C. elegans Enabled exhibits novel interactions with N-WASP, Abl, and cell-cell junctions

Abstract

Ena/VASP proteins are associated with cell-cell junctions in cultured mammalian cells [1] and Drosophila epithelia [2, 3], but they have only been extensively studied at the leading edges of migratory fibroblasts, where they modulate the protrusion of the leading edge [4]. They act by regulating actin-filament geometry, antagonizing the effects of actin-capping protein [5]. Embryos lacking the C. elegans Ena/VASP, UNC-34, display subtle defects in the leading edges of migrating epidermal cells but undergo normal epidermal morphogenesis. In contrast, embryos lacking both UNC-34 and the C. elegans N-WASP homolog have severe defects in epidermal morphogenesis, suggesting that they have parallel roles in coordinating cell behavior. GFP-tagged UNC-34 localizes to the leading edges of migrating epidermal cells, becoming redistributed to new junctions that form during epidermal-sheet sealing. Consistent with this, UNC-34 contributes to the formation of cadherin-based junctions. The junctional localization of UNC-34 is independent of proteins involved in Ena/VASP localization in other experimental systems; instead, junctional distribution depends upon the junctional protein AJM-1. We also show that Abelson tyrosine kinase, a major regulator of Enabled in Drosophila, is not required for UNC-34/Ena function in epithelia. Instead, our data suggest that Abelson kinase acts in parallel to UNC-34/Ena, antagonizing its function.

Figure 1

UNC-34 and WSP-1 Act Redundantly to Fulfill an Essential Role during Ventral Enclosure Projected confocal or multiphoton Z series, ventral views, with anterior to the left. (A)–(B′) and (F)–(G) show AJM-1::GFP in living embryos. (C)–(E) show DLG-1Δ7::GFP in living embryos. (F)–(G) show unc-34(gm104);wsp-1(RNAi) embryos with weaker enclosure phenotypes. The scale bar represents 10 μm. (A and A′) Wild-type enclosure. Leading cells (arrows) migrate ahead of posterior pocket cells (representative cells are marked with arrowheads). (B and B′) unc-34(gm104);wsp-1(RNAi) embryos fail to enclose properly. Leading cells ([B], arrows) show disrupted migration, and the epidermis ultimately retracts to the dorsal surface, ejecting pharyngeal tissue ([B′], arrow) and intestine to the surface of the embryo. (C and C′) Wild-type enclosure. Leading cells extend broad elaborate protrusions ([C], arrows) during migration toward the midline. (D and D′) unc-34(gm104);wsp-1(RNAi) embryos fail to enclose and have blunted leading cell protrusions ([D], arrows). (E) unc-34(gm104) embryos are not obviously disrupted, though they show subtle quantitative reduction in pocket cell protrusion dynamics. (F and F′) Embryos defective in cell migration and epithelial-sheet sealing, causing gaps to remain at the ventral midline junction ([F′], arrows). (G) Disrupted migration in ventral cells leads to hemi-junction formation (arrows).

Figure 2

UNC-34 localizes to the leading edge of cell protrusions and to apical junctions. (A and A′) Confocal projections of dorsolateral (A) and ventrolateral (A′) surfaces (anterior is to the left) of the same embryo stained at mid-enclosure with anti-UNC-34 show broad expression throughout the embryo with enrichment at apical epidermal junctions. (B–E) Time-lapse sequence of projected confocal Z series, visualizing UNC-34::GFP in the ventral leading cell region (anterior is to the left) during enclosure. Besides apical junction enrichment, UNC-34::GFP is also present at the leading edge (arrowheads) of leading cell protrusions (C). Junctions enriched in UNC-34::GFP become apparent along the anterior and posterior borders of migrating cells ([C], arrow). Protrusions in contralateral partners meet at the ventral midline ([D], arrow), where apical junctions enriched with UNC-34::GFP eventually form ([E], arrow). (F–H) Confocal images of a wild-type 3-fold stage embryo expressing UNC-34::GFP (F) costained for AJM-1 (G). A merged image is shown in (H). (I–K) Representative wide-field images of wild-type (I), ajm-1(ok160) (J), and hmr-1(zu389) maternal and zygotic loss (K) embryos expressing UNC-34::GFP. UNC-34::GFP at apical junctions is misdistributed into puncta in ajm-1 embryos ([J], arrowheads) but is largely normal in hmr-1 embryos. (L–O) Immunostaining of UNC-34 in embryos carrying a truncated ajm-1::gfp transgene lacking a putative consensus binding site for Ena/VASP proteins (ajm-1(102-868)::gfp). (L) and (M) show a wild-type embryo expressing AJM-1(102-868)::GFP. UNC-34 localizes to junctions ([M], arrow). UNC-34 is also prominently expressed in neurons of the nerve ring (asterisk). (N) and (O) show an ajm-1(ok160) embryo rescued by ajm-1(102-868)::gfp. Although the truncated AJM-1 localizes to junctions (N), it is insufficient to localize UNC-34 there (O), despite robust expression in neurons (asterisk). Scale bars represent 10 μm.

Figure 3

Loss of unc-34 Function Enhances the Penetrance and Severity of hmp-1(fe4) Morphogenetic Defects (A–C) Time-lapse images showing the morphogenesis of representative embryos for each genotype: hmp-1(fe4) (A), unc-34(gm104) (B), and unc-34(gm104) hmp-1(fe4) double mutant (C). Anterior is to the left in all panels. The leading cells initiate ventral enclosure in all three embryos (arrowheads), but in unc-34(gm104) hmp-1(fe4) double mutants, the leading cells fail to establish junctions at the ventral midline. By 38 min, the hmp-1(fe4) and unc-34(gm104) embryos have completed ventral enclosure and begun elongation. Ventral enclosure is incomplete in the unc-34(gm104) hmp-1(fe4) embryo and the internal tissues are extruded by the contraction of the epidermis ([C], asterisk). Note that the hmp-1(fe4) embryo undergoes ventral enclosure at the same rate as the unc-34(gm104) embryo, but its elongation rate is reduced relative to unc-34(gm104). Scale bar represents 10 μm. (D) Quantification of the morphological defects displayed by hmp-1(fe4) and unc-34(gm104) hmp-1(fe4) embryos. The bars show the proportion of embryos showing each class of defect. The following abbreviations are used: ventral enclosure defects (VE), embryos that arrest at less than twice the premorphogenetic length (Hmp), and embryos that elongate beyond 2-fold (Vab; this class is the only one to contain viable embryos able to reach fertile adulthood). At least 500 embryos were scored for each genotype.