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Figure 1. (.4) Sequence of XAC cDNAs and the encoded proteins. Regions at the Y-ends used for the probes for Southern and Northern
blots are underlined. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers U26270 (XAC1)
and U26269 (XAC2). (B) Comparison of the XAC1 and XAC2 protein sequences to those of chicken cofilin and ADF. The regulatory
phosphorylation site (V) and the region used for preparation of isoform specific antibodies (underline) are shown.
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Figure 2. (A) Purified GST-XAC fusion proteins from glutathione
column. Lanes: (M) Molecular mass markers (116, 97,
45, 31, 21, 14 kD); (1) 2 pug GST-XAC1; (2) 2 p~g GST-XAC2. (B)
pH-dependent F-actin binding of XAC1 and XAC2. Approximately
4 p~M XAC was mixed with 5 p~M G-actin (final concentrations)
in 30 mM Pipes, pH 6.8, or 30 mM Tris, pH 8.0 each containing
1 mM D'IT and 0.2 mM ATP. KC1 and MgCI 2 were added
to give 0.1 M and 2 mM final concentrations, respectively, in 50 p,i
final volume. After 60 min the samples were centrifuged at
170,000 g for 30 min. The supernate was removed, mixed, and a
40-~1 aliquot removed for analysis by SDS-PAGE. The pellet was
washed once with the buffer and salt mixture, and then solubilized
in SDS sample preparation buffer. Volumes loaded on the
gel represent an identical fraction of the supernatant (S) and pellet
(P) protein. Standard is thrombin-cleaved GST-XACI: G,
glutathione-S-transferase; X, XAC. (C) Depolymerization of
F-actin (3 Ixg) by GST-XAC1 (I) and GST-XAC2 (A) were
compared to recombinant chick cofilin (@) by measuring the
amount of G-actin released using the DNase I inhibition assay.
The DNase I was calibrated using muscle G-actin.
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Figure 3. (A) Southern blots of Xenopus genomic
DNA hybridized to XAC1 and XAC2
3'UTR DNA probes (see Fig. 1). Restriction
endonucleases used: E, EcoRI; Ba, BamHI;
P, PstI; H, HindlII; Bg, BgllI; A, ApaI. Arrowheads
show the XAC2 genomic DNA
bands which are cross-reactive with XAC1
probe. (B) Northern blot of total RNA (13
i~g/lane) from unfertilized eggs (Oc) and developing
embryos hybridized to coding region
DNA probe for XAC1. Two, four, and eight
cell embryos (2C, 4C, and 8C); other developmental
stages are shown by the stage number.
Positions of 18 S and 28 S ribosomal RNA remained
constant across the gel (visualized by
ethidium bromide staining) while those of the
XAC mRNAs decreased to the adult sizes.
Identical results were obtained using the
3'UTR DNA probes (not shown). Lanes $9-
$28 were from a separate gel (longer exposure)
that also contained oocyte RNA and
were aligned accordingly. Because of the
overlap in the signal with the rRNA bands
before stage 9, it is not possible to conclude
from this gel alone that the signals are specific.
However, immunoblotting and immunofluorescence
staining reported below support
the early expression seen here. (C) Northern
blot of total RNA (13 Ixg) from adult tissues
hybridized to 3'UTR DNA probes of XAC1
(left) and XAC2 (right). Ethidium bromide
staining (not shown) was used to verify that
similar amounts of RNA were loaded per
lane. These results demonstrate both the
specificity of probe hybridization to the 4.5-
kb mRNA, and the difference in size of the
smaller mRNA between oocyte and adult.
Identical results were obtained using the coding
region DNA probe for XAC1 used in Fig.
3 B (not shown). B, brain; SM, skeletal muscle;
CM, cardiac muscle; Oc, oocytes removed
from adult; Sto, stomach.
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Figure 4. In situ hybridization of Xenopus embryos with XAC riboprobes.
Identical patterns of expression were observed with
the specific probe to the 3'UTR of XAC2 and with the coding region
probe to XAC2 (cross-reacts with XAC1 gene fragments on
Southern blots). The anti-sense RNA probe used for a, b, d, e,
and fis from the XAC2 coding region, while the XAC1 3'UTR
probe was used for c (the dorsal view). Controls (g-k) use the
sense RNA probes made against the coding region of XAC2. Developmental
stages shown: 15/16 (a and g); 21 (b, c, and h); 26 (d
and i); 32 (e and j); 36 (fand k). The development of the reaction
product was stopped quickly to show the regions that contained
particularly high levels of the XAC mRNA. It is difficult to differentiate
specific regions within the head from these studies, but
immunofluorescence localization of the XAC was done on vertical
sections through the anterior end of the embryo (see below).
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Figure 5. (A) Specificity of
rabbit antiserum raised to
peptides from XAC1 and
XAC2 tested against different
amounts of GST-XAC1
and GST-XAC2 on Western
blots. Both antisera were
used at 1:1,000 dilutions and
the blots using XAC1 peptide
antibody were washed in
0.4 M MgC12 to improve
specificity. (B) Western blot
of extracts (2 wg protein)
from embryos made at 2, 4,
and 10 h postfertilization,
and from tadpoles 72 h after
fertilization probed with
XAC1 and XAC2 peptide
antibodies. TH, tadpole
head; TT, tadpole tail. To
show antibody specificity,
standards of thrombin-cleaved
GST-XAC1 and GSTXAC2
are loaded on the left
and right hand sides of the
gel, respectively, with the
amounts shown as rig/lane of
XAC. (C) Western blots,
probed with XAC1 and
XAC2 peptide antibodies, of
extracts from: Oc, unfertilized
eggs; SM, skeletal muscle;
Li, liver; Te, testis; Lu,
lung; St, stomach; Ht, heart;
In, intestine; Br, brain; SC,
spinal cord. All extracts contained
10 wg protein except
for brain and spinal cord
which contained 5 I~g protein.
Position of standards is
as in B with the amounts
shown as ng/lane of XAC.
(D) Specificity of monoclonal
antibodies (hybridoma
supernatants) 2F10 and 1All,
and polyclonal (PC) IgG
raised against GST-XAC,
tested against XAC1 stan-dard (S) and extracts of Xenopus stage 21 embryos (E) and adult brain (B). Coomassie blue-stained gel (Stain) and Western blots are shown. XAC1 standard was prepared by thrombin cleavage of GST-XAC1.
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Figure 6. Immunofluorescence localization of XAC (using monoclonal antibody 2F10) and actin in 5 p,m sections of Xenopus oocytes,
eggs, and embryos. Photographic and printing times were adjusted such that sections stained with secondary antibody alone (control for
monoclonal antibody) or nonimmune rabbit serum (control for actin polyclonal antibody) were black. (A) Oocytes (a-c), unfertilized
eggs (d-f), and zygotes 30 min after fertilization (g-/). Phase contrast micrographs showing the junction (arrowheads) between the animal
and vegetal hemisphere cortex (a, d, g, and j) and corresponding immunofluorescence (b, e, h, and k) showing both XAC (b, e, and
h) and actin (k). Immunofluorescence of the cortex within the vegetal hemisphere showing XAC (c, f, and i) and actin (/). (B) Sections
of zygotes at I h postfertilization (a-f) and during early first cleavage (g-/). Phase contrast micrographs showing the animal hemisphere
(a, d, g, and j) (arrowhead in a shows junction between hemispheres) and corresponding immunofluorescence for XAC (b and h) and actin (e and k). Immunofluorescence of vegetal hemisphere cortex stained for XAC (c and i) and actin (f and/) are also shown. (C)
Phase contrast micrographs (a and c) and corresponding immunofluorescence for XAC (b) and actin (d) in sections of the zygotes at
late first cleavage. Arrowhead in b shows staining of the XAC preceding the invagination of the cleavage furrow. Immunofluorescence
of XAC and actin in sections of the blastocysts at the end of first cleavage are shown in e and f, respectively. Staining of a midbody structure
for XAC is shown by arrowhead.
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Figure 7. Immunocytochemical staining for XAC (using the 2F10 monoclonal antibody) in whole mount (albino) Xenopus embryos during
development. Stages shown are (a) 2 cell, (b) 8 cell, and (c) stage 38. Development times were limited to allow visualization of the
regions of most intense staining. No staining was observed in embryos treated with secondary antibody alone. Immunofluorescence
staining of XAC in 5 ~m vertical sections of developing (wild type) Xenopus embryos. Stage 17 embryo (d) shows particularly strong
fluorescence for XAC in the region of the developing neural plate and neural fold. Cells within the neural tube (Nt), notochord (Nc) and
somites (S) are brightly stained at stage 24 (e). (f) An enlargement of the neural tube from a stage 24 embryo shows many brightly
stained cells. (g) The neural tube, epidermis (E), cells lining the lumen of the archenteron (L), and a layer of cells within the archenteron
(Ar) are brightly stained at stage 34. A more anterior vertical section through a stage 34 embryo (h) shows intense staining of
the neural tube, cells within the developing retina (R) and the neuronal cell bodies which constitute the base of the cement gland (C).
Immunofluorescent controls (secondary antibody only) for sections of oocyte and embryos were black (not shown).
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Figure 8. (A) Immunoblot of 2D gel (NEpHGE/SDS-PAGE) of
extracts from Xenopus zygote (1 h post fertilization) before and
after treatment of the denatured proteins with alkaline phosphatase
(Morgan et al., 1993). (B) Immunoblots of 2D gels of oocytes,
unfertilized egg, and fertilized eggs at 15, 30, 60 min, and 3 h
postfertilization. (C) pXAC as a percent of total immunoreactive
XAC quantified from 2D immunoblots of embryo extracts. Each
solid triangle represents values from at least three 2D gels of
pooled embryos (5-10) taken at the times shown after mass fertilization
of eggs. Open symbols represent values from individual
eggs or embryos at 0-, 15-, 30-, 60-, and 180-min time points to
show individual variability. In individual oocytes removed surgically
from the adult (O), pXAC was the only immunoreactive species
present (n = 8).
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Figure 9. Inhibition of XAC with antibody or introduction of constitutively
active GST-XAC inhibits cleavage of Xenopus embryos.
(a) Anti-XAC (PC-IgG) (10 mg/ml; 20 nl) was injected
into one blastomere of Xenopus embryos at the two cell stage.
Cleavage of the injected blastomere was inhibited as shown. Embryos
were allowed to develop for 3 h before photography. Injected
hemisphere is on the right side of embryo. (b) Embryo injected
with anti-XAC (PC-IgG) which had been neutralized with
GST-XAC before injection. Development is normal (16 cell
stage). (e) Phase contrast micrographs of 6 l~m cross-section of
animal hemisphere of embryo injected in one blastomere at the
two cell stage with anti-XAC (PC-IgG) (10 mg/ml; 20 nl). Embryos
were fixed 3.5 h postinjection. Lobed cleavage nucleus is
shown by an arrow and is magnified in d. Asters are shown by arrowheads
and magnified in e.
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Figure 10. (a) GST-XAC (5 mg/ml; 20 nl) was injected into one
blastomere of 34 embryos at the 2 cell stage. Cleavage of the injected
blastomere was inhibited in 68% of the embryos. Four representative
examples are shown with arrowheads pointing to the
injected blastomere. (b) Injection of GST-XAC (5 mg/ml; 20 nl)
into an egg undergoing first cleavage causes the regression of the
cleavage furrow (left, fixed in the process of regression) whereas
injection of buffer had no effect (right). (c-f) Phase contrast and
immunofluorescence photographs of paraffin sections of eggs microinjected
with GST-XAC during first cleavage and fixed after
furrow regression. (c and e) Phase contrast photographs show position
of regressed cleavage furrow (arrowheads). Pigment granules
were often depleted in region of regressed furrow with pigment
granules remaining deeper in cytoplasm. (d) Immunofluorescence
staining of actin in the same section of regressed furrow
shown in c. Actin fluorescence is diffuse in the cortex. (f) Immunofluorescence
staining of XAC (using 2F10 monoclonal antibody)
of the same section of regressed furrow shown in e. The
XAC is also diffuse in the cortex of the injected egg.
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Figure 11. GST-XAC is not
phosphorylated in Xenopus
eggs. Fertilized eggs were microinjected
with GST-XAC1
(4 mg/ml; 30 nl]egg) and 1 h
later the eggs were extracted,
proteins precipitated, and
half of the sample treated
with alkaline phosphatase as
described in Materials and
Methods. Samples and a standard
of uninjected protein
were separated by 2D gel
electrophoresis and immunoblotted using the 1All monoclonal antibody to XAC. (a) Recombinant GST-XAC1 before injection. (b)
Extracts from eggs injected with GST-XAC1 show the two endogenous XAC species (pXAC and XAC) and a single spot of 45 kD for
GST-XAC1. (c) Alkaline phosphatase treatment of this extract completely converted the endogenous pXAC to XAC, but the position
of the GST-XAC1 did not change.
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