Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 2000 Jun;123(2):645–654. doi: 10.1104/pp.123.2.645

Calcium-Calmodulin Suppresses the Filamentous Actin-Binding Activity of a 135-Kilodalton Actin-Bundling Protein Isolated from Lily Pollen Tubes

Etsuo Yokota 1,*, Shoshi Muto 1, Teruo Shimmen 1
PMCID: PMC59032  PMID: 10859194

Abstract

We have isolated a 135-kD actin-bundling protein (P-135-ABP) from lily (Lilium longiflorum) pollen tubes and have shown that this protein is responsible for bundling actin filaments in lily pollen tubes (E. Yokota, K. Takahara, T. Shimmen [1998] Plant Physiol 116: 1421–1429). However, only a few thin actin-filament bundles are present in random orientation in the tip region of pollen tubes, where high concentrations of Ca2+ have also been found. To elucidate the molecular mechanism for the temporal and spatial regulation of actin-filament organization in the tip region of pollen tubes, we explored the possible presence of factors modulating the filamentous actin (F-actin)-binding activity of P-135-ABP. The F-actin-binding activity of P-135-ABP in vitro was appreciably reduced by Ca2+ and calmodulin (CaM), although neither Ca2+ alone nor CaM in the presence of low concentrations of Ca2+ affects the activity of P-135-ABP. A micromolar order of Ca2+ and CaM were needed to induce the inhibition of the binding activity of P-135-ABP to F-actin. An antagonist for CaM, W-7, cancelled this inhibition. W-5 also alleviated the inhibition effect of Ca2+-CaM, however, more weakly than W-7. These results suggest the specific interaction of P-135-ABP with Ca2+-CaM. In the presence of both Ca2+ and CaM, P-135-ABP organized F-actin into thin bundles, instead of the thick bundles observed in the absence of CaM. These results suggest that the inhibition of the P-135-ABP activity by Ca2+-CaM is an important regulatory mechanism for organizing actin filaments in the tip region of lily pollen tubes.

In pollen tubes, actin filament bundles with parallel orientation to the long axis of a tube are well developed, and are involved in cytoplasmic streaming and the transport of vegetative nuclei and generative cells to the growing tip (Pierson and Cresti, 1992; Mascarenhas, 1993; Li et al., 1997; Taylor and Hepler, 1997). In the tip region, however, only a few thin bundles and short individual actin filaments in random orientation have been observed by electron microscopy in samples prepared using the rapid freeze fixation technique (Lancelle et al., 1987; Tiwari and Polito, 1988; Lancelle and Hepler, 1992; Miller et al., 1996). At the tip of actively growing pollen tubes, a tip-focused Ca2+ gradient has been reported that is indispensable for the tip growth (Rathore et al., 1991; Miller et al., 1992; Pierson et al., 1994, 1996) and tube orientation (Malhó et al., 1994, 1995; Malhó and Trewavas, 1996). An influx of external Ca2+ at the tip supports the tip-focused gradient of Ca2+ (Kuhtreiber and Jaffe, 1990; Pierson et al., 1994, 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997). The disorganization of actin-filament bundles in the tip region of growing tubes is believed to be due to the tip-focused Ca2+ gradient. When growing pollen tubes are treated with caffeine that diminishes the tip-focused Ca2+ gradient (Pierson et al., 1994), tip growth ceases and extensive actin-filament bundles extend into the tip (Miller et al., 1996; Lancelle et al., 1997). This observation also suggests that higher concentrations of Ca2+ at the tip region suppress actin filament bundle formation. For further insights into the regulation of tip growth of pollen tubes, it is important to elucidate how the organization of actin filaments is modulated by Ca2+ in the tip region of growing tubes.

Architecture of the actin cytoskeleton and polymerization-depolymerization of actin filaments in the cell are mediated by and modulated temporally and spatially by actin-binding proteins (Stossel et al., 1985; Pollard and Cooper, 1986). Recently, we have isolated an actin-binding protein (P-135-ABP) from lily (Lilium longiflorum) pollen tubes, which arranges filamentous actin (F-actin) into bundles in vitro (Yokota et al., 1998). F-actin filaments in the bundle formed by P-135-ABP in vitro showed uniform polarity (Yokota and Shimmen, 1999). Immunofluorescence and immunoelectron microscopy using an antibody against P-135-ABP showed colocalization of P-135-ABP with actin-filament bundles in lily pollen tubes (Yokota et al., 1998; Vidali et al., 1999). These observations strongly suggest that P-135-ABP is a factor responsible for bundling actin filaments in pollen tubes and its activity may be suppressed at the tip region of tubes. The activities of several kinds of actin-bundling proteins, including villin (Bretscher and Weber, 1980; Mooseker et al., 1980) and plastin/fimbrin (Glenney et al., 1981; Namba et al., 1992; Lin et al., 1994; Prassler et al., 1997), are inhibited by Ca2+ at physiological concentrations. In contrast, the activity of purified P-135-ABP was independent of Ca2+ (Yokota et al., 1998). It was suggested that some unknown factor(s) gave Ca2+ sensitivity to P-135-ABP. In the present study, we show that the binding activity of P-135-ABP to F-actin is significantly suppressed by calmodulin (CaM) in the presence of Ca2+.

RESULTS

Suppression of the Binding Activity of P-135-ABP to F-Actin by Ca2+-CaM

CaM had no effect on the binding activity of P-135-ABP to F-actin in the presence of EGTA. Most of P-135-ABP was coprecipitated with F-actin in a cosedimentation assay in the presence of EGTA and CaM (Fig. 1C). In contrast, 70% to 80% of P-135-ABP did not bind to F-actin and remained in the supernatant in the presence of Ca2+-CaM (Fig. 1D). As shown previously, Ca2+ alone did not affect the binding activity of P-135-ABP to F-actin in the cosedimentation assay (Fig. 1B; Yokota et al., 1998).

Figure 1.

Figure 1

Open in a new tab

Suppression of binding activity of P-135-ABP to F-actin by Ca2+-CaM. A mixture of 3.2 μg/mL P-135-ABP, 60 μg/mL F-actin, and 7.8 μm CaM was incubated in the absence (C) or the presence (D) of 0.5 mm CaCl2 at 20°C for 20 min. After centrifugation, the resulting supernatant (s) and pellet (p) were analyzed by SDS-PAGE on a 7.5% (w/v) acrylamide gel. As control experiments, a mixture of P-135-ABP and CaM without F-actin (A) or P-135-ABP and F-actin without CaM (B) was analyzed in the presence of CaCl2 as above. The arrowhead and the arrow indicate the 135-kD polypeptide of P-135-ABP (ABP) and actin, respectively. The molecular masses of standard proteins are indicated on the left in kD.

Dissociation of P-135-ABP from F-Actin by Ca2+-CaM

Next, we confirmed whether Ca2+-CaM possesses an ability to dissociate P-135-ABP from F-actin. At first P-135-ABP was mixed with F-actin in the presence of 0.2 mm EGTA for 20 min, and then with CaCl2 (final concentration at 0.5 mm) alone or with both CaCl2 and CaM (final concentration at 7.8 μm). In the case of the addition of CaCl2 alone, most of P-135-ABP was coprecipitated with F-actin (Fig. 2C). However, 70% to 80% of P-135-ABP was detected in the supernatant (Fig. 2D) when both CaCl2 and CaM were added to the mixture of F-actin and P-135-ABP. These results indicate that CaM caused dissociation of P-135-ABP from F-actin in the presence of Ca2+.

Figure 2.

Figure 2

Open in a new tab

Dissociation of P-135 from F-actin by Ca2+-CaM. P-135-ABP was mixed with F-actin. After a 20-min incubation at 20°C, samples were treated with no addition (A), with CaCl2 alone (B), with CaM alone (C), and with both CaCl2 and CaM (D). The mixtures were incubated for an additional 10 min at 20°C and then subjected to centrifugation. The resulting supernatant (s) and pellet (p) were analyzed by SDS-PAGE on a 7.5% (w/v) acrylamide gel. The final concentrations of P-135-ABP, F-actin, CaM, and CaCl2 were 3.2 μg/mL, 60 μg/mL, 7.8 μm, and 0.5 mm, respectively. The arrowhead and the arrow indicate the 135-kD polypeptide of P-135-ABP (ABP) and actin, respectively. The molecular masses of standard proteins are indicated on the left in kD.

Suppression of Bundling Activity of P-135-ABP by Ca2+-CaM

To examine the effect of Ca2+-CaM on the formation of actin bundles by P-135-ABP, a mixture of rhodamine-phalloidin (RP)-labeled F-actin and P-135-ABP was observed under an epifluorescence microscope in the presence of Ca2+ alone or Ca2+-CaM. In the presence of Ca2+ alone, RP-labeled actin filaments were arranged into thick bundles (2–12 μm in width) by P-135-ABP (Fig. 3B). This observation is consistent with the previous result showing the Ca2+-insensitive bundling activity of P-135-ABP (Yokota et al., 1998). On the other hand, only thin bundles with widths below 2 μm were seen in the presence of Ca2+-CaM (Fig. 3C). With these results, together with those obtained from the cosedimentation assay, it is concluded that the organization of F-actin into a bundle is suppressed by Ca2+-CaM due to inhibition of P-135-ABP binding to F-actin.

Figure 3.

Figure 3

Open in a new tab

Fluorescence micrographs of mixtures of P-135-ABP and RP-labeled F-actin. P-135-ABP (4.6 μg/mL) was mixed with RP-labeled F-actin (4.2 μg/mL) in a solution containing 0.5 mm CaCl2 with CaM (5.5 μm; C) or without it (B). The mixture was then observed under an epifluorescence microscope. A, RP-labeled F-actin alone in the presence of 0.5 mm CaCl2. The bar represents 30 μm.

Effect of Ca2+ and CaM Concentrations on the Binding Activity of P-135-ABP

The inhibitory effect of Ca2+-CaM was intimately dependent on the concentration of both Ca2+ and CaM. Figure 4A shows the relation between CaM concentrations and binding of P-135-ABP to F-actin in the cosedimentation assay. The amount of P-135-ABP associated with F-actin decreased remarkably as CaM concentrations increased up to 1 μm. Since the native molecular mass of P-135-ABP is 260 kD (Yokota et al., 1998), the molecular ratio of 3.2 μg/mL P-135-ABP:1 μm CaM is calculated to be 1:83.

Figure 4.

Figure 4

Open in a new tab

Dose-response relation of CaM and CaCl2 on the binding activity of P-135-ABP to F-actin. A, Effect of CaM concentrations on P-135-ABP binding to F-actin analyzed by a coprecipitation assay. The mixture of P-135-ABP (3.2 μg/mL) and F-actin (60 μg/mL) containing 0.5 mm CaCl2 in the presence of CaM at various concentrations was centrifuged and the supernatant and the pellet were electrophoresed on a 7.5% (w/v) acrylamide gel and subsequently analyzed by densitometry. The amount of P-135-ABP bound to F-actin was quantified and plotted using values relative to that for the mixture without CaM. The average values obtained from three separate preparations are shown. B, Effect of Ca2+ on the binding of RP-labeled F-actin on a glass surface coated with P-135-ABP. RP-labeled F-actin (0.5 μg/mL) and CaM (1.5 μm) in an assay medium containing Ca2+ of various concentrations were introduced into the flow chamber constructed using a P-135-ABP-coated coverslip. The amount of RP-labeled F-actin attached to the coverslip surface was quantified and plotted using values relative to that obtained in the presence of EGTA. The average values obtained from three separate preparations are shown.

Figure 4B shows the relation between Ca2+ concentrations and binding of RP-labeled F-actin to the glass surface coated with P-135-ABP. EGTA contamination from the P-135-ABP fraction can be avoided in this experiment, because the medium in the flow chamber is completely replaced by the assay medium, whose Ca2+ concentrations are controlled (see “Materials and Methods”). In randomly selected microscope fields (2,000 μm2 × 20), only a few RP-labeled actin filaments were attached at their one end to the glass surface not treated with P-135-ABP (data not shown), indicating a low level of non-specific binding. An RP-labeled F-actin attached to the P-135-ABP-coated glass surface along its whole length was judged as a bound F-actin. The number of F-actin bound to the glass surface coated with P-135-ABP in the presence of CaM significantly decreased by elevating Ca2+ concentration up to 2.5 μm (pCa 5.6). Further increase in the Ca2+ concentration induced only a gradual decrease in bound RP-labeled F-actin. In the absence of CaM, the number of F-actin bound to the glass surface coated with P-135-ABP was not affected by Ca2+ (data not shown).

In both assays, 20% to 30% of P-135-ABP remained bound to F-actin in a Ca2+-CaM-insensitive manner. For example, 28% of P-135-ABP was recovered in the F-actin pellet even in the presence of 0.5 mm Ca2+ and 7.8 μm CaM (Fig. 4A).

Ameliorating Effects of Antagonist for CaM on the Inhibitory Effect of Ca2+-CaM

To confirm that the inhibitory effect of Ca2+-CaM on the binding of P-135-ABP to F-actin is a specific phenomenon, we examined the influence of W-7 and W-5, antagonists for CaM, on the Ca2+-CaM-induced inhibition of binding activity of P-135-ABP (Fig. 5). Figure 6 shows the relation between concentrations of W-7 or W-5 and cosedimentation of P-135-ABP with F-actin in the presence of Ca2+-CaM. The amount of P-135-ABP associated with F-actin significantly increased when W-7 concentrations were elevated up to 10 μm. The addition of 30 μm W-7 increased the amount of P-135-ABP that coprecipitated with F-actin in the presence of Ca2+-CaM to a level similar to that in the presence of Ca2+ alone (compare lane Ap with lane Cp in Fig. 5). Thus, W-7 cancels the inhibitory effect of Ca2+-CaM on the binding activity of P-135-ABP to F-actin. W-5 also cancelled the inhibitory effect, but to a lesser extent (compare lane Dp with lane Cp in Figs. 5 and 6). The amount of P-135-ABP associated with F-actin was gradually increased with the elevation of the concentrations of W-5. However, only about 60% of P-135-ABP was coprecipitated with F-actin even in the presence of 30 μm W-5 (Fig. 6).

Figure 5.

Figure 5

Open in a new tab

Ameliorating effect of W-7 and W-5 on the inhibitory effect of Ca2+-CaM on P-135-ABP binding to F-actin in the presence of 0.5 mm CaCl2. A mixture of P-135-ABP (4.6 μg/mL), F-actin (60 μg/mL), and CaM (7.8 μm) was centrifuged in the presence of 30 μm W-7 (C) or W-5 (D). Resulting supernatants (s) and pellets (p) were analyzed by SDS-PAGE on a 7.5% (w/v) acrylamide gel. As a control experiment, a mixture of P-135-ABP and F-actin without CaM (A) or with CaM (B) in the absence of antagonists was treated with the same manner as above. The arrowhead and the arrow indicate the 135-kD polypeptide of P-135-ABP (ABP) and actin, respectively. The molecular masses of standard proteins are indicated on the left in kD.

Figure 6.

Figure 6

Open in a new tab

Dose-response relation of antagonists for CaM on the binding activity of P-135-ABP to F-actin in the presence of Ca2+-CaM. The mixture of P-135-ABP (4.6 μg/mL), F-actin (60 μg/mL), and CaM (7.8 μm), containing 0.5 mm CaCl2 in the presence of W-7 (○) or W-5 (●) at various concentrations, was centrifuged and the supernatant and the pellet were electrophoresed on a 7.5% (w/v) acrylamide gel and subsequently analyzed by densitometry. The amount of P-135-ABP bound to F-actin was quantified and plotted using values relative to that for the mixture without CaM and antagonists. The average values obtained from three analyses are shown.

These CaM antagonists also alleviated and cancelled the inhibitory effect of Ca2+-CaM on the arrangement of F-actin into bundles by P-135-ABP. Even in the presence of Ca2+-CaM, F-actin filaments were arranged into thick bundles (Fig. 7, C and D) whose diameters appeared to be similar to those in the presence of Ca2+ alone (Fig. 7A) when W-7 at concentrations above 10 μm was supplied to the mixture. In contrast, images of mixture of F-actin, P-135-ABP, and Ca2+-CaM in the presence of 10 μm W-5 (Fig. 7E) were identical with those in the absence of CaM antagonists (Fig. 7B). In the presence of 30 μm W-5, F-actin bundles became visible, (Fig. 7F) although the number and diameter of the bundles were significantly smaller than those observed in the presence of W-7.

Figure 7.

Figure 7

Open in a new tab

Ameliorating effect of W-7 and W-5 on the inhibitory effect of Ca2+-CaM on bundling activity of P-135-ABP in the presence of 0.5 mm CaCl2. P-135-ABP (4.6 μg/mL) was mixed with RP-labeled F-actin (4.2 μg/mL) in a solution containing 0.5% (v/v) DMSO (A), DMSO and 5.5 μm CaM (B), CaM and 10 μm W-7 (C), CaM and 30 μm W-7 (D), CaM and 10 μm W-5 (E), or CaM and 30 μm W-5 (F). The mixture was then observed under an epifluorescence microscope. The bar represents 30 μm.

DISCUSSION

In plant cells, it has been well known that CaM interacts with microtubules (Vantard et al., 1985; Wick et al., 1985; Fisher and Cyr, 1993) and that it is responsible for regulating the dynamics of cortical microtubules in a Ca2+-dependent manner (Cyr, 1991; Fisher et al., 1996). It is thought that the interaction between CaM and microtubules is mediated by microtubule-associated proteins (MAPs). Elongation factor-1α is one of these MAPs, whose Ca2+-CaM-dependent interaction with microtubules has been characterized in vitro: Ca2+-CaM inhibits the bundling formation and stabilization of microtubules induced by the elongation factor-1α (Durso and Cyr, 1994; Moore et al., 1998). In addition to MAPs, it has been reported that the motile and binding activity in vitro of microtubule-based motor protein, kinesin-like CaM binding protein (Reddy et al., 1996), which identified in plant cells, is suppressed by Ca2+-CaM through its binding to this protein (Song et al., 1997; Narasimhulu and Reddy, 1998).

In the present study, we have shown that the activity of an actin-binding protein from a higher plant is also modulated by CaM in a Ca2+-sensitive manner in vitro. Binding of P-135-ABP to F-actin was inhibited by Ca2+-CaM, but not by Ca2+ or CaM alone (Fig. 1). Moreover, Ca2+-CaM caused the dissociation of P-135-ABP from F-actin (Fig. 2). In general, two high-affinity and two low-affinity binding sites for Ca2+ are present in each CaM molecule. The dissociation constants (Kd) of these binding sites are about 10−6 and 10−5 m, respectively (Klee, 1988). The saturable concentration of Ca2+ and CaM for the inhibition of F-actin-binding of P-135-ABP was above 2.5 and 1.0 μm, respectively (Fig. 4). This Ca2+ concentration is comparable to the Kds for Ca2+ binding sites of the CaM molecule. Furthermore, W-7 cancelled the inhibitory effect of Ca2+-CaM on binding (Figs. 5 and 6) and bundling activities of P-135-ABP (Fig. 7). W-5 also alleviated the inhibitory effect of Ca2+-CaM, however, more weakly than W-7 (Figs. 5–7). It has been reported that W-5 interacted more weakly than W-7 with Ca2+-CaM and inhibited to a lesser extent the activation of Ca2+-CaM-dependent cyclic nucleotide phosphodiesterase in vitro (Tanaka et al., 1982). Together with the evidence showing that W-7 blocks the interaction of Ca2+-CaM with its target enzymes (Hidaka et al., 1980; Tanaka et al., 1982), these results support the specific interaction of Ca2+-CaM with P-135-ABP in the inhibition of P-135-ABP binding to F-actin.

Recently, it was revealed that P-135-ABP is a plant homolog of villin by cloning of a cDNA from a lily pollen library (Vidali et al., 1999). Villin is a well-characterized actin-bundling protein constructed from two domains: a gelsolin/severin domain and a head piece domain in COOH-terminal tail. At concentrations of Ca2+ below 10−6 m, villin bundles actin filaments through the head piece domain, whereas actin-filament severing and capping activities residing in the gelsolin/severin domain are expressed at Ca2+ concentrations above 10−6 m (Matsudaira and Janmey, 1988; Friederich et al., 1990; Otto, 1994). P-135-ABP also has these two domains (Vidali et al., 1999), however, the severing activity has not been demonstrated for isolated P-135-ABP even in the presence of Ca2+ at concentrations higher than 10−6 m (Yokota et al., 1998; this study). Furthermore, it has not been reported that non-plant villin thus far examined shows Ca2+-CaM sensitivity in its binding and bundling activities in vitro. Additional biochemical and molecular biological studies are needed to elucidate and confirm whether Ca2+-CaM sensitivity is characteristic for plant villin only and which regions or domains in the plant villin molecule provide this sensitivity.

Numerous actin-filament bundles are oriented parallel to the long axis of a pollen tube, from the grain into the subapical region in the growing tube. In contrast, only a few thin bundles are dispersed in random orientation at the tip, in which high concentrations of Ca2+ are present. In the case of lily pollen tubes, the Ca2+ concentration at the tip is estimated to be more than 3 μm (Pierson et al., 1994, 1996; Messerli and Robinson, 1997). This concentration is sufficient to induce inhibition of P-135-ABP binding to F-actin in the presence of 1.5 μm CaM (Fig. 4B). Furthermore, the tip focused distribution of CaM has been suggested in chemical fixed pollen tubes of tobacco and lily by using antibody against CaM (Tirlapur et al., 1994) and by fluphenazine that binds to CaM in a Ca2+-dependent manner (Hauβer et al., 1984), respectively. However, it was recently reported that the distribution of fluorescein-conjugated CaM microinjected into pollen tubes of Agapanthus umbellatus was uniform and that no tip-focused gradient was observed (Moutinho et al., 1998). The concentration of CaM in plant cells has been estimated to be 1.3 μm in barley aleurone cells (Schuurink et al., 1996), 4 μm in carrot cell lines (Fisher et al., 1996), and 11 μm in the cytoplasm of stamen hair cells of Tradescantia virginiana (Vos and Hepler, 1998). Therefore, even if CaM is dispersed uniformly throughout pollen tubes, as reported by Moutinho et al. (1998), it will be reasonable to deduce that the concentration of CaM in the tip region is in the micromolar order, which is sufficient to induce the inhibition of P-135-ABP binding to F-actin (Fig. 4A). Consequently, it is speculated that a high Ca2+ concentration is a cause for the lack of large actin-filament bundles at the tip region.

In the cosedimentation assay (Fig. 4A) and the binding assay of RP-labeled F-actin to glass surface coated with P-135-ABP (Fig. 4B), 20% to 30% of P-135-ABP remained bound to F-actin even in the presence of Ca2+-CaM. The thin bundles of RP-labeled F-actin in the presence of Ca2+-CaM appeared to be formed by Ca2+-CaM-insensitive P-135-ABP (Fig. 3C). The possibility is not excluded that a site or sites within a P-135-ABP molecule that interact with Ca2+-CaM are denatured during purification steps, making the actin-binding protein insensitive to Ca2+-CaM. However, it may also be that P-135-ABP that is insensitive to Ca2+-CaM is inherently present in lily pollen tubes and works to form thin bundles of actin filaments in the tip region containing high concentrations of Ca2+-CaM. This possibility remains unsolved.

In addition to P-135-ABP, other actin-binding proteins have been reported in pollen tubes such as a low Mr actin-sequestering and depolymerizing protein, profilin (Valenta et al., 1993; Mittermann et al., 1995; Huang et al., 1996; Vidali and Hepler, 1997; Gibbon et al., 1998), and an actin depolymerizing factor (ADF; Kim et al., 1993; Chung et al., 1995; Lopez et al., 1996). Recently, we isolated an actin-binding protein that is composed of a 115-kD polypeptide (Nakayasu et al., 1998). In addition, the presence of some factors fragmenting actin filaments in the presence of Ca2+ is suggested in lily pollen tubes. This is based on the observation that actin filaments are fragmented by the elevation of the intracellular Ca2+ concentration in pollen tubes (Kohno and Shimmen, 1987). Hence, the possibility must be considered that various actin-binding proteins and factors are also involved in controlling the architecture of actin filaments together with P-135-ABP in the tip region of pollen tubes.

MATERIALS AND METHODS

Purification of P-135-ABP and CaM from Lily Pollen Tubes

P-135-ABP was purified from the germinating pollen of lily (Lilium longiflorum) according to the method described previously (Yokota et al., 1998). Purified P-135-ABP was dialyzed against a solution containing 90 mm KCl, 0.2 mm EGTA, 2 mm MgCl2, 50 μg/mL leupeptin, 0.5 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm dithiothreitol (DTT), and 30 mm PIPES (1,4-piperazinediethanesulfonic acid)-KOH (pH 7.0). To remove any aggregate, dialysate was centrifuged at 300,000g for 20 min at 4°C. The resultant supernatant was used as P-135-ABP for various experiments described below.

CaM was also isolated from the germinating pollen of lily by the method described previously (Yokota et al., 1999). After dialysis against a solution containing 90 mm KCl, 50 μg/mL leupeptin, 0.5 mm PMSF, 1 mm DTT, and 30 mm PIPES-KOH (pH 7.0), CaM was stored at −80°C until use.

Cosedimentation Analysis of P-135-ABP with F-Actin

P-135-ABP was mixed with F-actin prepared from chicken skeletal muscle in an assay solution containing 90 mm KCl, 0.2 mm EGTA, 2 mm MgCl2, 50 μg/mL leupeptin, 0.5 mm PMSF, 1 mm DTT, and 30 mm PIPES-KOH (pH 7.0) and left standing for 10 min at 20°C. To examine the effect of Ca2+-CaM, CaCl2 (final concentration at 0.5 mm) and various concentrations of CaM were added to the assay solution. As a control, P-135-ABP alone was treated in the same manner. The samples were centrifuged at 150,000g for 20 min, and supernatants and pellets were analyzed by SDS-PAGE on a 7.5% (w/v) polyacrylamide gel (following the method of Laemmli [1970]). The amount of P-135-ABP bound to F-actin was determined quantitatively according to the method described in the previous paper (Yokota et al., 1998).

To examine the effect of Ca2+-CaM on the dissociation of P-135-ABP from F-actin, P-135-ABP was first mixed with F-actin. After a 20 min incubation at 20°C, CaCl2 (final concentration at 0.5 mm) alone or both CaCl2 and CaM were added to the mixture and left standing for 10 min at 20°C. The final concentrations of P-135-ABP, F-actin, and CaM were 3.2 μg/mL, 60 μg/mL, and 7.8 μm, respectively.

To examine the influence of W-7 and W-5, these chemicals (Sigma, St. Louis) dissolved in dimethylsulfoxide (DMSO) were added to a mixture of 4.6 μg/mL P-135-ABP, 60 μg/mL F-actin, and 7.8 μm CaM in the presence of 0.5 mm CaCl2. As a control, the same volume of DMSO (0.5% [v/v]) was added to the mixture.

Binding Assay of F-Actin on the Glass Surface Coated with P-135-ABP

RP-labeled F-actin was prepared by incubating F-actin with RP (Molecular Probes, Eugene, OR) according to the method of Kohno et al. (1991). A washing solution contained 30 mm KCl, 5 mm EGTA, 6 mm MgCl2, 5 mm DTT, 30 mm PIPES-KOH (pH 7.0), and various concentrations of CaCl2. To calculate actual Ca2+ concentrations from the Kds, a computer program was used (Kohno and Shimmen, 1988). Coverslips were coated with 0.2% (v/v) collodion dissolved in isopentyl acetate and then allowed to air dry. P-135-ABP was mixed with CaM and CaCl2 in the same solution used for the cosedimentation procedure and left standing on ice. The final concentrations of P-135-ABP, CaM, and CaCl2 were 4.1 μg/mL, 1.5 μm, and 0.5 mm, respectively. Fifty microliters of the mixture was placed on parafilm (American National Can, Neenah, WI) and a collodion-coated coverslip was laid on it. After 5 min at 25°C, a small amount of petroleum jelly was applied along the two opposite edges of the coverslip, and then this coverslip was placed on a glass slide to make a flow chamber with a volume of approximately 12 to 15 μL. The flow chamber was perfused with 100 μL of the washing solution and subsequently with 100 μL of the washing solution containing 0.5 μg/mL RP-labeled F-actin and 1.5 μm CaM. After 5 min, 100 μL of the washing solution was introduced into the chamber to remove unbound free RP-labeled F-actin.

To assess the binding activity of P-135-ABP, fluorescent images obtained by epifluorescence microscopy (BH2-RFC, Olympus, Tokyo) were recorded using a high-sensitivity television camera (C2400–08 SIT camera, Hamamatsu Photonics K. K., Hamamatsu, Japan) and a video tape recorder (model NV-FS65, National Co., Tokyo). For each condition, the number of RP-labeled F-actin bound to the glass surface coated with P-135-ABP was counted in 20 randomly selected areas of 2,000 μm2.

Observation of the Mixture of RP-Labeled F-Actin and P-135-ABP

RP-labeled F-actin was mixed with P-135-ABP in the presence of both CaM and CaCl2, or CaCl2 alone, under the same conditions as that in the cosedimentation assay. The final concentrations of RP-labeled F-actin, P-135-ABP, CaM, and CaCl2 were 4.2 μg/mL, 4.6 μg/mL, 5.5 μm, and 0.5 mm, respectively. To examine the influence of W-5 and W-7, these chemicals were added at final concentrations of 10 and 30 μm, respectively, to the mixture of RP-labeled F-actin, P-135-ABP, CaM, and CaCl2. The mixture was observed using the fluorescence microscope-video system described above.

Other Methods

SDS-PAGE was performed according to Laemmli (1970). Gels were stained with Coomassie Brilliant Blue. Protein concentrations were determined by the method of Bradford (1976). F-actin was prepared from acetone powder of chicken breast muscle according to Kohama (1981).

ACKNOWLEDGMENT

We thank the National Live Stock Breeding Center (Hyogo Station, Tatsuno, Japan) for the gift of chicken breast muscle.

LITERATURE CITED

  1. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Bretscher A, Weber K. Villin is a major protein of the microvillus cytoskeleton which binds both G and F actin in a calcium-dependent manner. Cell. 1980;20:839–847. doi: 10.1016/0092-8674(80)90330-x. [DOI] [PubMed] [Google Scholar]
  3. Chung Y-Y, Magnuson NS, An G. Subcellular localization of actin depolymerizing factor in mature and germinating pollen. Mol Cell. 1995;5:224–229. [Google Scholar]
  4. Cyr RJ. Calcium/calmodulin affects microtubule stability in lysed protoplasts. J Cell Sci. 1991;100:311–317. [Google Scholar]
  5. Durso NA, Cyr RJ. A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1α. Plant Cell. 1994;6:893–905. doi: 10.1105/tpc.6.6.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fisher DD, Cyr RJ. Calcium levels affect the ability to immunolocalize calmodulin to cortical microtubules. Plant Physiol. 1993;103:543–551. doi: 10.1104/pp.103.2.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fisher DD, Gilroy S, Cyr RJ. Evidence for opposing effects of calmodulin on cortical microtubules. Plant Physiol. 1996;112:1079–1087. doi: 10.1104/pp.112.3.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Friederich E, Pringault E, Arpin M, Louvard D. From the structure to the function of villin, an actin-binding protein of the brush border. Bioessays. 1990;12:403–408. doi: 10.1002/bies.950120902. [DOI] [PubMed] [Google Scholar]
  9. Gibbon BC, Zonia LE, Kovar DR, Hussey PJ, Staiger CJ. Pollen profilin function depends on interaction with poline-rich motifs. Plant Cell. 1998;10:981–993. doi: 10.1105/tpc.10.6.981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Glenney JR, Jr, Kaulfus P, Matsudaira P, Weber K. F-actin binding and bundling properties of fimbrin, a major cytoskeletal protein of microvillus core filaments. J Biol Chem. 1981;256:9283–9288. [PubMed] [Google Scholar]
  11. Hauβer I, Herth W, Reiss H-D. Calmodulin in tip-growing plant cells, visualized by fluorescing calmodulin-binding phenothiazines. Planta. 1984;162:33–39. doi: 10.1007/BF00397418. [DOI] [PubMed] [Google Scholar]
  12. Hidaka H, Yamaki T, Naka M, Tanaka T, Hayashi H, Kabayashi R. Calcium-regulated modulator protein interacting agents inhibits smooth muscle calcium-stimulated protein kinase and ATPase. Mol Pharmacol. 1980;17:66–72. [PubMed] [Google Scholar]
  13. Holdaway-Clarke TL, Feijó JA, Hackett GR, Kunkel JG, Hepler PK. Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium influx is delayed. Plant Cell. 1997;9:1999–2010. doi: 10.1105/tpc.9.11.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Huang S, McDowell JM, Weise MJ, Meagher RB. The Arabidopsis profilin gene family: evidence for an ancient split between constitutive and pollen-specific profilin genes. Plant Physiol. 1996;111:115–126. doi: 10.1104/pp.111.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kim S-R, Kim Y, An G. Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor. Plant Mol Biol. 1993;21:39–45. doi: 10.1007/BF00039616. [DOI] [PubMed] [Google Scholar]
  16. Klee CB. Interaction of calmodulin with Ca2+ and target proteins. In: Cohen P, Klee CB, editors. Calmodulin: Molecular Aspects of Cellular Regulation. Vol. 5. Amsterdam: Elsevier Biomedical Press; 1988. pp. 35–56. [Google Scholar]
  17. Kohama K. Amino acid incorporation rates into myofibrillar proteins of dystrophic chicken skeletal muscle. J Biochem. 1981;90:497–501. doi: 10.1093/oxfordjournals.jbchem.a133497. [DOI] [PubMed] [Google Scholar]
  18. Kohno T, Okagaki T, Kohama K, Shimmen T. Pollen tube extract supports the movement of actin filaments in vitro. Protoplasma. 1991;161:75–77. [Google Scholar]
  19. Kohno T, Shimmen T. Ca2+-induced fragmentation of actin filaments in pollen tubes. Protoplasma. 1987;141:177–179. [Google Scholar]
  20. Kohno T, Shimmen T. Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+ J Cell Biol. 1988;106:1539–1543. doi: 10.1083/jcb.106.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kühtreiber WM, Jaffe LF. Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J Cell Biol. 1990;110:1565–1573. doi: 10.1083/jcb.110.5.1565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lancelle SA, Cresti M, Hepler PK. Ultrastructure of the cytoskeleton in freeze-substituted pollen tubes of Nicotiana alata. Protoplasma. 1987;140:141–150. [Google Scholar]
  24. Lancelle SA, Cresti M, Hepler PK. Growth inhibition and recovery in freeze-substituted Lilium longiflorum pollen tubes: structural effects of caffeine. Protoplasma. 1997;196:21–33. [Google Scholar]
  25. Lancelle SA, Hepler PK. Ultrastructure of freeze-substituted pollen tubes of Lilium longiflorum. Protoplasma. 1992;167:215–230. [Google Scholar]
  26. Li Y-Q, Moscatelli A, Cai G, Cresti M. Functional interactions among cytoskeleton, membranes, and cell wall in the pollen tube of flowering plants. Int Rev Cytol. 1997;176:133–199. doi: 10.1016/s0074-7696(08)61610-1. [DOI] [PubMed] [Google Scholar]
  27. Lin C-S, Shen W, Chen ZP, Tu Y-H, Matsudaira P. Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney. Mol Cell Biol. 1994;14:2457–2467. doi: 10.1128/mcb.14.4.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lopez I, Anthony RG, Maciver SK, Jiang C-J, Khan S, Weeds AG, Hussey PJ. Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins. Proc Natl Acad Sci USA. 1996;93:7415–7420. doi: 10.1073/pnas.93.14.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Malhó R, Read ND, Pais MS, Trewavas AJ. Role of cytosolic free calcium in the reorientation of pollen tube growth. Plant J. 1994;5:331–341. [Google Scholar]
  30. Malhó R, Read ND, Trewavas AJ, Pais MS. Calcium channel activity during pollen tube growth and reorientation. Plant Cell. 1995;7:1173–1184. doi: 10.1105/tpc.7.8.1173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Malhó R, Trewavas AJ. Localized apical increases of cytosolic free calcium control pollen tube orientation. Plant Cell. 1996;8:1935–1949. doi: 10.1105/tpc.8.11.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mascarenhas JP. Molecular mechanisms of pollen tube growth and differentiation. Plant Cell. 1993;5:1303–1314. doi: 10.1105/tpc.5.10.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matsudaira P, Janmey P. Pieces in the actin-severing protein puzzle. Cell. 1988;54:139–140. doi: 10.1016/0092-8674(88)90542-9. [DOI] [PubMed] [Google Scholar]
  34. Messerli M, Robinson KR. Tip localized Ca2+ pulses are coincident with peak pulsatile growth rates in pollen tubes of Lilium longiflorum. J Cell Sci. 1997;110:1269–1278. doi: 10.1242/jcs.110.11.1269. [DOI] [PubMed] [Google Scholar]
  35. Miller DD, Callaham DA, Gross DJ, Hepler PK. Free Ca2+ gradient in growing pollen tubes of Lilium. J Cell Sci. 1992;101:7–12. [Google Scholar]
  36. Miller DD, Lancelle SA, Hepler PK. Actin microfilaments do not form a dense meshwork in Lilium longiflorum pollen tube tips. Protoplasma. 1996;195:123–132. [Google Scholar]
  37. Mittermann I, Swoboda I, Pierson E, Eller N, Kraft D, Valenta R, Heberle-Bors E. Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation. Plant Mol Biol. 1995;27:137–146. doi: 10.1007/BF00019185. [DOI] [PubMed] [Google Scholar]
  38. Moore RC, Durso NA, Cyr RJ. Elongation factor-1α stabilizes microtubules in a calcium/calmodulin-dependent manner. Cell Motil Cytoskelet. 1998;41:168–180. doi: 10.1002/(SICI)1097-0169(1998)41:2<168::AID-CM7>3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]
  39. Mooseker MS, Graves TA, Wharton KA, Falco N, Howe CL. Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells. J Cell Biol. 1980;87:809–822. doi: 10.1083/jcb.87.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Moutinho A, Love J, Trewavas AJ, Malhó R. Distribution of calmodulin protein and mRNA in growing pollen tubes. Sex Plant Reprod. 1998;11:131–139. [Google Scholar]
  41. Nakayasu T, Yokota E, Shimmen T. Purification of an actin-binding protein composed of 115-kDa polypeptide from pollen tubes of lily. Biochem Biophys Res Commun. 1998;249:61–65. doi: 10.1006/bbrc.1998.9088. [DOI] [PubMed] [Google Scholar]
  42. Namba Y, Ito M, Zu Y, Shigesada K, Maruyama K. Human T cell L-plastin bundles actin filaments in a calcium-dependent manner. J Biochem. 1992;112:503–507. doi: 10.1093/oxfordjournals.jbchem.a123929. [DOI] [PubMed] [Google Scholar]
  43. Narasimhulu SB, Reddy ASN. Characterization of microtubule binding domain in the Arabidopsis kinesin-like calmodulin binding protein. Plant Cell. 1998;10:957–965. doi: 10.1105/tpc.10.6.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Otto JJ. Actin-bundling proteins. Curr Opin Cell Biol. 1994;6:105–109. doi: 10.1016/0955-0674(94)90123-6. [DOI] [PubMed] [Google Scholar]
  45. Pierson ES, Cresti M. Cytoskeleton and cytoplasmic organization of pollen and pollen tubes. Int Rev Cytol. 1992;140:73–125. [Google Scholar]
  46. Pierson ES, Miller DD, Callaham DA, Shipley AM, Rivers BA, Cresti M, Hepler PK. Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell. 1994;6:1815–1828. doi: 10.1105/tpc.6.12.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pierson ES, Miller DD, Callaham DA, van Aken J, Hackett G, Hepler PK. Tip-localized calcium entry fluctuates during pollen tube growth. Dev Biol. 1996;174:160–173. doi: 10.1006/dbio.1996.0060. [DOI] [PubMed] [Google Scholar]
  48. Pollard TD, Cooper JA. Actin and actin-binding proteins: a critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
  49. Prassler J, Stocker S, Marriott G, Heidecker M, Kellermann J, Gerisch G. Interaction of a Dictyostelium member of the plastin/fimbrin family with actin filaments and actin-myosin complexes. Mol Biol Cell. 1997;8:83–95. doi: 10.1091/mbc.8.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rathore KS, Cork RJ, Robinson KR. A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes. Dev Biol. 1991;148:612–619. doi: 10.1016/0012-1606(91)90278-b. [DOI] [PubMed] [Google Scholar]
  51. Reddy ASN, Safadi F, Narasimhulu SB, Golovkin M, Hu X. A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain. J Biol Chem. 1996;271:7052–7060. doi: 10.1074/jbc.271.12.7052. [DOI] [PubMed] [Google Scholar]
  52. Schuurink RC, Chan PV, Jones RL. Modulation of calmodulin mRNA and protein levels in barley aleurone. Plant Physiol. 1996;111:371–380. doi: 10.1104/pp.111.2.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Song H, Golovkin M, Reddy ASN, Endow SA. In vitro motility of AtKCBP, a calmodulin-binding kinesin protein of Arabidopsis. Proc Natl Acad Sci USA. 1997;94:322–327. doi: 10.1073/pnas.94.1.322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stossel TP, Chaponnier C, Ezzell RM, Hartwig JH, Janmey PA, Kwiatkowski DJ, Lind SE, Smith DB, Southwick FS, Yin HL, Zaner KS. Nonmuscle actin-binding proteins. Annu Rev Cell Biol. 1985;1:353–402. doi: 10.1146/annurev.cb.01.110185.002033. [DOI] [PubMed] [Google Scholar]
  55. Tanaka T, Ohmura T, Hidaka H. Hydrophobic interaction of the Ca2+-calmodulin complex with calmodulin antagonists naphthalenesulfonamide derivatives. Mol Pharmacol. 1982;22:403–407. [PubMed] [Google Scholar]
  56. Taylor LP, Hepler PK. Pollen germination and tube growth. Annu Rev Plant Physiol Plant Mol Biol. 1997;48:461–491. doi: 10.1146/annurev.arplant.48.1.461. [DOI] [PubMed] [Google Scholar]
  57. Tirlapur UK, Scali M, Moscatelli A, Casino CD, Cai G, Tiezzi A, Cresti M. Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L. Zygote. 1994;2:63–68. doi: 10.1017/s0967199400001775. [DOI] [PubMed] [Google Scholar]
  58. Tiwari SC, Polito VS. Organization of the cytoskeleton in pollen tubes of Pyrus communis: a study employing conventional and freeze-substitution electron microscopy, immunofluorescence, and rhodamine-phalloidin. Protoplasma. 1988;147:100–112. [Google Scholar]
  59. Valenta R, Ferreira F, Grote M, Swoboda I, Vrtala S, Duchêne M, Deviller P, Meagher RB, McKinney E, Heberle-Bors E, Kraft D, Scheiner O. Identification of profilin as an actin-binding protein in higher plants. J Biol Chem. 1993;268:22777–22781. [PubMed] [Google Scholar]
  60. Vantard M, Lambert A-M, Mey JD, Picquot P, Van Eldik LJ. Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus. J Cell Biol. 1985;101:488–499. doi: 10.1083/jcb.101.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Vidali L, Hepler PK. Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum. Cell Motil Cytoskelet. 1997;36:323–338. doi: 10.1002/(SICI)1097-0169(1997)36:4<323::AID-CM3>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  62. Vidali L, Yokota E, Cheung AY, Shimmen T, Hepler PK. The 135-kDa actin-bundling protein from Lilium longiflorum pollen is the plant homologue of villin. Protoplasma. 1999;209:283–291. [Google Scholar]
  63. Vos JW, Hepler PK. Calmodulin is uniformly distributed during cell division in living stamen hair cells of Tradescantia virginiana. Protoplasma. 1998;201:158–171. [Google Scholar]
  64. Wick SM, Muto S, Duniec J. Double immunofluorescence labeling of calmodulin and tubulin in dividing plant cells. Protoplasma. 1985;126:198–206. [Google Scholar]
  65. Yokota E, Muto S, Shimmen T. Inhibitory regulation of higher-plant myosin by Ca2+ ions. Plant Physiol. 1999;119:231–239. doi: 10.1104/pp.119.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yokota E, Shimmen T. The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity. Planta. 1999;209:264–266. doi: 10.1007/s004250050631. [DOI] [PubMed] [Google Scholar]
  67. Yokota E, Takahara K, Shimmen T. Actin-bundling protein isolated from pollen tubes of lily: biochemical and immunocytochemical characterization. Plant Physiol. 1998;116:1421–1429. doi: 10.1104/pp.116.4.1421. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES