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100<br />
117 <br />
OEPMII<br />
Immunocytochemical and proteomic analysis of the γsecretase<br />
complex using the conformationspecific antiNicastrin antibody<br />
1,2 2 4 3 4 5 <br />
2<br />
1<br />
2 3 <br />
4 5 <br />
<br />
γSecretase is a membranespanning, multimeric protease that cleaves APP and<br />
is responsible for generating the Aβ peptides. Although the intracellular traffic is<br />
implicated in the regulation of the Aβ production, exact subcellular location where<br />
the enzyme functions remains controversial. Here we established a monoclonal<br />
antibody A5226A against Nicastrin, one of the γsecretase components, and found<br />
that A5226A specifically recognizes the active γsecretase. Immunocytochemistry<br />
using A5226A revealed that γsecretase localizes at the cell surface and late<br />
endosome. Moreover, we applied A5226A for the targeted proteomics of the<br />
γsecretase complex. We identified about 30 interacting proteins including<br />
tetraspanin family members such as CD81, which are known to form membrane<br />
microdomains. We found that knockdown of CD81 reduced the secretion of Aβ.<br />
Furthermore, the internalization rate of both the enzyme and APP was reduced<br />
by knockdown. These results suggest that CD81 affects the Aβ generation via<br />
regulating the traffic of the enzyme and the substrate from the cell surface.<br />
OFPMI<br />
Rho ROCK, ROCK<br />
1,2 2 2 1 1 <br />
1 2<br />
1<br />
2 <br />
GTP Rho <br />
Rho <br />
Rho Rho <br />
Rho <br />
Rho ROCK1, ROCK2<br />
<br />
Rho <br />
ROCK1 ROCK2 <br />
ROCK1 <br />
ROCK2 <br />
ROCK1 <br />
ROCK2 <br />
ROCK2 <br />
<br />
<br />
Rho <br />
<br />
OFPMI<br />
Photoactivatable Rac <br />
<br />
<br />
GTPase Rac1 <br />
<br />
<br />
phototropin LOV2 GTP Rac1 photo<br />
activatable PARac1 <br />
PARac1 <br />
RAW Rac1<br />
photo<br />
manipulation<br />
mCherry PARac1 <br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
Rac1 <br />
<br />
OFPMI<br />
GaAsP LIVE <br />
<br />
1 2 1 1 3 <br />
1<br />
1<br />
2 3 <br />
<br />
<br />
<br />
<br />
<br />
A1, A2, A5LIVE <br />
GaAsP<br />
<br />
<br />
<br />
GaAsP <br />
GFP <br />
BSC1<br />
LIVE <br />
GaAsP <br />
A1, A2, A4, A5 A7 <br />
<br />
<br />
OFPMI<br />
E <br />
CD<br />
<br />
1<br />
229EHCoV229E CD13 <br />
<br />
<br />
HCoV229E <br />
CD13 <br />
<br />
HCoV229E CD13 <br />
CD13 <br />
1 CD13 <br />
2<br />
mCherryβactin<br />
EGFPlifeact CD13 Factin <br />
CD13 Factin <br />
cytochalasin Blatrunculin A actin<br />
CD13 Factin <br />
<br />
CD13 Factin <br />
OFPMII<br />
Nphp <br />
<br />
<br />
<br />
650 <br />
<br />
Nphp3 Nphp3 1325 <br />
coiledcoilCC<br />
Nphp3 <br />
<br />
<br />
Nphp3GFP <br />
<br />
<br />
Nphp3 N 200 <br />
N N <br />
CC 2 Nphp3 <br />
Nphp3 <br />
Kif7, Tctn2, Arl6 <br />
Nphp3
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