process continues, the entire cell will be photobleached. These techniques
allow specific GFP to be identified and tracked in the cell without dis-
rupting protein pathways or creating protein imbalances in the cell. The
rates of fluorescence recovery or decay provide quantitative values for the
movement of proteins within the cell, the dynamics of protein–protein
complexes during different cellular states, and the interactions of proteins
with various cellular components.
Eukaryotic cells are organized into a complex network of compart-
ments with specialized function, separated by membranes. To understand
the function of proteins in eukaryotic cells, the location of proteins needs
to be identified and the role of each protein must be defined in each
cellular compartment. Localization studies can be performed by attaching
GFP as a fusion product to any given gene and monitoring the cellular
positions that show fluorescence. Although this can be performed on an
individual basis for any given protein, one of the goals of proteomics is to
perform such studies on every protein of the cell. The complete genome
has been sequenced for the yeast Saccharomyces cere 9 isiae, making this a
well-characterized model system for proteomics. The gene for GFP was
attached to the over 6000 open reading frames of S. cere 9 isiaeusing a library
that represents most of the proteome (Figure 19.9). The identification was
CHAPTER 19 MOLECULAR IMAGING 413
(a) Fluorescence recovery after photobleaching (FRAP) 100Photobleach40608020
0
0608020 40 100FluorescenceTime(b) Fluorescence loss in photobleaching (FLIP)Photobleach10040608020
0
0608020 40 100FluorescenceTimeFigure 19.8Kinetic microscopic technique for probing the movement of fluorescent molecules.
(a) In the top view, a region of the cell is selectively bleached and the recovery of the fluorescent
molecules into that region is assessed. (b) In the bottom view, a region of the cell is repeatedly
photobleached and movement of the photobleached molecules out of the region can be monitored.
Modified from Lippincott-Schwartz and Patterson (2003).