From the
ABP50, an F-actin bundling protein from Dictyostelium,
is also the protein synthesis co-factor, elongation factor 1
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 (
Surprisingly, cloning and sequencing
of ABP50 cDNA revealed that ABP50 is the Dictyostelium protein
synthesis co-factor, elongation factor 1
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
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
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
The present study was
undertaken to evaluate the influence of pH on the actin-binding
activity of Dictyostelium EF1
All chemicals were from Sigma unless otherwise noted.
The determination of Dictyostelium EF1
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
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
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
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
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
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
Interestingly, the partitioning of total cellular actin in response
to changes in intracellular pH parallels that of EF1
It was of concern that the technique used to manipulate
pH
To gain a sense of the vertical
co-localization of EF1
The amount of
fluorescence signal overlap between EF1
The major conclusions of this study show that binding of Dictyostelium EF1
The degree of F-actin binding by Dictyostelium EF1
Several actin-binding and cross-linking proteins, in
addition to EF1
Based upon the confocal immunofluorescence data (Fig. 5), much of the EF1
The control of the levels of EF1
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
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(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.
75 µM) implies that
it may have a significant influence on the structure of the actin
cytoskeleton in vivo.
(EF1
),(
)
with 75% identity to other
eukaryotic EF1
s(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.
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.
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) .
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 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.
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).
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.
, were obtained by treating the data as
transition curves and fitting by nonlinear least squares techniques
to
, 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
Right angle light scattering was employed to evaluate
the kinetics of F-actin bundle formation by EF1 Cross-linking of F-actin by Light
Scattering
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.
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 KH
PO
and
K
HPO
, 5 mM EGTA, 2 mM
MgSO
).
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.
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.
pH and Ionic Strength Can Influence Binding of EF1
Measurements of resting Dictyostelium pH
to F-actin
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
One consequence of the above results suggested that
the affinity of EF1 for F-actin Decreases as pH
Increases
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.
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
The issue of whether the dramatic
pH-induced changes in EF1 Content of
Triton-insoluble Cytoskeletons
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.
(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.
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.
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).
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.
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
Increasing pH or ionic
strength reduces the F-actin binding capacity of EF1 for F-actin
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).
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?
, 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.
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.
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.
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
, elongation factor 1
; DTT, dithiothreitol; Pipes,
1,4-piperazinediethanesulfonic acid; TBS, Tris-buffered saline.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.