1. FLUORESCENT RECOVERY AFTER PHOTOBLEACHING Analysis and Applications Zareen Butt Department of Chemistry and Biochemistry University of Windsor

2. OVERVIEW  Phenomenon of Photobleaching  Fluorescent Recovery After Photobleaching  Measure the mobility of nuclear proteins, macromolecular diffusion within cell membranes, the cytoplasm, nucleoplasm

3. HOW FRAP WORKS  Molecules covalently bound to a fluorophore  Inhomogenous fluorescent population  Spatial separation between fluorescent moleclues and photobleached molecules at time 0

4. Example of photobleaching in an indian muntjac fibroblast nucleus expressing ASF/SF2 GFP Photobleached Population

5. DATA COLLECTION 0s10s 20s30s 90s

6. FLUORESCENT RECOVERY CURVE Intensity Intensity Time (seconds)

7. Nucleoplasmic Topoisomerase I & Topoisomerase IIα & ß Chromatin associated Nucleosomal Histones Transcription Factor Estrogen Receptor Glucocorticoid Receptor Nuclear lamina/memebrane EmerinHA-95 List of some nuclear proteins investigated by FRAP

8. 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

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

10. 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

11. 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)

12. 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

13. FRAP analysis of gp130-YFP at the plasma membrane

14.  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

15. The fraction of mobile and immobile fractions remains constant after double bleaching

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

17. Rab-GFP FRAP.

18. 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

19. 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

20. 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

21.  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