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 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

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

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

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

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

(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

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

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

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