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dictyNews Volume 21 Number 12
Dicty News
Electronic Edition
Volume 21, number 12
October 17, 2003
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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Compromise of Clathrin Function and Membrane Association by Clathrin Light
Chain Deletion
Jingshan Wang, Valerie C. Virta, Kathryn Riddelle-Spencer, and
Theresa J. O'Halloran
Traffic, in press
While clathrin heavy chains from different species are highly conserved in
amino acid sequence, clathrin light chains are much more divergent. Thus
clathrin light chain may have different roles in different organisms. To
investigate clathrin light chain function, we cloned the clathrin light
chain, clcA, from Dictyostelium and examined clathrin function in clcA-
mutants. Phenotypic deficiencies in development, cytokinesis, and
osmoregulation showed that light chain was critical for clathrin function
in Dictyostelium. In contrast with budding yeast, we found the light chain
did not influence steady state levels of clathrin, triskelion formation, or
contribute to clathrin overassembly on intracellular membranes. Imaging
GFP-CHC in clcA- mutants showed that the heavy chain formed dynamic punctate
structures that were remarkably similar to those found in wildtype cells.
However, clathrin light chain knockouts showed a decreased association of
clathrin with intracellular membranes. Unlike wildtype cells, half of the
clathrin in clcA- mutants was cytosolic, suggesting that the absence of
light chain compromised the assembly of triskelions onto intracellular
membranes. Taken together, these results suggest a role for the
Dictyostelium clathrin light chain in regulating the self-assembly of
triskelions onto intracellular membranes, and demonstrate a crucial
contribution of the light chain to clathrin function in vivo.
Submitted by: Terry O'Halloran [t.ohalloran@mail.utexas.edu]
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Transfer RNA gene-targeted integration: an adaptation of retrotransposable
elements to survive in the compact Dictyostelium discoideum genome
Thomas Winckler(1), Karol Szafranski(2) and Gernot Gloeckner(2)
1 Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt/M.
(Biozentrum), D-60439 Frankfurt am Main, Germany
2 IMB Jena, Department of Genome Analysis, D-07745 Jena, Germany
Cytogenetic and Genome Research, in press
REVIEW
Almost every organism carries along a multitude of molecular parasites known
as transposable elements (TEs). TEs influence their host genomes in many ways
by expanding genome size and complexity, rearranging genomic DNA, mutagenizing
host genes, and altering transcription levels of nearby genes. The eukaryotic
microorganism Dictyostelium discoideum is attractive for the study of
fundamental biological phenomena such as intercellular communication,
formation of multicellularity, cell differentiation, and morphogenesis.
D. discoideum has a highly compacted, haploid genome with less than 1 kb of
genomic DNA separating coding regions. Nevertheless, the D. discoideum genome
is loaded with 10% TEs that managed to settle and survive in this inhospitable
environment. In depth analysis of D. discoideum genome project data has
provided intriguing insights into the evolutionary challenges that mobile
elements face when they invade compact genomes. Two different mechanisms are
used by D. discoideum TEs to avoid disruption of host genes upon
retrotransposition. Several TEs have invented the specific targeting of tRNA
gene-flanking regions as a means to avoid integration into coding regions.
These elements have been dispersed on all chromosomes, closely following the
distribution of tRNA genes. By contrast, TEs that lack bona fide integration
specificities show a strong bias to nested integration, thus forming large
TE clusters at certain chromosomal loci that are hardly resolved by
bioinformatics approaches. We summarize our current view of D. discoideum TEs
and present new data from the analysis of the complete sequences of
D. discoideum chromosomes 1 and 2, which comprise more than one third of the
total genome.
Submitted by: Thomas Winckler [winckler@em.uni-frankfurt.de]
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Actin-binding proteins required for reliable chromosome segregation in mitosis
Guenther GerischÊ1, Jan Faix 2, Jana Koehler 1,Êand
Annette Mueller-Taubenberger 1
1 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany;
2 Ludwig-Maximilians-Universitaet, Institut fuer Zellbiologie, D-80336
Muenchen, Germany.
Cell Motility and the Cytoskeleton, in press
While studying mitosis in Dictyostelium mutants with deficiencies in
actin-binding proteins, we found that two of these proteins, cortexillin and
Aip1, are required for the precise segregation of chromosomes. Atypical
spindles and nuclei with varying DNA content indicate that mutants lacking
cortexillin or Aip1 are genetically unstable. These aberrations are caused
by the detachment and irregular reattachment of centrosomes to the nuclear
surface. Live imaging showed how coalescing mitotic complexes give rise to
a multipolar spindle, and how excess centrosomes can be eliminated by
mitotic cleavage between anucleate and nucleated portions of a cell. We
hypothesize that mutations in regulatory proteins of the actin network
might be one cause of genetic instability of malignant tumor cells.
Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de]
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Dictyostelium discoideum: Cellular slime mold (Review)
Annette Mueller-Taubenberger 1, and Markus Maniak 2
1 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany;
2 Universitaet Gh Kassel, Department of Cell Biology, D-34132 Kassel,
Germany.
Accepted, Encyclopedia of Molecular Cell Biology and Molecular Medicine
(ed. R. A. Meyers), Wiley-VCH
Dictyostelium discoideum is a tractable system for the study of basic
processes in cell and developmental biology. These processes include
signal transduction, chemotaxis, motility, cytokinesis, phagocytosis,
and aspects of development such as cell sorting, pattern formation and
cell-type differentiation. Dictyostelium has unique advantages for
studying fundamental biological questions, since it is particularly
amenable to both genetic and biochemical analyses. Dictyostelium has
been chosen, along with several other organisms with known genomes, as a
model system to analyze the functions of the identified genes. Biological
questions in Dictyostelium have mainly addressed the function of individual
gene products, but more systematic approaches have now been launched based
on the knowledge derived from genome-, proteome- and cDNA-projects. Recent
studies on the genetics of Dictyostelium have opened new directions in
biomedical research. This review summarizes the current knowledge on the
Dictyostelium genome, updates the state of the Dictyostelium genome project
and concentrates on the molecular methods currently used to explore the
functions of individual gene products.
Submitted by: Annette Mueller-Taubenberger [amueller@biochem.mpg.de]
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Two components of a secreted cell-number counting factor bind to cells and
have opposing effects on cAMP signal transduction in Dictyostelium
Debra A. Brock1, Karen Ehrenman1, Robin Ammann1, Yitai Tang2, and
Richard H. Gomer1,2
1Howard Hughes Medical Institute and 2Department of Biochemistry and
Cell Biology, Rice University, Houston, TX
J. Biol. Chem., in press
A secreted 450 kDa complex of proteins called counting factor (CF)
is part of a negative feedback loop that regulates the size of the groups
formed by developing Dictyostelium cells. Two components of CF are countin
and CF50. Both recombinant countin and recombinant CF50 decrease group size
in Dictyostelium. countinø cells have a decreased cAMP-stimulated cAMP
pulse while recombinant countin potentiates the cAMP pulse. We find that
cf50ø cells have an increased cAMP pulse while recombinant CF50 decreases
the cAMP pulse, suggesting that countin and CF50 have opposite effects on
cAMP signal transduction. In addition, countin and CF50 have opposite
effects on cAMP-stimulated erk2 activation. However, like recombinant
countin, recombinant CF50 increases cell motility. We previously found
that cells bind recombinant countin with a Hill coefficient of
approximately 2, a KH of 60 pM and approximately 53 sites/ cells. We find
here that cells also bind 125I-recombinant CF50, with a Hill coefficient of
approximately 2, a KH of approximately 15 ng/ ml (490 pM), and
approximately 56 sites/ cell. Countin and CF50 require each other's presence
to affect group size, but the presence of countin is not necessary for CF50 to
bind to cells, and CF50 is not necessary for countin to bind to cells. Our
working hypothesis is that a signal transduction pathway activated by countin
binding to cells modulates a signal transduction pathway activated by CF50
binding to cells, and vice versa, and that these two pathways can be
distinguished by their effects on cAMP signal transduction.
Submitted by: Richard Gomer [richard@rice.edu]
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Dd-STATb, a Dictyostelium STAT protein with a highly aberrant SH2 domain,
functions as a regulator of gene expression during growth and early development
N. V. Zhukovskaya*, M. Fukuzawa*, M. Tsujioka, K. A. Jermyn, T. Kawata,
T. Abe, M. Zvelebil^ and
J. G. Williams+
School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street,
Dundee DD1 5EH, UK
^University College London, Ludwig Institute for Cancer Research,
The Cruciform Building, Gower Street, London WC1E 6BT, UK
ÊÊÊÊ * These authors contributed equally to this work
Development, in press
Dictyostelium, the only known non-metazoan organism to employ SH2
domain:phosphotyrosine signaling, possesses STATs (Signal Transducers and
Activators of Transcription) and protein kinases with orthodox SH2 domains.
Here, however, we describe a novel Dictyostelium STAT containing a remarkably
divergent SH2 domain. Dd-STATb displays a 15 amino acid insertion in its SH2
domain and the conserved and essential arginine residue, that interacts with
phosphotyrosine in all other known SH2 domains, is substituted by leucine.
Despite these abnormalities, Dd-STATb is biologically functional. It has a
subtle role in growth, so that Dd-STATb null cells are gradually lost from
the population when they are co-cultured with parental cells, and micro-array
analysis identified several genes that are either under-expressed or
over-expressed in the Dd-STATb null strain. The best characterised of these,
discoidin 1, is a marker of the growth-development transition and it is
over-expressed during growth and early development of Dd-STATb null cells.
Dimerisation of STAT proteins occurs by mutual SH2 domain:phosphotyrosine
interactions and dimerisation triggers STAT nuclear accumulation. Despite
its aberrant SH2 domain, the Dd-STATb protein sediments at the size expected
for a homo-dimer and it is constitutively enriched in the nucleus. Moreover,
these properties are retained when the predicted site of tyrosine
phosphorylation is substituted by phenylalanine. These observations suggest
a non-canonical mode of activation of Dd-STATb, that does not rely on
orthodox SH2 domain:phosphotyrosine interactions.
Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk]
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[End Dicty News, volume 21, number 12]
