Immunochemical identification of mung bean actin like ... - Springer Link

J. Biosci., Vol. 14, Number 3, September 1989, pp. 209-219. © Printed in India.

Immunochemical identification of mung bean actin like protein and its cellular involvement during germination G. GHOSH, A. PAL* and S. BISWAS Department of Biochemistry and *Laboratory of Tissue, Culture, Bose Institute, Centenary Building, Calcutta 700 054, India MS received 16 January 1989; revised 23 May 1989 Abstract. Actin like protein, extracted and purified from Vigna radiata (mung bean) seedling, has been found to give positive enzyme-linked immunosorbent assay with mouse monoclonal antiactin antibody. In vivo studies show that cytochalasin Β at sublethal dose inhibits the chromosomal movement at metaphase stage during germination. From in vitro studies it is found that the actin like protein isolated from mung bean seedling has a cytochalasin Β binding property with a Kd value 1·2 × 10–5 M. From these two specific observations it appears probable that the biological function of mung bean actin like protein is to take part in cell division process directly or indirectly during the time of seedling development. Keywords. Vigna radiata; ELISA; cytochalasin B.

Introduction Actin, which was once thought to be localized exclusively in muscle cells, has now been found to be associated with many types of non muscle motilities as for example ameboid movement, cytokinesis and cytoplasmic streaming (Forer, 1974; Pollard and Weighing, 1974; Sanger, 1975). Sanger in 1975 observed the presence of actin in the mitotic spindle of rat Kangaroo cells with the help of fluorescentlabelled heavy mero-myosin (HMM). According to him, the actin in the spindle is confined to the fibers, that connect the chromosome with the centriolar region. Moreover the notable fibrilar structure associated with cytokinesis is the contractile ring, whose short lived presence as a band or ring like array of microfilaments was observed under electron microscope (Schroeder, 1972). Their resemblance to actin was demonstrated by complexing with HMM and therefore it was concluded that contractile ring contains an actin like component (Perry et al., 1971). Furthermore, cytochalasin Β brings about the disruption of contractile ring microfilaments (Schmiedel and Schnepf, 1980). The specificity of these results strongly suggests that microfilaments of the contractile ring are closely related to muscle actin and hence have been considered as a mechanochemical organelle that cause cell cleavage. Antibodies against actin have been experimentally produced in animals in recent years (Fagraeus and Norberg, 1978). It has been reported in case of higher plants, for example soybean (Metcalf III et al., 1980) and wheat germ (Ilker et al., 1979), that the actin like protein cross reacts with anti-actin antibody produced against muscle actin. A major difficulty in the immunochemical approach to actin is the poor immunogenecity of this antigen. It has not been possible to raise antibodies Abbreviations used: HMM, Heavy mero-myosin; ABTS, 2,2'-azino-di (3-ethylbenzthazdline) sulphonate; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffer saline; SDS, sodium dodecyl sulphate; PAGE, polyacrylamide gel electrophoresis.

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against mung bean actin like protein. A quantitative study of the antigenic reactivity of mung bean actin like protein by following enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody raised against mouse actin has been presented here. In mung bean seedling, there is no apparent contractile event related to movement where the actin may participate actively. Hence the question may arise as to which particular cellular process involves the function of mung bean seedling actin. The fact that the mung bean seedling is growing fast during germination indicates that the actin like element present in this system may have some role during cell division. The present study describes the work which shows the antigenic reactivity of mung bean actin like protein and also its role during cell division. Materials and methods Chemicals and radiochemicals Conjugate of peroxidase (from horse radish) and protein A (from Staphylococcus aureus), 2,2'-azino-di(3-ethylbenzthazoline) sulphonate (ABTS), a tritiated cytochalasin Β [4(n)–3H] and mouse monoclonal anti-actin antibody were obtained from Radiochemical Centre, Amersham, England and Sephadex G-25 (particle size 20–80 µ) was purchased from Pharmacia Fine Chemicals, Upsala, Sweden. Orcein was purchased from E. Merck, Germany. ATP and cytochalasin Β were purchased from Sigma Chemical Co., St. Louis, Missouri, USA. Isolation of actin like protein The method used is that of Ghosh et al. (1988). ELISA Mouse monoclonal anti-actin antibody (Amersham, UK) was used in ELISA and a Standard curve was constructed with rabbit muscle actin (Sigma) following the protocol supplied by Amersham. Microtiter plates were coated with different concentrations of purified mung bean seedling actin in 100 µl volume overnight at 4°C in Tris-HCl (pH 9·5). Nonspecific absorption was blocked with 1% bovine serum albumin in phosphate buffer saline (PBS) of the following standard composition, 137 mM NaCl, 2·7 mM KCl, 1·5 mM KH2 PO4 , 8 mM Na2 HPO4. Anti-actin antibody diluted to 1:1000 in PBS was allowed to bind with the coated antigen at 37°C for 1 h. After thorough washing of the microtiter plates with PBS, protein A— enzyme conjugate at 1:10,000 dilution was allowed to hybridize at 37°C for 1 h. After washing ABTS was applied in each well (1 mM, 100 µl) as substrate. The colour development was monitored at 410 nm. All washings were done with PBS Tween-20. Microscopic observation of cell division in mung beqn seedling in the presence and absence of cytochalasin Β Mung bean seeds were allowed to germinate at 25°C for 24 h in a petridish on

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water saturated filter paper after surface sterilization with 1 % sodium hypochlorite solution for 10–15 min followed by washing with distilled water twice or thrice and swelling. The germination was performed in dark. Immediately after germination, the root tips of the seedlings were immersed separately in 5 ml of cytochalasin Β solution (2 and 50 µΜ respectively) for next 24 and 48 h, for the entry of the chemical inside the root-tip meristem. Three replicate sets were maintained for each treatment. The total length of each seedling was measured before and after cytochalasin Β treatment and the mean values were recorded. The root tips were then excised, washed with water and fixed in acetic acid and ethanol (1:2) for 3 h and finally stained with 2% orcein:HCl (9:1) for 45 min. The root tips were finally squashed on a slide and observed under transmitted light microscope. Binding of cytochalasin Β with mung bean seedling actin Binding of cytochalasin Β with mung bean actin was done according to the method of Hummel and Dreyer (1962). Protein concentration of each fraction was measured according to the method of Lowry et al. (1951). Radioactivity was determined by spotting 200 µl of each fraction on glass fibre filters (Whatman GF/C) and counting the filters in a Beckman LS-1800 liquid scintillation counter. Results An actin like protein purified by Sepharose 4B column chromatography from mung bean seedling (Ghosh et al., 1988) was analysed by the competitive ELISA and the results are depicted in table 1. A standard curve was constructed using pure actin from chicken gizzard (Sigma) (figure 1). Purified tubulin from goat brain was used as a nonspecific antigen. No activity could be measured when the same experiment was repeated with an antibody raised against the enzyme Inositol-1-phosphate dehydrogenase (obtained as gift from Mrs. M. Rudra, Biochemistry Department, Bose Institute). The lethal dose of cytochalasin Β was determined prior to studying its effect on cell division during germination of mung bean seeds. Pilot experiment using various amounts of cytochalasin Β demonstrated that germination was completely inhibited Table 1. ELISA for mung bean actin.

Assay was done as indicated in methods. The values are average of 5 determinations.

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Figure 1.

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at a lethal dose of 500 µΜ cytochalasin B. Insignificant germination was observed at sublethal doses, which were close to the lethal dose. Doses like 2 and 50 µΜ were chosen for this study; germination was poor at 50 µΜ concentration when compared to that of control. The normal cell division profile of the root tip cell of mung bean seedling is presented in figure 1B, C. Figure 1B shows the picture of an early anaphase, where chromosomes are separated from chromatids and movement of the chromosomes towards the pole has just started. Figure 1C represents the position of the chromosome in late anaphase where the chromosomes move closer to the poles. When the mung bean seedlings were treated with cytochalasin B, a significant variation in cell division profile was observed under the microscope (figure 2A, B). The haphazard distribution of chromatids at the equitorial plane is observed in figure 2A. Actually in the presence of 50 µΜ cytochalasin Β, the divisional frequency was less than the control set. Most of the (90%) dividing cells were in metaphase and less than 1% cells were in normal anaphase. Only 5% of the dividing cells showed chromatid separation, but no movement towards the pole was observed and chromatids were distributed randomly at the equitorial plane. After prolonged treatment (72 h) of cytochalasin Β, the spindle attachment was totally lost and the chromosomes were displaced from their original equitorial plane (figure 2B). The effect of the drug cytochalasin Β at 50 µm concentration on spindle attachment was found to be reversible as studied up to 24 h. In the presence of 2 µΜ cytochalasin Β, 75% of the mung bean seedling cells showed normal division and irregular spindle formation was observed in only 7% of the dividing cells. However less than 5% of the dividing cells showed chromatid separation, but without any movement. The length of the seedling before and after cytochalasin Β treatment is presented in table 2. The binding of actin (authentic) with cytochalasin Β is a reversible process and hence all the binding parameters are measured on basis of equilibrium dialysis (Lin and Lin, 1978) as the routinely used filter disc assay method is unsuitable for studying the reversible binding. However, the binding nature of plant actin with cytochalasin Β is still unknown. To study the binding of plant actin with cytochalasin Β, the method of Hummel and Dreyer (1962) is chosen because the method is (i) rapid, (ii) maintains the protein ligand complex in equilibrium conditions and (iii) specially suitable for the detection of relatively weak binding. In this method, mung bean seedling F-actin fraction was made 10 µΜ with [3H] cytochalasin Β and applied to a gel filtration column (Sephadex G-25), equilibrated with the same concentration (10 µΜ) of [3H] cytochalasin Β and the equilibrium value i. e., the base line level as evident from figure 3 are 10,000 cpm. The figure shows the elution profile of apparent cytochalasin binding, when actin was passed through the above mentioned column. As actin [3H] cytochalasin Β complex peak emerges at the excluded volume of the column, the total radioactivity in eluate rises above the equilibrium level at the position of actin elution. After the

Figure 1. (A) Standard curve for ELISA of the chicken gizzard actin (Sigma) used for coating the microtiter plates. Antibody (Amersham) was raised against chicken gizzard actin. (B) Control root tip cells showing early anaphase stage. In each case 3 different sets of experiments were done and 500 cells were counted. (C) Control root tip cells showing the position of chromosome in late anaphase stage.

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Figure 2. (A) 50 µM cytochalasin Β treated root tips (24 h). (B) 50 µM cytochalasin Β treated root tips (72 h). (C) Control root tip cells showing telophase.


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Table 2. Effect of cytochalasin Β on the growth of the mung bean seedling.

Figure 3. A Hummel-Dreyer column, Sephadex G-25 (25 × 1 cm) was used to measure the binding of cytochalasin Β with mung bean seedling actin. The column was equilibrated with 10 mM imidazole-chloride pH 7·5 containing 4 mM MgCl2 and 10 µΜ [3Η]cytochalasin B. 0·6 ml of mung bean seedling F-actin (5·71 mg/ml) mixed with column buffer containing 20 µM [3H]cytochalasin Β in a 1:1 ratio was applied to this column, followed by the elution with the column buffer. Protein concentration of the fractions (0·6 ml) was estimated by Lowry's method (Lowry et al., 1951). The radioactivity was determined by spotting 200 µl of each fraction on glass fibre filters (Whatman GF/C) and counting them in a Beckman LS-1800 liquid scintillation counter. All chromatography was performed at room temperature. Three different sets of experiments were done simultaneously. Mean value was plotted.

protein peak [3H] cytochalasin Β in the eluate decreases below the base line level to form a trough (which is equivalent to [3H] cytochalasin Β removed by protein actin). The amount of free [3H] cytochalasin Β removed from the column as displayed by this trough is equal to the amount of the drug found in the protein peak. The dissociation constant (Kd) of the actin cytochalasin Β complex was calculated from the following equation.

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In order to determine actin concentration, the peak fraction (figure 3) was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) along with purified rabbit actin in a parallel lane and finally stained with Commassie brilliant blue (R 250) (figure 4). After densitometric scanning, the peak area under actin region was measured and the actin concentration was calculated (table 3). The Kd value obtained in this case was 1·2 × 10–5 M (table 4).

Figure 4. 7·5% SDS-PAG electrophoretic pattern of purified actin. (a) Mung bean actin 67 µg. (b) Rabbit actin (Sigma) 10 µg. Table 3. Calculation of actin concentration from peak protein fraction by densitometric scanning.

Molecular weight of actin was considered as 42,000 Kd. Densitometrie scanning was performed using LKB 2202 ultrascan laser densitometer. Table 4. Binding of cytochalasin Β to actin like protein of mung bean seedling as measured by the Hummel-Dreyer method.

Discussion The results show evidences for the presence of actin like protein in mung bean seedling. Two independent means of studies based on immunological method and


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typical cytochalasin Β binding lead to the conclusion that this protein from higher plant has some similarity to actins from animal sources. However, studies on immunochemical characterization of actin from higher plants where also it has not been possible to raise antibody against plant actin have been reported (Metcalf III et al., 1980; Ilker et al., 1979). ELISA test with mung bean actin like protein shows that though some common antigenic sites exist between plant and animal actin, all the sites are not identical or available for the interactions. This actin like protein from mung bean seedling has a detectable cytochalasin Β binding capacity with a Kd value 1·2 × 10–5 M. The binding of muscle actin with cytochalasin Β was measured using equilibrium dialysis and the Kd value was 10–8 M, signifying much higher affinity. However, uptil now there is no other report about the binding of plant actin with cytochalasin Β to compare the binding characteristics of mung bean seedling actin with cytochalasin Β. The inhibitory effect of cytochalasin Β on in vitro actin polymerization from mung bean seedling was already shown (Ghosh et al., 1988). Furthermore, the in vivo effect demonstrates that cytochalasin Β at a sublethal dose (50 µM) blocks the chromosomal movement at metaphase stage and growth of the seedling is also affected. It has been reported that cytochalasin Β induces cessation of cytoplasmic streaming mediated by microfilament (Williamson, 1972; Bradley, 1973) and prevents normal telophase reorientation movement which leads to abnormal division planes in dividing guard mother cell in Allium (Palevitz, 1980). The presence of actin containing microfilaments in mitotic spindle in plants have been reported conclusively on the basis of binding specifically to fluorescently labelled phallotoxins (Wulf et al., 1979, Pesacreta et al., 1982; Clayton and Lloyd, 1985), but this does not prove beyond doubt its involvement during the process. The mild and delayed inhibition of onion root mitosis by cytochalasin Β even at 30 µg/ml (Thomas et al., 1973) is quite distinct from the rapid growth inhibition accompanied by tip swelling and polyploidy, induced by colchicine (Levan, 1938). Several studies (Hepler and Palevitz, 1974; Fonte et al., 1978; Griffith and Pollard, 1982; Traas et al., 1985) showed the possible involvement of actin in microtubule orientation most notably during differentiation of trachery elements in Zinnia (Falconer and Seagull, 1985) and in the cytokinetic apparatus in general (Clayton and Lloyd, 1985; Gunning and Wick, 1985; Palevitz, 1987). In fact, the effect of cytochalasin Β in arresting mitosis in the present case might be due to indirect effects such as involvement of actin in microtubule orientation or the many known effects of cytochalasin on plant cell functions, which might well contribute to an arrest in mitosis. Correlating all the above facts, it is indicative that for normal cell division, there must be some involvement of actin association with chromosomal movement. In this case if the drug cytochalasin Β enters inside the cell, it is expected to bind the actin molecules (confirmed from the binding study), eventually block the polymerization of actin protomer to the fibrous form (judged from the in vitro polymerization assay) and finally stop the chromosomal movement at metaphase stage (as observed in figure 2A). It is also observed that cytochalasin Β treated mung bean seedling cell extract is less potent in polymerizing ability, when compared with untreated cell extract (Ghosh et al., 1988). It is perhaps due to the capping of the actin molecule by cytochalasin Β. In dividing rat Kangaroo cell, actin was found to be present in discrete bundles connecting individual chromosomes to the poles (Sanger, 1975). Earlier,

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microtubules were thought to be the only contractile protein factor for responsible the movement of chromosome during cell division (Inoue and Sato, 1967). Since both actin and microtubules are connected to the chromosome, there may be some co-operation between the two proteins to produce the necessary contractile force for chromosome movement (Brown and Spudich, 1979). The idea that actin containing filament might interact with microtubules to disrupt the mitotic spindle has been considered by Forer (1974), Nicklas (1977) and Oakley and Heath (1978). Forer et al. (1979) have shown in Haemanthus endosperm that microtubules can interact with actin containing filaments which produce force during mitosis. It is relevant to point out that our observations are consistent with those of others (Forer et al., 1979; Forer and Jackson, 1979) in showing that actin is playing an active role in chromosome movement along with tubulin. Acknowledgements Thanks are due to the Council of Scientific and Industrial Research, New Delhi for the financial support and also to the Bose Institute for providing a research fellowship. References Brown, S. S. and Spudich, J. A. (1979) J. Cell Biol., 83, 657. Bradley, M. O. (1973) J. Cell Sci., 12, 327. Clayton, L. and Lloyd, C. W. (1985) Exp. Cell Res., 156, 231. Fagraeus, A. and Norberg, R. (1978) Curr. Top. Microbiol. Immunol., 82, 1. Falconer, Μ. Μ. and Seagull, R. W. (1985) Protoplasma, 125, 190. Fonte, V, G., Anderson, K. L., Wolosewich, J. J. and Porter, K. R. (1978) J. Cell Biol., 7, 74a (Abstract). Forer, A. (1974) in Cell cycle controls (eds G. M. Padilla, I. L. Cameron and A. M. Zimmerman) (New York: Academic Press) p. 319. Forer, A. and Jackson, W. T. (1979) J. Cell Sci., 37, 323. Forer, Α., Jackson, W. T. and Engberg, A. (1979) J. Cell Sci., 37, 349. Ghosh, G., Biswas, S. and Pal, A. (1988) J. Plant Physiol, Biochem., 15, 153. Griffith, L. M. and Pollard, T. D. (1982) J. Biol. Chem., 257, 9143. Gunning, B. E. S. and Wick, S. M. (1985) J. Cell Sci. (Suppl.), 2, 157. Hepler, P. K. and Palevitz, B. A. (1974) Annu. Rev. Plant Physiol., 25, 309. Hummel, J. P. and Dreyer, W. J. (1962) Biochim. Biophys. Acta, 63, 530. Ilker, R. Α., Breidenbach, R. W. and Murphy, T. M. (1979) Phytochemistry, 18, 1781. Inoue, S. and Sato, H. (1967) J. Gen. Physiol, 50, 259. Levan, A. (1938) Hereditas, 24, 471. Lin, D. C. and Lin, S. (1978) J. Biol. Chem., 253, 1415. Lowry, O. H., Rosebrough, A. L. and Randall, R. (1951) J. Biol. Chem., 193, 265. Metcalf III, T. N., Szabo, L. J., Schubert, K. R. and Wang, J. L. (1980) Nature (London), 285, 171. Nicklas, R. B. (1977) in Mitosis (eds M. Little, Ν. Paweletz, C. Petzelt, H. Ponstingl, D. Schroeter and H. P. Zimmerman) (Berlin, Heidelberg, New York: Springer-Verlag) p. 150. Oakley, B. R. and Heath, I. B. (1978) J. Cell Sci, 31, 53. Palevitz, B. A. (1980) Can. J. Bot., 58, 773. Palevitz, Β. Α. (1987) J. Cell Biol., 104,1515. Perry, Μ. Μ., John, Α. Η. and Thomas, Ν. S. Τ. (1971) Exp. Cell Res., 65, 249. Pesacreta, Τ. C, Carley, W. W., Webb, W. W. and Parthasarathy, M. V. (1982) Proc. Natl. Acad, Sci. USA, 79, 2898. Pollard, T. D. and Weighing, R. R. (1974) CRC Crit. Rev. Biochem., 2, 1. Sanger, J. W. (1975) Proc. Natl. Acad. Sci. USA, 72, 2451. Schmiedel, G. and Schnepf, E. (1980) Planta, 147, 405.


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