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dictyNews Volume 19 Number 03
Dicty News
Electronic Edition
Volume 19, number 3
August 3, 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
======================
Postdoctoral position in the Wellcome Trust Biocentre at the University
of Dundee
A postdoctoral position is available in Dr. Inke Nthke s laboratory to
join a team studying the function of the Adenomatous Polyposis Coli tumour
suppressor protein (APC). The project will involve using Dictyostelium
Discoideum to investigate the role of APC in cytoskeletal dynamics and
organisation. The general aim of work in the laboratory is to determine
how the diverse functions of APC relate to its role in cancer and how these
functions are co-ordinated and regulated. Experimental approaches include
general cell- and molecular biology techniques combined with high-resolution
fluorescence microscopy and use of Dictyostelium as a model organism.
The candidate will join a small, dynamic, highly interactive research team
with active collaborations worldwide.
A proven research ability and motivation are required and experience in
working with Dictyostelium is requisite. Experience in general cell biology
and molecular biology is essential. The position is available immediately
for 2 years in the first instance. For informal enquiries, please contact
Dr. Inke S. Nthke (+ 44 1382 345821; e-mail: i.s.nathke@dundee.ac.uk).
Applications should be directed to: Janette Cordiner, School Administrator,
School of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street,
Dundee, DD1 5EH; j.m.cordiner@dundee.ac.uk. Please cite reference
SC/763/1 on the application.
The Wellcome Trust Biocentre is part of the internationally renowned WTB/MSI
complex at the University of Dundee. The complex provides an extremely
interactive research environment and houses nearly 400 research staff.
Further information about research in the WTB/MSI complex can be obtained
at http://www.dundee.ac.uk/biocentre/.
=============
Abstracts
=============
Differential localization in cells of myosin II heavy chain kinases during
cytokinesis and polarized migration.
Wenchuan Liang, Lucila Licate, Hans Warrick, James Spudich, and Thomas Egelhoff
BMC Cell Biology 2002 3: 19
Abstract
Background
Cortical myosin-II filaments in Dictyostelium discoideum display enrichment
in the posterior of the cell during cell migration, and in the cleavage
furrow during cytokinesis. Filament assembly in turn is regulated by
phosphorylation in the tail region of the myosin heavy chain (MHC). Early
studies have revealed one enzyme, MHCK-A, which participates in filament
assembly control, and two other structurally related enzymes, MHCK-B and
-C. In this report we evaluate the biochemical properties of MHCK-C, and
using fluorescence microscopy in living cells we examine the localization
of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.
Results
Biochemical analysis indicates that MHCK-C can phosphorylate MHC with
concomitant disassembly of myosin II filaments. In living cells, GFP-MHCK-A
displayed frequent enrichment in the anterior of polarized migrating cells,
and in the polar region but not the furrow during cytokinesis. GFP-MHCK-B
generally displayed a homogeneous distribution. In migrating cells
GFP-MHCK-C displayed posterior enrichment similar to that of myosin II, but
did not localize with myosin II to the furrow during the early stage of
cytokinesis. At the late stage of cytokinesis, GFP-MHCK-C became strongly
enriched in the cleavage furrow, remaining there through completion of
division.
Conclusion
MHCK-A, -B, and -C display distinct cellular localization patterns
suggesting different cellular functions and regulation for each MHCK
isoform. The strong localization of MHCK-C to the cleavage furrow in the
late stages of cell division may reflect a mechanism by which the cell
regulates the progressive removal of myosin II as furrowing progresses.
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cAMP dependent protein kinase regulates Polysphondylium pallidum development
Satoru Funamoto1,3, Christophe Anjard2,4, Wolfgang Nellen2 and Hiroshi
Ochiai1,5
1Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo 060-0810, Japan; 2Department of Genetics, University of
Kassel, Heinrich-Plett-Str. 40 D-34132 Kassel, Germany; 3 Present address:
Department of Neuropathology, Faculty of Medicine, University of Tokyo,
Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan; 4Present address: Center for
Molecular Genetics, Department of Biology University of California, San Diego
9500 Gilman Drive, La Jolla, CA 92093-0634
Author for correspondence. E-mail: hochiai@sci.hokudai.ac.jp
Differentiation, in press.
Abstract
In eukaryotic cells the universal second messenger cAMP regulates various
aspects of development and differentiation. The primary target for cAMP is
the regulatory subunit of cAMP-dependent protein kinase A (PKA), which,
upon cAMP binding, dissociates from the catalytic subunit and thus activates
it. In the soil amoeba Dictyostelium discoideum the function of PKA in
growth, development and cell differentiation has been thoroughly
investigated and substantial information is available. To obtain a more
general view we investigated the influence of PKA on development of the
related species Polysphondylium pallidum. Cells were transformed to
overexpress either a dominant negative mutant of the regulatory subunit
(Rm) from Dictyostelium that cannot bind cAMP, or the catalytic subunit
(PKA-C) from Dictyostelium. Cells overexpressing Rm rarely aggregated and
the few multicellular structures developed slowly into very small fruiting
bodies without branching of secondary sorogens, the prominent feature of
Polysphondylium. Few round spores with reduced viability were formed. When
mixed with wild type cells and allowed to develop, the Rm cells were
randomly distributed in aggregation streams, but were later found in the
posterior region of the culminating slug or were left behind on the surface
of the substratum. The PKA-C overexpressing cells exhibited precocious
development and formed more aggregates of smaller size. Moreover,
expression of PKA-C under the control of the prestalk specific ecmB promoter
of Dictyostelium, lead to protrusions from aggregation streams. We conclude
that Dictyostelium PKA subunits introduced into Polysphondylium cells are
functional as signal components, indicating that a biochemically similar
PKA mechanism works in Polysphondylium.
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Element analysis of the Polysphondylium pallidum gp64 promoter
Naohisa Takaokaa, Masashi Fukuzawab, Atsushi Katoa, Tamao Saitoa and Hiroshi
Ochiaia, c
a. Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo, 060-0810 Japan; b. Wellcome Trust Building, Department
of Anatomy and Physiology, University of Dundee, Dow Street, Dundee, DD1 4HN, UK
Author for corresponding. E-mail: hochiai@sci.hokudai.ac.jp
BBA, in press.
Abstract
gp64 mRNA in Polysphondylium pallidum is expressed extensively during
vegetative growth, and begins to rapidly decrease at the onset of
development. To examine this unique regulation, 5 deletion analysis of the
gp64 promoter was undertaken, and two growth-phase activated elements have
been found: a food-dependent, upstream regulatory region (FUR, -222 to 170)
and a vegetatively activated, downstream region (VAD, -110 to 63). Here we
concentrate our analysis on an A1 and A2 sequences in the FUR region: A1
consists of a GATTTTTTTA sequence called a corresponding sequence and A2
consists of the direct repeat TTTGTTGTG. The cells carrying a combined
construct of A1 and A2 acted synergistically in a reporter activity. A
point mutation analysis in A1 indicates that a G residue is required for
the activation of A1. From analyses of promoter regulation in a liquid or
a solid medium, the promoter activity of the cells fed on bacteria in
A-medium (axenic medium for Polysphondylium) or grown in A-medium alone
was only one-fourth of that of the cells fed on bacteria.
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A novel Dictyostelium Cdk8 is required for aggregation, but dispensable for
growth
Kosuke Takeda, Tamao Saito, Hiroshi Ochiai*
Division of Biological Sciences, Graduate School of Science, Hokkaido
University Sapporo, Hokkaido 060-0810, Japan
Author for correspondence. E-mail: hochiai@sci.hokudai.ac.jp
Develop. Growth Differ., in press
Abstract
When Dictyostelium cells starve, they express genes necessary for
aggregation. Using insertional mutagenesis, we have isolated a mutant that
does not aggregate upon starvation and that forms small plaques on bacterial
lawns, thus indicating slow growth. The sequencing of the mutated locus
showed a strong similarity to the catalytic domain of cdc2-related kinase
genes. Phylogenetic analysis further indicated that the amino acid sequence
was more close to cyclin-dependent kinase 8 than to those of other cyclin-
dependent kinases. Thus we designated this gene as Ddcdk8. The Ddcdk8-null
cells do not aggregate and grow somewhat more slowly than parental cells
when being shaken in axenic medium or laid on bacterial plates. To confirm
whether these defective phenotypes were caused by disruption of this gene,
the Ddcdk8-null cells were complemented with DdCdk8 protein expressed from
an endogenous promoter but not an actin promoter, and when the complemented
cells were then allowed to grow on a bacterial lawn, they began to aggregate
as the food supply was depleted, and finally became fruiting bodies. The
results suggest that properly regulated DdCdk8 activity is essential for
aggregation. Since when starved, Ddcdk8-null cells do not express at all
the acaA transcripts required for aggregation, we deduce that Ddcdk8 is
epistatic for acaA expression, indicating that the Ddcdk8 products may
regulate expression of acaA and/or other genes.
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Cell adhesion during the migratory slug stage of Dictyostelium discoideum.
*Vivienne M. Bowers-Morrow, *Sinan O. Ali, and +Keith L. Williams
*Department of Biological Sciences, Macquarie University, Sydney, NSW, 2109
AUSTRALIA
+Proteome Systems Ltd, North Ryde, Sydney, NSW, 2113 AUSTRALIA
All correspondence to Vivienne Bowers-Morrow, email: vmorrow@rna.bio.mq.edu.au
CELL BIOLOGY INTERNATIONAL, in press
ABSTRACT
Prespore-specific Antigen (PsA) is selectively expressed on the surface of
prespore cells at the multicellular migratory slug stage of Dictyostelium
discoideum development. It is a developmentally regulated glycoprotein
which is anchored to the cell membrane through a glycosyl
phosphatidylinositol (GPI) anchor. We present the results of an in vitro
immunological investigation of the hypothesis that PsA functions as a cell
adhesion molecule (CAM), and of a ligand binding assay indicating that PsA
has cell membrane binding partner(s). This is the first evidence to
implicate a direct role for a putative CAM in cell-cell adhesion during the
multicellular migratory slug stage of D. discoideum development. Cell-cell
adhesion assays were carried out in the presence or absence of the monoclonal
antibody (mAb) MUD1 which has a single antigenic determinant: a peptide
epitope on PsA. These assays showed specific inhibition of cell-cell adhesion
by MUD1. Further, it was found that a purified recombinant form of PsA
(rPsA), can neutralize the inhibitory effect of MUD1; the inhibitory effect
on cell-cell adhesion is primarily due to the blocking of PsA by the mAb.
The resistance of aggregates to dissociation in the presence of 10mM EDTA
(ethylenediamintetraacetic acid) indicates that PsA mediates EDTA-stable
cell-cell contacts, and that PsA-mediated cell adhesion is likely to be
independent of divalent cations such as Ca 2+ or Mg 2+.
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The three-dimensional model of Dictyostelium discoideum racE based on the
human rhoA-GDP crystal structure
Madhavi Agarwala, Donald J. Nelsonb and Denis A. Larochellea
a Department of Biology, Clark University, 950 Main Street, Worcester,
MA 01610, USA; b Department of Chemistry, Clark University, 950 Main Street,
Worcester, MA 01610, USA
Corresponding author. Tel.: +1-508-793-7664; fax: +1-508-793-8861; email:
magarwal@clarku.edu
Journal of Molecular Graphics and Modelling 21, 3-18
Abstract
The three-dimensional structure of racE was modeled using several homologous
small G proteins, and the best model obtained using the human rhoA as
modeling template is reported. The three-dimensional fold of the racE model
is remarkably similar to the cellular form of human ras p21 crystal
structure. Its secondary structure consists of six -helices, six -strands
and three 310 helices. The model retains its secondary structure after a
300 K, 300 ps molecular dynamics (MD) simulation. Important domains of the
protein include its effector loop (residues 3446), the insertion domain
(residues 121136), and the polybasic motif (between 210 and 220) not
modeled in the current structure. The effector loop is inherently flexible
and the structure docked with GDP exhibits the effector loop moving
significantly closer to the nucleotide binding pocket, forming a tighter
complex with the bound GDP. The mobility of the effector loop is conferred
by a single residue `hinge' point at residue 34Asp, also allowing the
Switch I region, immediately preceding the effector loop, to be equally
mobile. In comparison, the Switch II region shows average mobility. The
insertion domain is highly flexible, with the insertion taking the form
of a helical domain, with several charged residues forming a complex
charged interface over the entire insertion region. While the GDP moiety
is loosely held in the active site, the metal cation is extensively
co-ordinated. The critical residue 38Thr exhibits high mobility, and is
seen interacting directly with the metal ion at a distance of 2.64 , and
indirectly via an intervening water molecule. 64Gln, a key residue involved
in GTP hydrolysis in ras, is seen facing the -phosphate group and the metal
ion. Certain residues (i.e. 51Asn, 38Thr and 65Glu) exhibit unique
characteristics and these residues, together with 158Val, may play important
roles in the maintenance of the protein's integrity and function. There is
strong consensus of secondary structural elements between models generated
using various templates, such as h-rac1, h-rhoA and h-cdc42 bound to RhoGDI,
all sharing only 5055% sequence identity with racE, which suggests that
this model is in all probability an accurate prediction of the true tertiary
structure of racE.
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A novel cGMP-signaling pathway mediating myosin phosphorylation and
chemotaxis in Dictyostelium
Leonard Bosgraaf#, Henk Russcher#, Janet L. Smith^, Deborah Wessels*, David
R. Soll* and Peter J.M. Van Haastert#
# Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747
AG Groningen, the Netherlands. ^ Boston Biomedical Research Institute, 64
Grove Street, Watertown, Massachusetts 02472-2829, U.S.A. * W.M. Keck Dynamic
Image Analysis Facility, Department of Biological Sciences, University of
Iowa, Iowa City, IA 52242, U.S.A.
EMBO J, in press
Chemotactic stimulation of Dictyostelium cells results in a transient
increase in cGMP levels, and transient phosphorylation of myosin II heavy
and regulatory light chains. In Dictyostelium, two guanylyl cyclases and
four candidate cGMP-binding proteins (GbpA-D) are implicated in cGMP
signaling. GbpA and GbpB are homologous proteins with a Zn2+-hydrolase
domain. A double gbpA/gbpB gene disruption leads to a reduction of cGMP-
phosphodiesterase activity and a ten-fold increase of basal and stimulated
cGMP levels. Chemotaxis in gbpA-B- cells is associated with increased myosin
II phosphorylation compared to wild type cells; formation of lateral
pseudopodia is suppressed resulting in enhanced chemotaxis. GbpC is
homologous to GbpD, and contains Ras, MAPKKK and Ras-GEF domains.
Inactivation of the gbp genes indicates that only GbpC harbours high
affinity cGMP-binding activity. Myosin phosphorylation, assembly of myosin
in the cytoskeleton as well as chemotaxis are severely impaired in mutants
lacking GbpC and D, or mutants lacking both guanylyl cyclases. Thus a novel
cGMP-signaling cascade is critical for chemotaxis in Dictyostelium, and
plays a major role in myosin II regulation during this process.
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An observation of the initial stage towards a symbiotic relationship
Masahiko Todoriki 1, Shinya Oki 1, Shin-Ichi Matsuyama1, Elizabeth P.
Ko-Mitamura1, Itaru Urabe 1 and Tetsuya Yomo 1,2,3*
1Department of Biotechnology, Graduate School of Engineering, Osaka
University, 2-1 Yamadaoka, Suita City, 2PRESTO, JST, 2-1 Yamadaoka, Suita
City, Osaka 565-0871, Japan and 3Department of Pure and Applied Sciences,
University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan.
*Corresponding author. Mailing address: Department of Biotechnology, Graduate
School of Engineering, Osaka University, 2-1 Yamadaoka, Suita City, Osaka
565-0871, Japan. E-mail: yomo@bio.eng.osaka-u.ac.jp; Tel. :+81-6-6879-7427;
Fax: +81-6-6879-7428
BioSystems 65 (2002) 105?112
Abstract
Two well-characterized and phylogenetically different species, Escherichia
coli and Dictyostelium discoideum, were used as the model organisms. When the
two species were mixed and allowed to grow on minimal agar plates at 22?C, the
two species remarkably achieved a state of coexistence at about two weeks. In
addition, the emerged colonies housing the coexisting species have a mucoidal
nature that was not observed from its origin. Moreover, the state of
coexistence was confirmed to be stable, and so does the mucoidal nature of
the emerged colonies. Comparing with the pure E.coli origin, mucoidal colony
showed a significant increase in higher molecular weight extracellular
components, with polysaccharide as the major constituent. Qualitative
analysis of the monosaccharide contents in the extracellular components of
the mucoidal colony revealed not only a significant increase in the glucose
content, but also significant amount of additional xylose and galactose.
The system permits the initial stages of the development of relationship
between two species be captured within a short period of time. This
feature, together with being simple and reproducible on laboratory
conditions, hence provides a new model system for the study of symbiosis,
especially when initial stages are concerned.
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[End Dicty News, volume 19, number 3]
