dictyNews
Electronic Edition
Volume 42, number 9
March 18, 2016
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Abstracts
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CP91 is a component of the Dictyostelium centrosome involved in
centrosome biogenesis
Sascha Putzler; Irene Meyer; Ralph Gräf
Eur. J. Cell Biol, in press
The Dictyostelium centrosome is a model for acentriolar centrosomes
and it consists of a three-layered core structure surrounded by a
corona harboring microtubule nucleation complexes. Its core structure
duplicates once per cell cycle at the G2/M transition. Through
proteomic analysis of isolated centrosomes we have identified CP91,
a 91- kDa coiled coil protein that was localized at the centrosomal
core structure. While GFP-CP91 showed almost no mobility in FRAP
experiments during interphase, both GFP-CP91 and endogenous CP91
dissociated during mitosis and were absent from spindle poles from
late prophase to anaphase. Since this behavior correlates with the
disappearance of the central layer upon centrosome duplication,
CP91 is a putative component of this layer. When expressed as
GFP-fusions, CP91 fragments corresponding to the central coiled coil
domain and the preceding N- terminal part (GFP-CP91cc and
GFP-CP91N, respectively) also localized to the centrosome but did
not show the mitotic redistribution of the full length protein suggesting
a regulatory role of the C-terminal domain. Expression of all GFP-fusion
proteins suppressed expression of endogenous CP91 and elicited
supernumerary centrosomes. This was also very prominent upon depletion
of CP91 by RNAi. Additionally, CP91-RNAi cells exhibited heavily
increased ploidy due to severe defects in chromosome segregation along
with increased cell size and defects in the abscission process during
cytokinesis. Our results indicate that CP91 is a central centrosomal
core component required for centrosomal integrity, proper centrosome
biogenesis and, independently, for abscission during cytokinesis.
submitted by: Ralph Gräf [[log in to unmask]]
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Src1 is a protein of the inner nuclear membrane interacting with the
Dictyostelium lamin NE81
Petros Batsios, Xiang Ren, Otto Baumann, Denis A. Larochelle,
and Ralph Gräf*
Cells, in press
The nuclear envelope (NE) consists of the outer and inner nuclear
membrane (INM), whereby the latter is bound to the nuclear lamina.
Src1 is a Dictyostelium homologue of the helix-extension-helix family
of proteins, which also includes the human lamin-binding protein MAN1.
Both endogenous Src1 and GFP-Src1 localized to the NE during the
entire cell cycle. Immuno electron microscopy and light microscopy
after differential detergent treatment indicated that Src1 resides
in the INM. FRAP experiments with GFP-Src1 cells suggested that at
least a fraction of the protein could be stably engaged in forming
the nuclear lamina together with the Dictyostelium lamin NE81. Both
a BioID proximity assay and mis-localization of soluble, truncated
mRFP-Src1 at cytosolic clusters consisting of an intentionally
mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and
NE81. Expression GFP-Src11-646, a fragment C-terminally truncated
after the first transmembrane domain, disrupted interaction of nuclear
membranes with the nuclear lamina, as cells formed protrusions of the
NE that were dependent on cytoskeletal pulling forces. Protrusions were
dependent on intact microtubules but not actin filaments. Our results
indicate that Src1 is required for integrity of the NE and highlight
Dictyostelium as a promising model for the evolution of nuclear
architecture.
submitted by: Ralph Gräf [[log in to unmask]]
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A High-Throughput, Multi-Cell Phenotype Assay for the Identification
of Novel Inhibitors of Chemotaxis/Migration
Xin-Hua Liao1,2, Netra Pal Meena2, Noel Southall3, Lunhua Liu4,
Manju Swaroop3, Arina Li Zhang1, Jan Jian Xiang1, Carole A. Parent4,
Wei Zheng3 & Alan R. Kimmel2
1Institute for Translational Medicine, School of Basic Medical Sciences,
Fujian Medical University, Fuzhou, Fujian 350108, China.
2Laboratory of Cellular and Developmental Biology, National Institute of
Diabetes and Digestive and Kidney Diseases, The National Institutes of
Health, Bethesda, MD 20892, USA.
3Therapeutics for Rare and Neglected Diseases, National Center for
Advancing Translational Sciences, The National Institutes of Health,
Bethesda, MD 20892, USA.
4Laboratory of Cellular and Molecular Biology, National Cancer Institute,
The National Institutes of Health, Bethesda, MD 20892, USA.
Scientific Reports, 2016
Chemotaxis and cell migration are fundamental, universal eukaryotic
processes essential for biological functions such as embryogenesis,
immunity, cell renewal, and wound healing, as well as for pathogenesis
of many diseases including cancer metastasis and chronic inflammation.
To identify novel chemotaxis inhibitors as probes for mechanistic studies
and leads for development of new therapeutics,we developed a unique,
unbiased phenotypic chemotaxis-dependent Dictyostelium aggregationassay
for high-throughput screening using rapid, laser-scanning cytometry.
Under defined conditions,individual Dictyostelium secrete chemoattractants,
migrate, and aggregate. Chemotaxis is quantifiedby laser-scanning
cytometry with a GFP marker expressed only in cells after chemotaxis/multi-
cellaggregation. We applied the assay to screen 1,280 known compounds
in a 1536-well plate format and identified two chemotaxis inhibitors. The
chemotaxis inhibitory activities of both compounds wereconfirmed in both
Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis
assay.The compounds were also shown to inhibit migration of two human
cancer cell lines in monolayerscratch assays. This test screen demonstrated
that the miniaturized assay is extremely suited for highthroughputscreening
of very large libraries of small molecules to identify novel classes of
chemotaxis/migratory inhibitors for drug development and research tools for
targeting chemotactic pathwaysuniversal to humans and other systems.
submitted by: Xin-Hua Liao [[log in to unmask]]
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The GATA transcription factor gene gtaG is required for terminal
differentiation in Dictyostelium
Mariko Katoh-Kurasawa*, Balaji Santhanam and Gad Shaulsky*
J. Cell. Sci, in press
The GATA transcription factor GtaG is conserved in Dictyostelids and
essential for terminal differentiation in Dictyostelium discoideum, but
its function is not well understood. Here we show that gtaG is expressed
in prestalk cells at the anterior region of fingers and in the extending
stalk during culmination. The gtaG– phenotype is cell-autonomous in
prestalk cells and non-cell-autonomous in prespore cells. Transcriptome
analyses reveal that GtaG regulates prestalk gene expression during cell
differentiation before culmination and is required for progression into
culmination. GtaG-dependent genes include genetic suppressors of the
Dd-STATa-defective phenotype as well as Dd-STATa target-genes, including
extra cellular matrix genes. We show that GtaG may be involved in the
production of two culmination-signaling molecules, cyclic di-GMP and the
spore differentiation factor SDF-1 and that addition of c-di-GMP rescues
the gtaG– culmination and spore formation deficiencies. We propose that
GtaG is a regulator of terminal differentiation that functions in concert
with Dd-STATa and controls culmination through regulating c-di-GMP
and SDF-1 production in prestalk cells.
submitted by: Mariko Katoh-Kurasawa [[log in to unmask]]
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[End dictyNews, volume 42, number 9]
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