/i‘~- tun-t!“ -‘-- '~-.‘D“ I» In 5" Emit/1R3? Michigan State University l‘ _ u}? mews This is to certify that the thesis entitled IDENTIFICATION AND PARTIAL ? CHARACTERIZATION OF ACTIN FROM GLYCINE MAX AND TRIFOLIUM REPENS presented by Leslie John Szabo has been accepted towards fulfillment of the requirements for M.S. degree in Biochemistry W Major professor Date_91ép“|d 9‘9]; [m 0-7639 OVERDUE FINES: 25¢ per day per in- mm LIBRARY MATERIAL : Place in book return to ram charge from circulation room ‘ ‘ ~»:«.‘lll_ll ~ : w\ "I, ' ‘ . IDENTIFICATION AND PARTIAL CHARACTERIZATION OF ACTIN FROM GLYCINE MAX AND TRIFOLIUM REPEHS By Leslie John Szabo A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1980 ABSTRACT IDENTIFICATION AND PARTIAL CHARACTERIZATION OF ACTIN FROM GLYCINE MAX AND TRIFOLIUM REPENS By Leslie John Szabo The leguminous plants, Glycine max (soybean) and Trifolium repens (clover), were examined for the presence of actin. Fluorescence and electron microscopy were used to identify and determine the distribution of microfilaments in these Species. Most tissues of seedlings and plants of both species contained filaments which could be decorated with heavy meromyosin. The decoration by heavy meromyosin was reversed by ATP. Biochemical evidence for the presence of actin in soybeans include: the ability of myosin to co-precipitate nuosin-binding material from 125I-labeled extracts of soybean, stimulation of the Mg-dependent ATPase activity of “wosin by a fraction eluting fron a Sepharose column to which uwosin had been linked and the partial purification by chroma- tography on DEAE-cellulose of an actin-like protein that cross-reacts with rabbit anti-actin antibodies raised against calf thymus actin and has the same molecular weight as that of rabbit muscle actin. DEDICATION To um parents, and my wife, Cheryl, for their abiding love. ii ACKNOWLEDGEMENTS I wish to thank Dr. K.R. Schubert for his guidance, criticism, and encouragement. Sincere appreciation goes to Dr. J.L. Wang for all that he has done. I would also like to thank Drs. P. Carlson and P. Filner for serving on my guidance committee. This work was done in collaboration with Tom N. Metcalf, whom I thank for sharing with me the successes and failures. Finally, I would like to thank Mary Tierney for her friendship and willingness to help. I acknowledge the financial support of the American Cancer Society (BC-277), the National Science Foundation (PCM-77-24683), the Department of Biochemistry at Michigan State University, the Michigan Agriculture Experiment Station, and the Biomedical Research Support Grants from Michigan State University, College of Osteopathic Medicine and College of Agriculture and Natural Resources. iii TABLE OF CONTENTS INTRODUCTION. 0 O O O O O O O O O O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . Properties of Monomeric Actin. . . . . . . . Polymerization of Actin. . . . . . . . . . Properties of Filamentous Actin. . . Effects of Cytochalasin B and Phalloidin . Location and Function of Microfilaments in Actin hmuunology . . . . . . . . . . . . . l nts. emu... 0‘00... P1ATERIALS Ai‘D METHODS O O O O O O O O O O O O O O O O O O Soybean Cell Culture . . . . . . . . . . . . . . . Growth Conditions for Soybean and Clover . . . . . Purification of Muscle Proteins. . . . . . . . . . . . Staining of Soybean Protoplasts with Fluorescent-DNase Myosin-Binding Assay . . . . . . . . . . . . . . . . . ChrdnatOgraphy on DNase I-Sepharose. . . . . . . . . . ChromatOgraphy on DEAE-Cellullose. . . . . . . . . hnnunochemistry. . . . . . . . . . . . . . . . . . Myosin and Heavy Meromyosin Affinity Chromatography. DNase I Inhibition Assay for Monomeric Actin . . . Measurement of ATPase Activity of Myosin . . . . . Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Electron Microscopic Examination of Microfilaments prOtein Deterlni nation. I O O O O O O O O O O O O 0 Material 5. O I O O O O O O O O O O 0 O O O O 0 O 0 RESULTS 0 O O O O O O O O O O O O O O O I O O O I O O O 0 Identification and Localization of Microfilaments by Electron Microscopy . . . . . . . . . . . . . . Myosin-Binding Protein in Soybeans . . . . . . . . Myosin-Binding Activity. . . . . . . . . . . . . Affinity Chromatography on Myosin-Sepharose and Heavy Meromyosin-Sepharose. . . . . . . . . . DNase 1 Binding Protein in Soybeans. . . . . . . . Staining of Protoplasts with fl-DNase I. . . Affinity Chromatography on DNase I-Sepharose Partial Purification and hnnunochemical Identification of Soybean Actin . . . . . DISCUSSIO'E. O O O O O O O O O O O O O O O O O 0 O O O 0 O REFEREI‘CESO O O O O O O O O O O O O O O O O O O O O O O 0 iv 0 o 0 o 0 o H. Page 00 0030101an 12 12 12 13 15 17 18 19 19 20 21 22 22 23 23 24 27 27 35 35 35 44 44 57 65 71 Figure 10 11 12 13 14 15 16 LIST OF FIGURES Page Identification of microfilaments by electron microsc0py. . 26 Schematic drawing of a seedling. . . . . . . . . . . . . . 29 Schematic drawing of a soybean plant . . . . . . . . . . . 31 Schematic drawing of a clover plant. . . . . . . . . . . . 33 Affinity ChromatOgraphy of soybean root extract on “LYOST n-Sepharose CO] [“1"]. I O O O O O O O O I I I O O O O O 38 SDS polyacrylamide gel electrOphoresis of material eluted with ATP from the myosin-Sepharose column. . . . . . . . . 4O Staining of soybean protoplasts with fl-DNase I. . . . . . 43 Affinity chromatography of calf thymus extract on DNase I-SEPharose C0] “Ill" 0 O O O O O O O O O O O O O O O O O 0 O 46 SDS polyacrylamide gel electrophoresis of calf thymus eluted from DNase I-Sepharose column . . . . . . . . . . . 48 Affinity chronatography of soybean cell extract on DNase I-Sepharose column . . . . . . . . . . . . . . . . . . . . 50 SDS polyacrylamide gel electrophoresis of the material eluted with 3 M guanidine°HCl from a DNase I—Sepharose column chronatographed with soybean cell extract . . . . . 52 Chromatography of soybean cell extract on Sepharose 48 CO] UNI" I O O O O O O O O O l O O O O O O O O O I O O 0 O O 54 Chromatography of soybean cell extract on BSA-Sepharose CO] “In" 0 O O O O O O O O O O O O O O O O O O O O I O O O O 56 Chromatography of soybean seedling extract on UEAE-cellulose C0] ”Inn 0 O O O O O O I O O O O O O O O O O O O O O I O O O 59 SDS polyacrylamide gel electrOphoresis of protein components obtained fron soybean cell extracts after fractionation by DEAE-cellulose chromatography and by affinity chromatography using rabbit anti-actin antibodies . . . . . . . . . . . . 6l SDS polyacrylamide gel electrophoresis of [IZSIJ-labeled proteins after inmunoprecipitation . . . . . . . . . . . . 63 LIST OF TABLES Table Page I Occurence of Microfilaments in Higher Plants. . . . . . . 8 2 Actin Purification. . . . . . . . . . . . . . . . . . . . 10 3 Distribution of Microfilaments. . . . . . . . . . . . . . 34 4 The Binding of Myosin to Proteins from Calf Thymus and Soybean Cell Extracts . . . . . . . . . . . . . . . . . . 36 vi ADP ATP ATPase BSA C DEAE DNA DNase I DTT EDTA EGTA F-actin .9 G-actin M9 SDS Tris LIST OF ABBREVIATIONS Adenosine 5'-diphosphate Adenosine 5'-triph05phate Adenosine 5'-triph05phatase Bovine serum albumin Temperature in degree Celsius Diethylaminoethyl Deoxyribonucleic acid Deoxyribonuclease I Dithiothreitol Ethylenediamine tetraacetic acid Ethyleneglycol-bis-(amino ethyl ether) tetraacetic acid Filamentous form of actin Force of activity Monomeric form of actin Magnesiwn (divalent form) Sodium dodecyl sulfate Tris (hydroxymethyl) aminomethane INTRODUCTION In the past few years, the muscle protein, actin, has been detected in a variety of non-muscle cells and it is now widely believed that actin is ubiquitous in all eukaryotic cells (47). Actin has been implicated in many diverse cellular activities such as nuclear migration, phagocytosis, cytoplasmic streaming, change in cell shape, and regulation of topographical distribution of membrane proteins (14,47). Most of these studies have been done exclusively in animal systems, but many of the same processes also occur in plants. Microfilaments (F-actin) have been detected in a number of plant species and a variety of cell types, including vascular bundles, root hairs, and parenchymacells (74). To date, most of the investigations concerning the possible function of actin in plants have dealt with studies of cytoplasmic streaming. This is only one of many processes in which plant actin may be involved. Another particularly intriguing system, and the genesis of this study, is the early infection process of the legume-Rhizobium symbiosis. The first step of the infection process is the recognition and bind- ing of the soil bacterium Rhizobium to the proximal end of the root hair of its leguminous host (for general review see reference 18). Once bind- ing has occurred, the root hair will curl to form a "shepherd's crook.“ The first sign of infection is the swelling of the root hair wall and an increase in cytoplasmic streaming. The nucleus enlarges, migrates to the site of infection and preceeds the infection thread as it elongates, apparently directing the growth of the thread. The infection thread is formed by an invagination of the plant membrane into the root hair cytoplasm, which usually grows towards the center of the root. The thin tube formed by this process contains Rhizobium in a mucopolysaccharide matrix. In the cortex tissue of the root, the site of nodule develOpment, the infection thread membrane fuses with the membrane of the cortex cell. The bacteria are encapsulated in a peribacteroid envelOpe by an endocytotic process. Many of the steps involved in the infection process are anal0gous to actin uediated processes in non-plant cells, such as nuclear migration, phagocytosis (encapsulation of bacteria) and alteration of cell shape (root hair curling). The first phase of the investigation of actin and its possible role in the infection process of legumes, has been the identification, localization and characterization of plant actin. In this study, two leguminous plants, Glycine max (soybean) and Trifolium repens (white clover), were examined for the presence of actin. LITERATURE REVIEW Actin has been isolated and characterized from a variety of non-- muscle animal cells (13,28,32,47,53,73,95) as well as other organisms of diverse evolutionary origin such as Mycoplasma (65,68), Acanthamoeba (30,75,96), Dictyostelium (39,91,102) and Saccharomyces cerevisiae (45,48). Actin can account for as much as 20-30% of the total cellular protein in Acanthamoeba (47,77,98) or as little as 1% in blood platelets (44,47). Actin from all sources examined are similar in composition and structure. Properties of Monomeric Actin The actin monomer (G-actin) is a single polypeptide chain with an approximate molecular weight of 42,000. The amino acid sequence is highly conserved (98). Only 6% of the amino acid residues Acanthamoeba in actin differ from those of rabbit muscle actin (44,47). All actins examined contain the unusual amino acid, N-3-methylhistidine (29,47) and bind 1 mole of ATP per mole of protein. There are significant chemical differences, however, in the proper- ties of actin isolated from different sources. Only actin from Acantha- moeba castellani, for example, contains the amino acid N-methyllysine, and all of the actins for which amino acid sequence data are available are different (47). At present, actin can be divided into three groups based upon differences in their respective isoelectric points. Alpha (a) actin which is obtained from muscle tissue is the most acidic form. Beta (8) and gamma ( ) are from non-muscle sources with gamma being the most basic. All non-muscle cells from vertebrates examined contain a mixture of both 8 and 1'actins (79,98). These isoforms of actin (8 and Y) are not the result of post-translational modification, but are coded for by different genes (25). Polymerization of Actin Under the appropriate ionic conditions purified actin polymerizes to form filamentous actin (F-actin). During polymerization, the ATP bound to actin is hydrolyzed to form free inorganic phosphate and bound ADP. In the process of depolymerization, the ADP is replaced with ATP. Although the specific role of ATP is unclear, ATP presumably serves as an energy source for conformational changes which occur during polymeriza- tion (87). The actual process of polymerization involves two steps, nucleation and elongation. Nucleation, the formation of a short actin polymer 6-8 units long, is the rate limiting step and depends on the concentration of free monomeric actin present in solution. When the concentration exceeds a critical concentration, polymerization of G-actin to F-actin occurs, establishing an equilibrium between the two forms. High concentrations of G-actin do occur in the cytoplasm of Phasarum (37), in bovine spleen cells and in the periacrosomal vesicle region of sperm (88,89). The presence of G-actin well above its critical concen- tration implies the presence of cellular control of polymerization. A 16,000 dalton protein (profilin) has been implicated as a regulatory molecular which controls polymerization of actin in spleen cells (10). Profilin forms a 1:1 crystalline complex with G-actin and inhibits poly- merization. Another protein known to interact with G-actin is DNase 1 (48,64) which also forms a 1:1 crystalline complex with actin. Whether this interaction is a regulatory mechanism for the control of actin polymerization, a regulator of DNase I enzymatic activity, or has no biological function, is unknown. PrOperties of Filamentous Actin The polymeric form of actin (F-actin) is comprised of a right handed, double stranded helix with a half pitch of 35 nm. The filaments upon electron microscopic examination exhibit a bead-like structure with a diameter of 5-7 an and form an arrowhead pattern Upon treatment with heavy meromyosin (19,33,36). Filament structure and decoration by heavy meromyosin are used as criteria for the identification of actin filaments. Heavy meromysin, the globular portion of the myosin molecule containing ATPase activity, is formed by tryptic digestion of myosin. Perphosphate or ATP inhibit or reverse the binding of heavy meromysin or myosin to F-actin. Moreover, F-actin activates the Mg-dependent ATPase activity of nwosin, which has been widely used as a quantitative assay for actin. Effects of Cytochalasin B and Phalloidin Two families of drugs appear to modulate the polymerization state of actin. The first is the cytochalasins which disrupt the actin filament structure; although their precise mode of action is still disputed. The second group includes the cyclic peptide, phalloidin, from the mushroom Amanita, which has been shown to enhance polymerization of actin. Cytochalasin B alters many processes in animal cells, including inhi- bition of phagocytosis (1), disruption of cell movement and severe alter- ations in cell shape (58). Cytochalasin B has been shown to inhibit the polymerization purified rabbit muscle actin, in vitro (54,59,63,84,85). Cytochalasin B also inhibits transmembrane glucose transport (3,52) although cytochalasin 0 does not (3). Furthermore, cytochalasin D is more potent in altering cell shape of cultured fibroblasts than the B analog (3). Cytochalasin B inhibits many plant processes which are believed to depend on microfilaments. These include cytoplasmic streaming (8,9,12,101), nuclear migration (32), and light-induced chloroplast movement in Mougeotia (94). In plants such as gga_mays and Avena sativa (9), treatment with cytochalasin 8 results in the cessation of cytoplasmic streaming and the aggregation of microfilaments into large bundles. In some cases, microfilaments can only be observed after the addition of cytochalasin B (9,99). Hepler gt_al, (32) suggested that in plants cytochalasins may affect actin structure by disruption of the membrane anchorage site for microfilaments. Phalloidin has been shown to cause an increase in the amount of actin microfilaments in liver cells (78). In jn_yitgg studies with rabbit muscle actin, phalloidin was shown to bind stoichiometrically, to enhance the rate of actin polymerization, and to stabilize the F-actin structure (17,52,60). The affect of phalloidin on plant cells has not been investigated. Location and Function of Microfilaments in Plants The occurrence of microfilaments in plant cells is now well docu- mented. The most intensive work has been done with the characean algal cells, Nitella and Chara. These cells along with Physarum have been used as models to study cytoplasmic streaming. Palevitz demonstrated that Nitella contained actin filaments (72,73) and these were often organized into large bundles. The polarity of the actin filaments within the bundles observed in these studies was parallel (44) and the orientation with reSpect to direction of movement was similar to that of nmscle fibers. In addition, organelles such as chlorOplasts are apparently attached to these microfilament bundles (44). These filaments are located in the stationary ectOplasm of the cell along the boundahy of the streaming endoplasmic layer (40). This boundary is thought to be the site of generation of motive force for cytoplasmic streaming (41). The actomyosin complex is believed to be involved in the generation of this force (42,82,100). Considerable cytological information is available about the distribu- tion and localization of microfilaments in higher plants (see reference 74 for review). Parthasarathy gt a1. (74) observed that filament bundles are most often found in elongating or elongated nucleated root cells of vascular or cambium tissue and rarely seen in isodiameteric cells. How- ever, Vahey gt a1. (92) reported that actin is present in the parenchyma cells of tomato fruit and O'Brien 32.21.159) observed microfilaments in the parenchyma cells of Avena sativa. Microfilaments are often orien- tated parallel to the long axis of the cell and are found either in the outer edge (69) or center of the cyt0plasm (74). In addition Forer and Jackson (21) observed microfilaments in the mitotic spindles of Haemanthus katherinae. Organelles such as endoplasmic reticulum, plas- tids, vesicles, chlorOplasts, and mitochondria are often found closely associated with these fibers (74). Actin filaments have been characteristically identified in higher plants by reversible binding of heavy meromyosin. The decoration is done either in droplets of cytoplasm (6,15) or in sections of tissue perfused Table 1 Occurrence of Microfilaments in Higher Plants* Source Reference Amaryllis belladonna Condeelis (1974) Cuscuta §p_ Bennett and Brown (1980) Gossypium hirsutum Bennett and Brown (1980) Haemanthus katherinae Forer and Jackson (1975) Hibiscus esculentus Bennett and Brown (1980) Impatiens sultanii Bennett and Brown (1980) Lemna minor Bennett and Brown (1980) Liriodendron tulipfera Bennett and Brown (1980) Mimosa sp Arraes-Hermans gt 31. (1976) Phaseolus aureus Bennett and Brown (1980) Phaseolus vulgaris Jackson and Doyle (1972) Pinus taeda Bennett and Brown (1980) Pueraria lobata Bennett and Brown (1980) Raphanus sativus Bennett and Brown (1980) Setraeasea purpurca Bennett and Brown (1980) my Ilker __t__a__l_. (1974) Xylosma congestum Ilker _t._l. (1974) ZEQ.M§X§ Bennett and Brown (1980) *Filaments were identified by electron microscopic examination of samples treated with heavy meromyosin. with heavy meromyosin in glycerol (35). A list of higher plants in which actin filaments have been identified by heavy meromyosin decoration is presented in Table I. In addition, Bennett and Brown (6) examined repre- sentative species fron each taxonomic class of the plant kingdon and found microfilaments were present in all but five. Although actin has been shown to be a cmnponent in numerous higher plant Species, very few biochemical studies have been conducted (Table II). The first biochemical report of an actomyosin-like complex was from vascular bundles of Nicotiana and Cucurbita (104). This material exhi- bited an ATP-dependent reduction in viscosity and ATPase activity.“ Little progress has been made in obtaining purified actin from higher plants although partial purification of actin from Phaselous vulgaris (38), Lyc0pesican esculentum (92), and Triticum aestivum (34) has been reported. Actin from higher plants still needs to be purified and characterized biochemically in terms of the kinetics of polymerization and the stimulation of Mg-dependent ATPase of myosin as well as amino acid composition and sequence. This would be an important initial step in understanding the role of actin in plants. Very little is known about the function of actin in plants, but progress is being made in studies of cytoplasmic streaming. There are many plant processes that involve movement, such as cytokinesis, opening of stomata, thigmotrophism, root hair curling and migration of chloroplasts and nuclei. With a clearer understanding of the biochemical prOperties of plant actin, the mechanism and regulation of these processes may be elucidated. 10 Table 2 Purification and Characterization of Actin from Plants (Ilker gt l., 1979) actomyosin ppt, gel filtration Source Procedure Characteristics Cucurbita and Nicotiana Enrichment by actomyosin ppt ATPase (Yen gt 91., 1965) Nitella Enrichment by polymerization dec. mf (Palevitz, 1976) Phaseclus vulgaris Enrichment by gel filtration, dec. mf (Jackson gt al., 1976) polymerization MW Lycopesicon esculentum Partial purification - ion dec. mf (Vahey gt al., 1978) exchange chromatography, MW polymerization Triticum aestivum Partial purification - MW ab Abbreviations used: Ppt ATPase dec. mf MW ab Precipitation Stimulation of nwosin ATPase activity Microfilaments decorated with heavy meromyosin Cross reacts with anti-actin antibodies Same molecular weight as actin from other sources 11 Actin Immunology Anti—actin antibodies are difficult to obtain, because actin is a ubiquitous protein and its structure is highly conserved. For these reasons a number of hmnunization methods are used. These include the injection of F-actin treated with glutaraldehyde (66), or SDS denatured actin (51). In addition, the amount of antigen used and the interval between injections vary widely (34,51). The properties of antisera produced also vary. Not all anti-actin antibodies fonn antigen-antibody complexes which produce precepetin lines in Ouchterlony double-diffusion tests. However, the presence of anti-actin antibodies is clearly demonstrable by double-antibody co-precipitation tests and radiohanunoassay (66). Moreover, not all antibodies react with all actins. Antiserum raised against chicken embryo brain actin cross-reacts with actins from bovine cardiac muscle and brain, rabbit skeletal muscle, and chicken embryo brain. 0n the other hand cardiac actin antiserum bound cardiac and skeletal muscle actin but did not bind actin (66) fron brain tissue of either source. MATERIALS AND METHODS Soybean Cell Culture SB-l cell line of soybean (Glycine max) cells was kindly provided by Dr. 0.L. Gamborg (Prairie Regional Laboratory, Saskatoon, Saskatchewan, Canada). Cultures were grown in 125 ml Erlenmeyer flasks containing 40 ml of solution at 25 to 30°C on a gyratory shaker. Liquid cultures were subdivided (25) every 3 to 4 days by transferring 10 ml of culture to 30 ml of fresh 1-B5 medium (26). Cultures of 58-1 cell lines were also maintained as callus which was grown on agar plates containing 0.5% (w/v) Bacto agar in l-BS medium. Callus cultures were transferred to new agar plates every month, sealed with parafilm and stored at room temperature in the dark. When necessary new liquid cultures were started by placing callus into flasks containing 20 mls of l-BS medium and incubated on a gyratory shaker. Initially spent medium was removed and replaced with fresh medium every week until a uniform cell su5pension was formed, at which time the culture was subdivided by mixing 20 ml of suspension with 20 ml of fresh medium until the culture was growing rapidly. Growth Conditions for Soybean and Clover Soybean (Glycine max (L.) Merr. var. Amsoy 71) and white clover (Trifolium repens L. var. Ladino) seeds were surface sterilized by immersing seeds first in 75% (v/v) ethanol and then in an acidified solu- tion of mecuric chloride (2 g HgClz and 5 ml concentrated HCl per liter) for 45 seconds each. The seeds were washed 8 to 10 times by soak- ing in sterile water for 5 minutes. Soybean seedlings were germinated in sterilized trays which were lined with paper towels moistened with water 12 13 and covered with aluminun foil. Soybean plants used in this study were grown in Perlite under greenhouse conditions. Clover seeds were ger- minated on 0.5% (w/v) water agar plates and seedlings were transferred and grown on Fahraeus slides (20), to which KNO3 was added to the Fahraeus medium to a final concentration of 2 mM. Purification of Muscle Proteins Acetone powder was prepared from rabbit back muscle by grinding 100 gm of tissue in a commercial meat grinder and washed with 10 mM sodium EDTA, 0.4% sodium bicarbonate, pH 7.0 for 15 minutes and rinsed with distilled water (5). The solution was decanted and the muscle tissue was added to 1 liter of acetone at -10°C. After stirring for 30 minutes, the suspension was filtered through a Buchner funnel and reextracted twice for 10 minutes with 500 ml of cold acetone. The residue was dried in a vacuum jar overnight under aspiration, ground and stored with dessicant at 4°C. Actin was isolated from 10 g of acetone powder by extraction with 200 mls of extraction buffer (2 mM Tris, 0.2 mM ATP, 0.5 mM s-mercapto- ethanol, 0.2 mM CaClz, pH 8.0) for exactly 10 minutes at 0°C (85). The suspension was filtered through 8 layers of cheesecloth and 1 layer of Miracloth and the filtrate was clarified by centrifugation at 10,000 xg for 1 hour. Actin was polymerized in the pooled supernatant fluids by adding 2 M KCl and 1 M MgClz to a final concentration of 50 mM and 2 mM, respectively, and was incubated for 2 hours. The concentration of KCl was adjusted to 0.8 M by adding solid KCl and the solution was gently stirred for 1.5 hours to dissociate actin from actin-binding proteins. The polymerized actin was pelleted by centrifugation at 80,000 xg for 3 14 hours. The pellet was resuspended in 30 mls of extraction buffer and dialyzed against 500 mls of the same buffer for 2 days with 2 changes of the buffer. After dialysis, the solution was centrifuged for 1.5 hours at 80,000 xg to remove any insoluble material and the supernatant fluid containing monomeric actin was stored at 4°C. To prevent bacterial growth, sodiwn azide was added to a final concentration of 0.05%. Myosin was isolated from fresh rabbit back muscle (45,89). The tissue was ground in a meat grinder and myosin was extracted with 3 volumes of buffer A (0.5 M KCl, 0.1 M KZHPO4, 1 mM DTT, 15 mM sodium EDTA, pH 6.8). After 20 minutes, the suspension was centrifuged for 30 minutes at 13,200 xg, and the supernatant fluid was filtered thrOUgh glass wool. Myosin was made insoluble and collected by dilution of the filtrate with 10 volumes of water, incubated for 30-45 minutes and centrifuged at 13,200 xg_for 15 minutes. The pellet containing myosin was resuspended in 100 mls of buffer B (0.5 M KCl, 50 mM KZHP04, 1 mM DTT, pH 6.8), and gently stirred for 20 minutes. Insoluble material was removed by centrifugation at 20,200 xg for 30 minutes. Actin was removed by precipitation of actomyosin by dilution of the supernatant fluid with 80 ml of water, incubation for 20 minutes and centrifugation for 90 minutes at 55,000 xg. The clear liquid was decanted, diluted with 7 volumes of water, incubated for 30 minutes, and centrifuged for 15 minutes at 13,200 xg to collect the “wosin. The process was repeated two more times as described above. The final myosin pellet was resuspended in 100 mls of buffer B and stored at 4°C. Sodium azide was added to a final concentration of 0.05% to prevent bacterial contamination. 15 Heavy meromyosin was prepared by tryptic digestion of nwosin (103). Myosin (4 mg/ml) was digested with 0.01 volume of TPCK-Trypsin (0.15 mg/ml in 1 mM HCl) for exactly 7.5 minutes at 25°C with constant stirring. The reaction was stopped by adding 0.1 volume of soybean trypsin inhibitor (1 mg/ml). Undigested myosin and light neromysin were precipitated by dialysis overnight of the digestion solution against 800 mls of 20 mM imidazole buffer, pH 6.6, with 2 Changes of buffer. The insoluble material was removed by centringation at 80,000 xg_for 1.5 hours. The supernatant fluid was fractionated by precipitation of heavy meromyosin with the addition of solid ammonium sulfate to a final concentration of 2.4 M and centrifugation at 25,000 xg for 30 minutes (72). The pellet was resuSpended in 10 mls of 5 nil Tris 0.5 nM EGTA, 1 mM DTT, pH 8.0, and dialyzed against 400 mls of the same buffer for 2 days with 3 changes of buffer. The final dialyzed solution was diluted with 1 volume of glycerol and stored at -20°C. All steps were carried out at 4°C unless noted otherwise. Staining of Soybean ProtOplasts with Fluorescent-DNase I Protoplasts were isolated by a modified procedure of Constabel (16). Actively growing SB-I cells (24-48 hours after transfer) were digested with an equal volume of enzyme solution containing 400 mg cellulysin, 200 mg pectinase and 2 g of D-sorbitol, pH 5.5 in 20 mls. After 2 hours, the protOplast suspension was filtered through a 48 DM nylon filter and pelleted by centrifugation in a clinical centrifuge for 2 minutes. The pelleted protoplasts were gently resuspended in 10 mls of protoplast medium (16) which was modified by substituting 20 g of D-sorbitol for sucrose. After 3 washes, protoplasts were resuspended in 1 ml of 16 protoplast medium, counted in a Thomas C-10 counting chamber and diluted to 5 x 105 cells per milliliter. The effects of the drugs cytochalasin B and colcochine on protoplasts were examined. To 5 mls of protoplast suspension, 50 pg of cytochalasin B (1 mg/ml), 20 pg of cholchicine (0.5 mg/ml) or 50 pl of modified proto- plast medium were added and incubated at room temperature. After 1 hour incubation, the protoplasts were pelleted and resuspended in formaldehyde and fixed. Soybean protoplasts were fixed by a modified procedure of Fowke (23). Protoplasts were pelleted and resuspended in modified protoplast medium containing 1% formaldehyde for 1 hour followed by 3% formaldehyde solu- tion for 2 hours at room temperature and washed 3 times with protoplast medium. The fixed protoplasts were incubated in 0.1% BSA solution in PBS (16 g NaCl, 0.4 9 KCl, 2.3 g NazHP04, 0.4 g KH2P04 per liter) to remove any remaining aldehyde groups and spread on microscope slides to air dry. Protoplasts bound to glass slides were dehydrated by incubation for 15 minutes each in an ethanol series of 10% to absolute ethanol, in increments of 10%. All solutions were at 0-4°C. After 2 treatments of absolute ethanol, the slides were air dried. Fluorescein labeled DNase I (fl-DNase) was used as a stain (11). To a DNase I solution (20 mg in 2 mls of 0.1 M NaC03, 0.1 mM CaClz, pH 8.2) 0.4 mg of fluorescein isothiocyanate was added, and stirred over~ night at 4‘C in the dark. The solution was dialyzed against 250 mls of PBS for 48 hours with 5 changes of buffer. The absorbance ratio (500 nm to 280 nm) of the final dialyzed solution was 1.5. The dehydrated protoplasts were stained by covering the slides with fl-DNase (diluted 1:10 with PBS) for 1 hour at room temperature in the 17 dark and excess stain was removed by washing the slides 3 times for 5 minutes with PBS. The slides were wet mounted in 50% glycerol and examined with a Leitz fluorescence nncrosc0pe. Micrographs were taken with Kodak Pan X-lOO film. Myosin-BindingyAssay Calf thymus extract was prepared by homogenizing 50 g of tissue with 150 mls of 10 11M Tris, 10 11M EDTA, 10 11M NaCl, 0.5% triton X-100, pH 7.5, in a Waring blender for 2 minutes (56). The homogenate was centrifuged for 10 minutes at 10,000 xg and the supernatant fluid recentrifuged for 2 hours at 100,000 xg. Extract of soybean cultured cells was prepared in a similar fashion except 200 g (fresh weight) of cells were ground with 200 mls buffer, 15 g of polyvinylpyrrolidone, and alumina. Bovine serum albumin (3 mg/ml) and extracts of calf thymus (13 mg/ml) 125 and soybean (2 mg/ml) were iodinated (24) with I and dialyzed against 1 liter 10 mM Tris, 5 mM CaClg, pH 7.5. Chicken muscle “wosin which was a generous gift from Dr. C. Suelter and Ms. D. Thonpson was 131 radiolabeled with I and dialyzed against 1 liter of precipitation buffer containing 10 M Tris, 1 mM sodium EDTA, 0.1 [M DTT, 0.1 M KCl, pH 7.5 (13). All samples were dialyzed for 2 days with 4 changes of buffer. Extracts of [IZSIJ-labeled calf thymus or soybeans (0.4 mls) were incubated with myosin (0.1 ml) for 30 minutes at roan temperature, and centrifuged for 4 minutes in an Eppendorf microfuge. Pellets were resuspended in 0.5 ml of precipitation buffer and transferred to fresh [125 tubes. Control experiments were performed with I]-labeled bovine serum albumin. Radioactivity was determined by ganmm counting 18 125 (Beckman Biogamma), with channel settings of 0-50 for I and 50-1000 for 131 I. Radioactivity was converted to micrograms of precipitated protein by the specific activity of each solution and was reported as such. Chromatography on DNase I-Sepharose Affinity columns were prepared by covalently coupling BSA or DNase I to Sepharose 4B (43,56). Cyanogen bromide (20 mls of 30 mg/ml solution) was added to Sepharose 4B (20 mls) and stirred for 6 minutes at room temperature while the pH was maintained between 11.0-11.3 by the addition of 2 N NaOH (4,77). The reaction was stopped by immediately washing the packing in 500 mls of ice cold water and 0.1 M NaHC03 and resuspended in 25 mls of 0.1 N NaHC03. A solution of DNase I or BSA (20 mg in 2 mls of 0.1 N NaHC03) was added to the activated Sepharose 4B, gently mixed overnight, and washed with a liter of water and 0.1 N NaHCO3 each. Columns (0.75 cm x 6.5 cm) were packed with BSA or DNase I-Sepharose and washed with 50 mls of 4 M guanidine HCl, 0.5 M sodiwn acetate, 30% glycerol followed by 500 mls of DNase column buffer (10 "M Tris, 5 mM CaClz, pH 7.5). Calf thymus extract was prepared by homogenizing 39 g of tissue with 100 mls of 10 mM Tris, 10 mM EDTA, 10 mM NaCl, 0.5% triton X-100, pH 7.5, in a Waring blender for 2 minutes (56). The homogenate was centrifuged at 12,000 xg_for 10 minutes to remove cell debris and was further clarified by centrifugation at 100,000 xg for 2 hours. An extract of soybean cells was prepared in a similar fashion except 200 g (fresh weight) of cells were ground with alumina, 15 g of polyvinylpyrrolidone and 200 mls of buffer. After centrifugation, the supernatant fluids were subjected to affinity chromatography. 19 Extracts of calf thymus (50 mls) or soybean (200 mls) were applied to 15 ml columns, washed with DNase column buffer and 0.75 M guanidine-HCl, 0.5 M sodium acetate, 30% glycerol, pH 6.5. Actin was eluted by washing the column with 3.0 M guanidine-HCl, 1.0 M sodium acetate, 30% glycerol, pH 6.5. Fractions from the column were pooled, dialyzed, lyophilized and analyzed by SDS gel electrophoresis. Chromatography on DEAE-Cellulose Ten-day-old soybean seedlings (350 g fresh weight) were homogenized for 2 minutes in a Waring blender in 700 mls of 3 mM imizadole buffer (3 mM imidazole, 0.5 mM ATP, 0.1 mM CaClz, 0.75 mM e-mercaptoethanol, pH 7.5 with 70 g of polyvinylpyrrolidone and 0.02 units'ml'l of the protease inhibitor, Trasylol. The homogenate was filtered through 8 layers of cheesecloth, 1 layer of Miracloth, centrifuged for 20 minutes at 16,300 x9, and further clarified by centrifugation at 100,000 xg for 90 minutes. The resulting supernatant fluid was chromatographed on a DEAE-cellulose colunn (4 cm x 25 cm) and eluted with a linear gradient from 0 to 0.25 M KCl, according to the procedure of Gordon 25.91: (30). Immunochemistry Rabbit anti-actin antibodies raised against calf thymus actin were a generous gift from Mr. T.N. Metcalf. The actin used as an antigen was purified according to the procedure of Gordon gt El: (30), subjected to SDS slab gel electrophoresis and extracted from mascerated gel sections. Rabbits were given primary injections of 0.8 mg denatured actin in complete Freund's adjuvant, followed by booster injections of 0.4 mg (in complete Freund's adjuvant) at 6 week intervals. Antisera was 2O collected bi-weekly. Ouchterlony immunodiffusion tests were performed as described by Munoz (67) and precipitin lines were usually observed after 1-2 weeks. Soybean material in fraction C and calf thymus extract were 125 labeled with I (31) and dialyzed against 3 mN imidazole buffer (see Chromatography on DEAE-cellulose section). Imnunoprecipitation was performed by mixing [IZSIJ-labeled calf thymus extract, calf thymus actin or fraction C with antisera and incubated for 30 minutes at 37°C, followed by overnight at 4°C. Goat antibodies directed against rabbit hmnunoglobulin were added, incubated for 30 minutes at 37°C and overnight at 4°C, and centrifuged for 5 minutes at 1,000 xg. The pellets were washed in 3 mM imidazole buffer, and analyzed by SDS gel electrophoresis. Myosin and Heavy Meromysin Affinity ChromatOgraphy Affinity columns were prepared by covalently coupling myosin and heavy meromysin to Sepharose 4B (7). Activation of Sepharose 4B was performed as previously described (see Chromatography on DNase I-Sepha- rose section). Activated packing (15 g) was washed and suspended in a total volume of 40 ml of coupling buffer (0.5 M KCl, 0.1 M KZHP04, pH 8.0). Solutions of myosin (40 mg) or heavy meromyosin (36 mg) in coupling buffer were added to the packing and gently mixed on a shaker overnight. The packing was washed in 500 mls of coupling buffer, 100 mls of 1 M Tris, pH 8.0, and incubated for 2 hours in 100 mls of 1 M Tris, pH 8.0, to remove any remaining active sites. Heavy meromyosin and myosin- Sepharose were equilibrated in phosphate buffer (20 mM KZHP04, 50 mM KCl, 5 mM MgClz, 5 mM EGTA, 2 mM DTT, pH 7.0) and triethanolamine buffer (10 mM triethanolamine, 50 mM KCl, 2.5 mM MgCl2, pH 7.5), respectively. 21 Extracts of soybean roots were prepared by homogenizing roots (132 g fresh weight) with 130 mls of equilibration buffer in a Waring blender for 2 minutes, and the homogenate was filtered thrOUgh 8 layers of cheesecloth and 1 layer of Miracloth. The filtrate was incubated for 1 hour at room temperature and centrifuged for 30 minutes at 22,000 xg. Extracts of rabbit muscle were prepared by stirring 2 g of acetone powder with 40 mls of extraction buffer (see section on Purification of Muscle Proteins) for exactly 10 minutes at 0°C. The tissue suspension was filtered through 8 layers of cheesecloth and 1 layer of Miracloth and, the filtrate was clarified by centrifugation at 10,000 xg_for 1 hour. The supernatant fluid was dialyzed against 250 ml of equilibration buffer overnight with 2 changes of buffer. Extracts of soybean or rabbit muscle were mixed with 15 mls of column packing, gently agitated for 1 hour at roon temperature, and poured into a 20 ml column (1.8 cm x 8.0 cm). The column was washed with equilibra- tion buffer, followed by equilibration buffer containing Zidn ATP. The elution of proteins was monitored by absorbance at 280 nm, and by stimu- lation of the Mg-dependent ATPase activity of myosin. Fractions were dialyzed against water, ly0philized, and analyzed by SDS gel electro- phoresis. DNase I Inhibition Assay for Monomeric Actin Monomeric actin was measured by inhibition of DNase I activity as described by Lindberg (55). Various amounts of inhibitor (0-100 pl) were added to 20 pl of DNase I solution (0.05 mg DNase I per ml of SOINH Tris, pH 7.5), and incubated for 30 seconds. Three milliliters of DNA solution (24 mg of double-stranded calf thymus DNA in 600 mls of 0.1 M Tris, 2.4 22 mM MgSO4, 1.7 mM CaClz, pH 7.5) was added, mixed, and the change in absorbance at 260 nm was monitored. One unit of inhibition activity was expressed as the amount of inhibitor needed to cause a decrease in DNase I activity of 0.01 absorbance units per minute. Measurement of ATPase Activity of Myosin Filamentous actin was detected by its ability to stimulate the Mg-dependent ATPase activity of myosin (14). Each assay mixture con- tained: 350 pl of buffer (15 mM KCl, 5 mM imidazole, 1 mM MgCl2, 1 mM ATP, pH 7.0), 100 pl of sample and 100,000-200,000 cpn of y-[32PJ-ATP. The reaction was initiated with the addition of 50 pl of myosin (1 mg/ml), incubated for 30 minutes at 37°C, and stOpped with the addition of 0.5 ml of cold 5% perchloric acid. The assay was a modification of the L32PJ-transfer assay used by Scnubert gt a1. (81). The amount of activity was measured by determining the quantity of non-charcoal absorbable radioactivity (80). Activities of myosin and heavy meromyosin were determined by the same method, except that the reaction mixture contained 450 pl of buffer (0.6 M KCl, 10 11M imidazole, 2 11M EDTA, 1 11M ATP, pH 7.0), 100,000-200,000 cpm of‘Y-[32Pj-ATP and 50 pl of sample (49). Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Discontinous polyacrylamide gel electrOphoresis in the presence of sodiwn dodecyl sulfate was carried out as described by O'Farrell (70). Gels were stained and fixed in 25% 150propanol, 10% acetic acid (v/v) and 0.125% Coomassie Blue (80). Gels were destained in 25% isoprOpanol and 10% acetic acid (v/v). 23 Electron Microscopic Examination of Microfilaments Squashes of plant material were prepared as described by Bennett and Brown (6). Small sections of plant material were minced in 2 to 5 drops of buffer (20 mM K2HPO4, 50 mM MgClz, 5 mM EGTA, 2 mM DTT, pH 7.0, containing 0.2-0.4 mg/ml heavy meromyosin) on a glass slide with a razor blade. A coverslip was placed over the minced tissue and pressure was applied until the cells ruptured. The liquid was removed with a Pasteur pipet and used inmediately. Copper grids (200 mesh) were coated with formvar/carbon. Films of Formvar were prepared glass slides dipped in a solution of 0.5% Formvar in chloroform and air dried. The film was removed by scraping the edges of the slide with a razor blade and by floating the film off on the surface of a water reservoir by slowly hnnersing the slide. Acetone washed grids were placed on the floating film, removed by inversion on an index card, and air dried. The Formvar coated grids were carbon coated with a Ladd carbon evaporator. Samples (1 drop) were applied to coated grids for 10 to 60 seconds, washed with 1 to 3 drops of buffer (20 mN K2HPO4, 50 mM MgClz, 5 mM EGTA, 2 nM DTT, pH 7.0) and stained with 2 to 3 drops of uranyl acetate (2% in water and stored in the dark). After 30 to 60 seconds, the stain was remomved with filter paper and air dried. Grids were examined with a Philips 201 C electron microscope at 60 KV. Electron micrographs were taken using Kodak electron image film 4463. Protein Determination The concentration of protein in a sample was measured either by the method of Lowry §t_al. (62) or by absorbance at 280 nm. The following 24 extinction coefficients were used: myosin 0.543 ml°cm-1°mg‘1 (61) and actin 1.15 ml°cm-1'mg"1 (19). Materials Cholchicine, cytochalasin B, fluorescein isothiocyanate, pectinase, soybean trypsin inhibitor, and Trasylol were purchased from Sigma. Bovine pancreatic DNase I and TPCK LL-(tosylamido-Z—phenyl) ethyl chloromethyl ketone] trypsin were obtained from Worthington. Uranyl acetate and 200 mesh c0pper grids were supplied by Ted Pella Co. (P.0.B. 510; Tustin, Ca. 92680). Cellulysin was purchased from Calbiochem. Polyvinylpyrrolidone, a gift of the G.A.F. Corporation, was acid washed as described by Lounis (57). Sepharose 4B and Sephadex G-150 were purchased from Pharmacia. Formvar was obtained from E.F. Fullman, Inc. DEAE-cellulose was suppled by Whatman. Highly polymerized, double- stranded calf thymus DNA was purchased from P-L Biochemicals, Inc. Iodogen (1,3,4,6-tetrachloro-3a-6a-diphenylglycoluril) was donated by Drs. P. Fraker and J. Speck. y-L32]-ATP was prepared by K.R. Schubert using a modified procedure of Glynn gt a1. (27). Goat anti-bodies directed against rabbit hnnunOglobulin were purchased from Miles Laboratories. 25 Figure 1. Identification of microfilaments by electron nflCrOSCOpy. Samples were treated with rabbit muscle heavy meromyosin, and stained with 2% uranyl acetate. a, rabbit muscle actin; b,c, microfilaments from roots of clover seedlings; d,e, microfilaments fron stems of soybean plants. The bar ( ) represents 100 nm. 26 FIGURE 1 RESULTS Identification and Localization of Microfilaments bnylectron Microscopy Microfilaments were observed in samples from soybean and clover plants that were examined by electron microscopy using the squash technique of Bennett and Brown (6). These filaments exhibited the classical arrowhead decoration with heavy meromyosin (Figure 1). When the grids were treated with 1 mM ATP prior to staining, decorated fila- ments were not observed. The heavy meromyosin decorated filaments showed a repeat of 30 to 34 nm for rabbit actin and 28 to 33 nm for soybean and clover microfilaments. The majority of microfilaments were quite short in length, ranging from 0.7 to 1.2 pM. Occassionally, longer filaments were observed (4 pM or larger), these often being in bundles with para- llel polarity. The longer microfilaments tended to be associated with or attached to organelles. The heavy meromyosin decoration of these fila- ments always pointed toward the site of attachment. As a control, grids were prepared with only heavy meromyosin and no decorated microfilaments were observed. The various anatomical structures of seedlings (Figure 2) and plants (Figure 3,4) were examined in both clover and soybean for the presence of filamentous actin. Microfilaments were observed in all regions except cotyledons, soybean petiols and clover leaves (Table 3). The frequency of microfilaments was found to be the highest in the root tip region, where one grid square may contain 15 to 30 filaments. Samples prepared from stems and leaves contained on the average 1 to 10 filaments per grid square. In some cases, several preparations were made and examined before decorated microfilaments were observed. 27 28 Figure 2. Schematic drawing of a typical seedling. Regions that were examined for the presence of microfilaments are indicated. 29 Cotyledon Hypocotyl Rodicle FIGURE 2 30 Figure 3. Schematic drawing of a soybean plant. Regions that were examined for the presence of microfilaments are indicated. 31 giolo / FIGURE 3 32 Figure 4. Schematic drawing of a clover plant. Regions that were examined for the presence of microfilaments are indicated. 33 (.55 7" 0 fig ‘36— Leaf 4—— Peiiole 0 / ) <——Secondary Root <5— Rooi Tip FIGURE 4 34 Table 3 Distribution of Microfilaments Source Glycine max Trifolium repens (soybean) (white clover) A. Seedlings radical ++ ++ hypocotyl + + cotyledons ND ND 8. Plants root tips ++ ++ mid section of secondary root ++ ++ stems + -- petioles N0 + leaves + N0 Symbols and Abbreviations ND - None detected ++ - 10 to 30 microfilaments were observed per grid square + - 1 to 10 microfilaments were observed per grid square No true stem structure in this plant (see Figure 4) 35 Both clover and soybeans contained microfilaments which decorated with heavy meromyosin. In order to confirm that these filaments were composed of actin, soybeans were exanined biochemically for the presence of the subunit protein, actin. Soybeans were selected because of the availability of a cell culture line and the ease in which large quantities of material could be obtained. Myosin-Binding Protein in Soybeans Myosin—Binding Activity The selective activity of myosin to bind actin and to be separated out as an actomyosin complex was used as another indication for the presence of an actin-like protein in soybean cells. BSA and extracts of 125 calf thymus and soybean cells were labeled with I, while chicken 131 125 myosin was labeled with I. Mixing [ IJ-labeled calf 131IJ-labeled myosin resulted in the co-PFECIP' 125 thymus extract with [ I]-labeled material (Table 4) 131 itation of myosin as well as [ presumed to be actin. Similarly, incubation of [ IJ-myosin with [IZSIJ-labeled extract of soybean cells caused co-precipitation of both [1251] and [131IJ-labeled material. In contrast, when [IZSIJ-labeled BSA was used, there was no precipitation of either [125 131 I]-labeled BSA or [ I]-labeled myosin. Affinity Chromatography on Myosin-Sepharose and Heavy Meromyosin-- Sepharose The selective ability of myosin to bind filamentous actin was also used as a purification technique. Affinity columns were initially 36 Table 4 The Binding of Myosin to Proteins from Calf Thymus and Soybean Cell Extracts 125 131 Conditions* I Protein I Protein Precipitated Precipitated 1251 (:1 + 1311 Myosin 10.5 a 2.5 0.9 a 0.1 1251 SB + 1311 Myosin 2.4 1 2.1 1.0 a 1.6 1251 BSA + 1311 Myosin 0 0 *Abbreviations: CT, calf thymus extract; SB, soybean cell extract; BSA, bovine serwn albumin; SEM, standard error of the mean. The specific 125 125 125 131 activities of I CT, I SB, I BSA, and I Myosin were: 60 mg/pg, 1100 cpm/pg, 1620 Cpm/pg and 100 Cpm/pg, respectively. 37 .»p_>_uoc mmeah< .Aqnlcv ms: owm moseDLOmoe .Ao...ov m.a Ia .ae< :5 H .~_umz :2 m.~ ._ue :5 om .a:25e_oee;aa_ca :2 OH :82; cap=_a ace m.~ Ia .~_umz :5 m.~ ._u¥ :5 om .a:_sa_oeeepa_cp 22 OH new: umzmmz we; Ago m x =0 Hv cs:_oo mcp .cE:_oo mmocozawnicwmoxs co puocpxm poo; :emnxom mo azaocmouesoczo »u_:_mm< .m mczm_m 38 (v—v) £_o|x quo Minnow asodyv Si 3 o l ' l ' l 2.4/<1 <1"-411; <\ 4‘ 1‘5 (1‘4 4 —-> . :40 f . O ._ If -————""°’0 ‘ 000.0? _.0 '0. o mu 099 eouoqmsqv 0.0 FIGURE 6 4l prepared using heavy meromyosin, because actin filaments in extracts of soybean plants decorated with heavy meromyosin. However, coupling of heavy meromyosin to Sepharose resulted in a substantial loss of ATPase activity and the ability to bind rabbit muscle actin. Therefore, affin- ity columns were prepared with myosin in the hope that the nmjority of coupling sites would be along the tail region of the molecule and thus leave an active head region. Although the amount of ATPase activity bound was not greatly enhanced, the capacity of the colunm to bind rabbit muscle actin increased approximately 60 fold upon replacing heavy meromyosin with myosin as the ligand. An extract of 34-day-old soybean roots were prepared and chroma- tographed on a myosin-Sepharose column (Figure 5). The column was monitored for protein by absorbance at 280 nm and for actin by the ability to stimulate the Mg-dependent ATPase activity of myosin. Stimu- latory activity was observed both in the fractions containing material not bound to the column and those eluted with ATP. The fractions eluted by ATP were pooled, dialyzed, lyOphilized and analyzed by SDS gel elec- tr0phoresis. This fraction contained 3 major bands (Figure 6) with the largest polypeptide having the same mobility as rabbit actin. In successive experiments, "actin activity“ was consistently detected in the pass through volume but there was no longer a peak of "actin activity" which was specifically eluted with ATP. Alteration of chromatographic conditions, variation in the age of the plant material used, and newly prepared affinity columns were tried but again, no peak of "actin activity" was detected. No significant loss of actin binding ability was observed when nwosin-Sepharose columns were tested for the ability to bind rabbit muscle actin before and after chromatOgraphy with soybean extracts. 42 Figure 7. Staining of soybean protoplasts with fl-DNase I. a,b, control; c,d, protoplasts incubated for 1 hour with 10 pg/ml of cytochalasin B; e,f, protoplasts incubated for 1 hour with 4 pg/ml of cholchicine. Frames a,c,e, and b,d,f, present results of fluorescence and phase-contrast microscopy, reSpectively. 43 FIGURE 7 44 DNase I-Binding Proteins in Soybeans Stainingyof Protoplasts with fl-DNase I Since the discovery of Lazarides and Lindberg (50) that actin was a Specific inhibitor of DNase I, this enzyme has become a useful tool for the identification and localization of actin within cells. In this study, fl-DNase was used to detect the presence and distribution of an actin-like protein in soybean prot0plasts. The following staining pattern was observed: the nuclear region was stained most intensely, sharply delineating the nucleus from the rest of the cell; the cytoplasm showed a more diffuse general staining pattern (Figure 7). No distinct bundles or "stress fibers" were observed. Control experiments showed that the protoplasts did not autofluoresce. In addition, pretreatment of the protoplasts with cytochalasin B (10 pg/ml) or cholchicine (4 pg/ml) for 60 minutes before fixation did not significantly alter the observed staining pattern (Figure 7). These results indicate that the enzyme DNase I does label soybeans, possibly by binding to actin-like protein. In order to characterize the DNase I binding material, this enzyme was used as a ligand for affinity chromatography. Affinity Chromatography on DNase I-Sepharose The specific ability of DNase I to bind actin served as the basis for affinity chromatography. Columns were prepared by covalently coupling DNase I to Sepharose 4B. This technique was used successfully to purify actin from calf thymus. The elution profile after chromatography of calf thymus extract on a DNase l-Sepharose column is presented in Figure 8. 45 .cE:_ou we» on emcee mg; _u:.mcwc_ce:m .zm co 2 9N.o mc_c_epcoo Lemmas mg» ;u_cz we mu:_oq ms» mumuvu:_ mzocce mch .m.o In ._ocmux_m mom .mpeumom cswcom z o.H ._u:.m:_c_:e:m z m xa cmzo__ow .m.o :q .Pocmume mom .mueumum E:_UOm z m.o ._uz.a:_e_ee=m z mN.o gap; oaa=_a ace .m.~ Ia .N_ua8 :5 m .m_Le 2: OH zuwz umcmmz we: A58 m.o x Eu m~.ov c5:_ou ash .cE:_ou mmocezammuH amaze :o uuecpxm mzsxzu m_eu $0 xzqecmoumeoczu xuwzwmm< .m bc:m_m 46 2:: cE:_o> cozgm 00 _ On 0 i olo \ololmlol/ wlolwo/ l 0 k z _ d l 0’ o l 0.. a Z m 5— who .. F - om u p mu 033 eouoqiosqv FIGURE 8 47 .m=_m mwmmeeoou saw: em:_mum we; Pam ace .Pmm mwsu co umUeOP mm; ewmpoca *o a: mu x_mumewxocaa< .xm.~ we: Pam ecu $0 cowu_moaeoo mvwEmFxcum wee .cE:_oo mmocmcammuH mmmzo 8 Sec» umpapm we: sows; :wuum msexzu cpmu mo mwmmcocaocuumpw Fem mqumpacumxroa mom .m mesmwd 48 0.0 3:322 «263m v.0 _ 000.0? 0. 0.N OJUOQQ aouoqmsqv FIGURE 9 49 .cE:_ou mcp Op emcee mm; .oz.mcwuwce:m z m co 2 mN.o mcvcwmucou Layman asp ;u_;; be mucwoa mcp mumo_u:_ mzocce och .m.o :Q ._ocmux_m flom .mpepmum Eswuom z o.fi ._u:.w:_cvcc:m z m use m.o In ._ocmox_m mom .mueumum Ezwuom z m.o ._ur.wcru_:e:m z mN.o +0 :o_u_uve pmwzqmum x2 eaa:_a we; _a_cabee meaaewn H amaze m.~ Ia .N_ue8 :2 m .mwcc 25 OH :90: umcmez we: Ase m.m x Eu mN.ov can—co asp .c2:_oo mmocmcammiH mmezo co puecuxm —_mo cemnxom mo xcaecmOmeoccu xu_:_wm< .oH mczmwd 50 26v oE:_o> cozsm on. 00. Rb O . ./ 4...,» a a 2n Znho .. v N‘ \I '9. o uiuoaz eouoqiosqv 0.. FIGURE 10 51 .mxu mcwxomcp e me new: we: m:_n _o:mcaosocm .cwpoe mashep 0_mu mo cowumcmw: mo :owp_moa mcu mmuou_ocw soccc map .xm.m we: _wm ecu mo coppwmoasoo mcwze_»cue mcp .uuecpxm __mu :meaOW saw; umgqmcmoue50ccu :E:_ou mmocmgqmmuH mmmzo w soc» Pu:.m:_u_:m:m z m :00: umuz_m we; cu_;; _cwcmuez any 00 mwmmcosaocuum_m Pwm wo_sm~>coox_oa mom .HH mczmwm 52 0.0 2:302 2:28. ed 000.0V 00.0 00.0 0.0 uJuogg aouoqaosqv FIGURE 11 53 .umuewuwcw we; .m.o Ia ._ocmoa_m mom .mumumum saruom z o.H ._u:.mcwuw:m:m z m co .n.m :3 ._ocmux_m xom .mpeumum szwuom z m.o ._o:.m:wuwce:m z mN.o x9 corps—w wmwzampm .mzoccm mzp an umumo_ucw u:_oq mg» u< .m.~ In .N_uwu z: m .m_cp 25 OH saw; umzmmz we; Asp m.o x =0 mm.ov :E:_ou wee .:53_ou me mmocecawm o co m__mo cemaxom *o poecpxm co mo xsqecmoumsoczu .NH mc:m_d 54 CF: 0830) c0335 08 on. 0%... o olololro'olonrrowlu u/# 0 .7. 4 ._. k -- 00 2F 2 who ’9 I F E1 o._ L _ 0&1 LOO 093 aouoqaosqv FIGURE 12 55 .czepoo mew o» emeee we: _u:.mcwe_:eem z m co 2 me.o mewcweucoe cme0ee wee :u_;3 we mpcwoq ecu mueuvecw mxocce esp .m.e Ia ._ocmo»_m «om .mpepmue Seweom z o.H ._ur.m:_e_ee:m z m ece .m.o IQ .Focmua_m mom .mpebeue seweom z m.o ._uz.mcwe_ce:m z mk.o Co eo_eceee am_zaaem AD ea83_a eee m.~ :1 .N_eee :2 m .mece :5 OH sew; emzmez we: Ase m.e x Eu mN.ov :5:_ou esp .cs:_ou emocecammi peaceecm _epop age .eaeewe_=w we; _ee 2 mm.o op o 5020 Auuiv peeveecm Lemcw— e .3occe ecu xe emueowecw pewoa we» u< .E: omm pe meowuuecm pcwe_mmm any we moceecomee esp mmpocme mew? empom use .m.m Ia .Foceeeaoeaeacae-a :5 m~.o .mpeee :5 H.o .aee :5 m.o .aPONeeaew :5 OH ;u_z emuecewrweem me: Ase mm x 50 ev caepou wee .cse_ou mmo_:__eu-w