Fig 1.
Nine evolutionarily conserved metazoan FH2 domain subtypes.
The evolutionary history for 100 FH2 domain amino acid sequences from representatives of eleven metazoan phyla was inferred by the ML method for 343 amino acid positions occupied in ≥ 95% of sequences. All bootstrap values are indicated, and the scale bar indicates the number of substitutions per site for branch lengths. Nine groups populated by formins from multiple species clustered behind nodes with bootstrap values ≥ 50, suggesting the presence of nine evolutionarily conserved subtypes. Seven of these conformed to the previously recognized DAAM, DIAPH, FHOD, FMN, FMNL, INF and GRID2IP subtypes, while two others, designated MWHF and PHCF, were novel. Asterisks (*) indicate formins for which a partial FH2 domain sequence was used for this analysis.
Fig 2.
PH domain-containing formins (PHCFs) in vertebrates and other metazoans.
(A) Domain organizations of PHCF homologs. Based on predicted structural domains, PH domain-containing formins are present in the opossum M. domestica (Md), zebrafish D. rerio (Dr), purple sea urchin S. purpuratus (Sp), Pacific oyster C. gigas (Cg), owl limpet L. gigantea (Lg), and sponge A. queenslandica (Aqu). The domain organization of the M. musculus (Mm) Drf-type DIAPH1 formin is shown for comparison. The scale bar indicates protein length in amino acid residue number. (B) Conserved synteny in the chromosomal neighborhood of vertebrate PHCF genes. The chromosomal neighborhood of the MC3R and CBLN4 genes is shown for human H. sapiens (Hs), common chimpanzee Pan troglodytes (Pt), rhesus macaque Macaca mulatta (Mmul), mouse M. musculus (Mm), rabbit Oryctolagus cuniculus (Oc), dog Canis familiaris (Cf), pig Sus scrofa (Ss), elephant Loxodonta africana (La), opossum M. domestica (Md), Tasmanian devil Sarcophilus harrisii (Sh), platypus Ornithorhynchus anatinus (Oa), zebra finch Taeniopygia guttat (Tg), flycatcher Ficedula ablicollis (Fab), chicken G. gallus (Gg), anole lizard Anolis carolinensis (Ac), Chinese turtle Pelodiscus sinesnsis (Ps), and coelacanth Latimeria chalumnae (Lc). Gene order is largely conserved, but a PHCF-coding gene (medium green) is present in this region only in coelacanth, birds, and marsupials. Distances are not drawn to scale, and white genes have no homologs. (C) Blocks of homology to the opossum PHCF coding sequence in the human genome. (Left) Six blocks of sequence in the human genome between the MC3R and CBLN4 genes can be aligned with parts of six predicted exons of the opossum PHCF-coding gene. (Right) Conceptual translations of the human sequences produce in-frame stop codons and shifts in reading frame relative to the opossum sequence, consistent with absence of a functional human PHCF-coding gene.
Fig 3.
Conserved DID-DD sequences in formins and MWH proteins.
(A) Predicted domain organization of DID- and DD-containing metazoan proteins. Mouse formins of the canonical subtypes shown for comparison are: Drf-type DIAPH1, DAAM1, and FMNL1; Drf-like INF2 with a comparatively truncated N-terminus; and non-Drf-type FHOD1 with a structurally distinct GTPase-binding domain (G2), GRID2IP with PDZ and Harmonin N-terminus-like (HN) domains, and FMN2 with a structurally divergent N-terminus. Also shown is the zebrafish non-Drf PHCF with N-terminal PH domains. The presence of C-terminal DAD/WH2-like motifs (red bars) generally correlates with the presence of an N-terminal DID. (Middle) Shown are predicted domain organizations for MWHF proteins identified in the polychaete worm C. teleta (Ct), the Pacific oyster C. gigas (Cg), the purple sea urchin S. purpuratus (Sp), and the sponge A. queenslandica (Aqu). Despite a Drf-type N-terminus, these proteins lack C-terminal DAD/WH2-like sequences. (Bottom) Drf-like DID and DD are also predicted for MWH of D. melanogaster (Dm), and related proteins in the water flea D. pulex (Dp), the horseshoe crab L. polyphemus (Lp), and the roundworms C. elegans (Ce) and S. ratti (Sr). Scale bar indicates protein lengths in amino acid residue number. (B) ML phylogenetic tree of metazoan DID and DD sequences. The evolutionary history for 56 DID-DD sequences from formins and MWH homologs was inferred by the ML method for 227 amino acid positions occupied in ≥ 90% of sequences. All bootstrap values are shown, and the scale bar indicates the number of substitutions per site for branch lengths. Proteins were selected for analysis from representatives of eleven phyla. Because no MWH-related protein could be detected in the representative nematode A. suum, the MWH-like protein F53B3.3 from C. elegans was also included in this analysis. For each formin, the subtype established based on FH2 domain (Fig 1) was recapitulated based on DID-DD. MWH DID-DD sequences grouped with a novel subtype of MWH-related formins (MWHFs). Note, ML trees generated without MWH proteins are otherwise essentially unchanged.
Fig 4.
Sequence similarities between MWHF, MWH, and FMNL proteins.
(A) DID and DD sequences. Shown are a subset of amino acid sequences used to estimate the DID-DD phylogenetic tree of Fig 3B, including those from the MWHF proteins of the polychaete worm C. teleta (Ct), the Pacific oyster C. gigas (Cg), and the purple sea urchin S. purpuratus (Sp), MWH from D. melanogaster (Dm), and the formins FMNL1, DAAM1, DIAPH1, and INF2 from the opossum M. domestica (Md). Green circles indicate amino acid positions for which MWH and two or more MWHFs are identical but distinct from other formins. (B) FH2 domain sequences. Shown are sequences from the same subset of formins used to estimate the FH2 domain phylogenetic tree of Fig 1. Red asterisks indicate conserved aromatic residues of the FH2 domain lasso. The second aromatic residue is phenylalanine in FMNL and MWHF proteins, but tryptophan in all other formins. A blue triangle indicates a position uniquely occupied by proline in FMNL and MWHF proteins.
Fig 5.
Divergent FH2 domains of nematode FOZI-1-like proteins belong to the FMNL subtype.
(A) Domain organizations of conventional nematode FMNL homologs and FOZI-1-related proteins. Predicted structural domains are shown for FMNL-subtype FH2 domain-containing proteins from M. musculus (Mm) and five nematodes, A. suum (As), C. elegans (Ce), S. ratti (Sr), R. culicivorax (Rc), and T. suis (Ts). The nematode proteins fall into two classes. The conventional FMNL proteins largely resemble M. musculus FMNL1 with a Drf-type N-terminal domain organization (G-DID-DD) and pair of WH2/DAD-like motifs (red bars) C-terminal to the FH2 domain. The second set of proteins, including C. elegans FOZI-1, have N-terminal zinc fingers (ZF) followed by a C-terminal FH2 domain and no additional formin homology. The scale bar indicates protein length in amino acid residue number. (B) ML phylogenetic tree of full-length nematode and other bilaterian FH2 domain sequences. The evolutionary history for 93 FH2 domain sequences from five nematodes belonging to two orders (chromadorea and enoplea), and from six non-nematode bilaterians, was inferred by the ML method for 280 amino acid positions fully occupied in all sequences. All bootstrap values are indicated, and the scale bar indicates the number of substitutions per site for branch lengths. All nematode FH2 domains fell within one of the nine subtypes. Within the FMNL subtype, the divergent FOZI-1-related proteins of the chromadorean nematodes formed a subgroup that was positioned closely to the chromadorean conventional FMNL formins.
Fig 6.
Patterns of formin subtype losses in metazoa.
The current disposition of formin subtypes among the metazoan phyla can be explained by the presence of all nine subtypes in the last common metazoan ancestor, and subsequent subtype losses (x) or partial losses (•) in different lineages. Because the evolutionary relationships among basal phyla (porifera, placozoa, and ctenophora) remain under debate [42–44], those phyla have each been drawn as independent branches. Five formin subtypes have been lost from placozoa (GRID2IP, FMNL, MWHF, DAAM, and PHCF), three from ctenophora (GRID2IP, FMNL, and FMN), and one from cnidaria (FMNL). Among the lophotrochozoans, one subtype has been lost from annelida (PHCF), and four from platyhelminthes (GRID2IP, MWHF, FMNL, and PHCF). Note, no helminth DAAM subtypes were displayed in phylogenetic trees, but the planaria Schmidtea mediterranea, which was not included in estimating those trees, encodes a DAAM-related formin. In the common ecdysozoan ancestor to arthropods and nematodes, two subtypes were lost (GRID2IP and PHCF), and the C-terminus of the MWHF subtype was lost, resulting in MWH proteins. In nematodes, an additional loss of the FMN subtype occurred. Among the deuterostomes, chordates lost a single subtype (MWHF). Porifera, mollusca and echinodermata retain homologs of all nine subtypes. Events along the same braches were positioned for visual clarity, and are not meant to imply relative timing.