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Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases1 Sharon Grabski, Eric Arnoys, Benjamin Busch, and Melvin Schindler* Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 plasm can be physically linked to coordinate and communicate changes in cell structure and secretion, which are required for cell growth, migration, and differentiation. Rearrangements of the actin network in animal cells and yeast have been shown to precede changes in topology and diffusion of transmembrane proteins (Edelman, 1976; Sheetz et al., 1980; Jacobson et al., 1987; Barbour and Edidin, 1992), cell shape (Sims et al., 1992), cell movement (Lauffenburger and Horwitz, 1996; Mitchison and Cramer, 1996), cell polarity (Quatrano, 1990; Drubin and Nelson, 1996), embryogenesis (Bonder et al., 1989), differentiation (Dahl and Grabel, 1989; Rodriguez-Fernandez and Ben Ze’ev, 1989), and secretion (Drubin and Nelson, 1996). Of particular interest are the recent observations that dynamic interconversions of G- and F-actin may play a significant role in the regulation of ionic channels in the plasma membrane and in this manner control cell volume and osmoregulation (Schwiebert et al., 1994; Tilly et al., 1996). Similarly, in plant cells these networks have been proposed to mediate such cellular activities as changes in the topology and movement of membrane proteins (Metcalf et al., 1983, 1986), cell growth and proliferation (Lloyd, 1989; Derksen et al., 1995), cell polarity (Quatrano, 1990), embryogenesis (Kropf et al., 1989), secretion (Picton and Steer, 1983) and migration/cell wall interactions (as proposed for pollen tube elongation) (Lord and Sanders, 1992), division plane formation (Lloyd, 1989), shape and movement of the ER (Quader et al., 1987), viral transport (Zambryski, 1995), and organelle movement and cytoplasmic streaming (Williamson, 1993; Staiger et al., 1994). The principal signaling agents demonstrated to initiate changes within the actin network of animal cells are calcium (Janmey, 1994) and lipids, e.g. polyphosphoinositides and lysophospholipids (Ridley and Hall, 1992; Janmey, 1994). These second messengers can trigger structural changes through interactions with actin-binding proteins, e.g. profilin (Goldschmidt-Clermont et al., 1991; Cao et al., 1992; Janmey, 1994; Staiger et al., 1994), or through alterations in phosphorylation mediated by calmodulin and protein kinases, particularly through the regulation of MLCK activity (Kolodney and Elson, 1993; Mobley et al., 1994; Goeckeler and Wysolmerski, 1995; Chrzanowska-
Changes in the organization and mechanical properties of the actin network within plant and animal cells are primary responses to cell signaling. These changes are suggested to be mediated through the regulation of G/F-actin equilibria, alterations in the amount and/or type of actin-binding proteins, the binding of myosin to F-actin, and the formation of myosin filaments associated with F-actin. In the present communication, the cell optical displacement assay was used to investigate the role of phosphatases and kinases in modifying the tension and organization within the actin network of soybean cells. The results from these biophysical measurements suggest that: (a) calcium-regulated kinases and phosphatases are involved in the regulation of tension, (b) calcium transients induce changes in the tension and organization of the actin network through the stimulation of proteins containing calmodulin-like domains or calcium/calmodulin-dependent regulatory proteins, (c) myosin and/or actin cross-linking proteins may be the principal regulator(s) of tension within the actin network, and (d) these actin cross-linking proteins may be the principal targets of calcium-regulated kinases and phosphatases.
Physical tension has been implicated as a vectorial regulator of actin dynamics, assembly, and organization within cells (Pasternak et al., 1989; Janson and Taylor, 1993; Kolodney and Elson, 1993; Heidemann and Buxbaum, 1994; Goeckeler and Wysolmerski, 1995; ChrzanowskaWodnicka and Burridge, 1996). These physical changes in actin filament organization and tension have been demonstrated to occur primarily through the regulation of G/Factin equilibria (Cao et al., 1992; Janmey, 1994; Staiger et al., 1994; Wyman and Arcaro, 1994), alterations in the amount and type of actin-binding proteins (Matsudaira, 1991; Janmey, 1994), and the assembly of myosin filaments and subsequent binding of filamentous myosin to F-actin (Citi and Kendrick-Jones, 1987; Giuliano et al., 1992; Kolodney and Elson, 1993; Cramer and Mitchison, 1995; Goeckeler and Wysolmerski, 1995; Chrzanowska-Wodnicka and Burridge, 1996). The binding of myosin results in the formation of contractile actomyosin strands with distinct polarities and connections between the plasma membrane, intracellular organelles, and transcytoplasmic actin strands (Giuliano et al., 1992; Drubin and Nelson, 1996; Mitchison and Cramer, 1996). In this manner, the plasma membrane and cell cyto-
Abbreviations: BDM, butanedione monoxime; CaMK, calcium/ calmodulin-dependent protein kinase; CDPK, calmodulin-like domain protein kinase; CODA, cell optical displacement assay; MLCK, myosin light-chain kinase; W-7, N-(6-aminohexyl)-5chloro-1-naphthalenesulfonamide.
1
This work was financially supported by the Rackham Foundation. * Corresponding author; e-mail
[email protected]; fax 1–517–353–9334. 279
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Wodnicka and Burridge, 1996), phosphatases (Fernandez et al., 1990; Inoue et al., 1990; Ferreira et al., 1993), and a recently described rho kinase and myosin phosphatase (Kimura et al., 1996). Modulation of the integrity of the actin network through the regulation of F-actin assembly, the amount and type of actin-binding proteins, and myosin binding and filament formation can, therefore, provide regulatory points for signal-mediated reorganizations of the actin network within specific domains of the cytoplasm. Such reorganizations may then promote topologically specific changes in the transport of ions and metabolites across the plasma membrane within those regions (Schweibert et al., 1994; Derksen et al., 1995; Tilly et al., 1996). During the past few years our laboratory has pursued measurements of tension within the actin network of soybean cells utilizing CODA (Grabski et al., 1994; Grabski and Schindler, 1995, 1996; Schindler, 1995). This method employs an optical trap (Ashkin and Dziedzic, 1989) to manipulate actin-containing strands. Measurements of strand displacement can be used to determine their viscoelastic properties. Such studies have provided evidence for the role of plant hormones and growth factors in modifying the tension within the actin network. These modifications in tension are proposed to result from the hormoneinduced formation of calcium gradients and lipid-signaling molecules, similar to the effectors of the actin network described in animal cells (Brownlee and Wood, 1986; Quatrano, 1990; Obermeyer and Weisenseel, 1991; Drøbak, 1993; Grabski et al., 1994; Janmey, 1994; Cote´, 1995; Grabski and Schindler, 1995, 1996). In the present communication we provide an initial identification of potential targets and regulatory molecules that are affected by secondary messengers and that are proposed to transduce the biophysical alterations in the elastic properties of the actin network. Results from these experiments provide support for the involvement of calciumregulated protein kinases (CDPK and/or CaMK) and phosphatases (calcineurin or calcineurin-type) as transducers of actin tension within the plant cell cytoskeleton, and suggest that myosin and/or actin cross-linking proteins may be the target of these regulatory molecules.
MATERIALS AND METHODS Reagents BDM, the ionophore A23187, staurosporine, W-7, calmidazolium (compound R24571), cyclosporin A, cytochalasin D, and phalloidin were obtained from Sigma. KT5926 (a kinase inhibitor), okadaic acid, 1-Nor-okadaone, cypermethrin, permethrin, allethrin, and N-(6-aminohexyl)-1napthalenesulfonamide were purchased from LC Laboratories (Woburn, MA). Aluminum chloride hexahydrate was purchased from Aldrich. Bodipy-phallacidin was obtained from Molecular Probes (Eugene, OR). All other reagents employed were of the highest purity obtainable.
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CODA
Drug Incubation Conditions Soybean (Glycine max [L.] Merr. cv Mandarin) cells (maintained in suspension culture and originally derived from roots) were grown in 1B5C media (Metcalf et al., 1983). For a typical incubation, 2- to 3-d-old cells (following split) were removed from the growth medium and washed with media (33). Cells were then incubated with the primary drug for 30 min at room temperature in a shaker and then examined. If necessary, the second drug was added and incubation was continued for an additional 15 min. Cells (2-mL suspension) from the incubation mixtures were placed on a slide and sealed under a coverslip using melted paraffin as the sealant. All CODA measurements were performed within 45 min following the last incubation time.
CODA Measurements Slides containing the incubation mixture were placed on a fluorescence interactive laser cytometer (ACAS 570; Meridian Instruments, Okemos, MI) (Wade et al., 1993) and viewed under phase illumination with an oil immersion objective 3100 (1.4 numerical aperture). In vivo measurements of tension within transvacuolar strands in soybean root cells was performed using the CODA technique, which is fully described in Grabski et al. (1994) and Schindler (1995). The assay uses a laser beam to trap and hold cellular structures at the focal plane of a focused beam of laser light. A laser trap (Ashkin and Dziedzic, 1989; Grabski et al., 1994; Grabski and Schindler, 1995, 1996; Schindler, 1995) was produced using an Ar ion laser beam (excitation wavelength 5 488 nm; at 1 mm in diameter) and focused onto a vesicle associated with a transvacuolar strand. The intensity of the laser beam is increased monotonically until the vesicle is trapped. The microscope stage is then moved through a defined distance at a constant velocity; this results in the displacement of the vesicle and associated actin-containing strand through the cytoplasm. The CODA quantifies these displacements in the following manner. Displacement curves are generated by performing 20 displacement attempts at a series of laser power settings. A parameter, termed the displacement threshold, is defined as the minimum laser power necessary to produce 20 out of 20 successful displacements. The displacement threshold did not vary between day-to-day measurements by more than 5 mW as recorded at the laser head (12% variation). The displacement threshold50, listed in Tables I and II, is the value in milliwatts of laser power that results in displacement of 10 out of 20 strands and it is used to compare tension values. A full explanation of the technique and the control experiments necessary to ensure that laser illumination does not damage the cell or the actincontaining strands is provided in Grabski et al. (1994) and Schindler (1995). All trapping experiments were recorded on videotape.
Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases Confocal Fluorescence Microscopy
Confocal Fluorescence Imaging Samples were prepared for confocal microscopy and quantitative imaging in the following manner.
Whole-Cell Analysis
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ological relevence of these observed changes in tension is inferred from the use of concentrations of plant hormones and calcium, which have previously been shown to affect the growth and physiology of plant cells. The following experiments were designed to determine the primary and regulatory targets of these effectors. Inhibition of Kinase Activity by KT5926 and Staurosporine
Cells (2–3 d following split) were incubated on a shaker with the primary drug for 30 min and the secondary drug (when applicable) for 15 min. An aliquot (250 mL) of the incubation mixture (1 mL) was then treated with an equal volume of actin stabilization/extraction buffer (0.03% NP40, 5% DMSO, 0.1 m mannitol, 100 mm Pipes, 10 mm EGTA, and 5 mm MgSO4, pH 6.9) (Traas et al., 1987) containing Bodipy phallacidin (15 mL, 300 units/1.5 in methanol). Cells were then further incubated at room temperature for 15 min on a shaker. Following incubation, the cells were washed 33 with a solution of media containing the particular drug (1 mL/wash with a 5-min incubation step in the wash media between washes). Cells were then analyzed in the media/drug solution within 30 min of the last wash. Individual optical sections of the fluorescence distribution of the Bodipy-phallacidin were acquired with an InSight Bilateral Laser Scanning Confocal microscope (Meridian Instruments), as previously described (Wade et al., 1993; Grabski et al., 1994). Cells shown in Figures 2 and 3 are characteristic for at least 80% of the sample under investigation.
Studies with animal cells have demonstrated that phosphorylation of myosin light chains by MLCK is the principal mechanism for the assembly and generation of tension within microfilaments (Kolodney and Elson, 1993; Mobley et al., 1994; Goeckeler and Wysolmerski, 1995; Chrzanowska-Wodnicka and Burridge, 1996; Mitchison and Cramer, 1996). These studies also provided biochemical evidence that the drug KT5926 was an extremely effective and specific inhibitor of MLCK (Kolodney and Elson, 1993; Goeckeler and Wysolmerski, 1995; ChrzanowskaWodnicka and Burridge, 1996). When added to cultured animal cells, KT5926 resulted in a decrease in myosin lightchain phosphorylation that correlated with actin filament disassembly and loss of tension (Goeckeler and Wysolmerski, 1995; Chrzanowska-Wodnicka and Burridge, 1996). As observed in Table I, incubation of soybean cells with KT5926 (50 nm) results in a significant decrease in the Table I. Regulation of tension within the actin network by calcium-regulated kinases and phosphatases Treatment
F-Actin Analysis F-actin filaments were prepared from rabbit muscle acetone powder (gift of Dr. Steven Heidemann, Michigan State University, East Lansing) following the procedure from Pardee and Spudich (1982). F-actin (120 mg/mL; in 2 mm Tris, pH 8.0, 0.2 mm Na2-ATP, 0.2 mm CaCl2, 0.5 mm 2-mercaptoethanol, 50 mm KCl, and 2 mm MgCl2) was stained with Bodipy-phallacidin (0.44 mm) (Molecular Probes) and deposited onto a slide. A cover-slip was placed on the sample and sealed with paraffin wax. Incubation of F-actin with BDM (10 mm) was performed as described for whole-cell experiments except the buffer employed was the F-actin buffer described above. The BDM was maintained in the incubation mixture throughout the confocal-imaging experiment. RESULTS In previous measurements using CODA, we demonstrated that plant hormones and growth factors (principal physiological regulators) can initiate rapid changes in the tension within the actin network of soybean cells grown in suspension culture (Grabski et al., 1994; Grabski and Schindler, 1996). We also showed that these changes may be transduced by the experimental formation of calcium transients following the addition of the ionophore A23187, by alterations in cytoplasmic pH, and by the direct addition of lipid second messengers (Grabski et al., 1994). The physi-
Displacement Threshold50a
Stability of Actin Filamentsb
mW
Control KT5926 (50 nM) A23187 (2 mM) KT5926 (50 nM) 1 A23187 (2 mM) Staurosporine (1 nM) Calmidazolium (3 mM) W-7 (15 mM) N-(6-aminohexyl)-1naphthalenesulfonamide (15 mM)c Calmidazolium (3 mM) 1 A23187 (2 mM) W-7 (15 mM) 1 A23187 (2 mM) Calmidazolium (15 mM) W-7 (75 mM) Okadaic acid (0.1 mM) 1-Nor-okadaonec (0.1 mM) Cyclosporin A (10 mM) Cypermethrin (0.1 nM) Permethrinc (0.1 nM) Allethrinc (0.1 nM) a
15–20 ,5 40 – 45 5–10 5 5 10 15–20
11 1 111 11 1 1 1 11
5
1
10 85 50 35– 40 20 –25 55– 60 40 – 45 15–20 15–20
1 111 111 111 11 111 1111 11 11
The displacement threshold50 is described in “Materials and b Methods” and Grabski et al. (1994). The stability of the actin filaments is semiquantitative and is derived from the examination by confocal fluorescence microscopy of fields of soybean cells (20 –30) stained with Bodipy-phallacidin, as described in “Materials and c Methods.” Inactive or less active analogues. 1, Indicates filamentous structures. The more filamentous the structures, the greater the number of 1.
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tension within the actin network, as indicated by a decrease in the displacement threshold below the control (Grabski et al., 1994). This suggests a lower resting tension for the actin filaments. Similar results are observed for a more general inhibitor of protein kinases, staurosporine (1 nm) (Table I). Staurosporine has previously been demonstrated to be an effective inhibitor of kinases in soybean cells (Chandra and Low, 1995). An effect of staurosporine on the actin network was also reported for fibroblasts. In these studies staurosporine caused the disassembly of the actin network (Mobley et al., 1994). We demonstrated earlier (Grabski et al., 1994) that incubation of soybean cells with A23187 increased the tension within the actin network (observed as an increase in the displacement threshold50) (Table I). Because a potential target for this tension-enhancing activity of calcium may involve the regulation of MLCK and/or a calciumdependent protein kinase, an experimental prediction would be that cells pretreated with KT5926 would no longer demonstrate enhanced tension within the actin strands following the addition of calcium. To test this prediction cells were first treated with KT5926 (50 nm) and then incubated with the ionophore A23187 (2 mm) in the presence of 1 mm calcium in the media. As shown in Table I, pretreatment of cells with KT5926 prevents the induction of tension caused by A23187 alone (Table I). Inhibition of Calmodulin Activity by Calmidazolium and W-7 Because MLCK, CDPK, and CaMK activity are regulated by calcium (Citi and Kendrick-Jones, 1987; Tan et al., 1992), we predicted that inhibitors of calmodulin stimulation of kinase activity would decrease the tension observed within the actin network and would also inhibit the tensionenhancing effect of calcium and A23187 (Table I). To determine whether calmodulin regulation was a component of the tension induction pathway in soybean cells, the drugs calmidazolium and W-7 were added to the cells in separate experiments. These drugs are chemically distinct but have been demonstrated to inhibit calmodulin and the activity of CDPKs in plants (Harper et al., 1991; Obermeyer and Weisenseel, 1991; Ling and Assman, 1992; Schaller et al., 1992; Shimazaki et al., 1992; Dasgupta, 1994; Estruch et al., 1994). Treatment of cells with calmidazolium (3 mm) or W-7 (15 mm) (both inhibitors used at the lowest concentration of effectiveness) resulted in a decrease in the resting tension within the actin network as was observed for treatment with KT5926 (Table I). N-(6-aminohexyl)-1naphthalenesulfonamide (W-5), a less-effective inhibitor of calmodulin or CDPK, normally utilized as a control, demonstrated little influence on the resting tension within the actin network (Table I). The changes observed with calmidazolium and W-7 are related to an inhibition of calmodulin activity and are not a result of a significant increase in cytosolic calcium induced by calmodulin inhibitors as reported by Gilroy et al. (1987), because at the concentration of calmidazolium employed in these experiments, Gilroy et al. (1987) observed only a slight increase in calcium. As predicted, pretreatment of cells with calmidazolium or W-7
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also prevented the increase in tension observed with A23187 alone (Table I).
Inhibition of Phosphatase Activity by Okadaic Acid, Cyclosporin A, and Cypermethrin The previous experiments provide evidence for the involvement of kinases in the modulation of tension within the actin network. The results also suggest that these kinases are regulated by calmodulin and/or calmodulin-like domains. This suggested a companion role for phosphatases in regulation. Recent experiments have shown in animal cells that the rho proteins can regulate actin filament assembly and organization (Ridley and Hall, 1992; Nobes et al., 1995). This regulation by rho is proposed to be mediated through the activity of a myosin phosphatase (Kimura et al., 1996). To determine whether phosphatases might be elements of a regulatory loop that modulates the physical properties and organization of the actin network in plant cells, we employed a number of inhibitors specific for phosphatases and determined their effect on the tension within the actin network. Okadaic acid, an effective phosphatase inhibitor (Cohen et al., 1990), was initially employed for CODA measurements. Incubation of cells with okadaic acid (0.1 mm) resulted in an increase in tension within the actin network (Table I). Such an increase in tension had previously been demonstrated in smooth muscle following the addition of okadaic acid (Obara et al., 1989). In contrast, incubation of cells with the inactive analog 1-Nor-okadaone (0.1 mm) resulted in no change (Table I). To determine whether a unique class of phosphatases is involved in regulation, we next used a more specific group of phosphatase inhibitors. Cyclosporin A and cypermethrin are inhibitors of the calmodulin-dependent protein phosphatase 2B, also called calcineurin (Enan and Matsumura, 1992; Luan et al., 1993; Cunningham and Fink, 1994). Incubation of cells with either cyclosporin A (10 mm) or cypermethrin (0.1 nm) resulted in a considerable increase in tension within the actin network (Table I). In sharp contrast, both permethrin and allethrin, less effective analogs of cypermethrin, demonstrated no similar increase in tension (Table I). Using higher concentrations of calmidazolium (15 mm) and W-7 (75 mm) than previously employed (Table I) resulted in an increase in tension. This would be predicted for inhibitors of calmodulin-dependent phosphatases and is consistent with the results obtained with phosphatase inhibitors (Table I). The interpretation of these results, however, must again be viewed in the context of Gilroy et al. (1987), whose results showed that the use of such higher concentrations of calmodulin inhibitors can result in a rise of intracellular calcium levels. Nevertheless, this indirect effect is considered a less likely interpretation of the data because of the observation that the addition of A23187 following treatment with inhibitors of MLCK and/or calmodulin or calmodulin-like domains prevents the induction of tension (Table I). These observations provide support that the enhanced tension observed using the higher concentrations of calmodulin inhibitors may be
Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases more related to the inhibition of phosphatases than an independent effect of calcium. Effect of Calmodulin and Calmodulin-Like Domain Inhibitors on Aluminum-Induced Tension We recently demonstrated that the addition of aluminum to soybean cells in suspension culture results in a significant increase in the tension within the transvacuolar actin network (Grabski and Schindler, 1995). These changes occur at a concentration of aluminum normally found to inhibit root growth and pollen tube extension (Konishi and Miyamoto, 1983; Baskin and Bivens, 1995; Delhaize and Ryan, 1995). The results suggested that aluminum toxicity in plants may occur through a direct effect on the actin/ actomyosin network. To determine if the observed effect of aluminum on the tension within the actin network is dependent on the activity of phosphatases and kinases, we examined the tension-inducing activity of aluminum in the presence of inhibitors of kinases and phosphatases. As mentioned earlier, inhibitors of calmodulin (W-7 and calmidazolium) and calmodulin-dependent kinases (KT5926), e.g. CDPK and MLCK, induced a decrease in the tension within the actin network of soybean cells (Table I). These measurements were consistent with a role for these enzymes in the regulation of tension. Preincubation of cells with the calmodulin inhibitors W-7 (15 mm) and calmidazolium (3 mm) prevented the ability of aluminum to enhance tension (displacement threshold505 170 mW in presence of aluminum and displacement threshold50 5 10–15 mW in the presence of calmodulin inhibitors and aluminum). The loss of the tension-enhancement activity mediated by aluminum was also observed if cells were preincubated with an inhibitor of MLCK, KT5926 (displacement threshold505 20 mW in the presence of MLCK inhibitor and aluminum). These effects were reversible following the removal of the reagents (Grabski and Schindler, 1995; data not shown).
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Effect of BDM on Actin Organization and Myosin Activity The previous measurements had demonstrated that incubation of soybean cells with inhibitors of calciumdependent regulatory molecules could modify the tension within the actin network. Because these regulators can function to control myosin activity and filament formation, BDM was used as a specific reagent for inactivating myosin. BDM is a drug that has previously been demonstrated to inhibit the ATPase activity of myosin (Higuchi and Takemori, 1989; Cramer and Mitchison, 1995; ChrzanowskaWodnicka and Burridge, 1996). Limited hydrolysis of ATP can occur following modification by BDM, but the products of hydrolysis, PO4, and ADP remain associated with the myosin in a nonproductive complex (McKillop et al., 1994; Zhao et al., 1995). This form of myosin can maintain a loose association with actin filaments (Zhao et al., 1995). BDM apparently has no effect on actin filaments, as shown by Cramer and Mitchison (1995) and in imaging experiments examining the structure of F-actin filaments in solution. Neither the organization nor the integrity of purified F-actin filaments is affected by BDM (Fig. 1). Addition of BDM (10 mm) to soybean cells resulted in a significant increase in actin tension (Table II). As previously observed in animal cells, removal of BDM from the media results in the normalization of tension to control levels (Table II). Pretreatment of cells with BDM followed by incubation with KT5926 (50 nm) prevented the increase in tension induced by BDM alone (Table II).
Stability of the Actin Network in the Presence of Aluminum and Inhibitors of CDPKs, CaMKs, and Phosphatases To determine whether the changes in tension observed with CODA were related to alterations in the stability and the state of assembly of the actin network, experiments were performed to image the organization of the actin
Figure 1. Confocal fluorescent images of F-actin filaments treated with BDM and stained with Bodipy-phallacidin. F-actin filaments were prepared as described in “Materials and Methods,” stained with Bodipy-phallacidin, and then examined utilizing confocal fluorescence microscopy. F-actin strands are shown in the absence (a) and presence (b) of BDM (20 mM). Bar represents 2 mm.
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Table II. Effect of BDM on the tension within the actin network Treatment
Displacement Threshold50a
Control BDM (10 mM) BDM (10 mM) recovery BDM (10 mM) 1 KT5926 (50 nM)
15 –20 80– 85 15 –20 15 –20
Stability of Actin Filamentsb
mW
11 1111 11 111
a
The displacement threshold50 is described in “Materials and b Methods” and Grabski et al. (1994). The stability of the actin filaments is semiquantitative and is derived from the examination by confocal fluorescence microscopy of fields of soybean cells (20 –30) stained with Bodipy-phallacidin, as described in “Materials and Methods.” 1, Indicates filamentous structures. The more filamentous the structures, the greater the number of 1.
network in the presence of inhibitors and aluminum under conditions described in Tables I and II. Cells were treated with the modulatory drugs and then permeabilized and stained with Bodipy-phallacidin (F-actin-binding fluorescent probe) (see “Materials and Methods”). Permeabilization and staining were performed in the presence of the drugs. Cells were then washed, as described in “Materials and Methods,” in the absence of actin stabilization buffer normally employed to image the stained actin network (Traas et al., 1987), and the integrity of the actin network was assessed (Wade et al., 1993). In previous studies in which the distribution of actin filaments within plant cells was examined, it was observed that the F-actin filaments were sensitive to fragmentation in the permeabilized cells in the absence of a stabilization buffer and fixative. This is best observed in Figure 2 in which soybean cells are labeled with Bodipy-phallacidin and are examined in the absence (Fig. 2a) and presence (Fig. 2b) of actin stabilization buffer. Because the inclusion of stabilization buffer in the analysis of the actin network is predicated on providing the most-pronounced retention of F-actin filaments, we felt that the use of the stabilization buffer in our whole-cell experiments could mask both steady-state organization of the actin network and alterations in filament structure induced by drugs or by changes in the activity of cell regulatory molecules. This masking could occur as a consequence of a shift in the G-/F-actin equilibria toward more F-actin assembly that could be promoted by the stabilization buffer either directly or indirectly by influencing the activity of myosin and/or actin-binding proteins. This is an example of a potentially serious experimental artifactual rearrangement that is not representative of the metabolic or signaling state of the cell, and can be introduced throughout such imaging experiments when utilizing stabilization buffer in whole cells in the absence of fixation. As can be seen in optical sections prepared with a laserscanning confocal microscope (Fig. 2), control cells in the absence of stabilization buffer demonstrated a fragmented actin network (Fig. 2a). Treatment of cells with calmidazolium (Fig. 2c) or KT5926 (Fig. 2d), both drugs shown to decrease the tension within the actin network (Table I), also resulted in disperse and fragmented staining. In marked
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contrast, cells treated with drugs that increased the tension within the actin network (Tables I and II), demonstrated a more assembled filamentous actin network. Examples of this type of staining are shown for the incubation of cells with BDM (Fig. 2e) and cypermethrin (Fig. 2f). This is in contrast with the absence of an effect of BDM on actin integrity, which we observed using purified F-actin filaments (Fig. 1), under incubation conditions in which F-actin filaments can be disrupted by Ca21-gelsonin and aluminum (E. Arnoys and M. Schindler, unpublished data). Confocal fluorescence imaging of the F-actin distribution in the presence of aluminum and inhibitors of CDPKs and/or CaMKs is illustrated in Figure 3. As observed in Figure 3, the addition of aluminum (50 mm) resulted in the stabilization of transvacuolar actin strands (Fig. 3a) in the absence of actin stabilization buffer (to be compared with the control in Fig. 2a). However, pretreatment of cells with calmidazolium (3 mm) followed by incubation with aluminum (50 mm) (Fig. 3b) demonstrated a disassembled actin network that was indistinguishable from that observed following treatment of cells with calmidazolium alone (Fig. 2c). The image of a disassembled actin network shown in Figure 2a is similar to images obtained with cells pretreated with W-7 (3 mm) and KT5926 (50 nm) and then exposed to aluminum (50 mm) (data not shown). DISCUSSION Signal-mediated changes in both the actin network and the activities of associated signal transduction proteins are primary events in reorganizing the structure, symmetries, polar functioning, and interactions of cells. In plant cells such cytoskeletal changes have been implicated in cell growth and proliferation, cell wall deposition, viral transport between cells, rhizobial-legume symbiosis, cytoplasmic streaming, nuclear migration, and response to environmental signals, e.g. growth factors, hormones, pathogens, and movement of membrane proteins (Metcalf et al., 1983; Picton and Steer, 1983; Quader et al., 1987; Kropf et al., 1989; Lloyd, 1989; Quatrano, 1990; Williamson, 1993; Staiger et al., 1994; Cote´, 1995; Derksen et al., 1995; Zambryski, 1995). Chemical signals demonstrated to initiate changes in the organization and physical properties of cytoskeletal networks in plant cells consist of topologically specific changes in: cytoplasmic calcium concentration (Brownlee and Wood, 1986; Derksen et al., 1995), proton concentration (Gibbon and Kropf, 1994), and synthesis of signal transduction molecules, e.g. lipids (Drøbak, 1993; Grabski et al., 1994; Janmey, 1994; Cote´, 1995). A number of molecules have been characterized as potential transducers and mediators of cytoskeletal signaling. Both actin and tubulin, as the principal structural elements of the microfilament and microtubule networks, have been shown to function in a manner analogous to their observed activities in animal cells (Tiwari et al., 1984; Traas, 1990). Homologs for myosins I, II, and V have been demonstrated in plant cells (Kinkema and Schiefelbein, 1994; Qiao et al., 1994; Miller et al., 1995). CDPKs and calmodulin in addition to different classes of phosphatases have been shown to be components of plant cell-signaling pathways (Harper et al.,
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Figure 2. Confocal optical sections of soybean cells treated with drugs affecting the stability of F-actin networks. Incubation conditions and fluorescence imaging are described in “Materials and Methods.” In the absence of actin-stabilization buffer, the F-actin network (as represented by Bodipy-phallacidin staining) is fragmented (a). In the presence of actin-stabilization buffer, F-actin filaments are observed within the cell (b). A more pronounced fragmentation is observed when cells were treated with calmidazolium (3 mM) (c), or KT5926 (50 nM) (d). In contrast, the actin network and F-actin fibers are stabilized in the absence of stabilization buffer when cells were incubated in BDM (10 mM) (e) or cypermethrin (0.1 nM) (f). Bar in a represents 1 mm.
1991; Ling and Assman, 1992; Schaller et al., 1992; Shimazaki et al., 1992; Estruch et al., 1994; Leung et al., 1994). Regulation of Tension within the Actin Network by Calcium-Dependent Kinases and Phosphatases Results from CODA experiments utilizing calmodulin inhibitors suggest that the effect of calcium on the tension within the actin network is calmodulin dependent and involves the coordinate regulation of both a CDPK and/or
CaMK and a calmodulin-dependent phosphatase. The results with the calmodulin inhibitors proved more complex than those observed with kinase and phosphatase inhibitors in that there was a concentration-dependent effect on tension (Table I). These dose-dependent results may be interpreted to suggest that there is a greater sensitivity to calmodulin inhibitors of calmodulin or calmodulin-like domains associated with MLCK and/or CDPK than the calmodulin associated with the calmodulin-dependent phosphatase. Results utilizing KT5926 (Table I) and BDM (Table II) provide some evidence that a principal target of kinase
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Figure 3. Confocal optical sections of soybean cells treated with aluminum in the absence and presence of calmidazolium. Incubation conditions and fluorescence imaging procedures are as described in “Materials and Methods.” Addition of aluminum (50 mM) to cells resulted in long, thick filaments that are stable in the absence of actin-stabilization buffer (a). Preincubation of cells with calmidazolium (3 mM) followed by the addition of aluminum (50 mM) prevented aluminum stabilization of the actin network (b).
activity may be either myosin or an actin-binding protein that is capable of bundling or cross-linking F-actin filaments. Although it is not known whether BDM can affect actin-bundling proteins, kinases have been shown to phosphorylate actin-binding proteins (Ohta and Hartwig, 1995). In vitro measurements from our laboratory using purified F-actin filaments, rabbit muscle myosin, or chicken gizzard filamin (an actin cross-linking protein) demonstrate that aluminum can enhance the viscosity of F-actin solutions only in the presence of myosin or filamin (E. Arnoys and M. Schindler, unpublished data). A decrease in F-actin viscosity occurs in the absence of cross-linking proteins (E. Arnoys and M. Schindler, unpublished data). These results provide evidence that cross-linking or bundling of F-actin filaments, whether through myosin, filamin, or other similar proteins, may provide a major site of regulation for mediating changes in actin tension. In the context of our present understanding of plant cell kinases, it would appear that calcium propagates the signal
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by (a) stimulating the activity of a CDPK (Harper et al., 1991; McCurdy and Harmon, 1992; Schaller et al., 1992) or (b) an independent calmodulin-activated kinase capable of phosphorylating myosin or actin-binding proteins. Stabilization and cross-linking of F-actin can then occur by enhanced phosphorylation of the myosin light chain, resulting in phosphorylated myosin filaments, as observed for animal cells (Giuliano et al., 1992; Kolodney and Elson, 1993; Goeckeler and Wysolmerski, 1995; ChrzanowskaWodnicka and Burridge, 1996) or possibly through the activation of F-actin-binding proteins. Our results further suggest that phosphatases may be particularly important in the control of tension and organization (Kimura et al., 1996). The inhibitory experiments support an interpretation that calmodulin-dependent phosphatase 2B (calcineurin) or another calcineurin-type phosphatase may provide the “off-switch” for kinasemediated increases in the tension within the actin network in plant cells. Both cyclosporin A and cypermethrin, two functionally and structurally distinct inhibitors of calmodulin-dependent phosphatase activity, enhanced the tension and stabilized the structure of the actin network. The use of both of these agents is significant in evaluating the specificity of these inhibitors because their mechanisms of inhibition are distinct. Cyclosporin A must interact with plant cyclophilins to form a terniary inhibitory complex with calcineurin (Breiman et al., 1992; Luan et al., 1993; Marivet et al., 1995), whereas cypermethrin functions directly to inhibit phosphatases (Enan and Matsumura, 1992). It is noteworthy that recent work with ABA and stomatal regulation has demonstrated that a calcineurin-type phosphatase is an essential element of the regulatory mechanism (Luan et al., 1993; Leung et al., 1994). The observed changes in K1 channel activity may depend on cytoskeleton rearrangements. Such a relationship between channel activity and cytoskeletal rearrangements was recently reported for a number of membrane channels in animal cells, in particular K1 and Cl2 (Schweibert et al., 1994; Martin et al., 1995; Ehrhart et al., 1996; Tilly et al., 1996). These reports are noteworthy in that both K1 and Cl2 are two ions that are the principal regulators of volume and osmoregulation in plant cells (Cote´, 1995). In earlier measurements it was shown that modification of the cytoplasmic pH could also trigger changes in the actin network (Grabski et al., 1994). Raising or lowering the pH in soybean root cells resulted in a decrease in tension within the actin network. Huang et al. (1994) demonstrated that changes in pH could affect the activity of calmodulin in stimulating signal transduction. It will be important to determine whether pH modification of the tension within the actin network is a result of modifying calmodulin activity rather than an independent pathway. In Figure 4 the results of the present study have been compiled in the form of a “road map” for signal-induced modification of actin assembly and tension within plant cells. The involvement of a particular cellular component in regulation and signal transduction is deduced from the changes in actin tension and network stability produced following the addition of specific inhibitors, which are listed beneath each component in Figure 4.
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Figure 4. Calcium-mediated signaling and the regulation of actin assembly and tension. A “road map” is presented for calcium signaling within the actin network. Regulatory molecules are presented with the inhibitory drugs used for identification listed beneath them. Other potential components of the regulatory pathway that have not been specifically tested, e.g. kinases, microtubule-associated proteins (MAPs), and actin-binding proteins (ABPs), are also included on the map. Arrows indicate putative targets for calmodulin, kinases, and phosphatases.
Regulation of Aluminum Activity by Calcium-Regulated Kinases and Phosphatases Evidence has been presented from work with animal cells that aluminum can affect the activity of protein kinases and phosphatases (Johnson et al., 1990; Yamamoto et al., 1990; Domenech et al., 1992; el Sebae et al., 1993; Strong and Jakowec, 1994). An interaction of aluminum with phosphatases has been speculated to result in the formation of highly phosphorylated b-amyloid plaques capable of assembly into large aggregates within neurons (Shin et al., 1995). These highly phosphorylated proteins are suggested to enhance the progression of Alzheimer’s disease. Work reported in this communication has provided support for the role of calmodulin-regulated kinases and phosphatases in the modification of the organization and tension of actin filaments within the actin network of soybean cells. In this context, we propose that aluminum toxicity may be related to the ability of aluminum to maintain the actin network in an assembled state. This induction of rigor has been proposed to require functional CaMK and/or CDPK (W-7, calmidazolium, and KT5926 measurements, Table II; Fig. 3). The results using KT5926 and BDM suggest that the kinase activity may be necessary for the phosphorylation of myosin light-chain or actin-binding proteins and the resultant formation and maintenance of F-actin interactions by either myosin filaments or actin-binding proteins (Craig et al., 1983; Citi and Kendrick-Jones, 1987). Enhanced kinase activity or decreased phosphatase activity resulting from the addition of aluminum would result in phosphorylated myosin light chains or actin-binding proteins, the maintenance and assembly of actin filaments, and increased tension. Such decreased phosphatase activity might result from either a direct inhibition of phosphatase activity or, alternatively, by aluminum binding to phosphorylated sites, masking them from phosphatase activity. In the context of our hypothesis, Gassmann and Schroeder (1994) showed that aluminum blocked inwardrectifying K1 channels at concentrations correlating with Al31 phytotoxicity and the observed effect of aluminum on tension (Grabski and Schindler, 1995). It was previously
demonstrated that these K1 channels are activated by a calcineurin-type calmodulin-dependent phosphatase (Luan et al., 1993). Inhibition of phosphatase activity would result in the inactivation of these channels. As shown in this communication, calcineurin or a calcineurin-type calciumdependent phosphatase may be a significant regulator of tension within the actin network. Inhibition of this phosphatase by either cypermethrin or cyclosporin A results in a significant increase in tension and a stabilization of the actin network (Table I). The stabilized actin network observed with confocal fluorescence microscopy following treatment with phosphatase inhibitors appears similar to the structures observed following the addition of aluminum (Fig. 3a). These results provide evidence for the hypothesis that phosphorylated sites on either myosin (phosphorylated by MLCK and/or CDPK) or potentially on other actin-binding proteins can bind aluminum and prevent phosphatase access, or a calcium-dependent phosphatase may be directly inhibited and is the physiological target for aluminum toxicity. Both mechanisms may act concurrently.
Topological Regulation of the Actin Network Although our measurements are global, in that signaling molecules are added to the whole cell, it is not difficult to imagine that more localized changes in the concentration or activity of signaling molecules, in conjunction with topologically specific interactions of the actin network, could provide the site-specific modifications in the assembly and physical properties of the actin network associated with polarized cellular function and vectorial responses. Recent work in our laboratory has demonstrated a connection between anion channel activity and the assembly state of the actin network in soybean cells (M. Schindler and S. Grabski, unpublished data). Future efforts will now be directed toward the structural characterization of candidate regulatory proteins and the biochemical elucidation of the patterns of phosphorylationdephosphorylation that are proposed to be the integral
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modifiers of both the physical properties and organization of the actin network in response to signaling molecules. Received May 9, 1997; accepted September 17, 1997. Copyright Clearance Center: 0032–0889/98/116/0279/12.
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