From the Department of Biophysics and Biophysical
Chemistry and the
Department of Cell Biology and Anatomy,
The Johns Hopkins University School of Medicine, Baltimore, Maryland
21205 and the ¶ Department of Chemical Engineering, The Johns
Hopkins University, Baltimore, Maryland 21218
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ABSTRACT |
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We used smooth muscle -actinin to evaluate the
contribution of cross-linker dynamics to the mechanical properties of
actin filament networks. Recombinant actin-binding domain (residues 2-269) binds actin filaments with a Kd of 1 µM at 25 °C, 20 times stronger than actin-binding
domain produced by thermolysin digestion of native
-actinin
(residues 25-257). Between 8 and 25 °C the rate constants for
recombinant actin-binding domain to bind to (0.8-2.7
µM
1 s
1) and dissociate from
(0.2-2.4 s
1) actin filaments depend on temperature. At
8 °C actin filaments cross-linked with
-actinin are stiff and
nearly solid, whereas at 25 °C the mechanical properties approach
those of actin filaments alone. In these experiments, high actin
concentrations kept most of the
-actinin bound to actin and
temperature varied a single parameter, cross-linker dynamics, because
the mechanical properties of pure actin filaments (a viscoelastic gel)
or biotinylated actin filaments cross-linked irreversibly by avidin (a
stiff viscoelastic solid) depend little on temperature. These results
show that the rate of exchange of dynamic cross-links between actin
filaments is an important determinant of the mechanical properties of
the networks.
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INTRODUCTION |
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Cytoplasm is viscoelastic, so that it can either deform or rebound
in response to force (1, 2). Deformation is essential for cellular
movement, whereas elastic responses allow cells to maintain their
shapes. Scientists have appreciated this duality of cytoplasm for more
than 100 years (see Ref. 3 for a review of early work). To explain
these complicated properties, Frey-Wyssling (4) proposed that cytoplasm
consists of a network of long thin filaments held together by dynamic
cross-links that can resist rapid deformation but allow slow
deformation in response to sustained force. He did not know the
molecular nature of the filaments or cross-links, because his
suggestion, originally made in the late 1930s, predated the discovery
of actin in muscle (5) and nonmuscle cells (6). Later Pollard and Ito
(7) identified actin filaments as major contributors to the cytoplasmic
gel, and Sato et al. (8) found that the mechanical
properties of actin filament networks cross-linked with
Acanthamoeba -actinin depend on the rate of deformation.
At low rates of deformation, a network containing
-actinin is no
stiffer than the actin filaments alone, but at high rates of
deformation, the network is far stiffer than actin alone. They
postulated that amoeba
-actinin forms dynamic cross-links between
actin filaments and suggested that the Frey-Wyssling model might
explain the ability of cells to resist rapid deformation but change
shape in response to a slowly imposed force.
This hypothesis raised the question of whether dynamic cross-linkers
are a general feature of actin filament networks. Janmey et
al. (9) reported that actin-binding protein
(ABP-280)1 from a human
smooth muscle tumor is not a dynamic cross-linker. ABP/actin gels have
a higher elastic modulus than gels of actin filaments alone at all
frequencies tested, similar to biotin-labeled actin filaments
cross-linked irreversibly by avidin (10). Much lower concentrations of
ABP than amoeba -actinin were needed to affect the modulus of actin
gels. They argued that if cytoplasmic actin filaments were cross-linked
by proteins like ABP-280, then rearrangement of cross-links could not
account for the properties of cytoplasm. This work emphasized the need
to explore the effects of cross-linkers with a range of affinities for
actin. The affinity of macrophage and smooth muscle ABP-280 for actin
is about 1 µM (11-13).
Wachsstock et al. (15) compared the mechanical properties of
actin filaments cross-linked with biotin-avidin (Kd = 1015 M; Ref. 10), smooth muscle
-actinin
(Kd = 0.6 µM; Ref. 14 and 15), or
Acanthamoeba
-actinin (Kd = 5 µM; Ref. 16). Biotin-actin/avidin gels are a viscoelastic solid at all frequencies, and concentrations of avidin >0.03
µM do not increase the stiffness. At 0.03 µM smooth muscle
-actinin increased the complex
modulus 10-fold less than biotin/avidin. At higher concentrations,
smooth muscle
-actinin makes bundles of actin filaments that behave
like a viscoelastic fluid (14, 16). High concentrations of amoeba
-actinin are required to increase the stiffness, and the complex
modulus depends dramatically on frequency.
The current work investigates actin filament cross-linking by
-actinin in more detail to determine the effect of cross-linker dynamics on the mechanical properties in a system with only one variable, the temperature. We used chicken smooth muscle
-actinin, because temperature affects its affinity for actin filaments (17, 18).
Previous studies examined the effect of temperature of gels of actin
and
-actinin (19-21), but this is the first to correlate rheological measurements and binding rate constants on the same preparations of proteins.
-Actinins consist of two identical polypeptides of 103 kDa with an N-terminal 30-kDa actin-binding domain,
a tail with triple helical repeats, and two C-terminal EF hands (22).
The rod-shaped tails associate to form an anti-parallel dimer with an
actin filament-binding site on each end. The bivalent protein
cross-links actin filament into three-dimensional gels or bundles
depending upon the conditions (16).
We used the monovalent actin-binding domain (ABD) for our kinetic
analysis to avoid complications arising from two binding sites. We
assume that the properties of isolated ABD are the same as the heads of
bivalent -actinin molecules. We initiated these experiments with ABD
produced from chicken smooth muscle
-actinin by proteolytic
digestion (14) but found that this fragment differs in size and
affinity for actin compared with full-length recombinant ABD, which
also provides a convenient fluorescence change when it binds actin
filaments (18).
We show that actin filament networks cross-linked by -actinin differ
fundamentally from biotin-actin/avidin gels. Biotin-actin/avidin gels
are viscoelastic solid at all frequencies and temperatures tested.
-Actinin/actin gels are less stiff, and the stiffness varies with
temperature, because cross-linker dynamics depend on the
temperature.
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MATERIALS AND METHODS |
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Protein Purification--
Actin was prepared from rabbit
skeletal muscle (23, 24) using Sephacryl S-300 instead of Sephadex
G-150 for gel filtration. -Actinin was purified from chicken smooth
muscle (25). Both actin and
-actinin were stored by dialysis against
daily changes of Buffer G (2 mM Tris, pH 8.0, at 25 °C,
0.2 mM ATP, 0.1 mM CaCl2, 0.5 mM dithiothreitol, 0.3 mM NaN3) and
used within 5 days of purification. Lyophilized avidin (Pierce) was
dissolved at 1 mg/ml in Buffer G. Actin was labeled with biotin as
described by Wachsstock et al. (15) and Janmey et
al. (9) by reacting polymerized actin with a 7-fold molar excess
of iodoacetyl-N'-biotinhexenediamine (from a 40 mM stock in dimethylformamide). After purification by
pelleting, depolymerization, and gel filtration, the extent of
biotinylation was measured by the displacement of
2-[4'-hydroxyazobenzene] benzoic acid from avidin with a kit from
Pierce. The actin was approximately 30% biotinylated. This actin was
diluted to 2% biotinylated actin with unlabeled actin for the
experiments.
Proteolytic Chicken Smooth Muscle -Actinin ABD--
The ABD
of chicken smooth muscle
-actinin was cleaved from the tails by
digestion with thermolysin coupled to agarose beads (Sigma) and
purified according to Wachsstock et al. (15), a modification
of the method of Pavalko and Burridge (26). The thermolysin beads were
filtered out and the soluble pABD was recovered in the void
volume of a 5-ml Econo-Q column (Bio-Rad) equilibrated with Buffer G. The column retains the 53-kDa rod domain. Dr. Wolfgang Fischer of The
Salk Institute for Biological Studies determined the N-terminal
sequence of this pABD by automated Edman degradation and
determined its mass by mass spectrometry.
Recombinant Chicken Smooth Muscle -Actinin ABD--
Dr.
D. R. Critchley (University of Leicester, UK) kindly provided an
NcoI-HincII restriction enzyme fragment encoding
the ABD of chicken smooth muscle
-actinin (residues 2-269, residue 1 = initiator met) cloned into expression vector pMW 172 (27). Recombinant ABD (rABD) was expressed in the BL21.de3 strain of Escherichia coli and purified by modifications of method
Kuhlman et al. (18). Cells were sonicated in 16 mM Na2HPO4, 4 mM
NaH2PO4, 200 mM NaCl, 1% Triton
X-100, pH 8.0, at 25 °C) and clarified. The supernatant was dialyzed
against Buffer T (10 mM Tris, pH 8.0, at 25 °C, 10 mM NaCl, 2 mM EDTA, 0.05%
-mercaptoethanol) for 3 h. rABD was eluted with 60 mM NaCl from a 2 × 16-cm DE-52 fast flow anion exchange column (Whatman International
Ltd., Maidstone, UK) equilibrated with Buffer T. After precipitation
with 60% ammonium sulfate, rABD was resuspended in 5 ml of Buffer G
and chromatographed on a 1 × 100-cm column of Sephadex G-75. The
rABD peak was run on a MonoQ column (Bio-Rad) equilibrated with Buffer
G and eluted with a linear gradient of 10-500 mM NaCl. The
concentration of ABD was determined by absorbance using the extinction
coefficient A280 = 23.8 µM·cm
1 (27) or by the Bradford (28)
method standardized with bovine serum albumin.
Stopped Flow Fluorescence Measurements-- The rate constant (kobs) for rABD binding to actin filaments was measured from the time course of the decrease in the intrinsic fluorescence after mixing equal volumes of the ABD and actin (18) in the stopped flow instrument described by Sinard and Pollard (29). Tryptophan fluorescence was excited at 289 nm with a short pass filter (Oriel, Stratford, CT), and emission was monitored using a 330-nm-long pass filter (Oriel, Stratford, CT). A thermocirculator controlled the temperature of the sample and the observation cell. Actin in Buffer G was polymerized for >2 h by adding one-tenth volume of 10× KME buffer (500 mM KCl, 10 mM MgCl2, 10 mM EGTA, 100 mM imidazole, pH 7.0). ABD was made up in the same buffer. Prior to loading the stopped flow syringes, proteins were diluted to desired concentrations with Buffer F (2 mM Tris, 10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.5 mM dithiothreitol, 0.3 mM NaN3). After rapid mixing (dead time, 3 ms), we collected 500 data points at a sampling interval of 0.1 or 1 ms. Transients were fitted to single exponentials. Association rate constants (k+) were determined from the dependence of kobs on rABD concentration. rABD concentrations were at least 4-fold higher than actin.
Rapid Dilution Fluorescence Measurements-- Rate constants for ABD dissociation from actin filaments were determined from the time course of the increase in intrinsic fluorescence after diluting the complex at least 20-fold with Buffer F. Measurements were made in a Photon Technology International Alphascan fluorescence spectrophotometer (Brunswick, NJ). The samples were diluted two ways: 1) syringe injection of 5 µl of complex into 3 ml of Buffer F in a 1 × 1-cm cuvette rapidly stirred with a magnetic stirring bar or 2) using a hand-driven model SFA-12 Rapid Kinetics Stopped Flow Accessory (Hi-Tech Scientific Ltd., Salisbury, UK) with a dead time of about 20 ms. The temperature of the sample handling system was maintained with circulating water. Actin and ABD were copolymerized by adding one-tenth volume of concentrated polymerized buffer 10× KME for at least 2 h. The time course was fitted with a single exponential.
Actin Filament Pelleting Experiments-- Various concentrations of actin with a fixed concentration of ABD were polymerized for 1 h at room temperature in 1× KME buffer and then centrifuged 200,000 × g for 40 min at 25 °C. The supernatants were concentrated by precipitation with chloroform/methanol, and the proteins were separated by polyacrylamide gel electrophoresis in SDS. After staining with Coomassie Blue, the gel was digitized using the program Adobe PhotoShop 3.0, and the integrated densities of the ABD bands were measured with the program NIH Image 1.6. These densities were converted to concentrations using ABD standards on the same gel, and the binding isotherm was determined by a nonlinear least squares fit of the data to a hyperbola.
Rheology--
The rheological measurements were made with a
parallel plate Rheometrics RFS II rheometer (Rheometrics Inc., NJ) in
the small amplitude (strain, 2%) forced oscillation mode (30).
Monomeric actin was mixed with one-tenth volume of 10× KME
polymerization buffer and immediately placed between the metal plates
of the rheometer to polymerize at desired temperature. The plates were sealed with mineral oil (Sigma) to prevent sample dehydration. Measurements of G' and G" were made every 30 s using time sweep mode to observe the gel formation. After
G' and G" reached a plateau, frequency sweep mode
was used to measure the rheological parameters: the magnitude of
complex modulus |G*|, where |G*| = (G'2 + G"2)1/2; and the phase shift
,
where
=
1(G"/G') (31).
A solid has a phase shift of 0. A viscous liquid has phase shift of 1.6 radians. Relaxation experiments measured stress relaxation
(
(t)) after a step input strain (
o) as a
function of time. The relaxation modulus G(t) =
(t)/
o. Unlike a dynamic mechanical assay,
a stress relaxation assay does not require deformation of the actin
network after the initial strain.
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RESULTS |
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Comparison of Recombinant and Proteolytic Actin-binding Domain of
Smooth Muscle -Actinin--
Both recombinant and proteolytic ABD
preparations bind actin filaments in pelleting experiments (Fig.
1), but the affinities for actin
filaments differ by an order of magnitude (Table
I). At room temperature, the
Kd values are 1.6 µM for rABD and 20 µM for pABD. This establishes that the wide range of
affinities reported in the literature (Table I) are due to the methods
of preparation and not differences in the assays.
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Kinetics of -Actinin ABD Binding to Actin Filaments--
We
followed the binding of rABD to actin filaments under pseudo-first
order conditions with a molar excess of rABD over polymerized actin
subunits. Following rapid mixing in the stopped flow instrument, rABD
binds to actin filaments accompanied by a quench in tryptophan fluorescence that follows an exponential time course (Fig.
2A), as first reported by
Kuhlman et al. (18). The pseudo-first order rate constant of
the fluorescence change depends linearly on rABD concentration up to a
rate of at least 80 s
1 (Fig. 2B). A simple
model for the reaction is a bimolecular binding event followed by a
first order change in fluorescence.
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Mechanical Properties--
Actin filament networks are
viscoelastic materials with mechanical properties that are strongly
influenced by cross-linking proteins. This study addresses the
hypothesis (8, 16) that the rate of cross-linker dissociation is a
major determinant of the mechanical properties of actin gels. The
particular test was to use temperature to vary the dissociation rate
constant of the well characterized cross-linker smooth muscle
-actinin and to measure how this single parameter influences the
mechanical properties of a cross-linked actin gel, under conditions
where network is isotropic and the actin concentration is high enough
that most
-actinin is bound at all temperatures.
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DISCUSSION |
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Comparison of Proteolytic and Recombinant ABD--
This and
previous work (14, 18) employed the isolated actin-binding domain of
chicken smooth muscle -actinin rather than the bivalent native
molecule to evaluate the rates of actin filament binding and
dissociation. In previous work on pABD of smooth muscle
-actinin, it
was assumed that thermolysin simply cleaves the ABD from the
-helical tail (14, 26), because Baron et al. (32) found
that isolated pABD did not yield any product upon Edman degradation, as
expected for a fragment with an intact, blocked N terminus (33).
However, Davison et al. (34) presented evidence that
thermolysin actually cleaves between Leu24 and
Leu25. We confirm this observation. By mass spectroscopy we
show that thermolysin also cleaves after Glu257, suggesting
that this is the end of the compactly folded head, rather than
Phe246, which begins the spectrin-like repeats of the tail
or Glu269, which seems to have been chosen as the end of
rABD simply because of a convenient restriction site (27). The last 12 residues of pABD were not included in the crystallized fimbrin ABD, but presumably link the
-actinin ABD to its tail.
Temperature Dependence of -Actinin Binding to Actin
Filaments--
Both the association and dissociation rate constants
for ABD-binding actin filaments depend on temperature (Table I). Our dissociation rate constants are smaller than those of Kuhlman et al. (18) using exactly the same proteins. Their values
were based on extrapolation to zero rABD concentration of plots of kobs versus rABD concentration in
association experiments, a procedure with some error. In a confirmatory
experiment they observed a rate constant of 15 s
1 in a
1:10 dilution of 5 µM complex but did not investigate the dissociation reaction in a much detail as we have. As expected for a
larger molecule, Goldman and Isenberg (13) found that intact smooth
muscle
-actinin binds and dissociates somewhat more slowly at
20 °C than ABD (Table I).
Cross-linker Dynamics Determine the Mechanical Properties of Actin
Filament Gels--
We assume that the two heads of -actinin bind
actin filaments with the same rate constants as rABD, with a small
correction for slower diffusion of the larger native protein (14), but several factors complicate the situation. First, actin filaments are
immobile on the time scale of the association reaction (39). Second, at
the concentrations employed most univalent reactions of
-actinin
with an actin filament will not position the second head appropriately
to interact with a neighboring (immobile) actin filament. Third,
prolonged dissociation of an
-actinin bound between two immobile
actin filaments will be slower than two independent heads, because
rebinding of a single dissociated head will be favored by being
anchored to the neighboring filament. This may not complicate
interpretation of mechanical properties, because dissociation of a
single head breaks cross-links between filaments. Finally, force
applied to a cross-link is likely to increase the rate of
dissociation.
Implications for the Cell-- The physical structure of the cytoplasm must adapt as a moving cell changes shape. There is little doubt that the actin cytoskeleton uses both filament assembly and disassembly as well as filament severing (and possibly annealing) to reconfigure. Because actin filaments themselves are quite stable kinetically (41), actin filament dynamics in the cell depend upon numerous actin-binding proteins that can sever, cap, nucleate, and cross-link the filaments.
Our current work provides support for the old idea that some of the adaptation of the actin cytoskeleton to change of shape is a passive response to force, allowed by the rapid exchange of some cross-links between the filaments. Our experiments confirm that the stiffness and phase shift of isotropic actin filament networks vary with the rate of dissociation of cross-links between the filaments. When force is applied slowly, dynamic cross-links allow the network to change shape. When force is applied suddenly, dynamic cross-links resist deformation and store part of the energy for elastic recoil. Such dynamic cross-links may contribute to the ability of the stiff actin network in the cell cortex to change shape slowly during movements such as cytokinesis. The known effect of Ca2+ on the affinity of some cytoplasmic ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. D. R. Critchley for the
cDNA of chicken smooth muscle -actinin ABD and Dr. Wolfgang
Fischer for help with protein chemistry. J. X. is very grateful to
Mike Ostap and Enrique De La Cruz for the technical advice and helpful
discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 26338 (to T. D. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Thomas C. Jenkins Fellowship.
** To whom correspondence should be addressed. Present address: Salk Inst. for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-453-4100, Ext. 1261; Fax: 619-546-0838; E-mail: pollard{at}salk.edu.
1 The abbreviations used are: ABP, actin-binding protein; ABD, actin-binding domain; rABD, recombinant ABD.
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REFERENCES |
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