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Xenopus actin-interacting protein 1 (XAip1) enhances
cofilin fragmentation of filaments by capping filament
ends
Kyoko Okada, Laurent Blanchoin, Hiroshi Abe, Hui Chen, Thomas D.
Pollard, James R. Bamburg
To cite this version:
Kyoko Okada, Laurent Blanchoin, Hiroshi Abe, Hui Chen, Thomas D. Pollard, et al.. Xenopus
actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends.
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2002, 277
(45), pp.43011-43016. �10.1074/jbc.M203111200�. �hal-02675680�
Xenopus Actin-interacting Protein 1 (XAip1) Enhances Cofilin
Fragmentation of Filaments by Capping Filament Ends*
Received for publication, April 1, 2002, and in revised form, June 2, 2002 Published, JBC Papers in Press, June 7, 2002, DOI 10.1074/jbc.M203111200 Kyoko Okada‡§, Laurent Blanchoin¶储, Hiroshi Abe§**, Hui Chen‡ ‡‡, Thomas D. Pollard¶§§,
and James R. Bamburgদ
From the ‡Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523, the §Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan, the¶Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, and the **Department of Cell Biology, Natural Institute for Basic Biology, Okazaki 444, Japan
Xenopus actin-interacting protein 1 (XAip1) is thought
to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured poly-mer lengths by fluorescence microscopy and the concen-tration of filament ends with an elongation assay. Cofi-lin creates ends by severing actin filaments. XAip1 alone does not sever actin filaments or prevent annealing/ redistribution of mechanically severed filaments and has no effect on the concentration of ends available for subunit addition. In the presence of XAip1, the apparent filament fragmentation by cofilin is enhanced, but XAip1 reduces rather than increases the concentration of ends capable of adding subunits. Electron microscopy with gold-labeled antibodies showed that a low concen-tration of XAip1 bound preferentially to one end of the filament. A high concentration of XAip1 bound along the length of the filament. In the presence of gelsolin-actin to cap filament barbed ends, XAip1 does not enhance cofilin activity. We conclude that XAip1 caps the barbed end of filaments severed by cofilin. This capping blocks annealing and depolymerization and allows more exten-sive severing by cofilin.
Actin dynamics underlie various cellular activities including cell motility, cytokinesis, endocytosis, and translocation of some intracellular organelles. Spatial and temporal regulation of actin assembly and disassembly as well as three-dimensional filament organization are required for these phenomena. Numerous actin-binding proteins coordinate these processes. Analysis of their mechanisms is required to understand com-plex processes such as actin assembly and filament turnover
during extension of lamellipodia at the leading edge of migrat-ing cells and the movement of intracellular bacterial pathogens (reviewed in Refs. 1 and 2). In vitro reconstitution of the actin-based motility of pathogenic bacteria demonstrated that five essential factors were required for sustained motility: actin, Arp2/3 complex activated by a bacterial surface protein or N-WASP, barbed end heterodimeric capping protein, and an
ADF1/cofilin protein (3). Motility was enhanced by adding
pro-filin, ␣-actinin, and VASP. Detailed analyses demonstrated
how interactions of these proteins affect the magnitude of the oriented polymerization and the stability of the actin filament network (4, 5).
Proteins of the ADF/cofilin family are ubiquitously expressed and highly conserved in eukaryotes (reviewed in Refs. 6 and 7). ADF/cofilins accelerate actin filament turnover either by sev-ering (8, 9) and/or promoting subunit dissociation from
fila-ment ends (10). ADF/cofilins also enhance Pi release from
ADP-Piactin filaments, which leads to debranching of actin
filament networks (5). Severing activity of ADF/cofilin proteins also contributes to the formation of new actin filament barbed ends in lamellipodia (11, 12). Although they have little effect on the concentration of polymerized actin, the combination of pro-filin and copro-filin increases the rate of subunit flux from polymer to monomer pool and back to filaments (8, 13). Phosphorylation inhibits the ability of vertebrate, protozoan and plant ADF/ cofilins (see Ref. 6 for review) to bind actin monomers and filaments (14 –17). Rapid cycling of phosphorylation and de-phosphorylation is another mechanism proposed to augment ADF/cofilins to release actin subunits and recycle for further filament disassembly (18) (reviewed in Ref. 19).
Actin-interacting protein 1 (Aip1) was first identified as a novel actin-binding protein using the yeast two-hybrid system (20). Aip1 is conserved in a variety of organisms including
Schizosaccharomyces pombe, Physarum polycephalum (21), Dictyostelium discoideum (22, 23), Caenorhabditis elegans (24), Xenopus laevis (25), and birds and mammals (26). Little Aip1
binds to pure actin filaments, whereas added cofilin creates more binding sites (22, 25, 27). Thus, Aip1 is an unusual, cofilin-dependent actin-binding protein. Two-hybrid analysis showed Aip1 interacts with both actin (20) and cofilin (27).
In vivo Aip1 is a multicopy suppressor of a cofilin
tempera-ture-sensitive mutant (28). An Aip1 null mutant of
Dictyoste-lium had prolonged cytokinesis, delayed phagocytosis and
macropinocytosis, and inhibited motility, although cofilin
* This work was supported by grants from the National Institutes of Health (GM35126 and GM54004 to J. R. B. and GM26338 to T. D. P.), the Japanese Ministry of Science (to H. A.), and a Japanese Society for Promotion of Science (JSPS) Research Fellowship for Young Scientists (to K. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
储Present address: Laboratoire de Physiologie Cellulaire Ve´ge´tale, Batiment C2, piece 435 De´partement de Re´ponse et Dynamique Cellu-laires, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France.
‡‡ Present address: Dept. of Biological Chemistry, The Johns Hop-kins University School of Medicine, Baltimore, MD 21205.
§§ Present address: Dept. of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520.
¶¶To whom correspondence should be addressed: Dept. of Biochem-istry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870. Tel.: 970-491-6096; Fax: 970-491-0494; E-mail: jbamburg@lamar.colostate.edu.
1The abbreviations used are: ADF, actin-depolymerizing factor; Aip1, actin-interacting protein 1; Xaip1, Xenopus Aip1; DBP, vitamin D-binding protein; DTT, dithiothreitol; XAC, Xenopus ADF/cofilin; PIPES, 1,4-piperazinediethanesulfonic acid.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org
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distribution was normal (23). Microinjection of excess Aip1 into
Xenopus embryos disturbed the cortical localization of both
actin and cofilin, suggesting that Aip1 is an activator of ADF/ cofilin, especially in cells where phosphorylation does not reg-ulate ADF/cofilin. In multicellular organisms expressing mul-tiple ADF/cofilins, Aip1 might cooperate with certain isoforms of ADF/cofilin that have weaker severing/depolymerizing activ-ities (29).
We used fluorescence microscopy to measure polymer lengths and an actin filament elongation assay to measure the concentration of polymer ends produced by treating filaments with XAip1 and cofilin. We found that Xenopus Aip1 (XAip1) promotes actin filament severing by cofilin but inhibits both elongation and depolymerization of the new barbed ends. Elec-tron micrographs of samples treated with gold-labeled antibod-ies revealed that XAip1 binds preferentially to one end of cofilin-actin filaments. These results suggest that XAip1 caps filaments severed by cofilin, preventing their annealing and elongation.
MATERIALS AND METHODS
Proteins—XAip1 was purified from Xenopus ovary as described (25)
with slight modification. Stepwise ammonium sulfate cuts at 45, 60, and 80% saturation were used. XAip1 is found in the 80% saturated ammonium sulfate precipitate. Recombinant chicken cofilin (30),
Xeno-pus ADF/cofilin (XAC) (31), gelsolin (32), and gelsolin-actin (1:1)
com-plex (33) were purified as described. Actin was purified from rabbit skeletal muscle acetone powder (34) and monomeric Ca-ATP-actin was isolated by Sephacryl S-300 chromatography (35) and labeled on Cys-374 with pyrene iodoacetamide (36, 37). Actin was polymerized by adding 1:9 (v/v) 10⫻ KME buffer (500 mMKCl, 10 mMMgCl2, 10 mM
EGTA, and 100 mMPIPES, pH 6.8, or 100 mMTris-HCl, pH 8.0).
Vitamin D-binding protein (DBP), also called Gc-globulin, was from Calbiochem-Novabiochem. Drs. James Casella and Susan Craig of Johns Hopkins Medical School kindly provided spectrin-actin-protein 4.1 complex.
Sedimentation Experiments—Effects of XAip1 and/or cofilin on the
concentration of polymerized actin were obtained by sedimentation of actin filaments at 436,000 ⫻ g for 20 min in a TLA100 rotor in a Beckman TL100 centrifuge. The total supernatant was removed, and the pellet was resuspended in SDS-containing buffer (0.25MTris, pH 6.8, 10% glycerol, 10% 2-mercaptoethanol and 1% SDS). An aliquot of the supernatant was prepared in the same buffer. Fractions from supernatant and pellet were subjected to SDS-PAGE (15% T, 2.67% C polyacrylamide gels), and amounts of the proteins in each fraction were quantified by densitometry following staining with Coomassie Blue R-250 or chemifluorescence detection following staining with Sypro-orange. In some experiments with XAip1 alone, filaments were pre-pared in the presence of ADP as well as ATP and were mechanically sheared by 50 passages through a yellow pipette tip.
Actin Filament Elongation Experiments—Actin filaments (2 M) were incubated in 1⫻ KME buffer with 2Mcofilin and various
con-centrations of XAip1 at room temperature for 20 min. The mixtures were diluted 3-fold in 2Mactin monomers (10% pyrenyl-labeled) and
KME buffer to initiate polymerization. Polymerization of actin was measured as the fluorescence change (excitation at 366 nm and emis-sion at 387 nm) with an Alphascan spectrofluorometer (Photon Tech-nology International, South Brunswick, NJ). Under these conditions, the rate of elongation from the added filaments is much faster than polymerization from spontaneously formed nuclei.
Concentrations of actin filaments were calculated from the initial rate of polymerization (V0) according to Equation 1,
V0⫽ k⫹关A兴 关N兴 ⫺ k⫺关N兴 (Eq. 1) where k⫹is the barbed end association rate constant for ATP-actin (11.4M⫺1s⫺1), [A] is the concentration of actin monomer, [N] is the concentration of free barbed ends and k⫺is the barbed end dissocia-tion rate constant for ATP-actin (1.4 s⫺1) (38). Except where we capped the barbed ends with gelsolin, we disregarded the pointed end elongation rate, because it contributes little to elongation under these conditions.
Actin Filament Depolymerization Experiments—The effect of XAip1
on filament depolymerization was measured in the presence of XAC according to the method of Moriyama and Yahara (33). Briefly,
recom-binant gelsolin at concentrations from 4 to 18 nMwas used to nucleate assembly of 3.3Mactin (5% pyrene-labeled) in buffer F (100 mMKCl, 2 mMMgCl2, 0.2 mMATP, 0.2 mMEGTA, 0.5 mMDTT, and 10 mMTris at pH 7.8). When the assembly reached steady state (at about 4 h), 0.12 MXAC (with or without 60 nMXAip1) and 150 nMgelsolin-actin (1:1) complex (to cap free barbed ends) were added to the actin and allowed to incubate 10 min. In control experiments the XAC was omitted. Changes in fluorescence intensity were monitored at 18 °C with an AVIV ATF 105 spectrofluorometer with excitation at 365 nm and emis-sion at 404 nm. DBP, a potent actin monomer-sequestering protein (KD ⫽ 1 nM(39)), was added in excess to sequester monomers and thus induce depolymerization from pointed ends of the filaments by main-taining the actin concentration below the critical concentration. Initial depolymerization rates were calculated from the fit of the data to the exponential rate equation (Vi⫽ k[N]), where N equals the number of free pointed ends. In control experiments to demonstrate complete capping by the gelsolin-actin (1:1) complex, filaments were nucleated from spectrin-actin-protein 4.1 seeds and were capped with gelsolin-actin prior to treatment with DBP.
Fluorescence Microscopy of Actin Filaments—Actin filaments were
labeled with 2Mrhodamine-phalloidin for 2 min at room temperature, diluted 50-fold with 1⫻ KME buffer, and then diluted into fluorescence buffer (50 mMKCl, 1 mMMgCl2, 0.1MDTT, 20g/ml catalase, 100 g/ml glucose oxidase, 3 mg/ml glucose, 0.5% methyl cellulose, 10 mM
imidazole, pH 7.0). An aliquot (2l) of the sample was applied to a nitrocellulose-coated coverslip, made by applying a 0.1% solution of nitrocellulose in amylacetate. Fluorescence images were observed with an Olympus IX-70 microscope using a mercury illumination source. Images were captured with a Hamamatsu digital camera. Filament lengths were measured manually with IPLab software.
Immuno-electron Microscopy—Protein solutions were placed on
carbon-coated Formvar grids, fixed with 2% formaldehyde and 0.1% glutaraldehyde in 60 mMKCl, 2 mMMgCl2, 1 mMDTT, and 50 mM
PIPES, pH 6.8, for 30 min, and washed with the same buffer without formaldehyde. The grids were treated with phosphate-buffered saline containing 1% bovine serum albumin for 1 h, incubated with anti-XAip1 monoclonal antibody, and treated with 10 nm gold-conjugated goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA) in 1% bovine serum albumin/phosphate-buffered saline. The antibody incubations were performed for 1 h at room temperature followed by washing thoroughly with phosphate-buffered saline. The specimens were negatively stained with 1.5% uranyl acetate and observed with a JOEL JEM 100CX electron microscope at an accelerating voltage of 80 kV.
RESULTS
Effects of Cofilin and XAip1 on Actin Filament Length
Treatment of 2 M polymerized actin with 2M cofilin
re-duced the mean length of the filaments from 5.39 (n⫽ 354) to
1.68m (n ⫽ 93) at pH 6.8 and to 2.19 m (n ⫽ 158) at pH 8.0
(Fig. 1). Treatment of actin filaments with XAip1 alone had no
effect on polymer length (mean ⫽ 5.38 m, n ⫽ 160), but
treatment with cofilin and XAip1 reduced the filament lengths beyond the effect of cofilin alone (Fig. 1A, c and d).
Effects of Cofilin and XAip1 on the Concentration of Poly-merized Actin—Samples of F-actin at pH 6.8 or 8.0 were
treated with equal molar cofilin and different concentrations of XAip1. After high-speed centrifugation to separate out the filamentous actin, actin in the supernatant and pellet frac-tions was quantified by SDS-PAGE. At pH 6.8, the combina-tion of the two proteins had no effect on the concentracombina-tion of actin that pelleted by ultracentrifugation (Fig. 2). At pH 8.0, cofilin plus XAip1 reduced the pelleted polymer concentra-tion only slightly. The difficulty of pelleting short filaments was strong evidence that the polymer concentration did not change. Both with and without cofilin, the concentration of
actin in the supernatant was about 0.15 M, close to the
critical concentration under these conditions (0.10M).
Effects of XAip1 on the Ability of Cofilin-fragmented Actin to Nucleate Actin Assembly—When used as seeds for elongation
by actin monomers, the cofilin-treated filaments elongated 2.5-fold faster than untreated actin filaments (Fig. 3A). Because the polymerization rate is proportional to the concentration of
Aip1 and Cofilin Sever and Cap Actin Filaments
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barbed ends, treatment with cofilin increased the concentra-tion of ends 2.5-fold, in reasonable agreement with the 3.1-fold reduction in length at constant actin polymer concentration. At
pH 8.0 cofilin increased the initial elongation rate⬃4-fold and
reduced the length 2.6-fold. The elongation rate is more likely than microscopy to give an accurate measurement of polymer ends in samples with cofilin, because short filaments are more difficult to detect than long ones. These observations confirm the severing activity of cofilin observed previously (reviewed in Refs. 6 and 40).
Treatment of actin filaments with XAip1 had no effect on the rate of elongation when the filaments were used as seeds at either pH 6.8 (Fig. 3A, h) or 8.0 (data not shown). Combined with our observation above that XAip1 had no effect on polymer length, these results confirm that XAip1 alone neither severs actin filaments nor caps their barbed ends.
Although treatment of filaments with cofilin plus XAip1 re-duced their lengths beyond the effect of cofilin alone (see Fig. 1), paradoxically the combination of cofilin and XAip1 also reduced the concentration of barbed ends capable of elongation
(Fig. 3). At pH 6.8 with 2M cofilin, mean filament lengths
were 0.6m (n ⫽ 241) with 20 nMXAip1 and 0.53m (n ⫽ 483)
with 200 nM XAip1. The concentration of ends produced by
cofilin depended on the concentration of XAip1 (Fig. 3). At pH
6.8 with 2MXAC, the minimum concentration of ends reached
FIG. 3. Effect of XAip1 and cofilin on elongation of actin
fila-ment seeds. A and B, time course of actin polymerization from
preex-isting filament ends. Conditions: 2Mpolymerized actin was mixed with 2Mrecombinant chicken cofilin and a range of concentrations of
XAip1 either at pH 6.8 (A) or 8.0 (B) and incubated at room temperature for 20 min. These filaments were diluted 3-fold into 2 M pyrenyl Mg-ATP-actin monomers with polymerization buffer to initiate elonga-tion. In A, XAip1 concentrations were: a, 0; b, 5 nM; c, 10 nM; d, 20 nM;
e, 50 nM; f, 100 nM; g, 200 nM; h, actin filaments plus 200 nMXAip1; i, actin filaments alone. In B, XAip1 concentrations were: a, 0 nM; b, 10 nM; c, 20 nM; d, 50 nM; e, actin filaments alone; f, actin filaments with cofilin 100 nMXAip1; g, actin filaments with cofilin and 200 nMXAip1.
C and D, dependence of filament length on the concentration of XAip1.
Concentration of filament ends is calculated from the elongation rate using Equation 1 (open circles). Filament lengths are calculated from the filament end concentration (open squares). Average filament length is measured by fluorescence microscopy from Fig. 1, A and B (filled
squares).
FIG. 1. Effects of cofilin and XAip1 on the length of actin
fila-ments observed by fluorescence microscopy after labeling with rhodamine-phalloidin. Samples of 2Mpolymerized actin⫾ 2M
recombinant chicken cofilin⫾ 20 or 200 nMXAip1 were incubated at
room temperature for 20 min in 50 mMKCl, 1 mMMgCl2, 1 mMEGTA, and 10 mMbuffer and then labeled with 2Mrhodamine-phalloidin for 2 min at room temperature. They were diluted 625-fold and viewed by fluorescence microscopy. A, at pH 6.8: a, actin filaments alone; b, actin with 200 nMXAip1; c, actin with cofilin; d, actin with cofilin and 20 nM
XAip1; e, actin with cofilin and 200 nMXAip1. B, at pH 8.0: a, actin with 200 nMXAip1; b, actin with cofilin; c, actin with cofilin and 200 nM
XAip1. C (pH 6.8) and D (pH 8.0), length distribution plots showing the percentage of filaments longer than X versus length X. Filled diamonds, actin alone; filled squares, actin with 200 nMXAip1; filled triangles, actin with cofilin; open circles, actin with cofilin and 20 nMXAip1; filled
circles, actin with cofilin and 200 nMXAip1.
FIG. 2. Effect of cofilin and XAip1 on the concentration of
polymerized actin. Samples of 2Mactin filaments in 50 mMKCl, 1 mMMgCl2, 1 mMEGTA, and 10 mMbuffer were treated with 2M
recombinant chicken cofilin and a range of concentrations of XAip1 and then centrifuged at 436,000⫻ g for 20 min to pellet actin filaments. Proteins in the supernatant (s) and pellet (p) were subjected to SDS-PAGE and stained with Sypro-orange. Upper panel (pH 6.8) and lower
panel (pH 8.0), from left to right in the presence of 0, 10, 20, 50, 100, 200
nMXAip1. The graph shows the concentrations of pelleted actin at pH 6.8 (filled circles) and pH 8.0 (open squares) over the range of XAip1 concentrations.
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a plateau at concentrations of XAip1 in the range of 100 nM, far
below the concentration of 2Mpolymerized actin (Fig. 3C). At
pH 8.0 XAip1 and cofilin reduced the concentration of ends even further, below that of untreated actin filaments (Fig. 3D). XAip1 and tissue-derived XAC gave similar results at pH 6.8. Given that the filaments were shorter but had fewer ends capable of elongation, the combination of cofilin and XAip1 must cap most barbed ends.
XAip1 Does Not Enhance F-actin Depolymerization by Cofilin
We measured the effect of cofilin in the presence and absence of XAip1 on the rate of depolymerization from filament pointed ends at pH 7.8 using DBP to sequester actin monomers as they dissociated spontaneously from polymer ends. Filaments with free barbed ends (grown from spectrin-actin-protein 4.1 seeds (41)) depolymerize rapidly upon addition of DBP but do not depolymerize when treated prior to addition of DBP with solin-actin (1:1) complex (Fig. 4A), demonstrating that the gel-solin-actin complex effectively caps the filament barbed ends. Actin filaments nucleated with different amounts of gelsolin depolymerized slowly in the presence of DBP, but the rate was
greatly enhanced by the addition of 0.12MXAC (Fig. 4B). The
addition of 60 nMXAip1 to the XAC did not alter the
depoly-merization kinetics, indicating that it did not increase the severing activity of XAC to generate more free pointed ends, nor did it alter the off-rates at the pointed ends already present.
Localization of XAip1 on Actin Filaments—We used
im-muno-electron microscopy to visualize XAip1 binding sites on
cofilin-decorated actin filaments. At 120 nMXAip1 was
local-ized with a higher frequency at one end than along the side of actin filaments (Fig. 5A). Gold beads were observed at one end of 82% of filaments, at both ends of 0% of filaments, and along
the sides of 38% of filaments (n ⫽ 133). At 960 nM XAip1
gold-labeled antibodies bound along the length of most fila-ments (Fig. 5B).
XAip1 Alone Does Not Inhibit Annealing/Redistribution of Mechanically Sheared F-actin—To determine whether XAip1
alone was able to prevent annealing/redistribution of F-actin,
we subjected samples of 5MF-actin in the presence or absence
of 1 M XAip1 to mechanical shear (50 passages through a
yellow pipette tip) and sedimented the sample immediately at
436,000⫻ g for 20 min or allowed it to recover for 40 min before
centrifugation. Supernatant and pellet fractions were then an-alyzed by SDS-PAGE (Fig. 6). Filaments were slow to anneal after shearing in the presence of ADP (compare lanes 11 and 12 with 15 and 16 in Fig. 6), as there was a larger fraction of actin in the supernatant of these samples. But even in the presence
FIG. 6. XAip1 alone does not bind F-actin nor does it prevent
the annealing/redistribution of mechanically sheared actin fil-aments. F-actin (5M), alone or in the presence of 1MXAip1, and in
the presence of either ATP or ADP (1 mM), was mechanically sheared by 50 passages through a yellow tip. Samples were centrifuged immedi-ately (lanes 13–16) or allowed to stand for 40 min before sedimentation at 436,000⫻ g for 20 min, and the proteins in the supernatant and pellet fractions were analyzed by SDS-PAGE. The amount of XAip1 in the pellet fraction was the same for all treatments. Filament annealing/ redistribution was rapid in ATP such that samples centrifuged imme-diately after shearing (lanes 14 and 15) showed no increase in super-natant actin over samples allowed to recover (lanes 5– 6 and 9 –10). Samples sheared in ADP and centrifuged immediately (lanes 15 and 16) showed a significant fraction (about 30%) of the actin in the superna-tant, whereas after 40 min recovery (lanes 7– 8 and 11–12), the actin distribution was the same as for the ATP samples. The presence of XAip1 did not prevent full recovery of the actin distribution as found in the control (lanes 1 and 2).
FIG. 4. XAip1 does not enhance severing or depolymerization
of XAC-treated F-actin when barbed ends are capped with gel-solin-actin. A, filaments were assembled in 100 mMKCl, 2 mMMgCl2, 0.2 mMEGTA, and 10 mMbuffer, pH 7.8, from 3.3M(5% pyrenyl) actin initiated by 60 nMspectrin-actin seeds (to block pointed ends) or 21 nM
gelsolin (to block barbed ends). Both produced 1.8 nM filaments (as determined by assembly rate assays to quantify filament ends). One sample of actin assembled from spectrin-actin seeds was treated with 200 nMgel-filtered gelsolin-actin (1:1) complex (33). These filaments were treated with 3.5MDBP to bind monomers and initiate depoly-merization, and the time course of the pyrene fluorescence was re-corded. a, filaments seeded by spectrin-actin and treated with gel-filtered 200 nM gelsolin-actin (both ends blocked); b, filaments nucleated from gelsolin (21 nM) with 200 nM gelsolin-actin added (barbed ends blocked, depolymerization from pointed ends); c, no treat-ment (pointed ends blocked, depolymerization from barbed ends). The
inset table shows the calculated depolymerization rates in fluorescence
units/min for the three curves. GA, gelsolin-actin. B, filaments were assembled as in A using different concentrations of gelsolin to nucleate assembly. After assembly was complete (about 4 h), filaments were incubated with 150 nMgelsolin-actin (1:1) complex alone (triangles), with the complex along with 0.12MXAC (diamonds), or with 0.12 mM
XAC plus 60 nMXAip1 (squares). After 10 min, 3.5 mMDBP was added, and the depolymerization was followed as described in A. Initial depo-lymerization rates were measured by a computer fit to the exponential fluorescence decrease; these rates were plotted versus the nucleating gelsolin concentration, which is assumed to be proportional to the initial filament concentration (33). XAip1 did not enhance the depoly-merizing activity of XAC when filament barbed ends were capped.
FIG. 5. Localization of XAip1 binding sites on actin filaments
by electron microscopy of negatively stained specimens. The
XAip1 monoclonal antibody was localized with a secondary anti-body conjugated to 10 nm gold. A, 4.7Mactin was polymerized with 5.2
Mrecombinant chicken cofilin and then incubated with 120 nMXAip1 for 5 min. B, 4.8Mactin was polymerized with equimolar XAC and then incubated with 960 nMXAip1 for 15 min. Bar in A⫽ 0.2m; bar in B⫽ 0.1m.
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of 1MXAip1, filament distribution between supernatant and pellet fractions recovered completely after 40 min.
DISCUSSION
The paradoxical effects of cofilin plus XAip1 in reducing both the length and number of filament ends capable of elongation under conditions in which polymer mass is constant is ex-plained most simply by these proteins capping many of the barbed ends produced by severing. We conclude from the strong inhibition of elongation and the inhibition of depolymerization that barbed ends are capped, because capping pointed ends would have a minimal effect on either the elongation or de-polymerization rates. Capping by XAip1 appears to depend on cofilin in two ways. First, severing by cofilin creates ends for capping by XAip1. Second, capping itself may also depend on the presence of cofilin, as we detected no capping of pure actin filaments by adding XAip1 at either pH 6.8 or 8.0. Thus, the observed capping was not due to contamination with animal capping protein or gelsolin, both of which cap F-actin alone.
Inhibition of annealing secondary to capping may explain why XAip1 reduces the length of fragments produced by cofilin. Other barbed end capping proteins inhibit not only elongation but also annealing of the fragments produced by severing or fragmentation. The steady state polymer length in the presence of ADF/cofilin depends on the balance between ongoing sever-ing and annealsever-ing (8, 9), and therefore inhibition of annealsever-ing with ongoing severing would produce shorter filaments without the necessity of invoking an increased rate of severing.
Many details remain to be investigated. For example, we do not yet know the affinity of XAip1 for barbed ends or the effectiveness of the capping. The XAip1 concentration depend-ence of the reduction in free ends (Fig. 3, C and D) suggests that
the affinity is high, with a Kd in the 25 nM range, but this
experiment is not a definitive method to measure affinity. The residual elongation of filaments saturated with XAip1 may be due to pointed end growth or to incomplete capping of barbed ends. Incomplete capping would explain why the combination of cofilin and XAip1 does not reduce the polymer concentration. Complete capping of barbed ends is required to increase the
critical concentration of Mg-ATP actin from 0.1 to 0.7M.
The pioneering work on Aip1 (22, 25, 27) concluded that Aip1 enhances the disassembly of actin filaments by cofilin. How-ever, these conclusions were based upon data from sedimenta-tion experiments in which it is difficult to separate short fila-ments from monomers. Indeed, Okada et al. (25) did examine the nonsedimentable pool of actin monomer using the DNase I inhibition assay and found no increase in G-actin when XAip1 was added to cofilin-actin mixtures. In retrospect all of the published data are consistent with severing and capping, which can reduce light scattering and pelleting of short actin fila-ments. These papers also concluded that Aip1 has a low affinity for actin filaments but could not distinguish between low af-finity and a low concentration of binding sites, as would occur if the high affinity binding site were at the end of a filament. In fact, the published data are consistent with high affinity bind-ing to a limited number of sites. For example, Rodal et al. (27)
detected no binding of 12–500 nMyeast Aip1 to 3.75Mactin
filaments (about 2 nMends, assuming a mean polymer length of
5m) but did detect binding of a low (but unspecified)
conctration in radioactive in vitro translated Aip1, which was en-hanced by cofilin. Okada et al. (25) demonstrated additional lower affinity binding sites by carrying out sedimentation
bind-ing studies to XAip1 concentrations of ⬎6 M, which likely
represent the lower affinity lateral binding observed here by electron microscopy.
The exchange of nucleotide at the barbed end of actin fila-ments is very rapid (42). When ADF/cofilins bind to G-actin
they inhibit nucleotide exchange (43– 45) and would likely do so as well at the barbed ends of filaments. Thus the barbed ends of unsevered filaments will consist of ATP-actin, whereas the barbed ends of ADF/cofilin severed filaments will consist of ADP-actin. Nucleotide exchange at these ends will be slow. Thus, our results suggest that XAip1 may have a specific af-finity for the ADF/cofilin-ADP-actin complex at the barbed end of newly severed filaments and little or no affinity for the ATP-actin barbed ends of unsevered filaments or mechanically severed filaments in which rapid nucleotide exchange at the barbed end will occur.
In vivo studies on Aip1 point to its role in regulating actin
dynamics and remodeling. Aip1 (Unc78) mutants in C. elegans show abnormal accumulation of actin aggregates in muscle (24), whereas in Aip1 null yeast, cofilin localizes abnormally to actin cables, which turn over inefficiently (27). Aip1 null mu-tants of Dictyostelium were defective in cytokinesis, phagocy-tosis, and motility (23). Overexpression of Aip1 in
Dictyoste-lium inhibited phagocytosis similarly to that of the actin
monomer binding compound, latrunculin A, suggesting that Aip1 increased the monomer pool (23). An increase in the disassembly of actin upon microinjection of Aip1 into Xenopus oocytes was measured directly (25). All of these effects can be explained by the in vitro activity of XAip1 reported here. Block-ing the annealBlock-ing and barbed end growth of ADF/cofilin frag-mented filaments will enhance the number of pointed ends from which ADF/cofilins can depolymerize actin. It is not yet known how XAip1 affects the ability of ADF/cofilins to depoly-merize the F-actin from the pointed end. However, the fact that XAip1 caps barbed filament ends and inhibits elongation may shift the state of cofilin-decorated actin filaments to disassem-bly, thus amplifying the ADF/cofilin function of increasing actin monomers that can be recycled rapidly for localized actin polymerization.
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Aip1 and Cofilin Sever and Cap Actin Filaments
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