dictyNews
Electronic Edition
Volume 37, number 1
July 8, 2011
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Abstracts
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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 [[log in to unmask]]
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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 Biomédicas 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 [[log in to unmask]]
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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 [[log in to unmask]]
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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 [[log in to unmask]]
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An amoeba host model to evaluate Streptococcus suis virulence
Laetitia Bonifait1,2, Steve J. Charette3,4,5, Geneviève Filion3,4,
Marcelo Gottschalk2,6 and Daniel Grenier1,2*
1Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine
Dentaire, Université Laval, Quebec City, Quebec, Canada;
2Centre de Recherche en Infectiologie Porcine (CRIP), Fonds Québécois
de la Recherche sur la Nature et les Technologies (FQRNT), Quebec City,
Quebec, Canada;
3Institut de Biologie Intégrative et des Systèmes (IBIS), Quebec City,
Quebec, Canada;
4Centre de Recherche de l’Institut Universitaire de Cardiologie et de
Pneumologie de Québec (IUCPQ), Hôpital Laval, Quebec City, Quebec,
Canada;
5Département de Biochimie, de Microbiologie et de Bio-Informatique,
Faculté des Sciences et de Génie, Université Laval, Quebec City,
Quebec, Canada;
6Groupe de Recherche sur les Maladies Infectieuses du Porc (GREMIP),
Faculté de Médecine Vétérinaire, Université de Montréal, 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 [[log in to unmask]]
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[End dictyNews, volume 37, number 1]
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