- Email: [email protected]
A lasp family protein of Ciona intestinalis
Available online at www.sciencedirect.com
Biochimica et Biophysica Acta 1779 (2008) 51 – 59 www.elsevier.com/locate/bbagrm
A lasp family protein of Ciona intestinalis Asako G. Terasaki a,⁎, Jin Hiruta a , Junko Suzuki a , Sachiko Sakamoto a , Tatsuji Nishioka b , Hiroshi Suzuki a , Kazuyo Ohashi b , Kaoru Azumi c , Michio Ogasawara b a
Graduate School of Science and Technology, Chiba University, Chiba 263-8522, Japan Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan Division of Innovative Research, Creative Research Initiative “Sousei”, Hokkaido University, Sapporo 001-0021, Japan b
c
Received 3 April 2007; received in revised form 23 August 2007; accepted 25 August 2007 Available online 3 December 2007
Abstract Lasp-1 and lasp-2 are actin-binding proteins that contain a LIM domain, nebulin repeats, and an SH3 domain and they are significantly conserved in mammalian and avian. Lasp-1 is widely expressed in nonmuscle tissues and lasp-2 is specifically expressed in the brain. Genes encoding proteins homologous to lasp-1 and lasp-2 were deposited in the genome/cDNA database of invertebrates such as sea urchins, nematodes, and insects; however, function of their proteins have not been studied in detail. In this study, we analyzed the gene structure, actin-binding activity, and expression of the lasp protein of the ascidian Ciona intestinalis (Ci lasp). A single gene encoding lasp protein was found in the ascidian, and the amino acid sequences of Ci lasp and other invertebrate lasp proteins exhibited similarity to vertebrate lasp-1 and lasp-2 to the same extent. A part of the exon–intron boundaries was conserved between the vertebrate lasp-1, the vertebrate lasp-2 and the invertebrate lasp genes. Ci lasp exhibited actin-binding activity in a co-sedimentation assay. In situ hybridization revealed that the expression of Ci lasp mRNA was apparent in nervous system of early embryos and was detected in various tissues in young adults. This suggests that the functions of invertebrate lasp proteins might include the functions of vertebrate lasp-1 and lasp-2. © 2007 Elsevier B.V. All rights reserved. Keywords: Ascidian; Lasp; Actin-binding; LIM domain; Nebulin repeat; SH3 domain
1. Introduction Lasp-1 (LIM and SH3 protein 1) is a 38 kDa actin-binding protein which was identified as a gene product (MLN50) amplified in human breast carcinoma [1]. Lasp-1 contains an Nterminal LIM domain and a C-terminal SH3 domain, each of which has the potential to interact with multiple binding partners [2]. Additionally, lasp-1 has two nebulin repeats that are thought to be responsible for its actin-binding activity [3]. Lasp-1 is expressed in various nonmuscle tissues and localizes in actin-rich subcellular regions such as focal complexes, cell– cell contacts, leading edges, and tips of lamellipodia, and the localization varies according to the cell types [3–6]. Lasp-1 is localized to chromosome 17q12 in humans [7] and to a pair of microchromosomes in chickens [8].
⁎ Corresponding author. Tel./fax: +81 43 290 3945. E-mail address: [email protected] (A.G. Terasaki). 1874-9399/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2007.08.001
We identified lasp-2 as a 34 kDa binding protein of F-actin affinity chromatography of chicken brain [9]. It was subsequently identified as a zyxin-binding protein [10]. The domain structure of lasp-2 is similar to lasp-1, but they are located on different chromosomes in humans and chickens [8,11]. In contrast to lasp-1, lasp-2 was highly expressed in chicken brain. The actin-binding activity and localization of green fluorescent protein (GFP)–lasp-2 in filopodia suggest that they play roles in cell morphology and movement via actin cytoskeleton [9]. Mammalian and avian species possess lasp-1 and lasp-2 genes and their chromosomal localization showed conserved linkage homology [8]; however, the mechanism/s by which they shared their functions during evolution is/are unknown. Analyzing whether various organisms have both lasp-1 and lasp-2, or other homologous proteins will be a key to understanding the functional differences in these proteins. In invertebrates, genes encoding the lasp family proteins of sea urchins (Strongylocentrotus purpuratus), nematodes (Caenorhabditis elegans), and fruit flies (Drosophila melanogaster)
52
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
53
homologous to both lasp-1 and lasp-2 have been deposited in current genome/cDNA databases. Their activity and localization have not been well investigated. A lasp mutant was reported exclusively in Drosophila where the oskar mRNA is absent in the posterior pole in mutant oocytes (http://flybase.bio.indiana.edu/). Here, we investigated the lasp family protein of ascidian, Ciona intestinalis because it is considered as a model animal of primitive chordates [12]. A draft genome sequence of C. intestinalis revealed that the genome size was approximately 160 Mb, and approximately 15,800 genes were predicted from the sequence [13]. C. intestinalis has a large integrated genome and cDNA database (“Ghost database”: http://ghost.zool.kyoto-u. ac.jp/), and expressed sequence tag (EST) clones were assembled into more than 20,000 independent clusters in the database [14]; thus, almost all the genes exist in the database. Whole-mount in situ hybridization (WISH) analysis has revealed the function of the genes in embryogenesis and morphogenesis. Ci lasp was identified as a single gene and the gene structure and expression pattern suggested that invertebrate lasp proteins might be the ancestral proteins of vertebrate lasp-1 and lasp-2. The actin-binding activity of Ci lasp suggested that it regulated developmental processes via interaction with the actin cytoskeleton.
numbers: CD337105, BG780452, and DN791585) that were obtained from NCBI sea urchin genome resources (http://www.ncbi.nlm.nih.gov/genome/ guide/sea_urchin/), by using amino acid sequences of chicken lasp-1 and lasp-2 as query sequences in BLAST searches. The mRNA sequence of ascidian (C. intestinalis) lasp was obtained from the Institute for Genome Research (http://compbio.dfci.harvard.edu/tgi/) and Ghost database (http://ghost.zool.kyoto-u.ac.jp) by using amino acid sequences of chicken lasp-1 and lasp-2 as query sequences in BLAST searches. Genomic sequences of the lasp genes in humans, fishes, sea urchins, ascidians, and nematodes were obtained from the Ensemble Genome Browser (http://www.ensembl.org/index.html). These sequences were inspected manually to analyze whether exon–intron boundaries were consistent with the multiple alignment. Multiple sequence files were assembled in FASTA formats for further analyses. Multiple sequence alignment of the amino acid sequences of lasp proteins was performed using Clustal X [15] and re-aligned manually using SeAl (Rambaut, A; http://evolve.zoo.ox.ac.uk/). Multiple alignments in GCGMSF format were used to prepare alignments for display with MacBoxShade (Baron, MD; http://www.isrec.isb-sib.ch/ftp-server/boxshade/MacBoxshade/). The data set of the LIM domain and nebulin repeats represented by Clustal X was analyzed by maximum parsimony analysis with bootstrap analysis consisted of 1000 replicates and displayed with TreeView (http://taxonomy.zoology.gla. ac.uk/rod/treeview.html). Domain searches of chicken lasp-1 and lasp-2 were performed using both the NCBI Conserved Domain Search (http://www.ncbi.nlm.nih.gov/Structure/ cdd/wrpsb.cgi) and Motif Scan (http://hits.isb-sib.ch/) as previously described [9]. Predictions for serine, threonine, and tyrosine phosphorylation sites were performed using NetPhos (http://www.cbs.dtu.dk/services/NetPhos/).
2. Materials and methods
2.3. Cloning and expression of Ci lasp
2.1. Biological materials
Full-length cDNA clones of C. intestinalis were obtained by PCR of the EST clone cieg029d09 (GenBank accession no. AV873594). The full-length of Ci lasp was amplified using forward primer (Ci lasp/5′BamHI: 5′-CGCGGATCCAACATGAATCCC-3′) and reverse primer (Ci lasp/3′EcoRI: 5′-CCGGAATTCTGTTAAACTTTCAGTACATAG-3′) with BamHI and EcoRI sites and KOD-Plus polymerase (TOYOBO, Tokyo, Japan). The resulting PCR products were cloned into a pBluescript vector and sequenced using an ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA). The clone was subcloned into a pGEX6P vector (GE Healthcare, Waukesha, WI) at BamHI and EcoRI sites and transfected into the E. coli BL21 strain. GST–Ci lasp fusion protein was expressed at 25 °C and purified using glutathione-Sepharose 4B (GE Healthcare).
Living animals of the ascidian C. intestinalis were collected at Port Chiba, Tokyo Bay, near Chiba University, Chiba, Japan. Adults were maintained under constant light to induce oocyte maturation. Eggs and sperm were obtained surgically from the gonoduct. After insemination, fertilized eggs developed into gastrulae, neurulae, and larvae, and then hatched at about 17 h of development. The larvae were allowed to undergo metamorphosis naturally. Juveniles that adhered to trays were cultured for about two weeks with the diatom Chaetoceros gracilis as their food source. They were fed every two days, and the seawater was changed every two days. Adult C. intestinalis survived up to four months.
2.4. Co-sedimentation of Ci lasp with F-actin 2.2. Sequence analysis Sequences of mRNAs coding human lasp-1 (Homo sapiens lasp-1 = Hs lasp1, GenBank accession no. NM_006148), human lasp-2 (Hs lasp-2, NM_213569), chicken lasp-1 (Gallus gallus lasp-1 = Gg lasp-1, BI394039), chicken lasp-2 (Gg lasp-2, AB114207), zebrafish lasp-1 (Danio rerio lasp-1 = Dr lasp-1, BC066574), frog lasp-1 (Xenopus laevis lasp-1 = Xl lasp-1, BC044681), and fruit fly lasp (D. melanogaster lasp = Dm lasp, AJ294538) were obtained from NCBI (http://www.ncbi.nlm.nih.gov). The nematode lasp protein was predicted and annotated as a LIM and SH3 protein (C. elegans lasp = Ce lasp, P34416) from the F42H10.3 region of the genome sequence (L08403). A lasp protein of sea urchins (S. purpuratus laspA = Sp laspA, XM_791992) was deposited in NCBI. The sequence of another sea urchin lasp protein (Sp laspB) was predicted from manually assembled cDNA clones (GenBank accession
Purified GST–Ci lasp was dialyzed against 20 mM Na-PO4 and 0.1 M KCl, pH 7.2. After centrifugation at 100,000 g for 1 h, the supernatants were mixed with G-actin (final concentration: 0.2 mg/ml G-actin, 20 mM Na-PO4, 0.1 M KCl, 5 mM MgCl2, and 0.2 mM ATP, pH 7.2) from rabbit back muscle prepared according to the method of Spudich and Watt [16]. The samples were incubated for 1 h at room temperature and then centrifuged at 100,000 g for 1 h. The supernatants and the pellets were analyzed by SDS-PAGE.
2.5. Sds-page SDS-PAGE was performed according to the method of Laemmli [17] by using 10% acrylamide gels. The gels were stained with Coomassie Brilliant Blue R-250.
Fig. 1. Lasp family proteins of vertebrate and invertebrate. A, Multiple sequence alignment of vertebrate and invertebrate lasp family proteins. Invariant residues are indicated in black and similar residues in light gray. Species: Hs, Homo sapiens (human); Gg, Gallus gallus (chicken); Xl, Xenopus laevis (frog); Dr, Danio rerio (zebrafish); Ci, Ciona intestinalis (sea squirt); Sp, Strongylocentrotus purpuratus (sea urchin); Dm, Drosophila melanogaster (fruit fly); Ce, Caenorhabditis elegans (nematode). The LIM domain, nebulin repeats, and SH3 domain predicted by alignment with human lasp-1 and lasp-2 are shown in boxes. Asterisks indicate phosphorylation sites reported in mammalian lasp-1. Wedges: exon–intron boundaries of Hs lasp-1. Arrowheads: exon–intron boundaries of Ci lasp. B, Phylogenetic tree of lasp family proteins. The data set represented by the Clustal X multiple sequence alignment of the N-terminal half (LIM, the first nebulin repeat, and the second nebulin repeat) of each protein was analyzed by maximum parsimony analysis and displayed as this tree. Bootstrap values are shown for each node. Nematode lasp (Ce lasp) was used as the outgroup in this unweighted tree.
54
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
Fig. 2. Genomic organization of lasp family proteins. A, Genomic organization of Ci lasp. B, Exon–intron boundaries of vertebrate lasp-1, lasp-2, and invertebrate lasp genes. Shaded regions correspond to the LIM domain, nebulin repeats, and SH3 domain. Connecting lines indicate the conserved exon–intron boundaries. Arrowheads indicate the boundary conserved between the vertebrate lasp-1, lasp-2, and Ci lasp genes.
2.6. In situ hybridization Embryonic samples at appropriate developmental stages and two-week-old C. intestinalis juveniles were fixed in 4% paraformaldehyde buffer for WISH as described in Ogasawara et al. [14]. Pieces of the neural complex dissected from a two-month-old adult were also fixed for whole-mount specimens. These specimens were kept at − 20 °C until use. WISH was preformed on the embryos, juveniles and neural complex as described in Ogasawara et al. [18]. After hybridization, specimens of the neural complex were embedded in polyester wax (BDH Chemicals, Dorset, UK) and sectioned at 10-μm intervals for observation at high magnification. The synthesis and purification of antisense digoxigenin (DIG)-labeled RNA probes were performed by the methods described in Ogasawara et al. [19].
To draw an expression profile for each gene during the life cycle, we computed the ratio of expression of each gene at adult stages by defining the expression level of each gene in the fertilized eggs as the reference “1” (0 in
2.7. Microarray analysis Microarray analysis using the C. intestinalis 22 K custom oligo DNA microarray was performed as described in Azumi et al. [20] and Ogasawara et al. [14]. Poly(A)+ RNAs of C. intestinalis specimens including fertilized eggs and 2-, 4-, 8-, 16-, 32-, and 64-cell embryos, and early gastrulae, late gastrulae, early neurulae, embryos at initial tailbud, early tailbud, middle tailbud, late tailbud, larvae, juveniles, 1.5-month-old adults, 2.5-month-old adults, and 4.0-month-old adults were prepared for hybridization. Poly(A)+ RNA of each stage was labeled with either Cy3 (reference sample) or Cy5 (test sample) using an Agilent Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA), and mixed together and hybridized to the oligo DNA microarrays. The microarrays were scanned with a GenePix 4000B DNA Microarray Scanner (Axon Instruments, Foster City, CA). Normalization and data extraction of the signals were performed using GenePix Pro4.0 Microarray Analysis Software (Axon Instruments), Bioconductor (R Foundation for Statistical Computing, Vienna, Austria; http:// www.R-project.org) and GeneSpring Software (Silicon Genetics, Redwood City, CA).
Fig. 3. Actin-binding activity of Ci lasp. G-actin (0.2 mg/ml) was polymerized under physiological conditions in the presence of GST–Ci lasp (molar ratio of actin and GST fusion protein is 5:1). s: supernatant; p: precipitant. Arrow indicates GST–Ci lasp.
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59 log scale). The expression patterns used are comprised of successively fluctuating patterns of the expression levels of each gene, from fertilized eggs to 4.0-month-old adults.
3. Results 3.1. Lasp family proteins of invertebrates mRNA sequences of Hs lasp-1, Hs lasp-2, Gg lasp-1, Gg lasp-2, Dr lasp-1, Xl lasp-1, Dm lasp, Ce lasp, and Sp laspA have been already deposited as LIM and SH3 proteins. The lasp-2 mRNAs of frogs (X. laevis) and fishes (D. rerio) were not deposited in the current databases (data not shown).
55
Additional BLAST searches were performed on the genome and cDNA databases of fishes, frogs, fruit flies, and nematodes by using amino acid sequences of chicken lasp-1 and lasp-2; however, no homologous proteins other than those already deposited in the current databases were detected. Only in the sea urchin EST database, we identified three overlapping clones (GenBank accession numbers: CD337105, BG780452, and DN791585), and manual sequence assembly predicted another lasp gene (Sp laspB). Several EST clones of Ci lasp were identified by a database search, and they were assembled in one cluster (CLSTR00855) in the Ciona cDNA database. According to the sequences, we designed PCR primers and obtained PCR products from a
Fig. 4. Temporal and spatial expression patterns of Ci lasp by whole-mount in situ hybridization. A–D, Distribution of Ci lasp transcripts in (A) fertilized egg, (B) 32cell embryo, (C) 44-cell embryo, and (D) 110-cell embryo, lateral view. Maternal transcripts of Ci lasp were distributed in the vegetal part of these embryos. The animal pole is at the top. E and F, Gastrula. (E) vegetal view, and (F) dorsal view. Zygotic expression of Ci lasp that begins in the neural lineage cells located in the anterior part of the embryos (arrowheads). Anterior is at the top. G and H, Neural plate embryos. (G) lateral view, and (H) dorsal view. Ci lasp was strongly expressed in the anterior part of the neural plate (arrowheads). Anterior is at the left. I and J, Middle tailbud embryos. (I) lateral view, and (J) dorsal view. Expression signals of Ci lasp were localized in some cells of the nerve cord (arrowheads). Anterior is at the left. K, A two-week-old juvenile; lateral view. Strong signals were detected in the neural complex (nc), endostyle (en), and stomach (st). Os, oral siphon; as, astrial siphon. L and M, Transverse section of the neural complex of a two-month-old adult. (L) Expression signals of Ci lasp were detected in both the neural ganglion (nga) and neural gland (ngl). (M) A high magnification image showed Ci lasp expression in some cells of the neural ganglion. Scale bars of A, I, and M are 100 μm, and the bar of K and L is 500 μm.
56
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
cDNA clone (cieg029d09) whose 3′ sequence covered the C-terminal of Ci lasp. The cDNA sequence of cDNA of 711 base pairs encoding 236 amino acids was deposited as GenBank accession no. AB182287. 3.2. Amino acid homology of Ci lasp A sequence alignment of lasp family proteins from vertebrates and invertebrates demonstrates the conservation in the LIM domain, the first and second nebulin repeats, and the SH3 domain (Fig. 1A). The amino acid identity of the LIM domain of Ci lasp to Hs lasp-1 is 74.2% and that of the SH3 domain is 61.4%. The SDXXYK motif of nebulin repeats is found in lasp-1 and lasp-2 in vertebrates (for example, SQVRYK in the first nebulin repeat and SNIKYH in the second nebulin repeat of Hs lasp-1 in Fig. 1A). The motif was weakly conserved in the first and second nebulin repeats of invertebrate lasp proteins (for example, SEVVYR and SNISYQ of Ci lasp). The possible third nebulin repeat predicted for lasp-2 (for example, SDAAYK of Hs lasp-2) [8] was not found in vertebrate lasp-1 and invertebrate lasp proteins. The linker sequence of vertebrate lasp-1 (except Dr lasp-1) and lasp-2, following the second nebulin repeat showed similarity in each group, but the sequences and length of the linker of the invertebrate lasp proteins varied between species. Phosphorylation sites of mammalian lasp-1 have been analyzed by in vitro phosphorylation, MS analysis, and mutant proteins and the sites varied across organisms and methods [5,6,21,22]. In mammalian, three serine sites, one tyrosine site (S61, S99, S146, and Y171 of human lasp-1, indicated by asterisks in Fig. 1) and one mouse-specific threonine site (156T, not present in human lasp-1) have been reported. In amphibian and avian lasp-1, all the serine and tyrosine sites are conserved. In mammalian and avian lasp-2, two serine and one tyrosine phosphorylation sites corresponding to those of lasp-1 have been predicted (S61, S99, and Y184 of Hs lasp-2 and Gg lasp-2); however, they have not been biochemically analyzed. In Ci lasp, the serine in the first phosphorylation site (S61 of Hs lasp-1) was not detected. The serine in the second site (99S of Hs lasp-1) was substituted with threonine (98T), and the tyrosine phosphorylation site of Ci lasp appeared to be conserved (152Y). Phosphorylation site search by NetPhos predicted several possible phosphorylation sites of Ci lasp, including 152Y. To illustrate the phylogenetic relationships among these proteins, the sequences of the LIM domain and two nebulin repeats were selected because these regions were reported to possess actin-binding activity [3]. They were subjected to maximum parsimony analysis, including bootstrap analysis (Fig. 1B) with Ce lasp as the outgroup. The phylogenetic tree indicated that invertebrate lasp proteins including Ci lasp might be ancestral proteins of lasp-1 and lasp-2. 3.3. Gene organization of Ci lasp A search of ensemble genome database suggested that Ci lasp was located in the 6.6 kb region of chromosome 1q and
comprised 11 exons (Fig. 2A). We compared the exon organization of representative available genomic loci (Fig. 2B). The human lasp-1 contains 7 exons in its coding regions, and all the exon–intron boundaries are conserved in chicken lasp-1 (data not shown). The exon organization of the Dr lasp-1 was also revealed to be conserved from amino acid alignment, indicating that the gene organization of lasp-1 in vertebrates has been almost completely conserved. The human lasp-2 also contains 7 exons and all the exon–intron boundaries are conserved between human and chicken lasp-2 genes [8]. We could not obtain sufficient genome data to analyze the exon– intron boundaries of Xl lasp-1 and Dm lasp. The exon–intron boundaries of vertebrate lasp-1 and lasp-2 are almost identical except the boundary between the fifth and sixth exons, which are located in the linker sequence (Fig. 2B). The first, second, and third exon–intron boundaries of vertebrate lasp-1 and lasp-2 (Hs lasp-1, Dr lasp-1, and Hs lasp-2) are conserved in the sea urchin S. purpuratus (Sp laspA and Sp laspB), and the second and third exon boundaries are also conserved in the nematodes C. elegans (Ce lasp). The exon structure of Ci lasp in the LIM domain and nebulin repeats is considerably different from that in the other organisms analyzed in this study; however, the last exon– intron boundary beginning from the SH3 domain is conserved in Ci lasp (Fig. 1A and Fig. 2B). 3.4. Actin-binding activity of recombinant Ci lasp We confirmed the actin-binding activity of Ci lasp in a cosedimentation assay, which is a common analysis method for actin-binding proteins. In the absence of actin, only a small part of GST–Ci lasp precipitated, with most of the protein remaining in the supernatant. When actin was added, most of the GST–Ci lasp co-precipitated with F-actin (Fig. 3). 3.5. Temporal and spatial expression patterns of Ci lasp The distribution of Ci lasp transcripts during the developmental stages of C. intestinalis was revealed by WISH. The transcripts were detected in the vegetal hemisphere of a fertilized egg as maternal transcripts (Fig. 4A). During the early cleavage stages, the signals in the vegetal hemisphere became weaker (Fig. 4B–D). From the gastrula stage onward, the maternal signal was low in intensity. The first zygotic expression of Ci lasp began at the gastrula stage, and strong expression signals of Ci lasp were detected in neural lineage cells located in the anterior part of the embryos (Fig. 4E and F). At the neural plate stage (Fig. 4G and H), Ci lasp expression was strongly detected in the brain, and the nerve cord precursor cells started the expression of Ci lasp. At the middle tailbud stage (Fig. 4I and J) the expression of Ci lasp became rather weak in the brain and strong expression signals were restricted to some cells of the nerve cord. After metamorphosis, twoweek-old C. intestinalis becomes juvenile and developed adult tissues/organs, including endostyle, gill slits, body wall muscle, esophagus, stomach, intestine, heart, neural complex, gonad, and so on. In the juvenile, Ci lasp was ubiquitously expressed in
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
Fig. 5. Temporal expression of Ci lasp analyzed by microarray analysis. The expression levels are relative values based on the fertilized egg as 0 and plotted on a logarithmic scale (base 2). FE, fertilized eggs; 2, 2-cell embryos; 4, 4-cell embryos; 8, 8-cell embryos; 16, 16-cell embryos; 32, 32-cell embryos; 64, 64cell embryos; EG, early gastrulae; LG, late gastrulae; EN, early neurulae; ITB, initial tailbud embryos; ETB, early tailbud embryos; MTB, mid tailbud embryos; LTB, late tailbud embryos; LV, larvae; JN, juveniles; 1.5M, 1.5month-old adults; 2.5M, 2.5-month-old adults; 4.0M, 4.0-month-old adults.
various tissues/organs, and clearly expressed in the neural complex (Fig. 4K). The sectioned specimens of the neural complex dissected from the two-month-old adult revealed that Ci lasp expression was detected in both the neural ganglion and neural gland (Fig. 4L). A high magnification image showed Ci lasp expression in some cells of the neural ganglion (Fig. 4M). The gene expression profile of Ci lasp was also analyzed using the microarray data at each stage of development, including adulthood (Fig. 5). The transcript level of Ci lasp was low during the early stages of embryogenesis, started to increase in early gastrula embryo, and was high during the juvenile and the adulthood. 4. Discussion The amino acid sequence, domain structure, and genome structure of mammalian and avian lasp-1 and lasp-2 proteins are highly conserved in each group [8]. Lasp-2 is thought to play any roles in nerve tissues because it is highly expressed in the brain [9]; however, the different roles of lasp-1 and lasp-2 in cytoskeletal regulation have not been characterized. Recent studies using model organisms have shown that conserved systems regulate cell polarity formation, cell movement, and actin dynamics. Sasakura et al. [23] analyzed genes involved in the pathways that establish cell polarity and cascades regulating actin dynamics in the genome of C. intestinalis, a basal chordate. They revealed that the Ciona genome contains orthologous genes of vertebrate actin-binding proteins such as the Arp2/3 complex, ADF/cofilin, and formins. This suggested that the conserved functions of pathways/ cascades might regulate the development of Ciona. In this study, we analyzed a gene in C. intestinalis that encodes a protein homologous to lasp-1 and lasp-2. Multiple alignment of vertebrate lasp-1, lasp-2, and invertebrate lasp proteins demonstrated that the amino acid sequences of the LIM domain, the first nebulin repeat, and SH3 domain are highly conserved (Fig. 1A). Phylogenetic analysis suggested that Ci lasp formed an independent group that showed homology to lasp-1 and lasp-2 to the same extent (Fig. 1B). The invertebrate
57
organisms analyzed in this study, with the exception of the sea urchin, appeared to have only a single lasp gene; the amino acid sequences of the other lasp proteins (including Sp laspA and Sp laspB) also shared homology with lasp-1 and lasp-2 to the same extent. The bootstrap values are consistent with the conventional phylogeny of these taxa (Fig. 1B). Therefore, it can be concluded that the lasp-1 and lasp-2 genes might have been generated during the evolution from invertebrates to vertebrates. With amino acid sequence and chromosome mapping, gene organization is believed to be a key to understand the molecular evolution of proteins [24]. We found that Ci lasp contains more exons than vertebrate lasp-1, lasp-2, and the other invertebrate lasp analyzed in this study (Fig. 2A and 2B). Additionally, it was notable that the exon–intron boundary located between the linker and the SH3 domain of the molecule is conserved between ascidians and vertebrates, while in nematodes and sea urchin, the exon–intron boundaries located in the LIM domain and nebulin repeats are conserved (Fig. 2B). Evolutionary relationships among invertebrate lasp genes might be revealed from the gene organization of other invertebrates. Based on domain structures similar to lasp-1 and lasp-2 (Fig. 1A), all the invertebrate lasp proteins are expected to have activities similar to lasp-1 and lasp-2. Schreiber et al. [3] revealed that the N-terminal half (LIM domain and two nebulin repeats) of the recombinant lasp-1 had actin-binding activity and suggested that the nebulin repeats are responsible for the interaction with actin. Nebulin repeats were first reported in nebulin, a giant protein (∼ 800 kDa) that is thought to be a “ruler” to specify the lengths of thin filament in striated muscle [25]. These repeats were also found in nebulette [26] and NRAP [27]. Bacterially expressed nebulin repeats of nebulin, nebulette, and N-RAP have actin-binding activities [27–29]. The actin-binding activity of Ci lasp (Fig. 3) supports the idea that the nebulin repeats in invertebrate lasp proteins may be actin-binding sites; however, further discussion on whether all the nebulin repeats in lasp-1, lasp-2, and invertebrate lasp proteins indeed possess actin-binding activity will be needed to resolve the outstanding questions. Zyxin binds to the SH3 domain of lasp-1 and lasp-2, and the interaction might regulate cell adhesions [10]. Dynamin binds to the SH3 domain of lasp-1 and they are thought to cooperate in endocytosis [30]. The linker sequences are considered to be essential for the functions of vertebrate lasp-1 and lasp-2 because the sequences are unique and wellconserved between mammalian and avian species. Similar binding proteins might regulate the function of invertebrate lasp proteins, and the linker sequences that are considerably different from each other might have different functions. Phosphorylation of mammalian lasp-1 has been thought to affect actin-binding activity and localization [5,6,28–31]. Ci lasp has putative phosphorylation sites and the activity of Ci lasp might be regulated by phosphorylation. Ci lasp transcripts were detected in the vegetal hemisphere of fertilized eggs as maternal transcripts (Fig. 4A). Nishikata et al. [32] reported various localization patterns of maternally expressed genes in the fertilized eggs of C. intestinalis. They suggested that the genes would contribute to the establishment
58
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
Fig. 6. Schematic structure of nebulin-related proteins. Molecular mass, domain structure, and tissue expression, and chromosomal assignment of proteins with nebulin repeats. Lasp-2 and nebulette are generated by alternative splicing events from a single gene. Shaded regions correspond to the LIM domain, nebulin repeats, and SH3 domain.
of the primitive body plan, and Ci lasp was expected to play roles in the process. Zygotic expression of Ci lasp at the gastrula stage (Fig. 4E and F) matches the expression profiles of Ciona microarray analysis data (Fig. 5). The strong signals of Ci lasp observed in the neural lineage cells in the gastrula, and the nerve cord at the neural plate stage (Fig. 4E, F, G, and H) are similar to the high lasp-2 expression in the chicken brain [9]. However, the expression of Ci lasp in the neural tissues became rather weak at the middle tailbud stage (Fig. 4I) and Ci lasp was ubiquitously expressed in the various tissues/organs in juveniles (Fig. 4K), similar to the wide lasp-1 expression in chicken tissues [9]. Among the tissues, the expression in the neural complex is comparatively higher (Fig. 4K), and Ci lasp expression was detected in both the neural ganglion and neural gland (Fig. 4L). The function of Ci lasp might include those of vertebrate lasp-1 and lasp-2. Morpholino treatment, which has been used to suppress gene expression in Ciona embryos [33], might reveal the function of Ci lasp in development. It would be interesting to investigate how the lasp-1 and lasp-2 genes were generated and how they shared their functions during evolution. Katoh and Katoh [11] reported that lasp-2 shares exons with nebulette on human chromosome 10 and some part of the gene locus around lasp-2/nebulette is paralogous to that of lasp-1 on chromosome 17 and nebulin on chromosome 2. They indicated the possibility that the human lasp-2/nebulette gene was generated through the homologous recombination of chromosome 17 and chromosome 2. However, it remained to be determined why the lasp-1 and nebulin genes in humans already have similar neighboring genes. The hypothesis that lasp-1, lasp-2, and other nebulin-related proteins might be generated by gene duplication from their ancestral genes could be derived from the phylogenetic analysis in this study. All the vertebrate proteins that have nebulin repeats, such as lasp-1, lasp-2, nebulette, N-RAP, and nebulin, have a LIM domain or an SH3 domain (or both) (Fig. 6). Additionally, the exon–intron structures of the nebulin repeats of these proteins are conserved [34–36]. All the abovementioned nebulin-related proteins were confirmed to exist in mammalian and avian species, and their chromosomal assignment has been revealed in human [1,26,36]; however, they have not been sufficiently
analyzed in invertebrates. We believe that chromosome mapping, genomic structure, and functional analysis of the lasp family proteins and other nebulin-related proteins in invertebrates will contribute to our understandings of the function and evolution of the proteins. Acknowledgments We thank Dr. Nori Satoh and Dr. Yutaka Satou of the Department of Zoology, Graduate School of Science, Kyoto University, for providing the C. intestinalis cDNA collection. This work was supported in part by the OM award from the Zoological Society of Japan. References [1] C. Tomasetto, C. Regnier, C. Moog-Lutz, M.G. Mattei, M.P. Chenard, R. Lidereau, P. Basset, M.C. Rio, Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11– q21.3 region of chromosome 17, Genomics 28 (1995) 367–376. [2] C. Tomasetto, C. Moog-Lutz, C.H. Regnier, V. Schreiber, P. Basset, M.C. Rio, Lasp-1 (MLN 50) defines a new LIM protein subfamily characterized by the association of LIM and SH3 domains, FEBS Lett. 373 (1995) 245–249. [3] V. Schreiber, C. Moog-Lutz, C.H. Regnier, M.P. Chenard, H. Boeuf, J.L. Vonesch, C. Tomasetto, M.C. Rio, Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions, Mol. Med. 4 (1998) 675–687. [4] C.S. Chew, J.A. Parente, Jr., X. Chen, C. Chaponnier, R.S. Cameron, The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia, J. Cell Sci. 113 (2000) 2035–2045. [5] C.S. Chew, X. Chen, J.A. Parente, Jr., S. Tarrer, C. Okamoto, H.Y. Qin, Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo, J. Cell Sci. 115 (2002) 4787–4799. [6] E. Butt, S. Gambaryan, N. Gottfert, A. Galler, K. Marcus, H.E. Meyer, Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146, J. Biol. Chem. 278 (2003) 15601–15607. [7] V. Schreiber, R. Masson, J.L. Linares, M.G. Mattei, C. Tomasetto, M.C. Rio, Chromosomal assignment and expression pattern of the murine Lasp1 gene, Gene 207 (1998) 171–175. [8] A.G. Terasaki, H. Suzuki, J. Ando, Y. Matsuda, K. Ohashi, Chromosomal assignment of LASP1 and LASP2 genes and organization of the LASP2 gene in chicken, Cytogenet Genome Res 112 (2006) 141–147.
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59 [9] A.G. Terasaki, H. Suzuki, T. Nishioka, E. Matsuzawa, M. Katsuki, H. Nakagawa, S. Miyamoto, K. Ohashi, A novel LIM and SH3 protein (lasp2) highly expressing in chicken brain, Biochem. Biophys. Res. Commun. 313 (2004) 48–54. [10] B. Li, L. Zhuang, B. Trueb, Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1, J. Biol. Chem. 279 (2004) 20401–20410. [11] M. Katoh, M. Katoh, Identification and characterization of LASP2 gene in silico, Int. J. Mol. Med. 12 (2003) 405–410. [12] N. Satoh, Y. Satou, B. Davidson, M. Levine, Ciona intestinalis: an emerging model for whole-genome analyses, Trends Genet. 19 (2003) 376–381. [13] P. Dehal, Y. Satou, R.K. Campbell, et al., The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins, Science 298 (2002) 2079–2270. [14] M. Ogasawara, N. Nakazawa, K. Azumi, E. Yamabe, N. Satoh, M. Satake, Identification of thirty-four transcripts expressed specifically in hemocytes of Ciona intestinalis and their expression profiles throughout the life cycle, DNA Res. 13 (2006) 25–35. [15] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) 4673–4680. [16] J.A. Spudich, S. Watt, The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of interaction of the tropomyosin–troponin complex with actin and proteolytic fragments of myosin, J. Biol. Chem. 246 (1971) 4866–4871. [17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [18] M. Ogasawara, A. Sasaki, H. Metoki, T. Shin-i, Y. Kohara, N. Satoh, Y. Satou, Gene expression profiles in young adult Ciona intestinalis, Dev. Genes Evol. 212 (2002) 173–185. [19] M. Ogasawara, M. Minokawa, Y. Sasakura, H. Nishida, K. Makabe, A large-scale whole-mount in situ hybridization system, Zool. Sci. 18 (2001) 187–193. [20] K. Azumi, M. Fujie, T. Usami, Y. Miki, N. Satoh, A cDNA microarray technique applied for analysis of global gene expression profiles in tributyltin-exposed ascidians, Mar. Environ. Res. 58 (2004) 543–546. [21] C. Keicher, S. Gambaryan, E. Schulze, K. Marcus, H.E. Meyer, E. Butt, Phosphorylation of mouse LASP-1 on threonine 156 by cAMP- and cGMP-dependent protein kinase, Biochem. Biophys. Res. Commun. 324 (2004) 308–316. [22] Y.H. Lin, Z.Y. Park, D. Lin, A.A. Brahmbhatt, M.C. Rio, J.R. Yates, R.L. Klemke, Regulation of cell migration and survival by focal adhesion targeting of Lasp-1, J. Cell Biol. 165 (2004) 421–432. [23] Y. Sasakura, L. Yamada, N. Takatori, Y. Satou, N. Satoh, A genomewide survey of developmentally relevant genes in Ciona intestinalis. VII.
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33] [34]
[35]
[36]
59
Molecules involved in the regulation of cell polarity and actin dynamics, Dev. Genes Evol. 213 (2003) 273–283. M.A. Senetar, R.O. McCann, Gene duplication and functional divergence during evolution of the cytoskeletal linker protein talin, Gene 362 (2005) 141–152. A.S. McElhinny, S.T. Kazmierski, S. Labeit, C.C. Gregorio, Nebulin: the nebulous, multifunctional giant of striated muscle, Trends Cardiovasc. Med. 13 (2003) 195–201. S. Millevoi, K. Trombitas, B. Kolmerer, S. Kostin, J. Schaper, K. Pelin, H. Granzier, S. Labeit, Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs, J. Mol. Biol. 282 (1998) 111–123. G. Luo, E. Leroy, C.A. Kozak, M.H. Polymeropoulos, R. Horowits, Mapping of the gene (NRAP) encoding N-RAP in the mouse and human genomes, Genomics 45 (1997) 229–232. G. Luo, J.Q. Zhang, T.P. Nguyen, A.H. Herrera, B. Paterson, R. Horowits, Complete cDNA sequence and tissue localization of N-RAP, a novel nebulin-related protein of striated muscle, Cell Motil. Cytoskelet. 38 (1997) 75–90. O. Ogut, M.M. Hossain, J.P. Jin, Interactions between nebulin-like motifs and thin filament regulatory proteins, J. Biol. Chem. 278 (2003) 3089–3097. C.T. Okamoto, R. Li, Z. Zhang, Y.Y. Jeng, C.S. Chew, Regulation of protein and vesicle trafficking at the apical membrane of epithelial cells, J. Control. Release 78 (2002) 35–41. C.S. Chew, J.A. Parente, Jr., C. Zhou, E. Baranco, X. Chen, Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell, Am. J. Physiol. 275 (1998) C56–C67. T. Nishikata, L. Yamada, Y. Mochizuki, Y. Satou, T. Shin-i, Y. Kohara, N. Satoh, Profiles of maternally expressed genes in fertilized eggs of Ciona intestinalis, Dev. Biol. 238 (2001) 315–331. Y. Satou, K.S. Imai, N. Satoh, Action of morpholinos in Ciona embryos, Genesis 30 (2001) 103–106. T. Arimura, T. Nakamura, S. Hiroi, M. Satoh, M. Takahashi, N. Ohbuchi, K. Ueda, T. Nouchi, N. Yamaguchi, J. Akai, A. Matsumori, S. Sasayama, A. Kimura, Characterization of the human nebulette gene: a polymorphism in an actin-binding motif is associated with nonfamilial idiopathic dilated cardiomyopathy, Hum. Genet. 107 (2000) 440–451. S.T. Kazmierski, P.B. Antin, C.C. Witt, N. Huebner, A.S. McElhinny, S. Labeit, C.C. Gregorio, The complete mouse nebulin gene sequence and the identification of cardiac nebulin, J. Mol. Biol. 328 (2003) 835–846. S.A. Mohiddin, S. Lu, J.P. Cardoso, S. Carroll, S. Jha, R. Horowits, L. Fananapazir, Genomic organization, alternative splicing, and expression of human and mouse N-RAP, a nebulin-related LIM protein of striated muscle, Cell Motil. Cytoskelet. 55 (2003) 200–212.
Biochimica et Biophysica Acta 1779 (2008) 51 – 59 www.elsevier.com/locate/bbagrm
A lasp family protein of Ciona intestinalis Asako G. Terasaki a,⁎, Jin Hiruta a , Junko Suzuki a , Sachiko Sakamoto a , Tatsuji Nishioka b , Hiroshi Suzuki a , Kazuyo Ohashi b , Kaoru Azumi c , Michio Ogasawara b a
Graduate School of Science and Technology, Chiba University, Chiba 263-8522, Japan Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan Division of Innovative Research, Creative Research Initiative “Sousei”, Hokkaido University, Sapporo 001-0021, Japan b
c
Received 3 April 2007; received in revised form 23 August 2007; accepted 25 August 2007 Available online 3 December 2007
Abstract Lasp-1 and lasp-2 are actin-binding proteins that contain a LIM domain, nebulin repeats, and an SH3 domain and they are significantly conserved in mammalian and avian. Lasp-1 is widely expressed in nonmuscle tissues and lasp-2 is specifically expressed in the brain. Genes encoding proteins homologous to lasp-1 and lasp-2 were deposited in the genome/cDNA database of invertebrates such as sea urchins, nematodes, and insects; however, function of their proteins have not been studied in detail. In this study, we analyzed the gene structure, actin-binding activity, and expression of the lasp protein of the ascidian Ciona intestinalis (Ci lasp). A single gene encoding lasp protein was found in the ascidian, and the amino acid sequences of Ci lasp and other invertebrate lasp proteins exhibited similarity to vertebrate lasp-1 and lasp-2 to the same extent. A part of the exon–intron boundaries was conserved between the vertebrate lasp-1, the vertebrate lasp-2 and the invertebrate lasp genes. Ci lasp exhibited actin-binding activity in a co-sedimentation assay. In situ hybridization revealed that the expression of Ci lasp mRNA was apparent in nervous system of early embryos and was detected in various tissues in young adults. This suggests that the functions of invertebrate lasp proteins might include the functions of vertebrate lasp-1 and lasp-2. © 2007 Elsevier B.V. All rights reserved. Keywords: Ascidian; Lasp; Actin-binding; LIM domain; Nebulin repeat; SH3 domain
1. Introduction Lasp-1 (LIM and SH3 protein 1) is a 38 kDa actin-binding protein which was identified as a gene product (MLN50) amplified in human breast carcinoma [1]. Lasp-1 contains an Nterminal LIM domain and a C-terminal SH3 domain, each of which has the potential to interact with multiple binding partners [2]. Additionally, lasp-1 has two nebulin repeats that are thought to be responsible for its actin-binding activity [3]. Lasp-1 is expressed in various nonmuscle tissues and localizes in actin-rich subcellular regions such as focal complexes, cell– cell contacts, leading edges, and tips of lamellipodia, and the localization varies according to the cell types [3–6]. Lasp-1 is localized to chromosome 17q12 in humans [7] and to a pair of microchromosomes in chickens [8].
⁎ Corresponding author. Tel./fax: +81 43 290 3945. E-mail address: [email protected] (A.G. Terasaki). 1874-9399/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2007.08.001
We identified lasp-2 as a 34 kDa binding protein of F-actin affinity chromatography of chicken brain [9]. It was subsequently identified as a zyxin-binding protein [10]. The domain structure of lasp-2 is similar to lasp-1, but they are located on different chromosomes in humans and chickens [8,11]. In contrast to lasp-1, lasp-2 was highly expressed in chicken brain. The actin-binding activity and localization of green fluorescent protein (GFP)–lasp-2 in filopodia suggest that they play roles in cell morphology and movement via actin cytoskeleton [9]. Mammalian and avian species possess lasp-1 and lasp-2 genes and their chromosomal localization showed conserved linkage homology [8]; however, the mechanism/s by which they shared their functions during evolution is/are unknown. Analyzing whether various organisms have both lasp-1 and lasp-2, or other homologous proteins will be a key to understanding the functional differences in these proteins. In invertebrates, genes encoding the lasp family proteins of sea urchins (Strongylocentrotus purpuratus), nematodes (Caenorhabditis elegans), and fruit flies (Drosophila melanogaster)
52
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
53
homologous to both lasp-1 and lasp-2 have been deposited in current genome/cDNA databases. Their activity and localization have not been well investigated. A lasp mutant was reported exclusively in Drosophila where the oskar mRNA is absent in the posterior pole in mutant oocytes (http://flybase.bio.indiana.edu/). Here, we investigated the lasp family protein of ascidian, Ciona intestinalis because it is considered as a model animal of primitive chordates [12]. A draft genome sequence of C. intestinalis revealed that the genome size was approximately 160 Mb, and approximately 15,800 genes were predicted from the sequence [13]. C. intestinalis has a large integrated genome and cDNA database (“Ghost database”: http://ghost.zool.kyoto-u. ac.jp/), and expressed sequence tag (EST) clones were assembled into more than 20,000 independent clusters in the database [14]; thus, almost all the genes exist in the database. Whole-mount in situ hybridization (WISH) analysis has revealed the function of the genes in embryogenesis and morphogenesis. Ci lasp was identified as a single gene and the gene structure and expression pattern suggested that invertebrate lasp proteins might be the ancestral proteins of vertebrate lasp-1 and lasp-2. The actin-binding activity of Ci lasp suggested that it regulated developmental processes via interaction with the actin cytoskeleton.
numbers: CD337105, BG780452, and DN791585) that were obtained from NCBI sea urchin genome resources (http://www.ncbi.nlm.nih.gov/genome/ guide/sea_urchin/), by using amino acid sequences of chicken lasp-1 and lasp-2 as query sequences in BLAST searches. The mRNA sequence of ascidian (C. intestinalis) lasp was obtained from the Institute for Genome Research (http://compbio.dfci.harvard.edu/tgi/) and Ghost database (http://ghost.zool.kyoto-u.ac.jp) by using amino acid sequences of chicken lasp-1 and lasp-2 as query sequences in BLAST searches. Genomic sequences of the lasp genes in humans, fishes, sea urchins, ascidians, and nematodes were obtained from the Ensemble Genome Browser (http://www.ensembl.org/index.html). These sequences were inspected manually to analyze whether exon–intron boundaries were consistent with the multiple alignment. Multiple sequence files were assembled in FASTA formats for further analyses. Multiple sequence alignment of the amino acid sequences of lasp proteins was performed using Clustal X [15] and re-aligned manually using SeAl (Rambaut, A; http://evolve.zoo.ox.ac.uk/). Multiple alignments in GCGMSF format were used to prepare alignments for display with MacBoxShade (Baron, MD; http://www.isrec.isb-sib.ch/ftp-server/boxshade/MacBoxshade/). The data set of the LIM domain and nebulin repeats represented by Clustal X was analyzed by maximum parsimony analysis with bootstrap analysis consisted of 1000 replicates and displayed with TreeView (http://taxonomy.zoology.gla. ac.uk/rod/treeview.html). Domain searches of chicken lasp-1 and lasp-2 were performed using both the NCBI Conserved Domain Search (http://www.ncbi.nlm.nih.gov/Structure/ cdd/wrpsb.cgi) and Motif Scan (http://hits.isb-sib.ch/) as previously described [9]. Predictions for serine, threonine, and tyrosine phosphorylation sites were performed using NetPhos (http://www.cbs.dtu.dk/services/NetPhos/).
2. Materials and methods
2.3. Cloning and expression of Ci lasp
2.1. Biological materials
Full-length cDNA clones of C. intestinalis were obtained by PCR of the EST clone cieg029d09 (GenBank accession no. AV873594). The full-length of Ci lasp was amplified using forward primer (Ci lasp/5′BamHI: 5′-CGCGGATCCAACATGAATCCC-3′) and reverse primer (Ci lasp/3′EcoRI: 5′-CCGGAATTCTGTTAAACTTTCAGTACATAG-3′) with BamHI and EcoRI sites and KOD-Plus polymerase (TOYOBO, Tokyo, Japan). The resulting PCR products were cloned into a pBluescript vector and sequenced using an ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA). The clone was subcloned into a pGEX6P vector (GE Healthcare, Waukesha, WI) at BamHI and EcoRI sites and transfected into the E. coli BL21 strain. GST–Ci lasp fusion protein was expressed at 25 °C and purified using glutathione-Sepharose 4B (GE Healthcare).
Living animals of the ascidian C. intestinalis were collected at Port Chiba, Tokyo Bay, near Chiba University, Chiba, Japan. Adults were maintained under constant light to induce oocyte maturation. Eggs and sperm were obtained surgically from the gonoduct. After insemination, fertilized eggs developed into gastrulae, neurulae, and larvae, and then hatched at about 17 h of development. The larvae were allowed to undergo metamorphosis naturally. Juveniles that adhered to trays were cultured for about two weeks with the diatom Chaetoceros gracilis as their food source. They were fed every two days, and the seawater was changed every two days. Adult C. intestinalis survived up to four months.
2.4. Co-sedimentation of Ci lasp with F-actin 2.2. Sequence analysis Sequences of mRNAs coding human lasp-1 (Homo sapiens lasp-1 = Hs lasp1, GenBank accession no. NM_006148), human lasp-2 (Hs lasp-2, NM_213569), chicken lasp-1 (Gallus gallus lasp-1 = Gg lasp-1, BI394039), chicken lasp-2 (Gg lasp-2, AB114207), zebrafish lasp-1 (Danio rerio lasp-1 = Dr lasp-1, BC066574), frog lasp-1 (Xenopus laevis lasp-1 = Xl lasp-1, BC044681), and fruit fly lasp (D. melanogaster lasp = Dm lasp, AJ294538) were obtained from NCBI (http://www.ncbi.nlm.nih.gov). The nematode lasp protein was predicted and annotated as a LIM and SH3 protein (C. elegans lasp = Ce lasp, P34416) from the F42H10.3 region of the genome sequence (L08403). A lasp protein of sea urchins (S. purpuratus laspA = Sp laspA, XM_791992) was deposited in NCBI. The sequence of another sea urchin lasp protein (Sp laspB) was predicted from manually assembled cDNA clones (GenBank accession
Purified GST–Ci lasp was dialyzed against 20 mM Na-PO4 and 0.1 M KCl, pH 7.2. After centrifugation at 100,000 g for 1 h, the supernatants were mixed with G-actin (final concentration: 0.2 mg/ml G-actin, 20 mM Na-PO4, 0.1 M KCl, 5 mM MgCl2, and 0.2 mM ATP, pH 7.2) from rabbit back muscle prepared according to the method of Spudich and Watt [16]. The samples were incubated for 1 h at room temperature and then centrifuged at 100,000 g for 1 h. The supernatants and the pellets were analyzed by SDS-PAGE.
2.5. Sds-page SDS-PAGE was performed according to the method of Laemmli [17] by using 10% acrylamide gels. The gels were stained with Coomassie Brilliant Blue R-250.
Fig. 1. Lasp family proteins of vertebrate and invertebrate. A, Multiple sequence alignment of vertebrate and invertebrate lasp family proteins. Invariant residues are indicated in black and similar residues in light gray. Species: Hs, Homo sapiens (human); Gg, Gallus gallus (chicken); Xl, Xenopus laevis (frog); Dr, Danio rerio (zebrafish); Ci, Ciona intestinalis (sea squirt); Sp, Strongylocentrotus purpuratus (sea urchin); Dm, Drosophila melanogaster (fruit fly); Ce, Caenorhabditis elegans (nematode). The LIM domain, nebulin repeats, and SH3 domain predicted by alignment with human lasp-1 and lasp-2 are shown in boxes. Asterisks indicate phosphorylation sites reported in mammalian lasp-1. Wedges: exon–intron boundaries of Hs lasp-1. Arrowheads: exon–intron boundaries of Ci lasp. B, Phylogenetic tree of lasp family proteins. The data set represented by the Clustal X multiple sequence alignment of the N-terminal half (LIM, the first nebulin repeat, and the second nebulin repeat) of each protein was analyzed by maximum parsimony analysis and displayed as this tree. Bootstrap values are shown for each node. Nematode lasp (Ce lasp) was used as the outgroup in this unweighted tree.
54
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
Fig. 2. Genomic organization of lasp family proteins. A, Genomic organization of Ci lasp. B, Exon–intron boundaries of vertebrate lasp-1, lasp-2, and invertebrate lasp genes. Shaded regions correspond to the LIM domain, nebulin repeats, and SH3 domain. Connecting lines indicate the conserved exon–intron boundaries. Arrowheads indicate the boundary conserved between the vertebrate lasp-1, lasp-2, and Ci lasp genes.
2.6. In situ hybridization Embryonic samples at appropriate developmental stages and two-week-old C. intestinalis juveniles were fixed in 4% paraformaldehyde buffer for WISH as described in Ogasawara et al. [14]. Pieces of the neural complex dissected from a two-month-old adult were also fixed for whole-mount specimens. These specimens were kept at − 20 °C until use. WISH was preformed on the embryos, juveniles and neural complex as described in Ogasawara et al. [18]. After hybridization, specimens of the neural complex were embedded in polyester wax (BDH Chemicals, Dorset, UK) and sectioned at 10-μm intervals for observation at high magnification. The synthesis and purification of antisense digoxigenin (DIG)-labeled RNA probes were performed by the methods described in Ogasawara et al. [19].
To draw an expression profile for each gene during the life cycle, we computed the ratio of expression of each gene at adult stages by defining the expression level of each gene in the fertilized eggs as the reference “1” (0 in
2.7. Microarray analysis Microarray analysis using the C. intestinalis 22 K custom oligo DNA microarray was performed as described in Azumi et al. [20] and Ogasawara et al. [14]. Poly(A)+ RNAs of C. intestinalis specimens including fertilized eggs and 2-, 4-, 8-, 16-, 32-, and 64-cell embryos, and early gastrulae, late gastrulae, early neurulae, embryos at initial tailbud, early tailbud, middle tailbud, late tailbud, larvae, juveniles, 1.5-month-old adults, 2.5-month-old adults, and 4.0-month-old adults were prepared for hybridization. Poly(A)+ RNA of each stage was labeled with either Cy3 (reference sample) or Cy5 (test sample) using an Agilent Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA), and mixed together and hybridized to the oligo DNA microarrays. The microarrays were scanned with a GenePix 4000B DNA Microarray Scanner (Axon Instruments, Foster City, CA). Normalization and data extraction of the signals were performed using GenePix Pro4.0 Microarray Analysis Software (Axon Instruments), Bioconductor (R Foundation for Statistical Computing, Vienna, Austria; http:// www.R-project.org) and GeneSpring Software (Silicon Genetics, Redwood City, CA).
Fig. 3. Actin-binding activity of Ci lasp. G-actin (0.2 mg/ml) was polymerized under physiological conditions in the presence of GST–Ci lasp (molar ratio of actin and GST fusion protein is 5:1). s: supernatant; p: precipitant. Arrow indicates GST–Ci lasp.
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59 log scale). The expression patterns used are comprised of successively fluctuating patterns of the expression levels of each gene, from fertilized eggs to 4.0-month-old adults.
3. Results 3.1. Lasp family proteins of invertebrates mRNA sequences of Hs lasp-1, Hs lasp-2, Gg lasp-1, Gg lasp-2, Dr lasp-1, Xl lasp-1, Dm lasp, Ce lasp, and Sp laspA have been already deposited as LIM and SH3 proteins. The lasp-2 mRNAs of frogs (X. laevis) and fishes (D. rerio) were not deposited in the current databases (data not shown).
55
Additional BLAST searches were performed on the genome and cDNA databases of fishes, frogs, fruit flies, and nematodes by using amino acid sequences of chicken lasp-1 and lasp-2; however, no homologous proteins other than those already deposited in the current databases were detected. Only in the sea urchin EST database, we identified three overlapping clones (GenBank accession numbers: CD337105, BG780452, and DN791585), and manual sequence assembly predicted another lasp gene (Sp laspB). Several EST clones of Ci lasp were identified by a database search, and they were assembled in one cluster (CLSTR00855) in the Ciona cDNA database. According to the sequences, we designed PCR primers and obtained PCR products from a
Fig. 4. Temporal and spatial expression patterns of Ci lasp by whole-mount in situ hybridization. A–D, Distribution of Ci lasp transcripts in (A) fertilized egg, (B) 32cell embryo, (C) 44-cell embryo, and (D) 110-cell embryo, lateral view. Maternal transcripts of Ci lasp were distributed in the vegetal part of these embryos. The animal pole is at the top. E and F, Gastrula. (E) vegetal view, and (F) dorsal view. Zygotic expression of Ci lasp that begins in the neural lineage cells located in the anterior part of the embryos (arrowheads). Anterior is at the top. G and H, Neural plate embryos. (G) lateral view, and (H) dorsal view. Ci lasp was strongly expressed in the anterior part of the neural plate (arrowheads). Anterior is at the left. I and J, Middle tailbud embryos. (I) lateral view, and (J) dorsal view. Expression signals of Ci lasp were localized in some cells of the nerve cord (arrowheads). Anterior is at the left. K, A two-week-old juvenile; lateral view. Strong signals were detected in the neural complex (nc), endostyle (en), and stomach (st). Os, oral siphon; as, astrial siphon. L and M, Transverse section of the neural complex of a two-month-old adult. (L) Expression signals of Ci lasp were detected in both the neural ganglion (nga) and neural gland (ngl). (M) A high magnification image showed Ci lasp expression in some cells of the neural ganglion. Scale bars of A, I, and M are 100 μm, and the bar of K and L is 500 μm.
56
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
cDNA clone (cieg029d09) whose 3′ sequence covered the C-terminal of Ci lasp. The cDNA sequence of cDNA of 711 base pairs encoding 236 amino acids was deposited as GenBank accession no. AB182287. 3.2. Amino acid homology of Ci lasp A sequence alignment of lasp family proteins from vertebrates and invertebrates demonstrates the conservation in the LIM domain, the first and second nebulin repeats, and the SH3 domain (Fig. 1A). The amino acid identity of the LIM domain of Ci lasp to Hs lasp-1 is 74.2% and that of the SH3 domain is 61.4%. The SDXXYK motif of nebulin repeats is found in lasp-1 and lasp-2 in vertebrates (for example, SQVRYK in the first nebulin repeat and SNIKYH in the second nebulin repeat of Hs lasp-1 in Fig. 1A). The motif was weakly conserved in the first and second nebulin repeats of invertebrate lasp proteins (for example, SEVVYR and SNISYQ of Ci lasp). The possible third nebulin repeat predicted for lasp-2 (for example, SDAAYK of Hs lasp-2) [8] was not found in vertebrate lasp-1 and invertebrate lasp proteins. The linker sequence of vertebrate lasp-1 (except Dr lasp-1) and lasp-2, following the second nebulin repeat showed similarity in each group, but the sequences and length of the linker of the invertebrate lasp proteins varied between species. Phosphorylation sites of mammalian lasp-1 have been analyzed by in vitro phosphorylation, MS analysis, and mutant proteins and the sites varied across organisms and methods [5,6,21,22]. In mammalian, three serine sites, one tyrosine site (S61, S99, S146, and Y171 of human lasp-1, indicated by asterisks in Fig. 1) and one mouse-specific threonine site (156T, not present in human lasp-1) have been reported. In amphibian and avian lasp-1, all the serine and tyrosine sites are conserved. In mammalian and avian lasp-2, two serine and one tyrosine phosphorylation sites corresponding to those of lasp-1 have been predicted (S61, S99, and Y184 of Hs lasp-2 and Gg lasp-2); however, they have not been biochemically analyzed. In Ci lasp, the serine in the first phosphorylation site (S61 of Hs lasp-1) was not detected. The serine in the second site (99S of Hs lasp-1) was substituted with threonine (98T), and the tyrosine phosphorylation site of Ci lasp appeared to be conserved (152Y). Phosphorylation site search by NetPhos predicted several possible phosphorylation sites of Ci lasp, including 152Y. To illustrate the phylogenetic relationships among these proteins, the sequences of the LIM domain and two nebulin repeats were selected because these regions were reported to possess actin-binding activity [3]. They were subjected to maximum parsimony analysis, including bootstrap analysis (Fig. 1B) with Ce lasp as the outgroup. The phylogenetic tree indicated that invertebrate lasp proteins including Ci lasp might be ancestral proteins of lasp-1 and lasp-2. 3.3. Gene organization of Ci lasp A search of ensemble genome database suggested that Ci lasp was located in the 6.6 kb region of chromosome 1q and
comprised 11 exons (Fig. 2A). We compared the exon organization of representative available genomic loci (Fig. 2B). The human lasp-1 contains 7 exons in its coding regions, and all the exon–intron boundaries are conserved in chicken lasp-1 (data not shown). The exon organization of the Dr lasp-1 was also revealed to be conserved from amino acid alignment, indicating that the gene organization of lasp-1 in vertebrates has been almost completely conserved. The human lasp-2 also contains 7 exons and all the exon–intron boundaries are conserved between human and chicken lasp-2 genes [8]. We could not obtain sufficient genome data to analyze the exon– intron boundaries of Xl lasp-1 and Dm lasp. The exon–intron boundaries of vertebrate lasp-1 and lasp-2 are almost identical except the boundary between the fifth and sixth exons, which are located in the linker sequence (Fig. 2B). The first, second, and third exon–intron boundaries of vertebrate lasp-1 and lasp-2 (Hs lasp-1, Dr lasp-1, and Hs lasp-2) are conserved in the sea urchin S. purpuratus (Sp laspA and Sp laspB), and the second and third exon boundaries are also conserved in the nematodes C. elegans (Ce lasp). The exon structure of Ci lasp in the LIM domain and nebulin repeats is considerably different from that in the other organisms analyzed in this study; however, the last exon– intron boundary beginning from the SH3 domain is conserved in Ci lasp (Fig. 1A and Fig. 2B). 3.4. Actin-binding activity of recombinant Ci lasp We confirmed the actin-binding activity of Ci lasp in a cosedimentation assay, which is a common analysis method for actin-binding proteins. In the absence of actin, only a small part of GST–Ci lasp precipitated, with most of the protein remaining in the supernatant. When actin was added, most of the GST–Ci lasp co-precipitated with F-actin (Fig. 3). 3.5. Temporal and spatial expression patterns of Ci lasp The distribution of Ci lasp transcripts during the developmental stages of C. intestinalis was revealed by WISH. The transcripts were detected in the vegetal hemisphere of a fertilized egg as maternal transcripts (Fig. 4A). During the early cleavage stages, the signals in the vegetal hemisphere became weaker (Fig. 4B–D). From the gastrula stage onward, the maternal signal was low in intensity. The first zygotic expression of Ci lasp began at the gastrula stage, and strong expression signals of Ci lasp were detected in neural lineage cells located in the anterior part of the embryos (Fig. 4E and F). At the neural plate stage (Fig. 4G and H), Ci lasp expression was strongly detected in the brain, and the nerve cord precursor cells started the expression of Ci lasp. At the middle tailbud stage (Fig. 4I and J) the expression of Ci lasp became rather weak in the brain and strong expression signals were restricted to some cells of the nerve cord. After metamorphosis, twoweek-old C. intestinalis becomes juvenile and developed adult tissues/organs, including endostyle, gill slits, body wall muscle, esophagus, stomach, intestine, heart, neural complex, gonad, and so on. In the juvenile, Ci lasp was ubiquitously expressed in
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
Fig. 5. Temporal expression of Ci lasp analyzed by microarray analysis. The expression levels are relative values based on the fertilized egg as 0 and plotted on a logarithmic scale (base 2). FE, fertilized eggs; 2, 2-cell embryos; 4, 4-cell embryos; 8, 8-cell embryos; 16, 16-cell embryos; 32, 32-cell embryos; 64, 64cell embryos; EG, early gastrulae; LG, late gastrulae; EN, early neurulae; ITB, initial tailbud embryos; ETB, early tailbud embryos; MTB, mid tailbud embryos; LTB, late tailbud embryos; LV, larvae; JN, juveniles; 1.5M, 1.5month-old adults; 2.5M, 2.5-month-old adults; 4.0M, 4.0-month-old adults.
various tissues/organs, and clearly expressed in the neural complex (Fig. 4K). The sectioned specimens of the neural complex dissected from the two-month-old adult revealed that Ci lasp expression was detected in both the neural ganglion and neural gland (Fig. 4L). A high magnification image showed Ci lasp expression in some cells of the neural ganglion (Fig. 4M). The gene expression profile of Ci lasp was also analyzed using the microarray data at each stage of development, including adulthood (Fig. 5). The transcript level of Ci lasp was low during the early stages of embryogenesis, started to increase in early gastrula embryo, and was high during the juvenile and the adulthood. 4. Discussion The amino acid sequence, domain structure, and genome structure of mammalian and avian lasp-1 and lasp-2 proteins are highly conserved in each group [8]. Lasp-2 is thought to play any roles in nerve tissues because it is highly expressed in the brain [9]; however, the different roles of lasp-1 and lasp-2 in cytoskeletal regulation have not been characterized. Recent studies using model organisms have shown that conserved systems regulate cell polarity formation, cell movement, and actin dynamics. Sasakura et al. [23] analyzed genes involved in the pathways that establish cell polarity and cascades regulating actin dynamics in the genome of C. intestinalis, a basal chordate. They revealed that the Ciona genome contains orthologous genes of vertebrate actin-binding proteins such as the Arp2/3 complex, ADF/cofilin, and formins. This suggested that the conserved functions of pathways/ cascades might regulate the development of Ciona. In this study, we analyzed a gene in C. intestinalis that encodes a protein homologous to lasp-1 and lasp-2. Multiple alignment of vertebrate lasp-1, lasp-2, and invertebrate lasp proteins demonstrated that the amino acid sequences of the LIM domain, the first nebulin repeat, and SH3 domain are highly conserved (Fig. 1A). Phylogenetic analysis suggested that Ci lasp formed an independent group that showed homology to lasp-1 and lasp-2 to the same extent (Fig. 1B). The invertebrate
57
organisms analyzed in this study, with the exception of the sea urchin, appeared to have only a single lasp gene; the amino acid sequences of the other lasp proteins (including Sp laspA and Sp laspB) also shared homology with lasp-1 and lasp-2 to the same extent. The bootstrap values are consistent with the conventional phylogeny of these taxa (Fig. 1B). Therefore, it can be concluded that the lasp-1 and lasp-2 genes might have been generated during the evolution from invertebrates to vertebrates. With amino acid sequence and chromosome mapping, gene organization is believed to be a key to understand the molecular evolution of proteins [24]. We found that Ci lasp contains more exons than vertebrate lasp-1, lasp-2, and the other invertebrate lasp analyzed in this study (Fig. 2A and 2B). Additionally, it was notable that the exon–intron boundary located between the linker and the SH3 domain of the molecule is conserved between ascidians and vertebrates, while in nematodes and sea urchin, the exon–intron boundaries located in the LIM domain and nebulin repeats are conserved (Fig. 2B). Evolutionary relationships among invertebrate lasp genes might be revealed from the gene organization of other invertebrates. Based on domain structures similar to lasp-1 and lasp-2 (Fig. 1A), all the invertebrate lasp proteins are expected to have activities similar to lasp-1 and lasp-2. Schreiber et al. [3] revealed that the N-terminal half (LIM domain and two nebulin repeats) of the recombinant lasp-1 had actin-binding activity and suggested that the nebulin repeats are responsible for the interaction with actin. Nebulin repeats were first reported in nebulin, a giant protein (∼ 800 kDa) that is thought to be a “ruler” to specify the lengths of thin filament in striated muscle [25]. These repeats were also found in nebulette [26] and NRAP [27]. Bacterially expressed nebulin repeats of nebulin, nebulette, and N-RAP have actin-binding activities [27–29]. The actin-binding activity of Ci lasp (Fig. 3) supports the idea that the nebulin repeats in invertebrate lasp proteins may be actin-binding sites; however, further discussion on whether all the nebulin repeats in lasp-1, lasp-2, and invertebrate lasp proteins indeed possess actin-binding activity will be needed to resolve the outstanding questions. Zyxin binds to the SH3 domain of lasp-1 and lasp-2, and the interaction might regulate cell adhesions [10]. Dynamin binds to the SH3 domain of lasp-1 and they are thought to cooperate in endocytosis [30]. The linker sequences are considered to be essential for the functions of vertebrate lasp-1 and lasp-2 because the sequences are unique and wellconserved between mammalian and avian species. Similar binding proteins might regulate the function of invertebrate lasp proteins, and the linker sequences that are considerably different from each other might have different functions. Phosphorylation of mammalian lasp-1 has been thought to affect actin-binding activity and localization [5,6,28–31]. Ci lasp has putative phosphorylation sites and the activity of Ci lasp might be regulated by phosphorylation. Ci lasp transcripts were detected in the vegetal hemisphere of fertilized eggs as maternal transcripts (Fig. 4A). Nishikata et al. [32] reported various localization patterns of maternally expressed genes in the fertilized eggs of C. intestinalis. They suggested that the genes would contribute to the establishment
58
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59
Fig. 6. Schematic structure of nebulin-related proteins. Molecular mass, domain structure, and tissue expression, and chromosomal assignment of proteins with nebulin repeats. Lasp-2 and nebulette are generated by alternative splicing events from a single gene. Shaded regions correspond to the LIM domain, nebulin repeats, and SH3 domain.
of the primitive body plan, and Ci lasp was expected to play roles in the process. Zygotic expression of Ci lasp at the gastrula stage (Fig. 4E and F) matches the expression profiles of Ciona microarray analysis data (Fig. 5). The strong signals of Ci lasp observed in the neural lineage cells in the gastrula, and the nerve cord at the neural plate stage (Fig. 4E, F, G, and H) are similar to the high lasp-2 expression in the chicken brain [9]. However, the expression of Ci lasp in the neural tissues became rather weak at the middle tailbud stage (Fig. 4I) and Ci lasp was ubiquitously expressed in the various tissues/organs in juveniles (Fig. 4K), similar to the wide lasp-1 expression in chicken tissues [9]. Among the tissues, the expression in the neural complex is comparatively higher (Fig. 4K), and Ci lasp expression was detected in both the neural ganglion and neural gland (Fig. 4L). The function of Ci lasp might include those of vertebrate lasp-1 and lasp-2. Morpholino treatment, which has been used to suppress gene expression in Ciona embryos [33], might reveal the function of Ci lasp in development. It would be interesting to investigate how the lasp-1 and lasp-2 genes were generated and how they shared their functions during evolution. Katoh and Katoh [11] reported that lasp-2 shares exons with nebulette on human chromosome 10 and some part of the gene locus around lasp-2/nebulette is paralogous to that of lasp-1 on chromosome 17 and nebulin on chromosome 2. They indicated the possibility that the human lasp-2/nebulette gene was generated through the homologous recombination of chromosome 17 and chromosome 2. However, it remained to be determined why the lasp-1 and nebulin genes in humans already have similar neighboring genes. The hypothesis that lasp-1, lasp-2, and other nebulin-related proteins might be generated by gene duplication from their ancestral genes could be derived from the phylogenetic analysis in this study. All the vertebrate proteins that have nebulin repeats, such as lasp-1, lasp-2, nebulette, N-RAP, and nebulin, have a LIM domain or an SH3 domain (or both) (Fig. 6). Additionally, the exon–intron structures of the nebulin repeats of these proteins are conserved [34–36]. All the abovementioned nebulin-related proteins were confirmed to exist in mammalian and avian species, and their chromosomal assignment has been revealed in human [1,26,36]; however, they have not been sufficiently
analyzed in invertebrates. We believe that chromosome mapping, genomic structure, and functional analysis of the lasp family proteins and other nebulin-related proteins in invertebrates will contribute to our understandings of the function and evolution of the proteins. Acknowledgments We thank Dr. Nori Satoh and Dr. Yutaka Satou of the Department of Zoology, Graduate School of Science, Kyoto University, for providing the C. intestinalis cDNA collection. This work was supported in part by the OM award from the Zoological Society of Japan. References [1] C. Tomasetto, C. Regnier, C. Moog-Lutz, M.G. Mattei, M.P. Chenard, R. Lidereau, P. Basset, M.C. Rio, Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11– q21.3 region of chromosome 17, Genomics 28 (1995) 367–376. [2] C. Tomasetto, C. Moog-Lutz, C.H. Regnier, V. Schreiber, P. Basset, M.C. Rio, Lasp-1 (MLN 50) defines a new LIM protein subfamily characterized by the association of LIM and SH3 domains, FEBS Lett. 373 (1995) 245–249. [3] V. Schreiber, C. Moog-Lutz, C.H. Regnier, M.P. Chenard, H. Boeuf, J.L. Vonesch, C. Tomasetto, M.C. Rio, Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions, Mol. Med. 4 (1998) 675–687. [4] C.S. Chew, J.A. Parente, Jr., X. Chen, C. Chaponnier, R.S. Cameron, The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia, J. Cell Sci. 113 (2000) 2035–2045. [5] C.S. Chew, X. Chen, J.A. Parente, Jr., S. Tarrer, C. Okamoto, H.Y. Qin, Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo, J. Cell Sci. 115 (2002) 4787–4799. [6] E. Butt, S. Gambaryan, N. Gottfert, A. Galler, K. Marcus, H.E. Meyer, Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146, J. Biol. Chem. 278 (2003) 15601–15607. [7] V. Schreiber, R. Masson, J.L. Linares, M.G. Mattei, C. Tomasetto, M.C. Rio, Chromosomal assignment and expression pattern of the murine Lasp1 gene, Gene 207 (1998) 171–175. [8] A.G. Terasaki, H. Suzuki, J. Ando, Y. Matsuda, K. Ohashi, Chromosomal assignment of LASP1 and LASP2 genes and organization of the LASP2 gene in chicken, Cytogenet Genome Res 112 (2006) 141–147.
A.G. Terasaki et al. / Biochimica et Biophysica Acta 1779 (2008) 51–59 [9] A.G. Terasaki, H. Suzuki, T. Nishioka, E. Matsuzawa, M. Katsuki, H. Nakagawa, S. Miyamoto, K. Ohashi, A novel LIM and SH3 protein (lasp2) highly expressing in chicken brain, Biochem. Biophys. Res. Commun. 313 (2004) 48–54. [10] B. Li, L. Zhuang, B. Trueb, Zyxin interacts with the SH3 domains of the cytoskeletal proteins LIM-nebulette and Lasp-1, J. Biol. Chem. 279 (2004) 20401–20410. [11] M. Katoh, M. Katoh, Identification and characterization of LASP2 gene in silico, Int. J. Mol. Med. 12 (2003) 405–410. [12] N. Satoh, Y. Satou, B. Davidson, M. Levine, Ciona intestinalis: an emerging model for whole-genome analyses, Trends Genet. 19 (2003) 376–381. [13] P. Dehal, Y. Satou, R.K. Campbell, et al., The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins, Science 298 (2002) 2079–2270. [14] M. Ogasawara, N. Nakazawa, K. Azumi, E. Yamabe, N. Satoh, M. Satake, Identification of thirty-four transcripts expressed specifically in hemocytes of Ciona intestinalis and their expression profiles throughout the life cycle, DNA Res. 13 (2006) 25–35. [15] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) 4673–4680. [16] J.A. Spudich, S. Watt, The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of interaction of the tropomyosin–troponin complex with actin and proteolytic fragments of myosin, J. Biol. Chem. 246 (1971) 4866–4871. [17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [18] M. Ogasawara, A. Sasaki, H. Metoki, T. Shin-i, Y. Kohara, N. Satoh, Y. Satou, Gene expression profiles in young adult Ciona intestinalis, Dev. Genes Evol. 212 (2002) 173–185. [19] M. Ogasawara, M. Minokawa, Y. Sasakura, H. Nishida, K. Makabe, A large-scale whole-mount in situ hybridization system, Zool. Sci. 18 (2001) 187–193. [20] K. Azumi, M. Fujie, T. Usami, Y. Miki, N. Satoh, A cDNA microarray technique applied for analysis of global gene expression profiles in tributyltin-exposed ascidians, Mar. Environ. Res. 58 (2004) 543–546. [21] C. Keicher, S. Gambaryan, E. Schulze, K. Marcus, H.E. Meyer, E. Butt, Phosphorylation of mouse LASP-1 on threonine 156 by cAMP- and cGMP-dependent protein kinase, Biochem. Biophys. Res. Commun. 324 (2004) 308–316. [22] Y.H. Lin, Z.Y. Park, D. Lin, A.A. Brahmbhatt, M.C. Rio, J.R. Yates, R.L. Klemke, Regulation of cell migration and survival by focal adhesion targeting of Lasp-1, J. Cell Biol. 165 (2004) 421–432. [23] Y. Sasakura, L. Yamada, N. Takatori, Y. Satou, N. Satoh, A genomewide survey of developmentally relevant genes in Ciona intestinalis. VII.
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33] [34]
[35]
[36]
59
Molecules involved in the regulation of cell polarity and actin dynamics, Dev. Genes Evol. 213 (2003) 273–283. M.A. Senetar, R.O. McCann, Gene duplication and functional divergence during evolution of the cytoskeletal linker protein talin, Gene 362 (2005) 141–152. A.S. McElhinny, S.T. Kazmierski, S. Labeit, C.C. Gregorio, Nebulin: the nebulous, multifunctional giant of striated muscle, Trends Cardiovasc. Med. 13 (2003) 195–201. S. Millevoi, K. Trombitas, B. Kolmerer, S. Kostin, J. Schaper, K. Pelin, H. Granzier, S. Labeit, Characterization of nebulette and nebulin and emerging concepts of their roles for vertebrate Z-discs, J. Mol. Biol. 282 (1998) 111–123. G. Luo, E. Leroy, C.A. Kozak, M.H. Polymeropoulos, R. Horowits, Mapping of the gene (NRAP) encoding N-RAP in the mouse and human genomes, Genomics 45 (1997) 229–232. G. Luo, J.Q. Zhang, T.P. Nguyen, A.H. Herrera, B. Paterson, R. Horowits, Complete cDNA sequence and tissue localization of N-RAP, a novel nebulin-related protein of striated muscle, Cell Motil. Cytoskelet. 38 (1997) 75–90. O. Ogut, M.M. Hossain, J.P. Jin, Interactions between nebulin-like motifs and thin filament regulatory proteins, J. Biol. Chem. 278 (2003) 3089–3097. C.T. Okamoto, R. Li, Z. Zhang, Y.Y. Jeng, C.S. Chew, Regulation of protein and vesicle trafficking at the apical membrane of epithelial cells, J. Control. Release 78 (2002) 35–41. C.S. Chew, J.A. Parente, Jr., C. Zhou, E. Baranco, X. Chen, Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell, Am. J. Physiol. 275 (1998) C56–C67. T. Nishikata, L. Yamada, Y. Mochizuki, Y. Satou, T. Shin-i, Y. Kohara, N. Satoh, Profiles of maternally expressed genes in fertilized eggs of Ciona intestinalis, Dev. Biol. 238 (2001) 315–331. Y. Satou, K.S. Imai, N. Satoh, Action of morpholinos in Ciona embryos, Genesis 30 (2001) 103–106. T. Arimura, T. Nakamura, S. Hiroi, M. Satoh, M. Takahashi, N. Ohbuchi, K. Ueda, T. Nouchi, N. Yamaguchi, J. Akai, A. Matsumori, S. Sasayama, A. Kimura, Characterization of the human nebulette gene: a polymorphism in an actin-binding motif is associated with nonfamilial idiopathic dilated cardiomyopathy, Hum. Genet. 107 (2000) 440–451. S.T. Kazmierski, P.B. Antin, C.C. Witt, N. Huebner, A.S. McElhinny, S. Labeit, C.C. Gregorio, The complete mouse nebulin gene sequence and the identification of cardiac nebulin, J. Mol. Biol. 328 (2003) 835–846. S.A. Mohiddin, S. Lu, J.P. Cardoso, S. Carroll, S. Jha, R. Horowits, L. Fananapazir, Genomic organization, alternative splicing, and expression of human and mouse N-RAP, a nebulin-related LIM protein of striated muscle, Cell Motil. Cytoskelet. 55 (2003) 200–212.