mTORC1 pathway activity biases cell fate choice.

Pluripotent stem cells can differentiate into distinct cell types but the intracellular pathways controlling cell fate choice are not well understood. The social amoeba Dictyostelium discoideum is a simplified system to study choice preference as proliferating amoebae enter a developmental cycle upon starvation and differentiate into two major cell types, stalk and spores, organised in a multicellular fruiting body. Factors such as acidic vesicle pH predispose amoebae to one fate.

Linking DNA repair and cell cycle progression through serine ADP-ribosylation of histones.

Although serine ADP-ribosylation (Ser-ADPr) by Poly(ADP-ribose)-polymerases is a cornerstone of the DNA damage response, how this regulates DNA repair and genome stability is unknown. Here, we exploit the ability to manipulate histone genes in Dictyostelium to identify that ADPr of the histone variant H3b at S10 and S28 maintains genome stability by integrating double strand break (DSB) repair with mitotic entry. Given the critical requirement for mitotic H3S10/28 phosphorylation, we develop separation of function mutations that maintain S10 phosphorylation whilst disrupting ADPr.

Moving the Research Forward: The Best of British Biology Using the Tractable Model System .

The social amoeba provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs.

Possible Involvement of the Nutrient and Energy Sensors mTORC1 and AMPK in Cell Fate Diversification in a Non-Metazoan Organism.

mTORC1 and AMPK are mutually antagonistic sensors of nutrient and energy status that have been implicated in many human diseases including cancer, Alzheimer's disease, obesity and type 2 diabetes. Starved cells of the social amoeba aggregate and eventually form fruiting bodies consisting of stalk cells and spores. We focus on how this bifurcation of cell fate is achieved.

as a Model to Assess Genome Stability Through DNA Repair.

Preserving genome integrity through repair of DNA damage is critical for human health and defects in these pathways lead to a variety of pathologies, most notably cancer. The social amoeba is remarkably resistant to DNA damaging agents and genome analysis reveals it contains orthologs of several DNA repair pathway components otherwise limited to vertebrates. These include the Fanconi Anemia DNA inter-strand crosslink and DNA strand break repair pathways.

Methylation-directed acetylation of histone H3 regulates developmental sensitivity to histone deacetylase inhibition.

Hydroxamate-based lysine deacetylase inhibitors (KDACis) are approved for clinical use against certain cancers. However, intrinsic and acquired resistance presents a major problem. Treatment of cells with hydroxamates such as trichostatin A (TSA) leads to rapid preferential acetylation of histone H3 already trimethylated on lysine 4 (H3K4me3), although the importance of this H3K4me3-directed acetylation in the biological consequences of KDACi treatment is not known.

Microbe Profile: : model system for development, chemotaxis and biomedical research.

The social amoeba is a versatile organism that is unusual in alternating between single-celled and multi-celled forms. It possesses highly-developed systems for cell motility and chemotaxis, phagocytosis, and developmental pattern formation. As a soil amoeba growing on microorganisms, it is exposed to many potential pathogens; it thus provides fruitful ways of investigating host-pathogen interactions and is emerging as an influential model for biomedical research.

Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events.

The amoeba Dictyostelium discoideum is a single-cell organism that can undergo a simple developmental program, making it an excellent model to study the molecular mechanisms of cell motility, signal transduction, and cell-type differentiation. A variety of human genes that are absent or show limited conservation in other invertebrate models have been identified in this organism. This includes ADP-ribosyltransferases, also known as poly-ADP-ribose polymerases (PARPs), a family of proteins that catalyze the addition of single or poly-ADP-ribose moieties onto target proteins.

Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks.

ADP-ribosyltransferases (ARTs) modify proteins with single units or polymers of ADP-ribose to regulate DNA repair. However, the substrates for these enzymes are ill-defined. For example, although histones are modified by ARTs, the sites on these proteins ADP-ribosylated following DNA damage and the ARTs that catalyse these events are unknown.

Emerging models for DNA repair: Dictyostelium discoideum as a model for nonhomologous end-joining.

DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends.

Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks.

ADP-ribosylation of proteins at DNA lesions by ADP-ribosyltransferases (ARTs) is an early response to DNA damage. The best defined role of ADP-ribosylation in the DNA damage response is in repair of single strand breaks (SSBs). Recently, we initiated a study of how ADP-ribosylation regulates DNA repair in Dictyostelium and found that two ARTs (Adprt1b and Adprt2) are required for tolerance of cells to SSBs, and a third ART (Adprt1a) promotes nonhomologous end-joining (NHEJ).

Investigation of DNA repair pathway activity.

DNA is constantly being damaged from endogenous and exogenous sources and efficient repair of different types of DNA lesions is essential for the survival of the organism. Dictyostelium is highly resistant to DNA damage and its genome sequence has revealed the presence of multiple repair pathways conserved with vertebrates but lost in other genetically tractable invertebrate models. As such, Dictyostelium is a powerful model organism to study selected human DNA repair pathways and may provide insights into the molecular basis of how cells become resistant to DNA damage.

Dynamic acetylation of lysine-4-trimethylated histone H3 and H3 variant biology in a simple multicellular eukaryote.

Dynamic acetylation of all lysine-4-trimethylated histone H3 is a complex phenomenon involved in Immediate-early gene induction in metazoan eukaryotes. Higher eukaryotes express repeated copies of three closely related H3 variants, inaccessible to genetic analysis. We demonstrate conservation of these phenomena in Dictyostelium which has three single-copy H3 variant genes.

The role of ADP-ribosylation in regulating DNA double-strand break repair.

ADP-ribosylation is the post translational modification of proteins catalysed by ADP-ribosyltransferases (ARTs). ADP-ribosylation has been implicated in a wide variety of cellular processes including cell growth and differentiation, apoptosis and transcriptional regulation. Perhaps the best characterised role, however, is in DNA repair and genome stability where ADP-ribosylation promotes resolution of DNA single strand breaks.

PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks.

Poly adenosine diphosphate (ADP)-ribosylation (PARylation) by poly ADP-ribose (PAR) polymerases (PARPs) is an early response to DNA double-strand breaks (DSBs). In this paper, we exploit Dictyostelium discoideum to uncover a novel role for PARylation in regulating nonhomologous end joining (NHEJ). PARylation occurred at single-strand breaks, and two PARPs, Adprt1b and Adprt2, were required for resistance to this kind of DNA damage.

DNA double-strand break repair pathway choice in Dictyostelium.

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium.

Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+.

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets.

Targets downstream of Cdk8 in Dictyostelium development.

Background: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production.

Control of cyclin C levels during development of Dictyostelium.

Background: Cdk8 and its partner cyclin C form part of the mediator complex which links the basal transcription machinery to regulatory proteins. The pair are required for correct regulation of a subset of genes and have been implicated in control of development in a number of organisms including the social amoeba Dictyostelium discoideum. When feeding, Dictyostelium amoebae are unicellular but upon starvation they aggregate to form a multicellular structure which develops into a fruiting body containing spores.

Differential roles of the PKC novel isoforms, PKCdelta and PKCepsilon, in mouse and human platelets.

Background: Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.

Methodology/principal Findings: In this study, we focus on the role of two novel PKC isoforms, PKCdelta and PKCepsilon, in both mouse and human platelets. PKCdelta is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220.

Distinct but critical roles for integrin alphaIIbbeta3 in platelet lamellipodia formation on fibrinogen, collagen-related peptide and thrombin.

Integrins are the major receptor type known to facilitate cell adhesion and lamellipodia formation on extracellular matrix proteins. However, collagen-related peptide and thrombin have recently been shown to mediate platelet lamellipodia formation when presented as immobilized surfaces. The aims of this study were to establish if there exists a role for the platelet integrin alpha(IIb)beta(3) in this response; and if so, whether signalling from the integrin is required for lamellipodia formation on these surfaces.

DNA damage signaling and repair in Dictyostelium discoideum.

Repair of DNA double strand breaks (DSBs) is critical for the maintenance of genome integrity. DNA DSBs can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). Whilst HR requires sequences homologous to the damaged DNA template in order to facilitate repair, NHEJ occurs through recognition of DNA DSBs by a variety of proteins that process and rejoin DNA termini by direct ligation.

DNA-PKcs-dependent signaling of DNA damage in Dictyostelium discoideum.

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer.

Developmental decisions in Dictyostelium discoideum.

Dictyostelium discoideum is an excellent system in which to study developmental decisions. Synchronous development is triggered by starvation and rapidly generates a limited number of cell types. Genetic and image analyses have revealed the elegant intricacies associated with this simple development system.