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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We have previously reported the isolation of threeZea maysgenes that encode actin-depolymerising factors/cofilins, a family of low molecular weight actin regulating proteins. In the present study, we have characterised one of these proteins, ZmADF3. We report that ZmADF3 binds G-actin with a 1:1 stoichiometry, and that the interaction with F-actin is pH-sensitive. ZmADF3 co-sediments mainly with F-actin at pH 6.0 and mainly with G-actin at pH 9.0. This response is more similar to that of vertebrate cofilin and ADF than to that ofAcanthamoebaactophorin which, although more similar in primary sequence to ZmADF3, is not pH sensitive. However, ZmADF3 requires a more basic environment to depolymerise actin relative to either vertebrate ADF or cofilin. Filaments decorated with ZmADF3 at low pH are very rapidly depolymerised upon raising the pH, which is consistent with a severing mechanism for the disruption of actin filaments. Also, we demonstrate that ZmADF3 binds specific polyphosphatidylinositol lipids, especially phosphatidylinositol 4,5-bisphosphate (PIP2), and we show that this binding inhibits the actin-depolymerising function of ZmADF3. Moreover, we show that a consequence of ZmADF3 binding PIP2 is the inhibition of the activity of polyphosphatidylinositol specific plant phospholipase C, indicating the possibility of reciprocal modulation of this major signalling pathway and the actin cytoskeleton.


Abbreviations
EDC

1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide;

Bis-Tris Propane

(1,3-bis[tris(Hydroxymethyl)methylamino]-propane;

PI

Phosphatidylinositol;

PIP

Phosphatidylinositol 4-monophosphate;

PIP2

Phosphatidylinositol 4,5-bisphosphate;

PS

Phosphatidylserine;

PE

Phosphatidyl ethanolamine;

PC

Phosphatidylcholine;

OAG

1-Oleoyl-2-acetylgylcerol;

IP3

Inositol 1,4,5-trisphosphate;

PLC

Phospholipase C.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The actin cytoskeleton is a highly dynamic structure that has the ability to reorganise to perform its essential functions, which include cell division and cytoplasmic streaming. This reorganisation of the actin cytoskeleton is mediated by a catalogue of actin binding proteins that serve to anchor, crosslink or regulate the network within the cell. One of the proteins that serves to regulate the dynamics of the actin network is actin-depolymerising factor (ADF) or cofilin. The ADF/cofilin group of proteins bind both G-actin and F-actin. They increase actin dynamics by severing the growing filaments thereby increasing the number of filament ends for polymerisation ( Maciver 1998) and/or by increasing the turnover of filaments by accelerating the depolymerisation rate at the pointed end ( Carlier et al. 1997 ;Theriot 1997).

In maize, three genes have been identified that encode actin depolymerising factor, ZmADF1, ZmADF2 and ZmADF3. These three genes are differentially expressed in the maize plant with ZmADF1 and ZmADF2 expressed exclusively in pollen and ZmADF3 expressed in every other maize tissue except pollen. The deduced amino acid sequences of the three proteins were found to show between 29% and 33% identity with vertebrate cofilin and ADF, respectively. Biochemical analysis of ZmADF3 showed that it possessed the G-actin and F-actin binding properties characteristic of the ADF/cofilin group ( Lopez et al. 1996 ). We recently described the intracellular localisation of ZmADF3 in the development of maize root hair cells and found that this protein redistributes to the growing tip as the actin cytoskeleton reorganises ( Jiang et al. 1997a ). In addition, ZmADF1 has been shown to cause the reorganisation of the actin cytoskeleton, presumably in co-operation with endogenous actin bundling proteins when microinjected into Tradescantia stamen hair cells ( Hussey et al. 1998 ). This localisation and effect of maize ADFs emphasises their role in the regulation of the actin network in vivo. Moreover, ZmADF3 can be phosphorylated on Ser 6 and a Ser 6 to Asp mutation causes loss of ADF activity, both facts indicating that at least one controlling influence on ZmADF3 is the reversible phosphorylation of this residue ( Smertenko et al. 1998 ). Vertebrate ADF/ cofilins possess a bipartite nuclear targeting motif which is reported to be important in redistribution of the protein into the nucleus of cells which have been subjected to heat shock or treatment with dimethylsulphoxide ( Nishida et al. 1987 ). Despite the fact that ZmADF3 does not have a recognisable nuclear targeting motif, this protein can also redistribute to the nucleus but only after treatment with cytochalasin D ( Jiang et al. 1997a ).

In this paper we describe the interaction of ZmADF3 with G- and F-actin and identify factors that modulate its actin depolymerising activity. We demonstrate that ZmADF3 activity is dependent on pH and that it is inhibited by specific phospholipids. The latter point is further emphasised by the fact that ZmADF3 inhibits the breakdown of PIP2 by plant phospholipase C presumably as a consequence of the high affinity of ZmADF3 for PIP2.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

ZmADF3 and actin monomers bind with a 1:1 stoichiometry

Previously, we had shown that ZmADF3 can form a complex with actin ( Lopez et al. 1996 ). Here, using two different methods, we show that ZmADF3 forms a 1:1 complex with actin monomers. First, chemical crosslinking of actin:ZmADF3 complexes by EDC produced a species with a molecular weight of approximately 60 kDa on one-dimensional SDS PAGE gels ( Fig. 1a). The electrophoretic size of this product is consistent with a 1:1 complex of actin and ZmADF3 (43 kDa plus 17 kDa). Second, two-dimensional electrophoresis of the actin:ZmADF3 complex (not chemically crosslinked) and subsequent quantification of the relative amounts of each protein present showed that the complex contains equimolar amounts of the two proteins ( Fig. 1b).

image

Figure 1. ZmADF3 and G-actin complex formation.

(a) Coomassie stained SDS PAGE gel loaded with EDC crosslinked ZmADF3 and G-actin. The crosslinking has resulted in the appearance of an actin:ZmADF3 complex at approximately 60 kDa.

(b) Coomassie stained two-dimensional gel of the actin:ZmADF3 complex (not crosslinked). A longitudinal strip was cut from a lane of a native gel which was loaded with the actin:ZmADF3 complex. This strip was laid across the top of an SDS PAGE gel. Known concentrations of actin and ZmADF3 are run as standards to compare the relative concentrations of the proteins.

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ZmADF3 preferentially binds ADP-actin monomers

Gel filtration of the actin:ZmADF3 complex in the presence of either ATP or ADP shows that ZmADF3 has a higher affinity for ADP-actin monomers than ATP-actin monomers. Figure 2 shows a series of S-200 gel filtration elution profiles of actin and ZmADF3, singly and together, in the presence of ATP or ADP. The profiles of actin or ZmADF3 alone in the presence of either ATP or ADP are unchanged; elution profiles shown in Fig. 2(a) are for actin or ZmADF3 alone in the presence of ATP. However, in the profiles of the equimolar mixtures of actin and ZmADF3 in the presence of ATP or ADP, the ZmADF3 elution peaks were shifted to co-migrate with actin which itself had shifted to a lower elution volume indicating that actin:ZmADF3 complex formation had occurred ( Fig. 2b,c). Moreover, the fact that a larger amount of ZmADF3 co-migrated with actin under ADP conditions (see Fig. 2b) compared to ATP conditions (see Fig. 2c) indicates that ZmADF3 binds ADP-actin monomers in preference to ATP-actin monomers.

image

Figure 2. Binding of ZmADF3 to ATP- and ADP-actin monomers.

S-200 gel filtration of actin (□) and ZmADF3 (▪) alone in the presence of ATP (a), as a mixture in the presence of ADP (b), as a mixture in the presence of ATP (c).

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Effects of ATP and ADP on ZmADF3-induced actin dynamics

Recently we showed that ZmADF3 increases in vitro actin dynamics ( Jiang et al. 1997a , 1997b;Smertenko et al. 1998 ). Light scattering has been used in this paper to examine the effect of ATP or ADP on the polymerisation of actin filaments. Figure 3(a) shows that under ATP conditions ZmADF3 accelerates the rate of spontaneous polymerisation of actin filaments much more so than under ADP conditions.

image

Figure 3. Effects of ATP and ADP on ZmADF3-induced actin dynamics.

(a) Actin polymerisation in the presence and absence of ZmADF3 under ATP or ADP conditions monitored over 30 min. □, actin (ATP);▪, actin/ZmADF3 (ATP);○, actin (ADP);•, actin/ZmADF3 (ADP). These data show that ZmADF3 accelerated actin dynamics is greater in the presence of ATP.

(b) Viscosity of polymerising actin solutions in either ATP (□) or ADP (▪) conditions in the presence of increasing concentrations of ZmADF3. These data show that ZmADF3 has a greater effect on the disruption of actin filaments in ATP conditions rather than in ADP conditions. For both (a) and (b) the standard deviations of three replicated experiments are shown.

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A viscosity assay has been used to examine the disruption of polymerising actin solutions by ZmADF3 in the presence of ATP or ADP. In the presence of ADP the ability of ZmADF3 to reduce the low shear viscosity of F-actin is markedly reduced compared to its ability in the presence of ATP, as demonstrated in Fig. 3(b).

ZmADF3 binds and depolymerisies F-actin in a pH sensitive manner

We have shown previously that ZmADF3 binds F-actin at pH 7.0 ( Lopez et al. 1996 ). Similar binding assays were carried out here but the sedimentations were performed at pH values ranging from 6.0 to 9.0. The quantity of actin and ZmADF3 in the pellets and the supernatants was quantified and plotted in Fig. 4(a). The results show that ZmADF3 co-sediments mainly with F-actin at pH 6.0 and mainly with G-actin at pH 9.0, indicating that the interaction of ZmADF3 and F-actin is sensitive to pH. The transition point, the point at which 50% each of actin and ZmADF3 is in the pellet, occurs at pH 7.7.

image

Figure 4. Effect of pH on ZmADF3 activity.

(a) Co-sedimentation of ZmADF3 with F-actin under different pH conditions (ranging from pH 6.0–9.0). Relative percentages of actin in the pellet (▪) and ZmADF3 in the supernatant (□) are shown. The standard deviations of three replicated experiments are shown.

(b) Actin (10 μM) was polymerised in the presence of ZmADF3 (1 μM, ▪; 10 μM, □) at pH 6.5 until the light scattering of the reaction mixture stabilised. After 75 sec the pH conditions were raised to pH 8.0. The standard deviations of two replicated experiments are shown. These data show that ZmADF3 mediates a pH sensitive destruction of actin filaments.

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Light scattering was used to measure the pH sensitive interaction of ZmADF3 with actin ( Fig. 4b). The addition of increasing concentrations of ZmADF3 to F-actin at pH 6.5 increases the light scattering, consistent with ZmADF3 binding to produce thicker filaments with higher light scattering ability. After 75 sec the pH was raised to 8.0. This pH switch caused an instantaneous drop followed by a progressive decline in light scattering indicating a rapid severing and depolymerisation of filaments.

ZmADF3 activity is inhibited by PIP and PIP2

Native gel electrophoresis was used to demonstrate that two phospholipids, phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4, 5-bisphosphate (PIP2), specifically bind to ZmADF3. ZmADF3 migrates as a distinct single band on the native gel in the absence of phospholipid and also in the presence of several different phospholipids with the exceptions of PIP and PIP2. Both PIP and PIP2, which do not bind actin ( Yonezawa et al. 1990 ), cause ZmADF3 to migrate anomalously in the gel which is indicative of a binding interaction ( Fig. 5a).

image

Figure 5. Effect of phospholipids on ZmADF3 activity.

(a) Native gel of mixtures of ZmADF3 and various phospholipids. Both PIP and PIP2 cause ZmADF3 to smear in the gel indicating a binding interaction.

(b) Co-sedimentation assays in which ZmADF3 (2 μM) was incubated with each of eight phospholipids (100 μM) prior to the addition of F-actin and subsequent sedimentation. The percentage of G-actin in the supernatant relative to control (–PL) is shown. Both PIP and PIP2 reduce the amount of G-actin in the supernatant indicating that these inhibit ZmADF3 activity.

(c) Co-sedimentation assay in which ZmADF3 (2 μM) was incubated with either PIP (□) or PIP2 (▪) in a range of molar ratios prior to the addition of F-actin and subsequent sedimentation. The relative percentage of G-actin in the supernatant is shown. Both PIP and PIP2 inhibit ZmADF3 activity.

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The effect of phospholipid on the actin depolymerising activity of ZmADF3 was demonstrated in sedimentation experiments at pH 8.0. ZmADF3 on its own is shown to depolymerise F-actin at pH 8.0 and is used as the standard in these experiments, i.e. showing 100% activity. The relative activity of ZmADF3 in the presence of several phospholipids was determined and it is shown that PIP and PIP2 reduce the activity of ZmADF3 by 83% and 98%, respectively, whilst the other phospholipids have little effect ( Fig. 5b). Similar sedimentation experiments were carried out using a range of PIP or PIP2 to ZmADF3 molar ratios. These data show that both PIP and PIP2 reduce the activity of ZmADF3 as the molar ratio increases with PIP2 having the greater effect of the two ( Fig. 5c).

ZmADF3 inhibits plant phosphoinositide phospholipase C activity

Figure 6 shows the effect of different concentrations of ZmADF3 on the activity of bean plasma membrane phosphoinositide phospholipase C (PLC) assayed in the presence of 50 μm PIP2. A strong and dose-dependent inhibition of PLC activity by ZmADF3 is evident, with the addition of 2 μm resulting in 50% inhibition of activity. Neither pre-incubations with control buffer nor ovalbumin in amounts equivalent to ZmADF3 had any effect on PLC activity (data not shown;Drøbak et al. 1994 ). These data demonstrate that ZmADF3 is capable of interfering with the utilisation of PIP2 by plant PLC – presumably by forming ZmADF3:PIP2 complexes, as suggested in Fig. 5, with stoichiometric ratios heavily biased towards PIP2.

image

Figure 6. Effect of ZmADF3 on plant phospholipase C activity.

The activity of plant PLC was measured in the presence of increasing concentrations of ZmADF3. These data show that ZmADF3 inhibits the hydrolysis of PIP2 by plant PLC. The standard deviations of three replicated experiments are shown.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Here we report that ZmADF3 binds G-actin with a 1:1 stoichiometry and that it preferentially binds ADP-actin rather than ATP-actin. We have shown that the binding of ZmADF3 to F-actin is pH sensitive. We have also shown that ZmADF3 is inhibited by specific polyphosphatidylinositol lipids, and that the interaction between ZmADF3 and these lipids inhibits the activity of plant phosphoinositide phospholipase C.

The fact that ZmADF3 binds ADP-actin in preference to ATP-actin is consistent with its role in severing actin filaments and increasing the dissociation rate constant at the pointed ends ( Carlier et al. 1997 ;Maciver 1998). ADF/cofilins are unique as actin binding proteins in their ability to alter the twist of actin filaments ( McGough et al. 1997 ). Although ADF/cofilins do not have bundling activity, they are known to enhance the bundling activity of microfilament bundling proteins in vitro ( Maciver et al. 1991a ). Bundles of actin are also seen in Dictyostelium amoebae induced to produce high levels of cofilin ( Aizawa et al. 1996 ) and in Tradescantia stamen hair cells injected with ZmADF1 ( Hussey et al. 1998 ), so this process may occur in vivo. ADF/cofilins are also highly co-operative in F-actin binding ( Hawkins et al. 1993 ;Hayden et al. 1993 ;McGough et al. 1997 ;Ressad et al. 1998 ) which is thought to be directly related to their altering of filament twisting ( McGough et al. 1997 ).

The pH dependent actin binding behaviour of ZmABP3 ( Fig. 4) is distinct from that of Acanthamoeba actophorin and vertebrate ADF/cofilin. The ZmADFs are more similar in amino acid sequence to Acanthamoeba actophorin than the vertebrate ADF/cofilins, but actophorin is not pH sensitive ( Maciver et al. 1998 ). The ADF/cofilins generally bind F-actin at low pH and G-actin at high pH values. A sharp transition between F- and G-actin binding takes place centred around pH 7.3 in the case of the vertebrate proteins ( Hawkins et al. 1993 ;Hayden et al. 1993 ), but we have found that with ZmADF3 this switch in behaviour occurs at higher pH values, pH 7.7. This may reflect the difference in resting pH in vertebrate cells and plant cells. In resting vertebrate cells the pH is close to neutral, while that of plant cells is more alkali, typically pH 7.5 ( Felle 1988;Gout et al. 1992 ;Saint-Ges et al. 1991 ). A more recent report measured the pH to be 7.1 in maize root hair cells ( Kosegarten et al. 1997 ) but these authors do point out that this is likely to be an underestimate due to the contribution of acidic vacuoles.

The fact that ZmADF3 exhibits a pH sensitive activity in vitro raises the possibility that such control may occur in vivo. Previously, we have shown that ZmADF3 redistributes to the tip of elongating root hair cells and based on these data we strongly suggested that ZmADF3 plays an active role in enhancing actin dynamics to promote tip growth ( Jiang et al. 1997a ). It is possible that localised changes in pH or pH gradients in the tip growing root hair cells regulate changes in maize ADF activity. For example, local alkalinization at or close to the tip could render ZmADF3 active resulting in an increase in actin polymerisation for cell elongation. Although there is evidence for proton transport across the plasma membrane of the root hair and that cytosolic pH can vary depending on the external environment, there is no evidence as yet for pH gradients or localised pH changes in root hairs ( Herrman & Felle 1995).

Recently, work with an actin-depolymerising factor (ADF1) from Arabidopsis thaliana showed that this protein increased the rate of actin dissociation from the pointed end of microfilaments ( Carlier et al. 1997 ). This has also been found to be the case for ZmADF3 ( Jiang et al. 1997a ), human ADF and actophorin from Acanthamoeba ( Maciver et al. 1998 ). Carlier and colleagues ( Carlier et al. 1997 ) argued that the effect of ADF1 on microfilaments could be explained solely by the 25-fold increase in the rate of dissociation from the pointed end and discounted altogether filament severing. However, others ( Theriot 1997) have pointed out that the two activities (increase in dissociation rate and filament severing) are not mutually exclusive. Moreover, the severing of actin filaments by actophorin has been observed directly in the light microscope ( Maciver et al. 1991b ). Furthermore, the very rapid disassembly observed by light scattering when ZmADF3 decorated filaments at pH 6.5 are switched to higher pH values ( Fig. 4b) is indicative of a severing mechanism. The following progressive decline in light scattering may indicate an increase in the rate of dissociation of monomer from the pointed end thus supporting the view that both filament severing and enhanced pointed end depolymerisation occur ( Maciver 1998;Theriot 1997).

The tertiary structure of mammalian ADF, yeast cofilin and Acanthamoeba actophorin have recently been deduced ( Fedorov et al. 1997 ;Hatanaka et al. 1996 ;Leonard et al. 1997 ). Despite the fact that the ADF/cofilin group share a very limited amino acid sequence similarity to gelsolin segment 1 or profilin ( Matsuzaki et al. 1988 ), they all share a common structural fold. In addition to binding actin, the three proteins, namely gelsolin, profilin and the ADF/cofilins bind PIP and PIP2 ( Quirk et al. 1993 ;Yonezawa et al. 1990 ). The ability of these proteins to bind inositide lipids may be an important mechanism whereby they are located close to the plasma membrane and are involved or regulated by the phosphoinositide signal transduction pathway ( Bamburg & Bray 1987;Jiang et al. 1997a ).

We have characterised the activity of the maize ADF, ZmADF3, and conclude that at least three controls influence its activity: phosphorylation ( Smertenko et al. 1998 ), pH and phosphoinositides. Perhaps it is the summation of these influences that serve to modulate the activity of ADF temporally and spatially within the cell so that the actin network can reorganise to perform its many different roles.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

G-and F-actin buffers

Buffer G: 2 m m Tris pH 8.0, 0.2 m m ATP or ADP where indicated, 0.2 m m CaCl2, 0.5 m m DTT and 1 m m NaN3. 10×KME: 0.5 m KCl, 10 m m MgCl2, 10 m m EGTA buffered with 0.1 m Tris pH 8.0 or 0.1 m Imidazole pH 6.5 or Bis-Tris Propane pH 6.0–9.0. Buffer F: Buffer G containing 1×KME buffered with one of the above.

Protein purification

Recombinant ZmADF3 protein was prepared as described in Lopez et al. (1996) . Actin was purified from rabbit skeletal muscle by the method of MacLean-Fletcher & Pollard (1980). Protein concentrations were determined spectrophotometrically. For actin solutions, the optical density was measured at 290 nm in order to lessen the interference from nucleotide absorbance. One optical density unit at 290 nm is equivalent to 38 μm ( Lehrer & Kerwar 1972). For ZmADF3, the deduced amino acid sequence ( Lopez et al. 1996 ) was used to calculate that one optical density unit at 280 nm is equivalent to 75.6 μm by the method of Gill & von Hippel (1989).

Electrophoresis

One-dimensional SDS-PAGE was as described in Laemmli (1970). Native polyacrylamide gel electrophoresis was performed as described in Maciver & Weeds (1994). The two-dimensional gel electrophoresis used to determine the stoichiometry of the actin:ZmADF3 complex was performed by mixing equimolar 10 μm concentrations of actin and ZmADF3 and fractionating this mixture on a native gel. A central longitudinal slice of this gel containing the actin:ZmADF3 complex was then placed across an SDS-PAGE gel and run alongside known standard concentrations of actin and ZmADF3 ( Fig. 1b)

We have modified the native gel system to investigate ZmADF3 binding to lipids. 2 μg of ZmADF3 was loaded with either PI, PIP, PIP2, PE, PC, PS, IP3 or OAG (Sigma, UK) at a molar ratio of 1:5 in 5% sucrose. Six per cent gels were prepared in half concentration gel buffer containing 0.2 m m ATP, 0.2 m m EGTA, and 0.5 m m DTT; the tank buffer was also half concentration. These gels were run at 10 mA for 40 min.

Chemical crosslinking

Crosslinking was carried out using EDC ( Doi et al. 1987 ). Equimolar 10 μM concentrations of G-actin and ZmADF3 were mixed under conditions favouring actin:ZmADF3 complex formation ( Lopez et al. 1996 ). 4.0 m m EDC was added and the mixture incubated for 1 h at room temperature. The reaction was stopped by the addition of SDS-PAGE sample buffer ( Laemmli 1970) and the products separated on a 12% gel.

Gel filtration

Actin and ZmADF3, each at 10 μM, in Buffer G containing either 0.2 m m ATP or ADP were run separately or as a mixture through an S-200 gel filtration column. Proteins were eluted in Buffer G (0.2 m m ATP or ADP) and monitored by absorbance at 280 nm. The actin and ZmADF3 in each fraction were separated by SDS-PAGE alongside standards of known concentration and then quantified by scanning the gels using a Joyce Loebl Chromoscan 3 scanning densitometer ( Fig. 2).

Light scattering measurements

The state of actin polymerization was assayed indirectly by light scattering. Light scattering was measured at an angle of 90° at a wavelength of 360 nm in a Perkin Elmer fluorescence spectrophotometer at 22°C. To assess the effect of ATP and ADP on the acceleration of actin polymerisation by ZmADF3, 100 μl of 10×KME pH 6.5 was added to a 1 ml mixture of 10 μM actin and 3 μM ZmADF3 in Buffer G containing either 0.2 m m ATP or ADP. The change in light scattering was measured immediately after the 10×KME pH 6.5 buffer was added ( Fig. 3a). To observe the effect of raising the pH of a polymerised actin solution (10 μM) containing ZmADF3 (1 μM or 10 μM) from 6.5 to 8.0 on the depolymerisation of actin filaments, 100 μl of 1 m Tris/HCl pH 8.0 was added to 1 ml of the polymerised actin mixture ( Fig. 4b)

Falling ball viscometry

Viscometry was performed by the method of Maclean-Fletcher & Pollard (1980). Actin at 10 μm was mixed with various concentrations (0–2 μM) of ZmADF3, volumes were made to 180 μl with Buffer G containing either 0.2 m m ATP or ADP. 20 μl of 10× KME pH 6.5 was added, the solution briefly vortexed and taken up in 100 μl capillaries (Fisons, Loughborough, UK) which were then sealed with ‘plasticene’. After approximately 3 h at room temperature the apparent viscosity was then measured by timing the fall of steel ball bearings (Atlas Ball-Bearing, Birmingham, UK) through the capillary tubes.

Sedimentation assays

The binding of ZmADF3 to F-actin was assessed by cosedimentation as described previously ( Lopez et al. 1996 ). To measure the extent of ZmADF3 binding to F-actin over a range of pH values F-actin and ZmADF3, each at 10 μM, were mixed in Buffer F in the pH range 6.0–9.0, centrifuged at 386 000 g andthe proteins in the pellets and supernatants separated by SDS-PAGE ( Fig. 4a). The amounts of protein in the supernatants and pellets were quantified by densitometric scanning of the SDS-PAGE gels. In Buffer F, Bis-Tris Propane (Sigma, UK) was used to buffer pH values at 6.0–9.0 making use of the fact that this buffer has two pKa values, 6.8 and 9.0. In additional experiments, imidazole was used for pH 6.5 and Tris/HCl was used at pH 8.0, these buffers gave similar results (data not shown).

To assess the effect of phospholipids on ZmADF3 activity, similar co-sedimentation experiments were performed in Buffer F at pH 8.0 with the following modifications. ZmADF3 (2 μm) was incubated with 100 μm of PI, PIP, PIP2, PE, PC, PS, IP3 or OAG in Fig. 5(b); 0–100 μM of PIP or PIP2 in Fig. 6(c) for 20 min at room temperature. F-actin was then added at a final concentration of 5 μm and incubated for 40 min at room temperature. The amount of G-actin in the supernatants after sedimentation was determined by densitometric scanning as described above.

Isolation and purification of plasma membrane vesicles

Preparation of plasma membrane vesicles from bean (Vicia faba) leaves was carried out as described in Drøbak et al. (1994) . The separation of plasma membranes from intracellular membranes was achieved by using the batch procedure to U4 described by Larssen et al. (1987) . The final upper phase containing the plasma membranes was diluted fivefold with 5 m m KH2PO4/K2HPO4 buffer at pH 7.8, 330 m m sucrose, 50 m m KCl, 0.1 m m EDTA, 1 m m DDT and centrifuged at 19 000 gfor 60 min. The plasma membrane pellets were then reconstituted in 5 m m KH2PO4/K2HPO4 buffer at pH 7.8, 330 m m sucrose, 50 m m KCl, 0.1 m m EDTA and gently homogenised and stored at –20°C until use. All steps in the extraction and purification procedures were carried out at 4°C. Enrichment of plasma membrane vesicles in the U4 fraction was determined by assay of membrane marker enzymes (1,3 b-glucan synthase, vanadate-inhibited ATPase, cytochrome c oxidase/reductase and NADH ferricyanide reductase) and silicotungstic acid staining ( Drøbak et al. 1994 ;Palmgren et al. 1990 ). Protein concentration was determined using the Bio-Rad Bradford protein assay (Bio-Rad, UK).

ZmADF3 inhibition of phospholipase C activity

Phosphoinositide specific phospholipase C activity was assayed as described by Drøbak et al. (1994) . After temperature equilibrium, PLC activity of plasma membranes (5 μg protein) was assayed at 25°C in 50 μl PLC buffer: 50 m m Tris/Malate pH 6.0, 10 μm CaCl2 containing a sonicated micellar suspension of PIP2 (50 μm) spiked with 0.86 kBq 3H-PIP2 (specific activity 325.6 GBq mmol–1). Various concentrations of ZmADF3 were pre-incubated with the PIP2 micelles in PLC-buffer (10 min at 25°C) and the assays were started by the addition of plasma membranes. After 6 min incubation, the reaction was stopped by the addition of 1 ml chloroform/methanol (2:1, v/v) and tubes were placed on ice for 5 min. 0.25 ml 0.6 N HCl was added to facilitate phase separation and the tubes were vortexed vigorously and centrifuged at 14000 gfor 2 min. Aliquots of the aqueous top phase were removed from each tube and radioactivity was determined by liquid scintillation spectrometry after addition of the scintillation fluid (Hionic-Fluor, Hewlett-Packard, UK).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We wish to thank P.A.C. Watkins for his excellent assistance in the PLC experiments. This work was funded by the Biotechnological and Biological Sciences Research Council.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Aizawa, H., Sutoh, K., Yahara, I. 1996 Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium. J. Cell Biol., 132, 335 344.
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