bret principle - cours de l'ue7 à...
TRANSCRIPT
BR
ET
pri
nci
ple
Rluc
Rluc
IFNGR1
IFNGR2
WT
TN
0
10
20
30
40
50
60
70
WT
TN
012345
IFN
GR
1-I
FN
GR
2 i
nte
ract
ion
s
Jak1
Jak2
Jak1
Jak2
)*+(),-./(0
"#$%&'(1(!
"2%32+(4$"5+5(6+7+4#5(
• D
ecre
ase
of
lipid
mic
rodom
ains
asso
ciat
ion
• A
bse
nce
of
IFN
GR
1-
IFN
GR
2 c
onfo
rmat
ional
chan
ges
aft
er I
FNγ
stim
ula
tion
• M
odifi
cati
on o
f Ja
k1
confo
rmat
ion a
nd a
ffinit
y
tow
ard I
FN
GR
1
Pa
uli
ne
Go
nn
ord
, Je
rem
y K
ag
an
Fro
m M
ayo
r a
nd
Pa
ga
no
, N
atu
re R
ev M
ol
Bio
l C
ell
20
07
IL2
-R,
Clo
stri
diu
m t
ox
ins
ST
xB
, C
Tx
B,
vir
us
ga
lect
ins
An
in
crea
sin
g d
egre
e o
f co
mp
lex
ity
….
La
bo
rato
ire
« T
rafi
c, S
ign
ali
sati
on
et
Cib
lag
e In
tra
cell
ula
ires
»
UM
R 1
44
CN
RS
Par
ton &
Sim
ons,
Nat
Rev
Mol
Cel
l B
iol.
2007
• 50
-80
nm
in
vag
inat
ion
s (1
95
3 P
alad
e, 1
95
5 Y
amad
a)
• ch
ole
ster
ol,
gly
cosp
hin
go
lip
ids,
den
sely
pac
ked
lip
ids
• Cav
eoli
n 1
, 2
: e
nd
oth
elia
l ce
lls,
ad
ipo
cyte
s
• Cav
-3 :
sk
elet
al a
nd
car
dia
c m
usc
le c
ells
• 10
0-1
50
cav
eoli
ns
per
cav
eola
• Min
imal
mo
bil
ity
of
cav
eoli
ns
ou
tsid
e o
f ca
veo
lae
Ca
ve
ola
e
Par
ton, S
cien
ce 2
001
Adi
pocy
te (
NIH
3T
3-L
1)
Sig
na
lin
g:
eNO
S,
ER
K ,
Ak
t ac
tiv
atio
n,
Ras
, E
GF
-R,
src
kin
ase,
Rh
o R
ac G
TP
ases
,
calc
ium
, co
ntr
adic
tory
ro
le o
f ca
veo
lae,
lac
k o
f co
nse
nsu
s.
Tu
mo
r p
rom
ote
r o
r su
pp
ress
or
Lip
id r
egu
lati
on
: M
ech
anis
m u
ncl
ear
En
do
cyto
sis:
to
xin
s, v
iru
ses,
in
teg
rin
s….
Reg
ula
ted
by
dy
nam
in,
PK
C,
tyro
sin
e
kin
ases
.
Car
go
can
tak
e al
tern
ativ
e p
ath
way
in
th
e ab
sen
ce o
f ca
veo
lae
Cav
-/- m
ice
hav
e n
orm
al e
nd
ocy
tosi
s.
Ca
v1
mu
tati
on
in
hu
ma
ns:
In
suli
n r
esis
tan
ce,
lack
of
bo
dy
fat
J C
lin
En
do
crin
ol
Met
ab
20
08
Ca
v3
mu
tati
on
in
hu
ma
ns:
mu
scu
lar
dy
stro
ph
ies
Ro
les o
f C
ave
ola
e
The
true
cell
ula
r fu
nct
ion o
f ca
veo
lae
rem
ains
deb
ated
A r
ole
for
caveo
lae
in c
ell
mec
han
ics?
Ab
un
dan
t in
ad
ipo
cyte
s, e
nd
oth
elia
l ce
lls,
mu
scle
cel
ls
(cel
ls t
ha
t ex
per
ien
ce r
epea
ted
mec
ha
nic
al
stre
ss)
Oh, M
cInto
sh &
Sch
nit
zer
JCB
1998
Hay
ashi
et a
l JC
I 2009
Roth
ber
g e
t al
Cel
l 1992
89$$'/%*-:',;$&<(*+=%&$)*$+/&,%$+)%&++$
:-'%&,+&+$)/&$,>*=-)$*?$',@&*;,&
Riz
zo e
t a
l. A
JP 2
00
3
Rat
Lu
ng
en
do
thel
ial
cell
s
6 h
Res
po
nd
to
sh
ear
stre
ss b
y c
han
gin
g
dis
trib
uti
on
of
cav
eoli
n e
xp
ress
ion
(Pa
rk e
t a
l., JB
C 1
99
8)
(Bo
yd
et a
l., A
m J
Ph
ysio
l H
ea
rt C
irc P
hysio
l, 2
003)
Bu
dd
ed m
emb
ran
e m
icro
do
mai
ns
as t
ensi
on
reg
ula
tors
Th
eore
tica
l p
rop
osa
l b
y S
ens
& T
urn
er
Ph
ys R
ev E
20
06
Cel
l st
ress
and m
embra
ne
tensi
on
" A*;&$*?$',@&*;,&$:-$%&+(*-+&$)*$,-$,'=)&$,-.$+=..&-$>
&'/,-:',;$+)%&++B$
*89*(#+'58&'(
2&:(#+'58&'(
restin
g te
nsio
n
;,<=-=,<=>/?0
$
46&;;$
+)%&)'/$
@+22(0
+0AB$'+(#+'58&'(*&0+&5#$585(
C*-D$)&%>
$%&+(*-+&$E9$>
:-=)&+F$G$%&D=;,H*-$)/%*=D/$&<*3&-.*8'7)*+:+$
Rap
id i
ncr
ease
of
mem
bra
ne
tensi
on b
y h
ypo-o
smoti
c sh
ock
CD3&=&50
&%4(5*&4E(
F5&=&50
&2$B8#D1(GG<0H50
(
CD3&=&50
&2$B8#D1(G<0H50
(
Z c
on
foca
l im
agin
g o
f H
eLa
Cav
1-G
FP
Ch
an
ges
in
cel
l v
olu
me
on
hy
po
sh
ock
~ 1
.4
IJ$'*-?*',;$:>
,D:-D$
- 5
0
5
1 0
1 5
2 0
2 5
3 0
3 5
0 . 7
0 . 8
0 . 9
1 . 0
1 . 1
1 . 2
1 . 3
1 . 4
Normalized volume
!+*$
K7(*$
Tim
e (m
in)
J&@&;*(&.$=+:-D$C,LM:&6N$M:+:*-$
0.0
30.0
k60.0
k90.0
k120.0
k150.0
k0
10
20
30
40
#
Cav
inte
nsi
ty
Counti
ng a
nd t
rack
ing c
aveo
lae
dynam
ics
• I+#+4%&'(($'6(2&4$28J$%&'(&7(&7($22(
4$K+&2$+(3+B(80
$9+((
• LK&2"%&'(&7('"0A+B(&7(4$K+&2$+(
3+B(4+22(:8#*(%0+(
• @$24"2$%&'(&7(#B$M+4#&BD(7&B($'D(
58'92+(4$K+&2$+(8'(%0+(
• N"$'%7D8'9(58J+O(8'#+'58#D(&7(58'92+(
4$K+&2$+(
• PODOJ(Q"4#"$%&'5(&7(+$4*(4$K+&2$+(
• !+$'(5R"$B+(68532$4+0+'#(SPO(D(
Q"4#"$%&'5T(&7(+$4*(4$K+&2$+($'6(
$K+B$9+6(&K+B(0"2%32+(
• L5%0$%&'(&7(6+9B++(&7(
4&'U'+0+'#O(3+B585#+'4+(
Cav
-1-G
FP
HeL
a ce
lls
OP&')$*?$/7(*)*-:'$+/*'Q$*-$',@&*;,&$-=>L&%$ A&'*@&%7$
VHWW(&7(5"B7$4+(4$K+&2$+(*-$/7(*)*-:'$)%&,)>
&-)$
K7(*8+/*'Q$
0
1 0 0
0
2 0 4 0 6 0 8 0
1 0 0
number of caveolae/cell
0
1 0 0
s e c o n d s
#*%>
,;$
Cel
l fo
otp
rin
t d
oes
no
t ch
ang
e
1-2
min
(no
im
agin
g)
seco
nds
Unia
xia
l st
retc
hin
g o
f ce
lls
RJS4$+/&&)$G$)/:'Q-&++$TU$V$WU$X>$
(&%>
,-&-);7$,Y,'/&.$)*$RJS4$+/&&)$*?$?&6$>
>$)/:'Q-&++$6:)/$
,$/*;&$,)$)/&$'&-)%&Z$$
L'$A2+5(*89*(B+5&2"%&'(80$98'9(AD()FXY$
S:'%*(,Y&%-&.$,./&+:@&$(,)'/&+$
E[L%*-&'H-F$*-$RJS4$?*%$%&D=;,H-D$'&;;$
+/,(&$,-.$*%:&-),H*-$
C+V$(4+225(5#$A2D(+P3B+558'9(@Z[,=\Y](
0
1 0
2 0
3 0
0 . 6
0 . 8
1 . 0
Percentage caveolae left
% S t r e t c h
Incr
ease
d s
tret
chin
g
# O-.*'7)*+:+$B$
$ 1'H-$.7-,>:'+$
$ 15R$.&(;&H*-$
Ca
veo
lae
dis
ap
pea
ran
ce i
s A
TP
an
d a
ctin
-in
dep
end
ent
! :&$$3;(%<
(0=&'3$<%#+3;('%>/%<
(<>+&'(%*('$31'%%
K*6$.*$',@&*;,&$.:+,((&,%$,)$)/&$RSB$
J7-,+*%&$)%&,)>
&-)$
- 1 0
0
1 0
2 0
3 0
4 0
5 0
0 , 6
0 , 7
0 , 8
0 , 9
1 , 0
1 , 1
1 , 2
1 , 3
Normalized number of caveolae
h y p o
Co
ntr
ol
Dy
nas
ore
(8
0µ
M)
Tim
e (m
in)
1
0
Iso
Hy
po
Hy
po
+ C
yto
D
Hy
po
no
AT
P
Normalized number of caveolae
# \/,)$/,((&-+$)*$)/&$',@&*;,%$+)%=')=%&B$$
" ?@##(#%0&;(1.&(%#3$&AA(&+%>/%B&C('3'-%1@*%
3'*1%*=(%A.&$<
&%<
(<>+&'(%
Hy
po
ton
ic
100 n
m
Mo
use
Lu
ng
En
do
thel
ial
Cel
ls (
ML
EC
)
Iso
ton
ic
100 n
m
Iso
Hy
po
0
1 0
2 0
3 0
4 0
5 0
6 0
% of gold
Mem
bra
ne
bound
caveo
lae
bound
?
Iso
Hy
po
Dee
p-e
tch
ed E
M
(N.
Mo
ron
e Ja
pa
n)
# ",)&$*?$',@&*;:-+B$$
$ !-'%&,+&.$>
*L:;&$?%,'H*-$*?$?%&&$',@&*;:-+$E=-.&%&+H>,)&.$
.=&$)*$>
&>L%,-&$&<(,-+:*-F$
T i m e
( s e c )
TIR
F-F
RA
P
0
4 0
8 0
1 2 0
0 . 0
0 . 1
0 . 2
Normalized Intensity
I s o
H y p o
* *
L&?*%&$
L;&,'/$
]W^U$+&'$
#*%>,;$ K7(*$
C*6$:-)&-+:)7$
K:D/$:-)&-+:)7$
free
cav
eoli
ns
(10
%)
free
cav
eoli
ns?
?
Cav
eola
e af
ter
retu
rn t
o i
so-o
smola
rity
?
• Cav
eola
e re
cover
y r
equir
es A
TP
• Dis
rupti
on o
f ac
tin d
ynam
ics
enhan
ces
the
rate
of
reco
ver
y
0
4 0
8 0
1 2 0
0 . 0
0 . 1
0 . 2
Fluorescence recovery
T i m e
( s e c )
I s o
H y p o
R e c
0
2
4
6
8
1 0
0
2 0
4 0
6 0
8 0
1 0 0
Number of Caveolae/cell
T i m e
( m i n u t e s )
c y t o D
A T P d e p
c o n t r o l
TIR
F
Normalized caveolae number
i s o
h y p o
r e c
0 . 0
0 . 5
1 . 0
1 . 5
Cav
1 H
eLa
Rec
ov
ery
t=5
min
150 n
m
ML
EC
Model
% C
ave
ola
e a
re r
ap
idly
dis
ass
emb
led
wh
en m
emb
ran
e te
nsi
on
in
crea
ses
(AT
P i
nd
epen
den
t).
Hypoto
nic
/ s
tret
ch
isoto
nic
% T
hey
are
ra
pid
ly r
eass
emb
led
wh
en t
he
ten
sio
n i
s re
du
ced
(A
TP
an
d a
ctin
dep
end
ent)
.
5/&$'7';&$*?$',@&*;,&$.:+,++&>L;73%&,++&>L;7$%&(%&+&-)+$)/&$
:-:H,;$'&;;$%&+(*-+&$)*$,'=)&$>
&'/,-:',;$+)%&++$
Rev
isit
ca
veo
lae
fun
ctio
ns
(sig
na
lin
g,
tra
ffick
ing
) th
rou
gh
th
is n
ov
el m
ech
an
ism
Cel
ls r
esp
on
d t
o m
ech
an
ica
l st
ress
by
rap
id d
isa
ssem
bly
of
cave
ola
e
Sin
ha
et a
l.,
Cel
l 2
011
_,@I$>
=),H*-+$,-.$>
=+'=;,%$.7+)%*(/:&+$
Ca
ve
olin
3
no
rma
l m
uscle
ce
lls
LG
MD
1 p
atie
nts
Du
chen
ne
My
op
ath
y a
sso
ciat
ed w
ith
in
crea
sed
lev
els
of
Cav
3 a
nd
LG
MD
-1C
wit
h l
ow
er l
evel
s
Wo
od
ma
n e
t a
l; (
20
04
) N
eu
rolo
gy
Ca
ve
olin
op
ath
ies: m
uta
tio
ns in
ca
ve
olin
-3 c
au
se
fo
ur
dis
tin
ct a
uto
so
ma
l d
om
ina
nt m
uscle
dis
ea
se
s
Par
ton e
t al
; JC
S 2
005
Wt
p2
8l
Hy
po
-osm
oti
c sh
ock
Hum
an F
HC
K (
Cav
3 P
28L
) m
yotu
bes
(Fam
ilil
al H
yp
erC
reat
ine
Kin
asem
ia)
IFNα
ST
AT
1 S
TA
T3
PR
OL
IFE
RA
TIV
E
AN
TI-
PR
OL
IFE
RA
TIV
E
Cav
eola
e an
d c
lath
rin-c
oat
ed p
its:
contr
ol
of
anti
pro
life
rati
ve
vs
pro
life
rati
ve
acti
vit
y o
f IF
Nα
CA
V1-/
- ce
lls:
incr
ease
d p
roli
fera
tion
Bre
ast
cance
r: C
AV
1 d
ow
nre
gula
tion
T l
ym
phom
as R
to I
FNα
: dec
reas
ed C
AV
1
Mec
han
ose
nsi
ng t
hro
ugh
caveo
lae
involv
es I
FNα
an
d
Sta
t3
Ba
lan
cin
g f
orc
es:
arc
hit
ectu
ral
con
tro
l o
f m
ech
an
otr
an
sdu
ctio
n
Va
leri
e W
eave
r U
CS
F N
atu
re R
ev M
ol
Cel
l B
iol
20
11
Cla
ud
ino
n,
Mo
nie
r a
nd
La
ma
ze,
Bio
ch
imie
20
07
- Pal
mit
oy
lati
on
of
IFN
AR
….
- Ro
le o
f ca
veo
lae…
- Dif
fere
nce
s IF
N-α
vs
IFN
-β
Stat/cancer:
19
97
: 1
60
ref
; 2
00
7:
18
00
ref
Med
lin
e
Th
e in
crea
sed
com
ple
xit
y o
f m
emb
ran
e tr
affi
ckin
g m
irro
rs t
he
com
ple
xit
y o
f
sign
ali
ng
!
Iden
tify
ing
reg
ula
tors
at
the
cro
ssro
ad
s o
f
tra
ffick
ing
an
d s
ign
ali
ng