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dictyNews Volume 28 Number 05
dictyNews
Electronic Edition
Volume 28, number 5
March 2, 2007
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 dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Diverse Cytopathologies in Mitochondrial Disease Are Caused by AMPK Signalling
Paul B. Bokko1, Lisa Francione1, Esther Bandala-Sanchez1, Afsar U. Ahmed1,
Sarah J. Annesley1, Xiuli Huang2, Taruna Khurana2, Alan R. Kimmel2 and
Paul R. Fisher1*
1Department of Microbiology, La Trobe University, Victoria 3086, Australia.
2National Institutes of Health, Bethesda, Maryland MD20892, USA.
Molecular Biology of the Cell, in press
The complex cytopathology of mitochondrial diseases is usually attributed to
insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive
cellular energy sensor that is stimulated by ATP-depleting stresses. By
antisense-inhibiting chaperonin 60 expression we produced mitochondrially
diseased strains with gene dose-dependent defects in phototaxis, growth and
multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene
dose-dependent manner by overexpressing a constitutively active AMPK alpha
subunit (AMPKalphaT). The aberrant phenotypes in mitochondrially diseased
strains were suppressed completely by antisense-inhibiting AMPKalpha expression.
Phagocytosis and macropinocytosis, although energy-consuming, were unaffected
by mitochondrial disease and AMPKalpha expression levels. Consistent with
AMPKÕs role in energy homeostasis, mitochondrial "mass" and ATP levels were
reduced by AMPKalpha antisense inhibition and increased by AMPKalphaT
overexpression, but near normal in mitochondrially diseased cells. We also
found that AICAR, a pharmacological AMPK activator in mammalian cells,
mimics mitochondrial disease in impairing Dictyostelium phototaxis and that
AMPKalpha antisense-inhibited cells were resistant to this effect. The
results show that diverse cytopathologies in Dictyostelium mitochondrial
disease are caused by chronic AMPK signalling not by insufficient ATP.
Submitted by Paul R. Fisher [P.Fisher@latrobe.edu.au]
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Role of PI3 kinases in Chemotaxis in Dictyostelium
Kosuke Takeda, Atsuo T. Sasaki, Hyunjung Ha, Hyun-A Seung, and
Richard A. Firtel
J. Biol. Chem., in press
Experiments in several cell types revealed that local accumulation of
phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] mediates the ability of
cells to migrate during gradient sensing. We took a systematic approach to
characterize the functions of the six putative Class I PI3Ks (PI3K1-6) in
Dictyostelium by creating a series of gene knockouts. These studies revealed
that PI3K1-PI3K3 are the major PI3Ks for chemoattractant-mediated PI(3,4,5)P3
production. We studied chemotaxis of the pi3k1/2/3 triple knockout strain
(pi3k1/2/3 null cells) to cAMP under two distinct experimental conditions, an
exponential gradient emitted from a micropipette and a shallow, linear
gradient in a Dunn chamber, using four cAMP concentrations ranging over a
factor of 10,000. Under all conditions tested, pi3k1/2/3 null cells moved
slower and had less polarity than wild-type cells. pi3k1/2/3 null cells
moved towards a chemoattractant emitted by a micropipette, although
persistence was lower than that of wild-type or pi3k1/2 null cells. In
shallow linear gradients, pi3k1/2 null cells had greater directionality
defects, especially at lower chemoattractant concentrations. Our studies
suggest that although PI3K is not essential for directional movement under
some chemoattractant conditions, it is a key component of the directional
sensing pathway and plays a critical role in linear chemoattractant gradients,
especially at low chemoattractant concentrations. The relative importance of
PI3K in chemotaxis is also dependant on the developmental stage of the cells.
Our data suggest that the output of other signaling pathways suffices to
mediate directional sensing when cells perceive a strong signal, but PI3K
signaling is crucial for detecting weaker signals.
Submitted by Rick Firtel [rafirtel@ucsd.edu]
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cAMP signaling in Dictyostelium: G-proteins activate separate Ras pathways
using specific RasGEFs
Helmut Kae, Arjan Kortholt, Holger Rehmann, Robert H. Insall,
Peter J.M. Van Haastert, George B. Spiegelman, and Gerald Weeks
EMBO Reports, In Press
In general, mammalian RasGEFs display little substrate specificity, even though
they are often thought to regulate specific pathways. Here we provide in
vitro and in vivo evidence that two RasGEFs can each act on specific Ras
proteins. During Dictyostelium development, RasC and RasG are activated in
response to cAMP, with each regulating different downstream functions; RasG
regulating chemotaxis, and RasC responsible for adenylyl cyclase activation.
RasC activation was abolished in a gefA- mutant, while RasG activation was
normal in this strain, indicating that RasGEFA activates RasC, but not RasG.
Conversely, RasC activation was normal in a gefR- mutant, whereas RasG
activation was greatly reduced, indicating that RasGEFR activates RasG. These
results were confirmed by the finding that RasGEFA and RasGEFR specifically
released GDP from RasC and RasG, respectively, in vitro. This RasGEF target
specificity provides a mechanism for one upstream signal to regulate two
downstream processes using independent pathways.
Submitted by: Helmut Kae [hkae@interchange.ubc.ca]
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Mitochondrial carrier family. Repertoire and peculiarities of the cellular
slime mould Dictyostelium discoideum
Michel Satre1, Sara Mattei2, Laurence Aubry1, Pascale Gaudet3, Ludovic
Pelosi1, Gerard Brandolin1, Gerard Klein1
1Laboratoire de Biochimie et Biophysique des Systemes Integres, iRTSV/BBSI,
UMR 5092, CNRS-CEA-UJF, CEA-Grenoble, Grenoble, France
2Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute,
UCSD, School of Medicine, 9500 Gilman Drive, CMM-W318, La Jolla, CA 92093-0668,
USA
3dictyBase, Center for Genetic Medicine, Northwestern University, Chicago,
IL, 60611, USA.
* To whom correspondence should be sent at the Laboratoire de Biochimie et
Biophysique des Systemes Integres, iRTSV/BBSI, CEA-Grenoble, 17 rue des
Martyrs, 38054 Grenoble cedex 9, France
tel : +33 438 784 661 fax : +33 438 784 499 e-mail : gklein@cea.fr
Biochimie, in press
Proteins of the mitochondrial carrier family (MCF) mediate the transport of a
large range ofcompounds, including metabolites and cofactors. They are
localized mainly in the innermitochondrial membrane, except for a few members
found in the membranes of peroxisomes. Similarity searches among Dictyostelium
discoideum protein sequences identified a total of 31MCF members. All these
are membrane proteins that possess three characteristic repeats of adomain
of approximately 100 residues. Among them, three proteins have
supplementarystructural domains consisting of Ca2+-binding motifs made up of
2 or 4 EF-hand unitslocalized on the N-terminal end, facing the mitochondrial
intermembrane space. The nature oftransported substrates is proposed on the
basis of sequence comparison with orthologscharacterized biochemically in
other organisms, of phylogenetic analysis, and of theconservation of
discriminating amino acid residues belonging to the substrate binding sites.
Carriers have been grouped in subclasses based on their specificity for the
transport ofnucleotides, amino acids or keto acids. Furthermore, we have
identified an iron carrier of themitoferrin type, an inorganic phosphate
carrier, and three carriers with similarity to uncouplerproteins. This study
provides a focus for mitochondrial carrier analysis in Dictyostelium discoideum.
Submitted by: Gerard Klein [ gerard.klein@cea.fr]
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[End dictyNews, volume 28, number 5]
