Vesicular Exocytosis “Neurotransmission and Catecholamines Release” Christian Amatore Ecole...
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Transcript of Vesicular Exocytosis “Neurotransmission and Catecholamines Release” Christian Amatore Ecole...
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/
Adapted from: http://www.abcam/neuroscience/
Adapted from: http://www.abcam/neuroscience/
Adapted from: http://www.mhhe.com/socscience/intro/ibank/set1.htm
The Chromaffin Cell
Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854.
Photographs adapted from: W. Almers et al., Nature 406, 2000, 849-854.
Photographs: release of insulin by pancreatic -cells. Robert Kennedy. Private communication. (2002). Left sketch adapted from: http://www.mhhe.com/socscience/intro/ibank/set1.htm
E.L. Ciolkowski, K.M. Maness, P.S. Cahill, R.M. Wightman, D.H. Evans, B. Fosset, C. Amatore. Anal. Chem., 66, 1994, 3611.
10 µm
Problems Associated with Ultrafast Electrochemistry
IC
IFItot = IF + IC
Problems associated with applying ultrafast electrochemical perturbations:
Ohmic Drop:
E(t) = ZF IF + RuItot(t)
Cell Time Constant:
cell = RuCd
IC
IFItot = IF + IC
Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant
IC
IFItot = IF + IC
Ru 1/r0
Cd r02
IC and IF r02
Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant
IC
IFItot = IF + IC
Ru Itot r0 0
Ru Cd r0 0
o For Planar Diffusion:
o For Any Diffusional Regime:
Compensation of Ohmic Drop and Time Constant
ZF IF = E(t) - RuItot)
IC = Cd(dE/dt) - RuCd(dItot/dt)
E(t) ZF IF IF Itot – Cd(dE/dt)
Ultramicroelectrode (measurement)
Living Cell
Micropipette(stimulation)
Release
Petri dish with PBS
10 µm
Principle of Electroanalytical Measurements at Single Cells
Preparation of Platinized Carbon Fiber Ultramicroelectrodes
o Sensitive detection of H2O2 ( "normal" [H2O2]cellular 10-9 to 10‑6 M )
o Sensitive detection of other expected species (NO°, etc.)
o Aerobic conditions ( [O2] 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 Requirementso Intrinsic Requirements
10-12 µm 1-5 µm
glass cases
insulatingpolymer
platinizedsurfaces
5 µm 5 µm
Qav = 0.9 pC Nav = 2.7 106 molecules
10 µm
Photographs adapted from: R. Fesce et al., Trends Cell Biol., 4, 1994, 1-4
0.
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, 1061-1068.
0
20
40
60
0 40 80 120cu
rren
t /pA
time /ms
I.
II.
III.
IV.
0. I. II. III. IV.
Full FusionFull FusionFusion PoreFusion PoreDockingDocking
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.
Docking Phase: Structure of SNAREs Protein Assembly
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.
Importance of SNAREs Assembling:
GFP alone (contro l 1 ; n=26)
GFP / S nap25 W T (contro l 2 ; n=21)
GFP / B otu lin A (n=21)GFP / S nap25 L203 (n=19)
0
20
40
60
Cu
mu
late
d S
ecr
etio
n E
ven
ts
0 40
time (s)
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.
Photographs adapted from: R. Fesce et al., Trends Cell Biol., 4, 1994, 1-4
0.
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, 1061-1068.
0
20
40
60
0 40 80 120cu
rren
t /pA
time /ms
I.
II.
III.
IV.
0. I. II. III. IV.
Full FusionFull FusionFusion PoreFusion PoreDockingDocking
Pore Formation: The Stalk Model
Regulating Exocytosis with Exogenous Bilipids
R R Wpore 22
.
C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.
2R
Surface tension Edge tension
Regulating Exocytosis with Exogenous Bilipids
Control
Regulating Exocytosis with Exogenous Bilipids
Control
LPC
AA
LPCNO P O
O
OO
H OH
O
AACO2H
LPCNO P O
O
OO
H OH
O
AACO2H
Regulating Exocytosis with Exogenous Bilipids
1400
Time (s)
0 50 100 150 200 250 300
# C
umu
late
d e
vent
s
0
200
400
600
800
1000
1200
AA
Control
LPC
Control
AA
(4 Hz)
(2.5 Hz)
(1 Hz)
Regulating Exocytosis with Exogenous Bilipids
Regulating Exocytosis with Exogenous Bilipids
U≠
pre - fusion
full fusion
U≠
Time (s)
0 50 100 150 200 250 300
Cum
ulat
ed e
vent
s
0
200
400
600
800
1000
1200
1400
LPC
AA
Control (U≠)LPC = kBT ln( ) - 1 kBT2.44
(U≠)AA = kBT ln( ) + 2 kBT 2.4 1
k = k0 exp(-U≠/kBT)
Regulating Exocytosis with Exogenous Bilipids
poregranulegranulediskfoot RCnFDii 4
Rpore /nm ≈ 0.3 x ifoot /pA
C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.
Release Through Initial Fusion Pore:
n = 2F = 96 500 Cb< Dgranule > = 4.8 10-8 cm2s-1
< Cgranule > = 0.6 M
Release Through Initial Fusion Pore:
Rpore /nm ≈ 0.3 x ifoot /pA
Rpore = (1.5 ± 0.5) nm
(patch-clamp measurements (Neher, Fernandez, etc.): Rpore between 1 and 3 nm)
0
20
40
60
0 40 80 120
i foot
= 6 pA
rpore
= 1.8 nm
curr
ent /
pA
time /ms
0
10
20
30
0 50 100
i foot
= 4 pA
rpore
= 1.2 nm
time /ms
0
15
30
0 40 80 120
i foot
= 3 pA
rpore
= 0.9 nm
time /ms
How Full Fusion May Follow Pore Release ?
R RW cellvespore 2 )( 2 .
C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.
R RW cellvespore 2 )( 2
How Full Fusion May Follow Pore Release ?
.
C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.
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
Photographs adapted from: R. Fesce et al., Trends Cell Biol., 4, 1994, 1-4
0.
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, 1061-1068.
0
20
40
60
0 40 80 120cu
rren
t /pA
time /ms
I.
II.
III.
IV.
0. I. II. III. IV.
Full FusionFull FusionFusion PoreFusion PoreDockingDocking
Rate of full fusion: surface area increases
Diffusion: control by Dt/Rvesicle2
.
C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.
Full Fusion: Two Phenomena Govern Spike Shapes:
0
1
0 50
0
0,05
0,1
0,15
0,2
0 1 2 3 4 5
0
1
0 50
0
0,05
0,1
0,15
0,2
0 1 2 3 4 5
t / ms
I(t) / Ipeak
or a(t)
0
1
0 50
0
0,05
0,1
0,15
0,2
0 1 2 3 4 5
I(t)
a(t) a(t) a(t)
I(t)I(t)
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.
Full Fusion: Two Phenomena Govern Spike Shapes:
Rate of full fusion: surface area increases
Diffusion: control by Dt/Rvesicle2
W. Almers et al., Nature 406, 2000, 849-854.
o Evanescent wave spectroscopy:
Full Fusion Kineticso Amperommetry:
0
1
0 50
0
0,05
0,1
0,15
0,2
0 1 2 3 4 5
0
1
0 50
0
0,05
0,1
0,15
0,2
0 1 2 3 4 5
t / ms
I(t) / Ipeak
or a(t)
0
1
0 50
0
0,05
0,1
0,15
0,2
0 1 2 3 4 5
I(t)
a(t) a(t) a(t)
I(t)I(t)
Area
Time (ms)
"Seeing" & "Measuring" :Fluorescence and Amperommetry
vesicle
cell
First Half of Full Fusion
R RW cellvesreleased 2 2 )(
)/( 4 dtdRRWreleased
o Energy released:(a)
o Dissipation of energy released:(b)
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.
0
0.2
0.4
0.6
0.8
0 0.25 0.5 0.75 1
(R /
Rve
sicl
e )
t / t 80%
stcellvesreleased c RW 2 )(
)/( 4 dtdRRWreleased
C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496.
First Half of Full Fusion:Dissipation of Cell and Vesicle Membrane High Tensions
vesicle
cell
0 cellvesicle
C. Amatore, Y. Bouret, E.R. Travis, R.M. Wightman, Biochim., 82, 2000, 481-496.
Second Half of Full Fusion: Dissipation of Line Tension Between Relaxed Membranes
R /
Rve
sicl
e
%66%98
%66
tt
tt
0.2
0.4
0.6
0.8
1
0 0.25 0.5 0.75 1
Testing Our Model
C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-154.
stcellvesreleased c RW 2 )(
)/( 4 dtdRRWreleased vesicle
cell
C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-
154.
fast
large
Testing Our Model
stcellvesreleased c RW 2 )(
)/( 4 dtdRRWreleased vesicle
cell
C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-
154.
fast slow
large
small
Testing Our Model
stcellvesreleased c RW 2 )(
)/( 4 dtdRRWreleased vesicle
cell
fast
large
Reducing , viz. the Driving Force, by Refraining Swelling
Photographs adapted from Geoffrey Fox:www.mpibpc.gwdg.de/inform/MpiNews/cientif/jahrg6/10.00/fig5.html
vesves
ves PR
2
10 pA10 s
La 3+ 10mMinjection
Electrode in contact with the cell
Reducing by Lanthanides Ions:
C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-154.
Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993, 8343.
stcellvesreleased c RW 2 )(
)/( 4 dtdRRWreleased
Increasing , viz. the Membrane Viscosity
stcellvesreleased c RW 2 )(
)/( 4 dtdRRWreleased
Control Hyperosmotic
Increasing with a Hyperosmotic Shock:
Increasing , viz. Membrane Viscosity, with Hyperosmotic Shock:
970
mO
sm
Q / pC
C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-154.
K.P. Troyer, R.M. Wightman, J. Biol. Chem., 277, 2002, 29101-29107.
Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993, 8343.
stcellvesreleased c RW 2 )(
)/( 4 dtdRRWreleased
Decreasing and Increasing by Cell Membrane Tension
Cell Membrane Tension Through a Hypoosmotic Shock
Control Hypoosmotic
excess
Cell Membrane Tension Through a Hypoosmotic Shock
0 50 100 150 200 250 300 3500
200
400
600
800
1000
Hypo.
Control
2.4 Hz
3.7 Hz
Time / s
# C
umu
late
d E
ven
ts