more transparency in bioanalysis of exocytosis: coupling ... · coupling of electrochemistry and...
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More Transparency in BioAnalysis of Exocytosis:
Coupling of Electrochemistry and Fluorescence Microscopy
at ITO Electrodes
Manon GUILLE-COLLIGNON
d
Laboratoire PASTEUR, Département de Chimie, Ecole Normale Supérieure, CNRS UMR 8640, Université Pierre & Marie Curie, PARIS, FRANCE
2015, 18-19th of November, EABS Workshop, Orsay, France
Exocytosis in Nerve and Humoral Transmission
Ph i i h ll di i l l h id b di h iPhenomenon in wich a cell directs materials release to the outside by dischargingthem as a membrane-bounded vesicles passing through the cell membrane.
Neuron bodyNeuron body
Synaptic cleft
http://lyrobossite.free.fr/Structure_II_Les synapses.htm
Example of exocytosis in synaptic transmission
Numerous questions under debate
p y y p
q
Exocytosis : Numerous Questions Under Debate
Transport and motion of vesicles (actin network, cytoskeleton, …)
Dynamics and stability of fusion pore (flickering, ...)Dynamics and stability of fusion pore (flickering, ...)
Nature of factors controlling fusion process (biological and physico-chemical ones)
Partial or full fusion
« Kiss and run » existence?
Bi l i l
Exocytosis : Regulation by Which Parameters?
BiologicalControl
SNAREs proteins, « key proteins » (Syntaxine, SNAP 25, VAMP, Munc 18…),Ca2+ ions, secretagogue…
Physico-chemical control
Membrane properties (nature of phospholipids, viscosity, membranetension, curvature…), pH, extracellular medium composition…
Main Used Analytical Tools
• Electrochemical amperometryElectrical recordings:
Time resolution
Tens of µs
• Fluorescence microscopyOptical recordings: ~ 50 ms
Electrochemical Detection
Ultramicroelectrode7 µm
« Artificial synapse » configuration
Minimization of the distance (<1µm) Volume
Carbon fiber
7 µm ( µ )Femtomoles emitted within ~ picolitre ~ [mM]Faradic current [Electroactive species]
Cell
Signal on Noise ratio (S/N 1/r0) Detection of weak currents : 1 pA à 1 nA
Response time ~ 1 ms ( r0) Study of fast biological phenomenon
Detected current proportional to the concentration i = 4nFDCr0
Advantages :
Single cell level Direct measurements Selectivity offered by the potential
Nature of the Monitored Signals
1 spike = 1 vesicle
)
50
60 Imax
Q (aire)t (pA
)
100
120
140p
Q (area)(pA
)
Microelectrode
A)
Imax
Cou
rant
(pA
20
30
40
t1/2C
oura
n40
60
80( )
Curr
ent(Cell
Curr
ent(
pA
Temps (ms)
0 20 40 60 80 1000
10
Temps de0 100 200 300 400 500
0
20
Chromaffin CellsTime (ms)
Micropipette
Temps de montéeTemps (s)
Oxydation of catecholamines at 650 mV vs. Ag/AgCl :
Rising timeTime (ms)
Time (s)
+ 2H+ + 2e-OH
OHOH
NH
CH3
H
O
OOH
NH
CH3
H
+ +
Amperometric Parameters:Events frequencyCharge (Q /fC)Intensity (I /pA)Half-width (t1/2 /ms)
PhysicoChemistry of Exocytosis : Effect of Membrane Curvature
Lysophosphatidylcholine “Inverted cone” LPC 2 µM
A hid i id
Short time incubation
Arachidonic acid “Cone” AA 20 µM
AALPC
Fusogenic Anti Fusogenic
BiophysJ, 1995, 69, 922 ; ChemBioChem, 2006, 7, 1998
Fusogenic
LPC
Membrane Curvature : Effects on Exocytosis Frequency
LPC
14001400
1000
1200
1400
LPC
men
ts
1000
1200
1400
LPC
men
ts FusogèneFusogenic
edev
ents
600
800Control
bre
d’év
ènem
600
800Control
bre
d’év
ènem
rof
dete
cte
200
400 AANom
b
200
400 AANom
bN
umbe
r
Temps / s0 50 100 150 200 250 300
0
Temps / s0 50 100 150 200 250 300
0 AA Anti-fusogèneAnti FusogenicTime (s)
Strong effect of membrane curvature on the secretion frequency
Membrane Curvature : Effects on Exocytosis Dynamics
30
40AAControlLPC 1400
1600
1800
AAC t l
e /
ms
20
30 LPC
800
1000
1200ControlLPC
e/ fC
Tim
e
10
200
400
600
800
Ch
arge
0t20-90 t1 t2
0
200
LPC favors vesicle / cell membranes fusion
Quantity of released catecholamines varies and can be rised with LPC
Better expansion of fusion pore? Fine regulation of exocytosis mechanism?
ChemBioChem, 2006, 7, 1998
Scientific Stake to Deploy a Coupling Methodology
P i iPriming Extracellular medium
Docking FusionCell
(diameter 10 µm)
Vesicle Intracellular medium
Cell membraneVesicle
(diameter 300 nm)
Is it possible to achieve a detection :
PennState Univ., USA
Is it possible to achieve a detection :- of the same exocytotic event- at the same place of the cell- with two different analytical techniques (optical and electrochemical)?y q ( p )
2) Total Internal Reflection Fluorescence-Microscopy = TIRF-M
1) Electrochemical amperometry
C rb n fib r
Main Used Analytical Tools
py
Objective lensCarbon-fiber
ultramicroelectrodeØ = 10 µm
WaterCell
Glass
Cell
Stimulatingcapillary
Laser beam
capillary
Principle:Excitation of fluorescent vesicles in cellby an evanescent waveof very low penetration depth (50-300 nm)
Intensity (pA)
Only vesicles near the plasmamembrane are monitored
Peak area Q 2 µm
membrane are monitored. Exocytotic events are seenas «flash» or extinction of fluorescence.
Time (s)
1) Electrochemical amperometry 2) TIRF-Microscopy
Analytical Tools
Advantages
Real-time detection of single events Quantitative information
- on kinetics
Real time detection of single events Vesicles motion observation before fusion
on kinetics- on number of released molecules
Drawbacks
Released molecules must be electroactive “Blind” technique before fusion pore No motion information of vesicles
No quantitative information Fluorescence of the vesicles is required
3 µm
Specific Devices for the CouplingRequired conditions:
Detection realizedat the bottom of the cell
Coupled detection at the same place of the cell
Choice of ITO: Indium Tin Oxide (90% In2O3 + 10% SnO2)
Transparent and conducting substrate
ITO band electrodes (200 id h)
Electrochemical limitation:(200 µm width)surface of ITO compromise between
a suitable electrical noise cells dimensions
Specific Devices for the Coupling
Technological process
4 independent working electrodes of ITO
CelluleCellule
Choice of Cells for the Coupling
Required conditions: Choice of enterochromaffin BON cells:Required conditions:
Optical detection: Expressing GFP-tagged neuropeptide-Yfluorescent probe
Electrochemical detection:
electroactive molecules
Releasing neurotransmitter serotonin(650 mV vs Ag/AgCl)
2 µm
electroactive molecules
‐2 H+ ; ‐2 e‐
Moderate frequency of secretion:
to assign to each amperometric peak
the corresponding optical signal Low frequency of exocytosis 0.1 Hz
the corresponding optical signal
Angew. Chem., 2011, 50 (22), 5081Biophys Chem 2012 162 14Biophys. Chem., 2012, 162, 14
Faraday Discussion , 2013, 164, 33Electrochimica Acta, 2014, 126, 74Electrochimica Acta, 2014, 140, 457
Experimental Set-Up
Ref Ag/AgCl
ITO
S l i
Cell
Selective
stimulation
of a single cell
Injection capillary(stimulation)
(ionomycin 5 µM)Cells
Same trigger for the optical and amperometrical recordings.Validation of the Combined Method
Extinction of fluorescence seen by TIRFy
Example 1
Amperometric spike
Fluorescence flash seen byflash seen by TIRF
Example 2
Amperometricspike
• ~ 4 coupled events by cell (n = 6)• Different temporal resolutions : TTIRF= 100 ms, TAmperometry = 10 µs
Conclusion…
Use of UME/electrochemistry for unraveling physico-chemical factorscontrolling exocytosis
Validation of a proof of conceipt :C li d TIRF l iCoupling amperometry and TIRF to analyze an exocytotic event
Analysis of events obtained : for n=6 cells (85 events)Analysis of events obtained : for n 6 cells (85 events)30% of coupled events
Acknowledgments
UMR CNRS-ENS-UPMC 8640 « PASTEUR » ENS PARIS« PASTEUR », ENS, PARIS
Christian AMATOREJérôme DELACOTTE
Rémy FULCRAND
UMR 8192, Institut de BiologiePhysico-Chimique, PARIS
Marine BRETOU
Université de Bordeaux 1, Institut des Sciences
Moléculaires UMR 5255 PessacRémy FULCRAND
Lihui HUFrédéric LEMAITRE
Xiaoqing LIU
Marine BRETOUFrançois DARCHEN
Isabelle FANGETOuardane JOUANNOT
UMR 5255, Pessac
Stéphane ARBAULT
Anne MEUNIERDamien QUINTON