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Vesicular Exocytosis “Neurotransmission and Catecholamines Release” Christian Amatore Ecole Normale Supérieure, Département de Chimie UMR CNRS-ENS-UPMC.

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Presentation on theme: "Vesicular Exocytosis “Neurotransmission and Catecholamines Release” Christian Amatore Ecole Normale Supérieure, Département de Chimie UMR CNRS-ENS-UPMC."— Presentation transcript:

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:

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4

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6 Adapted from:

7  The Chromaffin Cell

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

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10 Photographs: release of insulin by pancreatic  -cells. Robert Kennedy. Private communication. (2002). Left sketch adapted from:

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

12  Problems Associated with Ultrafast Electrochemistry ICIC IFIF I tot = I F + I C

13  Problems associated with applying ultrafast electrochemical perturbations:  Ohmic Drop: E(t) = Z F I F + R u I tot (t)  Cell Time Constant:  cell = R u C d ICIC IFIF I tot = I F + I C

14  Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant ICIC IFIF I tot = I F + I C R u  1/r 0 C d  r 0 2 I C and I F  r 0 2

15  Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant ICIC IFIF I tot = I F + I C R u I tot  r 0  0 R u C d  r 0  0 o For Planar Diffusion: o For Any Diffusional Regime:

16  Compensation of Ohmic Drop and Time Constant Z F I F = E(t) -  R u I tot ) I C = C d (dE/dt) - R u C d (dI tot /dt) E(t)  Z F I F I F  I tot – C d (dE/dt)

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

18  Preparation of Platinized Carbon Fiber Ultramicroelectrodes o Sensitive detection of H 2 O 2 ( "normal" [H 2 O 2 ] cellular  to 10 ‑ 6 M ) o Sensitive detection of other expected species (NO°, etc.) o Aerobic conditions ( [O 2 ]  0,23 mM at 25° C ) o Analysis medium: PBS o Microsensor dimensions: adapted to cell dimensions o Real-time detection of biological events. o Intrinsic Requirements µm 1-5 µm glass cases insulating polymer platinized surfaces 5 µm

19 Q av = 0.9 pCN av = molecules 10 µm

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

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

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 Ca 2 +, 2.5 mM. C. Amatore, S. Arbault, I. Bonifas, F. Darchen, M. Guille, JP. Henry, to be published.

24  Importance of SNAREs Assembling: Botulin + GFP Cells transfected through electroporation with modified plasmides / DNA. Secretion elicited 48 hrs later with Ca 2 +, 2.5 mM. C. Amatore, S. Arbault, I. Bonifas, F. Darchen, M. Guille, JP. Henry, to be published.

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

26  Pore Formation: The Stalk Model

27  Regulating Exocytosis with Exogenous Bilipids. C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, R Surface tension Edge tension

28  Regulating Exocytosis with Exogenous Bilipids Control

29  Regulating Exocytosis with Exogenous Bilipids Control LPC AA LPC N O P O O O O H OH O AA CO 2 H

30 LPC N O P O O O O H OH O AA CO 2 H  Regulating Exocytosis with Exogenous Bilipids

31 1400  Regulating Exocytosis with Exogenous Bilipids

32 U≠U≠ pre - fusion full fusion

33 U≠U≠  (  U ≠ ) LPC = k B T ln( )  - 1 k B T  (  U ≠ ) AA = k B T ln( )  + 2 k B T  k = k 0 exp(-  U ≠ /k B T)  Regulating Exocytosis with Exogenous Bilipids

34 R pore /nm ≈ 0.3 x i foot /pA C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999,  Release Through Initial Fusion Pore: n = 2 F = Cb = cm 2 s -1 = 0.6 M

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

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

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

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 Wiley NY, Photographs adapted from Geoffrey Fox:

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

40 Rate of full fusion: surface area increases Diffusion: control by Dt/R vesicle 2. C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999,  Full Fusion: Two Phenomena Govern Spike Shapes:

41 Release elicited by 10s BaCl 2, 2 mM, in Locke buffer with MgCl 2, 0.7 mM. C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999,  Full Fusion: Two Phenomena Govern Spike Shapes: Rate of full fusion: surface area increases Diffusion: control by Dt/R vesicle 2

42 W. Almers et al., Nature 406, 2000, o Evanescent wave spectroscopy:  Full Fusion Kinetics o Amperommetry: Area Time (ms)

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

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

45 C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000,  First Half of Full Fusion: Dissipation of Cell and Vesicle Membrane High Tensions

46 C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000,  Second Half of Full Fusion: Dissipation of Line Tension Between Relaxed Membranes R / R vesicle

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

48 fast   large  Testing Our Model

49 C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, fastslow   large   small  Testing Our Model fast   large

50  Reducing , viz. the Driving Force, by Refraining Swelling Photographs adapted from Geoffrey Fox:

51  Reducing  by Lanthanides Ions: C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003,

52 Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993,  Increasing , viz. the Membrane Viscosity

53 ControlHyperosmotic  Increasing  with a Hyperosmotic Shock:

54  Increasing , viz. Membrane Viscosity, with Hyperosmotic Shock: 970 mOsm Q / pC C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, K.P. Troyer, R.M. Wightman, J. Biol. Chem., 277, 2002,

55 Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993,  Decreasing  and Increasing  by Cell Membrane Tension

56  Cell Membrane Tension Through a Hypoosmotic Shock Control Hypoosmotic excess

57  Cell Membrane Tension Through a Hypoosmotic Shock Hypo. Control 2.4 Hz 3.7 Hz Time / s # Cumulated Events


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