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, AD
Trang 1migration in Caenorhabditis elegans
Shinji Ihara1 and Kiyoji Nishiwaki1,2
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.
Trang 2or ECM [7,13–15] We recently showed that MIG-17, an
ADAMTS in C elegans with a signal peptide,
prodo-main, MP doprodo-main, 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
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.
Trang 3the 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 Ni2+-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.
Trang 4forms 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
Fig 3 Characterization of antibodies
Poly-clonal and monoPoly-clonal 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.
Trang 5staining 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,
Cross-sections of wild-type animals express-ing 17–GFP (A, B) or expressexpress-ing 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.
Trang 6although 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
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.
Trang 7timing 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 gonongo-nadal
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· 106Sf21 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 Ni2+–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
Trang 8of 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 22C, 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 4C for 30 min, and
then centrifuged at 17 400 g for 20 min at 4C 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⁄ Pi contain-ing 3% Blockcontain-ing 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⁄ Pi 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⁄ Pi, 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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