1 Vrije Universiteit, Faculty of Earth and Life Sciences, Department of
Developmental Genetics, Section Molecular Plant Physiology and Biophysics, De
Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands
2 Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, and
Department of Developmental and Craniofacial Biology, Harvard School of Dental
Medicine, Boston, MA 02115, USA
Author for correspondence (e-mail:
mlevin{at}forsyth.org)
Accepted 1 July 2003
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SUMMARY |
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Key words: Left-right asymmetry, 14-3-3 protein, Fusicoccin, Xenopus
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Introduction |
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Fusicoccin (FC) is a metabolite of the plant pathogen Fusicoccum
amygdali Del. The toxin is remarkably effective in stimulating a number
of physiological processes in higher plants, including stomatal opening,
cellular respiration and radicle emergence from seed embryos
(Marre, 1979;
Kinoshita and Shimazaki,
1999
). FC binds with high affinity to a receptor that is formed
from an interaction of proteins from the 14-3-3 family with the plasma
membrane H+-ATPase (Korthout
and De Boer, 1994
; Olsson et
al., 1998
; Palmgren,
2001
). 14-3-3 proteins are a family of acidic, soluble proteins
with a number of key signaling roles in cell cycle, apoptosis and prion
diseases (Baldin, 2000
;
Fu et al., 2000
). Binding of
14-3-3 to the C-terminal (regulatory) domain of the H+ pump
releases its auto-inhibitory action
(Baunsgaard et al., 1998
;
Palmgren, 2001
), which results
in increased H+-extrusion along with changes in cytoplasmic pH
(Felle et al., 1986
). Despite
the considerable structural conservation of 14-3-3 proteins from plants and
animals (Fu et al., 2000
;
Sehnke et al., 2002
), and the
large number of 14-3-3 partners identified thus far in both kingdoms
(Van Hemert et al., 2001
), the
presence of FC binding sites in organisms other than higher plants has never
been demonstrated (Meyer et al.,
1993
).
Modulation of H+ currents is a hallmark of FC action; pH is a
key regulator of many cellular parameters, such as gap-junctional
communication between cells, which in turn is known to control a number of
physiological processes (Levin,
2001). These considerations prompted us to investigate whether FC
might be able to perturb embryonic patterning in a tractable vertebrate
embryonic model, the frog Xenopus laevis, and thus be a useful tool
to shed light on endogenous morphogenetic mechanisms. Interestingly, we found
that exposure to FC had one major effect on embryogenesis: randomization of
the left-right (LR) axis. The consistent asymmetry in the morphogenesis and
placement of the heart, gut, brain and other asymmetric organs in normal
individuals is very strongly conserved across evolution
(Neville, 1976
). Deviations
from the normal pattern of asymmetry (situs solitus) have significant
medical implications for human patients with laterality defects
(Kosaki and Casey, 1998
), and
this fascinating biological problem is now beginning to be understood in
molecular detail (Burdine and Schier,
2000
; Mercola and Levin,
2001
; Yost, 2001
).
However, very early LR patterning events are poorly understood and
considerable debate exists as to how early embryos of various species can
align the LR axis. Our characterization of FC signaling reveals that 14-3-3E
protein is asymmetrically localized in Xenopus at the first cell
cleavage and represents a novel, very early step in the LR pathway.
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Materials and methods |
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Homogenization of embryos for biochemical analysis
Stage 5/6 or stage 10 embryos (5 g) were ground under liquid nitrogen in a
mortar until a fine powder was obtained. 2 ml of homogenization buffer [20 mM
Tris-HCl (pH 7.4), 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 1 mM PMSF and 1 complete
protease inhibitor tablet (Roche Biochemicals) per 50 ml buffer] was then
added and the suspension ground until liquid. All subsequent steps were
carried out at 4°C. The suspension was centrifuged for 10 minutes at 2500
g and the pellet (which consists mainly of residual yolk
protein) was discarded. Subsequently, the supernatant was centrifuged at
10,000 g for 15 minutes and the pellet discarded. Finally, the
supernatant was centrifuged for 60 minutes at 100,000 g and
the resulting pellet (microsomal fraction) and supernatant (cytoplasmic
fraction) collected.
Fusicoccin binding assay
Binding assays were performed in glass tubes in 20 mM Mes-Tris (pH 6.5), 1
mM CaCl2, 5 mM MgSO4, 2.3 mM DTT, 1 mM PMSF and
5x108 M
[3H]9'-nor-fusicoccin-8'-alcohol at 30°C. The final
incubation volume was 0.9 ml and reactions were incubated for 3 hours. The
incubation was started by the addition of 4 mg microsomal or cytoplasmic
protein extract. The reaction was terminated by the addition of 1 ml ice-cold
wash buffer (25 mM glycine-KOH, pH 9.5) to the tubes and adding this, together
with another 4 ml of wash buffer, on a Millipore filtration manifold. Rapid
filtration was performed through polyethyleneimine (1% w/w) pre-treated
Whatman GF/B filters, which were washed with 5 ml distilled water and 5 ml
wash buffer just before filtration. The filters were then washed twice with 5
ml of wash buffer. The whole procedure of filtering and washing took about 30
seconds. The filters were then transferred to 5 ml Pharmacia Optiphase HiSafe
3 cocktail. After 24 hours extraction, the radioactivity was measured for 5
minutes with a LKB Wallac Rackbeta 1219 liquid scintillation counter.
Non-specific binding was measured in saturation experiments by the inclusion
of 10 µM unlabelled FC. The binding data of the saturation and competition
experiments were analyzed with Sigmaplot 5.0. P-peptide was used at a final
concentration of 1 µM. Further deviations from the protocol are mentioned
in the text and figures.
Chromogenic in situ hybridization
In situ hybridization was performed according to a standard protocol
(Harland, 1991). Briefly,
Xenopus embryos were collected and fixed in MEMFA. Prior to in situ
hybridization, embryos were washed in PBS+0.1% Tween-20 and then transferred
to methanol through a 25%/50%/75% series. Probes for in situ hybridization
were generated in vitro from linearized templates using DIG-labeling mix
(Roche). Chromogenic reaction times were optimized for signal: background
ratio.
Drug exposure
FC was obtained from Professor G. S. Muromtsev (Moscow), and tested using
the lettuce seed germination assay (Lado
et al., 1974) before use with embryos, as the FC obtained from
Sigma was found to be of variable quality. FC was used at 50 µM in
0.1xMMR medium in systemic application experiments. Consistent with
previous biochemical studies, which found optimal FC binding to receptors at
lower pH (Drabkin et al.,
1997
), we observed that the maximal effect was achieved with FC
dissolved in 0.1xMMR at pH<6 (embryos raised in medium with pH in the
range 5 to 8 exhibit the normal control levels of heterotaxia: <1%,
n=180).
Microinjection
For microinjection, peptides and mRNA were dissolved in water and injected
into embryos in 3% Ficoll using standard methods (100 millisecond pulses in
each injected cell with borosilicate glass needles calibrated for a bubble
pressure of 55-62 kPa in water). Peptide stock was 2.6 mM in water;
approximately 2.7 nL were injected into each cell. After 30 minutes, embryos
were washed in 0.75xMMR for 30 minutes and cultured in 0.1xMMR
until desired stages.
Immunohistochemistry
Embryos were fixed overnight in MEMFA and stored at 4°C in PBTr
(1xPBS + 0.1% Triton X-100). They were embedded in gelatin/albumin
medium and sectioned at 40 µm on a Leica vibratome. The sections were
washed three times in PBTr, blocked with 10% goat serum and incubated with
primary antibody at 1:200 in PBTr overnight. They were then washed six times
with PBTr and incubated with an alkaline-phosphatase-conjugated secondary
antibody overnight. After six washes in PBTr, detection was carried out using
NBT and BCIP (X-Phos). Detection times were optimized for signal:noise
ratio.
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Results |
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14-3-3-blocking peptide disrupts FC binding in vitro and randomizes
laterality in embryos
To test the hypothesis that 14-3-3 proteins participate in the formation of
the Xenopus FC receptor, as they do in plants, we took advantage of
one of the 14-3-3 hallmarks: the formation of a tight complex with
phosphorylated ligands containing either of the two sequence motifs
R[S/Ar]XSPXP or RX[S/Ar]XSPXP, where Ar denotes aromatic
residues and SP denotes phosphorylated Ser/Thr
(Yaffe et al., 1997). The snug
fit of such ligands into the highly conserved amphipathic groove of the 14-3-3
protein specifically and effectively disrupts the interaction between 14-3-3
and partners, in vivo as well as in vitro
(Ballio et al., 1981
;
Yaffe et al., 1997
;
Booij et al., 1999
;
Bunney et al., 2001
). We used
a phosphopeptide modeled on the phosphorylated hinge 1 region of nitrate
reductase (NR), which contains a `strong' 14-3-3 interaction motif
(RSXSPXP) that is also found in many mammalian 14-3-3 partners
(e.g. Raf-kinase, Cdc25, PKC and BAD)
(Yaffe et al., 1997
), and
binds to a domain fully conserved in Xenopus 14-3-3 proteins
(Rittinger et al., 1999
;
Aitken et al., 2002
;
Wurtele et al., 2003
).
Addition of this phosphorylated NR peptide (1 µM) to the FC-binding assay
(using 4 nM radioligand) resulted in a 75% reduction in FC-binding to the
oocyte receptor as compared with the control (bound [3H]FC was,
respectively, 1.010±0.024 and 0.260±0.013
pmol.mg1 protein). In light of the specificity and
effectiveness of this peptide (Bunney et
al., 2001
), these data suggest that 14-3-3 proteins are essential
components of the embryo FC receptor.
On the basis of the FC-induced heterotaxia and the involvement of 14-3-3
proteins in in vitro FC-binding, we formulated the hypothesis that endogenous
14-3-3 proteins are instrumental in correct LR asymmetry. We tested this model
by complementing the FC gain-of-function data with a competitive
loss-of-function experiment using the same NR-peptide as was utilized in the
FC-binding assay. Microinjection of the NR-peptide (which is expected to act
as a dominant negative by interfering with FC/14-3-3 complex formation) into
embryos at the one-cell stage resulted in a 31% incidence of heterotaxia
(Table 2;
2=58.27, P<2.2·1014). By
contrast, injections of a control peptide (which is identical in amino acid
sequence, except that the serine in the binding motif is not phosphorylated)
did not induce heterotaxia. These results are consistent with the hypothesis
that 14-3-3 proteins mediate the randomizing effect of FC, and suggest the
possibility that 14-3-3 proteins are endogenously involved in the patterning
of the LR axis.
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The localization of 14-3-3E protein was markedly different from 14-3-3Z (demonstrating lack of cross-over of the two antibodies). Prior to fertilization, 14-3-3E was localized as a fairly tight spot in the center of the egg (Fig. 4E). Strikingly, after the first cleavage, 14-3-3E was localized to only one of the two blastomeres (Fig. 4F). This pattern continues at the second cell cleavage, when dorsoventral (DV) pigment differences allow DV and LR orientation of the embryo, showing that localization is present in the right blastomeres (Fig. 4G). This asymmetric localization of 14-3-3E protein strongly supports the hypothesised endogenous role for 14-3-3E in LR asymmetry, and is the earliest LR-asymmetric molecular localization described in any species to date. By gastrulation, zygotic 14-3-3E is detected in the endodermal yolk mass, in a domain that is more restricted than that of 14-3-3Z.
In light of the asymmetry of 14-3-3E localization in the first two cleavages, we next examined the results of the 14-3-3E overexpression. In embryos that were injected with 14-3-3E mRNA (using the same conditions as in the above experiments that induced subsequent heterotaxia), very high levels of 14-3-3E protein were detected throughout each blastomere during the two- and four-cell stages (Fig. 4I,J). These results confirmed that the injections of 14-3-3E mRNA indeed resulted in high levels of protein that was present ubiquitously, and also served as a cross-check for the fact that the 14-3-3E antibody does recognize 14-3-3E.
Fusicoccin exposure abolishes the asymmetry in 14-3-3E
localization
The fact that FC exposure randomizes asymmetry
(Table 1) could be due to an
alteration of 14-3-3 protein function or localization. We investigated the
mechanism underlying the destabilizing effects of FC by examining the
localization of 14-3-3E proteins in embryos exposed to levels of FC that cause
heterotaxia. Control embryos at the two-cell stage exhibit the asymmetric
pattern of 14-3-3E localization (Fig.
5A,B). Interestingly, embryos exposed to FC from fertilization
showed bilaterally-symmetrical localization of 14-3-3E
(Fig. 5C,D). These data
demonstrate that ectopic early FC exposure can abolish the normal asymmetry in
14-3-3E protein localization.
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Discussion |
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Elements of Fusicoccin signaling are shown to be conserved to vertebrates.
This conclusion is confirmed by the biochemical data demonstrating the
existence of a FC receptor in Xenopus embryos
(Fig. 2). The existence of an
FC receptor complex suggests the possibility that endogenous FC-like compounds
exist in vertebrates and remain to be characterized. This new aspect of FC
action also warrants further study into possible subtle teratological effects
of certain food products in the light of reports that plants may produce
FC-like molecules (Muromtsev et al.,
1994) (A.H.D.B. and M. Wang, unpublished).
Interestingly, the Xenopus FC receptor is cytoplasmic, which is
consistent with the localization patterns of 14-3-3 proteins we described
(e.g. Fig. 4A,G). There may
well be other differences between the receptor as it exists in plant compared
with in animal cells. The ability of the dominant-negative NR peptide to
compete with in vitro FC binding to 14-3-3
(Fig. 2), and to induce
heterotaxia in embryos (Table
2), suggests that 14-3-3 proteins are components of the endogenous
FC receptor in Xenopus. 14-3-3 proteins have known roles in key
processes, such as the cell cycle, apoptosis and prion diseases, and are thus
crucial biomedical targets (Baldin,
2000; Fu et al.,
2000
; Van Hemert et al.,
2001
; Baxter et al.,
2002
). Higher doses of the NR peptide induced non-specific
teratogenic defects in embryos (e.g. exogastrulation; data not shown), which
is consistent with known important later roles for various 14-3-3 proteins in
Xenopus development (Wu and
Muslin, 2002
). The amount of 14-3-3-blocking peptide used in our
studies specifically induced heterotaxia. Left-right patterning is thus a new
role for this important and versatile family of signaling proteins.
The NR peptide targets the binding of all 14-3-3 proteins (it does not
distinguish between the isoforms). By contrast, misexpression of specific
mRNAs allowed us to address differential activities of the various 14-3-3
family members. Injection of 14-3-3E mRNA at the one-cell stage
causes ubiquitous expression of high levels of 14-3-3E protein at the two- and
four-cell stages (Fig. 4I,J),
confirming the specificity of our mRNA and antibody reagents, and
demonstrating that high levels of 14-3-3E protein can saturate and overwhelm
the normal 14-3-3E protein localization machinery
(Fig. 4F,G). Such
overexpression of 14-3-3E, but not of 14-3-3Z, causes heterotaxia
(Table 3). These data
demonstrate that different 14-3-3 isoforms may have distinct developmental
roles, and that the E isoform but not the Z can interact with mechanisms
involved in LR asymmetry. The misexpression experiments further allowed us to
probe the timing of 14-3-3E involvement in LR asymmetry. 14-3-3E function is
upstream of the expression of XNR1, as its localization is randomized
by 14-3-3E overexpression (Table
4). Moreover, the fact that injections after the first cell
division have a far smaller effect on LR asymmetry than do injections
immediately after fertilization suggests that processes occurring during the
first 1.5 hours after fertilization involve 14-3-3E. As, like most exogenous
mRNAs, 14-3-3E mRNA is efficiently translated within about an hour of
microinjection (Fig. 4I,J), it
is likely that 14-3-3E-mediated LR patterning mechanisms function during the
first two cell divisions. This is consistent with a lack of laterality
phenotypes reported from loss-of-function studies that introduced
14-3-3-blocking reagents after the first cleavage
(Wu and Muslin, 2002).
The proposed LR pathway function of 14-3-3E protein during the first two
cell cleavages is consistent with the endogenous protein localization data.
Maternal protein for both 14-3-3E and 14-3-3Z exists in unfertilized eggs, and
exhibits different localization patterns, suggesting that 14-3-3 family
proteins interact with different components of the subcellular localization
machinery and, thus, can be targeted to different locales within the
blastomeres (Muslin and Xing,
2000). Whereas 14-3-3Z is expressed at a low level and exhibits no
significant asymmetries, 14-3-3E is asymmetric at the first two cell cleavages
(Fig. 4F,G). Although the
maternal mRNA for 14-3-3E is also asymmetric at these stages
(Fig. 6G-J), the asymmetries in
protein and mRNA expression overlap temporally. The 2-cell stage lasts about
20 minutes at room temperature; this time period is probably too brief to
allow significant levels of protein to be translated from the mRNA and
correctly localized. Thus, the mRNA asymmetry is unlikely, by itself, to
account for the observed protein localization. The asymmetric protein
localization is probably caused by movement of maternal protein, which exists
even prior to fertilization. The reason for asymmetrically localizing both
mRNA and protein is unknown, but may represent redundancy in
as-yet-uncharacterized later roles that depend on mRNA localization.
In Xenopus, the first cell cleavage bisects the dorsal and ventral
progenitor blastomeres (which become obvious when pigment differences enable
the identification of dorsal versus ventral blastomeres at the second cell
division). When one of two blastomeres is injected with any reagent, the other
blastomere gives rise to the contralateral side of the embryo and thus serves
as a convenient internal control in many studies (e.g.
Warner et al., 1984;
Harvey and Melton, 1988
;
Vize et al., 1991
;
Louie et al., 2000
). In our
experience with microinjections of lineage markers, the first cleavage plane
corresponds very well to the LR midline of the tadpole. Whether the first cell
division truly gives rise to left and right blastomeres has been questioned
(Danilchik and Black, 1988
).
The plane of first cleavage can be experimentally repositioned independently
of the plane of bilateral symmetry by lateral compression of the uncleaved egg
(Black and Vincent, 1988
).
However, the first cell division in unperturbed embryos appears normally to
occur along the LR midline in most Xenopus embryos
(Klein, 1987
;
Masho, 1990
), and, in
Triturus, separation of blastomeres at the two-cell stage results in
randomization of one of the twins (Ludwig,
1932
), thus supporting the view that the first cleavage results in
left and right blastomeres in some amphibians.
The identification of LR asymmetry in 14-3-3E protein localization at the first cell cleavage is of significance for several reasons. First, this is the earliest LR-asymmetric molecular localization shown to date in any species. Second, this new entry point into the LR pathway can be used to address upstream mechanisms of LR asymmetry. These data constrain the first step of asymmetry to about the 1.5 hours following fertilization; by utilizing the reagents described in this study, we are currently characterizing the machinery that establishes asymmetric 14-3-3E protein localization, in efforts to identify the molecular nature of `Step 1' of LR asymmetry.
The specific mechanisms underlying the asymmetric localization of 14-3-3E
protein and mRNA are unknown. They may involve differential degradation,
anchoring or directed transport by motor proteins. We favor the latter
possibility because of a number of studies linking motor protein function with
LR asymmetry (Supp et al.,
1997; Takeda et al.,
1999
; Vogan and Tabin,
1999
; Hirokawa,
2000b
; Brueckner,
2001
; Levin,
2003b
). Animal-vegetal asymmetries in mRNA localization have been
well-studied in Xenopus (Deshler
et al., 1997
; Mowry and Cote,
1999
). Although LR-asymmetric mRNA localization has recently been
found (Levin et al., 2002
),
the observed localization for 14-3-3E protein demonstrates the existence of
previously uncharacterized localization mechanisms. Moreover, aside from the
LR-relevant asymmetry, the complex subcellular localization seen for the
various members of the 14-3-3 family (Fig.
6D-F) shows that novel patterns of mRNA destinations exist in
early embryonic cells (Oleynikov and
Singer, 1998
). 14-3-3 proteins are known to participate in
directed intracellular localization events in a number of species
(Brunet et al., 2002
). The
mechanisms that direct messenger molecules to spatially-complex locales within
cells, as well as the developmental significance of these patterns in the
large blastomeres of early embryos, remain to be characterized.
The asymmetry in 14-3-3E localization provided a way to probe the mechanism
of FC action on LR patterning. The simplest prediction would have been that
the randomization of laterality by FC would be due to an alteration of 14-3-3
protein binding to its target. Surprisingly, we found that FC exposure
abolished the asymmetry of 14-3-3E protein localization. Taken together, all
these data suggest the following model of this new aspect of LR asymmetry. We
propose (Fig. 7A) that the
14-3-3E protein is normally asymmetrically localized at the two-cell stage and
provides differential signaling to the left and right sides, which eventually
feeds into the asymmetric gene cascade
(Levin and Nascone, 1997;
Levin, 2003c
). Overexpression
of 14-3-3E protein prior to the first cell division overwhelms the endogenous
localization machinery and 14-3-3E accumulates in both blastomeres, thus
providing equal signal to both lineages and randomizing asymmetry
(Fig. 7B). FC exposure
randomizes asymmetry by virtue of abolishing the differential 14-3-3E
localization (Fig. 7C). This
may be owing to competition of excess exogenous FC for 14-3-3 binding by
whatever localization machinery maintains the tight asymmetric distribution of
14-3-3E protein. Such mechanisms may involve FC-like molecules, further
suggesting that compounds related to FC occur endogenously in animal embryos.
In this model, the dominant-negative NR peptide induces heterotaxia by virtue
of interference with the binding of 14-3-3E to its downstream target
(Fig. 7D).
|
Another possible model involves interactions between gap junctions and
14-3-3 proteins. Gap junctions composed of connexin proteins have been shown
to be an important step in LR patterning in Xenopus
(Levin and Mercola, 1998b) and
chick (Levin and Mercola,
1999
) embryos. Connexin proteins undergo serine phosphorylation,
which affects their gating properties
(Lampe et al., 2000
), and are
upregulated in certain tissues together with 14-3-3 proteins
(Villaret et al., 2000
).
Connexin 43 binds directly to tubulin
(Giepmans et al., 2001
) and
14-3-3 might regulate its function in the same fashion that it regulates other
tubulin-binding proteins such as CLIC4
(Suginta et al., 2001
). Thus,
perhaps 14-3-3 proteins bind connexins or a connexin-regulating protein in a
phosphorylation dependent manner, thereby affecting LR asymmetry through
modulation of the junctional flow that initiates at the fourth cell
cleavage.
In our view, the most likely mechanism involves the regulation of ion flux
across cell membranes. 14-3-3 proteins are known to control a variety of
H+ pumps (Bunney et al.,
2001; Bunney et al.,
2002
) and ion channels (De
Boer, 2002
). Proton flux may indirectly affect permeability states
of connexin-based gap junctions through changes in cytosolic pH
(Ek-Vitorin et al., 1996
).
Furthermore, we recently showed that K+ and H+ flux is
asymmetric in early embryos and controls LR asymmetry
(Levin et al., 2002
). 14-3-3
proteins (including 14-3-3E) have recently been shown to be able to modulate
K+ currents in Xenopus oocytes
(Chan et al., 2000
;
Benzing et al., 2002
). In light
of the differential LR subcellular localization of ion pumps, such as the
H+/K+-ATPase (Levin
et al., 2002
), and of the ability of 14-3-3 proteins to control
the localization of their binding partner
(Muslin and Xing, 2000
), we
propose that 14-3-3E protein functions in the LR pathway by differentially
regulating the endogenous activity and/or localization of LR-relevant ion
channels or pumps on each side of the midline. Other (non-LR) roles for 14-3-3
proteins clearly exist in Xenopus (such as in mesoderm induction),
and have been described in loss-of-function studies
(Wu and Muslin, 2002
).
Recently, it was reported that, in Caenorhabditis elegans, a
14-3-3 protein (PAR-5) is required for cellular asymmetry in the early embryo
(Morton et al., 2002). PAR5
likewise functions in axial asymmetry in Drosophila
(Benton et al., 2002
).
Analogously to the suggested role of the FC/14-3-3 receptor in
Xenopus, PAR-5 in C. elegans acts at an early step in
establishing polarity, but its precise role is unclear. It was suggested that
PAR-5 binds to and blocks recruitment of one or more PAR proteins (notably
PAR-2 and PAR-3) to the cell cortex
(Morton et al., 2002
). The
involvement of 14-3-3 proteins in cellular asymmetry in early cleavages of
both C. elegans and Xenopus is further evidence of a deep
and fundamental underlying similarity in the mechanisms by which asymmetry and
polarity, whether on the cellular level, or on the scale of the organism, is
established. The finding that elements of FC signaling are conserved from
plants to animals presents a new perspective from which to investigate novel
aspects of large-scale morphogenetic control in vertebrates, and is likely to
make manipulation of 14-3-3 protein signaling a powerful tool for addressing
aspects of asymmetry at every scale.
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ACKNOWLEDGMENTS |
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Footnotes |
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