FLUORESCENT RECOVERY AFTER

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


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

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

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Photobleached Population
Example of photobleaching in an indian muntjac
fibroblast nucleus expressing ASF/SF2 GFP
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DATA COLLECTION
0s
10s
20s
30s
90s
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FLUORESCENT RECOVERY CURVE
Intensity
Intensity
Time (seconds)
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Nucleoplasmic Topoisomerase I Topoisomerase IIa ß
Chromatin associated Nucleosomal Histones
Transcription Factor Estrogen Receptor Glucocorticoid Receptor
Nuclear lamina/memebrane Emerin HA-95
List of some nuclear proteins investigated
by FRAP


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• 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

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

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• 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)

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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-ß2 activity and mode of
• memebrane interactions in living cells

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FRAP analysis of gp130-YFP at the plasma
membrane
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FRAP ANALYSIS OF gp-130YFP 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

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The fraction of mobile and immobile fractions
remains constant after double bleaching
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FRAP recovery curves demonstarting that Rac
2(12V) reduces fluorescent recovery rate of
GFP-PLCß2
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Rab-GFP FRAP.
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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-ß2 activity and mode of
• memebrane interactions in living cells

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• 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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• 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

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• 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