* Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030; and Medical
Research Council Laboratory of Molecular Biology, Cambridge, CB2 2QH, United Kingdom
Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter `actin crossovers' (~75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.
THE actin-based cytoskeleton plays an important role
in cell locomotion. As cells move in response to external signals, the cytoskeleton is continuously remodeled: actin filament networks are formed at the frontal
lamella, which attaches to the underlying substratum through
focal adhesions, thereby providing adhesive contacts for
the traction necessary for retraction of the cell body. For
this process to continue, actin subunits must be continuously recycled to the leading edge (for review see Small,
1995 ADF (also called destrin) was first isolated as an actin
depolymerizing factor in chick embryo brain and is widely
distributed in animal and plant tissues (Bamburg et al.,
1980 The mechanism by which these proteins accelerate actin
filament dynamics is still controversial. Actophorin and
ADF were classified as actin filament-severing proteins
based on visualization by light microscopy (Maciver et al.,
1991 Here we report our studies on the interaction of cofilin
with F-actin by electron cryomicroscopy and image reconstruction. Our analysis shows that cofilin binds cooperatively between two actin subunits along the filament. The
most startling finding of this work is that cofilin induces a
substantial change in the twist of F-actin. One ramification
of this structural change is to alter the phalloidin-binding
site, thereby preventing its binding and making it ineffective as a probe for F-actin. This is the first demonstration
of an actin-binding protein that "competes" for binding by
altering F-actin's twist. The implications of this structure
on cofilin's function in the cell and the structure of the actin filament will be discussed.
Protein Preparations and Binding Studies
Recombinant human cofilin was expressed in Escherichia coli as previously described for ADF (Hawkins et al., 1993 Rabbit muscle actin was prepared as described previously (Harris and
Weeds, 1983 Sample Preparation for Electron Microscopy
For structural studies, fully decorated actin filaments were produced by
incubating 2.38 µM platelet F-actin with 11.9 µM cofilin in F-buffer (50 mM NaCl, 2 mM MgCl2, 0.2 mM CaCl2, 1 mM DTT, 0.2 mM ATP), containing 15 mM Pipes, pH 6.6 ("low pH buffer"), for 30-90 min on ice.
Samples were plunged in a room maintained at 18°C and <30% humidity.
Approximately 7 µL of filaments was placed on 400 mesh copper grids
prepared with holey carbon films, blotted with filter paper, rapidly frozen
in ethane slush cooled with liquid nitrogen, and maintained in liquid nitrogen until use. Plunged samples were pelleted for 15 min at 100,000 g in an
airfuge (Beckman Instr.) and SDS-PAGE gels run to confirm cofilin binding to actin. Partial decoration of muscle F-actin was achieved using 2 µM
F-actin with 6 µM cofilin in "low pH buffer" for 30-90 min at room temperature. The effect of cofilin binding at higher pH was examined using 2 µM F-actin and 6 µM cofilin in F-buffer containing 10 mM Tris, pH 8.2, for 30-90 min at room temperature. Grids of F-actin were prepared as
controls.
Electron Microscopy
Transmission electron microscopy of frozen-hydrated samples was performed at 100 kV using an electron microscope (1200EX; Jeol, Ltd., Tokyo, Japan) with a Gatan anti-contaminator cooled to Image Processing and Analysis
Electron cryomicrographs with a good distribution of filaments and exhibiting minimal drift or charging were scanned on a densitometer (Perkin-Elmer Corp., Norwalk, CT) at 5.3 Å per pixel. The defocus of the micrographs
used for the structural analysis was determined by incoherent averaging of
calculated diffraction patterns obtained from either regions of adjacent
carbon or protein embedded in ice (Zhou et al., 1996 Particle Indexing
Computed power spectra of decorated filaments revealed a movement
from the usual position of the first layerline for F-actin at 1/(365 ± 15) Å Layerline Collection, Merging, and Calculation of
Three-dimensional Reconstructions
Layerline collection, merging, and helical reconstructions were done by
standard methods (DeRosier and Moore, 1970 Datasets showing disagreement in polarity assigned to the two sides,
low (<10°) up/down difference in phase residuals, high (>50°) phase residuals for both sides, or uncharacteristically large tilts and/or shifts during
correction for phase origin were excluded. The mean phase residuals calculated from the F-actin and cofilin/F-actin datasets during the last round
of alignment were 29.4° (up/down difference = 25.6°) and 38.6° (up/down
difference = 20.5°), respectively. The cofilin/F-actin reconstruction included 13 layerlines (highest l = 42; largest n = 7) averaged from 38 datasets representing the contributions of ~4,380 actin/cofilin subunits.
The F-actin reconstruction was calculated from an average of 20 datasets
(16 layerlines; highest l = 20; largest n = 7) containing a total of 3,294 actin subunits. The two reconstructions were brought to a common phase
origin by aligning preliminary averages of each against a reconstruction of
rabbit muscle F-actin. The polarity of the filament was determined by
alignment against a reconstruction of myosin S1-decorated F-actin (Whittaker et al., 1995a Visualization of Maps
Three-dimensional density maps were calculated by Fourier-Bessel inversion of averaged layerline data. The F-actin reconstruction was calculated
at both 54/25 and 20/9 symmetries for comparison to the cofilin/F-actin reconstruction. The significance of features within the reconstructions was
assessed by generating t-maps between near- and far-side averages of each
reconstruction (Milligan and Flicker, 1987 Appearance of Actin Filaments Decorated with Cofilin
Fig. 1 a shows a muscle actin filament of approximately
five crossovers. The crossovers are ~365 Å in length,
which is typical for F-actin. Actin filaments decorated with
human cofilin and ADF are shown in Fig. 1, b and c, respectively. At pH 6.5, both cofilin and ADF bind filaments
without destabilizing them. The decorated filaments are
thicker, owing to the presence of cofilin. Surprisingly,
however, they have substantially shorter crossover lengths
(~270 Å on average). Therefore, a major effect of binding by either cofilin or ADF to actin is to change the helical
twist.
Fig. 2 shows cryomicrographs of platelet actin at pH 6.6, in the presence and absence of cofilin. A single computationally straightened filament without cofilin, ~100 Å diam, is shown in Fig. 2 a. The fine features are difficult to
discern owing to the imaging conditions used to produce
these cryomicrographs, but the calculated diffraction pattern (Fig. 2 c) shows that the filament is highly ordered.
The strongest layerlines have been labeled with their helical index parameters: n (Bessel order) and l (layerline
number). (These values are analogous to the indices used
to describe discrete reflections in the diffraction patterns of crystalline objects.) The spacings of layerlines n = 2, l = 4 and n =
The filament in Fig. 2 b was obtained after incubating
platelet F-actin with a 4-fold excess of human cofilin at pH
6.6. Under these conditions all of the filaments were fully
decorated with cofilin. They differ from F-actin in two major respects: (a) they are ~20 to 30% larger in diameter,
presumably owing to the additional mass of cofilin; (b)
they differ in the length of the "crossover," which is a characteristic of F-actin. This is more apparent in the computed diffraction patterns (Fig. 2 d). The height of layerline n = 2, l = 2 indicates that the crossover length of this
filament is 272 Å. This movement relative to the equivalent layerline in the undecorated filament (n = 2, l = 4)
dramatically illustrates that cofilin changes the twist of
nonmuscle actin also. The position of the first meridional
shows that the axial rise per subunit is virtually unchanged
by cofilin binding. Thus, the effect of cofilin is to reduce
the rotation per subunit (running along the short-pitched,
left-handed helix) by about 4 to 5° while causing little or
no change in the rise per subunit (for a review of actin's helical symmetry see Aebi et al., 1986 It has been reported that ADF binds muscle F-actin in a
highly cooperative manner in vitro (Hawkins et al., 1993
Fig. 4 presents cryomicrographs of muscle actin incubated with a 2-fold excess of cofilin at pH 6.6. The lower
cofilin to actin ratio was used to prepare a mixture of both
decorated and undecorated filaments to be imaged in the
same micrograph (Fig. 4 a). The diffraction patterns in Fig.
4, d and e were calculated from the boxed filaments in Fig.
4 a. The heights of the n = 2 layerlines (at 1/352 and 1/275
Å
We also obtained images of partially decorated filaments. This is a very rare event at a 3:1 cofilin to actin ratio
(the conditions used to produce the micrographs in Fig. 4)
probably because of the highly cooperative nature of cofilin binding to filaments. Fig. 4 b shows a partially decorated filament that was computationally divided into its
undecorated (upper) and decorated (lower) halves. Calculated diffraction patterns revealed that the undecorated half has a mean crossover length of 371 Å, whereas the
decorated half has a crossover length of 278 Å (Fig. 4, f
and g). Another example of a partially decorated filament
is shown in Fig. 4 c. In this case the undecorated half has a
mean crossover length of 356 Å, and the decorated half
has a mean crossover length of 278 Å. These results indicate that the change in twist occurs only in those regions of
the filament where cofilin is bound and is not propagated
along the rest of the filament.
F-actin from rabbit muscle was also incubated with a 2-fold
excess of cofilin at pH 8, to see whether filaments were
also decorated under conditions in which depolymerization is strongly favored. We found a dramatic difference in
the appearance and distribution of the filaments in the cryomicrographs (results not shown). From 93 micrographs
taken at 30,000×, 77 had few or no filaments in the fields.
In some cases (16 micrographs) we found short actin filaments that were laterally aggregated into disorganized
"bundles," which ranged in size from 60 to 120 nm diam
and 0.75 to 2.1 µm in length. In contrast, at pH 6.6, only one of these lateral aggregates was observed (out of 68 micrographs taken). Some single filaments were also observed at the higher pH. These appeared to be decorated
based on their diameters and short crossover lengths.
Thus, the change in twist is induced by cofilin and is independent of pH.
Features in the Reconstructions
Our structural analysis of cofilin/actin was pursued further
using the images obtained from platelet actin. Decorated
filaments diffracted strongly compared to their undecorated counterparts (Fig. 2, c and d). In addition to the
change in twist that affected the heights of various layerlines, other differences were clearly visible relative to undecorated F-actin. The radial positions of the peaks in the
diffraction pattern for the first n = 2 and 4 were moved inward owing to the increased filament diameter, and their
intensities were stronger. This was expected based on the
enhanced appearance of the crossovers which correspond
to the two-start actin helix. In addition, the intensity of the
n = The final mean phase residuals calculated from the controls and decorated actin datasets were 29.4 and 38.6°, respectively. These phase residuals compare very favorably
to those reported for other actin structures calculated in a
similar way (that is, calculated using the entire layerlines
rather than just the peaks; McGough et al., 1994 Three-dimensional density maps were computed by
Fourier-Bessel inversion of the averaged layerline data after truncation of the layerlines at 1/30 Å
Identification of the Cofilin-binding Site
When we brought the F-actin layerline data to the twist of
the cofilin/F-actin, we saw little change in the actin subunit. Fig. 6 a presents the reconstruction with the approximate locations of the four subdomains of actin indicated
on the filament. The surface view of platelet F-actin is
equivalent to reconstructions of muscle actin. Comparison
of the reconstruction of the decorated filament (Fig. 6 b)
with that for F-actin shows several structural features in
common. Both filaments display two twisting strands of
the long-pitch helix that are half staggered relative to each other and connected by a bridge of density running along
the one-start, left-handed helix, connecting subdomain 4 of one subunit with subdomain 1 of the next. The cofilin-decorated filament differs from F-actin because of the extra density at the junction between two longitudinally associated actin subunits.
A difference map was computed in real space after
aligning the reconstructions to a common phase origin.
Contouring the positive differences to enclose the nominal
molecular volume of cofilin reveals a single rugby ball-shaped mass of dimensions 32 × 37 × 43 Å (copper-colored mass in Fig. 6 c). These values agree well with the
overall dimensions of destrin, which is closely related to
cofilin (Hatanaka et al., 1996 Analysis of Filament Structures Rendered at Different
Contour Levels
Because of the limited resolution of most EM reconstructions, there is some ambiguity in assigning the precise
boundary separating the particle from the embedding medium. Nonetheless, reconstructions are generally displayed as volume renderings having definite molecular boundaries. There are a number of ways to choose the
boundary (for review see Frank, 1996 Cofilin Changes the Twist of Actin Filaments
The most startling finding of this work is that cofilin induces a substantial change in the twist of F-actin. This is
the first protein found to do so. Cofilin brings filaments to
a new mean twist of 2.218 units/turn resulting in a shorter
crossover length of 269 Å, ~72% that of F-actin. Our micrographs showing both decorated and undecorated filaments in the same field (Fig. 4) clearly demonstrate that
the difference in crossover length is not attributable to
large variations in the imaging conditions. This structural
change probably accounts for cofilin's cooperative binding
to F-actin (Fig. 3).
It has long been noted that F-actin exhibits a natural
variation in twist (Hanson et al., 1967). The range in mean
twists normally observed is from 2.154 to 2.167 subunits
per turn, resulting in mean crossover lengths ranging from
385 to 358 Å. The two prevailing models describing actin's
natural variation in twist are the "angular disorder" model
(Egelman et al., 1982 Whatever the source of the disorder, it has been suggested that there is a biological advantage in having an actin filament with variable twist, particularly with regards to
actin's ability to incorporate into macromolecular assemblies such as bundles (Trachtenberg et al., 1986 It has been reported that binding of ADF to F-actin is
inhibited by phalloidin in vitro (Hayden et al., 1993 The binding of cofilin to F-actin is highly co-operative,
which we believe reflects its nonrandom association with
F-actin. Recently, Carlier et al. (1997) The ability of cofilin and ADF to bind ADP-actin subunits and alter the twist of F-actin may explain its acceleration of the dissociation rate of subunits from the pointed
ends of filaments (Maciver, S.K., B. Pope, and A. Weeds,
unpublished observations; Carlier et al., 1997 Relationship to Other Actin-binding Proteins
Electron microscopy has been used to study the binding
sites of a variety of actin-binding proteins including myosin, tropomyosin, Towards Building a Model for
Cofilin/F-Actin Interaction
We first examined whether the cofilin-dependent change
in rotation introduced bad contacts in the atomic model of
F-actin. A model of F-actin at the new twist was built using
the Lorenz model of actin (Lorenz et al., 1993 The cofilin/F-actin reconstruction provides the first direct structural framework into which cofilin's interactions
with actin may be modeled. As a first step in this process,
we have interactively fit the actin and destrin models to
the molecular envelope of F-actin/cofilin. An interesting
feature of all three of the atomic structures from members
of the cofilin family (Hatanaka et al., 1996 The results of this manual fitting are shown in Fig. 7. Cofilin is positioned to make interactions with two actin
subunits at the cleft between subdomains 1 and 3 of the
upper and at subdomains 2 and 1 of the lower subunits.
Residues in the upper actin monomer that fall in or near
the cofilin-binding site include: Tyr143-Thr149, Ile345-
Leu346, Leu349-Thr351, and Gln354. Residues in the lower actin monomer include: Phe21, Arg28, Gly36-Pro38,
His40-Val43, Gln49-Val54, Glu57, Lys61, His87-His88,
Phe90-Val96, and His101. Unfortunately, unlike actin or
myosin subfragment 1 which are both readily fitted into
EM maps (Schroder et al., 1993
One property of cofilin that might be explained from its
location on F-actin is the pH sensitivity of filament binding. After positioning the destrin coordinates on the actin
filament, we investigated the resulting placement of histidine residues at the putative interface, reasoning that
these might be important in the pH-mediated switch. Histidines are not conserved in the cofilin family, and comparison of the locations of the two histidines in destrin with
the three in actophorin, when mapped onto the structure
shown in Fig. 7, shows that none is at the actin interface. On the other hand, there are four actin histidines near the
cofilin-binding site (40, 87, 88, 101) that might be important in cofilin/actin interactions. Both cofilin and ADF/destrin bind preferentially to filaments <pH 7.3, and depolymerize actin at higher pH values (Yonezawa et al., 1985 Another property of cofilin that might be explained by
the cofilin/F-actin structure is cofilin's sensitivity to the nucleotide state of actin (Maciver and Weeds, 1994 Conclusions
Our reconstruction provides the first direct information
about how the cofilin family of proteins binds actin. Cofilin binds F-actin between adjacent subunits along the two-start helix. Binding by both cofilin and ADF (destrin) results in a substantial reduction in the crossover length of
the filament. This structural change probably accounts for
the co-operative nature of binding and represents a novel
mechanism to prevent filament binding by other actin
binding proteins. In addition, the cofilin-induced twisting
of actin filaments may introduce strains in the actin filament that contributes to the increase in filament dynamics
and fragmentation observed by others.
; Welch et al., 1997
). Although the assembly rates of
actin in vitro are extremely rapid (Drenckhahn and Pollard, 1986
), the pointed end disassembly rate constant
measured in vitro (~0.3 s
1) is far too slow to recycle the
monomers necessary for this process to continue. Moreover, rates nearly 100 times slower than this have been
measured under physiological conditions (Coluccio and Tilney, 1983
). This compares with required rates in excess
of 7 s
1 for the turnover of actin filaments in the lamellipods of macrophages and 10-fold higher rates that might
be necessary to account for the locomotion of leukocytes
at 30 µm/min (Zigmond, 1993
). The most obvious way to
increase the disassembly rate is to sever filaments to expose new barbed ends, where the dissociation rate constant for ADP-actin subunits is much higher (>7 s
1; Pollard, 1986
). The cofilin/ADF family of proteins are the
most obvious candidates to do this, particularly because of
their apparent ability to sever filamentous (F)1-actin without capping (Hawkins et al., 1993
; Hayden et al., 1993
).
; Bamburg and Bray, 1987
; Lopez et al., 1996
). A
highly homologous protein, cofilin (70% identical to ADF
in sequence), has been shown to modulate actin filament
disassembly in a pH-dependent manner (Yonezawa et al.,
1985
). Related proteins have since been identified in a
number of other organisms (Cooper et al., 1986
; Moon et
al., 1993
; McKim et al., 1994
; Gunsalus et al., 1995
). Actin
binding is inhibited by phosphorylation of Ser3 (Agnew et
al., 1995
) and by binding to phosphatidyl inositol 4,5-bis
phosphate (Yonezawa et al., 1990
). Taken together these
results suggest that the ADF/cofilin family of proteins is
composed of stimulus-responsive modulators of actin dynamics (for review see Moon and Drubin, 1995
; Theriot, 1997
).
; Hawkins et al., 1993
). However, high concentrations
of protein gave minimal severing, and attempts to quantitate the severing activity by biochemical methods suggested that it is <0.1% that of gelsolin. Like cofilin, ADF
binds preferentially to filaments <pH 7.3, and promotes
disassembly at higher values (Hawkins et al., 1993
; Hayden et al., 1993
). Because these proteins consist of a single
domain (Hatanaka et al., 1996
; Federov et al., 1997
; Leonard et al., 1997
), the switch from G- to F-actin binding
must involve subtle differences in the cofilin/actin interface at the two pH values. Like actophorin (Maciver and
Weeds, 1994
), ADF binds preferentially to ADP-actin monomers, which may in part explain its preferential binding to filament subunits (Maciver, S.K., B. Pope, and A. Weeds, unpublished observations). Recent work has cast
doubt on the idea that disassembly is due solely to filament severing: rather ADF appears to modulate the rate
constants of assembly and disassembly at the ends of filaments (Carlier et al., 1997
; Maciver, S.K., B. Pope, and A. Weeds, unpublished observations).
Materials and Methods
). It was purified from the
bacterial lysate on DEAE-cellulose at pH 8.0, where it was not retarded
(Hawkins et al., 1993
). After adjusting the pH to 6.6, the protein was further purified on CM-cellulose (10 × 2.5 cm) in 10 mM sodium succinate,
pH 6.6, 0.5 mM dithiothreitol, 1 mM NaN3 and eluted with a gradient to
0.2 M NaCl. The purified protein was dialyzed into 10 mM Tris-HCl, pH
8.0, 0.2 mM EGTA, 2 mM dithiothreitol, 1 mM NaN3, concentrated by
Centricon ultrafiltration and stored at
80°C after drop freezing into liquid nitrogen.
) or obtained from Cytoskeleton (Denver, CO) and stored at
10 mg/ml in 2.0 mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM Na2ATP,
0.005% NaN3 at
80°C. Human platelet actin was also obtained from Cytoskeleton. Binding to F-actin was measured by cosedimentation in an ultracentrifuge (TL; Beckman Instr., Fullerton, CA) at 220,000 g as described previously (Hawkins et al., 1993
).
179°C with liquid
nitrogen. Grids were maintained at
167°C using a Gatan cryotransfer
system cooled with liquid nitrogen. Images were recorded on Kodak SO-163 film at a nominal magnification of 30,000 and an electron dose of ~8
e
/Å2. Electron microscopy of filaments negatively stained with uranyl acetate was performed using a transmission electron microscope (301; Philips, Eindhoven, The Netherlands, CT) at 80 kV.
). Images of cofilin-decorated platelet actin were recorded at 1.4-1.6 µm underfocus (first
node of the contrast transfer function, 1/22.5-1/24.5 Å
1) and those of undecorated platelet actin at 1.5-2.1 µm underfocus (first node, 1/24-1/29
Å
1). Scanned images were selected for their straightness, distribution, and length (minimum of 8 crossovers). Image preparation for reconstruction was performed using Phoelix run on a Silicon Graphics (Mountain View, CA) workstation (Whittaker et al., 1995b
; Schroeter and Bretaudiere, 1996
). Regions of cofilin-decorated filaments analyzed ranged in
length from 0.22 to 1.41 µm (mean = 0.58 µm). Those from F-actin ranged
from 0.29 to 2.01 µm (mean = 0.82 µm).
1
to 1/(270 ± 6) Å
1. Some of the best preserved particles had visible reflections in the computed diffraction patterns past 1/27.5 Å
1. Cofilin-decorated filaments were indexed manually after computationally straightening the particles and computing their Fourier transforms. Layerline
heights (l) and radial positions of the first peaks (R) were measured from
the calculated diffraction patterns. In addition, layerlines were identified
and their positions measured from synthetic "phase display" patterns,
which incorporate information about the phase differences across the meridians of the particles weighted by the amplitudes (display-phase program courtesy of M. Schmid, Baylor College of Medicine, Houston, TX).
Values of l and R for layerlines for which there was clear phase behavior were used to index the patterns (DeRosier and Moore, 1970
; Stewart, 1988
). The simplest selection rule most consistent with the particles was
found to be 20 units in 9 turns (twist of 2.222 subunits per turn), assuming
that the hand of the actin filament was unchanged. Small variations in the
exact symmetries of the filaments studied were observed as is usual for actin structures. The mean twist for the population of filaments included in
the reconstruction was 2.218, which corresponds to a selection rule of 264 units in 119 turns. However, all layerlines collected were brought to the
simpler selection rule before merging and performing the three-dimensional reconstruction.
; Amos, 1975
; Whittaker et
al., 1995b
). After correcting for the phase origin and particle tilt, layerlines
were separated into near- and far-side datasets and subjected to three
rounds of alignment against successive averages. For the final round of
alignment the following layerlines (n [Bessel order], l) were used from F-actin: (2,4), (4,8), (
5,17), (
3,21), (
1,25), (1,29), (3,33), and (5,37).
The equivalent layerlines were used for the cofilin-decorated F-actin
dataset. All data points along the layerlines (excluding those at the meridian) up to a resolution of 1/26 Å
1 for the decorated filaments and 1/30
Å
1 for the actin filaments were used in the alignments.
).
). These maps revealed no significant differences between the near- and far-side averages for either reconstruction, indicating that the features in the maps are reliable. Atomic models and electron density maps were displayed and manipulated using
IRIS Explorer (Numerical Algorithms Group, Ltd., Oxford, UK) and O
version 5.9 (Jones et al., 1991
). Ribbon diagrams were generated using
Ribbons 2.65 (Carson and Bugg, 1986
), saved as Inventor format files, and
displayed in IRIS Explorer.
Results
Fig. 1.
Cofilin and ADF
change the twist of rabbit
muscle F-actin. Electron micrographs of negatively stained
F-actin (a) and filaments decorated with cofilin (b) and
ADF (c) at pH 6.5. Both cofilin and ADF induce a 25%
reduction in crossover length.
White bars indicate crossover positions. Bar, 0.1 µm.
[View Larger Version of this Image (91K GIF file)]
1, l = 27 occur at 1/366 Å
1 and 1/59 Å
1, consistent with their positions in diffraction patterns of muscle F-actin, in which they correspond to the periodicities of
the long pitch double helix of actin and the left-handed,
short-pitch helix, respectively. Thus, muscle and nonmuscle actin filaments are indistinguishable at this resolution.
Fig. 2.
Cofilin changes the twist of platelet F-actin. (a) Portion
of a computationally straightened F-actin filament; (b) portion of
a computationally straightened actin filament decorated with cofilin. The defocus of both micrographs shown was 1.5 µm. Decorated filaments are ~120-130 Å diam with accentuated `crossovers' relative to control actin. (c) Computed diffraction pattern
calculated from the actin filament shown in (a). The length of the
filament used to calculate this pattern was 1.44 µm. (d) Computed diffraction pattern calculated from the cofilin-decorated
actin filament shown in (b). The length of the filament used to
calculate this pattern was 1.41 µm. Prominent layerlines in each
diffraction pattern are labeled with values of n and l. The axial
position of layerline n = 2, l = 4 in the F-actin diffraction pattern
is 1/366 Å1 and that of n = 2, l = 2 in the cofilin/F-actin diffraction pattern is 1/272 Å
1.
[View Larger Version of this Image (60K GIF file)]
; Trachtenberg et al.,
1986
). Therefore, although the crossover length is reduced, the change in twist does not affect the overall
length of the filaments. The range of crossover lengths observed for cofilin-decorated filaments was between 264 and 276 Å (n = 30), well below the normal range for muscle actin and the range of mean crossover lengths we observed for platelet actin (347 to 370 Å; based on 70 measurements).
;
Hayden et al., 1993
). Cosedimentation experiments with
F-actin demonstrate that cofilin binds to F-actin in a similar manner (Fig. 3). The Hill constant calculated by nonlinear least squares fitting of 6.4 confirms the high degree
of cooperativity.
Fig. 3.
Cooperativity of cofilin binding to rabbit muscle F-actin
at pH 6.5. 6 µM F-actin was cosedimented with cofilin (0-35 µM). Supernatant and pellets were analyzed by SDS-PAGE and concentrations of cofilin and actin estimated by densitometry. Bound
cofilin is expressed as a molar ratio to actin subunits. Nonlinear
least-squares fitting shows a maximum binding at 1.16 cofilin/actin and a Hill constant of 6.4.
[View Larger Version of this Image (13K GIF file)]
1, respectively) confirm that this field contains both
types of filament, lending support to the idea that cofilin
binds cooperatively.
Fig. 4.
Cooperativity of cofilin binding to rabbit muscle F-actin
shown by electron cryomicroscopy. (a) Micrograph of frozen-
hydrated filaments decorated with cofilin at subsaturating conditions. (b and c) Micrographs of partially decorated filaments: the
top region of each filament is thinner, bare F-actin, whereas the lower region is decorated with cofilin. (d and e) Computed diffraction patterns calculated from the bare F-actin and decorated
F-actin images in a. (f and g) Computed diffraction patterns from
the undecorated and decorated halves of filament b. (h and i)
Computed diffraction patterns from the undecorated and decorated halves of filament c. In this case the decorated region of the
filament is only about four crossovers long. This results in a
broadening of the layerline data i. Arrowheads indicate the positions of equivalent (J2) layerlines.
[View Larger Version of this Image (122K GIF file)]
1 was dampened while other layerlines (n =
3, 3,
2, and 0), which are difficult to see in F-actin diffraction
patterns, were strengthened in the decorated filaments.
Ultimately, we were able to reconstruct the cofilin-decorated filaments to higher resolution (27.5 Å axially) than
that attained from undecorated filaments (35 Å axially).
Nevertheless, the major differences that exist between the
two reconstructions were still present when the resolution of the cofilin/actin reconstruction was truncated to match
the F-actin map (results not shown).
; Orlova et
al., 1994
; Owen and DeRosier, 1993
; Bremer et al., 1994
;
Lehman et al., 1994
). The somewhat higher phase residual
obtained for the decorated filaments may be a function of
the smaller defocus used to obtain these images and the inclusion of higher resolution data during the alignment.
1 (for F-actin)
and 1/25 Å
1 (for cofilin/actin) to take into account the position of the first node in the contrast transfer function.
Fig. 5 presents surface renderings of the platelet F-actin
and cofilin/F-actin reconstructions showing several crossovers of each. Each filament shown contains 40 actin subunits to emphasize that although the crossover length has
been reduced by cofilin binding, the overall length of the
filament is unchanged.
Fig. 5.
Reconstructions of F-actin and cofilin/actin filaments
demonstrating the change in crossover length. (a) Platelet F-actin exhibiting 54/25 symmetry, equivalent to 2.160 subunits per turn. (b) Cofilin/F-actin exhibiting 20/9 symmetry (2.222 subunits per turn). Arrows mark the crossover length. There are 40 actin subunits in each filament.
[View Larger Version of this Image (30K GIF file)]
Fig. 6.
Identification of the cofilin-binding site by difference mapping. (a) Platelet actin reconstruction brought to the twist of the cofilin/actin filament (2.222 subunits per turn). The subdomains of actin and the phalloidin-binding site (yellow asterisk) are indicated. (b)
Cofilin/actin reconstruction at the same orientation as in (a). (c) Positive difference density (copper) calculated by subtracting a from b.
The contour level chosen encloses 100% (nominal) molecular volume. (d) Effects of contouring the cofilin/actin reconstruction using
high thresholds. The actin reconstruction is shown as a transparent surface (silver) and the cofilin-decorated filament (gold) at two different molecular volumes (40 and 10%) to emphasize the strongest densities in the map.
[View Larger Version of this Image (49K GIF file)]
). The major axis of cofilin makes a 30° angle with the plane normal to the helical axis
of the filament. The difference map shows that cofilin is
centered (axially) at about the position of subdomain 2 of
the lower actin subunit and radially at the cleft between
subdomains 1 and 3 of the upper actin subunit.
). We have found it
helpful in our analysis to display the reconstructions at a
variety of contour levels as has been suggested and done
by others (DeRosier and Moore, 1970
; Milligan and
Flicker, 1987
; Bremer et al., 1994
). Fig. 6 a presents the
F-actin reconstruction contoured at a level that encloses
the Lorenz model of the actin filament (nominal molecular volume = 150%). The cofilin/F-actin reconstruction in Fig. 6 b is contoured at a molecular volume (nominally
145%) at which the actin portion of the decorated filament
best matches that of the undecorated filament. In the composite map (Fig. 6 d) the F-actin reconstruction is shown as
a transparent surface (silver) and the cofilin-decorated filament (gold) at two different molecular volumes (40 and
10%) to emphasize the strongest densities in the map. This
reveals that the strongest feature corresponds to cofilin,
while the next strongest corresponds to subdomains 1 and
4 of actin, respectively. The strongest intrafilament contacts are actin-cofilin-actin contacts along the two-start
helix, followed by the actin-actin contacts running along
the one-start, left-handed helix. The longitudinal actin-
actin contacts are the weakest. The same type of exercise on
F-actin (results not shown) reveals that the strongest contact is along the one-start helix and the strongest density in
subdomain 1. Subdomain 4 is not resolvable as a separate
mass in the F-actin reconstruction. It is important to bear in
mind, however, that the nominal resolution of the F-actin and cofilin/F-actin reconstructions differ. In addition, the
F-actin reconstruction contained images that were further
from focus than the cofilin/F-actin images. Therefore, it is
not clear if there is any biological significance to the relative strengths of these contacts (Bremer et al., 1994
).
Discussion
; Egelman and DeRosier, 1992
) and
the "lateral slipping" model (Aebi et al., 1986
; Bremer et al., 1991
). These models differ in a number of key respects,
including whether changes in twist are coupled with
changes in diameter and whether they are cumulative or
compensatory. Analysis of the cofilin/F-actin filaments may
shed light on this issue, since these filaments represent an
unprecedented shift in actin twist. We have compared the
positions of subdomains 1 and 4 (using high contour levels) for the cofilin/F-actin reconstruction with those from
reconstructions of
A1-2/F-actin (McGough et al., 1994
)
and S1-decorated actin (Whittaker et al., 1995a
). We
found them to be at identical radii from the filament axis
(results not shown). This suggests that changes in F-actin
twist are not coupled with changes in diameter. In addition, the observation that the crossover lengths in bare regions of actin adjacent to stretches of decorated filament
are not unusually long (Fig. 4), would tend to argue against a compensatory mechanism underlying F-actin
twist. It is interesting to note that the change in rotation
per subunit induced by cofilin binding to actin (~4 to 5°)
falls within the estimates of the magnitude of the angular
component in the random angular disorder model (Egelman and DeRosier, 1992
).
). Some actin-binding proteins, including myosin and scruin, have
been shown to reduce this disorder (Trachtenberg et al.,
1986
; Stokes and DeRosier, 1987
) as also does the fungal toxin phalloidin (Bremer et al., 1991
). However, in none of
these cases is the crossover length outside the normal
range for actin.
; Carlier et al., 1997
), and rhodamine-phalloidin will not bind to
cofilin/actin rods in vivo (Moriyama et al., 1987
; Moon et
al., 1993
). One interpretation of this is that ADF/cofilin
and phalloidin compete for the same site on actin. The
Lorenz model of F-actin proposed that phalloidin is positioned to contact 3 actin subunits (Lorenz et al., 1993
; Fig.
6 a, asterisk). Our work shows that the phalloidin-binding site is 15 to 20 Å away from cofilin on the filament; thus
the mutual exclusion of binding cannot be attributed to
overlapping sites. Rather, the results suggest that the
change in twist induced by cofilin alters the phalloidin-binding site and thereby prevents its binding. Conversely,
once phalloidin has bound and stabilized the filament
structure, cofilin is no longer able to interact. This is analogous to the effect of allosteric regulators, which act mainly
by altering the affinity for the binding of substrates. The
fact that cofilin-actin rods (induced by heat shock and
other stresses to the cell) fail to bind phalloidin (Moriyama
et al., 1987
; Moon et al., 1993
) suggests that this change in
twist is also present inside cells. This supports the idea that
the twist change induced by cofilin is biologically relevant.
have argued that
the apparent co-operativity of binding of ADF from Arabidopsis thaliana to rabbit muscle actin is a consequence of
the relative affinities of this protein for G-ADP actin over
F-ADP. The balance of these affinities was such that only
partial depolymerization was achieved at saturating ADF
concentrations, varying little with pH. Our results with human ADF and cofilin have shown complete depolymerization at saturating ADF concentrations at pH 8.0, indicating a much higher affinity for G-ADP actin than F-ADP
actin as compared to the Arabidopsis ADF (Hawkins et
al., 1993
). By contrast, there is very little depolymerization
by ADF or cofilin at pH 6.5, suggesting a reversal of the
relative affinities at the lower pH value. Thus we would attribute the high degree of co-operativity to the structural transition rather than to the relative affinities for monomeric and polymeric actins.
). If the
strongest intrafilament contacts are between actin-cofilin-
actin contacts along the two start helix, then labeled, terminal subunits or adjacent, unlabeled subunits may be less
tightly bound. This may also explain the apparent weak
severing activity (Hawkins et al., 1993
; Hayden et al., 1993
;
Maciver, S.K., B. Pope, and A. Weeds, unpublished observations). Light microscopic observations showed that
curved segments of actin had a higher probability of
breaking in the presence of actophorin (Maciver et al.,
1991
). It seems reasonable that the change in twist increases the distortion in the filaments, which might result
in fragmentation. It has recently been shown that actin filaments are more easily broken when they are twisted: when 10-µm filaments were turned through 90°, the breaking force was reduced from 600 to 320 pN (Tsuda et al.,
1996
). Thus, although much of the depolymerizing effect
of these proteins might arise from an increase in the dissociation rate constant at the pointed end (Maciver, S.K., B. Pope, and A. Weeds, unpublished observations; Carlier et
al., 1997
), the low levels of fragmentation observed by microscopy could be an adventitious consequence of the
structural changes induced in the filaments.
-actinin, gelsolin, and scruin. The cofilin-binding site appears to overlap the sites for myosin
(Rayment et al., 1993
; Schroder et al., 1993
), tropomyosin
(Vibert et al., 1993
; Lehman et al., 1994
, 1995
), caldesmon-containing thin filaments (Hodgkinson et al., 1997
), and
-actinin (McGough et al., 1994
). This is in agreement with published biochemical data indicating competition
between cofilin and these proteins (Bernstein and Bamburg, 1982
; Nishida et al., 1984
; Yonezawa et al., 1988
).
Gelsolin also binds F-actin in a similar location as cofilin
(McGough, A., W. Chiu, and M. Way, unpublished observations). With the possible exception of myosin, which is
still a matter of some debate (Rayment et al., 1993
; Schroder et al., 1993
), all F-actin-binding proteins have been
found to interact with at least two subunits in the filament. We propose that it is this feature that confers filament
specificity on F-actin-binding proteins. Thus, any change
in the filament geometry could modulate the binding of
other proteins and thereby control the hierarchy of interactions that must exist in cells where many actin-binding proteins occur together. As such, one previously overlooked
function of cofilin may be its action as an "F-actin sequestering protein," providing the cell with a pool of polymerized actin that is distinct from the actin cytoskeleton in that
this pool might not bind cross-linking or motor proteins.
) and applying a rotation between adjacent monomers of
162° about
the z-axis. Examination of the model at the new twist revealed only two possibly bad contacts (atoms <2 Å apart),
both of which occur at the interface between subdomain 2 of the lower and 3 of the upper subunit (Val45 with Lys291; Arg39 with Glu167). Residues 39 and 45 are located in the DNase I-binding loop, which is postulated to
be a variable domain of actin (Orlova and Egelman, 1992
;
Lorenz et al., 1993
; Bremer et al., 1994
; Tirion et al., 1995
).
Because no changes in the actin subunits were required to
accommodate the new symmetry, we have interpreted our
results using a simple rigid body rotation to the Lorenz
model. However, it is possible (although not necessary) that the actin subunits undergo conformational changes in
addition to the large change in twist. Unfortunately, differences in the imaging conditions used to obtain the micrographs of F-actin and cofilin/F-actin are likely to produce
changes in the molecular envelopes of the two maps;
therefore, it would not be appropriate to use these structures for a detailed investigation of domain movements.
We are currently working to improve the resolution of the
F-actin reconstruction to match that of the cofilin/F-actin structure.
; Federov et al.,
1997
; Leonard et al., 1997
) is that the helix that has been
proposed to bind to G-actin (Hatanaka et al., 1996
) is distorted. One consequence of this distortion in the helix is
that the destrin molecule is now able to fit more readily into the pocket in the atomic model of F-actin that is
formed between the two longitudinally associated actin
subunits. (This is in stark contrast to the clashes that would
exist if, for example, the gelsolin domain 1 structure is
placed in the same location.) Our reconstruction and difference mapping show that this pocket is the region on
F-actin with which cofilin interacts (Fig. 6 c). We rotated
and translated the destrin coordinates to both fit this
pocket on F-actin and match the molecular envelope from our reconstruction. Additional biochemical data concerning residues involved in the interaction were also considered, in particular the involvement of two lysine residues
at the NH2-terminal end of the helix (Yonezawa et al.,
1991
; Moriyama et al., 1992
). After completing the fit, two
loops in the destrin structure (Ser24-Ile29 and Gly61-
Ile64) were not accounted for by the electron density map
of cofilin/F-actin. In addition, a region of the so-called hydrophobic plug (Ser265-Glu270) is not enclosed by the cofilin/F-actin structure.
; Rayment et al., 1993
;
Bremer et al., 1994
; McGough et al., 1994
), cofilin (ADF/
destrin) is a relatively symmetrical object at low resolution. Therefore, although the long axis of the molecule can
be oriented with a fair amount of confidence, the surface
of destrin that actually contacts actin is far from certain,
based on the electron cryomicroscopy data alone. Our
model is intended to serve as a starting point from which
experiments may be designed to determine precise information about the actin/cofilin interface. Further electron
cryomicroscopy studies in which cofilin has been labeled at specific residues should also aid in this process. Residues in destrin that could be involved in the interaction
with actin based on the placement shown in Fig. 7 b include: Ser1-Val5, Cys12-Lys22, and Leu111-Lys132.
Fig. 7.
Atomic modeling of cofilin binding to F-actin. (a) Stereo images showing the placement of the difference density calculated from the cofilin/F-actin structure on the model filament.
Magenta asterisks show the positions of His 40, 87 with 88, and
101. (b) Stereo images showing the model placement of the destrin coordinates on the filament based on the cofilin/F-actin reconstruction. This model can be generated by starting with the
proposed placement of destrin on G-actin (Hatanaka et al., 1997)
and applying the following matrix and vector translation:
The NH2 terminus of destrin is oriented at ~2 o'clock in this figure (near His 40 of the lower actin monomer).
0.8515
0.5072
0.1331
0.3293
0.7147
0.6170
0.4081
0.4815
0.7756
1.7062
12.3144
12.5306
[View Larger Version of this Image (93K GIF file)]
;
Hawkins et al., 1993
; Hayden et al., 1993
). By contrast, actophorin cosedimentation with F-actin is not pH-dependent (Maciver, S.K., B. Pope, and A. Weeds, unpublished
observations). This dichotomy argues against a simple explanation of the pH switch being mediated solely by histidines.
). There is
growing evidence that the nucleotide state of actin affects
the position of subdomain 2 (Orlova and Egelman, 1992
,
1993
; Strzelecka-Golaszewska et al., 1993; Tirion et al.,
1995
). Our work shows that the cofilin-binding site includes subdomain 2. In addition, analysis of the atomic model of F-actin shows that the twist change introduces a
strain at the longitudinal actin-actin contact (between subdomains 2 and 3 of two actin subunits). This strain might
be more easily accommodated in ADP-F-actin because of
increased flexibility in this form of actin (Orlova and Egelman, 1993
). One or both of these factors might provide the
structural basis for cofilin's sensitivity to the nucleotide
state of actin. However,
-actinin also binds near subdomain 2 (McGough et al., 1994
), and there is no evidence
for its binding being regulated in this way.
Received for publication 29 April 1997 and in revised form 19 June 1997.
Please address all correspondence to Dr. Amy McGough, Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: (713) 798-6989; Fax: (713) 796-9438.We thank Sue Whytock (MRC-LMB, Cambridge, UK) for the negatively stained images in Fig. 1; S. Almo (Albert Einstein College of Medicine, New York, NY), H. Hatanaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), P. McLaughlin (University of Edinburgh, Edinburgh, Scotland), and K. Holmes (Max-Planck Institut für Medizinsche Forshung, Heidelberg, Germany) for providing atomic coordinates; and R. Milligan (Scripps Research Institute, La Jolla, CA) for the actoS1 layerline data. We also thank D. DeRosier (Brandeis University, Waltham, MA), R. Diaz (Florida State University, Tallahassee, FL), M. Sherman (Baylor College of Medicine, Houston, Texas), P. Thuman-Commike (Rice University, Houston, Texas), M. Stewart, and T. Bullock (MRC-LMB, Cambridge, UK) for helpful discussions.
This work was supported by a Grant-in-Aid from the American Heart Association (to A. McGough), grants from National Institutes of Health (RR02250) and the National Science Foundation (BIR9413229 and BIR9412521) to W. Chiu, and the W.M. Keck Center for Computational Biology (Houston, TX).
F-actin, filamentous actin; G-actin, globular actin; l, layerline height; n, Bessel order; R, radial position of the first peak.
1. | Aebi, U., R. Millonig, H. Salvo, and A. Engel. 1986. The three-dimensional structure of the actin filament revisited. Ann. N.Y. Acad. Sci. 483: 100-119 |
2. |
Agnew, B.J.,
L.S. Minamide, and
J.R. Bamburg.
1995.
Reactivation of phosphorylated actin depolymerizing factor and identification of the regulatory
site.
J. Biol. Chem.
270:
17582-17587
|
3. | Amos, L.A.. 1975. Combination of data from helical particles: correlation and selection. J. Mol. Biol. 99: 65-73 |
4. | Bamburg, J.R., and D. Bray. 1987. Distribution and cellular localization of actin depolymerizing factor. J. Cell Biol. 105: 2817-2825 [Abstract]. |
5. | Bamburg, J.R., H.E. Harris, and A.G. Weeds. 1980. Partial purification and characterization of an actin depolymerizing factor from brain. FEBS (Fed. Eur. Biochem. Soc.) Lett. 121: 178-182 . |
6. | Bernstein, B.W., and J.R. Bamburg. 1982. Tropomyosin binding to F-actin protects the F-actin from disassembly by brain actin depolymerizing factor (ADF). Cell Motil. 2: 1-8 |
7. | Bremer, A., R.C. Millonig, R. Sutterlin, A. Engel, and T. Pollard. 1991. The structural basis for the intrinsic disorder of the actin filament: the "lateral slipping" model. J. Cell Biol. 115: 689-703 [Abstract]. |
8. | Bremer, A., C. Henn, K. Goldie, A. Engel, P.R. Smith, and U. Aebi. 1994. Towards atomic interpretation of F-actin filament three-dimensional reconstructions. J. Mol. Biol. 741: 683-700 . |
9. |
Carlier, M.,
J. Santolini,
V. Laurent,
D. Didry,
H. Yan,
N. Chua, and
D. Pantaloni.
1997.
Actin depolymerizing factor (ADF/cofilin) enhances the rate of
filament turnover: implication in actin-based motility.
J. Cell Biol.
136:
1307-1323
|
10. | Carson, M., and C.E. Bugg. 1986. Algorithm for ribbon models of proteins. J. Mol. Graphics. 4: 121-122 . |
11. | Coluccio, L., and L. Tilney. 1983. Under physiological conditions actin disassembles slowly from the nonpreferred end of an actin filament. J. Cell Biol. 97: 1629-1634 [Abstract]. |
12. |
Cooper, J.A.,
J.D. Blum,
R.C.J. Williams, and
T.D. Pollard.
1986.
Purification
and characterization of actophorin, a new 15,000-Dalton actin-binding protein from Acanthamoeba castellanii.
J. Biol. Chem.
261:
477-485
|
13. | DeRosier, D.J., and P.B. Moore. 1970. Reconstruction of three-dimensional images from electron micrographs of structures with helical symmetry. J. Mol. Biol. 52: 355-369 |
14. |
Drenckhahn, D., and
T. Pollard.
1986.
Elongation of actin filaments is a diffusion-limited reaction at the barbed end and is accelerated by inert macromolecules.
J. Biol. Chem.
261:
12754-12758
|
15. | Egelman, E.H., and D.J. DeRosier. 1992. Image analysis shows that variations in actin crossover spacings are random, not compensatory. Biophys. J. 63: 1299-1305 [Abstract]. |
16. | Egelman, E.H., N. Francis, and D.J. DeRosier. 1982. F-actin is a helix with a random variable twist. Nature (Lond.). 298: 131-135 |
17. | Federov, A., P. Lappalainen, E. Federov, D. Drubin, and S. Almo. 1997. Structure determination of yeast cofilin. Nat. Struct. Biol. 4: 366-369 |
18. | Frank, J. 1996. Three-Dimensional Electron Microscopy of Macromolecular Assemblies. Academic Press, San Diego. 342 pp. |
19. | Gunsalus, K.C., S. Bonaccorsi, E. Williams, F. Verni, M. Gatti, and M.L. Goldberg. 1995. Mutations in twinstar, a Drosophila gene encoding a cofilin/ADF homologue, result in defects in centrosome migration and cytokinesis. J. Cell Biol. 131: 1243-1259 [Abstract]. |
20. | Hanson, J.. 1967. Axial period of actin filaments. Nature (Lond.). 213: 353-356 . |
21. | Harris, H.E., and A.G. Weeds. 1983. Plasma actin depolymerizing factor has both calcium-dependent and calcium-independent effects on actin. Biochemistry. 22: 2728-2741 |
22. | Hatanaka, H., K. Ogura, M. Moriyama, S. Ichikawa, I. Yahara, and F. Inagaki. 1996. Tertiary structure of destrin and structural similarity between two actin-regulating protein families. Cell. 85: 1047-1055 |
23. | Hawkins, M., B. Pope, S. Maciver, and A.G. Weeds. 1993. Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments. Biochemistry. 32: 9985-9993 |
24. | Hayden, S.M., P.S. Miller, A. Brauweiler, and J.R. Bamburg. 1993. Analysis of the interactions of actin depolymerizing factor (ADF) with G- and F-actin. Biochemistry. 32: 9994-10004 |
25. | Hodgkinson, J.L., M. El-Mezgueldi, S. Marston, R. Craig, P. Vibert, and W. Lehman. 1997. 3D reconstruction of smooth muscle thin filaments: contribution of caldesmon and calponin to filament structure. Biophys. J. 72: 2398-2404 [Abstract]. |
26. | Holmes, K.C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Atomic model of the actin filament. Nature (Lond.). 347: 44-49 |
27. | Jones, T.A., J.Y. Zou, C. Cowan, and M. Kjeldgaard. 1991. Improved methods for the building of protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47: 110-119 . |
28. | Lehman, W., R. Craig, and P. Vibert. 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature (Lond.). 368: 65-67 |
29. | Lehman, W., P. Vibert, P. Uman, and R. Craig. 1995. Steric-blocking by tropomyosin visualized in relaxed vertebrate muscle thin filaments. J. Mol. Biol. 251: 191-196 |
30. | Leonard, S., A. Gittis, E. Petrulla, T. Pollard, and E. Lattman. 1997. Crystal structure of the actin-binding protein actophorin from Acanthamoeba. Nat. Struct. Biol. 4: 369-373 |
31. |
Lopez, I.,
R. Anthony,
S. Maciver,
C.-J. Jiang,
S. Khan,
A.G. Weeds, and
P. Hussey.
1996.
Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins.
Proc. Natl. Acad. Sci. USA.
93:
7415-7420
|
32. | Lorenz, M., D. Popp, and K.C. Holmes. 1993. Refinement of the F-actin model against X-ray fiber diffraction data by the use of a directed mutation algorithm. J. Mol. Biol. 234: 826-836 |
33. | Maciver, S.K., and A.G. Weeds. 1994. Actophorin preferentially binds monomeric ADP-actin over ATP-bound actin: consequences for cell locomotion. FEBS (Fed. Eur. Biochem. Soc.) Lett. 347: 251-256 . |
34. | Maciver, S.K., H.G. Zot, and T.D. Pollard. 1991. Characterization of actin filament severing by actophorin from Acanthamoeba castellanii. J. Cell Biol. 115: 1611-1620 [Abstract]. |
35. |
McGough, A.,
M. Way, and
D. DeRosier.
1994.
Determination of the ![]() |
36. | McKim, K., C. Matheson, M. Marra, M. Wakarchuk, and D. Baillie. 1994. The Caneorhabditis elegans unc-60 gene encodes proteins homologous to a family of actin-binding proteins. Mol. Gen. Genet. 242: 346-357 |
37. | Milligan, R.A., and P.F. Flicker. 1987. Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy. J. Cell Biol. 105: 29-39 [Abstract]. |
38. | Moon, A., and D. Drubin. 1995. The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics. Mol. Biol. Cell. 6: 1423-1431 |
39. | Moon, A.L., P.A. Janmey, K.A. Louie, and D. Drubin. 1993. Cofilin is an essential component of the yeast cortical cytoskeleton. J. Cell Biol. 120: 421-435 [Abstract]. |
40. | Moriyama, K., E. Nishida, N. Yonezawa, H. Sakai, S. Matsumoto, E. Nishida, K. Iida, N. Yonezawa, S. Koyasu, I. Yahara, et al . 1987. Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells. Proc. Natl. Acad. Sci. USA. 84: 5262-5266 [Abstract]. |
41. |
Moriyama, K.,
N. Yonezawa,
H. Sakai,
I. Yahara, and
E. Nishida.
1992.
Mutational analysis of chimeric proteins between cofilin and destrin.
J. Biol.
Chem.
267:
7240-7244
|
42. | Nishida, E., S. Maekawa, and H. Sakai. 1984. Cofilin, a protein in porcine brain that binds to actin filaments and inhibits their interactions with myosin and tropomyosin. Biochemistry. 23: 5307-5313 |
43. | Orlova, A., and E.H. Egelman. 1992. Structural basis for the destabilization of F-actin by phosphate release following ATP hydrolysis. J. Mol. Biol. 227: 1043-1053 |
44. | Orlova, A., and E.H. Egelman. 1993. A conformational change in the actin subunit can change the flexibility of the actin filament. J. Mol. Biol. 232: 334-341 |
45. | Orlova, A., X. Yu, and E.H. Egelman. 1994. Three-dimensional reconstruction of a complex of F-actin with antibody Fab fragments to actin's amino terminus. Biophys. J. 66: 276-285 [Abstract]. |
46. | Owen, C., and D.J. DeRosier. 1993. A 13 Å map of the actin-scruin filament from the Limulus acrosomal process. J. Cell Biol. 123: 337-344 [Abstract]. |
47. | Pollard, T.D.. 1986. Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J. Cell Biol. 103: 2747-2754 [Abstract]. |
48. | Rayment, I., H.M. Holden, M. Whittaker, M. Yohn, M. Lorenz, K.C. Holmes, and R.A. Milligan. 1993. Structure of the actin-myosin complex and its implications for muscle contraction. Science (Wash. DC). 261: 58-65 |
49. |
Rosenblatt, J.,
B.J. Agnew,
H. Abe,
J.R. Bamburg, and
T.J. Mitchison.
1997.
Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the
turnover of actin filaments in Listeria monocytogenes tails.
J. Cell Biol.
136:
1323-1332
|
50. | Schroder, R.R., D.J. Manstein, W. Jahn, H. Holden, I. Rayment, K.C. Holmes, and J.A. Spudich. 1993. Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1. Nature (Lond.). 364: 171-174 |
51. | Schroeter, J.P., and J.-P. Bretaudiere. 1996. SUPRIM: easily modified image processing software. J. Struct. Biol. 116: 131-137 |
52. | Small, J.V.. 1995. Getting the actin filaments straight: nucleation-release or treadmilling. Trends Cell Biol. 5: 52-55 . |
53. | Stewart, M.. 1988. Computer image processing of electron micrographs of biological structures with helical symmetry. J. Electron Micros. Tech. 9: 325-358 |
54. | Stokes, D.L., and D.J. DeRosier. 1987. The variable twist of actin and its modulation by actin-binding proteins. J. Cell Biol. 109: 1005-1017 . |
55. |
Theriot, J.A..
1997.
Accelerating on a treadmill: ADF/cofilin promotes rapid actin filament turnover in the dynamic cytoskeleton.
J. Cell Biol.
136:
1165-1168
|
56. | Tirion, M.M., D. ben-Avraham, M. Lorenz, and K.C. Holmes. 1995. Normal modes as refinement parameters for the F-actin model. Biophys. J. 68:5-12. |
57. | Trachtenberg, S., D. Stokes, E. Bullitt, and D. DeRosier. 1986. Actin and flagellar filaments: two helical structures with variable twist. Ann. N.Y. Acad. Sci. 483: 89-99 . |
58. |
Tsuda, Y.,
H. Yasutake,
A. Ishijima, and
T. Yanagida.
1996.
Torsional rigidity
of single actin filaments and actin-actin bond breaking force under torsion
measured directly by in vitro micromanipulation.
Proc. Natl. Acad. Sci. USA.
93:
12937-12942
|
59. | Vibert, P., R. Craig, and W. Lehman. 1993. Three-dimensional reconstruction of caldesmon-containing smooth muscle filaments. J. Cell Biol. 123: 313-321 [Abstract]. |
60. | Welch, M., A. Mallavarapu, J. Rosenblatt, and T. Mitchison. 1997. Actin dynamics in vivo. Curr. Opin. Cell Biol. 9: 54-61 |
61. | Whittaker, M., E.M. Wilson-Kubalek, J.E. Smith, L. Faust, R.A. Milligan, and H.L. Sweeney. 1995a. A 35-Å movement of smooth muscle myosin on ADP release. Nature (Lond.). 378: 748-751 |
62. | Whittaker, M., B.O. Carragher, and R.A. Milligan. 1995b. PHOELIX: a package for automated helical reconstruction. Ultramicroscopy. 58: 245-250 |
63. |
Yonezawa, N.,
E. Nishida, and
H. Sakai.
1985.
pH control of actin polymerization by cofilin.
J. Biol. Chem.
260:
14410-14412
|
64. |
Yonezawa, N.,
E. Nishida,
K. Iida,
I. Yahara, and
H. Sakai.
1990.
Inhibition of
the interactions of cofilin, destrin, and deoxyribonuclease-i with actin by
phosphoinositides.
J. Biol. Chem.
265:
8382-8386
|
65. | Yonezawa, N., E. Nishida, S. Maekawa, and H. Sakai. 1988. Studies on the interaction between actin and cofilin purified by a new method. Biochem. J. 251: 121-127 |
66. |
Yonezawa, N.,
E. Nishida,
K. Iida,
H. Kumagai,
I. Yahara, and
H. Sakai.
1991.
Inhibition of actin polymerization by a synthetic dodecapeptide patterned
on the sequence around the actin-binding site of cofilin.
J. Biol. Chem.
266:
10485-10489
|
67. | Zhou, Z.H., S. Hardt, B. Wang, M.B. Sherman, J. Jakana, and W. Chiu. 1996. CTF determination of images of ice-embedded single particles using a graphics interface. J. Struct. Biol. 116: 216-223 |
68. | Zigmond, S.H.. 1993. Recent quantitative studies of actin filament turnover during cell locomotion. Cell Motil. Cytoskeleton. 25: 309-316 |