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dictyNews Volume 19 Number 04
Dicty News
Electronic Edition
Volume 19, number 4
August 10, 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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Abstracts
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MECHANISTICS OF AMOEBOID LOCOMOTION: SIGNAL TO FORCES
Yoshio Fukui
Cell Biology International, accepted for publication, 08/08/2002
Dictyostelium serves as an ideal model system for studying the molecular
and structural properties of the actomyosin and microtubule systems. This
organism also has been the vehicle on which the gene-targeting technique
was pioneered. Dictyostelium also represents a small number of organisms
whose chemotactic ligand-receptor mechanism has been well characterized.
This article reviews recent advances in studies of the actin-based
cytoskeletal system in Dictyostelium, focusing on the mechanistic aspects
of the amoeboid motion. Special emphasis is placed on the recently
identified cell-substrate-anchoring structures eupodia , and the
measurement of single-cell migration forces. The recent advances in signal
transduction cascade is also discussed with relevance to the mechanistics
in amoeboid locomotion.
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Identification of a novel type of cGMP phosphodiesterase that is defective
in the chemotactic stmF mutants
Marcel E. Meima, Ricardo M. Biondi and Pauline Schaap.
School of Life Sciences, University of Dundee, MSI/WTB complex,
Dow Street, Dundee DD1 5EH, UK.
Mol. Biol. Cell, in press.
ABSTRACT
StmF mutants are chemotactic mutants that are defective in a cGMP
phosphodiesterase (PDE) activity. We identified a novel gene, PdeD, which
harbours two cyclic nucleotide binding domains and a metallo-$-lactamase
homology domain. Similar to stmF mutants, pdeD null mutants displayed
extensively streaming aggregates, prolonged elevation of cGMP levels after
chemotactic stimulation and reduced cGMP-PDE activity. PdeD transcripts
were lacking in stmF mutant NP377, indicating that this mutant carries a
PdeD lesion. Expression of a PdeD-YFP fusion protein in pdeD null cells
restored the normal cGMP response and showed that PdeD resides in the
cytosol. When purified by immunoprecipitation, the PdeD-YFP fusion protein
displayed cGMP-PDE activity, which was retained in a truncated construct
that contained only the metallo-$-lactamase domain.
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A secreted cell-number counting factor represses intracellular glucose
levels to regulate group size in Dictyostelium
Wonhee Jang, Binh Chiem, and Richard H. Gomer
Howard Hughes Medical Institute and Department of Biochemistry and Cell
Biology, Rice University, Houston, TX 77005-1892
Journal of Biological Chemistry, in press
Summary
Developing Dictyostelium cells form evenly sized groups of ~2 x 104 cells.
A secreted 450 kDa protein complex called counting factor (CF) regulates
group size by repressing cell-cell adhesion and myosin polymerization, and
increasing cAMP-stimulated cAMP production, actin polymerization, and cell
motility. We find that CF regulates group size in part by repressing
internal glucose levels. Transformants lacking bioactive CF and wild-type
cells with extracellular CF depleted by antibodies have high glucose levels,
while transformants oversecreting CF have low glucose levels. A component
of CF, countin, affects group size in a manner similar to CF, and a 1-minute
exposure of cells to countin decreases glucose levels. Adding 1 mM
exogenous glucose negates the effect of high levels of extracellular CF on
group size and mimics the effect of depleting CF on glucose levels, cell-
cell adhesion, cAMP pulse size, actin polymerization, myosin assembly, and
motility. These results suggest that glucose is a downstream component in
part of the CF signaling pathway, and may be relevant to the observed role
of the insulin pathway in tissue size regulation in higher eukaryotes.
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Identification and Characterization of Two Unusual cGMP-stimulated
Phoshodiesterases in Dictyostelium
Leonard Bosgraaf, Henk Russcher, Helena Snippe, Sonya Bader, Joyce Wind, and
Peter J.M. Van Haastert
Department of Biochemistry, University of Groningen,Nijenborgh 4, 9747 AG
Groningen, the Netherlands
Molecular Biology of the Cell, in press
Recently we recognized two genes, gbpA and gbpB, encoding putative cGMP-
binding proteins with a Zn2+-hydrolase domain and two cyclic nucleotide
binding (cNB) domains. The Zn2+-hydrolase domains belong to the superfamily
of beta-lactamases, also harboring a small family of class II
phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation
and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated
cGMP-phosphodiesterase that was characterized biochemically previously, and
shown to be involved in chemotaxis. cAMP neither activates nor is a
substrate of GbpA. The gbpB gene is expressed mainly in the multicellular
stage and appears to encode a dual specificity phosphodiesterase with
preference for cAMP. The enzyme hydrolyses cAMP about 9-fold faster than
cGMP, and is activated by cAMP and cGMP with a Ka of about 0.7 and 2.3 m M,
respectively. Cells with a deletion of the gbpB gene have increased basal
and receptor stimulated cAMP levels, and are sporogeneous. We propose that
GbpA and GbpB hydrolyze the substrate in the Zn2+-hydrolase domain, while
the cNB domains mediate activation. The human cGMP-stimulated cAMP/cGMP
phosphodiesterase has similar biochemical properties, but a completely
different topology: hydrolysis takes place by a class I catalytic domain
and GAF domains mediate cGMP-activation.
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TEMPERATURE-SENSITIVE INHIBITION OF DEVELOPMENT IN DICTYOSTELIUM DUE TO A
POINT MUTATION IN THE piaA GENE
Barbara Pergolizzi, Barbara Peracino, James Silverman#, Adriano Ceccarelli,
Angelika Noegel*, Peter Devreotes# and Salvatore Bozzaro
ABSTRACT
The Dictyostelium mutant HSB1 is temperature-sensitive for development,
undergoing aggregation and fruiting body formation at temperatures below 18C
but not above. In vivo G protein-linked adenylylcyclase activation is
defective in HSB1 and the enzyme is not stimulated in vitro by GTPgS;
stimulation is restored upon addition of wild-type cytosol. Transfection
with the gene encoding the cytosolic regulator PIA rescued the mutant. We
excluded the possibility that HSB1 cells fail to express PIA and show that
the HSB1 piaA gene harbours a point mutation, resulting in the aminoacid
exchange G917D. Both wild-type and HSB1 cells were also transfected with
the HSB1 piaA gene. The piaAHSB1 gene product displayed a partial inhibitory
effect on wild-type cell development. We hypothesize that PIA couples the
heterotrimeric G protein to adenylyl cyclase via two binding sites, one of
which is altered in a temperature-sensitive way by the HSB1 mutation. When
overexpressed in the wild-type background, PIAHSB1 competes with wild-type
PIA via the non-mutated binding site, resulting in dominant-negative
inhibition of development. Expression of GFP-fused PIA shows that PIA is
homogeneously distributed in the cytoplasm of chemoctically moving cells.
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[End Dicty News, volume 19, number 4]
