Program of the 2001 International Worm Meeting - Sternberg Lab ...

725
725. Characterization of C. elegans
claudins
Akira ASANO 1 , Kimiko ASANO 1 ,
Mikio FURUSE 2 , Hiroyuki SASAKI 3 ,
Shoichiro TSUKITA 1,2
1 Tsukita Cell Axis Project, ERATO, Kyoto
Res. Park, Kyoto 600-8813, Kyoto, JAPAN
2 Faculty of Med., Kyoto Univ., Kyoto
606-8501, Kyoto, JAPAN
3 KAN Res. Inst., Kyoto Res. Park, Kyoto
600-8815, Kyoto, JAPAN
Homologues of claudin, major integral protein
of mammalian tight junction with four span
transmembrane structure, were found in the
genomic data base of C. elegans. Among them,
CLC-1(C09F12.1) was discovered by a blast
search with mouse claudin-6, it was found later
that homology is also present with mouse
claudin-7 and human claudin-14. It has in its
first loop two Cys residues that are conserved
among mammalian claudins. CLC-2
(T05A10.2) was found by a blast search with
CLC-1, and has 33% identity (70% homology)
with CLC-1, although no significant homology
was found with vertebrate claudins so far
known. CLC-3 (ZK563.4) has homology with
mouse claudin-10 and cattle claudin-16. CLC-4
was found with a blast search with mouse
claudin-6, but it was thought to be an eight span
transmembrane protein at that time. Recently, it
was found that this gene (C01C10.1) encodes
two separate proteins, and the down-stream
operon (C01C10.1b) was reported to encode a
Gas3/PMP-22 homlogue (Agostoni E.et al.,
Gene, 234, 267-274, 1999). Product of the other
operon (C01C10.1a) was not characterized well.
Homology with claudin-6, -7, and -9 was found
with this product, therefore named as CLC-4.
These four claudin homologues have either
PMP-22/EMP/MP20 motif or transmembrane
four signature or both, and most of them have
srg integral membrane protein motif.
Furthermore, two conserved Cys residues are
present in all C. elegans claudins. Interestingly,
many of vertebrate claudins have one or two of
these motifs or signature. Although most of
vertebrate claudins has CLAUDIN3 signature,
no such signature was found in the nematode
claudins.
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All of coding sequences of the nematode
claudins were isolated from cDNA libraries,
therefore they are all expressed in the worm.
Some ESTs were reported for CLC-1 and -3,
previously. Expression of these claudins was
studied with GFP-tagged molecules. All of
claudins is expressed in spermatheca, intestine
and hypodermis. CLC-1 and -4 were expressed
strongly in pharynx, and sometimes localized at
cell-cell junctions. They are also seems to be
expressed in excretory-secretory system.
CLC-1::GFP is also expressed at cell-cell
junction of vulva. Localization of CLC-1 is
under study with HA-tagged molecule.
Preparation of antibodies against the nematode
claudins was very difficult so far. But, affinity
purified antibodies raised against loop 1 of
CLC-1 seem to be useful for CLC-1 detection in
the worm. Results with these antibodies were
very similar to those obtaind with GFP-tagged
CLC-1. To see if these claudins function as
mammalian claudins do, i.e. barrier function,
penetration of TRITC-dextran (MW=10,000)
was checked after injection of dsRNA’s
(RNAi). Experiments with full length CLC-1
dsRNA showed that barriers for the high
molecular weight dye were damaged by RNAi,
in other words, penetration of the dye to
pharynx and some other tissues were observed.
Similar experiments with other claudins did not
detect any barrier damage. This is because the
dye only goes into entrance of intestine under
normal condition, therefore, we tried weak
osmotic shock to deliver the dye to entire
intestinal lumen and excretory-secretory duct
system. Under this condition, control injection
of dsGFP did not result in penetration of the dye
to other area of the body. On the other hand,
RNAi with the combination of CLC-1 and -4
RNA’s resulted in penetration of the dye to 72%
of the worm from pharynx, intestine and vulva
(or from excertory-secretory system) to the
body, whereas barrier was damaged only 40%
of worm with a combination of CLC-3 and -4.
RNAi effects with other combination of CLC’s
will be reported, and effects of RNAi to the
retention of the sperm in spermatheca will be
studied. Accordingly, claudins of C. elegans
seems to function as barrier at least partly. Other
functions, if any, will be surveyed. We would
like to thank excellent technical assistance of
Miss. Akiko Kamamoto, without her help this
project could not be completed.