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