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. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= 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] -------------------------------------------------------------------------------- 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] -------------------------------------------------------------------------------- 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] -------------------------------------------------------------------------------- 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] ============================================================== [End dictyNews, volume 34, number 1]