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
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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