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dictyNews Volume 18 Number 12
Dicty News
Electronic Edition
Volume 18, number 12
June 22, 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.
======================
Position Available
======================
Scientific Curator, DictyBase
A position is available for a scientific curator to work with DictyBase.
This is a great opportunity for someone experienced with Dictyostelium
as an experimental system, who is also interested in bioinformatics.
The individual occupying this position will become part of the DictyBase
team which includes other scientific curators and bioinformatics support.
Responsibilities will include curating entries in DictyBase,
working closely with the sequencing centers involved in sequencing the
Dictyostelium genome as well as the Dictyostelium research community,
to integrate the scientific knowledge derived from research using
Dictyostelium with genome sequence and the scientific literature.
The DictyBase scientific curators will also interact with scientific
curators from other model organism databases such as FlyBase, WormBase,
SGD (Sacchromyces Genome Database) and MGI (Mouse Genome Informatics).
Desired experience includes research using Dictyostelium, familiarity
with genome informatics, and the use of computers in research. This
position will be located in the Northwestern University Center for
Genetic Medicine in Chicago. Appointment will be on the research
scientist track at Northwestern University and includes a generous
salary and full benefits.
Interested candidates should submit a current CV and a brief description
of research experience and interest in bioinformatics (preferably by email)
to:
Rex L. Chisholm (r-chisholm@northwestern.edu)
Center for Genetic Medicine
Northwestern University
303 E. Chicago Ave.
Chicago, IL 60611
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Abstracts
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Regulated expression of the MADS-box transcription factor SrfA mediates
activation of gene expression by PKA during Dictyostelium sporulation
Ricardo Escalante* and Leandro Sastre
Instituto de Investigaciones Biomdicas C.S.I.C/U.A.M. C/Arturo Duperier,
4, 28029 Madrid, Spain.
*Author for correspondence: Fax: +34-91-585-4587.
E-mail: rescalante@iib.uam.es
Mechanisms of Development (in press)
Abstract
Cell differentiation and morphogenesis are tightly regulated during
sporulation in the lower eukaryote Dictyostelium discoideum. The control of
the cAMP-dependent protein kinase (PKA) is essential to coordinate these
processes. Several signal transduction pathways are being recognized that
lead to the regulation of intracellular cAMP levels. However, very little is
known about the events lying downstream of PKA that are essential to
activate late gene expression and terminal differentiation of the spores.
We have studied the relationship between PKA and the MADS-box transcription
factor SrfA, essential for spore differentiation. Constitutive activation of
PKA was not able to rescue sporulation in a strain that lacks srfA suggesting
the possibility that srfA functions downstream of PKA in a signal
transduction pathway leading to spore maturation. A distal promoter region
regulates the induction of srfA expression in the prespore region during
culmination. We found that this promoter can be induced precociously by
activating PKA with 8-Br-cAMP suggesting a transcriptional regulation by
PKA. Moreover, precocious sporulation and expression of the spore marker
spiA in a strain that over-expresses PKA correlates with a precocious
induction of srfA expression. The temporal and spatial pattern of expression
was also studied in a mutant strain lacking the main adenylyl cyclase that
functions during culmination, ACR. This strain is expected to have lower
PKA activity and consistently, the level of srfA expression was reduced.
Moreover, the temporal induction of srfA in the prespore region was also
delayed during culmination. Our results strongly suggest that PKA activation
during culmination leads to the induction of the expression of srfA. The
correct temporal and spatial pattern of srfA expression appears to be part
of a mechanism that ensures the adequate coordination of gene expression
and morphogenesis.
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Cells respond to and bind countin, a component of a multisubunit cell-number
counting factor
Tong Gao1, Karen Ehrenman1, Lei Tang2, Matthias Leippe3, Debra A. Brock1,
and Richard H. Gomer1, 2
1Howard Hughes Medical Institute and 2Department of Biochemistry and Cell
Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892
and 3Molecular Parasitology Group, Research Center for Infectious Diseases,
Rntgenring 11, 97070 Wuerzburg, Germany
Journal of Biological Chemistry, in press
Summary
In Dictyostelium discoideum counting factor (CF), a secreted ~450kDa
complex of polypeptides, inhibits group and fruiting body size. When the
gene encoding countin (a component of CF) was disrupted, cells formed large
groups. We find that recombinant countin causes developing cells to form
small groups with an EC50 of ~3 ng/ ml, and affects cAMP signal transduction
in the same manner as semipurified CF. Recombinant countin increases cell
motility, decreases cell-cell adhesion, and regulates gene expression in a
manner similar to the effect of CF. However, countin does not decrease
adhesion or group size to the extent that semipurified CF does. A 1 minute
exposure of developing cells to countin causes an increase in F-actin
polymerization and myosin phosphorylation, and a decrease in myosin
polymerization, suggesting that countin activates a rapid signal
transduction pathway. 125I-labelled countin has countin bioactivity, and
binding experiments suggest that vegetative and developing cells have ~53
cell-surface sites which bind countin with a KD of approximately 1.5 ng/ ml
or 60 pM. We hypothesize that countin regulates cell development through
the same pathway as CF and that other proteins within the complex may modify
the activity of countin and/or have independent size-regulating activities.
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A single cell-density sensing factor stimulates distinct signal transduction
pathways through two different receptors
William J. Deery, Tong Gao, Robin Ammann, and Richard H. Gomer
Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology,
MS-140, Rice University, 6100 S. Main Street Houston, TX 77005-1892
Journal of Biological Chemistry, in press
Summary
In Dictyostelium discoideum, cell density is monitored by levels of a secreted
protein, conditioned-medium factor (CMF). CMFR1 is a putative CMF receptor
necessary for CMF-induced G protein-independent accumulation of the SP70
prespore protein, but not for CMF-induced G protein-dependent IP3 production.
Using recombinant fragments of CMF, we find that stimulation of the IP3
pathway requires amino acids 170 - 180 whereas SP70 accumulation does not,
corroborating a two-receptor model. Cells lacking CMFR1 do not aggregate,
due to the lack of expression of several important early developmentally
regulated genes, including gp80. Although many aspects of early
developmental cAMP-stimulated signal transduction are mediated by CMF,
CMFR1 is not essential for cAMP-stimulated cAMP and cGMP production or
Ca++ uptake, suggesting the involvement of a second CMF receptor.
Exogenous application of antibodies against either the region between a
first and second or a second and third possible transmembrane domain of
CMFR1 induces SP70 accumulation. Antibody- and CMF-induced gene expression
can be inhibited by recombinant CMFR1 corresponding to the region between
the first and third potential transmembrane domains, indicating that this
region is extracellular and likely contains the CMF binding site. These
observations support a model where a one- or two-transmembrane CMFR1
regulates gene expression and a G protein-coupled CMF receptor mediates
cAR1 signal transduction.
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A second UDP glucose pyrophosphorylase is required for differentiation and
development in Dictyostelium discoideum
John D. Bishop1, Byoung C. Moon2, Faith Harrow2, Richard H. Gomer1, Robert P.
Dottin2, and Derrick T. Brazill2
1Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology,
MS-140, Rice University, Houston, TX 77251-1892 2Hunter College, Department
of Biological Sciences, 695 Park Avenue, New York, NY 10021
J. Biological Chemistry, in press.
Summary
Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a
developmentally regulated enzyme in Dictyostelium discoideum, which is
involved in trehalose, cellulose and glycogen synthesis. Two independent
UDPGP proteins are believed to be responsible for this activity. To
determine the relative contributions of each protein, the genes encoding
them were disrupted individually. Cells lacking the udpgp1 gene exhibit
normal growth and development and make normal levels of cellulose. In
agreement with these phenotypes, udpgp1- cells still have UDPGP activity,
although at a reduced level. This supports the importance of the second
UDPGP gene. This newly identified gene, ugpB, encodes an active UDPGP as
determined by complementation in E. coli. When this gene is disrupted,
cells undergo aberrant differentiation and development ending with small,
gnarled fruiting bodies. These cells also have decreased spore viability
and decreased levels of glycogen, whose production requires UDPGP activity.
These phenotypes suggest that UgpB constitutes the major UDPGP activity
produced during development. Sequence analysis of the two UDPGP genes shows
that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1.
This includes the presence of 5 conserved lysine residues. Udpgp1 only has
1 of these lysines.
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RabD, a Dictyostelium Rab14-related GTPase, Regulates Phagocytosis and
Homotypic Phagosome and Lysosome Fusion
Edward Harris and James Cardelli
Department of Microbiology and Immunology and Feist-Weiller Cancer Center
LSU Health Sciences Center Shreveport, LA
J. Cell Science (in press)
Abstract
RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal
pathway and contractile vacuole system of membranes, and cell-lines
expressing dominant negative RabD were defective in endocytosis, endosomal
membrane flow and homotypic lysosome fusion. In support of support of a role
in fusion, cells overexpressing constitutively active RabDQ67L accumulated
enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent
with RabD directly regulating lysosome fusion. To determine if RabD also
regulated phagocytosis and/or homotypic phagosome fusion (a process
stimulated by many intracellular pathogens), cells overexpressing dominant
active (RabDQ67L+ cells) or dominant negative (RabN121I+ cells) RabD were
analyzed microscopically and biochemically. The rate of phagocytosis was
increased 2-fold in RabDQ67L+ cells and reduced by 50% in RabDN121I+ cells
as compared to control cells. To examine the role of RabD in the formation
of multi-particle phagosomes, we performed a series of pulse-chase
experiments using fluorescently labeled bacteria and fluorescent latex
beads. The rate of fusion of newly formed phagosomes was 5 times higher
in the RabDQ67L+ cells and reduced over 50% in RabDN121I+ cells as compared
to control cells. GFP-RabDQ67L+ was found to ring multi-particle spacious
phagosomes, supporting a direct role for this protein in regulating fusion.
Inhibition of PI 3-kinase activity, known to regulate phagosome fusion in
the wild-type cells, reduced the rate of phagosome fusion in RabDQ67L+ cells,
indicating that RabD acted upstream of or parallel with PI 3-kinase. We
hypothesize that RabD and, possibly, Rab14, a related GTPase that associates
with phagosomes in mammalian cells, are important regulators of homotypic
phagosome and endo-lysosome fusion
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Expression Pattern of Alkaline Phosphatase in Dictyostelium
Muatasem Ubeidat, Bradley R. Joyce and Charles L. Rutherford*
Biology Department, Molecular and Cellular Biology Section, Virginia
Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA
Mechanisms of Development, In Press
Abstract
We used two different methods to study the expression pattern of alkaline
phosphatase (alp) in Dictyostelium. In situ staining of the endogenous
enzyme activity at different stages of development showed that the enzyme
was active early in the aggregation stage and localized to the area where
the tip of the first finger was initiated. The activity was localized to
the anterior region of developing slugs, then became restricted to the
region between the prestalk and prespore cells at the culmination stage.
In the complete fruiting body, the activity was confined to the lower and
upper cup. A second method to study alp expression utilized a beta-
galactosidase reporter gene under the control of the alp promoter. A low
level of (-galactosidase activity was observed in vegetative cells, then
increased during development. Reporter gene activity was restricted to
PstO cells at the slug stage. At the culmination stage, the expression
was restricted to prestalk cells at the interface between the prestalk
and prespore cells. In the completed fruiting body, the expression was
observed in the upper and lower cup.
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[End Dicty News, volume 18, number 12]
