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dictyNews Volume 16 Number 12
Dicty News
Electronic Edition
Volume 16, number 12
June 23, 2001
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.
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Abstracts
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Inhibition of multicellular development switches cell death of Dictyostelium
discoideum towards mammalian-like unicellular apoptosis.
Irène Tatischeff1) a, Patrice Xavier Petit b, Alain Grodet c, Jean-Pierre
Tissier d, Isabelle Duband-Goulet e, Jean-Claude Ameisen f
a: Laboratoire de Physicochimie Biomoléculaire et Cellulaire, CNRS ESA 7033,
Université Pierre et Marie Curie, Paris/France
b: Institut Cochin de Génétique Moléculaire, INSERM U129 - CHU Cochin
Port-Royal, Paris/France
c: Unité 82 INSERM, Faculté de Médecine Xavier Bichat, Paris/France
d: INRA LGPTA, Villeneuve d'Ascq/France
e: Institut Jacques Monod, Laboratoire de Biologie Structurale du Noyau
Cellulaire, Paris/France
f: INSERM EPI 9922, CHU Bichat, Université Paris VII, Paris/France
Eur. J. Cell Biol. (2001) 80, (6), 428-441
Abstract
The multicellular development of the single celled eukaryote Dictyostelium
discoideum is induced by starvation and consists of initial aggregation of
the isolated amoebae, followed by their differentiation into viable spores
and dead stalk cells. These stalk cells retain their structural integrity
inside a stalk tube that support the spores in the fruiting body. Terminal
differentiation into stalk cells has been shown to share several features
with programmed cell death (Cornillon et al (1994), J. Cell Sci. 107, 2691-
2704). Here we report that, in the absence of aggregation and
differentiation, D. discoideum can undergo another form of programmed cell
death that closely ressembles apoptosis of most mammalian cells, involves
mitochondrial transmembrane potential loss, phosphatidylserine surface
exposure, and engulfment of dying cells by neighboring D. discoideum cells.
This death has been studied by various techniques (light microscopy and
scanning- or transmission-electron microscopy, flow cytometry, DNA
electrophoresis), in two different conditions inhibiting D. discoideum
multicellular development. The first one, corresponding to an induced
unicellular cell death, was obtained by starving the cells in a
"conditioned" cell-free buffer, prepared by previous starvation of another
D. discoideum cell population in potassium phosphate buffer (pH 6.8). The
second one, corresponding to death of D. discoideum after axenic growth in
suspension, was obtained by keeping stationary cells in their culture
medium. In both cases of these unicellular-specific cell deaths, microscopy
revealed morphological features known as hallmarks of apoptosis for higher
eukaryotic cells and apoptosis was further corroborated by flow cytometry.
The occurrence in D. discoideum of programmed cell death with two different
phenotypes, depending on its multicellular or unicellular status, is further
discussed.
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A non-Golgi a1,2-Fucosyltransferase that Modifies Skp1 in the Cytoplasm
of Dictyostelium
Hanke van der Wel@, Howard R. Morris§, Maria Panico§, Thanai Paxton§, Simon J.
North§, Anne Dell§, J. Michael Thomsoný and Christopher M. West@
@ Dept. of Anatomy & Cell Biology, University of Florida College of Medicine,
Gainesville, FL 32610-0235 USA, §Dept. of Biochemistry, Imperial College,
London SW7 2AY United Kingdom
J. Biol. Chem., in press
Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle
and other regulatory factors for degradation. In Dictyostelium, Skp1 is
modified by a pentasaccharide containing the type 1 blood group H
trisaccharide at its core. To address how the third sugar, fucose a1,2-
linked to galactose, is attached, a proteomics strategy was applied to
determine the primary structure of FT85, previously shown to copurify
with the GDP-Fuc:Skp1 a1,2-fucosyltransferase. Tryptically-generated
peptides of FT85 were sequenced de novo using Q-TOF tandem mass
spectrometry. Degenerate primers were used to amplify FT85 genomic
DNA, which was further extended by a novel linker-PCR method to yield
an intronless open reading frame of 768 amino acids. Disruption of the
FT85 gene by homologous recombination resulted in viable cells which had
altered light scattering properties as revealed by flow cytometry. FT85
was necessary and sufficient for Skp1-fucosylation, based on biochemical
analysis of FT85-mutant cells and E. coli expressing FT85 recombinantly.
FT85 lacks sequence motifs that characterize all other known a1,2-
fucosyltransferases, and lacks the signal-anchor sequence that targets
them to the secretory pathway. The C-terminal region of FT85 harbors
motifs found in inverting family 2 glycosyltransferase domains, and its
expression in FT85-mutant cells restores fucosyltransferase activity
toward a simple disaccharide substrate. Whereas most prokaryote and
eukaryote family 2 glycosyltransferases are membrane-bound and oriented
toward the cytoplasm where they glycosylate lipid-linked or polysaccharide
precursors prior to membrane translocation, the soluble, eukaryotic Skp1-
fucosyltransferase modifies a protein that resides in the cytoplasm and
nucleus.
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Expression Patterns of Cell-type Specific Genes in Dictyostelium
Negin Iranfar, Danny Fuller, Roman Sasik@, Terence Hwa@, Michael Laub* and
William F. Loomis#
Division of Biology, and @ Department of Physics, U.C.S.D. La Jolla, CA 92093
* Department of Cell and Developmental Biology, Stanford Medical School,
Stanford, CA
Molecular Biology of the Cell, in press
Abstract
Cell-type specific genes were recognized by interrogating
microarrays carrying Dictyostelium gene fragments with probes prepared from
fractions enriched in prestalk and prespore cells. Cell-type specific
accumulation of mRNA from 17 newly identified genes was confirmed by
Northern analyses. DNA microarrays carrying 690 targets were used to
determine expression profiles during development. The profiles were fit to
a biologically based kinetic equation to extract the times of
transctription onset and cessation. While the majority of the genes that
were cell type enriched at the slug stage were first expressed as the
prespore and prestalk cells sorted out in aggregates, some were found to be
expressed earlier before the cells had even aggregated. These early genes
may have been initially expressed in all cells and then preferentially
turned over in one or the other cell type. Alternatively, cell type
divergence may start soon after the initiation of development.
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The protein kinase YakA regulates G-protein linked signaling responses
during growth and development of Dictyostelium
Saskia van Es, Karin E. Weening, and Peter N. Devreotes
Department of Cell Biology and Anatomy, Johns Hopkins University, School of
Medicine, Baltimore, MD 21205
J. Biol. Chem. In press.
Abstract
A genetic screen for Dictyostelium mutants that phenotypically resemble
cells lacking the G-protein b-subunit yielded the protein kinase YakA.
Like gb null cells, yakA null cells fail to enter development and display
slow growth on bacterial lawns. We created a temperature sensitive YakA
mutant and showed that YakA activity is required not only at the onset but
also during development. The yakA null cells have strong defects in folic
acid induced responses, such as actin polymerization and cGMP accumulation,
indicating that they play a role in G-protein mediated signaling responses.
We propose that YakA acts downstream of G-proteins, because cAMP receptors
still couple to G-proteins in the yakA mutant. In addition, the previously
observed growth arrest induced by overexpression of YakA also occurs in gb
mutants. We localized YakA-GFP to the cytosol suggesting that YakA may be
a functional homologue of its mammalian counterparts Dyrk2 and Dyrk3, a
subclass of Dual-specificity Yak Related Kinases (Dyrk) with unknown function.
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Sphingosine-1-phosphate lyase has a central role in the development of
Dictyostelium discoideum
Guochun Li, Christopher Foote, Stephen Alexander and Hannah Alexander
Division of Biological Sciences, University of Missouri, Columbia, MO
65211-7400
Development, in press.
SUMMARY
Sphingosine-1-phosphate, a product of sphingomyelin degradation, is an
important element of signal transduction pathways regulating cell
proliferation and cell death. In this work we demonstrate additional roles
for sphingosine-1-phosphate in growth and multicellular development. The
specific disruption in Dictyostelium discoideum of the sphingosine-1-phosphate
lyase gene, which encodes the enzyme catalyzing sphingosine-1-phosphate
degradation, results in a mutant strain with aberrant morphogenesis as well
as an increase in viability during stationary phase. The absence of
sphingosine-1-phosphate lyase affects multiple stages throughout development
including the cytoskeletal architecture of aggregating cells, the ability
to form migrating slugs, and the control of cell-type specific gene
expression and terminal spore differentiation. This pleiotropic effect
due to the loss of sphingosine-1-phosphate lyase establishes sphingolipids
as pivotal regulatory molecules in a wide range of processes in multicellular
development.
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Technical innovation
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[Editor's note: Although not yet in press, many of you may be interested in
the following abstract of what promises to be a valuable technical advance]
Making ts mutants in essential genes in Dictyostelium
Mark S Bretscher and Chris Thompson,
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England.
We decided to try to make a ts mutant in an essential gene in Dictyostelium
discoideum Ax2, and selected the single gene encoding NSF (the NEM-sensitive
factor which appears to be required in all membrane fusion processes) that
exists in Dicty (Weidenhaupt et al, 1998) as a target. Our strategy was
basically to adapt the current procedure used for gene knockouts, hoping
to replace the endogenous gene with one putatively encoding a temperature
sensitive allele. There are several temperature-sensitive mutants in NSF
in Drosophila and two of these, comatose 4 and 6, occur in highly conserved
regions. We therefore constructed a Dicty NSF vector which contained, in the
linear mutagenic portion: about 300bp upstream of the gene, the gene itself
(including a 100 bp intron, ~2200bp in all) containing the comatose 4 site
(marked with a BssHII site, at position1270 from the 5' end of the vector)
or 6 site (marked with an AflII site, at position1640), followed by 200bp
trailer, a blasticidin casette (1350bp) and a further 1300bp of 3' genomic
sequence.
These constructs were electroporated into Ax2 in the same manner as
for gene knockouts. The cells were then parcelled out into wells and clones
selected for drug resistance. These were recloned and grown up andthe DNA
from 18 of each mutant subjected to Southern blotting. This showed that in
each case, 17/18 clones had arisen by homologous recombination. In the case
of comatose 4, none of the clones contained the desired mutation; conversely,
in the case of comatose 6, all of them contained the desired site. Furthermore,
in clones in which an in-frame termination codon had been placed just after
the comatose 6 site, all 12 clones examined had recombined homologously, but
none now carried the comatose 6 mutation. This shows that the gene is essential
and suggests that when it contains the comatose 4 site, the encoded NSF is
non-functional. The comatose 6 clones had, unfortunately, no new phenotype.
We therefore made a mutagenic library in which the last 1000bp of coding
sequence was made by pcr mutagenesis. Ax2 cells were transformed as before and
positive wells recloned. About 1500 such clones were generated and each was
screened for growth at 22°C and 26.5°C. From this, 11 ts mutants were
recovered; they all arose by homologous recombination. Their properties are
under study.
Reference
Weidenhaupt,M., Bruckert,F. and Satre, M. (1998) Gene 207, 53-60.
We have been greatly assisted in this by David Traynor and, especially, Rob
Kay.
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[End Dicty News, volume 16, number 12]
