s for Z V ) (n=2). The A Z appeared more sensitive to A l exposure 2+ when isolated and A l toxicity was exacerbated in low [Ca ] e x (0.025 m M ) . Tips bathed in media containing high concentrations of calcium (8 mM) exhibited a 3 fold rise in R F I values from that of baseline (Figure 47). Cell tips bathed in solutions containing low external calcium (0.025 mM) demonstrated a steady loss in fluo-3 fluorescence after the A l -induced rise (Figure 48). In these experiments, approximately 90% of cell tips lysed within 2 minutes of addition of A l to the system (n=2). Amelioration of Al-Induced Cessation of Chloroplast and Mitochondria Translocation by the Addition of Exogenous Calcium In the Z V , the onset of Al-induced cessation of cytoplasmic streaming could be delayed or prevented by the addition of calcium to the medium (Tables 3 and 4). The addition of calcium (6.0 m M or 8 mM) soon (i.e. 5 seconds) after exposure to A l completely ameliorated its inhibitory effects on chloroplast and mitochondria translocation. However, addition of the same concentration of calcium 30 seconds after exposure to A l resulted in a reduction of amelioration effects on toxicity (Table 3). This is more apparent in Table 4 where the addition of calcium (8.0 mM) reduced Al-induced cessation of chloroplast and mitochondria translocation only i f it was added within 30 seconds of exposure to A l . 96 Table 3. Effects of TCa I P V added post A l (80 uM) exposure on cytoplasmic streaming of chloroplasts and mitochondria 2+ Solutions of varying [Ca ] e x were added to cells incubated in A S W containing 80 p ,M 2+ A l and 2.5 m M [Ca ] e x at p H 4.5. Calcium added sooner (5 seconds) after exposure to A l ameliorates toxicity (as defined by rates of cytoplasmic streaming) at lower concentrations. Values represent average rate + S.D. (n=3). fCa^+lpx Cytoplasmic Streaming Rate* (mM) (pm min'1) + Ca at 5 seconds post +Ca at 30 seconds post Al exposure Al-exposure Chp Mitoch. C h p . Mitoch 0 1.0 2.0 3.0 4.0 6.0 8.0 0 0 0 0 0 0 0 0 0 0 0 0 Control 2 . 3 ± 4 . 8 9.3 ± 8.4 16.2 +6.8 38.0+9.9 24.3+1.8 56.1+7.0 3 7 . 1 ± 8 . 2 58.6+7.7 3 6 . 3 ± 4 . 8 65.4+2.7 38.0+9.2 60.9+7.3 4.7+8.1 13.2+6.8 12.3+4.9 20.7+3.7 16.7+7.4 35.9+6.2 34.1+4.8 62.9+7.6 *Rates measured 60 seconds after addition of calcium. Note: 80 u M A I C I 3 prepared in A S W at p H 4.5 97 Table 4. Effects of time of addition of 8.0 m M T C a 2 + I P V post A l (80 pM) exposure on cytoplasmic streaming of chloroplasts and mitochondria 2+ Ameliorative effects decrease with delayed addition of [Ca ] e x after exposure to A l . Values represent average rates + S.D. (n=3). Time Post Al Exposure (seconds) Mitochondria 5 15 25 35 45 55 65 Control Cytoplasmic Streaming Rate* (fim min'1) Chloroplasts 29.2 24.1 18.4 3.9 0 0 0 34.7 ± 4 . 8 + 10.0 +2.2 + 1.8 +3.9 59.7 56.3 26.0 12.7 0 0 0 61.0 +7.9 +4.4 ± 3 . 8 +2.1 +5.5 *Rates measured 60 seconds after addition of calcium. Note: 80 p M A I C I 3 prepared in A S W at p H 4.5 99 Figure 40 2+ C T C localization of Ca in proximity to hydrophobic (membrane) surfaces, (a) Control cell stained with C T C showing fluorescence restricted to most apical portion of the cell. Bar= 15 pm. (b) Brightfield image of portion of a vegetative filament showing growing 2+ tip. Bar=35 um. (c) Control cell stained with Ca -insensitive analog of C T C , O T C , 2+ demonstrating affinity of C T C for C a . Bar= 15 um. (d) Control cells showing C T C fluorescence restricted to apical portions of cells and absent from distal regions as well as 2+ intracellular membranes. Bar= 60 pm. (e) Al-treated cell in 2.5 m M [Ca ] e x- C T C staining appears more diffuse and occurs in regions posterior to the most apical portion of the cell. Note tip swelling. Bar=35 pm. (f) Al-treated cell in 8 m M [ C a 2 + ] e x - C T C staining appears more diffuse. Tip swelling occurs in less than 20 % of cells. Bar= 12 um 2+ (g). Al-treated cell in 0.025 m M [Ca ] ex- C T C staining appears diffuse though, in cells observed, does not extend as far distally. Note severe swelling of the cell tip. Bar= 15 pm. 101 Figure 41 2+ 2+ Fluo-3 imaging of Ca (Cyt) in the apical zone. (a). Resting levels of C a (Cyt) in the A Z are higher than in the Z V . N o fluorescence is visible in the Z V at the same gain setting. 2+ (b). A l treatment produces a rise in Ca (Cyt) within 60 seconds after exposure, (c). 2+ Ca (cyt) levels decrease slowly but remain higher than in control cell. loa 103 Figure 42 2+ Fluo-3 imaging of Ca (Cyt) in the Zone of Vacuolation. The apical portion of the cell is toward the upper left hand corner of each image, (a-b), Responsiveness of fluo-3 dye is demonstrated by artificially modulating the signal using the calcium ionophore, ionomycin 2+ in 2.5 m M [Ca ] e x • The arrowhead represents the point of contact of the solution added 2+ to cells. Bar=65 pm. (c-e), Ca (Cyt) levels in response to A l treatment, (c) Resting 2+ levels of C a (cyt) in the Z V . Dye is excluded from vacuole as evidenced by lack of 2+ fluorescence in this region, (d). Addition of 80 p M A l causes an increase in Ca (Cyt) in a spatially heterogeneous manner throughout a portion of the cell (image taken approximately 45 seconds after addition of A l ) . Variations in fluo-3 signal may arise due 2+ to changes in optical path and should be considered a source of error, (e) Ca (Cyt) levels approximately 90 seconds after exposure to A l . Bar=65 pm. 105 Figure 43 2 + Fluo-3 imaging of Ca (Cyt) in the Z V . The apical portion of the cell is toward the upper left-hand corner of each image, (a-d), Images obtained by photographing monitor screen directly using a 35 mm camera mounted on a tripod. The arrowhead indicates the point of addition of the A I C I 3 solution which was added using a picospritzer (See Materials and Methods), (a), A small rise in Ca 2 + ( C yt) occurs in the immediate region of A l addtion. (b), (c) and (d), Ca 2 + ( C yt) continues to increase (b and c) and spreads throughout a localized portion of the cell (d). This event occurs within 2 minutes of exposure to A l and demonstrates that A l induces a spatially heterogeneous rise in Ca 2 + ( C yt) . 106 Time (seconds) 107 Figure 44 Representative graph of a typical response of a population of cells whose Z V s have been 2+ isolated using the gelatin embedding method. Responses of cells in 8.0 m M [Ca ] e x 2+ (high) and 2.5 m M [Ca ] e x (experimental) were identical. After the addition of 80 p M A l , an initial lag period is noted during which fluo-3 signal (RFI values) does not change (Section "a"). This is followed by a brief drop in the RFI values (Section "b") and a rise which achieves RFImax approximately 45 seconds after addition of A l to the system (Section "c"). Following RFImax, a decrease in R F I values is noted(Section "d"). This 2+ demonstrates that varying [Ca ] e x does not appear to affect calcium dynamics. 108 109 Figure 45 Typical response of a population of cells whose regions containing the vacuole (ZV) have been isolated using the gelatin embedding method. Observations were done in 0.025 m M 2+ (low) [Ca ]ex- After the addition of 80 p,M A l a lag period is noted (Section "a") prior to a subsequent large increase in the fluo-3 signal (Section "c") which occurs sooner than 2+ in cells treated in medium and high [Ca ] ex- However the overall R F I profile is similar 2+ to the medium and high [Ca ] e x treatments. There is a subsequent decrease in R F I values and the establishment of an erratic baseline (Section "d"). 110 I l l Figure 46 Representative graph of a typical response of a population of cells whose cell tips (AZ) have been isolated using the gelatin embedding method. Addition of A l in medium (2.5 2+ m M ) [Ca ]ex results in a short lag period during which no changes in the fluo-3 signal are noted. Following this, a brief drop in R F I values and concommitant lysis of approximately 20% of cell tips occurs (n=2). Remaining cells exhibit a sustained increase in fluo-3 signal and a subsequent, erratic decrease in RFI levels. Time (seconds) 113 Figure 47 Typical response of a population of cells whose cell tips (AZ) have been isolated using the 2+ gelatin embedding method. Addition of A l to cells bathed in high (8.0 mM) [Ca ] e x results in a decrease in fluo-3 intensity followed by a transient rise in R F I values. This is followed by another drop in RFI values which stabilize but remain erratic. 114 115 Figure 48 Typical response of a population of cells whose tips (AZ) have been isolated using the 2+ gelatin embedding method. Addition of A l to cells bathed in low [Ca ] e x results in a decrease in the fluo-3 signal and the subsequent lysis of 40% of the cell tips. Remaining cells exhibit an increase in fluo-3 values and a subsequent, steady decrease in the fluo-3 signal. Lysis occurs in approximately 90% of cells (n=2). 116 5.3. Discussion Calcium appears to be ubiquitous in its presence both in the external and internal environments of cells yet it can produce specific responses to stimuli which may be varied 2+ in both strength and secondary responses. The once suspected central importance of C a signaling in plants is now well established (Miller et al. 1992). However, although the measurement of free cytosolic calcium concentrations, spatial distributions and dynamics is becoming routine in animal cells, it remains a problematic challenge in intact, walled plant cells. Plant scientists have encountered severe problems when attempting to load plant cells with fluorescent calcium dyes. These problems are also complicated by the fact that, contrary to animal cells, a great deal of variation in technique is required depending on the species and even cell type used (Read et al. 1993). The technique of ester loading (Tsien and Tsien, 1990), most used by animal biologists, has provided poor and inconsistent results with plant cells which possess cell wall-associated esterases (Read et al. 1993). The use of protoplasts has facilitated the use of electroporation and microinjection. However, it has been strongly argued that the removal of the cell wall alters the responses of the cell to its environment and is a poor comparison to in vivo systems (Pont-Lezica, personal communication). This study has utilized a modified ion-trapping procedure which does not affect cell viability. This procedure exploits the robust p H range in which vegetative filaments of V. longicaulis grow. Despite these advantages, problems were encountered which included compartmentalization of fluo-3 particularly into the vacuole, E R and chloroplasts upon exposure to U V light. Theoretically, a dye molecule which is introduced into the cytosol using the acid loading protocol loses the protonating hydrogen ion from its calcium binding group upon encountering physiological pHs (Read et al. 1993). Although the charged form of fluo-3 117 should not be able to cross hydrophobic membranes such as those of organelles or the tonoplast, practical observation provides evidence otherwise. It has been suggested that there may be an active mechanism by which the free dye is modified by a glutathione-S-transferase (GST) and the conjugated product is actively taken up by a glutathione-dependent uptake system (Ehrhardt, personal communication). Despite the fact that the usual problems and caveats which exist in plant cell calcium imaging and measurements in general also exist in this system, controls and bioassays ensure that these data reflect events which may be attributed to Al-treatment. The differences in gain settings between the two regions of the cell make the separate visualization of each portion necessary. The advantage of the gelatin embedding protocol is that cell tips may be physically isolated from the rest of the cell, treated and imaged separately from the Z V . Physical isolation entails overlaying portions of the cell (e.g. the older, vacuolated region) while leaving (e.g.) the tip exposed. As a whole, the cell is not visibly perturbed but the differences in calcium dynamics in response to A l can be resolved between these two distinct regions. Furthermore, use of the gelatin embedding protocol allows one of the fundamental criticisms of calcium imaging, particularly acute in plant cells, to be addressed. This arises from concerns that any experimental manipulations of cell during the course of handling may cause a change in intracellular calcium from the "steady state". In this system, by including controls which have been similarly handled as well as by immobilizing cells in phytagel, the data are validated and may be attributed to a single variable, that of A l . Vaucheria longicaulis provides a system which allows experimental manipulations to be minimized and the whole cell may be used as opposed to a protoplast or excised tissue. Hence, the description of calcium distribution in the control and experimental systems represents a more accurate, albeit entirely qualitative, picture of events which are occurring in vivo. 118 The use of single cells versus cell populations There are a number of advantages in examining calcium at the single cell level. Clearly, this is the only approach that can yield information about the spatial organization 2+ of C a (cyt) changes within the cell. Furthermore, imaging at the single cell level can circumvent problems associated with a heterogeneous cell population and asynchronous 2+ C a (cyt) response, allowing data to be obtained from an identifiable cell type. The main advantage of cell population measurements is that these are simpler to carry out and the signal-to-noise ratio is often better, especially with high temporal resolution (Read et al. 1993). Problems which may arise when using populations, such as the presence of dead cells which may exclude or accumulate the dye may be reduced by monitoring individual cells throughout the course of the experiment. Correlation of the rise in cytosolic calcium and cytological changes during exposure to A l Exposure to A l causes an increase in cytosolic calcium as indicated by an increase in the fluorescence intensity of fluo-3 (e.g. Figures 41, 42 and 43). This increase is noted in both the A Z and the Z V with minor variations. However, the response to A l in the A Z differs with respect to the concentration of external calcium supplied in the bathing media (Figures 46, 47 and 48). Exposure to A l at low external calcium concentrations results in an initial drop in RFI values and concommitant tip lysis. In the Z V , this initial drop in R F I values is not apparent but A l does cause a rise in cytosolic calcium (Figures 42, 43 and 44). This increase occurs within a time frame which may be correlated with the slowing and cessation of cytoplasmic streaming of chloroplasts and mitochondria (in the Z V ) . Such a clear statement is more difficult to make in the apical zone. In this region it is apparent 2+ that a similar rise occurs in Ca (cyf> apparently slightly slightly sooner after exposure to A l than is observed in the Z V but a direct correlation to a visible cellular parameter such as cytoplasmic streaming, which does not occur in this region, cannot be made. 119 In the A Z , the effects of A l on fluo-3 fluorescence vary depending on the concentration of externally supplied calcium. This suggests that the degree of A l toxicity may be ameliorated by exogenous (apoplasmic) calcium. Although the A Z appears to be able to mobilize some form of internal calcium store this source may be limited and/or dependent on either synchronous or subsequent calcium entry from the external environment in a manner similar to that which has been demonstrated in Lilium pollen tubes (Hepler, 1997). However, unequivocal evidence for this is not provided by this study. Aluminum has been shown to reduce net rates of calcium uptake into roots, as expressed per unit of root absorbing surface after prolonged exposure to A l (Godbold, 1991; Rengel and Robinson, 1989, 1990). It has also been shown to inhibit adsorption of calcium to the negative charges in the Donnan free space (Cronan, 1991; Jentschke et al. 45 2+ 1991), to competitively reduce short term net Ca uptake into intact roots of various tree and crop species (Asp and Breggen, 1990; Lindberg, 1990). Using a range of 2+ calcium channel blockers as well as inhibitors of Ca -ATPase, calmodulin and G T P 2+ proteins it was concluded that A l ions act as a Ca -channel blocker and inhibits net 45 2+ accumulation of C a in Amaranthus tricolour protoplasts (Rengel and Elliot, 1992a). 45 2+ Aluminum affects net Ca uptake by binding to the verapamil-specific channel receptor site as well as by interfering with the action of GTP-binding proteins (Rengel and Elliot, 1992 b). Another study conducted using fluo-3 and Al-tolerant and Al-sensitive wheat cultivars shows that calcium influx is reduced while the efflux remained the same as in control plants. Despite the decrease in calcium influx into the root hair cells of these plants, fluo-3 fluorescence increases in Al-treated hairs compared to controls and this increase is greater in the Al-sensitive cultivar (Nichol and Oliveira, 1995). Furthermore, a recent 2+ study using Triticum aestivum indicates that 50 u M A l causes an increase in C a (cyt) 120 within 1 minute of exposure which is preceeded by a brief, transient drop in C a (Cyt) levels (Lindberg and Strid, 1997). In agreement with these studies, A l treatment in V . 2+ longicaulis vegetative filaments results in a rise in Ca (cyt)-It has been shown that an increase in the activity of divalent cations (especially 2+ 2+ Ca and M g ) in solutions bathing roots ameliorates Al-induced rhizotoxicity (Kinraide and Parker, 1987). The addition of exogenous calcium results in variations in the time of onset of the initial symptoms of A l toxicity in V. longicaulis. This is reflected in the recovery of the rate of cytoplasmic streaming of chloroplasts and mitochondria which varies depending on the concentration of calcium added following A l treatment (Table 4). The remediation is sensitive not only to the concentration of added calcium but also to the time of addition (Table 4). The sooner that exogenous calcium is added, the more pronounced the amelioration effects. These observations support the hypothesis that interaction of A l and C a 2 + at the apoplasmic site of a calcium channel may occur since the ameliorative effectiveness of A l toxicity by divalent cations is shown to follow the sequence C a » M g ~ S r (Kinraide and Parker, 1987). Sensitivity of the Apical Zone to extracellular calcium Evidence has been obtained from pollen tube systems that a tip-directed inward current of extracellular calcium exists which is not present in older portions of the cell 2+ (Pierson et al. 1994). Treatments which eliminate the influx of Ca cause an inhibition of growth, swelling and/or lysis of the cell tip (Pierson et al. 1994). It has also been demonstrated that A l toxicity varies between the root apex and mature root regions in wheat (Huang et al. 1993). The present study supports these findings since exposure to A l in low external calcium solutions results in exacerbated and irreversible toxicity. This may be reflective of a disruption of an influx of calcium from the environment via a (putative) high concentration of calcium channels present in this portion of the plasma membrane (refer to Figure 1.1, page 13). The fact that RFI profiles in the A Z are dependent on the 121 concentration of external calcium suggests that entry of external calcium may occur. In high external calcium, entry of calcium into the cell may be favoured thus providing a source of calcium other than intracellular stores (Figure 47). These observations are in agreement with the suggestion that calcium efficiency is at least one of the features of plant cells which exhibit A l tolerance (Kochian, 1995). The vacuole as a source of intracellular calcium during aluminum exposure In the A Z the increase in fluo-3 fluorescence which follows A l exposure may be varied depending on the concentration of calcium present in the external media whereas dynamics in the Z V appear to occur independently of the concentration of externally supplied calcium. When observing the comparative morphologies of each portion of the cell it is noted that neither the longitudinal E R or the vacuole extend into the cell tip (See Chapter 3). Hence, the potential sources of intracellular calcium (i.e. stored but not necessarily available calcium) are theoretically restricted to the dense cortical E R network, chloroplasts and other putative calcium sequestering organelles present in the cell tip (Cai et al. 1997). Conversely, the Z V may contain not only the sub-cortical E R and calcium sequestering organelles but also the cortical E R as well as the relatively unlimited supply of calcium contained in the vacuole. The vacuole is considered to be the largest stimulus-releasable source of calcium available to the plant cell. Not only does it act in sequestering cytosolic calcium such that free calcium concentrations are maintained at low levels, it also acts as a single most abundant source of messenger calcium which may be mobilized during signal transduction (Shumaker and Sze, 1989). It has been shown that A l interacts with phosphoinositol metabolism in rat cerebral cortical membranes to stimulate cerebral phosphoinositide metabolism (Audus et al. 1988). The release of inositol 1,4,5-triphosphate (InsP3) into the cytoplasm could result in the release of calcium from the vacuole and/or the E R . Such events could possibly explain the differences in the response of the A Z versus the Z V during Al-treatment. 122 Use of fluorescent dyes in plant calcium imaging The primary obstacle to using fluorescent indicators of calcium in plant cells is usually dye uptake. Calcium sensitive dyes are charged and do not readily cross biological membranes. The ester loading technique used by animal cell biologists to successfully load cells with calcium indicators has limited applicability to plant cells becaue of extracellular hydrolysis (e.g. Hahm and Saunders, 1991), incomplete removal of ester groups in the cytosol or sequestration of the dyes by organelles (Read et al. 1993). The acid loading method utilizes the proton binding properties of the dyes to produce an uncharged molecule at low p H (in this case, 4.5) that is l ipid soluble. Once in the cytosol, where the p H is 6.8 or higher, the dye dissociates and is trapped as the charged anion, the fluorescence of which indicates cytoplasmic calcium concentrations. This technique has been successfully applied to plant cells, however, compartmentalization remains a problem (e.g. Kiss et al. 1991; Russ et al. 1991; Hahm and Saunders, 1991). Furthermore, it must be stated that the use of single wavelength dyes is not ideal since these dyes do not allow adjustments for optical path length or variations in dye concentration in the cytosol due to sequestration (Hepler, 1997). However, although the problems of interpreting data obtained from single wavelength measurements are acknowledged, they still provide valid information on calcium dynamics in plant cells so long as their limitations are stated and data are collected in a consistent manner. The interpretations of data in this system are purely qualitative and no statements may be made 2+ on long term Ca (Cyt) dynamics. Conclusions These data support the hypothesis that A l interferes with calcium homeostasis in 2+ plant cells. Treatment with A l results in a rise in Ca (cyt) within a time frame that may be correlated to the cessation of chloroplast and mitochondria translocation. It is noted that R F I profiles of the young, apical portion of the cell differ from the older, vacuolated 123 portion with respect to sensitivity to [Ca^+ ] e x . Apical tips are more sensitive to treatment with A l and toxicity is exacerbated in low [ C a ^ + ] e x whereas this is not the case for the Z V . 124 6.0. Calcium modulator Studies 6.1. Introduction Like many other cell types, plants respond to environmental stimuli via a sensitive and complex series of internally produced signal transduction mechanisms. Changes in cytoplasmic calcium are central to these processes (Bush, 1995). Aluminum toxicity manifests itself in V. longicaulis within 60 seconds after exposure and causes visible perturbations in processes and structures such as cytoplasmic streaming of chloroplasts and mitochondria as well as the F-actin cytoskeleton, which appear to be calcium dependent 2+ (Bush, 1995). In the previous chapter, Al-induced changes in C a (Cyt) were demonstrated. In this chapter, an attempt wi l l be made to determine whether or not perturbation of calcium homeostasis, in an Al-free system, mimics the cytomorphological symptoms observed in response to A l throughout this study. Calcium modulators are used to explore the differences in calcium dynamics observed in the apical zone (AZ) and the zone of vacuolation (ZV) of cell populations. In animal cells, thapsigargin acts upon sarcoplasmic endoplasmic reticulum type calcium pumps (Lytton et al. 1991). It also may facilitate calcium entry across the plasma membrane under conditions of high exogeneous calcium and during early stages of cell development (Takemura et al. 1991). T M B - 8 is an intracellular calcium release antagonist whose mechanism of action is largely unknown. It has been widely used to block cellular function dependent on calcium messenger systems in animal cells and in cells of organisms such as Funaria and Dictyostelium (Saunders and Hepler, 1983). Early work showed that T M B - 8 blocks receptor-mediated cellular processes and that its effects may be largely overcome by the addition of exogenous calcium (Smith and Iden, 1979). The spatial and temporal calcium events which may be blocked by T M B - 8 are not yet clear. In sea urchin eggs, T M B - 8 prevents the release of calcium which follows fertilization (Stapleton et al. 1985) and in 125 permeabilized eggs prevents IP3-induced calcium release from the E R (Clapper and Lee, 1985). The available information on plant material is consistent with T M B - 8 inhibiting receptor-mediated calcium mobilization from internal stores. T M B - 8 inhibits, in a dose-45 2+ 45 2+ dependent manner, the InsP3-induced release of Ca from C a -loaded tonoplast vesicles of oat root (Schumaker and Sze, 1986). In cells of the moss Funaria, T M B - 8 enhances the accumulation of calcium in internal membrane-bound compartments, suggesting that efflux of calcium from internal stores is blocked but influx from the cytoplasm and external environment of the cell is not affected (Saunders and Jones, 1988). T M B - 8 strongly inhibits auxin stimulated growth and cell wall synthesis in Pisum sativum and its effects may be reversed by the addition of exogenous calcium (Brummell and Maclachlan, 1989). 126 Ionomycin Ionomycin is used as the calcium ionophore in this study as Br-A23187 is an efficient proton exchanger while also acting as a calcium ionophore and it has been documented that changes in cytosolic p H can affect calcium homeostasis and the F-actin array in plant cells (Bush et al. 1993). Ionomycin wi l l be used to test the hypothesis that A l toxicity is mediated, at least in part, by affecting calcium homeostasis at the level of the plasma membrane. If this is true, 2+ altering Ca (cyt) directly, using ionomycin, should result in similar changes in the F-actin array and rates of cytoplasmic streaming. The use of T M B - 8 and thapsigargin wi l l test the 2+ hypothesis that the source of the Al-induced rise in Ca (Cyt) is intracellular. If this is true, then blocking the release of calcium (using T M B - 8 ) upon exposure to A l should block the observed effects of toxicity. Conversely, stimulating the release of calcium from intracellular stores (using thapsigargin) should result in changes similar to those observed upon Al-treatment. 127 6.2. Results Observations in the Apical Zone Demonstration of stimulus release of calcium from an internal source 2+ Thapsigargin in Varied TCa Ipy 2+ Thapsigargin in the presence of low [Ca ] e x (0.025 mM) produced an increase in fluo-3 fluorescence intensity, of which a representative example is shown in Figure 49. The addition of E G T A did not abolish this rise suggesting that the source of calcium was 2+ internal (Figure 49, inset). In low [Ca ] e x , RFI values continued to increase to a maximum value which was followed by a decrease in R F I values to below those of baseline. Lysis of the cell tip occurred concommitantly. 2+ Cel l tips treated with thapsigargin in medium (2.5 mM) and high (8 mM) [Ca ] e x exhibited R F I profiles which stabilized at a higher level (compared to baseline) or continued to increase until the dye became compartmentalized (Figure 49). After approximately 90 s, the addition of E G T A to the media containing 2.5 m M calcium resulted in the abolision of this increase (Figure 49, inset). Staining with FiTC-phalloidin revealed F-actin patterns similar to those observed in Al-treated cells in that they displayed bundled F-actin rather than diffuse staining in the A Z . However, it was interesting to note that whereas the bundles appeared disorganized (i.e. at angles to each other) in the Al-exposed tips, the thapsigargin-treated tips exhibited a more axially aligned array of thick bundles which extended to the apical plasma membrane (Figure 50, compare with Figure 20). Cells which possessed this F-actin pattern in their cell tips ceased to grow. Thapsigargin treatment produced C T C staining which occurred distal to the most apical portion of the tip instead of remaining localized as was noted in controls (Figure 50). 128 Is the source of calcium which is released upon A l exposure internal or external? Pre-treatment with thapsigargin and subsequent treatment with A l 2+ In an attempt to investigate whether the Al-induced rise in C a (Cyt) in the A Z occurred via the release of intracellular stores, isolated cell tips were pre-treated with the 2+ drug and subsequently exposed to A l . The results for the medium and high [Ca ] e x wre 2+ similar and hence only the high and low [Ca ] e x data are represented in Figure 51. In both cases, A l treatment resulted in a drop in RFI values and concornmitant lysis of the cell 2+ tip. Cel l tips survived longer in media containing high [Ca ] e x whereas they lysed 2+ sooner when bathed in media containing low [Ca ] ex-Treatment with T M B - 8 in varied r C a 2 + W Cel l tips were treated with T M B - 8 in low (0.025 mM) , medium (2.5 m M ) and high 2+ (8 m M ) [Ca ] ex- The drug did not appear to have an effect on fluo-3 fluorescence values at the medium and high external calcium concentrations but treatment in low external calcium levels resulted in the gradual decrease of RFI values to nearly undetectable levels (Figure 52). 2+ Pre-treatment with T M B - 8 and subsequent treatment with A l in varied [Ca ]ex The hypothesis that A l caused the release of calcium from intracellular stores was explored by pre-treating cell tips with T M B - 8 and subsequently exposing them to A l . A typical response is shown in Figure 53. The response of the cell tips to A l treatment in 2+ medium and high [Ca ] e x were similar. A gradual decrease in R F I values was observed 2+ which continued to nearly undetectable levels. In most cell tips, treatment in low [Ca ] e x 129 resulted in R F I values which dropped rapidly until no signal was detectable and concomrnitant tip lysis occurred. Observations in the Zone of Vacuolation 2+ Is the Zone of Vacuolation sensitive to [Ca ] e x? 2+ Treatment with thapsigargin in varied[Ca ]ex Treatment with thapsigargin in the zone of vacuolation resulted in a transient increase in R F I values (Figure 54). Cells were repeatedly treated with thapsigargin, every ten minutes for 20 minutes each time, up to 20 times, in an attempt to deplete intracellular calcium stores. After each treatment, chloroplast and mitochondria translocation ceased temporarily within the time frame of the transient rise in RFI values. Organelle streaming resumed within approximately 10 minutes after treatment with thapsigargin and the R F I 2+ profile did not vary notably in any of the concentrations of [Ca ] e x used. The DIC-visible cytoplasmic strands remained unchanged in morphology compared to controls. Results are shown for the high (8.0 mM) and low (0.025 mM) calcium values. Relative fluorescence intensity values began to increase to a maximum value after which a gradual decrease occurred and eventually stabilized around baseline levels. This was also the case 2+ for low [Ca ] e x levels. Addition of E G T A did not affect the rise in R F I values nor did 2+ the addition of 8 m M exogenous calcium to media containing low [Ca ] e x regardless of the concentration or time of addition (data not shown). 2+ Pre-treatment with thapsigargin and subsequent treatment with A l in varied [Ca ]ex Cessation of cytoplasmic streaming of the chloroplasts and mitochondria occurred in cells which were pre-treated with thapsigargin and subsequently treated with A l . The 2+ RFI profile for these cells indicated that A l treatment resulted in a rise in Ca (Cyt) which was not visibly different from that observed in cells treated with A l alone (Figure 55). 130 Repeated treatment with thapsigargin did not appear to diminish or alter that pattern of A l -2+ induced increase in Ca (cyt)-Treatment with T M B - 8 in varied external concentrations of calcium 2+ Treatment with T M B - 8 in low, medium or high concentrations of [Ca ] e x did not appear to have an effect on the RFI profile in the Z V (Figure 56). Cytoplasmic streaming of chloroplasts and mitochondria continued unabated. 2+ Pre-treatment with T M B - 8 and subsequent treatment with A l in variedfCa ]ex Subsequent addition of 80 p M A l to cells which were pre-treated with T M B - 8 resulted in an increase in R F I values (Figure 57). This increase was consistently lower than in cells which were exposed to A l only. The rise in calcium levels occurred coincidently with the slowing down and cessation of chloroplast and mitochondrial streaming. The response of the Z V to A l exposure after treatment with T M B - 8 did not 2+ differ between cells bathed in low, medium or high [Ca ] ex-F-actin Pre-treatment with thapsigargin or T M B - 8 and subsequent treatment with A l resulted in the disorganization of the F-actin array. Visualization of the F-actin array in the Z V after treatment with thapsigargin, T M B - 8 and ionomycin is shown in Figures 58, 59 and 60). The most notable change occurred upon treatment with ionomycin (Figure 60). It is interesting to note that the pattern observed with this treatment appearred consistently throughout the cell. In the A Z , the only calcium modulator treatment in which visualization of F-actin was successful was that of thapsigargin. Treatment with this drug resulted in a change from the normally diffuse F-actin staining to distinctly bundled F-actin which extended to the cell tip (Figure 50 a). This was noted in almost all cells observed. Time (seconds) 132 Figure 49 Typical response of a population of cells whose cell tips (AZ) have been isolated using the 2+ gelatin embedding method and treated with 500 n M thapsigargin in varied [Ca ] e x- (a). 2+ Cel l tips treated in 0.025 m M [Ca ] e x exhibit a steady rise in R F I values followed by a rapid decrease in the fluo-3 signal and subsequent lysis of 100% of cell tips. Inset: Early addition of E G T A (2 mM) does not abolish the rise in R F I values suggesting an internal 2+ source of calcium, (b). Cell tips treated in 2.5 m M [Ca ] e x exhibit an increase in R F I values and subsequently level off at R F I values higher than those observed prior to treatment. Inset: Addition of E G T A results in a drop in RFI values, suggesting an external 2+ source of calcium, (c). Ce l l tips treated in 8 m M [Ca ] e x exhibit a steady and sustained increase in R F I values which continue to increase up until the fluo-3 signal becomes unreliable. 133 134 Figure 50 2+ FITC-phalloidin and C T C staining in a cell tip (AZ) to visualize F-actin and C a ions in proximity to hydrophobic membranes respectively. Bottom panel, (a), cell tip showing diffuse F-actin staining in the most apical portion of the cell, (b) C T C staining pattern showing restriction of fluorescence to cell tip. Top panel, (a), cell tip treated with 500 n M thapsigargin showing distinctly bundled F-actin which is seen to extend into the most apical portion of the cell tip in a similar pattern to that which is observed upon A l treatment, (b) Corresponding C T C staining pattern showing derealization of fluorescence to regions distal from the most apical cell tip much as is observed upon A l treatment. Bar=30 pm. 135 £-onu ( J J H ) .MisuajuT aDuaosaionu a.\nE|a^ 136 \ Figure 51 Typical response of a population of cell tips (AZ) isolated using the gelatin embedding 2+ method. Cel l tips are pre-treated with 1 p:M thapsigargin for 10 minutes in 8 m M [Ca ] e x prior to imaging and subsequently treated with 80 p M A l . Aluminum causes a steady decrease in the fluo-3 signal (I). Cell tips are pretreated with 500 n M thapsigargin for 10 2+ minutes and subsequently treated with 80 p,M A l in 0.025 m M (low) [Ca ] e x- A decrease occurs in RFI values concommitantly with lysis of the cell tips (II). 137 5 u Si CA c <u u C o IIS a a a ul > •JS 13 Time (seconds) 138 Figure 52 Typical response of a population of cell tips (AZ) isolated using the gelatin embedding 2+ method. Ce l l tips are treated with 2 p M T M B - 8 in varied [Ca ] e x for 10 minutes. The drug does not appear to have an effect on Ca 2 + ( C yt) at medium and high [Ca ] e x but 2+ results in a gradual decrease m RFI values when treatment takes place in low [Ca ] ex-139 140 Figure 5 3 Typical response of a population of cell tips (AZ) isolated using the gelatin embedding method. Cell tips are pretreated with 2 pJVI TMB-8 for 10 minutes and subsequently 2+ treated with 80 p.M Al . A drop in RFI values is observed for all three [Ca ]ex 2+ experiments however, cell tips incubated in low [Ca ]ex demonstrate a rapid decrease in the fluo-3 signal and concommitant lysis. The RFI profiles for the 2.5 mM and 8 mM 2+ [Ca ] ex trials are similar to each other. 141 u u C 01 O) u c (A 41 I i O u-01 > m f9 'Z Time (seconds) 142 Figure 5 4 Typical response of a population of cells whose vacuolated regions (ZV) were isolated using the gelatin embedding method. Repeated treatment with 1 pM thapsigargin results in a transient increase in the fluo-3 signal (during each treatment) and a subsequent return to 2+ baseline. Responses for 8 mM and 0.025 mM [Ca ] ex were similar. 143 Time (seconds) 144 Figure 55 Typical response of a population of cells whose vacuolated regions (ZV) were isolated using the gelatin embedding method and repeatedly treated with 1 p M thapsigargin for varying times (see text). Subsequent treatment with 80 p M A l results in a rise in R F I values followed by a decrease and stabilization at an erratic baseline which is consistently higher than that observed in control cells. 145 £ - ° n U (IJCI) A i^suaiuT aousosaionij S A I ^ T ^ T 146 Figure 56 Typical response of a population of cells whose vacuolated regions (ZV) were isolated using the gelatin embedding method. Treatment with 2 p M T M B - 8 (for times, refer to 2+ text) in any [Ca ] e x (i-e. low, medium or high) did not result in visible changes from baseline. 148 Figure 57 Typical response of a population of cells whose vacuolated regions (ZV) were isolated using the gelatin embedding method. Pre-treatment with 2 p M T M B - 8 did not cause any changes in rates of cytoplasmic streaming of chloroplasts and mitochondria (for times, refer to text). Subsequent addition of 80 p M A l resulted in rise in R F I values which was lower than that observed in cells treated with A l only. This increase in Ca^ + ( C y t ) was sufficient to cause a transient cessation of chp/mitoch. streaming. Note sampling interval = 5 seconds. 150 Figure 58 FITC-phalloidin staining to visualize the F-actin arrray. (a) Control cell showing ordered, longitudinal orientation of F-actin. (b) 1 hour pre-treatment with 2 p M T M B - 8 and subsequent treatment with 80 p M A l results in a disorganization of the array. Cell visualized 60 minutes after exposure to A l . Bar=60 pm. Figure 59 FITC-phalloidin staining to visualize the F-actin array, (a) Control cell showing ordered longitudinal orientation of F-actin. (b) 1 hour pre-treatment with 1 p M thapsigargin and subsequent treatment with 80 p M A l results in the disorganization of the array. Cell visualized 60 minutes after exposure to A l . Bar=55 pm Figure 60 FITC-phalloidin staining to visualize the F-actin array, (a) Control cell showing ordered longitudinal orientation of F-actin. Treatment with ionomycin results in a the rapid disorganization of the array (b) and (c). Bar=50 pm. Figure 61 FITC-phalloidin staining to visualize the F-actin array in an Al-only treated cell (as a control for Figures 58-60). Bar=35 pm. 151 6.3. Discussion The interpretation of data obtained using various calcium "modulators" must be done with the caveat that perturbing processes as ubiquitous and crucial as calcium fluxes may give rise to "abnormal" cellular events which are difficult to interpret. The use of thapsigargin in animal systems is well documented and produces, in most cases, a 2+ consistent rise in Ca (Cyt) (Berridge, 1993). In plant cells, thapsigargin has been used with variable results and has evoked different responses in different species and even cell types (Ehrhardt, personal communication). T M B - 8 has been used effectively to block the release of calcium from internal stores in plants (Saunders and Hepler, 1983) although the mechanism of its action is poorly understood. Free calcium concentrations in plant cells, as in most eukaryotic cells, is maintained -6 -7 at low (10 to 10 mM) levels by active extrusion at the plasma membrane and active transport into the vacuole, E R and organelles (Bush, 1995). Extracellular concentrations -4 -3 of calcium in the range of 10 to 10 M are common for the plant cell and similarly, -3 intracellular compartments have been shown to contain calcium concentrations of up to 10 M (Bush, 1995). With such a large gradient of calcium between the external environment and the cytosol as well as between intracellular compartments, it is easy to envisage how A l , interacting with various components at the plasma membrane, could cause perturbations of the calcium homeostat. Furthermore, it seems apparent from these data that the A Z and Z V differ in their response to external calcium levels. The A Z appears sensitive to this parameter whereas the Z V does not. Apical Zone 2+ Thapsigargin Studies: The Apical Zone is sensitive to [Ca ] e x The putative action of thapsigargin as a mediator of capacitative calcium entry from the external environment is supported when observing the RFI profiles in Figure 49. Treatment in low external calcium results in a loss of fluo-3 signal and lysis of the cell tip 152 whereas treatment in media containing sufficient external calcium results in the continued elevation of RFI values. This is further supported by the fact that addition of E G T A results in a rapid drop in RFI values. Furthermore, it may be speculated that thapsigargin may, in fact, initially trigger the release of intracellular stores (i.e. the rise in R F I values is not abolished by the early addition of E G T A ) and then possibly facilitates calcium entry from the bathing media (i.e. the " E G T A sensitive" phase). This could explain differences 2+ observed between the low and high [Ca ] e x responses. It appears that the A Z of tip-growing cells contains the highest concentrations of free cytosolic calcium, up to 1.39 p M 2+ for Lilium longiflorum pollen tubes (Hepler, 1997). Thus, the C a (Cyt) levels in tips of these cells may be contributed to, not only by intracellular stores, but also by exogenous calcium. Predictions using the Nernst equation Some predictions of the effects of varying external calcium may be made using the Nernst equation. This takes into consideration the membrane potential (which wi l l be assumed based on data obtained from other plant cells), the internal concentration of calcium (which wi l l also be assumed based on data from other plant cells) and the external concentration of calcium. The Nernst equation states that it is not only the chemical potential of an ion (i.e. concentration) which determines its net movement into or out of the cell but also its electrical potential (i.e. charge) and the electrical potential difference across the membrane. I am assuming that the membrane potential for Vaucheria longicaulis is similar to that measured for Nitella (i.e. -98 mV) and the cytosolic calcium concentration in 6 7 the Z V is similar to that of other vacuolated plant cells (i.e. 10" to 10" mM) . In the A Z , I am assuming the internal calcium concentration is similar to that found in other tip elongating cells (i.e. 1.39 p M ) . Thus, for both the Z V and the A Z , the Nernst equation predicts that the direction of the passive (electrochemical) gradient should be into the 153 cytosol for each concentration of external calcium used (0.025 m M , 2.5 m M and 8.0 m M ) . 2+ However, under conditions of low [Ca ] e x , cell tips experience lysis upon treatment with A l as well as different RFI profiles from those observed in medium and high 2+ 2+ [Ca ]ex- In the Z V , varying [Ca ] e x does not appear to influence RFI profiles. Tip-elongating cells appear to possess a high concentration of calcium channels at their apices. Data obtained from Lilium pollen tubes indicates that calcium entry occurs only in the first 2.5 pm of the cell's tip and no longer occurs away from the apex of the tip. 2+ This could explain the insensitivity of the Z V to varied [Ca ] e x- The type of calcium channel favoured in the tip region of the cell is primarily stretch activated (Cosgrove and Hedrich, 1991; Ding and Pickard, 1993; Bush, 1995). Such mechano-sensitive calcium channels are set at a high open-close threshold such that only those channels undergoing maximal stretching wi l l be open (Pierson et al. 1994). This follows the fact that tip elongating cells have a hemispherical shape at their tip and the displacement of membrane would follow a cosine function, with a maximum at the apex, falling to zero at the base of the dome (Green, 1974). With this model, open channels wi l l be restricted to the extreme apex of the cell and wi l l close as they move away due to tip elongation. This membrane displacement process occurs within 5 seconds in pollen tubes of Lilium longiflorum. The process of rapid calcium channel inactivation may help explain why many treatments which block growth lead to a rapid dissipation of the tip-focused gradient. In experiments using Utomyces, treatment with latrunculin, a competitive calcium channel blocker, results in the 2+ rapid dissipation of tip-focused calcium influx when [Ca ] e x is less than 0.48 m M (Zhou, 1991). 2+ In the A Z of V. longicaulis, treatment with A l in low [Ca ] e x results in a drop in RFI profiles and concommitant tip lysis. This suggests that cytosolic calcium is decreasing rapidly even though influx should occur. A possible reason for this observation is that A l 154 may be blocking calcium channels. If this blockage is competitive, then conditions of low calcium may favour A l binding to tip localized calcium channels and inhibit influx whereas 2+ raising [Ca ] e x by a factor of 10 or greater may be sufficient to ameliorate this inhibition. Another speculation is that, i f these calcium channels are stretch-activated, a reduction in growth would rapidly prevent them from being fully open. This could reduce their effectiveness in facilitating calcium influx. However, this may only become apparent in 2+ low [Ca ]ex- Obviously, a great deal of further study is required to justify such speculation. Pre-treatment with thapsigargin and subsequent treatment with A l The standard concentration of thapsigargin for whole cells (1 p M ) and the Z V could not be used on gelatin-isolated cell tips for unknown reasons. Isolated cell tips could not be treated with 1 p M thapsigargin without lysis or exhibiting a drop in growth rates. When 500 n M thapsigargin is used a greater number of cells remain viable during treatment. Regardless, subsequent treatment with A l results in cell tip lysis in the majority 2+ 2+ of cells tested irrespective of the concentration of [Ca ] ex- However, [Ca ] e x affects the time before tip lysis occurs following Al-treatment. This extreme sensitivity to A l treatment after exposure to thapsigargin is notable. If thapsigargin results in the depletion of intracellular stores as well as facilitating external calcium entry, it is not unreasonable to speculate that A l may be preventing entry of calcium into the cell tip. Aluminum has been 45 2+ shown to result in the inhibition of Ca uptake in a variety of cell types, it has also been suggested that that Al :Ca ratio in the environment is a indicator of the severity of A l 2+ toxicity. The fact that higher [Ca ] e x results in the delay of cell tip lysis and loss of fluo-3 signal supports the hypothesis that A l may competitively inhibit calcium uptake (Lindberg, 1990). 155 T M B - 8 Studies T M B - 8 (2 p M ) treatment does not cause changes in growth rates or morphology of 2+ the cell tips unless cells are bathed in media containing low [Ca ] ex- T M B - 8 does not 2+ affect the R F I profile of cells in medium or high [Ca ] e x but does have an effect of those 2+ in low [Ca ]ex (Figure 52). A possible reason for this observation is that T M B - 8 may 2+ prevent the release of C a required in the region of most active growth. This may result in a requirement for extracellular sources of calcium (Picton and Steer, 1995). It is unclear why the cell is unable to access the high amounts of calcium bound in the cell wal l . It is well documented that these sources are fairly tightly bound (Pierson et al. 1994). The addition of A l to cell tips which have been pre-treated with T M B - 8 results in greatly exacerbated toxicity as evidenced by tip lysis as well as the R F I profile shown in Figure 53. These results are not entirely consistent with the observations of Brummell and Maclachlan (1989) whose data indicated that T M B - 8 does not appear to prevent calcium uptake into pea stem cells. If this is the case, then another variable may be preventing an influx of calcium into V. longicaulis. RFI values continue to decrease, even in high external concentrations. This observation may be rectified i f A l acts as an inhibitor of calcium uptake. Thus the difference in the time required to cause cell tip lysis between the 2+ low, medium and high [Ca ] e x may be explained. However, since the complete spectrum of effects of T M B - 8 are poorly characterized, this interpretation remains entirely speculative. 156 Zone of Vacuolation Thapsigargin Studies: The Zone of Vacuolation is insensitive to [ C a 2 + ] e x Repeated treatment of the Z V region of vegetative filaments with 1 u M thapsigargin results in a transient increase in fluo-3 fluorescence intensity and a subsequent return to baseline R F I values. Cytoplasmic streaming of chloroplasts and mitochondria slows down and ceases within a time-frame consistent with the rise in R F I values caused by this treatment. However, cytoplasmic organization remains unchanged and streaming resumes shortly after R F I values return to baseline levels. Repeated treatments are conducted because thapsigargin is metabolized fairly quickly by most eukaryotic cells. Hence, a single treatment would not be expected to deplete putative intracellular calcium stores, particularly that of the vacuole. It is interesting that thapsigargin causes a rise in calcium in this region of the cell but it is impossible to determine which source of intracellular calcium this modulator is acting upon. However, the fact that treatment with thapsigargin in both 2+ low and high [Ca ] e x results in similar effects in this region of the cell does suggest that the source of calcium may be internal. Subsequent addition of A l to cells which have been pre-treated with thapsigargin results in an R F I profile similar to that observed in cells which are treated with A l alone in all concentrations of exogenous calcium. Thus, it appears that thapsigargin pre-treatment does not exhaust the supply of intracellular calcium stores in the Z V . Cytoplasmic streaming of chloroplasts and mitochondria ceases upon A l treatment and visible cytoplasmic disorganization occurs within 30 minutes. Visualization of the F-actin array with FTTC-phalloidin reveals that disorganization occurs in a manner similar to those cells which are treated with A l only (Figure 59, compare with Figure 61). Thus, the vacuole 157 may be thapsigargin sensitive and repeated exposures to the drug could result in a rise in 2+ C a (cyt) followed by a return to baseline as the drug is metabolized by the cell (as is noted in animal systems) (Shi et al. 1993). The fact that repeated treatments with the drug 2+ do not diminish the Al-induced rise in C a (Cyt) supports data which indicate that the vacuole provides an extensive source of calcium in plant cells (Schumaker and Sze, 1986). There is also a possibility that thapsigargin is acting upon the E R , either the longitudinal cortical type or that o f the sub-cortical network, however, it appears that thapsigargin-sensitive calcium pumps are not usually localized to plant E R except in developmentally young tissue (DuPont et al. 1990). T M B - 8 Studies 2+ The Al-induced rise in Ca (Cyt) is not completely abolished by T M B - 8 . Hence, these data do not fully support the hypothesis that A l may be triggering the release of intracellular calcium stores. However, an alternative possibility may be that T M B - 8 does not inhibit the type of calcium pumps found in this region of the cell. Another possibility is that, i f it is inhibitory, it may not be present in sufficient amounts to cause an effect. This latter scenario may be more likely since treatment with A l results in a rise in R F I values which are consistently lower than those observed in cells treated with A l only. The fact 2+ that the Z V appears insensitive to the concentration of [Ca ] e x used in these experiments suggests that it may rely on internal stores. This is consistent with the hypothesis that vacuolated cells possess extensive sources of accessible calcium which are retained in the vacuole (Miller et al. 1992). Furthermore, it has been shown that the vacuole of plant cells is IP3 sensitive (Schumaker and Sze, 1986; Kochian 1995). Thus T M B - 8 , which has been shown to inhibit IP3 mediated intracellular calcium release (Brummell and Machlachlan, 1989) may possess activity in this system but perhaps not be present in sufficient quantities to fully suppress calcium release from the vacuole. Use of higher 158 concentrations of T M B - 8 (in an. attempt to provide the drug in sufficient amounts) were lethal to the cell. 2+ What is the source of the Al-induced rise in Ca (cyt)? Results obtained using calcium modulators such as thapsigargin and T M B - 8 2+ provide interesting but inconclusive results as to the source of the rise in free C a (Cyt) upon treatment with A l . Heparin, a specific inhibitor of the IP3 pathway in plant cells is not able to cross membranes and requires loading by microinjection which was unsuccessful in this system. Treatment with verapamil, a potent inhibitor of calcium uptake in plant cells results in rapid cell death (i.e. lysis) within 30 seconds in 100% of vegetative filaments when used at the lowest concentrations recommended (Ehrhardt, personal communication). However, it is apparent from the data that the two regions of the 2+ cell, the A Z and Z V respond differently to [Ca ] ex- The fact that the Z V appears 2+ relatively insensitive to [Ca ] e x may be due to the cell's ability to access the large supply of calcium in the vacuole. Comparatively, the A Z may not possess large stores of intracellular calcium and may also rely on exogenous calcium as appears to be the case in other tip-growing systems (Picton and Steer, 1995). Thus, the degree of toxic effects of A l in this system may depend on a) the degree of vacuolation, b) the concentration of calcium present in the external environment and c) the age of the region of the cell which A l comes into contact with (i.e. young apical regions versus older vacuolated regions). F-actin The effects of A l on the F-actin array may be mimicked, to some extent, by calcium modulators. It is noted that these drugs produce more consistent patterns of F-actin disorganization than does Al-treatment alone. This pattern may be described as a change from an axially aligned, linear array consisting of long F-actin bundles to one consisting of 159 transversely arranged, thickened bundles of F-actin (Figure 60). The latter pattern is fairly consistent throughout the Z V and is similar in each cell observed (approximately 25 cells, n=3). Similar results have been reported for calcium-induced fragmentation in pollen tubes of Lilium longiflorum using the calcium ionophore Br-A23187-Br (Kohno and Shimmen, 1987) (Note: See caveat on use of Br-A23187 in Materials and Methods). In Al-treated cells, although the end result is always the disorganization and fragmentation of the F-actin array, the patterns observed through this process vary. Furthermore, regions where different patterns are observed are noted throughout the Z V , for instance, where F-actin disorganization appears more or less advanced. Two hypotheses are forwarded to explain the differences in the effects of A l and ionomycin. In the case of the ionophore, one speculates that, under similar experimental conditions, the major source of variation which exists is that which is inherent in the population of cells being observed. Conversely, treatment with A l is subject to a number of possible variables which may not always be controlled for. For example, the spatial distribution of A l may not be consistent throughout the media during each trial and throughout the course of the experiment. Therefore, depending of the extent of contact of A l with a portion of the cell at a given time, the observed toxicity effects of A l may vary. This is plausible given the large size of the vegetative filaments and could explain the spatially resolved regions of inhibition of organelle translocation (Chapter 3) as well as spatial variations in the disorganization of the F-actin array (Chapter 4). Ultimately, modulation of calcium, alone, does not entirely mimick the effects of A l on the microfilament array but does suggest that the former variable may play a central role in Al-induced changes in F-actin. Conclusions It has been demonstrated that modulation of calcium, using ionomycin, in an A l -free system wi l l result in the disorganization of the F-actin in a manner similar to but not exactly like those observed with Al-treatment alone. These data support the hypothesis that A l toxicity may be mediated, at least in part, by the perturbation of calcium homeostasis at 160 the level of the plasma membrane. Experiments with T M B - 8 and thapsigargin do not yield 2+ conclusive data on whether the source of the Al-induced rise in C a (Cyt) is internal or external. However, they do support the hypothesis that intracellular calcium stores may play a role the rapid rise in cytosolic calcium which occurs upon treatment with A l . 161 7.0. Conclusions The present studies have focused on documenting the toxic effects of Al in a tip-growing, model algal system. Overall, these data have demonstrated that the effects of Al occur rapidly following exposure (within seconds to minutes) suggesting that it acts at the level of the plasma membrane. Furthemore, Al affects processes and structures which are calcium sensitive, such as microfilament-dependent organelle translocation and the F-actin array. Consistent with these observations, Al causes a rise in cytosolic calcium within 2 minutes of exposure. Thus, this study provides evidence which supports the hypothesis that Al toxicity is initiated in the apoplasm, at least in part, via a perturbation of calcium homeostasis rather than by entry into the cytosol. Specific conclusions are as follows: I. Aluminum affects microfilament-dependent cytoplasmic streaming and cytoplasmic organization a) Aluminum inhibits cytoplasmic streaming of chloroplasts and mitochondria, within seconds, in a dose-dependent manner supporting the hypothesis that Al may initally act at the level of the plasma membrane. b) Exposure to Al causes the disorganization of the DIC-visible cytoplasmic strands as well as the ER, structures whose integrity are dependent on the F-actin cytoskeleton. This suggests that Al affects the organization of the F-actin array. II. Aluminum causes the fragmentation of the cortical F-actin array and the formation of stable F-actin aggregates a) In the cell tip, the diffuse F-actin array, noted in control cells, is replaced by bundled F-actin which extends to the apical plasma membrane. In the ZV, the cortical F-actin array undergoes fragmentation and is replaced by stable F-actin aggregates, which appear to self-assemble in vivo. 162 b) The response of F-actin to A l in this alga is similar to those of other plant cells in response to a diverse forms of stress. This suggests that the toxic effects of A l on F -actin, in this system, may be a conserved stress response. III. Treatment with A l causes a rise in cytosolic calcium. a) Aluminum results in a rise in calcium levels within 2 minutes of exposure. This occurs within a time-frame that is consistent with the slowing down and cessation of calcium-dependent organelle translocation. b) Calcium dynamics in the cell apex differ from those in the vacuolated portion. The cell tip is particularly sensitive to A l treatment and toxicity is exacerbated in low external calcium concentrations. c) Localized treatment with A l produces a relatively localized elevation in calcium. This suggests that the Al-induced rise in cytosolic calcium may not occur homogeneously throughout the whole cell. This is consistent with the observations of the heterogeneous effects of A l exposure on cytoplasmic streaming, cytoplasmic organization and the F-actin array. I V . The effects of A l exposure can be mimicked in an A l free system a) The use of the calcium ionophore, ionomycin, results changes in cytoplasmic streaming and the F-actin cytoskeleton which closely resemble those observed during treatment with A l alone. However, spatially and temporally, the pattern of fragmentation is more consistent throughout the cell than is observed with Al-treatment. This is consistent with the hypothesis that A l toxicity is mediated, at least in part, by a perturbation of calcium dynamics in this system. Furthermore, these data raise the possibility that Al-induced fragmentation of the F-actin array may vary according the spatial distribution of A l in the 163 media. It does not reject the hypothesis that unequal rates of entry of A l into the cytosol may also produce heterogeneous patterns of fragmentation. This latter hypothesis is not favoured since A l does not appear to be present in the cytosol within 30 minutes of exposure (Taylor, personal communication). b) Data obtained using the calcium modulators thapsigargin and T M B - 8 do not provide conclusive information regarding the identity of the intracellular source of calcium which is released during Al-treatment. These data do provide preliminary evidence, however, that intracellular stores may play a role in A l toxicity. Furthermore, their use further supports the sensitivity of the A Z and the relative insensitivity of the Z V to external calcium concentrations. 164 Literature Cited Alessa, L . and Oliveira, L . (1997a). Aluminum toxicity studies in Vaucheria longicaulis vai.macounii. I: Effects on Cytoplasmic Organization. J. Plant Res. In press. Alessa, L . and Oliveira, L . (1997b). 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