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