The C. elegans zonula occludens ortholog cooperates with the cadherin complex to recruit actin during morphogenesis

Abstract

The dramatic cell-shape changes necessary to form a multicellular organism require cell-cell junctions to be both pliable and strong. The zonula occludens (ZO) subfamily of membrane-associated guanylate kinases (MAGUKs) are scaffolding molecules thought to regulate cell-cell adhesion [1-3], but there is little known about their roles in vivo. To elucidate the functional role of ZO proteins in a living embryo, we have characterized the sole C. elegans ZO family member, ZOO-1. ZOO-1 localizes with the cadherin-catenin complex during development, and its junctional recruitment requires the transmembrane proteins HMR-1/E-cadherin and VAB-9/claudin, but surprisingly, not HMP-1/alpha-catenin or HMP-2/beta-catenin. zoo-1 knockdown results in lethality during elongation, resulting in the rupture of epidermal cell-cell junctions under stress and failure of epidermal sheet sealing at the ventral midline. Consistent with a role in recruiting actin to the junction in parallel to the cadherin-catenin complex, zoo-1 loss of function reduces the dynamic recruitment of actin to junctions and enhances the severity of actin filament defects in hypomorphic alleles of hmp-1 and hmp-2. These results show that ZOO-1 cooperates with the cadherin-catenin complex to dynamically regulate strong junctional anchorage to the actin cytoskeleton during morphogenesis.

Figure 1. ZOO-1 junctional recruitment is dependent on HMR-1/E-cadherin and VAB- 9/Claudin, but not on HMP-2/β-catenin or HMP-1/α-catenin

(A–L) Confocal images of elongating embryos stained for ZOO-1 (A, D, G, J, M, green), AJM-1 as a junctional marker (B, E, H, K, N, red), and the merged image (C, F, I, L, O). Wild-type (A–C), dlg-1(RNAi) (D–F), and hmp-1(RNAi) (G–I) embryos display proper junctional localization of ZOO-1, despite disruption of AJM-1 localization in the case of dlg-1(RNAi) (E). (J–L) hmr-1(RNAi) embryo exhibits abrogated junctional ZOO-1 staining, though staining persists in sarcomeres (J, arrow). (M–O) vab-9(ju6) embryo lacks junctional ZOO-1 staining, though ZOO-1 localization in sarcomeres in unaffected (M, arrow). Scale bar, 10 µm.

Figure 2. Loss of zoo-1 function causes embryonic lethality

Nomarski images of representative embryos undergoing elongation are shown. t = 0 correlates with 90 minutes post ventral enclosure. (A–C) Wild-type embryo. (D–F) rrf-3(pk1426) embryo. (G–I) zoo-1(RNAi);rrf-3(pk1426) embryo exhibiting failed elongation with pronounced body shape defects. (J–L) zoo-1(RNAi);rrf-3(pk1426) embryo that has ruptured from the posterior region. Note the delayed elongation of the zoo-1(RNAi);rrf-3(pk1426) embryos relative to wildtype. (M–O). zoo-1(RNAi);mel-11(it26) embryo. Note the ventral rupture (N, arrow). Scale bar, 10 µm.

Figure 3. Loss of zoo-1 function disrupts actin accumulation at cell-cell junctions

(A,B) Ventral views of a wild-type (A) and zoo-1(RNAi);rrf-3 (B) embryo at the end of ventral enclosure expressing a gfp-tagged fragment of vab-10 that binds F-actin in epidermal cells [41]. In the wild-type embryo, two pairs of anterior cells have accumulated dense actin at the midline (A, arrows), whereas only small actin puncta (B, left arrow) or detached actin filaments (B, right arrow) remain at the same position in the zoo-1(RNAi);rrf-3 embryo (B, arrows). (C) Robust actin cables are visible at cell-cell borders in epidermal cells in a comma stage embryo (arrows). (D) Actin is less evenly distributed at junctions in epidermal cells of comma stage zoo-1 knockdown embryos (arrows). (E,F) Embryos at the two-fold stage of elongation stained for F-actin. (E) Wild-type embryo. (F) zoo-1(RNAi) embryo. Note the abnormal clustering of circumferential actin filament bundles in the zoo-1(RNAi) embryo (arrow). Scale bars, 5 µm.

Figure 4. zoo-1(RNAi) enhances the elongation defects of hmp-1(fe4) mutants

(A–C) Nomarski images at 90-minute time intervals of representative embryos undergoing elongation. (B) Wild-type embryo. (B) hmp-1(fe4) embryo with visible body shape defects. (C) zoo-1(RNAi);hmp-1(fe4) embryo that has ruptured from the ventral surface (arrow). (D) Distribution of embryonic lethal phenotypes of hmp-1(fe4) and zoo-1(RNAi);hmp-1(fe4) animals. (E, F) Representative confocal images of F-actin staining in a hmp-1(fe4) (E) and zoo-1(RNAi);hmp-1(fe4) (F) embryo. The organization of circumferential actin filaments is consistently and markedly disrupted in zoo-1(RNAi);hmp-1(fe4) embryos. Scale bar, 10 µm.