Cell-specific association of heat shock-induced proton flux with actin ring formation in Chenopodium cells: comparison of auto- and heterotroph cultures

Anchalee Chaidee & Ilse Foissner & Wolfgang Pfeiffer

Abstract

A comparison of the responses of extracellular pH, buffering capacity and actin cytoskeleton in autotroph and heterotroph Chenopodium rubrum cells to heat shock revealed cell-specific reactions: alkalinization caused by the heat shock at 25–35°C was higher in heterotroph cells and characterized by heat shock-induced changes in the actin cytoskeleton and ring formation at 35–37°C. Rings (diameter up to 3 μm) disappeared and extracellular pH recovered after the heat-shocked cells were transferred into control medium. At 41°C, no rings but a network of coarse actin filaments were induced; at higher temperatures, fragmentation of the actin cytoskeleton and release of buffering compounds occurred, indicating sudden membrane leakage at 45–47°C. The calcium chelator EGTA [ethylene-glycol-bis(β-aminoethyl-ether)-N,N,N’,N’-tetraacetic-acid] increased the frequency of heat shock-induced rings. Ionophore (10 µM nigericin) and the sodium/proton antiport blocker [100 µM 5(N-ethyl-N-isopropyl)-amiloride] mimicked the effect of the 37°C heat shock. The cytoskeleton inhibitors latrunculin B, cytochalasin D and 2,3-butanedione monoxime inhibited ring formation but not alkalinization. In autotroph cells, the treatment with nigericin (10 µM) produced rings, although the actin cytoskeleton was not affected by temperatures up to 45°C. We conclude that Chenopodium cells express a specific temperature sensor that has ascendancy over the organization of the actin cytoskeleton; this is probably a temperature- and potential-sensitive proton-transporting mechanism that is dependent on the culture conditions of the heterotroph cells.

Keywords Actin . Chenopodium rubrum L. . EIPA . Extracellular pH . Heat shock . Latrunculin B

Introduction

The number of physical and chemical responses that can be used to describe the effect of heat shock on a living plant cell is constantly increasing as more and more genes, metabolites and proteins are discovered. One such example is the progress which has been made in protein purification, revealing the presence of protein isoforms or isoenzymes of
the vacuolar proton pumping pyrophosphatase (Kranewitter et al. 1999). The heat shock proteins and heat shock factors are ideal target materials for the investigation of long-term effects of heat shock (Vierling 1991; Schöffl et al. 1998; Wang et al. 2003). The enzymatic and membrane-associated responses can be used to investigate the immediate effect of heat shock on plant cells (Chaidee and Pfeiffer 2006). Ion fluxes respond rapidly to most types of stimulation; of these, proton fluxes play a central role, especially in plants, where transport is driven mostly by antiport and symport mechanisms, depending on the presence of proton gradients (Lüttge and Smith 1984; Bremberger et al. 1988; Pfeiffer and Hager 1993; Pfeiffer 1998), and where pH is known to signal a variety of environmental and hormonal stimuli (Fasano et al. 2001; Felle 2001). A high sensitivity to temperature has been reported for proton channels of animal cells (DeCoursey and Cherny 1998), and a general role of extracellular alkalinization in plant cell signaling was suggested (Felle 2001). The suspension-cultured cells of Chenopodium rubrum have been established as a model system for studying cellular signaling and plant membrane biology (Bentrup et al. 1986; Bille et al. 1992; Kranewitter et al. 1999; Pfeiffer and Höftberger 2001). A study on the effects of heat shock on membrane-associated parameters showed that extracellular pH signals temperature in heterotroph Chenopodium cells (Chaidee and Pfeiffer 2006). The extracellular alkalinization observed after heat shock can be driven by cation proton exchangers, as has been described for potassium (Atkinson et al. 1993) and sodium (Denker and Barber 2002). In addition, extracellular alkalinization can be mediated by the activation of separate proton and cation channels.
Various environmental stimuli regulate the activity of ion channels during stomatal opening and closing, such as light, humidity, abscisic acid, CO2 and temperature (Staiger 2000). There have been several reports of the actin cytoskeleton playing an important role in sensing and mediating stimuli. Abscisic acid has been reported to activate K+ channels mediating stomatal closing (Li et al. 1998; Nick 1998), and closed and open stomata were found to show different distributions of actin filaments (Eun and Lee 1997). Moreover, actin filaments were shown to regulate the activity of potassium channels in guard cells (Hwang et al. 1997). In root hair cells, abscisic acid and Nod factors were found to induce cytoskeletal rearrangements within a few minutes (Eun and Lee 1997; de Ruijter et al. 1999), while fusicoccin and changes in pH are known to affect actin arrays in plant cells (Nick 1999; Staiger 2000; Lovy-Wheeler et al. 2006). The effect of fusicoccin is mediated by the activation of the plasma membrane proton pump (Oecking et al. 1997). Interestingly, V-ATPase, which is primarily responsible for the regulation of extracellular pH in mouse osteoclasts, binds actin to the amino-terminal region of the B subunit (Chen et al. 2004), indicating a linkage between the actin cytoskeleton and extracellular pH in animal cells. Moreover, the large negatively charged surface of the cytoskeleton (Janmey 1998) is able to sense changes of intracellular pH, which may be linked to the extracellular pH. In suspension-cultured cells of C. rubrum, heat shock induced pronounced changes of extracellular pH (Chaidee and Pfeiffer 2006). Temperature-dependent effects on the actin cytoskeleton have been described in other cells as well (e.g. Iida et al. 1986; Benjamin and McMillan 1998; Holubářova et al. 2000; Volkov et al. 2003; Müller et al. 2007). These findings and the fact that actin can regulate proton export from osteoclasts (Chen et al. 2004) points to some degree of coupling between the actin cytoskeleton and extracellular pH.
We therefore have compared heat shock responses of extracellular pH and the actin cytoskeleton in autotroph and heterotroph Chenopodium cells. We also characterized the effects of proton flux on actin signaling with specific drugs. Nigericin is a cation proton exchanger, and its effect can indicate the presence of ion gradients, which drive extracellular alkalinization following heat shock (Pressman 1976; Bevensee et al. 1999). 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) is a more specific derivative of amiloride that is known to inhibit Na+/H+-antiport in plant cells (Spickett et al. 1993; Viehweger et al. 2002). We also used inhibitors for actin- and myosin-mediated processes, such as latrunculin B, cytochalasin D and 2,3-butanedione monoxime (BDM) (Yokota et al. 2000) to elucidate the mechanism of actin reorganization.
Briefly, we searched for cell-specific effects of heat shock on proton flux and the actin cytoskeleton by comparing autotroph and heterotroph suspension cells of C. rubrum. The involvement of proton flux was investigated by two approaches: measurement of extracellular pH and buffering capacity and by a study of the effects of nigericin. We characterized cell-specific effects of heat shock on the actin structure and proton flux in terms of temperature dependence, time kinetics and inhibitors.

Materials and Methods

Plant material

A red, betalain-containing heterotrophic suspension culture of Chenopodium rubrum L. established by Harms et al. (1977) was maintained at room temperature (RT, 22°C). The general conditions for the culture of the green, autotrophic cells have been described by Berlin et al. (1986).

Heat shock treatment and determination of pH and temperature

The heat shock treatment was carried out in a water bath (5l polypropylene water bath; Minitherm 2, Dinkelberg, NeuUlm, Germany) adjusted to 27.5, 30, 32.5, 35, 40, 45, 48, 50, 55, 60, 65 or 70°C. A reaction vessel (2.2-ml polypropylene, type 623201; Greiner, Kremsmünster, Austria) containing 1 ml of a 9-day-old heterotroph cell suspension was incubated for 5 min unless indicated otherwise. Autotroph cell suspensions (8 weeks old) were used in some experiments. The temperature of the cell suspension during a 30-min equilibration period in the reaction vessel on a shaker (110 rpm) prior to incubation was 22°C. At the end of the incubation, extracellular pH and the temperature of the cell suspension were measured with a pH-meter (digital pH-meter 525 with temperature sensor; Wissenschaftlich Technische Werkstätten WTW, Weilheim, Germany) and microelectrode (Schott, Hofheim, Germany). The following temperatures were measured in the cell suspension at the end of the heat shock treatment (5 min): 24.6, 26.5, 27.6, 28.5, 32.3, 35.1, 37, 38.2, 41.3, 44.7, 46.6 or 50°C. These temperature values of the cell suspension were used to indicate the temperature of the heat shock treatment. The medium for the heterotroph, betalaincontaining cell culture contained Murashige and Skoog (1962) salts supplemented with 21 mM ammonium nitrate, 0.1 mM boric acid, 3 mM calcium chloride, 0.1 µM cobalt chloride, 0.1 µM cupric sulfate, 0.1 mM EDTA sodium salt, 0.1 mM ferrous sulfate, 1.5 mM magnesium sulfate, 0.1 mM manganese sulfate, 0.1 µM sodium molybdate, 5 µM potassium iodide, 19 mM potassium nitrate, 1.25 mM potassium phosphate, 30 µM zinc sulfate and with the following additives: 3% sucrose, 0.15 µM 2,4-dichlorphenoxy acetic acid, 1.2 µM kinetin, 4 µM nicotinic acid, 0.3 µM thiamine, 2.5 µM pyridoxine, 27 µM glycine and 0.56 mM myo-inositol. Red cell suspensions sub-cultured for 9 days in this medium were used for the heat shock experiments. In some experiments red cells were incubated after a heat shock at 22°C for 3 h prior to repetition of the heat shock treatment.
The autotroph, green cells were cultured in double-floor Erlenmeyer flasks. The lower floor contained 0.5 M KHCO3 and 1.5 M K2CO3 as a source of carbon dioxide. The upper floor contained green cells in a modified Linsmaier-Bednar and Skoog medium (Linsmaier-Bednar and Skoog 1965) with 0.1 mM Fe-EDTA. The modified Linsmaier-Bednar and Skoog medium was composed of 21 mM ammonium nitrate, 0.1 mM boric acid, 2 mM calcium chloride, 0.1 µM cobalt chloride, 0.1 µM cupric sulfate, 0.1 mM EDTA disodium salt, 0.1 mM ferrous sulfate, 1.95 mM magnesium sulfate, 0.1 mM manganese sulfate, 1 µM sodium molybdate, 5 µM potassium iodide, 19 mM potassium nitrate, 1.25 mM potassium dihydrogenphosphate, 37 µM zinc sulphate, with the following additives: 1.2 µM thiamine, and 0.55 mM myo-inositol. The green cell suspensions used for heat shock experiments were sub-cultured for 8 weeks in this medium.

Chemicals

The effect of heat shock (heating from 22 to 37°C in 5 min) on extracellular pH and the actin cytoskeleton of Chenopodium cells was characterized using EGTA [ethylene-glycolbis(β-aminoethyl-ether)-N,N,N’,N’-tetraacetic-acid], latrunculin B, cytochalasin D, BDM, nigericin, amiloride and EIPA. The stock solutions were: latrunculin B 2.5 mM in DMSO, cytochalasin D 10 mM in DMSO, BDM 1 M freshly dissolved in bi-destilled water, nigericin 10 mM in ethanol and amiloride hydrochloric acid and EIPA 100 mM in DMSO. Equivalent concentrations of solvent were used in the control experiments. All chemicals were from SigmaAldrich (Vienna, Austria).

Determination of buffering capacity and proton flux

The supernatant of the Chenopodium cell suspension culture was separated by filtration with a 20-µm frit immediately after the heat shock treatment. The buffering capacity of the supernatant was determined by titration with HCl and NaOH. The addition of titrant resulted in final concentrations of 0–4000 µM, associated with a maximum change of volume of 5%. The heat shock experiments for proton flux were run in duplicate with aliquots from one glass of cell suspension culture. The data for the calculation of heat shock-induced proton flux are means of four to six independent experiments. The mean of the heat shock-induced proton flux was calculated from the mean pH change using a titration curve for the corresponding temperature range. Means of pH are typical values that can be used for the characterization of heat shock effects, as repeated pH measurements show a Gaussian normal distribution (Metcalf 1987). The temperature of the cell suspension at the end of the heat shock incubation (5 min) is indicated in all graphs. Calculations and graphs were made with SIGMA PLOT 2001 (Jandel Scientific, Erkrath, Germany).

Actin staining

The actin cytoskeleton was stained in heterotroph Chenopodium cells using a simultaneous fixation and labeling method described by Foissner et al. (2002). The fixation solution contained 1.5% (w/v) formaldehyde, 0.05% (v/v) glutaraldehyde, 0.05% (w/v) Triton-X-100, 10 mM EGTA, 5 mM MgSO4 and 100 mM PIPES, pH 6.9 (Grolig 1990). It was freshly prepared every 2 h, and Alexa Fluor 488 phalloidin (Invitrogen, Carlsbad, CA) was added to a final concentration of 0.16 μM immediately before fixation from a stock solution (6.6 µM in methanol). The concentrations of triton and fluorescent dye were doubled in some experiments with heterotroph and autotroph cells in order to improve staining, in particular for autotroph cells. Cells were fixed and stained within 10 min after treatment in a perfusion chamber, as described by Foissner et al. (2002). Images were taken without washing and within 30 min to avoid increased background fluorescence.

Confocal laser scanning microscopy and determination of the frequency of actin rings

The confocal laser scanning microscopy equipment was from Zeiss (Zeiss Axiovert IM 35 and Zeiss LSM 510; Zeiss, Oberkochen, Germany) and included an Argon ion laser (excitation wavelength 488 nm) and a band-pass filter (505–550 nm). Images were taken with a high numerical aperture oil immersion objective (Plan-Apochromat 63x, NA 1.4) and at minimum laser intensity and pixel time to avoid bleaching.
Actin fluorescence was visible up to 15 µm from the cell surface, which allowed the counting of actin rings in a partial volume of the cell covering the space between nucleus and surface. The volume was a cap of the selected heterotrophic Chenopodium cells with a spherical shape and variable radius r (17–28 µm). The average radius of the cells investigated for a given treatment was 22–25 µm. The height (h) of the cap was 15 µm for all cells investigated. The volume (V) was given by V=π 3−1 h2 (3r-h). This volume was used for the determination of the frequency of actin rings and sectioned along its height into a stack of 27–43 slices (thickness 0.35–0.55 µm). Threedimensional images were necessary for the recognition of individual actin rings. The frequency of actin rings per volume was obtained by animating pictures along the zaxis because it cannot be estimated from single or overlaid two-dimensional images, where the resolution for the third dimension is missing. The frequency of the actin rings for a given treatment was estimated in 9–15 cells from three to five independent experiments, depending on the number of cells of a similar size and shape found in differently treated samples. Images shown in this study are projections of at least 21 single optical sections or single sections and are representative of at least two independent experiments.

Results

Effect of heat shock on extracellular pH of heterotroph and autotroph Chenopodium cells

The effects of heat shock on the pH of autotroph and heterotroph cell suspensions are shown in Fig. 1. The initial pH was 4.6 in the autotroph and 4.8 in the heterotroph cultures (Fig. 1A,B). In this context, it should be mentioned that all cell cultures were grown at the same temperature (22°C). The maximum extracellular alkalinization was found in both cell cultures at 47°C, corresponding to maximum heat shock-induced proton flux of 520 nmol H+ g−1min−1 in heterotroph and 440 nmol H+ g−1min−1 in autotroph cells (Fig. 1C,D). These values show that heterotroph cells have an 18% higher capacity for heat shock-induced extracellular proton flux than autotroph cells.
The determination of heat shock-induced proton fluxes in heterotroph and autotroph Chenopodium cell cultures required an estimation of the effect of the heat shock on the buffering capacity of the culture medium in both cell cultures. Heat shock-induced extracellular alkalinization in heterotroph Chenopodium cell cultures covers the range of pH 4.5–5.5 (Chaidee and Pfeiffer 2006). Thus, the buffering capacity within this interval was used to measure the effect of heat shock on different cell cultures (Table 1). The titration curves of the culture supernatant at pH 3–10 after different heat shock treatments of autotroph and heterotroph Chenopodium cells are shown in Fig. 2A and B. Up to 41°C, there was little detectable effect of heat shock (Table 1, Fig. 2). The maximum effect of heat shock on the buffering capacity of the medium was found at 50°C in autotroph and heterotroph cell cultures. At 50°C, autroph cells released 2635 nmol g (Table 1). At 47°C, autotroph cells released 91% and heterotroph cells 92% of the maximum (Table 1). The ratio of the released buffering capacities from heteroand autotroph cells shows a higher accumulation of metabolites under heterotroph conditions (Table 1). At high temperatures in particular, heterotroph cells released more buffering capacity than autotroph cells: 5.35-fold at 47°C and 5.17-fold at 50°C; the comparison with values at 41°C (1.27-fold) and at 45°C (1.10-fold) reveals a sudden increase between 45 and 47°C.

Repetition of heat shock and extracellular alkalinization

The effect of repeated heat shock on the extracellular pH of heterotroph cells was investigated. Prior to repetition of the heat shock, the cells were incubated 3 h at 22°C. Thus, a long-term effect of heat shock was measured after 3 h (Fig. 3) in addition to the immediate effect already described (Fig. 1B). A comparison (Figs. 1B and 3) shows a dependence on the heat shock temperature of the transience, persistence or amplification of the heat shockinduced extracellular alkalinization. After 3 h, cells recovered from the immediate change of pH, which was induced by heat shock at 22–35°C, to control values (pH 4.0–4.3), whereas they amplified the immediate change of pH induced at 38–41°C (38°C: pH 5.1; 41°C: pH 5.8). Thus, within 3 h at 22°C, the initially gradual response of extracellular pH (Fig. 1B) was changing to an all-or-none response (Fig. 3), with a threshold at 35–38°C for the induction of long-term extracellular alkalinization resulting in 0.8 and 1.5 pH increases at 38 and 41°C, respectively. The repetition of heat shock after a 3-h incubation at 22°C resulted in a second alkalinization at 28–41°C (Fig. 3).

Effect of heat shock on the actin cytoskeleton of heterotroph and autotroph Chenopodium cells

Chenopodium cells were characterized under standard conditions by differential interference contrast (DIC) images and actin staining (Fig. 4A–D). The actin cytoskeleton of control cells consisted of long, often bundled filaments, randomly orientated in the cortical cytoplasm (Figs. 4B,5D,K), in the transvacuolar strands and in the cytoplasm around the nucleus (not shown). The measurements of extracellular pH and buffering capacity revealed a physiological range for heat shock temperature up to 45°C (Figs. 1, 2 and 3). This range of temperature was therefore used for studying the effect of heat shock on the actin cytoskeleton in heterotroph and autotroph Chenopodium cells (Fig. 5). The most pronounced effect was obtained at 37°C. At this temperature, actin rings were induced specifically in heterotroph cells (Fig. 5J). Few rings were induced at 35°C (Fig. 5F), whereas other heat shock treatments at 32, 41 and 45°C did not change the frequency of actin rings in heterotroph cells (Fig. 5E,K,L) relative to that in the control cells kept at 22°C (Fig. 5D). The actin cytoskeleton of cells treated with heat shock at 41°C was similar to that of the control cells (Fig. 5D). At a high temperature (45°C, Fig. 5L) irreversible fragmentation of the actin filaments, but no actin rings, were observed. No actin reorganization was found in autotroph cells after heat shock at 32, 35, 37, 41 and 45°C (Fig. 5B,C,G,H,I). In total, the actin structure of autotroph cells (Fig. 5A,B,C,G, H,I) was inert against heat shock at 32–45°C.
Actin rings (maximum diameter 3 µm) were occasionally observed in control cells at a frequency of 0.30 per picoliter protoplasm (Fig. 5D, Table 2). They were found in the cortical cytoplasm and near the surface of the nucleus (Fig. 6A). Heat shock at 37°C increased the frequency of actin rings twenty-fold (Table 2). It is noteworthy that the increase in the frequency of actin rings per volume (Table 2) can often not be seen in a single two-dimensional figure consisting of overlaid projections (see Materials and Methods). The actin rings had a flat or a distorted shape and seemed not to be associated with organelles visualized by bright field or DIC (not shown). The formation of actin rings occurred within 10 min, which was the shortest handling time for our protocol of actin visualization (e.g. Fig. 5F,J). The frequency of heat shock-induced actin rings was not increased by prolongation of the incubation time after heat shock from 10 min to 3 h (Fig. 6B,C). The repetition of the heat shock (37°C, 5 min) after 3 h of recovery also did not increase the frequency of actin rings (Fig. 6D). The heat shock-induced actin rings disappeared completely after an incubation period of 6 h in fresh culture medium (Fig. 6E,F), associated with the recovery of extracellular pH (not shown), and they persisted at least for 9 h without medium exchange (not shown).
The comparison of Fig. 3 with Fig. 5 shows that the temperature for the induction of actin rings (35–37°C) is close to the minimum heat shock temperature (38°C) for long-term extracellular alkalinization. This may indicate a relationship between changes in extracellular pH and the formation of actin rings. This relationship was characterized further by the effects of different chemicals, including inhibitors of ion transport and inhibitors of actin and myosinmediated processes, as described in the following sections. Effect of EGTA on the formation of actin rings
Calcium is known to play a central role in the regulation of many physiological processes in plant cells. We tested the effect of a decrease in free extracellular calcium ions on the formation of heat shock-induced actin rings. The culture medium contained 3 mM CaCl2. Thus, the formation of actin rings was induced by heat shock at 37°C in the presence of excess EGTA (5 mM) to chelate free calcium ions (Table 2). The addition of EGTA to the culture medium increased the frequency of heat shock-induced actin rings by 1.5-fold (Table 2). Relative to control cells, the combined treatment with EGTA and heat shock at 37°C increased the frequency of actin rings twenty-fold (Table 2). The enhancing effect of EGTA on the formation of heat shock-induced actin rings was compensated for by the addition of 6 mM CaCl2 to the medium (Table 2). The effect of heat shock on extracellular pH disappeared in the presence of 5 mM EGTA; in contrast to the effect on actin organization it could not be restored by the addition of 6 mM CaCl2 (Table 2). This finding is explained by the increase in buffering capacity following the addition of EGTA to the culture medium (Table 2). Thus, EGTA did not allow any investigation of the calcium dependence of the heat shock-induced extracellular alkalinization.

Nigericin- and EIPA-induced actin rings and extracellular alkalinization

The extracellular alkalinization induced by heat shock can be mediated by cation proton exchangers (Atkinson et al. 1993; Denker and Barber 2002). Therefore, we used nigericin as an artificial potassium/proton antiporter to induce ion flux and to investigate whether this ion flux can affect the actin cytoskeleton. Nigericin (10 µM) affected extracellular pH and actin cytoskeleton in both auto- and heterotroph cells (Fig. 7). In heterotroph cells, nigericin increased extracellular pH from 1.5 to 5.6 within 1 h and induced the formation of actin rings (Fig. 7C,E). A high concentration of nigericin (100 µM) did not induce actin rings, although it raised extracellular pH to 6.8 in 20 min (Fig. 7F). Thus, 10 µM nigericin can be used to induce physiological responses (Fig. 7C) such as that induced by heat shock (Fig. 1), which results in a final extracellular pH around 5.5. The application of a high concentration of nigericin (100 µM) allows an estimation of the maximum capacity for extracellular alkalinization.
Responses to the artificial potassium/proton antiporter nigericin (Pressman 1976; Bevensee et al. 1999) in Chenopodium cells can indicate the involvement of cellular antiporters in the heat shock-induced extracellular alkalinization (Figs. 1, 3) and the formation of actin rings (Fig. 5). Therefore, we studied the effects on heterotroph cells of the inhibitors of cellular antiporters for monovalent cations, amiloride and EIPA. Amiloride and EIPA have been reported to inhibit sodium/proton antiporters in plant cells (Spickett et al. 1993; Viehweger et al. 2002). We found that amiloride (100 µM) had no effect on the extracellular pH (Table 3). The more specific compound EIPA (100 µM) increased pH in a time-dependent manner (Fig. 8A). EIPA-induced actin rings were detected after 20 and 60 min (Fig. 8B–D). The results obtained with EIPA suggest the involvement of antiporters for monovalent cations in the heat shock response.

Effects of actin and myosin inhibitors (latrunculin B, cytochalasin D, BDM) on extracellular pH and actin (re-) organization

The finding that ion transport inhibitors can induce extracellular alkalinization and the formation of actin rings (Figs. 7 and 8) raised the question of whether or not actin and myosin inhibitors can also induce or affect both extracellular alkalinization and the formation of actin rings. Latrunculins bind actin monomers and, depending on the concentration, disrupt or reorganize the actin cytoskeleton (Yokota et al. 2000 for references). The treatment of heterotroph Chenopodium cells with 50–500 nM latrunculin B at 22°C did not change the pH of the culture medium (Table 3). Moreover, the extracellular alkalinization response to heat shock (heating from 22°C to 37°C in 5 min) was not affected by the pre-treatment with latrunculin B (Table 4). In contrast to extracellular alkalinization, the heat shockinduced formation of actin rings was reduced by 50 nM latrunculin B, and the actin cytoskeleton consisted of short, randomly oriented actin filaments and bundles (Fig. 9B). It is noteworthy that the treatment with 50 nM latrunculin B did not affect the actin organization in cells incubated at 22°C (Fig. 9A). Thus, the effect of 50 nM latrunculin B was dependent on cellular stimulation by heat shock.
Cytochalasins inhibit cytoplasmic streaming and reorganize the actin cytoskeleton of plant cells (e. g. Foissner and Wasteneys 2007). Similar to latrunculin B, cytochalasin D (1 h, 1–20 µM) did not change the pH of the heterotroph Chenopodium cell suspensions (Table 3), and the heat shock-induced extracellular alkalinization was not affected by the pre-treatment with cytochalasin D (Table 4). However, in cells treated with 10 µM cytochalasin D for 30 min, the actin cytoskeleton was reorganized into short actin fragments and dots at a constant temperature of 22°C (Fig. 9C), and in cells treated with heat shock at 37°C (Fig. 9D), the heat shock-induced formation of actin rings in Chenopodium cells was completely inhibited (Fig. 9D). The effect of cytochalasin D was not dependent on heat shock, which is in contrast to that of latrunculin B. Therefore, latrunculin B can detect more specifically the heat shock-dependent signaling mediated by actin.
The myosin inhibitor BDM reduces the activity of isolated myosin from pollen tubes (Tominaga et al. 2000). In contrast to latrunculin B and cytochalasin D, BDM (10– 60 mM) affected both actin organization and extracellular pH. 2,3 Butanedione monoxime induced the alkalinization of Chenopodium cell suspensions within 3 h (Table 3). During shorter incubation times (10 min), BDM did not affect the extracellular pH (Table 3). Therefore, 10-min pretreatments with 10 and 60 mM BDM were used to study the effect on the heat shock-induced formation of actin rings: the lower concentration (10 mM BDM) had no effect on cell suspensions treated with constant temperature or heat shock (data not shown), while the higher concentration (60 mM of BDM) induced short actin filaments in cells treated at a constant temperature of 22°C (Fig. 9E) or heat shock and prevented the heat shock-induced formation of actin rings (Fig. 9F).

Discussion

The major results described here reveal specific conditions for the induction of actin rings in Chenopodium cells. The cell-specific induction of actin rings by heat shock in combination with their general induction by 10 µM nigericin (Figs. 5 and 8) represents a promising basis for further investigations of the cell-specific association of proton flux with the formation of actin rings. The results of our study of extracellular pH (Figs. 1, 2, and 3), effects of temperature (Fig. 5, Table 1), time kinetics (Fig. 6), calcium (Table 2), nigericin (Fig. 7), drugs and cytoskeleton inhibitors (Tables 3, 4, Figs. 8 and 9) suggest the involvement of a specific, proton-transporting and potential-sensitive mechanism upstream of the heat shock-induced physiological formation of actin rings. This mechanism seems to depend on heterotroph culture conditions and to contribute to a variable fraction of extracellular proton flux. The separate findings of this composite result are discussed in the following sections.

Sensitivity to heat shock of extracellular pH in heterotroph and autotroph cells

In order to measure the width of the physiological range for heat shock temperatures, we used temperatures up to 50°C. The release of buffering capacity increased suddenly at 45–47°C, indicating the loss of membrane integrity and death of cells (Table 1). At 47°C, a fivefold higher release of buffering capacity was induced in heterotroph cells (2435 nmol g−1) than in autotroph cells (455 nmol g−1; Table 1). This temperature is close to the onset of cell death reported in mammalian cells after heat shock at 45°C (Gerweck et al. 1983) and close to the limit of the lethal temperature for long-term exposure of most Mediterranean evergreen plants (48–55°C; Larcher 2000). In total, proton flux and the release of buffering capacity were found to respond in different proportions to extreme heat shock at 47°C in auto- and heterotroph Chenopodium cells (Table 1, Figs. 1, 2). The different release of buffering capacity seems to be associated with the different culture conditions: the presence (heterotroph) and absence (autotroph) of sucrose inducing different metabolic activities and growth rates, as reflected by the periods for subculturing, i.e. 2 weeks for heterotroph cells and 8 weeks for autotroph cells, necessary to obtain increases of fresh weight in a similar range.
After heat shock at 25–35°C, heterotroph cells showed a higher proton flux than autotroph cells (Fig. 1C,D). Thus, responses of extracellular buffering capacity at 41–50°C (Table 1), indicating the release of buffering compounds such as organic or amino acids, were separate from that of proton flux (Fig. 1C,D). This finding can indicate the activation of a distinct proton channel or proton transport system in heterotroph cells at 25–35°C. At higher temperatures, other channels or processes may be activated in autotroph cells, leading to similar responses of proton flux in both cell types at 37–50°C (Fig. 1C,D).

Cell-specific effect of heat shock on the actin cytoskeleton

Theactincytoskeletonplaysa central role in cellularresponses to external and internal stimulation (e.g. Staiger 2000; Gerthoffer and Gunst 2001). In animal and yeast cells, the organization of the actin cytoskeleton is known to be sensitive to heat shock (Iida et al. 1986; Benjamin and McMillan 1998; Holubářová et al. 2000). Moreover, there are investigations on the role of small heat shock proteins in thermo-tolerance and the regulation of actin filament polymerization (Liang and MacRae 1997; Mounier and Arrigo 2002). In plants, the role of the actin cytoskeleton in growth, development and signaling has been described (Gibbon et al. 1999; Staiger 2000; Ketelaar et al. 2002; Baluska et al. 2000; Fu et al. 2001). Under temperature stresses, rearrangement of the actin cytoskeleton was found to occur downstream of the changes in membrane fluidity (Sangwan et al. 2002). Recently, heat stress-mediated (41°C, 10 min) dramatic changes of microtubular and actin cytoskeletons have been revealed in Arabidopsis epidermal root cells (Müller et al. 2007). In addition to structural changes, heat shock has been reported to induce a decrease in the amount of actin mRNA (At-ACT2) and an increase in the amount of heat shock protein mRNA (Volkov et al. 2003) and Latrunculin B-activated stress-induced mitogen-activated protein kinases (Samaj et al. 2002). In total, the finding that heat shock can cause extensive changes in the cytoskeleton of animal and plant cells suggests that the temperature sensitivity of actin structures allows cells to sense changes in the environmental temperature.
We found that the formation of actin rings in heterotroph Chenopodium cells was induced by a specific temperature treatment at 35–37°C (5 min). The size of the heat shockinduced actin rings in Chenopodium cells was 0.5–3 μm, with a mean of 1.7 μm (Figs. 5, 6). This size is similar to that of actin rings described from other plant cells under various conditions (Hasezawa et al. 1988; Tiwari and Polito 1988; Kadota and Wada 1992; Frost and Roberts 1996). In the heterotrophic, chloroplast-free Chenopodium cells, the disappearance of heat shock-induced actin rings together with the recovery of extracellular pH within 6 h after heat shock (Fig. 6F) suggest that actin rings have a transient physiological function or, at least, indicate a special physiological state during the period of homeostatic disturbance after heat shock.
Microtubules of tobacco cells are disrupted at 42°C but not at 38°C (Smertenko et al. 1997). A similar temperature was found to cause the depolymerization of actin and the microtubule cytoskeleton in Arabidopsis root cells within 10 min (Müller et al. 2007). Unlike microtubules, the actin cytoskeleton showed a non-cell-specific response to heat shock in Arabidopsis (Müller et al. 2007). We found that the heat shock-induced formation of actin rings at 35–37°C was specific for heterotroph Chenopodium cells (Fig. 5) and that the formation of actin rings can be correlated to an event downstream of proton flux. The irreversible disassembly of actin filaments and bundles into short fragments was observed only in heterotroph cells at 45°C (Fig. 5I,L), which is near to the threshold temperature for the release of buffering capacity (Table 1). Thus, cell-specific responses of the actin organization can occur within the temperature range up to unspecific perturbations of biological membranes in Chenopodium above 45°C (Chaidee and Pfeiffer 2006). We consider cell type and culture conditions to explain our observation that the heat shock-induced actin rings appeared only in heterotroph but not in autotroph cells. Both, the autotroph and heterotroph cell cultures contained meristematic, interphase cells, not synchronized by cell cycle. The culture conditions over about 30 years included the absence of 2,4-D in autotroph cell cultures and the presence of 2,4-D in heterotroph cell cultures. Therefore, we suggest that the sensitivity to heat shock of the actin cytoskeleton may be caused by heterotroph culture conditions or by 2,4-D-induced somaclonal variation.

Non-cell-specific effect of nigericin on the actin cytoskeleton

The binding of actin to a proton pump responsible for extracellular pH regulation (Chen et al. 2004) and the cellular acidosis after heat shock (Kregel 2002) have been reported in animal cells. Moreover, the extreme sensitivity of ion fluxes to temperature has been documented (DeCoursey and Cherny 1998; Chaidee and Pfeiffer 2006). Thus, a linkage between actin responses and that of pH may be assumed, which was further investigated by the use of ion transport affecting drugs, i.e. nigericin and EIPA. The heat shock-induced formation of actin rings was found only in heterotroph cells (Fig. 5). In contrast to heat shock, the effect of nigericin, an artificial potassium/ proton antiporter, was not cell-specific (Fig. 7). At 10 µM, nigericin mimicked the effect of heat shock and induced the formation of actin rings and a change in the extracellular pH in both heterotroph and autotroph cells. Interestingly, an extreme concentration of nigericin (100 µM), just like higher temperature (41°C), could not induce actin rings. Thus, specific ion fluxes and gradients are necessary for the induction of actin rings, which probably involves the activity of antiporters for monovalent cations.
In addition to the similar effect that a moderate dose of heat shock and nigericin have on the induction of actin rings in heterotroph cells, the finding that all five heat shock temperatures between 25 and 35°C induced higher proton fluxes in heterotroph cells than in autotroph cells (Fig. 1C,D) suggests the presence of a specific proton transporting mechanism in heterotroph cells. At higher temperatures additional mechanisms may contribute to extracellular alkalinization and superimpose or suppress via a potential-sensitive mechanism the effect visible at 25–35°C (Fig. 5). Alternatively, the proton transporting mechanism may be coupled specifically to a temperature sensor in heterotroph cells. In this context, reports on an animal temperature-sensitive proton channel (DeCoursey and Cherny 1998) point to the existence of a similar sensor in heterotroph cells, which may trigger the specific proton flux mediating the formation of actin rings. This temperature sensor can be a potential-sensitive proton channel (DeCoursey and Cherny 1998). It has been suggested that the induction of actin rings by both nigericin (10 µM) and EIPA (Figs. 7, 8), drugs which act differently on ion transport mechanisms, may be mediated by a potentialsensitive process. Many transporters affect extracellular pH (Felle 2001). Therefore, pharmacological and physiological data are necessary to characterize a specific proton flux. In this context, it is of interest that a wide range of doses of heat and nigericin induced extracellular alkalinization, whereas a narrow range of intermediate doses only induced actin rings. This indicates that the association of a specific fraction of proton flux with the formation of actin rings contributes to extracellular alkalinization in a variable proportion (Figs. 1, 3, 5) depending on temperature and time. The effects of latrunculin B, cytochalasin D and 2,3butanedione monoxime (Fig. 9) on the heat shock response of the cytoskeleton, but not on heat shock-induced extracellular alkalinization (Tables 3 and 4), are also in line with the suggestion of a proton channel as a temperature sensor.

Pharmacological characterization of heat shock-induced changes in extracellular pH and actin ring formation in heterotroph cells

Interestingly, the formation of actin rings was suppressed in heat-shocked cells pretreated with 50 nM latrunculin B, 10 μM cytochalasin D or 60 mM BDM (Fig. 9, Table 4). At these concentrations, BDM and cytochalasin B disorganized the actin cytoskeleton of the control cells, which impedes any interpretation of heat shock data. However, 50 nM latrunculin B did not affect the organization of the actin cytoskeleton in control cells treated at 22°C (Fig. 9A, Table 3). Short actin fragments were present instead of actin rings in heat shock-treated cells (Fig. 9B), indicating that the effect of 50 nM latrunculin B is dependent on cellular stimulation by heat shock and suggesting that the formation of heat shock-induced actin rings is not due to the winding up of existing filaments but, rather, requires de- and repolymerization of F-actin. Latrunculins have been used widely to destabilize the actin cytoskeleton and to demonstrate the role of actin organization in various cellular activities, in particular polarized growth (e.g. Gibbon et al. 1999; Kost et al. 1999; Samaj et al. 2002; Ketelaar et al. 2002). The pharmacological data obtained with these inhibitors also indicate that although actin may be involved in the sensing of high temperature, the formation of actin rings is not required for heat shock-induced proton fluxes and, on the other hand, that unspecific actin reorganization is not automatically correlated with extracellular alkalinization. The frequency of actin rings depended on the polymerization state of the actin cytoskeleton, as indicated by the effects of cytochalasin D and latrunculin B on the frequency of heat shock-induced actin rings (Figs. 5, 6, 7, 8, 9).
Latrunculin B, cytochalasin D and BDM were also found to inhibit the calyculin A-induced formation of actin rings in root hairs and pollen tubes (Yokota et al. 2000; Foissner et al. 2002). Foissner et al. (2002) discussed the possibility that actin ring formation may occur by selforganization via the binding of two-headed myosin and the translocation of motile actin filaments relative to each other. The effect of the myosin ATPase inhibitor (60 mM BDM) in this study may support this concept (Fig. 9). However, the fact that at this concentration (60 mM) some unspecific effects on different cellular activities affecting actin organization are likely to occur should be taken into consideration (Tominaga et al. 2000). This is also consistent with the extracellular alkalinization of Chenopodium cells observed after a 3-h incubation with BDM (Table 3).
We also investigated the effect of extracellular calcium on the induction of actin rings by heat shock in Chenopodium cells (Table 2). The frequency of heat shock-induced (37°C) actin rings could be enhanced by lowering the free extracellular calcium level with EGTA (Table 2). After the addition of excess calcium to the EGTA-containing culture medium, heat shock induced a similar frequency of actin rings as that found in cell cultures with no chemicals added (Table 2). These data indicate that calcium can protect Chenopodium cells against stress induced by heat shock, which is in agreement with results reported elsewhere (Klein and Ferguson 1987; Gong et al. 1998; Liu et al. 2003). The investigation of parameters for cellular viability and membrane function in Chenopodium cells showed that heat shock up to 45°C does not cause nonspecific changes of the permeability of the cell membrane (Chaidee and Pfeiffer 2006). This was confirmed by our data on the release of buffering compounds (Table 1, Fig. 2). Based on this result, it is possible that extracellular calcium could protect the cell against heat shock at 37°C (Table 2) without increasing intracellular calcium and stimulating the disruption of actin filaments, as described by Drøbak et al. (2004). Briefly, EGTA amplified the heat shock-induced formation of actin rings to a maximum (Table 2). The determination of the maximum of the heat shock-induced frequency of actin rings is helpful in terms of calibrating other effects.

Temperature sensing by the actin cytoskeleton?

The actin cytoskeleton has been suggested as a temperature sensor (Benjamin and McMillan 1998; Holubářova et al. 2000; Volkov et al. 2003; Müller et al. 2007). The effect of heat shock on extracellular pH in heterotroph Chenopodium cells indicated that extracellular protons are also involved in temperature sensing, probably via a channel-mediated pathway (Chaidee and Pfeiffer 2006). Therefore, we investigated the relation of proton flux with responses of the actin cytoskeleton in autotroph and heterotroph Chenopodium cells (Tables 1, 2, 3, 4). At 37°C, a cell-specific response of the actin cytoskeleton was found (Fig. 5). Nigericin induced a response which was similar, but not cell-specific (Fig. 7). Together with the pharmacological characterization (Tables 2, 3, 4), these results enable us to draw some conclusions on temperature sensing and signaling in Chenopodium cells. The finding that nigericin-induced actin rings appeared in both heterotroph and autotroph cells indicates that actin ring formation is downstream of a specific proton flux and suggests that actin reorganization reflects specific cell conditions but does not participate in transmitting temperature signals to ion channels. In total, the specific conditions for the induction of actin rings, in terms of cell type, temperature (37°C), extracellular pH and effect of nigericin, revealed an ascendancy of proton flux over the organization of the actin cytoskeleton. This observation is in agreement with the suggestion of potentialsensitive proton channels being temperature sensors in animal cells (DeCoursey and Cherny 1998) and reports on rearrangements of the actin cytoskeleton in plant cells downstream of changes in membrane fluidity (Sangwan et al. 2002). Changes in membrane fluidity may also act on calcium channels, which have been reported to show a high sensitivity to temperature (Peloquin et al. 2008). Therefore, the effects of EGTA and calcium described here (Table 2) may, together with the effects of nigericin (Fig. 7) and EIPA (Fig. 8), point to an interconnection of temperature- and potential-sensitive cation fluxes affecting the organization of the actin cytoskeleton.

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