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dictyNews Volume 19 Number 12
Dicty News
Electronic Edition
Volume 19, number 12
November 30, 2002
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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An RGS-Containing Kinase is Important for Chemotaxis and Multicellular
Development in Dictyostelium
Binggang Sun and Richard A. Firtel
in press: Molecular Biology of the Cell
ABSTRACT
We have identified a gene encoding RCK1, a novel RGS domain-containing
protein kinase. RCK1 mutant strains exhibit strong aggregation and
chemotaxis defects. rck1 null cells chemotax ~50% faster than wild-type
cells, suggesting RCK1 plays a negative regulatory role in chemotaxis.
Consistent with this finding, overexpression of wild-type RCK1 reduces
chemotaxis speed by ~40%. Upon cAMP stimulation, RCK1 transiently
translocates to the membrane/cortex region with membrane localization
peaking at ~10 sec, similar to the kinetics of membrane localization of
the PH domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase
activity also increases dramatically. The RCK1 kinase activity does not
rapidly adapt, but decreases after the cAMP stimulus is removed. This is
particularly novel considering that most other chemoattractant-activated
kinases (e.g. Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation.
Using site-directed mutagenesis, we further show that both the RGS and
kinase domains are required for RCK1 function and that RCK1 kinase activity
is required for the delocalization of RCK1 from the plasma membrane.
Genetic evidence suggests RCK1 function lies downstream from Ga2, the
heterotrimeric G protein that couples to the cAMP chemoattractant receptors.
We suggest that RCK1 might be part of an adaptation pathway that regulates
aspects of chemotaxis in Dictyostelium.
submitted by: Rick Firtel [rafirtel@ucsd.edu]
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THE ROLE OF BEACH PROTEINS IN DICTYOSTELIUM.
Arturo De Lozanne
Section of Molecular Cell & Developmental Biology
and Institute for Cellular and Molecular Biology,
University of Texas at Austin, Austin, TX 78712.
TRAFFIC, in press.
ABSTRACT
The BEACH family of proteins is a novel group of proteins with diverse roles
in eukaryotic cells. The identifying feature of these proteins is the BEACH
domain named after the founding members of this family, the mouse beige and
the human Chediak-Higashi Syndrome (CHS) proteins. Although all BEACH
proteins share a similar structural organization they appear to have very
distinct cellular roles ranging from lysosomal traffic, to apoptosis and
cytokinesis. Very little is currently known about the function of most of
these proteins, few binding-partner proteins have been identified and no
molecular mechanism for any of these proteins has been discovered. Thus,
it is important to establish good model systems for the study of these
novel proteins. Dictyostelium contains six BEACH proteins that can be
classified into four subclasses. Two of them, LvsA and LvsB, have clearly
distinct roles in the cell. LvsA is localized on the contractile vacuole
membrane and is essential for cytokinesis and osmoregulation. LvsB is
most similar in sequence to the mammalian beige/CHS proteins and shares
with them a common function in lysosomal trafficking. Structural and
functional analysis of these proteins in Dictyostelium will help elucidate
the function of this enigmatic novel family of proteins.
submitted by: (a.delozanne@mail.utexas.edu)
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The Dictyostelium prestalk cell inducer DIF regulates nuclear accumulation
of a STAT protein by controlling its rate of export from the nucleus
Masashi Fukuzawa+, Tomoaki Abe+ and Jeffrey G. Williams*,
School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street,
Dundee DD1 5EH, UK
+ These two authors contributed equally to this work
DEVELOPMENT IN PRESS
Summary
Dd-STATc becomes tyrosine phosphorylated, dimerises and accumulates in the
nuclei of Dictyostelium cells exposed to DIF, the chlorinated hexaphenone
that directs prestalk cell differentiation. By performing cytoplasmic
photobleaching of living cells, we show that DIF inhibits the nuclear
export of Dd-STATc. Within Dd-STATc there is a 50 amino acid region
containing several consensus CRM1 (exportin 1)-dependent nuclear export
signals (NESs). Deletion of this region causes Dd-STATc to accumulate in
the nucleus constitutively and, when coupled to GFP, the same region directs
nuclear export. We show that the N terminal-proximal 46 amino acids are
necessary for nuclear accumulation of Dd-STATc and sufficient to direct
constitutive nuclear accumulation when fused to GFP. Combining the
photobleaching and molecular analyses, we suggest that DIF-induced
dimerisation of Dd-STATc functionally masks the NES-containing region
and that this leads to nett nuclear accumulation, directed by the N
terminal-proximal import signals. These results show that the regulated
nuclear accumulation of a STAT protein can be controlled at the level of
nuclear export and they also provide a better understanding of the mechanism
whereby DIF directs cell type divergence.
submitted by: Jeff Williams [j.g.williams@dundee.ac.uk]
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Mitochondrial membrane dynamics are altered in cluA- mutants of
Dictyostelium.
Fields, S.D., Arana, Q., Heuser, J., and Clarke, M.
Musc. Res. Cell Motil. (special Dictyostelium issue), in press.
In cluA- mutants of Dictyostelium, mitochondria are clustered near the cell
center rather than being dispersed throughout the cytoplasm. We have examined
two possible mechanisms that could account for this phenotype. First, we
sought evidence that the cytoskeleton or a presumptive mitochondrion-
cytoskeleton linkage was altered in mutant cells. We found that cytoskeletal
structures in cluA- cells appeared normal by immunostaining, and that the
distribution of peroxisomes in mutant cells was indistinguishable from that
in wild type cells. Treatment of wild type cells with drugs that disrupted
microtubules or actin filaments did not mimic the cluA- phenotype. Thus,
cytoskeletal defects seemed unlikely to account for the mitochondrial
clustering in cluA- cells. Observation of the movement of GFP-tagged
mitochondria in wild type cells suggested that mitochondria are transported
along microtubules, as in mammalian cells, rather than along actin filaments,
as in budding yeast. Therefore, the similar phenotypes of cluA- Dictyostelium
cells and clu1D yeast cells argued against CluA/Clu1p acting as a
mitochondrion-cytoskeleton linker. We next examined the ultrastructure of
mitochondria in freeze-substituted, thin-sectioned cells. We found that
the clustered mitochondria in cluA-cells are interconnected. Often, adjacent
mitochondria are linked by narrow membranous strands, although sometimes the
mitochondria are partially merged. The presence of narrow constrictions at
presumptive division sites argues that the constriction step of division
proceeds normally. Our data suggest that cluA- cells may be blocked at a
very late step in fission of the outer mitochondrial membrane.
submitted by: Margaret Clarke, PhD [Margaret-Clarke@mail.omrf.ouhsc.edu]
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Chaperonin 60 and mitochondrial disease in Dictyostelium.
Martha Kotsifas, Christian Barth, Arturo de Lozanne, Sui T. Lay, and Paul
R. Fisher
Journal of Muscle Research and Cell Motility. In Press.
Abstract
The single Dictyostelium chaperonin 60 gene, hspA, was cloned, sequenced
and characterized. Sequence comparisons and a 3-D model for the structure
of the encoded protein showed that it exhibits the conserved sequence and
structural features expected for its role as the Dictyostelium mitochondrial
chaperonin 60. Dictyostelium hspA contains two introns and, unusually for
a member of this major heat shock gene family, is not stress-inducible in
response to heat, cold or cadmium ions. Although transcription of hspA is
down regulated during early Dictyostelium development in response to
starvation, the levels of the chaperonin 60 protein remain constant
throughout the life cycle. Consistent with the essential role of
chaperonin 60 in mitochondrial biogenesis, we were unable to isolate
mutants in which the hspA gene had been disrupted. However, transformants
were isolated that exhibited differing levels of antisense inhibition of
chaperonin 60 expression, depending upon the number of copies of the
antisense-expressing plasmid in the genome. Orientation in phototaxis
(and thermotaxis) was severely impaired in all antisense transformants,
while growth and morphogenesis were markedly defective only in
transformants with higher levels of antisense inhibition. This pattern
of phenotypes is similar to that reported previously to result from
targeted disruption of the mitochondrial large subunit rRNA gene in a
subpopulation of mitochondria. This suggests that, regardless of the
nature of the underlying genetic defect, mitochondrial deficiency
impairs signal transduction more sensitively than other cellular
activities.
submitted by: Paul Fisher [SMTP:fisher@lumi.micro.latrobe.edu.au]
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Multiple Signalling Pathways Connect Chemoattractant Receptors and Calcium
Channels in Dictyostelium
Thomas Nebl1, Martha Kotsifas1, Pauline Schaap2? and Paul R. Fisher1,*
Journal of Muscle Research and Cell Motility. In Press.
SUMMARY
Dictyostelium mutants expressing aequorin were used to study and compare
the roles of heterotrimeric G-proteins and the second messengers IP3 and
cGMP in regulating folate- and cAMP receptor-activated [Ca2+]i signals.
The calcium responses of vegetative cells to folate were dramatically
impaired in Gb and Ga4 null mutants but were restored with altered kinetics
and temperature-sensitivity in Gb null mutants overexpressing wild type
and temperature-sensitive Gb isoforms. Folic acid receptors thus mediate
changes in [Ca2+]i via a Ga4bg-dependent pathway. Neither folate nor cAMP-
induced [Ca2+]i signals were significantly altered in PLC null
transformants, but [Ca2+]i changes elicited by both attractants were
significantly prolonged in two stmF mutants lacking cGMP-specific
phosphodiesterase activity. This confirms an important role of cGMP in
regulating receptor-activated Ca2+ uptake and/or extrusion systems. This
cGMP-dependent part of the Ca2+ response to cAMP stimuli was developmentally
down-regulated and all but disappeared by the time the cells reached full
aggregation competence after 8 hours of starvation. The results suggest
that folate and cAMP receptor-activated [Ca2+]i signals are regulated in a
complex manner via multiple signalling pathways, one that is G-protein-
and cGMP-dependent (present at the vegetative and early poststarvation
stage) and another that is G-protein-independent (dominant in fully
aggregation-competent cells at ~ 8 hours post starvation).
submitted by: Paul Fisher [SMTP:fisher@lumi.micro.latrobe.edu.au]
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[End Dicty News, volume 19, number 12]
