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Townley A.K.,University of Bristol | Schmidt K.,University of Bristol | Schmidt K.,Max F Perutz Laboratories | Hodgson L.,University of Bristol | Stephens D.J.,University of Bristol
Journal of Cell Science | Year: 2012

Epithelial morphogenesis is directed by interactions with the underlying extracellular matrix. Secretion of collagen and other matrix components requires efficient coat complex II (COPII) vesicle formation at the endoplasmic reticulum. Here, we show that suppression of the outer layer COPII component, Sec13, in zebrafish embryos results in a disorganized gut epithelium. In human intestinal epithelial cells (Caco-2), Sec13 depletion causes defective epithelial polarity and organization on permeable supports. Defects are seen in the ability of cells to adhere to the substrate, form a monolayer and form intercellular junctions. When embedded in a three-dimensional matrix, Sec13-depleted Caco-2 cells form cysts but, unlike controls, are defective in lumen expansion. Incorporation of primary fibroblasts within the three-dimensional culture substantially restores normal morphogenesis. We conclude that efficient COPIIdependent secretion, notably assembly of Sec13-Sec31, is required to drive epithelial morphogenesis in both two- and three-dimensional cultures in vitro, as well as in vivo. Our results provide insight into the role of COPII in epithelial morphogenesis and have implications for the interpretation of epithelial polarity and organization assays in cell culture. © 2012.


Yang H.,Chonnam National University | Tong J.,Chonnam National University | Leonard T.A.,Max F Perutz Laboratories | Im Y.J.,Chonnam National University
FEBS Letters | Year: 2013

Sec14 family homologs are the major yeast phosphatidylinositol/ phosphatidylcholine transfer proteins regulating lipid metabolism and vesicle trafficking. The structure of Saccharomyces cerevisiae Sfh3 displays a conserved Sec14 scaffold and reveals determinants for the specific recognition of phosphatidylinositol ligand. Apo-Sfh3 forms a dimer through the hydrophobic interaction of gating helices. Binding of phosphatidylinositol leads to dissociation of the dimer into monomers in a reversible manner. This study suggests that the substrate induced dimer-monomer transformation is an essential part of lipid transfer cycles by Sfh3. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.


Doetsch M.,Max F Perutz Laboratories | Furtig B.,Max F Perutz Laboratories | Gstrein T.,Max F Perutz Laboratories | Stampfl S.,Max F Perutz Laboratories | Schroeder R.,Max F Perutz Laboratories
Nucleic Acids Research | Year: 2011

The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono-and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing-both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal. © 2011 The Author(s).


Breuker K.,University of Innsbruck | Bruschweiler S.,Max F Perutz Laboratories | Tollinger M.,University of Innsbruck | Tollinger M.,Max F Perutz Laboratories
Angewandte Chemie - International Edition | Year: 2011

One, two, three, whee! After transfer into the gas phase, the three-helix bundle protein KIX is sufficiently stabilized by electrostatic interactions to compensate for the loss of hydrophobic bonding. It thus retains its global fold on a timescale of more than 4 s. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Ewing G.,University of Vienna | Ewing G.,Max F Perutz Laboratories | Hermisson J.,University of Vienna | Hermisson J.,Max F Perutz Laboratories
Bioinformatics | Year: 2010

Motivation: We have implemented a coalescent simulation program for a structured population with selection at a single diploid locus. The program includes the functionality of the simulator ms to model population structure and demography, but adds a model for deme-and time-dependent selection using forward simulations. The program can be used, e.g. to study hard and soft selective sweeps in structured populations or the genetic footprint of local adaptation. The implementation is designed to be easily extendable and widely deployable. The interface and output format are compatible with ms. Performance is comparable even with selection included. © The Author(s) 2010. Published by Oxford University Press.


Matuszewski S.,University of Vienna | Hermisson J.,University of Vienna | Hermisson J.,Max F Perutz Laboratories | Kopp M.,Aix - Marseille University
Genetics | Year: 2015

Adaptation lies at the heart of Darwinian evolution. Accordingly, numerous studies have tried to provide a formal framework for the description of the adaptive process. Of these, two complementary modeling approaches have emerged: While socalled adaptive-walk models consider adaptation from the successive fixation of de novo mutations only, quantitative genetic models assume that adaptation proceeds exclusively from preexisting standing genetic variation. The latter approach, however, has focused on short-term evolution of population means and variances rather than on the statistical properties of adaptive substitutions. Our aim is to combine these two approaches by describing the ecological and genetic factors that determine the genetic basis of adaptation from standing genetic variation in terms of the effect-size distribution of individual alleles. Specifically, we consider the evolution of a quantitative trait to a gradually changing environment. By means of analytical approximations, we derive the distribution of adaptivesubstitutions from standing genetic variation, that is, the distribution of the phenotypic effects of those alleles from the standing variation that become fixed during adaptation. Our results are checked against individual-based simulations. We find that, compared to adaptation from de novo mutations, (i) adaptation from standing variation proceeds by the fixation of more alleles of small effect and (ii) populations that adapt from standing genetic variation can traverse larger distances in phenotype space and, thus, have a higher potential for adaptation if the rate of environmental change is fast rather than slow. © 2015 by the Genetics Society of America.


Bruschweiler S.,University of Innsbruck | Bruschweiler S.,Max F Perutz Laboratories | Konrat R.,Max F Perutz Laboratories | Tollinger M.,University of Innsbruck
ACS Chemical Biology | Year: 2013

The KIX domain of the transcriptional coactivator CREB binding protein (CBP) co-operatively mediates interactions between transcription factors. Binding of the transcription factor mixed-lineage leukemia (MLL) induces the formation of a low-populated conformer of KIX that resembles the conformation of the KIX domain in the presence of a second transcription factor molecule. NMR spin relaxation studies have previously shown that allosteric coupling proceeds through a network of hydrophobic core residues that bridge the two binding sites. Here we describe high-resolution NMR solution structures of the binary complex of KIX with MLL and the ternary complex of KIX formed with MLL and phosphorylated kinase inducible domain of CREB (pKID) as a second ligand. We show that binding of pKID to the binary complex of KIX with MLL is accompanied by a defined repacking of the allosteric network in the hydrophobic core of the protein. Rotamer populations derived from methyl group 13C chemical shifts reveal a dynamic contribution to the repacking process that is not captured by the structural coordinates and exemplify the dynamic nature of allosteric communication in the KIX domain. © 2013 American Chemical Society.


Conduit P.T.,University of Oxford | Feng Z.,University of Oxford | Richens J.H.,University of Oxford | Baumbach J.,University of Oxford | And 6 more authors.
Developmental Cell | Year: 2014

Centrosomes are important cell organizers. They consist of a pair of centrioles surrounded by pericentriolar material (PCM) that expands dramatically during mitosis-a process termed centrosome maturation. How centrosomes mature remains mysterious. Here, we identify a domain in Drosophila Cnn that appears to be phosphorylated by Polo/Plk1 specifically at centrosomes during mitosis. The phosphorylation promotes the assembly of a Cnn scaffold around the centrioles that is in constant flux, with Cnn molecules recruited continuously around the centrioles as the scaffold spreads slowly outward. Mutations that block Cnn phosphorylation strongly inhibit scaffold assembly and centrosome maturation, whereas phosphomimicking mutations allow Cnn to multimerize invitro and to spontaneously form cytoplasmic scaffolds invivo that organize microtubules independently of centrosomes. We conclude that Polo/Plk1 initiates the phosphorylation-dependent assembly of a Cnn scaffold around centrioles that is essential for efficient centrosome maturation in flies. © 2014 The Authors.


Klug H.,Max Planck Institute of Immunobiology and Epigenetics | Xaver M.,Max F Perutz Laboratories | Chaugule V.K.,Max Planck Institute of Immunobiology and Epigenetics | Koidl S.,Max Planck Institute of Immunobiology and Epigenetics | And 3 more authors.
Molecular Cell | Year: 2013

Posttranslational modification with the small ubiquitin-relatedmodifier SUMO depends on the sequential activities of E1, E2, and E3 enzymes. While regulation by E3 ligases and SUMO proteases is well understood, current knowledge of E2 regulation is very limited. Here, we describe modification of the budding yeast E2 enzyme Ubc9 by sumoylation (Ubc9*SUMO). Although less than 1% of Ubc9 is sumoylated at Lys153 at steady state, a sumoylation-deficient mutant showed significantly reduced meiotic SUMO conjugates and abrogates synaptonemal complex formation. Biochemical analysis revealed that Ubc9*SUMO is severely impaired in its classical activity but promoted SUMO chain assembly in the presence of Ubc9. Ubc9*SUMO cooperates with charged Ubc9 (Ubc9~SUMO) by noncovalent backside SUMO binding and by positioning the donor SUMO for optimal transfer. Thus, sumoylation of Ubc9 converts an active enzyme into a cofactor and reveals a mechanism for E2 regulation that orchestrates catalytic (Ubc9~SUMO) and noncatalytic (Ubc9*SUMO) functions of Ubc9. © 2013 Elsevier Inc.


Tobler R.,Institute For Populationsgenetik | Franssen S.U.,Institute For Populationsgenetik | Kofler R.,Institute For Populationsgenetik | Orozco-Terwengel P.,Institute For Populationsgenetik | And 5 more authors.
Molecular Biology and Evolution | Year: 2014

Experimental evolution in combination with whole-genome sequencing (evolve and resequence [E&R]) is a promising approach to define the genotype-phenotype map and to understand adaptation in evolving populations. Many previous studies have identified a large number of putative selected sites (i.e., candidate loci), but it remains unclear to what extent these loci are genuine targets of selection or experimental noise. To address this question, we exposed the same founder population to two different selection regimes-a hot environment and a cold environment-and quantified the genomic response in each. We detected large numbers of putative selected loci in both environments, albeit with little overlap between the two sets of candidates, indicating that most resulted from habitat-specific selection. By quantifying changes across multiple independent biological replicates, we demonstrate that most of the candidate SNPs were false positives that were linked to selected sites over distances much larger than the typical linkage disequilibrium range of Drosophila melanogaster. We show that many of these mid-to long-range associations were attributable to large segregating inversions and confirm by computer simulations that such patterns could be readily replicated when strong selection acts on rare haplotypes. In light of our findings, we outline recommendations to improve the performance of future Drosophila EandR studies which include using species with negligible inversion loads, such as D. mauritiana and D. simulans, instead of D. melanogaster. © 2013 The Author.

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