Biology Reference
In-Depth Information
Oligomerization
human histamine1 receptor complexes in living HeLa cells using line-scanning fluo-
rescence cross-correlation spectroscopy.
INTRODUCTION
Diffusion and oligomerization are essential processes controlling the activity of nu-
merous membrane-localized receptor proteins that are involved in signaling cas-
cades. Various biochemical and biophysical approaches have been used to
quantify these processes in vitro using artificial or reconstituted membrane systems.
Recently, techniques have been developed to study these in living cells, and in this
chapter, the application of fluorescence fluctuation techniques will be discussed.
Fluorescence correlation spectroscopy (FCS) is the most well-known member of
the family of fluorescence fluctuation spectroscopy (FFS) techniques analyzing tem-
poral changes of the fluorescence intensity and relating these fluctuations to physical
parameters of the observed fluorescent molecules. The high sensitivity of the tech-
nique allows the detection of single molecules and obtaining information about con-
centration levels, diffusion rates, and interactions with other molecules ( Haustein &
Schwille, 2007 ). Using a confocal microscope and illumination with a focused laser
beam in combination with a pinhole, fluorescence is detected from a subfemtoliter
volume element ( 1 m m 3 ). Photons emitted by the fluorescent molecules present in
this element pass through a pinhole and are detected by a sensitive detector. During
the measurement, molecules will, due to their (Brownian) motion, move into and out
of the volume element and emit photons in a burst-type manner. The observed fluc-
tuations can be used to determine the average time a molecule requires passing
through the volume element. Due to the high signal-to-noise ratio that could be
achieved, measurements can be performed at the single-molecule level. In practice,
FFS operates in the nanomolar concentration range, which is close to the physiolog-
ical expression levels of many membrane receptors.
The mobility of a membrane receptor is related, among other factors, to the size of
the molecule. Therefore, it is possible to examine if receptors are moving freely or
are part of a large, more slowly moving complex. Meseth, Wohland, Rigler, and
Vogel (1999) examined the resolving power of FCS to distinguish between different
sized molecules. In case of an unchanged fluorescence yield upon binding, the dif-
fusion coefficients of the free and complexed form have to differ at least 1.6-fold,
corresponding to a fourfold mass increase for globular particles, which is required
to discriminate both species without prior knowledge of the system. This will not
suffice to discriminate receptor monomers from dimers, which involves a doubling
of the mass. However, the distribution of the detected fluorescence intensities can
still give information about dimerization, since dimers are twice as bright as mono-
mers, as is exploited in photon counting histogram (PCH) ( Chen, Johnson,
Macdonald, Wu, & M¨ ller, 2010; Hink, Shah, Russinova, de Vries, & Visser,
2008; Herrick-Davis, Grinde, Lindsley, Cowan, & Mazurkiewicz, 2012 ;
Chapter 10 ) or using number and brightness (N&B) analysis ( Crosby et al., 2013;
Digman, Dalal, Horwitz, & Gratton, 2008 ).
Search WWH ::




Custom Search