XB-ART-18505J Cell Biol 1996 Mar 01;1325:871-85.
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Xenopus laevis actin-depolymerizing factor/cofilin: a phosphorylation-regulated protein essential for development.
Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.
PubMed ID: 8603919
PMC ID: PMC2120733
Species referenced: Xenopus laevis
Genes referenced: actb actl6a cfl1 diras1 dstn nsd1 tbx2 uqcc6
Article Images: [+] show captions
|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.|
|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.|
|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.|
|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).|
|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.|
|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.|
|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).|
|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).|
|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.|
|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.|
|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.|
References [+] :
Abe, Sequence of cDNAs encoding actin depolymerizing factor and cofilin of embryonic chicken skeletal muscle: two functionally distinct actin-regulatory proteins exhibit high structural homology. 1990, Pubmed