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The Premetazoan Ancestry of Cadherins

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Science  15 Feb 2008:
Vol. 319, Issue 5865, pp. 946-948
DOI: 10.1126/science.1151084

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Abstract

Cadherin-mediated cell adhesion and signaling is essential for metazoan development and yet is absent from all other multicellular organisms. We found cadherin genes at numbers similar to those observed in complex metazoans in one of the closest single-celled relatives of metazoans, the choanoflagellate Monosiga brevicollis. Because the evolution of metazoans from a single-celled ancestor required novel cell adhesion and signaling mechanisms, the discovery of diverse cadherins in choanoflagellates suggests that cadherins may have contributed to metazoan origins.

The evolution of animals (metazoans) from their single-celled ancestors required genomic innovations that allowed cells to adhere and communicate (13). Cadherins are critical mediators of metazoan cell adhesion and signaling and provide the structural basis for vital developmental processes, including tissue morphogenesis and maintenance, cell sorting, and cell polarization (48). However, despite their importance for metazoan multicellularity, cadherins are apparently lacking from all nonmetazoan multicellular organisms (e.g., plants and fungi). Indeed, cadherins have only been found in metazoans and their closest single-celled relatives, the choanoflagellates (9, 10), which suggests that the study of choanoflagellate cadherins may illuminate the transition from single-celled organisms to multicellular metazoans.

Choanoflagellates are unicellular and colony-forming organisms that use a single apical flagellum surrounded by a collar of actin-filled microvilli to swim and capture bacterial prey. Both the cell morphology and feeding strategy of choanoflagellates are nearly indistinguishable from those of feeding cells (choanocytes) in sponges (11, 12). In contrast to sponges and other metazoans, however, all choanoflagellates have a unicellular stage in their life history. Furthermore, choanoflagellates are not metazoans and did not evolve from sponges or more recently derived metazoan phyla (1316). Thus, the common ancestor of choanoflagellates and metazoans was probably unicellular or, at most, capable of forming simple colonies.

Given the absence of overt cell adhesion in M. brevicollis, one might expect choanoflagellates to have fewer cadherin genes than metazoans. We identified cadherins in the recently sequenced M. brevicollis genome and compared them with those in four metazoan genomes: Nematostella vectensis (phylum Cnidaria), Drosophila melanogaster (phylum Arthropoda), Ciona intestinalis (phylum Chordata), and Mus musculus (phylum Chordata) (17). Cadherin gene numbers range from 17 in D. melanogaster to 127 in M. musculus (representing from 0.12 to 0.39% of the gene catalog; Table 1). Similarly, 23 putative cadherin genes (representing 0.25% of the gene catalog) are present in M. brevicollis, revealing that the absolute and relative abundances of cadherins in choanoflagellates are comparable to those of diverse metazoan genomes despite a lesser degree of morphological complexity (Table 1 and table S1 and fig. S1). In contrast, the numbers of extracellular cadherin (EC) repeats (the defining domain of cadherins) in M. brevicollis and N. vectensis cadherins exceed those of more recently derived metazoans (Table 1).

Table 1.

Cadherin abundance and number of EC repeats in M. brevicollis and diverse eukaryotes. Atha, Arabidopsis thaliana; Ddis, Dictyostelium discoideum; Scer, Saccharomyces cerevisiae; Mbre, Monosiga brevicollis; Nvec, Nematostella vectensis; Dmel, Drosophila melanogaster; Cint, Ciona intestinalis; Mmus, Mus musculus. Normalized cadherin abundance is the percentage of EC repeat–encoding genes in the draft gene catalog.

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Cadherins containing epidermal growth factor (EGF), laminin G (LamG), and transmembrane domains linked to EC repeats [proteins with these four linked domains are classified as Fat cadherins (18)] are observed in M. brevicollis, sponges, sea urchins, and humans, among others (Fig. 1, A and B), which suggests that cadherins with these physically linked domains are ubiquitous and predate metazoan origins. Additionally, M. brevicollis, the sponge Amphimedon queenslandica, and N. vectensis (19) share cadherins with EC repeats linked to the Src homology 2 (SH2), Hedgehog N-terminal peptide (N-hh), immunoglobulin (Ig), and von Willebrand type A domains, which suggests that cadherins containing these domains evolved before metazoans. Because SH2 domains bind sites of tyrosine phosphorylation (20), choanoflagellate and cnidarian cadherins containing cytoplasmic SH2 domains [MBCDH1 and 2 and NvHedgling (19); Fig. 1, A and C] could connect extracellular cues to intracellular processes such as cell cycle regulation and cellular metabolism. The presence of a protein tyrosine phosphatase domain in two M. brevicollis cadherins, MBCDH21 and MBCDH7 (Fig. 1, A and B, and fig. S1), provides further evidence of a connection between choanoflagellate cadherins and tyrosine kinase signaling. Likewise, the presence of an N-hh domain at the amino termini of cadherins in choanoflagellates, sponges, and cnidarians (Fig. 1, A and C) suggests an ancestral connection between cadherins and hedgehog signaling components important in metazoan development (21).

Fig. 1.

(A) Venn diagram analysis of domains linked to EC repeats in M. brevicollis, N. vectensis, and M. musculus cadherins. (B) Representative composition of Fat-related cadherins from M. brevicollis and diverse metazoan taxa. The cladogram depicts relations among metazoan phyla (34, 35). Green boxes highlight clusters of EGF and LamG domains, and EC repeats are shown in blue. (C) Protein domains shared by M. brevicollis MBCDH1 and 2, MBCDH10, and MBCDH11; the sponge cadherin AmqHedgling; and the cnidarian cadherin NvHedgling. These domains are absent from M. musculus cadherins. Blue boxes contain SH2 domains, yellow boxes contain immunoglobulin domains, and red boxes contain N-hh and VWA domains. (Key) Symbols used in (B) and (C). LCA, last common ancestor. LamN and LamG domains in MBCDH21 are below the SMART e-value threshold but above the Pfam threshold (36, 37). See fig. S1 for the complete domain structure of MBCDH21, table S2 for protein identifiers and species names, and table S3 for domain abbreviations.

The linkage of EC repeats to SH2 and N-hh domains in M. brevicollis and N. vectensis appears to be absent in more recently derived lineages, which suggests that this ancient protein architecture was lost relatively early in metazoan evolution. In contrast, the connection between metazoan cadherins and β-catenin, part of the Wnt developmental signaling pathway (6, 22) and an important regulator of cadherin-mediated adhesion (23, 24), seemingly represents a metazoan innovation that evolved after the divergence of choanoflagellate and metazoan lineages. Unlike M. brevicollis, metazoan classical cadherins contain a highly conserved cadherin cytoplasmic domain (CCD) with binding sites for β-catenin. N. vectensis has five CCDs (fig. S2), and we found that a previously identified sponge cadherin also contains a cadherin CCD domain (Fig. 1B) (17, 25). This indicates that CCD-containing cadherins evolved before the origin of Bilateria.

Monosiga brevicollis leads a unicellular lifestyle and is not known to form cell-cell contacts. Therefore, the biological processes mediated by choanoflagellate cadherins remain enigmatic. We investigated the subcellular localization of MBCDH1 and MBCDH2, two nearly identical cadherins (note S1 in supporting online text and fig. S3) with domain contents resembling those of inferred ancestral cadherins (Fig. 1C). Antibodies raised against an extracellular portion of MBCDH1, which has 95% sequence identity to MBCDH2, recognized a single protein band of the predicted size (∼191 kD; figs. S3 and S4) that presumably represents both MBCDH1 and MBCDH2. Subcellular cadherin localization was revealed by probing cells with the antibody either before or after cell membrane permeabilization. MBCDH1 and MBCDH2 were detected in four regions of the choanoflagellate cell: the apical collar of actin-filled microvilli, the basal pole of the cell, an unidentified structure at the apical end of the cell, and puncta within the cell body (Fig. 2, A to J). Antibodies bound to extracellular MBCDH1 and MBCDH2 colocalized with polymerized actin, most strikingly in the apical collar and, to a lesser extent, near the basal end of the cell (Fig. 2, F, G, and I). The colocalization of actin filaments and M. brevicollis cadherins suggests that associations between cadherins and actin filaments predate the diversification of choanoflagellates and metazoans. Metazoan SH2 domain–containing proteins, such as mammalian Nck, interact with actin-binding proteins through phosphorylated tyrosines (26, 27), which suggests that the cytoplasmic tails of MBCDH1 and MBCDH2 might indirectly interact with the underlying actin cytoskeleton.

Fig. 2.

Subcellular localization of MBCDH1 and MBCDH2 (A and F), compared with polymerized actin stained with rhodamine-phalloidin (B and G), or antibodies against β-tubulin (C and H). Cells were exposed to antibodies against MBCDH1 after (A to E) or before (F to J) permeabilization. Overlay of MBCDH1 and MBCDH2 (green), actin (red), and β-tubulin (blue) reveals colocalization of MBCDH1 and MBCDH2 with actin (yellow) on the collar and at the basal pole (D and I). Differential interference contrast microscopy shows cell morphology (E and J). Brackets, collar of microvilli; arrow, apical organelle; arrowhead, basal pole; asterisk, cluster of autofluorescent bacterial detritus.

On the basis of the shared domain content of M. brevicollis and metazoan cadherins (Fig. 1), cadherins in the last common ancestor of choanoflagellates and metazoans had, among others, SH2, N-hh, LamG, EGF, immunoglobulin, and transmembrane domains. The functions of these domains suggest that some choanoflagellate cadherins may mediate intracellular signaling. For example, the SH2 domain interacts with targets of tyrosine kinase phosphorylation, one of the few metazoan-type signaling networks found in choanoflagellates (9, 28). In metazoan epithelial cells, recruitment of β-catenin facilitates essential interactions between classical cadherins and the actin cytoskeleton to establish and maintain cell shape and polarity (29, 30). If M. brevicollis cadherins associate with the local actin cytoskeleton, as suggested by their colocalization, this interaction merits further investigation as MBCDH1 and MBCDH2 lack the CCD and the M. brevicollis genome lacks a β-catenin ortholog.

Metazoan E-cadherins and flamingo cadherins are bound by pathogenic bacteria which exploit them as extracellular tethers during host cell invasion (3133). It is possible that choanoflagellate cadherins fill an equivalent role in binding bacterial prey for recognition or capture, functions consistent with the enrichment of MBCDH1 and MBCDH2 on the feeding collar (Fig. 2). If ancient cadherins bound bacteria in the unicellular progenitor of choanoflagellates and metazoans, cadherin-mediated cell adhesion in metazoans may reflect the co-option of a class of proteins whose earliest function was to interpret and respond to cues from the extracellular milieu. Indeed, the transition to multicellularity likely rested on the co-option of diverse transmembrane and secreted proteins to new functions in intercellular signaling and adhesion.

Supporting Online Material

www.sciencemag.org/cgi/content/full/319/5865/946/DC1

Materials and Methods

SOM Text

Figs. S1 to S4

Tables S1 to S5

References

References and Notes

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