Download More Transparency in BioAnalysis of Exocytosis: Coupling of

More Transparency in BioAnalysis of Exocytosis:
Coupling of Electrochemistry and Fluorescence Microscopy
at ITO Electrodes
d
Manon GUILLE-COLLIGNON
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
Phenomenon in
Ph
i wich
i h a cellll directs
di
materials
i l release
l
to the
h outside
id by
b discharging
di h i
them as a membrane-bounded vesicles passing through the cell membrane.
Neuron body
Synaptic cleft
http://lyrobossite.free.fr/Structure_II_Les synapses.htm
Example
p of exocytosis
y
in synaptic
y p transmission
Numerous q
questions under debate
Exocytosis : Numerous Questions Under Debate
Transport and motion of vesicles (actin network, cytoskeleton, …)
 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?
Exocytosis : Regulation by Which Parameters?
Biological
Bi
l i l
Control
SNAREs proteins, « key proteins » (Syntaxine, SNAP 25, VAMP, Munc 18…),
Ca2+ ions, secretagogue…
Physico-chemical control
Membrane properties (nature of phospholipids, viscosity, membrane
tension, curvature…), pH, extracellular medium composition…
Main Used Analytical Tools
Time resolution
Electrical recordings: • Electrochemical amperometry
Optical recordings:
• Fluorescence microscopy
Tens of µs
~ 50 ms
Electrochemical Detection
« Artificial synapse » configuration
Ultramicroelectrode
Minimization of the distance ((<1µm)
µ )  Volume 
Femtomoles emitted within ~ picolitre  ~ [mM]
7 µm
Carbon fiber
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
Advantages :
 Single cell level
 Direct measurements
 Selectivity offered by the potential
i = 4nFDCr0
Nature of the Monitored Signals
1 spike
p = 1 vesicle
Cell
Micropipette
Chromaffin Cells
120
60
100
50
Current
(pA
A)
Courant (pA)
Microelectrode
Current
Couran((pA)
t (pA)
140
80
60
40
Imax
Imax
Q
Q (aire)
((area))
40
30
t1/2
20
20
10
0
0
0
0
100
200
300
400
20
500
60
80
100
Temps (ms)
Temps de
Temps
(s)
Time (s)
40
Rising
time
montée
Time (ms)
Oxydation of catecholamines at 650 mV vs. Ag/AgCl :
O
HO
OH
OH
HO
Amperometric Parameters:
Events frequency
Charge (Q /fC)
Intensity (I /pA)
Half-width (t1/2 /ms)
+ CH3
N
H
H
O
+ CH3
N
H
H
+ 2H+ + 2e-
PhysicoChemistry of Exocytosis : Effect of Membrane Curvature
LPC 2 µM Lysophosphatidylcholine
Short time incubation
AA 20 µM
A hid i acid
Arachidonic
id
LPC
Fusogenic
BiophysJ, 1995, 69, 922 ; ChemBioChem, 2006, 7, 1998
“Inverted cone”
“Cone”
AA
Anti Fusogenic
Membrane Curvature : Effects on Exocytosis Frequency
LPC
Nomb
bre
ments
Number
r ofd’évènem
detecteed
events
1400
1200
Fusogenic
 Fusogène
LPC
1000
800
Control
600
400
AA
200
0
0
50
100
150
200
250
300
Time (s)
Temps
/s
Strong effect of membrane curvature on the secretion frequency
AA
 Anti-fusogène
Anti Fusogenic
Membrane Curvature : Effects on Exocytosis Dynamics
1800
40
30
AA
Control
LPC
1600
AA
C t l
Control
LPC
1400
Chargee/ fC
Timee /ms
1200
20
10
1000
800
600
400
200
0
t20-90
t1
t2
0
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
Pi i
Priming
Docking
Cell
(diameter 10 µm)
Vesicle
(diameter 300 nm)
Vesicle
Extracellular
medium
Fusion
Intracellular
medium
Cell membrane
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
y
techniques
q
((optical
p
and electrochemical)?
)
Main Used Analytical Tools
2) Total Internal Reflection
Fluorescence-Microscopy
py = TIRF-M
1) Electrochemical amperometry
Carbon-fiber
C
rb n fib r
ultramicroelectrode
Ø = 10 µm
Objective lens
Cell
Stimulating
capillary
Water
Cell
Glass
Laser beam
Principle:
Excitation of fluorescent vesicles in cell
by an evanescent wave
of very low penetration depth
(50-300 nm)
Intensity (pA)
Peak area Q
Time (s)
2 µm
 Only vesicles near the plasma
membrane are monitored.
monitored
 Exocytotic events are seen
as «flash» or extinction of fluorescence.
Analytical Tools
1) Electrochemical amperometry
2) TIRF-Microscopy
Advantages
 Real time detection of single events
 Real-time detection of single events
 Vesicles motion observation before fusion
 Quantitative information
- 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 Coupling
Required conditions:
 Coupled detection at the same place of the cell
 Detection realized
at the bottom of the cell
 Transparent and conducting substrate
 Choice of ITO:
Indium Tin Oxide
(90% In2O3 + 10% SnO2)
 Electrochemical limitation:
surface of ITO compromise between
 a suitable electrical noise
 cells dimensions
 ITO band electrodes
(200 µm width)
id h)
Specific Devices for the Coupling
Technological process
4 independent
working electrodes of ITO
Cellule
Choice of Cells for the Coupling
Choice of enterochromaffin BON cells:
Required conditions:
 Optical detection:
fluorescent probe
 Expressing GFP-tagged neuropeptide-Y
2 µm
 Releasing neurotransmitter serotonin
 Electrochemical detection:
electroactive molecules
(650 mV vs Ag/AgCl)
‐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
Angew. Chem., 2011, 50 (22), 5081
Biophys Chem.,
Biophys.
Chem 2012,
2012 162
162, 14
Faraday Discussion , 2013, 164, 33
Electrochimica Acta, 2014, 126, 74
Electrochimica Acta, 2014, 140, 457
Experimental Set-Up
Ref Ag/AgCl
ITO
Cell
Cells
Injection capillary
(stimulation)
Selective
S
l i
stimulation
of a single cell
(ionomycin 5 µM)
Validation of the Combined Method
 Same trigger for the optical and amperometrical recordings.
Extinction of
fluorescence
seen byy TIRF
Example 1
Amperometric spike
Fluorescence
flash seen by
TIRF
Example 2
Amperometric
spike
•
•
~ 4 coupled events by cell (n = 6)
Different temporal resolutions : T TIRF= 100 ms, TAmperometry = 10 µs
Conclusion…
 Use of UME/electrochemistry for unraveling physico-chemical factors
controlling exocytosis
 Validation of a proof of conceipt :
C
Coupling
li amperometry and
d TIRF to analyze
l
an exocytotic
i event
n=66 cells (85 events)
 Analysis of events obtained : for n
30% of coupled events
Acknowledgments
UMR CNRS-ENS-UPMC 8640
« PASTEUR »,
» ENS
ENS, PARIS
Christian AMATORE
Jérôme DELACOTTE
Rémy FULCRAND
Lihui HU
Frédéric LEMAITRE
Xiaoqing LIU
Anne MEUNIER
Damien QUINTON
UMR 8192, Institut de Biologie
Physico-Chimique, PARIS
Marine BRETOU
François DARCHEN
Isabelle FANGET
Ouardane JOUANNOT
Université de Bordeaux 1,
Institut des Sciences
Moléculaires
UMR 5255
5255, Pessac
Stéphane ARBAULT