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dictyNews Volume 19 Number 02
Dicty News
Electronic Edition
Volume 19, number 2
July 27, 2002
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
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Positions Available
=======================
A Postdoc position funded by the Deutsche Forschungsgemeinschaft
(Epigenitcs Program) is available in the Genetics Group at Kassel
University. The aims of the project are
1. to understand the mechanisms of RNA interference and
2. the mechanisms and function of DNA methylation.
In both cases, Dictyostelium provides an excellent model system which is
both "lean" (in terms of the small genome) and "general" (in that apparently
all or most components found in higher eukaryotes also exist in Dictyostelium).
Some more details are outlined on our web page
(http://www.uni-kassel.de/fb19/genetics/projects/projects.html).
Within the DFG Schwerpunkt Programm, we closely cooperate with other groups
working on epigenetics in plants, flys, and mammals. Applicants should have
obtained their Ph.D in a relevant field of molecular biology or biochemistry.
Applications containing CV, a brief description of research experience, research
interests and the names and e-mail address of two potential referees may be sent
by e-mail to Nellen@hrz.uni-kassel.de
A position for a Ph.D. student (funded by an OBF stipend) is available in the
Genetics Group at Kassel University in the new laboratory of Christian Hammann.
The project is aimed at understanding the role of different RNA directed RNA
polymerases (RdRP) in posttranscriptional gene silencing. Biochemical, biophysical
and molecular biology methods will be applied. The project will be carried out in
close cooperation with the Epigenetics Program in the Genetics group and with the
Center for Interdisciplinary Nanostructure Science and Technology (CINSaT).
Applicants should have a diploma or equivalent qualification in biology,
biochemistry or biophysics. Applications containing CV, a brief description of
research experience, research interests and the names and e-mail address of two
potential referees may be sent by e-mail to c.hammann@uni-kassel.de
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Abstracts
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A Rho GDP-dissociation inhibitor is involved in cytokinesis of
Dictyostelium
Keita Imai,a Toshirou Kijima,b,c Yoichi Noda,a Kazuo Sutoh,b Koji Yoda,a and
Hiroyuki Adachi a,*
a Department of Biotechnology, Graduate School of Agricultural and Life
Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,
Japan and b Department of Life Sciences, Graduate School of Arts and
Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 Japan
*Corresponding author. Fax: +81-3-5841-8139.
E-mail address: adachih@mail.ecc.u-tokyo.ac.jp (H. Adachi).
c On leave from Department of Applied Chemistry, Kogakuin University,
Shinjuku-ku, Tokyo 163-8677, Japan.
Biochem. Biophys. Res. Commun. in press
Abstract
Homology searches toward the EST databases of Dictyostelium discoideum
identified two putative Rho GDP-dissociation inhibitors (RhoGDIs), RhoGDI1
and RhoGDI2. In this study, the roles of RhoGDI1 in cytokinesis were
examined. The RhoGDI1-null Dictyostelium strains produced by homologous
recombination were viable but generated multinucleate giant cells in
suspension culture, suggesting that RhoGDI1 is involved in cytokinesis.
The expression of green fluorescent protein (GFP)-tagged RhoGDI1
complemented the defects of the RhoGDI1-null cells, and the GFP-RhoGDI1
is predominantly present in cytoplasm of the cell like yeast RhoGDI. Of
fifteen Rho family GTPases in Dictyostelium currently known, Dictyostelium
versions of Rac1 proteins (Rac1A, Rac1B and Rac1C) and RacE that are
reportedly involved in Dictyostelium cytokinesis, showed two-hybrid
interactions with RhoGDI1 as well as human and yeast Cdc42. These results
suggest that RhoGDI1 is involved in cytokinesis of Dicytostelium through
the regulation of Rho family GTPases Rac1s and/or RacE.
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Defects in cytokinesis, actin reorganization and the contractile vacuole in
cells deficient in RhoGDI
Francisco Rivero(1), Daria Illenberger(2), Baggavalli P. Somesh(1), Heidrun
Dislich(1), Nicola Adam(2) and Ann-Kathrin Meyer(1)
(1)Institut fr Biochemie I, Medizinische Fakultt, University of Cologne.
Joseph-Stelzmann-Strasse 52, D-50931 Kln, Germany
(2)Department of Pharmacology and Toxicology, University of Ulm, Albert-
Einstein-Allee 11, D-89081 Ulm, Germany
EMBO J., in press
Rho GDP-dissociation inhibitors (RhoGDIs) modulate the cycling of Rho GTPases
between active GTP-bound and inactive GDP-bound states. We identified two
RhoGDI homologues in Dictyostelium. GDI1 shares 51-58% similarity to RhoGDIs
from diverse species. GDI2 is more divergent (40-44% similarity) and lacks
the N-terminal regulatory arm characteristic for RhoGDI proteins. Both are
cytosolic proteins and do not relocalize upon reorganization of the actin
cytoskeleton. Using a two-hybrid approach we identified Rac1a/b/c, RacB,
RacC and RacE as interacting partners for GDI1. Cells lacking GDI1 are
multinucleate, grow slowly and display a moderate pinocytosis defect, but
rates of phagocytosis are unaffected. Mutant cells present prominent actin-
rich protrusions, and large vacuoles that are continuous with the
contractile vacuole system. The actin polymerization response upon
stimulation with cAMP was reduced but the motile behavior toward the
chemoattractant was unaffected. Our results indicate that GDI1 plays a
central role in the regulation of signal transduction cascades mediated
by Rho GTPases.
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Endosome fusion and microtubule-based dynamics in the early endocytic
pathway of Dictyostelium
Margaret Clarke1*, Jana Khler2, John Heuser3, and Gnther Gerisch2
1Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation,
Oklahoma City, Oklahoma; 2Max-Planck-Institut fr Biochemie, D82152
Martinsried, Germany; 3Washington University School of Medicine, St. Louis,
Missouri.
*Author for correspondence. E-mail: clarkem@omrf.ouhsc.edu
Traffic, in press.
Abstract
Dictyostelium amoebae, like mammalian macrophages, take up fluid by
macropinocytosis. The present study used fluorescent fluid phase markers
and GFP-labeled microtubules to visualize the uptake, dynamics, and fusion
of early endosomes in Dictyostelium. Consecutive labeling with two
fluorescent fluid phase markers demonstrated that within the first few
minutes after uptake, new macropinosomes underwent fusion with pre-existing
endosomes. The fusing endosomes, which represent the mixing compartment,
displayed extreme shape changes and rapid transport about the cell in
association with microtubules. The great plasticity of endosomes at this
stage of maturation was also evident by electron microscopy. The constant
undulatory motion of microtubules was implemental in establishing contact
with endosomes. Treatment of cells with agents that selectively disrupted
either actin filaments or microtubules confirmed that endosome dynamics
were microtubule-based. Further maturation of endosomes led to loss of
pleiomorphy in favor of a spherical shape, inability to fuse with new
macropinosomes, and diminished motility.
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Evidence for a role for the Dictyostelium monomeric GTPase, Rap1, in cell
viability and the response to osmotic stress
Rujun Kang, Helmut Kae, Hermia Ip, George B. Spiegelman and Gerald Weeks
Department of Microbiology and Immunology, University of British Columbia,
300 - 6174 University Blvd., Vancouver, BC V6T 1Z3, Canada
J. Cell Science, in press.
Summary
The Dictyostelium genome contains a single gene, rapA, encoding a
Rap1 monomeric G protein. Since attempts at generating rapA null Dictyostelium
cells had been unsuccessful, expression of antisense RNA from the rapA gene
under control of the folate repressible discoidin promoter was used to reduce
cellular levels of the Rap1 protein. As Rap1 levels gradually decreased
following antisense rapA RNA induction, growth rate and cell viability also
decreased, a result consistent with the idea that rapA is an essential gene.
The Rap1 depleted cells exhibited reduced viability in response to osmotic
shock. The accumulation of cGMP in response to 0.4M sorbitol was reduced
after rapA antisense RNA induction and was enhanced in cells expressing the
constitutively activated Rap1(G12V) protein, suggesting a possible role for
Rap1 in the generation of cGMP. Dictyostelium Rap1 was shown to complex to
the Ras binding domain of RalGDS only when it was in a GTP-bound state.
This assay was used to demonstrate that activation of Rap1 in response to
0.4M sorbitol occurred with initial kinetics similar to those observed for
the accumulation of cGMP. Furthermore, the addition of 2mM EDTA to
osmotically shocked cells, a treatment that enhances cGMP accumulation,
also enhanced Rap1 activation. These results suggest a direct role for
Rap1 in the activation of guanylyl cyclase during the response to
hyperosmotic conditions. Rap1 was also activated in response to low
temperature, but was not activated in response to low osmolarity or high
temperature.
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OSBPa, a predicted oxysterol binding protein of Dictyostelium, is required
for regulated entry into culmination
Masashi Fukuzawa and Jeffrey G. Williams*
School of Life Sciences, University of Dundee, Wellcome Trust Biocentre,
Dow Street, DUNDEE, DD1 5EH, UK
FEBS Letters, in press
Abstract
The oxysterol binding (OSBP) proteins are believed to control cholesterol
homeostasis but their precise mechanism of action is not well understood.
The Dictyostelium osbA gene encodes a predicted OSBP, OSBPa, which lacks the
PH domain that in most other OSBPs directs targetting to the Golgi. OSBPa
instead localises selectively to the cell periphery and also, in some cells,
to the peri-nuclear region. OSBPa null strains form normal fruiting bodies
but are defective in the regulation of the transition from slug migration
to culmination. Thus a plasma membrane-enriched OSBP family member is
essential for correct regulation of the slug-fruiting body switch.
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[End Dicty News, volume 19, number 2]
