1. Flow Cytometric Analysis of FRET to Study the Interaction Between CFP- and YFP-Tagged Proteins
David Stepensky

2. Classical pathway of major histocompatibility complex (MHC) class I antigen processing, loading, and presentation
Groothuis et al, Immunol Rev 2005

3. 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

4. Fluorescence Resonance Energy Transfer
transfer of excited state energy from one fluorophore to another
CFP HC
Tapasin
CFP
YFP
Excitation & emission spectra
YFP HC
Tapasin
CFP
YFP
CFP excitation CFP & FRET signals
YFP excitation YFP signal
%FRET
extent of interaction

5. 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 (n=15-20 cells)
FRET, %
Tapasin-YFP
Tapasin C95A-YFP
w/o Tapasin
Tapasin
HLA-A2.1-CFP
HLA-A2.1 T134K-CFP
HLA-B44-CFP
HLA-A2.1-
CFP
HLA-A2.1-YFP-CFP

6. 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
CFP
488
FRET/YFP

7. FACS-FRET: the raw data
Negative
control
Exper.
sample
FRET
CFP
Positive
control
positive control
experimental sample
negative control
FRET
cells
CFP
YFP

8. FACS-FRET: the results
experimental cell lines
controls
FACS-FRET results are consistent with the confocal data
both techniques seem to quantify correctly the interaction between the constructs
Tapasin-YFP
Tapasin C95A-YFP
w/o Tapasin
Tapasin
FRET/CFP ratio, normalized
HLA-A2.1-CFP
HLA-A2.1 T134K-CFP
HLA-B44-CFP
HLA-A2.1-
CFP
HLA-A2.1-YFP-CFP

9. (using the applied setup) individual organelles
FRET assessment using FACS or confocal microscope:
selected characteristics
FACS
(using the applied setup)
Confocal microscopy
Acquisition speed
high (~103 cells/s)
low (~102 cells/h)
Origin of FRET, CFP & YFP signals
different cells
the same cell
the whole cell
individual organelles
FRET quantification
relative value
absolute value
Sorting of cell populations
possible
impossible

10. Alternative setups for FACS-FRET
Dye, Clin Appl Immunol Rev, 2005
He et al, Cytometry Part A, 2003
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

11. FACS Analysis of FRET simple setup
combination of FACS with Confocal analysis
possibility of cell sorting

12. Thanks Prof. Peter Cresswell and the group Cell Sorter Facility
Geoff Lyon Tom Taylor Don Foster