The basics of FRET Fluorescence Resonance Energy Transfer

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Flow Cytometric Analysis of FRET to Study the
Interaction Between CFP- and YFP-Tagged Proteins
David Stepensky
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Classical pathway of major histocompatibility
complex (MHC) class I antigen processing,
loading, and presentation
Groothuis et al, Immunol Rev 2005
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Objectives
to study the interactions within the MHC I
loading complex using fluorescently-tagged
components

• the kinetics and sequence of association and
  dissociation of the loading complex
• effects of the individual interactions on the
  loading of the MHC class I molecules with peptides

Cresswell et al, Immunol Rev 2005
Approach

• generation of fluorescently tagged components of
  the loading complex
• investigation of functioning of the tagged
  proteins

fluorescence-based techniques (FRET, FRAP,
etc.) biochemical techniques
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Fluorescence Resonance Energy Transfer
transfer of excited state energy from one
fluorophore to another
CFP
HC
Tapasin
CFP
YFP
Excitation emission spectra
YFP
Tapasin
HC
CFP
YFP
CFP excitation CFP FRET signals YFP
excitation YFP signal
extent of interaction
FRET
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Experimental setup
experimental cell lines
controls

• interaction between MHC class I HC Tapasin
• M553 tapasin deficient melanoma
• stable transfection with
• Tapasin-YFP
• MHC I heavy chain-CFP
• multiclonal cell lines
• interaction (FRET efficiency) was measured using
  confocal microscope (n15-20 cells)

FRET,
Tapasin-YFP Tapasin C95A-YFP Tapasin-YFP Tapasin
C95A-YFP Tapasin-YFP Tapasin C95A-YFP w/o
Tapasin w/o Tapasin Tapasin Tapasin-YFP Tapasin
C95A-YFP
HLA-B44-CFP
HLA-A2.1-YFP-CFP
HLA-A2.1- CFP
HLA-A2.1-CFP
HLA-A2.1 T134K-CFP
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Flow Cytometric Analysis of FRET
Objective to obtain statistically robust
measurement of FRET efficiency in the studied
cell lines
CFP
FACS setup

• FACSAria
• Violet laser 405 nm

Excitation emission spectra
CFP (450/40 nm) FRET (530/30 nm)
YFP

• Blue laser 488 nm

YFP (530/30 nm)

• one laser at a time, sequential acquisition of
  the same sample

405
488
CFP
FRET/YFP
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FACS-FRET the raw data
Negative control
Exper. sample
FRET
CFP
Positive control
positive control experimental sample negative
control
FRET
cells
CFP
YFP
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FACS-FRET the results

• FACS-FRET results are consistent with the
  confocal data
• both techniques seem to quantify correctly the
  interaction between the constructs

experimental cell lines
controls
Tapasin-YFP Tapasin C95A-YFP Tapasin-YFP Tapasin
C95A-YFP Tapasin-YFP Tapasin C95A-YFP w/o
Tapasin w/o Tapasin Tapasin Tapasin-YFP Tapasin
C95A-YFP
FRET/CFP ratio, normalized
HLA-B44-CFP
HLA-A2.1-YFP-CFP
HLA-A2.1- CFP
HLA-A2.1-CFP
HLA-A2.1 T134K-CFP
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FRET assessment using FACS or confocal
microscope selected characteristics
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Alternative setups for FACS-FRET
He et al, Cytometry Part A, 2003
Dye, Clin Appl Immunol Rev, 2005

• FACSVantage SE
• spatial separation of the laser lines
• optional laser
• nonstandard mirrors/filters

• FACSVantage SE
• laser tuning to 458 nm
• simultaneous excitation of CFP YFP
• nonstandard mirrors/filters

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FACS Analysis of FRET

• simple setup
• combination of FACS with Confocal analysis
• possibility of cell sorting

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Thanks
Prof. Peter Cresswell and the group
Cell Sorter Facility
Geoff LyonTom TaylorDon Foster