Attenuation of insulin signalling contributes to FSN‐1‐mediated regulation of synapse development

A neuronal F‐box protein FSN‐1 regulates Caenorhabditis elegans neuromuscular junction development by negatively regulating DLK‐mediated MAPK signalling. In the present study, we show that attenuation of insulin/IGF signalling also contributes to FSN‐1‐dependent synaptic development and function. The aberrant synapse morphology and synaptic transmission in fsn‐1 mutants are partially and specifically rescued by reducing insulin/IGF‐signalling activity in postsynaptic muscles, as well as by reducing the activity of EGL‐3, a prohormone convertase that processes agonistic insulin/IGF ligands INS‐4 and INS‐6, in neurons. FSN‐1 interacts with, and potentiates the ubiquitination of EGL‐3 in vitro, and reduces the EGL‐3 level in vivo. We propose that FSN‐1 may negatively regulate insulin/IGF signalling, in part, through EGL‐3‐dependent insulin‐like ligand processing.     

Regulation of β-catenin stabilization in human platelets

The Wnt/β-catenin pathway controls developmental processes and homeostasis; however, abnormal activation of this pathway has been linked to several human diseases. Recent reports have demonstrated regulation of platelet function by canonical and non-canonical Wnt signalling. Platelet aggregation plays a crucial role in haemostasis and thrombosis. Here we report for the first time that, induction of sustained aggregation of platelets by a strong agonist in the presence of calcium was associated with nearly complete proteolysis of β-catenin, which was abrogated upon depletion of calcium from platelet suspension. β-catenin cleavage was disallowed in absence of aggregation, thus implicating integrin α_IIbβ₃ engagement in β-catenin proteolysis. Degradation of β-catenin was blocked partially by inhibitors of either proteasome or calpain and completely when cells were exposed to both the inhibitors. Protein kinase C inhibition, too, abolished β-catenin degradation. Thus activities of proteasome, calpain and protein kinase C regulate stabilization of β-catenin in aggregated human platelets.     

Enhanced adhesion and chemoattraction of zoospores of the fouling alga enteromorpha to some foul‐release silicone elastomers

Significantly higher numbers of zoospores of the fouling, green alga Enteromorpha adhered to silicone elastomers cured by dibutyltin dilaurate (DBTDL) than to identical polymers cured by dibutyltin diacetate (DBTDA). Enhanced zoospore adhesion was shown to be due to the presence of DBTDL and the effect was concentration‐dependent. Further experiments revealed that enhanced zoospore adhesion also occurred to glass coverslips coated with lauric acid (C₁₂) and other saturated fatty acids. The possibility that fatty acids may act as chemical cues (chemoattractants), guiding motile zoospores to the substratum for settlement in the natural environment is discussed. Settlement of other fouling organisms to DBTDL‐cured silicone elastomers is currently being investigated.     

How calcium signals in myocytes and pericytes are integrated across in situ microvascular networks and control microvascular tone

The microcirculation is the site of gas and nutrient exchange. Control of central or local signals acting on the myocytes, pericytes and endothelial cells within it, is essential for health. Due to technical problems of accessibility, the mechanisms controlling Ca²⁺ signalling and contractility of myocytes and pericytes in different sections of microvascular networks in situ have not been investigated. We aimed to investigate Ca²⁺ signalling and functional responses, in a microcirculatory network in situ. Using live confocal imaging of ureteric microvascular networks, we have studied the architecture, morphology, Ca²⁺ signalling and contractility of myocytes and pericytes. Ca²⁺ signals vary between distributing arcade and downstream transverse and precapillary arterioles, are modified by agonists, with sympathetic agonists being ineffective beyond transverse arterioles. In myocytes and pericytes, Ca²⁺ signals arise from Ca²⁺ release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca²⁺ release and not via ryanodine receptors or Ca²⁺ entry into the cell. The responses in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. Myocytes and pericytes are electrically coupled, transmitting Ca²⁺ signals between arteriolar and venular networks dependent on gap junctions and Ca²⁺ entry via L-type Ca²⁺ channels. Endothelial Ca²⁺ signalling inhibits intracellular Ca²⁺ oscillations in myocytes and pericytes via L-arginine/nitric oxide pathway and intercellular propagating Ca²⁺ signals via EDHF. Increases of Ca²⁺ in pericytes and myocytes constrict all vessels except capillaries. These data reveal the structural and signalling specializations allowing blood flow to be regulated by myocytes and pericytes.     

The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK)

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.     

Why did eukaryotes evolve only once? Genetic and energetic aspects of conflict and conflict mediation

According to multi-level theory, evolutionary transitions require mediating conflicts between lower-level units in favour of the higher-level unit. By this view, the origin of eukaryotes and the origin of multicellularity would seem largely equivalent. Yet, eukaryotes evolved only once in the history of life, whereas multicellular eukaryotes have evolved many times. Examining conflicts between evolutionary units and mechanisms that mediate these conflicts can illuminate these differences. Energy-converting endosymbionts that allow eukaryotes to transcend surface-to-volume constraints also can allocate energy into their own selfish replication. This principal conflict in the origin of eukaryotes can be mediated by genetic or energetic mechanisms. Genome transfer diminishes the heritable variation of the symbiont, but requires the de novo evolution of the protein-import apparatus and was opposed by selection for selfish symbionts. By contrast, metabolic signalling is a shared primitive feature of all cells. Redox state of the cytosol is an emergent feature that cannot be subverted by an individual symbiont. Hypothetical scenarios illustrate how metabolic regulation may have mediated the conflicts inherent at different stages in the origin of eukaryotes. Aspects of metabolic regulation may have subsequently been coopted from within-cell to between-cell pathways, allowing multicellularity to emerge repeatedly.     

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

G-protein coupled receptors (GPCRs) form a ternary complex of agonist, receptor and G-proteins during primary signal transduction at the cell membrane. Downstream signalling is thought to be preceded by the process of dissociation of Gα and Gβγ subunits, thus exposing new surfaces to interact with downstream effectors. We demonstrate here for the first time, the dissociation of heterotrimeric G-protein subunits (i.e., Gα and Gβγ) following agonist-induced GPCR (α_2A-adrenergic receptor; α_2A-AR) activation in a cell-free assay system. α_2A-AR membranes were reconstituted with the G-proteins (±hexahistidine-tagged) Gα_i1 and Gβ₁γ₂ and functional signalling was determined following activation of the reconstituted receptor:G-protein complex with the potent agonist UK-14304, and [³⁵S]GTPγS. In the presence of Ni²⁺-coated agarose beads, the activated his-tagged Gα_i1his-[³⁵S]GTPγS complex was captured on the Ni²⁺-presenting surface. When his-tagged Gβ₁γ₂ (Gβ₁γ₂his) was used with Gα_i1, the [³⁵S]GTPγS-bound Gα_i1 was not present on the Ni²⁺-coated beads, but rather, it was separated from the β₁γ₂(his)-beads, demonstrating receptor-induced dissociation of Gα and Gβγ subunits. Treatment of the reconstituted α_2A-AR membranes containing Gβ₁γ₂his:Gα_i1 with imidazole confirmed the specificity for the Ni²⁺:G-protein surface dissociation of Gα_i1 from Gβ₁γ₂his. These data demonstrate for the first time, the complete dissociation of the G-protein subunits and extend observations on the role of G-proteins in the assembly and disassembly of the ternary complex in the primary events of GPCR signalling.