dictyNews Electronic Edition Volume 32, number 1 January 9, 2009 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. ========= Abstracts ========= ReASH- another viable option for in vivo protein labeling in Dictyostelium Ran-Der Hwang, Chin-Chi Chen and David A. Knecht Journal of Microscopy, in press Biarsenical-tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to GFP or RFP. However, there are no reports of the tetracysteine tagging system being used in Dictyostelium. In order to establish this tagging system in Dictyostelium, the filamin gene (FLN) was modified to express a C-terminal tetracysteine sequence and then transfected into cells. After addition of either FlAsH-EDT2 or ReAsH-EDT2, the fluorescence intensity of cells increased in a time dependent manner and reached a plateau after three hours of incubation. ReAsH had a much stronger and more specifically localized fluorescent signal compared to FlAsH. After removal of the ReAsH-EDT2 reagent, the fluorescence signal remained detectable for at least twenty-four hours. The localization of filamin labeled by ReAsH was similar to that of an mRFP-filamin fusion protein, but the fluorescence signal from the ReAsH labeled protein was stronger. Our findings suggest that the ReAsH-tetracysteine tagging system can be a useful alternative for in vivo protein tagging in Dictyostelium. Submitted by: Dave Knecht [david.knecht@uconn.edu] -------------------------------------------------------------------------------- Autophagy contributes to degradation of Hirano bodies Dong-Hwan Kim, Richard C. Davis, Ruth Furukawa and Marcus Fechheimer Autophagy, Vol 5;1 P: 44 - 51 Hirano bodies are actin-rich inclusions reported most frequently in the hippocampus in association with a variety of conditions including neurodegenerative diseases, and aging. We have developed a model system for formation of Hirano bodies in Dictyostelium and cultured mammalian cells to permit detailed studies of the dynamics of these structures in living cells. Model Hirano bodies are frequently observed in membrane-enclosed vesicles in mammalian cells consistent with a role of autophagy in the degradation of these structures. Clearance of Hirano bodies by an exocytotic process is supported by images from electron microscopy showing extracellular release of Hirano bodies, and observation of Hirano bodies in the culture medium of Dictyostelium and mammalian cells. An autophagosome marker protein Atg8-GFP, was co-localized with model Hirano bodies in wild type Dictyostelium cells, but not in atg5- or atg1-1 autophagy mutant strains. Induction of model Hirano bodies in Dictyostelium with a high level expression of 34 kDa DeltaEF1 from the inducible discoidin promoter resulted in larger Hirano bodies and a cessation of cell doubling. The degradation of model Hirano bodies still occurred rapidly in autophagy mutant (atg5-) Dictyostelium, suggesting that other mechanisms such as the ubiquitin-mediated proteasome pathway could contribute to the degradation of Hirano bodies. Chemical inhibition of the proteasome pathway with lactacystin, significantly decreased the turnover of Hirano bodies in Dictyostelium providing direct evidence that autophagy and the proteasome can both contribute to degradation of Hirano bodies. Short term treatment of mammalian cells with either lactacystin or 3-methyl adenine results in higher levels of Hirano bodies and a lower level of viable cells in the cultures, supporting the conclusion that both autophagy and the proteasome contribute to degradation of Hirano bodies. Submitted by: Ruth Furukawa [furukawa@cb.uga.edu] -------------------------------------------------------------------------------- Steroids initiate a signaling cascade that triggers rapid sporulation in Dictyostelium Christophe Anjard, Yongxuan Su and William F. Loomis* Center for Molecular Genetics, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0368 Development, in press Encapsulation of prespore cells of Dictyostelium discoideum is controlled by several intercellular signals to ensure appropriate timing during fruiting body formation. Acyl CoA binding protein, AcbA, is secreted by prespore cells and processed by the prestalk protease TagC to form the 34 amino acid peptide SDF-2. The SDF-2 receptor is a constitutive histidine kinase, DhkA, which no longer stimulates the cAMP phosphodiesterase, RegA, when SDF-2 is bound. The subsequent increase in cAMP and PKA triggers rapid encapsulation. AcbA is secreted when gamma-aminobutyric acid (GABA) is released from prespore cells and binds to GrlE, a G protein coupled receptor (GPCR). Analysis of SDF-2 production in a series of mutant strains lacking Galpha subunits and GPCRs, either as pure populations or when mixed with other mutant strains, uncovered the non-cell autonomous roles of GrlA, a membrane localized GPCR, Galpha4 and Galpha7. We found that Galpha7 is essential for the response to GABA and is likely to be coupled to GrlE. GrlA and Galpha4 null cells respond normally to GABA but fail to secrete it. We found that they are necessary for response to a small, hydrophobic molecule, SDF-3, which is released late in culmination. Pharmacological inhibition of steroidogenesis during development blocked the production of SDF-3. Moreover, the response to SDF-3 can be blocked by the steroid antagonist mifepristone, while hydrocortisone and other steroids mimic the effects of SDF-3 when added in the nanomolar range. It appears that SDF-3 is a steroid that elicits rapid release of GABA by acting through GrlA coupled to G protein containing the Galpha4 subunit. It may either stimulate the prespore specific glutamate decarboxylase, GadA, that synthesizes GABA or inhibit the GABA transaminase, GabT, that degrades GABA. SDF-3 is at the head of the cascade that amplifies the signal for encapsulation to ensure rapid, synchronous formation of spores. Submitted by: Bill Loomis [wloomis@ucsd.edu] -------------------------------------------------------------------------------- DNA Passage to Nuclei: Role of Endo/lysosomal circuit in Eukaryotic Dictyostelium. Bhavesh Vats#, Harish Padh* Department of Cell and Molecular Biology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej- Gandhinagar Highway, Thaltej, Ahmedabad – 380054, INDIA. Telephone Number: +91-79-27439375, Fax number: +91-79-27450449. Email: perd@perdcentre.com *Corresponding author #Present affiliation: Analytical Development, INTAS Biopharmaceuticals Limited, Sarkhej- Bavla Highway, Ahmedabad- 382 210, INDIA. Telephone number: +912717660100, Fax number: +912717251189 Email: bhavesh.vats@intasbiopharma.co.in Canadian Journal of Microbiology, in press The understanding of DNA passage in eukaryotic cells is still very ambiguous. The route to the nucleus is difficult due to the barriers- metabolic as well as membranous, posed by the eukaryotic cells. Endocytosis appears to be the most likely process responsible for the transport but is also the major culprit of low transfection efficiencies. Here, we report a study on a eukaryotic amoeba, Dictyostelium discoideum, where by disruption of the endocytic process at the opportune moment, the transformant number increased. We have observed by disruption of fluid phase uptake of calcium phosphate DNA nanoparticles, the number of clones increased with probable increase in number of foreign genes integrating in the host genome. The method described here leads to the possibility of safe and inexpensive methods for transfer of genes required for heterologous recombinant protein production as well as generation therapeutic recombinant cells. Submitted by: Harish Padh [hpadh@yahoo.com] ============================================================== [End dictyNews, volume 32, number 1]

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