Chemistry Reference
In-Depth Information
7.3.2
Techniques
Classical techniques for investigating the lateral diffusion of
fluorescently-labeled
lipids and proteins are
fluorescence recovery after photobleaching (FRAP or FRP)
and post-electrophoresis relaxation (PER) that measure the local translational diffu-
sion coef
cient and the mobile fraction of diffusing species on surfaces of living
cells [50]. More recently, the trajectories of individual molecules on cellular mem-
branes have been monitored using a variety of reporting labels such as colloidal
particles and latex beads by nanovideomicroscopy [51
-
53],
fluorescent organic
probes [54, 55] and quantum dots [44, 56, 57] by
fluorescence imaging, and gold
nanoparticles by photothermal interference contrast [58, 59]. Tracking of various
membrane receptors on different cell types or organelles revealed the heterogeneity
of molecular motions on cell membranes as re
ected by the presence of various
diffusion modes and the broad distribution of (restricted) mobilities in length and
time scales [60, 61].
Another complementary approach to the investigation of the rapid single-molecule
dynamics in living cells is
fluorescence correlation spectroscopy (FCS). Although
the principles of FCS were formulated in the 1970s, only recent technological
advances providing high sensitivity and speci
c protein labeling allowed non-
invasive applications to live cells (reviews: [62, 63]). Diffusion of molecules across
a diffraction-limited focal volume introduces
fluctuations in the
fluorescence inten-
sity characteristic of their velocities (Figure 7.1A, B). The autocorrelation function G
(
) that describes the two-dimensional Brownian diffusion of multiple membrane
components is given by:
t
N
X
n
1
y
i
G
ðtÞ¼
1
þ
þ
t
i
;
ð
7
:
1
Þ
1
i
¼
1
where y
i
and
t
i
are the fraction and the diffusion time constant of the species i,
respectively, and N is are the average number of molecules in the detection volume.
The diffusion coef
cient Di
i
of the species i is related to the diffusion time by:
w
xy
4
D
i
¼
;
ð
7
:
2
Þ
t
i
where w
xy
is the radius of the detection volume in the focal plane. Complex
diffusional behaviors that originate from non-planar membrane topology or interac-
tion networks can also be investigated by FCS.
Therefore, the effects of ligand binding to its membrane receptor can be observed
as changes in the diffusional behavior of the active receptor along the different
molecular events of the signaling pathway. In the Table 7.1, we present some
examples that stress the utility of single particle/molecule tracking and FCS to better
understand the mechanism of signal transductionmediated by membrane receptors
on cultured cells and to address the question of the functional implication of the
receptor mobility.
