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dictyNews Volume 31 Number 15
dictyNews
Electronic Edition
Volume 31, number 15
November 14, 2008
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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Differentiation inducing factor-1 (DIF-1) induces gene and protein expression
of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin
Danton H. O’Day1,2,4, Yekaterina Poloz2, Michael A. Myre3
1Department of Biology, University of Toronto at Mississauga, Mississauga,
Ontario L5L 1C6, CANADA
2Department of Cell & Systems Biology, University of Toronto, Toronto,
Ontario M5S 3G5, CANADA
3Center for Human Genetic Research, Massachusetts General Hospital,
Harvard Medical School, 185 Cambridge St. , Boston, MA 02114, USA
4danton.oday@utoronto.ca
Cellular Signalling, accepted, http://dx.doi.org/10.1016/j.cellsig.2008.10.019
The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain,
calmodulin binding protein that regulates nuclear number. To gain insight into
the regulation of numA, we assessed the effects of the stalk cell differentiation
inducing factor-1 (DIF-1), an extracellular signalling molecule, on the
expression of numA1 RNA and protein. For comparison, the extracellular
signalling molecules cAMP (mediates chemotaxis, prestalk and prespore
differentiation) and ammonia (NH3/NH4+; antagonizes DIF) were also
studied. Starvation, which is a signal for multicellular development, results
in a greater than 80% decrease in numA1 mRNA expression within 4 hours.
Treatment with ammonium chloride led to a greater than 90% inhibition of
numA1 RNA expression within 2 hours. In contrast, the addition of DIF-1
completely blocked the decrease in numA1 gene expression caused by
starvation. Treatment of vegetative cells with cAMP led to decreases in
numA1 RNA expression that were equivalent to those seen with starvation.
Western blotting after various morphogen treatments showed that the
maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells
was reflected in significantly increased numA1 protein levels. Treatment
with cAMP and/or ammonia led to decreased protein expression and each
of these morphogens suppressed the stimulatory effects of DIF-1. Protein
expression levels of CBP4a, a calcium-dependent binding partner of
numA1, were regulated in the same manner as numA1 suggesting this
potential co-regulation may be related to their functional relationship.
NumA1 is the first calmodulin binding protein shown to be regulated by
developmental morphogens in Dictyostelium being upregulated by
DIF-1 and down-regulated by cAMP and ammonia.
Submitted by: Danton O'Day [danton.oday@utoronto.ca]
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An extrachromosomal, inducible expression system for
Dictyostelium discoideum.
Douwe M. Veltman*, Ineke Keizer-Gunnink and Peter J.M. Van Haastert
* corresponding author
Plasmid, in press
Inducible expression systems are essential for the expression of toxic proteins
and are very convenient for proteins that induce strong side effects such as
retardation of growth or development. Currently available systems for use in
Dictyostelium either do not have a very tight control over expression levels
or use a combination of an integrating and an extrachromosomal vector. We
designed a new vector in which all components of the available 2-plasmid
tetracycline-inducible system were combined onto a single extrachromosomal
vector. Two types of inducible plasmids are presented, in which transcription
is induced by adding or removing doxycycline respectively. The location and
orientation of the components was optimized in order to obtain a low
background expression combined with high inducibility. The resulting vectors
have a very low expression in the uninduced state (>1,000-fold lower
expression compared to that resulting from the act15 promoter), show a
10,000-fold induction of gene expression in a doxycycline
concentration-dependent manner and are comparatively small (8.5 kb).
With these new vectors, inducible gene expression is as easy as
constitutive gene expression.
Submitted by: Douwe M. Veltman [d.veltman@beatson.gla.ac.uk]
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Efficient cell lysis method for isolation of total RNA from slime mold Dictyostelium:
Applicability in preparation of cDNA
Bhavesh Vats and Harish Padh
Department of Cell and Molecular Biology
B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre,
Thaltej- Gandhinagar Highway, Thaltej, Ahmedabad – 380054, INDIA.
Telephone Number: +91-79-27439375, Fax number: +91-79-27450449.
Email: perd@perdcentre.com
International Journal of Biotechnology and Biochemistry, in press
Dictyostelium is a model organism for understanding of many cellular processes,
evolutionary studies, behavioral analysis, gene expression and now even drug
metabolism. RNA analysis is vital to most studies at molecular level. Extracting
RNA is no easy task due to the omnipresence of robust RNases. Attaining full
length mRNAs is the chief task in isolation of pure RNA. Stable ribonucleases
are the major obstacle in isolating RNA of any kind. Many methods and kits are
available for different organisms with differing efficiency, utility and expense.
For isolation of RNA from Dictyostelium, Blumberg and Lodish devised a simple,
in-expensive yet effective method, though for identification of mRNA use of
DNase and poly-dT columns were required. We modified the cell
homogenization process and isolated total RNA with the method. The
modification enables us to detect specific mRNA from the total RNA pool
without purifying through poly-dT columns or processing for DNA removal. The
integrity of RNA isolated by the modified method was similar to that of RNA
extracted with Qiagen RNAasy Mini kit indicative of the quality of the total RNA
extracted. With this modification, we have been able to identify three mRNA
transcripts and specifically amplify the cDNA using sequence specific primers.
Submitted by: Harish Padh [hpadh@yahoo.com]
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Multiple Mechanisms for Accumulation of Myosin II Filaments at the Equator
During Cytokinesis
Shigehiko Yumura, Masahiro Ueda, Yasushi Sako, Toshiko Kitanishi-Yumura,
Toshio Yanagida
Traffic, in press
Total internal reflection fluorescence microscopy revealed how individual bipolar
myosin II filaments accumulate at the equatorial region in dividing Dictyostelium
cells. Direct observation of individual filaments in live cells provided us with
much convincing information. Myosin II filaments accumulated at the equatorial
region by at least two independent mechanisms: (i) cortical flow, which is driven
by myosin II motor activities and (ii) de novo association to the equatorial cortex.
These two mechanisms were mutually redundant. At the same time, myosin II
filaments underwent rapid turnover, repeating their association and dissociation
with the actin cortex. Examination of the lifetime of mutant myosin filaments in
the cortex revealed that the turnover mainly depended on heavy chain
phosphorylation and that myosin motor activity accelerated the turnover. Double
mutant myosin II deficient in both motor and phosphorylation still accumulated
at the equatorial region, although they displayed no cortical flow and
considerably slow turnover. Under this condition, the filaments stayed for a
significantly longer time at the equatorial region than at the polar regions,
indicating that there are still other mechanisms for myosin II accumulation
such as binding partners or stabilizing activity of filaments in the equatorial
cortex.
Submitted by: Shigehiko Yumura [yumura@yamaguchi-u.ac.jp]
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Identification of a target for CudA the transcription factor that directs formation
of the Dictyostelium tip organiser
Hong Yu Wang and Jeffrey G. Williams
University of Dundee
College of Life Sciences
University of Dundee
Dow St.
Dundee DD1 5EH
U. K.
Int J Dev Biol, in press
The tip of the Dictyostelium slug functions much like an embryonic organiser; when
grafted onto the flank of a recipient slug it recruits a mass of prespore cells and
leads them away as part of a secondary slug. CudA is a nuclear protein that is
expressed in prespore cells where it acts as a specific transcription factor. CudA
is also expressed in an anteriorly located group of cells, the tip-organiser, that
is believed to constitute the functional tip. We identify an expansin-like gene,
expl7, that is expressed within the tip-organiser region and that is not expressed
in a cudA null strain. The expl7promoter contains a region that binds to CudA in
vitro and this region is necessary for expression in the tip-organiser. These
results provide an end-point for a previously defined signal transduction pathway;
wherein regionalised expression of the ACA adenylyl cyclase within the
tip-organiser leads to localised cAMP-induced activation of STATa and consequent
binding of STATa to the cudA promoter. STATa then induces expression of cudA and
cudA directs the transcription of target genes such as expl7.
Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk]
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Dictyostelium Dock180-related RacGEFs Regulate the Actin Cytoskeleton
during Cell Motility
Alessia Para*, Miriam Krischke*, Sylvain Merlot, Zhouxin Shen, Michael Oberholzer,
Susan Lee, Steven Briggs, and Richard A. Firtel
* These two authors contributed equally to the work
Section of Cell and Developmental Biology
Division of Biological Sciences
University of California, San Diego
9500 Gilman Drive
La Jolla, CA 92093-0380
Molecular Biology of the Cell, in press
Cell motility of amoeboid cells is mediated by localized F-actin polymerization
that drives the extension of membrane protrusions to promote forward movements.
We show that deletion of either of two members of the Dictyostelium Dock180
family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing
cells. The phenotype is enhanced in the double mutant and expression of DockA
or DockD complements the reduced speed of randomly moving DockD null cells’
phenotype, suggesting that DockA and DockD are likely to act redundantly and
to have similar functions in regulating cell movement. In this regard, we find that
overexpressing DockD causes increased cell speed by enhancing F-actin
polymerization at the sites of pseudopod extension. DockD localizes to the cell
cortex upon chemoattractant stimulation and at the leading edge of migrating
cells and that this localization is dependent on PI3K activity, suggesting that
DockD might be part of the pathway that links PtdIns(3,4,5)P3 production to
F-actin polymerization. Using a proteomic approach, we found that DdELMO1
is associated with DockD and that Rac1A and RacC are possible in vivo DockD
substrates. In conclusion, our work provides a further understanding of how
cell motility is controlled and provides evidence that the molecular mechanism
underlying Dock180-related protein function is evolutionarily conserved.
Submitted by: Rick Firtel [rafirtel@ucsd.edu]
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Autophagy contributes to degradation of Hirano bodies
Kim, D. H., Davis R. C., Furukawa R. , Fechheimer M.
Department of Cellular Biology, University of Georgia, Athens, Georgia,
USA
Autophagy, in press.
Hirano bodies are actin-rich inclusions reported most frequently in the
hippocampus in association with a variety of conditions including
neurodegenerative diseases and aging. We have developed a model
system for formation of Hirano bodies in Dictyostelium and cultured
mammalian cells to permit detailed studies of the dynamics of these
structures in living cells. Model Hirano bodies are frequently observed
in membrane-enclosed vesicles in mammalian cells consistent with a
role of autophagy in the degradation of these structures. Clearance of
Hirano bodies by an exocytotic process is supported by images from
electron microscopy showing extracellular release of Hirano bodies,
and observation of Hirano bodies in the culture medium of Dictyostelium
and mammalian cells. An autophagosome marker protein Atg8-GFP was
colocalized with model Hirano bodies in wild-type Dictyostelium cells, but
not in atg5(-) or atg1-1 autophagy mutant strains. Induction of model Hirano
bodies in Dictyostelium with a high-level expression of 34 kDa DeltaEF1
from the inducible discoidin promoter resulted in larger Hirano bodies
and a cessation of cell doubling. The degradation of model Hirano bodies
still occurred rapidly in autophagy mutant (atg5(-)) Dictyostelium,
suggesting that other mechanisms such as the ubiquitin-mediated
proteasome pathway could contribute to the degradation of Hirano bodies.
Chemical inhibition of the proteasome pathway with lactacystin significantly
decreased the turnover of Hirano bodies in Dictyostelium, providing direct
evidence that autophagy and the proteasome can both contribute to
degradation of Hirano bodies. Short-term treatment of mammalian cells
with either lactacystin or 3-methyl adenine results in higher levels of
Hirano bodies and a lower level of viable cells in the cultures, supporting
the conclusion that both autophagy and the proteasome contribute to
degradation of Hirano bodies.
Submitted by: Chandra Jack [chanj@rice.edu]
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[End dictyNews, volume 31, number 15]
