Tetraspanin Is Required for Generation of Reactive Oxygen Species by the Dual Oxidase System in Caenorhabditis elegans

Reactive oxygen species (ROS) are toxic but essential molecules responsible for host defense and cellular signaling. Conserved NADPH oxidase (NOX) family enzymes direct the regulated production of ROS. Hydrogen peroxide (H2O2) generated by dual oxidases (DUOXs), a member of the NOX family, is crucial for innate mucosal immunity. In addition, H2O2 is required for cellular signaling mediated by protein modifications, such as the thyroid hormone biosynthetic pathway in mammals. In contrast to other NOX isozymes, the regulatory mechanisms of DUOX activity are less understood. Using Caenorhabditis elegans as a model, we demonstrate that the tetraspanin protein is required for induction of the DUOX signaling pathway in conjunction with the dual oxidase maturation factor (DUOXA). In the current study, we show that genetic mutation of DUOX (bli-3), DUOXA (doxa-1), and peroxidase (mlt-7) in C. elegans causes the same defects as a tetraspanin tsp-15 mutant, represented by exoskeletal deficiencies due to the failure of tyrosine cross-linking of collagen. The deficiency in the tsp-15 mutant was restored by co-expression of bli-3 and doxa-1, indicating the involvement of tsp-15 in the generation of ROS. H2O2 generation by BLI-3 was completely dependent on TSP-15 when reconstituted in mammalian cells. We also demonstrated that TSP-15, BLI-3, and DOXA-1 form complexes in vitro and in vivo. Cell-fusion-based analysis suggested that association with TSP-15 at the cell surface is crucial for BLI-3 activation to release H2O2. This study provides the first evidence for an essential role of tetraspanin in ROS generation.

The activity of the catalytic core of NOX enzymes is posttranslationally controlled by the recruitment of regulatory subunits to the plasma membrane [5,31,32].In contrast to NOX isozymes, the current understanding of the regulation of DUOX proteins is unclear, despite the identification of maturation factors (DUOXA) [33].Dual oxidase maturation factors (DUOXA1 and DUOXA2) heterodimerize with DUOX and contribute to its intracellular trafficking [34][35][36].In the absence of DUOXA, DUOX is not recruited to the plasma membrane and is inactive [37,38].DUOX1 preferentially dimerizes with DUOXA1, while DUOX2 preferentially forms dimers with DUOXA2 to achieve maximum activity [35,36].Similar to DUOX2, missense mutations in the DUOXA2 gene were found in patients with congenital hypothyroidism [39].In addition to regulation by DUOXA, DUOX contains EF-hand motifs in the cytoplasmic region, and calcium (Ca 2+ ) stimulation is essential for H 2 O 2 production.
Tetraspanins are integral membrane proteins defined by conserved secondary structures including four transmembrane regions, short cytoplasmic tails at the N-and C-termini, and small and large extracellular loops containing conserved cysteine residues [40,41].They constitute a widely expressed protein superfamily with 33 members in humans.Tetraspanin acts as a molecular facilitator by association and orchestrates a number of other proteins and tetraspanins in specialized membrane microdomains, termed tetraspanin-enriched microdomains (TEMs).TEMs are a distinct class of membrane microdomains with their own biochemical characteristics.TEMs are reportedly a new type of signaling platform involved in cell-cell communication [42][43][44].Numerous studies have shown the functional relevance of tetraspanins in cell adhesion, motility, membrane fusion and antigen presentation.Additionally, tetraspanins are implicated in pathological processes such as tumor malignancy and infectious diseases [45,46].In addition to modification of integrin-mediated cellular functions [47], tetraspanins are important for the proteolytic regulation of bamyloid precursor protein (b-APP) and Notch, and the specificity of Norrin/b-catenin signaling by regulating its receptor, Frizzled-4 [48][49][50].
Evidence from model organisms and inherited human diseases has provided insight into tetraspanin functions in vivo.Previously we demonstrated that a reduction in tetraspanin tsp-15 function, led to exoskeletal deficiencies and lesions in the maintenance of barrier function [51].The exoskeleton (cuticle) of C. elegans is mainly composed of collagen, synthesized and secreted from the apical surface of underlying epidermal cells (hypodermis) [52].In the current study, we have identified a series of mutations in genes that are components of the nematode DUOX system.Based on our evidence, we propose that tetraspanin is a newly identified regulatory component of the DUOX system for H 2 O 2 production.

Identification of DUOX system mutants resembling the tsp-15 mutant
The splicing error mutation, sv15, within tsp-15 causes a reduction in function of tsp-15 [51].We characterized other tsp-15 mutants and found that those with deletions within tsp-15 coding regions were lethal to embryos (Table 1, Figure S1, Table S1, Video S1).We screened for novel mutants similar to the tsp-15 hypomorph mutant to obtain clues for the tsp-15 mutant phenotype.We have shown that tsp-15(sv15) mutants have a distinct blister phenotype compared with classical bli mutants, that were classified by dpy-7p::gfp expression (Figure 1B) [51].Both N2 and OB43 imIs1[dpy-7p::gfp] strains were mutated and screened to exclude typical bli mutants [53].We isolated thirteen alleles classified in four independent complementation groups (CG1-CG4; Table S2).Through single nucleotide polymorphism (SNP) mapping, DNA sequencing, RNAi, and complementation assays, we identified five mutations (Table S2).All identified responsible genes encoded DUOX or related proteins (Figure 1).The im10 mutation in CG1 is a missense mutation in the F56C11.1 gene encoding bli-3/CeDuox-1, a homologue of mammalian dual oxidases (Figure 1A) [17].A conserved proline at position 1311 in the NOX domain was changed to leucine (P1311L) in im10 mutants.The gk141 was thought to have a deletion in the bli-3 region, and hT2-balanced heterozygotes produced gk141/hT2 adults, indicating that gk141/gk141 homozygotes were embryonically lethal (Table 1, Figure S2A).The im21 and im32 mutations in CG3 were located in the splicing site of the C06E1.3gene (Figure 1A), possibly causing premature termination.Amino acid comparisons implied that C06E1.3 is a nematode homologue of DUOXA and essential for maturation and membrane targeting of DUOX (Figure S3) [33]; we named this gene doxa-1.Both im38 and im39 in CG4 were identified as missense mutations in ZK430.8, reported as mlt-7 (Figure 1A, Figure S2C) [25].Mutations im38 and im39 caused a change in the conserved isoleucine at 343 to serine, and phenylalanine at 375 to serine in the peroxidase domain.MLT-7 is a heme peroxidase and essential for cuticle biogenesis in combination with BLI-3 [25].

Involvement of tsp-15 in bli-3 pathway
To investigate the genetic relationship between tsp-15 and newly isolated mutants, we performed mutant rescue assays (Figure 2, Table 1).Over-expression of tsp-15 did not restore the defects of the bli-3 and doxa-1 mutants.Over-expression of bli-3 as well as doxa-1 alone did not rescue the tsp-15 mutant.Co-expression of bli-3 and doxa-1 in the tsp-15 hypomorph mutant effectively rescued the cuticle deficiency (Figure 2, Table 1).In contrast, bli-3 and doxa-1 co-expression in the tsp-15 null mutant resulted in partial rescue of the lethal phenotype.In approximately 1.5% of transgenic animals, embryonic lethality was recovered and the larvae showed a tsp-15 hypomorph mutant-like morphology (Figure 2), which has not been observed in tsp-15 null mutants previously.
Decreased dityrosine levels in tsp-15 mutants BLI-3 is reported as a key enzyme for the generation of H 2 O 2 for tyrosine cross-linking in the cuticle since the level of di-and Author Summary ROS are highly reactive molecules, which can be inappropriately produced during aerobic metabolism or by exogenous stresses such as exposure to UV light and radiation.ROS interact with cellular components including nucleic acids, lipids, and proteins and irreversibly inhibit their functions.However, ROS are essential for innate host defense and multiple physiological processes and are generated by conserved NADPH oxidase (NOX) family enzymes.The release of ROS by ROS generator enzymes must be properly controlled, as chronic oxidative stress can cause an imbalance of the redox state and is often associated with disease and aging.Using C. elegans as a model, we identified a tetraspanin (TSP-15) protein as a new key component of the ROS generation system controlled by dual oxidase (BLI-3), a unique NOX isozyme in C. elegans.Mutants of both bli-3 and tsp-15 developed the same defects in extracellular matrix cross-linking.Using a combination of genetics and reconstitution experiments in mammalian cells, we have demonstrated a novel requirement of tetraspanin for dual oxidase-dependent ROS generation via complex formation at the cell surface.
tri-tyrosine formation is reduced in the cuticle of bli-3(RNAi) animals [17].To examine distribution of cross-linked tyrosine in the exoskeleton of tsp-15 mutants, we carried out immunohistochemical analysis using anti-di-tyrosine antibody.We also checked the endogenous distribution of DPY-7 (a nematode collagen) in tsp-15 mutants by immunostaining.In the normal embryo, di-tyrosine was distributed over the entire cuticle representing the body surface structure, whereas di-tyrosine formation was severely reduced in tsp-15 null embryos (Figure 3A).In contrast, the level of collagen found in tsp-15 null embryos, examined via DPY-7, was comparable to tsp-15(+) embryos, although distribution was severely disturbed (Figure 3B).Thus, cuticle collagen is likely synthesized and secreted from the hypodermis correctly, but a failure of cross-linking of secreted collagen results in fragility of the cuticle in tsp-15 mutants.DUOX was originally identified as a hydrogen peroxide generator for thyroid hormone synthesis [9,11] and DUOXA is essential for DUOX targeting to the plasma membrane [33].We produced stable transfectants of TSP-15, BLI-3 and DOXA-1 in human HT1080 cells (Figure 4A) to confirm the roles of TSP-15 and DOXA-1 for BLI-3.Release of H 2 O 2 into the culture medium was measured in the absence of other C. elegans proteins.Extracellular H 2 O 2 from HT1080 B and HT1080 TB cells was almost equivalent to basal activity (see Materials and Methods for description of stable transfectants).Unlike the mammalian DUOX system, BLI-3 was only slightly activated by co-expression of DOXA-1 in HT1080 DB cells.In addition to DOXA-1, concomitant expression of TSP-15 strongly enhanced production of H 2 O 2 in HT1080 TDB cells (Figure 4B).The generation of H 2 O 2 was blocked by the flavoprotein inhibitor diphenyleneiodonium (DPI), indicating that DUOX was involved in enhanced H 2 O 2 production in TSP-15-transduced cells.We concluded that BLI-3 requires TSP-15 and DOXA-1 for proper function.BLI-3 P1311L and BLI-3 G246D identical to the im10 or e767 mutation, respectively, resulted in decreased H 2 O 2 production (Figure 4B).The same results were observed in other independently established stable cell lines, and by transient expression in COS-7 and HeLa cells (data not shown).Regulation of Ca 2+ characteristically elicits DUOX activity as a thyroid hormone synthesizer.BLI-3 did not require calcium stimulation to produce H 2 O 2 in HT1080 TDB cells, and HT1080 DB cells were not activated by calcium stimulation either (Figure 4C).This may be due to the fact that the critical amino acid residues for Ca 2+ -binding are poorly conserved in the EF-hand motifs of BLI-3 proteins [17].Furthermore, BLI-3 was not activated by forskolin (fsk) and phorbol 12-myristate 13-acetate (PMA), which have previously been reported to be mammalian DUOX stimulators (Figure 4C) [54].

BLI-3 activation by TSP-15 at the cell surface
The molecular requirement of TSP-15 in the BLI-3 system is possibly due to formation of a complex at the plasma membrane, therefore BLI-3 could be activated by TSP-15 localized at the cell surface.We assessed this hypothesis through cell fusion-based analysis (Figure 6A).TSP-15-expressing cells (HT1080 T ) and DOXA-1/BLI-3-expressing cells (HT1080 DB ), both of which did not produce H 2 O 2 (Figure 4B), were fused utilizing Sendai virus (HVJ).After the cell fusion reaction, extracellular H 2 O 2 production from fused cells was measured.The fused cells (T::DB) produced H 2 O 2 , and the production was inhibited by DPI treatment (Figure 6B).In contrast, BLI-3 carrying the P1311L mutation did not result in H 2 O 2 production.Inhibition of de novo protein synthesis by cycloheximide (CHX) resulted in a slight decrease in H 2 O 2 producing activity in T::DB fusion cells (Figure 6B).These results indicate that TSP-15 did not promote BLI-3 protein expression, but existing TSP-15 at the cell surface was sufficient to activate BLI-3 for H 2 O 2 production.The capability of H 2 O 2 production was rapidly acquired after cell fusion.H 2 O 2 was immediately detectable in T::DB fusion cells 15 min after the fusion reaction (Figure 6C), indicating that alternatively derived TSP-15, BLI-3 and DOXA-1 rapidly formed functional units at the cell surface.

Discussion
Organisms have developed regulatory systems to control ROS generation in host defense and cellular signaling.For mammalian DUOX proteins, association with a maturation factor (DUOXA) for targeting to the plasma membrane, Ca 2+ regulation via EFhand motifs, and PKA-or PKC-mediated phosphorylation were identified as regulatory systems.We propose that the tetraspanin protein is a novel component of the DUOX system for ROS generation (Figure 7).Using C. elegans as a model organism, we identified a series of genes for ROS generation in which mutants exhibited a phenotype resembling the tetraspanin tsp-15 mutant.The genes bli-3, doxa-1, and mlt-7 were, respectively, the homologues of mammalian DUOX, DUOXA and peroxidase, and mutants of these displayed the same cuticle deficiency (Figure 1, Figure 2, Figure S1, Figure S2).The reason for this cuticle disorganization is deterioration of tyrosine cross-linking in cuticle development as shown in bli-3 knockdown-animals (Figure 3) [17].We showed that the tsp-15 mutant was rescued by simultaneous over-expression of bli-3 and doxa-1, implying that these three genes are part of the same genetic pathway (Figure 1, Figure 2).Reconstitution of BLI-3, TSP-15 and DOXA-1 in mammalian cells demonstrated that H 2 O 2 generation by BLI-3 was dependent on TSP-15 as well as DOXA-1 (Figure 4).
It was hypothesized that TSP-15 might enhance BLI-3 protein levels by elevating protein expression or promoting targeting to the cell surface; however, BLI-3 and DOXA-1 protein expression levels were comparable, with or without TSP-15 expression.TSP-15 expression did not affect BLI-3 expression at the cell surface, and did not enhance the association of BLI-3 and DOXA-1.This implies that the role of TSP-15 in the BLI-3 system is not just augmentation of its expression.Our observations support this notion, since over-expression of bli-3 and doxa-1 resulted in incomplete rescue of tsp-15 null mutants.In the sv15 hypomorph mutant, tsp-15 expression was reduced, and expressed at 10% of wild-type levels (data not shown), therefore the BLI-3 system recovered to produce adequate H 2 O 2 by concomitant overexpression of bli-3 and doxa-1.If the up-regulation of BLI-3 activity by TSP-15 is quantitative, tsp-15 null mutants should be completely rescued by BLI-3/DOXA-1, however this was not the case.The molecular role of tetraspanin in the DUOX system is likely qualitative.We believe that TSP-15 up-regulates the activity of BLI-3 at the plasma membrane.Cell fusion-based analysis strongly supports this idea, since cells that acquired TSP-15 from other cells rapidly produced H 2 O 2 even when protein synthesis was inhibited.During the HVJ-mediated fusion process, intracellular organelles were morphologically altered and repaired within 30 min [55].We observed that H 2 O 2 generation was initiated 15 min after recovery, suggesting that individually derived BLI-3/ DOXA-1 and TSP-15 rapidly assembled at the cell surface, forming functional complexes.The lipid raft marker protein, flotillin, was rapidly assembled during cell fusion [56].Inhibition of de novo protein synthesis did not affect H 2 O 2 production in fusion cells, indicating that the existing TSP-15 at the cell surface is sufficient for facilitation of BLI-3 activity.
The molecular mechanisms of up-regulation are still unclear, but we showed that TSP-15, BLI-3 and DOXA-1 form complexes in vitro and in vivo (Figure 5).BLI-3 directly associates with DOXA-1, as demonstrated in mammalian DUOX and DUOXA.We also demonstrated the association between BLI-3 and TSP-15, and that this was independent of DOXA-1 expression.It is known that tetraspanin associates with a number of membrane proteins and forms large protein complexes at certain membrane microdomains.We speculate that TSP-15 may establish or maintain a specialized membrane microdomain that facilitates generation of H 2 O 2 in conjunction with BLI-3.As reported for other NOX isozymes and their subunits, association with TSP-15 might induce a conformational change in BLI-3 to function properly.Alternatively, TSP-15 may support the recruitment of unknown factors at the membrane microdomain that are essential for BLI-3 activity.Although DOXA-1 is essential for H 2 O 2 production by BLI-3, the role of DOXA-1 in the BLI-3 system is unclear.Unlike mammalian DUOX, BLI-3 was unexpectedly recruited to the plasma membrane in the absence of DOXA-1.DOXA-1 might not regulate BLI-3 trafficking in the DUOX system in C. elegans, but we cannot exclude the possibility that expression of C. elegans proteins in mammalian cells may cause dysregulation in BLI-3 trafficking.Further investigation is needed to clarify the molecular functions of DOXA-1 in the BLI-3 system.In addition, unlike the mammalian DUOX system, BLI-3 did not respond to various stimuli when reconstituted in mammalian cells (Figure 4C).Absence of negative regulatory factors may explain the constitutive active state of BLI-3 in the heterologous system.For instance, NOXA1 has an inhibitory role in stabilizing the inactive state of mammalian DUOX1 [57].No NOXA1-like sequence has been found in C. elegans, but further investigation would clarify this hypothesis.
Reciprocal homology searches suggested that several human tetraspanins are related to TSP-15 with CD151 (TSPAN24) and TSPAN11 being the most closely related.However, we have not identified any mammalian tetraspanins that could be functionally substituted for TSP-15 in the tsp-15 mutant [51], or for H 2 O 2 production in the BLI-3/DOXA-1 reconstitution system (data not shown).It is also uncertain whether mammalian tetraspanins have a pivotal role in mammalian DUOX system.Mutations in tetraspanin genes have not been identified in patients suffering from congenital hypothyroidism.In contrast to other NOX isozymes, understanding the regulation of DUOX proteins is emerging.Our data clearly shows that tetraspanin is a new component for directing DUOX activity, contributing to greater understanding of the molecular mechanisms of ROS generation and disorders caused by impairment of ROS generation systems [58,59].

Worm strains and culture
C. elegans was grown at 20uC on NGM plates as described previously [60].The Bristol N2 strain was used as the wild type.Strains and their genotype used in this study are listed in Table S1.

Mutant screening and identification
N2 or OB43 were mutated with 50 mM ethyl methanesulfonate or 5 mM ethyl nitrosourea for 4 h.F2 recessive mutants showing a sv15 mutant-like phenotype were screened.SNPs between N2 and Hawaiian CB4856 strains were used for physical mapping of the alleles [61].Mutations were determined by further DNA sequencing and confirmed by complementation tests, rescue assays by DNA transformation, and RNAi analyses (see Text S1).Mutants were outcrossed at least five times with N2.

Immunohistochemistry and microscopic imaging of embryos
Embryos were collected from gravid hermaphrodites and were immunostained as previously described [63].Mouse anti-dityrosine (1C3; Nikken Seil Co., Ltd.Shizuoka, Japan), mouse anti-DPY-7 (a gift from Iain L. Johnstone, University of Glasgow) [64] were used at 1:200 and 1:500 dilutions, respectively.Alexa Fluor 488 or Alexa Fluor 546 (Invitrogen) conjugated anti-mouse IgG antibodies were used as secondary antibodies at 1:500 dilutions.Confocal images were acquired with a LSM5 Pascal microscope (Zeiss, Germany).The three-dimensional projections were reconstructed using images of serial Z-section (1-1.5 mm).Micrographs of fluorescence microscopy were captured using a BX50 microscope (Olympus, Tokyo, Japan) equipped with a VB7010 digital CCD camera (Keyence, Osaka, Japan).Image processing and movie construction was performed with Adobe Photoshop CS4 and Image J 1.34, respectively.
Cell culture and transfection HT1080, HeLa, and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum.Transfection for transient expression was performed using FuGENE HD reagent (Roche, Germany) according to the manufacturer's protocol.Stable transfectants were obtained by retrovirus transfection [62].Plasmids were transfected into Plat-E packaging cells with FuGENE 6 reagent (Roche).Culture supernatant was added to HT1080 ecoR, HeLa ecoR and COS-7 ecoR cells which are stable transfectants of the ecotropic retrovirus receptor, mCAT-1 (gifts from Hiroto Mizushima, Osaka University).Transfected cells were selected by incubation with a combination of 1 mg/ml puromycin, 10 mg/ml blastcidine S, and/ or 300 mg/ml zeocin for at least two weeks.Stable transfectants of C. elegans genes were named after the transgene that was transduced.T, D, and B refer to tsp-15, doxa-1, and bli-3, respectively (e. g.HT1080 TDB cells express tsp-15, doxa-1, and bli-3).

Monitoring H 2 O 2 production in mammalian cells
The H 2 O 2 release into culture supernatants was measured using Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine; Invitrogen), which reacts with H 2 O 2 and is transformed into fluorescent resorufin in the presence of peroxidase [65].Stable or transient transfectants were plated on 96-well plates at 1-5610 4 cells/well, and cultured for 24-48 h.Cells were incubated in 100 ml Hanks' balanced salt solution containing 50 mM Amplex Red, and 0.1 U/ml HRP (Nacalai Tesque, Kyoto, Japan), with or without 10 mM diphenyleneiodonium (DPI), 1 mM ionomycin, 1 mM forskolin (Fsk), or 1 mM phorbol 12-myristate 13-acetate (PMA) at 37uC for 1 h.The fluorescence (530 nm excitation, 590 nm emission) was measured by a Power Scan HT (DS Pharma Biomedicals, Osaka, Japan).Foldincrease was compared with the basal activity of non-transfected HT1080 cells and was determined from least four independent experiments.

HVJ-mediated cell fusion
HVJ (Sendai virus)-mediated cell fusion was performed using GenomONE-CF (Ishihara Sangyo Co. Ltd., Osaka, Japan).Cells Venus-tagged DOXA-1 was analyzed with anti-GFP antibody.BLI-3 was co-immunoprecipitated with endogenous and Xpress-tagged TSP-15 and Venus-tagged DOXA-1 in 1% Triton-X100 cell lysates.Endogenous TSP-15 also associated with DOXA-1::Venus.Normal rat and mouse IgG was used as a negative control.doi:10.1371/journal.pgen.1002957.g005were harvested using trypsin.HT1080 DB or HT1080 DBP1311L cells (8610 5 ) were mixed with HT1080 ecoR or HT1080 T cells (8610 5 ) in a total volume of 200 ml reaction buffer.A 1 ml volume of inactivated HVJ was then added to the cell mixture.After incubation for 15 min at 37uC, 1 ml of DMEM was added and further incubated for 1 h at 37uC for recovery.Cells were treated with 10 mg/ml cycloheximide (Sigma-Aldrich) during incubation for inhibition of protein synthesis.Cells were washed, and H 2 O 2 production was measured as described above.For the time-course experiment, recovery incubation was examined from 0-120 min, and H 2 O 2 production was measured after a 30 min incubation.
For visualization of the fusion event, HT1080 DB cells were prestained with 10 mM Cell Tracker Orange (Invitrogen) for 20 min at 37uC, and then fused with GFP-expressing HT1080 cells.Each experiment was tested in duplicate and performed at least three times.

Statistical analysis
Data are presented as mean value and error bars indicate the standard error of the mean (SEM) from multiple independent assays.Significance was determined using a two-tailed Student's ttest.

Figure 1 .
Figure 1.bli-3 and doxa-1 mutants are similar to the tsp-15 mutant.(A) The structure of the gene/proteins related to tsp-15 function.Schematic representation of the BLI-3 and MLT-7 protein with functional domain, and the genomic structure of the doxa-1 gene.The im10, im21, im32, im38 and im39 mutations are indicated.Previously identified missense mutations in the bli-3 gene including e767 (glycine to aspartic acid at 246) and n529 (aspartic acid to asparagine at 392) are shown.The bold line indicates the region of the gk141 deletion allele.The im10 mutation has a leucine instead of a proline at position 1311 within the NOX domain.TM and NOX refer to the transmembrane and NOX domains, respectively.The im21 mutation is characterized by a G to A transition in the splice donor site at the fifth intron.The im32 mutation is a G to T transversion in the splice acceptor site at the fourth intron.The im38 and im39 alleles are indicated in the MLT-7 protein.Both alleles contain the missense mutations in the peroxidase domain.(B) bli-3(im10) and doxa-1(im21), but not bli-2(e768) are similar to tsp-15(sv15).Hypodermal expression of GFP driven by dpy-7p::gfp in the mutants revealed an unusual accumulation of cellular materials in the blisters of bli-3, doxa-1 and tsp-15 mutants (indicated by black arrows), but not in bli-2 mutants (indicated by the white arrow).The scale bars represent 50 mm.doi:10.1371/journal.pgen.1002957.g001

Figure 3 .
Figure3.Deterioration of dityrosine in the tsp-15 mutant.(A) Representative immunofluorescent images showing the distribution of dityrosine in embryos of the tsp-15(ok854) null mutant.Embryos were obtained from the OB129 strain, which was the tsp-15(ok854) mutant rescued by a tsp-15p::(His)6 Xp::tsp-15 extrachromosomal array.Nuclear GFP fluorescence by sur-5::gfp defined the rescued (tsp-15(+)) or spontaneously arraylost (null; tsp-15(0)) embryo.Micrographs on the left show merged Nomarski images exhibiting GFP and dityrosine immunolocalization.Right panels show the reconstruction of confocal images for dityrosine distribution in the same embryo that is displayed on the left.In tsp-15(+) normal embryos, dityrosine localization showed a regular pattern representing the cuticle surface structure.Fluorescence intensity was severely deteriorated in tsp-15(0) embryos.Scale bars indicate 10 mm.(B) DPY-7 localization was compared under the same conditions as in (A).In tsp-15(+) embryos, DPY-7 localized as regular bands in the cuticle.In the tsp-15(0) embryo, the expression of DPY-7 was comparable to the normal embryo despite its disorganized pattern.Scale bars indicate 10 mm.doi:10.1371/journal.pgen.1002957.g003

Figure 6 .
Figure 6.Requirement of TSP-15 for reconstitution of BLI-3 function at cell surface.(A) HVJ-mediated cell fusion.GFP-expressing HT1080 cells and HT1080 DB cells labeled with Cell Tracker Orange were fused with HVJ.Under HVJ(+) conditions, fused cells were large compared with HVJ(2) cells and exhibited yellow/orange fluorescence.Scale bar indicates 50 mm.(B) HT1080 DB cells fused with HT1080 T cells (T::DB) produced H 2 O 2 , which was inhibited by DPI.Mock::DB and T::DB P1311L fusion cells did not produce H 2 O 2 .Treatment of T::DB fusion cells with 10 mg/ml cycloheximide (CHX) did not inhibit H 2 O 2 production.The graph shows the means 6 SEM.The number of independent experiments is indicated.*P,10 26 .(C) Rapid H 2 O 2 production from T::DB fusion cells.The recovery time after the fusion event was examined to determine when fusion cells acquired the ability to produce H 2 O 2 .Maximum H 2 O 2 production was observed at 30-60 min, although production was observed at 15 min post-fusion.The graph shows the means 6 SEM (n = 3).doi:10.1371/journal.pgen.1002957.g006

Figure 7 .
Figure 7. Molecular regulation of BLI-3 by TSP-15 and DOXA-1.TSP-15 and DOXA-1 are essential for H 2 O 2 production by BLI-3.TSP-15 associates with BLI-3 at the cell surface or during trafficking.The role of DOXA-1 in BLI-3 targeting to the plasma membrane remains elusive.BLI-3/ DOXA-1 complexes at the cell surface are inactive, but recruiting to the tetraspanin-microdomain facilitates the formation of a functional unit for generation of H 2 O 2 that is utilized by innate host immunity, and cross-linking of extracellular matrix with peroxidase (MLT-7).doi:10.1371/journal.pgen.1002957.g007