M embranes N anotubes P ulled C ooperatively by M olecular M otors

transcript

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

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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Membrane Tubes

Membrane Nanotubes

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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

E tube = 2πLκ

2R+ 2πRσL − fL

κσπ 220 =f

R0 =κ

Dérényi et al, PRL 88 (2002) 238101

κ: bending rigidity

σ: membrane tension

x -> FTension σ

Experimental confirmation

Optical Tweezers+

Micropipette

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Results

f0=18 pN

= 8. 10-5 N.m-1

Theory

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

VSVG-GFP

J. Lippincott Schwartz (CBMB-NIH)

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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

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)

Roux A. et al PNAS (2002) 99, 5394

Minimal System

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

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

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 %∞

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

nb: number of bound motors at the tip

Jb: incoming flux of bound motors Ju: incoming flux of unbound motors

Motors bound to MT

Motors unbound to MT

bbubb nnkJ

dn)(−=

exp( 00

bBuu nTK

afkk =

fVV −=

Theoretical analysis O. Campàs, J.-F. Joanny and J. Prost

C. Leduc et al, PNAS (2004) 101, 17096

Short time scales

Fluxes equilibrium & V>0:

Bifurcation diagramAnalytical solutions

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

0min =

∞ > e2

⎝ ⎜

⎠ ⎟

2kb + ku

V0≡ ρ∞

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

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

One parameter fit

kb = 4.7 ± 2.4 s-1

Experiments vs. Theory

λ =ku

2kB V0−V( )1+ 1+

V0 −V( )2

⎜ ⎜

⎠ ⎟ ⎟

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)

Dynamical Diagram (O. Campàs)

Stable states Oscillatory regime

Kinetic Montecarlo simulations

Experiments

Theory

Collective oscillationsStops

Dynamical Diagram (O. Campàs)

distance ( m)

Fluore

scence

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)

M embranes N anotubes P ulled C ooperatively by M olecular M otors

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