dictyNews Electronic Edition Volume 37, number 1 July 8, 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 ========= Nucleocytoplasmic transfer of cyclin dependent kinase 5 and its binding to puromycin-sensitive aminopeptidase in Dictyostelium discoideum Robert J. Huber1 and Danton H. O'Day1,2 1Department of Cell & Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5 2Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6 Histochemistry and Cell Biology, in press The Dictyostelium discoideum homologue of mammalian cyclin-dependent kinase 5 (Cdk5) has previously been shown to be required for optimal growth and differentiation in this model organism, however the subcellular localization of the protein has not previously been studied. In this study, immunolocalizations and a GFP-fusion construct localized Cdk5 predominantly to the nucleus of vegetative cells. Western blots showed that Cdk5 was present in both nuclear and non-nuclear fractions suggesting a functional role in both cellular locales. During the early stages of mitosis, Cdk5 gradually moved from a punctate nucleoplasmic distribution to localize adjacent to the inner nuclear envelope. During anaphase and telophase, Cdk5 localized to the cytoplasm and was not detected in the nucleoplasm. Cdk5 returned to the nucleus during cytokinesis. Proteolytic activity has been shown to be a critical regulator of the cell cycle. Immunoprecipitations coupled with immunolocalizations, identified puromycin- sensitive aminopeptidase A (PsaA) as a potential Cdk5 binding partner in Dictyostelium. Immunoprecipitations also identified two phosphotyrosine proteins (35 kDa and 18 kDa) that may interact with Cdk5 in vivo. Together, this work provides new insight into the localization of Cdk5, its function during cell division, and its binding to a proteolytic enzyme in Dictyostelium. Submitted by: Danton O'Day [danton.oday@utoronto.ca] -------------------------------------------------------------------------------- Protocol manuscript: A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum Javier Calvo-Garrido, Sergio Carilla-Latorre, Ana Mesquita and Ricardo Escalante Instituto de Investigaciones Biomedicas Alberto Sols. C.S.I.C./U.A.M. Calle Arturo Duperier 4, 28029 Madrid. Spain. Autophagy, in press Abstract Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hinders the assessment of new mutants or drugs. One of these techniques, which has been used successfully in yeast and mammalian cells, but has not yet been described in Dictyostelium, is based on the presence of proteolytic fragments derived from autophagic degradation of expressed fusion proteins. Lysosomotropic agents such as NH4Cl penetrate acidic compartments and raise their pH, thus allowing the accumulation and measurement of these cleaved fragments, which otherwise would be rapidly degraded. We have used this property to detect the presence of free GFP fragments derived from the fusion protein GFP-Tkt-1, a cytosolic marker. We demonstrate that this proteolytic event is dependent on autophagy and can be used to detect differences in the level of autophagic flux among different mutant strains. Moreover, treatment with NH4Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8. Submitted by Ricardo Escalante [rescalante@iib.uam.es] -------------------------------------------------------------------------------- The putative bZIP transcripton factor BzpN slows proliferation and functions in the regulation of cell density by autocrine signals in Dictyostelium Jonathan E. Phillips1 , Eryong Huang2, Gad Shaulsky2, and Richard H. Gomer3 1Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 3Department of Biology, Texas A&M University, College Station, TX, USA. PLoS One, in press The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation. Submitted by Jonathan Phillips [JPhillips@mail.bio.tamu.edu] -------------------------------------------------------------------------------- Heteroplasmic mitochondrial disease in Dictyostelium discoideum. Lisa M. Francione and Paul R. Fisher. Department of Microbiology, La Trobe University, VIC 3086, Australia Biochemical Pharmacology, in press. The bewildering complexity of the relationship between genotype and phenotype in human mitochondrial diseases has delayed an understanding of the related cytopathological mechanisms. To explore the relationship between mitochondrial dysfunction in Dictyostelium discoideum and the related cytopathologies, we determined whether the phenotypic outcomes were similar regardless of which D. discoideum mitochondrial gene was targeted for disruption. The disruption of the mitochondrial genes resulted in a similar pattern of phenotypes to those caused by other mitochondrial defects. These include impairment of phototaxis, multicellular development and growth on plates and in liquid medium. As the reduced growth rates could have been due to defective phagocytic or macropinocytic nutrient uptake, these processes were tested but found to be unaffected. Since mitochondria have been associated with Legionella pathogenesis of human macrophages, it was also determined if mitochondrially diseased Dictyostelium strains were better or worse than healthy cells at supporting the growth of Legionella pneumophila. The results revealed that the mitochondrially diseased strains supported greater L. pneumophila growth than the wild type Dictyostelium strain (AX2). Quantitative northern blotting showed a significant reduction in the level of expression of the entire mitochondrial genome, regardless of which mitochondrial gene was targeted for disruption, suggesting a generalized deficiency in mitochondrial gene expression and function. The phenotypic outcomes were the same as those shown previously to result from chronic hyperactivity of the energy-sensing protein kinase, AMPK, after knockdown of mitochondrial chaperonin 60. Submitted by Paul Fisher [P.Fisher@latrobe.edu.au] -------------------------------------------------------------------------------- An amoeba host model to evaluate Streptococcus suis virulence Laetitia Bonifait1,2, Steve J. Charette3,4,5, Genevieve Filion3,4, Marcelo Gottschalk2,6 and Daniel Grenier1,2* 1Groupe de Recherche en ecologie Buccale (GREB), Faculte de Medecine Dentaire, Universite Laval, Quebec City, Quebec, Canada; 2Centre de Recherche en Infectiologie Porcine (CRIP), Fonds Quebecois de la Recherche sur la Nature et les Technologies (FQRNT), Quebec City, Quebec, Canada; 3Institut de Biologie Integrative et des Systmes (IBIS), Quebec City, Quebec, Canada; 4Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Quebec (IUCPQ), Hpital Laval, Quebec City, Quebec, Canada; 5Departement de Biochimie, de Microbiologie et de Bio-Informatique, Faculte des Sciences et de Genie, Universite Laval, Quebec City, Quebec, Canada; 6Groupe de Recherche sur les Maladies Infectieuses du Porc (GREMIP), Faculte de Medecine Veterinaire, Universite de Montreal, Ste-Hyacinthe, Quebec, Canada. Applied and Environmental Microbiology (AEM), in press The Gram-positive bacterium Streptococcus suis is a major swine pathogen worldwide that causes meningitis, septicemia, and endocarditis. In this study, we demonstrated that the amoeba Dictyostelium discoideum can be a relevant alternative system to study the virulence of S. suis. Submitted by Steve Charette [Steve.Charette@bcm.ulaval.ca] ============================================================== [End dictyNews, volume 37, number 1]