Characterization of the Cadherin-Catenin Complex of the Sea Anemone Nematostella vectensis and Implications for the Evolution of Metazoan Cell-Cell Adhesion

Abstract

The cadherin-catenin complex (CCC) mediates cell-cell adhesion in bilaterian animals by linking extracellular cadherin-based adhesions to the actin cytoskeleton. However, it is unknown whether the basic organization of the complex is conserved across all metazoans. We tested whether protein interactions and actin-binding properties of the CCC are conserved in a nonbilaterian animal, the sea anemone Nematostella vectensis We demonstrated that N. vectensis has a complete repertoire of cadherin-catenin proteins, including two classical cadherins, one α-catenin, and one β-catenin. Using size-exclusion chromatography and multi-angle light scattering, we showed that α-catenin and β-catenin formed a heterodimer that bound N. vectensis Cadherin-1 and -2. Nematostella vectensis α-catenin bound F-actin with equivalent affinity as either a monomer or an α/β-catenin heterodimer, and its affinity for F-actin was, in part, regulated by a novel insert between the N- and C-terminal domains. Nematostella vectensis α-catenin inhibited Arp2/3 complex-mediated nucleation of actin filaments, a regulatory property previously thought to be unique to mammalian αE-catenin. Thus, despite significant differences in sequence, the key interactions of the CCC are conserved between bilaterians and cnidarians, indicating that the core function of the CCC as a link between cell adhesions and the actin cytoskeleton is ancestral in the eumetazoans.

Keywords: adherens junction; cadherin; catenin; cell adhesion; cnidarians; evolution.

Fig. 1

Nematostella vectensis has a full complement of CCC components. (A) Predicted domain composition of N. vectensis cadherins, and α- and β-catenin examined in this study. Identified pFam motifs are annotated. Numbers for NvCad-1 and -2 and NvDachsous indicate the number of extracellular cadherin repeats. (B and C) Gene trees for N. vectensis cadherins and α-catenin. Nematostella vectensis genes are in red. Asterisks indicate the proteins examined in this study. Numbers indicate the posterior probability of each branch. Abbreviations for species names are as follows: Aq, A. queenslandica; Bf, B. floridae; Ce, C. elegans; Cg, C. gigas; Ci, C. intestinalis; Ct, C. teleta; Dd, D. discoideum; Df, D. fasciculatum; Dm, D. melanogaster; Dp, D. purpureum; Dr, D. rerio; Lg, L. gigantea; Mb, M. brevicollis; Ml, M. leiydi; Mm, M. musculus; Nv, N. vectensis; Oc, O. carmela; Sk, S. kowalevskii; Sp, S. purpuratus; Sr, S. rosetta; Ta, T. adhaerens; Xl, X. laevis. (B) A consensus phylogeny of the N. vectensis cadherins that cluster within known cadherin sub-types from bilaterian organisms generated using FastTree2. (C) A phylogeny of α-Catenin and Vinculin orthologs from metazoan organisms and related unicellular eukaryotes.

Fig. 2

Domain organization is partially conserved between N. vectensis α-catenin and bilaterian orthologs. (A) Mus musculus αE-catenin is composed of 5 four-helix bundles, a hinge region, and a C-terminal five-helix bundle. Nematostella vectensis α-catenin has regional homology to the N- and C-terminal helical bundle regions (blue) of M. musculus αE-catenin, but also includes a novel insertion into the hinge region (green). β-catenin (β-cat) binding/dimerization, modulation (M), and F-actin binding (ABD) domains in M. musculus αE-catenin are annotated. Regions of homology in N. vectensis α-catenin are indicated by dashed lines. (B–D) Limited proteolysis of full-length N. vectensis α-catenin (B), a variant lacking the insert (α-NM-ABD; C), and the insert alone (α-I; D). Predicted MW of variants are 139, 98, and 57 kDa for full-length α-catenin, α-NM-ABD, and α-I, respectively. Protein schematics are included for reference to domains. Coomassie-stained SDS–PAGE of proteins incubated for the indicated times with 0.05 mg/ml trypsin. The insert (residues 663–902) and M-domain (residues 392–642), as identified by Edman degradation N-terminal sequencing, are marked with arrows. (E) Native-PAGE of 20 μM full-length N. vectensis α-catenin (α-WT), α-NM-ABD and α-I (from B–D).

Fig. 3

Nematostella vectensis α- and β-catenin bind to form a heterodimer. (A) Schematic of the α-catenin·β-catenin complex based on crystal structures of mammalian orthologs. The interacting surface is highlighted (red). β-catenin helices β1 and β2 displace the N-terminal α-catenin α1 helix from the N_I domain in order to form 2 four-helix bundles with α-catenin helices α1–α4 (Pokutta et al. 2014). The loop connecting β-catenin helices β1 and β2 interacts with the N_II bundle of α-catenin. Percent Identity and similarity between N. vectensis β-catenin and a bilaterian consensus sequence is compared for the interacting β1 and β2 helices and loop region, and the entire binding region. (B) A multiple alignment of the α-catenin binding region of N. vectensis β-catenin with the corresponding region of β-catenin orthologs from representative bilaterian species. β1 and β2 helices and loop region (boxes), as well as the entire binding region (red bar), are annotated. Abbreviations used for species names are as follows: B. flo.—B. floridae; C. gig.—C. gigas; D. mel.—D. melanogaster; D.rer.—D. rerio; M. mus.—M. musculus; N. vec.—N. vectensis; P. dum.—P. dumerilii; S. kow.—S. kowalevskii; S. pur.—S. purpuratus. (C) SEC-MALS elution profiles for N. vectensis α- and β-catenin (yellow and blue, respectively) run independently, and following co-incubation at 18 °C for 30 min (green). MW measurements corresponding to the UV peaks for each run are plotted in darker shades of the same color. (D) SDS–PAGE of fractions collected from (C).

Fig. 4

Nematostella vectensis Cadherin-1 and -2, but not Dachsous, bind to the α·β-catenin heterodimer. (A–C) SEC-MALS elution profiles for the cytoplasmic tails of N. vectensis Cadherin-1 (A, yellow), Cadherin-2 (B, orange), and Dachsous (C, green) run independently, and following incubation at 18 °C for 30 min with either β-catenin (blue) or a pre-assembled α·β-catenin heterodimer (red). MW measurements corresponding to the UV peaks for each run are plotted in darker shades of the same color. (C) SDS–PAGE analysis (below) of fractions collected from the elution of N. vectensis Dachsous incubated with the α·β-heterodimer (red) indicate that the high-molecular-weight peak observed (asterisk) consists only of α·β-heterodimer, and does not contain Dachsous, which elutes independently as a second, low-molecular-weight peak (see table 2 for observed MWs).

Fig. 5

Nematostella vectensis Cadherin-1 and -2 bind to N. vectensis β-catenin with high affinity. (A) Alignment of the cytoplasmic domain of N. vectensis Cadherin-1 and -2 with those of M. musculus E-cadherin and C. elegans HMR1. Conserved residues shown to be critical for the mammalian E-cadherin·β-catenin interaction are highlighted (grey). Regions II–V as identified in (Huber and Weis 2001) are annotated above with colored bars; the thickened rectangles indicate helices. Known phosphorylated residues in mammalian E-cadherin are highlighted (yellow boxes), and the conserved phosphoserine residue affecting β-catenin affinity is indicated (red arrow). Acidic residues within the phosphorylation site of N. vectensis cadherins that are not present in mammalian E-cadherin are annotated (blue boxes, asterisks). (B and C) Nematostella vectensis Cadherin-1 and -2 binding to N. vectensis β-catenin was quantified using ITC. (B) Nematostella vectensis Cadherin-1 was titrated into N. vectensis β-catenin. The ratio of heat released (Kcal) per mole of N. vectensis Cadherin-1 injected into N. vectensis β-catenin was plotted against the molar ratio of the two proteins, and the K_d calculated from these measurements is indicated. (C) Nematostella vectensis Cadherin-2 was titrated into N. vectensis β-catenin as in (B).

Fig. 6

Nematostella vectensis α-catenin and a N. vectensis α·β-catenin heterodimer bind to F-actin. (A–E) High-speed cosedimentation assays of full-length N. vectensis α-catenin (A), the α-NM-ABD variant lacking the insert (B), and the α·β-catenin heterodimer (C) with F-actin. Proteins were incubated at the final concentrations indicated with either 2 μM F-actin (+ Actin) or no-actin control (− Actin). Samples were analyzed by SDS–PAGE and CBB staining (A–C), and bound protein was plotted against free protein and fit with a hyperbolic function (D—red line; K_d and B_max are listed). (E) Mean K_d values are reported.

Fig. 7

Nematostella vectensis α-catenin, but not a N. vectensis α·β-catenin heterodimer, inhibits Arp2/3 complex-mediated nucleation of F-actin. (A and B) Effect of N. vectensis α-catenin (A) or N. vectensis α·β-catenin heterodimer (B) on Arp2/3-mediated actin polymerization in a pyrene-actin assay. Reactions contained 2.32 μM of a 10% pyrene-actin mix (− control, black circles) plus 12.5 nM Arp2/3 complex and 23.2 nM WASp-VCA (+ control, grey diamonds), and the indicated concentrations of protein. Examples shown are each representative of at least three independent experiments.