Biology Reference
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region by unbleached molecules, it cannot follow the fate of the bleached
molecules themselves. In contrast, FLAP does allow the bleached molecules to
be tracked. This is facilitated by comparison of the distribution of remaining
unbleached molecules with that of another reference fluorophore. For
example, to visualize the steady-state dynamics of actin turnover, YFP-actin
and CFP-actin fusion proteins were co-expressed in the same cell (Dunn et al.,
2002). The YFP-actin was then bleached in a predefined region of the cell. The
CFP was not photobleached and acted as a reference for the YFP-actin, since
statistically the two molecules would have co-localized under the observed
resolution. Subtraction of the YFP signal from the CFP signal resulted in a
difference image reflecting the position of the bleached YFP molecules. When
this approach was used to observe actin dynamics in the membrane rues at
the leading edge of a cell, labelled actin was seen rapidly to pervade the ru e
system (Dunn et al., 2002). In contrast, actin was found to be extremely stable
in the perinuclear region of the same cell. The approach has also been used to
observe actin dynamics in actin bundles or stress fibres and in the cytokinetic
ring formed during the later stages of mitosis. In both cases these actin bundle
structures were surprisingly unstable and showed rapid interchange of actin
with surrounding structures - a process that we believe to occur by rapid
depolymerization and repolymerization.
FLAP: Image acquisition and image processing
In experiments that analyse actin dynamics, transformed rat fibroblasts (a gift
from Dr Pavel Vesely) are microinjected with DNA constructs encoding
yellow and cyan N-terminal fluorescent proteins fused to b-actin (the b-actin
cDNA was a gift from Dr John Copeland) (Figure 7.4A). Vectors encoding
pECFP-C1 and pEYFP-C1 were obtained from ClonTech. Cells are visualized
using a Zeiss LSM 510 laser scanning confocal microscope and a 636 Plan-
Apochromat NA 1.4 PH 3 oil objective. Temperature is stabilized using a
378C temperature-controlled hood, since minor changes can cause focus drifts
that result in artefacts due to chromatic aberration of the objective. The CFP
and YFP channels use the 458 nm and 514 nm argon lines respectively for
excitation. Simultaneous phase contrast is collected with the CFP channel,
whilst simultaneous interference reflection is collected with the YFP channel
to enable visualization of focal adhesions (Figure 7.4B and 7.4C). The two
emission channels are split by a 545 nm dichroic mirror followed by a
bandpass 475-525 nm filter (CFP) and a longpass 530 nm filter (YFP). The
pinholes are set to give 3 mm depth of focus. Each image line is scanned as
rapidly as possible, first by the 514 nm laser line with the YFP channel
detector and interference reflection detector active followed by the 458 nm
laser line with the CFP detector and the phase contrast detector active.
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