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dictyNews Volume 22 Number 04
Dicty News
Electronic Edition
Volume 22, number 4
February 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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Chimeric analysis of the small GTPase RacE in cytokinesis signaling in
Dictyostelium discoideum
Madhavi Agarwal, Nicholas A Guerin, Denis A Larochelle
Department of Biology, Clark University, Worcester, MA 01610, USA.
Experimental Cell Research, in press
RacE is a small GTPase required for cytokinesis in Dictyostelium discoideum.
To investigate RacEâs potential binding and signaling interfaces that allow
its function in cytokinesis, 10 different chimeras were created between RacE
and the closely related small GTPase, RacC. RacE/RacC chimeras, containing
various combinations of four RacE regions, EI-IV: E-I (aa 1-67), E-II
(aa 68-124), E-III (aa 125-184), and E-IV (aa 185-224), were tested in their
ability to rescue the multinucleated, cytokinesis-defective phenotype of
RacE null cells grown in suspension. Regions E-II and E-IV were essential but
not sufficient for the rescue of RacE null cells. These two regions, in
combination with either region E-I or E-III, resulted in rescue. Results
presented here suggest that region E-II contains a crucial, yet incomplete,
binding site. Regions E-I or E-III separately provide additional, necessary
elements for RacEâs function. The extended E tail of RacE (E-IV) may act as
a Îsensorâ of the bound nucleotide state of RacE and facilitate GDP to GTP
exchange (possibly through interactions with a GEF molecule), thereby
resulting in activation of RacE. This study provides new evidence for small
GTPases engaging several distinct protein interfaces to mediate signaling
in various cellular processes.
Submitted by: Madhavi Agarwal [magarwal@clarku.edu]
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Global change in Escherichia coli gene expression in initial stage of
symbiosis with Dictyostelium cells
Shin-Ichi Matsuyama1*, Chikara Furusawa2*, Masahiko Todoriki3, Itaru Urabe3
and Tetsuya Yomo1,,3,4,5
* Both authors equally contributed to this work.
1 Department of Bioinformatic Engineering, Graduate School of Information
Science and Technology, Osaka University, 2-1 Yamada-oka, Suita City, Osaka
565-0871 Japan
2 Center for Developmental Biology, The Institute of Physical and Chemical
Research (RIKEN), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, JAPAN
3 Department of Biotechnology, Graduate School of Engineering, Osaka
University, 2-1 Yamada-oka, Suita City, Osaka 565-0871 Japan
4 Intelligent Cooperation and Control Project, PRESTO, JST, 2-1 Yamada-oka,
Suita City, Osaka 565-0871 Japan
5 Department of Pure and Applied Sciences, University of Tokyo, Komaba,
Meguro-ku, Tokyo, 153-8902 Japan
Biosystems, in press
Genome-wide gene expression profiling was performed to investigate the early
formation of symbiosis using an artificial symbiosis of Escherichia coli and
Dictyostelium discoideum. We have previously reported that when these two
species were allowed to grow on minimal agar plates, they achieved a stable
state of coexistence, in which the emerging E. coli colonies housing
Dictyostelium cells were of a mucoidal nature that was not observed
originally. We used this microbiological system as a model to study the
initial stages of the development of the symbiotic relationship. The E. coli
gene expression profiles of symbiotic cells and non-symbiotic cells captured
using GeneChip technology were compared. It was clearly shown that the gene
expression profile was substantially altered in E. coli cells undergoing
symbiotic transition. The genes responsible for central energy metabolism as
well as those responsible for translation apparatus were down-regulated in
symbiotic E. coli. The transcriptional patterns of genes coding for the
E. coli cell surface structure were drastically altered, and this alteration
may determine the mucoidal nature and unique structure of coexisting colonies.
General stress inducible genes were expressed at low levels in symbiotic
E. coli. These observed changes in the transcription profile indicate that
the central metabolism of symbiotic E. coli is attenuated as a whole, and
the cells are probably under less stress because of the benefits brought by
coexistence with the symbiotic counterpart Dictyostelium.
Submitted by: Shin-Ichi Matsuyama [sxm67@bio.eng.osaka-u.ac.jp]
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Structure-Function Analysis of the Beach Protein LvsA
Wei-I Wu, Juhi Yajnik, Michael Siano, and Arturo De Lozanne
Section of Molecular Cell & Developmental Biology and Institute for Cellular
and Molecular Biology, University of Texas at Austin, Austin, TX 78712.
Traffic, in press
ABSTRACT
Most eukaryotes have several members of the BEACH family of proteins but the
molecular function of these large proteins remains unknown. The
Dictyostelium BEACH protein LvsA is essential for cytokinesis and contractile
vacuole activity. The functional contribution of different portions of LvsA
was tested here by deletion analysis. The C-terminal WD domain was important
for protein stability and C-terminal deletions resulted in loss of LvsA
function. In contrast, N-terminal deletions yielded abundant protein
expression that could be assayed for function. Despite very low sequence
conservation of the N-terminal portion of LvsA, this region is important
for its function in vivo. Deletion of 689 N-terminal amino acids produced
a protein that was functional in cytokinesis but partially functional in
osmoregulation. Further deletions resulted in the complete loss of LvsA
function. Using in vitro fractionation assays we found that LvsA
sedimented with membranes but this association does not require the
N-terminal portion of LvsA. Interestingly, the association of LvsA with
the contractile vacuole was perturbed by the loss of drainin, a protein
important for vacuole function. In drainin-null cells LvsA bound
irreversibly to engorged contractile vacuoles that fail to expel water.
These experiments help delineate the biochemical and physiological
requirements for function of one important BEACH protein, LvsA.
Submitted by: Arturo De Lozanne [a.delozanne@mail.utexas.edu]
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RasC Plays a Role in the Transduction of Temporal Gradient Information in the
cAMP Wave of Dictyostelium
Deborah Wessels1, Rebecca Brincks1, Spencer Kuhl1, Vesna Stepanovic1,
Karla J. Daniels1, Gerald Weeks2, Chinten J. Lim2, George Spiegelman2,
Danny Fuller3, Negin Iranfar3, William F. Loomis3 and David R. Soll1
W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences,
The University of Iowa, Iowa City, IA 522421; Department of Microbiology and
Immunology,
The University of British Columbia, Vancouver, British Columbia2; Department of
Biology,
University of California, San Diego, La Jolla, California 920393
Eukaryotic Cell, in press
To define the role RasC plays in motility and chemotaxis, the behavior of a
rasC null mutant, rasC -, was analyzed in buffer and in response to the
individual spatial, temporal and concentration components of a natural cAMP
wave, using computer-assisted 2D and 3D motion analysis systems. These
quantitative studies reveal that rasC - cells translocate at the same
velocity and chemotax up spatial gradients of cAMP with the same efficiency
as control cells. However, rasC - cells exhibit defects in maintaining
anterior-posterior polarity along the substratum and a single anterior
pseudopod when translocating in buffer in the absence of attractant.
rasC - cells also exhibit defects in their responses to both the
increasing and decreasing temporal gradients of cAMP in the front and back
of a wave. These defects result in the inability of rasC - cells to
chemotax in a natural wave of cAMP. The inability to respond normally to
temporal gradients of cAMP results in defects in the organization of the
cytoskeleton, most notably in higher levels of coritcal F-actin in both
buffer and the front of the wave, and the failure of myosin II to exit the
cortex in response to the decreasing temporal gradient of cAMP in the back
of the wave. The behavioral defect in the front of the wave is similar to
that of the myosin I double mutant myoA -/myoF -, while the behavioral and
cytoskeletal defects in the back of the wave are similar to those of the
myosin II regulatory light chain phosphorylation mutant S13A. Expression
array data support the premise that the behavioral defects exhibited by
the rasC - mutant are the immediate result of the absence of RasC function.
Submitted by: Deborah Wessels [deborah-wessels@uiowa.edu]
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Identification of genes dependent on the MADS-boxtranscription factor SrfA in
Dictyostelium development
R. Escalante, N. Iranfar, L. Sastre, and W.F. Loomis
Eukaryotic Cell, in press
Analysis of microarrays containing 6345 Dictyostelium discoideum genes has
identified twenty one whose expression is dependent on the MADS-box
transcription factor SrfA. In wild type cells, all these genes are induced
late in development. At least four of them are necessary for proper spore
differentiation, stability and/or germination.
Submitted by: Leandro Sastre [lsastre@iib.uam.es]
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[End Dicty News, volume 22, number 4]
