©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification, Characterization, and Intracellular Distribution of Cofilin in Dictyostelium discoideum(*)

Hiroyuki Aizawa , Kazuo Sutoh (2), Satoshi Tsubuki (1), Seiichi Kawashima (1), Ai Ishii , Ichiro Yahara (§)

From the (1) Department of Cell Biology and Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113 and the (2) Department of Pure and Applied Science, College of Arts and Science, University of Tokyo, Komaba, Tokyo 153, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that D COF1 is expressed both in vegetative and differentiating cells and that D COF2 is not expressed under any conditions examined. Gene disruption experiments suggested that D COF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of D COF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.


INTRODUCTION

Actin filaments are a major constituent of the cytoskeleton in eukaryotic cells which are composed of monomeric actin molecules. The structure of actin filaments was regulated by various types of actin-binding proteins, such as cross-linking proteins, actin filament-severing proteins, end-capping proteins, and monomer-sequestering proteins (Stossel et al., 1985; Pollard and Cooper, 1986; Vandekerckhove and Vancompernolle, 1992).

Cofilin was originally identified as an actin monomer-binding protein using DNase I column chromatography from mammalian brain extracts (Maekawa et al., 1984). Cofilin binds to actin molecule in a 1:1 molar ratio and increases critical concentration of actin for polymerization in vitro (Nishida et al., 1985; Moriyama et al., 1992; Hayden et al., 1993). Cofilin also exhibits actin filament severing activity in a calcium ion-independent manner (Nishida et al., 1985; Moriyama et al., 1992; Hayden et al., 1993). Phosphatidylinositides inhibit the depolymerizing activity of cofilin (Yonezawa et al., 1990). Conversely, cofilin protects these phosphatidylinositides from hydrolysis by phospholipase C (Yonezawa et al., 1991). Thus it would be possible that actin cytoskeletons are affected by signal transduction pathways in a cofilin-dependent manner and that cofilin modulates conversely these signal transductions, although little is known about interactions of cofilin with cell membranes.

Now cofilin and its related proteins are known to be ubiquitously distributed among eukaryotes including porcine cofilin (Nishida et al., 1984) and destrin (Nishida et al., 1985), human actin depolymerizing factor (Hawkins et al., 1993), murine cofilin (Moriyama et al., 1990), chicken cofilin and actin depolymerizing factor (Adams et al., 1990; Abe et al., 1990), starfish depactin (Mabuchi, 1983), Acanthamoeba actophorin (Cooper et al., 1986), lily actin depolymerizing factor (Kim et al., 1993), and yeast cofilin (Iida et al., 1993; Moon et al., 1993). In the budding yeast Saccharomyces cerevisiae, disruption of the COF1 gene caused cell lethality (Iida et al., 1993; Moon et al., 1993). The expression of mammalian cofilin or destrin rescued yeast cells from the lethality (Iida et al., 1993). These results indicate that the function of cofilin is conserved among eukaryotes from yeast to mammals.

Dictyostelium discoideum exhibits a number of cellular processes depending upon dynamic movements of actin filaments in various stages of spore formation induced under starvation stress. Those include cytokinesis, phagocytosis, intracellular vesicular transport, ruffling membrane formation, substrate adhesion, pseudopod formation, chemotactic migration toward cyclic AMP, and cell-cell adhesion to form a multicellular slug (Loomis, 1982). Thus, this organism is a powerful laboratory system for investigation on the actin-based cellular processes. In this study, we have newly identified cofilin in D. discoideum and cloned its genes. Furthermore, we have investigated dynamic behavior of cofilin during processes leading to spore formation.


EXPERIMENTAL PROCEDURES

D. discoideum Cell Line and Vectors

A shuttle vector (pBIG) between D. discoideum and Escherichia coli (Uyeda et al., 1994) was a generous gift from Dr. J. Spudich (Stanford University of Medicine). A plasmid vector pDNeo2 was a generous gift from Dr. A. Noegel (Max Planck Institute for Biochemistry, Martinsried, Germany). Axenical D. discoideum cells (Ax2 cell line) were generally grown in HL5 medium (Sussman, 1987) in a cell density between 4 10 cells/ml and 2 10 cells/ml except when cells were used for large scale preparation of proteins (1 10 cells/ml). Passage did not extend beyond 6 weeks from germination of stocked spores.

Chemicals and Enzymes

Restriction enzymes, T4 DNA ligase, E. coli DNA polymerase I large fragment, taq polymerase, reverse transcriptase, and calf intestine alkaline phosphatase were purchased from Takara Shuzo Co. Ltd. (Kyoto, Japan). A plasmid vector pBluescript SK(-), a phage vector gt10, and a packaging extract Giga Pack Gold were from Stratagene Co. (La Jolla, CA). A plasmid pMC-1871 was from Pharmacia LKB Biotechnology (Uppsala, Sweden). Blasticidin S was from Funakoshi Co. (Tokyo, Japan). DNase I was from Boehringer Mannheim (Mannheim, Germany). MES, HEPES, phenylmethylsulfonyl fluoride), aprotinin, leupeptin, dithiothreitol, neomycin, phosphatidylinositol (PI),() phosphatidylinositol monophosphate (PIP), phosphatidylinositol bisphosphate (PIP), phosphatidylserine (PS), phosphatidylcholine (PC), inositol 1,4,5-triphosphate (IP), and 1-oleoly-2-acetyl-glycerol were from Sigma. The lipids and IP were dissolved in water at 1 mM and sonicated three times for 10 s with a sonicator just before use. ATP, lysylendopeptidase, and other chemicals were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Antibodies

Monoclonal antibody against actin (clone C4) was from ICN Biomedicals Inc. (Costa Mesa, CA). Fluorescein-conjugated goat anti-rabbit IgG antibody and rhodamine-conjugated goat anti-mouse IgG were from Organon Teknika Co. (Durham, NC). Alkaline phosphatase-conjugated goat anti-rabbit IgG was from Jackson Immuno Research Laboratories (West Grove, PA).

Protein Purification

Rabbit muscle actin was prepared as described (Spudich and Watt, 1971) and further purified by HiLoad 16/60 Superdex 200 preparation grade (Pharmacia LKB Biotechnology, Uppsala, Sweden) gel filtration column equilibrated with G-buffer (2 mM HEPES, 0.1 mM CaCl, 0.2 mM ATP, 0.1 mM dithiothreitol, 0.01% NaN, pH 7.8) using a Pharmacia fast protein liquid chromatography control system (Pharmacia LKB Biotechnology, Uppsala, Sweden). D. discoideum actin was purified as described (Sutoh et al., 1991). Actin concentration was determined by UV absorption measurement based on absorbance of 1% actin solution at 290 nm to be 6.5.

The 15-kDa protein was purified from D. discoideum cells as follows. Ax2 cells were grown to a cell density of 1 10 cells/ml in 10 liters of HL5 medium. Cells were harvested by centrifugation at 1,000 g for 5 min, and the packed cells were resuspended in 250 ml of phosphate-buffered saline (PBS; 10.6 mM NaHPO, 1.7 mM KHPO, 140 mM NaCl, 2.7 mM KCl). After centrifugation at 1,000 g for 5 min, packed cells (25 g) were resuspended in 250 ml of MEM buffer (20 mM MES, 2 mM EGTA, 1 mM MgCl, pH 6.9) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1% aprotinin. The suspension was sonicated four times using a Branson Sonifier for 10 s at range 4. The disruption of cells were confirmed microscopically. The extracts were clarified by ultracentrifugation at 100,000 g for 1 h. The crude extracts (28 mg protein/ml, 100 ml) were mixed with DNase I beads (50 ml in wet volume) for 1 h at 4 °C with occasional shaking. DNase I beads were prepared as described previously (Iida et al., 1993). The beads were packed in a glass tube (16 mm 250 mm), and adsorbed proteins were eluted by a linear gradient of NaCl from 0 to 600 mM in 100 ml of MEM buffer. An aliquot (4 µl) of each fraction (5 ml) was analyzed by SDS-PAGE, and fractions (30 ml) containing the 15-kDa protein were collected, concentrated to 8 ml by OMEGACELL (Filtron Technology Co., Northbotough MA), and dialyzed against 10 mM sodium phosphate buffer, pH 7.0. After centrifugation at 100,000 g for 15 min, the dialysate was applied to a hydroxylapatite column (6 ml, 10 76 mm), after which the adsorbed materials were eluted by a linear gradient of sodium phosphate concentration from 10 to 100 mM in 30 ml of solution. An aliquot (4 µl) of each fraction (2 ml) was analyzed by SDS-PAGE, and fractions containing the 15-kDa protein were concentrated to 0.4 ml, and dialyzed against MEM buffer. The protein concentration of the final fraction was 1.4 mg/ml, and the purity as determined by SDS-PAGE was more than 95%.

Biochemical Characterization for Cofilin Activity

Sedimentation analysis was performed as follows. D. discoideum actin (17.8 µM) was polymerized in 10 mM MES, pH 6.9, 10 mM KCl, 5 mM MgCl, 0.2 mM ATP at 25 °C for 2 h. The polymerized actin was diluted into a solution containing various concentrations of cofilin and incubated at 25 °C for 15 min. Then, the solutions (50 µl) were centrifuged at 400,000 g for 15 min, and the resultant supernatants and pellets were analyzed by SDS-PAGE. Pyrene-labeled actin was prepared from rabbit muscle actin as described (Brenner and Korn, 1983). The amount of polymerized rabbit actin was estimated by fluorescence intensity of pyrene-labeled actin at 25 °C with a Hitachi F-200 fluorescence spectrophotometer (Hitachi Co., Tokyo, Japan). The excitation and emission wavelengths were 365 and 407 nm, respectively. Low shear rate apparent viscosity was measured with a miniature falling ball system at 25 °C using 50-µl capillary tubes using rabbit actin (Clay Adams, Parsippany, NJ). Actin monomers were polymerized at 25 °C by adding 0.1 volume of 10F-buffer (100 mM MES, 500 mM KCl, 10 mM EGTA, 5 mM ATP, 10 mM MgCl, pH 6.9).

Partial Amino Acid Sequence Analysis

Samples were completely digested by lysylendopeptidase, followed by separation by reverse-phase Vydac C column chromatography as described (Aizawa et al., 1989). Amino-terminal amino acid sequences of the peptides were determined by a gas-phase sequencer (470A Protein Sequencer, Applied Biosystems Japan, Tokyo).

Molecular Cloning of the 15-kDa Protein

Molecular biological techniques followed methods described in the text (Maniatis et al., 1982) if otherwise indicated. In order to get a partial DNA fragment of the 15-kDa protein, we used reverse-transcriptase-polymerase chain reaction (RT-PCR, Takara Shuzo Co. Ltd., Kyoto, Japan) using total RNA purified from Ax2 cells as a template. We synthesized two degenerated oligonucleotides TA(T,C)GG(G,A,T,C)GG(G,A,T,C)AT(A,T,C)AT(A,T,C)TA and C(T,G)(A,G)CA(C,T)TC(A,G)TT(C,T)TC(G,A,T,C)GG which corresponded to the two determined amino acid sequences of the 15-kDa protein, YGGIIY and LPENECR, respectively, with a DNA synthesizer (391 DNA Synthesizer, Applied Biosystems). RT-PCR with the two primers amplified a cDNA fragment of 119 base pares (bp), which encoded a part of the 15-kDa protein (nucleotides 510-628 in Fig. 4 C). EcoRI-digested genomic DNA library was constructed in phage vector gt10 and packaged into bacteriophage particles using the packaging extract Giga-Pack Gold, and grown on E. coli strain C600Hfl. The genomic library consisting of 40,000 independent clones was screened using the amplified cDNA as a probe, and three different positive clones were obtained; COF1 (2.3 kbp), COF2 (1.8 kbp), and COF3 (0.3 kbp). The inserts of COF1, COF2, and COF3 were subcloned into the EcoRI site of plasmid vector pBluescript SK(-) to construct pCOF1, pCOF2, and pCOF3, respectively. The nucleotide sequences of both strands were determined by the dideoxy chain-termination method (Sanger et al., 1977).


Figure 4: Genomic structure of D. discoideum cofilin. A, schematic genomic structures of the two cofilin genes D COF1 and D COF2. Exon regions are indicated by thick lines. An intron region and 5`- and 3`-nontranscribed regions are indicated by thin lines. Dotted lines indicate the corresponding regions of the two cofilin genes. Cleavage sites of several restriction enzymes are denoted as E ( EcoRI), H ( HincII), K ( KpnI), and N ( NdeI). An amplified region by PCR (nucleotides 510-628 in C) is also shown at the bottom ( thick bar). B, genomic Southern analysis of cofilin genes. Genomic DNA was completely digested with BamHI ( lane 1), EcoRI ( lane 2), HindIII ( lane 3), KpnI ( lane 4), or PstI ( lane 5). The digested DNA (1 µg) was electrophoresed in 0.8% agarose gel. The amplified PCR fragment (nucleotides 510-628 in C) was used as a hybridization probe. Positions of size markers, 23, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.6 kbp, are indicated on the right side from top to bottom. C, nucleotide and predicted amino acid sequences of D. discoideum cofilin. The nucleotide residues are numbered beginning at the first 5`-nucleotide residues presented. Conserved nucleotide residues between D COF1 and D COF2 genes are presented in large characters. Non-conserved residues are presented in small characters. 2, the first 60 nucleotide sequenced of D COF2 gene. 1, 910 nucleotide sequence of D COF1 gene. The predicted initial ATG codon was double-underlined. Amino acid sequences determined by a gas-phase sequencer are indicated by underlines below the amino acid residues. The nucleotide sequence data of D COF1 and D COF2 will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with the following accession numbers D37980 and D37981, respectively.



Plasmid Construction

In order to determine the promoter activity of D COF1 and D COF2, we constructed plasmid vectors pDCOF1.BIG and pDCOF2.BIG, respectively, as follows. A DNA fragment was obtained from the pMC-1871 plasmid by digestion with SmaI and SalI. This fragment encodes whole bacterial -galactosidase except that the amino-terminal 9 residues (MTMITDSLA) were substituted by 3 residues (GDP). The fragment was inserted in plasmids pCOF1 and pCOF2, which had been pretreated successively with SalI and HincII, to construct pDCOF1 and pDCOF2, respectively. Both of the plasmids encode a fusion protein consisting of amino-terminal 40 residues of cofilin, three artificial residues (GDP), and carboxyl-terminal 1,015 residues of -galactosidase. The inserts of pDCOF1 and pDCOF2 were cut off by XbaI and XhoI digestions, and the inserts were ligated with pDNeO2 which had been treated with XbaI and XhoI. Both plasmids (termed pDCOF1T and pDCOF2T, respectively) contained an actin8 terminator just after the coding region of the cofilin-galactosidase fusion protein. The pDCOF1T and pDCOF2T were digested with HindIII and then treated with DNA polymerase I large fragment to make blunt ends. The linearized plasmids were further digested with XbaI, and produced insert DNA fragments were ligated with the shuttle vector pBIG which had been pretreated sequentially with BamHI, DNA polymerase I large fragment, and XbaI. The finally constructed plasmids were named pDCOF1.BIG and pDCOF2.BIG, respectively.

Construction of plasmid vectors for gene replacement experiments were performed as follows. The pCOF1 was digested with SalI and NdeI and self-ligated by T4 DNA ligase after both ends were blunted by DNA polymerase I large fragment. The produced plasmid was named pCOF1. The pCOF2 was digested by BstXI and BamHI and then treated with Exonuclease III. After both ends were blunted, the linear plasmid was self-ligated to construct pCOF2. This plasmid lacks 200 bp of 5`-nucleotides of pCOF2 insert. The pCOF3 insert was cut off by EcoRI digestion and inserted into an EcoRI site of pCOF1 and pCOF2 to construct pDCOF1 and pDCOF2, respectively. The pDCOF2 was treated by SalI and DNA polymerase I large fragment and then self-ligated by T4 DNA ligase to produce pDCOF2. The bsr gene fragment was cut off from pBsr2 (Sutoh, 1993) by XbaI and HindIII digestions, and both ends were blunted. The fragment was inserted into HincII site of pDCOF1 and pDCOF2 to produce pDCOF1:BSR and pDCOF2:BSR, respectively. The pDCOF1:BSR was digested by BssHII and PstI, and pDCOF2:BSR was digested by BssHII and EcoRV. The fragments were introduced into cells by electroporation.

Electroporation

Electroporation of DNA into Ax2 cells was performed as described (Egelhoff et al., 1991) with minor modifications. Briefly, Ax2 cells were grown in HL5 medium to 3 10 cells/ml. Cells (15 ml) were mixed with chilled 10 mM sodium phosphate buffer, pH 6.1, containing 50 mM sucrose (35 ml), and centrifuged at 120 g for 4 min. Pelleted cells were resuspended with 1 ml of chilled 10 mM sodium phosphate buffer, pH 6.1, containing 50 mM sucrose. DNA solution (5 µg in 5 µl) was mixed with 0.2 ml of suspended cells, and electroporation was performed at 0.45 kV and 3 microfarads in an E. coli Pulser Cuvette with 0.2-cm electrode gap (Bio-Rad) by Gene Pulser (Bio-Rad). After electroporation, cells were immediately mixed with 12 ml of HL5 medium and incubated at 22 °C for 16 h in a 9-cm dish. To select stable transformants, the medium was changed at every 3 days with a new HL5 medium containing 10 µg/ml neomycin or 10 µg/ml blasticidin S.

Preparation of Antiserum

Antiserum against D. discoideum cofilin was raised in a virgin female Japan white rabbit. The most pure cofilin fraction (500 µg) was further purified by excision of gels containing cofilin protein after staining the SDS-polyacrylamide gel electrophoresis (PAGE) gels with Coomassie Brilliant Blue (CBB). The excised gels (1.2 g) were washed twice with PBS, and homogenized in 2.4 ml of PBS (total 3.6 ml). The homogenate was sonicated four times at range 3 for 3 min. Freund's complete adjuvant (1.5 ml) was added to 1.5 ml of the sonicated cofilin solution, and after sonication the mixture was injected into a rabbit. Freund's incomplete adjuvant (0.75 ml) was mixed with the cofilin solution (0.75 ml) and injected at days 27 and 45. Sera collected at days 52 and 56 had sufficient and specific reactivity. For immunocytochemistry, we used affinity-purified antibodies with immobilized cofilin as described (Olmsted, 1981).

Sporulation on a Filter

Ax2 cells were grown to cell density of 2 10 cells/ml in HL5 medium in the presence or absence of selection materials. Cells were harvested by a centrifugation at 1,000 g for 5 min and washed once with 20 mM sodium phosphate buffer, pH 6.1. Cells were suspended in a cell density of 2 10 cells/ml, and 1 ml of the suspension was applied on a membrane filter HA (pore size 0.45 µm, 13 mm in diameter, Nihon Millipore Ltd., Tokyo, Japan) which was put on an AP10 Absorbent Pad (Nihon Millipore Ltd., Tokyo, Japan). The HA filter was then placed on a 9-cm dish containing 2% agarose in 20 mM sodium phosphate buffer and incubated in a dark chamber at 22 °C.

Indirect Immunostaining

Ax2 cells were grown on a circular glass coverslip (14 mm in diameter) in 24-well cell culture dish (Nunc A/S. Roskilde, Denmark) containing 1 ml of HL5 medium to a final cell density of 2 10 cells/mm (5 10 cells/well). Starvation stress was given by changing medium from HL5 medium to MCG solution (20 mM MES, 0.2 mM CaCl, 2 mM MgCl, pH 6.9) at time 0. At the indicated periods, cells were fixed by adding 0.1 volume of 37% formaldehyde solution to the medium and incubated for 15 min at 25 °C. After washing twice with PBS, cells were permeabilized and post-fixed by cold methanol at -20 °C for 5 min. After washing with PBS, the fixed cells were incubated in the blocking solution (10% goat serum in PBS) for 20 min at 25 °C and treated with the first antibodies for 16 h at 4 °C. For the first antibodies, affinity-purified anti-cofilin antibody, anti-cofilin antiserum, and monoclonal antibody against actin were used at 5, 100, and 8 dilution, respectively. The cells were subsequently treated with the second antibodies for 2 h at 25 °C. As the second antibodies, we used fluorescein-conjugated goat anti-rabbit IgG antibody and rhodamine-conjugated goat anti-mouse IgG antibody at 100 dilution. The cells were then washed with PBS, further washed briefly with distilled water, and mounted with mountant PermaFluor (Lipshaw Co., Pittsburgh, PA). For staining cells treated with dimethyl sulfoxide (MeSO), cells were grown on poly-L-lysine-coated cover glasses, and fixed by cold ethanol containing 1% formaldehyde at -20 °C for 5 min. The stained cells were observed and recorded under a confocal laser scanning microscopy MRC600 (Bio-Rad Laboratories, Tokyo, Japan) equipped with an Argon ion (25 milliwatt) and Helium/Neon (0.3 milliwatt) dual laser system, Nikon optiphot-2, and super high pressure Mercury lamp power supply, and a Nikon Plan Apo60 oil immersion objective (Nikon Co., Tokyo, Japan).

Other Methods

Protein concentrations were determined by the Bio-Rad protein assay system (Bio-Rad) with goat -globin as a standard. SDS-PAGE was carried out according to the method of Laemmli (1970) with 10-20% gradient gel, Multigel 10/20 (Daiichi Pure Chemicals Co., Tokyo, Japan). Molecular mass markers for SDS-PAGE were purchased from Bio-Rad; myosin (200 kDa), -galactosidase (116 kDa), phsophorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Gels were stained by CBB R-250. Western blotting was performed as previously reported (Towbin et al., 1979). Proteins were transferred from gels to Clear Blot Membrane-p (ATTO Co., Tokyo, Japan) by semi-wet Transfer System (ATTO Co.). The membranes were treated with rabbit antiserum against D. discoideum cofilin at 2,000 dilution with blocking solution (5% skim milk in PBS). Alkaline phosphatase-conjugated goat anti-rabbit IgG was then used as a second antibody. Reacted proteins were visualized by adding nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate as substrates. -Galactosidase activity was determined using the -Galactosidase Enzyme Assay System with Reporter Lysis Buffer (Promega Co., Madison, WI).


RESULTS

Identification and Purification of an Actin Monomer-binding Protein with M 15,000

Since the cofilin family was first identified biochemically as an actin monomer-binding protein using DNase I affinity chromatography, we sought for actin monomer binding proteins in cell extracts of D. discoideum as described under ``Experimental Procedures'' (Fig. 1 A). Crude cell extracts contain about 12% (w/w) actin, and most of the actin molecules were adsorbed by a DNase I affinity column (Fig. 1, lanes 1 and 2). The adsorbed materials were eluted with 0.6 M NaCl in MEM buffer and subsequently with 2 M urea in MEM buffer. In the 0.6 M NaCl eluent, we detected five major proteins with 95, 70, 55, 33, and 15 kDa together with a number of minor components (Fig. 1, lanes 3-6). The 15-kDa protein appeared to be eluted relatively slowly compared to the other eluted proteins. Subsequent treatment with 2 M urea dissociated 40-, 33-, and 30-kDa proteins as well as 42-kDa actin from the column (Fig. 1, lanes 7-10). In order to purify the 15-kDa protein, we eluted the adsorbed materials with a linear gradient of NaCl from 0 to 600 mM. Fractions containing the 15-kDa protein (Fig. 1 B, lanes 11-16) were collected and applied onto a hydroxyapatite column and fractionated with a linear gradient of sodium phosphate (10-100 mM) (Fig. 1 C). The purity of the 15-kDa protein in final fractions was higher than 95% (Fig. 1 C, lanes 9 and 10).


Figure 1: Identification and purification of actin monomer-binding protein with M 15,000 form D. discoideum. A, identification of actin monomer-binding proteins by DNase I column chromatography. Crude extracts ( lane 1) were prepared by centrifugation from sonicated cell homogenates at 400,000 g for 1 h as described under ``Experimental Procedures.'' The extracts were loaded on a DNase I column, and unbounded materials ( lane 2) were washed through the column. Proteins adsorbed to the DNase I column were eluted sequentially by 0.6 M NaCl solution ( lanes 3-6) and 2 M urea solution ( lanes 7-10). Molecular weights of marker proteins ( lane M) are indicated on the right side in thousands. B, DNase I column chromatography. Crude extracts were fractionated by DNase I column chromatography with a liner gradient of NaCl from 0 to 600 mM. Fraction numbers are indicated on the top of each lane. Fractions containing the 15-kDa protein ( lanes 10-15) were collected for the next purification step. M, marker proteins. C, hydroxylapatite column chromatography. The DNase I column fractions were further fractionated by hydroxylapatite column chromatography with a linear gradient of sodium phosphate buffer from 10 to 100 mM. Fraction numbers are indicated on the top of each lane. Fractions 9 and 10 were collected and used as a purified 15-kDa protein fraction. All of the fractions were analyzed by SDS-PAGE using 10-20% gradient gel.



Biochemical Characterization of the 15-kDa Protein

Cofilin has biochemical activities to sequester actin monomers and to sever actin filaments, and it was reported that cofilin depolymerized actin filaments and decreased low shear viscosity of actin solutions (Stephen et al., 1993). Before starting molecular cloning of the 15-kDa protein, we investigated a few biochemical activities of the protein in order to confirm that the protein is a candidate of D. discoideum cofilin. First, we examined actin filament-depolymerizing activity of the protein. When the 15-kDa protein was added to actin filaments, it depolymerized the filaments in a dose-dependent manner (Fig. 2 A). We also demonstrated that the fluorescence intensity of pyrene-labeled actin filaments decreased by addition of the 15-kDa protein in a dose-dependent manner (Fig. 2 B). These results suggested that the 15-kDa protein has a monomer sequestering activity. Next, we revealed that the 15-kDa protein decreased the apparent viscosity of actin filaments in a dose-dependent manner (Fig. 2 C). These biochemical activities of the 15-kDa protein resemble those of cofilin proteins in other organisms, although we cannot definitively conclude that the 15-kDa protein severs actin filaments only from our data. It was reported that an interaction of porcine cofilin or destrin with actin was inhibited by phosphoinositides (Yonezawa et al., 1990). We investigated effects of various phospholipids on the activity to decrease the low shear viscosity of the 15-kDa protein. Among phospholipids tested, only phosphatidylinositides inhibited the activity (Fig. 3 A). Two hydrolyzed products of PIP by phospholipase C, IP, and 1-oleoly-2-acetylglycerol had no effect on the activity. PI, PIP, and PIP inhibited the severing activity of the 15-kDa protein (4 µM) to 50% at 200, 45, and 30 µM, respectively (Fig. 3 B).


Figure 2: Actin filament depolymerizing activity of the 15-kDa protein. A, effects of the 15-kDa protein on the amount of polymerized actin. Polymerized actin (17.8 µM) was diluted into solutions containing various concentrations of the 15-kDa protein. Final ionic conditions were 33.8 mM MES, 81.8 mM KCl, 2.9 mM MgCl, 36 µM ATP for pH 6.8, and 3.8 mM MES, 81.8, mM KCl, 2.9 mM MgCl, 36 µM ATP, 30 mM Tris for pH 8.3. Final actin concentration was 3.2 µM in all experiments. Critical concentrations of actin were 0.08 µM, pH 6.8, and 0.2 µM, pH 8.3. Calculated amounts of sedimented actin ( closed symbols) and 15-kDa protein ( open symbols) at pH 6.8 ( squares) and pH 8.3 ( circles) are plotted. B, effects of the 15-kDa protein on the fluorescence of pyrene-labeled actin filaments. Polymerized actin (7.5% pyrene-labeled actin) was mixed with the 15-kDa protein at zero time, and the amount of polymerized actin was monitored by fluorescence at 25 °C. The assay buffers contained 10 mM MES, 50 mM KCl, 1 mM EGTA, 0.5 mM ATP, 1 mM MgCl, pH 6.9. The actin concentration was 4.0 µM in all experiments. The concentrations of the 15-kDa proteins are 0 µM (), 1 µM (), 2.4 µM (), 4.7 µM (), 9.4 µM (), and 18.8 µM (). C, effects of the 15-kDa protein on low shear viscosity of actin solutions. Actin monomer (5 µM) was polymerized by adding 0.1 volume of 10F buffer at 25 °C for 2 h in the presence of various amounts of the 15-kDa protein. Low shear viscosity was determined by falling ball tests.




Figure 3: Effects of phospholipids on the reduction in low shear viscosity of actin solutions by the 15-kDa protein. A, various phospholipids were mixed with actin in the presence ( hatched column) or absence ( white column) of the 15-kDa protein. Actin was polymerized by adding of 0.1 volume of 10xF buffer at 25 °C for 2 h. Final concentrations of phospholipids, actin, and the 15-kDa protein were 200 µM, 5 µM, and 4 µM, respectively. B, Dose dependence of the phospholipid effects. Various concentrations of PI (), PIP (), and PIP () were mixed with actin (5 µM) and cofilin (4 µM). Actin was polymerized by adding 0.1 volume of 10F buffer at 25 °C for 2 h. , actin only. Low shear viscosity was determined by falling ball tests.



Isolation of D. discoideum Genomic DNA Clones Encoding the 15-kDa Protein

In order to confirm that the 15-kDa protein is a member of the cofilin family, we isolated genomic DNA clones encoding the 15-kDa protein as follows. First, we determined amino acid sequences of two proteolytic fragments derived from the protein. The obtained sequences are YGGIIYRISDDSK and XLPENECRYVVLDYQYK, in which the letter X denotes an unidentified amino acid residue. Next, we synthesized two degenerated oligonucleotide primers corresponding to the two determined sequences, respectively. RT-PCR with the two primers amplified a cDNA fragment of 119 bp which encoded a part of the 15-kDa protein (Fig. 4 A, nucleotides 510-628 in Fig. 4 C). The amino acid sequence of 40 residues deduced from the amplified cDNA showed 38.5% identity to a part of yeast cofilin sequence (Fig. 5). This sequence similarity indicated that the 15-kDa protein is D. discoideum cofilin. Genomic Southern analysis using the amplified fragment as a probe revealed that complete digestion of genomic DNA by EcoRI produced three reactive fragments of 2.3, 1.8, and 0.3 kbp. Since the amplified fragment itself contains a single restriction site of EcoRI, D. discoideum genome is considered to contain more than one gene for cofilin. Using this amplified cDNA fragment as a probe, we screened an EcoRI genomic library of D. discoideum for entire genes encoding cofilin. We isolated three different positive clones possibly encoding cofilin, COF1 (2.3 kbp), COF2 (1.8 kbp), and COF3 (0.3 kbp). Nucleotide sequence analysis revealed that both COF1 and COF2 have a common nucleotide sequence corresponding to the 5` part of amplified cDNA EcoRI fragment, and COF3 has a sequence corresponding to the 3` part of the amplified cDNA EcoRI fragment. These results suggested the presence of two genes possibly encoding cofilin. We termed the two genes D COF1 and D COF2, respectively. Sequence analysis revealed that the two genes have identical nucleotide sequences except for their promoter and intron regions (Fig. 4, A and C). No different nucleotide sequence was detected even in the 5`- and 3`-noncoding regions. We synthesized an oligonucleotide M1 (AAAAAACTATATATAAAAAATG; nucleotides 55-76 in Fig. 4 C), and an oligonucleotide M2 (TCAAATTATTTAGATTTTGG; complementary sequence of nucleotides 844-863 in Fig. 4 C). The PCR of genomic DNA using M1 and M2 oligonucleotides as primers amplified two DNA fragments of 809 and 442 bp, respectively. Nucleotide sequence analysis revealed that the two fragments were derived from D COF1 and D COF2 genes, respectively. These results confirmed the predicted gene structures of D COF1 and D COF2 shown in Fig. 4 A. D COF1 gene contained a predicted intron region just after the initiation methionine codon. We amplified cofilin mRNA from total RNA by RT-PCR using the M1 and M2 oligonucleotides as primers, and nucleotide sequence analysis of an amplified 442-bp fragment revealed that the predicted intron region in D COF1 gene was really cut off in the mRNA (data not shown). Both of the two cofilin genes encode the same protein consisting of 137 amino acid residues, and the predicted molecular weight and calculated isoelectric point are 15,224 and 6.37, respectively. The predicted amino acid sequence contained both of the two partial amino acid sequences of the 15-kDa protein determined by a gas-phase amino acid sequencer (Fig. 4 C). The amino acid sequence of D. discoideum cofilin shows a significant homology, throughout its entire molecule, to those of proteins belonging to the cofilin family (Fig. 5). The two regions that were putatively identified as actin-binding regions were also well conserved in the D. discoideum cofilin. The percent identity/similarity of Acanthamoeba actophorin, yeast cofilin, pig destrin, and starfish depactin to the D. discoideum cofilin was 38/60%, 42/61%, 25/44%, and 15/30%, respectively.


Figure 5: Comparison of D. discoideum cofilin sequence with other cofilin family members. The amino acid sequences of D. discoideum cofilin ( D. Cof), Acanthamoeba actophorin ( A. Act), yeast cofilin ( Y. Cof), pig destrin ( P. Des), and starfish depactin ( S. Dep) were compared. Residue numbers are indicated on right side. Residues conserved among all of the five members in Dayhoff's criteria are boxed. Among them, completely identical residues among the 5 are indicated by shading. Two actin-binding sites determined by cross-linking experiments and synthetic peptide experiments are also denoted by horizontal double lines. All the sequence data except for D. Cof were obtained from Genbank.



Expression and Disruption of the Two Cofilin Genes

The expression of the D COF1 and D COF2 was examined. Since the two COF genes are identical in their coding and noncoding sequences, we determined the promotor activities of the two genes. We made chimeric genes which encode an in-frame bacterial -galactosidase gene adjacent to the HincII restriction site of each COF gene (Fig. 6 A). These chimeric genes were separately inserted into the extrachromosomal plasmid vector, pBIG, and introduced into D. discoideum cells by electroporation. Stable transformants containing the extrachromosomal plasmids were selected in medium containing neomycin. The promoter activities of D COF1 and D COF2 genes were estimated by measuring the activities of -galactosidase expressed in both vegetative and differentiating D. discoideum cells carrying the plasmids (Fig. 6 B). The D COF1 promoter was active in vegetative cells. When cells were induced to enter the spore formation process on a filter, the activity transiently increased and then gradually decreased during spore formation. Little -galactosidase activity was detected in mature spores. In contrast, we did not detect any promoter activity of D COF2 under the experimental conditions examined. This suggests that D COF2 might be a pseudogene although it does not contain any stop codon.


Figure 6: Promoter activities of the cofilin genes. A, construction of reporter genes for promoter assay. Plasmids (pDCOF1.BIG and pDCOF2.BIG) were constructed as described under ``Experimental Procedures.'' Briefly, bacterial -galactosidase gene ( shaded box) was fused to the HincII site of each of the cofilin genes (presented as in Fig. 4 A), and a termination signal ( A8-T, dashed lines) was added to its 3`-flanking region. The linear DNAs were inserted in an extrachromosomal vector plasmid, pBIG, respectively. The copy number of pBIG is reported to be 50-100 copies/cell. The pBIG also contains a resistant gene against neomycin. B, -galactosidase activity of Ax2 cells carrying the reporter plasmids. Ax2 cells carrying extrachromosomal reporter plasmid pDCOF1.BIG () or pDCOF2.BIG () were selected in HL5 medium containing 10 µg/ml neomycin after electroporation. The selected cells (2 10 cells) were induced to differentiate on a Millipore filter, harvested at indicated times, and lysed in 200 µl of lysis buffer. As a background, endogenous -galactosidase activity was also determined at each differentiation point with Ax2 cells carrying pBIG. No significant endogenous -galactosidase activity was detected with the control cells in all experiments.



Next, we examined whether disruption of either or both of the COF genes caused abnormal phenotypes. Gene targeting vectors were constructed using a recently developed bsr gene cassette as a selection marker (Fig. 7 A). After electroporation of the targeting vectors, cells harboring the replaced genes were selected by a medium containing blasticidin S. After several passages, each of the cloned cell lines was subjected to genomic Southern analysis to check whether or not the endogenous COF gene was replaced by the exogenous targeting vector (Fig. 7, B and C). We randomly picked up 20 independently selected cell lines and found that the endogenous D COF2 gene was replaced by the exogenous targeting vector in all of them (Fig. 7 B). This result suggested that homologous recombination occurred efficiently under experimental conditions employed. This also suggested that D COF2 is not an essential gene for viability of D. discoideum slime. Western blot analysis using anti-cofilin antibodies (see below) revealed that the content of cofilin was not significantly altered by the disruption of D COF2 (data not shown). This result is consistent with the above observation that the D COF2 promoter was not active (Fig. 6 B). No abnormal phenotype was associated with the D COF2-disruptants in either vegetative or sporulation stages. These results indicated that all of the cofilin protein was derived from D COF1 gene in vegetative and differentiating cells.


Figure 7: Gene targeting of cofilin genes. A, schematic representation of DNA fragments of pDCOF1:BSR and pDCOF2:BSR for gene replacement experiments. Both of the plasmids contain a selection marker gene for blasticidin S in their single HincII site and lack the endogenous EcoRI site at the 5` border region (see Fig. 4 A). Arrows indicate the bsr gene cassette and the direction of transcription. The cofilin genes are presented as in Fig. 4 A. B, genomic Southern analysis of cloned stable transformants with pDCOF2:BSR. DNA fragments of pDCOF2:BSR were electroporated into Ax2 cells. After selection with blasticidin S, transformant clones were picked up randomly. DNA (1 µg) prepared from each clone was digested completely by EcoRI, electrophoresed in 0.8% agarose gel, and hybridized with a DNA probe (nucleotides 444-596 in Fig. 5 C). The results with eight transformants ( lanes 1-8) and a control Ax2 cell ( lane 9) are shown. Homologous recombination was expected to insert the bsr gene into the HincII site of D COF2 resulting in a shift of hybridized band from 1.8 to 3.2 kbp. The sizes of positive bands corresponding to D COF1, D COF2, and D COF2::bsr are indicated on the right side, respectively. C, genomic Southern analysis of cloned stable transformants with pDCOF1:BSR. DNA fragments of pDCOF1:BSR were electroporated into Ax2 cells. Then genomic Southern blotting was performed as in B. Six clonal transformants were picked up randomly ( lanes 1-6) and subjected to Southern analysis. The result with a control Ax2 cell ( lane 7) is also shown. D COF1::bsr was expected to be 3.7 kbp.



Four days after electroporation of D COF1 targeting vector, we identified as many colonies each of which consisted of about 100 cells as in the case of D COF2 targeting experiments. But in the case of D COF1 gene targeting experiments, almost all the observed colonies disappeared by day 6 after electroporation, and less than 3% of the colonies were able to survive as stable transformants. In the survived transformants, no homologous recombination for D COF1 was detected (Fig. 7 C). We obtained only one homologous recombinant event using the D COF1 targeting vector but found that the replaced endogenous gene was D COF2 but not D COF1 (Fig. 7 C, lane 6). The D COF1 targeting vector has homologous regions, which consist of 160 and 360 nucleotides at the 5`- and 3`-bordering regions of the bsr cassette, respectively, for D COF2 gene as well as D COF1 gene. These results are consistent with the notion that D COF1 is essential for the viability of this organism as is the case for yeast COF1 (Iida et al., 1993; Moon et al., 1993). However, we could not completely rule out the possibility that the region of D COF1 gene in the D. discoideum chromosomes is not available for homologous recombination.

Intracellular Localization of Cofilin

We examined the intracellular distribution of the protein by indirect immunofluorescence microscopy. A rabbit antiserum was raised against purified D. discoideum cofilin. This antiserum specifically reacted to cofilin on Western blotting (Fig. 8 A). We determined the content of cofilin in cells undergoing spore formation on a nylon filter. The total amount of cofilin in cells of D. discoideum was constant throughout the sporulation processes on a filter (Fig. 8 B).


Figure 8: Western blotting of cofilin protein in D. discoideum Ax2 cells. A, the specificity of a polyclonal antiserum against cofilin. Purified cofilin (0.2 µg, lanes 1 and 2) and total Ax2 cell proteins (30 µg, lanes 3 and 4) were resolved by SDS-PAGE. Lanes 1 and 3, CBB staining. Lanes 2 and 4, Western blotting with 2,000 diluted anti-cofilin antiserum. B, quantitative analysis of cofilin at various stages of sporulation. Ax2 cells (2 10 cells) on Millipore filters were incubated under sporulation-inducing conditions for 0 h ( lanes 1 and 8), 4 h ( lanes 2 and 9), 8 h ( lanes 3 and 10), 12 h ( lanes 4 and 11), 16 h ( lanes 5 and 12), 20 h ( lanes 6 and 13), and 24 h ( lanes 7 and 14), respectively. Samples prepared from 6 10 cells were analyzed by SDS-PAGE and Western blotting with 2,000 diluted anti-cofilin antiserum. Lanes 1-7, CBB staining; lanes 8-14, Western blotting. M, marker proteins.



It has been known that starvation stress induces cell-shape change and tight substrate adhesion of D. discoideum cells. We examined intracellular distribution of cofilin along this response in comparison with that of actin by indirect fluorescence immunocytochemistry. In vegetative cells, the majority of cofilin was distributed uniformly in the cytoplasm although tiny dots of the distribution were also observed (Fig. 9 A). Unlike cofilin, actin was localized at cell cortex especially at the crown-like structures as well as at cytoplasmic region (Fig. 9 B). Four h after starting starvation, the crown-like structures disappeared and almost all the cells were flattened and tightly attached to substratum. In these flattened cells, actin and cofilin were co-localized in peripheral regions (Fig. 10, K and L), which periodically extend thin sheet-like processes known as lamellipodia from the leading edge. Optical sectioning observation of the cell clearly indicated that cofilin and actin co-localized at the lamellipodium (Fig. 10, I and J). Confocal sectioning images also revealed that these flattened cells are about 3 µm thick while vegetative cells are about 12 µm thick. In the flattened cell, actin also accumulated at the close contact site with substratum (Fig. 10 B), which located at about 1.5 µm inside from the advancing edge of the cell (Fig. 10 L). We revealed that cofilin did not localize at the focal adhesion site (Fig. 10 A).


Figure 9: Intracellular localization of cofilin and actin in vegetative cells. Ax2 cells were grown to 2 10 cells/mm in a 24-well dish containing HL5 medium. The cells were fixed and doubly stained with affinity-purified anti-cofilin antibody ( A) and anti-actin monoclonal antibody ( B). Optically sectioned images of fluorescein and rhodamine stainings were simultaneously recorded by laser scanning confocal microscopy. Optically sectioned images at 0.8 µm intervals along the vertical axis ( z axis) were collected as Z-series. Each sectioned figure at 6.4, 4.0, and 1.6 µm above the glass substrate are shown from the top to the bottom. We also demonstrated the reconstituted figures by projecting all the Z-series at the bottom panels. Arrows indicate crown-like structures. Bar, 10 µm.




Figure 10: Intracellular localization of cofilin and actin in Ax2 cells under starvation stress. Cells were grown and developed for 4 h as in Fig. 9 and doubly stained with affinity-purified anti-cofilin antibody ( A, C, E, G, I, and K) and anti-actin monoclonal antibody ( B, D, F, H, J, and L). Optical sectioned images were recorded by laser scanning confocal microscopy at 0.5 µm intervals along the vertical axis. Sectioned images at 0 µm ( A and B), 1 µm ( C and D), 2 µm ( E and F), 3 µm ( G and H), and 4 µm ( I and J) from the glass substrate are shown. Reconstituted figures by projecting all the Z-series are also shown ( K and L). Arrows indicate lamellipodia. Arrowheads indicate focal adhesion sites. Bar, 10 µm.



Intranuclear Actin-Cofilin Rods in D. discoideum

It has been previously reported that MeSO treatment induced reorganization of actin structures and formed intranuclear actin rods in D. discoideum cells (Fukui, 1978). In mammalian cells, the rods induced by various stresses contain cofilin as a major component in addition to actin (Nishida et al., 1987). Thus we examined whether D. discoideum cofilin is redistributed to the nucleus and forms the rods in response to the MeSO treatment of growing cells (Fig. 11). Indirect immunofluorescence experiments revealed that both cytoplasmic cofilin and cortical actin were redistributed into the nucleus after treatment with 10% (v/v) MeSO (Fig. 11).


Figure 11: Intracellular localization of cofilin and actin in cells treated with MeSO. Ax2 cells were grown in HL5 medium ( A and B) and treated with 10% (v/v) MeSO in HL5 medium for 30 min ( C and D). Cells were fixed and immunofluorescently stained with affinity-purified anti-cofilin antibody ( A and C) and anti-actin monoclonal antibody ( B and D). Bar, 25 µm.




DISCUSSION

In this paper, we described identification, biochemical characterization, primary structure, gene structure, and intracellular localization of D. discoideum cofilin. In addition, we demonstrated that starvation stress leading to cell flattening, which was associated with reorganization of actin structures, induced translocation of cofilin from cytosol to ruffling membranes. Cofilin was also shown to translocate to the nucleus when the slime was treated with MeSO.

Mammalian cofilin and destrin have been reported to interact with actin monomer at a 1:1 molar ratio (Nishida et al., 1984). On the assumption that D. discoideum cofilin also interact with actin at 1:1 molar ratio, we estimated the dissociation constant of cofilin and actin monomer to be 0.55 µM, pH 6.8, and 0.74 µM, pH 8.3, from the data in Fig. 2 A. We also estimated the dissociation constant of cofilin and actin filaments to be 20.2 µM, pH 6.8, and 9.6 µM, pH 8.3. Mammals have at least two distinct members of cofilin family, cofilin and destrin (Nishida et al., 1985). The mammalian cofilin can bind to actin filaments as well as actin monomer, and its sequestering and severing activities are pH-dependent. On the other hand, mammalian destrin does not bind to actin filaments, and its sequestering and severing activities are pH independent in vitro. D. discoideum cofilin was able to bind to actin filaments, and the activity was pH-dependent. The lethality of yeast cells caused by the disruption of the yeast COF1 gene was complemented by the expression of either mammalian cofilin or destrin (Iida et al., 1993). This result indicated that the two proteins share common in vivo functions essential for the viability of the mutant yeast cells. It is unclear, therefore, that differences in the biochemical properties of cofilin and destrin in vitro are biologically significant.

The total actin concentration in D. discoideum cells was calculated to be 300 µM, and about a half of them are polymerized in vivo (Hall et al., 1988; Dharmawardhane et al., 1991). Thus, the concentration of unpolymerized actin is apparently 150 µM in vivo, while the critical concentration of purified actin is 0.08 and 0.2 µM at pH 6.8 and 8.3, respectively, in vitro. We demonstrated that D. discoideum cofilin sequestered actin monomers from polymerization in vitro. We estimated the cofilin concentration in Ax2 cells to be 100 µM by densitometric analysis of SDS-PAGE gels.() We observed that cofilin was diffusely distributed in the cytoplasm in vegetative cells but little in cell cortex or crown-like structures that are rich in filamentous actin. These results suggest that cofilin is one of major factors which sequester actin monomers for polymerization in vegetative cells. Another actin monomer sequestering protein, profilin, has been previously identified in D. discoideum (Haugwitz et al., 1994). The concentration of profilin was estimated to be about 100 µM in D. discoideum cells. Thus, it would be possible that cofilin and profilin might be co-operatively involved in regulation of the monomer-polymer transition of actin in vivo.

We revealed that D. discoideum cells contain two cofilin genes, D COF1 and D COF2. Gene disruption of D COF2 did not cause any change in protein amount of cofilin, and the mutant cells showed completely the same phenotype as wild cells. This is consistent with the result that the promoter activity of D COF2 was not detected in our experiments. D COF2 gene lacks an intron which exists in D COF1 gene. These are well-known common features of processed pseudogenes (Lee et al., 1983), and it is considered that D COF2 gene is a pseudogene. However, we cannot completely rule out the possibility that the D COF2 gene might express during processes, which we did not examine in this study, such as macrocyst formation and mating processes.

Cofilin accumulated in the lamellipodia of migrating cells exposed to starvation stress. We also revealed that cofilin localized to the filopodia which appeared in the head and tail regions of aggregating cells for cell-cell association. Some of these lamellipodia and filopodia adhered to the substratum or to other cells, while others failed to adhere, curled back over the top of the cell, and were swept backward as ``ruffles.'' In these regions, actin filaments are supposed to be assembled, contract, and disappear dynamically according to the cell movement. We suggest that cofilin plays an important role in the reorganization of actin filaments in these highly moving regions of cells. This is consistent with the facts that members of the cofilin family localized at lamellipodia of Acanthamoeba (Quirk et al., 1993), ruffling membranes of cultured fibroblasts (Yonezawa et al., 1987), filopodia of neuronal growth cones (Bamberg and Bray, 1987), and cortical actin patches of yeast (Moon et al., 1993), all of which move dynamically. We also revealed for the first time that cofilin did not localize at the focal adhesion sites where rigid actin meshwork architectures were constructed. It is suggested that cofilin does not involved in the formation and maintenance of the rigid actin structures.

When mammalian cultured cells were exposed to various stresses such as heat-shock or MeSO treatment, cofilin and actin cotranslocated to the nucleus and formed actin-cofilin rods (Nishida et al., 1987). Mammalian cofilin has a short stretch of basic amino acid residues (KKRKK) at residues 39-43 which is similar to the SV40 large T-antigen nuclear localizing signal (Matsuzaki et al., 1988). Furthermore, the KKRKK sequence has been proven essential for translocation of cofilin to the nucleus in mammalian cells (Iida et al., 1992). The corresponding region of D. discoideum cofilin contains the sequence GRKYG that are homologous only in 2 out of 5 amino acid residues to the nuclear localization signal of mammalian cofilin. We demonstrated here, however, that MeSO treatment of D. discoideum cells induced translocation of cofilin into the nucleus and formation of actin-cofilin rods. This prompts us to re-examine the previously identified nuclear localization signal sequence in mammalian cofilin. The formation of intranuclear actin-cofilin rod appears to be a well-conserved response against extracellular stress signals among eukaryotic cells, including D. mucoroides (Fukui, 1978) and tetrahymena (Katsumaru and Fukui, 1982) although actin rod was not induced in yeast (Moon et al., 1993).()


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-3-3823-2101; Fax: 81-3-5685-2932.

The abbreviations used are: PI, phosphatidylinositol; CBB, Coomassie Brilliant Blue; IP inositol 1,4,5-triphosphate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PCR, polymerase chain reaction; PIP, phosphatidylinositol monophosphate; PIP, phosphatidylinositol bisphosphate; PS, phosphatidylserine; RT, reverse transcriptase; MES, 4-morphoinepropanesulfonic acid; bp, base pair(s); kbp, kilobase pair(s).

H. Awzawa, K. Sutoh, S. Tsubuki, S. Kawashima, A. Ishii, and I. Yahara, unpublished results.

K. Iida, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. J. Spudich and Dr. A. Noegel for their generous gifts of a D. discoideum strain and vectors. We thank Dr. Y. Fukui, Dr. K. Iida, Dr. S. Matsumoto, Dr. Y. Miyata, and M. K. Moriyama for various technical advises and fruitful suggestions.


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