dictyNews
Electronic Edition
Volume 39, number 3
January 25, 2013
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Abstracts
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Stress and development in Dictyostelium discoideum: the involvement
of the catalytic calcineurin A subunit.
Sascha Thewes, Sebastian K. Schubert, Kyuhyeon Park, and
Rupert Mutzel
Journal of Basic Microbiology, in press
Calcium signaling is one of the most important signaling-pathways in all
eukaryotes. One important target activated by an increased intracellular
calcium concentration via calmodulin is the protein phosphatase calcineurin,
which is composed of a catalytic subunit (calcineurin A) and a regulatory
subunit (calcineurin B). The importance of calcium and calcineurin for the
differentiation and development of the social amoeba Dictyostelium
discoideum has already been shown by pharmacological approaches.
However, so far only a RNAi-silenced calcineurin B mutant has been
investigated on a molecular level. Here we describe the construction and
phenotypic investigation of a RNAi-silenced calcineurin A mutant.
Phenotypic aberrations during development resemble those produced by
silencing of calcineurin B with ectopic tip formation of the fruiting
bodies. Additionally we tested the response of the mutants under various
stress conditions in liquid culture as well as during development. Both,
calcineurin A and B RNAi-mutants, are hypersensitive during development
towards cation stress. Besides its role in development calcineurin is thus also
involved in the stress response in D. discoideum. Further, our data imply that
many functions of calcineurin are conserved among the eukaryotes.
Submitted by Sascha Thewes [[log in to unmask]]
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Conserved gene-regulatory function of the carboxy-terminal domain
of dictyostelid C-module-binding factor
Anika Schmith, Marco Groth, Josephine Ratka, Sara Gatz, Thomas
Spaller, Oliver Siol, Gernot Glöckner and Thomas Winckler
Eukaryotic Cell, in press
C-module-binding factor (CbfA) is a jumonji-type transcription regulator
that is important for maintaining the expression and mobility of the
retrotransposable element TRE5-A in the social amoeba Dictyostelium
discoideum. CbfA-deficient cells have lost TRE5-A retrotransposition,
are impaired in the ability to feed on bacteria, and do not enter
multicellular development due to a block in cell aggregation. In this
study, we performed Illumina RNA sequencing on growing CbfA mutant
cells to obtain a list of CbfA-regulated genes. We demonstrate that the
carboxy-terminal domain of CbfA alone is sufficient to mediate the
majority of CbfA-dependent gene expression. The carboxy-terminal
domain of CbfA from the distantly related social amoeba Polysphondylium
pallidum restored the expression of CbfA-dependent genes in the
D. discoideum CbfA mutant, indicating a deep conservation in the gene
regulatory function of this domain in the dictyostelid clade. The CbfA-like
protein CbfB displays ~25% sequence identity with CbfA in the amino-
terminal region, which contains a JmjC domain and two zinc finger regions
and is thought to mediate chromatin-remodeling activity. In contrast to
CbfA proteins, where the carboxy-terminal domains are strictly conserved
in all dictyostelids, CbfB proteins have completely unrelated carboxy-
terminal domains. Outside the dictyostelid clade, CbfA-like proteins with
the CbfA-archetypical JmjC/zinc finger arrangement and individual carboxy-
terminal domains are prominent in filamentous fungi but are not found in
yeasts, plants, and metazoans. Our data suggest that two functional
regions of the CbfA-like proteins evolved at different rates to allow the
occurrence of species-specific adaptation processes during genome
evolution.
Submitted by Thomas Winckler [[log in to unmask]]
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Matricellular Signal Transduction Involving Calmodulin in the Social
Amoebozoan Dictyostelium
Danton H. O’Day1,2,* and Robert J. Huber3
1 Department of Biology, University of Toronto Mississauga, 3359
Mississauga Road North, Mississauga, ON, Canada L5L 1C6
2 Department of Cell and Systems Biology, University of Toronto,
25 Harbord Street, Toronto, ON, Canada M5S 3G5
3 Center for Human Genetic Research, Massachusetts General Hospital,
Harvard Medical School, Richard B. Simches Research Center, 185
Cambridge Street, Boston, MA, USA 02114
Genes, in press
Abstract: The social amoebozoan Dictyostelium discoideum undergoes a
developmental sequence wherein an extracellular matrix (ECM) sheath
surrounds a group of differentiating cells. This sheath is comprised of
proteins and carbohydrates, like the ECM of mammalian tissues. One of
the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL)
repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The
first EGFL repeat of CyrA increases the rate of random cell motility and
cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63kDa)
releases two major EGFL repeat-containing fragments (~45kDa and ~40kDa)
in an event that is developmentally regulated. Evidence for an EGFL repeat
receptor also exists and downstream intracellular signaling pathways
involving CaM, Ras, protein kinase A and vinculin B phosphorylation have
been characterized. In total, these results identify CyrA as a true matricellular
protein comparable in function to tenascin C and other matricellular proteins
from mammalian cells. Insight into the regulation and processing of CyrA has
also been revealed. CyrA is the first identified extracellular CaMBP in this
eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been
shown to be present in the ECM sheath where it binds to CyrA and inhibits its
cleavage to release the 45kDa and 40kDa EGFL repeat-containing fragments.
The presence of extCaM and its role in regulating a matricellular protein during
morphogenesis extends our understanding of CaM-mediated signal transduction
in eukaryotes.
Submitted by Danton H. O’Day ([log in to unmask])
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A SAP-domain containing protein shuttles between the nucleus and cell
membranes and plays a role in adhesion and migration in D. discoideum.
Jessica S. Kelsey and Daphne D. Blumberg
Department of Biological Sciences, University of Maryland, Baltimore County,
Baltimore, Maryland 21250
BiologyOpen, accepted
The AmpA protein reduces cell adhesion, thereby influencing cell migration in
Dictyostelium. To understand how ampA influences cell migration, second site
suppressors of an AmpA overexpressing cell line were created by REMI
mutagenesis. Mutant candidates were identified by their ability to suppress the
large plaques that the AmpA overexpressing cells form on bacterial lawns as a
result of their increased rate of migration. One suppressor gene, sma, encodes
an uncharacterized protein which contains a SAP DNA binding domain and a
PTEN like domain. Using sma gene knockouts and Sma-mRFP expressing cell
lines, a role for sma in influencing cell migration was uncovered. Knockouts of
the sma gene in a wildtype background enhanced chemotaxis. An additional role
for Sma in influencing cell-cell adhesion was also demonstrated. Sma protein
transitions between cytosolic and nuclear localizations as a function of cell
density. In growing cells migrating to folic acid it is localized to regions of actin
polymerization and absent from the nucleus. A role for Sma in influencing ampA
mRNA levels is also demonstrated. Sma additionally appears to be involved in
ampA pathways regulating cell size, actin polymerization, and cell substrate
adhesion. We present insights to the SAP domain-containing group of proteins
in Dictyostelium and provide evidence of a role for a SAP domain-containing
protein shuttling from the nucleus to sites of actin polymerization during
chemotaxis to folic acid and influencing the efficiency of migration.
Submitted by Daphne D. Blumberg [[log in to unmask]]
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[End dictyNews, volume 39, number 3]
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