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dictyNews Volume 24 Number 17
Dicty News
Electronic Edition
Volume 24, number 17
June 24, 2005
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 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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Release of Ca2+ from the endoplasmic reticulum contributes to Ca2+
signalling in Dictyostelium.
Zofia Wilczynska1, Kathrin Happle2, Annette Mller-Taubenberger3,
Christina Schlatterer2, Dieter Malchow2 and Paul R. Fisher1,*
1 Department of Microbiology, La Trobe University, Victoria 3086,
Australia.
2 Universitt Konstanz, 78464 Konstanz, Federal Republic of Germany.
3 Max-Planck-Institut fr Biochemie, 82152 Martinsried bei Mnchen,
Federal Republic of Germany.
Eukaryotic Cell, in press
Ca2+ responses to two chemoattractants (folate and cAMP) were assayed in
Dictyostelium mutants deficient in one or both of two abundant Ca2+-binding
proteins of the endoplasmic reticulum (ER) - calreticulin and calnexin.
Mutants deficient in either or both proteins exhibited enhanced cytosolic
Ca2+ responses to both attractants. Not only were the mutant responses
greater in amplitude, but they also exhibited earlier onsets, faster rise
rates, earlier peaks and faster fall rates. Correlations amongst these
kinetic parameters and the response amplitudes suggested that key events in
the Ca2+ response are autoregulated by the magnitude of the response itself
ie. by cytosolic Ca2+ levels. This autoregulation was sufficient to explain
the altered kinetics of the mutant responses - larger responses are faster in
both mutant and wild type cells in response to both folate (vegetative cells)
and cAMP (differentiated cells). Searches of the predicted Dictyostelium
proteome revealed 3 putative Ca2+ pumps and 4 putative Ca2+ channels. All
but one contained sequence motifs for Ca2+- or calmodulin-binding sites,
consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+
responses in the calnexin and calreticulin mutants are enhanced, the influx
of Ca2+ from the extracellular medium into the mutant cells was smaller.
Compared to wild type cells, Ca2+ release from the ER in the mutants thus
contributes more to the total cytosolic Ca2+ response while influx from the
extracellular medium contributes less. These results provide the first
molecular genetic evidence that release of Ca2+ from the ER contributes to
cytosolic Ca2+ responses in Dictyostelium.
Submitted by: Paul Fisher [fisher@lumi.latrobe.edu.au]
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Engineered gene over-expression as a method of drug target identification
Christopher J. Sugden +, Janine R. Roper + and Jeffrey G. Williams*
School of Life Sciences
University of Dundee
Wellcome Trust Biocentre
Dow Street
DUNDEE, DD1 5EH, UK
+contributed equally to this work
*Corresponding Author
Professor Jeffrey Williams,
School of Biological Sciences, University of Dundee
MSI/WTB Complex, Dow Street
DUNDEE, DD1 5EH
Fax (44) 01382 345386
Email j.g.williams@dundee.ac.uk
Biochem. and Biophys. Res. Comm., in press
The proposed target of aminobisphosphonate (aBP) bone resorption inhibitors,
both in mammalian osteoclasts and in Dictyostelium, is the enzyme farnesyl
diphosphate synthase (FDP synthase). The genetic evidence, obtained with
Dictyostelium, derives from variant strains that over-express FDP synthase
and that are relatively resistant to aBPs. We show that forced FDP synthase
over-expression also leads to aBP resistance; by placing FDP synthase under
control of a semi-constitutive promoter, transforming it into Dictyostelium
cells and selecting with the aBP alendronate. This combination of drug and
dominant selectable marker provides a novel selection system for
transformation. We further show that, when a population of Dictyostelium
cells expressing an entire growth stage cDNA library is placed under
alendronate selection, FDP synthase is the only cDNA insert that confers
drug resistance. This confirms FDP synthase as the primary target of aBPs
and suggests a general method of drug target identification based upon
engineered gene over-expression.
Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk]
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Loss of SMEK, a novel, conserved protein, suppresses mek1 null cell polarity,
chemotaxis, and gene expression defects
Michelle C. Mendoza, Fei Du, Negin Iranfar, Nan Tang, Hui Ma, William F.
Loomis, and Richard A. Firtel
Molecular and Cellular Biology, in press.
MEK/ERK MAP kinase signaling is imperative for proper chemotaxis.
Dictyostelium mek1- (MEK1 null) and erk1- cells exhibit severe defects in
cell polarization and directional movement, but the molecules responsible
for the mek1- and erk1- chemotaxis defects are unknown. Here, we describe a
novel, evolutionarily conserved gene, smkA (suppressor of mek1-), whose
loss partially suppresses the mek1- chemotaxis phenotypes. SMEK also has
MEK1-independent functions: SMEK,but not MEK1, is required for proper
cytokinesis during vegetative growth, timely exit from the mound stage
during development, and myosin II assembly. SMEK localizes to the cell
cortex through an EVH1 domain at its N-terminus during vegetative growth.
At the onset of development, SMEK translocates to the nucleus via an NLS
(nuclear localization signal) at its C-terminus. The importance of SMEKâs
nuclear localization is demonstrated by our findings that a mutant lacking
the EVH1 domain complements SMEK deficiency, whereas a mutant lacking the
NLS does not. Microarray analysis reveals that some genes are precociously
expressed in mek1- and erk1- cells. The mis-expression of some of these
genes is suppressed in the smkA deletion. These data suggest that loss of
MEK1/ERK1 signaling compromises gene expression and chemotaxis in a
SMEK-dependent manner.
Submitted by: Rick Firtel [rafirtel@ucsd.edu]
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The effect of the disruption of a gene encoding a PI4 kinase on the
developmental defect exhibited by Dictyostelium rasC- cells
Meenal Khosla, George B. Spiegelman and Gerald Weeks
Department of Microbiology and Immunology
University of British Columbia
Vancouver, BC, V6T 1Z3
Canada
Developmental Biology, in press
The disruption of the gene encoding the Dictyostelium Ras sub-family
protein, RasC results in a strain that fails to aggregate with defects in
both cAMP signal relay and chemotaxis. Restriction Enzyme Mediated
Integration disruption of a second gene in the rasC- strain resulted in
cells that were capable of forming multicellular structures in plaques on
bacterial lawns. The disrupted gene, designated pikD1, encodes a member
of the phosphatidyl-inositol-4-kinase °ñ subfamily. Although the
rasC-/pikD1 cells were capable of progressing through early development,
when starved on a plastic surface under submerged conditions, they did not
form aggregation streams or exhibit pulsatile motion. The rasC-/pikD1
cells were extremely efficient in their ability to chemotax to cAMP in a
spatial gradient, although the reduced phosphorylation of PKB in response
to cAMP observed in rasC- cells, was unchanged. In addition, the activation
of adenylyl cyclase, which was greatly reduced in the rasC- cells, was only
minimally increased in the rasC-/pikD1 strain. Thus, although the
rasC-/ pikD- cells were capable of associating to form multicellular
structures, normal cell signaling was clearly not restored. The disruption
of the pikD gene in a wild type background resulted in a strain that was
delayed in aggregation and formed large aggregation streams, when starved on
a plastic surface under submerged conditions. This strain also exhibited a
slight defect in terminal development. In conclusion, disruption of the
pikD gene in a rasC- strain resulted in cells that were capable of forming
multicellular structures, but which did so in the absence of normal
signaling and aggregation stream formation.
Submitted by: Gerald Weeks [gerwee@interchange.ubc.ca]
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[End Dicty News, volume 24, number 17] 
