Continental collision and slab break-off: numerical models and surface observables

Collision zones worldwide show a vast array of complexity that is difficult to relate to simple collision dynamics. Here we address the problem of linking surface observables to collision dynamics through the use of two and three-dimensional numerical models. We will focus on two specific observables, topography and the presence of ultra-high pressure metamorphic terranes. Our model topography predictions are used to explain the uplift and subsidence history for the Arabia-Eurasia collision. This allows us to relate the post-collisional marine deposits, found on the overriding plate, to a steepening of the subduction interface. Our three-dimensional model is used to explore possible exhumation mechanisms for the Western Gneiss Complex in Norway. From these models we show how an asymmetric collision can help drive exhumation of material that has experienced comparable conditions to the Western Gneiss Complex. The linking of upper mantle and lithosphere dynamics to both topography observation and exhumation patterns allow better understanding of the subduction and collision process

High-energy collision-induced dissociation of macromolecules using tandem double-focusing/time-of-flight mass spectrometry

The first part of this study involves the adaptation of a matrix-assisted laser desorption/ionisation (MALDI) ion source for a tandem double-focusingltime-offlight instrument (MAG-TOF). Ion trajectory modelling was carried out for defining the optimum ion optical configuration for a new extraction region and associated ion optics that were designed and constructed. Installation of the new ion source resulted in increased sensitivity and no loss of resolution. The second part of this study involves the analysis of fullerenes and fullerene derivatives by high-energy collision-induced dissociation (CID). The structure of fullerenes formed by coalescence under the conditions of laser desorption were shown to be that of a single fullerene closed-cage structure. The dissociation of exohedral fullerene hydride derivatives was investigated. The third part of this study investigates the high-energy collision-induced dissociation of polyglycol polymer ions generated by MALDI. Mechanisms have been proposed for the dissociation of poly(ethylene glycol) and poly(propylene glycol). High-energy CID has been shown to be particularly useful for the determination of polymer end-group structure

Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses

Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure–function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H+-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses

N-methyl-D-aspartate receptors mediate the phosphorylation and desensitization of muscarinic receptors in cerebellar granule neurons.

Changes in synaptic strength mediated by ionotropic glutamate N-methyl-D-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the G(q/11)-coupled M(3)-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M(3)-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity

Differential G-protein-coupled receptor phosphorylation provides evidence for a signaling bar code.

G-protein-coupled receptors are hyper-phosphorylated in a process that controls receptor coupling to downstream signaling pathways. The pattern of receptor phosphorylation has been proposed to generate a "bar code" that can be varied in a tissue-specific manner to direct physiologically relevant receptor signaling. If such a mechanism existed, receptors would be expected to be phosphorylated in a cell/tissue-specific manner. Using tryptic phosphopeptide maps, mass spectrometry, and phospho-specific antibodies, it was determined here that the prototypical G(q/11)-coupled M(3)-muscarinic receptor was indeed differentially phosphorylated in various cell and tissue types supporting a role for differential receptor phosphorylation in directing tissue-specific signaling. Furthermore, the phosphorylation profile of the M(3)-muscarinic receptor was also dependent on the stimulus. Full and partial agonists to the M(3)-muscarinic receptor were observed to direct phosphorylation preferentially to specific sites. This hitherto unappreciated property of ligands raises the possibility that one mechanism underlying ligand bias/functional selectivity, a process where ligands direct receptors to preferred signaling pathways, may be centered on the capacity of ligands to promote receptor phosphorylation at specific sites

The structure of the core NuRD repression complex provides insights into its interaction with chromatin

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromati

<i>Plasmodium </i>Condensin Core Subunits SMC2/SMC4 Mediate Atypical Mitosis and Are Essential for Parasite Proliferation and Transmission

Condensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle

Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC

Mycobacterium tuberculosis (Mtb) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: ΔuspC or the entire transport complex: ΔuspABC. Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an Nin-Cin orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and ΔuspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways

Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

Sam68, also known as KHDRBS1, is a member of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins and its role is modulated by post-translational modifications, including serine/threonine phosphorylation, that differ at various stages of the cell cycle. However, the molecular basis and mechanisms of these modulations remain largely unknown. Here, we combined mass spectrometry, nuclear magnetic resonance spectroscopy and cell biology techniques to provide a comprehensive post-translational modification mapping of Sam68 at different stages of the cell cycle in HEK293 and HCT116 cells. We established that Sam68 is specifically phosphorylated at T33 and T317 by Cdk1, and demonstrated that these phosphorylation events reduce the binding of Sam68 to RNA, control its cellular localization and reduce its alternative splicing activity, leading to a reduction in the induction of apoptosis and an increase in the proliferation of HCT116 cells

Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs

Copyright @ 2011 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs