http://orcid.org/0000-0001-7294-1465
Figures
Abstract
Ran (ras-related nuclear protein) is a small GTPase belonging to the RAS superfamily that is specialized in nuclear trafficking. Through different accessory proteins, Ran plays key roles in several processes including nuclear import-export, mitotic progression and spindle assembly. Consequently, Ran dysfunction has been linked to several human pathologies. This work illustrates the high degree of amino acid conservation of Ran orthologues across evolution, reflected in its conserved role in nuclear trafficking. Moreover, we studied the evolutionary scenario of the pre-metazoan genetic linkage between Ran and Stx, and we hypothesized that chromosomal proximity of these two genes across metazoans could be related to a regulatory logic or a functional linkage. We studied, for the first time, Ran expression during amphioxus development and reported its presence in the neural vesicle, mouth, gill slits and gut corresponding to body regions involved in active cell division.
Citation: Coppola U, Caccavale F, Scelzo M, Holland ND, Ristoratore F, D’Aniello S (2018) Ran GTPase, an eukaryotic gene novelty, is involved in amphioxus mitosis. PLoS ONE 13(10): e0196930. https://doi.org/10.1371/journal.pone.0196930
Editor: Jr-Kai Sky Yu, Academia Sinica, TAIWAN
Received: November 10, 2017; Accepted: April 23, 2018; Published: October 9, 2018
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Funded by FP7 People: Marie Curie Actions PCIG09-GA-2011-293871 to Dr Salvatore D'Aniello.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Ras-related nuclear protein (Ran) is an eukaryotic evolutionary conserved small GTPase representing the sole Ras superfamily member active in nucleus and traditionally considered a master regulator of nuclear trafficking [1,2]. Like other Ras proteins, it interacts with many cellular proteins comprising GTPase-activating protein (RanGAP), guanine-nucleotide-exchange factor (RanGEF) and GTP-Ran binding proteins, such as RanBP1 [3]. RanGEF catalyzes the GDP/GTP exchange while RanGAP promotes the GTP hydrolysis of Ran protein. In contrast, the cytoplasmic RanBP1 is an accessory protein that accelerates GTP hydrolysis carried out by RanGAP when other GTP-Ran binding proteins are absent [4]. A further relevant Ran accessory protein implicated in GTP hydrolysis is the importin β3, also named RanBP5, able to bind nuclear pore complexes [5]. Ran activity is crucial for nuclear import-export, and the presence of a GTP-Ran gradient across the nuclear envelope has been implicated in nucleo-cytoplasmic transport [6]. In addition, RanBP1 overexpression in murine fibroblasts and Xenopus laevis egg extracts has demonstrated the involvement of Ran pathway in mitotic progression [7,8]. The decrease of GTP-Ran levels in frog eggs resulted in disrupted spindle assembly, potentially connected to tubulin polymerization microtubule activities [8] and to DNA replication [9]. Moreover, it has been shown that Ran is involved in the stability of centrosomes [10].
In mouse embryos Ran is expressed in the nervous system with a strong accumulation in neural crest cells and sensory pits, as well as in the hematopoietic system with prevalence in blood islands [11]. The expression in mammalian nervous tissues is consistent with that observed in Drosophila and Xenopus [12,13]. In the prawn Penaeus monodon Ran transcripts are present in several body tissues and especially in developing ovary [14]. Recently, Ran has been described as a fundamental player in retinal cell proliferation and differentiation in zebrafish [15].
Ran has been associated to several human pathologies, like the rare X-linked neuron Kennedy’s disease caused by the expansion of the polyglutamine repeated region inside the androgen receptor, resulting in spinal and bulbar muscular atrophy [16]. Ran promotes bacteria infection through a modification of vacuole motility in Legionnaires’ disease, an atypical pneumonia caused by bacteria belonging to Legionella genus [17]. Moreover, Ran has been identified as a marker for the development of melanoma [18] and it is involved in teratocarcinoma, the most common testicular germ cell tumours [19], in serous epithelial ovarian tumour [20] and in lung cancer [21].
The present study reveals high conservation in the organization of the Ran locus during metazoan evolution as well as in the amino acid sequences of Ran proteins in a spectrum of eukaryotes. Special attention is given to the structure and function of Ran in the European amphioxus, Branchiostoma lanceolatum, in light of its key phylogenetic position within the phylum Chordata. Importantly, amphioxus Ran is conspicuously expressed in the developing neural and endodermal regions undergoing intensive cell division. This pattern is consistent with a key role for amphioxus Ran in mitotic dynamics in agreement with previous studies of the involvement of vertebrate Ran in cell proliferation during normal development and neoplastic growth.
Results
Ran protein sequence conservation
In order to study the evolution of nuclear-specific Ran, we carried out a detailed search in representatives of several available genomes and we did not find any Ran protein in Eubacteria and Archaea sequenced until now. This could suggest that Ran genes are an exclusive feature of Eukaryotes. We retrieved Ran proteins from available genomes encompassing: unicellular eukaryotes (Monosiga brevicollis, Capsaspora owczarzaki, Fusarium migrarium, Saccharomyces cerevisiae, Puccinia striiformis, Fragilariopsis cylindrus), plants (Arabidopsis thaliana, Oryza sativa), sponges (Amphimedon queenslandica), placozoans (Trichoplax adhaerens), ctenophores (Mnemiopsis leidyi), cnidarians (Exaiptasia pallida, Nematostella vectensis), arthropods (Drosophila melanogaster, Limulus poliphemus, Daphnia magna), nematodes (Caenorhabditis elegans), mollusks (Lottia gigantea, Crassostrea gigas, Octopus bimaculoides), annelids (Capitella teleta), brachiopods (Lingula anatina), priapulids (Priapulus caudatus), echinoderms (Strongylocentrotus purpuratus), hemichordates (Saccoglossus kowalevskii), cephalochordates (Branchiostoma floridae, Branchiostoma lanceolatum), urochordates (Oikopleura dioica, Ciona robusta, formerly known as Ciona intestinalis type A [22]), and vertebrates (Lethenteron japonicum, Callorhinchus milii, Latimeria chalumnae, Lepisosteus oculatus, Scleropages formosus, Danio rerio, Salmo salar, Xenopus tropicalis, Gallus gallus, Anolis carolinensis, Homo sapiens) (S1 File).
Most of the surveyed species possess a single-copy gene, except the yeast S. cerevisiae, the demosponge A. queenslandica, the fly D. melanogaster, the spotted gar L. oculatus and the Asian Arowana S. formosus, in which we found a second protein (Fig 1). Moreover, we found four Ran proteins in salmon S. salar. Given that some vertebrates possess more than one Ran, we performed a phylogenetic tree suggesting the presence of two distinct Ran paralogues in gnathostomes (S1 Fig), that we called Ran1 and Ran2. The unique Ran of tetrapods, coelacanth and ghost shark belongs to Ran2 clade, whereas eutelosts (zebrafish, salmon) possess only Ran1. On the other hand, spotted gar and Asian arowana retain both Ran genes (S1 Fig).
This picture shows the alignment of Ran proteins from unicellular eukaryotes to human, focused on the three main Rab domains: P-Loop (green), Switch I (blue) and Switch II (magenta). Asterisks have been used to point out the total amino acid conservation (81%, with ≤ 3 residues change). We highlighted in red the gnathostome-specific amino acid changes.
The alignment of 48 proteins from 40 species (Fig 1) revealed a high degree of Ran primary structure conservation across eukaryotes. Our analysis has been focused on a region of 79 amino acids comprising well-known functional Rab domains (P-Loop, Switch I, Switch II) with a very high sequence homology from unicellular eukaryotes to vertebrates (81%, with ≤ 3 residues change). Furthermore, we found that most of gnathostomes Ran proteins exhibit three conserved amino acid residues (QVQ) upstream to the P-Loop domain, which could confer an additional cellular function. The only exception was observed in coelacanth Ran and in salmon Ran1a and Ran1c, which have DVQ in place of QVQ.
Our findings indicate a strong degree of Ran conservation during evolution and a scenario of genome duplications and independent losses in vertebrates.
Ran-Syntaxin microsyntenic pair evolution
In order to gain insights on Ran evolutionary history, we investigated its genomic locus in selected eukaryotic genomes. Irimia et al. [23] have identified an ultra-conserved gene duplet formed by Ran and Syntaxin (Stx), an important family of Q-SNARE proteins implicated in exocytosis [24]. Here, we updated the evolutionary scenario of this genomic locus by surveying more vertebrate species (gar L. oculatus, arowana S. formosus, and salmon S. salar). The microsyntenic pair in analysed invertebrates (sea anemone N. vectensis, limpet L. gigantea, acorn worm S. kowalevskii and amphioxus B. lanceolatum) is formed by Ran and Stx1 (Fig 2). On the other hand, gnathostomes exhibit a different genomic combination of Ran and Stx genes. In order to clarify the origin of Stx and Ran duplet, we performed a phylogenetic analysis of Stx proteins (S2 Fig) and a syntenic survey in jawed vertebrates (S3 Fig, S4 Fig). The Maximum-Likelihood phylogenetic reconstruction showed the expansion of Stx family in vertebrates and the common evolutionary origin of Stx members flanking Ran genes (Stx1, Stx2 and Stx3) (Fig 2). Stx proteins included in the tree have been listed in the S2 File.
The scheme demonstrates the ultra-conserved genomic neighborhood between distinct Ran (pink boxes) and Stx (blue box) genes among distantly related species, showing how the ancestral genomic locus results conserved from protostomes to vertebrates. The Ran genomic loci of Callhorhincus milii (NW_006890249.1, Ran2) and Latimeria chalumnae (Scaffold JH126570.1, Ran2) show the same organization of human (data not shown).
As previously shown [23], Ran in invertebrates is always found associated to Stx1, while human gene is linked with Stx2 (Fig 2). Additionally, we have found that the unique Ran of zebrafish Danio rerio is coupled with Stx3a (Fig 2). In the genomes of L. oculatus [25] and S. formosus [26] we found that both Ran genes are coupled on distinct scaffolds with Stx2 and Stx3a, respectively (Fig 2). The analysis of Ran genomic loci in different vertebrates (human, spotted gar, Asian arowana, zebrafish, salmon) revealed a high degree of synteny (Fig 2, S3 Fig, S4 Fig) clarifying the orthology among multiple Ran and Stx genes (Fig 2): in particular we discovered that Ran1 is always associated with Stx3, while Ran2 with Stx2 (S3 Fig) and they probably derive from a duplicative event at the stem of vertebrates [27,28]. Moreover, the syntenic survey found four Ran1 loci in S. salar genome (S4 Fig), as an effect of teleost and salmonid extra genome duplications [29,30] and the loss of Ran2 in euteleosts (S3 Fig and S4 Fig). Altogether, our data suggest the existence of the ancient chromosomal linkage between Ran and Stx genes, which in vertebrates has been affected by distinct genome duplications.
Ran expression pattern in amphioxus
The Ran expression pattern during B. lanceolatum development was analysed by whole mount in situ hybridization (Fig 3). At middle neurula stage (24 hpf), Ran mRNA has been detected prevalently in endoderm and mesoderm (Fig 3A). At late neurula (30 hpf), labeled cells are still present in endoderm in the ventral part of the embryos (Fig 3B, sections in Fig 3E, 3F and 3G), and Ran expressing cells have also been found close to neural groove (Fig 3B). Pre-mouth larvae (48 hpf) present a very faint Ran expression in brain vesicle (Fig 3C), in oral endoderm corresponding to the pharyngeal area, (Fig 3C and section in 3H) in gill slits and in the mid- and hindgut (white arrowheads, Fig 3C and section in 3I). At larval stage (72 hpf), Ran is expressed in pre-oral pit (white arrow), mouth and first gill slit, in midgut plus the rostral part of hindgut (white arrowheads) (Fig 3D). The region of the tailbud is also positively labeled as shown in Fig 3D’.
A) middle neurula; B) late neurula; C) pre-mouth larva; D) 3 dpf larva; D’) enlargement of the 3 dpf larva’s tail. E) section of the late neurula in B at the level “e”. F) section of the late neurula in B at the level “f”. G) section of the late neurula in B at the level “g”. H) section of the pre-mouth in C at the level “h”. I) section of the pre-mouth in C at the level “i”. Abbreviation: ng, neural grove; bv, brain vesicle; o-en, oral endoderm; op, pre-oral pit; mo-gs, mouth and gill slits. White arrowheads indicate the anterior and posterior limits of Ran expression in gut. In all images the anterior is to the left and dorsal to the top. Scale bars: 60 μm in A-B-C-D; 15 μm in E.
Since a key role of Ran in cell divisions has been proposed, we performed immunostaining using PHH3 [Phospho-Histone H3 (Ser10)] antibody as a mitosis-specific marker in amphioxus to understand if there is a positive correlation of its expression pattern with cellular mitotic activity during development (Fig 4). PHH3 is expressed in proliferating cells and is important for the proper initial chromosomal condensation and segregation [31]. At middle neurula, the endoderm and somitic mesoderm cells are PHH3-positive, while ectodermal cells showed no evidence of proliferation (Fig 4A). Embryos at late neurula stage showed several positive cells in the endoderm, in the neural groove and in the posterior neural tube (Fig 4B). Later in development, at pre-mouth larvae, mitotic cells are visible in the oral endoderm region and in the neural tube (Fig 4C). At mouth-larva, PHH3 positive cells are present in the mouth, in the gill slits region and also along the gut (Fig 4D). Furthermore, few mitotic cells are visible in the most caudal part of the neural tube, and in the tailbud region, and residual signal is also detectable in the brain vesicle (Fig 4D). We observed that PHH3 expression profile varies slightly at different amphioxus specimens at the same developmental stage, as can be seen in additional pictures in S5 Fig. PHH3-positive cells pattern seemed to be faithfully superimposable to the dividing cells profile obtained in a previous study by using Bromodeoxyuridine [32], a synthetic analogue of thymidine, commonly used for detection of proliferating cells in vivo. In summary, we here described the Ran expression profile in embryos and larvae by whole mount in situ hybridizations, and compared it with PHH3 in order to inquire its involvement in cellular division in amphioxus.
A) middle neurula; B) late neurula; C) pre-mouth larva; D) 3 dpf larva. Abbreviation: ng, neural grove; nt, neural tube; bv, brain vesicle; o-en, oral endoderm; mo-gs, mouth and gill slits; tb, tailbud. In all images the anterior is to the left and dorsal to the top. Scale bars: 60 μm.
Discussion
Ran is an atypical small GTPase that is localized exclusively in nucleus and usually linked to macromolecular transport through nuclear pores. This protein plays a pivotal role in diverse mitotic steps in mammalian cells, like spindle formation and chromosome separation [33], and has been implicated in diverse human pathologies (detailed above in introduction).
Since Ran is so important for aspects of eukaryotic cell behaviour, we inquired into its molecular history broadly in Eukarya domain (Fig 1). Except few unrelated duplications, we found a single Ran from unicellular eukaryotes to higher plants and vertebrates, with a high degree of amino acid identity across tree of life. All the analysed taxa, in fact, possessed a Ran protein with an outstanding sequence conservation suggesting the importance of the evolutionary preservation of Ran role in cellular physiology. On the other hand, the fact that this protein has never been described in bacteria, despite extensive searches, speaks in favour of Ran advent in unicellular eukaryotes, consistently with a series of roles strictly related with nuclear functioning. The protein alignment hinted at some retained amino acid residue changes (QVQ) outside the fundamental domains in gnathostome Ran proteins, which indicates that there may be an unknown functional protein adaptation in this clade. Interestingly, the Ran of coelacanth and two Ran proteins of S. salar (Ran1a, Ran1c) show partial conservation of this amino acid stretch (DVQ), suggesting a possible diversification during evolution.
Additionally, if one considers that gene order on chromosomes is non-random [34], gene clusters can provide important insights into the evolution of genomes and their regulatory mechanisms. The physical linkage between Ran and Stx, two unrelated genes, belongs to a list of duplets unchanged across over 600 million years during evolution [23]: they form a conserved ancestral microsyntenic pair already present in the cnidarian-bilaterian ancestor. Our phylogeny evidences the common evolutionary origin for Syntaxins genes, i.e. Stx1, Stx2 and Stx3, which flank Ran genes in different animal lineages; moreover Stx1 is the unique gene present in non-vertebrate metazoans (S2 Fig).
Our phylogenetic and syntenic approach demonstrated that Ran1 and Ran2 of the gar and the arowana are paralogues deriving from whole-genome duplication at the root of vertebrate radiation [27,28]. The analysis of genomic loci of Ran genes showed that chondrichthyes and sarcopterygians have lost the Ran1, while zebrafish has lost Ran2 (Fig 2, S1 Fig, S2 Fig). In light of the Osteoglossomorpha and Holostea data we inferred the actinopterygian ancestor possessed both the Ran genes, and Ran2 was subsequently lost before the emergence of euteleosts (S1 Fig, S3 Fig). We found four Ran genes in S. salar, which previously was thought to have only two Ran [35] and this is probably due to teleost-specific (3R) and salmonid-specific fourth vertebrate whole-genome duplication (Ss4R) (S1 Fig) [29,36] [30]. The phylogeny and synteny demonstrated that salmon possess only Ran1 paralogues (S1 Fig, S4 Fig): this fits well with the concept of Ran1 as the unique gene retained in the ancestor of euteleosts. Since in extant analysed species Ran1 is always associated with Stx3 and Ran2 with Stx2 we hypothesized the existence of an ancestral linkage between a Ran1/2 and a Stx2/3 (S3 Fig, S4 Fig). Nevertheless, this gene duplet in gnathostomes has been strongly influenced by genome duplications and distinct gene losses whose impact should be further investigated [37].
The conserved Ran-Stx microsynteny might be due to regulatory logics: for instance, this linkage could be based on cis-Acting Transcriptional Repression [38]. Conversely, it has been proposed that Stx3 binds to RanBP5 in the nucleus interacting with several transcription factors [39], suggesting that Ran-Stx genomic proximity subtend a functional connection between Ran and Stx.
The amphioxus expression profile during embryogenesis could help our understanding of Ran evolutionary landscape in chordates, since cephalochordates are the closest living relatives of the ancestral chordate [40]. Our in situ hybridizations, in fact, showed Ran expression in several amphioxus tissues as mouth, gill slits, gut, and nervous system, whereas it is absent in epidermal cells: probably this is correlated with its multiple roles in the cell physiology. In particular, Ran is known to orchestrate multiple stages of the cell cycle including spindle assembly and nuclear envelope rearrangements, congruent with its important role in cell proliferation. Immunostaining with the proliferation marker PHH3 showed that cells of different embryonic structures are in active proliferation. It is also interesting to note that Ran mRNA expression is confined to structures known for their high proliferating rate: this suggests its possible function in cell division during amphioxus development. Interestingly, Ran absence in amphioxus epidermis could be associated to the strong expression of Dral proliferation suppressor in epidermal cells of late embryonic and early larval stages [41]. Moreover, Ran expression pattern in amphioxus is closely comparable to expression profiles previously observed in other animal models, supporting the concept of its determinant role in mitosis during evolution.
Conclusions
Phylogenetic and syntenic survey shed light on the conservation of Ran from unicellular eukaryotes to vertebrates, with the preservation of protein sequence possibly linked to its role in mitosis. Moreover, we highlighted the presence of Ran in amphioxus proliferating tissues during embryogenesis. Inter alia, we expanded the current knowledge about the retained pre-metazoan genetic linkage between Ran and Stx genes, likely under strong stabilizing selection in distantly related lineages. Our findings unravelled a complex evolutionary scenario for Ran-Stx duplet, which has been heavily impacted by genome duplications and gene losses in vertebrates that needs further investigations in future.
Materials and methods
Molecular evolution analysis
The sequences employed for the evolutionary survey were retrieved from the NCBI, Ensembl and JGI databases. Human RAN was the initial query sequence employed for tBlastn searches [42] from unicellular eukaryotes to vertebrates, and reciprocal Blasts were carried out. The identity of Ran proteins was further assessed by using the domain bank of PROSITE database [43] and manually aligned as in Coppola et al. [44]. Synteny between Ran and Stx was mapped manually through the examination of public databases (Ensembl, NCBI).
The Stx and Ran proteins were aligned employing Clustal Omega [45] and the phylogenetic reconstructions were computed with the Maximum Likelihood (ML) estimation using MEGA6 with 1,000 replicates and the WAG matrix (γ = 4 and proportion of invariable sites = 0.4) [46] and the graphical representation was performed with Dendroscope [47]. B. lanceolatum sequences and genomic loci were annotated using the genome draft, kindly provided by the Branchiostoma lanceolatum genome consortium.
Ethics statement
Branchiostoma lanceolatum (amphioxus) were collected in the Gulf of Napoli, Italy (latitude 40°48’33”N and longitude 14°12’55”E) from a location that is not privately-owned or protected, according to the authorization of Marina Mercantile (DPR 1639/68, 09/19/1980 confirmed by D. Lgs. 9/01/2012 n.4). The field studies did not involve endangered or protected species. All animal procedures were in compliance with the guidelines of the European Union.
Amphioxus embryo collection
Adult amphioxus (Branchiostoma lanceolatum) were maintained in an open seawater circulation aquaculture system under a 14 hours light/10 hours dark cycle. Animals were reared in tanks at 16–20°C with 10 cm of sand from the collection site and fed daily using a mix of two unicellular algae: Rhodomonas baltica and Isochrysis galbana. Spawning events were induced indoor in late spring by employing a thermic shock as described by Fuentes et al. [48]. After in vitro fertilization, embryos were cultured in 0.22 μm filtered seawater at 18°C and at desired developmental stages were fixed with 4% paraformaldehyde (PFA) in MOPS buffer overnight at 4°C, and then stored in 70% ethanol at -20°C.
Whole-mount in situ hybridization
Whole-mount in situ hybridization experiments were performed essentially as described in Annona et al. [49]. Briefly, embryos were rehydrated in phosphate buffered saline solution containing 0.1% Tween-20 (PBT) and treated with Proteinase K (5 μg/ml) to facilitate riboprobe penetration; the reaction was stopped by adding 4 μl of 10% glycine and then washed with 2 mg/ml glycine in PBT. The embryos were re-fixed in PBT containing 4% of PFA for 1 h at room temperature, subsequently washed in 0.1M triethanolamine and then in 0.1 M triethanolamine plus acetic anhydride, to prevent the formation of non-specific background. Embryos were washed with PBT several times and hybridized with the probe by shaking at 65°C overnight in DEPC-H2O hybridization buffer (50% deionized formamide; 100 μg/ml Heparin; 5X SSC; 0.1% Tween-20; 5 mM EDTA; Denhardt’s 1 mg/ml; yeast RNA 1 mg/ml). The day after, several washes were performed with decreasing concentration of SSC in 50% formamide/dH2O at hybridization temperature (from 5X to 2X), then a RNAse digestion was conducted at 37°C. Then, additional washes were carried out at room temperature using decreasing concentrations of SSC in dH2O (from 2X to 0.2X). Embryos were incubated overnight in primary antibody (anti-DIG AP, Roche), pre-adsorbed at 1:3000, with rocking at 4°C. The signal was revealed at room temperature using BM-Purple substrate (Roche). When the signal was deemed appropriate, several washes in PBT were performed before the post-fixation of embryos in PFA 4% for 20 minutes. Embryos were mounted in 80% glycerol in PBS, and photographed as whole mounts under the imaging system LEICA DMI6000B. Subsequently the embryos were counterstained in Ponceau S, embedded in Spurr’s resin, and prepared as sections for light microscopy [50].
Whole-mount immunostaining
Embryos, fixed as in the preceding paragraph, were rehydrated in PBT and blocked for 1 h at room temperature in 2 mg/mL BSA, 10% goat serum in PBT 1X (Blocking solution). The embryos were incubated overnight at 4°C in rat Phospho anti-Histone H3 (PHH3) (Ser10) antibody (Upstate Biotechnology), diluted 1:250 in blocking solution. After labeling with the primary antibody, the specimens were rinsed in seven 20-min changes of PBT. Then a 1:500 dilution of the secondary antibody [Alexa Fluor 555 goat anti-rat IgG heavy and light chain (Thermo Fisher Scientific)] for 2 h at 4°C. The labeled specimens were rinsed in seven 20-min changes of PBT, mounted in 80% glycerol in PBS, and imaged by Axio Imager with ApoTome (Zeiss).
Supporting information
S1 Fig. Phylogenetic reconstruction of Ran proteins.
The Maximum Likelihood (ML) tree indicates the existence in gnathostomes of Ran1 (orange box) and Ran2 (red box). The Ran of Latimeria chalumnae (Ran2) has been excluded from the tree for its divergence. Values at the branches indicate replicates obtained using the ML estimation method.
https://doi.org/10.1371/journal.pone.0196930.s001
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S2 Fig. Phylogenetic reconstruction of Syntaxin family.
In particular, the Maximum Likelihood (ML) tree evidences a common evolutionary origin for Stx1 (blue box), Stx2 (light blue box) and Stx3 (violet box). Values at the branches indicate replicates obtained using the Maximum Likelihood estimation method.
https://doi.org/10.1371/journal.pone.0196930.s002
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S3 Fig. Analysis of Ran genomic loci in four vertebrates: Homo sapiens (chromosome 11 and chromosome 12), Lepisosteus oculatus (LG9 and LG20), Scleropages formosus (NW_017371567 and NW_017371527), Danio rerio (chromosome 14 and chromosome 8).
A color code has been used to represent orthologue genes.
https://doi.org/10.1371/journal.pone.0196930.s003
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S4 Fig. Ran genomic loci in Salmo salar (ssa04, ssa05, ssa08, ssa09, ssa24).
https://doi.org/10.1371/journal.pone.0196930.s004
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S5 Fig.
Collection of two series of embryos (A’-A”, B’-B”, C’-C”, D’-D” represent distinct specimens) showing slightly different PHH3 immunolocalization signals (red). Scale bars: 60 μm. Embryos orientation: anterior to the left, dorsal to the top.
https://doi.org/10.1371/journal.pone.0196930.s005
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S1 File. Database of sequences employed in Ran protein alignment of Fig 1 and Ran phylogeny of S1 Fig.
https://doi.org/10.1371/journal.pone.0196930.s006
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S2 File. Database of sequences used in Stx phylogeny of S2 Fig.
https://doi.org/10.1371/journal.pone.0196930.s007
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Acknowledgments
We thank MaRe unit of Stazione Zoologica Anton Dohrn Napoli for the help with the amphioxus culture. PHH3 antibody was a gift of Dr. Paolo Sordino. Ugo Coppola and Filomena Caccavale were supported by SZN OU PhD fellowships.
References
- 1. Melchior F, Paschal B, Evans J, Gerace L (1993) Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol 123: 1649–1659. pmid:8276887
- 2. Moore MS, Blobel G (1993) The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365: 661–663. pmid:8413630
- 3. Sazer S, Dasso M (2000) The ran decathlon: multiple roles of Ran. J Cell Sci 113 (Pt 7): 1111–1118.
- 4. Bischoff FR, Krebber H, Smirnova E, Dong W, Ponstingl H (1995) Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1. EMBO J 14: 705–715. pmid:7882974
- 5. Deane R, Schafer W, Zimmermann HP, Mueller L, Gorlich D, Prehn S, et al. (1997) Ran-binding protein 5 (RanBP5) is related to the nuclear transport factor importin-beta but interacts differently with RanBP1. Mol Cell Biol 17: 5087–5096. pmid:9271386
- 6. Izaurralde E, Kutay U, von Kobbe C, Mattaj IW, Gorlich D (1997) The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J 16: 6535–6547. pmid:9351834
- 7. Battistoni A, Guarguaglini G, Degrassi F, Pittoggi C, Palena A, Di Matteo G, et al. (1997) Deregulated expression of the RanBP1 gene alters cell cycle progression in murine fibroblasts. J Cell Sci 110 (Pt 19): 2345–2357.
- 8. Kalab P, Pu RT, Dasso M (1999) The ran GTPase regulates mitotic spindle assembly. Curr Biol 9: 481–484. pmid:10322113
- 9. Hughes M, Zhang C, Avis JM, Hutchison CJ, Clarke PR (1998) The role of the ran GTPase in nuclear assembly and DNA replication: characterisation of the effects of Ran mutants. J Cell Sci 111 (Pt 20): 3017–3026.
- 10. Lavia P (2016) The GTPase RAN regulates multiple steps of the centrosome life cycle. Chromosome Res 24: 53–65. pmid:26725228
- 11. Lopez-Casas PP, Lopez-Fernandez LA, Krimer DB, del Mazo J (2002) Ran GTPase expression during early development of the mouse embryo. Mech Dev 113: 103–106. pmid:11900983
- 12. Koizumi K, Stivers C, Brody T, Zangeneh S, Mozer B, Odenwald WF (2001) A search for Drosophila neural precursor genes identifies ran. Dev Genes Evol 211: 67–75. pmid:11455416
- 13. Onuma Y, Nishihara R, Takahashi S, Tanegashima K, Fukui A, Asashima M (2000) Expression of the Xenopus GTP-binding protein gene Ran during embryogenesis. Dev Genes Evol 210: 325–327. pmid:11180838
- 14. Zhou F, Zheng L, Yang Q, Qiu L, Huang J, Su T, et al. (2012) Molecular analysis of a ras-like nuclear (Ran) gene from Penaeus monodon and its expression at the different ovarian stages of development. Mol Biol Rep 39: 3821–3827. pmid:21748319
- 15. Lin CY, Huang HY, Lu PN, Lin CW, Lu KM, Tsai HJ (2015) Ras-Related Nuclear Protein is required for late developmental stages of retinal cells in zebrafish eyes. Int J Dev Biol 59: 435–442. pmid:26864484
- 16. Hsiao PW, Lin DL, Nakao R, Chang C (1999) The linkage of Kennedy's neuron disease to ARA24, the first identified androgen receptor polyglutamine region-associated coactivator. J Biol Chem 274: 20229–20234. pmid:10400640
- 17. Hilbi H, Rothmeier E, Hoffmann C, Harrison CF (2014) Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection. Small GTPases 5: 1–6.
- 18. Caputo E, Wang E, Valentino A, Crispi S, De Giorgi V, Fico A, et al. (2015) Ran signaling in melanoma: implications for the development of alternative therapeutic strategies. Cancer Lett 357: 286–296. pmid:25444926
- 19. Bischoff FR, Ponstingl H (1991) Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature 354: 80–82. pmid:1944575
- 20. Caceres-Gorriti KY, Carmona E, Barres V, Rahimi K, Letourneau IJ, Tonin PN, et al. (2014) RAN nucleo-cytoplasmic transport and mitotic spindle assembly partners XPO7 and TPX2 are new prognostic biomarkers in serous epithelial ovarian cancer. PLoS One 9: e91000. pmid:24625450
- 21. Palmieri D, Scarpa M, Tessari A, Uka R, Amari F, Lee C, et al. (2016) Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells. Oncotarget 7: 18371–18383. pmid:26943034
- 22. Brunetti R, Gissi C, Pennati R, Caicci F, Gasparini F, Manni L (2015) Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis. Journal of Zoological Systematics and Evolutionary Research 53: 186–193.
- 23. Irimia M, Tena JJ, Alexis MS, Fernandez-Minan A, Maeso I, Bogdanovic O, et al. (2012) Extensive conservation of ancient microsynteny across metazoans due to cis-regulatory constraints. Genome Res 22: 2356–2367. pmid:22722344
- 24. Bennett MK, Garcia-Arraras JE, Elferink LA, Peterson K, Fleming AM, Hazuka CD, et al. (1993) The syntaxin family of vesicular transport receptors. Cell 74: 863–873. pmid:7690687
- 25. Amores A, Catchen J, Ferrara A, Fontenot Q, Postlethwait JH (2011) Genome evolution and meiotic maps by massively parallel DNA sequencing: spotted gar, an outgroup for the teleost genome duplication. Genetics 188: 799–808. pmid:21828280
- 26. Austin CM, Tan MH, Croft LJ, Hammer MP, Gan HM (2015) Whole Genome Sequencing of the Asian Arowana (Scleropages formosus) Provides Insights into the Evolution of Ray-Finned Fishes. Genome Biol Evol 7: 2885–2895. pmid:26446539
- 27. Abi-Rached L, Gilles A, Shiina T, Pontarotti P, Inoko H (2002) Evidence of en bloc duplication in vertebrate genomes. Nat Genet 31: 100–105. pmid:11967531
- 28. Dehal P, Boore JL (2005) Two rounds of whole genome duplication in the ancestral vertebrate. PLoS Biol 3: e314. pmid:16128622
- 29. Kuraku S, Meyer A (2009) The evolution and maintenance of Hox gene clusters in vertebrates and the teleost-specific genome duplication. Int J Dev Biol 53: 765–773. pmid:19557682
- 30. Lien S, Koop BF, Sandve SR, Miller JR, Kent MP, Nome T, et al. (2016) The Atlantic salmon genome provides insights into rediploidization. Nature 533: 200–205. pmid:27088604
- 31. Ladstein RG, Bachmann IM, Straume O, Akslen LA (2012) Prognostic importance of the mitotic marker phosphohistone H3 in cutaneous nodular melanoma. J Invest Dermatol 132: 1247–1252. pmid:22297638
- 32. Holland ND, Holland LZ (2006) Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development. Evol Dev 8: 142–149. pmid:16509893
- 33. Ciciarello M, Mangiacasale R, Lavia P (2007) Spatial control of mitosis by the GTPase Ran. Cell Mol Life Sci 64: 1891–1914. pmid:17483873
- 34. Hurst LD, Pal C, Lercher MJ (2004) The evolutionary dynamics of eukaryotic gene order. Nat Rev Genet 5: 299–310. pmid:15131653
- 35. Lundin MH, Mikkelsen B, Gudim M, Syed M (2000) The structure and expression of the Salmo salar Ran gene. DNA Seq 11: 41–50. pmid:10902908
- 36. Taylor JS, Braasch I, Frickey T, Meyer A, Van de Peer Y (2003) Genome duplication, a trait shared by 22000 species of ray-finned fish. Genome Res 13: 382–390. pmid:12618368
- 37. Albalat R, Canestro C (2016) Evolution by gene loss. Nat Rev Genet 17: 379–391. pmid:27087500
- 38. Imai KS, Daido Y, Kusakabe TG, Satou Y (2012) Cis-acting transcriptional repression establishes a sharp boundary in chordate embryos. Science 337: 964–967. pmid:22923581
- 39. Giovannone AJ, Winterstein C, Bhattaram P, Reales E, Low SH, Baggs JE, et al. (2018) Soluble syntaxin 3 functions as a transcriptional regulator. J Biol Chem. pmid:29475951
- 40. Putnam NH, Butts T, Ferrier DE, Furlong RF, Hellsten U, Kawashima T, et al. (2008) The amphioxus genome and the evolution of the chordate karyotype. Nature 453: 1064–1071. pmid:18563158
- 41. Schubert M, Holland ND, Holland LZ (1998) Amphioxus AmphiDRAL encoding a LIM-domain protein: expression in the epidermis but not in the presumptive neuroectoderm. Mech Dev 76: 203–205. pmid:9767167
- 42. Gertz EM, Yu YK, Agarwala R, Schaffer AA, Altschul SF (2006) Composition-based statistics and translated nucleotide searches: improving the TBLASTN module of BLAST. BMC Biol 4: 41. pmid:17156431
- 43. de Castro E, Sigrist CJ, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, et al. (2006) ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res 34: W362–365. pmid:16845026
- 44. Coppola U, Annona G, D'Aniello S, Ristoratore F (2016) Rab32 and Rab38 genes in chordate pigmentation: an evolutionary perspective. BMC Evol Biol 16: 26. pmid:26818140
- 45. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7: 539. pmid:21988835
- 46. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 2725–2729. pmid:24132122
- 47. Huson DH, Scornavacca C (2012) Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst Biol 61: 1061–1067. pmid:22780991
- 48. Fuentes M, Benito E, Bertrand S, Paris M, Mignardot A, Godoy L, et al. (2007) Insights into spawning behavior and development of the European amphioxus (Branchiostoma lanceolatum). J Exp Zool B Mol Dev Evol 308: 484–493. pmid:17520703
- 49. Annona G, Caccavale F, Pascual-Anaya J, Kuratani S, De Luca P, Palumbo A, et al. (2017) Nitric Oxide regulates mouth development in amphioxus. Sci Rep 7: 8432. pmid:28814726
- 50.
Holland LZ, Holland PWH, Holland ND (1996) Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: amphioxus versus vertebrates. In: Ferraris JD, Palumbi SR, editors. Molecular zoology: advances, strategies, and protocols. New York, Chichester etc: Wiley-Liss. pp. 267–282.