Chemistry Reference
In-Depth Information
loaded with Alx-Fab
0
through the pores. Finally, ATP was added to initiate the reaction.
Three types of the
fluorescent spots were observed on the cell surface; 20% were Rh-
EGF binding sites that were not colocalized with Alx-Fab
0
, another 10% were Rh-EGF
binding sites that were colocalizedwithAlx-Fab
0
, and the other were attributable toAlx-
Fab
0
without colocalization of Rh-EGF. The last population is responsible for amplify-
ing EGFsignals. Overall, the time-course of ampli
cation of EGFR activation observed
in semi-intact cells was similar to that observed in intact cells (Figure 5.4A).
The
fluorescence intensities of EGF, activated receptors, and activated receptors
co-localized with EGF were examined at different time points after the addition of
ATP (Figure 5.4B). Cluster size distributions of EGF were not changed with time
while activated receptors formed clusters whose size increased with time. This
increase was not caused by the clustering of receptors bound with EGF since the
distribution of EGF did not change. From the histograms representing activated
receptors colocalized with EGF, it can be seen that clustering was more rapid and
evident at the colocalization. This is probably because the binding sites of EGFare the
leading spots for activation. Thus, secondary activated receptors without EGF binding
formed clusters around the receptors primarily activated by EGF. Some of the
secondarily activated receptors diffused out of the clusters.
Figure 5.4C shows a model for signal ampli
cation using dynamic clustering and
lateral mobility of receptors. Receptors primarily activated by EGF binding exchange
the pair of dimers and activate other receptors unoccupied by EGF. Activation of the
receptor was propagated on the cell surface by reorganization of the receptor clusters
and lateral mobility. Fusion and splitting of the receptor clusters moving around the
cell surface by thermal diffusion were actually observed using single-molecule
visualization.
5.5
Dynamics of the NGF/NGFR Complex
NGF inducesmorphological and functional changes of the rat pheonochromocytoma
cell line PC12 to a neuron-like cell [12]. NGF (2.5S NGF) labeled with a single Cy3 or
Cy3.5 dye per molecule was prepared to investigate movements of NGF/NGFR
complexes on the PC12 cell surface in single molecules [13].
Typical trajectories of the lateral diffusion movements of Cy3-NGF/NGFR com-
plexes showed periods of mobility and immobility with abrupt switching between the
two (Figure 5.5A). The mobile and immobile phases were separated according to the
method described by Simson et al. [14]. In brief, for every segment of the trajectory,
the local diffusion coef
cient and the maximum radius of the segment were
obtained, and the probability of a molecule diffusing randomly with the local
diffusion coef
cient to remain within the maximum radius was calculated. In the
case of NGF on PC12, when a segment longer than 270 ms showed probability less
than 10
4
, the segment was de
ned to be immobile. These parameters were
determined from a comparison between experiments and simulations of random
walks.
