Biology Reference
In-Depth Information
An alternative approach to monitor oligomerization has been developed by
Schwille, Meyer-Almes, and Rigler (1997)
who applied dual-color fluorescence
cross-correlation spectroscopy (FCCS). In these studies, each of the two types of
interacting molecules is tagged by a spectrally different fluorescent group, for exam-
ple, a green or a red fluorescent dye. The different fluorescent dyes can be excited
either with different lasers or with the same laser. The emission light is split into two
different detectors so that the fluorescence of the two fluorophores can be monitored
over time simultaneously. In contrast to single-color FCS, the discriminating factor is
not the increase of the molecular mass or increased brightness upon complex forma-
tion but the simultaneous detection of fluorescence bursts in both detection channels.
Although most FCCS studies published use two dyes to monitor interactions between
two types of molecules, also higher-order complexes could be monitored using more
than two fluorophores (
Heinze, Jahnz, & Schwille, 2004; Shcherbakova, Hink,
Joosen, Gadella, & Verkhusha, 2012
).
Classical FCS techniques as described earlier are based on point illumination by
parking the laser beam at a preferred location within the cell, like the plasma mem-
brane, and illuminate this point during the data acquisition. However, membrane
movement or fluorophore photobleaching, caused by a low mobility of membrane
receptors or their complexes, often results in artifacts in the FCS curves. Photo-
bleaching can be reduced by exciting the molecules less frequently, for example,
by moving the laser beam, thus scanning lines or even whole images. FFS approaches
that combine temporal and spatial information are referred to as image correlation
spectroscopy techniques (
Kolin & Wiseman, 2007
). One of these techniques, line
FCS, has the advantage that movement of the cell and or the plasma membrane dur-
ing the measurement can easily be corrected for after acquisition and that the
obtained diffusion time is independent on the z-focus (
Ries, Yu, Burkhardt,
Brand, & Schwille, 2009
).
In this chapter, we describe the use of dual-color line-scan FCCS in order to study the
dimerization and diffusion of fluorescently labeled human histamine1 receptors (H
1
R)
in nonstimulated living HeLa cells. Typically, these experiments are complemented by
PCH or N&B analysis to monitor the stoichiometry of the complexes being formed.
The application of PCH is being discussed in
Chapter 10
in this topic.
11.1
MATERIALS
11.1.1
Solutions, DNA constructs, and cells
1.
Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM sodium
phosphate dibasic, 2 mM potassium phosphate monobasic (pH 7.4).
2.
Microscopy medium: 20 mM HEPES (pH
ΒΌ
7.4), 137 mM NaCl, 5.4 mM KCl,
1.8 mM CaCl
2
, 0.8 mM MgCl
2
, and 20 mM glucose.
3.
Purified mTurquoise2 and sYFP2 in PBS pH 7.4. The fluorescent proteins (FPs)
are purified using the IMPACT (New England Biolabs) system, which
utilizes inducible self-cleavage activity of an intein splicing element to separate
the FP from the affinity tag. Transformed bacteria are collected by