©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
pH Regulation of the F-actin Binding Properties of Dictyostelium Elongation Factor 1 (*)

Brian T. Edmonds (§) , John Murray , John Condeelis

From the (1)Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

ABP50, an F-actin bundling protein from Dictyostelium, is also the protein synthesis co-factor, elongation factor 1 (EF1). Concomitant with cAMP stimulation in Dictyostelium is a cytoplasmic alkalinization (Aerts, R. J., DeWit, R. J. W., and Van Lookeren Campagne, M. M.(1987) FEBS Lett. 220, 366-370) and a redistribution of EF1 (Dharmawardhane, S., Demma, M., Yang, F., and Condeelis, J.(1991) Cell Motil. Cytoskel. 20, 279-288). In addition, others have shown a correlation between intracellular pH and the level of protein synthesis in Dictyostelium (Aerts, R. J., Durston, A. J., and Moolenaar, W. H.(1985) Cell 43, 653-657). The present study investigates the relationship between pH and the F-actin binding properties of EF1. We found that increasing pH over the physiological range 6.2-7.8 causes a loss of EF1-mediated F-actin bundling and single filament binding, with corresponding increases in the amount of free EF1 in vitro. Similar results also were obtained by cell fractionation and confocal immunofluorescence microscopy. The EF1 binding constant (K) for F-actin is increased from 0.2 µM to > 2.2 µM over the same pH range. In addition, EF1-induced actin bundle formation is freely reversible by changes in pH. Thus, pH may be a potent modulator of cytoarchitecture in Dictyostelium and may also influence mRNA translation rates by modifying the interactions between the protein synthetic machinery and the actin cytoskeleton.


INTRODUCTION

ABP50 is a 50-kDa actin-binding protein first identified by its co-sedimentation with filamentous actin (F-actin) from Dictyostelium cell extracts(1) . An unique feature of ABP50 is the in vitro cross-linking of actin filaments into square-packed bundles whose organization would tend to exclude other known actin bundling proteins(2) . Quantitative analysis shows that ABP50 comprises 1% of total cell protein(1) . This high cellular concentration of ABP50 (75 µM) implies that it may have a significant influence on the structure of the actin cytoskeleton in vivo.

Surprisingly, cloning and sequencing of ABP50 cDNA revealed that ABP50 is the Dictyostelium protein synthesis co-factor, elongation factor 1 (EF1),() with 75% identity to other eukaryotic EF1s(3) . The current model of eukaryotic translation portrays an EF1-mediated binding of aminoacyl-tRNA to the ribosome through a GTP-dependent mechanism(4) . Functionally, ABP50 is indistinguishable from rabbit EF1 in an in vitro translation assay; rabbit EF1, as well as that from carrot, brine shrimp, and mouse, binds F-actin (5).()()Thus, it appears that actin binding is a universal property of eukaryotic EF1 and may imply that actin is important for EF1 function.

The actin cytoskeleton potentially plays a role in mRNA translation (for review, see Ref. 6). For example, many translational co-factors associate with actin filaments(7, 8, 9, 10) , and translation of mRNAs associated with the cytoskeleton appears to be more efficient(11) . While the significance of these observations is unclear, they raise the issue of the physiological relevance of an association between the actin cytoskeleton and the mRNA translational machinery(12) . Do translational components affect the dynamics and organization of the actin cytoskeleton? Conversely, does the cytoskeleton exist merely as a passive framework for the translational apparatus or does it serve a more active function? The actin cytoskeleton may provide a mechanism to help segregate specific translational components to distinct intracellular compartments resulting in localized protein synthesis (10, 13). How actin might mediate this segregation is unknown.

In addition to regulating mRNA translation EF1 also may regulate the organization of the actin cytoskeleton. The intracellular association of Dictyostelium EF1 with the cytoskeleton changes in response to cyclic AMP, a natural Dictyostelium chemoattractant. Whereas the distribution of Dictyostelium EF1 is diffuse in resting cells, as revealed by immunofluorescence, in cAMP-stimulated cells EF1 becomes localized to de novo surface projections(14) . The relevant second messenger pathways in Dictyostelium mediating this redistribution of EF1 have not been identified.

Intracellular pH could serve as one potential link between the cytoskeleton and protein synthesis. Cytoplasmic alkalinization follows cAMP stimulation of Dictyostelium with a time course similar to that for the observed EF1 redistribution; the mean Dictyostelium resting pH increases 0.2-0.4 units within 90 s after stimulation(15, 16) . In addition, the rate of translation of some mRNAs increases within minutes of the onset of Dictyostelium development(17) .

Cytoplasmic alkalinization appears to be a common reaction to stimulation in many cell types and may serve as the signal for proliferation (for review, see Ref. 18). In this vein, there is a direct relation between pH and the level of protein synthesis in many cell types. Typically, pH increases just precede increases in protein synthesis. In Dictyostelium, artificially raising the pH causes an increase in the rates of protein and DNA synthesis(19) . Additionally, changes in pH and protein synthesis are synchronous with the cell cycle in many cell types (reviewed in Ref. 20), including Dictyostelium(19) , and cytoplasmic alkalinization is a primary signal for the stimulation of protein synthesis after fertilization(21) .

The present study was undertaken to evaluate the influence of pH on the actin-binding activity of Dictyostelium EF1. The results indicate that the association of EF1 with actin is greatly affected in vitro and in vivo by pH within the physiological range measured in Dictyostelium and suggest that pH may be a potent modulator of actin organization in these cells.


MATERIALS AND METHODS

All chemicals were from Sigma unless otherwise noted.

Protein Purification

Dictyostelium EF1 (also known as ABP50) was purified by a modified protocol from Demma et al.(1) . Dictyostelium discoideum strain AX3 cell cultures were washed in Buffer W (5 mM Tris, pH 7.0) and then harvested in Buffer L (5 mM Tris, pH 7.5, 5 mM EGTA, 1 mM DTT). Cells were lysed by N cavitation in a Parr Bomb (Parr, Moline, IL) with protease inhibitors aprotinin (0.3 ml/ml) and chymostatin, leupeptin, and pepstatin (10 µg/ml each). Following lysis the pH was adjusted to 9.0 with KOH. Lysates then were centrifuged for 100,000 g for 1 h at 4 °C, and the resultant supernatants were loaded onto a DE52 (Whatman) anion exchange column equilibrated with Buffer 52 (20 mM Tris, pH 9.0, 1 mM DTT, 0.1 mM EDTA, 25% glycerol (v/v)). EF1 elutes in the flow-through volume and is immediately loaded onto a Poros II HS (PerSeptive Biosystems, Cambridge, MA) cation exchange column equilibrated with Buffer 52. The column is washed with 175 mM NaCl in Buffer 52, and then EF1 is eluted with a step to 1 M NaCl in Buffer 52. This eluent is dialyzed at 4 °C against Buffer HTP (10 mM Pipes, pH 6.8, 10 mM KPO, 1 mM DTT, 25% glycerol) and then loaded onto an hydroxylapatite (Bio-Rad) column equilibrated with Buffer HTP. EF1 is eluted with a 0-1 M NaCl gradient. EF1-containing fractions are vacuum-concentrated at pH 9.0 and stored under liquid N in Buffer S (10 mM Pipes, pH 7.0, 1 mM DTT, 0.1 mM EDTA, 25% glycerol).

Evaluation of F-actin Binding by EF1

F-actin binding by Dictyostelium EF1 was assessed by co-polymerization sedimentation. Dictyostelium or rabbit monomeric actin (G-actin; 5 µM) was incubated with 1 µM EF1 in Sedimentation Assay Buffer (20 mM Pipes, 2 mM MgCl, 2 mM EGTA) for >18 h at 0 °C. To distinguish between actin bundling versus single filament binding, EF1-actin mixtures were centrifuged differentially in an Airfuge (Beckman). Bundle structures are pelleted preferentially at 50,000 g for 1.5 min (low speed pellet)(1) . The low speed supernatants were then spun at 130,000 g for 30 min thereby pelleting single actin filaments (high speed pellet). Polypeptide mixtures were then separated by SDS-PAGE and the amounts of protein quantitated by densitometry: either (a) gel imaging with an Ektron camera system (Eastman Kodak Co.) and image analysis with the public domain software NIH-Image (version 1.47; written by Wayne Rasband and available via Internet by anonymous ftp from zippy.nimh.nih.gov); or (b) laser scanning imaging and analysis using a Molecular Dynamics Computing Densitometer system and software (version 3.3; Sunnyvale, CA). Nonlinear least squares curve fitting was performed with Origin 3.5 software (MicroCal Software, Northampton, MA).

The determination of Dictyostelium EF1 binding affinity for F-actin was performed by mixing varying amounts of EF1 with 1 µM preassembled Dictyostelium F-actin in Sedimentation Assay Buffer at pH 6.5 or 7.8. The mixture was incubated for 1 h at 0 °C and then spun at 130,000 g in an Airfuge. Separation and quantification of the amounts of free and bound EF1 and actin were as above.

All parameters for equilibrium binding were estimated using methods of nonlinear least squares analysis (22) that determine the best-fit parameter values corresponding to a minimum in the variance using a variation of the Gauss-Newton procedure that searches the parameter space along a vector dependent on the parameter guesses. Values for the equilibrium dissociation constant, K, were obtained by treating the data as transition curves and fitting by nonlinear least squares techniques to

On-line formulae not verified for accuracy

where

On-line formulae not verified for accuracy

where K = dissociation constant, n = Hill coefficient, x = concentration of free EF1, m = 1/(UL - LL), and b = LL/(LL - UL), where UL and LL are the upper and lower end points, respectively, of the transition curve (23). For pH 6.5 (LL = 0.001 µM; UL = 1.34 µM) and for pH 7.8 (LL = 0.001 µM; UL = 6.9 µM).

Evaluation of EF1 Cross-linking of F-actin by Light Scattering

Right angle light scattering was employed to evaluate the kinetics of F-actin bundle formation by EF1 in real time. A Hitachi F-2000 fluorescence spectrophotometer was used with 600-nm emission/excitation wavelengths combined with a slit width of 5 nm. Dictyostelium preassembled F-actin (1.5 µM) was mixed with Dictyostelium EF1 (0.5 µM) in Assay Buffer at 11 different pH levels over the range 6.2-7.8. Prior to EF1 addition base-line readings of buffer and F-actin alone were obtained. Data were collected and analyzed by SpectraCalc and GRAMS/386 software (Galactic Industries Corp., Salem, NH).

Cell Fractionation

The effect of pH on EF1 binding to Dictyostelium F-actin in situ was determined by a cell fractionation strategy. Cells were grown to a density of 5 10 cell/ml. After pelleting and washing with 20 mM Na/K/PO buffer, cells were resuspended to a final density of 1 10 cells/ml and incubated with shaking for 1-4 h to start the cells into development. To clamp intracellular pH at known values, aliquots of cells were pelleted and resuspended in an equal volume of 20 mM Tris-maleate buffer (pH 6.0-8.0) containing 100 µM amiloride and 40 mM potassium acetate or ammonium chloride(24, 25) . Experiments using 5 mM propionic acid or methylamine gave similar results. After 15 min of incubation with shaking, a 200-µl aliquot was added to 1 ml of ice-cold lysis buffer at a corresponding pH (40 mM Pipes, 20 mM KCl, 2 mM MgSO, 5 mM EGTA, 5 mM DTT, 1 mM ATP, 0.5% Triton X-100, 5 µg/ml chymostatin, leupeptin, and pepstatin A). The total ionic strength of the lysis buffer was within the range of amoeboid cytoplasm (see ``Results'' and Ref. 26). To separate Triton-insoluble material, cell suspensions were immediately centrifuged for 3 min at 8730 g with a Beckman Microfuge. The supernatant from this low speed sample was immediately transferred to a Beckman TL-100 centrifuge and spun at 400,000 g for 20 min. The EF1 and actin contents of supernatants and resuspended pellets were analyzed by SDS-PAGE and laser densitometry of either Coomassie Blue-stained gels or Western blots.

For Western blots, proteins were transferred from polyacrylamide gels using a semidry blotter (Bio-Rad) according to the manufacturer's instructions. The amount of EF1 was determined by ECL chemiluminescence (Amersham Corp.) and an affinity-purified antibody raised against Dictyostelium EF1(1) .

Immunofluorescence Microscopy

The co-distribution of EF1 and F-actin as a function of cytoplasmic pH in whole cell Dictyostelium preparations was assessed by confocal immunofluorescence microscopy. Cells in HL-5 medium were allowed to settle onto glass coverslips and then washed with excess 20 mM Na/K/PO at pH 6.0-8.0. Coverslips were then incubated at the corresponding pH for 5 min with Na/K/PO buffer containing 5 mM propionic acid or methylamine. Experiments using 40 mM potassium acetate or ammonium chloride gave similar results. Fixation was carried out at the corresponding pH with 1% glutaraldehyde (Fluka) and 0.1% Triton X-100 in general buffer (10 mM Pipes, 20 mM KHPO and KHPO, 5 mM EGTA, 2 mM MgSO).

Autofluorescence was quenched with 2 mg/ml sodium borohydride, and nonspecific binding sites were blocked with 1% bovine serum albumin and fetal calf serum. Coverslips were incubated with rhodamine-phalloidin (Molecular Probes) and an affinity-purified anti-EF1 antibody (see above) in TBS (20 mM Tris, pH 8.0; 150 mM NaCl, 1% bovine serum albumin). After extensive washing with TBS containing 0.02% saponin, coverslips were incubated with a fluorescein-conjugated goat anti-rabbit IgG (Cappel) preabsorbed against glutaraldehyde-fixed Dictyostelium cells. After washing with TBS-saponin, coverslips were mounted in TBS containing 50% glycerol and n-propyl gallate.

Cells were viewed with a Bio-Rad MRC600 scanning confocal microscope equipped with a krypton/argon laser to ensure complete separation of the fluorescein and rhodamine channels. Optical sections (0.3 µM thick) were imaged with a Nikon 60 flat field objective (NA = 1.4) on a Nikon Diaphot microscope. Three-dimensional image reconstruction and quantitative analysis was performed with VoxelView (Vital Images, Fairfax, IA), NIH-Image (version 1.55), and Origin 3.5 software.

Video Microscopy

To assess the gross morphological and behavioral effect of manipulating cytoplasmic pH, Dictyostelium amoebae were viewed with time-lapse video microscopy during treatment. Cells growing in HL-5 suspension culture were washed, resuspended to a density of 10 cells/ml, and starved in 20 mM Na/K/PO buffer for 1 h at 22 °C. Aliquots of the cell suspension were allowed to settle for 45 min on uncoated glass coverslips incorporated into the bottom of 35-mm culture dishes (MatTek). The spread cells were then flooded with 2 ml of 20 mM Na/K/PO buffer adjusted to pH 6.0, 7.0, or 8.0 and placed on the stage of an inverted Nikon microscope, which incorporates a long working distance condenser with a Nikon PlanApo 60 DM objective (NA = 1.4 oil). Cells were viewed for 5 min before and after the addition of 4 ml of buffer containing: 5 mM propionic acid, pH 6; no addition control, pH 7; or 5 mM methylamine, pH 8. Images were collected with a Hamamatsu C2400 camera containing a Newvicon tube and recorded with a Panasonic AG-6720 time-lapse video recorder where time was compressed by a factor of 12.


RESULTS

pH and Ionic Strength Can Influence Binding of EF1 to F-actin

Measurements of resting Dictyostelium pH by a variety of independent techniques indicate a broad range between 6.0 and 8.2 with a median of 7.2(27, 28) . These resting values can increase by 0.2-0.4 units within 90 s after stimulation by cyclic AMP, a natural Dictyostelium chemoattractant(15, 16) . Because the intracellular distribution of Dictyostelium EF1 changes within this same time frame after cAMP stimulation(14) , it was of interest to ascertain if changes in pH could influence binding of EF1 to actin in vitro. Fig. 1shows the effect of increasing pH at constant ionic strength (35 mosM titrated with KCl) on binding of EF1 to Dictyostelium (toppanel) and rabbit (middlepanel) F-actin in a co-polymerization sedimentation assay. At low pH (6.2-6.6) 100% of total EF1 is found in actin-containing structures pelleted at low g force. Previous studies demonstrated that such structures are actin filaments cross-linked into bundled arrays(1) . As the pH is increased (6.6-7.5), a sharp loss of bundles occurs and is accompanied by an increase in the association of EF1 with actin filaments that pellet only at high g force. EF1 is not detected in the supernatant until the pH is raised even further (>pH 7.2). These results suggest that bundles of F-actin cross-linked by EF1 are more susceptible to pH changes than the binding of EF1 to uncross-linked actin filaments, and that actin filament cross-linking and filament binding by EF1 are separate events, perhaps indicative of multiple actin binding sites with different pH sensitivities. EF1 alone was freely soluble at all pH values.


Figure 1: Effect of pH and ionic strength on F-actin binding by Dictyostelium EF1. 5 µMDictyostelium (upper) or rabbit (middle) G-actin was co-polymerized with 1 µMDictyostelium EF1 for 18 h at 11 different pH ranging from 6.2 to 7.8. EF1-bundles (, low speed pellet (LSP)) were separated from EF1 bound to single actin filaments (, high speed pellet (HSP)) and free EF1 () by differential centrifugation. Plots are representative examples from six experiments. Lower, effect of ionic strength on binding of Dictyostelium EF1 to Dictyostelium F-actin. The conditions were as above except pH was 6.7. Plot is a representative example from three experiments.



Further evidence that the interaction between EF1 and actin is charge-dependent is provided by a reduction in actin binding by EF1 as total ionic strength is increased (Fig. 1, bottompanel). The total ionic strength of amoeboid cytoplasm has been estimated to lie within the range of 70-120 mosM(26) . When G-actin and EF1 are copolymerized at pH 6.7 under low ionic strength conditions (<80 mosM), the majority of EF1 is found in bundles pelleted at low g force. However, similarly to increasing pH, as the ionic strength is increased by the addition of either KCl or NaCl, there is a progressive loss of bundled EF1 accompanied by an increase in EF1 bound to uncross-linked filaments and free EF1. Thus, even under pH conditions where maximal EF1-mediated F-actin cross-linking occurs, bundle formation can be perturbed by increased salt concentration, implying the presence of charge screening.

Affinity of EF1 for F-actin Decreases as pH Increases

One consequence of the above results suggested that the affinity of EF1 for F-actin changes as a function of pH. This was directly determined by quantitative co-sedimentation assays at various pH. Fig. 2shows the binding relationships between EF1 and preassembled Dictyostelium F-actin after one hour at pH 6.5 and 7.8. Both curves show binding saturation approaching a molar ratio of 1.0 in agreement with previous observations(1) . The K as determined from fitting to the Langmuir isotherm incorporating the Hill coefficient (see ``Materials and Methods'') is 0.2 µM at pH 6.5 and 2.2 µM at pH 7.8. Thus, the affinity of EF1 for F-actin changes 10-fold over the pH range observed in vivo. The Hill coefficients at each pH were greater than 1 (pH 6.5 = 2 ± 0.8; pH 7.5 = 1.2 ± 0.4), but the large standard error obfuscates an accurate determination of the number of binding sites from the present data.


Figure 2: Changes in binding affinity of EF1 for Dictyostelium F-actin with pH. , binding curve for pH 6.5; , binding curve for pH 7.8. Moles of bound EF1 are expressed per mol of total bound F-actin.



Increasing pH Inhibits Bundling of F-actin by EF1

The initial observations of reduced F-actin binding at elevated pH were obtained under equilibrium conditions after >18 h of co-polymerization of EF1 with monomeric actin. This time scale is far longer than the minute time scale in vivo when EF1 redistributes after cAMP stimulation (14) or for the cAMP-induced alkalinization(15, 16) . To ascertain on the minute time scale the influence of pH on the rate and extent of F-actin cross-linking by EF1, right angle light scattering studies were conducted using preassembled Dictyostelium F-actin. Fig. 3A shows the changes in scattered light intensity of F-actin solutions in the pH range 6.2-7.8 when EF1 is added. After obtaining intensity levels for buffer and F-actin alone, EF1 is added at a 1:3 molar ratio to F-actin in approximation to the ratio measured in vivo. EF1 alone had no significant light scatter above the buffer alone control. The regions marked by an asterisk display no signal during mixing due to the closed emission shutter. Upon opening the shutter, a dramatic increase in scattered light intensity is present that clearly develops within the 30 s required for mixing. These results agree well with the previous observation of rapid formation of F-actin bundles visible with the light microscope(1) . These results demonstrate that EF1 can affect the organization of F-actin in vitro within the time course of cAMP-induced cytoplasmic alkalinization and the redistribution of EF1 observed in vivo.


Figure 3: Effect of pH on right angle light scattering of Dictyostelium EF1-F-actin solutions. A, 1.5 µM actin that was preassembled for 18 h was mixed with 0.5 µM EF1 in the pH range 6.2-7.8. The pH values of each curve are: 1st (top), 6.2; 2nd, 6.3; 3rd, 6.6; 4th, 6.68; 5th, 7.0; 6th, 7.13; 7th, 7.23; 8th, 7.41; 9th, 7.54; 10th, 7.73; 11th (bottom), 7.81. B, initial rate of cross-linking as a function of pH. EF1 (0.1 µM) was mixed with F-actin (1.5 µM) at the indicated pH. C, light scattering intensity 400 s after EF1 addition as a function of solution pH. D, reversibility of EF1-mediated cross-linking of Dictyostelium F-actin. F-actin (1.5 µM) was mixed with EF1 (0.5 µM) at the indicated pH. Tris was added to a final pH 8.2. IU, arbitrary intensity units; *, emission shutter closed during mixing.



The magnitude of the increase in light scattering intensity is inversely proportional to the pH. The extent of cross-linking after 400 s decreases with increasing pH (Fig. 3C). Note the great similarity of the pH transition point of bundling in Fig. 3C to that in Fig. 1. Additionally, at lower EF1-F-actin molar ratios, the initial rate of cross-linking is reduced but still displays an inverse sensitivity to pH (Fig. 3B). In summary, as the pH is increased, F-actin cross-linking by EF1 is reduced and remains low. These data, obtained over a 6-min period, are in good agreement with the results of the 18-h co-sedimentation experiments.

Above pH 7.0 a significant decrease in the secondary rate of bundle formation occurs, such that at pH 7.2-7.3 there is no net bundle formation beyond that of the initial mix. Above pH 7.4 there is a decrease in the scattered intensity, suggesting a disassembly of cross-linked structures. We speculate that this reproducible decrease in light scatter at higher pH may be a mixing artifact; the added EF1 solution is at pH 7 and a brief period of time may be required to equilibrate to the final experimental pH. Immediately after addition, the reaction mix pH may be lower than the final pH and some reversible cross-linking may occur (see below).

Bundle Formation Is Reversed by Increasing pH

The changes in pH of Dictyostelium cytoplasm following cAMP stimulation are transient(15) , as is the redistribution of EF1(14) . The issue of whether cross-linking of F-actin by EF1 is reversible in response to changes in pH was also addressed by light scattering (Fig. 3D). EF1 and preassembled Dictyostelium F-actin were mixed at different pH as in Fig. 3A. After a stable base line was achieved (150 s), Tris was added to a final pH of 8.2. At all pH values, there is a rapid decrease in the light scatter intensity after Tris addition that stabilizes to the intensity of F-actin alone. Similar results were obtained by sedimentation assay (data not shown). These data demonstrate that the formation of F-actin/EF1 bundles is reversible and that bundle disassembly is as rapid as formation. Thus, EF1-F-actin structures could form and dissipate within the temporal parameters of transient pH changes measured in Dictyostelium, i.e. an alkalinization within 90 s after cAMP stimulation followed by a return to resting pH within 5 min(15, 16) .

Increasing pH Decreases the EF1 Content of Triton-insoluble Cytoskeletons

The issue of whether the dramatic pH-induced changes in EF1 binding to F-actin in vitro occurs in situ was addressed by cell fractionation experiments and immunofluorescence microscopy. It has been demonstrated that the pH of Dictyostelium can be clamped to that of the extracellular bathing medium by the inclusion of weak acids or bases (reviewed in Refs. 20, 25, and 27). This technique was used to stabilize pH at known values, after which the cells were lysed in buffer at the same pH and the cytoskeletal and cytosolic fractions were quickly separated by differential centrifugation. Fig. 4(upper) shows the pH-dependent partitioning of EF1 in whole cell lysates prepared in this manner. As the pH is increased from 6.0 to 7.0, there is a total loss of EF1 associated with the Triton-insoluble fraction pelleted by low g force. This low speed fraction contains cross-linked cytoskeletal elements(29) . Over the same pH interval, there is a corresponding increase in the amount of cytosolic EF1 and little change associated with the high speed pellet where single actin filaments are found. At cytoplasmic pH greater than 7.0, increases in the cytosolic pool of EF1 are correlated with losses from the high speed pellet fraction. Under these alkaline conditions, EF1 is undetectable in low speed pellets by Western blotting.


Figure 4: Effect of intracellular pH on the distribution of total cellular EF1 (upper) and actin (lower) between cytosolic and cytoskeletal compartments. The intracellular pH of cells was clamped at the indicated pH. Following lysis, cell extracts were separated into low speed pellet (LSP, ), high speed pellet (HSP, ), and high speed supernatant (HSS, ) fractions by differential centrifugation. The relative amounts of protein was quantitated by laser scanning densitometry of either Coomassie Blue-stained SDS-PAGE gels (actin and EF1) or chemiluminescent Western blots (EF1; see ``Materials and Methods'').



These results indicate that the pH-sensitive binding of EF1 to F-actin observed under simple bimolecular in vitro conditions is also demonstrable in the presence of other resident cytoplasmic components. However, note that above pH 6.5 the majority of cellular EF1 does not pellet with actin, in contrast to the observations in vitro (see Fig. 1). The actual amount of EF1 in the cytoskeleton in vivo is probably lower due to competition between EF1 and other actin-binding proteins for actin substrate.

Interestingly, the partitioning of total cellular actin in response to changes in intracellular pH parallels that of EF1 (Fig. 4, lower). There is a 20% loss of actin pelleted by low g force as pH increases from 6.5 to 7.0. Similar to EF1, this loss of actin from the low speed fraction is correlated with an increase of actin in the cytosolic pool. It is likely that this material appearing in the cytosolic fraction is not monomeric G-actin that was entrapped at lower pH, but rather short F-actin oligomers that are too small to be sedimented under the centrifugation conditions employed(30) . Above pH 7.0 there is little change in the distribution of actin between the three fractions. The pH sensitivity of EF1 binding to G-actin and short F-actin oligomers has not been determined.

It was of concern that the technique used to manipulate pH could be deleterious to cell viability and thereby create spurious morphological artifacts. To address this concern, cells were viewed with time-lapse video microscopy while pH was clamped at acidic or alkaline values. When exposed to 5 mM propionic acid, highly motile cells become arrested within 2 min and all intracellular organelle movements stop. This condition of stasis is reversed within 20 min by washing out of the acid-containing medium, or within 1 min by the addition of 5 mM methylamine to the washout buffer. Methylamine alone does not have any gross effect on morphology or motility. From these experiments we conclude that, while dramatic behavioral changes are produced by cytoplasmic acidification, overall cell viability is unaffected. Thus, any changes in the distribution of EF1 and/or F-actin are probably related to changes in intracellular pH.

Fluorescence Microscopy Shows a pH-sensitive Co-distribution of F-actin and EF1

Previous immunofluorescence studies showed that the intracellular distribution of EF1 was altered upon cAMP stimulation(14) ; therefore, it was of interest to determine if changes in pH could produce a redistribution of EF1. Consistent with the cell fractionation experiments described above, a dramatic increase in the co-localization of EF1 and F-actin as determined by confocal immunofluorescence microscopy results after a reduction of pH. Fig. 5(panelsA-H) shows the distribution of EF1 and F-actin in representative single optical sections after treatment with propionic acid at pH 6 or as controls, untreated cells fixed at pH 7. In untreated cells (panels E-H), EF1-fluorescein staining is generally diffuse with a slightly stronger signal associated with cortical regions (panelG). No staining is associated with the nucleus. In contrast, the strongest F-actin staining (rhodamine-phalloidin) is primarily found in surface projections (panelF). When the EF1 (shown in green) and F-actin (red) channels are merged (panelH), the regions of overlap (yellow) are principally in the cortical cytoplasm at the base of surface projections.


Figure 5: Immunofluorescent images acquired by confocal microscopy of pH-induced changes in EF1 and F-actin staining in Dictyostelium. A-H, single optical sections: A and E, phase contrast; B and F, F-actin visualized with rhodamine-phalloidin; C and G, fluorescein-EF1; D and H, images with merged rhodamine and fluorescein channels (red, rhodamine-phalloidin; green, fluorescein-EF1; yellow, pixels that contain both red and green signals). A-D, pH 6; E-H, pH 7. I-P, digitally reconstructed z-series: I and M, phase contrast images in x-y plane; J and N, reconstructed image in x-y plane; K and O, oblique view in x-y-z planes; L and P, vertical cross-section through x axis at indicated value. I-L, pH 6; M-P, pH 7. One tick mark on axis = 10 µm.



In cells where the pH was clamped to pH 6 (Fig. 5, panels A-D), a striking redistribution of EF1 and F-actin occurs. EF1 staining (panelC) is associated with condensed arrays that extend throughout the cytoplasm. There is also an increase in the staining within the cortical compartment (compare panelsG and C). The F-actin pattern (panelB) is also altered in that the strongest staining is still associated with the cortical cytoplasm, yet can be also observed in certain optical planes (see lower cell) as punctate specks throughout the entire cytoplasm. In the merged image (panelD), the region of strongest signal overlap is cortical; however, from inspection it appears that there is more overlap of the EF1 and F-actin staining at pH 6 compared to pH 7.

To gain a sense of the vertical co-localization of EF1 and F-actin, digitally reconstructed images of z-series were taken through entire cells with the green and red channels merged (Fig. 5, panels I-P). At pH 7, as can be seen in whole cells (panels M-O) or in vertical cross-section (panelP), the majority of F-actin staining is associated with the cortical cytoplasm and surface projections, whereas EF1 fluorescence is generally diffuse. Overlapping areas of EF1 and F-actin staining generally are observed only at the base of surface projections. As pH is reduced to pH 6 (panels I-L), there is a significant increase in the degree of fluorescence overlap associated with the cortical cytoplasm over the entire height of the cell (compare panelsL and P).

The amount of fluorescence signal overlap between EF1 and F-actin staining patterns in single cells was quantitated by analysis of digitized images acquired by the confocal microscope. A total of 3 cells at each pH were optically sectioned, and the resultant x-y coordinate slices were serially stacked into a three-dimensional reconstruction. Individual sections (25-51/cell) then were analyzed separately for pixel overlap and averaged to obtain a range of overlap for the whole cell at all possible pixel intensities ( Fig. 6and ). The total amount of EF1 and F-actin staining, represented by the mean percentages of red or green pixels, did not change significantly with pH (). Thus, changes in pixel overlap are due to protein redistribution rather than extraction during fixation. Overall, the results demonstrate that the percentage of image pixels that contain signals from both the red (F-actin) and green (EF1) channels increases as the pH decreases, i.e. the amount of EF1 and F-actin overlap increases.


Figure 6: Effect of intracellular pH on the degree of co-localization for F-actin staining and EF1 immunofluorescence. Three whole cells at each pH were optically sectioned and analyzed as described under ``Materials and Methods.'' Each curve represents the mean pixel overlap for one whole cell over the full range of pixel threshold intensities.



The results from the cell fractionation and immunofluorescence experiments are directly comparable (). For example, at a pixel threshold intensity of 25, the percentage of pixel overlap for both pH 6 and pH 7 is identical to the amount of total cellular EF1 bound to the cytoskeleton (see Fig. 4, upper). These data suggest that the immunofluorescence images are an accurate reflection of the degree of association between EF1 and F-actin.


DISCUSSION

The major conclusions of this study show that binding of Dictyostelium EF1 to F-actin is regulated by pH both in vitro and in vivo over the physiological pH range as measured in Dictyostelium. With increasing pH, the binding affinity of EF1 for F-actin is reduced 10-fold in vitro and by a similar amount in vivo. Additionally, the cross-linking of actin filaments by EF1 is more sensitive to pH compared to the binding of EF1 to single actin filaments. The observations are consistent with the idea that pH serves as a modulator of cytoskeletal architecture in Dictyostelium and also suggest that protein synthesis potentially may be influenced by interactions with the actin-based cytoskeleton.

Affinity of EF1 for F-actin

Increasing pH or ionic strength reduces the F-actin binding capacity of EF1 by severalfold (Fig. 1). At higher pH, more EF1 is bound to Dictyostelium actin compared to rabbit actin, indicating that the affinity for Dictyostelium actin is higher. The major difference in amino acid sequence between actins lies at the NH terminus(31) , suggesting that the EF1-binding site is located within this part of the actin molecule; however, other divergent regions of the actin sequence cannot be ruled out. The NH terminus of actin appears to be a ``hot spot'' for the interaction with several actin-binding proteins (for discussion, see Ref. 32).

The degree of F-actin binding by Dictyostelium EF1 is very sensitive to pH in the range between 6.8 and 7.4 ( Fig. 1and 4A). The implication of this observation, considering the abundance of EF1 in cytoplasm (75 µM), is that changes in pH over a narrow range could produce dramatic EF1-mediated reorganizations of the F-actin cytoskeleton, especially in the structural integrity of filament cross-links. For example, cross-links between F-actin and EF1 form and break very rapidly in response to changes in pH (Fig. 3). This structural plasticity may be important for the cytoskeletal remodeling necessary for cell locomotion during chemotaxis. In addition, the variable amount of cytosolic EF1 in vivo (Fig. 4) indicates a pH-sensitive shuttling between the cytoskeleton and cytosol. Thus, pH can potentially serve as a potent modulator of cytoarchitecture and also influence the spatial and temporal localization of an important protein synthesis cofactor.

Significance of pH to Actin-based Cell Motility

Dictyostelium is a well accepted model system for the study of molecular aspects of cell chemotaxis, motility, and differentiation during development(33) . Amoeboid movement is thought to consist of extensions at the leading cell edge coordinated with contractions at the cell posterior. Inherent in this model is a regulated assembly and disassembly of the actin-based cytoskeleton (for reviews, see Refs. 34 and 35). Upon stimulation with cAMP, Dictyostelium amoebae increase their speed of locomotion concomitant with cytoplasmic alkalinization(15, 16, 36) . This effect on motility can be mimicked in Dictyostelium as well as in other cell types by artificially raising pH(16, 37, 38) . How might changes in pH influence cell motility related to the actin cytoskeleton?

Several actin-binding and cross-linking proteins, in addition to EF1, show reduced actin binding with increasing pH over the range used in this study: hisactophilin(39) , -actinin(24, 40) , actin depolymerizing factor(41) , and talin(42) . The predicted net effect on the overall actin cytoskeletal organization by an increase in pH would be to increase the dynamic instability of the actin cytoskeleton and to favor the fragmentation of preexisting actin-containing structures formed by these proteins. In fact, this phenomena was observed as a solation of Dictyostelium motile extracts when pH was increased above 7.0(43) . Perhaps in this plastic state the necessary remodeling of the cytoskeleton required for optimal locomotion and myosin-mediated filament sliding can occur. After stimulation, as pH returns to resting levels and cells become quiescent, actin filaments would be cross-linked back into more stable arrays.

Based upon the confocal immunofluorescence data (Fig. 5), much of the EF1 localization is in close juxtaposition to the plasma membrane. This observation is particularly relevant due to the presence in the plasma membrane of an outwardly directed proton pump that is responsible for the cytoplasmic alkalinization following cAMP stimulation(44) . Therefore, within the cytosol of a single Dictyostelium cell, microcompartments of pH may exist that reflect the range of pH values measured for whole cells. It is probably not coincidental that such an intracellular microcompartment contains a high concentration of a pH-sensitive actin cross-linking protein. As a consequence the cortical actin cytoskeleton may be characterized by dynamic instability due to localized pH transients near the inner surface of the plasma membrane.

Significance of pH to Protein Synthesis

At present, it is unknown how pH might exert an influence on the level of protein synthesis in Dictyostelium. Artificially raising pH alone is sufficient to increase protein synthesis(19) . One provocative hypothesis is that EF1 and other translational cofactors are complexed with F-actin in specific regions of the cell, and a pH-sensitive transition in cytoskeletal association increases translational efficiency. For example, as pH is increased, EF1-mediated cross-links between actin filaments are broken and EF1 remains either bound to single filaments or is released into the cytosolic pool. Thus the efficiency of EF1 activity in translation may change between each of these cytoskeletal-bound states.

The control of the levels of EF1 is important for normal cell function. It has been demonstrated that increased EF1 expression is related to increased cell proliferation(45) , oncogenic transformation (46, 47), and delayed cell senescence(48) . Therefore, it is important to note that the total amount of free EF1 in Dictyostelium cells increases by 20% with alkalinization from pH 7.0 to 8.0, and by 60% from pH 6.0 to 8.0 (Fig. 4). It seems reasonable that the location in the cell where the association of EF1 with actin is altered may be as important as the quantity of protein. If other components of the translational machinery located in the same compartments as EF1 also are activated by changes in pH, perhaps through differential association with the cytoskeleton, then a large effect on the overall level of protein synthesis can be expected.

Developmentally, intracellular pH can influence the determination of cell fate of Dictyostelium cell subpopulations; acidic pH leads to prestalk cell formation, whereas alkaline pH leads to prespore differentiation(49) . Clearly, cellular differentiation can be linked to regulation at the level of gene transcription; however, coincident scenarios might involve pH-sensitive regulation of the amount or rate of translation of specific developmentally important mRNAs(17, 50, 51) or pH-mediated alterations in cell motility that place differentially affected cells into distinct locations within a gradient of morphogen (52).

Various secreted compounds can influence the developmental fate of free-living Dictyostelium amoebae as well as aggregated cells within the motile pseudoplasmodium stage (reviewed in Ref. 53). It is hypothesized that gradients of such morphogens are responsible for the stereotypic patterning that characterizes the later stages of the Dictyostelium developmental cycle(52) ; cells that are exposed to a certain concentration of a certain morphogen will follow a defined developmental program. Furukawa and co-workers (28) have shown that pH of a significant subpopulation of Dictyostelium amoebae becomes acidified 3-4 h (preaggregation stage) into the developmental cycle. The explanation as to why the cytoplasm of some cells becomes acidified compared to neighboring cells is unclear, but may be related to the phase in each cell's mitotic cycle at which starvation begins(54) . As shown here, one potential effect of such a selective acidification is a reduced motility with respect to other aggregating amoebae and a change in the cellular distribution of EF1. Evidence exists that Dictyostelium amoebae with reduced motility become localized to specific areas of aggregating streams and migrating slugs when mixed with normally moving cells(55) . Thus, these less motile cells will be exposed to morphogenetic gradients to a different degree than their more motile neighbors, may exhibit different levels of translation, and as a result may show a proclivity for specific cell fates. More information is required to clarify any direct correlation between cytoplasmic acidification, reduced motility, EF1 localization, and cell fate determination in Dictyostelium.

  
Table: Effect of cytoplasmic pH on percentage of EF1 bound to cytoskeleton



FOOTNOTES

*
This work was supported by National Institutes of Health Grants NS08864 (to B. T. E.) and GM25813 (to J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4113; Fax: 718-518-7236.

The abbreviations used are: EF1, elongation factor 1; DTT, dithiothreitol; Pipes, 1,4-piperazinediethanesulfonic acid; TBS, Tris-buffered saline.

J. Sanders, M. Brandsma, G. M. C. Janssen, J. Dijk, and W. Möller, submitted for publication.

G. Liu, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Wim Möller and Dave Knecht for sharing data prior to publication. We also thank Dr. Michael Brenowitz for help with analysis of binding, Michael Cammer for help with gel densitometry and confocal microscopy analysis in the Albert Einstein College of Medicine Image Analysis Facility, and Dr. Gang Liu and other members of the Condeelis laboratory for helpful discussions and critical review of the manuscript. Laser scanning densitometry was performed in the Albert Einstein College of Medicine Cancer Research Center Scientific Computing Facility.


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