Download Molecular Evolution and Structure of a

Molecular Evolution and Structure of a-Actinin
Ana Virel and Lars Backman
Department of Biochemistry, Umeå University, SE-901-87 Umeå, Sweden
The N-terminal actin-binding domain of a-actinin is connected to the C-terminal EF-hands by a rod domain. Because of
its ability to form dimers, a-actinin can cross-link actin filaments in muscle cells as well as in nonmuscle cells. In the
prototypic a-actinins, the rod domain contains four triple helical bundles, or so-called spectrin repeats. We have found
some atypical a-actinins in early diverging organisms, such as protozoa and yeast, where the rod domain contains one
and two spectrin repeats, respectively. This implies that the four repeats present in modern a-actinins arose after two
consecutive intragenic duplications from an a-actinin with a single repeat. Further, the evolutionary gene tree of aactinins shows that the appearance of four distinct a-actinin isoforms may have occurred after the vertebrate-invertebrate
split. The topology of the tree lends support to the hypothesis that two rounds (2R) of genome duplication occurred early
in the vertebrate radiation. The phylogeny also considers these atypical isoforms as the most basal to a-actinins of
vertebrates and other eukaryotes. The analysis also positioned a-actinin of the fungi Encephalitozoo cuniculi close to the
protozoa, supporting the suggestion that microsporidia are early eukaryotes. Because a-actinin is considered the basal
member of the spectrin family, our studies will improve the understanding of the origin and evolution of this superfamily.
Introduction
a-Actinin is a ubiquitous actin cross-linker belonging
to the spectrin superfamily (Blanchard, Ohanian, and
Critchley 1989; Dubreuil 1991). It has been identified in
most eukaryotic organisms, from human (Beggs et al.
1992; Mills et al. 2001) and mouse (Mills et al. 2001) to fly
(Drosophila melanogaster) and worm (Caenorhabditis
elegans) (Fyrberg et al. 1990; Barstead, Kleiman, and
Waterston 1991). Although fission yeast (Schizosaccharomyces pombe) appears to express an a-actinin (or at least
an a-actinin–like protein) baker’s yeast (Saccharomyces
cerevisiae) does not (Wu, Bahler, and Pringle 2001).
The structure of the a-actinin has been determined in
a great detail. Depending on the source, this elongated
protein has a molecular mass of 93 to 103 kDa (Blanchard,
Ohanian, and Critchley 1989). The actin-binding site is
located at the N-terminus, which comprises two calponin
homology domains, where only the most N-terminal one
binds actin (Norwood et al. 2000). At the C-terminus, there
are two EF-hands, where the second EF-hand is crucial for
calcium binding (Witke et al. 1993; Janssen et al. 1996).
These two domains are connected by the rod domain. This
domain is formed by triple-helical repeats or so-called spectrin repeats, where each repeat contains approximately 106
amino acid residues (Djinovic-Carugo et al. 1999). The rod
domain of a-actinin is important for dimerization (DjinovicCarugo et al. 1999; Ylanne et al. 2001). There are usually
four spectrin repeats in the rod domain, but a-actinin from
some organisms appears to have only one or two repeats.
Because the rod domain determines the distance between cross-linked actin filaments and serves as an interaction site for receptors and adaptor proteins, the number
of repeats has implications for actin bundling as well as
for cytoskeleton organization (Djinovic-Carugo et al. 1999;
Ylanne et al. 2001; Djinovic-Carugo et al. 2002).
a-Actinins are classified into four different main
isoforms (Dixson, Forstner, and Garcia 2003). Humans
and probably other vertebrates express all four isoforms,
whereas invertebrates and protista seem to express only
one isoform. The muscle isoforms, a-actinin 2 and of aactinin 3 are localized to the Z-disc of the sarcomeres
(Blanchard, Ohanian, and Critchley 1989; Mills et al.
2001). These two isoforms are insensitive to calcium
because their EF-hands are nonfunctional (Beggs et al.
1992). In contrast, the nonmuscle isoforms, a-actinin 1 and
of a-actinin 4 are sensitive to calcium (Tang, Taylor, and
Taylor 2001). a-Actinin 1 and of a-actinin 4 can be found
at the leading edge of motile cells, at cell adhesion sites
and focal contacts, and along actin stress fibers in
migrating cells (Barstead, Kleiman, and Waterston 1991).
To better understand the structural and evolutionary
relationships between the a-actinin isoforms, we have
characterized a-actinin from the urochordate Ciona
intestinalis, as well as several other primitive eukaryotes.
Our results demonstrate that the general structural pattern
of a-actinin is not applicable to the most primitive
eukaryotes, as these isoforms are shorter because of fewer
spectrin repeats in the rod domain.
The phylogenic analysis implies that the multiple aactinin isoforms found in modern vertebrates arose after
the divergence of the vertebrate and urochordate lineages.
The determined phylogeny displayed an ((A, B) (C, D))
topology, implying that the calcium-insensitive a-actinin
isoforms 2 and 3 evolved together as did the calciumsensitive isoforms 1 and 4.
It is believed that an a-actinin–like precursor has given
rise to members of the spectrin superfamily (i.e., a-actinins,
spectrin, and dystrophin) by gene duplications and gene
rearrangements (Pascual, Castresana, and Saraste 1997;
Thomas et al. 1997; Viel 1999; Baines 2003). Therefore,
a better understanding of the evolution and structure of aactinins may provide a better insight into the evolution of
this superfamily.
Key words: a-actinin, phylogeny, evolution, spectrin superfamily,
spectrin repeat.
Materials and Methods
E-mail: [email protected].
Mol. Biol. Evol. 21(6):1024–1031. 2004
DOI:10.1093/molbev/msh094
Advance Access publication March 10, 2004
A Ciona k-Zap cDNA library (generously made
available by Dr. Nori Satoh) made from tail-bud embryos
was screened using the following a-actinin–specific
oligonucleotides: N-term forward: 59-CAGGAGGAG-
Molecular Biology and Evolution vol. 21 no. 6 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
Molecular Evolution of a-Actinin 1025
Table 1
Accession Number, Protein Name, and Phylum of a-Actinins Used in the Phylogenetic
Analysis
Phylum
Accession Number
Protein
Protozoa
Dictyostelium discoideum
Encephalitozoon cunculi
Enthamoeba histolytica
Trichomonas vaginalis
P05095
CAD27000
Q9U3Z8
AAC72899
a-Actinin
Similarity to nonmuscle a-actinin
Actinin-like protein [fragment]
a-Actinin
Arthropoda
Anopheles gambia
Drosophila melanogaster
EAA00884
FAFFAA
a-Actinin
Nemata
Caenorhabditis elegans
NP_506127.1
ATN-1
Tunicata
Ciona intestinalis
TC14429
Similar to Drosophila a -actinin
Fungus
Schizosaccharomyces pombe
Neurospora crassa
Z97208
EAA28084
Hypothetical protein
P12814
P35609
Q08043
AA43707
Q9JI91
O88990
P57780
Q9Z1P2
Q8R4I6
Q9QXQ0
P05094
P20111
Q90734
AAN77132
SINFRUP00000054951
AAH43995
a-Actinin 1
a-Actinin 2
a-Actinin 3
a-Actinin 4
a-Actinin 2
AAC3_MOUSE (a-actinin 3)
a-Actinin 4
a-Actinin 1
a-Actinin 3
a-Actinin 4
a-Actinin 1
a-Actinin 2
a-Actinin 4
a-Actinin
Gene: SINFRUG00000054669
Similar to a-actinin 1
Vertebrata
Homo sapiens (human)
Mus musculus (mouse)
Ratus norvergicus (rat)
Gallus gallus (chicken)
Danio rerio (zebrafish)
Takifugu rubripes (puffer fish)
Xenopus laevis (frog)
GAGTGGGACCGCG-3 and N-term reverse: 59-CCCATACTAGACCTGGTAGTAGG-39 (corresponding to actin-binding domain of human a-actinin); forward I:
59-GGTGGCTTGACAAGGAAACTAG-39 and forward
II: 59-GATGGTTTGGCTTTCTGTGCC C-39; and reverse
I: 59-CTCCTGGTTCAAACCAAGAACCTTC-39 and reverse II: 59-GCCTTGACT TCTTGGAAGGTGC-39 (all
specific to the downstream region of Ciona a-actinin). M13specific primers (M13 forward: 59-CGTTGTAAAACGACGGCCAGTG-39 and M13 reverse: 59-GCATT-39
AGTACCAGTATCGACAAAGGAC-39) were also used.
PCR Master mix (Promega) was used for the PCR reactions.
Obtained PCR products were purified by QIAquick PCR
purification kit (Qiagen) and ligated into pGEM-T vector
(Promega). After the ligation, TG1 E. coli cells were
transformed by heat shock and cultured on agar plates
overnight at 378C. Isolated clones were sequenced using the
ABI PRISM BigDye terminator cycle sequencing kit
(Applied Biosystems).
and Sanger Institute. Retrieved sequences are summarized
in table 1.
Sequences were aligned in ClustalX (Thompson et al.
1997), using default parameters, and refined manually.
Phylogenetic analysis was conducted using maximumparsimony, neighbor-joining, and Bayesian inference; they
all resulted in very similar results, both in topology and in
significance. The analyses were done using the PHYLIP
(Felsenstein 1989), MEGA2 (Kumar et al. 2001), and
MrBayes software packages (Huelsenbeck and Ronquist
2001).
One-thousand bootstrapped data sets were created
with the program SEQBOOT, and the phylogeny estimate
for each data was calculated using PROTPARS, selecting
1,000 data sets. The input order of the sequences was
randomized with a jumble number of 10. The majority-rule
consensus tree was created by CONSENSUS.
Phylogenetic Analysis
A Ciona cDNA library was screened by PCR for the
presence of a-actinin. The primers used for this purpose
were based on the 59-end of the human a-actinin nucleotide sequence, corresponding to the highly conserved
Available nucleotide and amino acid sequences of aactinins were retrieved from NCBI, TIGR, Swiss Protein
Results
Ciona intestinalis a-Actinin
1026 Virel and Backman
FIG. 1.—Pairwise sequence identity of a-actinin isoforms.
N-terminal domain. Subcloning and subsequent sequencing of obtained PCR products identified several overlapping DNA fragments. The translated amino acid
sequence was highly similar to a-actinin of vertebrates.
During the course of this work, part of the Ciona
genome was published (Dehal et al. 2002; Satou et al.
2002). Alignment of our sequence with the TIGR Ciona
intestinalis Gene Index, pulled out TC14429, identified as
a partial fragment of the a-actinin (97%) similar to
Drosophila a-actinin. We observed that the sequence we
obtained was extended upstream of the probable start
codon, completing the sequence. From this sequence new
specific primers for the Ciona a-actinin were designed, and
a longer sequence was obtained that overlapped completely
with the sequence in the TIGR database.
As our PCR results imply, Ciona appears to have only
a single gene for a-actinin. Previous comparative studies of
gene families in Ciona have show that most genes that are
present in multiple copies in vertebrates only have a single
representative in Ciona (Dehal et al. 2002).
sequences (table 1). These sequences were first aligned using
ClustalX, with default settings, and then refined manually.
It is obvious that throughout the vertebrates, all aactinins are very similar; 93% (or 837 of 892) of the amino
acid residues are identical in human and frog a-actinin. The
isoforms of a single species are also highly similar; of the
approximately 890 amino acid residues in the four isoforms
of human a-actinins 73% or more are identical (fig. 1).
Although the unicellular a-actinins are less similar, the
degree of identity is still very high, particular in the Nterminus and C-terminus. The largest sequence differences
are found in the rod domain. In all vertebrate sequences, it is
possible to locate four spectrin repeats, whereas the rod
domains of Schizosaccharomyces pombe and Neurospora
crassa contain two spectrin repeats, and those of Entamoeba histolytica and Encephalitozoo cuniculi have one
repeat.
a-Actinin from Other Organisms
S. pombe and N. crassa are both ascomycetes
belonging to the fungus kingdom (Keeling, Luker, and
Palmer 2000). As expected, the a-actinins of these two
organisms display a very high degree of identity. When
compared with other a-actinins, it is obvious that the
calponin homology (actin-binding) domain in the Nterminus and the calcium-binding domain in the Cterminus have been preserved during evolution. However,
the rod domain in both S. pombe and N. crassa a-actinins
differs. This part is much shorter than in other a-actinins
and comprises only two spectrin repeats. These repeats are
Available databases (e.g., NCBI, SwissProt, TIGR,
and Sanger Institute) were searched for sequences similar to
Ciona a-actinin, as well as for annotated a-actinin sequences. We retrieved sequences of a-actinin from 17
different species, mostly from the animal kingdom. Several
of the vertebrates have more than one isoform. In humans
and mouse, four different isoforms have been identified,
whereas in rat and chicken, only three distinct isoforms have
been identified so far. In total, we retrieved 26 a-actinin
Schizosaccharomyces pombe and Neurospora
crassa a-Actinins
Molecular Evolution of a-Actinin 1027
FIG. 2.—Phylogenetic tree of a-actinins. The most-parsimonious tree shows the relation between a-actinins of different organism, as well as of
different isoforms of the same organism. Bootstrap values in percentage were calculated from 1,000 data sets.
similar to the first repeat (27% identity) and fourth repeat
(21% identity), respectively, of Drosophila a-actinin (Wu,
Bahler, and Pringle 2001).
Entamoeba histolytica a-actinin–like Protein
Entamoeba histolytica is considered one of the most
primitive protozoa. Although it is a eukaryotic organism,
it lacks mitochondria and several other characteristic
organelles. E. histolytica is parasitic and infects predominantly humans and other primates (Nickel et al.
2000). A protein that cross-reacts with antibodies to aactinin has been identified (Bailey et al. 1992). From the
alignment of this a-actinin–like protein, the typical domain
structure is not obvious. When the sequence (Nickel et al.
2000) was submitted either to InterPro scan or SMART, an
actin-binding motif and two calponin homology domains
in the N-terminus as well as two, or possibly three EFhand motifs in the C-terminus were returned. Using the
ProteinPredict server, the actin-binding domain could be
modeled on the coordinates of the actin-binding domain of
human dystrophin (1dxx) and the calcium-binding domain
of porcine calponin (1alv).
The rod domain of this protein is much shorter than
that of the prototypic a-actinin. It comprises only approximately 125 amino acid residues, which should
correspond to a single spectrin repeat. This repeat was
found to be most closely related to the first repeat of chicken
a-actinin.
Encephalitozoo cuniculi a-Actinin
Encephalitozoo cuniculi is an obligate intracellular
parasite, belonging to the microsporidia (Weiss 2001).
Similar to E. histolytica, microsporidia are characterized
by the lack of normal mitochondria and peroxisomes
(Roger and Silberman 2002; Williams et al. 2002). The
taxonomy of this group has been reclassified several times
and is still uncertain (Germot, Philippe, and Le Guyader
1997; Peyretaillade et al. 1998; Keeling, Luker, and
Palmer 2000; Keeling and Fast 2002; Keeling 2003).
Based on phylogenetic analysis of small subunit ribosomal
RNA as well as the lack of mitochondria, it has been
suggested that microsporidia are one of the earliest
eukaryote lineages (Leipe et al. 1993). More recent
analysis indicate that microsporidia are related to fungi
and not to early diverging eukaryotes (Hirt et al. 1999;
Keeling, Luker, and Palmer 2000; Keeling 2003).
Similar to the fungi isoforms, the a-actinin of E.
cuniculi is short; it is only 537 amino acid residues long.
The similarity to the prototypic a-actinin is low (fig. 1).
When submitted to the InterPro Scan and SMART, an
actin-binding motif and two calponin homology domains
in the N-terminal domain were identified. Further analysis
of the primary structure indicated the highest similarity
with sequences from fungi and amoebae (fig. 1).
Trichomonas vaginalis a-Actinin
Trichomonas vaginalis is a sexually transmitted
human parasite. When it adheres to the host cell, it
undergoes a transformation from a flagellate to an amoeboid
form. Previous analysis of the a-actinin of T. vaginalis has
suggested the presence of five repeats in the rod domain in
addition to the common motifs in both termini (Addis et al.
1998; Bricheux et al. 1998). Interesting to note is that only
the first of these repeats shows some similarity with other aactinins, and then only with the first repeat of other aactinins. It was suggested that the other four repeats have
1028 Virel and Backman
FIG. 3.—Phylogenetic analysis of spectrin repeats in chicken, yeast, and amoeba. In the most-parsimonious tree, the single repeat of Enthamoeba
histolytica (entamoeba) appears closest to the first repeat of chicken (SR1) a-actinin. Similarly, the first repeat of the Schizosaccharomyces pombe
isoform (pombe SR1) appears most related to the first repeat in the chicken isoform (SR1), whereas the second repeat of yeast a-actinin (pombe SR2)
seems to be closest to the fourth chicken repeat (SR4). The bootstrap values in percentage were calculated from 1,000 data sets.
evolved because of intragenic duplication that has not
occurred in other sequences (Bricheux et al. 1998).
Phylogenetic Analysis
The aligned sequences were analyzed using the
PHYLIP, MEGA2, and MrBayes packages. The program
SEQBOOT was used to create 1,000 bootstrapped data set
and PROTPARS was used to calculate the phylogeny
estimate. The maximum-parsimony tree obtained is shown
in figure 2. Phylogenetic analysis based on neighborjoining and Bayesian inference resulted in trees with very
similar topologies as well as significance.
There are three main branches in the tree; two
vertebrate branches and one invertebrate and unicellular
organisms branch. One of the vertebrate branches includes
the calcium-insensitive isoforms 2 and 3, and the other
branch includes the calcium-sensitive isoforms 1 and 4,
thus giving rise to a phylogenetic tree with an ((A, B) (C,
D)) topology. That a-actinins of each isoforms are grouped
together implies a higher degree of similarity between
a particular isoform of all organisms than between the
isoforms of a single organism. Most branches in the maximum-parsimony consensus tree are supported by bootstrap values of 90% or higher.
The phylogeny was also determined on a subset of the
a-actinin sequences. Analyzing the amino acid sequences
of the actin-binding domain of the vertebrate a-actinins
resulted in very similar tree topologies. However, when the
nucleotide sequences were used for the analysis instead, an
(A, (B, (C, D))) topology was obtained.
In the invertebrate branch Ciona a-actinin appears to
be closest related to a-actinins of the arthropod branch
(Drosophila and Anopheles) and the nematode C. elegans.
The placing of unicellular a-actinins at the end of this branch
indicates that these have branched off early in evolution.
Analysis of the Spectrin Repeats
We also analyzed the phylogenies of the spectrin
repeats. All methods tested placed the single spectrin
repeat of E. histolytica a-actinins closest to the first
spectrin repeat (SR1), as shown in figure 3. The two
repeats of S. pombe and N. crassa are most similar to SR1
and SR4, respectively. This suggests a possible evolutionary pathway (fig. 4). In the primordial ancestor to presentday a-actinins, the rod domain contained only a single
spectrin repeat that became the first repeat (SR1) in
modern a-actinins. A first intragenic duplication gave rise
to the second repeat, which became the fourth repeat (SR4)
in modern a-actinins. A subsequent second intragenic
duplication added two more repeats (SR2 and SR3).
Discussion
The existence of a notochord in the larvae stage
implies that the urochordate Ciona share a common
ancestor with vertebrates before the vertebrate radiation
(Dehal et al. 2002; Gee 2002; Pennisi 2002). Since Ciona
appears to have only a single a-actinin gene in contrast to
higher vertebrates such as human, mouse, and chicken, it
seems likely that the appearance of the different a-actinin
isoforms arose after the vertebrate radiation.
This theory is substantiated by the topology of the
phylogenetic tree, which indicated one invertebrate and two
major vertebrate branches. One of the vertebrate branches
encompasses the calcium-insensitive a-actinin 2 and aactinin 3, and the other branch encompasses the calciumsensitive isoforms 1 and 4. This ((A, B) (C, D)) tree
topology lends support to the suggestion that two rounds of
genome duplication occurred early in vertebrate evolution
(the 2R hypothesis) (Hughes 1999; Durand 2003).
However, the observed phylogeny can also be explained
by independent duplication of individual genes or gene
segments.
A phylogenetic analysis using the nucleotide sequences corresponding to the actin-binding domain of
vertebrate a-actinins resulted in a different tree topology
(Dixson, Forstner, and Garcia 2003). In this case, the tree
displayed a (A, (B, (C, D))) topology, suggesting that the
calcium-insensitive a-actinin 2 evolved initially, followed
first by the calcium-insensitive a-actinin 3 and then by the
calcium-sensitive a-actinins 1 and 4. When we repeated
Molecular Evolution of a-Actinin 1029
FIG. 4.—Schematic structure of ancestral and modern a-actinins. All a-actinins have preserved the two calponin homology domains and, in most
cases, the calcium-binding domain, whereas the rod domain differs significantly between different isoforms. Basal organisms, such as Entamoeba
histolytica and Schizosaccharomyce pombe with one and two spectrin repeats, respectively, in the rod domain indicate that the two repeats in S. pombe
and the four repeats present in other organisms are the result of two consecutive intragenic duplications from the single spectrin repeat of a primordial aactinin, like the one present in E. histolytica.
the phylogenetic analysis on this subset of sequence data,
using the amino acid sequence of the actin-binding
domain, we obtained tree topologies very similar to those
obtained for the full data set. However, when we repeated
the analysis using the nucleotide sequences, we obtained
an (A, (B, (C, D))) tree topology, similar to that reported
by Dixson, Forstner, and Garcia (2003). Thus, depending
on whether amino acid or nucleotide sequences are used in
the phylogenetic analysis, different tree topologies may be
obtained.
It has been suggested that phylogenetic analysis
based on amino acid sequences are more reliable than
analysis based on nucleotide sequences because of
compositional bias in the DNA sequences (Foster and
Hickey 1999). This, together with the high bootstrap
values, gives substantial support to our phylogenetic
analysis.
Also frog (X. laevis) and fish (D. rerio and F. rubripes)
appear to have a single a-actinin isoform. The frog and
puffer fish a-actinins are related to isoform 1, whereas the aactinin of zebrafish is related to a-actinin 3. The appearance
of only a single isoforms in each of these organisms does not
exclude the possibility of other isoforms, as their genomes
have not yet been completely sequenced.
All tested phylogeny models placed the isoform of E.
histolytica as the earliest diverging a-actinin, followed by
the a-actinin of E. cuniculi. Together with the isoform of
T. vaginalis, these two a-actinins are also those that are
least similar to the consensus sequence. Despite the low
similarity, these proteins display certain of the typical
hallmarks of a-actinins and can be regarded as a-actinins
or at least as a-actinin–like proteins.
The fungi S. pombe and N. crassa also branched off
early in evolution. The fungi a-actinins are more similar to
the present day a-actinins, despite the short rod domain
with its two spectrin repeats.
When aligning the sequence of E. cuniculi a-actinin
with a-actinins of E. histolytica and S. pombe, it is
apparent that the sequence is similar not only to the S.
pombe isoform but also to the E. histolytica a-actinin,
again placing microsporidia between the fungus and
protozoa branches.
In all bacterial and plant genomes available to date,
we have not been able to identify any genes for a a-actinin
or a a-actinin–like protein. This implies that the primordial
a-actinin appeared in a primitive unicellular organism
belonging to the protozoan kingdom.
a-Actinin is composed of three major domains: an
N-terminal actin-binding domain, consisting of two
calponin homology domains; a rod domain of spectrin
repeats, and a C-terminal calcium-binding domain. These
domains are highly conserved throughout the animal
kingdom. However, in basal eukaryotes and, in particular,
in unicellular organisms, this domain structure is less well
preserved. The evolutionary best-preserved domain is the
N-terminal domain with its two calponin homology
domains, which is present in all a-actinins and aactinin–like proteins. This is not surprising, as the major
function of a-actinin is believed to be binding actin
filaments (Blanchard, Ohanian, and Critchley 1989).
Although it was not possible to identify a calciumbinding motif in all sequences, it is apparent that this
domain is also well conserved among the different aactinins. It should be noticed that not all isoforms of aactinin are sensitive to calcium ions because of structural
changes in this region during evolution (Dixson, Forstner,
and Garcia 2003).
The most variable part is the rod domain, which is
composed of one, two, or four spectrin repeats. In aactinins able to form antiparallel dimers, the function of
the rod domain is mainly to separate the actin-binding
domains of each molecule (Ylanne et al. 2001). Therefore,
the rod part could be built from any amino acid sequence
that can form a coiled-coil or rodlike structure. The aactinin of the protozoa Trichomonas vaginalis seems to
support this conclusion, as only one spectrin repeat can be
identified in the rod domain in addition to a long coiledcoil region. Also, the rod domain of E. histolytica a-actinin
contains only a single repeat unit, but in this case, there is
no additional coiled-coil region.
1030 Virel and Backman
The rod domain of the isoforms of E. histolytica and
E. cuniculi are much shorter, and only a single spectrin
repeat could be identified. Although the rod domain of
T. vaginalis a-actinin is longer, also in this case, only
a single repeat could be identified.
The rod domain of a-actinins of all vertebrates, as
well as of invertebrates, has four spectrin repeats. This
suggests a likely scenario for the evolution of the rod
domain. In the primordial a-actinin, the rod domain was
short, spanning a single spectrin repeat. An intragenic
duplication added a second repeat, followed by yet another
intragenic duplication. As the single repeat in E. histolytica
appears most related to the first repeat (SR1), it is likely
that this repeat also was the first to evolve. Likewise, as the
two repeats of fungi are closest to repeat 1 (SR1) and 4
(SR4), this suggests that repeat 4 aroused through intragenic duplication. The second intragenic duplication
added repeats 2 (SR2) and 3 (SR3). Thus, our data do not
support the conclusion that the ancestor of a-actinin should
possess four spectrin repeats (Viel 1999).
It has been demonstrated that SR2 and SR3 are
essential for dimerization of a-actinin (Djinovic-Carugo et
al. 1999). In the dimer, SR2 and SR3 pair with the SR3
and SR2 of the other antiparallel molecule. Therefore, it is
possible that these primitive a-actinins are unable to form
dimers, which would exclude any function involving actin
cross-linking. The function of these short and atypical aactinins is still unknown.
Supplementary Material
The final alignment of all retrieved a-actinin sequences is available online at www.mbe.oupjournals.org.
Acknowledgments
This work was supported by grants from EU HPRNCT-2000–00096, Åke Wibergs Stiftelse, Magn. Bergvalls
Stiftelse and O.E. och Edla Johansson Stiftelse.
The TIGR Ciona intestinalis Gene Index updated
February 2, 2004, now contains clone TC35693, which is
identical to the Ciona a-actinin sequence determined in
this work.
Literature Cited
Addis, M. F., P. Rappelli, G. Delogu, F. Carta, P. Cappuccinelli,
and P. L. Fiori. 1998. Cloning and molecular characterization
of a cDNA clone coding for Trichomonas vaginalis alphaactinin and intracellular localization of the protein. Infect.
Immun. 66:4924–4931.
Bailey, G. B., P. S. Shen, M. J. Beanan, and N. E. McCoomer.
1992. Actin associated proteins of Entamoeba histolytica.
Arch. Med. Res. 23:129–132.
Baines, A. J. 2003. Comprehensive analysis of all triple helical
repeats in beta-spectrins reveals patterns of selective evolutionary conservation. Cell. Mol. Biol. Lett. 8:195–214.
Barstead, R. J., L. Kleiman, and R. H. Waterston. 1991. Cloning,
sequencing and mapping of an alpha-actinin gene from the
nematode Caenorhabditis Elegans. Cell Motil. Cytoskelet.
20:69–78.
Beggs, A. H., T. J. Byers, J. H. M. Knoll, F. M. Boyce, G. A. P.
Bruns, and L. M. Kunkel. 1992. Cloning and characterization
of two human skeletal muscle alpha-actinin genes located on
chromosomes 1 and 11. J. Biol. Chem. 267:9281–9288.
Blanchard, A., V. Ohanian, and D. Critchley. 1989. The structure
and function of alpha-actinin. J. Muscle Res. Cell Motil.
10:280–289.
Bricheux, G., G. Coffe, N. Pradel, and G. Brugerolle. 1998.
Evidence for an uncommon a-actinin protein in Trichomonas
vaginalis. Mol. Biochem. Parasitol. 95:241–249.
Dehal, P., Y. Satou, R. K. Campbell et al. (87 co-authors) 2002.
The draft genome of Ciona intestinalis: insights into chordate
and vertebrate origins. Science. 298:2157–2167.
Dixson, J. D., M. J. Forstner, and D. M. Garcia. 2003. The a-actinin
gene family: A revised classification. J. Mol. Evol. 56:1–10.
Djinovic-Carugo, K., M. Gautel, J. Ylanne, and P. Young. 2002.
The spectrin repeat: a structural platform for cytoskeletal
protein assemblies. FEBS Lett. 513:119–123.
Djinovic-Carugo, K., P. Young, M. Gautel, and M. Saraste. 1999.
Structure of the a-actinin rod: molecular basis for crosslinking of actin filaments. Cell. 98:537–546.
Dubreuil, R. R. 1991. Structure and evolution of the actin
crosslinking proteins. Bioessays. 13:219–226.
Durand, D. 2003. Vertebrate evolution: doubling and shuffling
with a full deck. Trends Genet. 19:2–5.
Felsenstein, J. 1989. PHYLIP (phylogeny inference package)
Version 3.2. Caldistics. 5:164–166.
Foster, P. G., and D. A. Hickey. 1999. Compositional bias may
affect both DNA-based and protein-based phylogenetic
reconstructions. J. Mol. Evol. 48:284–290.
Fyrberg, E., M. Kelly, E. Ball, C. Fyrberg, and M. C. Reedy.
1990. Molecular genetics of Drosophila alpha-actinin: mutant
alleles disrupt Z disc integrity and muscle insertions. J. Cell
Biol. 110:1999–2011.
Gee, H. 2002. Genomics: return of a little squirt. Nature.
420:755–756.
Germot, A., H. Philippe, and H. Le Guyader. 1997. Evidence for
loss of mitochondria in Microsporidia from a mitochondrialtype HSP70 in Nosema locustae. Mol. Biochem. Parasitol.
87:159–168.
Hirt, R. P., J. M. Logsdon, Jr., B. Healy, M. W. Dorey, W. F.
Doolittle, and T. M. Embley. 1999. Microsporidia are related
to Fungi: evidence from the largest subunit of RNA
polymerase II and other proteins. Proc. Natl. Acad. Sci.
USA 96:580–585.
Huelsenbeck, J. P., and F. Ronquist. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:
754–755.
Hughes, A. L. 1999. Phylogenies of developmentally important
proteins do not support the hypothesis of two rounds of
genome duplication early in vertebrate history. J. Mol. Evol.
48:565–576.
Janssen, K. P., L. Eichinger, P. A. Janmey, A. A. Noegel, M.
Schliwa, W. Witke, and M. Schleicher. 1996. Viscoelastic
properties of F-actin solutions in the presence of normal and
mutated actin-binding proteins. Arch. Biochem. Biophys.
325:183–189.
Keeling, P. J. 2003. Congruent evidence from alpha-tubulin and
beta-tubulin gene phylogenies for a zygomycete origin of
microsporidia. Fungal. Genet. Biol. 38:298–309.
Keeling, P. J., and N. M. Fast. 2002. Microsporidia: biology and
evolution of highly reduced intracellular parasites. Annu. Rev.
Microbiol. 56:93–116.
Keeling, P. J., M. A. Luker, and J. D. Palmer. 2000. Evidence
from beta-tubulin phylogeny that microsporidia evolved from
within the fungi. Mol. Biol. Evol. 17:23–31.
Molecular Evolution of a-Actinin 1031
Kumar, S., K. Tamura, I. B. Jakobsen, and M. Nei. 2001.
MEGA2: molecular evolutionary genetics analysis software.
Bioinformatics. 17:1244–1245.
Leipe, D. D., J. H. Gunderson, T. A. Nerad, and M. L. Sogin.
1993. Small subunit ribosomal RNA1 of Hexamita inflata
and the quest for the first branch in the eukaryotic tree. Mol.
Biochem. Parasitol. 59:41–48.
Mills, M., N. Yang, R. Weinberger, D. L. Vander Woude, A. H.
Beggs, S. Easteal, and K. North. 2001. Differential expression
of the actin-binding proteins, alpha-actinin-2 and -3, in
different species: implications for the evolution of functional
redundancy. Hum. Mol. Genet. 10:1335–1346.
Nickel, R., T. Jacobs, B. Urban, H. Scholze, H. Bruhn, and M.
Leippe. 2000. Two novel calcium-binding proteins from
cytoplasmic granules of the protozoan parasite Entamoeba
histolytica. FEBS Lett. 486:112–116.
Norwood, F. L. M., A. J. Sutherland-Smith, N. H. Keep, and
J. Kendrick-Jones. 2000. The structure of the N-terminal actinbinding domain of human dystrophin and how mutations in
this domain may cause Duchenne or Becker muscular
dystrophy. Struct. Fold Des. 8:481–491.
Pascual, J., J. Castresana, and M. Saraste. 1997. Evolution of the
spectrin repeat. BioEssays. 19:811–817.
Pennisi, E. 2002. Comparative genomics: tunicate genome shows
a little backbone. Science. 298:2111–2112.
Peyretaillade, E., V. Broussolle, P. Peyret, G. Metenier,
M. Gouy, and C.P. Vivares. 1998. Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of
mitochondrial evolutionary origin. Mol. Biol. Evol. 15:
683–689.
Roger, A. J., and J. D. Silberman. 2002. Cell evolution:
mitochondria in hiding. Nature. 418:827–829.
Satou, Y., L. Yamada, Y. Mochizuki et al. (14 co-authors) 2002.
A cDNA resource from the basal chordate Ciona intestinalis.
Genesis. 33:153–154.
Tang, J., D. W. Taylor, and K. A. Taylor. 2001. The threedimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca21-dependent actin
binding. J. Mol. Biol. 310:845–858.
Thomas, G. H., E. C. Newbern, C. C. Korte, M. A. Bales, S. V.
Muse, A. G. Clark, and D. P. Kiehart. 1997. Intragenic
duplication and divergence in the spectrin superfamily of
proteins. Mol. Biol. Evol. 14:1285–1295.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin,
and D. G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment
aided by quality analysis tools. Nucleic Acids Res. 25:
4876–4882.
Viel, A. 1999. a-Actinin and spectrin structures: an unfolding
family story. FEBS Lett. 460:391–394.
Weiss, L. M. 2001. Microsporidia: emerging pathogenic protists.
Acta Trop. 78:89–102.
Williams, B. A., R. P. Hirt, J. M. Lucocq, and T. M. Embley.
2002. A mitochondrial remnant in the microsporidian
Trachipleistophora hominis. Nature. 418:865–869.
Witke, W., A. Hofmann, B. Koeppel, M. Schleicher, and A. A.
Noegel. 1993. The Ca21-binding domains in non-muscle type
a-actinin: biochemical and genetic analysis. J. Cell Biol.
121:599–606.
Wu, J. Q., J. Bahler, and J. R. Pringle. 2001. Roles of a fimbrin
and an a-actinin-like protein in fission yeast cell polarization
and cytokinesis. Mol. Biol. Cell. 12:1061–1077.
Ylanne, J., K. Scheffzek, P. Young, and M. Saraste. 2001.
Crystal structure of the alpha-actinin rod reveals an extensive
torsional twist. Structure (Camb). 9:597–604.
Michele Vendruscolo, Associate Editor
Accepted January 5, 2004