Phase transitions in the assembly of multivalent signalling proteins

Cells are organized on length scales ranging from ?ngstr?m to micrometres. However, the mechanisms by which ?ngstr?m-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid-liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott-Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.     

Deciphering a neuronal circuit that mediates appetite

Hypothalamic neurons that co-express agouti-related protein (AgRP), neuropeptide?Y and γ-aminobutyric acid (GABA) are known to promote feeding and weight gain by integration of various nutritional, hormonal, and neuronal signals. Ablation of these neurons in mice leads to cessation of feeding that is accompanied by activation of Fos in most regions where they project. Previous experiments have indicated that the ensuing starvation is due to aberrant activation of the parabrachial nucleus (PBN) and it could be prevented by facilitating GABA(A) receptor signalling in the PBN within a critical adaptation period. We speculated that loss of GABA signalling from AgRP-expressing neurons (AgRP neurons) within the PBN results in unopposed excitation of the PBN, which in turn inhibits feeding. However, the source of the excitatory inputs to the PBN was unknown. Here we show that glutamatergic neurons in the nucleus tractus solitarius (NTS) and caudal serotonergic neurons control the excitability of PBN neurons and inhibit feeding. Blockade of serotonin (5-HT(3)) receptor signalling in the NTS by either the chronic administration of ondansetron or the genetic inactivation of Tph2 in caudal serotonergic neurons that project to the NTS protects against starvation when AgRP neurons are ablated. Likewise, genetic inactivation of glutamatergic signalling by the NTS onto N-methyl D-aspartate-type glutamate receptors in the PBN prevents starvation. We also show that suppressing glutamatergic output of the PBN reinstates normal appetite after AgRP neuron ablation, whereas it promotes weight gain without AgRP neuron ablation. Thus we identify the PBN as a hub that integrates signals from several brain regions to bidirectionally modulate feeding and body weight.     

A complete insect from the Late Devonian period

After terrestrialization, the diversification of arthropods and vertebrates is thought to have occurred in two distinct phases, the first between the Silurian and the Frasnian stages (Late Devonian period) (425-385?million years (Myr) ago), and the second characterized by the emergence of numerous new major taxa, during the Late Carboniferous period (after 345?Myr ago). These two diversification periods bracket the depauperate vertebrate Romer's gap (360-345?Myr ago) and arthropod gap (385-325?Myr ago), which could be due to preservational artefact. Although a recent molecular dating has given an age of 390?Myr for the Holometabola, the record of hexapods during the Early-Middle Devonian (411.5-391?Myr ago, Pragian to Givetian stages) is exceptionally sparse and based on fragmentary remains, which hinders the timing of this diversification. Indeed, although Devonian Archaeognatha are problematic, the Pragian of Scotland has given some Collembola and the incomplete insect Rhyniognatha, with its diagnostic dicondylic, metapterygotan mandibles. The oldest, definitively winged insects are from the Serpukhovian stage (latest Early Carboniferous period). Here we report the first complete Late Devonian insect, which was probably a terrestrial species. Its 'orthopteroid' mandibles are of an omnivorous type, clearly not modified for a solely carnivorous diet. This discovery narrows the 45-Myr gap in the fossil record of Hexapoda, and demonstrates further a first Devonian phase of diversification for the Hexapoda, as in vertebrates, and suggests that the Pterygota diversified before and during Romer's gap.     

Atomic model of the type III secretion system needle

Pathogenic bacteria using a type III secretion system (T3SS) to manipulate host cells cause many different infections including Shigella dysentery, typhoid fever, enterohaemorrhagic colitis and bubonic plague. An essential part of the T3SS is a hollow needle-like protein filament through which effector proteins are injected into eukaryotic host cells. Currently, the three-dimensional structure of the needle is unknown because it is not amenable to X-ray crystallography and solution NMR, as a result of its inherent non-crystallinity and insolubility. Cryo-electron microscopy combined with crystal or solution NMR subunit structures has recently provided a powerful hybrid approach for studying supramolecular assemblies, resulting in low-resolution and medium-resolution models. However, such approaches cannot deliver atomic details, especially of the crucial subunit-subunit interfaces, because of the limited cryo-electron microscopic resolution obtained in these studies. Here we report an alternative approach combining recombinant wild-type needle production, solid-state NMR, electron microscopy and Rosetta modelling to reveal the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. We show that the 80-residue subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellar filament of S. typhimurium. In contrast to established models of the needle in which the amino terminus of the protein subunit was assumed to be α-helical and positioned inside the needle, our model reveals an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen.     

Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2

Autism spectrum disorders comprise a range of neurodevelopmental disorders characterized by deficits in social interaction and communication, and by repetitive behaviour. Mutations in synaptic proteins such as neuroligins, neurexins, GKAPs/SAPAPs and ProSAPs/Shanks were identified in patients with autism spectrum disorder, but the causative mechanisms remain largely unknown. ProSAPs/Shanks build large homo- and heteromeric protein complexes at excitatory synapses and organize the complex protein machinery of the postsynaptic density in a laminar fashion. Here we demonstrate that genetic deletion of ProSAP1/Shank2 results in an early, brain-region-specific upregulation of ionotropic glutamate receptors at the synapse and increased levels of ProSAP2/Shank3. Moreover, ProSAP1/Shank2(-/-) mutants exhibit fewer dendritic spines and show reduced basal synaptic transmission, a reduced frequency of miniature excitatory postsynaptic currents and enhanced N-methyl-d-aspartate receptor-mediated excitatory currents at the physiological level. Mutants are extremely hyperactive and display profound autistic-like behavioural alterations including repetitive grooming as well as abnormalities in vocal and social behaviours. By comparing the data on ProSAP1/Shank2(-/-) mutants with ProSAP2/Shank3αβ(-/-) mice, we show that different abnormalities in synaptic glutamate receptor expression can cause alterations in social interactions and communication. Accordingly, we propose that appropriate therapies for autism spectrum disorders are to be carefully matched to the underlying synaptopathic phenotype.     

Global variation in copy number in the human genome

Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.