Biology Reference
In-Depth Information
colors are spatially corrected and then overlaid. Receptors must be expressed at low
density in cell plasma membranes because high-density expression (
2 molecules/
m
m
2
) creates difficulty for tracking individual fluorescent spots. In addition, the recep-
tors should be labeled with highly photostable fluorophores at high efficiency because
short photobleaching lifetimes and low labeling efficiency of receptors reduce the
probability of detecting dimers and oligomers. In this chapter, we describe methods
for observing and detecting colocalization of the individual fluorescent spots of recep-
tors labeled with fluorophores via small tags and the estimation of true dimer and olig-
omer lifetimes after correction with photobleaching lifetimes of fluorophores.
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INTRODUCTION
Receptor clustering after ligand binding is often the first key step for the induction of
intracellular signaling (
Kondo et al., 2012; Veatch et al., 2012
). Even before ligation,
many receptors, including epidermal growth factor receptors (
Chung et al., 2010
),
several G protein-coupled receptors (GPCRs) (
Hern et al., 2010; Kasai et al.,
2011
), and several glycosylphosphatidylinositol (GPI)-anchored receptors
(
Brameshuber et al., 2010; Seong, Wang, Kinoshita, & Maeda, 2013; Sharma
et al., 2004; Suzuki et al., 2012
), have been proposed to form dimers.
Understanding the signal transduction mechanisms of these receptors requires in-
vestigation into how these receptors are organized in resting cells and how upon li-
gation they are incorporated into signaling complexes.
Single-molecule imaging techniques are very important and are powerful tools to
observe receptor clustering and interactions of receptors with signaling molecules in
cell plasma membranes (
Jaqaman et al., 2011, 2008; Nagata, Nakada, Kasai,
Kusumi, & Ueda, 2013; Nguyen, Kamio, & Higuchi, 2003; Opazo et al., 2010;
Sako, Minoghchi, & Yanagida, 2000; Yamagishi, Shirasaki, & Funatsu, 2013
). Re-
cent single-molecule imaging studies revealed that receptor-receptor interactions are
very dynamic in steady-state cells. For example,
Kasai et al. (2011)
found that in live
cells at 37
C, N-formyl peptide receptor (FPR), a chemoattractant GPCR, formed
transient homodimers with an equilibrium constant of 3.6 copies/
m
m
2
and dissocia-
tion and two-dimensional association rate constants of 11.0 s
1
and 3.1 copies/
m
m
2
s
1
, respectively (see
Fig. 20.1
A).
Furthermore,
Suzuki et al. (2012)
have discovered that CD59, a GPI-anchored
protein (GPI-AP), forms mobile transient homodimers induced by ectodomain pro-
tein interactions and stabilized by raft-lipid interactions (
Fig. 20.1
B). They also
found that CD59 did not form heterodimers with prolonged lifetimes, but under
higher physiological expression conditions, homodimers coalesced to form hetero-
and homo-GPI-anchored receptor oligomers through raft-based lipid interactions.
After ligation, CD59 forms stable homo-oligomers, which induce intracellular
Ca
2
þ
responses dependent on GPI anchorage and cholesterol, suggesting that tran-
sient homodimers play a key role (
Suzuki, 2012; Suzuki, Fujiwara, Edidin, &
