From the
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.
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.
The 15-kDa protein was purified from D. discoideum cells as follows. Ax2 cells were grown to a cell density of 1
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
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 Me
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
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 Me
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
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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 Na
HPO
, 1.7
mM KH
PO
, 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 10
F-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 (M
TMITDSLA
) 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 pDCOF1
T and pDCOF2
T, respectively) contained
an actin8 terminator just after the coding region of the
cofilin-galactosidase fusion protein. The pDCOF1
T and pDCOF2
T
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.
. 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 (Me
SO), 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).
Identification and Purification of an Actin
Monomer-binding Protein with M
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).
15,000
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 10
F
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 10
F 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 Me
SO 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) Me
SO
(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)
Me
SO 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.
SO.
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.
SO 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 Me
SO 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).
(
)
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).
. K. Moriyama for various technical advises and
fruitful suggestions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.