FLUORESCENT RECOVERY AFTER PHOTOBLEACHING

Analysis and Applications

Zareen Butt Department of Chemistry and Biochemistry University of Windsor

OVERVIEW

Phenomenon of Photobleaching

Fluorescent Recovery After Photobleaching

Measure the mobility of nuclear

proteins, macromolecular diffusion within cell

membranes, the cytoplasm, nucleoplasm

HOW FRAP WORKS

Molecules covalently bound to a fluorophore

Inhomogenous fluorescent population

Spatial separation between fluorescent moleclues

and photobleached molecules at time 0

Example of photobleaching in an indian muntjac fibroblast nucleus expressing ASF/SF2 GFP

Photobleached Population

DATA COLLECTION

0s 10s

20s 30s 90s

FLUORESCENT RECOVERY CURVE

Intensity

Intensity

Time (seconds)

NucleoplasmicNucleoplasmic Topoisomerase I &Topoisomerase I &

Topoisomerase IITopoisomerase IIαα & ß& ß

Chromatin Chromatin associatedassociated

Nucleosomal Nucleosomal HistonesHistones

Transcription Transcription FactorFactor

Estrogen ReceptorEstrogen Receptor

Glucocorticoid Glucocorticoid ReceptorReceptor

Nuclear Nuclear lamina/memebranelamina/memebrane

EmerinEmerin

HA-95HA-95

List of some nuclear proteins investigated by FRAP

PREFRAP ANALYSIS

Steady-State distribution in living cells

Artifactual Distributions

Diffused distribution

Formation of large spherical aggregates

Examples of these distributions illustrated for

histone deactylase-GFP fusion proteins

Abnormal distribution of GFP-tagged histone deacetylases in mouse 10t1/2 cells transfected with HDAC4-GFP (left) and HDAC3-GFP (right).

DATA NORMALIZATION

The raw data must be normalized in order to compensate for:

1) the background signal in the image 2) the loss of total cellular fluorescence due to photobleaching a subregion of the cell 3) any loss of fluorescence that occurs during the course of collection of recovery time series

DATA ANALYSIS

Diffusion coefficient (measures the rate of movement and represents the mean squared displacement of proteins over time)

Effective diffusion coefficient (does not take into consideration any interaction the proteins might undergo in the process of diffusion)

RECENT ENZYMATIC STUDIES USING FRAP ANALYSIS

gp130/Jak 1 interaction Kinetics of association and the state of activation of GTPases in phagosomes

Mobility of Glucocorticoid Receptor in the nucleus

Phospholipase C-β2 activity and mode of memebrane interactions in living cells

FRAP analysis of gp130-YFP at the plasma

membrane

Cos-7 cells transfected with a gp130-YFP

containing expression vector

Region of interest with a diameter of 1.3µm is

photobleached

As a result of double bleaching, the mobile and

immobile fractions remains constant

FRAP ANALYSIS OF gp-130YFP AT THE PLASMA MEMBRANE

The fraction of mobile and immobile fractions

remains constant after double bleaching

FRAP recovery curves demonstarting that Rac 2(12V) reduces fluorescent recovery rate of GFP-PLCß2

Rab-GFP FRAP.

RECENT ENZYMATIC STUDIES USING FRAP ANALYSIS gp130/Jak 1 interaction Kinetics of association and the state of activation of GTPases in phagosomes

Mobility of Glucocorticoid Receptor in the nucleus

Phospholipase C-β2 activity and mode of memebrane interactions in living cells

CONCLUSION

In the future, FRAP combined with useful mathematical analysis, and use of engineered proteins will serve as an important tool to study the mobility of molecules in living cells

REFERENCES

Carrero, G., Macdonald, D., Crawford, E., Vries de., and Hendzel, M. (2003) Methods. 29, 14-28

Giese, B., Au-Yeung, C., Herrmann, A., Diefenbach, S., Haan, C., Kuster,A., Wortmann S., Roderburg, C., Heinrich P., Behrmann, I., and Muller-Newen, G. (2003) The journal of biochemistry. 278, 39205-39213

Illenberger, C., Walliser, C., Strobel, J., Gutman, O., Niv, H., Gaidzik, V., Kloog Y., Gierschik, P., and Henis, Y. (2003) The journal of biochemistry. 278, 8645-8652

Schaaf, M., and Cidlowski, J. (2003) Molecular and Cellular Biology. 23, 1922-1934

Vieira, O., Bucci, C., Harrison, R., Trimble, W., Lanzetti, L., Greunberg J., Schreiber, A., Stahl, P., and Grinstein, S. (2003) Molecular and Cellular Biology. 23, 2501-2514