Department of Pathology, Emory University, Whitehead IO5N, Atlanta, Georgia 30322, USA
* Author for correspondence (e-mail: sono{at}emory.edu)
Accepted 7 February 2003
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Summary |
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Key words: Myofibrils, Thin filaments, Actin dynamics, Embryogenesis, Cytokinesis
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Introduction |
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Actin depolymerizing factor (ADF)/cofilins are a family of actin-binding
proteins that promote rapid turnover of the actin cytoskeleton
(Bamburg, 1999;
Bamburg et al., 1999
;
Carlier et al., 1999
;
Maciver and Hussey, 2002
).
ADF/cofilins preferentially bind to ADP-actin
(Carlier et al., 1997
;
Maciver and Weeds, 1994
) and
enhance the turnover of actin filaments by increasing the rate of
depolymerization from their pointed ends
(Carlier et al., 1997
;
Maciver et al., 1998
) and by
severing filaments (Du and Frieden,
1998
; Hawkins et al.,
1993
; Hayden et al.,
1993
; Ichetovkin et al.,
2000
; Maciver et al.,
1991
; Nishida et al.,
1984
; Nishida et al.,
1985
). Filament binding by ADF/cofilin changes the twist of the
actin filaments (McGough et al.,
1997
) and weakens lateral contacts in the filaments
(McGough and Chiu, 1999
).
ADF/cofilin binds to F-actin in a cooperative manner and generates an unstable
population of filaments (McGough et al.,
1997
; Ressad et al.,
1998
).
Multicellular organisms have multiple ADF/cofilin genes that are often
expressed in tissue-specific patterns, whereas simple organisms, such as
yeast, have a single essential gene for cofilin
(Iida et al., 1993;
Lappalainen and Drubin, 1997
;
Lappalainen et al., 1997
;
Moon et al., 1993
).
Vertebrates have two or three ADF/cofilin genes that are designated as ADF
(also known as destrin) (Abe et al.,
1990
; Adams et al.,
1990
; Moriyama et al.,
1990
), non-muscle-type cofilin/cofilin-1
(Matsuzaki et al., 1988
) and
muscle-type cofilin/cofilin-2 (Gillett et
al., 1996
; Ono et al.,
1994
; Thirion et al.,
2001
). In mice, cofilin-1 is expressed in most cell types, whereas
cofilin-2 is predominant in muscle cells and ADF is specific for epithelia and
endothelia (Mohri et al.,
2000
; Ono et al.,
1994
; Vartiainen et al.,
2002
). These ADF/cofilin isoforms exhibit different
actin-regulatory activities. ADF is more potent in depolymerization than
cofilin (Abe and Obinata, 1989
;
Giuliano et al., 1988
;
Nishida et al., 1985
), whereas
cofilin apparently remains bound to filaments
(Abe et al., 1989
;
Nishida et al., 1984
).
Detailed comparisons of ADF and cofilin revealed that they have nearly
identical activities to accelerate depolymerization and filament severing, but
are different in their nucleating activities to initiate polymerization when
they make complexes with actin (Yeoh et
al., 2002
). The cofilin-actin complex initiates spontaneous
polymerization more efficiently than the ADF-actin complex. The difference is
significant at a high pH and confers their pH-sensitive activities
(Yeoh et al., 2002
;
Yonezawa et al., 1985
).
Cofilin-2 is more effective in enhancing polymerization than ADF and cofilin-1
(Vartiainen et al., 2002
),
suggesting that the critical concentration of the cofilin-2-actin complex is
very low.
In cells where multiple ADF/cofilin isoforms are expressed, the isoforms
behave very similarly in localizing to stress-induced intranuclear actin rods
(Ono et al., 1993) and in
translocating to the lamellipodia after growth factor stimulation
(Meberg et al., 1998
). Also,
many extracellular stimuli regulate phosphorylation levels of both ADF and
cofilin in common pathways (Kanamori et
al., 1995
; Meberg et al.,
1998
; Saito et al.,
1994
). However, ADF and cofilin respond differently to changes in
some of the cellular states. Colocalization of ADF with monomeric actin is
enhanced upon an increase in intracellular pH, whereas that of cofilin is less
sensitive to pH changes (Bernstein et al.,
2000
), which is consistent with their in vitro properties
(Yeoh et al., 2002
).
Expression of ADF, but not cofilin, is downregulated by an increase in the
actin monomer pool (Minamide et al.,
1997
). In contrast, expression of cofilin, but not ADF, is
upregulated in dystrophic muscles
(Hayakawa et al., 1993
;
Nagaoka et al., 1996
). Thus,
ADF and cofilin might have both redundant and non-redundant functions.
There are some functional differences among ADF/cofilin isoforms from
studies using model organisms in which mutations in ADF/cofilin genes have
been isolated. The slime mold Dictyostelium discoideum has two
cofilin genes, cofilin-1 and cofilin-2
(Aizawa et al., 2001). A null
mutant of cofilin-1 has not been isolated in previous attempts of gene
knockout, suggesting that cofilin-1 is essential for cell viability
(Aizawa et al., 1995
), whereas
cofilin-2-null cells develop normally
(Aizawa et al., 2001
). The
nematode Caenorhabditis elegans expresses two ADF/cofilins from the
unc-60 gene (McKim et al.,
1994
; Ono and Benian,
1998
). Point mutations of UNC-60B, a muscle-specific isoform,
cause defects in actin organization specifically in body wall muscle
(Ono et al., 1999
). A lethal
unc-60 mutation has been isolated, but the responsible isoform for
lethality has not been identified (McKim
et al., 1994
). Multiple ADF/cofilin isoforms have also been
identified in Drosophila (Edwards
et al., 1994
; Goldstein and
Gunawardena, 2000
; Gunsalus et
al., 1995
) and plants (Dong et
al., 2001a
; Dong et al.,
2001b
; Lopez et al.,
1996
), but functional characterization and comparison of the
isoforms are still limited. In this study, we investigated the function of the
two ADF/cofilin isoforms in C. elegans using isoform-specific RNA
interference and gene knockout. We found that each isoform was required for
distinct actin-dependent processes. The results suggest that multicellular
organisms have functionally different ADF/cofilin isoforms to support the
complexity of tissue organization.
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Materials and Methods |
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Northern blot
A northern blot containing total RNA from different developmental stages
was kindly provided by Edward Kipreos (University of Georgia, Athens,
Georgia). This blot was first hybridized with a probe for 18S rRNA as
described previously (Kipreos et al.,
1996), then stripped and re-hybridized with a probe specific for
unc-60A, stripped and re-hybridized with a probe specific for unc-60B. unc-60
probes were created by PCR from a cDNA library (kindly provided by Robert
Barstead, Oklahoma Medical Research Foundation, Oklahoma City, OK), and 50 ng
was labeled with 32P by random primer method. Hybridization was
conducted overnight at 42°C in 150 µg/ml single-stranded salmon sperm
DNA, 5xSSC (0.75 M NaCl, 0.075 M trisodium citrate, pH 7.0),
1xBlocking Quencher (Molecular Research Center, Inc.), 0.2% SDS and 50%
formamide. Final washes were at 65°C in 0.5x SSC, 1% SDS and
2xDenhardts solution (0.04% Ficoll 400, 0.04% polyvinylpyrrolidone,
0.04% bovine serum albumin).
Immunofluorescence microscopy
Worm embryos were obtained by cutting gravid adults on poly-lysine-coated
slides, freeze-cracked as described previously
(Epstein et al., 1993) and
fixed with methanol at 20°C for 5 minutes. They were washed with
phosphate-buffered saline (PBS) for 10 minutes and stained with antibodies
diluted in 1% bovine serum albumin in PBS. Gonads were dissected by cutting
adult worms at the level of the pharynx on poly-lysine-coated slides,
freeze-cracked, fixed and stained in the same manner as embryo staining.
Antibody staining of adult worms was performed as described elsewhere
(Finney and Ruvkun, 1990
).
Rabbit polyclonal anti-UNC-60A and UNC-60B antibodies were described
previously (Ono et al., 1999).
An anti-UNC-60B antibody was further absorbed with acetone-fixed powder of the
unc-60 (su158) (unc-60B-null) mutant worms to remove
non-specific reactivity (Miller and
Shakes, 1995
). Mouse monoclonal anti-myoA antibody (clone 5.6)
(Miller et al., 1983
) was
provided by Henry Epstein (Baylor College of Medicine, Houston, Texas). Mouse
monoclonal anti-vinculin antibody (MH24) and mouse monoclonal
anti-
-actinin antibody (MH40)
(Francis and Waterston, 1985
)
were provided by Michelle Hresko (Washington University School of Medicine,
St. Louis, MO). Mouse monoclonal anti-actin antibody (C4) was purchased from
ICN Biomedicals. Rabbit polyclonal anti-actin antibody was purchased from
Cytoskeleton Inc. Mouse monoclonal anti-
-tubulin antibody was purchased
from Amersham Biosciences. Secondary antibodies used were Alexa488-labeled
goat anti-mouse IgG (Molecular Probes) and Cy3-labeled goat anti-rabbit IgG
(Jackson ImmunoResearch Laboratories). To stain DNA,
4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (Sigma-Aldrich)
was included in the solution of secondary antibodies at 0.1 µg/ml.
Samples were viewed by epifluorescence using a Nikon Eclipse TE2000 inverted microscope with a 40x CFI Plan Fluor objective. Images were captured by a SPOT RT Monochrome CCD camera (Diagnostic Instruments) and processed by the IPLab imaging software (Scanalytics, Inc.) and Adobe Photoshop 6.0.
RNA interference experiments
The cDNAs containing entire coding regions of UNC-60A or UNC-60B were cut
out of bacterial protein expression vectors pET-UNC-60A or pET-UNC-60B
(Ono and Benian, 1998) and
cloned into L4440 (provided by Andrew Fire, Carnegie Institution of
Washington, Baltimore, Maryland) at the cloning site between two oppositely
oriented T7 promoters (Timmons and Fire,
1998
). E. coli HT115 (DE3), an RNase III-deficient strain
(Timmons et al., 2001
), was
transformed with the plasmids and used for RNA interference (RNAi) by feeding
worms essentially as described previously
(Ono and Ono, 2002
). For
control experiments, HT115 (DE3) was transformed with L4440 with no insert and
used to feed worms. For scoring brood size and phenotypes, a single L4 larva
was placed in an RNAi-feeding plate (60 mm diameter) and transferred to a new
plate after 24 hours when they became an adult. The adult worm was transferred
to a new plate every 24 hours and the progeny was examined 1 day after the
adult had been removed. For time-lapse recording of embryogenesis, 20 L4
larvae were cultured in an RNAi-feeding plate (100 mm diameter) for 24 hours,
transferred to a new plate and cultured for 24 hours. Then, the adults were
dissected by a pair of 26G needles in egg salts (118 mM NaCl, 48 mM KCl, 2 mM
MgCl2, 2 mM CaCl2, 10 mM HEPES-NaOH, pH 7.4) and the
released embryos subjected to time-lapse differential interference contrast
(DIC) microscopy.
Time-lapse DIC microscopy
Embryos were mounted on a 2% agarose pad with egg salts and covered with a
glass coverslip. They were set on a Nikon Eclipse TE2000 inverted microscope
and observed with a 40x CFI Plan Fluor objective with DIC optics. Images
were captured by a SPOT RT Monochrome CCD camera (Diagnostic Instruments) and
recorded every 15 seconds using the IPLab imaging software (Scanalytics,
Inc.).
Sequencing of genomic DNA
Total DNA was prepared from unc-60 homozygotes as described
previously (Sulston and Brenner,
1974) and used as a template for PCR reactions. Several primer
sets were used to amplify 1 to 2 kb genomic DNA fragments from different
regions of the unc-60 gene by REDTaq DNA polymerase
(Sigma-Aldrich) to determine the location of deletions in the unc-60
alleles. DNA fragments with deletions were sequenced with an ABI PRIZM dye
terminator cycle sequencing kit and an ABI310 genetic analyzer (Applied
Biosystems) to determine precise deletion sites.
Western blot
Total worm lysates were prepared as described previously
(Ono and Ono, 2002). Protein
concentrations of the lysates were determined by a filter paper dye-binding
assay (Minamide and Bamburg,
1990
). 10 µg of proteins from each lysate was separated on a
15% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride
membrane (Immobilon-P: Millipore) using a Genie Blotter (Idea Scientific). The
membranes were blocked in 5% non-fat milk in PBS containing 0.1% Tween 20 and
incubated for 1 hour with anti-UNC-60A, anti-UNC-60B or anti-actin (C4; ICN
Biomedicals) antibodies followed by treatment with peroxidase-labeled goat
anti-rabbit IgG or goat anti-mouse IgG (Pierce Chemical Co.). The reactivities
were detected with a SuperSignal chemiluminescence reagent (Pierce Chemical
Co.).
Motility assay
A motility assay was performed as described previously
(Epstein and Thomson, 1974).
Briefly, adult worms were placed in M9 buffer (22 mM
KH2PO4, 42 mM Na2HPO4, 85.5 mM
NaCl, 1 mM MgSO4). Then, one beat was counted when a worm swung its
head to either left or right. The total number of beats in 30 seconds was
recorded.
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Results |
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|
Expression and localization of UNC-60A and UNC-60B were characterized by
immunofluorescent staining of wild-type C. elegans with specific
antibodies. UNC-60A was maternally expressed and present in most of embryonic
cells throughout embryogenesis (Fig.
2). At the one-cell stage, UNC-60A was diffuse in the cytoplasm
and localized to no particular structure
(Fig. 2a,d). Actin was
localized throughout the cortex as well as in the diffuse cytoplasm
(Fig. 2b) and assembled at the
early cleavage site in the telophase of the first mitosis
(Fig. 2e), whereas UNC-60A did
not significantly accumulate in these structures
(Fig. 2c,f). During progression
of cytokinesis and after the two-cell stage, association of UNC-60A with the
cortical regions, especially at the cell-cell boundaries became evident
(Fig. 2g). In addition, UNC-60A
was colocalized with actin to dot-like structures at the anterior cortex of
each blastomere (Fig. 2g-i).
These are believed to be remnants of cell division
(Waddle et al., 1994). In the
later stages, staining of UNC-60A in the nerve ring became intense
(Fig. 2j). In contrast, we have
previously demonstrated that UNC-60B is not expressed in early embryos and
become detectable from the comma to 1.5-fold stages (approximately 290 minutes
after first cell division) only in the body wall muscle cells
(Ono et al., 1999
;
Ono and Ono, 2002
). UNC-60B is
diffusely localized in the cytoplasm of the embryonic body wall muscle in the
later stages as demonstrated previously
(Ono et al., 1999
;
Ono and Ono, 2002
).
|
In adult worms, UNC-60A was highly expressed in the intestine (data not
shown), germ cells in the distal gonad, oocytes and spermatheca
(Fig. 3a) but not in body wall
muscle (data not shown). By contrast, UNC-60B was expressed in body wall
muscle (Ono et al., 1999),
vulva (data not shown) and spermatheca
(Fig. 3b). Both ADF/cofilin
isoforms were not detected in the smooth-muscle-like myoepithelial sheath
cells (Fig. 3c,d). Localization
of the nuclei of the gonad by DAPI indicated that both UNC-60A
(Fig. 3a,e,g) and UNC-60B
(Fig. 3b,f,h) were mostly
localized to the cytoplasm. Also, they were undetectable in sperm, which does
not have an actomyosin system (Nelson et
al., 1982
) (data not shown).
|
UNC-60A is essential for early embryonic development
To characterize the in vivo roles of UNC-60A and UNC-60B, we suppressed
expression of each ADF/cofilin isoform by RNAi and characterized the resultant
phenotypes. When worms were fed with bacteria expressing dsRNA for
unc-60A, they produced many dead embryos and a greatly reduced number
of developed progeny (Table 1).
However, treatment with dsRNA for unc-60B caused did not alter brood
size (Table 1), morphology or
motility (data not shown). Immunostaining of the unc-60A (RNAi)
embryos showed remarkable reduction of the UNC-60A protein to somewhat
variable extents (Fig. 4). In
control embryos (Fig. 4a-c),
UNC-60A was distributed in the same patterns as in wild-type embryos under
standard culture conditions (Fig.
2). The unc-60A (RNAi) treatment significantly reduced
the cytoplasmic staining of UNC-60A (Fig.
4d-f), whereas staining of some structures that resembled
cell-cell boundaries or premature cleavage furrows was observed
(Fig. 4d, arrowheads),
suggesting that UNC-60A was not completely depleted. Western blot analysis
indicated that the UNC-60B protein was not decreased by feeding with the
unc-60B dsRNA (data not shown). Therefore, we concluded that RNAi was
successful in demonstrating the requirement of UNC-60A but not UNC-60B in
embryonic development.
|
|
Time-lapse recording of the developmental process of the unc-60A
(RNAi) embryos showed that there were variable defects in their early
development (Fig. 5). Among 36
embryos examined, 39% had a defect in cytokinesis, 28% showed abnormal
positioning of the blastomeres and 33% were apparently normal up to the
eight-cell stage. There was a tendency for the cytokinesis-defective embryos
to have a greater reduction in UNC-60A immunostaining than the
positioning-defective or apparently normal embryos (data not shown),
suggesting that the cytokinesis defect is a stronger phenotype than the
positioning defect. The cytokinesis- and patterning-defective embryos were
also defective in pseudocleavage (Fig.
5a-c). In the cytokinesis-defective embryos
(Fig. 5b,e,h,k,n), a cleavage
furrow was formed and progressed (Fig.
5h) but regressed or disassembled before completion of cell
division (Fig. 5k, Movie 1).
The furrow in this embryo progressed at 1 µm/minutes (Movie 2), which
is one tenth of the speed of furrow progression in control embryos (
10
µm/minute) (Movie 1). However, regression of the furrow (
6
µm/minute) was relatively rapid (Movie 2). Embryos still underwent multiple
rounds of nuclear division and attempts at cytokinesis, which resulted in
multinucleated cells or irregularly compartmentalized embryos
(Fig. 5n). In a representative
positioning-defective embryo (Fig.
5c,f,i,l,o), first and second rounds of cytokinesis were
apparently normal except that the cleavage of the P1 blastomere occurred
slightly earlier than the control (Fig.
5l). However, subsequent positioning of the blastomeres at the
four-cell stage became abnormal (compare
Fig. 5m with o). In control
embryos, the cleavage plane of the AB blastomere occurs parallel to the long
axis of the embryo, whereas that of the P1 blastomere is set perpendicular to
the long axis (Fig. 5j, Movie
1). The resultant ABp and EMS blastomeres slide to the sides, and ABa and P2
are located at anterior and posterior poles, respectively
(Fig. 5m). However, in some of
the unc-60A (RNAi) embryos, although the orientation of the spindles
and cleavage planes was normal (Fig.
5l), both ABa and ABp remained at the anterior side and EMS was
squeezed into the center of the embryos
(Fig. 5o, Movie 3). These
embryos continued to divide but were not successful at completing the
following morphogenesis.
|
In addition, we observed a defect in extrusion of the polar body and
abnormal cortical activity in the unc-60A (RNAi) embryos
(Fig. 6a,b). In 71%
(n=17) of the unc-60A (RNAi) embryos, the polar body was not
extruded after meiosis, which resulted in the appearance of the third
pronucleus during pronuclear migration
(Fig. 6a, arrow). This is
likely to be due to a cytokinesis defect in meiosis. Also, 41% of the
unc-60A (RNAi) embryos showed abnormally active membrane protrusion
and retraction after the two-cell stage
(Fig. 6b, arrowheads, Movie 3,
see the lower side of cell-cell boundary at the two-cell stage), suggesting
that the cell cortex became unstable, as observed with RNAi of the Arp2/3
complex (Severson et al.,
2002).
|
Actin filaments are important for establishment of the anterior-posterior
(A-P) polarity at the one-cell stage (Hill
and Strome, 1988; Hill and
Strome, 1990
). However, the A-P polarity is not severely disturbed
by the unc-60A (RNAi) treatment
(Fig. 6c-e). We measured
positions of pronuclear meeting (Fig.
6c) and the first mitotic spindle
(Fig. 6d), which are normally
posteriorly localized (Golden,
2000
). The position of pronuclear meeting was more variable in
unc-60A (RNAi) embryos than in control embryos
(Fig. 6e). In some unc-60A
(RNAi) embryos, the pronuclei met near the center of the embryos
(Fig. 5c), whereas in other
embryos, it occurred at a posterior region
(Fig. 5b). However, the average
value of the meeting position in unc-60A (RNAi) embryos
(65±8.9% egg length from the anterior pole) was not significantly
different from that of control (67±4.1%) (P=0.54 by a
t-test). Similarly, positions of the anterior and posterior spindle
poles were slightly more variable in unc-60A (RNAi) embryos than
control embryos, but the differences were not statistically significant
(Fig. 6e). These results
suggest that the A-P polarity might be slightly unstable but not severely
disturbed when UNC-60A was suppressed.
In control embryos, actin was uniformly localized to the cortex (Fig. 7a). However, in the unc-60A (RNAi) embryos with the multinucleated phenotype, cortical actin was unevenly distributed and enriched at one end of the cell cortex (Fig. 7d,g) and sometimes accumulated at a cleavage-furrow-like structure (Fig. 7g; indicated by an arrow). These phenotypes suggest that UNC-60A is required for even distribution of cortical actin and proper progression of a cleavage furrow. Mitotic spindles were often found around nuclei (Fig. 7e), indicating that separation of the spindle poles and spindle assembly were not significantly affected by depletion of UNC-60A.
|
A null mutation of unc-60B causes specific defects in actin assembly
in body wall muscle Previously characterized mutations of unc-60B
were all point mutations that resulted in expression of mutant UNC-60B
proteins (Ono et al., 1999).
Therefore, although these mutants were homozygous viable and showed
disorganization of actin filaments in body wall muscle, we were not able to
exclude the possibility that the mutant UNC-60B proteins function sufficiently
in some cellular activities other than myofibril assembly. To clearly
determine the requirement of unc-60B in vivo, we need to characterize
a null phenotype of unc-60B.
We collected unc-60 mutant strains from other researchers,
determined their sequence alterations and found that two unc-60
alleles contain deletions in the unc-60B region
(Fig. 8A). unc-60
(s1586) is homozygous lethal at a late larval stage
(McKim et al., 1994). We found
that this allele had a deletion that completely removes exon 2B
(Fig. 8A). However, the
deletion also extends to the unc-60A region and removes approximately
250 bp of the 3'-untranslated region in exon 5A that contains a putative
polyadenylation signal for unc-60A. Therefore, this deletion is
likely to affect expression of both ADF/cofilin isoforms, which may be a cause
of the lethal phenotype. In contrast, we found that unc-60 (su158)
(Zengel and Epstein, 1980
) had
a deletion of 600 bp that completely removed exons 3B and 4B and did not
disturb the unc-60A region (Fig.
8A). Even if exons 2B and 5B are connected by an aberrant splicing
event, exons 1 and 2B encodes only 30 amino acids from the N-terminus of
UNC-60B and the coding region in exon 5B will be out of frame. The unc-60
(su158) mutant is homozygous viable and shows a more severe motility
defect than a strong loss-of-function mutant unc-60 (e677)
(Fig. 8B). In the unc-60
(su158) homozygotes, the UNC-60B protein was not detected by western
blotting, whereas UNC-60A was present at an equivalent amount to that found in
wild-type worms (Fig. 8C).
Thus, unc-60 (su158) is a null allele of unc-60B that does
not interfere with unc-60A.
|
The unc-60B null mutants were homozygous viable and showed a muscle-specific defect in actin organization. During embryonic development, morphological observation by DIC microscopy revealed no abnormalities (data not shown). However, staining with an anti-actin antibody revealed that, although early embryogenesis from the one-cell to 1.75-fold stages appeared to be normal (Fig. 9Ad), actin became discontinuous in the body wall muscle after the two-fold stage (Fig. 9Bd,Cd, indicated by arrows). The defect in actin organization was specifically detected in the body wall muscle cells. In addition, other myofibrillar components, myoA myosin heavy chain (Fig. 9A-Cb,e) and vinculin (Fig. 9A-Cc,f), appeared undisturbed, suggesting that initial assembly of the myosin thick filaments and adhesion structures are independent of UNC-60B-mediated actin dynamics. Nonetheless, as a result of the muscle defects, movement of the mutant embryos in the egg shell was significantly slower than wild-type embryos (data not shown). In addition, RNAi of unc-60A on the unc-60B-null mutant resulted in similar cytokinesis and patterning defects (40% and 27%, respectively, n=15) to wildtype, and no enhancement of the phenotype was observed, suggesting that UNC-60B does not function in early embryogenesis.
|
When the unc-60B-null mutants grew into adult worms, they were
nearly paralyzed and large actin aggregates were formed in the body wall
muscle (Fig. 10). The extent
of actin disorganization was slightly more severe than a strong
loss-of-function mutant unc-60 (e677)
(Ono et al., 1999). However,
unlike the embryos, the organization of the myosin filaments and dense bodies
in the adults was disturbed (Fig.
10). In wildtype, myosin was clearly arranged in a striated
pattern (Fig. 10Aa). In the
unc-60B-null mutant, myosin was assembled into wide and uneven bands
with very obscure striation (Fig.
10Ad). These myosin bands were spatially segregated from the actin
aggregates (Fig. 10Af). Dense
bodies are adhesion structures that appear as discrete spots in the center of
the I-bands, as shown by staining for
-actinin and vinculin
(Fig. 10B,C). In the
unc-60B null mutant, both
-actinin and vinculin were aligned
in striation, but the staining patterns were often continuous as if several
dense bodies were fused together (Fig.
10Bd,Cd). In addition, vinculin was somewhat diffuse in some
regions (Fig. 10Cd). Double
staining of the dense body components with actin showed that
-actinin
and vinculin were not the components of actin aggregates in the mutant
(Fig. 10Bf,Cf). These results
suggest that UNC-60B-dependent actin dynamics are required for proper
alignment of thick filaments and dense bodies during post-embryonic
development, and that the actin-bundling activity by myosin,
-actinin
or vinculin does not contribute to formation of the actin aggregates in the
unc-60B mutants. In addition, the expression pattern of UNC-60A in
the unc-60 (su158) mutant was not significantly different from
wild-type (data not shown), suggesting that there is no compensatory mechanism
to upregulate UNC-60A in the absence of UNC-60B.
|
![]() |
Discussion |
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Role of UNC-60A in embryogenesis
We found that UNC-60A was required for cytokinesis in early C.
elegans embryos. Similarly, ADF/cofilin has been reported to be required
for cytokinesis in Drosophila
(Gunsalus et al., 1995;
Somma et al., 2002
) and
Xenopus eggs (Abe et al.,
1996
), indicating that ADF/cofilin is a conserved regulator of
cell division. Our immunostaining indicated that UNC-60A was not concentrated
in the early accumulation of actin at the cleavage site. Rather, it was
localized to progressing cleavage furrows in the late phase of cytokinesis and
remained associated with cortical actin at cell-cell contacts. This is also
consistent with the observations that cofilin is localized to the cleavage
furrow in cultured cells (Nagaoka et al.,
1995
) and Xenopus eggs
(Abe et al., 1996
). This
localization pattern suggests that the role of UNC-60A is in late cytokinesis
and explains the cytokinesis defect by RNAi of unc-60A. In the
unc-60A (RNAi) embryos, a cleavage furrow was often formed and
progressed, but prematurely disassembled, suggesting that UNC-60A-mediated
actin dynamics are required for efficient completion of cytokinesis. Also
intriguingly, progression of a cleavage furrow in cytokinesis-defective
embryos was much slower than that in control embryos, suggesting that
UNC-60A-dependent actin dynamics might be a driving force for furrow
progression. The contractile ring in dividing cells has been shown to be an
active site of actin polymerization
(Noguchi and Mabuchi, 2001
;
Pelham and Chang, 2002
).
Therefore, actin depolymerization and severing by ADF/cofilin would be
required to maintain dynamic actin turnover during cytokinesis.
Similar phenotypes in late cytokinesis have been described for mutants or
RNAi suppression of the formin-like protein CYK-1
(Swan et al., 1998), the
kinesin-like protein ZEN-4/CeMKLP1 (Powers
et al., 1998
; Raich et al.,
1998
; Severson et al.,
2000
), CYK-4 Rho-GAP
(Jantsch-Plunger et al.,
2000
), the aurora-related kinase AIR-2
(Kaitna et al., 2000
;
Schumacher et al., 1998
;
Severson et al., 2000
) and the
syntaxin SYN-4 (Jantsch-Plunger and
Glotzer, 1999
). Formin has been shown to nucleate actin
polymerization (Pruyne et al.,
2002
; Sagot et al.,
2002
), but it is not known how ADF/cofilin affects this process.
ZEN-4 and CYK-4 physically interact and are required for assembly of the
central spindle, which is implicated in completion of cytokinesis
(Mishima et al., 2002
).
Syntaxin mediates vesicle fusion and may be required for membrane addition at
the cleavage furrow (Skop et al.,
2001
). However, the mechanism by which these proteins promote the
completion of cytokinesis is poorly understood. How UNC-60A might be involved
in these processes remains to be determined.
Defects in early embryonic patterning in the unc-60A (RNAi)
embryos are probably weaker phenotypes than the cytokinesis defects, because
embryos with patterning defects were successful in multiple rounds of cell
division. Nonetheless, the observed phenotype is novel. Embryonic polarity and
asymmetric cell division are essential for determining cell fate and embryonic
patterning. Actin and myosin are required for polarized localization of the
products of polarity genes (Rose and
Kemphues, 1998). However, the unc-60A (RNAi) embryos
showed an apparently normal pattern of spindle orientation and cleavage
pattern at the second cell division, whereas subsequent positioning of the
blastomeres was defective. It is still possible that the embryonic polarity is
partially disrupted so that the blastomeres were not able to locate at
appropriate positions. Alternatively, cortical rigidity or activity might be
disturbed by unc-60A (RNAi). UNC-60A is colocalized with cortical
actin at the cell-cell contacts, and unc-60A (RNAi) alters cortical
distribution of actin and induces irregular membrane activity. Therefore, the
cell cortex might have lost its rigidity and, thus, might be unable to push or
to be pushed by the neighboring cells.
Specific requirement of UNC-60B in myofibril assembly and
development
Phenotypic analysis of the unc-60B-null mutant confirms our
previous observations that UNC-60B is specifically required for assembly of
actin (Ono et al., 1999). In
addition, our extended analysis demonstrates that UNC-60B is not required for
early development and that it is required for organized assembly of other
myofibrillar components during postembryonic development. This indicates that
the presence of the UNC-60A isoform is sufficient for viability of worms.
UNC-60A is expressed in every embryonic cell but not in adult body wall muscle
cells. The expression of UNC-60A in muscle was already low in larval stages,
and so we were not able to determine when UNC-60A is downregulated (K.O. and
S.O., unpublished). Although the antibody failed to detect UNC-60A in adult
muscle, it is possible that a low level of UNC-60A is expressed in adult
muscle and supports cell viability.
Phenotypic characterization of the unc-60B-null mutant showed that
actin was disorganized from the embryonic stages onwards, whereas myosin thick
filaments and dense bodies were disorganized only in adults. This suggests
that UNC-60B is primarily required for actin assembly and that the effects on
myosin and dense bodies are secondary and are due to disrupted thin filament
organization. Therefore, the initial assembly of thick filaments and dense
bodies may not require UNC-60B-mediated actin dynamics. This supports early
observations that myosin and actin are initially assembled into nascent
structures that are distinct from myofibrils
(Epstein et al., 1993),
suggesting that the assembly processes of myosin and actin are independent.
However, the subsequent development and maintenance of these structures may
need an organized myofibril structure that can generate contractile forces. In
vertebrate striated muscle cells, inhibition of muscle contraction causes
disorganization of myofibrils (De Deyne,
2000
; Soeno et al.,
1999
). These observations suggest that a regulated actin-myosin
interaction facilitates proper alignment of other myofibrillar components.
Functional significance of ADF/cofilin isoforms
We demonstrated that the two C. elegans ADF/cofilin isoforms have
different functions in vivo. This is consistent with the different
actin-regulating activities of UNC-60A and UNC-60B
(Ono and Benian, 1998). We
also observed that UNC-60A accelerates subunit dissociation from F-actin more
rapidly than UNC-60B (S. Yeoh, S.O. and A. Weeds, unpublished). Our results
are consistent with biochemical studies using different methods showing that
UNC-60A is ADF-like, whereas UNC-60B is cofilin-like (H. Chen and J. Bamburg,
personal communication). UNC-60A is a more potent depolymerizing agent than
UNC-60B, suggesting that UNC-60A is suitable in cells where actin filaments
are dynamic, whereas UNC-60B is adapted to cells, such as muscle, in which
actin filaments are relatively stable. In addition, we have recently found
that UNC-78/actin-interacting protein 1
(Ono, 2001
) disassembles actin
filaments more efficiently in the presence of UNC-60B than UNC-60A (K. Mohri,
A. G. Weeds and S. Ono, unpublished), indicating that isoform-specific
interactions with other cytoskeletal proteins are also important determinants
of actin dynamics.
The other mechanism that differentiates these isoforms is their
differential expression in different cell types. RNAi suppression or mutations
of each isoform causes a specific phenotype in which the expression of the
target isoform is predominant. However, it is still possible that the two
ADF/cofilin isoforms have a redundant function in some biological aspects when
both isoforms co-exist. UNC-60A and UNC-60B are produced by alternative
splicing of the unc-60 gene
(McKim et al., 1994).
Therefore, the tissue-specific splicing machinery seems to be involved in the
regulation of expression of the ADF/cofilin isoforms. It would be interesting
to examine if misexpression of the ADF/cofilin isoforms causes a dominant
phenotype or if forced expression of UNC-60B in early embryos or UNC-60A in
muscle suppresses the RNAi or mutant phenotypes. The lethal phenotype of the
yeast cofilin-null mutant can be rescued by expression of vertebrate
ADF/cofilins (Iida et al.,
1993
) or Dictyostelium cofilin-1, but not cofilin-2
(Aizawa et al., 2001
). Thus,
further functional characterization of the ADF/coflin isoforms is important to
understand how evolution of actin-regulatory proteins has contributed to
increasing the diversity in actin cytoskeletal structure and function.
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Acknowledgments |
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