dictyNews Electronic Edition Volume 40, number 1 January 3, 2014 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 ========= The inverse BAR-domain protein IBARa drives membrane remodelling to control osmoregulation, phagocytosis and cytokinesis Joern Linkner (1), Gregor Witte (2), Hongxia Zhao (3), Alexander Junemann (1), Benjamin Nordholz (1), Petra Runge-Wollmann (2), Pekka Lappalainen (3) and Jan Faix (1) 1) Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany; 2) Gene Center and Department of Biochemistry at the Ludwig-Maximilians-University, 81377, Munich, Germany; 3) Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Helsinki 00014, Finland. Journal of Cell Science, in press Here, we analyzed the single I-BAR family member IBARa from D. discoideum. The X-ray structure of the N-terminal I-BAR domain solved at 2.2  resolution revealed an all-alpha helical structure that self-associates into a 165  zeppelin-shaped antiparallel dimer. The structural data are consistent with its shape in solution obtained by small-angle X-ray-scattering. Cosedimentation, fluorescence-anisotropy as well as fluorescence and electron microscopy revealed the I-BAR domain to bind preferentially to phosphoinositide-containing vesicles and drive the formation of negatively curved tubules. Immunofluorescence labelling further showed accumulation of endogenous IBARa at the tips of filopodia, the rim of constricting phagocytic cups, in foci connecting dividing cells during the final stage of cytokinesis, and most prominently at the osmoregulatory contractile vacuole (CV). Consistently, IBARa-null mutants displayed defects in CV formation and discharge, growth, phagocytosis and mitotic cell division, whereas filopodia formation was not compromised. Of note, IBARa-null mutants were also strongly impaired in cell spreading. Together, these data suggest IBARa to constitute an important regulator of numerous cellular processes intimately linked with the dynamic rearrangement of cellular membranes. Submitted by Jan Faix [faix.jan@mh-hannover.de] --------------------------------------------------------------------------- A PIP5Kinase essential for efficient chemotactic signalling Louise Fets, John Nichols and Robert R. Kay Current Biology, in press In neutrophils and Dictyostelium, chemoattractant gradients generate directed cell migration by eliciting signalling events that bias intrinsic motility and favour the production and retention of up-gradient pseudopods[1, 2]. Phosphoinositides are actively regulated during chemotaxis in these cells, most iconically in the production of PI(3,4,5)P3 gradients within the plasma membrane[3, 4]. Although it is now known that PI(3,4,5)P3 signalling is non- essential for gradient sensing[5, 6], the role of the related phosphoinositide PI(4,5)P2 is little understood, despite its clear importance in many cell- biological processes[7]. We describe a PIP5kinase, PikI, which produces PI(4,5)P2 and is essential for efficient chemotaxis of Dictyostelium cells. Without PikI, PI(4,5)P2 levels are reduced by 90%, and while pikI- cells move at normal speeds, they are highly disorientated in cAMP gradients. Following chemotactic stimulation, Ras is efficiently activated in pikI- cells, yet Ras- dependent responses (including activation of PKB) are severely impaired. PikI is phosphorylated by PKB[8], and in vitro studies of a phospho-mimic mutant suggest that this phosphorylation increases PikI activity. We propose that adequate PI(4,5)P2 levels are required to couple activated Ras to its downstream effectors and that these levels are regulated by PikI, making it a crucial player in gradient sensing. Submitted by Louise Fets [lfets@nimr.mrc.ac.uk] --------------------------------------------------------------------------- A Retinoblastoma orthologue is required for the sensing of a chalone i n Dictyostelium Deenadayalan Bakthavatsalam,* Michael J. V. White, Sarah E. Herlihy, J onathan E. Phillips, and Richard H. Gomer Department of Biology, Texas A&M University, College Station, Texas, USA *Current address: Texas Heart Institute, Houston, Texas, USA Eukaryotic Cell, In press Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium Retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblAř cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and after starvation have a lower spore viability compared to wild-type cells, possibly because in shaking culture rblAř cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblAř cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells, but not that of rblAř cells, whereas CfaD inhibits the proliferation of both wild type cells and rblAř cells. Similar to aprAř cells, rblAř cells have a normal mass and protein accumulation rate on a per nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through Retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors. Submitted by Richard Gomer [rgomer@tamu.edu] --------------------------------------------------------------------------- Regulation of transcriptional bursting by a naturally oscillating signal Adam M. Corrigan and Jonathan R. Chubb MRC Laboratory for Molecular Cell Biology, Department of Cell and Developmental Biology, University College London, Gower Street, London, WC1E 6BT Current Biology, in press Transcription is highly stochastic, occurring in irregular bursts[1-3]. For temporal and spatial precision of gene expression, cells must somehow deal with this noisy behaviour. To address how this is achieved, we investigated how transcriptional bursting is entrained by a naturally oscillating signal, by direct measurement of transcription together with signal dynamics in living cells. We identify a Dictyostelium gene showing rapid transcriptional oscillations with the same period as extracellular cAMP signalling waves. Bursting approaches anti-phase to cAMP waves, with accelerating transcription cycles during differentiation. While coupling between signal and transcription oscillations was clear at the population level, single cell transcriptional bursts retained considerable heterogeneity, indicating transcription is not solely governed by signalling frequency. Previous studies imply burst heterogeneity reflects distinct chromatin states [4-6]. Here we show heterogeneity is determined by multiple intrinsic and extrinsic cues, and is maintained by a transcriptional persistence. Unusually for a persistent transcriptional behaviour, the lifetime was only 20 minutes, with rapid randomisation of transcriptional state by the response to oscillatory signalling. Linking transcription to rapid signalling oscillations allows reduction of gene expression heterogeneity by temporal averaging, providing a mechanism to generate precision in cell choices during development. Submitted by Jonathan Chubb [j.chubb@ucl.ac.uk] ============================================================== [End dictyNews, volume 40, number 1]