Biomedical Engineering Reference
In-Depth Information
that mediates endosomal transport of mRNAs and ribosomes
most likely to guarantee translation throughout the entire length
of the hypha [
13
-
15
]. Importantly, Rrm4 also mediates endosome-
coupled translation of specifi c mRNAs such as
cdc3
mRNA for
loading of endosomes with septin. The translation product is
delivered by these endosomes to the hyphal tip for correct assem-
bly into higher-order septin fi laments [
14
]. Thus, these endo-
somes are versatile multipurpose carriers transporting not only
obvious cargo such as lipids but also cytoskeletal components,
ribosomes, and mRNAs.
The ascomycete
A. nidulans
is an excellent model system for
fi lamentous fungi. It completes the asexual life cycle, starting from
a single conidiospore, within 24 h and is well suitable for genetic
screens [
16
]. These resulted in the discovery of important eukary-
otic factors such as the fi rst eukaryotic tubulin genes—including
ʳ
-tubulin [
17
,
18
]. Furthermore, the kinesin motor
bimC
was the
founding member for a whole class of mitotic kinesins, and, last
but not least, the isolation of nuclear distribution mutants (
nud
)
revealed important insights into the human disease lissencephaly
[
19
,
20
]. In recent years,
A. nidulans
became an attractive model
to study polar growth. The multinucleated hyphal tip compart-
ment grows remarkably fast (about 0.5
m/min) in an actin- and
microtubule-dependent manner [
21
]. This growth speed depends
on the predominant secretion of vesicles at the apex and also on
endocytosis, which is required for membrane recycling. Its essen-
tial role was shown with different mutants of the endocytotic
machinery, e.g., fi mbrin (an F-actin cross-linking protein that plays
a role in endocytotic internalization) and ArfB (a small GTPase)
that leads to polarity defects [
22
,
23
]. Shuttling Rab5-positive
endosomes can be labeled with two paralogues RabA and
RabB. These endosomes show characteristic long-distance bidirec-
tional movement on microtubules, driven by the corresponding
motor proteins dynein and most likely a kinesin-3 type motor
UncA [
10
,
24
,
25
].
Here, we describe important techniques for live cell imaging
of endosomal traffi cking such as (1) millisecond alternating laser
excitation (msALEX) for dual-color acquisition applied during
in vivo colocalization studies on moving endosomes. msALEX
allows for the alternating excitation of different fl uorophores with
a minor time shift, but with the advantage of a longer observation
time because of reduced bleaching of the respective fl uorophores.
(2) RNA live imaging to demonstrate endosomal mRNA trans-
port [
13
,
14
,
26
] relies on the insertion of binding sites for a
specifi c peptide derived from a heterologous RNA-binding pro-
tein like
ʼ
untranslated region (UTR) of the mRNA of
interest. Thereby, the localization of these mRNAs can be visual-
ized by expressing a fusion protein consisting of
ʻ
N in the 3
′
N-Gfp, because
the fl uorescent protein is specifi cally recruited to these mRNAs.
(3) FRAP (fl uorescence recovery after photobleaching) is used to
ʻ
