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Vesicular Exocytosis “Neurotransmission and Catecholamines Release”

Vesicular Exocytosis “Neurotransmission and Catecholamines Release”. Christian Amatore Ecole Normale Supérieure, Département de Chimie UMR CNRS-ENS-UPMC 8640 "PASTEUR" Paris - France. Adapted from: http://www.abcam/neuroscience/. Adapted from: http://www.abcam/neuroscience/.

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Vesicular Exocytosis “Neurotransmission and Catecholamines Release”

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  1. Vesicular Exocytosis “Neurotransmission and Catecholamines Release” Christian Amatore Ecole Normale Supérieure, Département de Chimie UMR CNRS-ENS-UPMC 8640 "PASTEUR" Paris - France

  2. Adapted from: http://www.abcam/neuroscience/

  3. Adapted from: http://www.abcam/neuroscience/

  4. Adapted from: http://www.abcam/neuroscience/

  5. Adapted from: http://www.abcam/neuroscience/

  6. Adapted from: http://www.mhhe.com/socscience/intro/ibank/set1.htm

  7. The Chromaffin Cell

  8. Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854.

  9. Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854.

  10. Photographs: release of insulin by pancreatic b-cells. Robert Kennedy. Private communication. (2002). Left sketch adapted from: http://www.mhhe.com/socscience/intro/ibank/set1.htm

  11. 10 µm E.L. Ciolkowski, K.M. Maness, P.S. Cahill, R.M. Wightman, D.H. Evans, B. Fosset, C. Amatore. Anal. Chem., 66, 1994, 3611.

  12. IC Itot = IF + IC IF • Problems Associated with Ultrafast Electrochemistry

  13. IC Itot = IF + IC IF • Ohmic Drop: • E(t) = ZFIF + RuItot(t) • Cell Time Constant: • tcell = RuCd • Problems associated with applying ultrafast electrochemical perturbations:

  14. Ru 1/r0 Cd r02 IC and IF r02 • Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant IC Itot = IF + IC IF

  15. IC Itot = IF + IC IF • For Planar Diffusion: RuItot  r0  0 • For Any Diffusional Regime: Ru Cd r0  0 • Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant

  16. E(t) ZFIF IF Itot – Cd(dE/dt) • Compensation of Ohmic Drop and Time Constant ZF IF = E(t) - (RuItot) IC= Cd(dE/dt) - RuCd(dItot/dt)

  17. Ultramicroelectrode (measurement) Release Micropipette (stimulation) Living Cell Petri dish with PBS 10 µm • Principle of Electroanalytical Measurements at Single Cells

  18. glass cases platinized surfaces insulating polymer 5 µm 5 µm 1-5 µm 10-12 µm • Preparation of Platinized Carbon Fiber Ultramicroelectrodes • Intrinsic Requirements • Sensitive detection of H2O2 ( "normal" [H2O2]cellular  10-9 to 10‑6 M ) • Sensitive detection of other expected species (NO°, etc.) •  Aerobic conditions ( [O2]  0,23 mM at 25° C ) •  Analysis medium: PBS •  Microsensor dimensions: adapted to cell dimensions • Real-time detection of biological events.

  19. 10 µm Qav = 0.9 pC Nav = 2.7 106 molecules

  20. Five Independent Physicochemical Stages Govern Exocytosis: 0. I. II. III. IV. Docking Fusion Pore Full Fusion III. 0. 0. II. IV. I. III.  IV. I. Photographs adapted from: R. Fesce et al., Trends Cell Biol., 4, 1994, 1-4 T.J. Schroeder, R. Borges, K. Pihel, C. Amatore, R.M. Wightman. Biophys. J., 70, 1996, 1061-1068.

  21. Docking Occurs at Specifically Structured Areas in Cell Membrane: Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854. Sketchs adapted from: Y. Humeau, F. Doussau, N.J. Grant, B. Poulain, Biochim., 82, 2000, 427-446.

  22. Docking Phase: Structure of SNAREs Protein Assembly

  23. Blocking Docking by Altering SNAREs Assembling with Botulin: Cells transfected through electroporation with modified plasmides / DNA. Secretion elicited 48 hrs later with Ca2+, 2.5 mM. C. Amatore, S. Arbault, I. Bonifas, F. Darchen, M. Guille, JP. Henry, to be published.

  24. 60 40 Cumulated Secretion Events 20 0 0 40 time (s) • Importance of SNAREs Assembling: Botulin + GFP Cells transfected through electroporation with modified plasmides / DNA. Secretion elicited 48 hrs later with Ca2+, 2.5 mM. C. Amatore, S. Arbault, I. Bonifas, F. Darchen, M. Guille, JP. Henry, to be published.

  25. Five Independent Physicochemical Stages Govern Exocytosis: 0. I. II. III. IV. Docking Fusion Pore Full Fusion III. 0. 0. II. IV. I. III.  IV. I. Photographs adapted from: R. Fesce et al., Trends Cell Biol., 4, 1994, 1-4 T.J. Schroeder, R. Borges, K. Pihel, C. Amatore, R.M. Wightman. Biophys. J., 70, 1996, 1061-1068.

  26. Pore Formation: The Stalk Model

  27. 2R • Regulating Exocytosis with Exogenous Bilipids Surface tension Edge tension . C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

  28. Control • Regulating Exocytosis with Exogenous Bilipids

  29. LPC Control AA O H O H LPC P O O O O N O C O H 2 AA • Regulating Exocytosis with Exogenous Bilipids

  30. O H O H LPC P O O O O N O C O H 2 AA • Regulating Exocytosis with Exogenous Bilipids

  31. LPC (4 Hz) 1200 1000 Control 800 (2.5 Hz) Control 600 # Cumulated events AA 400 (1 Hz) AA 200 0 0 50 100 150 200 250 300 Time (s) • Regulating Exocytosis with Exogenous Bilipids 1400

  32. DU≠ pre - fusion full fusion • Regulating Exocytosis with Exogenous Bilipids

  33. 1400 DU≠ 1200 LPC n k = k0 exp(-bDU≠/kBT) 1000 800 Control 2.4 d(DU≠)LPC = kBT ln( )  - 1 kBT 4 600 Cumulated events 400 AA 200 2.4 d(DU≠)AA = kBT ln( )  + 2 kBT 0 1 0 50 100 150 200 250 300 Time (s) • Regulating Exocytosis with Exogenous Bilipids

  34. Release Through Initial Fusion Pore: n = 2 F = 96 500 Cb <Dgranule>=4.8 10-8 cm2s-1 <Cgranule> = 0.6 M Rpore /nm ≈ 0.3x ifoot /pA C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

  35. Release Through Initial Fusion Pore: Rpore /nm ≈ 0.3x ifoot /pA Rpore= (1.5 ± 0.5) nm (patch-clamp measurements (Neher, Fernandez, etc.): Rpore between 1 and 3 nm)

  36. How Full Fusion May Follow Pore Release ? . C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

  37. How Full Fusion May Follow Pore Release ? . C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

  38. Full Fusion: Driving Force = Granule Swelling upon Release Concept based on de Gennes’ "Blob Theory« , see e.g.: J.L. Barrat, J.F. Joanny, in Adv. Chem. Phys. (I. Prigogine & S. Rice, eds.). Vol 44, pp. 37-33. Wiley NY, 1996. Photographs adapted from Geoffrey Fox: www.mpibpc.gwdg.de/inform/MpiNews/cientif/jahrg6/10.00/fig5.html

  39. Five Independent Physicochemical Stages Govern Exocytosis: 0. I. II. III. IV. Docking Fusion Pore Full Fusion III. 0. 0. II. IV. I. III.  IV. I. Photographs adapted from: R. Fesce et al., Trends Cell Biol., 4, 1994, 1-4 T.J. Schroeder, R. Borges, K. Pihel, C. Amatore, R.M. Wightman. Biophys. J., 70, 1996, 1061-1068.

  40. Diffusion: control by Dt/Rvesicle2 Rate of full fusion: surface area increases • Full Fusion: Two Phenomena Govern Spike Shapes: . C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

  41. Diffusion: control by Dt/Rvesicle2 Rate of full fusion: surface area increases • Full Fusion: Two Phenomena Govern Spike Shapes: Release elicited by 10s BaCl2, 2 mM, in Locke buffer with MgCl2, 0.7 mM. C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

  42. Full Fusion Kinetics • Amperommetry: Area Time (ms) • Evanescent wave spectroscopy: W. Almers et al., Nature 406, 2000, 849-854.

  43. "Seeing" & "Measuring" :Fluorescence and Amperommetry

  44. Energy released:(a) • Dissipation of energy released:(b) • First Half of Full Fusion C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496. (a) : Energy of a membrane pore: Taupin and de Gennes (b) : Rate law for viscous dissipation: F. Brochard-Wyart & colls., PNAS, 96, 1999,10591-10596.

  45. 0.8 0.6 (R / R ) 0.4 vesicle 0.2 0 0 0.25 0.5 0.75 1 t / t 80% • First Half of Full Fusion: • Dissipation of Cell and Vesicle Membrane High Tensions C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496.

  46. 1 0.8 R / Rvesicle 0.6 0.4 0.2 0 0.25 0.5 0.75 1 • Second Half of Full Fusion: Dissipation of Line Tension Between Relaxed Membranes C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496.

  47. Testing Our Model C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-154.

  48. Ss large h • Testing Our Model fast C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-154.

  49. fast slow Ss Ss Ss large small large h h h • Testing Our Model fast C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-154.

  50. Reducing Ss, viz. the Driving Force, by Refraining Swelling Photographs adapted from Geoffrey Fox: www.mpibpc.gwdg.de/inform/MpiNews/cientif/jahrg6/10.00/fig5.html

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