Förster Resonance Energy Transfer of Cerulean and Venus Fluorescent Proteins In Live Drosophila melanogaster Eyes

Saki MihoriDr. Donald Ready’s Lab

What is FRET• Physical phenomenon used in

biology to observe and quantify protein-protein interaction

• Donor protein- Cerulean Fluorescent Protein (CFP)

• Acceptor protein- Venus Fluorescent Protein (YFP)

• How FRET works– Energy absorbed by donor is

transferred to acceptor, which then fluoresces

• Only works if distance between proteins is within 10nm, which is the realm of protein-protein interaction distance.

"FRET (Fluorescence Resonance Energy Transfer)." Semrock. N.p., n.d. Web. <https://www.semrock.com/fret.aspx>.

Model Organism: Drosophila melanogaster

• 3 transgenic flies made by Kirk Mecklenburg• C5V

– Cerulean- 5 amino acid linker-Venus– Spacing of 5.0 nm

• C17V– Spacing of 5.3 nm

• C32V– Spacing of 5.7 nm

• Established FRET efficiency standards:– Vogel

• 43% C5V• 38% C17V• 31% C32V

– Roszik• 45% C5V• 37.3% C17V• 29.9% C32V

Procedure• Microscope: Nikon

Eclipse TE200 – Dual View camera

• 2 channel imaging system

• Simultaneous view of CFP (donor) channel and YFP (acceptor) channel

– Cooled CCD camera• Fluorescence

microscopy applications

MetaMorph• Image analysis and microscopy automation

software• Split view

– Simultaneously record CFP and YFP• Acceptor emission on donor excitation is raw

FRET– But has contamination

• Isolate donor (CFP), transfer (FRET) and acceptor (YFP) image

• Using FRET app, correct FRET image by subtract spectral bleed through or artifactual contributions to raw FRET

• Obtain ratiometric image of FRET image of corrected FRET over donor

• Warmer colors indicate higher FRET efficiency• Only gives indication of comparison of what

the FRET efficiency is (no quantitative value)

L to R: C5V, C17V, C32V

RiFRET• ImageJ plugin• Calculates FRET efficiency pixel by

pixel• Subtact background for noise on 3

images, set threshold so only calculate FRET on eye

• False coloration to help identify FRET efficiency

L to R: C5V, C17V, C32V

Histogram:Isolated peaks at mean ratiometric values of different constructs

Conclusion• Established FRET efficiency standards:

– Vogel• 43% C5V• 38% C17V• 31% C32V

– Roszik• 45% C5V• 37.3% C17V• 29.9% C32V

• My values• 44.5% C5V• 37.3% C17V• 29.2% C32V

Conclusion

• Demonstrated accurately measured FRET in Drosophila and calibrated FRET standards

• Next step: work with actin-CFP and actin-YFP– Blue light

Questions?