Membranes Nanotubes
Pulled Cooperatively by Molecular Motors
Organelles in Cells
Intracellular Membrane Traffic
Kirschhausen T.,
Nature reviews (2000)
Formation of “transport intermediates”
Budding - Fission - Transport - Fusion
Transport Intermediates:
Small Vesicles
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Long Tubes
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Trafficking of P2X4-GFP receptors in neuron
R. D. Murrell-Lagnado, Cambridge, UK
Trafficking of Rab6 in HeLa cell
(White & al. JCB 147, 743-760)
Microtubules: Rails for Membrane Transport
Bar = 5 µm
Tubulin dimer
Plus end
• Tubulin dimers self-assembled
in parallel protofilaments
• Polarized hollow rigid
cylinders
Minus end
The Cell, Alberts et al, (2002)
Kinesin: Molecular Motor Moving on Microtubules
Lippincott-Schwartz et al, JCB (1995)
Hirokawa, Science (1998)
tail
thread
Kinesin-1
Microtubule
Bar = 5 µm
Motor
domains
Bar = 50 nm
• Transport of membrane intermediates ADP
• Mechano-enzyme:
ATP hydrolysis
• Steps = 8 nm
-
Barre = 10 nm
ATP
+
Dynamics of Kinesins
Bead assay
_
+
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V0: velocity of kinesin in
absence of external load
V0 = 0.6 ± 0.1 µm/s
In presence of applied force
ku increases
ku 
 f0a 
0
ku exp

K
T
 B 
ku0: unbinding rate
at zero
• V decreases
withload
ku0 = 0.42 s-1
applied force
Vale et al., Nature (1996)
• Stall force:
kFB : =
binding
6 pNrate of
S
kinesin onto MT

Block et al., PNAS (2003)
Membrane Tubes
Force
Membrane Nanotubes
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Outlines
• Physics of membrane tubes : tube formation
• Pulling on membrane with molecular motors
• Different dynamical regimes
1.Tube Formation
D. Cuvelier
A. Roux
P. Nassoy
Physics of Membrane Tubes
: bending rigidity
L
f
2R

E tube  2L
 2RL  fL
2R
: membrane
tension
f 0  2 2

R0 
2
Dérényi et al, PRL 88 (2002) 238101
Experimental confirmation
Optical Tweezers
+
Micropipette
DP
Tension 
Dx -> F
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Results
EPC
Theory
f0=18 pN
 = 8. 10-5 N.m-1

f0  2 2 
Bending rigidity measurements
Vesicles : lipids +
5%DOPE-Peg2000 /
DOPE - peg2000 -biotin (1/1000)
 (kBT)
EPC
13.6 ± 1.3
50% DOPC + 50% cholesterol
(liquid disordered)
30 ±3.0
50% sphingomyelin +
50% cholesterol (liquid ordered)
65 ± 6
Roux et al EMBO J. 24 (2005) 1537
2. Pulling Tubes with Molecular Motors
Tubular structures in living cells
Very dynamic tubular structures in living cells (GFP)
Endoplasmic Reticulum, Golgi, Endosomes
E.R.
Golgi
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VSVG-GFP
J. Lippincott Schwartz (CBMB-NIH)
Bar = 1 µm
Waterman-Storer & Salmon, Curr. Biol. (1998)
Microtubules
RE
Microtubules depolymerization
or Kinesin inhibition
NO TUBE
Required :
Microtubules
+ Motors
HYPOTHESIS
Molecular Motors (kinesins) in contact with Microtubules
bound to Membrane of Giant Unilamellar Vesicles (GUVs)
can extract membrane tubes
+ ATP
Kinesin
Membrane
• How many motors required to pull tubes ?
f0 >10 pN
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
• 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
Binding motors to the membrane
Streptavidin
coated BEADS
(100nm)
+
Biotinylated
lipids (5%)
+
Biotinated
kinesins
Minimal System
+ ATP
(1 mM)
kinesins
Vesicle
TUBE
microtubule
Roux A. et al PNAS (2002) 99, 5394
Transmission Electronic Microscopy
d=40±10 nm
microtubules
membrane nanotubes
d2 
2
Bars:
5mm
500 nm
Coll. J. Cartaud (Inst. J. Monod, Paris)
 5.105 N m
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X 40
(total = 15 min.)
Microtubules
Membrane tubes
Bar = 5 µm
Tubes
WITHOUT Beads
Cécile Leduc (Exp)
Otger Campàs (Theory)
Motors individually bound to lipids
TUBES !!!!!
C. Leduc et al, PNAS (2004) 101, 17096
Parameters regulating tube extraction

force necessary for extracting tubes F0.
F02(2)1/2
F0 ~ 28 pN
r∞
number of motors pulling the tube
Conditions for Tube Extraction
• Fixed motor concentration r∞ :
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Higher tension
 ,
F0
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Low tension
Threshold in tension for a given motor concentration
C. Leduc et al, PNAS (2004) 101, 17096
• Fixed membrane tension 
Quantitative measurements
NO
TUBE
0
For  = 2.10-4 N/m,
TUBE
r∞min = 200 motors/µm2
0,01 %
r∞min
0,1 %
1%
r∞
Threshold in motor concentration for a given tension
• Theoretical analysis effectively predicts:
r∞min = cste . max
System Geometry
Side view
(3D Reconstruction)
Bar = 5 µm
Dynamical recruitment
of motors
"Physical" clusters
G. Koster et al, PNAS (2003)100, 15183
C. Leduc, O. Campàs et al, PNAS (2004) 101, 17096
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Theoretical analysis O. Campàs, J.-F. Joanny and J. Prost
Tip
|Ju|
Motors unbound to MT
Motors bound to MT
kb
ku0
V0
Jb
nb
V
nb: number of bound motors at the tip
Jb: incoming flux of bound motors
Ju: incoming flux of unbound motors
dnb
 J b  ku (nb )n b
dt
ku  ku0 exp(
C. Leduc et al, PNAS (2004) 101, 17096
V  V0 (1 
f0a 1
)
K BT nb
f0 1
)
f S nb
Short time scales
Theoretical analysis
O. Campàs, J.-F. Joanny and J. Prost
Ju
Jb
Ju
nb
Jb
Fluxes equilibrium & V>0:
nb


~
r
J b( x  0;V [nb ])  ku (nb )nb
Analytical solutions
Bifurcation diagram
Short time scales
Condition
for tube formation at the threashold
O. Campàs, J.-F. Joanny and J. Prost
Conditions for tube extraction
2


e
a
k b  k 0u k 0u
min
r  fS 

r


2 K BT  k b V0
2
nb
min

At the
threashold:
f0a

2 K BT
rmin, th  400  200 motors/µm2
rmin, exp  200  100 motors/µm2
nbmin ~ 5 motors
Theory
Experiments
Motor Distribution Along the Tubes
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Biotinylated and Fluorescent Lipid
(L. Bourel, Lille)
Motor accumulation at the tip
x 60
Bar : 1 mm
Instantaneous motor distribution
Experiments
Theory
1.0
0.8
0.6
0.4
0.2
0.0
0
10
20
30
40
Experiments vs. Theory
Experiments
Theory
Exponential distribution

2
4kB V0  V  
1  1 



0
0
2k BV0  V 
ku
kuD 

0
k uD
k0u = 0.42 s-1
control
With
D = 1,0 ± 0.5 µm2/s
(FRAP)
V0 = 0.6 ±0.1 µm/s
One parameter fit
nB≈ 20 motors
k = 4.7 ± 2.4 s-1
3. Other Dynamical Regimes
Long Tubes
f 0  2 2
Constant
tension:
Constant Force
Non-fixed
tension:

Entropic regime
Elastic regime
Increasing Force
Cuvelier et al Europhys. Lett (2005)
Dynamical Diagram
Floppy vesicles
(O. Campàs)

Dynamical Diagram
(O. Campàs)
Stops
Stable
states
Collective
oscillations
Oscillatory
regime
Theory
Experiments
Kinetic Montecarlo simulations
Experiments
Fluorescence Intensity
distance (mm)
Large Scale Traffic Phenomena
time (s)
distance (mm)
Tip
Conclusions
• Minimal system mimicking transport intermediates
• Formation of dynamical clusters (physics origin)
• Molecular parameter of the motors (kB) 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
Maturation of dentritic cells
Reorganisation of multivesicular bodies (late endosomes)
Tension= switch ?
Before activation
After activation
M. Kleijmeer et al JCB (2001)
Perspectives
Modeling :
• Oscillations
• Traffic jams
• Motors with different dynamic characteristics
• Tubes pulled by non-processive motors
• Plus-end and Minus-end motors. Competition?
• Pulling tubes in living cells
The People :
Collaborations
Curie Institute
Cécile Leduc
Aurélien Roux
Damien Cuvelier
Pierre Nassoy
Biology
Bruno GOUD
• J. Cartaud (IJM, Paris)
• G.Koster, M.Van Duijn,
M.Dogterom
(AMOLF Amsterdam)
• P.Joliemaitre and L. Bourrel
(Pasteur Inst.,Lille)
• F. Nédélec (EMBL, Heidelberg)
Theory
O. Campas,
I.Dérényi, C. Storm, F. Jülicher,
J-François Joanny,
Jacques Prost