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First published online February 18, 2009
doi: 10.1242/10.1242/jcs.030205

Journal of Cell Science 122, 636-643 (2009)
Published by The Company of Biologists 2009
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Research Article

Ancient animal ancestry for nuclear myosin

Wilma A. Hofmann1, Thomas A. Richards2,* and Primal de Lanerolle1,*

1 Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
2 Centre for Eukaryotic Evolutionary Microbiology, School of Biosciences, University of Exeter, Devon EX4 4QD, UK

* Authors for correspondence (e-mail: T.A.Richards{at}exeter.ac.uk; primal{at}uic.edu)

Accepted 3 November 2008


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The identification of nuclear myosin I (NMI) has raised the possibility that myosin might have had an early functional role in the eukaryotic nucleus. To investigate this possibility, we examined the molecular evolution of the vertebrate myosin-I proteins. We found that myosin I has undergone at least five duplication events in the common ancestor of the vertebrates (vertebrate-specific duplications), leading to nine myosin-I vertebrate gene families, followed by two additional myosin-I duplication events in the lineage leading to modern fish. This expansion suggests a large-scale adaptive radiation in myosin-I function in an early phase of vertebrate evolution. The branching order of the evolutionary tree suggests that the functional role of NMI predates this expansion. More specifically, in the tunicate Ciona intestinalis, we found a myosin-I protein that localizes to the nucleus, but that branches on phylogenetic trees before the duplication that led to vertebrate myosin IC and myosin IH. This relationship suggests that the common ancestor of these three proteins encoded a nuclear isoform and that the localization of myosin I to the nucleus predates the origin of the vertebrates. Thus, a functional role for NMI appears to have been present at an early stage of animal evolution prior to the rise of both myosin IC and the vertebrates, as NMI was present in the last common ancestor of vertebrates and tunicates.

Key words: Evolution, Transcription, Nuclear myosin I, Molecular motors, Myosin gene family


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The evolution of molecular motors, including myosin, was one of the most important steps in the origin of the eukaryotic cell, giving rise to much of the diversity and biological complexity now present on Earth. The myosin gene family has undergone a large number of duplication events, leading to sequence diversification and a multitude of functional roles in eukaryotic cells (Richards and Cavalier-Smith, 2005Go; Thompson et al., 1997Go). However, the primordial function of the first myosin is unclear. Although myosins are generally considered to be cytoplasmic motors, the recent discovery of nuclear myosin I (NMI) (Pestic-Dragovich et al., 2000Go) and the demonstration that it plays an active role in transcription (Fomproix and Percipalle, 2004Go; Pestic-Dragovich et al., 2000Go; Philimonenko et al., 2004Go) suggest that myosins formed an important component of the nucleus during ancient phases of eukaryotic-cell evolution.

Previous comparisons of myosin-I diversity in vertebrates have led to the classification of eight vertebrate paralogs, MYO1A to MYO1H (Gillespie et al., 2001Go). Myosin-I genes encode a peptide with an N-terminal molecular-motor head domain followed by a TH1 domain (Richards and Cavalier-Smith, 2005Go; Thompson and Langford, 2002Go), a domain composed of highly basic residues that functions in some proteins to bind phospholipids and membranes (Wagner et al., 1992Go) and/or interact with actin (Lee et al., 1999Go; Liu et al., 2000Go). Furthermore, many identified myosin-I genes possess an additional C-terminal SH3 domain (Richards and Cavalier-Smith, 2005Go). Recent studies have demonstrated that an isoform of myosin IC (NMI) is present in vertebrate nuclei (Pestic-Dragovich et al., 2000Go). NMI contains a unique N-terminal peptide, because the transcription start site for NMI is in an exon that is located upstream to the start site for cytoplasmic myosin IC (Pestic-Dragovich et al., 2000Go). Functionally, NMI colocalizes with RNA polymerase I (Fomproix and Percipalle, 2004Go; Nowak et al., 1997Go) and RNA polymerase II (Fomproix and Percipalle, 2004Go; Nowak et al., 1997Go; Pestic-Dragovich et al., 2000Go), and co-purifies and co-immunoprecipitates with both polymerases (Pestic-Dragovich et al., 2000Go; Philimonenko et al., 2004Go). Additionally, NMI-specific antibodies inhibit transcription by these polymerases both in vivo and in vitro (Fomproix and Percipalle, 2004Go; Pestic-Dragovich et al., 2000Go; Philimonenko et al., 2004Go).

Myosins usually work in concert with actin, which has also been shown to associate with polymerases I (Philimonenko et al., 2004Go), II (Hofmann et al., 2004Go) and III (Hu et al., 2004Go), suggesting that actin and NMI work together in transcription. Recent work has also linked NMI to chromatin remodeling (Percipalle et al., 2006Go), and to topoisomerase II (Smukste et al., 2006Go), and has implicated NMI in the movement of chromosomal regions in the nuclei of mammalian cells (Chuang et al., 2006Go). DNA replication and transcription are essential for life, and ribosomal RNA genes are likely to have evolved very early because they are necessary for all prokaryotic and eukaryotic RNA-to-protein translation systems (Woese, 1998Go). By contrast, myosins are present in all the major eukaryotic lineages; only a few taxa appear to have lost all myosin homologs (Richards and Cavalier-Smith, 2005Go). Consequently, at some point between the origin of the eukaryotic cell and the rise and diversification of vertebrates, myosins with nuclear functions evolved. Here, we investigate the evolution of the NMI subfamily to define the evolutionary history of NMI in animals.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Comparative genomics and phylogenetics demonstrate that the myosin-I gene family has undergone numerous gene-duplication events (Fig. 1A; Fig. 2). It is likely that phylogenetic results underestimate the total number of gene-duplication events because of absence of sampling from some evolutionary branches that currently do not have genome-project representation, meaning that some patterns of gene duplication and loss might remain unidentified. Accounting for this possible source of error, we pinpointed five myosin-I duplication events that occurred at the base of the vertebrate evolutionary radiation, producing nine myosin-I ortholog sets (individual gene subfamilies). These included the following sister paralogs: MYO1A-MYO1B, MYO1D-MYO1G, MYO1C-MYO1H and MYO1E-MYO1F, which have been previously annotated (Gillespie et al., 2001Go), and an additional myosin-I vertebrate ortholog set produced by a gene-duplication event located in the ancestral branch of the MYO1E subfamily (Fig. 2).


Figure 1
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  		Fig. 1. Myosin-I phylogeny. (A) Subsection of the myosin-I phylogeny (see Fig. 2 for the rest of the tree). The topology shown is a PHYML tree. Posterior probability/PHYML bootstrap (100 replicates)/SH test values are marked on nodes that are directly discussed in the text. All other topology support values are marked using black or white circles depending on topology support (see key). The latest possible acquisition of NMI phenotype is marked (blue triangle). Vertebrate ortholog sets are marked with gray blocks and labeled according to the annotation convention established by Gillespie et al. (Gillespie et al., 2001Go). Orange triangles and lines mark duplications that occurred in the ancestral vertebrate branch. Branches of the phylogenetic tree are labeled with species followed by a combination of GenBank accession number or DOE JGI gene annotation code (given in rounded parentheses) and/or followed by the annotation name given by Odronitz and Kollmar (Odronitz and Kollmar, 2007Go) (given in square parentheses) if available. Non-equivalent higher taxonomic groupings are labeled. Red ovals marked `N' are sequences with two alternative putative start sites, suggesting the presence of an N-terminal candidate nuclear-retention peptide. (B) Alignment of putative NMI N-terminal-extension peptides. The putative NMI N-terminal-extension peptide identified here in Ciona (DOE JGI identifier 240514–chr_04q) is aligned with additional NMI isoforms identified by Kahle et al. (Kahle et al., 2007Go) in Homo (XP_0238385), Bos (NP_776821), Mus (AAG02570), Gallus (NP_001006220), Xenopus (ENSXETP00000049503), Danio (XP_695924) and Tetraodon (GSTENT00022181001).

 

Figure 2
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  		Fig. 2. Subsection of the myosin-I phylogeny showing additional vertebrate-specific duplications, bringing the total to nine vertebrate myosin-I paralogs. Phylogeny is labeled as described in Fig. 1A. We have extended the vertebrate ortholog annotation convention established by Gillespie et al. (Gillespie et al., 2001Go) to include one additional ortholog group (MYO1I), labeled using a gray box.

 

The five vertebrate-specific myosin-I gene-duplication events are marked on Fig. 1A and Fig. 2; in all cases, their placement has strong topology support values: a bootstrap support value in excess of 90%, a MrBayes posterior probability of 1 (the highest possible score) and a Shimodaira-Hasegawa-like (SH) test support in excess of 0.98 (2% significance level). We have temporarily annotated this newly identified vertebrate ortholog family MYO1I, to be consistent with the annotations of the myosin community (Gillespie et al., 2001Go). However, this additional vertebrate-specific myosin-I ortholog family was also detected by Odronitz and Kollmar (Odronitz and Kollmar, 2007Go) but was given a range of different designations depending on the species (Fig. 2). Although MYO1I has only been detected in amphibians and fish at present, it is likely to have arisen in an early vertebrate ancestor, because resolved multi-gene phylogenies (Delsuc et al., 2006Go) pinpoint mammals and birds as an evolutionary branch within the amphibian and fish clades. This suggests that MYO1I was present in the common ancestor of all vertebrates but then lost in the mammals and birds sampled in this study.

Both our analyses (Fig. 1A) and the analyses of Odronitz and Kollmar (Odronitz and Kollmar, 2007Go) pinpointed two additional vertebrate duplications that, according to the genomes sampled in both analyses, are specific to the fish lineage (with moderate-to-strong tree topology support values in excess of 79% bootstrap support, MrBayes posterior probability of 1, and SH-test values of 0.99) (Fig. 1A).

All four major vertebrate myosin-I clades, which contain the nine vertebrate myosin-I gene subfamilies, were monophyletic, forming a branch on the phylogenetic tree to the exclusion of all other sequences, with >90% bootstrap support (as shown in Fig. 1A and Fig. 2). This suggests that the duplications that we detected are specific to the vertebrate lineage and occurred in the last common ancestor of the vertebrates sampled here. This pinpoints a large-scale diversification in the myosin-I gene family early in the vertebrate lineage and suggests that a series of myosin gene innovations occurred prior to the diversification of the vertebrate fauna.

Until very recently it was unclear which group of animals formed the phylogenetic sister group to the vertebrates. Delsuc et al. (Delsuc et al., 2006Go) used large-scale gene sampling and sophisticated phylogenetic methods to demonstrate that the sister group to the vertebrates are the tunicates, such as the sea squirt Ciona intestinalis (Delsuc et al., 2006Go). This evolutionary relationship has been named the Olfactores hypothesis (Delsuc et al., 2006Go). NMI-like transcripts have been identified in all the major vertebrate lineages (Kahle et al., 2007Go). The results of the Delsuc et al. study and the premise of the Olfactores hypothesis is important for understanding the evolution of NMI because it demonstrates that the tunicates are the closest non-vertebrate relative to the vertebrates and therefore the best candidate model organisms for investigating NMI evolution below the vertebrate radiation. In all four vertebrate myosin-I phylogenetic groups (Fig. 1A; Fig. 2), we consistently recovered a vertebrate/tunicate (olfactores) clade in the top-scoring topology with a bootstrap support of 88% (Fig. 2: MYOIE, MYOII and MYOIF), 61% (Fig. 1A: MYOIC and MYOIH), 57% (Fig. 1A: MYOID and MYOIG) and 20% (Fig. 1A: MYOIA and MYOIB). Although these branching relationships are moderately-to-weakly supported, the best-scoring tree topology is consistent with the Olfactores hypothesis (Delsuc et al., 2006Go) and suggests that the tunicates represent a good non-vertebrate model organism for investigating the evolution of NMI prior to the radiation of the vertebrates.

NMI was initially identified (Pestic-Dragovich et al., 2000Go) as being encoded by the MYO1C gene in Mus musculus (Dumont et al., 2002Go). Although model organisms from four major vertebrate lineages (mammals, fish, amphibians and birds) express orthologs to the mouse gene that encodes NMI (Fig. 1B) (Kahle et al., 2007Go), we found that the MYO1C ortholog family does not predate the vertebrates, because it forms an exclusive sister-group relationship with the vertebrate-specific MYO1H gene family, with 100% bootstrap support (Fig. 1A). Consequently, the NMI phenotype, if restricted to the MYO1C gene family, appears to be vertebrate-specific and, therefore, the NMI phenotype is potentially only as old as the vertebrates. On the premise of the Olfactores hypothesis and using the tunicate C. intestinalis as the closest available non-vertebrate model organism, we found a myosin-like gene that branches next to the MYO1C-MYO1H vertebrate clade with moderate topology support of 61% bootstrap support and 0.86 SH test support (Fig. 1A). This putative myosin I branches below the MYO1C-MYO1H duplication, suggesting that the C. intestinalis gene was directly derived from the single parent gene that later underwent duplication to form the MYO1C and MYO1H vertebrate gene families (paralogs) (Fig. 1A). On the basis of the phylogenetic analyses, this Ciona protein represents our best candidate for a non-vertebrate NMI.

To test the hypothesis that the NMI phenotype is older than the MYO1C-MYO1H duplication and the vertebrate lineage, we investigated possible nuclear localization of myosin I in C. intestinalis. The protein with the closest similarity to the protein predicted from the C. intestinalis myosin-I sequence is the M. musculus NMI protein found on chromosome 4q at location 4,160,077 to 4,171,961 and is 56.6% identical to M. musculus NMI at the amino-acid level. Similar to M. musculus NMI, myosin I of C. intestinalis has two alternative putative start sites, one of which encodes an N-terminal extension of 15 amino acids (Fig. 1B). In mouse cells, a similar N-terminal extension leads to expression of a myosin-IC isoform that localizes to the nucleus (Pestic-Dragovich et al., 2000Go).


Figure 3
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  		Fig. 3. Myosin-I isoforms in C. intestinalis. (A) Sequence alignment of the myosin-I-tail-region epitope that is recognized by the anti-pan-myosin-I-tail antibody. (B) Western blot analysis of total cell extract from C. intestinalis and NIH-3T3 cells using the anti-pan-myosin-I-tail antibody (left panel) and the anti-NMI-peptide antibody (right panel). Both antibodies recognize a protein with the appropriate molecular mass (~120 kD) in NIH-3T3 and C. intestinalis whole-cell extracts. Molecular-mass markers are shown in kD on the left. (C) Confocal images of C. intestinalis hemocytes showing predominantly nuclear staining by the anti-NMI-peptide antibody, a combination of distinctive cytoplasmic staining with punctate nuclear staining by the anti-pan-myosin-I-tail antibody, and exclusively cytoplasmic staining by the anti-{alpha}-tubulin antibody. Left column: cells stained with the indicated antibodies. Middle column: cells stained with DAPI to visualize nuclei. Right column: merged images of the first and second columns. When the cell shape was not obvious (top and bottom rows), phase-contrast images were included into the merged images. The size of the scale bar is indicated in each panel individually.

 
To test whether there are multiple isoforms of this myosin-I gene in C. intestinalis and whether the predicted myosin I with the N-terminal extension also localizes to the nucleus, we first checked whether our anti-NMI antibodies recognize myosin I in C. intestinalis. We used two different antibodies. One was an anti-pan-myosin-IC antibody that recognizes an epitope in the tail of bovine myosin IC (formerly known as myosin Iβ) (Wagner et al., 1992Go). Sequence comparison showed that this epitope is highly conserved in vertebrates and that the corresponding sequences in M. musculus and C. intestinalis myosin I have 60% identity (Fig. 3A). The second antibody was an IgM antibody that recognizes the NMI-specific N-terminal peptide in M. musculus (Pestic-Dragovich et al., 2000Go). Comparison of the N-terminal peptide in mouse and C. intestinalis revealed 31.2% sequence identity. The reactivity of this antibody with the N-terminal peptide was investigated by performing an ELISA on synthetic peptides representing the M. musculus and C. intestinalis N-terminal peptides and the first eight amino acids of the cytoplasmic myosin I. Table 1 shows that the IgM peptide antibody recognizes the C. intestinalis N-terminal peptide almost as well as the M. musculus N-terminal peptide. Table 1 also shows that this antibody does not recognize the last eight amino acids alone of the same peptides. Furthermore, probing whole-cell extracts from NIH-3T3 cells and C. intestinalis cells showed that both antibodies predominantly recognize a single band in the C. intestinalis extract that correlates with the molecular weights of myosin I and NMI from NIH 3T3 cells (Fig. 3B). Thus, these experiments indicate the expression of a myosin-I protein in C. intestinalis that contains an N-terminal extension similar to the N-terminal extension found in mouse NMI.


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  		Table 1. Reactivity of IgM peptide antibody with M. musculus and C. intestinalis N-terminal peptides (aligned to match the mouse NMI sequence)

 

Next, we investigated the presence of myosin I in the nuclei of C. intestinalis cells by purifying hemocytes and immunostaining with the NMI-specific IgM peptide antibody, the anti-pan-myosin-IC-tail antibody and an antibody to {alpha}-tubulin. Confocal microscopy (Fig. 3C) showed that the staining patterns of the NMI IgM peptide and anti-pan-myosin-I-tail antibodies differ. The NMI peptide antibody mainly recognizes the nucleus, and optical slices through the cell demonstrate intranuclear staining. The anti-pan-myosin-I-tail antibody gives strong punctate staining of the cytoplasm and weaker staining in the nucleus. By contrast, the anti-{alpha}-tubulin antibody stains centrosomes and microtubules, which are exclusively cytoplasmic. These data indicate that indeed two isoforms of myosin I seem to exist in C. intestinalis. One isoform, recognized by the NMI-specific IgM peptide antibody, seems to localize predominantly to the nucleus, whereas the anti-pan-myosin-I-tail antibody seems to recognize both the nuclear and cytoplasmic isoforms of myosin I.

To corroborate these results, we separated nuclei and cytoplasm from C. intestinalis using sucrose gradient centrifugation (Hinegardner, 1962Go) (Fig. 4A). Fractions from the gradient were analyzed with antibodies to RNA polymerase II, β-actin and {alpha}-tubulin to identify the major nuclear and cytoplasmic fractions, respectively. Fig. 4B shows that fractions 4 and 5 contained RNA polymerase II, whereas {alpha}-tubulin as well as the majority of β-actin was found in fraction 6, which contains cytoplasmic debris (Hinegardner, 1962Go). Consistent with other data (Hofmann et al., 2004Go), fraction 5 (intact nuclei) also contained actin (Fig. 4B). However, the absence of {alpha}-tubulin in fractions 4 and 5 shows that they indeed contain highly purified nuclei without apparent cytoplasmic contamination. In agreement with immunofluorescence microscopy, the NMI-specific peptide antibody recognized a protein with the appropriate molecular weight in the fractions containing nuclei (Fig. 4B). By contrast, the anti-pan-myosin-I-tail antibody recognized a protein with the correct molecular weight in all fractions, with the strongest signal in the cytoplasmic fraction (layer 6, Fig. 4B).


Figure 4
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  		Fig. 4. Identification of a myosin-I isoform in nuclei of C. intestinalis cells. (A) Sucrose gradient showing isolation of C. intestinalis nuclei. Layer 4, broken nuclei; layer 5, intact nuclei; layer 6, cytoplasmic debris. For a complete description of each layer, see Hinegardner (Hinegardner, 1962Go). (B) Western blot analysis of individual layers using the indicated antibodies. Relevant molecular-mass markers are shown in kD on the left. (C) Identification of NMI in C. intestinalis by immunoprecipitation. C. intestinalis nuclear extract was incubated with antibodies to the myosin-I tail or with nonspecific IgG. Proteins bound to the antibody were precipitated and analyzed by immunoblotting using either the NMI-specific peptide antibody (left panel) or the anti-pan-myosin-I-tail antibody (right panel). Both antibodies recognize the same two bands in the fraction precipitated with the anti-pan-myosin-I-tail antibody. No bands were recognized when nonspecific IgG was used. The relevant molecular-mass marker is indicated in kD on the right.

 
Finally, we used an immunoprecipitation assay to confirm that the protein recognized by the NMI-specific IgM peptide antibody is indeed an isoform of myosin I. For this, C. intestinalis nuclear extract was incubated with the anti-pan-myosin-I-tail antibody. The immunoprecipitated proteins were then analyzed by immunoblotting using the anti-pan-myosin-I-tail antibody as well as the NMI-specific IgM peptide antibody. Fig. 4C shows that both antibodies recognize the same protein, indicating that the protein immunoprecipitated with the anti-pan-myosin-I-tail antibody is indeed a nuclear isoform of myosin I. The second band, which appears at a lower molecular weight, seems to be a degradation product because, in the whole-cell extract (see Fig. 3B), both antibodies recognize only one band at this molecular weight.


   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
The data presented above demonstrate the presence of myosin-I protein with an N-terminal extension in many different animal species representing distant evolutionary lineages. One of these organisms, C. intestinalis, expresses a myosin-I protein that is encoded by the Ciona homolog of the vertebrate gene encoding NMI, myosin IC. The myosin I in C. intestinalis contains an N-terminal extension that shares identity with a similar extension in mouse myosin I. Previous work has shown that this extension is responsible for the nuclear localization of the mouse protein (Pestic-Dragovich et al., 2000Go). Alignment of the putative N-terminal sequences showed that they have varying lengths and degrees of homology with the mouse sequence (Fig. 1B). Because of this variability we cannot be sure that the individual N-terminal extensions target all the respective myosin-I proteins to the nucleus. However, identity with the extension in mouse myosin I and the nuclear localization of C. intestinalis myosin I suggest nuclear functions for myosin-I proteins with a similar N-terminal extension in distantly related organisms. Moreover, the nuclear localization of myosin I in the tunicate C. intestinalis suggests that NMI is likely to have arisen prior to the evolution of the vertebrates and prior to the myosin-I duplication that gave rise to the MYO1C-MYO1H gene families.

Discounting the possibility that nuclear-functioning myosin I evolved separately and convergently from the same parental myosin I in the vertebrate and the tunicate lineage, these data suggest that NMI is at least as old as the last common ancestor of the tunicates and the vertebrates (as shown in Fig. 1A). These analyses also demonstrate that the NMI gene family has undergone further evolutionary diversification, duplicating to give rise to the MYO1H and MYO1C paralogs, and duplicating further in the branch leading to the fish lineage to give rise to additional myosin-I paralogs (Fig. 1A). Post-duplication, the MYO1C paralog became a functional component of the vertebrate ear (Dumont et al., 2002Go). In addition, within the MYO1C-MYO1H gene subfamily, a nuclear-functioning myosin-I isoform (NMI) encoded by both the parental form and the MYO1C vertebrate daughter paralog was maintained. However, these analyses do not rule out a much earlier ancestry to NMI. Our phylogenetic analyses pinpointed myosin-I genes from additional animal taxa and from the choanoflagellate protozoa, known to be close relatives of the animals (Lang et al., 2002Go), that grouped close to the NMI phylogenetic group (Fig. 1A). Therefore, other taxa might possess candidate NMI isoforms, suggesting the possibility that NMI dates back to the earliest evolutionary branches of the animals.

The evolution of molecular motors, including myosin, was one of the most important steps in the origin and diversification of the eukaryotic cell. Traditionally, all eukaryotic motors are thought of as cytoplasmic proteins responsible for a diverse array of cytoplasmic functions. Nevertheless, there is increasing evidence that myosins and actins are associated with transcription and other nuclear functions. For example, myosin Va (Pranchevicius et al., 2008Go), myosin VI (Vreugde et al., 2006Go), myosin XVIb (Cameron et al., 2007Go) and actin, in addition to NMI, are found in eukaryotic nuclei. NMI interacts with topoisomerase II (Smukste et al., 2006Go), and MreB, an actin-like protein, interacts with RNA polymerase in bacteria (Kruse et al., 2006Go). Indeed, myosin VI, a distant relative to NMI that travels in the opposite direction to NMI on actin filaments, has also been shown to function in RNA-polymerase-II-dependent transcription (Vreugde et al., 2006Go).

Thus, a deep evolutionary acquisition of myosin as a nuclear protein is logical and intellectually pleasing because transcription and other nuclear processes undoubtedly preceded and diversified before the evolution of many cytoplasmic motor functions, especially in complex multicellular animal forms. Furthermore, the data suggest two possible hypotheses regarding the origin of nuclear myosins: (1) at least two independent and convergent evolutionary acquisitions of nuclear myosins I and VI and associated transcriptional activities; (2) ancestrally, myosin functioned in the nucleus as a factor in transcription or other nuclear process. In support of the second hypothesis, we demonstrate here that myosin-I localization to the nucleus predates the vertebrate and tunicate radiation and the MYO1C-MYO1H gene-duplication event, suggesting an early evolution of nuclear myosin in animals. Future work should therefore consider the possibility that myosin functioned within the nucleus at a very early point in eukaryotic-cell evolution, potentially predating the diversification of the majority of cytoplasmic myosin motors.


   Materials and Methods
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Comparative genomics and phylogenetics
Using the M. musculus NMI sequence (AAG02570) as a BLAST seed, BLASTp and tBLASTn searches were used to sample myosin I across a range of eukaryotic genomes representing diverse evolutionary branches from gene data archived in the NCBI GenBank nr database and additional eukaryote genome databases. These additional eukaryote genome databases included genome projects hosted at: the Institute of Genomic Research (www.tigr.org), Genoscope (www.cns.fr), the Baylor College of Medicine (www.hgsc.bcm.tmc.edu) and the Department of Energy Joint Genome Initiative (www.jgi.doe.gov). Taxa with only EST projects were not analyzed to avoid comparisons of partial sequences. Comparisons of nucleotide and amino-acid sequences in the N-terminal region were performed using SE-AL (http://evolve.zoo.ox.ac.uk/software.html?id=seal) to identify multiple putative start codons in the N-terminal region indicative of an NMI N-terminal extension. We then compared our myosin-I datasets with the recently published and curated myosin gene models to check our sequence sampling (Odronitz and Kollmar, 2007Go). Because our study focused on patterns of gene evolution among invertebrate animal taxa, we reduced our dataset to include the span of higher taxonomic groups that were available, but excluded closely related species with similar myosin complements and representing very similar myosin amino-acid sequences (see Fig. 1A and Fig. 2). This reduction was necessary because it enabled us to use sophisticated and computationally intensive phylogenetic methods that accounted for complex models of sequence evolution.

All candidate myosin-I genes were aligned using CLUSTAL-X (Thompson et al., 1997Go) through the SEAVIEW platform (Galtier et al., 1996Go). The alignment was then extensively corrected manually as the CLUSTAL analysis produced numerous alignment errors. Because phylogenetic analyses based in CLUSTAL only use uncorrected distance methods, which have been shown to produce artifactual phylogenetic results (Huelsenbeck et al., 1996Go) and appear to be inappropriate for myosin analysis when compared to more-sophisticated methods (Foth et al., 2006Go), we adopted computationally complex approaches that used both Bayesian and maximum likelihood (ML) methods and that accounted for the fact that sites have evolved at different rates across the myosin-protein motor domain.

Prior to phylogenetic analyses, the alignment was masked to remove all positions that were either `gappy' or for which an unambiguous alignment was not possible, leaving an alignment of 158 sequences and 575 amino-acid characters. The alignment was subject to MODELGENERATOR analyses (Keane et al., 2006Go) to obtain the most appropriate substitution matrix (Rt-REV) and model of site rate variation ({Gamma}=8, {alpha}=0.93 and I=0.04). These model parameters were entered into PHYML for a 100-replicate bootstrap analysis (Guindon et al., 2005Go). MrBayes analysis (Ronquist and Huelsenbeck, 2003Go) was conducted for 1,000,000 generation samples, using a substitution matrix and model of site rate variation as before, but allowing the MCMCMC to search alternative site rate variation model parameter values. The tree search included two MCMCMC searches with four chains each (three heated, heat parameters set to default) with a sampling frequency of 250 generations. The likelihood values of the two MCMCMC searches were compared to check whether they had converged, a `burnin' of 400 generation samples was excluded and the remaining plateau sampled for the consensus Bayesian tree. To further test the support for contentious branching points (labeled with actual values on Fig. 1A and Fig. 2), we performed nonparametric branch support tests based on a Shimodaira-Hasegawa-like procedure (SH test) using PHYML to test the statistical significance of specific topological relationships over a collapsed version of the same branching relationship.

Biological materials
Adult specimens of C. intestinalis were maintained in seawater. NIH-3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium plus 10% calf serum and antibiotics at 37°C in an atmosphere containing 5% CO2.

Antibodies
An affinity-purified monoclonal mouse IgM to the 16-amino-acid N-terminal peptide specific to mouse NMI was used. Formerly we used a peptide antibody made in rabbits raised against the same peptide (Pestic-Dragovich et al., 2000Go). To avoid confusion, the antibody used here was called the NMI-specific IgM peptide antibody. The anti-pan-myosin-I-tail antibody (mT2) is a monoclonal antibody that recognizes a peptide in the tail region (Wagner et al., 1992Go). Monoclonal antibodies to β-actin and the C-terminal domain of RNA polymerase II (8WG16) were obtained from Sigma (St Louis, MO) and to {alpha}-tubulin from BAbCO (Richmond, CA). Secondary antibodies were acquired from Jackson ImmunoResearch Laboratories (West Grove, PA).

Isolation of C. intestinalis nuclei and preparation of nuclear extract
Nuclei from C. intestinalis were isolated following essentially the method described for isolating nuclei from the sea urchin, with some modifications (Hinegardner, 1962Go). Nuclear extract from C. intestinalis and NIH-3T3 cells was prepared essentially as described (Dignam et al., 1983Go). Briefly, one or two live, adult specimens were immersed in isotonic magnesium chloride. After complete relaxation, the tunic was removed and the specimens were cut to remove the stomach, intestine and rectum. All subsequent steps were performed at 4°C. The remaining tissue was cut into small pieces and homogenized in 2M dextrose solution containing 2 mM MgCl2 using a Polytron homogenizer at slow speed. The homogenate was centrifuged at 1000 g for 30 minutes. The supernatant was discarded and the pellet was washed twice in buffer A (10 mM HEPES, 2 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF, pH 7.9) and collected by centrifugation at 1000 g for 10 minutes. Finally, the pellet was resuspended in three pellet volumes of buffer A. After incubation for 20 minutes on ice, the suspension was passed ten times through an 18G needle. After centrifugation at 1000 g for 20 minutes, the supernatant was discarded and the pellet was resuspended in three volumes of a 30% 2.5 M sucrose solution. The nuclei were then separated from residual debris by high-speed gradient centrifugation at 56,000 g for 45 minutes (Beckman SW28 swinging bucket rotor). For this, the nuclei were layered over a gradient that consisted of 3.5 ml each of 50%, 60%, 70%, 80% and 95% of a 2.5 M sucrose solution. The nuclei collect at the interphase of the 95% to 80% solutions. This layer was analyzed by phase-contrast microscopy for the purity of the isolated nuclei and by western blot for the presence of RNA polymerase II, actin, {alpha}-tubulin, NMI and myosin I. The layer containing nuclei was diluted 1:1 with 2 mM MgCl2 and concentrated by centrifugation at 300 g for 10 minutes. For further purification, the nuclei were resuspended in 10 ml of sucrose buffer containing 0.35 M sucrose, 2 mM MgCl2, 10 mM Tris-HCl, pH 7.5, and layered over a 20 ml sucrose cushion consisting of 1.8 M sucrose, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 10 mM Tris-HCl, pH 7.5 and centrifuged at 30,000 g for 45 minutes in an SW 28 swinging bucket rotor. To obtain C. intestinalis nuclear extract, the pellet containing the nuclei was resuspended in two pellet volumes of buffer C [20 mM HEPES, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, pH 7.9] and homogenized using a Dounce homogenizer. The suspension was stirred gently for 30 minutes using a magnetic stirrer and then centrifuged at 25,000 g for 30 minutes in a JA 20 rotor. The resulting clear supernatant was then diluted to a final concentration of 150 mM NaCl using buffer D [20 mM HEPES, 0.5 mM DTT, 20% (v/v) glycerol, 0.5 mM PMSF, pH 7.9] and frozen as aliquots in liquid nitrogen.

Isolation of C. intestinalis hemocytes
Live, adult specimens were washed in 0.2-µm filtered artificial seawater (FSW) (Instant Ocean, Aquarium Systems, Mentor, OH) and blotted dry to remove any excess seawater. The specimens were then placed onto a sterilized surface and the tunic was cut carefully without injuring the internal organs. The perivisceral fluid that exuded from the animal was collected and placed in an equal volume of ice-cold anticoagulant (FSW containing 0.38% sodium citrate, pH 7.2). The hemolymph from the heart was then drained with a sterile 25G needle into a syringe that contained ice-cold anticoagulant. The hemocytes were then washed by centrifugation at 400 g for 10 minutes and resuspended in FSW containing anticoagulant. The cells were plated on glass coverslips that were coated with 50 mg/ml poly-L-lysine (Sigma, St Louis, MO) in FSW and allowed to attach by incubating the slides in a moist environment for 30 minutes at 4°C.

Immunoprecipitation experiments
C. intestinalis nuclear extract (80 µg) was diluted in ten volumes of IP buffer (10 mM HEPES, 100 mM potassium glutamate, 2.5 mM MgCl2, 3.5% glycerol, 1 mM PMSF, pH 7.9) and incubated overnight with 8 µg anti-pan-myosin-I-tail antibody at 4°C. Protein-G Sepharose (GE Healthcare Bio-Sciences, Uppsala, Sweden) was added and the mixture was incubated for 2 hours at 4°C. The beads were washed extensively in IP buffer, eluted by boiling in SDS and analyzed by protein immunoblotting using the NMI-peptide antibody.

Light microscopy
C. intestinalis hemocytes, isolated as described (Cammarata and Parrinello, 1995Go) with minor modifications as described above, were fixed with 3% paraformaldehyde in FSW for 7 minutes at room temperature. Cells were then washed in FSW, permeabilized by incubating in 0.1% Triton X-100, 0.1% deoxycholate in FSW for 7 minutes at room temperature and pre-blocked in 5% bovine serum albumin for 30 minutes. After several washes, cells were stained with 0.5 µg/ml anti-{alpha}-tubulin, 3 µg/ml anti-NMI-peptide, or 2 µg/ml anti-pan-myosin-I-tail antibodies followed by Texas-red-conjugated antibodies to mouse IgM or Cy2-conjugated antibodies to mouse IgG. Coverslips were mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and examined using a Leica TCS SP laser scanning confocal microscope system.

Enzyme-linked immunosorbant assay (ELISA)
Synthetic peptides were suspended in water and diluted to 2 mM. Aliquots of 50 µl of each peptide were added to 96-well plates in duplicate and incubated overnight at 4°C. The wells were blocked with 1% BSA in PBS–Tween-20. The bound peptides were incubated with the IgM anti-NMI peptide antibody and horseradish-peroxidase-labeled secondary antibody. The wells were then washed extensively and the color reactions developed. The absorbance was measured at 405 nm and the data were expressed as a percentage of the absorbance of the wells with the mouse NMI peptide. The mouse and Ciona cytoplasmic myosin I peptides were used as negative controls.


   Footnotes
 
We thank William Smith and the Santa Barbara Ascidian Stock Center (NIH/R24GM075049) at the University of California, Santa Barbara for providing us with C. intestinalis; Peter Gillespie, Oregon Health Sciences University, for providing the mT2 clone for the anti-pan-myosin-I-tail antibody; and Loriano Ballarin, University of Padova, for advice on isolating C. intestinalis hemocytes. We thank The Institute of Genomic Research (www.tigr.org), Genoscope (www.cns.fr), The Baylor College of Medicine (www.hgsc.bcm.tmc.edu) and the Department of Energy Joint Genome Initiative (www.jgi.doe.gov) for making their genome data available for public use. We thank Nicholas J. Talbot (University of Exeter) and Holly Goodson (University of Notre Dame) for comments. Supported in part by a grant from the US National Science Foundation (0517468) and the National Institutes of Health (GM 080587) to P.d.L. T.A.R. thanks the Leverhulme Trust for fellowship support. The authors certify that they have no competing interests. Deposited in PMC for release after 12 months.


   References
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 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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W. A. Hofmann, T. A. Richards, and P. de Lanerolle
Ancient animal ancestry for nuclear myosin
Development, March 15, 2009; 136(6): e607 - e607.
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