Plant Physiol. (1998) 116: 279-290

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

	ABSTRACT

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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.

	INTRODUCTION

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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; Chrzanowska-Wodnicka and Burridge, 1996). These physical changes in actin filament organization and tension have been demonstrated to occur primarily through the regulation of G/F-actin 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 cytoplasm 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-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 hormone-induced 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; Coté, 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 calcium-regulated 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

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Reagents

CODA

Drug Incubation Conditions

CODA Measurements

Confocal Fluorescence Microscopy

Confocal Fluorescence Imaging

Whole-Cell Analysis

View larger version (168K): [in this window] [in a new window]   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 µm) (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 µm.

View larger version (101K): [in this window] [in a new window]   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 µm) to cells resulted in long, thick filaments that are stable in the absence of actin-stabilization buffer (a). Preincubation of cells with calmidazolium (3 µm) followed by the addition of aluminum (50 µm) prevented aluminum stabilization of the actin network (b).

F-Actin Analysis

	RESULTS

Top Abstract Introduction Methods Results Discussion References

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 physiological 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

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 calcium-dependent 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 µm) 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

Inhibition of Phosphatase Activity by Okadaic Acid, Cyclosporin A, and Cypermethrin

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 µm) 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 µm) and W-7 (75 µm) 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 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

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 µm) and calmidazolium (3 µm) prevented the ability of aluminum to enhance tension (displacement threshold₅₀= 170 mW in presence of aluminum and displacement threshold₅₀ = 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 threshold₅₀= 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).

Effect of BDM on Actin Organization and Myosin Activity

View larger version (91K): [in this window] [in a new window]   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 µm.

Stability of the Actin Network in the Presence of Aluminum and Inhibitors of CDPKs, CaMKs, and Phosphatases

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 laser-scanning 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 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 Ca²⁺-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 µm) 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 µm) followed by incubation with aluminum (50 µm) (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 µm) and KT5926 (50 nm) and then exposed to aluminum (50 µm) (data not shown).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

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; Coté, 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; Coté, 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., 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

In the context of our present understanding of plant cell kinases, it would appear that calcium propagates the signal 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; Chrzanowska-Wodnicka 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 kinase-mediated 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 K⁺ 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 K⁺ and Cl (Schweibert et al., 1994; Martin et al., 1995; Ehrhart et al., 1996; Tilly et al., 1996). These reports are noteworthy in that both K⁺ and Cl are two ions that are the principal regulators of volume and osmoregulation in plant cells (Coté, 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.

View larger version (42K): [in this window] [in a new window]   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

In the context of our hypothesis, Gassmann and Schroeder (1994) showed that aluminum blocked inward-rectifying K⁺ channels at concentrations correlating with Al³⁺ phytotoxicity and the observed effect of aluminum on tension (Grabski and Schindler, 1995). It was previously demonstrated that these K⁺ 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 calcium-dependent 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

Future efforts will now be directed toward the structural characterization of candidate regulatory proteins and the biochemical elucidation of the patterns of phosphorylation-dephosphorylation that are proposed to be the integral modifiers of both the physical properties and organization of the actin network in response to signaling molecules.

	FOOTNOTES

   Received May 9, 1997; accepted September 17, 1997.

	ABBREVIATIONS

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)-5-chloro-1-naphthalenesulfonamide.

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