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dictyNews Volume 23 Number 06
Dicty News
Electronic Edition
Volume 23, number 6
August 13, 2004
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
Back issues of Dicty-News, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Identification of new modes of Dd-STATa regulation of gene expression in
Dictyostelium by in situ hybridisation
NAO SHIMDA1, MINEKO MAEDA2, HIDEKO URUSHIHARA3 and TAKEFUMI KAWATA1, 4
1Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama,
Funabashi, Chiba 274-8510, Japan
TEL & FAX: +81-47-472-5156, E-mail: tkawata@bio.sci.toho-u.ac.jp
2 Department of Biology, Graduate School of Science, Osaka University
, Toyonaka, Osaka 560-0043, Japan
3 Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572,
Japan
4 Corresponding author
Int. J. Dev. Biol., in press.
STATs (signal transducers and activators of transcription) are
transcription factors, which lie at the end of cytokine and growth signal
transduction pathways. Dictyostelium Dd-STATa is a functional homologue of
metazoan STATs. It is activated by cAMP and, at the slug stage, it
translocates into the nuclei of the tip cells : a sub-set of the anterior,
prestalk A (pstA) cells. Here we searched for novel Dd-STATa regulated
genes by in situ hybridisation. A set of 54 cDNA clones, whose gene
expressions patterns are known to be prestalk-specific (Maeda et al.,
2003), were chosen as probes and we compared their expression patterns in
parental and Dd-STATa-null strains. We identified 13 genes that are
candidates for direct induction by Dd-STATa. In the parental strain, most
of these genes are expressed in the cone shaped mass of pstAB cells that
is located within the prestalk region region. These cDNAs show little or
no expression in the Dd-STATa-null strain. This contrasts markedly with
the paradigmatic, ecmB gene which is expressed in pstAB cells in parental
cells but which is expressed throughout the prestalk zone in the Dd-STATa
null strain. We also identified several genes that are normally expressed
in the pstA cells, or throughout the prestalk region, but whose expression
is markedly down-regulated in the null mutant. Again, this contrasts with
markers derived from the paradigmatic, ecmA gene which are expressed
normally in the Dd-STATa-null strain. The identification of these novel
genes provides valuable tools to investigate the role of Dd-STATa.
Submitted by: Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp]
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Developmental control of cAMP-induced Ca2+-influx by cGMP: Influx is delayed
and reduced in a cGMP-phosphodiesterase D deficient mutant of Dictyostelium
discoideum
Daniel F. Lusche and D. Malchow
Cell Calcium, in press
It was previously shown that cGMP enhances cAMP-induced Ca2+-influx in
Dictyostelium discoideum. This finding is based on experiments done with
strains defective in cGMP-hydrolysis, the streamer F cells. In this work,
we show that these chemically mutagenized cells display different
properties in their cAMP-induced light-scattering response and cAMP-induced
Ca2+-influx compared with a cGMPphosphodiesterase knock-out strain,
pdeD KO, generated by homologous recombination. PdeD KO cells possess a
reduced Ca2+-influx that is developmentally regulated. This finding
contradicts the result of streamer F cells, where cAMP-induced Ca2+-influx
is prolonged and elevated. Both mutants, however, showed a three to
four-fold delayed response to cAMP at 3ö4 h of starvation. Thus, the
consequence of an elevated cGMP concentration is a delay and an inhibition
of Ca2+-influx and not an enhancement. Results obtained with streamer F
cells should therefore be interpreted with caution because the mutation(s)
responsible for the divergent phenotype to pdeD KO cells has not been
identified. We show by the use of membrane-permeant cGMP-analogues in wild
type (wt) cells, permeabilized cells and measurements on isolated vesicles
that the cause for the reduced Ca2+-influx seems to be due to
developmentally regulated Ca2+-channel inhibition by cGMP.
Submitted by: Daniel Lusche [Daniel.Lusche@uni-konstanz.de]
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Multichannel apparatus for parallel monitoring of light scattering in
Dictyostelium discoideum cell suspensions
Daniel F. Lusche*, Hanni Rtzer*, Rudolf Merz, Hubert Fink, Rupert Mutzel+,
Christina Schlatterer
Faculty for Biology, University of Konstanz, 78457 Konstanz, FRG
* these authors contributed equally to the work
+ Institute of Biology-Microbiology, Free University of Berlin,
Knigin-Luise-Strasse 12-16, 14195 Berlin, FRG
BioTechniques, in press
Suspensions of Dictyostelium discoideum amoebae display free-running light
scattering oscillations at the onset of development. We describe a device
to monitor these oscillations in several samples in parallel. The apparatus
consists of a thermostatted cuvette holder where up to eight cuvettes
containing cell suspension are inserted. Cells are aerated and kept in
suspension via an airlift. Infrared light emitted from a five-diode array
passes through the suspension and is detected by an array of five light
detecting diodes. The resulting signal is digitized and recorded with a
sampling rate of two measuring points/sec. The parallel analysis approach
allows determination of the effects of adding of agents or of variations
in the external conditions in the same batch of amoebae at the same
developmental time point. This represents an advantage over the conventional
single cuvette approach as oscillation characteristics themselves are
developmentally regulated. Moreover, as the new experimental setup enables
simultaneous analyses of up to eight samples the behaviour of wild type and
several mutant strains can be compared under identical experimental
conditions.
Submitted by: Christina Schlatterer [Christina.Schlatterer@uni-konstanz.de]
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Translocation of the Dictyostelium TRAP1 homologue to mitochondria induces a
novel prestarvation response
Tsuyoshi Morita*, Aiko Amagai and Yasuo Maeda
Department of Developmental Biology and Neurosciences, Graduate School of
Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan
* Present address: Department of Neuroscience (D13), Osaka University
Graduate School of Medicine, Yamadaoka 2-2, Suita City, Osaka 565-0871,
Japan
J. Cell Science, in press
Dd-TRAP1 is a Dictyostelium homologue of tumor necrosis factor receptor
associated protein 1 (TRAP-1). Dd-TRAP1 is located in the cortex of cells
growing at a low density, but was found to be translocated to mitochondria
with the help of a novel prestarvation factor that was accumulated in growth
medium along with increased cell densities. The knockdown mutant of Dd-TRAP1
(TRAP1-RNAi cells) exhibited a significant defect in prestarvation response.
Although TRAP1-RNAi cells showed normal expressions of classical
prestarvation genes (discoidin I and car1), the expressions of
differentiation-associated genes (dia1 and dia3) induced by prestarvation
response were markedly repressed. On the other hand, transformants
overexpressing Dd-TRAP1 showed a precocious prestarvation response and also
augmented expressions of dia1 and dia3 in a cell density-dependent manner.
Importantly, introduction of Dd-TRAP1 antibody into D. discoideum Ax-2 cells
by electroporation inhibited the translocation of Dd-TRAP1 from the cortex
to mitochondria and greatly inhibited the initiation of differentiation.
Taken together, these results indicate that Dd-TRAP1 is translocated to
mitochondria by sensing the cell density in growth medium and enhances the
early developmental program through a novel prestarvation response.
Submitted by: Y. Maeda [ymaeda@mail.tains.tohoku.ac.jp]
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Cell shape regulation and co-translocation of actin and adenosyl
homocysteinase in response to intermediate hypertonicity
Yohko Yamada1.*Û and Masazumi Sameshima1,2
1 The Tokyo Metropolitan Institute of Medical Science, Electron Microscopy
Center
2 Department of Biofunctional Science, Faculty of Agriculture and Life
Science, Hirosaki University,
FEMS Microbiol Lett, in press
Hypertonic stimulation induced association of S-adenosyl-L-homocysteine
hydrolase (SAHH) with the F-actin-rich cell cortex in Dictyostelium. At
intermediate, but not higher, levels of hypertonicity, SAHH further
translocated from the cortex to the cytosol in company with a fraction of
actin and cofilin. At the same time the cells rounded up. Acidification of
the cells stimulated both the cell rounding and the translocation of actin
and SAHH, whereas alkalinization retarded these responses, suggesting that
cellular pH is involved in their control. On the other hand, mutant analysis
suggested that neither cGMP signaling nor conventional myosin is required.
Submitted by: Yoko Yamada [yyamada@chaos.bio.sci.osaka-u.ac.jp]
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[End Dicty News, volume 23, number 6]
