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BACKGROUND: The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive.
RESULTS: Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects.
CONCLUSION: Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development.
Figure 7. Mis-expression of Eps8 results in gastrulation defects. (A) RT-PCR analysis of the developmental expression of XEps8. ODC serves as a control for RNA isolation and reverse transcription. (B) Western blot probed with anti-XEps8 antibodies show that XEps8 protein is provided maternally and is present in gastrula, neurula, and tailbud stage embryos. (C) Control and (D) Eps8-injected embryos at stage 12. In control embryos the blastopore is well formed and has progressed vegetally (arrowheads). In contrast, Eps8-injected embryos display severe buckling of tissue above the blastopore (arrowheads) and a disorganized blastopore lip that is delayed and malformed. (E) Control stage 37/38 embryos. (F) Eps8-injected embryos show a range of phenotypes including microcephaly, cyclopia, and shortening and arching of the A-P axis. Top = low dose (50 pg); middle = intermediate dose (200 pg); bottom = high dose (1 ng). (G,H) Histological analysis of Eps8-injected embryos shows that Eps8 expression causes a broadening of the notochord (no) and disorganization of the neural tube (nt) and somites (so).
Figure 1. Eps8 induced actin remodeling in cultured cells. Phalloidin staining of the actin cytoskeleton in untransfected (A) B16F1, (B) MDA-MB231, and (C) MDA-MB231BO cells. Cells possess few filopodia-like structures extending from lateral and dorsal surfaces and do not possess cytoplasmic actin cables. Actin structures induced by Eps8 in (D-I) B16F1, (J-L) MDA-MB231, and (M-O) MDA-MB231BO cells. Distribution of Eps8-myc (D,G,J,M) revealed by 9e10 anti-c-myc antibody and actin (E,H,K,N) revealed by phalloidin staining in fixed cells. Right column (F,I,L,O) shows merged images with Eps8-myc in red and actin in green. The boxed region in (F) is enlarged in the inset. Eps8 induces the formation of filopodia-like structures in B16F1 and MDA-MB231 cells and localizes to filopodia-like structures, ruffles, and actin cables in MDA-MB231BO cells. In B16F1 cells, Eps8 localizes along the length of the filopodia-like structures (arrowheads in G-I) and is enriched at their tips (arrowheads, inset in F). Scale bar is equal to 10 μm in (A-F) and (J-O) and 5 μm in (G-I).
Figure 2. Abi1 modulates the morphology of actin structures induced by Eps8. Actin structures induced by Eps8 and Abi1 in (A-C) B16F1, (D-F) MDA-MB231, and (G-I) MDA-MB231BO cells. Distribution of Eps8-myc (A,D,G,J) revealed by 9e10 anti-c-myc antibody, Abi1-GFP (B,E,H), and Abi1DY-GFP (K) revealed by GFP, and actin (C,F,I,L) revealed by phalloidin staining in fixed cells. (A-I) Simultaneous expression of Eps8 and Abi1 induces the formation of actin cables in B16F1, MDA-MB231, and MDA-MB231BO. Eps8 and Abi1 co-localize in association with actin cables (arrows) but Abi1 is not enriched with Eps8 in filopodia (arrowheads in A-C). (J-L) Formation of actin cables in B16F1 cells is dependent on the interaction of Eps8 and Abi1. Abi1DY does not co-localize with Eps8 and does not induce actin cable formation. Scale bar is equal to 10 μm.
Figure 3. Eps8-induced actin remodeling in Xenopus embryos. Distribution of actin (A,C,D,E,G,H) revealed by phalloidin staining and Eps8-myc (B,D,F,H) revealed by 9e10 anti-c-myc antibody in animal cap cells of Xenopus embryos. In control animal caps, actin filaments are enriched at cell-cell junctions in superficial epithelial cells (A) and the cortex of deep cells (E). (B-D) In superficial epithelial cells, Eps8 expression causes an enrichment of actin filaments at cell-cell junctions (arrowheads). (F-H) In deep cells facing the blastocoel, Eps8 expression induces the formation of actin cables (arrowheads). (D,H) Eps8 is red and actin is green in merged images. Scale bar is equal to 10 μm.
Figure 4. Abi1 modulates Eps8-induced actin remodeling in a Rac-independent manner. Distribution of Eps8-myc (A,D), Abi1GFP (B), actin (C) revealed by phalloidin staining, and Rac (E,F) in Xenopus animal cap cells. (A-C) Simultaneous expression of Eps8 and Abi1 induce the formation of actin stars at the apical surface of superficial epithelial cells. (D-F) Endogenous Rac is not recruited to Eps8/Abi1-induced actin stars. (F) Eps8 is red and Rac is green in the merged image. Scale bar is equal to 10 μm in A,B and D-F and to 5 μm in C.
Figure 5. Regulation of Eps8/Abi1-induced actin remodeling in Xenopus. Distribution of Eps8-myc (A,E,I,M; red in D,H,L,P), actin (B,F,J,N; green in D,H,L,P), CP-GFP (C, blue in D), N-WASP-GFP (G, blue in H), FP4-mito-GFP (K, blue in L), and Xvasp-GFP (O, blue in P). (A-D) CP does not block formation of actin stars. (E-H) N-WASP is recruited to Eps8/Abi1-induced actin stars. Actin star formation is not altered in response to inhibition of Ena/VASP activity (I-L) or increased levels of Xvasp (M-P). Scale bar is equal to 10 μm in A-L and 5 μm in M-P.
Figure 6. Dsh is recruited to the plasma membrane and actin filaments in response to Eps8 expression. Distribution of Dsh-GFP (A,B,D, green in F), actin (C), Eps8-myc (E,G, red in F), Abi1-GFP (H), and Dsh-flag (I) in Xenopus animal cap cells. (A) Localization of Dsh-GFP in control animal caps. (B,C) In response to Eps8 expression, Dsh is recruited to the membrane and cell-cell junctions in superficial epithelial cells where it co-localizes with actin (arrows). (D-F) In deep cells facing the blastocoel, Eps8 induced the recruitment of Dsh to cytoplasmic actin cables and the cell cortex where Dsh co-localized with Eps8 (co-localization appears yellow). (G-I) Co-localization of Eps8, Abi1, and Dsh in animal caps cells (arrows mark site of co-localization). Scale bars are equal to 10 μm.
Figure 8. Eps8 blocks elongation of activin treated animal caps. (A,B) Control and Eps8-injected animal cap explants remain rounded in the absence of activin. (C,D) Control explants elongate extensively in the presence of activin, whereas Eps8 expression inhibits elongation. (E) RT-PCR analysis shows that Eps8 expression does not block activin-mediated induction of the mesodermal markers Xbra and MyoD. ODC is a control for mRNA isolation, reverse transcription, and gel loading.
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