m embranes n anotubes p ulled c ooperatively by m olecular m otors
TRANSCRIPT
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Membranes Nanotubes
Pulled Cooperatively by Molecular Motors
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Organelles in Cells
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Kirschhausen T.,Nature reviews (2000)
Intracellular Membrane Traffic
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Budding - Fission - Transport - Fusion
Formation of “transport intermediates”
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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 TransportBar = 5 µm
Tubulin dimer
Plus end
Minus end
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Bar = 50 nm
Hirokawa, Science (1998)Lippincott-Schwartz et al, JCB (1995)
Bar = 5 µm
-
+
MicrotubuleKinesin-1
Kinesin: Molecular Motor Moving on Microtubules
ATPADP
Motor domains
thread
tail
Barre = 10 nm
• Transport of membrane intermediates
• Mechano-enzyme: ATP hydrolysis
• Steps = 8 nm
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Block et al., PNAS (2003)
Dynamics of Kinesins
kB : binding rate of kinesin onto MT
• V decreases with applied force
• Stall force:
FS = 6 pN
V0: velocity of kinesin in absence of external load
Bead assay
V0 = 0.6 ± 0.1 µm/s
ku0: unbinding rate
at zero load
ku0 = 0.42 s-1
Vale et al., Nature (1996)
_+
In presence of applied forceku increases
€
ku = ku0 exp
f0aKBT
⎛
⎝ ⎜
⎞
⎠ ⎟
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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
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1.Tube Formation
D. CuvelierA. RouxP. Nassoy
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Physics of Membrane Tubes
Lf
2R
€
E tube = 2πLκ
2R+ 2πRσL − fL
κσπ 220 =f
€
R0 =κ
2σ
Dérényi et al, PRL 88 (2002) 238101
κ: bending rigidity
σ: membrane tension
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P
x -> FTension σ
Experimental confirmation
Optical Tweezers+
Micropipette
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Results
f0=18 pN
= 8. 10-5 N.m-1
Theory
EPC
€
f0 =2π 2κ σκ
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Vesicles : lipids +
5%DOPE-Peg2000 /
DOPE - peg2000 -biotin (1/1000)
κ (kBT)
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
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2. Pulling Tubes with Molecular Motors
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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 µm
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 depolymerizationor Kinesin inhibition
NO TUBE
Required :Microtubules
+ Motors
Membrane
Kinesin
+ ATP
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1 kinesin ≈ 6 pN max (stall force)
A few kinesins should be sufficientbut
MORE THAN ONE kinesin required
Small Motor CLUSTERS should be necessary
• How many motors required to pull tubes ?
f0 >10 pN
• Tube extraction : Combination of the membrane physical properties and of the dynamical properties of the motors
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"Chemical" Clusters
of Motors
pulling Membrane Tubes
A. Roux
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Streptavidin coated BEADS
(100nm)
+
Biotinylated lipids (5%)
+
Biotinated kinesins
Binding motors to the membrane
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microtubule
kinesins
Vesicle
+ ATP(1 mM)
TUBE
Roux A. et al PNAS (2002) 99, 5394
Minimal System
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Transmission Electronic MicroscopyTransmission Electronic Microscopy
€
d=2 κ2σ
€
σ≈5.10−5 NmBars: 5m 500 nm
Coll. J. Cartaud (Inst. J. Monod, Paris)
microtubules
membrane nanotubes
d=40±10 nm
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X 40(total = 15 min.)
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Bar = 5 µmMicrotubules
Membrane tubes
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TubesWITHOUT Beads
Cécile Leduc (Exp)Otger Campàs (Theory)
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Motors individually bound to lipids
TUBES !!!!!
C. Leduc et al, PNAS (2004) 101, 17096
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Parameters regulating tube extraction
F02π(2σκ)1/2
F0 ~ 28 pN
∞ number of motors pulling the tube
σ force necessary for extracting tubes F0.
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Conditions for Tube ExtractionConditions for Tube Extraction
• Fixed motor concentration ∞ :
Higher tension Low tension
σ F0
Threshold in tension for a given motor concentration
C. Leduc et al, PNAS (2004) 101, 17096
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• Fixed membrane tension σ
0 ∞ 0,01 %∞
min
0,1 % 1 %
NO TUBE TUBE
Quantitative measurements
For σ = 2.10-4 N/m,
∞min = 200 motors/µm2
• Theoretical analysis effectively predicts:
∞min = cste . σmax
Threshold in motor concentration for a given tension
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Side view
(3D Reconstruction)Bar = 5 µm
System Geometry
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Dynamical recruitment of motors
G. Koster et al, PNAS (2003)100, 15183
"Physical" clusters
C. Leduc, O. Campàs et al, PNAS (2004) 101, 17096
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V
nb: number of bound motors at the tip
Jb: incoming flux of bound motors Ju: incoming flux of unbound motors
nb
Motors bound to MT
Motors unbound to MT
ku0kb
Jb
|Ju|
V0
bbubb nnkJ
dt
dn)(−=
)1
exp( 00
bBuu nTK
afkk =
)1
1( 00
bS nf
fVV −=
Theoretical analysis O. Campàs, J.-F. Joanny and J. Prost
Tip
C. Leduc et al, PNAS (2004) 101, 17096
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Short time scales
Fluxes equilibrium & V>0:
Bifurcation diagramAnalytical solutions
nb
Ju
Jb nb
Ju
Jb
Theoretical analysis O. Campàs, J.-F. Joanny and J. Prost
bbubb nnknVxJ )(])[;0( ==∞
∝σν ~
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Conditions for tube extraction
Short time scales Condition for tube formation at the threashold
O. Campàs, J.-F. Joanny and J. Prost
At the threashold:
nbmin ~ 5 motors
200 400 min, ±=∞th
100 200exp min, ±=∞
motors/µm2
motors/µm2
TK
afn
Bb 2
0min =
€
∞ > e2
2fS
aKBT
⎛
⎝ ⎜
⎞
⎠ ⎟
2kb + ku
0
kb
ku0
V0≡ ρ∞
min
Theory
Experiments
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Motor Distribution Along the Tubes
Biotinylated and Fluorescent Lipid (L. Bourel, Lille)
Motor accumulation at the tipx 60
Bar : 1 m
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Instantaneous motor distribution
Theory1.0
0.8
0.6
0.4
0.2
0.0
403020100
Experiments
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control
Theory
Exponential distribution
k0u = 0.42 s-1
D = 1,0 ± 0.5 µm2/s (FRAP)
V0 = 0.6 ±0.1 µm/s
With
One parameter fit
kb = 4.7 ± 2.4 s-1
Experiments vs. Theory
€
λ =ku
0D
2kB V0−V( )1+ 1+
4kB
ku0
V0 −V( )2
ku0D
⎛
⎝
⎜ ⎜
⎞
⎠ ⎟ ⎟
Experiments
nB≈ 20 motors
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3. Other Dynamical Regimes
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Entropic regime
Elastic regime
Long Tubes
Constant
tension:
€
f0 = 2π 2κσ
Constant Force
Non-fixed
tension:
Increasing ForceCuvelier et al Europhys. Lett (2005)
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Flo
pp
y
vesic
les
Dynamical Diagram (O. Campàs)
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Stable states Oscillatory regime
Kinetic Montecarlo simulations
Experiments
Experiments
Theory
Collective oscillationsStops
Dynamical Diagram (O. Campàs)
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Tip
distance ( m)
Fluore
scence
In
tensi
tydis
tan
ce
(m
)
time (s)
Large Scale Traffic Phenomena
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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 :expressionregulation of the fixation sites
- More efficient : modulation of the membrane tension
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Reorganisation of multivesicular bodies (late endosomes)
Tension= switch ?
Maturation of dentritic cells
Before activation After activation
M. Kleijmeer et al JCB (2001)
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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
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The People :
Curie Institute
Cécile LeducAurélien RouxDamien CuvelierPierre Nassoy
O. Campas, I.Dérényi, C. Storm, F. Jülicher,J-François 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. Nédélec (EMBL, Heidelberg)