Cell News 01/2017
33
Viewing synthesis and decay: Simultaneous detection
of mRNA transcription and decay intermediates by dual
colour single mRNA FISH on subcellular resolution
Susanne Kramer
Presenting author: Susanne Kramer
University of Würzburg
The detection of mRNAs undergoing transcription or decay
is challenging, because both processes are fast. However, the
relative proportion of an mRNA in synthesis or decay increases
with mRNA size and decreases with mRNA half-life. Based
on this rationale, I have exploited a 22 200 nucleotide-long,
short-lived endogenous mRNA as a reporter for mRNA metabo-
lism in trypanosomes. The extreme 5' and 3' ends were labelled
with red- and green-fluorescent Affymetrix® single mRNA
FISH probes, respectively. In the resulting fluorescence images,
yellow spots represent intact mRNAs; red spots are mRNAs in
transcription or 3'-5' decay, and green spots are mRNAs in 5'-
3' degradation. Most red spots were nuclear and insensitive to
transcriptional inhibition and thus likely transcription inter-
mediates. Most green spots were cytoplasmic, confirming that
the majority of cytoplasmic decay in trypanosomes is 5'-3'. The
system showed the expected changes at inhibition of tran-
scription or translation and RNAi depletion of the trypanosome
homologue to the 5'-3' exoribonuclease Xrn1. The method
allows to monitor changes in mRNA metabolism both on
cellular and on population/tissue wide levels, but also to study
the subcellular localization of mRNA transcription and decay
pathways. The system is applicable to mammalian cells.
OTHER TOPICS
Keratins regulate actin cortex formation
and stabilize cell-cell junctions
Fanny Loschke, Melanie Homberg, Thomas M. Magin
Presenting author: Fanny Loschke
SIKT Leipzig & Biology, Division of Cell and Developmental
Biology, University of Leipzig, Philipp-Rosenthal-Straße 55,
04103 Leipzig, Germany
Maintenance of epithelial cell adhesion is crucial for epidermal
morphogenesis, homeostasis and barrier integrity and relies
predominantly on the interaction of keratins with desmosomes.
Desmosome stability in turn influences adherens junctions
and the actin cytoskeleton. While the importance of desmo-
somes to epidermal coherence and keratin organization is well
established, the significance of specific keratin isotypes in
desmosome and actin organization has not been fully resolved.
We have recently shown that keratinocytes lacking all keratins
show elevated, PKC
α
-mediated desmoplakin phosphorylation
and subsequent destabilization of desmosomes. Re-expression
of the keratin pair K5/14, inhibition of PKC
α
activity or block-
ing of endocytosis reconstituted both desmosome localization
at the plasma membrane and epithelial adhesion, whereas ke-
ratinocytes expressing “stress”-keratins K6/K17 show elevated
PKC
α
-mediated desmosome disassembly (Kröger&Loschke et
al. JCB 2013, Loschke et al. JID 2016).
In subsequent experiments, we realized that in keratin-de-
ficient keratinocytes, actin organization and maturation of
adherens junctions were affected, implying a role of keratins
in these processes. Impaired adherens junction maturation
and actin reorganization were correlated with increased
Rac1 activity downstream of PI3K-activity in the absence of
keratins. Our current data suggest that sustained PI3K-activity
and increased Rac1-activity prevent maturation of adherens
junctions and reorganization of the actin cytoskeleton in
the absence of keratins. Whether keratins primarily regulate
desmosome turnover, which subsequently regulate adherens
junctions, or also influence the organization of the actin cyto-
skeleton and adherens junctions independently of desmosomes
remains to be shown.
