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dictyNews Volume 18 Number 01
Dicty News
Electronic Edition
Volume 18, number 1
January 12, 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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A Novel Dictyostelium Gene Encoding Multiple Repeats of Adhesion Inhibitor-
Like Domains has Effects on Cell-Cell and Cell-Substrate Adhesion.
Timothy R. Varney, Elisabeth Casademunt, Hoa N. Ho, Chere' Petty, Jayne
Dolman, and Daphne D. Blumberg*
Department of Biological Sciences, University of Maryland Baltimore County,
1000 Hilltop Circle, Baltimore, Maryland 21250
Developmental Biology in press
Abstract
The Dictyostelium protein AmpA (Adhesion modulation protein A) is
encoded by the gene originally identified by the D11 cDNA clone. AmpA
contains repeated domains homologous to a variety of proteins that influence
cell adhesion. The protein accumulates during development, reaching a
maximal level at the finger stage. Much of the AmpA protein is found
extracellularly during development, and in culminants AmpA is found in
association with Anterior-Like Cells. Characterization of an ampA- strain
generated by gene replacement reveals a significant increase in cell-cell
clumping when cells are starved in non-nutrient buffer suspensions.
Developing ampA- cells are also more adhesive to the underlying substrate
and are delayed in developmental progression, with the severity of the delay
increasing as cells are grown in the presence of bacteria or on tissue
culture dishes rather than in suspension culture. Reintroduction of the
ampA gene rescues the developmental defects of ampA- cells however
expression of additional copies of the gene in wild type cells results
in more severe developmental delays and decreased clumping in suspension
culture. We propose that the AmpA protein functions as an anti-adhesive
to limit cell-cell and cell-substrate adhesion during development and thus
facilitate cell migration during morphogenesis.
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A Gene Encoding a Novel Anti-Adhesive Protein is Expressed in Growing Cells
and Restricted to Anterior-Like Cells During Development of Dictyostelium.
Elisabeth Casademunt, Timothy R. Varney, Jayne Dolman, Chere' Petty and
Daphne D. Blumberg*
Department of Biological Sciences, University of Maryland Baltimore County,
1000 Hilltop Circle, Baltimore, Maryland 21250
Differentiation, in press
Abstract
The Dictyostelium gene ampA, initially identified by the D11 cDNA,
encodes a novel anti-adhesive-like protein. The ampA gene product inhibits
premature cell agglutination during growth and modulates cell-cell and cell-
substrate adhesion during development. Analysis of the promoter indicates
that cap site-proximal sequence directs ampA expression during both growth
and early development. Expression following tip formation is controlled by
more distal sequence, which contains TTGA repeats known to regulate prestalk
cell gene expression in other promoters.
Comparison of reporter gene expression and endogenous mRNA accumulation
indicate that during growth the ampA gene is expressed in an increasing
number of cells as a function of density. The number of cells expressing
the ampA gene drops as development initiates, but the cells that continue
to express the gene do so at high levels. These cells are initially
scattered throughout the entire aggregate. By the tip formation stage
however, the majority of ampA-expressing cells are localized to the mound
periphery, with only a few cells remaining scattered in the upper portion
of the mound. In the final culminant, ampA is expressed only in the upper
cup, lower cup and basal disc. Although reporter expression is observed
in cells that migrate anteriorly to a banded region just posterior to the
tip, expression is rarely observed in the extreme tip. AmpA protein
however, is localized to the tip as well as to ALCs during late
development. The results presented here suggest that ampA gene expression
is shut off in ALCs that continue along the prestalk differentiation
pathway before they are added to the primordial stalk.
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Deducing the Origin of Soluble Adenylyl Cyclase, a Gene Lost
in Multiple Lineages
Jeroen Roelofs and Peter J.M. Van Haastert
Department of Biochemistry, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, the Netherlands
Molecular Biology and Evolution, in press
The family of eukaryotic adenylyl cyclases consists of a very
large group of twelve transmembrane adenylyl cyclases and a
very small group of soluble adenylyl cyclase (sAC). Orthologs of
human sAC are present in rat, Dictyostelium and bacteria, but
absent from the completely sequenced genomes of Drosophila
melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and
Saccharomyces cereviciae. sAC consists of two cyclase
domains and a long ~1000 amino acid C-terminal (sCKH)
region. This sCKH region and one cyclase domain have been
found in only four bacterial genes; the sCKH region was also
detected in bacterial Lux transcription factors and in complex
bacterial and fungal kinases. The phylogenies of the kinase and
cyclase domains are identical to the phylogeny of the
corresponding sCKH domain, suggesting that the sCKH region
fused with the other domains early during evolution in bacteria.
The amino acid sequences of sAC proteins yield divergence
times from the human lineage for rat and Dictyostelium that are
close to the reported divergence times of many other proteins in
these species. The combined results suggest that the sCKH
region was fused with one cyclase domain in bacteria, and a
second cyclase domain was added in bacteria or early
eukaryotes. The sAC was retained in a few bacteria and during
the entire evolution of the human lineage, but lost independently
from many bacteria and in the lineages to plants, yeast, worms
and flies. We conclude that within the family of adenylyl
cyclases, soluble AC was poorly fixed during evolution while
membrane bound AC has expanded to form the subgroups of
prevailing adenylyl and guanylyl cyclases.
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Characterization of Two Unusual Guanylyl cyclases from Dictyostelium
Jeroen Roelofs and Peter J.M. Van Haastert
GBB, Department of Biochemistry, University of Groningen
J. Biol. Chem., in press
GCA and sGC encode guanylyl cyclases (GC) in Dictyostelium, and have a
topology similar to twelve- transmembrane and soluble adenylyl cyclase,
respectively. We demonstrate that all detectable GC activity is lost in
a cell line in which both genes have been inactivated. Cell lines with one
gene inactivated were used to characterize the other guanylyl cyclase
(i.e. GCA in sgc- null cells, and sGC in gca- null cells). Despite the
different topologies, the enzymes have many properties in common. In vivo,
extracellular cAMP activates both enzymes via a G-protein coupled receptor.
In vitro, both enzymes are activated by GTPgS (Ka = 11 and 8 mM for GCA and
sGC, respectively); addition of GTPgS leads to a 1.5-fold increase of
Vmax and a 3.5-fold increase of the affinity for GTP. Ca2+ inhibits
both GCA and sGC with Ki of about 50 and 200 nM,
respectively. Other biochemical properties are very different;
GCA is mainly expressed during growth and multicellular
development, while sGC is mainly expressed during cell
aggregation. Folic acid and cAMP activate GCA maximally
about 2.5-fold, whereas sGC is activated about 8-fold. Osmotic
stress strongly stimulates sGC, but has no effect on GCA
activity. Finally, GCA is exclusively membrane bound and mainly
active with Mg2+, while sGC is predominantly soluble and more
active with Mn2+.
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A Transcriptional Profile of Multicellular Development in Dictyostelium
discoideum
Nancy Van Driessche 1,2,10, Chad Shaw 1,10, Mariko Katoh 6,10, Takahiro
Morio 6, Richard Sucgang 3, Miroslava Ibarra 1, Hidekazu Kuwayama 6, Tamao
Saito 7, Hideko Urushihara 6, Mineko Maeda 8, Ikuo Takeuchi 9, Hiroshi
Ochiai 7, William Eaton 5, Jeffrey Tollett 1,4, John Halter 5, Adam
Kuspa 1,2,3, Yoshimasa Tanaka 6, and Gad Shaulsky 1,2,11.
1 Department of Molecular and Human Genetics, 2 Graduate Program in
Developmental Biology, 3 Department of Biochemistry and Molecular Biology,
4 DNA Array Core Facility, 5 Department of PM&R and Division of Neuroscience,
Baylor College of Medicine, Houston TX 77030, USA; 6 Institute of Biological
Sciences, University of Tsukuba, Tsukuba, 7 Division of Biological Sciences,
Hokkaido University, Sapporo, 8 Department of Biology, Osaka University,
Osaka, 9 Novartis Foundation for the Promotion of Science, Takarazuka, Japan.
10 These authors contributed equally to this work
Development, in press.
SUMMARY
A distinct feature of development in the simple eukaryote Dictyostelium
discoideum is an aggregative transition from a unicellular to a multicellular
phase. Using genome-wide transcriptional analysis we show that this
transition is accompanied by a dramatic change in the expression of more
than 25% of the genes in the genome. We also show that the transcription
patterns of these genes are not sensitive to the strain or the nutritional
history, indicating that Dictyostelium development is a robust physiological
process that is accompanied by stereotypical transcriptional events.
Analysis of the two differentiated cell types, spores and stalk cells, and
their precursors revealed a large number of differentially expressed genes
as well as unexpected patterns of gene expression that shed new light on
the timing and possible mechanisms of cell-type divergence. Our findings
provide new perspectives on the complexity of the developmental program
and the fraction of the genome that is regulated during development.
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Cytoplasmic Dynein-Associated Structures Move Bidirectionally in vivo
Shuo Ma and Rex L. Chisholm
Department of Cell and Molecular Biology, Robert H. Lurie Comprehensive
Cancer Center, and Center for Genetic Medicine, Northwestern University
Medical School, Chicago, Illinois 60611
J. Cell Sci., in press.
Summary
Intracellular organelle transport is driven by motors that act upon
microtubules or microfilaments. Microtubule-based motors, cytoplasmic
dynein and kinesins, are believed to be responsible for retrograde and
anterograde transport of intracellular cargo along microtubules. Many
vesicles display bidirectional movement, however, the mechanism regulating
directionality is unresolved. Directional movement might be accomplished
by alternate binding of different motility factors to the cargo.
Alternatively, different motors could associate with the same cargo and
have their motor activity regulated. While several studies have focused
on the behavior of specific types of cargos, little is known about the
traffic of the motors themselves and how it correlates with cargo movement.
To address this question, we studied cytoplasmic dynein dynamics in living
Dictyostelium cells expressing dynein intermediate chain-green fluorescent
protein (IC-GFP) fusion in an IC-null background. Dynein-associated
structures display fast linear movement along microtubules in both minus-end
and plus-end directions, with the velocities similar to that of dynein and
kinesin-like motors. In addition, dynein puncta often rapidly reverse
direction. Dynein stably associates with cargo moving in both directions
as well as with those that rapidly reverse their direction of movement,
suggesting that directional movement is not regulated by altering motor-
cargo association, but rather by switching activity of motors associated
with the cargo. These observations suggest that both plus- and minus-end
directed motors associate with a given cargo and that coordinated
regulation of motor activities controls vesicle directionality.
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[End Dicty News, volume 18, number 1]
