dictyNews Electronic Edition Volume 36, number 3 Jan 28, 2011 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 ========= Bio-electrospraying and aerodynamically assisted bio-jetting the model eukaryotic Dictyostelium discoideum: assessing stress and developmental competency post-treatment Nicholl K. Pakes, Suwan N. Jayasinghe and Robin S. B. Williams Journal of the Royal Society Interface, in press Bio-electrospraying (BES) and aerodynamically assisted bio-jetting (AABJ) have recently been established as important novel biospray technologies for directly manipulating living cells. To elucidate their potential in medical and clinical sciences, these bio-aerosol techniques have been subjected to increasingly rigorous investigations. In parallel to these studies, we wish to introduce these unique biotechnologies for use in the basic biological sciences, for handling a wide range of cell types and systems, thus increasing the range and the scope of these techniques for modern research. Here, the authors present the analysis of the new use of these biospray techniques for the direct handling of the simple eukaryotic biomedical model organism Dictyostelium discoideum. These cells are widely used as a model for immune cell chemotaxis and as a simple model for development. We demonstrate that AABJ of these cells did not cause cell stress, as defined by the stress-gene induction, nor affect cell development. Furthermore, although BES induced the increased expression of one stress-related gene (gapA), this was not a generalized stress response nor did it affect cell development. These data suggest that these biospray techniques can be used to directly manipulate single cells of this biomedical model without inducing a generalized stress response or perturbing later development. Submitted by Robin Williams [Robin.williams@rhul.ac.uk] -------------------------------------------------------------------------------- eIF2alpha Kinases Control Chalone Production In Dictyostelium discoideum Robert L. Bowman*, Yanhua Xiong, Janet H. Kirsten and Charles K. Singleton Department of Biological Sciences, Vanderbilt University, VU Station B 351634, Nashville, TN 37235 Eukaryotic Cell, in press Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high molecular weight complex that reversibly inhibits cell proliferation but not growth via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2 type eIF2alpha kinases, proteins that phosphorylate the translational initiation factor eIF2alpha and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead it is due to an enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a non- phosphorylatable eIF2alpha. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2alpha phosphorylation. Both proteins are upregulated during culmination of development, and this enhanced production is lacking in a strain that possesses a non- phosphorylatable eIF2alpha. Submitted by Charles Singleton [charles.k.singleton@Vanderbilt.Edu] -------------------------------------------------------------------------------- Atg1 allows second-signalled autophagic cell death in Dictyostelium Marie-Franoise Luciani,1,2,3 Corinne Giusti,1,2,3 Birthe Harms,1,2,3 Yoshiteru Oshima,4 Haruhisa Kikuchi,4 Yuzuru Kubohara,5 and Pierre Golstein1,2,3,* 1Centre dÕImmunologie de Marseille-Luminy (CIML), FacultŽ des Sciences de Luminy, Aix-Marseille UniversitŽ, Marseille F-13288, France; 2INSERM U631, Marseille F-13288, France; 3CNRS UMR6102, Marseille F-13288, France; 4Laboratory of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan; 5Institute for Molecular & Cellular Regulation, Gunma University, Maebashi 371-8512, Japan Autophagy 2011, in press We investigated the role of atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that atg1 was required for ACD. Thus, in the same cells atg1 was required in two apparently opposite ways, upon first-signalling for cell survival and upon second-signalling for ACD. Our findings strongly suggest that atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signalling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts. Submitted by Pierre Golstein [golstein@ciml.univ-mrs.fr] -------------------------------------------------------------------------------- Origin and function of the stalk-cell vacuole in Dictyostelium Toru Uchikawa(a), Akitsugu Yamamoto(b), Kei Inouye(a) (a) Department of Botany, Graduate School of Science, Kyoto University (b) Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology Dev. Biol., in press Large vacuoles are characteristic of plant and fungal cells, and their origin has long attracted interest. The cellular slime mould provides a unique opportunity to study the de novo formation of vacuoles because, in its life cycle, a subset of the highly motile animal-like cells (prestalk cells) rapidly develop a single large vacuole and cellulosic cell wall to become plant-like cells (stalk cells). Here we describe the origin and process of vacuole formation using live-imaging of Dictyostelium cells expressing GFP-tagged ammonium transporter A (AmtA-GFP), which was found to reside on the membrane of stalk-cell vacuoles. We show that stalk-cell vacuoles originate from acidic vesicles and autophagosomes, which fuse to form autolysosomes. Their repeated fusion and expansion accompanied by concomitant cell wall formation enables the stalk cells to rapidly develop turgor pressure necessary to make the rigid stalk to hold the spores aloft. Contractile vacuoles, which are rich in H+-ATPase as in plant vacuoles, remained separate from these vacuoles. We further argue that AmtA may play an important role in the control of stalk-cell differentiation by modulating the pH of autolysosomes. Submitted by Kei Inouye [inoue@cosmos.bot.kyoto-u.ac.jp] -------------------------------------------------------------------------------- Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and alpha-N-acetylglucosaminyltransferase enzymes involved in O2-signaling in Dictyostelium Hanke van der Welą, Jennifer M. Johnsoną, Yuechi Xuą, Chamini V. Karunaratne¤, Kyle D. Wilsoną, Yusuf Vohra¦, Geert-Jan Boons¦, Carol M. Taylor¤, Brad Bendiak#, and Christopher M. Westą ąDepartment of Biochemistry and Molecular Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 USA; ¤Department of Chemistry, 742 Choppin Hall, Louisiana State University, Baton Rouge, LA 70803 USA; ¦Dept. of Chemistry and Complex Carbohydrate Research Center, 315 Riverbend Road, University of Georgia, Athens, GA 30602 USA; #Department of Cell and Developmental Biology and Structural Biology and Biophysics Program, University of Colorado Denver, Anschutz Medical Campus, Mail Stop 8108, RC-1 South Bldg., L18-12120, 12801 East 17th Avenue, Aurora, CO 80045 USA Biochemistry, in press. The social amoeba Dictyostelium expresses a hypoxia inducible factor- alpha (HIFalpha)-type prolyl 4-hydroxylase (P4H1) and an alpha-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein)-class of E3 ubiquitin-ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O2-regulation of development, suggesting the existence of an ancient O2-sensing mechanism related to modification of the transcription factor HIFalpha by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O2-signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. 1H-NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFalpha, and highly purified P4H1 was inhibited by Krebs cycleintermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFalpha by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3SCFubiquitin-ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily-related E3VBCubiquitin-ligases. Submitted by: Chris West [Cwest2@ouhsc.edu] ============================================================== [End dictyNews, volume 36, number 3]