Biology Reference
In-Depth Information
was a “mere” morphological characterization. In addition, if quan-
titative information is to be extracted from image data, appropriate
protocols and software are needed.
A variety of fl uorescent protein-based markers has been used
to trace cytoskeletal structures in living cells, including those of
plants. Plant microtubules have been successfully visualized using
both GFP-tubulin fusions [
7
,
8
] and GFP-tagged ortho- or heter-
ologous microtubule-associated proteins (MAPs) such as mamma-
lian MAP4 [
9
] or several isoforms of Arabidopsis MAP65 [
10
]. In
addition to labeling microtubules along their whole length, micro-
tubule ends can be specifi cally marked by tags based on end-
binding proteins such as EB1 preferring minus ends [
11
] or
mammalian CLIP170 for plus ends [
12
]. For actin visualization,
fl uorescent protein-tagged mammalian talin [
13
] or constructs
based on the C-terminal actin-binding domain of Arabidopsis fi m-
brin (FABD, refs. [
14
,
15
]) can be used. A very promising actin
marker is the 17 amino acid actin-binding peptide known as
LifeAct, which has been successfully used to target fl uorescent pro-
teins to actin fi laments also in plant cells [
16
].
It has to be stressed that any experiments including (over)expres-
sion of tagged (i.e., modifi ed) and possibly heterologous proteins
have to be interpreted with caution, as (1) only a subset of the
relevant cytoskeletal structures may be labeled, as shown, e.g., for
the various MAP65 isoforms [
10
], and (2) the tag itself may affect
cytoskeletal structure and dynamics. Both talin and MAP4-based
markers cause visible phenotypic alteration on the whole plant level
[
17
], and in particular GFP-tagged talin was shown to interfere
with actin dynamics and aggravate the effects of some treatments
and mutations affecting the actin cytoskeleton [
18
,
19
].
A suitable high-resolution fl uorescence microscopy and micro-
photography equipment is required to make full advantage of in
vivo cytoskeletal labeling. Conventional fl uorescence microscopy,
although useful, is limited by spatial resolution, interfering back-
ground (auto)fl uorescence, and usually also by long exposure
times. However, advanced microscopy techniques, such as confo-
cal laser scanning microscopy (CLSM), can be used to improve
spatial resolution. Very thin samples can be observed with supreme
spatial and temporal resolution using the total internal refl ection
microscopy (TIRFM) technique; however, TIRFM can only reach
up to some 200 nm from the cover slip. Nevertheless, TIRFM
hardware can also be used in variable angle epifl uorescence micros-
copy (VAEM) mode with a reasonable trade-off between lateral
resolution and imaging depth, allowing thus visualization of a thin
cortical layer of the cytoplasm through the cell wall [
20
-
25
].
However, it is possible that in plant cells, the evanescent wave
might be initiated between the cell wall and plasmalemma, the cell
wall thus being a part of the optical system, and even true TIRFM
may thus work [
22
].
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