Ancient origin of the integrin-mediated adhesion and signaling machinery Arnau Sebé-Pedrósa, Andrew J. Rogerb, Franz B. Langc, Nicole Kingd, and Iñaki Ruiz-Trilloa,e,1 aDepartament de Genètica and Institut de Recerca en Biodiversitat, Universitat de Barcelona, 08028 Barcelona, Spain; bCentre for Comparative Genomics and Evolutionary Bioinformatics, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada B3H 1X5; cDepartment of Biochemistry, Université de Montréal, Montréal, QC, Canada H3C 3J7; dDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and eInstitució Catalana per a la Recerca i Estudis Avançats at Parc Científic de Barcelona, 08028 Barcelona, Spain Edited* by W. Ford Doolittle, Dalhousie University, Halifax, NS, Canada, and approved April 28, 2010 (received for review February 24, 2010) The evolution of animals (metazoans) from their unicellular Integrins are heterodimeric transmembrane proteins composed ancestors required the emergence of novel mechanisms for cell of one α and one β subunit (17). The integrin-mediated process of adhesion and cell–cell communication. One of the most important linking the extracellular matrix to the intracellular actin cyto- cell adhesion mechanisms for metazoan development is integrin- skeleton is made in concert with several cytoskeletal proteins that mediated adhesion and signaling. The integrin adhesion complex form adhesion-triggered signaling complexes (22): α-actinin and mediates critical interactions between cells and the extracellular talin [both of which directly bind to the integrin β subunit (23– matrix, modulating several aspects of cell physiology. To date this 25)]; and paxillin and vinculin [both of which are scaffolding machinery has been considered strictly metazoan specific. Here we proteins that indirectly bind to integrin-β via talin and α-actinin report the results of a comparative genomic analysis of the integ- (26, 27)]. An important element of the integrin adhesion ma- rin adhesion machinery, using genomic data from several unicel- chinery is the heterotrimer IPP complex, which is composed of lular relatives of Metazoa and Fungi. Unexpectedly, we found that ILK (integrin-linked kinase), PINCH (particularly interesting core components of the integrin adhesion complex are encoded in Cys-His–rich protein), and parvin (28, 29). This complex plays an the genome of the apusozoan protist Amastigomonas sp., and important role in integrin-mediated signaling, regulating apo- therefore their origins predate the divergence of Opisthokonta, ptosis, and cell dynamics (29). Finally, integrin-mediated signaling the clade that includes metazoans and fungi. Furthermore, our occurs mainly via two kinases known to be concentrated at the analyses suggest that key components of this apparatus have integrin adhesion machinery, namely c-Src tyrosine kinase and been lost independently in fungi and choanoflagellates. Our data FAK (focal adhesion kinase) (22, 30, 31). Many other proteins highlight the fact that many of the key genes that had formerly are indirectly involved with the integrin adhesion complex (32), been cited as crucial for metazoan origins have a much earlier but here we focus on those most directly involved in the clustering origin. This underscores the importance of gene cooption in the of integrins into the adhesion complex (22). unicellular-to-multicellular transition that led to the emergence of The recent completion of genome sequences for five close rel- the Metazoa. atives (some strictly unicellular, some colonial) of metazoans and fungi provides the opportunity to reconstruct the evolution of cell adhesion | lateral gene transfer | metazoan origins | multicellularity proteins required for integrin-mediated cell adhesion (ref. 33; see also http://www.broadinstitute.org/annotation/genome/multi- ittle is known about how multicellular animals (metazoans) or cellularity_project/MultiHome.html). By examining the genomes Lfungi evolved from their single-celled or colonial ancestors. of the amoeba C. owczarzaki, two basal fungi, Allomyces macro- Cell adhesion and cell signaling are two important features of gynus and Spizellomyces punctatus, the apusozoan Amastigomonas the multicellular metazoan lifestyle that were likely critical to the sp., and a choanoflagellate, Proterospongia sp., we find that the origin of Metazoa (1, 2). Recent data have shown that many of the integrin adhesion and signaling machinery evolved in unicellular major metazoan signaling pathways and cell adhesion systems are progenitors of apusozoan protists and opisthokonts (i.e., Fungi, ubiquitous across the metazoan kingdom, including nonbilaterian choanoflagellates, and Metazoa). Integrin α and β and several lineages [sponges, placozoans, and cnidarians (3–6)]. These other components of the integrin adhesion complex are absent findings indicate that cell adhesion and cell signaling genes might from choanoflagellates and fungi and were presumably lost in- have evolved before the origin of Metazoa. Consistent with this dependently in these lineages. By comparing genome data from view, choanoflagellates, the unicellular putative sister group of a broad sampling of unicellular taxa, we have been able to clarify Metazoa (7–11), have been shown to possess some genes involved the dynamic evolutionary history of the integrin adhesion complex. in cell signaling and adhesion, such as tyrosine kinases and cad- Results herins (1, 12–14). Expressed sequence tag surveys of other uni- β cellular relatives of metazoans, such as Capsaspora owczarzaki Integrins. Outside Metazoa, we found four integrin and four α β α and Ministeria vibrans, also yielded homologs of genes involved in integrin genes in C. owczarzaki, and one and one in Amas- β metazoan cell adhesion and cell signaling (9, 15). tigomonas sp. Interestingly, we found one of the integrin domains Here we report a comparative genomic survey of integrin- (the extracellular domain) in the cyanobacterium Trichodesmium mediated adhesion machinery, a critical cell–matrix adhesion mechanism in metazoans that also plays a vital role in cell sig- naling (16–18). Integrin-mediated signaling occurs in two ways: Author contributions: A.S.-P. performed research; A.S.-P., F.B.L., and I.R.-T. analyzed data; A.J.R., F.B.L., and N.K. contributed new reagents/analytic tools; I.R.-T. designed research; as an “inside-out” signaling modulated through intracellular and A.S.-P., A.J.R., F.B.L., N.K., and I.R.-T. wrote the paper. “ ” events, and as outside-in signaling that reacts via binding of The authors declare no conflict of interest. a ligand to the receptor (17, 19, 20). Thus, integrins are involved *This Direct Submission article had a prearranged editor. in diverse cellular processes, including embryogenesis, cell spread- Data deposition: The sequences reported in this paper have been deposited in the Gen- ing, cell migration, and proliferation (16–18). However, integrin Bank database (accession nos. GU320672–GU320675). adhesion and signaling seems to be absent from other multicellular 1To whom correspondence should be addressed. E-mail: [email protected]. organisms (e.g., plants and fungi) and is generally considered to be This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. metazoan specific (2, 5, 21). 1073/pnas.1002257107/-/DCSupplemental. 10142–10147 | PNAS | June 1, 2010 | vol. 107 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1002257107 Downloaded by guest on September 29, 2021 erythraeum (34), which lacks all of the other integrin β domains. We next analyzed whether β integrins from C. owczarzaki and Integrins were not detected in any other examined eukaryote. Amastigomonas sp. and the integrin β extracellular domain from Interestingly, although an integrin α ortholog was thought to be T. erythraeum have conserved the functional domains and motifs present in the choanoflagellate M. brevicollis (12), we failed to present in metazoan integrins (Fig. 1 and Fig. S3). The cation- detect a bona fide integrin α in either Monosiga brevicollis or Pro- binding motifs MIDAS, ADMIDAS, and LIMB, which are lo- terospongia sp. The putative integrin α from M. brevicollis (XP_ cated in the extracellular domain (35, 36), are well conserved in β 001749484) did not pass any of our criteria (for example, reverse the different nonmetazoan integrin , except for C. owczarzaki β β β blast did not give integrin α hits; Methods). The M. brevicollis gene integrin 4 (Fig. 1A). Moreover, C. owczarzaki integrin 1, 2, β β XP_001749484 shares with integrin α homologs the presence of and 3 and Amastigomonas sp. integrin have a clear expansion some FG-GAP repeats domains, which are not specifictointegrinα of the cysteine-rich stalk (Fig. 1A and Fig. S3), which accounts for their longer size relative to metazoan integrin β proteins. and are found in other nonintegrin proteins. A phylogeny made Other key motifs in metazoan integrin β proteins are the cyto- from FG-GAP repeats shows the M. brevicollis putative integrin α plasmic integrin α-interacting motif and the NPXY motif, which homolog clustering with nonintegrin bacterial proteins but not with plays a key role in protein interactions (17, 20, 37, 38). Both integrin α (Fig. S1). On the other hand, our phylogenetic analysis of β –β β motifs are well conserved in C. owczarzaki integrin 1 3 and integrin (Fig. S2) shows the four C. owczarzaki integrins clustering Amastigomonas sp. integrin β (Fig. 1A). Finally, both C. owc- together with a bootstrap