636
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
Centre for Eukaryotic Evolutionary Microbiology, School of Biosciences, University of Exeter, Devon EX4 4QD, UK
2
*Authors for correspondence (e-mail: [email protected]; [email protected])
Journal of Cell Science
Accepted 3 November 2008
Journal of Cell Science 122, 636-643 Published by The Company of Biologists 2009
doi:10.1242/jcs.030205
Summary
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 myosinI 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
Introduction
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, 2005; Thompson et al., 1997). 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., 2000)
and the demonstration that it plays an active role in transcription
(Fomproix and Percipalle, 2004; Pestic-Dragovich et al., 2000;
Philimonenko et al., 2004) 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., 2001). Myosin-I genes encode a peptide
with an N-terminal molecular-motor head domain followed by a
TH1 domain (Richards and Cavalier-Smith, 2005; Thompson and
Langford, 2002), a domain composed of highly basic residues that
functions in some proteins to bind phospholipids and membranes
(Wagner et al., 1992) and/or interact with actin (Lee et al., 1999;
Liu et al., 2000). Furthermore, many identified myosin-I genes
possess an additional C-terminal SH3 domain (Richards and
Cavalier-Smith, 2005). Recent studies have demonstrated that an
isoform of myosin IC (NMI) is present in vertebrate nuclei (PesticDragovich et al., 2000). 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 (PesticDragovich et al., 2000). Functionally, NMI colocalizes with RNA
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
polymerase I (Fomproix and Percipalle, 2004; Nowak et al., 1997)
and RNA polymerase II (Fomproix and Percipalle, 2004; Nowak
et al., 1997; Pestic-Dragovich et al., 2000), and co-purifies and coimmunoprecipitates with both polymerases (Pestic-Dragovich et al.,
2000; Philimonenko et al., 2004). Additionally, NMI-specific
antibodies inhibit transcription by these polymerases both in vivo
and in vitro (Fomproix and Percipalle, 2004; Pestic-Dragovich et
al., 2000; Philimonenko et al., 2004).
Myosins usually work in concert with actin, which has also been
shown to associate with polymerases I (Philimonenko et al., 2004),
II (Hofmann et al., 2004) and III (Hu et al., 2004), suggesting that
actin and NMI work together in transcription. Recent work has also
linked NMI to chromatin remodeling (Percipalle et al., 2006), and
to topoisomerase II (Smukste et al., 2006), and has implicated NMI
in the movement of chromosomal regions in the nuclei of
mammalian cells (Chuang et al., 2006). 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, 1998). 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, 2005). 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
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
Evolution of nuclear myosin I
A
duplication specific
to fish lineage
Bos taurus (NP_776821 [BtMyo1C])
N
NM1 phenotype
Mus musculus (AAG02570 [MmMyo1C])
N
MYO1C confirmed
Homo sapiens (XP_028385 [HsMyo1C])
N
Gallus gallus (NP_001006220 [GgMyo1C])
N
Xenopus tropicalis (Xentr_266159 [XtMyo1C])
N
O
Danio rerio (XP_695924 [BrMyo1G])
N
L
Tetraodon nigroviridis (CAG02935 [TnMyo1G]) N
Latest evolutionary
F
Tetraodon nigroviridis [TnMyo1F]
point that NM1
A
Danio rerio (XP_694821 [BrMyo1F])
100/1/1
Vertebrata C
evolved
Homo sapiens (NP_001094891 [HsMyo1H])
79/1/1
T
Mus musculus (XP_977982 [MmMyo1H])
MYO1H
Bos taurus (XP_604335 [BtMyo1H])
O
Gallus gallus (XP_415190 [GgMyo1H])
R
61/1/0.86
Xenopus tropicalis (NP_001008063 [XtMyo1H])
E
Danio rerio (XP_001337167 [BrMyo1H])
S
Tetraodon nigroviridis (CAF99250 [TnMyo1H])
Tetraodon nigroviridis (CAG08533 [TnMyo1I])
100/1/0.99
Danio rerio (CAM15181 [BrMyo1I])
NM1 phenotype
Ciona intestinalis (Ci240514 - chr_04q [CiMyo1C]) N
Tunicata
confirmed
Ciona savignyi [CisMyo1C]
Branchiostoma floridae (Br 280254)
Cephalochordata
Lottia gigantea (Lg238422)
Mollusca
Nematostella vectensis (Nemve 10540 [NvMyo1C])
Cnidaria
Anopheles gambiae [AngMyo1B]
Aedes aegypti (XP_001662030 [AeaMyo1B])
Insecta
Drosophila melanogaster (AAL39189 [DmMyo1B])
Tribolium castaneum (Tc 11553 [TicMyo1B])
Apis mellifera (XP_394436 [AmMyo1B])
Daphnia pulex (Dp 199690 [DapMyo1B])
Crustacea
Capitella sp. (Cs 120713)
Annelida
Monosiga brevicollis (XP_001745332 [MbMyo1E])
Choanoflagellata
Monosiga brevicollis (XP_001744998 [MbMyo1D])
Trichoplax adhaerens (Tr 49825)
Placozoa
Monosiga brevicollis (XP_001747996 [MbMyo1F])
Choanoflagellata
Homo sapiens (NP_056009 [HsMyo1D])
MYO1D/MYO1G
Mus musculus (CAI26107 [MmMyo1D])
duplication specific
Bos taurus (NP_001069306 [BtMyo1D])
to vertebrata
O
Gallus gallus (XP_418085 [GgMyo1D])
MYO1D
Xenopus tropicalis (Xentr 340728 [XtMyo1D])
L
Danio rerio (XP_688008 [BrMyo1E])
F
Vertebrata A
Tetraodon nigroviridis (CAF87192 [TnMyo1E])
100/1/0.99
Homo sapiens (NP_149043 [HsMyo1G])
C
Bos taurus (XP_594668 [BtMyo1G])
T
Mus musculus (NP_848534 [MmMyo1G])
MYO1G
57/0.87/0.57
O
Xenopus tropicalis (XtMyo1G)
R
Gallus gallus (NP_001026132 [GgMyo1G])
E
Danio rerio (NP_001038686 [BrMyo1D])
S
Ciona intestinalis (Ci 380757 [CiMyo1B])
Tunicata
Ciona savignyi [CisMyo1B]
Branchiostoma floridae (Br 89089)
Cephalochordata
Strongylocentrotus purpuratus (XP_780215 [StpMyo1B])
Echinodermata
Anopheles gambiae [AngMyo1A]
Aedes aegypti (XP_001662758 [AeaMyo1A])
Insecta
Drosophila melanogaster (NP_523538 [DmMyo1A])
Apis mellifera (XP_624678 [AmMyo1A])
Tribolium castaneum (XP_966392 [TicMyo1A])
Daphnia pulex (Dp 192228 [DapMyo1A])
Crustacea
Nematostella vectensis (XP_001637898 [NvMyo1B])
Cnidaria
Helobdella robusta (Hr 187944)
Capitella sp. (Cs 167519)
Annelida
Helobdella robusta (Hr 186525)
Lottia gigantea (Lg 143556)
Mollusca
Schistosoma mansoni (ScmMyo1A)
Nematoda
Caenorhabditis elegans (CAA53244 [CeMyo1B])
Monosiga brevicollis [MbMyo1C]
Choanoflagellata
Bos taurus (NP_001095669 [BtMyo1B])
MYO1A/MYO1B
Mus musculus (AAH54786 [MmMyo1B])
O
duplication specific
Homo sapiens (NP_036355 [HsMyo1B])
L
MYO1B
to vertebrata
Gallus gallus (XP_421901 [GgMyo1B])
F
Danio rerio (XP_001920959 [BrMyo1J])
Vertebrata A
Tetraodon nigroviridis (TnMyo1J)
100/1/1
C
Homo sapiens (NP_005370 [HsMyo1])
Bos taurus (NP_776820 [BtMyo1A])
T
20/--/-Mus musculus (NP_001074688 [MmMyo1A])
O
MYO1A
Gallus gallus (NP_990494 [GgMyo1A])
R
Xenopus tropicalis (NP_001008063 [XtMyo1A])
E
Ciona intestinalis (Ci 2499735 [CiMyo1D])
S
Tunicata
Ciona savignyi [CisMyo1D]
Branchiostoma floridae [Br 233021]
Cephalochordata
Cnidaria
Nematostella vectensis (XP_001641578 [NvMyo1D])
Aedes aegypti (XP_001663112 [AeaMyo1C])
Anopheles gambiae [AngMyo1C]
Insecta
Drosophila melanogaster (NP_001027208 [DmMyo1C])
Tribolium castaneum (XP_971077 [TicMyo1C])
Crustacea
Daphnia pulex (Dp 57481 [DapMyo1C])
Helobdella robusta (Hr 193971)
Annelida
Capitella sp. (Cs 166394)
Mollusca
Lottia gigantea (Lg 212414)
Placozoa
Trichoplax adhaerens (Tr 20417)
Monosiga brevicollis (XP_001749663 [MbMyo1B])
Choanoflagellata
Dictyostelium discoideum (XP_636359 [DdMyo1F])
Amoebozoa
Dictyostelium discoideum (XP_636580 [DdMyo1E])
Journal of Cell Science
MYO1C/MYO1H
duplication specific
to vertebrata
Fig. 2 Outgroup of myosin I genes
including MYO1E, MYO1F, MYOI, and
paralogs from fungi and protozoa
0.1 Subs/site
85/0.95 + phyML bootstrap / MrBayes posterior probability
N
55/0.8 + phyML bootstrap / MrBayes posterior probability
N-terminal NM1 like extention identified
Branch lengths reduced by 50%
B
Homo
Bos
Mus
Gallus
Xenopus
Danio
Tetraodon
Ciona
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., 2001), 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).
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
M R YR AS AL G S DG - V RV TM
M R YR AS AL G S DG - V RV TM
M R YR AS AL G S DG - V RV TM
M K YR GA GA G T NG - V RL TM
M K YR AA AP A I DG - I RV TM
M K YR RR EV G V EG G V RL MM
M K YH GR EV D I EG R V RL VM
M R LY YC CL Q R YS - V -S TM
MYO1C
637
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., 2001).
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, 2007) (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 Nterminal candidate nuclear-retention
peptide. (B) Alignment of putative
NMI N-terminal-extension peptides.
The putative NMI N-terminalextension 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., 2007) in
Homo (XP_0238385), Bos
(NP_776821), Mus (AAG02570),
Gallus (NP_001006220), Xenopus
(ENSXETP00000049503), Danio
(XP_695924) and Tetraodon
(GSTENT00022181001).
newly identified vertebrate ortholog family MYO1I, to be consistent
with the annotations of the myosin community (Gillespie et al.,
2001). However, this additional vertebrate-specific myosin-I
ortholog family was also detected by Odronitz and Kollmar
(Odronitz and Kollmar, 2007) 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., 2006) 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, 2007) 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,
638
Journal of Cell Science 122 (5)
Journal of Cell Science
Fig. 1A Myosin I genes including
MYO1A, MYO1B, MYO1C,
MYO1D, MYO1G, MYO1H
Naegleria gruberi [NgMyo1B]
Naegleria gruberi [NgMyo1D] Excavata
Naegleria gruberi [NgMyo1E]
Naegleria gruberi [NgMyo1C]
Haptophyta
Emiliania huxleyi (Em 75288)
Phytophthora ramorum [PhrMyo1A]
Oomycota
Hyaloperonospora parasitica [HypMyo1A]
Dictyostelium discoideum (XP_641363 [DdMyo1A])
Amoebozoa
Dictyostelium discoideum (XP_643446 [DdMyo1D])
Acanthamoeba castellanii (AAC98089 [AcMyo1C])
Excavata
Naegleria gruberi [NgMyo1F]
Dictyostelium discoideum (XP_636382 [DdMyo1B])
Acanthamoeba castellanii (P19706 [AcMyo1B])
Amoebozoa
Entamoeba histolytica (XP_654280 [EhMyo1])
Dictyostelium discoideum (AAO51841 [DdMyo1G])
Naegleria gruberi [NgMyo1A]
Excavata
Homo sapiens (NP_004989 [HsMyo1E])
MYO1E/MYO1I
100/1/0.97
Mus musculus (AAH51391 [MmMyo1E])
duplication specific
Bos taurus (XP_601785 [BtMyo1E])
to vertebrata
O
MYO1E
Gallus gallus (XP_413782 [GgMyo1E])
Xenopus tropicalis [XtMyo1E]
L
MYO1E-I/MYO1F
Tetraodon nigroviridis (CAF99593 [TnMyo1A])
F
duplication specific
Danio rerio (XP_682849 [BrMyo1A])
A
to vertebrata
Tetraodon nigroviridis [TnMyo1C]
C
Vertebrata
Danio rerio (NP_956930 [BrMyo1C])
MYO1I
T
Xenopus tropicalis (NP_001011082 [XtMyo1I])
99/1/1
O
90/1/0.98
Homo sapiens (NP_036467 [HsMyo1F])
R
Bos taurus (XP_612193 [BtMyo1F])
E
Mus musculus (NP_444444 [MmMyo1F])
88/1/0.95
MYO1F
S
Gallus gallus (NP_990585 [GgMyo1F])
Xenopus tropicalis [XtMyo1F]
Tetraodon nigroviridis (CAG00732 [TnMyo1B])
Danio rerio (XP_001921060 [BrMyo1B])
Ciona intestinalis (Ci 248730 [CiMyo1A])
Tunicata
Helobdella robusta (Hr 165806)
Capitella sp. (Cs 95339)
Annelida
Helobdella robusta (Hr 116401)
Lottia gigantea (Lg 103140)
Mollusca
Branchiostoma floridae (Br 58680)
Cephalochordata
Strongylocentrotus purpuratus (AAF71717 [StpMyo1A])
Echinodermata
Nematostella vectensis [NvMyo1A]
Cnidaria
Caenorhabditis elegans (NP_492393 [CeMyo1A])
Nematoda
Daphnia pulex (DapMyo1D)
Crustacea
Nematostella vectensis (XP_001627107 [NvMyo1E])
Cnidaria
Batrachochytrium dendrobatidis (BDEG_06627 [BadMyo1])
Chytridiomycota
Saccharomyces cerevisiae (NP_012793 [Sc_cMyo1A])
Saccharomyces cerevisiae (NP_013827 [Sc_cMyo1B])
Candida glabrata (XP_448551 [CglMyo1B])
Candida glabrata (XP_448376 [CglMyo1A])
Candida albicans (XP_710973 [Ca_aMyo1Alpha[)
Ascomycota
Candida albicans (XP_710973 [Ca_aMyo1Beta])
Debaryomyces hansenii (XP_458067 [DehMyo1])
Fungi
Yarrowia lipolytica (XP_503442 [YlMyo1])
Neurospora crassa (XP_964105 [NcMyo1])
Magnaporthe grisea (MgMyo1)
Aspergillus niger (XP_001396984 [AnMyo1])
Ustilago maydis (XP_760259 [Um aMyo1])
Basidiomycota
Ustilago maydis [Um bMyo1]
Coprinopsis cinerea (A8N2Y6 [CpcMyo1])
Ascomycota
Schizosaccharomyces pombe (NP_595402 [SpMyo1])
Dictyostelium discoideum (XP_643060 [DdMyo1C])
Amoebozoa
Acanthamoeba castellanii (AAC35357 [AcMyo1A])
Trypanosoma brucei (Xp_844488 [TbMyo1])
Kinetoplastida
Leishmania major (CAJ07807 [LemMyo1])
Phytophthora ramorum [PhrMyo1B]
Oomycota
Hyaloperonospora parasitica [HypMyo1B]
0.1 Subs/site
85/0.95 + phyML bootstrap /MrBayes posterior probability
55/0.8 phyML bootstrap / MrBayes posterior probability
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., 2006) 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., 2006). This evolutionary relationship has been named
the Olfactores hypothesis (Delsuc et al., 2006). NMI-like transcripts
have been identified in all the major vertebrate lineages (Kahle et
al., 2007). 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
Branch lengths reduced by 50%
Fig. 2. Subsection of the myosin-I
phylogeny showing additional vertebratespecific 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., 2001) to include one additional
ortholog group (MYO1I), labeled using a
gray box.
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., 2006) 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., 2000) as
being encoded by the MYO1C gene in Mus musculus (Dumont et
al., 2002). 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., 2007),
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 vertebratespecific 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 nonvertebrate 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
Evolution of nuclear myosin I
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., 2000).
To test whether there are multiple isoforms of this myosin-I gene
in C. intestinalis and whether the predicted myosin I with the Nterminal 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-panmyosin-IC antibody that recognizes an epitope in the tail of bovine
myosin IC (formerly known as myosin Iβ) (Wagner et al., 1992).
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., 2000).
Journal of Cell Science
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
639
Fig. 3. Myosin-I isoforms in C. intestinalis.
(A) Sequence alignment of the myosin-I-tailregion epitope that is recognized by the anti-panmyosin-I-tail antibody. (B) Western blot analysis
of total cell extract from C. intestinalis and NIH3T3 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-panmyosin-I-tail antibody, and exclusively
cytoplasmic staining by the anti-α-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.
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Journal of Cell Science 122 (5)
Table 1. Reactivity of IgM peptide antibody with M. musculus and C. intestinalis N-terminal peptides
(aligned to match the mouse NMI sequence)
Peptide
M. musculus NMI peptide
M. musculus CMI start sequence
C. intestinalis NMI peptide
C. intestinalis CMI start sequence
Sequence
MRYRASALGSDGVRVTMESALTAR
MESALTAR
MRLYYCCLQRYSVS TMEGALTAR
MEGALTAR
% maximum
100
0
80
0
Journal of Cell Science
Peptide synthesis and ELISA determination of antibody reactivity are described in the Materials and Methods. Peptides representing the first eight amino acids
of the mouse and Ciona cytoplasmic myosin I (CMI) were used as negative controls.
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.
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-myosinIC-tail antibody and an antibody to α-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-Itail antibody gives strong punctate staining of the cytoplasm and
weaker staining in the nucleus. By contrast, the anti-α-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-myosinI-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, 1962) (Fig. 4A). Fractions from the gradient were
analyzed with antibodies to RNA polymerase II, β-actin and αtubulin to identify the major nuclear and cytoplasmic fractions,
respectively. Fig. 4B shows that fractions 4 and 5 contained RNA
polymerase II, whereas α-tubulin as well as the majority of β-actin
was found in fraction 6, which contains cytoplasmic debris
(Hinegardner, 1962). Consistent with other data (Hofmann et al.,
2004), fraction 5 (intact nuclei) also contained actin (Fig. 4B).
However, the absence of α-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).
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 NMIspecific IgM peptide antibody. Fig. 4C shows that both antibodies
recognize the same protein, indicating that the protein
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, 1962).
(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.
Evolution of nuclear myosin I
Journal of Cell Science
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
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 Nterminal 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 (PesticDragovich et al., 2000). 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., 2002). 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., 2002), 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.,
2008), myosin VI (Vreugde et al., 2006), myosin XVIb (Cameron
et al., 2007) and actin, in addition to NMI, are found in eukaryotic
nuclei. NMI interacts with topoisomerase II (Smukste et al., 2006),
641
and MreB, an actin-like protein, interacts with RNA polymerase in
bacteria (Kruse et al., 2006). 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-IIdependent transcription (Vreugde et al., 2006).
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 myosinI 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 eukaryoticcell evolution, potentially predating the diversification of the
majority of cytoplasmic myosin motors.
Materials and Methods
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, 2007). 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.,
1997) through the SEAVIEW platform (Galtier et al., 1996). 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., 1996) and appear to be inappropriate for
myosin analysis when compared to more-sophisticated methods (Foth et al., 2006),
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., 2006) to obtain the
most appropriate substitution matrix (Rt-REV) and model of site rate variation (Γ=8,
α=0.93 and I=0.04). These model parameters were entered into PHYML for a 100replicate bootstrap analysis (Guindon et al., 2005). MrBayes analysis (Ronquist and
Huelsenbeck, 2003) 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
642
Journal of Cell Science 122 (5)
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.
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.
Biological materials
Light microscopy
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.
C. intestinalis hemocytes, isolated as described (Cammarata and Parrinello, 1995)
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-α-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.
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., 2000). 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., 1992). Monoclonal antibodies to β-actin
and the C-terminal domain of RNA polymerase II (8WG16) were obtained from Sigma
(St Louis, MO) and to α-tubulin from BAbCO (Richmond, CA). Secondary antibodies
were acquired from Jackson ImmunoResearch Laboratories (West Grove, PA).
Journal of Cell Science
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, 1962).
Nuclear extract from C. intestinalis and NIH-3T3 cells was prepared essentially as
described (Dignam et al., 1983). 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,
α-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,
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-peroxidaselabeled 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.
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 antipan-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.
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