The simple phosphoinositide phosphatidylinositol 3-phosphate (PI(3)P) has been known to have important functions in endocytic and phagocytic traffic, and to be required for the autophagic pathway. In all of these settings, PI(3)P appears to create platforms that serve to recruit specific effectors for membrane trafficking events. In autophagy, PI(3)P may form the platform for autophagosome biogenesis.

Members of the widespread rhomboid family of intramembrane proteases cleave transmembrane domain (TMD) proteins to regulate processes as diverse as EGF receptor signaling, mitochondrial dynamics, and invasion by apicomplexan parasites. However, lack of information about their substrates means that the biological role of most rhomboids remains obscure. Knowledge of how rhomboids recognize their substrates would illuminate their mechanism and might also allow substrate prediction. Previous work has suggested that rhomboid substrates are specified by helical instability in their TMD. Here we demonstrate that rhomboids instead primarily recognize a specific sequence surrounding the cleavage site. This recognition motif is necessary for substrate cleavage, it determines the cleavage site, and it is more strictly required than TM helix-destabilizing residues. Our work demonstrates that intramembrane proteases can be sequence specific and that genome-wide substrate prediction based on their recognition motifs is feasible.

The molecules on mammalian spermatozoa that mediate recognition and binding to the zona pellucida of the egg are still not understood. Current concepts favour their assembly into multimolecular complexes in the plasma membrane in response to cholesterol efflux, an important step during sperm capacitation. Here, we track in real time diffusion of cross-linked clusters containing zona-binding molecules and GM1 gangliosides in the plasma membrane of live boar spermatozoa before and after cholesterol reduction. Both GM1 gangliosides and zona-binding molecules partition into a low density Triton X100 resistant phase suggesting their association with lipid rafts. Initially, GM1 and zona-binding molecules localize to the apical ridge on the acrosome but following cholesterol efflux with methyl-beta-cyclodextrin, clusters containing zona-binding molecules diffuse randomly over the acrosomal domain. Diffusing clusters of either type do not access the postacrosome. Spermatozoa agglutinated head-to-head show contact-induced coalescence of GM1 gangliosides (but not zona-binding molecules) suggestive of a specific mechanosensitive response. Thus, cholesterol efflux initiates diffusion (and possibly formation) of novel lipid raft-like structures containing zona-binding molecules over the sperm acrosome. We hypothesise that in combination with contact coalescence, these mechanisms concentrate important molecules to the appropriate site on the sperm surface to mediate zona binding.

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant-negative inactive mutant of Myotubularin-related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy-related PtdIns3P-binding proteins, GFP-DFCP1 and GFP-WIPI-1alpha (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock-down of MTMR3 increased autophagosome formation, and overexpression of wild-type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3-kinase and PI 3-phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.

Biological signal transduction commonly involves cooperative interactions in the binding of ligands to their receptors. In many cases, ligand concentrations in vivo are close to the value of the dissociation constant of their receptors, resulting in the phenomenon of ligand depletion. Using examples based on rotational bias of bacterial flagellar motors and calcium binding to mammalian calmodulin, we show that ligand depletion diminishes cooperativity and broadens the dynamic range of sensitivity to the signaling ligand. As a result, the same signal transducer responds to different ranges of signal with various degrees of cooperativity according to its effective cellular concentration. Hence, results from in vitro dose-response analyses cannot be applied directly to understand signaling in vivo. Moreover, the receptor concentration is revealed to be a key element in controlling signal transduction and we propose that its modulation constitutes a new way of controlling sensitivity to signals. In addition, through an analysis of the allosteric enzyme aspartate transcarbamylase, we demonstrate that the classical Hill coefficient is not appropriate for characterizing the change in conformational state upon ligand binding to an oligomeric protein (equivalent to a dose-response curve), because it ignores the cooperativity of the conformational change for the corresponding equivalent monomers, which are generally characterized by a Hill coefficient . Therefore, we propose a new index of cooperativity based on the comparison of the properties of oligomers and their equivalent monomers.

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110delta, and class IB, p110gamma catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110delta, but not p110gamma. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein-coupled receptor signaling in beta-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of beta-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond beta-selection.

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins.

Axon degeneration is an early event in many neurodegenerative disorders. In some, the mechanism is related to injury-induced Wallerian degeneration, a proactive death program that can be strongly delayed by the neuroprotective slow Wallerian degeneration protein (Wld(S)) protein. Thus, it is important to understand the Wallerian degeneration mechanism and how Wld(S) blocks it. Wld(S) location is influenced by binding to valosin-containing protein (VCP), an essential protein for many cellular processes including membrane fusion and endoplasmic reticulum-associated degradation. In mice, the N-terminal 16 amino acids (N16), which mediate VCP binding, are essential for Wld(S) to protect axons, a role which another VCP binding sequence can substitute. In Drosophila, the Wld(S) phenotype is weakened by a similar N-terminal truncation and by knocking down the VCP homologue ter94. Neither null nor floxed VCP mice are viable so it is difficult to confirm the requirement for VCP binding in mammals in vivo. However, the hypothesis can be tested further by introducing a Wld(S) missense mutation, altering its affinity for VCP but minimizing the risk of disturbing other aspects of its structure or function. We introduced the R10A mutation, which weakens VCP binding in vitro, and expressed it in transgenic mice. R10AWld(S) fails to co-immunoprecipitate VCP from mouse brain, and only occasionally and faintly accumulates in nuclear foci for which VCP binding is necessary but not sufficient. Surprisingly however, axon protection remains robust and indistinguishable from that in spontaneous Wld(S) mice. We suggest that either N16 has an additional, VCP-independent function in mammals, or that the phenotype requires only weak VCP binding which may be driven forwards in vivo by the high VCP concentration.

The discovery of interchromosomal interactions in higher eukaryotes points to a functional interplay between genome architecture and gene expression, challenging the view of transcription as a one-dimensional process. However, the extent of interchromosomal interactions and the underlying mechanisms are unknown. Here we present the first genome-wide analysis of transcriptional interactions using the mouse globin genes in erythroid tissues. Our results show that the active globin genes associate with hundreds of other transcribed genes, revealing extensive and preferential intra- and interchromosomal transcription interactomes. We show that the transcription factor Klf1 mediates preferential co-associations of Klf1-regulated genes at a limited number of specialized transcription factories. Our results establish a new gene expression paradigm, implying that active co-regulated genes and their regulatory factors cooperate to create specialized nuclear hot spots optimized for efficient and coordinated transcriptional control.

Expression of the nuclear orphan receptor gene Nur77 in neuronal cells is induced by activity-dependent increases in intracellular Ca2+ ions. Ca2+ responsiveness of the Nur77 gene has been attributed to two distinct DNA regulatory regions that recruit the transcription factors cAMP response element binding protein (CREB) and myocyte enhancer factor-2 (MEF2). Here we used dominant interfering and constitutively active mutants of CREB and MEF2 proteins to assess their relative contribution to depolarization-induced Nur77 expression in undifferentiated PC12 cells and hippocampal neurons. We show that while CREB is necessary for Ca2+-activated Nur77 expression MEF2 functions to modulate CREB-dependent Nur77 expression by acting as a repressor in quiescent cells.

Cohesin is a chromatin-associated protein complex that mediates sister chromatid cohesion by connecting replicated DNA molecules. Cohesin also has important roles in gene regulation, but the mechanistic basis of this function is poorly understood. In mammalian genomes, cohesin co-localizes with CCCTC binding factor (CTCF), a zinc finger protein implicated in multiple gene regulatory events. At the imprinted IGF2-H19 locus, CTCF plays an important role in organizing allele-specific higher-order chromatin conformation and functions as an enhancer blocking transcriptional insulator. Here we have used chromosome conformation capture (3C) assays and RNAi-mediated depletion of cohesin to address whether cohesin affects higher order chromatin conformation at the IGF2-H19 locus in human cells. Our data show that cohesin has a critical role in maintaining CTCF-mediated chromatin conformation at the locus and that disruption of this conformation coincides with changes in IGF2 expression. We show that the cohesin-dependent, higher-order chromatin conformation of the locus exists in both G1 and G2 phases of the cell cycle and is therefore independent of cohesin's function in sister chromatid cohesion. We propose that cohesin can mediate interactions between DNA molecules in cis to insulate genes through the formation of chromatin loops, analogous to the cohesin mediated interaction with sister chromatids in trans to establish cohesion.

Chromatin regulates many key processes in the nucleus by controlling access to the underlying DNA. SNF2-like factors are ATP-driven enzymes that play key roles in the dynamics of chromatin by remodelling nucleosomes and other nucleoprotein complexes. Even simple eukaryotes such as yeast contain members of several subfamilies of SNF2-like factors. The FUN30/ETL1 subfamily of SNF2 remodellers is conserved from yeasts to humans, but is poorly characterized. We show that the deletion of FUN30 leads to sensitivity to the topoisomerase I poison camptothecin and to severe cell cycle progression defects when the Orc5 subunit is mutated. We demonstrate a role of FUN30 in promoting silencing in the heterochromatin-like mating type locus HMR, telomeres and the rDNA repeats. Chromatin immunoprecipitation experiments demonstrate that Fun30 binds at the boundary element of the silent HMR and within the silent HMR. Mapping of nucleosomes in vivo using micrococcal nuclease demonstrates that deletion of FUN30 leads to changes of the chromatin structure at the boundary element. A point mutation in the ATP-binding site abrogates the silencing function of Fun30 as well as its toxicity upon overexpression, indicating that the ATPase activity is essential for these roles of Fun30. We identify by amino acid sequence analysis a putative CUE motif as a feature of FUN30/ETL1 factors and show that this motif assists Fun30 activity. Our work suggests that Fun30 is directly involved in silencing by regulating the chromatin structure within or around silent loci.

Embryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.

Exchanging and sharing scientific results are essential for researchers in the field of computational modelling. BioModels.net defines agreed-upon standards for model curation. A fundamental one, MIRIAM (Minimum Information Requested in the Annotation of Models), standardises the annotation and curation process of quantitative models in biology. To support this standard, MIRIAM Resources maintains a set of standard data types for annotating models, and provides services for manipulating these annotations. Furthermore, BioModels.net creates controlled vocabularies, such as SBO (Systems Biology Ontology) which strictly indexes, defines and links terms used in Systems Biology. Finally, BioModels Database provides a free, centralised, publicly accessible database for storing, searching and retrieving curated and annotated computational models. Each resource provides a web interface to submit, search, retrieve and display its data. In addition, the BioModels.net team provides a set of Web Services which allows the community to programmatically access the resources. A user is then able to perform remote queries, such as retrieving a model and resolving all its MIRIAM Annotations, as well as getting the details about the associated SBO terms. These web services use established standards. Communications rely on SOAP (Simple Object Access Protocol) messages and the available queries are described in a WSDL (Web Services Description Language) file. Several libraries are provided in order to simplify the development of client software. BioModels.net Web Services make one step further for the researchers to simulate and understand the entirety of a biological system, by allowing them to retrieve biological models in their own tool, combine queries in workflows and efficiently analyse models.

Two main approaches in exploring causal relationships in biological systems using time-series data are the application of Dynamic Causal model (DCM) and Granger Causal model (GCM). These have been extensively applied to brain imaging data and are also readily applicable to a wide range of temporal changes involving genes, proteins or metabolic pathways. However, these two approaches have always been considered to be radically different from each other and therefore used independently. Here we present a novel approach which is an extension of Granger Causal model and also shares the features of the bilinear approximation of Dynamic Causal model. We have first tested the efficacy of the extended GCM by applying it extensively in toy models in both time and frequency domains and then applied it to local field potential recording data collected from in vivo multi-electrode array experiments. We demonstrate face discrimination learning-induced changes in inter- and intra-hemispheric connectivity and in the hemispheric predominance of theta and gamma frequency oscillations in sheep inferotemporal cortex. The results provide the first evidence for connectivity changes between and within left and right inferotemporal cortexes as a result of face recognition learning.

Cardiac hypertrophy is associated with profound remodeling of Ca(2+) signaling pathways. During the early, compensated stages of hypertrophy, Ca(2+) fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca(2+) homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca(2+) release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca(2+) store are primarily responsible for the Ca(2+) flux that induces myocyte contraction, a role for Ca(2+) release via the inositol 1,4,5-trisphosphate receptor (InsP(3)R) in cardiac physiology has also emerged. Specifically, InsP(3)-induced Ca(2+) signals generated following myocyte stimulation with an InsP(3)-generating agonist (e.g., endothelin, ET-1), lead to modulation of Ca(2+) signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca(2+) release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP(3)R (InsP(3)R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca(2+) release via InsP(3)Rs serves to sensitize neighboring RyRs causing an augmentation of Ca(2+) fluxes during ECC as well as an increase in non-triggered Ca(2+) release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP(3)R expression during hypertrophy, the spontaneous Ca(2+) release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP(3)R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP(3)R expression may be a general phenomenon that underlies Ca(2+) changes during hypertrophy.

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

Ribosomal protein S6 (rpS6) is a key component of the translational machinery in eukaryotic cells and is essential for ribosome biogenesis. rpS6 is phosphorylated on evolutionarily conserved serine residues, and data indicate that rpS6 phosphorylation might regulate cell growth and protein synthesis. Studies in cell lines have shown an important role for the serine kinase mammalian target of rapamycin (mTOR) in rpS6 phosphorylation, further linking rpS6 to control of cellular metabolism. rpS6 is essential in T cells because its deletion in mouse double-positive thymocyte cells results in a complete block in T cell development; however, the signaling pathway leading to rpS6 phosphorylation downstream of TCR stimulation has yet to be fully characterized. We show that maximal TCR-induced rpS6 phosphorylation in CD8 T cells requires both Lck and Fyn activity and downstream activation of PI3K, mTOR, and MEK/ERK MAPK pathways. We demonstrate that there is cross-talk between the PI3K and MAPK pathways as well as PI3K-independent mTOR activity, which result in differential phosphorylation of specific rpS6 serine residues. These results place rpS6 phosphorylation as a point of convergence for multiple crucial signaling pathways downstream of TCR triggering.

Mice can learn a food preference from odor cues transmitted on the breath of a conspecific, even if the "demonstrator" is anesthetized. To our knowledge there are no studies examining the effect of anesthetizing the "observer" on development of memory for socially transmitted food preferences (STFP). In Experiment 1 we found that 2-4 month-old F2 C57Bl/6x129sv male and female mice demonstrated a STFP after a 5min exposure to an anesthetized demonstrator mouse when tested 24h later. In Experiment 2, observer mice anesthetized with Sagatal (60 mg/kg) prior to the "social interaction" preferentially avoided the cued food when tested 24h later. This aversion was not due to any overt aversive effects of this dose of Sagatal because mice that ate the food and were then anesthetized, or could only smell the food for 5 min while anesthetized, showed no preference or aversion. In a third experiment we found that the Sagatal-induced aversion was not a general property of anesthesia because there were varied results produced by observer mice treated with anesthetic drugs with different mechanisms of action. Vetalar (200mg/kg) and Rompun (10 mg/kg) treated animals ate similar amounts of cued and non-cued food at test, indicating an absence of learning. Hypnorm (0.5 ml/kg) treated animals showed a preference for the cued food whereas those treated with Hypnovel (2.5 ml/kg) showed an aversion to the cued food. These results show that the food aversion observed with Sagatal is not a general property of anesthetic agents, but appears to be restricted to those acting primarily on the GABAergic system. Thus, we have shown that under certain conditions it is possible for an anesthetized observer mouse to learn a preference or aversion of a socially-linked olfactory cue.

Fibroblast growth factors (FGFs) acting through their cognate receptors (FGFRs) play vital roles in development and de-regulation of FGF/FGFR signalling is associated with many developmental syndromes. In addition there is much interest in inhibiting FGF/FGFR signalling as a therapeutic approach to cancer. FGF/FGFR signalling is certainly important in tumour angiogenesis but studies in the last few years have uncovered increasing evidence that FGFRs are driving oncogenes in certain cancers and act in a cell autonomous fashion to maintain the malignant properties of tumour cells. These observations make FGFRs increasingly attractive as targets for therapeutic intervention in cancer. In this article, we review FGFR signalling and describe recent advances in cancer genomics and cancer cell biology that demonstrate that specific tumour types are dependent upon or addicted to de-regulated FGFR. We also describe the range of therapeutic strategies currently employed or in development to antagonise de-regulated FGFRs including antibodies and small molecule tyrosine kinase inhibitors.

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3'-5' cyclic guanosine monophosphate (cGMP). Ca(2+)-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC(50) values of 11, 80 and 180 microM respectively. Here we sought to elucidate the underlying mechanism(s).

Lymphocyte migration is crucial for immunological surveillance. A better understanding of the mechanisms that regulate lymphocyte migration will help appreciate how lymphocytes deliver specific functions to appropriate anatomical sites. Phosphoinositide-3 kinases (PI3Ks) are lipid kinases that regulate numerous cellular responses, including cell motility and chemotaxis. Here we discuss how PI3K isoforms differentially regulate lymphocyte migration and trafficking, with an emphasis on natural killer cells.