Cell News | Issue 01, 2017 - page 22

Cell News 01/2017
Quantitative cell cycle analysis based on an endogenous
all-in-one reporter for cell tracking and classification
Thomas Zerjatke, Igor A. Gak, Dilyana Kirova, Markus Fuhrmann, Katrin Daniel, Magdalena
Gonciarz, Doris Müller, Ingmar Glauche and Jörg Mansfeld
Presenting author: Jörg Mansfeld
Cell Cycle, Biotechnology Center, Technische
Universität Dresden, 01307 Dresden, Germany
Cell cycle dynamics are crucial to cell-fate decisions. Although
live imaging has provided extensive insights into this rela-
tionship at the single-cell level, limitations on the number of
fluorescent markers that can be used in a single experiment
have hindered efforts to link individual protein dynamics
responsible for decision-making directly to cell-cycle pro-
gression. Here, we present endogenously tagged PCNA as an
all-in-one cell cycle reporter that allows simultaneous analysis
of cell cycle progression and the dynamics of individual fate
determinants. We provide an accompanying image analysis
pipeline allowing segmentation, tracking and classification of
all cell cycle phases including the transition into quiescence in
an automated manner. Combing the all-in-one reporter with
endogenously labeled cyclins and p21 as prime examples of cell
cycle-regulated fate determinants, we show how cell cycle and
quantitative protein dynamics can be simultaneously extract-
ed to gain insights into G1 phase regulation and responses to
Keratin-dependent regulation of thymic stromal
lymphopoietin mediated by ERK1/2 kinases: a major
role of keratinocytes in immune signaling
Scheffschick A, Kiritsi D, Behr M, Kumar V, Bruckner-Tuderman L, Magin TM
Presenting author: Andrea Scheffschick
Institute of Biology, Division of Cell and Developmental
Biology, University of Leipzig
Epidermal barrier defects can cause the inflammatory skin
disease atopic dermatitis (AD) frequently accompanied by
allergies and asthma. The proinflammatory cytokine thymic
stromal lymphopoietin (TSLP) is strongly increased in lesional
skin of AD patients and is involved in itch and inflammatory
processes by activating dendritic cells and inducing T help-
er type 2 cytokines. According to current hypotheses, TSLP
upregulation occurs upon barrier defects in combination with
endogenous and exogenous factors, however, the mechanisms
remain incompletely understood. Patients suffering from the
skin blistering disease epidermolysis bullosa simplex (EBS),
caused by keratin (K) 5 or 14 mutations frequently suffer
from itch and inflammation. This raises the question whether
keratins contribute to inflammatory responses in EBS by upreg-
ulating TSLP. We therefore investigated a link between keratins
and TSLP expression.
We identified strongly increased TSLP levels in the serum and
the epidermis of mice lacking all keratins. Corresponding kera-
tinocytes and mutants expressing an EBS-type K14 mutation
show highly elevated TSLP expression. Re-expression of wild-
type K14 reduced TSLP levels suggesting a cell-intrinsic and
keratin dependent mechanism being responsible for TSLP up-
regulation. Of note, a subset of EBS patients showed strongly
elevated TSLP levels, further supporting involvement of keratins
in TSLP upregulation. Moreover, we identified an EGFR-ERK1/2
signaling axis downstream of keratin loss which is involved in
mediating TSLP increase. We hypothesize that elevated TSLP
levels might explain the high prevalence of itch in EBS. The
further elucidation of keratin-dependent TSLP regulation will
enable to identify novel pathways responsible for itch in EBS
and other EB entities. Our data support the hypothesis that
mutations in epithelial keratinocytes modify local and systemic
immune responses by affecting interaction with and signaling
to innate and adaptive immune cells.
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