M embranes N anotubes P ulled C ooperatively by M olecular M otors
description
Transcript of M embranes N anotubes P ulled C ooperatively by M olecular M otors
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 TransportBar = 5 µm
Tubulin dimer
Plus end
Minus end
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
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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⎝ ⎜
⎞
⎠ ⎟
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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
1.Tube Formation
D. CuvelierA. RouxP. Nassoy
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
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
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
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 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
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
"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 MicroscopyTransmission Electronic Microscopy
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d=2 κ2σ
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σ≈5.10−5 NmBars: 5m 500 nm
Coll. J. Cartaud (Inst. J. Monod, Paris)
microtubules
membrane nanotubes
d=40±10 nm
X 40(total = 15 min.)
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Bar = 5 µmMicrotubules
Membrane tubes
TubesWITHOUT 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
F02π(2σκ)1/2
F0 ~ 28 pN
∞ number of motors pulling the tube
σ force necessary for extracting tubes F0.
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
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
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
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 ~
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
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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
Instantaneous motor distribution
Theory1.0
0.8
0.6
0.4
0.2
0.0
403020100
Experiments
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
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Experiments
nB≈ 20 motors
3. Other Dynamical Regimes
Entropic regime
Elastic regime
Long Tubes
Constant
tension:
€
f0 = 2π 2κσ
Constant Force
Non-fixed
tension:
Increasing ForceCuvelier et al Europhys. Lett (2005)
Flo
pp
y
vesic
les
Dynamical Diagram (O. Campàs)
Stable states Oscillatory regime
Kinetic Montecarlo simulations
Experiments
Experiments
Theory
Collective oscillationsStops
Dynamical Diagram (O. Campàs)
Tip
distance ( m)
Fluore
scence
In
tensi
tydis
tan
ce
(m
)
time (s)
Large Scale Traffic Phenomena
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
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
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)