Biology Reference
In-Depth Information
10.3
DISCUSSION
The most important considerations in performing FCS molecular brightness exper-
iments in living cells are instrument alignment and calibration, choice of controls,
fluorescence labeling of the receptor, and proper positioning of the sample in the ob-
servation volume. These topics are reviewed in more detail in the succeeding text.
Begin every FCS recording session by performing a pinhole adjustment using a
control sample of the fluorescent probe used to label the receptor of interest to ensure
proper alignment of the light path. Control samples, such as GPF or YFP expressed in
the cytosol or a fluorescent dye in solution, provide a means to monitor day-to-day
variability in instrument performance through reproducibility of molecular bright-
ness values at a given laser setting. Likewise, end each session with additional con-
trol samples to monitor instrument stability throughout the session. If studying
plasma membrane receptors, suitable plasma membrane controls are essential. Mo-
nomeric and dimeric receptors, such as CD-86 and CD-28, are a good choice for
“decoding” the oligomeric composition of unknown samples by molecular bright-
ness analysis. Be sure that the selected control and receptor of interest have their
fluorescent tags in similar position and orientation with respect to the plasma mem-
brane. Avoid the use of GPI-anchored or short myristoyl/palmitoyl chains to target
GFP variants to the plasma membrane as they can enhance clustering within micro-
domains (
Zacharias, Violin, Newton, & Tsein, 2002
). We have tested such constructs
and found them to be several times brighter than other monomeric controls. The use
of the A206K/L221K mutations to eliminate GFP aggregation can be used, but re-
ceptor levels studied in FCS experiments are well below the concentration at which
aggregation occurs (
Zacharias et al., 2002
). It is critical to use the lowest laser power
possible while still maintaining a good signal to noise ratio, as photobleaching will
result in artificially fast diffusion times and decreased molecular brightness.
AGFP variant attached to the C-terminus of the receptor is the most common method
for receptor labeling. It has the advantage of ensuring an exact 1:1 labeling of the recep-
tor, critical for molecular brightness analysis. A fluorescent ligand can be used if it has
very slow dissociation kinetics. However, if the receptor of interest forms dimers, for
example, experiments must be performed to confirm that the ligand binds with slow dis-
sociation kinetics to both protomers of the dimer. A case in point is risperidone, which
binds in a pseudo-irreversible manner to only one protomer of the 5-HT
7
receptor homo-
dimer (
Teitler, Toohey, Knight, Klein, & Smith, 2010
). Alternatively, a monoclonal,
monovalent Fab fragment recognizing the native conformation of the receptor could
be used provided each Fab has exactly one fluorescent tag. A singly labeled Fab, such
as Fab cloned into a GFP expression plasmid, would provide the ultimate tag as it could
be used to label native receptors endogenously expressed in primary cultures.
The most critical parameter in molecular brightness analysis is proper positioning
of the sample within the observation volume. The observation volume is not illumi-
nated homogenously, and the detected photon counts decrease as a fluorescent mol-
ecule moves away from the center of the observation volume. Since a plasma
membrane is approximately 1/200th of the axial dimension, optimal positioning
