. w. A Afix v .3 Vuwa ‘3! V 1. A :v,‘ Iaflmfifi‘n‘awumw *v-O-Q'H *-().36:1 found in 3T3 cells (5A,55), and/or the substitu- tion of stigmasterol, sitosterol, and campesterol f2n~<flnolesterol as the major steroids in plant membranes (53). An increased sterol content will induce a more rigid (gel-like) structure (less ordered below and more ordered above the transition temperature) which would have a direct effect on the lateral mobility of components present in such domain. Because these conditions are not as prevalent in mammalian cells, restricted mobilities in small domains are most likely averaged when measured by FRAP, resulting in a single diffusion coefficient. 17 On the basis of the above results, it was proposed that the plasma membrane of soybean protoplasts is comprised of two distinct, immiscible gel- and fluid-like lipid domains at 18°C. In the context of the previous analysis of cell surface compo- nents in soybean cells (38,A0,A5), these results suggest that certain plasma membrane receptors, such as those for WGA, have a higher affin- ity for more fluid domains, while receptors for soybean agglutinin prefer a more aggregated or ordered lipid environment. This is supported by the observation of a 6- to 7-fold variation in lipid mobility between the two mobile species. The physiological role of the lipid domains and their possible role in the process of ligand- receptor mediated endocytosis remain to be determined. 18 Cytoskeletal Structures in Plant Cells Microfilaments are composed of double helical polymerscfl’the monomer actin, whose molecular weight varies slightly from species to species but generally ranges from A2,000 to A5,000 daltons. lkfidn microfilaments can be readily identified under the electron microscope as filaments of 6-10 nm in average diameter.- The interaction of these microfilaments with myosin proteolytic subfragments bearing the heavy chain, yields a characteristic complex with the appearance of arrowheads along the length of the filament (56,57, for reviews on microfilaments see 58-60). Primary observations of a microfilament network in plants came from analyses of thin sections which revealed fine filamentous struc- tures having an average diameter of 7 nm (61,62). These structures were subsequently confirmed to be actin microfilaments by the revers- ible binding of heavy meromyosin (HMM) and the subfragment 1 of myosin (S-1), which yielded the characteristic arrowhead decoration of the filament (63-72). lbs addition, the specific binding of phallotoxins to filmmnmous actin (F-actin) (73) has been used as a tool in the identification of microfilaments in plant cells (7A-79). Characean cells carry out extensive cytoplasmic motions (cytoplas- mic streaming) that are responsible for the movement of particles and organelles (80). It has been shown that this phenomenon is brought about by the movement of myosin molecules over acthinutmofilaments (81). Ihibfitella, the presence of fibrillar structures attached to the moving chloroplasts were visualized by light (82)enuielectron microscopy (83). These structures were located parallel to the direc- 19 tion in which the streaming occurs. These fibrils were shown to consist of bundles of microfilaments of 5-6 nm in diameter (83), and they were composed of F-actin, as jugded by their ability to form arrowhead complexes with HMM and 8-1 (63,8A,85). Furthermore, the addition of cytochalasin B to the cells caused the streaming to cease (86). Similarly, in higher land plants actin microfilaments have been found to be more abundant in tissues where cytoplasmic streaming occurs extensively (7A). Parthasararhy and Mulethaler (61) examined elongating parenchyma cells in twelve species of plants and found that microfilaments were present at the periphery of these particular cells. In addition, they were oriented parallel to the longitudinal axis in the direction in which growth occurs. Metcalf et al. (72) found an increased amount of actin microfilaments in radicles and root tips of soybean seedlings and mature plants as compared with the cotyledons and petioles. Pesacreta et a1. (7A) found microfilament bundles in vascular parenchyma cells of conifer roots using a fluorescently- labeled phallotoxin. In contrast, other tissue types from the same plants such as the cortex, where cytoplasmic streaming was non- existent, did not possess the microfilament bundles. The presence of actin microfilaments has also been demonstrated in Mg (67). Amaryllis (68), Haemanthus (69,70), Lycopersicon (71), and Trifolium (72). In algae, cytoplasmic streaming and organelle orientation (87) occurs via an actomyosin system (81). In higher land plants, an analogous system may exist, although direct evidence of such a complex 20 has not been reported; only one report exists on the isolation of a myosin-like molecule from tomato endocarp tissue (71, 88). A role of actin microfilaments in the mitotic process has also been proposed. Rhodamine-phalloidin staining has revealed actin microfilaments associated with the preprophase band of onion (75) . An intricate array of microfilaments was observed in cycling suspension cultures of Medicago sativa and Vicia hajastana (76) . Three types of microfilament populations interconnected with each other during interphase were observed: a) a peripheral network close to the plasma membrane: b) large subcortical cables; and c) a meshwork surrounding the nucleus. All these networks disappeared at the onset of mitosis and reappeared after cytokinesis. It was proposed that the nuclear associated meshwork (thereby termed as "nuclear basket") could be a center for microfilament initiation and/or organization. Traas et al. (77) observed similar arrays of F-actin in interphase cells of Daucus carrota suspension cultures. They demonstrated that the actin network does not disappear during mitosis, but rather organizes differently. The microfilament network was observed associated with the preprophase band, the mitotic spindle, and the phragmoplast. Schmit and Lambert (78) further showed that a cortical actin microfilament network is present throughout mitosis in endosperm cells of Haemanthus. It was proposed that this microfilament network acts as a deformable cage, and may help in the guidance of cytoplasmic vesicle transport to the nucleus during mitosis in higher plant cells. Microtubules, the other main component of the cytoskeleton, are long hollow structures of 25 nm in diameter (for reviews see refer- ences 60.79.89). They are composed of the protein tubulin, which is 21 éultl’B heterodimer of 110,000 molecular weight. Each subunit is found in a one-to-one ratio and are almost identical in molecular weight (Mr = 55,000 daltons). Tubulin-like proteins have been isolated from a variety of plant tissues such as liggg seedlings (90), fern sperm flagella (91). cultured tobacco cells, (92), and Paul's Scarlett Rose (93). The protehiieolated in the latter case was capable of assembling into microtubular structures in vitro. Although it appears that the biochemical properties of plant tubulin closely resemble those of animal tubulin, it has been reported that tubulins from different plant species are immunologically unrelated among their'cr-1and B-subunits, with the o-subunits having the most divergence (9A). In addition, they showed differential binding of colchicine with respect to each other and, in general, less binding than that observed with bovine brain tubulin. The observatitni<3f microtubules in plant cells both by electron and immunofluorescence microscopy has been documented extensively (reviewed ir11M3). Electron microscopic analyses of plant tissue have revealed arrays of microtubules underlying the plasma membrane and running parallel to cellulose microfibrils (95-97). This and the fact that upon treatment with anti-microtubule reagents this orientation is lost have led to the proposal that microtubules play'a role in the guidance and orientation of cellulose microfibril deposition. This Inale however, has not been conclusively demonstrated. Besides corti- cal microtubules, cytoplasmic microtubules in higher plant cells have also been observed by indirect immunofluorescence (98-100). Cytoplas- mic microtubules at different stages of timzrnitotic cycle have been 22 observed associated with the preprophase band, interphase arrays, the mitotic spindle, and the phragmoplast (60,79). Anti-microtubule reagents have been shown to induce abnormalities in cell division and differentiation in higher plants (60,101,102). Thus, they appear to play a role in these cellular processes as well. Microtubules have also been proposed to be involved in cell elongation and growth. A current hypothesis on their organization at interphase proposes that these assemblies form dynamic helices. These helices could exist in a number of orientations according to the particular cellular stage (79). Support for this hypothesis arises from immunofluorescently-stained microtubules in the form of right- and left-handed helices along the length of cells. These arrays have been observed in onion and Urtica root hairs (10A), cortical Raphanus cells (105), and cotton fibers (106). In addition, the disruption of the orientation of the helical pattern has been shown to result UT abnormal growth (107). Microfilaments and microtubules, the main cytoskeletal compo- nents, and their respective protein components actin and tubulin, have unquestionably been identified in plant cells. A detafhxicharac- terizathnmcfi'their biochemical properties should improve our understanding of cytoskeletal functions in plant cells. 23 Actin in Plant Cells The monomeric component of microfilaments is the protein actin. Monomeric actin (G-actin) occurs mainly in three isoforms (a, B, and Y) which are identified by their isoelectric points (108). a-Actin (pl-5.6) is found exclusively in muscle cells, whereas 8" (pl-5.8) and Y- (pl-5.9) actins are present in all non-muscle cells. Although these are the isoforms typically found in many species examined (109,110), other isotypes have also been reported. Chicken gizzard actin has been shown to have a slightly more basic pI than that of chicken brain Y-actin, whereas the main actin species from Physarum polycephalum was found to migrate with a slightly more acidic pI than that of rabbit muscle actin (109). When a high resolution two dimensional gel electrophoresis system was used to analyze actin from Physarum, it was found that the previously reported main species consisted of three closely spaced isoforms. In addition, a different new more basic isoform was found that differed by 0.A pH units from the main actin triplet (111). Actins with more basic isoelectric points than the main a-, B-, and Y-isotypes have also been reported to occur in BS nerve cultured cells (112). They were observed as unstable species with a lifetime of less than 2 hours, although it was acknowledged that they may be non-obligate precursors of the usual 8- and Y-isoforms which lack the N-terminal acetylation. Finally, a single actin species from Tetrahymena pyriformis was reported to have a pI 0.2 pH units more basic than that of rabbit muscle actin, and a slightly higher molecular weight on sodium dodecyl sulfate polyacryla- mide gels (Mr = A8,000) (113). 24 The isoelectric point of actin from plants has not been reported so far. Although it is well documented that actin is present in plants (60-79) , the isolation and characterization of the actin poly- peptide from plant tissues has been a difficult task. This is due to the low amounts present and/or the presence of potent proteases which are released as soon as the tissue is broken. Only a few reports describing a partial characterization of the molecule from plant tissue exist in the literature. Ilker et a1. (11A) isolated a mole- cule ffiwmn wheat germ by gel filtration chromatography. Their material was identified as actin by the following criteria: a) solubility properties; b) co-migration of the polypeptide with rabbit muscle actin in SDS polyacrylamide gels; c) immunological cross-reactivity with anti-turkey gizzard actin antiserum; and d) ability to form needle-shaped fibrils similar to those observed from sea urchin actin. A molecule with the biochemical characteristics of actin has been isolated from tomato endocarp tissue (71,88) by ammonium sulfate frac- tionaticnianid gel filtration chromatography. The protein exhibited a molecular weight of A2,000 daltons in SDS polyacrylamide gels. Before the colimn1.fractionation step, tomato extracts were shown to assemble into filaments of 6 nm in diameter capable of ATP-reversible binding with the S-1 subfragment of rabbit myosin. These extracts were also capable of activating the Mg2+ ATPase activity of the rabbit S-1 subfragments by 10-fold. After the column fractionation however, this ATPase activation decreased to 2.5-fold and the microfilament pnwepara- tions were reported to be very labile. In a later report, Vahey (115) described the isolation of a protein of 72,000 daltons from tomato which inhibited the ability of F-actin to stimulate the low ionic 25 strength Mg2+ ATPase activity of the rabbit S-1 subfragment of myosin. This protein was hypothesized to be responsible for the low levels of ATPase activation by tomato actin found previously (71). Metcalf et al. (116) isolated a protein from soybean seedlings after extraction in a low ionic strength buffer and ion exchange chromatography. The fraction containing material reactive with anti-calf thymus actin antibodies was further purified by affinity chromatography on a Sepharose column coupled with the same antibodies. The purified polypeptide comigrated with calf thymus actin in SDS polyacrylamide gels. In addition, 125I-labeled fractions of anti-actin reactive material from the ion exchange column were immunoprecipitated with the same antibodies. The immunoprecipitated material was shown to contain a single major polypeptide with an electrophoretic mobility identical to that of calf thymus actin. Although the purified polypeptide was not used to prepare microfila- ments, Metcalf et a1. (72) have described in a different report the observation by electron microscopy of 7 nm diameter microfilaments from soybean seedling squashes. These microfilaments were capable of ATP-reversible binding with HMM to yield arrowhead complexes. Taken together these results suggest that actin from higher plants shares the structural and functional properties of actin from animal cells and other eukaryotic organisms. Despite the above-mentioned biochemical similarities of plant actin with other organisms, an increased divergence has been found between the deduced amino acid sequences obtained from genomic clones of soybean and maize actin, and the amino acid sequences of other actins (117,118). Nagao et al. (119) first reported the existence of 26 a small multigene family of actin-related sequences in soybean. Subsequently, Shah et al. (117,118) isolated actin genes from genomic libraries of maize and soybean, and their nucleotide sequences were determined. The deduced amino acid sequences from two soybean actin genes (named Sac1 and SacB), and from one maize actin gene (Mac1) showed a high degree of homology but increased divergence with those of other eukaryotic actins such as mold, protist, and animal actins. For example, a recent report describing the deduced amino acid sequence of actin from Tetrahymena pyriformis showed a sequence homology of only 73% with soybean actin (113), the highest divergence for an inter-species actin reported so far. Previous to this report the highest divergence found in species other than plants was between yeast and Drosophila actin (86.A% amino acid sequence homology) (117). Two members of the soybean actin gene family showed a difference of 27% in their nucleotide sequences. This intra-family divergence is also high when compared with the highest divergence reported previ- ously for an actin gene family (15% divergence in nucleotide sequences for Drosophila actin genes (120). A distinct feature that was found in the plant actin genes analyzed was that their first nine amino acids at the amino terminal end were highly conserved even between soybean, a dicot, and maize, a monocot (118). The corresponding sequences in distant animal actin genes are much less conserved. These data suggest that the actin genes from higher plants diverged from a common ancestral gene. The actin(s) arising from these genes may have a certain unique function in the plant. The increased differences in plant actin gene structure and their unique N-terminal sequences, prompted investigators to look for a 27 differential expression of these genes (121). Hybridization studies on the six soybean actin genes SAc1, SAc2, SAc3, SAcA, SAc6, and SAc7 indicated the following: a) 2 genes (SAc6 and SAc7) were the most highly expressed accounting for 80% of the actin mRNA; 6) SAc3 and SAc1 were found to be expressed at intermediate and low levels, respectively, whereas SAcZ and SAcA were found to be expressed at extremely low levels; and c) SAc6 was expressed at the same level in root, shoot, and hypocotyl, whereas SAcB and 3A0? were more highly expressed in shoot than in root and hypocotyl. In all three organs analyzed, SAc3 and SAc1 produced 1.7-kb size transcripts. In Drosophila (122), at least three actin genes produced 1.7-kb size transcripts, two genes produced 1.6- and 1.75-kb size transcripts, and one gene produced 1.7-, 1.95-, and 2.2-kb size transcripts. This indicated that in all cases, the transcripts contained a significant amount of flanking sequences (-1.1-kb are needed for the actin polypeptide alone). Root, shoot, and hypocotyl contain vascular, cortical, and epidermal tissues, that are continuously undergoing cytoplasmic streaming, and actin has been found in more abundance in these tissues (61,7A). It was proposed that the two soybean actin genes that represented the majority of the expressed mRNA (SAc6 and SAc7) might be specific for vascular tissue although no evidence was provided. Therefore, it is still unknown how each of the actin genes relate to the different plant functions in which actin has been implicated such as structure, mitosis, cytoplasmic streaming, and receptor-cytoplasmic interactions. 28 In conclusion, the actin molecule from plants remains poorly characterized. Although it is similar to its animal counterpart, it is one of the most divergent forms of actin. Based on fragmentary evidence, at least some of its biochemical properties parallel those of animal actin; i.e. similar molecular weight, morphology1of assembled microfilaments, specific binding of these microfilaments by phallotoxins and myosin fragments, and immunological cross-reactivity. A more complete characterization of the molecule‘will shed more light into its similarities and/or differences with animal actin. Further- more, if indeed the similarities are greater than the differences, the search for its regulatory proteins (reviewed in 123) can be initiated. 10. 11. 12. 13. 1A. 15. 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Cell Biol. 29, 1761-1765 Metcalf, T.N., III, Szabo, L.J., Schubert, K.R., and Wang, J.L. 1980. Nature (Lond.) 995, 171-172 Shah, D.M., Hightower, R.C., and Meagher, R.B. 1982. Proc. Natl. Acad. Sci. U.S.A. 19, 1022-1026 Shah, D.M., Hightower, R.C., and Meagher, R.B. 1983. J. Mol. Appl. Genet. 9, 111-126 Nagao, R.T., Shah, D.M., Eckenrode, V.K., and Meagher, R.B. 1983. DNA 1, 1-9. Fryberg, B.A., Bond, B.J., Hershey, N.D., Mixter, K.S., and Davidson, N. 1981. Cell 99, 107-116 Hightower, R., and Meagher, R.B. 1985. EMBO J. 9, 1-8 Fryberg, B.A., Mahaffey, J.W., Bond, B.J., and Davidson, N. 1981. Cell 99, 107-116 Pollard, T.D., and Cooper, J.A. 1986. Ann. Rev. Biochem. 55, 987-1035 CHAPTER 2 LATERAL MOBILITY OF SURFACE-BOUND MONOCLONAL ANTIBODY MVS-l ON SOYBEAN PROTOPLASTS 37 ABSTRACT Monoclonal antibodies were prepared against protoplasts from soybean (Glycine max) suspension cultures (SB-1 line). One particular antibody, termed MVS-1, bound to a defined protein (Mr = A80,000) on the outer surface of the plasma membrane of the protoplast. The diffusion coefficient of the monoclonalantibody was determined by fluorescence redistribution after photobleaching (D = 3.2 x 10'10 cmZ/s). THua binding of soybean agglutinin to the protoplasts reduced the lateral mobility of antibody MVS-1 ten-fold (D a u.1 x 10’11 cmZ/s). Soybean agglutinin did not interact with the monoclonal anti- body or its antigenic target, ruling out cross-linking between these molecules and the lectin to a set of immobile receptors. When cyto- chalasin B or colchicine were added along with the soybean agglutinin, time lectin-induced modulation was partially reversed. When both drugs were added together, the lectin-induced modulation of nmflaility of the antibody-antigen complex was totally reversed. These data are similar to our previous observations on the modulation by soybean agglutinin of the mobility of wheat germ agglutinin receptors. The present study represents a more refined analysis to the level of a single defined receptor on the membrane. These results further suggest that the binding of soybean agglutinin to the plasma membrane of the soybean protoplast induces an altered cytoskeletal structure which, in turn, restricts the mobility of other plasma membrane molecules. 38 INTRODUCTION The binding of soybean agglutinin (SBA) to protoplasts derived from a cultured cell line, SB-1, resulted in a decrease in the lateral diffusion coefficient of fluorescently-labeled wheat germ agglutinin (WGA) bound to the plasma membrane of the same cells (1). 'The lateral mobility of the plasma membrane components was determined using the technique of fluorescence redistribution after photobleaching (FRAP). Because lectins bind to a heterogeneous population of receptors on the cell surface, the lateral diffusion coefficients (D values) obtained for WGA-receptor complexes probably reflect ensemble averages rather than the mobility of a single diffusing species on the membrane. Thus, it was not known whether the decrease in mobility was due to: a) a decrease in the number of fast-moving receptors; b) an increase in the number of slow-moving receptors; or c) an intrinsic change in the mobility of all the receptors. We have refined these studies on the effect of SBA by the use of a monoclonal antibody designated MVS-1, that binds to a defined cxmnpo- nent (Mr - A80,000) on the outer surface of soybean protoplasts (2). we now report the analysis of the lateral mobility, and its modulation by SBA, of this antibody-antigen complex in the plasma membrane1of soybean probopLasts. This lectin-induced modulation appears to be mediated by the cytoskeletal components of the plant cell. 39 MATERIALS AND METHODS Cell Culture and Protoplast Isolation The SB-1 cell line of soybean (3) was kindly provided by Dr. G. Lark (Department of Biology, University of Utah, Salt Lake City, UT) and was grown in the dark as suspension cultures. Protoplasts were prepared by resuspending a certain volume of 2-day-old cells with fresh 1BS-C culture medium (A), and diluting this suspension with an equal volume of a solution containing 1 mg/ml pectinase (Sigma, St. Louis,1KD, 2 mg/ml Cellulysin (Calbiochem, La Jolla, CA), and 100 mg/ml sorbitol (Sigma), pH 5.5. This mixture was incubated for 2 h at 25°C and filtered through a A8 um nlen mesh. The filtered protoplasts were washed by centrifugation (A60 g for 5 min) and resus- pension in Modified Gamborg's buffer (A). Binding of Lectins and Antibody Probes to Protoplasts The generation and characterization of the monoclonal antibody MVS-1 and its Fab fragment have been described in detail (2,5). The preparathm1anmilabeling of the fluorescent reagents have also been described. Protoplasts (106/ml) were incubated for 1 h at 25°C with the fluorescent probe in 50 mM Tris, 10 mM CaClz, 0.55 M Sorbitol, {fli'7.5 (buffer A), followed by centrifugation and resuspension in equal volumes of the same buffer. The sequential binding of lectin and fluorescent EUHlibOdy was done by pretreatment of the protoplasts with 50-250 ug/ml SBA or 250 ug/ml WGA for 1 h at 25°C. Washing and labeling with the fluorescent antibody were performed as above. 41 For indirect immunofluorescence, 5 x 108 protoplasts resuspended in buffer A were incubated in separate test tubes for 5 h at 25°C with one of the following: a) 50 ug/ml rhodamine-conjugated antibody MVS-1; b) a 1:1 dilution of the culture supernatant from clone 5 (2), followed by a 1:30 dilution of rhodamine-conjugated rabbit anti-mouse IgG (Cappel Laboratories, Cochranville, PA); or c) a 1:30 dilution of rhodamine-conjugated rabbit anti-mouse IgG (Cappel Laboratories). The incubations were followed by washing of the protoplasts A times in the same buffer. The fluorescent antibody-treated protoplasts were deposited on microscope slides and visualized under a Leitz (Wetzlar, FRG) fluorescence microscope equipped with a Leitz KP580 dichrdic mirror. Human platelets were coated with SBA (5) using paraformaldehyde as the fixative. For photobleaching experiments, 2 X 108 SBA-coated platelets were incubated with 5 X 105 protoplasts for 1 h at 25°C, followed by washing and labeling with the fluorescent antibody as described above. Drungreatments Protoplasts were also pretreated with certain drugs that affect cytoskeletal structures. The cells (106) were preincubated with 1 uM colchicine (COL, Sigma Chemical Co.), and/or 10 ug/ml cytochalasin B (CB, Sigma Chemical Co.), for 30 min at 25°C. After washing, the protoplasts were treated with lectin or antibody as described above but the concentration of the drugs was maintained throughout. Binding of the fluorescent lipid, 1-acyl-2-(N-A-nitrobenzo-2-oxa-1,3-diazole)- 42 aminocaproyl phosphatidylcholine (NBD-PC, Avanti Polar Lipids) to soybean protoplasts was carried out as described (5). 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