Stage-specific activation of MIG-17 ⁄ ADAMTS controls cell migration in Caenorhabditis elegans Shinji Ihara 1 and Kiyoji Nishiwaki 1,2 1 RIKEN Center for Developmental Biology, Hyogo, Japan 2 Department of Bioscience, Kwansei-Gakuin University, Hyogo, Japan The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family is a group of zinc- dependent metalloproteases that mediate a wide variety of extracellular proteolytic events, including degrada- tion of extracellular matrix (ECM) components, such as proteoglycans and collagens [1–6]. The ADAMTS family consists of 19 genes in mammalian genomes and of five in the Caenorhabditis elegans genome. ADAMTS members contain a signal peptide, a prodomain, a metalloprotease (MP) domain, a disintegrin (DI) domain, a variable number of thrombospondin type I (TS) motifs, and additional domains near the C-termi- nus [7–9]. The prodomain maintains enzymatic latency of the proenzyme and is proteolytically removed to produce the active mature enzyme [10]. Although several ADAMTSs are activated by proteolytic processing in the trans-Golgi network by serine proteases such as furin [11,12], at least proportions of the ADAMTS-1, ADAMTS-7B, ADAMTS-9 and ADAMTS-10 enzymes are secreted as proforms and localize to the cell surface Keywords activation; ADAMTS protease; antibody; Caenorhabditis elegans; MIG-17 Correspondence K. Nishiwaki, Department of Bioscience, Kwansei-Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan Fax: +81 79 565 9077 Tel: +81 79 565 7639 E-mail: nishiwaki@kwansei.ac.jp (Received 18 April 2008, revised 9 June 2008, accepted 25 June 2008) doi:10.1111/j.1742-4658.2008.06573.x The activation of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family proteases depends on removal of the prodomain. Although several studies suggest that ADAMTS activities play roles in development, homeostasis and disease, it remains unclear when and where the enzymes are activated in vivo. MIG-17, a Caenorhabditis elegans glycoprotein belonging to the ADAMTS family, is secreted from the body wall muscle cells and localizes to the gonadal basement membrane to control the migration of gonadal distal tip cells. Here, we developed a monoclonal antibody that recognizes the N-terminal neo-epitope of the activated MIG-17. In western blotting, the antibody specifically detected the activated form, the signal for which dramatically increased during the third and fourth larval stages, when MIG-17 is required to direct distal tip cell migration. In in situ staining, the mono- clonal antibody recognized the activated form in the basement membrane, whereas it failed to detect a processing-resistant mutant form localized to the basement membrane. MIG-17 was activated in the basement mem- branes of the muscle, intestine and gonad in the third larval stage, and downregulated in nongonadal basement membranes in young adults and in gonadal basement membranes in older adults. Thus, the activation of MIG-17 is regulated in a spatiotemporal manner during C. elegans development. This is the first report demonstrating the regulated activation of an ADAMTS protein in vivo. Our results suggest that monoclonal anti- bodies against neo-epitopes have potential as powerful tools for detecting activation of ADAMTSs during development and in disease pathogenesis. Abbreviations ADAMTS, a disintegrin and metalloprotease with thrombospondin motifs; DI, disintegrin; DTC, distal tip cells; ECM, extracellular matrix; GFP, green fluorescent protein; KLH, keyhole limpet hemocyanin; L, larval stage; MP, metalloprotease; PLAC, protease and lacunin; TS, thrombospondin type I. 4296 FEBS Journal 275 (2008) 4296–4305 ª 2008 The Authors Journal compilation ª 2008 FEBS or ECM [7,13–15]. We recently showed that MIG-17, an ADAMTS in C. elegans with a signal peptide, prodo- main, MP domain, DI domain and protease and lacunin (PLAC) domain [16] (Fig. 1A), is also secreted as a pro- form and localizes to the basement membrane of the developing gonad [17]. Conceivably, these secreted pro- form enzymes can be activated in the extracellular envi- ronment to function in target tissues. However, the timing of activation of secreted pro-ADAMTSs during organogenesis has not been explored. The gonad of the C. elegans hermaphrodite consists of two symmetrical U-shaped arms formed by direc- tional migration of gonadal leader cells called distal tip cells (DTCs) (Fig. 1B). The DTCs are formed at the anterior and posterior ends of the gonad primordium of the C. elegans hermaphrodites in the first larval (L1) stage, and start to migrate on the ventral body wall muscle in L2. They make two 90° turns during L3 and L4, generating symmetrical U-shaped gonad arms [18] (Fig. 1B). The spatial and temporal patterns of DTC migration are strictly regulated by extracellular environmental cues involving various secreted and membrane-bound proteins [19–26]. MIG-17 is secreted from the body wall muscle cells and localizes to the gonadal basement membrane, where it is required for directional migration of DTCs [16]. In mig-17 mutants, the DTCs migrate aberrantly, causing the gonad arms to be deformed (Fig. 1B, right bottom). The MIG-17-dependent control of gonad develop- ment offers an excellent system with which to study the function of ADAMTSs during organogenesis in vivo. In this study, we isolated recombinant MIG-17 and determined the amino acid sequence of the N-ter- minal neo-epitope of the activated form, which is generated by prodomain removal. A monoclonal anti- body raised against this neo-epitope specifically recog- nized the activated MIG-17 in vivo. We demonstrate that MIG-17 is activated in basement membranes in worms and that the activation is spatially and tempo- rally regulated during development. Results MIG-17 is activated during development in C. elegans We previously showed that activation of MIG-17, which controls DTC migration, requires removal of A B CD Fig. 1. MIG-17 is activated during larval de- velopment. (A) Domain structure of MIG-17. (B) Developmental stages and gonad mor- phogenesis in C. elegans hermaphrodites. The cartoon at the bottom right shows abnormal gonad morphology in mig-17 mutant adults. The sizes of the animals do not correlate with the actual sizes. (C, D) Activation of MIG-17 during larval develop- ment. Cell lysates from each developmental stage were prepared from L1 to the adult stage. Cell lysates (20 lg) from staged wild-type worms expressing MIG-17–GFP or MIG-17(E303Q)–GFP were subjected to SDS ⁄ PAGE followed by immunoblotting with anti-GFP IgG. Arrow, proform; arrowhead, activated form; SP, signal peptide. S. Ihara and K. Nishiwaki Regulated activation of MIG-17 ⁄ ADAMTS FEBS Journal 275 (2008) 4296–4305 ª 2008 The Authors Journal compilation ª 2008 FEBS 4297 the prodomain [17]. Using an MIG-17–green fluores- cent protein (GFP) fusion protein, we examined the levels of the activated form (prodomain removed) during larval development. We found that the level of activated MIG-17–GFP was substantially increased in L3 and L4 as compared to L1, L2 and the adult stage (Fig. 1C), suggesting that the process- ing activity of MIG-17 is specifically upregulated during L3 and L4, when DTCs actively migrate. Consistent with our in vitro experiments [17], when MIG-17–GFP with a mutation in the catalytic active site [MIG-17(E303Q)–GFP] was expressed in wild- type animals, no activated form was produced, even in L3 and L4 (Fig. 1D), indicating that MIG-17 prodomain processing depends on MIG-17 activity and is mediated by autocatalytic activation. Identification of the prodomain cleavage site A C-terminally histidine-tagged MIG-17 (MIG-17– 6His) was expressed in Sf21 cells infected with recombinant baculovirus and was efficiently secreted into the culture medium. MIG-17–6His was purified from the culture medium using an Ni 2+ -chelating Sepharose FF column, and two protein bands of 70 and 43 kDa, which appeared to correspond to the proform and activated form, were detected by SDS ⁄ PAGE (Fig. 2A). Immunoblot analysis with an antibody against polyhistidine revealed the same-sized bands, suggesting that the C-terminal histidine tag remained in both the proform and activated form in the recombinant enzyme after its secretion into the culture medium (Fig. 2B). To determine the N-terminal sequence of the acti- vated form of MIG-17–6His, we transferred it onto a poly(vinylidene difluoride) membrane and performed N-terminal Edman degradation sequencing. The obtained peptide sequence, FVDIT, matched with the MIG-17 sequence from residues 207 to 211, indicating that the cleavage occurred between Lys206 and Phe207 (Fig. 2C). Generation of antibodies against the proform and activated form of MIG-17 We previously showed that MIG-17 is recruited to the gonad surface in a prodomain-dependent manner (prodomain targeting) and that the prodomain must be removed for MIG-17 to be activated to control DTC migration [17]. These observations suggested that the activation (prodomain removal) of MIG-17 occurs on the gonad surface. To explore the activation of MIG-17 in vivo, we developed monoclonal antibodies against the potential N-terminal neo-epitope – the FVDITLEE peptide observed at the N-terminus of MIG-17–6His secreted from Sf21 cells (Fig. 2C). A polyclonal antibody was also raised using the poly- peptide of the MIG-17 activated form expressed in Escherichia coli (Fig. 2C). The polyclonal antibody recognized both the proform and activated form of MIG-17–GFP expressed in worms and MIG-17–6His expressed from Sf21 cells (Fig. 3A). Two hybridomas were selected on the basis of their ability to bind the neo- epitope peptide but not the peptide spanning the processing site of MIG-17–6His using ELISA (Fig. 2C). We produced two monoclonal antibodies, Monoclonal 1 and Monoclonal 2, that recognized the activated form of MIG-17 (Fig. 3B,C). We tested their specificities against activated forms derived from either MIG-17–GFP or MIG-17–6His. Mono- clonal 1 predominantly recognized the activated A C B Fig. 2. Purification of MIG-17–6His and production of antibodies. (A, B) Detection of purified MIG-17–6His. (A) Purified MIG-17–6His (300 ng) was separated on a 10% SDS ⁄ PAGE gel and stained with Coomassie Brilliant Blue (CBB). (B) Recombinant MIG-17–6His was detected by immunoblotting using antibody to polyhistidine. (C) Antigens used for antibody production. The regions used to pro- duce polyclonal antibodies against the prodomain (antigen 1) [17] and the activated form (antigen 2) are indicated. The amino acid sequence of the neo-epitope (underlined) used to produce the monoclonal antibodies is shown with surrounding sequences. The sequence determined by Edman degradation is in bold. The neo- epitope and the spanning peptide used for selection of hybridomas are shown. SP, signal peptide. Regulated activation of MIG-17 ⁄ ADAMTS S. Ihara and K. Nishiwaki 4298 FEBS Journal 275 (2008) 4296–4305 ª 2008 The Authors Journal compilation ª 2008 FEBS forms of MIG-17–His and MIG-17–GFP but also detected a faint band of the MIG-17–6His proform and an additional lower molecular weight band in worm lysates (Fig. 3B). Monoclonal 2 specifically rec- ognized the activated forms of MIG-17–GFP and MIG-17–6His without detecting the proforms (Fig. 3C). When we examined MIG-17–GFP expres- sion along developmental stages using Monoclonal 2, we detected strong bands corresponding to the acti- vated form during L3 and L4, weak bands in L2 and the adult stage, and no bands in L1 (Fig. 3D). These results indicated that Monoclonal 2 specifically recognized the neo-epitope produced in the processed ⁄ activated form of MIG-17, and suggested that the in vivo processing of MIG-17–GFP occurred at the same site where MIG-17–6His was processed. MIG-17 activation is regulated spatially and temporally during development Using an antibody against the prodomain, we previ- ously demonstrated that the MIG-17 proform localizes to the surfaces of the gonad, intestine and hypodermis corresponding to the basement membranes [17]. To determine the sites of activation of MIG-17 and tissue distribution of activated MIG-17 in vivo, we performed immunostaining using Monoclonal 2. When we tried to detect endogenous MIG-17, the antibody showed no signal in nontransgenic worms, suggesting that endogenous activated MIG-17 is expressed below the level of detection of the antibody. Thus, we stained animals expressing the MIG-17–GFP fusion protein, which can rescue mig-17 mutants [16,17]. Immuno- A D BC Fig. 3. Characterization of antibodies. Poly- clonal and monoclonal antibody specificities evaluated by western blotting. (A–C) Protein samples were immunoblotted with poly- clonal antibody to activated MIG-17 (A), Monoclonal 1 (B), and Monoclonal 2 (C). Lysates prepared from wild-type worms expressing MIG-17–GFP were incubated at room temperature for the indicated periods and immunoblotted (left two lanes). Purified MIG-17–6His, including both the proform and the activated form, was immunoblotted (right lane). Single asterisk, proform of MIG- 17–GFP; double asterisk, activated form of MIG-17–GFP; arrow, proform of MIG-17– 6His; arrowhead, activated form of MIG-17– 6His. (D) Lysates prepared from staged wild-type worms expressing MIG-17–GFP were immunoblotted with Monoclonal 2. Arrowhead, activated form of MIG-17–GFP. S. Ihara and K. Nishiwaki Regulated activation of MIG-17 ⁄ ADAMTS FEBS Journal 275 (2008) 4296–4305 ª 2008 The Authors Journal compilation ª 2008 FEBS 4299 staining of cross-sections of L3 larvae with antibody to GFP revealed signals in the cytoplasm of muscle cells, the source of MIG-17 expression, and on the surface of the gonad and the intestine (Fig. 4A). The poly- clonal antiactivated form antibody showed a similar staining pattern (data not shown). In contrast, Mono- clonal 2 detected signals only on the surfaces of muscles, intestines, and gonads (Fig. 4B). To assess the specificity of the monoclonal antibody, we stained animals expressing MIG-17(KK202LL)–GFP, in which Lys202 and Lys203 were replaced with two leucines so that the MIG-17 prodomain could not be processed [17]. Although we obtained a similar staining pattern as for MIG-17–GFP using antibody to GFP (Fig. 4C), Monoclonal 2 gave no signal (Fig. 4D). We obtained the same results using Monoclonal 1 (data not shown), indicating that these monoclonal antibodies specifically detected the processed ⁄ activated form. Coimmuno- staining of worms expressing MIG-17–GFP with Monoclonal 2 and the antibody to the prodomain revealed that the signal colocalized to the basement membrane of the muscle and the gonad (Fig. 4E). These results indicate that activation of MIG-17 occurred in the basement membranes of the muscle, intestine and gonad in the L3 larvae. We next studied the activation of MIG-17 in adult animals expressing MIG-17–GFP. Both antibody to GFP and antibody to the prodomain labeled the muscle cell cytoplasm and the surfaces of the muscle, intestine and gonad in young adults, as in L3 larvae (Fig. 5A,B). Although Monoclonal 2 recognized the gonadal basement membrane, the staining of intestinal and muscular basement membranes was often faint and discontinuous (Fig. 5C). In the older adults, AB CD EF Fig. 4. MIG-17 activation in L3 larvae. (A–D) Cross-sections of wild-type animals express- ing MIG-17–GFP (A, B) or expressing MIG- 17(KK202LL)–GFP (C, D) were stained with anti-GFP IgG (pink), Monoclonal 2 (orange), fluorescein–phalloidin (green) and DAPI (blue). Fluorescein–phalloidin labeled actin in the outer surface of body wall muscle cells. (E) Coimmunostaining of anti-prodomain (pink) and Monoclonal 2 (green). Overlap of the two antibody stains appears white. (F) Schematic presentation of a cross-section of an L3 larva. Scale bar, 10 lm. Top, dorsal. Regulated activation of MIG-17 ⁄ ADAMTS S. Ihara and K. Nishiwaki 4300 FEBS Journal 275 (2008) 4296–4305 ª 2008 The Authors Journal compilation ª 2008 FEBS although antibody to GFP still detected most of the basement membranes, the signal from Monoclonal 2 became very faint even in the gonadal basement mem- brane (Fig. 5D,E). Taken together, these results indi- cated that although MIG-17 was activated in various basement membranes during the L3 and L4 stages, activation lessened in nongonadal tissues in the young adult stage, and the activation in the gonad mostly ceased in older adults. Discussion In the present study, we expressed the MIG-17 cDNA in Sf21 cells and detected the proform and activated form of MIG-17–6His secreted into the medium. N-terminal sequencing of the activated MIG-17–6His revealed the sequence FVDIT, indicating that proteo- lytic processing occurs between Lys206 and Phe207. This was unexpected, because in a previous study we found that replacement of Arg205–Lys206 with Leu– Leu did not affect the processing, whereas replacement of Lys202–Lys203 with Leu–Leu strongly inhibited it [17]. It is possible that MIG-17 with Leu–Leu in place of Arg205–Lys206 is cleaved at a different site. Alter- natively, MIG-17 might be first processed near Lys202–Lys203 and subsequently between Lys206 and Phe207, although the latter processing would not be essential for activation. Using the N-terminal peptide information from the activated MIG-17–6His, we produced a monoclonal antibody that specifically recognizes the activated MIG-17 in vivo. Western blot analysis revealed that the activated form of MIG-17 is markedly increased during L3 and L4 when DTCs actively migrate, indi- cating that MIG-17 activation in C. elegans is linked to the developmental stages in which leader cells migrate. Consistent with these results, activation of MIG-17 was extensive in the basement membranes of muscle, intestine and gonad in L3. Although MIG-17 is secreted from muscle cells of late embryos, it begins to accumulate at the gonadal basement membrane soon after the first turn of DTCs in mid-L3. This AB C DE F Fig. 5. MIG-17 activation in the adult stage. Cross-sections of young adults (A–C) and 1-day-old adults (D, E) expressing MIG-17–GFP were stained with anti-GFP IgG (pink), antibody to prodomain (pink), Monoclonal 2 (orange), fluorescein–phalloidin (green), and DAPI (blue). (F) Schematic presentation of a cross-section of an adult worm. Scale bar, 10 lm. Top, dorsal. S. Ihara and K. Nishiwaki Regulated activation of MIG-17 ⁄ ADAMTS FEBS Journal 275 (2008) 4296–4305 ª 2008 The Authors Journal compilation ª 2008 FEBS 4301 timing of MIG-17 localization coincides well with the appearance of DTC migration defects in mig-17 mutants [16]. DTC migration in mig-17 mutants mean- ders from the first turn (mid-L3) to the cessation of migration (late L4). Thus, it is reasonable that the MIG-17 localized to the gonadal basement membrane is activated during L3 and L4 to support the direc- tional migration of DTCs. The question then arises as to what activates MIG-17. MIG-17 localization to the gonadal basement membrane requires the prodomain, and we have suggested that prodomain cleavage⁄ acti- vation is autocatalytic [17]. Therefore, perhaps binding of the prodomain to the gonadal basement membrane results in a conformational change in the pro-MIG-17 so that the catalytic site is apposed to the processing site. The receptor for MIG-17 in the gonadal basement membrane remains unknown. However, identification of this receptor and subsequent in vitro binding studies with MIG-17 may clarify the hypothesis of autocata- lytic activation. MIG-17 is also activated in the intestinal and mus- cular basement membranes in L3. Although no clear defects are found in these tissues in mig-17 mutants, we have often observed that the DTCs detach from the muscle, adhere abnormally to the intestine, and migrate over the intestine. Therefore, activation of MIG-17 in these tissues may allow proper adhesiveness between the basement membranes of the gonad and the muscle or the intestine. Activation of MIG-17 was downregulated in nongo- nadal tissues in the young adult stage and in gonadal tissue in older adults. Although MIG-17 expression in DTCs is sufficient to promote normal DTC migration in mig-17 mutants [16], we have sometimes observed small bulges in the gonad arms. Because the gonad grows substantially during the early adult stages, due to proliferation of the germline, the gonadal basement membrane is probably continuously remodeled and expanded even after cessation of DTC migration. We speculate that MIG-17 may be activated for proper remodeling of the gonadal basement membrane as it is expanded to support the integrity of the gonad. The downregulation of MIG-17 activation might involve expression of protease inhibitors in the basement mem- branes. In mammals, tissue inhibitor of proteinases-3 inhibits ADAMTS proteases [27,28]. Moreover, the extracellular glycoprotein papillin inhibits an ADAMTS in Drosophila [29]. Genes encoding related proteins can be found in the C. elegans genome, but the tissue distri- butions of these proteins remain to be investigated. This article presents the first evidence of in vivo activation of an ADAMTS protein that is essential for correct organ morphogenesis. Monoclonal antibod- ies against neo-epitopes in ADAMTS proteins may prove to be useful diagnostic markers to determine which ADAMTS proteins are activated in particular pathogenic conditions, such as destruction of the ECM in rheumatism. Experimental procedures Strains and culture conditions C. elegans was cultured and handled according to standard methods [30]. The following strains were used: N2 (wild-type) and unc-119(e2498) [31]. The transgenic lines for mig-17:: GFP, mig-17(E303Q)::GFP and mig-17(KK202LL)::GFP have been described previously [16,17]. Plasmid construction The mig-17 cDNA yk151f6 lacked the first 29 nucleotides starting from the initiation codon. These were added by PCR, and the resultant full-length cDNA was cloned into the EcoRI and XhoI sites of a baculovirus transfer vector, pBAC-1 (Novagen, Madison, WI, USA), having a six-histi- dine tag, and the resulting plasmid (mig-17::6His) was puri- fied using a Qiagen Plasmid Mini kit (Qiagen, Valencia, CA, USA). Preparation of recombinant virus mig-17::6His DNA (100 ng) was cotransfected with 500 ng of BacVector-1000 Triple Cut Virus genome DNA (Nov- agen) into 1 · 10 6 Sf21 cells. The transfection was carried out using Cell-Fectin (Gibco-BRL, Rockville, MD, USA). The culture medium containing the recombinant virus generated by homologous recombination was collected 3 days after transfection. The titers of recombinant viruses were further amplified by several rounds of infection prior to use. Preparation of recombinant MIG-17–His Sf21 cells were infected with the recombinant baculovirus carrying mig-17::6His, and the culture medium was harvested 2 days after infection to purify the recombinant protein that had been secreted by the infected cells. The culture medium was applied to an Ni 2+ –nitrilotriacetic acid agarose column (Qiagen) equilibrated with 20 mm phos- phate buffer (pH 7.5) containing 10 mm imidazole. The column was then washed thoroughly with phosphate buffer (pH 7.5) containing 500 mm NaCl and 10 mm imidazole. The MIG-17–6His protein was eluted from the column with 20 mm phosphate buffer (pH 7.5) containing 500 mm NaCl and 500 mm imidazole. Each fraction was analyzed by SDS ⁄ PAGE followed by silver staining to monitor elution Regulated activation of MIG-17 ⁄ ADAMTS S. Ihara and K. Nishiwaki 4302 FEBS Journal 275 (2008) 4296–4305 ª 2008 The Authors Journal compilation ª 2008 FEBS of MIG-17–6His and its purity. Protein concentrations were determined using a bicinchoninic acid kit (Pierce, Rockford, IL, USA) with BSA as a standard. N-terminal Edman degradation sequencing The purified proform and activated form of MIG-17–6His were separated by 10–20% SDS ⁄ PAGE and transferred to a poly(vinylidene difluoride) membrane. After staining with Ponceau Red, the activated form was excised and sequenced by Edman N-terminal degradation. Preparation of a polyclonal antibody against the activated form of MIG-17 To generate an antigen comprising activated MIG-17 con- taining histidines, the coding sequence for residues 271–509 of MIG-17 was inserted into the pET-19b fusion vector (Novagen). The tagged protein was isolated from E. coli transformed with this pET-19b MIG-17–His fusion vector. The rabbit antiserum was purified on a column fixed with the antigen. Production of monoclonal antibodies against the neo-epitope The neo-epitope peptide (FVDITLEE) and the spanning peptide were conjugated to keyhole limpet hemocyanin (KLH). C57BL6 mice were immunized with the neo- epitope–KLH conjugate emulsified in Freund’s complete adjuvant. Lymphocytes isolated from mesenteric lymph nodes were fused with P3-X63 Ag8.U1 (P3U1) mouse mye- loma cells 2 weeks after immunization. Hybridomas with reactivity against the neo-epitope peptide–BSA but not against the spanning peptide–BSA were screened by ELISA. Of 480 hybridomas, six were selected. The immu- noreactivity of two of the six hybridomas was inhibited by addition of the neo-epitope. These two antibodies were named Monoclonal 1 and Monoclonal 2. Western blot analysis For correcting staged worms, eggs were harvested from gravid adults by the alkaline bleaching method as previ- ously described [32]. The eggs were incubated at 22 °C, and the hatched larvae were corrected after 5, 15, 24, 32 and 44 h for L1, L2, L3, L4 and adult samples, respectively. Worms were disrupted by glass beads using Micro Smash MS-100 (Tomy, Tokyo, Japan) in 100 mm Tris ⁄ HCl (pH 7.4), 150 mm NaCl and 1% (w ⁄ v) Triton X-100. After disruption, the lysates were rotated at 4 °C for 30 min, and then centrifuged at 17 400 g for 20 min at 4 ° C. The super- natants were boiled in SDS ⁄ PAGE sample buffer, separated by SDS ⁄ PAGE (7.5% gel), and then transferred to a nitro- cellulose membrane. After blocking with NaCl ⁄ P i contain- ing 3% Blocking One (Nacalai, Kyoto, Japan) for 2 h at room temperature, the membrane was immunoblotted with rabbit anti-GFP IgG (2 lgÆmL )1 ; Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. The membrane was washed three times with NaCl ⁄ P i containing 0.05% (w ⁄ v) Tween-20 for 10 min, and this was followed by incu- bation with peroxidase-conjugated anti-(rabbit IgG) (0.4 lgÆmL )1 ; Amersham, Piscataway, NJ, USA) at room temperature for 1 h. After washing with the same proce- dure, the membrane was developed using the ECL kit (Amersham) according to the manufacturer’s protocol. Microscopy Nomarski and fluorescence microscopy were performed using a Zeiss Axioplan 2 microscope equipped with both optical systems. Images were captured with an Axiocam MRm camera (Zeiss, Oberkochen, Germany) connected to a Windows computer. Plan-Neofluar ·40, 0.75 numerical aperture and C-Apochromat ·63 W objectives (Zeiss) were used. The localization of MIG-17–GFP proteins in cross- section was analyzed with the laser-scanning confocal microscope, LSM5 PASCAL v. 3.2 (Zeiss). In situ staining Frozen sections were prepared as previously described [33]. After blocking of the sections with 1% BSA in NaCl ⁄ P i , samples were incubated with rabbit anti-(activated MIG-17) IgG (2 lgÆmL )1 ), antibody to prodomain (2 lgÆmL )1 ) and rabbit anti-GFP IgG (Invitrogen) for 2 h, tetramethyl rho- damine isothiocyanate donkey anti-(rabbit IgG) (1 : 100; Jackson, Westgrove, PA, USA) for 1 h, fluorescein–phalloi- din (2 UÆmL )1 ; Invitrogen) for 1 h, and 4¢,6-diamidino- 2-phenylindole (DAPI) (2 lgÆmL )1 ; Wako, Osaka, Japan) for 10 min at room temperature. To detect the activated form of MIG-17–GFP, sections were incubated with Mono- clonal 2 for 2 h, and then with tetramethyl rhodamine isothiocyanate donkey anti-(mouse IgG) (1 : 100; Jackson) for 1 h. Images were overlaid using Adobe photoshop 9.0. Acknowledgements We thank Andy Fire (Stanford University Medical Center, CA, USA) for GFP fusion vectors, and members of our laboratory for critical reading of the manuscript. 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ADAMTS members contain a signal peptide, a prodomain, a metalloprotease (MP) domain, a disintegrin (DI) domain, a variable number of thrombospondin. results indicate that activation of MIG-17 occurred in the basement membranes of the muscle, intestine and gonad in the L3 larvae. We next studied the activation of MIG-17 in adult animals expressing. staining of intestinal and muscular basement membranes was often faint and discontinuous (Fig. 5C). In the older adults, AB CD EF Fig. 4. MIG-17 activation in L3 larvae. (A–D) Cross-sections of