Biology Reference
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Magenau, & Gaus, 2012; Rossy, Owen, Williamson, Yang, & Gaus, 2012; Sengupta
et al., 2011
) and stochastic optical reconstruction microscopy (
Bates, Huang,
Dempsey, & Zhuang, 2007
), although these modalities are outside the scope of this
chapter. Along with single-fluorescent-molecule imaging, single-particle tracking of
gold (
Fujiwara, Ritchie, Murakoshi, Jacobson, & Kusumi, 2002; Murase et al., 2004;
Suzuki, Ritchie, Kajikawa, Fujiwara, & Kusumi, 2005
), latex beads (
Suzuki and
Sheetz, 2001; Suzuki, Sterba, & Sheetz, 2000
), and quantum dots (
Chen,
Veracini, Benistant, & Jacobson, 2009; Lidke et al., 2004
) attached to receptors have
unraveled the mechanisms through which receptor diffusion is regulated by cell
plasma membrane structures such as meshes of cytoskeletal actin filaments
(
Kalay, Fujiwara, & Kusumi, 2012; Kusumi, Fujiwara, Chadda, et al., 2012;
Kusumi, Fujiwara, Morone, et al., 2012; Morone et al., 2006
), lipid rafts
(
Kusumi, Koyama, & Suzuki, 2004; Kusumi & Suzuki, 2005; Tanaka et al.,
2010
), and synapses (
Bannai et al., 2009
). However, these bulky probes would dis-
turb associations between receptors, and the low efficiency of receptor labeling cre-
ates challenges for the detection of receptor-receptor interactions. Therefore, this
chapter focuses on single-molecule imaging of fluorophores attached to receptors
via small protein tags.
20.1
RECEPTOR EXPRESSION AND FLUORESCENT LABELING
This section describes a method for expressing receptors in cell plasma
membranes and labeling them with fluorophores for the observation of receptor-
receptor interactions using single-molecule imaging. As mentioned in the introduc-
tion, receptor-receptor interactions can be detected by observing the colocalization
of individual fluorescent spots, which represent receptor monomers. Observing the
colocalization requires that conditions be optimized for receptor expression and fluo-
rescent labeling. If the expression of receptors is high (
Fig. 20.2
A), the individual spots
of the attached fluorophores are indistinguishable, precluding single-molecule imag-
ing. By contrast, if the fluorescent labeling efficiency of the receptors is low and the
photobleaching lifetime of the fluorophores short (
Fig. 20.2
B), dimerization and olig-
omerization of receptors are barely detectable. Therefore, observing receptor-receptor
interactions using single-molecule imaging requires that the expression density of re-
ceptors be low enough to observe single molecules and fluorescent labeling efficiency
of receptors be high enough to observe receptor clustering (
Fig. 20.2
C). If cells endog-
enously express receptors at a low density (
2.0 molecules/
m
m
2
), single molecules of
receptors in the cell membranes can be observed via fluorescently labeled Fab frag-
ments or ligands attached to most of the receptors. However, this scenario is a rare
one. For example, the literature reports that the physiological expression levels of
GPI-APs are 3.0 and 8.2 copies/
m
m
2
for urokinase receptors in bovine microvascular
endothelial cells and U937 cells, respectively (
Mandriota et al., 1995
); 10 copies/
m
m
2
for phospholipase A2-activating protein in HeLa TCRC-1 cells (
Jemmerson, Shah,
Takeya, & Fishman, 1985
); 40 copies/
m
m
2
for decay-accelerating factor in HeLa cells
<
