SAHANA V

Fluorescence(Forster) Resonance Energy Transfer

Fluorescence

FRAP(Fluorescence Recovery After Photobleachin

g)

FLIM(Fluorescence

Lifetime imaging

Microscopy)

FRET(Fluorescence

Resonance Energy

Transfer)

FRET

CONDITIONS

Donor Emission = Acceptor Excitation

Close proximity of donor and acceptor (1-10 nm)

Fluorescence Lifetime of the donor should be sufficient

Measurement

FRET-FLIMAcceptor

Photobleaching

Sensitized Emission

FRET Data

Population average (dist

between 2 points)

Single Molecule

(distribution and kinetics

of transmission)

APPLICATIONS

Structure studies

Conformational analysis

Interaction between

molecules

Live cell imaging

VADIM DEGTYAR et a l , .

J NEUROSCI .  2013; 33(13): 5507–5523.

Dance of the SNAREs: Assembly and rearrangements detected with FRET at neural synapses

BACKGROUND

MATERIALS AND METHODS

Scheme 1 Cerulean: N termini

VAMP Citrine: SNAP-25 Changes in their

separation and orientation

Scheme 2 Cerulean: Syntaxin Citrine: C termini

VAMP trans-cis

conformational change in SNAREs on vesicle fusion

VAMP: Vesicle Associated Membrane ProteinSNAP: Synaptosomal associated protein of 25kD

ULTIMATE AIM

1. Detection of resting SNARE complexes

2

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3. Disassembly prior to endocytosis of vesicular protein

4.SNARE assembly at newly docked vesicles

OUTCOME

Reoreintation of the

SNARE motif upon

exocytosis

SNARE disassembly in

the active zone

periphery

SNARE assembly in the

newly docked vesicle

s

Trans-cis conformational

changes in

SNAREs on

vesicle fusion

SPECIFIC AIM 1Labeling with fluorophores

Expression and correct targeting of VAMP and SNAP-25-FM4-64 staining

Schematic of assembled SNARE complex comprising VAMP labeled with cerulean, SNAP-25 labeled with citrine, and syntaxin

Continued…

No complete block of neurosecretion by Botulinum E and tetanus toxin

Normal release by co-transfected cells

Transfection procedure-not fluorophores reduces the release probability

SPECIFIC AIM 2Resting FRET of pre-assembled SNAREs

Donor dequench on acceptor photobleach

FRET-FLIM: FRET ratio of donor:

2.7%(4.9% uncertainty) With acceptor: 4.6%

(5.9% uncertainty) Control:

FRET ration: 2.4%Average: 2.4 -4.6 = 3%FRET not from protein

crowding or un-complexed donor or acceptor

SPECIFIC AIM 3Dynamic FRET technique

Sensitized emission“3 cube method”Image splitter:Emission

of both fluorophores measured simultaneously

Polychroic mirror- reflects both emission bands

Quadruple- separate donor, acceptor, bright-field and FM4-64 images

SPECIFIC AIM 4N-terminal FRET signals

Continued…

1 •Signals from non-transmitting boutons did not fit this pattern

2 •No consistent signals with donor or acceptor alone

3 •No signals, when exocytosis was blocked by omitting calcium- signals dependency of the calcium

4 •Signals dependent on intact SNAREs- Botulinum toxin

5 •Signals also dependent on disassembly and reassembly of SNAREs- Nethylmaleimide (NEM)

SPECIFIC AIM 5Dispersion of SNARE complex

VAMP is deposited on the plasma membrane on exocytosis and recovered by endocytosisSimultaneous lateral dispersion of both SNAP-25 and syntaxin.

Continued…

Changes in FRET signal is due to dispersion of SNAREs .

SPECIFIC AIM 6C- Terminal FRET

SNARE cis-trans transformation.

Scheme 2 was followed.

synaptopHluorin effect.

To reduce spill over and contamination.

Continued…

Increase in acceptor fluorescence

Decrease in donor fluorescence

FRET increaseReports trans-cis

conformation

The SNARE cycle

Continued…

DISCUSSION

Dynamic changes in pre assembled SNARE

Dispersion as an intact complex

Disassembly

Assembly of newly docked and primed vesicles

References

S. R. Swift and L. Trinkle-Mulcahy. Basic principles of FRAP, FLIM and FRET.

S. A. Hussain et. al. An Introduction to Fluorescence Resonance Energy Transfer (FRET)

Janos Szollosi et al. Application of Fluorescence Resonance Energy Transfer in the Clinical Laboratory: Routine and Research.

Thank you