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 0s10s 20s30s 90s

FLUORESCENT RECOVERY CURVE Intensity Intensity Time (seconds)

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

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

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