I. Exocytose

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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.mhhe.com/socscience/intro/ibank/set1.htm



The Chromaffin Cell



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



Photographs: release of insulin by pancreatic β -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

I C

I F I tot = I F + I C



Problems associated with applying ultrafast electrochemical perturbations:

Ohmic Drop:

E(t) = ZF I F + RuI tot (t)

Cell Time Constant:

τ cell = RuCd

I C

I FI tot = I F + I C



Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant

I C


Ru ∝ 1/r 0

Cd ∝ r 02

I C and I F ∝ r 02



Using Ultramicroelectrodes to Decrease Ohmic Drop and Cell Time Constant

I C


Ru I tot ∝ r 0 →0

Ru Cd ∝ r 0 →0

o For Planar Diffusion:

o For Any Diffusional Regime:



Compensation of Ohmic Drop and Time Constant

ZF I F = E (t) - (RuI tot )

I C

= Cd

(dE /dt) - RuCd(dItot /dt)

E (t) # ZF I F I F # I tot – 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 H

2 O

2 ( "normal" [H

2 O

2 ]

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 Requirements

10-12 µm 1-5 µm

glass cases

insulating

polymer

platinized

surfaces

5 µm 5 µm



Qav = 0.9 pC N av = 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 80 120

c u r r e n t

/ p A

time /ms

I.

II.

III.

IV.

0. I. II. III. IV.

Full FusionFusion PoreDocking



Docking Occurs at Specifically Structured Areas in Cell Membrane:


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:

G F P a l o n e ( c o n t r o l 1 ; n = 2 6 )

G F P / S n a p 2 5 W T ( c o n t r o l 2 ; n = 2 1 )

G F P / B o t u l i n A ( n = 2 1 )

G F P / S n a p 2 5 L 2 0 3 ( n = 1 9 )

0

20

40

60

Cum

ulate

dSecretionE

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.





0.

I.

0.

III. → IV.



0

20

40

60

0 40 80 120

c u r r e n t

/ p A

time /ms

I.

II.

III.

IV.

0. I. II. III. IV.




Pore Formation: The Stalk Model



Regulating Exocytosis with Exogenous Bilipids

R RW pore π ρ π σ 22 +−=

.

C. Amatore, Y. Bouret, L. Midrier, Chem. Eur. J., 5, 1999, 2151-2162.

2R

Surface tension Edge tension




Control




Control

LPC

AA

LPC N O

P

OO

O

O

H OH

O

AACO2H



LPC N O

P

OO

O

O

H OH

O

AACO2H




1400

Time (s)

0 50 100 150 200 250 300

#C

umulated

eve

nts

0

200

400

600

800

1000

1200

AA

Control

LPC

Control

AA

(4 Hz)

(2.5 Hz)

(1 Hz)


R l ti E t i ith E Bili id




∆ U≠

pre - fusion

full fusion

R l ti E t i ith E Bili id



∆ U≠

Time (s)

0 50 100 150 200 250 300

Cumulatedev

en

ts

0

200

400

600

800

1000

1200

1400

LPC

AA

Controlδ (∆ U≠)

LPC= k BT ln( ) ≅ - 1 k BT

2.4

4

δ (∆ U≠)AA

= k BT ln( ) ≅ + 2 k BT2.4

1

ν ∝ k = k0 exp(-β ∆ U≠/k BT)




pore granule granuledisk foot RC nFDii 4==

R pore /nm ≈ 0.3 x i foot /pA


Release Through Initial Fusion Pore:

n = 2

F = 96 500 Cb

< D granule > = 4.8 10-8 cm2s-1

< C granule > = 0.6 M



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)

0

20

40

60

0 40 80 120

i foot

= 6 pA

r pore

= 1.8 nm

c u r r e n t

/ p A

t ime /ms

0

10

20

30

0 50 100

i foot

= 4 pA

r pore

= 1.2 nm

time /ms

0

15

30

0 40 80 120

i foot

= 3 pA

r pore

= 0.9 nm

time /ms



How Full Fusion May Follow Pore Release ?

R RW cell ves pore 2)( 2π ρ+σ+σπ−=

.




R RW cell ves pore 2)( 2π ρ+σ+σπ−=

How Full Fusion May Follow Pore Release ?

.




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





0.

I.

0.

III. → IV.



0

20

40

60

0 40 80 120

c u r r e n t / p A

time /ms

I.

II.

III.

IV.

0. I. II. III. IV.


Full Fusion: Two Phenomena Govern Spike Shapes:



Rate of full fusion:

surface area increases

Diffusion: control by Dt/R vesicle2

.






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) / I peak

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 BaCl 2 , 2 mM, in Locke buffer with MgCl 2 , 0.7 mM.



Rate of full fusion:

surface area increases

Diffusion: control by Dt/R vesicle2

Full Fusion Kinetics



W. Almers et al., Nature 406, 2000, 849-854.

o Evanescent wave spectroscopy:

Full Fusion Kinetics

o 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) / I peak

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



Seeing & Measuring :Fluorescence and Amperommetry



vesicleσ

cell σ

First Half of Full Fusion

R RW cell vesreleased 22π ρ σ σ π −+= )(

)/(4 dt dR RW released ηπ=

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/R

vesicle

)

t / t 80%

st

cell vesreleased

c RW +σ+σπ=2 )(

)/(4 dt dR RW released π η=

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

vesicleσ

cell σ



0≅+cell vesicle

σ σ

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/R

vesicle

%66%98

%66

t t

t t

−

−

0.2

0.4

0.6

0.8

1

0 0.25 0.5 0.75 1

)/( dt dR R RW fold released 42 πη πρ =−≈

dt dR fold )/(2/ ηρ−≈

O



Testing Our Model

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

st

cell vesreleased

c RW +σ+σπ=2 )(


vesicleσ

cell σ

T ti O M d l



C. Amatore, S. Arbault, I. Bonifas, Y. Bouret, M. Erard, M. Guille, ChemPhysChem, 4, 2003, 147-

154.

fast

Σ ση

large

Testing Our Model

st

cell vesreleased

c RW +σ+σπ=2 )(


vesicleσ

cell σ

T ti O M d l




154.

fast slow

Σ ση

large Σ ση

small

Testing Our Model

st

cell vesreleased

c RW +σ+σπ=2 )(


vesicleσ

cell σ

fast

Σ ση

large

Reducing Σ σ viz the Driving Force by Refraining Swelling



Reducing Σ σ , viz. the Driving Force, by Refraining Swelling

Photographs adapted from Geoffrey Fox:

www.mpibpc.gwdg.de/inform/MpiNews/cientif/jahrg6/10.00/fig5.html

ves

ves

ves P R

∆=σ 2

Reducing Σ σ by Lanthanides Ions:



10 s

La3+

10mM injection

Electrode in contact with the cell

Reducing Σ σ by Lanthanides Ions:

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

Increasing η viz the Membrane Viscosity



Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993, 8343.

st cell vesreleased c RW +σ+σπ=

2 )(


Increasing η , viz. the Membrane Viscosity

Increasing η with a Hyperosmotic Shock:



st

cell vesreleased c RW +σ+σπ=2

)(


Control Hyperosmotic

Increasing η with a Hyperosmotic Shock:

Increasing η viz Membrane Viscosity with Hyperosmotic



Increasing η , viz. Membrane Viscosity, with Hyperosmotic

Shock:

970mOsm

Q / pC


154.K.P. Tro er R.M. Wi htman J. Biol. Chem. 277 2002 29101-29107.

Decreasing η and Increasing σ by Cell Membrane Tension



Molecular dynamic simulations adapted from: H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem., 97, 1993, 8343.

st cell vesreleased c RW +σ+σπ=

2 )(


Decreasing η and Increasing σ by Cell Membrane Tension

Cell Membrane Tension Through a Hypoosmotic Shock




Control Hypoosmotic

excess





0 50 100 150 200 250 300 3500

200

400

600

800

1000

Hypo.

Control

2.4 Hz

3.7 Hz

Time / s

#Cum

ulate

dEvents

I. Exocytose

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