dictyNews Electronic Edition Volume 39, number 31 November 8, 2013 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 dictyBase has a job opening! http://dictybase.org/dictybase_jobs.html ========= Abstracts ========= Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in Dictyostelium David J. Schwebs, Hoai-Nghia Nguyen, Jamison A. Miller, Jeffrey A. Hadwiger Cellular Signalling, in press Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well- understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Galpha-2 and Galpha-4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP- specific phosphodiesterase RegA. The regA gene was disrupted in galpha-2- and galpha-4- cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2- mutants. The regA- mutation had no major effects on developmental morphology but enriched the distribution of the Galpha- mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Galpha-4- mediated folate chemotaxis. However, the regA gene disruption in galpha-4- cells, but not in galpha-2- cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA- strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from galpha-4-regA- strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Galpha-4HC (Galpha-4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Galpha-4 subunit expression can mask regA- associated phenotypes even when provided with wild-type intercellular signaling. These finding indicate that the Galpha-2 and Galpha-4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development. Submitted by Jeff Hadwiger [jeff.hadwiger@okstate.edu] --------------------------------------------------------------------------- Control of cell differentiation by mitochondria, typically evidenced in Dictyostelium development Yasuo Maeda1* and Junji Chida2 1Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan; E-mail: kjygy352@ybb.ne.jp; 2Division of Molecular Neurobiology, Institute for Enzyme Research, The University of Tokushima, Kuramoto-cho, Tokushima 770-8503, Japan; E-mail: jchida@tokushima-u.ac.jp. *Author to whom all correspondence should be addressed: E-mail: kjygy352@ybb.ne.jp; Tel. & Fax: +81-22-224-2863 Biomolecules [Special Issue "Focus Update in Biomolecules"] (MDPI), in press Review In eukaryotic cells, mitochondria are self-reproducing organelles with their own DNA and they play a central role in adenosine triphosphate (ATP) synthesis by respiration.Increasing evidence indicates that mitochondria also have critical and multiple functions in the initiation of cell differentiation, cell-type determination, cell movement, and pattern formation. This has been most strikingly realized in development of the cellular slime mold Dictyostelium. For example, the expression of the mitochondrial ribosomal protein S4 (mt-rps4) gene is required for the initial differentiation. The Dictyostelium homologue (Dd-TRAP1) of TRAP-1 (tumor necrosis receptor- associated protein 1), a mitochondrial molecular chaperone belonging to the Hsp90 family, allows the prompt transition of cells from growth to differentiation through a novel prestarvation factor (PSF-3) in growth medium. Moreover, a cell-type-specific organelle named a prespore- specific vacuole (PSV) is constructed by mitochondrial transformation with the help of the Golgi complex. Mitochondria are also closely involved in a variety of cellular activities including CN-resistant respiration and apoptosis. These mitochondrial functions are reviewed in this article, with special emphasis on the regulation of Dictyostelium development. Keywords: differentiation; mitochondria; mitochondrial ribosomal protein S4 (mt-RPS4); tumor necrosis receptor-associated protein 1 (TRAP-1); CN-resistant respiration; prespore-specific vacuole (PSV); Dictyostelium; ESC Submitted by Yasuo Maeda [kjygy352@ybb.ne.jp] --------------------------------------------------------------------------- Identification of a Farnesol Analogue as a Ras Function Inhibitor using both an in vivo Ras activation sensor and phenotypic screening approach. Kamalakkannan Srinivasan, Thangaiah Subramanian, H. Peter Spielmann, and Chris Janetopoulos* Molecular and Cellular Biochemistry, in press Mutation in Ras isoforms such as K-Ras, N-Ras, H-Ras contributes to roughly 85%, 15% and 1% of human cancers, respectively. Proper membrane targeting of these Ras isoforms, a prerequisite for Ras activity, requires farnesylation or geranylgeranylation at the C-terminal CAAX box. We devised an in vivo screening strategy based on monitoring Ras activation and phenotypic physiological outputs for assaying synthetic Ras function inhibitors (RFI). Ras activity was visualized by the translocation of RBDRaf1-GFP to activated Ras at the plasma membrane. By using this strategy, we screened one synthetic farnesyl substrate analog (AGOH) along with nine putative inhibitors and found that only m-CN-AGOH inhibited Ras activation. Phenotypic analysis of starving cells could be used to monitor polarization, motility and the inability of these treated cells to aggregate properly during fruiting body formation. Incorporation of AGOH and m-CN-AGOH to cellular proteins was detected by western blot. These screening assays can be incorporated into a high throughput screening format using Dictyostelium discoideum and automated microscopy to determine effective RFIs. These RFI candidates can then be further tested in mammalian systems. Submitted by Chris Janetopoulos [c.janetopoulos@vanderbilt.edu] --------------------------------------------------------------------------- A microfluidic-enabled mechanical microcompressor for the immobilization of live single- and multi-cellular specimens. Yingjun Yan, Liwei Jiang, Karl J. Aufderheide, Gus A. Wright, Alexander Terekhov, Lino Costa, Kevin Qin, W. Tyler McCleery, John J. Fellenstein, Alessandro Ustione, J. Brian Robertson, Carl Hirschie Johnson, David W. Piston, M. Shane Hutson, John P. Wikswo, William Hofmeister, Chris Janetopoulos* Microscopy and Microanalysis, in press A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes, The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila. Submitted by Chris Janetopoulos [c.janetopoulos@vanderbilt.edu] ============================================================== [End dictyNews, volume 39, number 31]