Biology Reference
In-Depth Information
photobleach a small spot on the cell surface. Originally the light source was a broad spec-
trum mercury or xenon lamp and was used in conjunction with a color filter to assure the
spot would receive light that matched the absorbance of the fluorescent probe. Tunable
lasers have now replaced the older light sources and filters. The intense laser pulse photo-
bleaches a spot of predetermined size and shape. This spot appears dark on a highly fluo-
rescent, bright, unbleached background. The laser destroys the fluorescence of the probe
but has no effect on the membrane component (lipid or protein) to which the tag is associ-
ated. With time, the still fluorescent-labeled membrane components diffuse into the photo-
bleached spot, while the non-fluorescent (bleached) components diffuse out of the spot.
Eventually the once bleached spot recovers most of its fluorescence, although never fully
recovering.
A detailed plot of data obtained from a typical FRAP experiment is shown in
Figure 9.14
[20]
. The initial fluorescence background for the unbleached cell is indicated by line (1). Pho-
tobleaching (arrow) decreases fluorescence in the spot (2). The net loss in fluorescence is X.
Brownian motion allows for recovery of fluorescence into the spot (3). Eventually fluores-
cence returns in the spot to a new baseline (4) that is lower than the pre-bleached fluorescence
(1). The fluorescence recovery is Y (Y
100 and
represents the percent of the fluorescent-tagged membrane component that is diffusible. The
percent of the membrane component that is non-diffusible is:
100
ð
<
X). The percent recovery is therefore Y/X
100
Þ
Lateral mobility is directly related to the slope of the recovery process. A steeper slope
indicates a faster recovery and higher diffusion rate. The diffusion rate D can be obtained
from the following equation:
Y
=
X
W
2
=
4
D
¼
t
1
=
2
where w is the width of the photobleaching beam and t
1/2
is the time required for the bleach
spot to recover half of its initial intensity.
Single Particle Tracking (SPT)
While FRAP clearly has advantages over the original Frye-Edidin method, it still just gives
an averaged diffusion rate for an ensemble of membrane residents. Single particle tracking
(SPT), as the name implies, can follow diffusion of one molecule at a time
[21]
. Akiharo
Kusumi (
Figure 9.15
) of the Kyoto University School of Medicine is recognized as the world
leader in this emerging field
[22]
. In his laboratory, the temporal resolution of SPT has been
pushed down to 25
s and spatial resolution down to nanometers. As might be expected, this
complex technology has resulted in a completely new paradigm in our understanding of
membrane structure. STP development was driven by a basic observation that confused
scientists for decades. If movement in a membrane is the result of free Brownian motion,
why do membrane components diffuse 10
m
100 fold slower in a cell (plasma) membrane
than they do in an artificial lipid bilayer membrane? In 1983 Michael Sheetz
[23]
proposed
that trans-membrane proteins might be contained by the cytoskeleton ('fence' theory).
However, membrane technology was not sufficiently advanced to confirm this hypothesis.
Confirmation had to await the development of SPT.
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