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dictyNews Volume 20 Number 06
Dicty News
Electronic Edition
Volume 20, number 6
March *, 2003
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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Abstracts
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RacB regulates cytoskeletal function in Dictyostelium
Eunkyung Lee1+, David J. Seastone2, Ed Harris2 James A. Cardelli2 and
David A. Knecht1*
1Department of Molecular and Cell Biology, University of Connecticut,
Storrs, CT 06269
2Department of Microbiology and Immunology, Louisiana State University
Medical Center, 1501 Kings Highway, Shreveport, LA 71130
*to whom correspondence should be addressed
phone-860-486-2200
fax- 860-486-4331
Email knecht@uconn.edu
Eukaryotic Cell, in press
ABSTRACT
Thus far 14 homologues of mammalian Rac proteins have been identified in
Dictyostelium. It is unclear whether each of these genes has a unique
function or to what extent they play redundant roles in actin cytoskeletal
organization. In order to investigate the specific function of RacB, we
have conditionally expressed wild type (WT-RacB), dominant negative
(N17-RacB) and constitutively activated (V12-RacB) versions of the
protein. Upon induction, cells expressing V12-RacB stopped growing,
detached from the surface and formed numerous spherical surface
protrusions, while cells overexpressing WT-RacB became flattened on the
surface. In contrast, cells overexpressing N17-RacB did not show any
significant morphological abnormalities. The surface protrusions seen
in V12-RacB cells appear to be actin driven protrusions because they
were enriched in F-actin and were inhibitable by cytochalasin A treatment.
The protrusions in V12-RacB cells did not require myosin II activity, which
distinguishes them from blebs formed by wild type cells under stress.
Finally, we examined the functional consequences of expression of wild
type and mutant RacB. Phagocytosis, endocytosis and fluid phase efflux
rates were reduced in all cell-lines expressing RacB proteins but the
greatest decrease was observed for cells expressing V12-RacB. From these
results, we conclude that like other members of the Rho family, RacB
induces polymerization of actin, but the consequences of activation
appears to be different from other Dictyostelium Rac proteins so far
investigated, resulting in different morphological and functional changes
in cells.
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A STAT regulated, stress-induced signalling pathway in Dictyostelium
(Dictyostelium STAT and the stress response)
Tsuyoshi Araki*, Masatsune Tsujioka*, Tomoaki Abe, Masashi Fukuzawa, Marcel
Meima, Pauline Schaap, Takahiro Morio+, Hideko Urushihara+, Mariko Katoh+,
Mineko Maeda^, Yoshimasi Tanaka+, Ikuo Takeuchi@ and Jeffrey G. Williams
School of Life Sciences, University of Dundee, Wellcome Trust Biocentre
Dow Street, DUNDEE, DD1 5EH, UK
+ Institute of Biological Sciences, University of Tsukuba,Tsukuba, Ibaraki
305-8572, JAPAN
^Department of Biology, Osaka University, Machikaneyama 1-16, Toyonaka,
Osaka 560-0043 JAPAN
@Novartis Foundation (Japan) for the Promotion of Science, Roppongi,
Minato-ku, Tokyo 106-0032, JAPAN
*These authors contributed equally to this work
Author for correspondence (e-mail address j.g.williams@dundee.ac.uk)
J. Cell Science, in press.
SUMMARY
The Dictyostelium stalk cell inducer DIF directs tyrosine phosphorylation
and nuclear accumulation of the STAT (Signal Transducer and Activator of
Transcription) protein Dd-STATc. We show that hyper-osmotic stress, heat
shock and oxidative stress also activate Dd-STATc. Hyper-osmotic stress
is known to elevate intracellular cGMP and cAMP levels and the membrane
permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, while 8-bromo-
cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc
remains stress activatable in null mutants for components of the known
cGMP-mediated and cAMP-mediated stress-response pathways and in a double
mutant affecting both pathways. Also, Dd-STATc null cells are not
abnormally sensitive to hyper-osmotic stress. Micro-array analysis
identified two genes, gapA and rtoA, that are induced by hyper-osmotic
stress. Osmotic stress induction of gapA and rtoA is entirely dependent
upon Dd-STATc. Neither gene is inducible by DIF but both are rapidly
inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent
inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a
transcriptional activator in a stress response pathway and the
pharmacological evidence, at least, is consistent with cGMP acting as
second messenger.
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Synergistic control of cellular adhesion by TM9 proteins
Mohammed Benghezal, Sophie Cornillon, Leigh Gebbie, Laeticia Alibaud, Franz
Brckert, Franois Letourneur and Pierre Cosson
Molecular Biology of the Cell (in press)
ABSTRACT
The transmembrane 9 (TM9) family of proteins contains numerous members in
eukaryotes. Although their function remains essentially unknown in higher
eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently
reported to be essential for cellular adhesion and phagocytosis. Here the
function of Phg1a and of a new divergent member of the TM9 family called
Phg1b was further investigated in Dictyostelium discoideum. The phenotypes
of PHG1a, PHG1b and PHG1a/PHG1b double knockout cells revealed that Phg1a
and Phg1b proteins play a synergistic but not redundant role in cellular
adhesion, phagocytosis, growth and development. Complementation analysis
supports a synergistic regulatory function rather than a receptor role for
Phg1a and Phg1b proteins. Altogether these results suggest that Phg1
proteins act as regulators of cellular adhesion, possibly by controlling
the intracellular transport in the endocytic pathway and the composition
of the cell surface.
submitted by Benghezal@athelas.com
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Dictyostelium discoideum transformation by oscillating electric field
electroporation
Laeticia Alibaud, Pierre Cosson, and Mohammed Benghezal
Biotechniques (in press)
ABSTRACT
Dictyostelium discoideum has been used as a genetically tractable model
organism to study many biological phenomena. High efficiency transformation
is a prerequisite for successful genetic screens such as mutant
complementation, identification of suppressor genes or insertional
mutagenesis. Although exponential decay electroporation is the standard
transformation technique for Dictyostelium discoideum, its efficiency is
relatively low and its reproducibility weak. In this study we optimized
the oscillating electroporation technique for Dictyostelium discoideum
transformation and compared it to the exponential decay electroporation.
A twenty-fold increase of the efficiency was reproducibly achieved. This
alternative electroporation technique should facilitate future genetic
approaches in Dictyostelium discoideum.
submitted by Benghezal@athelas.com
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[End Dicty News, volume 20, number 6]
