Membranes Nanotubes

Pulled Cooperatively by Molecular Motors

Organelles in Cells
Kirschhausen T.,
Nature reviews (2000)
Intracellular Membrane Traffic
Budding - Fission - Transport - Fusion
Formation of transport intermediates
Transport Intermediates:
Small Vesicles
Trafficking of P2X4-GFP receptors in neuron
R. D. Murrell-Lagnado, Cambridge, UK
(White & al. JCB 147, 743-760)
Long Tubes
Trafficking of Rab6 in HeLa cell
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The Cell, Alberts et al, (2002)
Tubulin dimers self-assembled
in parallel protofilaments

Polarized hollow rigid
cylinders

Microtubules: Rails for Membrane Transport
Bar = 5 m
Tubulin dimer
Plus end
Minus end
Bar = 50 nm
Hirokawa, Science (1998)
Lippincott-Schwartz et al, JCB (1995)

Bar = 5 m
-
+
Microtubule
Kinesin-1
Kinesin: Molecular Motor Moving on Microtubules
ATP
ADP
Motor
domains
thread
tail
Barre = 10 nm
Transport of membrane intermediates
Mechano-enzyme:
ATP hydrolysis
Steps = 8 nm
Block et al., PNAS (2003)
Dynamics of Kinesins
k
B
: binding rate of
kinesin onto MT

V decreases with
applied force

Stall force:
F
S
= 6 pN
V
0
: velocity of kinesin in
absence of external load
Bead assay
V
0
= 0.6 0.1 m/s
k
u
0
:

unbinding rate
at zero load
k
u
0
= 0.42 s
-1

Vale et al., Nature (1996)
_
+
In presence of applied force
k
u
increases

k
u
= k
u
0
exp
f
0
a
K
B
T
|
\

|
.
|
Quic kTime et u n
d c o mp r es s eu r
s on t r eq u is p ou r vis ion n er c ette ima g e.
Membrane Tubes
Membrane Nanotubes
Force
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Physics of membrane tubes : tube formation
Pulling on membrane with molecular motors
Different dynamical regimes
Outlines
1.Tube Formation
D. Cuvelier
A. Roux
P. Nassoy
Physics of Membrane Tubes
L
f
2R

E
tube
= 2tL
k
2R
+2tRoL fL
ko t 2 2
0
= f

R
0
=
k
2o
Drnyi et al, PRL 88 (2002) 238101
k: bending rigidity
o: membrane
tension
AP
Ax -> F
Tension o
Experimental confirmation
Optical Tweezers
+
Micropipette
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Results
f
0
=18 pN
o = 8. 10
-5
N.m
-1

Theory
EPC

f
0
= 2t 2k o
k


Vesicles : lipids +
5%DOPE-Peg
2000
/
DOPE - peg
2000
-biotin (1/1000)
k (k
B
T)
EPC
13.6 1.3
50% DOPC + 50% cholesterol
(liquid disordered)
30 3.0
50% sphingomyelin +
50% cholesterol (liquid ordered)
65 6
Bending rigidity measurements
Roux et al EMBO J. 24 (2005) 1537
2. Pulling Tubes with Molecular Motors
Very dynamic tubular structures in living cells (GFP)
Endoplasmic Reticulum, Golgi, Endosomes
Tubular structures in living cells
Waterman-Storer & Salmon, Curr. Biol. (1998)
Microtubules
RE
Bar = 1 m
Golgi
VSVG-GFP
J. Lippincott Schwartz (CBMB-NIH)
E.R.
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HYPOTHESIS
Molecular Motors (kinesins) in contact with Microtubules
bound to Membrane of Giant Unilamellar Vesicles (GUVs)
can extract membrane tubes
Microtubules depolymerization
or Kinesin inhibition
NO TUBE
Required :
Microtubules
+ Motors
Membrane
Kinesin
+ ATP
1 kinesin 6 pN max (stall force)

A few kinesins should be sufficient
but
MORE THAN ONE kinesin required
Small Motor CLUSTERS should be necessary
How many motors required to pull tubes ?
f
0
>10 pN
Tube extraction :
Combination of the membrane physical properties
and of the dynamical properties of the motors
"Chemical" Clusters
of Motors
pulling Membrane Tubes
A. Roux
Streptavidin
coated BEADS
(100nm)
+
Biotinylated
lipids (5%)
+
Biotinated
kinesins
Binding motors to the membrane
microtubule
kinesins
Vesicle
+ ATP
(1 mM)
TUBE
Roux A. et al PNAS (2002) 99, 5394
Minimal System
Transmission Electronic Microscopy

d=2
k
2o

o~5.10
5
Nm
Bars: 5m 500 nm
Coll. J. Cartaud (Inst. J. Monod, Paris)
microtubules
membrane nanotubes
d=4010 nm
X 40
(total = 15 min.)
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Bar = 5 m
Microtubules
Membrane tubes
Tubes
WITHOUT Beads
Ccile Leduc (Exp)
Otger Camps (Theory)
Motors individually bound to lipids
TUBES !!!!!
C. Leduc et al, PNAS (2004) 101, 17096
Parameters regulating tube extraction
F
0
=2t(2ok)
1/2

F
0
~ 28 pN


number of motors pulling the tube
o force necessary for extracting tubes F
0
.
Conditions for Tube Extraction
Fixed motor concentration

:
Higher tension
Low tension
o , F
0

Threshold in tension for a given motor concentration
C. Leduc et al, PNAS (2004) 101, 17096
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Fixed membrane tension o
0

0,01 %

min
0,1 % 1 %
NO
TUBE
TUBE
Quantitative measurements
For o = 2.10
-4
N/m,

min
= 200

motors/m
2

Theoretical analysis effectively predicts:

min
= cste . o
max

Threshold in motor concentration for a given tension
Side view
(3D Reconstruction)
Bar = 5 m
System Geometry
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Dynamical recruitment
of motors
G. Koster et al, PNAS (2003)100, 15183
"Physical" clusters
C. Leduc, O. Camps et al, PNAS (2004) 101, 17096
V
n
b
: number of bound motors at the tip
J
b
: incoming flux of bound motors
J
u
: incoming flux of unbound motors
n
b

Motors bound to MT
Motors unbound to MT
k
u
0

k
b

J
b
|J
u
|
V
0

b b u b
b
n n k J
dt
dn
) ( =
)
1
exp(
0
0
b B
u u
n T K
a f
k k =
)
1
1 (
0
0
b S
n f
f
V V =
Theoretical analysis O. Camps, J.-F. Joanny and J. Prost
Tip
C. Leduc et al, PNAS (2004) 101, 17096
Short time scales
Fluxes equilibrium & V>0:
Bifurcation diagram
Analytical solutions
n
b

J
u
J
b
n
b

J
u
J
b
Theoretical analysis
O. Camps, J.-F. Joanny and J. Prost
b b u b b
n n k n V x J ) ( ]) [ ; 0 ( = =

o
v
~
Conditions for tube extraction
Short time scales
Condition for tube formation at the threashold
O. Camps, J.-F. Joanny and J. Prost
At the
threashold:
n
b
min
~ 5 motors
200 400
min,
=

th

100 200
exp min,
=

motors/m
2
motors/m
2
T K
a f
n
B
b
2
0
min
=

>
e
2
2
f
S
a
K
B
T
|
\

|
.
|
2
k
b
+k
u
0
k
b
k
u
0
V
0

min
Theory
Experiments
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Motor Distribution Along the Tubes
Biotinylated and Fluorescent Lipid
(L. Bourel, Lille)
Motor accumulation at the tip
x 60
Bar : 1 m
Instantaneous motor distribution
Theory
1.0
0.8
0.6
0.4
0.2
0.0
40 30 20 10 0
Experiments
control
Theory
Exponential distribution
k
0
u
= 0.42 s
-1

D = 1,0 0.5 m
2
/s
(FRAP)
V
0
= 0.6 0.1 m/s
With
One parameter fit
k
b
= 4.7 2.4 s
-1

Experiments vs. Theory

=
k
u
0
D
2k
B
V
0
V
( )
1 + 1 +
4k
B
k
u
0
V
0
V
( )
2
k
u
0
D
|
\


|
.
|
|
Experiments
n
B
20 motors
3. Other Dynamical Regimes
Entropic regime
Elastic regime
Long Tubes
Constant
tension:

f
0
= 2t 2ko
Constant Force
Non-fixed
tension:
Increasing Force
Cuvelier et al Europhys. Lett (2005)

F
l
o
p
p
y

v
e
s
i
c
l
e
s

Dynamical Diagram
(O. Camps)
Stable states Oscillatory regime
Kinetic Montecarlo simulations
Experiments
Experiments
Theory
Collective oscillations Stops
Dynamical Diagram
(O. Camps)
Tip
distance (m)
F
l
u
o
r
e
s
c
e
n
c
e

I
n
t
e
n
s
i
t
y

d
i
s
t
a
n
c
e

(

m
)

time (s)
Large Scale Traffic Phenomena
Conclusions
Minimal system mimicking transport intermediates
Formation of dynamical clusters (physics origin)
Molecular parameter of the motors (k
B
) deduced from
macroscopic measurements
Membrane tubes: perfect system for studying motor
collective behavior
Threshold (motor concentration - membrane tension) for tube
formation
Regulation of tube formation :
- Forming proteins assemblies (coats) to fix the motors
- Regulating the number of motors on the membrane :
expression
regulation of the fixation sites
- More efficient : modulation of the membrane tension
Reorganisation of multivesicular bodies (late endosomes)
Tension= switch ?
Maturation of dentritic cells
Before activation After activation
M. Kleijmeer et al JCB (2001)
Perspectives
Motors with different dynamic characteristics
Tubes pulled by non-processive motors
Plus-end and Minus-end motors. Competition?
Pulling tubes in living cells
Modeling :
Oscillations
Traffic jams
The People :
Curie Institute
Ccile Leduc
Aurlien Roux
Damien Cuvelier
Pierre Nassoy
O. Campas,
I.Drnyi, C. Storm, F. Jlicher,
J-Franois Joanny,
Jacques Prost
Theory
Collaborations
Bruno GOUD
Biology
J. Cartaud (IJM, Paris)
G.Koster, M.Van Duijn,
M.Dogterom
(AMOLF Amsterdam)
P.Joliemaitre and L. Bourrel
(Pasteur Inst.,Lille)
F. Ndlec (EMBL, Heidelberg)