Flow Cytometric Analysis of FRET
to Study the Interaction Between
CFP- and YFP-Tagged Proteins
David Stepensky
Classical pathway of major histocompatibility complex
(MHC) class I antigen processing, loading, and presentation
Groothuis et al, Immunol Rev 2005
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
Fluorescence Resonance Energy Transfer
transfer of excited state energy from one fluorophore to another
HC
Tapasin
CFP
YFP
Tapasin
YFP
HC
Excitation & emission spectra
CFP
CFP YFP
CFP excitation
YFP excitation
CFP & FRET signals
YFP signal
%FRET
extent of interaction
Experimental setup
experimental cell lines
• interaction between MHC
30
class I HC & Tapasin
melanoma
25
FRET, %
• M553 tapasin deficient
controls
20
15
10
was measured using confocal
microscope (n=15-20 cells)
HLAA2.1CFP
HLAA2.1
T134K
-CFP
HLAB44CFP
HLAA2.1CFP
HLA-A2.1YFP-CFP
Tapasin C95A-YFP
Tapasin-YFP
Tapasin
w/o Tapasin
w/o Tapasin
Tapasin-YFP
Tapasin C95A-YFP
• interaction (FRET efficiency)
Tapasin C95A-YFP
• multiclonal cell lines
-5
Tapasin-YFP
MHC I heavy chain-CFP
0
Tapasin C95A-YFP
Tapasin-YFP &
5
Tapasin-YFP
• stable transfection with:
Flow Cytometric Analysis of FRET
Objective:
to obtain statistically robust
in the studied cell lines
FACS setup:
• FACSAria
• Violet laser 405 nm
CFP (450/40 nm)
FRET (530/30 nm)
• Blue laser 488 nm
Excitation & emission spectra
measurement of FRET efficiency
CFP
YFP
YFP (530/30 nm)
• one laser at a time, sequential
acquisition of the same sample
405 CFP 488 FRET/
YFP
FACS-FRET: the raw data
300
Negative
control
10
5
10
4
10
3
10
2
200
100
0
0
0
10
2
10
3
10
4
10
5
10 5
10
10
0
10 2
10
3
10
4
10
5
50
40
4
30
FRET
Exper.
sample
2
0
10 3
20
10
2
10
0
0
0
10 2
10 3
10 4
10 5
10 3
10 4
10 5
CFP
10 5
Positive
control
100
10 4
positive control
experimental sample
negative control
80
60
10 3
10
20
0
0
CFP
10 2
10 3
10 4
10 5
cells
FRET
40
2
0
YFP
0
10 2
10 3
10 4
10 5
FACS-FRET: the results
• FACS-FRET results are
consistent with the
confocal data
controls
100
80
• both techniques seem to
quantify correctly the
interaction between the
constructs
60
40
20
experimental cell lines
0
controls
10
5
HLAA2.1CFP
HLAA2.1
T134K
-CFP
HLAB44CFP
HLAA2.1CFP
HLA-A2.1YFP-CFP
Tapasin C95A-YFP
Tapasin-YFP
Tapasin
w/o Tapasin
w/o Tapasin
Tapasin C95A-YFP
-5
Tapasin-YFP
0
Tapasin C95A-YFP
FRET, %
Tapasin C95A-YFP
Tapasin-YFP
w/o Tapasin
Tapasin
HLA-A2.1YFP-CFP
15
Tapasin-YFP
HLAA2.1CFP
20
Tapasin-YFP
HLAB44CFP
w/o Tapasin
Tapasin C95A-YFP
Tapasin-YFP
Tapasin C95A-YFP
HLAA2.1
T134K
-CFP
25
Tapasin C95A-YFP
HLAA2.1CFP
Tapasin-YFP
Tapasin C95A-YFP
30
Tapasin-YFP
FRET/CFP ratio, normalized
experimental cell lines
FRET assessment using FACS or confocal microscope:
selected characteristics
FACS
(using the applied setup)
Acquisition speed
Confocal microscopy
high (~103 cells/s)
low (~102 cells/h)
different cells
the same cell
the whole cell
individual organelles
relative value
absolute value
possible
impossible
Origin of FRET, CFP &
YFP signals
FRET quantification
Sorting of cell populations
Alternative setups for FACS-FRET
Dye, Clin Appl Immunol Rev, 2005
He et al, Cytometry Part A, 2003
• FACSVantage SE
• FACSVantage SE
• spatial separation of the laser lines
• laser tuning to 458 nm
• optional laser
• simultaneous excitation of CFP & YFP
• nonstandard mirrors/filters
• nonstandard mirrors/filters
FACS Analysis of FRET
• simple setup
• combination of FACS with Confocal analysis
• possibility of cell sorting
FACS setup:
• FACSAria
CFP
450/40 nm
FRET
530/30 nm
• Blue laser 488 nm
FRET
• Violet laser 405 nm
CFP
YFP
530/30 nm
• one laser at a time, sequential
acquisition of the same sample
positive control
experimental sample
negative control
Thanks
Prof. Peter Cresswell and the group
Cell Sorter Facility
Geoff Lyon
Tom Taylor
Don Foster