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Cell Reports, Volume 9
Supplemental Information
The C. elegans HGF/Plasminogen-like Protein SVH-1
Has Protease-Dependent and -Independent Functions
Naoki Hisamoto, Chun Li, Motoki Yoshida, and Kunihiro Matsumoto
Supplemental Experimental Procedures, relates to Experimental
Procedures.
C. elegans strains.
C. elegans strains used in this study are listed below. All strains were maintained
on nematode growth medium (NGM) plates and fed with bacteria of the OP50
strain, as described previously (Brenner, 1974). Mapping of the kmDp1
duplication by a SNP mapping method (Wicks et al., 2001) revealed that kmDp1
is translocated to the right end on LGX. PCR analysis revealed that kmDp1
carries the intact length of the svh-1 gene with at least the 1.7 kb promoter.
The kmDp1 duplication can be maintained only in heterozygotes, as kmDp1
homozygotes are embryonic or early-larval lethal.
Bristol N2 (WT)
CB4856: Hawaiian strain (for SNP mapping)
KU21: kgb-1(km21) IV.
KU251: svh-1(ok2531) IV; kmDp1/+ X.
KU252: svh-1(ok2531)/nT1[qIs51] (IV, V).
KU253: svh-1(tm2646)/nT1[qIs51] (IV, V).
KU255: svh-2(tm737) X.
KU256: juIs76 II; svh-1(ok2531)/nT1[qIs51] (IV, V).
KU260: juIs76 II; svh-1(ok2531) IV; kmEx505.
KU261: juIs76 II; svh-1(ok2531)/nT1[qIs51] (IV, V); kmEx520.
KU262: juIs76 II; svh-1(ok2531)/nT1[qIs51] (IV, V); kmEx517.
KU263: juIs76 II; svh-1(ok2531) IV; kmEx201.
KU264: juIs76 II; svh-1(ok2531) IV; kmEx202.
KU265: kmEx203 (generated transiently).
KU267: unc-119(e2498) Ⅲ; svh-1(ok2531)/nT1[qIs51] (IV, V); Ex[fbl-1c::venus +
unc-119].
KU268: fbl-1(hd43)/nT1[qIs51] (IV, V).
KU269: svh-1 fbl-1(hd43)/nT1[qIs51] (IV, V).
KU501: juIs76 II.
KU502: juIs76 II; svh-1(ok2531) IV; kmDp1/+ X.
KU508: juIs76 II; svh-1(ok2531) IV; kmDp1/+ X; kmEx505.
KU533: juIs76 II; svh-1(ok2531) IV; kmDp1/+ X; kmEx520.
KU534: juIs76 II; svh-1(ok2531) IV; kmDp1/+ X; kmEx517.
NF1077: unc-119(e2498) Ⅲ; Ex[fbl-1c::venus + unc-119].
Transgenic animals.
Transgenic animals were obtained as described (Li et al., 2012). The
Pjkk-1::svh-1Δcl (25 ng/µl), Pjkk-1::svh-1Δk (25 ng/µl), Pjkk-1::svh-1Δpan (25
ng/µl) and Pmyo-2::DsRed monomer (25 ng/µl) plasmids were used in
kmEx201 (Pjkk-1::svh-1Δcl + Pmyo-2::DsRed monomer), kmEx202
(Pjkk-1::svh-1Δk + Pmyo-2::DsRed monomer), and kmEx203
(Pjkk-1::svh-1Δpan + Pmyo-2::DsRed monomer), respectively. The kmEx505
(wild-type svh-1), kmEx517 [svh-1(h755a)] and kmEx520 (svh-1Δss) transgenes
were described previously (Li et al., 2012).
Suppression of the svh-1 growth defect by fbl-1 RNAi.
RNA interference experiments were conducted by feeding as described in a
previous study (Li et al., 2012). Animals of the svh-1(ok2531)/nT1[qIs51]
genotype were grown from the L4 larval stage, allowed to lay eggs on NGM
plates seeded with bacteria of the E. coli HT115 strain carrying plasmids
expressing a double-stranded RNA targeting fbl-1, and incubated for 5 days.
GFP-negative animals that grew to the adult stage were confirmed to have the
ok2531 deletion by PCR.
Microscopy and laser ablation.
Standard fluorescent images of transgenic animals were observed under a 60 X
objective of a Nikon Eclipse E800 fluorescent microscope and photographed
with a Hamamatsu ORCA 3CCD camera. Movies were taken using the same
system except under a 40 X objective lens. Confocal fluorescent images were
taken on an Olympus FV500 confocal laser scanning microscope with 60 X or
100 X objectives. Laser microsurgery for determining axon regeneration was
performed as described previously (Li et al., 2012).
Phalloidin staining.
Pharyngeal muscles were stained with Rhodamine-conjugated phalloidin based
on the methods described elsewhere (Strome, 1986).
Plasmids.
The Pjkk-1::svh-1(h755a) and Pjkk-1::svh-1Δss plasmids were described
previously (Li et al., 2012). Mutations and deletions were introduced into
Pjkk-1::svh-1 by PCR using PhusionTM DNA polymerase (NEB).
High-speed video imaging of pharyngeal pumping.
High-speed video images were taken using a Nikon Eclipse E800 microscope
(60 X objective) fitted with a Hamamatsu ORCA 3CCD camera.
Homology search, phylogenetic analysis, identification of domains and
alignments of amino acids.
Homology search, identification of conserved domains, signal peptides,
transmembrane regions and alignments of amino acids were executed by the
NCBI BLAST, NCBI CD-search, CBS SignalP4.1, CBS TMHMM2.0 and
Genetyx-Mac programs, respectively. The evolutionary relationships among
candidate genes were determined by constructing neighbor-joining phylogenetic
trees using MEGA5.2 software (Tamura et al., 2011).
Supplemental references
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
Li, C., Hisamoto, N., Nix, P., Kanao, S., Mizuno, T., Bastiani, M., and Matsumoto,
K. (2012). The growth factor SVH-1 regulates axon regeneration in C. elegans
via the JNK MAPK cascade. Nat. Neurosci. 15, 551-557.
Strome, S. (1986). Fluorescence visualization of the distribution of
microfilaments in gonads and early embryos of the nematode Caenorhabditis
elegans. J. Cell. Biol. 103, 2241–2252.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S.
(2011). MEGA5: molecular evolutionary genetics analysis using maximum
likelifood, evolutionary distance, and maximum parsimony methods. Mol. Biol.
Evol. 28, 2731-2739.
Wicks, S.R., Yeh, R.T, Gish W.R., Waterston, R.H., and Plasterk, R.H.A. (2001).
Rapid Gene mapping in Caenorhabditis elegans using a high density
polymorphism map. Nat. Genet. 28, 160-164.
A
TM
Human
SEMA
Amphioxus
P
SEMA
Sea urchin
P
SEMA
I
I
I
SEMA
C. elegans
kinase
I
I
I
I
I
kinase
I
kinase
SEMA
Oyster 2
kinase
I
I
P
SEMA
Oyster 1
I
kinase
P
P
I
kinase
I
B
C. elegans
Water flea
C
Pea aphid
C
Ant
C
Bee
C
Oyster
Sea urchin
C
C
C
C
C
C
C
CL
SCR
K
CL
K
L N
L SCR
SP
SCR L SCR
SP
SCR
CL
K
L
N L
SCR
CL
K
L
N L SCR L SCR
SP
SCR
CL
K
L N L SCR L SCR
SP
SCR
CL
K L N L SCR L SCR
SP
K
N L SCR SCR SCR SCR SCR
SP
K
SP
N
L
N
L
N L SCR SCR
Amphioxus 1
SCR K K L N L SCR L K L K
SP
Amphioxus 2
SCR K SCR
SP
Figure S1, relates to Figure 1. Structure of the SVH-1-like and SVH-2-like proteins
(A) Schematic diagrams of Met-like receptor tyrosine kinases from Homo sapiens (Met), sea urchin
(Strongylocentrotus purpuratus XP_793763.3), oyster (Crassostrea gigas EKC24814.1 and EKC24815.1),
amphioxus (Branchiostoma floridae XP_002597380.1) and C. elegans (SVH-2) are shown. Domains are
shown as follows: a signal peptide (black box), a Sema domain (Sema), a PSI-like domain (P), an IPT-like
domain (I), a transmembrane domain (TM), and a tyrosine kinase domain (kinase). The predicted oyster
Met-like-1 protein is probably incomplete, as it lacks a signal peptide. (B) Schematic diagrams of SVH-1like proteins from C. elegans (SVH-1), water flea (Daphnia pulex EFX82507), pea aphid (Acyrthosiphon
pisum LOC100160261), ant (Camponotus floridanus EFN70973.1), honeybee (Apis mellifera LOC724971),
oyster (Crassostrea gigas EKC30355), sea urchin (Strongylocentrotus purpuratus LOC578177), amphioxus
Bf-SVH-1-1 (Branchiostoma floridae XP_002601961.1 + XP_002601959.1) and Bf-SVH-1-2
(Branchiostoma floridae XP_002610402.1) are shown. Domains are shown as follows: a signal peptide
(black box), a chitin-binding domain (C), a C-type lectin domain (CL), a kringle domain (K), an LDL
receptor-like domain (L), a PAN domain (N), a scavenger receptor domain (SCR), and a serine proteaselike domain (SP). Gray color indicates a SP domain lacking a catalytic triad. The predicted oyster and
amphioxus SVH-1 like proteins are probably incomplete, as they lack signal peptides.
C. elegans
713
Water flea
1216
Pea aphid
1733
Ant
2036
Bee
2033
Oyster
1242
Sea urchin
1274
Amphioxus 1 1031
Amphioxus 2
387
Human
Plg 581
Zebrafish Plg 589
Human
HGF 490
Zebrafish HGF 477
Human
MSP 498
Zebrafish MSP 482
VVGGFETVP-GAFPWTAALR-------NKATKAHHCGASILDKTHLITAAHCFEEDER-VSSYEVVVGDWDNNQTDGNEQIFYLQ
VVKGEPTKP-GAYPWQVGVR----VRNSGKSDNHWCGATIISEHFILTAAHCMEDFPK--GLYVLRVGDYNTEDSDVEEEQFTVE
IASGFNTER-GDHPWQASIR----STTPAGQTEHVCGAVIISKYHVLTAAHCVRDLDK--DFYYVRIGDYNMGTLEDSEQDIYID
VVHGSIAPK-GTYPWQASIR----VRGHS-RSSHWCGAVMISPIYLLTAAHCLEGYNK--GTYFVRAGDYNTDIDEGTEAEANIE
VVRGNIAPK-GSYPWQASIR----VRGYS-KSNHWCGAVIISPLHVLTAAHCLEGYNK--KTYFVRAGDYNTEIDEGTEIEANIE
IFGGLNADY-GMFPWQVAIRKVIYRTQTRKIDAQHCGGIVLSRFWILSAAHCFDDEVK--SDLIIRVGDLNNKEPDSDEEEFEVE
IIGGSSAKR-GNWPWQAQLI--------LRGSGHYCGGTLIDETHVLTAAHCFQRYGK--NSFKVRLGEHHQHINESSEQDFRIS
PLSGDVAVK-GEHPWQALLL--------TKGVHHACSGTLVHDCWVVTAAHCMDG-SK--DAYVIRLGEYNTVLGEQTEQDFGID
IVGGVLGER-GRWPWVAEVR--------LNGYGHWCGGALIRDCWVLSAAHCFYDYSK--SSFTVRLGEYNLSSAESGEQVFSIE
VVGGCVAHP-HSWPWQVSLR--------TRFGMHFCGGTLISPEWVLTAAHCLEKSPRP-SSYKVILGAHQEVNLEPHVQEIEVS
IVGGCVSKP-HSWPWQISLR--------TRGKIHFCGGTLIDPQWVVTAAHCLERSDSP-SAYKIMLGIHTERATESSKQERDVT
VVNGIPTRT--NIGWMVSLR--------YRN-KHICGGSLIKESWVLTARQCFPS--RDLKDYEAWLGIHDVHGRGDEKCKQVLN
IVGGMRVQRAEDGSWVVSIQ--------KGN-RHWCGGSLIREEWVLTDQQCFSTCVPDLSEYTVQVGLLHLNASAG---TQALR
VVGGH---P-GNSPWTVSLR--------NRQGQHFCGGSLVKEQWILTARQCFSSCHMPLTGYEVWLGTLFQNPQHGEPSLQRVP
IVGGT---P-GNSPWTVSLR--------DRKGNHFCGGSLVSSEWVISTKQCFSSCYVDLTGYTAMMGTLFRDPKEGEPDLQRIS
788!
1293!
1810!
2112!
2109!
1323!
1347!
1100!
460!
651!
659!
561!
549!
570!
554
C. elegans
789
Water flea
1294
Pea aphid
1811
Ant
2113
Bee
2110
Oyster
1324
Sea urchin
1348
Amphioxus 1 1101
Amphioxus 2
461
Human
Plg 652
Zebrafish Plg 660
Human
HGF 567
Zebrafish HGF 550
Human
MSP 571
Zebrafish MSP 555
RIHFYPLYKD--IFSHDIAILEIP----YPGIEFNEYAQPICLPSKDFVYTPGRQCVVSGWGSM---GLRYAERLQAALIPIINR
RMHFHEEFGQGGHLNNDIALIRIKK-KSNQGIRFGSHVQPICLPSPSTEYVAGMNCTIAGWGSPGQPGAAFAIKLQSATVPILSD
KIYIHENFEVNVKLNNDIAVIKLKT--SGAGIKFNQYVQPICLPSEVIKLKSNMNCTITGWGSDGSIGSSFAKTMRSATVPIIDM
DYYVHEEFRKGHRMNNDIALILLK----GHGIPLGKDIMPICLPSENTDYSPGLNCTISGFGSIETGKTTQSKNLRYGWVPLLDQ
DYYIHEEFRKGHRMNNDIALVLLK----GRGIPLGKNVMPICLPSERIEYPAGLNCTISGFGSIETGKSTHSKDLRYGWIPLLDQ
EIFMHEGYDS-ETYDNDIALIKITP-KDGRGIMAGMYVQPACLPSDNTRYTEDLDCYISGWGATSLGSPN---KLKYAEVPIIDR
CIYKHPDYDS-RTTNNDIAVLRLDR-----PAHITSFVTPACLPTDGE-FAADHQCWISGWG--NTGNDNYPSRLQEARVPLLPR
KAVIHPGYDV-GTGQHDIALLKLRK-KNGACARTTNFVRPICLPDAEMTFPVGTQCDVSGWGRTDTDRSLRPTTLMKARIPLLHD
RMFLHPDYHP-ITNHNDIVLVRLREHADGTCARTGPYVRPACLPTPGETLQPGSNCSIVGWGKANSSDTSFSDILMEASVPFIPR
RLFLEP-------TRKDIALLKLSS-----PAVITDKVIPACLPSPNYVVADRTECFVTGWG--ETQGTFGAGLLKEAQLPVIEN
KIIKGP-------AGTDIALLKLDR-----PALINDKVSPVCLPEKDYIVPSNTECYVTGWG--ETQDTGGEGYLKETGFPVIEN
VSQLVYGPEG-----SDLVLMKLAR-----PAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWG--YTGLINYDGLLRVAHLYIMGN
IAHVVCGPEG-----SNLALLKLTT-----PAPLSEHVRTVQLPVAGCAVAEGTLCLMYGWG--DTKGTGHEGSLKMVGLPIVSN
VAKMVCGPSG-----SQLVLLKLER-----SVTLNQRVALICLPPEWYVVPPGTKCEIAGWG--ETKGTGNDTVLNVALLNVISN
LTKIVCGPSE-----SHLVMLQLET-----PAQFNERVSQICLPPERYIVPDGTICEIAGWG--ETKGKGDETVLNVAQMPVLSN
864!
1377!
1893!
2193!
2190!
1403!
1423!
1186!
544!
726!
734!
634!
621!
641!
627!
C. elegans
865
Water flea
1378
Pea aphid
1894
Ant
2194
Bee
2191
Oyster
1404
Sea urchin
1424
Amphioxus 1 1187
Amphioxus 2
545
Human
Plg 727
Zebrafish Plg 735
Human
HGF 635
Zebrafish HGF 622
Human
MSP 642
Zebrafish MSP 628
FDCVNSSQIY--SSMSRSAFCAGYLEGGIDSCQGDSGGPFACRREDGAFVLAGVISWGDGCAQKKQPGIYTMVAPYLSWISAII
DTC-KAPYVYGPDRIKVGMFCAGLLEGGVDACQGDSGGGLVCL-VDGRPTLMGVISWGFGCGRPNRPGVYTRVVHYLPWIYSKL
KIC-NAAYVYGKQTISSGMFCAGNLDGGADACQGDSGGPMVCSTELGE-TVMGITSWGYGCGRANKPGVYTNVQHYEDWLSKTL
SVC-RASYVYGEGAISEGMMCAGYLDEGIDTCDGDSGGPLACY-HNGAFTLYGITSWGQHCGKANKPGVYVRVAHYRRWIDQKI
SVC-RAGHVYGERAISDGMVCAGYLNEGIDTCDGDSGGPLVCL-HNGVFTLYGLTSWGQHCGKMNKPGVYVRVSYYRQWIDKKI
NIC-R--NVY--KRLTPSMFCAGYLKGGIDSCQGDSGGPLVCK-VGGKYTVLGVTSWGRGCGDPNSPGVYTVVKSHLQWIQDKL
STCTR-QNVYG-NKLTPQMLCAGYLRGGIDSCDGDSGGPLVCENSNSVWKVVGVTSWGYGCAQPNAPGVYAVVTRYLGFINEKM
GLC---QHIYG-DKLLPGMLCAGHVRGGNNACKGDIGGPLTCK-LDGKWVLWGVTSWGFGCGEPKTPGVYTRVAEYTDWLQRVM
QEC-R-DRAYG-NMVTDRMTCAGFWEGGVDTCQGDSGGPLLCQ-QDDRWTVWGVTSWGDGCARPNWPGVYTAVEEFLGWIQETI
KVCNRYEFLNG--RVQSTELCAGHLAGGTDSCQGDSGGPLVCF-EKDKYILQGVTSWGLGCARPNKPGVYVRVSRFVTWIEGVM
KVCNRPSFLNG--RVKDHEMCAGNIEGGNDSCQGDSGGPLVCY-AQNTFVLQGVTSWGLGCANAMKPGVYTRVSKFVDWIERSI
EKCS--QHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCE-QHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII
KRCS--QSHNGILPITETKICAGGKRD-QGVCEKDYGGPLVCQ-EGESKVIVGVSINGRGCAVARRPAVFVNVAFYSEWIRKVF
QECN--IKHRG--RVRESEMCTEGLLAPVGACEGDYGGPLACF-THNCWVLEGIIIPNRVCARSRWPAVFTRVSVFVDWIHKVM
KDCN--QYFKG--RVRENEMCTMAFQAGVGACERDYGGPLACQ-NSDCWVLEGVIIPMRRCGHAGQPNIFIRVSVYVDWIKKVM
Figure S2, relates to Figure 1. Comparisons of protease-like domains
Amino acid alignments of protease-like domains are shown. Identical residues are highlighted with
gray shading. The Asp-His-Ser catalytic triads are indicated by red characters.
946!
1459!
1975!
2275!
2272!
1481!
1505!
1265!
624!
807!
815!
715!
702!
722!
706!
human HGF*
HumHGF
zebrafish HGF*
ZebraHGF
HumMSP
human MSP*
HGF/MSP
zebrafish MSP*
ZebraMSP
HumPlasminogen
human Plasminogen
Plasminogen
ZebraPlasminogen
zebrafish Plasminogen
BfSVH-1PN
amphioxus SVH-1-1*
BfEEN66412
amphioxus SVH-1-2
SeaurchinSVH-1
sea urchin SVH-1
OysterSVH-1
oyster SVH-1
Apis
SVH-1
bee
SVH-1
SVH-1!
antSVH-1
ant SVH-1
DaphniaSVH-1
water flea SVH-1
peaSVH-1
pea aphid SVH-1
CelegansSVH-1
C. elegans SVH-1
human Thrombin
Thrombin
Thrombin!
zebrafish thrombin
zebraThrombin1
BfEEN52030
XP_002596018
BfEEN52035
XP_002596023
kringle-!
proteases!
#
BfEEN52019
XP_002596007
Humelastase
human elastase 1
Chymotrypsin
0.2
Figure0.2S3, relates to Figure 1. Phylogenetic tree depicting genetic relationships
A phylogenetic tree was constructed using MEGA5.2 software. The sequences of the protease-like
domains in the indicated proteins were compared. Human chymotrypsin was used as an outgroup.
Asterisks indicate “dead proteases” lacking intact catalytic triads. A sharp indicates a compressed
amphioxus-specific branch including XP_002590082, XP_002590083, XP_002596021 and
XP_002596022. The scale bar represents the evolutionary distance.
A
wild type
B
***
svh-1(ok2531)
C
vector RNAi
svh-1(ok2531)
(Terminal bulb/whole head)
Ratio of fluorescence
1.5!
1!
0.5!
0!
wild type
fbl-1 RNAi
svh-1 !
(ok2531)
Figure S4, relates to Figure 4. Effect of the svh-1 mutation on the localization of FBL-1C::VENUS.
(A) Confocal images of animals expressing FBL-1C::VENUS are shown in the top panels. The quantified
areas of whole head regions and terminal bulbs are indicated by white and read lines, respectively. (B)
Ratio of mean fluorescence intensities of FBL-1C::VENUS (the terminal bulbs/the whole head regions).
The data (n = 10 animals) are quantified by the Welch’s t test. ***, P < 0.001. Error bars represent SEM.
Scale bar = 10 µm. (C) Suppression of the svh-1 phenotype by fbl-1 RNAi.Light microscopy of animals
grown for 5 days are shown. Suppression of the svh-1 growth defect was examined as described in
Experimental Procedures. In svh-1 mutants treated with control RNAi, no animals grew to the adult stage
on 40 plates. In svh-1 mutants treated with fbl-1 RNAi, animals grew to the adult stage on 7 plates out of
37 plates. Anterior is to the left. Scale bar = 50 µm.
Movies S1 and S2. Time-lapse video microscopy of pharyngeal pumping.
Movies S1 and S2 show time-lapse images of pharyngeal pumping in wild-type (Movie S1) and
svh-1 mutant (Movie S2) animals, respectively. The movies were taken by using a Hamamatsu
ORCA system at 20 frames/sec.

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