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dictyNews Volume 26 Number 02
dictyNews
Electronic Edition
Volume 26, number 2
January 13, 2006
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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Cleavage with phospholipase of the lipid anchor in the cell adhesion
molecule, csA, from Dictyostelium discoideum
Motonobu Yoshida*, Naoya Sakuragi, Ken Kondo, Eiji Tanesaka
Department of Agriculture, Kinki University, Nakamachi, Nara 631-8505,
Japan
Comp. Biochem. Physiol., in press
A cell adhesion molecule, 80-kDa csA, is involved in EDTA-resistant cell
contact at the aggregation stage of Dictyostelium discoideum. A 31-kDa
csA was isolated from the 80-kDa csA by treatment with Achromobacter
protease I. Results from thin-layer chromatography and MALDI-TOF MS analysis
indicated that the 31-kDa csA contains ceramide as a component of
glycosylphosphatidyl-inositol (GPI). Comparison between the 80-kDa csA and
the 31-kDa csA treated with phosphatidylinositol-specific phospholipase C
(PI-PLC) or GPI-specific phospholipase D (GPI-PLD) was carried out. Our
results indicated that the GPI-anchor of the 31-kDa csA was more sensitive
to PI-PLC treatment than that of the 80-kDa csA, and that the anchor in both
was easily cleaved by GPI-PLD treatment. The results of the 80-kDa csA and
the 31-kDa csA treated with sphingomyelinase were almost the same as those
with PI-PLC treatment. In the presence of 1,10-phenanthroline, a GPI-PLD
inhibitor, development of Dictyostelium was markedly inhibited, suggesting
that GPI-PLD is functional in developmental regulation.
Submitted by: Motonobu Yoshida [yoshida_m@nara.kindai.ac.jp]
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A secondary disruption of the lmpA gene encoding a large membrane protein
allows aggregation defective Dictyostelium rasC- cells to form multicellular
structures
Meenal Khosla1, Paul Kriebel2, Carole A. Parent2, George B. Spiegelman1 and
Gerald Weeks1
1Department of Microbiology and Immunology, University of British Columbia,
Vancouver, BC, V6T 1Z3, Canada and
2The Laboratory of Cellular and Molecular Biology, National Cancer
Institute, NIH, MD, USA
Corresponding author; G. Weeks
Tel: 604-822-6649. Fax: 604-822-6041
Mailing address: 2350 Health Sciences Mall., Vancouver, BC, V6T 1Z3, Canada.
Email: gerwee@interchange.ubc.ca
Developmental Biology, in press
The disruption of the gene encoding the Dictyostelium Ras sub-family protein,
RasC, results in a strain that does not aggregate and has defects in both
cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the
rasC- strain by Restriction Enzyme Mediated Integration produced cells that
were capable of forming multicellular structures in plaques on bacterial
lawns. The disrupted gene (lmpA) encoded a novel large membrane protein
that was designated Lmp1. Although the rasC-/lmpA- cells progressed through
early development, they did not form aggregation streams on a plastic surface
under submerged starvation conditions. Phosphorylation of PKB in response to
cAMP, which is significantly reduced in rasC- cells, remained low in the
rasC-/lmpA- cells. However, in spite of this low PKB phosphorylation, the
rasC-/lmpA- cells underwent efficient chemotaxis to cAMP in a spatial
gradient. Cyclic AMP accumulation, which was greatly reduced in the rasC-
cells, was restored in the rasC-/lmpA- strain, but cAMP relay in these cells
was not apparent. These data indicate that, although the rasC-/lmpA- cells
were capable of associating to form multicellular structures, normal
aggregative cell signaling was clearly not restored. Disruption of the
lmpA gene in a wildtype background resulted in cells that exhibited a slight
defect in aggregation and a more substantial defect in late development.
These results indicate that in addition to the role played by Lmp1 in
aggregation, it is also involved in late development.
Submitted by: Gerald Weeks [gerwee@interchange.ubc.ca]
-----------------------------------------------------------------------------
A secondary disruption of the lmpA gene encoding a large membrane protein
allows aggregation defective Dictyostelium rasC- cells to form multicellular
structures
Meenal Khosla1, Paul Kriebel2, Carole A. Parent2, George B. Spiegelman1 and
Gerald Weeks1
1Department of Microbiology and Immunology, University of British Columbia,
Vancouver, BC, V6T 1Z3, Canada and
2The Laboratory of Cellular and Molecular Biology, National Cancer Institute,
NIH, MD, USA
Corresponding author; G. Weeks
Tel: 604-822-6649. Fax: 604-822-6041
Mailing address: 2350 Health Sciences Mall., Vancouver, BC, V6T 1Z3, Canada.
Email: gerwee@interchange.ubc.ca
Developmental Biology, in press
The disruption of the gene encoding the Dictyostelium Ras sub-family protein,
RasC, results in a strain that does not aggregate and has defects in both
cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the
rasC- strain by Restriction Enzyme Mediated Integration produced cells that
were capable of forming multicellular structures in plaques on bacterial
lawns. The disrupted gene (lmpA) encoded a novel large membrane protein that
was designated Lmp1. Although the rasC-/lmpA- cells progressed through early
development, they did not form aggregation streams on a plastic surface under
submerged starvation conditions. Phosphorylation of PKB in response to cAMP,
which is significantly reduced in rasC- cells, remained low in the
rasC-/lmpA- cells. However, in spite of this low PKB phosphorylation, the
rasC-/lmpA- cells underwent efficient chemotaxis to cAMP in a spatial
gradient. Cyclic AMP accumulation, which was greatly reduced in the rasC-
cells, was restored in the rasC-/lmpA- strain, but cAMP relay in these cells
was not apparent. These data indicate that, although the rasC-/lmpA- cells
were capable of associating to form multicellular structures, normal
aggregative cell signaling was clearly not restored. Disruption of the
lmpA gene in a wildtype background resulted in cells that exhibited a slight
defect in aggregation and a more substantial defect in late development.
These results indicate that in addition to the role played by Lmp1 in
aggregation, it is also involved in late development.
Submitted by: Gerald Weeks [gerwee@interchange.ubc.ca]
-----------------------------------------------------------------------------
HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not
antisense mediated gene silencing.
Blagovesta Popova1, Markus Kuhlmann1 Andrea Hinas2, Fredrik Söderbom2 and
Wolfgang Nellen1*
1Abt. Genetik, Universität Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel,
Germany
2Department of Molecular Biology, Biomedical Center, Swedish University of
Agricultural Sciences, Box 590, S-75124 Uppsala, Sweden
*corresponding author:
Wolfgang Nellen
Abt. Genetik, Universität Kassel,
Heinrich-Plett-Str. 40,
D-34132 Kassel,
Germany
Nucleic Acids Res. in press
We have identified a putative RNA helicase from Dictyostelium that is
closely related to drh1, the “dicer-related-helicase” from C. elegans and
that also has significant similarity to proteins from vertebrates and
plants. GFP-tagged HelF protein was localized in speckles in the nucleus.
Disruption of the helF gene resulted in a mutant morphology in late
development. When transformed with RNAi constructs, HelF- cells displayed
enhanced RNA interference on four tested genes. One gene that could not be
knocked down in the wild type background was efficiently silenced in the
mutant. Furthermore, the efficiency of silencing in the wild type was
dramatically improved when helF was disrupted in a secondary transformation.
Silencing efficiency depended on transcription levels of hairpin RNA and
the threshold was dramatically reduced in HelF- cells. However, the amount
of siRNA did not depend on hairpin transcription.
HelF is thus a natural nuclear suppressor of RNA interference. In contrast,
no improvement of gene silencing was observed when mutant cells were
challenged with corresponding antisense constructs. This indicates that RNAi
and antisense have distinct requirements even though they may share parts
of their pathways.
Submitted by: Wolfgang Nellen [nellen@uni-kassel.de]
-----------------------------------------------------------------------------
Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in
Dictyostelium:
A quantitative in vivo analysis by inhibition of PI3-kinase
Harriët M. Loovers, Marten Postma, Ineke Keizer-Gunnink, Yi Elaine Huang,
Peter N. Devreotes, and Peter J.M. van Haastert
Department of Molecular Cell Biology, University of Groningen, Kerklaan 30,
9751NN Haren, the Netherlands
Department of Cell Biology, Johns Hopkins University School of Medicine,
725 N. Wolfe St., 114 WBSB, Baltimore, MD 21205
Molecular Biology of the Cell, in press
The role of PI(3,4,5)P3 in Dictyostelium signal transduction and chemotaxis
was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells.
The increase of PI(3,4,5)P3 levels after stimulation with the
chemoattractant cAMP was blocked >95% by 60 ?M LY294002 with half-maximal
effect at 5 ?M. This correlated well with the inhibition of the membrane
translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce
cAMP-mediated cGMP production, but significantly reduced the cAMP response
up to 75% in wild type and completely in pi3k-null cells. LY294002-treated
cells were round, not elongated as control cells. Interestingly, cAMP
induced a time and dose-dependent recovery of cell elongation. These
elongated LY294002-treated wild type and pi3k-null cells exhibited
chemotactic orientation towards cAMP that is statistically identical to
chemotactic orientation of control cells. In control cells, PHcrac-GFP and
F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases
does not affect the first phase of the actin polymerization at a wide range
of chemoattractant concentrations. Our data show that severe inhibition of
cAMP-mediated PI(3,4,5)P3 accumulation leads to inhibition of cAMP relay,
cell elongation and cell aggregation, but has no detectable effect on
chemotactic orientation, provided that cAMP had sufficient time to induce
cell elongation.
Submitted by: Peter van Haastert [P.J.M.van.haastert@rug.nl]
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[End dictyNews, volume 26, number 2] 
