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Sekiguchi Lesion Gene Encodes a Cytochrome P450 Monooxygenase That Catalyzes Conversion of Tryptamine to Serotonin in Rice*

Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the Gα subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.

Plants have developed various immune responses to protect themselves from pathogen attack (1). One of them is the hypersensitive response, which is often accompanied by programmed cell death, and is effective to prevent the growth of biotrophic pathogens (2). The hypersensitive response is induced coordinately with production of reactive oxygen species, lignification, production of phytoalexins, and expression of defense-related genes.
To understand the roles of hypersensitive response cell death in immune responses, a large number of lesion mimic mutants, in which cell death is spontaneously induced, have been inves-tigated in many plant species, including rice, Arabidopsis, and maize (3). Most of lesion mimic mutants are known to activate immune responses in the absence of pathogens (2). A rice lesion mimic mutant, Sekiguchi lesion (sl), exhibits unique orange colored lesions that are induced by inoculation of pathogens, including Magnaporthe grisea (4,5). In the sl mutants, increased activities of tryptophan decarboxylase and monoamine oxidase are correlated with lesion formation (5). However, the identity of the SL gene and its function in cell death and disease resistance remain to be studied.
Plant Rac/Rop GTPases constitute a unique subfamily of the Rho family of small GTPases and are highly conserved in plant species. In rice, OsRac1 regulates a series of immune responses, including cell death, reactive oxygen species production, activation of pathogenesis-related genes, liginification, and phytoalexin production (6). Recently, it was found that OsRac1 forms a complex with RAR1, HSP90, HSP70, OsMAPK6, OsrbohB, Sti1/Hop, and RACK1 (7)(8)(9)(10), which play important roles in rice innate immunity. Heterotrimeric G protein is also involved in innate immunity through OsRac1. In fact, rice dwarf1 (d1) mutants lacking a G␣ subunit of heterotrimeric G protein reduces elicitor-induced defense gene expression and resistance to M. grisea (11).
Serotonin is known as neurotransmitter in mammals, which regulates mood, sleep, and anxiety in human (12). Although serotonin was found in many plant species (13), the serotonin biosynthesis and its function are unclear. Recently, serotonin has been reported to be accumulated in rice during senescence and defense response (14,15). In addition, serotonin is incorporated into the cell wall at disease lesions infected with Bipolaris oryzae and M. grisea, suggesting that serotonin plays an important role in control of the strength of the cell wall for the mechanical barrier against pathogens (15).
To understand how the SL gene regulates cell death and disease resistance in rice, we have isolated the SL gene by a map-based approach. We found that the SL gene encodes cytochrome P450 monooxygenase, which catalyzes 5-hydroxylation of tryptamine to synthesize serotonin. In addition, our results suggest that serotonin contributes to innate immunity by activating intracellular signaling of immune responses. CM1019, and CM2229), induced by N-methyl-N-nitrosourea in the Kinmaze background, were provided by the Japanese National BioResource Project of Rice. The CM265 mutant was used in a cross with a substitution line (SL239) carrying a segment of chromosome 12 from an indica rice (cv. Kasalath) in a japonica rice background (cv. Koshihikari). The F 2 and F 3 plants were analyzed using a sequence-tagged site and cleaved amplified polymorphic sequence markers to construct a physical map of the region containing the SL gene (supplemental Fig. S1). Details of the mapping are provided in the supplemental "Experimental Procedures".
Plasmid Constructs and Rice Transformation-The SL cDNA was cloned into the p2K1 vector containing the maize Ubq1 promoter (16). For the RNA interference construct of Sti1a/Hop, a 350-bp 3Ј-untranslated region of the Sti1a cDNA was PCR-amplified and cloned in the reverse orientation behind the Ubp1 promoter in the pANDA vector (16). The RNA interference construct of OsRac1 was described previously (10). Details of plasmid construction are provided in supplemental "Experimental Procedures". Transgenic plants were generated from Agrobacterium-transformed rice calli (17).
Reverse Transcription-PCR and Real Time PCR-Total RNAs were isolated from leaves and suspension cultures using TRIzol reagent (Invitrogen) and treated with DNase I (Invitrogen). cDNA was synthesized using SuperScriptII reverse transcriptase (Invitrogen) and used for reverse transcription-PCR and real time PCR with gene-specific primers (see supplemental Table S1).
Rice Blast Infection-Rice seedlings were inoculated at the four-to six-leaf stage by spraying with aqueous spore suspensions containing 10 5 spores/ml of the compatible rice blast races 003 and 007. Inoculated seedlings were kept in a dark chamber with a moisture-saturated atmosphere at 24°C for 20 h and then maintained in a greenhouse. Inoculated leaves were harvested at the indicated time points and used for real time PCR. To examine the effect of serotonin on rice blast resistance, rice seedlings were watered with 400 g/ml serotonin for 22 h and then inoculated with race 007 as described above. Lesion development was scored on rice leaves at 6 days after inoculation by measuring lesion length with a ruler.
Chitin, Serotonin, and Tryptamine Treatments-Rice suspension cultures were treated with 2 g/ml N-acetylchitooligosaccharide elicitor, 400 g/ml (1.88 mM) serotonin (5-hydroxytryptamine hydrochloride (M r 212.68)), or 400 g/ml (2.03 mM) tryptamine (tryptamine hydrochloride (M r 196.68)), and then incubated for various lengths of time. As reported previously (15), less than 30% of applied serotonin is considered to be incorporated by the feeding experiment. Total RNAs were extracted from each sample and subjected to real time PCR. For were purified from rice suspension cultured cells either mock-treated or treated with 2 g/ml N-acetylchitooligosaccharide elicitor and used for real time PCR. Ubiquitin (Ubq) was used as an internal control. Error bars indicate standard deviations. E, expression of SL in seedlings after mock inoculation or inoculation with compatible rice blast fungus (race 003 and 007). Real time PCR was carried out using total RNAs prepared from the seedlings. Ubq was used as an internal control. Error bars indicate S.D. the detection of cell death, suspension-cultured cells were treated with serotonin or tryptamine for 24 h, stained for 10 min with 0.05% (w/v) Evans Blue, and then washed four times with distilled water (18). Dye bound to dead cells was dissolved in a solution of 50% methanol and 1% SDS for 30 min at 50°C and quantified by absorbance at 595 nm.
Measurement of SL Activities-The SL and SL-G455D (CM265) cDNAs were cloned as SpeI-PstI fragments in the pFastBac1 vector (Invitrogen) and used to transform E. coli strain DH10Bac (Invitrogen). Preparation of the cDNAcontaining recombinant baculovirus DNAs and transfection of Spodoptera frugiperda 9 (Sf9) cells were carried out according to the manufacturer's instructions (Invitrogen). Heterologous expression in Sf9 cells and spectrophotometric analysis of P450s were carried out as described previously (19).
Microsomal fractions of insect cells expressing either SL or SL-G455D were obtained as follows. Infected suspensioncultured cells (300 ml) were washed with phosphate-buffered saline buffer and suspended in buffer A, consisting of 20 mM potassium phosphate (pH 7.25), 20% (v/v) glycerol, 1 mM EDTA, and 1 mM dithiothreitol. The cells were sonicated, and cell debris was removed by centrifugation at 10,000 ϫ g for 15 min. The supernatant was further centrifuged at 100,000 ϫ g for 1 h, and the pellet was homogenized with buffer A to provide the microsomal fractions. The microsomal fractions were stored at Ϫ80°C before the enzyme assays were performed. SL activity was reconstituted by mixing the microsomes with purified Arabidopsis NADPH-P450 reductase (20). The reaction mixture consisted of 20 mM potassium phosphate (pH 7.25), 50 pmol/ml recombinant P450 protein, 0.1 unit/ml NADPH-P450 reductase, 1 mM NADPH, and 100 M tryptamine. Reactions were initiated by the addition of NADPH, and were carried out at 30°C for 30 min. The samples were diluted with 20 mM phosphate (pH 2.5) and subjected to HPLC 4 as follows: column, COSMOSIL 5C 18 -PAQ column (150 mm length ϫ 4.6 mm inner diameter; Nacalai Tesque); solvent, 20 mM phosphate (pH 2.5); flow rate, 1.0 ml/min; detection, a fluorescence detector with an excitation wavelength at 285 nm and an emission wavelength at 340 nm. Retention times of authentic samples were 6.5 min for serotonin and 14.5 min for tryptamine. The identity of the reaction product with tryptamine was examined using HPLC equipped with an API-3000 triple stage quadrupole mass spectrometer (Applied Biosystems) as described previously (15). For kinetic analysis, SL activity was assayed using the substrate tryptamine at concentrations ranging from 1 to 40 M. Reactions were initiated by the addition of NADPH and carried out at 30°C for 10 min. The kinetic constants were calculated from triplicate data sets. K m and k cat values were calculated from a double-reciprocal plot of the initial velocity (v 0 ) versus substrate concentration.
Immunoblotting-Microsomal fractions of the insect cells, expressing either SL or SL-G455D, were subjected to immunoblotting using a purified anti-SL antibody as described in supplemental "Experimental Procedures."

Isolation of SL Gene by Map-based
Cloning-To identify a gene responsible for the sl phenotype, we used four sl mutants (CM265, CM2229, CM998, and CM1019; supplemental Fig.  S1), which were generated by mutagenesis using N-methyl-Nnitrosourea (21). The sl locus was mapped to a 350-kb genomic region on chromosome 12, where 7 candidate genes are located (supplemental Fig. S1). PCR experiments indicated that the CM998 mutant has a 30-kb deletion containing genes for a P450 and a zinc finger protein. 5 Sequence analyses in the other mutants revealed that SL encodes a P450 monooxygenase (Fig.  1A). SL gene expression levels were significantly reduced in CM2229. CM998 showed no SL expression because of the deletion of the entire P450 gene. CM1019 had two altered transcripts as follows: an unspliced transcript resulting from a point mutation at the 3Ј splice site of the single intron, and another generated by cryptic splicing at a site near the 3Ј splice site (Fig.  1B). Overexpression of the P450 mRNA complemented the sl phenotype (supplemental Fig. S2).
SL Belongs to the CYP71 Family-The SL protein possesses motifs conserved in P450 proteins, including the heme-binding motif, the EXXR motif, and the proline-rich motif (supplemental Fig. S3). The G455D mutation in CM265 is in the heme-binding motif and is likely to disturb the heme coordination structure. The threonine residue at position 314, which is mutated in CM2229, is important for the oxygen activation process in the P450 catalytic cycle (supplemental Fig. S3). The SL protein shares 41% identity with CYP71A1 from avocado fruit (GenBank TM accession number AAA32913). In rice there are 90 genes and 28 pseudogenes in the CYP71 family; however, the SL protein shows less than 45% identity with any other members of the family. Hence, the SL protein is a unique member of the CYP71 family and is designated CYP71P1.
The intracellular location of the SL protein was investigated using the transient expression of a GFP-fused protein in rice protoplasts (Fig. 1C). The SL protein, with GFP fused to the C terminus, co-localized with a marker protein that was designed to localize to the endoplasmic reticulum (ER). The marker protein was constructed by adding the N-terminal signal peptide of pro-2S albumin, which localizes to the ER (22), and the C-terminal HDEL ER retention sequence, to SECFP (23). The results indicate that SL localizes to the ER.
SL Gene Is Expressed during Innate Immune Responses-Expression of the SL gene in rice suspension cultures was analyzed after treatment with a chitin elicitor that is known to induce a series of defense responses in rice (Fig. 1D) (24). A clear increase in SL mRNA levels was observed at 1 h after chitin treatment (Fig. 1D). We also analyzed SL expression after infection with M. grisea. Infection by two races of compatible blast fungus caused the activation of SL gene expression in rice plants 4 The abbreviations used are: HPLC, high pressure liquid chromatography; TDC, tryptophan decarboxylase; ER, endoplasmic reticulum; GFP, green fluorescent protein; SECFP, super enhanced cyan fluorescent protein. 5 M. Isshiki, unpublished results.
at 48 h after infection, and expression was further increased at 72 h (Fig. 1E). These results suggest that SL has a role in rice innate immunity. SL Protein Possesses Tryptamine 5-Hydroxylase Activity-It was shown previously that tryptamine levels were higher in an sl mutant than in wild type plants and that serotonin levels were very low (4,5,15). These results suggest that SL converts tryptamine to serotonin. To test this possibility, we generated recombinant SL protein with or without the G455D mutation found in the CM265 allele, using a baculovirus expression system in insect cells. The presence of the recombinant SL and SL-G455D proteins in the microsomal fractions was confirmed by immunoblot analysis (Fig. 2A). Fractions containing the wild type SL protein had a cytochrome P450-specific reduced CO difference spectrum with a clear absorption peak at 450 nm (Fig. 2B). In contrast, the fractions containing the SL-G455D protein exhibited no reduced CO difference spectrum (Fig. 2B).
These results indicate that SL protein is indeed a cytochrome P450 enzyme. The microsomal fractions from insect cells expressing the recombinant SL proteins were used in enzyme assays with tryptamine as the potential substrate. HPLC analysis of the reaction products showed a specific peak at the same retention time as that of serotonin when fractions containing the wild type SL protein, but not the SL-G455D mutant protein, were used (Fig. 2C). The SL protein catalyzed the 5-hydroxylation of tryptamine with a high affinity and efficiency (K m ϭ 7.3 Ϯ 0.8 M and k cat ϭ 45 Ϯ 1.5 min Ϫ1 ), although the SL protein did not metabolize tryptophan, indole 3-acetic acid, or tyramine. Thus, it is likely that the SL protein is tryptamine 5-hydroxylase that catalyzes the conversion of tryptamine to serotonin.
Serotonin Plays a Role in Innate Immune Responses-Serotonin has been reported to be accumulated at a high level (1.9 mM) in disease lesions infected with B. oryzae and M. grisea (15), whereas the high accumulation of serotonin is abolished in the sl mutant. In addition, the sl mutant is more susceptible to infection of B. oryzae as compared with wild type, and application of serotonin suppresses the fungal growth in the leaves of the sl mutant (15). Thus, it is possible that serotonin is involved in innate immunity. To elucidate how serotonin regulates immunity responses, we analyzed the expression of four defense genes, probenazol 1 (PBZ1), phenylalanine ammonia-lyase 1 (PAL1), chitinase 1 (Cht1), and chitinase 3 (Cht3) in rice cell suspension cultures. These four genes are induced by chitin elicitor  from fungal pathogens (25). 6 Expression of these defense genes was clearly induced at 12 h after serotonin treatment, but no induction was found with tryptamine ( Fig. 3, A-D). Serotonin also induced cell death in cultured rice cells, as shown by Evans Blue staining (Fig. 3E), although reduced levels of cell death occurred after tryptamine treatment.
Because serotonin was able to induce immune responses in rice cell cultures, we examined the possibility that known components of rice innate immunity are involved in serotonin-induced defense gene induction. It has been shown that the small GTPase OsRac1 and its signaling partners, including Sti1/ Hop and a heterotrimeric G protein, are involved in rice innate immunity (8,10,26). For these experiments, we used rice cell cultures expressing RNA interference knockdown constructs for OsRac1 and Sti1/Hop, and a cell culture of the dwarf1 (d1) mutant, which lacks a functional G␣ subunit of the heterotrimeric G protein (11). The results clearly showed that these proteins are involved in serotonin-induced defense gene induction (Fig. 4, A-D).
Because our results suggest that serotonin plays a role in rice innate immunity, we examined whether serotonin treatment of rice seedlings (ϳ1 month old) had any effect on infection with a compatible race of M. grisea. The results indicate that seedlings treated with serotonin are significantly more resistant to fungal infection than mock-treated seedlings (Fig. 5), suggesting that serotonin has the ability to induce resistance against rice blast infection.

DISCUSSION
Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans (27). In mammals, serotonin is synthesized from tryptophan in two steps involving L-tryptophan 5-hydroxylase and aromatic L-amino acid decarboxylase (Fig. 6) (28). Tryptophan hydroxylase belongs to a family of aromatic amino acid hydroxylases and requires iron and the coenzyme tetrahydropterin for catalytic activity (29). It is interesting that the SL gene encodes CYP71P1 in the P450 superfamily, which is different from the tetrahydropterin-dependent hydroxylase family in mammals (Fig. 6). Serotonin has been found in more than 42 plant species (13), suggesting that enzymes for serotonin biosynthesis are conserved in plants. However, the SL orthologs have not been found in other plants, including Arabidopsis. Among the Arabidopsis P450s, CYP83B1/SUR2 shows the highest identity (39%) with the SL protein. CYP83B1/SUR2 catalyzes the N-hydroxylation of indole-3-acetaldoxime to synthesize indole glucosinolate (30).
It has been reported that some serotonin derivatives such as coumaroylserotonin and feruloylserotonin, which are hydroxycinnamic acid amides, are involved in plant defense responses against pathogen infection (31). The accumulation of hydroxycinnamic acid amides in the cell wall could provide an unde-   Rice seedlings were inoculated with the compatible blast fungus race 007. Disease lesion development was scored at 6 days after inoculation by measuring lesion length. Error bars indicate standard error (n ϭ 78). An asterisk indicates a significant difference by the t test at p Ͻ 0.01. gradable mechanical barrier for pathogens (31,32). In fact, it was shown that serotonin itself is polymerized and incorporated into cell walls in rice, leading to the suppression of growth of the fungus B. oryzae (15). It was also shown that hydroxycinnamic acid amides possess anti-fungal activity (33). These studies suggest that serotonin may act as a phytoalexin, an antimicrobial compound that is produced by plant cells in response to pathogen infection. However, we found no effect of serotonin on the germination of M. grisea spores (supplemental Table S2). In this study, we found that serotonin induces immune responses including defense gene expression and cell death through the pathway involved in G␣, OsRac1, and Sti1. Taken together, it is suggested that serotonin may have dual functions that regulate the strength of the cell wall for the physical barrier against pathogens as well as activate intracellular signaling for immune responses.
We have identified SL as tryptamine 5-hydroxylase enzyme for serotonin biosynthesis, and we showed that in rice, serotonin is synthesized in different pathways found in animals. Recently, it was reported that serotonin is accumulated during senescence in rice (14). However, whether SL is involved in senescence response remains to be analyzed. Two genes, tryptophan decarboxylase and SL, required for serotonin synthesis from tryptophan have now been identified in plants. It is therefore possible to control the serotonin levels in plants by manipulating the expression of these genes.


extfieldsofstudy:
Medicine
Computer Science
Biology

text:
CaSNP: a database for interrogating copy number alterations of cancer genome from SNP array data

Cancer is known to have abundant copy number alterations (CNAs) that greatly contribute to its pathogenesis and progression. Investigation of CNA regions could potentially help identify oncogenes and tumor suppressor genes and infer cancer mechanisms. Although single-nucleotide polymorphism (SNP) arrays have strengthened our ability to identify CNAs with unprecedented resolution, a comprehensive collection of CNA information from SNP array data is still lacking. We developed a web-based CaSNP (http://cistrome.dfci.harvard.edu/CaSNP/) database for storing and interrogating quantitative CNA data, which curated ∼11 500 SNP arrays on 34 different cancer types in 104 studies. With a user input of region or gene of interest, CaSNP will return the CNA information summarizing the frequencies of gain/loss and averaged copy number for each study, and provide links to download the data or visualize it in UCSC Genome Browser. CaSNP also displays the heatmap showing copy numbers estimated at each SNP marker around the query region across all studies for a more comprehensive visualization. Finally, we used CaSNP to study the CNA of protein-coding genes as well as LincRNA genes across all cancer SNP arrays, and found putative regions harboring novel oncogenes and tumor suppressors. In summary, CaSNP is a useful tool for cancer CNA association studies, with the potential to facilitate both basic science and translational research on cancer.

INTRODUCTION
Cancer is a complex genetic disease, whose initiation and progression are often accompanied by genome alterations. A great amount of copy number alterations (CNAs) is known to occur in the malignant neoplasm at full scale of human genome. Of the different types of genome variations, CNA has been the most implicated in oncogenesis and cancer progression, and many CNAs are known to be characteristic of specific types of cancers (1)(2)(3). There is a growing demand to understand the nature of CNA in cancer, as CNAs not only serve as biomarkers to predict cancer malignancy and prognosis, but also often harbor tumor suppressors and oncogenes (4,5), the studies of which could shed light on the sequence and mechanism of oncogenesis. In addition, there is increasing evidence that some CNAs could target noncoding RNA (ncRNA) genes such as miRNAs (6), suggesting ncRNAs might be extensively involved in oncogenesis.
Array comparative genomic hybridization (aCGH) has long been the standard platform to investigate the relative gains and losses of genomic DNA by measuring the relative signal ratios of the differentially labeled array hybridization between tumor and normal samples. Several repositories focusing on CNAs detected from aCGH are already publicly available (7)(8)(9). However, most of these aCGH studies use BAC or cDNA probes, which have a coarse resolution for CNA detection.
In the last few years, single nucleotide polymorphism (SNP) arrays have gradually become the major platform for SNP genotyping and CNA detection (10,11). SNP array has probes on known SNPs that are densely distributed in the human genome, and allows accurate SNP genotyping at these loci for individual biological samples. In addition, by comparing the signal intensities at the SNP loci between cancer and normal reference samples, one can also gain high-resolution CNA knowledge about the cancer of interest. It has been reported that SNP arrays outperform traditional aCGH in CNA detection resolution (12), enabling high-resolution SNP and CNA detection at individual gene level (13)(14)(15). Currently, some studies use websites to display the results of their own (e.g. http://www.broadinstitute.org/ tumorscape). However, a comprehensive resource of CNA data from all cancer SNP array experiments is still unavailable.
We present CaSNP as a comprehensive collection of CNA information inferred from cancer SNP array data. We analyzed 11 500 Affymetrix SNP arrays on 34 different cancer types in 104 studies to profile the genome-wide CNAs. This includes all the publicly available cancer SNP profiles using Affymetrix SNP arrays, mostly from Gene Expression Omnibus (GEO) (16). We also developed a data extraction and annotation schema to interrogate copy number on user-specified genomic region by cancer type and across different array platforms (from SNP 10K to 6.0) and studies. CaSNP is available at http://cistrome.dfci.harvard.edu/CaSNP/.

Data analysis and curation
Among the 104 studies collected, 100 are from GEO, one is from GlaxoSmithKline (https://cabig.nci.nih.gov/tools/ caArray_GSKdata) and three are from individual publication's supplementary websites (17,18). The raw data (.cel file) of array experiments and accompanying genotype files (if available) for samples were collected. dCHIP-SNP (19), a widely used and referenced SNP array analysis algorithm (cited by 238 accordingly to Google Scholar), was applied to each data set. Array raw data within each study were normalized in Figure 1. Overview of internal structure of CaSNP. The input for genome regions, whatever of its kind, will be uniformly translated into genomic coordinates, by querying coordinate tables of miRNA or refSeq gene, and then sent to the query engine. The input for cancer type will be checked against sample information table, to extract the names of samples qualifying this cancer type, which will further be used by the query engine to search the CNA data tables. The CNA data are stored in tables of each series and grouped by platform type. After having been extracted from data tables, relevant copy number data are combined and grouped by the output engine to calculate average copy numbers and the percentage of threshold-passing samples, which will be further displayed on the result page. Besides, a graphic display is available within which the signals of each series on the region of query will be represented as heatmaps. In addition, the returned CNA data are coordinated and written to .bed files for users to download. Detailed information for each study could be viewed on the 'Browse Data' page by linking to their corresponding annotations on GEO. dCHIP-SNP with invariant set normalization, and signal values for individual SNP loci were further computed with the model-based expression index method (20). Relative copy number value for each SNP was calculated as the signal ratio of tumor samples versus the average of normal reference samples within the same data set, and was exported and stored in CaSNP. For data sets with no normal reference samples, the average 'normal reference' was calculated for each SNP from the tumor samples bearing the middle 50% of signals (i.e. 25% outlier signals from both sides were excluded). We did not choose normal samples from other experiments of the same array type as reference to avoid potential microarray batch effect.
To treat and query copy number data from different array platforms in a unified manner, we updated the genome coordinate system to the latest human genome assembly (UCSC hg19). In addition, all SNP IDs were converted to dbSNP129. We also manually extracted and curated information on sample clinical background and organized them at two levels: the top level on the tissue origin (e.g. lung cancer), while the second on cancer subtypes (e.g. small cell lung carcinoma).

Query parameters
The only required field in a user query is the genome region where a user inputs a genomic coordinate range (limited to 2-MB size), a gene name, a RefSeq ID or an miRNA name-all of which will be internally converted to a genomic coordinate range. The user could optionally specify the cancer type and subtype to limit the query. Alternatively, one could also go to the 'Browse Data' page to select a subset of the data sets/series for analysis. The 'Browse Data' option allows the user to focus on specific studies or conduct joint analysis in two or more studies and/or across multiple cancer types. When the user specifies a cancer type or subtype or data sets/ series, CaSNP will consult the sample information table to extract the matching samples for analysis. In addition, the user can specify the upper and lower CNA thresholds (default 2.2 and 1.8, respectively) for CaSNP to calculate the percentage of samples beyond the thresholds within each study. A flowchart depicting the internal table schema of CaSNP is shown in Figure 1.

Data output and visualization
A screenshot of CaSNP's result output page is shown in Figure 2. The most important results from a CaSNP query is the average copy number of the queried region for each of the series involved, and the percentage of samples exceeding the copy number thresholds. This value is calculated as the mean of all biological samples in each series. If there are multiple array platforms for a sample, all data for the sample will be combined before the calculation. If user specifies the upper or lower CNA threshold at input, the frequency of threshold-passing samples will also be displayed for each series. This could help the user to determine whether an observed CNA is prevalent in many samples or only caused by outlier ones. The percentage values of threshold-passing samples at the SNP loci in the region are also coded in the bedGraph file format, which is the standard for displaying continuous-valued data as a track in the UCSC genome browser. The bed files generated could be directly viewed in UCSC genome browser (21) via a link or downloaded. Also displayed on the result page are statistics of sample and SNP number for each series, links to their corresponding GEO entries at NCBI and other relevant information.
A graphic display of the results is also provided through the 'HeatMap' query page (Figure 3). The series returned are grouped by array platforms, with CNAs (loss to gain) expressed in color gradient (blue to red), and white for normal diploid (copy number 2) which gives users a comprehensive view of the copy number data in the queried region and cancer types. The heatmaps are dynamically generated from the data in the database.

Database implementation
CaSNP is running on an Apache web server and the data resides in a MySQL server. The scripts for query processing and data analysis are written in Python and the user interface is based on a django frame.

A CASE STUDY USING CaSNP
As an example of how CaSNP can be used for cancer biomarker or oncogene/tumor suppressor detection, we systematically exctracted the copy number of all 20 221 RefSeq genes from CaSNP. We then calculated a G-score, which is a component of the GISTIC methodology (22) for each gene to summarize both the frequency and amplitude of its copy number alteration in all 11 500 cancer samples. When comparing with known annotated database of oncogenes (http://www.sanger.ac.uk/genetics/ CGP/Census/) and tumor suppressor genes (http://cbio .mskcc.org/CancerGenes/), we found that regions of highest or lowest G-scores often harbor known oncogenes and tumor suppressor genes, respectively (Figure 4). This partially validated the quality of the data and the accuracy of our copy number estimation. Interestingly, we observed that many chromosome ends show strong deletions in cancer, and harbor some of the well-known tumor suppressors such as STK11, TSPAN32, MAPK9 and PTGES.
A very striking exception is a strong amplified region on the left tip of chromosome 5, with no previously annotated tumor suppressors and oncogenes. The region was implicated in breast cancer risk (23), and a recent cancer CNA study (24) identified the putative target amplification gene as TERT, but did not experimentally validate its function in breast caner. Checking Oncomine (25), we found that TERT is not highly expressed in breast cancers. Instead, a nearby gene IRX2 not only shows gene amplification and enhanced expression in breast cancers, but also has some literature support for playing a role in mammary gland neoplasia (26). Alternatively, the oncogene in the Chr5 left tip might be an ncRNA, so we investigated the CNAs of all 4013 newly identified LincRNAs (27) in mammalian genomes (Supplementary Figure S1). Although gene expression of LincRNA in breast cancers is still lacking, our analysis did generate interesting leads for potential follow up validations of LincRNAs as tumor suppressors and oncogenes and demonstrate the value of CaSNP.

DISCUSSION
Here, we have presented the CaSNP database for identifying and visualizing CNAs in cancers at any specific region within the human genome. CaSNP stores pre-computed raw copy numbers, and dynamically generates viewable and downloadable summaries of CNA status in response to user queries. A schema for uniformly processing, storing, annotating and presenting data sets across different data sets or platforms was successfully implemented, making CaSNP a useful tool for cancer genomic meta-study. The query results contain numerical values of cancer copy numbers and the frequencies of CNA events, which are well suited for more detailed analysis by other software or methods. Besides the tabular display, the heatmap view displays SNP copy numbers in colors, enabling users to intuitively and comprehensively visualize the results and facilitating finding novel CNA regions in subset of samples. Besides, we provided a scenario of using CaSNP to explore cancer biomarkers or genes through a meta-analysis, and proved CaSNP's ability in suggesting novel oncogenes/tumor suppressors, whether a protein coding gene or a ncRNA.
Benefited from the abundance of SNP array data sets in recent years, CaSNP is the largest repository of SNP array-oriented CNA data among all the databases of the similar type. The amount of public-accessible SNP array data on cancer is still expanding, so will be the data collection in CaSNP. Such a large-scale analysis will be extremely valuable when correlating CNA data with a genomic location with specific diagnostic, prognostic or therapeutic value found in other studies, or to reduce noise from individual studies via meta-analysis. Nowadays, when high-throughput methods as ChIPchip or ChIP-seq could generate hundreds of thousands of regions of interest in a single run, CaSNP will be powerful for independent validation purpose, such as screening the regions which might be related to oncogenesis and might go unnoticed in ChIP experiments alone. Besides collecting more data, we will commit our work to make better use of them. The loss-of-heterozygosity (LOH) information deduced from genotype data will be added, and the CNA status will be compared across different cancer types for specified regions and across the genome.


extfieldsofstudy: Medicine Biology

text:
Cytohesin-2/ARNO, through Its Interaction with Focal Adhesion Adaptor Protein Paxillin, Regulates Preadipocyte Migration via the Downstream Activation of Arf6*

The formation of primitive adipose tissue is the initial process in adipose tissue development followed by the migration of preadipocytes into adipocyte clusters. Comparatively little is known about the molecular mechanism controlling preadipocyte migration. Here, we show that cytohesin-2, the guanine-nucleotide exchange factor for the Arf family GTP-binding proteins, regulates migration of mouse preadipocyte 3T3-L1 cells through Arf6. SecinH3, a specific inhibitor of the cytohesin family, markedly inhibits migration of 3T3-L1 cells. 3T3-L1 cells express cytohesin-2 and cytohesin-3, and knockdown of cytohesin-2 with its small interfering RNA effectively decreases cell migration. Cytohesin-2 preferentially acts upstream of Arf6 in this signaling pathway. Furthermore, we find that the focal adhesion protein paxillin forms a complex with cytohesin-2. Paxillin colocalizes with cytohesin-2 at the leading edges of migrating cells. This interaction is mediated by the LIM2 domain of paxillin and the isolated polybasic region of cytohesin-2. Importantly, migration is inhibited by expression of the constructs containing these regions. These results suggest that cytohesin-2, through a previously unexplored complex formation with paxillin, regulates preadipocyte migration and that paxillin plays a previously unknown role as a scaffold protein of Arf guanine-nucleotide exchange factor.

Adipose tissue plays a key role in energy storage and in the secretion of regulatory lipid hormones such as leptin, adiponectin, and tumor necrosis factor-␣ (1-3). The initial stage of adipocyte differentiation begins with the proliferation of multipotent mesenchymal stem cells, which then enter the first stage of differentiation into preadipocytes. These fibroblast-like imma-ture adipocytes still have proliferative activity; they migrate to various sites to undergo further adipogenesis. They eventually enter into the second differentiation stage, during which they become spherical mature adipocytes containing droplets of fat. In contrast to the normal development of adipose tissue, chronic overnutrition of the adipose tissue leads to macrophage infiltration, resulting in local inflammation that potentiates insulin resistance (4). Many signaling molecules, especially those for transcriptional regulatory proteins, participate in the process of adipocyte differentiation and in the formation of pathological conditions (5), but the intracellular signals responsible for cellular morphological changes and for the migration of preadipocytes before initiation of their differentiation into adipocytes remain largely unknown.
Cell migration is essential for many aspects of developmental processes and requires several spatially and temporally coordinated changes in cytoskeletons and cellular membranes. Dynamic structures critical to cell migration include filopodia, lamellipodia, and actin stress fibers. The cellular morphological changes involved in cell migration are regulated by Rho family GTPases (6), the ADP-ribosylation factors (Arfs), and the other small GTP-binding proteins. Arf proteins primarily regulate intracellular vesicular transport to control endocytic and secretory pathways and may be involved in morphological changes. In mammalian cells the Arf family consists of five or six isoforms (7,8) that are grouped into three classes: class I (Arf1 and Arf2 and/or Arf3), class II (Arf4 and Arf5), and class III (Arf6). As this classification suggests, Arf6 is unique in important ways. It can regulate peripheral actin cytoskeleton as well as vesicle transport by controlling lipid-modifying enzymes such as phosphatidylinositol-4-phosphate 5-kinases and phospholipase D isozymes and other downstream effectors (8 -10).
Like other small GTP-binding proteins, Arfs act as molecular switches and are active when bound to GTP and inactive when bound to GDP. Guanine-nucleotide exchange factors (GEFs) 3 catalyze the intrinsically slow exchange reaction of GDP with free cytoplasmic GTP to generate active conformations, whereas GTPase-activating proteins (GAPs) inactivate GTPbinding proteins. The former reaction is a very important ratelimiting step, as GEFs define the specificity of small GTP-binding protein activation by integrating intracellular signals. Arf GEFs are divided into five families based on overall structure and domain organization (11,12): Golgi brefeldin A (BFA)resistance factor 1/BFA-inhibited GEF (GBF/BIG) (13), Arf nucleotide binding site opener (ARNO)/cytohesin, exchange factor for Arf6 (EFA6) (14,15), BFA-resistant Arf GEF (BRAG) (16), and F-box only protein 8 (FBX8) (17). The large number of GEFs relative to the number of Arf proteins suggests that GEF activities are controlled under extensive regulatory conditions in various types of cells.
Paxillin is cytoskeletal adaptor/scaffold protein involved in the dissemination of signals from extracellular cell adhesion proteins such as integrins and growth factor receptors (18,19). Paxillin contains only protein-protein interaction domains: five leucine-rich repeat (LD) domains and four lin-11, isl-1, mec-3 (LIM) domains. Through these, paxillin intracellularly mediates interactions with actin cytoskeleton regulatory proteins. Thus, paxillin is crucial in changing the morphology of cells and promoting cell migration (18, 20 -23); for example, it binds to regulator and effector complexes of Rho GTPases and regulates their spatiotemporal actions. These facts suggest that there may be additional links between paxillin and small GTP-binding proteins and/or their regulatory proteins.
In this study we report that the cytohesin-2 and Arf6 signaling unit mediates migration of mouse preadipocyte 3T3-L1 cells. Furthermore, we find that paxillin directly involves cytohesin-2 and Arf6 in cell migration before the initiation of adipocyte differentiation, adding a unique binding partner to the components of the cellular paxillin complex.
FLAG-tagged cytohesin-2 PHϩb protein was purified from 293T cells transiently transfected with p3ϫFLAG-cytohesin-2 PHϩb using the CalPhos transfection reagent (Clontech, Mountain View, CA) according to the manufacturer's protocols (23). In brief, cells were lysed in lysis buffer A and centrifuged. The supernatant was mixed with protein G resin (GE Healthcare) that was preadsorbed with an anti-FLAG antibody. Bound FLAG-tagged PHϩb protein was extensively washed with lysis buffer A containing 500 mM NaCl and subsequently with lysis buffer A containing 500 mM NaCl and 50 mM EDTA and eluted with lysis buffer A containing 20 mM FLAG peptide (Sigma) according to the manufacturer's protocols. The buffer contained in elution fractions was exchanged with reaction buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5 mM MgCl 2 , 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 g/ml leupeptin, and 1 mM EDTA). The aliquot was stored at Ϫ80°C until use.
RT-PCR-Total RNA was extracted from 3T3-L1 cells using a Trizol reagent (Invitrogen). The cDNAs were prepared from 1 g of total RNA with Superscript III (Invitrogen) according to the manufacturer's instructions. PCR amplification was performed with ExTaq polymerase (Takara Bio) in 30 cycles, each cycle consisting of denaturation at 94°C for 1 min, annealing at 58.5-61.5°C (depending on the primer pair's Tm value) for 1 min, and extension at 72°C for 1 min.
Cell Culture-Unless otherwise described, mouse preadipocyte 3T3-L1 cells and human embryonic kidney 293T cells were cultured at 37°C in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 mg/ml streptomycin. For the migrating assay, cells were preincubated with or without 10 M cytohesin inhibitor SecinH3 (Compound ID 1029232; CAS no. 853625-60-2; Merck) in the presence of 10 M AraC. Less than 5% of cells incorporated trypan blue after at least 10 h of culture in the presence of 10 M SecinH3 and/or 10 M AraC.
Plasmid Transfection-For 3T3-L1 cells, the plasmids encoding fluorescence proteins were transfected using the Lipofectamine 2000 reagent (Invitrogen) or the cell line Amaxa Nucleofector Kit (Lonza Group Ltd, Basel, Switzerland), each according to its manufacturer's protocols. For lipofection and electroporation, the medium was replaced 4 and 24 h after transfection, respectively. Unless otherwise indicated, cells were cultured in medium containing fetal bovine serum for an additional 24 or 44 h. For 293T cells, plasmid DNAs were transfected into cells using the CalPhos transfection reagent (Clontech). The fluorescent images were captured using an Eclipse TE-300 microscope system (Nikon, Tokyo, Japan) and analyzed with AxioVision software (Carl Zeiss, Oberkochen, Germany).
siRNA Transfection-3T3-L1 cells were transfected with siRNA oligonucleotides using a CrediaTF reagent (Credia, Kyoto, Japan) according to the manufacturer's protocols. The medium was replaced 4 h after transfection, and cells were cultured in medium containing fetal bovine serum for an additional 44 h.
Immunofluorescence-Cultured cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), blocked with 20% heat-inactivated fetal bovine serum in PBS, 0.05% Tween 20, incubated with each primary antibody for 12 h at 4°C, and treated with fluorescence-labeled secondary antibodies and/or fluorescence-labeled phalloidin (Invitrogen) in PBS, 0.1% Tween 20 for 0.5 h at room temperature. The coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA) onto slides for confocal microscopic observation. The confocal images were collected with an IX81 microscope with a Laser-Scanning FV500 System (Olympus, Tokyo, Japan) and analyzed with FluoView software (Olympus) and Adobe Photoshop (contrast value, 50; Adobe, San Jose, CA).

Evaluation of Localization on the Plasma Membrane and the Focal Adhesion Region in the Cytoplasm-
The cellular localizations of cytohesin-2, paxillin, vinculin, and actin were determined as described previously (27,28). The localization of each of these molecules was evaluated by means of fluorescence intensity profiling of a typical line scan using ImageJ 1.42q software.
Immunoprecipitation-Cells were lysed with lysis buffer B. The cell extracts were mixed with protein G-Sepharose CL-4B (GE Healthcare) absorbed with the primary antibody. The immune complexes were precipitated by means of centrifugation and washed three times with lysis buffer B. The immunoprecipitates were boiled in sample buffer and then separated on SDS-polyacrylamide gels. The bound proteins were detected through immunoblotting.
In Vitro Arf1 and Arf6 Activity-3T3-L1 cells were plated on 60-mm dishes and stimulated after being scratched. The Arf1 and Arf6 activities were measured as described (28,29). Briefly, cells were lysed in ice-cold lysis buffer. Samples were incubated for 10 min at 4°C and spun at 14,000 rpm for 10 min at 4°C. GST-GGA3 protein coupled to glutathione-Sepharose 4B was added to each supernatant, and samples were rotated at 4°C for 1 h. Proteins were eluted by being heated to 95°C in Laemmli buffer (0.4 M Tris-HCl (pH 6.8), 0.2 M dithiothreitol, 0.2% bromphenol blue, 4% SDS) for 5 min, subjected to SDS-PAGE, and immunoblotted.
Wound Closure Migration Assay-3T3-L1 cells were cultured in 35-or 60-mm dishes and incubated at 37°C for 0, 6, or 10 h with or without SecinH3 in the presence of 10 M AraC. The monolayers of confluent cells were scraped with the narrow end of a micropipette tip to generate wounds ϳ50 m in width (30). The rate of wound healing was quantified as follows: rate of healing (%) ϭ (width of wound at 0 h minus width of wound at 0, 6, or 10 h/width of wound at 0 h) ϫ 100.
In Vitro Binding Assay-Immobilized recombinant cytohesin-2 PHϩb protein was mixed with recombinant GST-paxillin LIM2 for 2 h at 4°C. The complex was washed three times with lysis buffer B, subjected to SDS-PAGE, and immunoblotted with an anti-GST antibody. The K d value was calculated according to a Scatchard analysis.
Statistical Analysis-Values shown represent the means Ϯ S.D. of data from separate experiments. ANOVA was followed by Fisher's protected least significant difference post hoc comparison (*, p Ͻ 0.01).

Involvement of the Cytohesin Family in Migration of Mouse
Preadipocyte 3T3-L1 Cells-Preadipocytic cell lines such as the 3T3-L1 line are very useful in vitro models for studying hormonal regulation of adipocyte differentiation and metabolism (31). The signaling mechanisms underlying preadipocyte migration, however, remain largely unknown. In an attempt to clarify these, we searched for endogenous intracellular proteins that can regulate actin cytoskeleton/focal adhesion assembly and turnover. In this search we used SecinH3, a newly identified inhibitor specific to the cytohesin family of GEFs for Arf proteins, on 3T3-L1 cells (32,33).
In our wound healing assay the movement of 3T3-L1 cells into artificial wounds was measured at 0, 6, and 10 h in the presence or absence of SecinH3 (10 M (32, 33)). As shown in Fig. 1, A and B, the migration distances traveled by control cells over periods of 6 and 10 h were 43 Ϯ 3 and 57 Ϯ 5 m, respec-

FIGURE 2. Suppression of cytohesin-2, but not cytohesin-3, inhibits migration of 3T3-L1 cells.
A, in the wound healing assay 3T3-L1 cells were transfected with an siRNA for control (luciferase), cytohesin-2, or cytohesin-3. After siRNA treatment, 3T3-L1 cell monolayers were wounded by scraping and observed microscopically at 0 and 6 h. The rate of healing was calculated based on the width of the wound. Data were evaluated with one-way ANOVA (*, p Ͻ 0.01). B, RT-PCR of mouse cytohesin-2, cytohesin-3, and actin (as the control) was performed using total RNA of 3T3-L1 cells treated with each type of siRNA.

Cytohesin-2 Interacts with Paxillin
tively, whereas those of SecinH3-treated cells were 16 Ϯ 4 and 23 Ϯ 2 m. At both 6 and 10 h, SecinH3-treated cells had traveled approximately half as far as control cells had. This phenomenon is likely due to changes in the rate of cell migration rather than to alterations in cell proliferation, considering the fact that the experimental period was only 12 h. The experiment was deliberately kept short to minimize the potential effects of proliferation in the presence of AraC, an anti-mitotic reagent. AraC treatment did not exert a significant effect on the incorporation of trypan blue (less than 5% of treated cells incorporated trypan blue).
The effect of SecinH3 on cell migration was dependent on its concentration (Fig. 1C), suggesting that this cytohesin family GEF regulates 3T3-L1 cell migration through certain signaling pathways. Less than 5% of cells incorporated trypan blue under treatment with 0, 0.1, and 10 M SecinH3. These results suggest that the cytohesin-Arf protein signaling unit may regulate 3T3-L1 migration.
The cytohesin family is composed of cytohesin-1, cytohesin-2/ARNO, cytohesin-3/Grp1/ARNO3, and cytohesin-4 (11). To determine which of these cytohesins is responsible for the inhibitory effect of SecinH3, we performed RT-PCR using total RNA of 3T3-L1 cells. RT-PCR analysis showed that 3T3-L1 cells express cytohesin-2, cytohesin-3, and, weakly, cytohesin-1. Any expression of cytohesin-4 was below the detection  . Cytohesin-2, acting through Arf6 but not through Arf1, regulates migration of 3T3-L1 cells. A, after 0-30 scratches, cells were stimulated for 6 h, and Arf1 activity levels were measured. Cells were lysed then affinity-precipitated with a recombinant GST-GGA3 (200 ng) and immunoblotted with an anti-Arf1 antibody. Total Arf1 was detected using an anti-Arf1 antibody. In a parallel experiment, applied GST-GGA3 was stained with Coomassie Brilliant Blue. B, after 0 -30 scratches, cells were stimulated for 6 h, and Arf6 activity levels were measured. Cells were lysed and affinity-precipitated with a recombinant GST-GGA3 and immunoblotted with an anti-Arf6 antibody. Total Arf6 was detected using an anti-Arf6 antibody. In a parallel experiment, applied GST-GGA3 was stained with Coomassie Brilliant Blue. C, cells were transfected with an siRNA for control (luciferase) or for cytohesin-2. After 0 or 30 scratches, cells were incubated for 6 h, and Arf1 activity levels were measured, respectively. Cells were lysed and affinityprecipitated with a recombinant GST-GGA3 and immunoblotted with the corresponding antibody. Using aliquots of total lysates, total Arf1 was detected with anti-Arf1 antibody. To confirm the effect of cytohesin-2 siRNA on its knockdown specificity, RT-PCR of cytohesin-2 was performed using total RNA of 3T3-L1 cells that had been treated with each siRNA. In a parallel experiment, applied GST-GGA3 was stained with Coomassie Brilliant Blue. Data were evaluated with oneway ANOVA (*, p Ͻ 0.01). D, cells were transfected with an siRNA for control (luciferase) or for cytohesin-2. After 0 or 30 scratches, cells were incubated for 6 h, and Arf6 activity levels were measured, respectively. Cells were lysed and affinityprecipitated with a recombinant GST-GGA3 and immunoblotted with the corresponding antibody. Using aliquots of total lysates, total Arf6 were detected with anti-Arf6 antibody. To confirm the effect of cytohesin-2 siRNA on its knockdown specificity, RT-PCR of cytohesin-2 was performed using total RNA of 3T3-L1 cells that had been treated with each siRNA. In a parallel experiment, applied GST-GGA3 was stained with Coomassie Brilliant Blue. Data were evaluated with oneway ANOVA (*, p Ͻ 0.01).
level (supplemental Fig. S1). Because the cytohesin family is the GEF for Arf proteins, we next sought to determine which Arf protein is expressed in 3T3-L1 cells. RT-PCR analysis showed that all Arf proteins exist in 3T3-L1 cells (supplemental Fig. S2), indicating that 3T3-L1 cells possess the cytohesin-2/3-Arf protein signaling unit and that this unit may be responsible for the ability of SecinH3 to inhibit migration.
Cytohesin-2 Regulates Cell Migration through Arf6 in 3T3-L1 Cells-Because cytohesin-2 and cytohesin-3 are primarily expressed in 3T3-L1 cells, both or either may regulate cell migration. Accordingly, we transfected siRNA oligonucleotides specific for cytohesin-2, cytohesin-3, and control luciferase into cells. The expression levels of cytohesin-2 and cytohesin-3 were specifically and markedly reduced by transfection with siRNAs for cytohesin-2 and cytohesin-3, respectively, whereas the expression level of actin was unaffected, as revealed by RT-PCR (Fig. 2B). In the wound healing assay, we observed that knockdown of cytohesin-2 significantly inhibited migration after 6 h. In contrast, knockdown of cytohesin-3 did not have an obvious inhibitory effect on migration (Fig.  2A). These results suggest that cytohesin-2 is the primary mediator of the migration of 3T3-L1 cells.
Cytohesin-2 as a GEF is preferential for Arf1 and Arf6 (11). Accordingly, we tested whether the suppression of Arf1 or Arf6 by each isoform siRNA had an effect on cell migration. The expression levels of Arf1 and Arf6 were specifically reduced by transfection with Arf1 siRNA and Arf6 siRNA, respectively, whereas the expressions of other proteins were unaffected, as revealed by immunoblotting analysis (Fig. 3B). Knockdown of Arf1 or Arf6 inhibited migration (Fig. 3A), suggesting that Arf1 and Arf6 may act downstream of cytohesin-2.
We next examined bona fide Arf protein activity in 3T3-L1 cells. We performed affinity precipitation using the recombinant GST-tagged N-terminal domain of GGA3, which binds to active GTP-bound forms of Arf1 and Arf6. Endogenous active Arf1 and Arf6 were present in a scratch-number-dependent manner 6 h after scratching (Fig. 4, A  and B).
Next, we confirmed that Arf1 and Arf6 are regulated by cytohesin-2. Knockdown of cytohesin-2 inhibited scratch-induced Arf6 activation (Fig. 4D) but not Arf1 activation (Fig. 4C). These results indicate that cytohesin-2 is the primary upstream regulator for Arf6 in this signaling pathway.
Interaction of Cytohesin-2 with Paxillin in 3T3-L1 Cells-Cell migration is a complex process consisting of multiple cell signaling steps, each of which requires cytoskeletal recognition and the coordination of membrane trafficking. The migration process is controlled by the regulated activities of many proteins; one of these is paxillin, which modulates multipotent associations between various signaling molecules and cytoskeletal proteins (18,19). Previously, we studied the relationship between paxillin and small GTP-binding protein signals in cell morphological changes (20 -22, 26). These studies prompted us FIGURE 5. Cytohesin-2, acting through Arf6, cooperates with paxillin at the leading edge of migrating 3T3-L1 cells. A, the plasmid encoding GFP-tagged cytohesin-2 was electroporated into 3T3-L1 cells, and cells were immunostained using anti-GFP antibody and anti-paxillin antibody 6 h after scratching. Representative confocal images of GFP-tagged cytohesin-2 (green) and endogenous paxillin (red) were collected with a confocal microscope system. Staining intensities measured according to pixel brightness were evaluated through a fluorescence intensity (F.I.) profile of a typical line scan (lines 1 and 2). The lower panels (a) are high magnification images of the areas boxed with white lines in the corresponding upper panels. The scale bar indicates 50 m. B and C, 3T3-L1 cells were transfected with an siRNA for control (luciferase) or paxillin. B, cell monolayers were wounded by scraping and observed microscopically at 0 and 6 h. The rate of healing was calculated based on the width of the wound. C, after 0 or 30 scratches, the Arf6 GTP form was measured with GST-GGA3, respectively. Cell lysates were immunoblotted with an antibody against paxillin, small GTP-binding protein, or actin. Data were evaluated with one-way ANOVA (*, p Ͻ 0.01). JULY 30, 2010 • VOLUME 285 • NUMBER 31 to investigate whether cytohesin-2 interacts with paxillin in 3T3-L1 cell migration. As shown in Fig. 5A, immunofluorescence labeling revealed that GFP-tagged cytohesin-2 was partially colocalized with paxillin in the cell peripheral regions at 6 h after scratching. These colocalization data are consistent with the finding that paxillin localizes to a greater degree in membrane-ruffling surfaces or cell peripheral regions in migrating cells (34,35). In addition, knockdown of paxillin inhibited migration of 3T3-L1 cells (Fig. 5B) and scratch-induced Arf6 activation (Fig. 5C). The siRNA knockdown of paxillin slightly decreased the basal Arf6 activity. This reason may be because paxillin is involved in regulation of the network of signaling molecules, including small GTPbinding proteins. The expression level of paxillin was specifically reduced by transfection with siRNA for paxillin, whereas the expression levels of other proteins were unaffected, as revealed by immunoblotting. Thus, cytohesin-2, acting through paxillin, may regulate Arf6 in 3T3-L1 cells.

Cytohesin-2 Interacts with Paxillin
The C-terminal Polybasic Region of Cytohesin-2 Is Required for Binding to the LIM2 Domain of Paxillin-Because cytohesin-2 partially colocalizes and interacts with paxillin in migrating 3T3-L1 cells, we assessed the direct interaction between paxillin and cytohesin-2. We first performed co-immunoprecipitation of endogenous paxillin with endogenous cytohesin-2 from 3T3-L1 cell lysates and found that cytohesin-2 formed an immune complex (Fig.  6A). It is possible that in 3T3-L1 cells cytohesin-2 may be the primary partner of paxillin among the cytohesin family.
Next, to determine which domain of cytohesin-2 interacts with paxillin, we constructed four FLAGtagged truncated mutants of cytohesin-2: the CC domain (amino acids 1-59), the catalytic Sec7 domain (amino acids 60 -254), the phosphoinositide binding PH domain plus C-terminal polybasic region (PHϩb) (amino acids 255-400), and the isolated PH domain (amino acids 255-386) (Fig. 6B). We found that the FLAG-tagged PHϩb domain of cytohesin-2 was responsible for interaction with GFP-paxillin (Fig. 6C). Interestingly, the cytohesin-2 PHϩb domain also binds to Arl4D, the other type of Arf protein, whereas the isolated PH domain does not (28,36); in a similar manner, the interaction between cytohesin-2 and paxillin may require the C-terminal polybasic region in addition to the PH domain. Thus, we tested the possibility that the isolated C-terminal polybasic region binds paxillin. As shown Fig. 6D, the GFP-FIGURE 6. Specific interaction of cytohesin-2 with paxillin in cells. A, endogenous paxillin was immunoprecipitated from 3T3-L1 cell lysates using anti-paxillin antibody and immunoblotted with an anti-cytohesin-2 antibody. The total lysates were also used for immunoblotting with an anti-paxillin antibody or an anti-cytohesin-2 antibody. B, the schematic structures of full-length cytohesin-2 and its domains are illustrated. C, 293T cells were transfected with plasmids coding either FLAG-tagged cytohesin-2 domain (CC, Sec7, PHϩb, PH) or GFP-tagged full-length paxillin. After 48 h, cells were lysed, immunoprecipitated with an anti-FLAG antibody, and immunoblotted with an anti-GFP antibody. The total lysates were also used for immunoblotting with an anti-FLAG antibody or anti-GFP antibody. D, 293T cells were transfected with plasmids coding GFP-tagged isolated C-terminal polybasic region of cytohesin-2 and RFP-tagged full-length paxillin. After 48 h, cells were lysed, immunoprecipitated with an anti-RFP antibody, and immunoblotted with an anti-GFP antibody. The total lysates were also used for immunoblotting with an anti-GFP antibody or anti-RFP antibody. E, the schematic structures of full-length paxillin, and its domains are illustrated. F and G, 293T cells were transfected with plasmids coding both FLAG-tagged full-length cytohesin-2 and one of the RFP-tagged paxillin domains. After 48 h, cells were lysed, immunoprecipitated with an anti-FLAG antibody, and immunoblotted with an anti-RFP antibody. The total lysates were also used for immunoblotting with an anti-FLAG antibody or anti-RFP antibody. The squares with dotted lines indicate specific binding (F) compared with the negative control that does not express FLAG-cytohesin-2 (G). Asterisks indicate nonspecific binding. IP, immunoprecipitation; IB, immunoblotting.

Cytohesin-2 Interacts with Paxillin
tagged isolated polybasic region of cytohesin-2 weakly coimmunoprecipitated with RFP-tagged paxillin, suggesting that it is the minimum interaction site with paxillin.
To assess the interaction between E. coli produced, GST-tagged paxillin LIM2, and the 293T cell-produced FLAG-tagged cytohesin-2 PHϩb domain before further analysis, we performed an in vitro binding assay. The dissociation constant (K d ) was ϳ0.2 M (Fig. 7C), indicating that the association of paxillin with cytohesin-2 is direct and significant. This association constant is comparable with that of the GIT1 paxillin binding domain with paxillin LD2 (25.1 Ϯ 0.2 M) or LD4 motifs (7.0 Ϯ 0.2 M) (38). Together with the results shown in Figs. 6 and 7, this indicates that cytohesin-2 binds to paxillin and that the interaction occurs through the PHϩb domain and the LIM2 domain.
The LIM2 Domain of Paxillin Plays an Important Role in Arf6 Activation-We next checked whether the LIM2 domain of paxillin is required for Arf6 activation. Transfection with the plasmid encoding the isolated LIM2 domain (381-440) (Fig. 8B) into 3T3-L1 cells inhibited scratch-induced Arf6 activation (Fig. 8A), as did transfection with the LIM2 domain-deficient paxillin mutant (Fig. 8, B and  D), which has no ability to bind to cytohesin-2 (Fig. 8C). These results further support the idea that the LIM2 domain structurally participates in the binding of paxillin to cytohesin-2 and the regulation of Arf6 GEF activity in cells.
Migration of 3T3-L1 Cells Is Meditated through the Region Containing the Polybasic Amino Acid Sequence of Cytohesin-2 and the LIM2 Domain of Paxillin-To investigate whether paxillin interacts functionally with cytohesin-2, we electroporated plasmids encoding each RFP-tagged domain of paxillin into 3T3-L1 cells. The number of migrating RFP-positive cells was counted 6 h after scratching and compared with the number of migrating cells expressing only control RFP. Cells within the healing area were inhibited by transfection with the RFP-tagged LD4/5, LIM1, and LIM2 domains of paxillin (Fig. 9, A-D) but not by transfection with the RFP-tagged LD1, LD2, or LD3 domains (Fig. 9, A and B). The RFP-tagged LIM3/4 domain modestly inhib- ited migration (Fig. 9, C and D). These inhibitory effects exhibited by domains other than the LIM2 domain may be caused by the existence of many other paxillin-interacting proteins (18,19).
We next transfected plasmids encoding each GFP-tagged domain of cytohesin-2. The number of migrating GFP-positive cells was counted 6 h after scratching and compared with the number of migrating cells expressing only control GFP. Cells within the healing area were inhibited by transfection with the GFP-tagged PHϩb domain of cytohesin-2 and modestly by transfection with the isolated PH domain of cytohesin-2 but not by transfection with the GFP-tagged CC or Sec7 domains of cytohesin-2 (Fig. 10, A and B). The PHϩb domain is also known to be a minimally functional domain that participates in the interaction of cytohesin-2 with the Arf family small GTP-binding protein Arl4D and that acts upstream of cytohesin-2 in migrating cells (28,36). Taken together, these data indicate that the paxillin-cytohesin-2 interaction is involved in the migration of 3T3-L1 cells and that this is mediated mostly by the LIM2 domain of paxillin and possibly through the region containing the polybasic amino acid sequence of cytohesin-2.

DISCUSSION
Preadipocytes differentiate from multipotent mesenchymal stem cells, then migrate to various sites where they undergo adipogenesis into mature adipocytes (3,39). Although an array of transcription factors are known to control adipocyte differentiation, the mechanism underlying preadipocyte migration remains poorly understood. Here, using the preadipocyte 3T3-L1 cell line as a model, we have discovered a key signaling pathway through Arf protein, the small GTP-binding protein involved in cellular morphological changes essential for cell migration.
A large part of the signaling mechanism underlying 3T3-L1 cell migration is mediated by the Arf GEF cytohesin-2 and Arf6. This conclusion is supported by our observation that inhibition of cytohesin-2 or Arf6 with a pharmacological inhibitor or knockdown of the specific molecule through RNA interference attenuates migration and by our observation that cytohesin-2-dependent Arf6 activity is stimulated in a scratch-dependent manner. Importantly, 3T3-L1 cell migration requires the interaction of paxillin with cytohesin-2. This interaction occurs between the LIM2 domain of paxillin and the C-terminal PH domain in tandem with a short polybasic amino acid sequence of cytohesin-2. Expression of the LIM domain or C-terminal region containing the PH domain in cells results in an inhibitory effect on migration. These results demonstrate that migration of 3T3-L1 cells FIGURE 8. Role of paxillin LIM2 domain in interaction with cytohesin-2. A, 3T3-L1 cells were electroporated with RFP and RFP-paxillin LIM2 domain. After 0 or 30 scratches, cells were incubated for 6 h, and Arf6 activity was measured, respectively. Cells were lysed, affinity-precipitated with GST-GGA3, and immunoblotted with the corresponding antibody. B, the schematic structures of paxillin wild type, isolated paxillin LIM2 domain, and paxillin ⌬LIM2 domain are shown. C, 293T cells were transfected with various combinations of plasmids of pEGFP-paxillin, pEGFP-paxillin ⌬LIM2 domain, and p3ϫFLAG-cytohesin-2. After 48 h, cells were lysed, and supernatants were incubated with an anti-FLAG antibody and collected with protein G-Sepharose CL-4B. After washing the resin with ice-cold lysis buffer, bound proteins as well as total lysate were separated by SDS-PAGE and detected by Western blotting with antibodies against FLAG tag and GFP-tag. IP, immunoprecipitation; IB, immunoblotting. D, 3T3-L1 cells were electroplated with RFP and RFP-paxillin ⌬LIM2 domain. After 0 or 30 scratches, cells were incubated for 6 h, and Arf6 activity was measured, respectively. Cells were lysed, affinityprecipitated with GST-GGA3, and immunoblotted with the corresponding antibody.
involves a previously unknown functional signaling unit, namely, the cytoskeletal scaffold protein paxillin-Arf GEF cytohesin-2 complex.
It is clear that cytohesin-2, acting through Arf6, regulates migration of 3T3-L1 cells, but other GEFs for Arf6 may also participate in migration. EFA6 and BRAG family proteins as well as cytohesin family proteins are also GEFs for Arf6 (11). All of these GEFs consist of similar domains organized in a similar manner; the catalytic Sec7 domain is in tandem with the PH domain and the coiled-coil region. The EFA6 family of proteins is composed of four members: EFA6A, EFA6B, EFA6C, and EFA6D. EFA6B and EFA6D are widely expressed in tissues, whereas EFA6A and EFA6C are pre-dominantly expressed in brain tissues (14,15). The BRAGs include three members, among which BRAG2 shows ubiquitous expression; in contrast, BRAG1 and BRAG3 display primal expression in the brain (16). It is conceivable that EFA6A, EFA6E, and/or BRAG2 could act together with cytohesin-2 in 3T3-L1 cell migration.
It is worth noting that cytohesin family proteins also act as GEFs for Arf1, as migration also involves Arf1. Moreover, 3T3-L1 cells weakly express other cytohesin family proteins in addition to cytohesin-2. These GEFs may act upstream of Arf1 and control migration, at least in part. Alternatively, the well characterized brefeldin A-sensitive GEFs may be upstream regulators of Arf1 in this signaling pathway. In fact, BIG1 mediates migration of HepG2 cells (40), and BIG2 promotes neural progenitor cell migration in the cerebral cortex (41). It will be interesting to study how the GEFs for Arf1 and/or Arf6 cooperatively regulate 3T3-L1 cell migration.
Paxillin plays crucial roles in cytoskeletal organization, cell adhesion, migration, and cellular morphological changes (18,19). It is a multidomain adaptor or scaffold protein that links extracellular signals from cell adhesion proteins and growth factor receptors to intracellular signaling proteins through direct interaction and/or phosphorylation-dependent association. Herein, we identify the Arf GEF cytohesin-2 as a novel binding partner of paxillin. Paxillin is already known to form a complex with some Arf GAPs, including G protein-coupled receptor kinase-interacting protein 1 (GIT1; also called Cat-1/p95-APP1), GIT2 (also called Cat-2/ p95-APP2/PKL), Arf GAP with Src homology 3 domain, ankyrin repeats, and PH domains 2 (ASAP2; also called PAP/ AMAP2/DEF1/PAG3) (42,43). GIT2 displays nearly ubiquitous expression in tissues, whereas GIT1 appears to be expressed only in endothelial cells. GIT1/2 binds to the LD4 domain of paxillin (42). ASAP2, which ubiquitously expresses in tissues, binds to the N-terminal region involving all LD domains (44). Because the paxillin-binding sites of Arf GAPs GIT1/2 and ASAP2 are different from that of cytohesin-2, these Arf GAPs and cytohesin-2 can bind to paxillin simultaneously. Interestingly, GIT1/2 also exists as a tight physiological com-

Cytohesin-2 Interacts with Paxillin
plex with p21-activated kinase-interacting exchange factor (PIX)/Cool proteins, the GEFs for Rac and Cdc42, members of the Rho family of small GTPases (45). In addition, CdGAP, a GAP for Rho GTPases, interacts with paxillin (46). Cell migration involves dynamic cytoskeletal changes and intracellular membrane traffic and is mediated by organized cycles of on/off switch mechanisms in signaling molecules. Paxillin may pro-vide a platform for small GTP-binding protein regulators, including the GEFs and GAPs for the Arf and Rho proteins; this idea is consistent with our observation that expression of domains other than the LIM2 domain in cells partially inhibits migration of 3T3-L1 cells.
It is well established that Arf6 directly activates phosphatidylinositol-4-phosphate 5-kinases and phospholipase D isoenzymes in many types of cells (6,8,10). Phosphatidylinositol-4-phosphate 5-kinases and phospholipase D isoenzymes participate in actin cytoskeletal changes that generate phosphoinositides and phospholipids as the products. These lipidmodifying enzymes may, therefore, act as the functional effectors downstream of Arf6 in this signaling pathway. Alternatively, cytohesin-2 and Arf6 may regulate Rac, which contributes to actin cytoskeletal changes. Arf6 activation of Rac is proposed to be mediated by the Rac-GEF Dock180-engulfment and cell motility (ELMO) complex (54). Montagnac et al. (55) have recently reported that c-Jun N-terminal kinase (JNK)interacting protein (JIP) 3 and JIP4 are targets of Arf6. JIP3/4 may mediate migration of 3T3-L1 cells, as it forms a complex with kinesin-1 and dynactin to control the microtube-dependent endosome trafficking required for migration. Because these Arf6 effectors are involved in basic cellular functions such as phosphoinositide cycling and intracellular trafficking, it is conceivable that they also function coordinately in this signaling pathway.
In this study we show for the first time that paxillin forms a functional complex with the Arf-GEF cytohesin-2 to mediate scratch-induced migration of 3T3-L1 preadipocytes before the initiation of differentiation to adipocytes. It is known that adipogenesis is regulated by activities of small GTP-binding proteins (56,57). For example, RhoA regulates adipogenesis (37) and maintains the cell morphology of primary mesenchymal stem cells. Inhibition of RhoA and the downstream Rho kinase leads to differentiation to adipocytes. Similarly, our preliminary data show that inhibition of cytohesin family proteins by means of the pharmacological inhibitor SecinH3 promoted differentiation of 3T3-L1 cells (data not shown). As in the case of RhoA, Arf6 activity may support the preservation of the undifferentiated phenotype as well as migration. Further studies on the role of the cytohesin-2-Arf6 signaling pathway in 3T3-L1 cells and also in primary cells will advance our understanding of the involvement of small GTP-binding protein activity in adipocyte differentiation. In addition, because inhibition of cytohesin-2 in vivo results in hepatic insulin resistance (32,33) and insulin resistance is a hallmark of type 2 diabetes, inhibition of this signaling pathway through cytohesin-2 is closely associated with pathological changes. Conversely, if this pathway is properly regulated, it may present a means for improving type 2 diabetes. Elucidation of the details of this pathway molecular mechanism and of the cytohesin-2 role therein may contribute to the development of drug target-specific medicines for type 2 diabetes.


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A New Perspective on Intercalated Disc Organization: Implications for Heart Disease

Adherens junctions and desmosomes are intercellular adhesive junctions and essential for the morphogenesis, differentiation, and maintenance of tissues that are subjected to high mechanical stress, including heart and skin. The different junction complexes are organized at the termini of the cardiomyocyte called the intercalated disc. Disruption of adhesive integrity via mutations in genes encoding desmosomal proteins causes an inherited heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC). Besides plakoglobin, which is shared by adherens junctions and desmosomes, other desmosomal components, desmoglein-2, desmocollin-2, plakophilin-2, and desmoplakin are also present in ultrastructurally defined fascia adherens junctions of heart muscle, but not other tissues. This mixed-type of junctional structure is termed hybrid adhering junction or area composita. Desmosomal plakophilin-2 directly interacts with adherens junction protein alphaT-catenin, providing a new molecular link between the cadherin-catenin complex and desmosome. The area composita only exists in the cardiac intercalated disc of mammalian species suggesting that it evolved to strengthen mechanical coupling in the heart of higher vertebrates. The cross-talk among different junctions and their implication in the pathogenesis of ARVC are discussed in this review.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease estimated to affect approximately 1 in 5,000 individuals [1]. The prominent features are myocytes loss, fibro-fatty tissue replacement, and life-threatening ventricular arrhythmias [2][3][4]. Approximately a third of patients with ARVC have one or more mutations in genes encoding cardiac desmosomal proteins; hence ARVC is referred to as "a disease of the desmosome" [5,6].
Desmosomes and adherens junctions are intercellular adhesive junctions that anchor intermediate filaments and actin cytoskeleton, respectively, at the plasma membrane of adjoining cells, thereby provide mechanical attachment between the cells, and support the structural and functional integrity of the tissues. Desmosomes consist of three families of proteins, desmosomal cadherins, armadillo proteins, and plakins (Table 1) [7]. Desmosomal cadherins, desmogleins (DSGs), and desmocollins (DSCs) form the extracellular connections by homophilic and heterophilic binding. The cytoplasmic tails of desmosomal cadherins bind to the armadillo protein plakoglobin (PG) and plakophilins (PKP), which in turn bind to the plakin protein, desmoplakin (DP). Desmoplakin links desmosomes to intermediate filament protein. Different desmosomal proteins are differentially expressed in a tissue-specific as well as differentiationdependent manner. Unlike DSG-1, -3, and -4 and DSC-1 and 3, which are predominantly expressed in the skin epidermis, DSG-2 and DSC-2 are highly expressed in the myocardium of the heart. PKP-1 and PKP-3 are the major epidermal PKPs, whereas PKP-2 is the sole PKP present in cardiac tissue. Desmosomal proteins DP, PG, and plectin are shared by epidermis and myocardium (Table 1). Consistent with differential expression pattern of desmosomal components between heart and skin, human mutations causing ARVC in the genes encoding PG, DP, and DSC-2 are associated with the cardiocutaneous syndrome [8]. In addition to ARVC, patients exhibit woolly hair and palmoplantar keratoderma, and they may also have skin blistering.
The classical cadherins are calcium-dependent cell adhesion receptors, located in the adherens junction. The 2 Dermatology Research and Practice extracellular domain of cadherins interacts homophilically mediating strong cell-cell adhesion and plays a key role in the maintenance of tissue structure. In the classic model for adherens junction, the cytoplasmic tail of cadherin interacts in a mutually exclusive manner with either β-catenin or PG. β-catenin or PG links cadherins to α-catenin, and αcatenin interacts with the actin cytoskeleton. Gap junction is another intercellular junction in the cell responsible for cell-cell communication and electrical coupling by mediating small molecules and ion transfer between the cells. Each gap junction is composed of two hexameric structures called connexons or hemichannels that dock across the extracellular space and form a permeable pore. Each connexon consists of six transmembrane proteins called connexin (Cx). Cx43 is the most abundant connexin isotype in the heart. The different junctional complexes must be properly organized in the intercalated disc (ICD) of the myocardium to preserve normal mechanical and electrical function of the heart.
Plakoglobin, also known as γ-catenin, is the only linker protein present in both desmosomes and adherens junctions in skin and heart [9]. Plakoglobin was also the first component of the desmosome to be implicated in the pathogenesis of ARVC. Studies of individuals from the Greek island of Naxos identified an autosomal recessive inherited ARVC with palmoplantar keratoderma and woolly hair. Gene sequencing revealed a homozygous 2 bp deletion (2157-2158delGT) in the junction plakoglobin gene (JUP) in affected individuals [10]. A study of a German family recently reported the first dominantly inherited JUP mutation (S39 K40insS) to cause ARVC without cutaneous abnormalities [11]. Importantly, reduced immunoreactive signal of PG at the ICD is a consistent feature in patients with ARVC making it an important diagnostic tool for ARVC in affected individuals [12]. Most recently, studies on cardiac restricted deletion of PG in adult mice have shown similar features to ARVC, including myocytes loss, inflammation, fibrosis, and cardiac dysfunction [13].
The mixing of junctional components in the heart was first described in PG null mice. The homozygous PG null animals die between embryonic days 12-16 due to ventricular rupture and hemorrhaging into the pericardial cavity. Desmosomes are not detected in the mutant hearts. Instead extended adherens junctions develop, which contain desmosomal proteins such as desmoplakin, forming "mixed type" adhering junction [14,15]. In the heart of PKP-2 null embryos, the desmosome-like structures are also not present, and two morphotypes of desmosomes and adherens junctions are found difficult to distinguish [16]. These observations in mutant mice suggest that the formation of hybrid adhering junctions may be compensatory response to weakened adhesion due to loss of desmosomal proteins PG or PKP-2 in the myocardium.
The typical morphological appearance of cardiac intercalated disc at the ultra structural level in the mice is submembranous plaques with electron dense material adjacent to intercellular space between the myocytes (Figure 1(A)). A relatively large junction that anchors primarily bundles of actin myofilaments is fascia adhaerens-like junction, and junction that anchors primarily intermediate filaments is a Plectin Periplakin desmosomal-like junction (Figure 1(A)). For desmosomes, distinct electron dense material is often observed in the intercellular space. During embryonic development, the shape of the individual cardiomyocyte changes from more polygonal to more elongate with alignment of the myofibrils to the longitudinal axis of the cell. Accompanying the morphological change, the junctional components distribute from all round the cell to the sites of myofibril attachment and eventually restrict to the cell-cell contact of polarized adult myocytes (i.e., ICD) [17].
By comprehensive immunoelectron microscopy with immunogold DP antibody labeling, Franke et al. observed that in normal heart muscle, DP is located in all plaques of both the desmosome-like and fascia adhaerens-type junctions. Very intensely labeled junctions with DP are seen in the more desmosome-like junctions (Figure 1(B)), whereas equal label intensity of continuous DP is seen in mixedtype junctions or hybrid adhering junctions (Figure 1(C)). Using various antibodies to desmosomal plaque proteins, Franke et al. further found that other desmosomal molecules, PG, PKP-2, DSC-2, and DSG-2, are also not restricted to the desmosome-like junctions but also can be detected in adherens junction structures. This large plaque-coated hybrid structure therefore has been termed an "area composita" [18] (Figure 2). By light and electron microscopy, the molecules known as typical components of fascia adhaerens, including N-cadherin, α-catenin, and β-catenin, also have been shown to colocalize with desmosomal proteins in the majority of the area composita junctions [19]. These studies suggest that the area composita is an unusual high molecular complexity and the elements that exist in this hybrid structure are intimately associated [19]. Consistent with late maturation of the ICD structure during development, the formation of the extended area composita junction is also a late, primarily postnatal process in mammalian heart [20]. By contrast, in nonmammalian species (fishes, amphibia, birds), adherens junctions and desmosomes remain separate and distinct structures in these adult hearts, suggesting that the formation of the area composita is not only a relatively late process in mammalian ontogenesis but also in vertebrate evolution [20,21].
αT-catenin is a recently identified member of α-catenin family with restricted expression in testis, brain, and cardiac muscle [22]. Extensive studies from Goossens et al. provided Note that DP immunogold label is enriched in plaques of the junctions. Higher label intensity of DP is desmosome-like structure (the right hand junction in Figure 1(B)), and the fascia adhaerens-like junction is shown in less intensity of DP labeling (the left hand junction in Figure 1(B)). A continuous and equal intensity of DP labeling shows the hybrid junctions (Figure 1(C)) [16]. Figures 1(B Figure 2: Model for cadherin-based area composita in the heart. αT-catenin recruits desmosomal protein, plakophilin-(PKP-) 2 to hybrid adhering junction (left drawing), thereby forming, together with desmosome (right drawing), an area composita, which is an enforced, mixed-type junctional structure attached to both the actin cytoskeleton and the intermediate filament (PG: plakoglobin; DP: desmoplakin). molecular evidence that αT-catenin, a unique molecule of adherens junction, functions as cytoskeletal linker protein that specifically brings the desmosomes and adherens junctions together in the intercalated disc of the heart [23]. Using yeast two-hybrid and co-immunoprecipitation, αTcatenin was shown to interact specifically with desmosomal PKP-2 implicating a novel molecular linkage between the adherens junction and desmosome. By double αTcatenin/PKP-2 immunolabeling electron microscopy, αTcatenin is observed to colocalize with other molecules of cadherin/catenin complex, β-catenin, and N-cadherin at the fascia adhaerens-like junctions, but also with desmosomal proteins such as PKP-2, DSG-2, and DP. By contrast, no localization of either αE-catenin or β-catenin could be seen at desmosome-like junctions of the ICD. Based on the biochemical and morphological studies, αT-catenin is thought to recruit desmosomal proteins to hybrid adhering junctions, forming a mixed-type, reinforced junction at the ICD that is attached to both the intermediate filaments and actin cytoskeletons ( Figure 2). Accordingly, interfering with αT-catenin function may impaire intercellular coupling between αT-catenin, PKP-2, and the cytoskeleton, which may subsequently result in destabilization of the ICD structure, cardiac dysfunction, as well as cardiac arrhythmia [23]. αTcatenin is coexpressed with a closely related family member, αE-catenin, in the heart. Most importantly, αE-catenin lacks the PKP-2 binding domain; therefore mutations in αTcatenin are predicted to have an adverse affect on the organization of the area composita since αE-catenin is not able to interact with PKP-2. Human αT-catenin gene CTNN3 has been mapped to chromosome 10q21, a region that links to autosomal dominant familial dilated cardiomyopathy (DCM) [24]. Although genetic screening has not detected any DCM-linked CTNN3 mutations to date, αT-catenin is considered a candidate gene and may be the potential cause of DCM or ARVC [24].
It is estimated that as many as 70% of the desmosomal mutations linked to familial ARVC are in the gene coding for PKP-2 [25]. Abnormal expression of gap junction protein Cx43 has been observed in heterozygous human PKP-2 mutations [26,27]. Similar observations have also been made in patients with mutations in plakoglobin (Naxos disease) or desmoplakin (Carvajal syndrome) genes [5,[28][29][30]. These studies suggest that abnormalities in intercellular adhesion caused by mutations in desmosomal proteins may promote remodeling of gap junctions, which, in turn, alters cardiac conduction and potentially leads to ventricular arrhythmogenic phenotype in this disease [5]. This hypothesis has been supported in a cellular model in which PKP-2 was knockdown by shRNA [31]. Loss of PKP-2 expression in the cells leads to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intercellular space, and a decrease in dye coupling between cells. GST-pulldown assays have shown that PKP-2 and Cx43 coexist in the same macromolecular complex. Recently, siRNA-mediated reduction of PKP-2 has been shown to disintegrate area composita junction structures in cardiac myocytes [32]. These results provide a possibility that PKP-2 is directly involved in the stabilization of Cx43 within the gap junction plaques [31].
The presence of stable mechanical coupling mediated by N-cadherin/catenin adhesion complex is of paramount importance for maintaining the structural integrity of the heart. We demonstrated that cardiac-specific deletion of N-cadherin in mice (N-cad CKO) causes dissolution of the adherens junction, desmosomes, and area composita resulting in absence of ICD structure in the N-cadherin mutant heart [33]. Gap junction protein Cx43 is also markedly decreased from the ICD in the N-cad CKO mice, leading to spontaneous ventricular tachycardia and sudden cardiac death [34]. In contrast, induced deletion of Cx43 in the adult heart did not affect the structure of the ICD with respect to the spatial organization of adherens junction and desmosome [35]. To our knowledge, N-cad CKO is the first animal model with such a dramatic structural phenotype affecting all the junctional complexes in the heart. Our studies demonstrated that the integrity of ICD structure including the area composita is dependent on N-cadherin function in the adult myocardium [33].
In conclusion, the idea that anchoring junctions of the ICD are segregated into distinct domains performing independent functions needs to be reevaluated based on recent data demonstrating novel protein interactions between components from different junction complexes; for example, αT-catenin/PKP-2 and PKP-2/Cx43. The different junctional complexes must be properly localized in the ICD to mediate normal mechanical and electrical coupling between cardiomyocytes. The mixed-type, reinforced junction at the ICD may have evolved to maintain the exceptionally large gap junction plaques found in the mammalian heart, which is under high mechanical stress. Given the large number of PKP-2 mutations identified in ARVC patients, it will be imperative to understand the function of this plakophilin in maintaining electrical synchrony in the heart. Recent studies from human genetics and animal models suggest crosstalk between intercellular junctions constituting the ICD, including adherens junction, desmosome, area composita, and gap junctions. Loss-of-function studies in mice have provided important insight into the hierarchical relationship between the different junction complexes; however subtle mutations in ICD proteins will be necessary to understand the molecular mechanisms underlying ARVC. In the future, it will be interesting to know how the area composita is affected in ARVC patients and its implication in the pathogenesis of the disease.