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dictyNews Volume 34 Number 01
dictyNews
Electronic Edition
Volume 34, number 1
January 8, 2010
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.
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=========
Abstracts
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Mitochondrial processing peptidase activity is controlled by the processing
of alpha-MPP during development in Dictyostelium discoideum
Koki Nagayama a),b),† and Tetsuo Ohmachi b)
a) Science of Bioresources, United Graduate School of Agricultural Sciences,
Iwate University, Morioka, Iwate 020-8551, Japan
b) Department of Biochemistry and Biotechnology, Faculty of Agriculture and
Life Science, Hirosaki University, Hirosaki, 036-8561, Japan
† Present address: Faculty of Life Sciences, University of Manchester,
Manchester, M13 9PT, UK
Microbiology, in press
We investigated the expression of the a subunit of the Dictyostelium
mitochondrial processing peptidase (Dda-MPP) during development.
Dda-MPP mRNA is expressed at highest levels in vegetatively growing
cells and during early development, and is markedly down-regulated
after 10 h of development. The Dda-MPP protein is expressed as two
forms, designated a-MPPH and a-MPPL, throughout the Dictyostelium l
ife cycle. The larger form, a-MPPH, is cleaved to produce the functional
a-MPPL form. We were not able to isolate mutants in which the a-mpp
gene had been disrupted. Instead, an antisense transformant aA2
expressing a-MPP at a lower level than the wild-type AX-3 was isolated
to examine the function of the a-MPP protein. Development of the aA2
strain was normal until the slug formation stage, but the slug stage was
prolonged to ~24 h. In this prolonged slug stage, only a-MPPH was
present, and a-MPPL protein and MPP activity were not detected. After
28 h, a-MPPL and MPP activity reappeared, and normal fruiting bodies
were formed after a delay of approximately 8 h compared with normal
development. These results indicate that MPP activity is controlled by the
processing of a-MPPH to a-MPPL, during development in Dictyostelium.
Submitted by Tetsuo Ohmachi [tohmachi@cc.hirosaki-u.ac.jp]
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Genetic heterogeneity in wild isolates of cellular slime mould social groups
Santosh Sathe1*, Sonia Kaushik1, Albert Lalremruata2, Ramesh K. Aggarwal2,
James C. Cavender3 and Vidyanand Nanjundiah1
1Centre for Ecological Sciences, Indian Institute of Science, Bangalore
560012, India
2Centre for Cellular and Molecular Biology, Hyderabad 500007, India;
3Department of Environmental and Plant Biology, Ohio University,
Athens, Ohio 45701, USA.
Microbial Ecology, in press
Knowledge of the genetic structure of social groups in the wild is necessary
for explaining the evolutionary origin and maintenance of sociality in the
cellular slime moulds (CSMs). This study addresses the issues of spatial
distribution, dispersal and genetic heterogeneity in social groups of CSMs.
We confirm that CSMs are widespread in undisturbed forest soil from South
India. They are also seen in the dung of a variety of large mammals. The
species found by us include Dictyostelium discoideum, D. purpureum,
D. giganteum, D. rosareum, D. polycephalum, D. minutum, D. macrocephalum,
Dsp (bifurcating – awaiting further description), Polysphondylium pallidum and
P. violaceum; others await identification. Consistent with the mode of dispersal
that this implies, most social groups in the two species examined for detailed
study, Dictyostelium giganteum and D. purpureum, are multi-clonal. 9 out of
11 D. giganteum and 6 out of 6 D. purpureum fruiting bodies that were
examined, meaning 15 out of 17 fruiting bodies in all, were chimeras (8 of
those fruiting bodies were from animal dung and 9 from soil). The minimum
number of distinct genotypes in a single fruiting body was 3 to 7 (animal dung)
and 1 to 9 (soil). Taken together with earlier findings on functional heterogeneity
between Dictyostelids found in the same microhabitat, these observations have
implications for models of the evolution of social behaviour in the CSMs.
Submitted by Santosh Sathe [santosh_sathe@ces.iisc.ernet.in]
--------------------------------------------------------------------------------
Digital nature of the immediate-early transcriptional response
Michelle Stevense, Tetsuya Muramoto, Iris Müller and Jonathan R. Chubb
Development, in press
Stimulation of transcription by extracellular signals is a major component of cells’
decision making. Yet the quantitative relationship between signal and acute
transcriptional response is unclear. One view is that transcription is directly graded
with inducer concentration. In an alternative model the response occurs only above
a threshold inducer concentration. Standard methods for monitoring transcription
lack continuous information from individual cells or mask immediate-early
transcription by measuring downstream protein expression. We have therefore
used a technique for directly monitoring nascent RNA in living cells, to quantify
the direct transcriptional response to an extracellular signal in real time, in single
cells. At increasing doses of inducer, increasing numbers of cells displayed a
transcriptional response. However, over the same range of doses, the change in
cell response strength, measured as the length, frequency and intensity of
transcriptional pulses, was small, with considerable variation between cells. These
data support a model where cells have different sensitivities to developmental
inducer and respond in a digital manner above individual stimulus thresholds.
Biased digital responses may be necessary for certain forms of developmental
specification. Limiting bias in responsiveness is required to reduce noise in
positional signalling.
Submitted by Jonathan Chubb [j.chubb@dundee.ac.uk]
--------------------------------------------------------------------------------
Methylation of H3K4 is required for inheritance of active transcriptional states
Tetsuya Muramoto, Iris Müller, Giles Thomas, Andrew Melvin and
Jonathan R. Chubb
University of Dundee, United Kingdom
Current Biology, in press
Background
Maintenance of differentiation programmes requires stability, when
appropriate, of transcriptional states. However, the extent to which
inheritance of active transcriptional states occurs from mother to
daughter cells has not been directly addressed in unperturbed cell
populations.
Results
By live imaging of single gene transcriptional events in individual
cells, we have directly recorded the potential for mitotic inheritance
of transcriptional states down cell lineages. Our data showed strong
similarity in frequency of transcriptional firing between mother and
daughter cells. This memory persisted for complete cell cycles. Both
transcriptional pulse length and pulsing rate contributed to overall
inheritance, and memory was determined by lineage, not cell environment.
Analysis of transcription in chromatin mutants demonstrated the histone
H3K4 methylase Set1, and Ash2, a component of the methylase complex,
were required for memory. The effects of Set1 methylation may be
mediated directly by chromatin, as loss of memory also occurred when
endogenous H3K4 was replaced by alanine. Although methylated H3K4
is usually associated with active transcriptional units, the modification
was not required for gene activity, but stabilised transcriptional
frequency between generations.
Conclusions
Our data indicate methylated H3K4 can act as a chromatin mark reflecting
the original meaning of “epigenetic”.
The University of Dundee is a registered Scottish charity, No: SC015096
Submitted by Tetsuya Muramoto [t.muramoto@dundee.ac.uk]
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A new Dictyostelium prestalk cell sub-type
Yoko Yamada1, Robert R. Kay3, Gareth Bloomfield2,3, Susan Ross1,
AlasdairIvens2, and Jeffrey G. Williams1*
School of Life Sciences1,University of Dundee, Dow St., Dundee DD3 5EH, UK
WellcomeTrust Sanger Institute2, Hinxton,UK
MRC Laboratory of Molecular Biology3, Hills Road, Cambridge, CB2 2QH, UK
*Author for correspondence
e-mail:j.g.williams@dundee.ac.uk
Dev. Biol., in press
The mature fruiting body of Dictyostelium consists of stalk and spore
cells but its construction, and the migration of the preceding slug
stage, requires a number of specialized sub-types of prestalk cell whose
nature and function are not well understood. The prototypic
prestalk-specific gene, ecmA, isinducible by the polyketide DIF-1 in a
monolayer assay and requires the DimB and MybE transcription
factors for full inducibility. We perform genome-wide micro-array
analyses, on parental, mybE-and dimB- cells, and identify many
additional genes that depend on MybE and DimB for their DIF-1
inducibility. Surprisingly, an even larger number of genes are only
DIF-inducible in mybE- cells, some genes are only inducible in DimB-
cells and some are inducible when either transcription factor is absent.
Thus in assay conditions where MybE and DimB function as inducers of
ecmA these genes fall under negative control by the same two
transcription factors. We have studied in detail rtaA, one of the MybE and
DimB repressed genes. One especially enigmatic group of prestalk cells
is the anterior-like cells (ALCs), which exist intermingled with
prespore cells in the slug. A promoter fusion reporter gene, rtaA:galu,
is expressed in a sub-set of the ALCs that is distinct from the ALC
population detected by a reporter construct containing ecmA and ecmB
promoter fragments. At culmination, when the ALC sort out from the
prespore cells and differentiate to form three ancillary stalk cell
structures: the upper cup, the lower cup and the outer basal disc, the
rtaA:galu expressing cells preferentially populate the upper cup region.
This fact, and their virtual absence from the anterior and
posterior regions of the slug, identifies them as a new prestalk
sub-type: the pstU cells.PstU cell differentiation is, as expected,
increased in a dimB- mutant during normal development but, surprisingly,
they differentiate normally in a mutant lacking DIF. Thus genetic removal
of MybE or DimB reveals an alternate DIF-1 activation pathway, for pstU
differentiation, that functions under monolayer assay conditions but that
is not essential during multicellular development.
Submitted by Jeff Williams [j.g.williams@dundee.ac.uk]
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Dual regulation of a Dictyostelium STAT by cGMP and calcium signalling
Tsuyoshi Araki1, Wouter N.van Egmond2,Peter J. M. van Haastert2, and
Jeffrey G.Williams1+
University of Dundee1, College of Life Sciences, DowStreet, Dundee DD1 5EH, UK
University of Groningen2, Kerklaan 30, 9751NN Haren, NL
+ author for correspondence:
j.g.williams@dundee.ac.uk
J. Cell Sci., in press
When cells are exposed to hyper-osmotic stress the Dictyostelium STAT
orthologue STATc is rapidly tyrosine phosphorylated. Previous
observations suggest a non-paradigmatic mode of STAT activation, whereby
stress-induced serine phosphorylation of the PTP3 protein tyrosine
phosphatase inhibits its activity towards STATc. We show that two serine
residues in PTP3, S448 and S747, are rapidly phosphorylated after osmotic
stress. cGMP is a second messenger for hyper-osmotic stress responses and
8-bromo-cGMP, a membrane permeant form of cGMP, is a known activator of
STATc. GbpC, a cGMP binding Ras-GEFprotein, is a founder member of a
protein family that includes LRRK2, the gene commonly mutated in familial
Parkinsons disease. Genetic ablation of gbpC prevents STATc activation
by 8-bromo-cGMP. However, osmotic stress-induced activation of STATc
occurs normally in the gbpC null mutant. Moreover, 8-bromo-cGMP does
not stimulate phosphorylation of S747 and S448 of PTP3.
These facts imply redundant activation pathways and we present evidence
that intracellular calcium is a parallel second messenger; by showing
that agents that elevate intracellular calcium are potent STATc
activators that do stimulate phosphorylation of S747 and S448. We propose
that stress-induced cGMP signalling may exert its stimulatory effect by
potentiating the activity of a semi-constitutive tyrosine kinase that
phosphorylates STATc while parallel, stress-induced calcium signalling
represses STATc dephosphorylation via its inhibitory effect on PTP3.
The University of Dundee is a registered Scottish charity, No: SC015096
Submitted by Jeff Williams [j.g.williams@dundee.ac.uk]
--------------------------------------------------------------------------------
Retrieval of the vacuolar H-ATPase from phagosomes revealed by
live cell imaging.
Clarke M, Maddera L, Engel U, Gerisch G.
PLoS ONE. 2010; 5(1):e8585.
The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit
enzyme that transports protons across membranes at the expense of ATP.
The resulting proton gradient serves many essential functions, among them
energizing transport of small molecules such as neurotransmitters, and
acidifying organelles such as endosomes. The enzyme is not present in
the plasma membrane from which a phagosome is formed, but is rapidly
delivered by fusion with endosomes that already bear the V-ATPase in
their membranes. Similarly, the enzyme is thought to be retrieved from
phagosome membranes prior to exocytosis of indigestible material,
although that process has not been directly visualized. To monitor
trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium
discoideum, we fed the cells yeast, large particles that maintain their
during trafficking. To track pH changes, we conjugated the yeast with
fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a
fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel
with stage-specific endosomal markers or in combination with mRFP-tagged
cytoskeletal proteins. We find that the V-ATPase is commonly retrieved from
the phagosome membrane by vesiculation shortly before exocytosis.
However, if the cells are kept in confined spaces, a bulky phagosome may
be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole
coated with actin typically separates from the acidic phagosome shortly
before exocytosis. This vacuole is propelled by an actin tail and soon
acquires the properties of an early endosome, revealing an unexpected
mechanism for rapid recycling of the V-ATPase. Any V-ATPase that
reaches the plasma membrane is also promptly retrieved. Thus, live cell
microscopy has revealed both a usual route and alternative means of
recycling the V-ATPase in the endocytic pathway.
Submitted by Margaret Clarke [clarkem@omrf.ouhsc.edu]
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[End dictyNews, volume 34, number 1]
