Modeling chemotaxis reveals the role of reversed phosphotransfer and a bi-functional kinase-phosphatase

Understanding how multiple signals are integrated in living cells to produce a balanced response is a major challenge in biology. Two-component signal transduction pathways, such as bacterial chemotaxis, comprise histidine protein kinases (HPKs) and response regulators (RRs). These are used to sense and respond to changes in the environment. Rhodobacter sphaeroides has a complex chemosensory network with two signaling clusters, each containing a HPK, CheA. Here we demonstrate, using a mathematical model, how the outputs of the two signaling clusters may be integrated. We use our mathematical model supported by experimental data to predict that: (1) the main RR controlling flagellar rotation, CheY6, aided by its specific phosphatase, the bifunctional kinase CheA3, acts as a phosphate sink for the other RRs; and (2) a phosphorelay pathway involving CheB2 connects the cytoplasmic cluster kinase CheA3 with the polar localised kinase CheA2, and allows CheA3-P to phosphorylate non-cognate chemotaxis RRs. These two mechanisms enable the bifunctional kinase/phosphatase activity of CheA3 to integrate and tune the sensory output of each signaling cluster to produce a balanced response. The signal integration mechanisms identified here may be widely used by other bacteria, since like R. sphaeroides, over 50% of chemotactic bacteria have multiple cheA homologues and need to integrate signals from different sources.

Phosphoinositide (PI) 3-kinase assays

The regulation of phosphoinositide (PI) 3-kinase activities has been linked to many normal and disease-related processes, including cell survival, cell growth and proliferation, cell differentiation, cell motility, and intracellular vesicle trafficking. However, as the family of enzymes has now grown to include eight true members, in three functional classes, plus several related protein kinases that are also inhibited by the widely used PI 3-kinase selective inhibitors, wortmannin and LY294002, extended methodologies are required to identify which type of kinase is involved in a particular cellular process, or protein complex, under study. A robust in vitro PI 3-kinase assay, suitable for use with immunoprecipitates, or purified proteins, is described here together with a series of modifications of substrate and assay conditions that will aid researchers in the identification of the particular class and isoform of PI 3-kinase that is involved in a signaling process under investigation.

A novel parallel approach to the likelihood-based estimation of admixture in population genetics

Inferring population admixture from genetic data and quantifying it is a difficult but crucial task in evolutionary and conservation biology. Unfortunately state-of-the-art probabilistic approaches are computationally demanding. Effectively exploiting the computational power of modern multiprocessor systems can thus have a positive impact to Monte Carlo-based simulation of admixture modeling. A novel parallel approach is briefly described and promising results on its message passing interface (MPI)-based C implementation are reported.

Hepatitis C virus NS5A protein binds the SH3 domain of the Fyn tyrosine kinase with high affinity: mutagenic analysis of residues within the SH3 domain that contribute to the interaction

Background: The hepatitis C virus (HCV) non-structural 5A protein (NS5A) contains a highly conserved C-terminal polyproline motif with the consensus sequence Pro-X-X- Pro-X-Arg that is able to interact with the Src-homology 3 (SH3) domains of a variety of cellular proteins. Results: To understand this interaction in more detail we have expressed two N-terminally truncated forms of NS5A in E. coli and examined their interactions with the SH3 domain of the Src-family tyrosine kinase, Fyn. Surface plasmon resonance analysis revealed that NS5A binds to the Fyn SH3 domain with what can be considered a high affinity SH3 domain-ligand interaction (629 nM), and this binding did not require the presence of domain I of NS5A (amino acid residues 32-250). Mutagenic analysis of the Fyn SH3 domain demonstrated the requirement for an acidic cluster at the C-terminus of the RT-Src loop of the SH3 domain, as well as several highly conserved residues previously shown to participate in SH3 domain peptide binding. Conclusion: We conclude that the NS5A: Fyn SH3 domain interaction occurs via a canonical SH3 domain binding site and the high affinity of the interaction suggests that NS5A would be able to compete with cognate Fyn ligands within the infected cell.

A dual role for integrin-linked kinase in platelets: regulating integrin function and alpha-granule secretion

Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with beta 1- and beta 3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLC gamma 2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in alpha-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for alpha-granule secretion and therefore may play a central role in the regulation of platelet function. (Blood. 2008; 112: 4523-4531)

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

100 years on: a century of genetics

In 2006 we celebrated the centenary of a remarkable year that saw the birth of genetics as a scientific discipline. This birth had its origins in horticulture and was supervised by a remarkable Cambridge academic, accompanied by a loyal group of female colleagues who worked together in underfunded conditions with little institutional support. Despite this deprivation, they established the foundations of an ongoing revolution, with huge academic and commercial consequences that we can recognize today in the shape of genomics and its application to biomedicine.

Development of a reverse genetics system enabling the rescue of recombinant avian influenza virus A/Turkey/England/50-92/91 (H5N1)

We previously described the use of an established reverse genetics system for the generation of recombinant human influenza A viruses from cloned cDNAs. Here, we have assembled a set of plasmids to allow recovery of the avian H5N1 influenza virus A/Turkey/England/50-92/91 entirely from cDNA. This system enables us to introduce mutations or truncations into the cDNAs to create mutant viruses altered specifically in a chosen gene. These mutant viruses can then be used in future pathogenesis studies in chickens and in studies to understand the host range restrictions of avian influenza viruses in humans.

Domestication of plants in the americas: Insights from mendelian and molecular genetics

Background Plant domestication occurred independently in four different regions of the Americas. In general, different species were domesticated in each area, though a few species were domesticated independently in more than one area. The changes resulting from human selection conform to the familiar domestication syndrome, though different traits making up this syndrome, for example loss of dispersal, are achieved by different routes in crops belonging to different families. Genetic and Molecular Analyses of Domestication Understanding of the genetic control of elements of the domestication syndrome is improving as a result of the development of saturated linkage maps for major crops, identification and mapping of quantitative trait loci, cloning and sequencing of genes or parts of genes, and discoveries of widespread orthologies in genes and linkage groups within and between families. As the modes of action of the genes involved in domestication and the metabolic pathways leading to particular phenotypes become better understood, it should be possible to determine whether similar phenotypes have similar underlying genetic controls, or whether human selection in genetically related but independently domesticated taxa has fixed different mutants with similar phenotypic effects. Conclusions Such studies will permit more critical analysis of possible examples of multiple domestications and of the origin(s) and spread of distinctive variants within crops. They also offer the possibility of improving existing crops, not only major food staples but also minor crops that are potential export crops for developing countries or alternative crops for marginal areas.

PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5) P). We show that PtdIns(5) P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns(5) P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5) P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns(5) P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns(5) P production that plays an important role in host cell responses such as survival.

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2β). Both PtdIns-3-P and PI3K-C2β are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2β as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2β- PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.

The Bayesian revolution in genetics

Bayesian statistics allow scientists to easily incorporate prior knowledge into their data analysis. Nonetheless, the sheer amount of computational power that is required for Bayesian statistical analyses has previously limited their use in genetics. These computational constraints have now largely been overcome and the underlying advantages of Bayesian approaches are putting them at the forefront of genetic data analysis in an increasing number of areas.