Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202
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Abstract |
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Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.
Key words: actin; cytoskeleton; budding yeast; twinfilin; cofilin ![]() |
Introduction |
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THE actin cytoskeleton plays an essential role in multiple cellular processes, including polarized cell
growth, cell motility, cytokinesis, secretion, and endocytosis. All of these processes rely on the capacity of the
actin cytoskeleton to respond to cellular signals and reorganize spatially and temporally into a variety of specific
structures. To regulate the organization and turnover of
the actin cytoskeleton, a number of actin filament- and actin monomer-binding proteins have evolved in eukaryotic
organisms (for recent review, see Carlier and Pantaloni,
1997). Most families of actin-binding proteins are highly
conserved throughout evolution and are found in organisms as diverse as humans and yeast. This suggests that
these proteins existed previously in a common eukaryotic ancestor and that the basic mechanisms for the regulation
of the actin cytoskeleton dynamics are conserved among
diverse organisms and cell types.
Several different families of actin-binding proteins can
directly regulate the nucleation and elongation of actin filaments. The evolutionarily conserved Arp2/3 complex is
composed of two actin-related proteins and five to six other
subunits (Machesky et al., 1994). This protein complex nucleates actin polymerization by serving as a template for the
formation of new actin filaments (Welch et al., 1997). On
the other hand, actin filament barbed-end capping proteins
of the capZ family regulate elongation of actin filaments by
blocking the addition of actin monomers to the barbed-end of the filament (Hug et al., 1995). Actin filament depolymerizing proteins of the actin-depolymerizing factor
(ADF)1/cofilin family regulate actin filament depolymerization and turnover by increasing the dissociation rate of actin
monomers from the pointed end in vitro and in vivo (Carlier
et al., 1997; Lappalainen and Drubin, 1997; Rosenblatt et al.,
1997). Cofilin, together with the actin monomer binding
proteins thymosin-
-4 and profilin, helps to maintain a
pool of actin monomers that is available for new filament
assembly (Carlier and Pantaloni, 1997
). Thymosin-
4 functions as a strong actin monomer-sequestering protein,
whereas cofilin and profilin do not sequester subunits, but
appear to promote subunit addition at the barbed-end
(Pantaloni and Carlier, 1993
; Carlier et al., 1997). Although cofilin and profilin are highly conserved in evolution from yeast to mammals, thymosin-
-4 has to date only
been found in animal cells (Sun et al., 1995), and is absent
in the S. cerevisiae genome (P. Lappalainen and B.L.
Goode, unpublished data). Thus, a high affinity actin-sequestering protein that is evolutionarily conserved has
not yet been identified.
Here we describe the identification and characterization of a novel, highly conserved actin monomer binding protein. This protein is composed of two cofilin-like regions and was therefore named twinfilin. Biochemical characterization of twinfilin shows that this protein sequesters actin monomers by forming a 1:1 molar complex with actin. Furthermore, in vivo analyses of the TWF1 gene in budding yeast showed that twinfilin is involved in the regulation of the cortical actin cytoskeleton.
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Materials and Methods |
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TWF1 Deletion
The TWF1 gene was deleted from the wild-type diploid strain DDY1102 (Table I) using a PCR-based one-step gene replacement technique (Baudin et al., 1993). The URA3 gene was amplified from pRS316 plasmid (Sikorski and Hieter, 1989) using oligonucleotides that introduce 50-bp TWF1 5' and 3' flanking sequences to the ends of the URA3 gene. The PCR products were gel purified and transformed into DDY1102 to yield integrative recombinants that were selected for by growth on medium lacking uracil. Correct integration was confirmed by PCR amplification of genomic DNA from URA+ cells with primers external to the TWF1 gene.
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Immunofluorescence and Localization of a GFP-Twinfilin Fusion Protein in Yeast
Cells were prepared for immunofluorescence as described by Ayscough
and Drubin (1998). The guinea pig anti-yeast actin serum was used at
1:2,000 dilution and the rabbit anti-GFP serum, a gift from Pam Silver
(Dana Farber Cancer Institute, Harvard University), was used at 1:10,000
dilution. Cells were viewed using a Zeiss Axioskop fluorescence microscope with a 100 W mercury lamp and a Zeiss 100X Plan-NeoFluar oil immersion objective. Images were captured electronically using a 200-E
CCD camera (Sony Electronics Inc., San Jose, CA) and displayed on a
Micron 133 computer (Micron Electronics Inc., Nampa, ID) using Northern Exposure software (Phase 3 Imaging Systems, Milford, MA).
A GFP-TWF1 gene fusion was generated by PCR amplifying the TWF1
coding region using primers that introduce HindIII and XbaI sites at the
5' and 3' ends, respectively. The PCR product was ligated into the GFP
vector, pTS408 (a gift from Tim Stearns, Stanford University, Stanford,
CA), which had been digested with HindIII and XbaI.The resulting plasmid, pGFP-TWF1, was transformed into haploid (DDY757; Mat a) and
diploid (DDY759; Mat a/) yeast strains (Table I). To induce expression
of the GFP-twinfilin fusion protein, cultures were grown in selective synthetic medium plus glucose to log phase (OD600 = 0.1), pelleted, and
transferred to synthetic selective medium plus 2% galactose for 12 h at
25°C. Live cells were mounted on slides and imaged by fluorescence microscopy.
Bud-Scar Staining and Endocytosis Assay
The budding patterns of TWF1/TWF1 (DDY1102) and twf1/
twf1
(DDY1436) diploid cells were scored after calcofluor staining as described
in Yang et al. (1997)
. For each strain, >200 cells with 3 or more visible
scars were scored. Fluid phase endocytosis was assayed as described
(Dulic et al., 1991) by monitoring uptake of the dye lucifer yellow to the
vacuole.
Overexpression of Twinfilin in Yeast
To overexpress twinfilin in yeast, the entire TWF1 gene coding region was amplified by PCR using primers that introduce HindIII and KpnI sites at the 5' and 3' ends, respectively. The PCR product was ligated in frame into the gal-inducible CEN/URA3 vector, pRB1438 (a gift from the Botstein lab, Stanford University) digested with HindIII and KpnI, and the resulting plasmid, pGAL-TWF1, transformed into DDY757 cells (Table I). To induce overexpression of twinfilin, cultures were grown in selective synthetic medium plus glucose to log phase (OD600 = 0.1), pelleted, and transferred to synthetic selective medium plus 2% galactose for 24 h at 25°C. The effects of overexpression on the actin cytoskeleton were examined by actin immunofluorescence in parallel with cells transfected with vector alone.
Protein Expression and Purification
To express full-length twinfilin and each of its two cofilin-like repeats
(amino acids 1-162 and 163-332, respectively) separately in Escherichia
coli, appropriate fragments of the TWF1 coding region were amplified
from S. cerevisiae genomic DNA by PCR using oligonucleotides that introduce NcoI and HindIII sites at the 5' and 3' ends of the fragments, respectively. In full-length twinfilin and repeat-1 constructs, the 5' oligonucleotide
results in substitution of Ala for Ser at position 2. The PCR-fragments were ligated into NcoI-HindIII-digested pGAT2 plasmid (Peränen et al.,
1996). All constructs were sequenced by the chain-termination method
and the clones containing undesired mutations were discarded. Full-length
twinfilin and each of its cofilin-like repeats were expressed as glutathione-
S-transferase fusion proteins in E. coli BL21(DE3) cells under control of
the Plac promoter. Cells were grown in 2,000 ml of Luria broth to an optical
density of 0.5 at 600 nm and then the expression was induced with 0.2 mM isopropyl-thio--D-galactoside (IPTG). Cells were harvested 3 h after induction, washed with 50 ml of 20 mM Tris (pH 7.5) and resuspended in 10 ml of PBS and 0.2 PMSF. Cells were lysed by sonication followed by a
centrifugation for 15 min at 14,000 g. GST-fusion proteins were enriched
from the supernatant using glutathione-agarose beads as described by
Ausubel et al. (1990)
. GST-fusion proteins were incubated overnight at
4°C with thrombin (5 U/ml) to cleave twinfilin proteins away from GST.
The beads were washed 3× with 50 mM Tris (pH 7.5) and 150 mM NaCl
and the supernatants were concentrated to ~1.5 ml using Centricon 10-kD
cutoff devices. Concentrated supernatants were loaded onto a Superdex-75
HiLoad gel-filtration column (Pharmacia Biotech, Inc., Piscataway, NJ) equilibrated with 10 mM Tris (pH 7.5), 50 mM NaCl to remove thrombin and further purify the twinfilin proteins. The peak-fractions containing
twinfilin and repeat-1 (eluted from the column at 56 and 67 ml, respectively) were pooled, concentrated in Centricon 10-kD cutoff devices to a
final concentration of 30-100 µM, frozen in liquid N2, and then stored at
80°C. The full-length twinfilin and repeat-1 were >90% pure, based on
Coomassie-stained SDS-polyacrylamide gels. Yeast actin and yeast cofilin
were purified as described previously by Lappalainen et al. (1997)
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Actin Cosedimentation Assays
For the first set of F-actin cosedimentation assays (see Fig. 2), 40-µl aliquots of 0/2.5/5/7.5/10 µM yeast actin were polymerized for 45 min in F-buffer (10 mM Tris, pH 7.5, 0.7 mM ATP, 0.2 mM CaCl2, 100 mM KCl, 0.2 mM DTT, and 2 mM MgCl2). After polymerization, 10 µl of 10 µM twinfilin or repeat-1 (in 10 mM Tris, pH 7.5, and 50 mM NaCl) were mixed with actin and incubated at room-temperature for 15 min. For the second set of cosedimentation assays (see Fig. 3), 40-µl aliquots of 2.5 µM yeast actin were polymerized in F-buffer for 45 min, mixed with 10 µl of 0, 2.5, 5, 10, 20, 30, and 40 µM purified twinfilin or repeat-1, and incubated at room temperature for 15 min. The actin filaments were sedimented by centrifugation at 90,000 rpm for 20 min at 20°C in a TLA-100 rotor (Beckman Instruments, Inc., Fullerton, CA), and equal portions of the pellets and the supernatants were loaded onto 10 or 12% SDS gels. The gels were Coomassie-stained and the intensity of twinfilin and actin bands was quantified using an IS-1000 densitometer (Alpha Innotech Corporation, San Leandro, CA).
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Monomer Binding Assays
Interactions of twinfilin and repeat-1 with actin monomers were monitored by native-gel electrophoresis and by the inhibition of actin nucleotide exchange. For native gel electrophoresis, the twinfilin and yeast actin were mixed to a final concentration of 15 µM each and electrophoresis
was carried out as described by Safer (1989). For nucleotide exchange assays, 40 µl of G-buffer (10 mM Tris, pH 7.5, 2 mM CaCl2, 2 mM DTT, and
25 mM ATP) containing 2.5 µM yeast actin and 0, 2, and 4 µM twinfilin or
repeat-1 were mixed with 10 µl of 1 mM etheno-ATP. The nucleotide exchange reaction was followed at 20°C in a F-4010 fluorescence spectrophotometer (Hitachi Instruments, Inc., San Jose, CA) at an excitation of 360 nm and emission of 410 nm.
Depolymerization Assay
Kinetics of actin filament disassembly were monitored by pyrene fluorescence with excitation at 365 nm and emission at 407 nm. 6 µM actin (5:1 ratio of yeast actin/pyrene-labeled rabbit skeletal muscle actin) was polymerized in F-buffer for 45 min in the presence of 5 nM human platelet gelsolin. Depolymerization of the F-actin was induced by mixing 40 µl of the F-actin with 10 µl of 250 µM latrunculin-A, 30 µM Twf1, 30 µM yeast cofilin, or 10 µM yeast cofilin plus 250 µM latrunculin-A, and monitored for 10 min by the decrease in fluorescence at 407 nm in the fluorescence spectrophotometer.
In Vitro Kinase Assay
Purified full-length twinfilin (80 ng) was mixed with 5 µg of myelin basic
protein (Sigma Chemical Co., St. Louis, MO) or poly(Glu-Tyr) 4:1 (Sigma
Chemical Co.) in 30 µl of kinase reaction buffer (50 mM Hepes, pH 7.5, 60 mM KAc, 10 mM MgCl2, 5 mM MnCl2, 50 µM ATP). 5 µCi of -[32P]ATP
(Amersham Corp., Arlington Heights, IL) was added on each reaction.
After incubation for 15 min at room temperature, 10 µl of 4× SDS gel
sample buffer (Laemmli, 1970
) was added, and the proteins were resolved
on a 10% SDS-polyacrylamide gel. 32P-labeled bands were visualized by
autoradiography after exposure of dried gels to X-ray film at
70°C.
Miscellaneous
PAGE in the presence of sodium dodecyl sulfate was carried out using the
buffer system of Laemmli (1970). The concentrations of twinfilin and repeat-1 were determined by comparison to standard curves of known
amounts of purified yeast cofilin and actin on Coomassie-stained SDS-
polyacrylamide gels. Protein concentrations were determined using this
method, rather than the calculated extinction coefficient, to avoid the possible contributions from small amounts of contaminating protein to absorbance at 280 nm.
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Results |
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Identification of a Novel Actin-binding Protein in Yeast
In a search of the yeast Saccharomyces cerevisiae genome
database for proteins with sequence homology to the actin
filament depolymerizing protein cofilin, we discovered a
previously uncharacterized open reading frame (YGR080W)
encoding a novel cofilin-like protein. This gene, predicted
to encode a 332-amino acid protein, is composed of not
one, but two cofilin-like sequences with 21% and 19%
amino acid sequence identity to yeast cofilin (Lappalainen
et al., 1998). These cofilin-like repeats also show ~15% sequence identity to each other, and therefore we named this
protein twinfilin (TWF1). The protein encoded by TWF1
also shows homology to the human and mouse A6 proteins,
previously identified by a screen of an embryonic cDNA expression library using a anti-phospho-tyrosine antibody (Beeler et al., 1994) and to a sequence in the C. elegans
genome (these data are available from GenBank/EMBL/
DDBJ under accession number U46668). Biochemical analyses in this earlier report suggested that human A6 protein
might be a protein tyrosine kinase. However, A6 protein
lacks any sequence homology to known protein kinases.
Furthermore, we did not detect any kinase activity for purified yeast twinfilin using identical substrates and conditions to those described by Beeler et al. (1994; data not shown;
see Materials and Methods).
A sequence alignment of yeast cofilin and twinfilin repeats 1 and 2 from yeast, human, and mouse (Fig. 1) shows
that the positions of cofilin secondary structure elements
identified from the yeast cofilin crystal structure (Fedorov
et al., 1997) are relatively well conserved between cofilin
and twinfilins. Furthermore, the sequence insertions in the
twinfilins are located in regions predicted to form loops,
suggesting that each twinfilin repeat has a tertiary structure
similar to the ADF/cofilin proteins. Each twinfilin repeat
also has an ~20-amino acid extension at its COOH-terminal region not found in any of the known ADF/cofilin proteins (Lappalainen et al., 1998).
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The residues of cofilin that have been shown to be essential for interactions with actin monomers and actin filaments (indicated by asterisks above the sequences in Fig. 1) also are relatively well conserved in each repeat of twinfilins (see Fig. 1). However, the residues in yeast cofilin that have been shown to be essential for binding to actin filaments are less conserved in twinfilins than residues implicated in monomer binding. The overall structural conservation as well as the conservation of the actin-binding residues between cofilin and twinfilins suggests that twinfilins might bind directly to actin, and that their interactions with actin may be similar to ADF/cofilin proteins. In support of the hypothesis that twinfilin is an actin-binding protein, we also have identified twinfilin as a protein enriched from yeast extracts on an actin affinity column (Goode, B.L., D. Shieltz, J. Yates, and D.G. Drubin, unpublished observations).
Twinfilin Sequesters Actin by Forming a 1:1 Complex with Actin Monomers
To test directly whether twinfilin binds to actin filaments and/or actin monomers, we expressed full-length twinfilin in E. coli as a glutathione-S-transferase fusion protein. The twinfilin GST-fusion proteins were purified from E.coli extracts using glutathione-agarose beads. Twinfilin was subsequently cleaved from GST by digestion with thrombin. Purification was finalized by gel-filtration chromatography on a Superdex-75 column. The majority (70- 80%) of the full-length twinfilin eluted from the column as a single peak at the expected position for a monomer (at ~ 54 ml). However, a small fraction of twinfilin (20-25%) eluted in the void volume, suggesting some aggregation under these conditions. After freezing and thawing of the monomeric fraction of twinfilin, we again observed that 20-25% of the twinfilin sedimented on its own upon ultracentrifugation for 20 min at 90,000 rpm in a TLA-100 rotor (Fig. 2, lane 2). In the pelleting assays described below, the presence of the insoluble twinfilin fraction that pellets on its own is subtracted from the results. In monomer binding gel shift assays (see Fig. 4), the insoluble twinfilin fraction appears to not enter the gel and therefore should not effect on the results.
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To study the interactions of the purified twinfilin with actin filaments, we first carried out actin filament cosedimentation assays using a constant concentration (2 µM) of twinfilin and variable concentrations (0-8 µM) of purified yeast F-actin. As shown in Fig. 2, addition of twinfilin to the actin filaments leads to a significant increase in the amount of actin present in the supernatant. Whereas ~90% of the actin is normally found in the pellet fraction under these conditions, addition of equimolar amounts of twinfilin to F-actin decreases the amount of actin in the pellet to ~25% (compare Fig. 2, lane 1 with 3). At higher actin concentrations twinfilin appears to shift actin to the supernatant in an ~1:1 molar ratio (i.e., for every molecule of twinfilin added to the reaction, one molecule of actin shifts to the supernatant). Several different mechanisms of action by twinfilin could underlie these observations, including actin monomer sequestration and/or capping of the barbed end of the filaments to prevent new subunit addition; however, the 1:1 stoichiometry of actin and twinfilin in the supernatant strongly suggests monomer sequestration. Fig. 2 also shows that only a small increase in the amount of twinfilin in the pellet occurs upon addition of increasing concentrations of F-actin, suggesting that twinfilin does not bind tightly to actin filaments.
To further examine the ability of twinfilin to depolymerize actin filaments, we carried out a cosedimentation assay using a constant (2 µM) concentration of actin and variable twinfilin concentrations (0 and 6 µM). As shown in Fig. 3, a and c, addition of twinfilin causes an equimolar amount of actin to redistribute from the pellet fraction to the supernatant. Because ~25% of twinfilin pellets on its own, the addition of 2 µM twinfilin to actin filaments can shift a maximum of 1.5 µM actin to the supernatant. The observation that twinfilin-actin monomer binding saturates at 2 µM twinfilin and 2 µM actin (see Fig. 2) strongly suggests that twinfilin forms a tight 1:1 complex with actin monomers.
To further test the ability of twinfilin to form a complex
with actin monomers, we used native gel electrophoresis.
Reactions containing a final concentration of 15 µM twinfilin and 15 µM yeast actin in G-buffer (see Materials and
Methods) were fractionated on a 7.5% native polyacrylamide gel run at 100 V for 120 min. The motilities of yeast
actin and recombinant twinfilin alone are shown in Fig. 4
(lanes 1 and 2, respectively). Lane 3 in Fig. 4 shows that
addition of twinfilin to actin at a 1:1 molar ratio causes in a
dramatic shift in the motility of actin, resulting in the formation of a smear between the twinfilin and actin bands. These results suggest that the complex that forms between
twinfilin and actin is relatively stable in vitro. As an independent test of monomer binding, we also investigated
whether addition of twinfilin could inhibit nucleotide exchange that normally occurs for actin monomers in solution.
Inhibition of nucleotide exchange has been demonstrated
previously for ADF/cofilin proteins (Nishida, 1985). As
shown in Fig. 5, twinfilin inhibits nucleotide exchange of ATP-actin monomers in a concentration-dependent manner similar to that described for cofilin (Nishida, 1985
).
Taken together, these data (the shift in F-actin steady state
towards monomer in a 1:1 molar ratio, the detection of
twinfilin/actin monomer complexes on native gels, and the
inhibition of nucleotide exchange by actin when twinfilin is
bound) demonstrate that twinfilin functions in vitro like a
bona fide actin monomer-sequestering protein.
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ADF/cofilin proteins have been shown to promote the
rapid depolymerization of actin filaments from their pointed
ends (Carlier et al., 1997). Because twinfilin has sequence
homology to ADF/cofilin proteins, we tested whether it
also effects actin filament depolymerization kinetics in a
similar manner to ADF/cofilin proteins. To specifically measure the filament depolymerization from the pointed
end, actin filaments capped at their barbed ends were prepared by polymerizing 6 µM yeast actin in the presence of
5 nM gelsolin as described by Carlier et al. (1997). Depolymerization was then induced by the addition of twinfilin,
cofilin or the actin monomer-sequestering drug latrunculin-A. As shown in Fig. 6, twinfilin and latrunculin-A have
similar effects in this assay, consistent with the conclusion that twinfilin sequesters actin monomers without stimulating filament depolymerization. In contrast, cofilin stimulates rapid filament depolymerization. Because the stoichiometric cofilin-induced depolymerization was too rapid to
monitor using our experimental system (Fig. 6 D), we also
performed this assay using substoichiometric cofilin and a
sufficient concentration of latrunculin A to sequester depolymerized actin monomers (Fig. 6 C). These conditions
allowed detection of the cofilin-induced rapid actin filament depolymerization. It is important to note that the
rapid decrease in signal may be in part due to fluorescence
quenching of the pyrene actin upon cofilin binding (Carlier et al., 1997
). However, it is clear from our results that,
despite its sequence homology with ADF/cofilin proteins,
twinfilin has little or no effect on filament depolymerization rate at the pointed end. One potential caveat of these
experiments is that the recombinant twinfilin used in these
assays could be improperly folded. However, the recombinant twinfilin eluted as a single peak on a gel filtration column and showed strong activities on actin, suggesting that
these preparations are homogeneous and active. On the
other hand, these results do not rule out the possibility that native yeast twinfilin might have additional activities
not detected here for the recombinant protein.
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Both Cofilin-like Repeats in Twinfilin Are Required for Strong Actin Monomer Binding
Because twinfilin is composed of two ADF homology domains (repeats 1 and 2), we investigated whether individual repeat domains might have activities similar to those of full-length twinfilin. Each repeat was expressed in E. coli as a GST-fusion protein and purified as described above for full-length twinfilin. As shown in Table II, both repeats were expressed at high levels in E. coli, but only repeat-1 was soluble after cleavage from GST with thrombin. All of the repeat-2 eluted in void volume from the Superdex-75 gel-filtration column after cleavage from glutathione- S-transferase and sedimented on its own upon ultracentrifugation for 20 min at 90,000 rpm in TLA-100 rotor. Therefore, only repeat-1 was used for the assays described below.
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In cosedimentation assays using a range of F-actin concentrations (0-8 µM) and a constant repeat-1 concentration (2 µM), repeat-1 did not exhibit copelleting with actin filaments (data not shown). However, repeat-1 was able to increase the amount of actin in the supernatant in a concentration-dependent manner (Fig. 3, b and c). This activity was significantly weaker than that observed for full-length twinfilin, which suggests that both repeats are necessary for strong actin monomer sequestering. We also tested directly the interaction of repeat-1 with actin monomers by native gel electrophoresis and by the inhibition of nucleotide exchange on actin monomers. As shown in Fig. 4 (lanes 4 and 5) addition of repeat-1 to G-actin results in the formation of a weak smear between the repeat-1 and the actin bands, suggesting that these two proteins form a low affinity molecular complex with each other. As shown in Fig. 5, Repeat-1 also inhibits nucleotide exchange by actin monomers in a concentration dependent manner. Together, these results suggest that repeat-1 forms a complex with actin monomers similar in nature, but weaker in strength to the one formed by cofilin and full-length twinfilin.
A GFP-Twinfilin Fusion Protein Localizes to the Cytoplasm and the Cortical Actin Cytoskeleton in Yeast Cells
To investigate the localization of twinfilin protein in yeast,
a GFP-twinfilin fusion protein was expressed and its localization observed in living cells. As shown in Fig. 7 a, the
majority of cells expressing the GFP-twinfilin fusion protein showed strong cytoplasmic staining with additional
cortical punctate staining. The cortical spot structures
moved in real time, with some of the patches holding a stable position and others dramatically translocating over the
span of seconds (data not shown). Since these movements
are very similar to those of cortical actin patches described
in previous reports (Doyle and Botstein, 1996; Waddle et al., 1996), we used double immunofluorescence with
anti-actin and anti-GFP antibodies to address whether the
GFP-twinfilin patches correspond to actin patches. In the
majority of cells examined, the anti-GFP staining localized
primarily to the cytoplasm, but many cells also showed
patch-like staining. Examples of cells with clear patch
staining are shown in Fig. 7 b. In these cells, the GFP-staining patches overlapped with a subset of the cortical actin patches. Taken together, these results suggest that
twinfilin localizes primarily to the cytoplasm, but also to
the cortical actin cytoskeleton. However, it is important to
remember that this localization was carried out in cell
overexpressing GFP-twinfilin fusion protein and may
therefore not fully represent the localization of twinfilin in
wild-type cells. While the cytoplasmic localization of twinfilin is consistent with its activities as an actin monomer-sequestering protein, the patch-like staining raises intriguing possibilities about the regulation of twinfilin function.
One possibility is that a fraction of twinfilin is associated
with cortical actin patches through binding interactions
with patch components other than actin. This also could
explain the above mentioned isolation of twinfilin from
yeast extracts on actin filament affinity columns.
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Deletion of the TWF1 Gene Results in Synthetic Lethality with a Cofilin Mutant
To investigate the in vivo functions of twinfilin, we generated
a strain in which the TWF1 gene is deleted and replaced by the URA3 gene. Haploid twf1 cells exhibit normal
growth and have normal morphologies over a temperature
range of 20-37°C (data not shown). The growth of twf1
cells also was indistinguishable from wild-type cells on a
variety of stressful media, including media produced with
low and high pH, high NaCl, KCl, MgCl2, CaCl2, and formamide (data not shown). Furthermore, twf1
cells have no detectable defects in fluid phase endocytosis (data not
shown; see Materials and Methods) and their actin cytoskeletons appear normal by immunofluorescence except
for consistently brighter actin patch staining (Fig. 8).
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The absence of a strong detectable phenotype in twf1
cells suggests that there may be functional redundancy between twinfilin and other proteins in yeast. Because the actin cytoskeleton is characterized by a high complexity of
protein components, and by many examples of genetic redundancy, gene disruption of one actin binding protein often has no significant effects on growth rate of cells or the
overall appearance of the actin cytoskeleton by immunofluorescence. However, such gene disruptions can lead to
strong synergistic defects in combination with other mutations in genes that encode actin-binding proteins (e.g.,
Holtzman et al., 1993). To test the possibility that functional
redundancy explains the lack of pronounced defects in
twf1
mutants, we crossed twf1
mutants with mutants of
other genes encoding actin binding proteins, concentrating
on genes that encode proteins with ADF-homology domains (COF1 and ABP1; Lappalainen et al., 1998
) and on
genes that encode known actin monomer binding proteins
(profilin/PFY1, cofilin/COF1, and SRV2). As shown in Table III, twf1
demonstrates a strong and specific synthetic
phenotype with the cofilin allele cof1-22. This cofilin mutant
has been shown to have significant defects in F-actin binding and depolymerization both in vivo and in vitro, and it
results in lethality at the temperatures >30°C. However, at
20°C cof1-22 cells show normal morphology and exhibit growth rates similar to wild-type cells (Lappalainen and
Drubin, 1997
). After 3 d at 20°C, none of the twf1
cof1-22
double mutants formed visible colonies. However, after
prolonged incubation (5-7 d at 20°C), tiny twf1
cof1-22
colonies appeared. The cells in these colonies were abnormally large. To visualize the actin cytoskeletons in such
twf1
cof1-22 cells, the segregants were inoculated into a
small volume of YPD and grown at 20°C for 48 h. Fig. 8
shows a comparison of the morphologies of the actin
cytoskeletons in wild-type, twf1
cells, cof1-22, and twf1
cof1-22 double mutant cells grown at 20°C. Whereas twf1
and cof1-22 cells show some increase in the brightness
(= size) of the cortical actin structures compared with wild-type cells, most twf1
cof1-22 double mutant cells have
completely depolarized cortical actin cytoskeletons and abnormally large and chunky actin patches. These results suggest that TWF1 and COF1 genes may share a function required for the regulation of actin-based processes.
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Deletion of the TWF1 Gene Causes Random Budding Pattern and Bumpy Surface Morphology in Diploid Yeast Cells
We also examined the morphology of diploid yeast cells
homozygous for the twf1 gene deletion (DDY1436). Fig.
9 a shows that twf1
/twf1
cells appear to form normal
buds, but the cells have large bumps on their surfaces.
Similar phenotypes have been reported previously for a
subset of actin alleles that have defects in bipolar bud patterning (Drubin et al., 1993; Yang et al., 1997). Calcofluor
staining of the twf1
/twf1
cells revealed that each bump
is marked by a bud scar, suggesting that the bumps represent sites of past bud formation and cytokinesis. In normal
diploid yeast, the first bud to emerge from a daughter cell
is usually formed at the pole opposite to the birth scar, and
subsequent buds form at sites that are either at the same
pole as the birth scar or the opposite pole (Chant and Pringle, 1995
). In wild-type cells, this leads to a bipolar budding pattern (the accumulation of multiple bud scars positioned at either pole). It has been shown that disruption of
the actin cytoskeleton does not affect the position of the
first bud to emerge from the daughter cell, but subsequently results in a random budding pattern in diploid cells (Yang et al., 1997). Diploid-specific bud pattern defects also have been observed in actin-binding protein mutants, including sla2
, rvs167
, and sac6
(Drubin et al.,
1993; Yang et al., 1997). We found that 56% of twf1
/
twf1
cells exhibit random bud scar patterning (examples
are shown in Fig. 9 b), compared with only 2% of wild-type cells. Such frequencies of random budding are similar
to those reported previously for actin and actin-binding protein mutants, and support the model that TWF1 is involved in actin cytoskeletal functions in vivo.
|
Overexpression of Twinfilin Causes Depolarization of the Cortical Actin Cytoskeleton
Finally, we examined the effects of overexpressing twinfilin in yeast cells. One might predict that increased levels of an actin monomer-sequestering protein would lead to reduced polymer levels, and the build up of a larger pool of sequestered actin monomers in the cytoplasm. Recently it has been shown that the overexpression of previously identified actin monomer-binding proteins in yeast have different effects on the actin cytoskeleton depending on the actin-binding protein (Hofmann, C., and D.G.Drubin, unpublished results). While overexpression of Srv2p has no detectable effects, overexpression of profilin and cofilin both lead to a partial depolarization of the cortical actin cytoskeleton and the formation of cytoplasmic actin bars (aberrant structures that are not likely to be composed of filamentous actin since they do not stain with rhodamine-phalloidin). As shown in Fig. 10, overexpression of twinfilin leads to a complete depolarization of the actin cytoskeleton and the accumulation of cytoplasmic actin bars. Both of these effects support the conclusion that twinfilin functions as an actin monomer-sequestering protein in vivo.
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Discussion |
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Twinfilin Is a Novel and Widely Conserved Actin Monomer-binding Protein
In this study, we have identified and characterized a novel
actin monomer-binding protein in budding yeast. This
protein, which we named twinfilin, is composed of two
cofilin-like regions and appears to be evolutionarily conserved. Our database searches identified genes in C. elegans,
humans, and mice that encode homologous proteins
(~20% identical). Because the ADF/cofilin-like repeats 1 and 2 of twinfilin are more closely related across species
than they are to each other within a given species, we can
predict that twinfilin in human, mouse, and yeast cells
evolved from a common ancestral twinfilin and, therefore,
twinfilin represents a single protein family (Lappalainen et
al., 1998).
In vitro and in vivo data in this paper strongly suggest
that twinfilin functions as an actin monomer-sequestering
protein. Previous biochemical analyses of the human homologue of twinfilin (named A6) suggested a different
function. Human twinfilin/A6 was originally identified as a
phosphoprotein from an embryonic cDNA expression library, and studies using recombinant human twinfilin/A6 suggested that this protein might be a novel protein tyrosine kinase (Beeler et al., 1994). However, twinfilin/A6
lacks any of the sequence motifs found in protein kinases,
and we have been unable to detect any kinase activity for
recombinant yeast twinfilin using identical substrates and
conditions to those reported by Beeler et al. (1994). On
the other hand, our biochemical and genetic data support
the conclusion that twinfilin is an actin monomer-sequestering protein.
So far, only three classes of actin monomer-binding proteins that are conserved across species as diverse as yeast
and mammals have been identified. These proteins are
Srv2/CAP, ADF/cofilin and profilin (Freeman et al., 1995, Carlier and Pantaloni, 1997). Although the biochemical
properties of Srv2/CAP are relatively poorly understood,
the activities of ADF/cofilin and profilin from a variety of
organisms have been characterized extensively. Our data
show that the activities of twinfilin are distinct from those
of ADF/cofilin and profilin, indicating that twinfilin makes a unique contribution to the regulation of the cytoskeleton. Whereas ADF/cofilin interacts with both actin monomers and filaments and induces rapid dissociation of actin
monomers from the pointed ends of filaments (Carlier et
al., 1997), twinfilin interacts primarily with actin monomers and has no significant effect on dissociation of subunits from filament ends. Furthermore, twinfilin-bound actin monomers are blocked from reassembly into actin
filaments, whereas ADF/cofilin-bound actin monomers
are readily added at the barbed ends of actin filaments
(Carlier et al., 1997). Thus, at the protein concentrations
used in this study, both twinfilin and ADF/cofilin bind to
actin monomers, but only twinfilin sequesters monomers.
These activities of twinfilin contrast even more sharply with those of profilins, which stimulate nucleotide exchange on actin (Perelroizen et al., 1995) and promote actin assembly at the barbed end (reviewed by Carlier and
Pantaloni, 1997
). Thus, amongst all of the actin-binding
proteins widely conserved in eukaryotes, twinfilin is
unique in its ability to sequester actin monomers.
Despite the functional differences between cofilin and
twinfilin mentioned above, several lines of evidence suggest that cofilin and twinfilin may interact with actin
monomers through a similar interface. First, the overall sequence homology between these proteins suggests that
they may have similar tertiary structures. Second, the high
conservation of the residues that have been shown to be
essential for actin monomer-binding in yeast cofilin suggests that twinfilin and cofilin may have similar actin interactions (Lappalainen et al., 1997, see Fig. 1). Finally, the
similarity in their inhibition of the nucleotide exchange reaction on actin suggests that twinfilin and cofilin may bind
to actin monomers at a common interface. From the inhibition of nucleotide exchange activities of twinfilin (Fig. 5)
and cofilin (Lappalainen et al., 1997), it also appears that
twinfilin and ADF/cofilin may bind to ATP-actin monomers with similar affinities (<1 µM). To more thoroughly
understand the role of twinfilin in regulating actin filament
turnover, in the future it will be important to measure directly the affinity of twinfilin for ADP- and ATP-actin
monomers.
What then accounts for the distinct activities of ADF/ cofilin and twinfilin on actin? We speculate that the presence of two covalently attached ADF-homology domains could stabilize twinfilin/actin monomer complexes by decreasing the dissociation rate of actin monomers and/or by masking sites on the actin monomer surface that are required for subunit addition at the barbed end. Consistent with this model, we have shown that strong actin monomer-sequestering activity of twinfilin requires both of its ADF homology domains.
A Role for Twinfilin in Regulation of the Yeast Cortical Actin Cytoskeleton
Several lines of evidence resulting from in vivo analyses of twinfilin function suggest a role in regulating assembly of the actin cytoskeleton. Deletion of the TWF1 gene leads to an increase in the intensity of cortical actin patch staining, suggesting that twinfilin acts to limit actin filament assembly in vivo. Moreover, overexpression of twinfilin causes depolarization of the actin cytoskeleton and formation of cytoplasmic actin bars, and diploids homozygous for the twinfilin null mutation show a random budding pattern. These are signature phenotypes for yeast with defects in the cortical actin cytoskeleton.
More specifically, twinfilin may be involved in regulating levels of free actin monomer in cells. The twinfilin deletion mutation shows a pronounced negative synergism
when combined with the cof1-22 allele of the yeast cofilin
gene. This cofilin allele exhibits both diminished F-actin
binding and defects in actin filament depolymerization in
vivo and in vitro (Lappalainen and Drubin, 1997; Lappalainen et al., 1997). The cells carrying cof1-22 have enlarged, irregularly shaped actin patches, suggesting that
these cells may have elevated levels of filamentous actin
(Lappalainen and Drubin, 1997
). Cells carrying the
cof1-22 allele are predicted to have an unusually low actin
monomer pool due to defects in actin filament depolymerization. We suggest that the synthetic phenotype observed
between twf1
and cof1-22 arises from a synergistic depletion of unassembled actin. Thus, through its monomer-sequestering activity, twinfilin may function with cofilin to
maintain a pool of actin monomers available for assembly.
Given the strong monomer-sequestering activity displayed by twinfilin in vitro, it is likely that twinfilin activity
is tightly regulated in vivo. Possible mechanisms for such
regulation include phosphorylation of twinfilin, physical
interactions between twinfilin and other proteins (possibly
other actin-binding proteins), and regulation by association with phospholipids. Phosphorylation and PIP2 binding
both have been shown previously to regulate actin-related activities of ADF/cofilin proteins (for review se; Moon and
Drubin, 1995), and multiple protein interactions have
been implicated in the regulation of actin-binding proteins
in yeast (Lila et al., 1997). The observation that a fraction
of GFP-twinfilin localizes to the cortical actin supports the
model that twinfilin may interact with other proteins associated with cortical actin filaments. Consistent with this
hypothesis, we have also isolated twinfilin from yeast extracts on an F-actin affinity column (B.L. Goode, D. Shieltz, J. Yates, and D.G. Drubin, unpublished observations). In future experiments, it will be important to identify twinfilin-interacting proteins. Such proteins might account for the partial colocalization of twinfilin with
cortical actin patches. In particular, we wish to understand
how one or more of the above-mentioned regulatory mechanisms may stimulate the release of actin monomer
from twinfilin in cells, providing a stimulus-responsive
pool of actin monomers.
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Footnotes |
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Address correspondence to Dr. Lappalainen at his present address, Institute of Biotechnology, University of Helsinki, P.O. Box 56, FIN-00014, Helsinki, Finland. Tel.: 358 9 708 59 499. Fax: 358 9 708 59 366. E-mail: pekka.lappalainen{at}helsinki.fi
Received for publication 24 March 1998 and in revised form 24 June 1998.
We thank Paul Janmey (Harvard University) for a generous gift of human platelet gelsolin.
This study was supported by grants from National Institutes of Health to B.L. Goode (GM-17715), American Cancer Society to D.G. Drubin (GM-42759) and Human Frontier Science Program to P. Lappalainen.
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Abbreviations used in this paper |
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ADF, actin-depolymerizing factor; GFP, green fluorescent protein.
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