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

M embranes N anotubes P ulled C ooperatively by M olecular M otors. O rganelles in C ells. I ntracellular M embrane T raffic. Kirschhausen T., Nature reviews (2000). Formation of “transport intermediates”. Budding - Fission - Transport - Fusion. T ransport I ntermediates:.

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

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  1. Membranes NanotubesPulled Cooperatively by Molecular Motors

  2. Organelles in Cells

  3. Intracellular Membrane Traffic Kirschhausen T.,Nature reviews (2000)

  4. Formation of “transport intermediates” Budding - Fission - Transport - Fusion

  5. Transport Intermediates: Small Vesicles Long Tubes Trafficking of P2X4-GFP receptors in neuron Trafficking of Rab6 in HeLa cell R. D. Murrell-Lagnado, Cambridge, UK (White & al. JCB147, 743-760)

  6. 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)

  7. Kinesin:MolecularMotor Moving on Microtubules - + Lippincott-Schwartz et al, JCB (1995) Hirokawa, Science (1998) tail thread Bar = 5 µm Kinesin-1 Motor domains Microtubule Barre = 10 nm Bar = 50 nm ADP • Transport of membrane intermediates • Mechano-enzyme: • ATP hydrolysis • Steps = 8 nm ATP

  8. Bead assay _ V0:velocity of kinesin in absence of external load + 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 force ku increases • V decreases with applied force • Stall force: • FS = 6 pN kB :binding rate of kinesin onto MT Block et al., PNAS (2003) Dynamics of Kinesins

  9. Membrane Tubes Force Membrane Nanotubes

  10. Outlines • Physics of membrane tubes : tube formation • Pulling on membrane with molecular motors • Different dynamical regimes

  11. 1.Tube Formation D. Cuvelier A. Roux P. Nassoy

  12. L f 2R Physics of Membrane Tubes k: bending rigidity s: membrane tension Dérényi et al, PRL 88 (2002) 238101

  13. DP Tension s Dx -> F Experimental confirmation Optical Tweezers + Micropipette

  14. Results Theory s= 8. 10-5 N.m-1  EPC f0=18 pN

  15. Bending rigidity measurements Roux et al EMBO J. 24 (2005) 1537

  16. 2.Pulling Tubes with Molecular Motors

  17. Microtubules RE Tubular structures in living cells Very dynamic tubular structures in living cells (GFP) Endoplasmic Reticulum, Golgi, Endosomes E.R. Golgi VSVG-GFP Bar = 1 µm J. Lippincott Schwartz (CBMB-NIH) Waterman-Storer & Salmon, Curr. Biol. (1998)

  18. HYPOTHESIS Molecular Motors(kinesins) in contact with Microtubules bound to Membrane ofGiant Unilamellar Vesicles (GUVs) can extract membrane tubes + ATP Kinesin Required : Microtubules + Motors Membrane Microtubules depolymerization or Kinesin inhibition NO TUBE

  19. Small Motor CLUSTERS should be necessary • 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 • Tube extraction : • Combination of the membrane physical properties • and of the dynamical properties of the motors

  20. "Chemical" Clusters of Motors pulling Membrane Tubes A. Roux

  21. Binding motors to the membrane Streptavidin coated BEADS(100nm) + Biotinylated lipids (5%) + Biotinated kinesins

  22. + ATP (1 mM) kinesins Vesicle microtubule Minimal System TUBE Roux A. et al PNAS (2002) 99, 5394

  23. Transmission Electronic Microscopy d=40±10 nm microtubules membrane nanotubes Bars: 5mm 500 nm Coll. J. Cartaud (Inst. J. Monod, Paris)

  24. X 40 (total = 15 min.)

  25. Microtubules Bar = 5 µm Membrane tubes

  26. Tubes WITHOUT Beads Cécile Leduc (Exp) Otger Campàs (Theory)

  27. Motors individually bound to lipids TUBES !!!!! C. Leduc et al, PNAS (2004) 101, 17096

  28. F0 ~ 28 pN Parameters regulating tube extraction s force necessary for extracting tubesF0. F0=2p(2sk)1/2 r∞number of motors pulling the tube

  29. Conditions for Tube Extraction • Fixed motor concentration r∞ : s, F0 Higher tension Low tension Threshold in tension for a given motor concentration C. Leduc et al, PNAS (2004) 101, 17096

  30. Fixed membrane tension s Quantitative measurements For s = 2.10-4 N/m, r∞min= 200motors/µm2 NO TUBE TUBE r∞ 0,01 % r∞min 0,1 % 1 % 0 Threshold in motor concentration for a given tension • Theoretical analysis effectively predicts: • r∞min = cste . smax

  31. System Geometry Dynamical recruitment of motors "Physical" clusters G. Koster et al, PNAS (2003)100, 15183 C. Leduc, O. Campàs et al, PNAS (2004) 101, 17096 Side view (3D Reconstruction) Bar = 5 µm

  32. Theoretical analysis O. Campàs, J.-F. Joanny and J. Prost Tip |Ju| Motors unbound to MT Motors bound to MT kb ku0 nb V0 Jb V nb:number ofboundmotors at the tip Jb:incoming flux of bound motors Ju: incoming flux ofunbound motors C. Leduc et al, PNAS (2004) 101, 17096

  33. Theoretical analysis O. Campàs, J.-F. Joanny and J. Prost Short time scales Ju Ju nb nb Jb Jb Fluxes equilibrium & V>0: ~ Analytical solutions Bifurcation diagram

  34. Condition for tube formation at the threashold O. Campàs, J.-F. Joanny and J. Prost Short time scales Conditions for tube extraction motors/µm2 Theory At the threashold: Experiments motors/µm2 nbmin ~ 5 motors

  35. x 60 Bar : 1 mm Motor Distribution Along the Tubes Biotinylated and Fluorescent Lipid(L. Bourel, Lille) Motor accumulation at the tip

  36. Instantaneous motor distribution Experiments Theory

  37. Experiments vs. Theory k0u = 0.42 s-1 D = 1,0 ± 0.5 µm2/s (FRAP) V0 = 0.6 ±0.1 µm/s With control nB≈ 20 motors Experiments Theory Exponential distribution One parameter fit kb = 4.7 ± 2.4 s-1

  38. 3.Other Dynamical Regimes

  39. Constant tension: Constant Force Entropic regime Non-fixed tension: Elastic regime Increasing Force Cuvelier et al Europhys. Lett (2005) Long Tubes

  40. Dynamical Diagram (O. Campàs) Floppy vesicles 

  41. Dynamical Diagram (O. Campàs) Collective oscillations Stops Oscillatory regime Stable states Theory Experiments Kinetic Montecarlo simulations Experiments

  42. Large Scale Traffic Phenomena distance (m) time (s) Tip Fluorescence Intensity distance (m)

  43. 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

  44. Maturation of dentritic cells Reorganisation of multivesicular bodies (late endosomes) Tension= switch ? Before activation After activation M. Kleijmeer et al JCB (2001)

  45. 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

  46. The People : Curie Institute Collaborations Cécile Leduc Aurélien Roux Damien Cuvelier Pierre Nassoy • 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) Biology Bruno GOUD Theory O. Campas, I.Dérényi, C. Storm, F. Jülicher,J-François Joanny, Jacques Prost

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