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]