Biology Reference
In-Depth Information
fluorescent probes is indiscernible. In addition, fluorophore-conjugated Fab is appli-
cable to the observation of receptor-receptor interactions only when Fab has high
affinity to receptors, because high labeling efficiency of receptors with fluorophores
is required for this experiment. Furthermore, for high-efficiency fluorescent labeling
of receptors, cells must be incubated with fluorescent Fab fragments with a concen-
tration much higher than the
K
d
value.
Another method is to label receptors with fluorophores via small protein tags. Of
course, we can observe the colocalization of individual fluorescent spots of receptors
fused with monomeric GFP (mGFP, A206Kmutant). However, photobleaching must
be carefully taken into account because the photobleaching lifetime of mGFP is
shorter than 1 s when single molecules of mGFP are observed at a high signal-
to-noise ratio. The short photobleaching lifetime makes precise estimation of
colocalization lifetimes difficult. Therefore, instead of mGFP, we often used protein
tags, especially Halo7-tag (Promega) (
Keuning, Janssen, & Witholt, 1985
) and
acyl carrier protein (ACP)-tag (New England Biolabs) (
Meyer et al., 2006; Rock
& Cronan, 1979; Sielaff et al., 2006
), which are fused with receptors and
covalently conjugated with photostable organic dye molecules. These tags can be
labeled with fluorophores at a precise 1:1 mol ratio. Furthermore, neither Halo7
nor ACP form dimers and therefore do not induce receptor clustering. Notably, how-
ever, when these tags are inserted at the extracellular side of receptors, the signal
peptides of the receptors sometimes need to be replaced by others for expression
of the receptors in cell plasma membranes.
Table 20.1
shows the characteristics
for these tags.
Halo7-tag (molecular weight
33 kDa) is a modified haloalkane dehalogenase
designed to bind covalently to synthetic ligands that are suicide substrates.
¼
Table 20.1
Characteristics of Tag Proteins Suitable for Single-Molecule Imaging of
Receptor-Receptor Interactions
Acyl Carrier
Protein (ACP)
Characteristics
Halo7
Molecular weight
33 kDa
8 kDa
Reaction with
ligand
The ligand is a suicide substrate for
the tag
The ligand is covalently
bound to the tag with
enzyme
Fluorophores for
labeling at
cytoplasmic site
Rhodamine 110
Tetramethylrhodamine
None
Fluorophores for
labeling at
extracellular site
ATTO488, Alexa488
Cy3, Dy547
ATTO594, Alexa594, etc.
Same as Halo7
Signal peptide at
N-terminus
The original signal peptide of
receptors sometimes needs to be
replaced by others
Same as Halo7
