bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation
Authors: Ma Wenzhe , Chen Maorong , Kang Hong , Steinhart Zachary , Angers Stephane , He Xi , Kirschner Marc W. 
Published: May 2019

AbstractDvl (Dishevelled) is one of several essential non-enzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits other components of the destruction complex leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for this process is lacking. In this study, we tagged mEGFP to C-terminus of dishevlled2 gene using CRISPR/Cas9 induced homologous recombination and observed its dynamics directly at the single molecule level with Total Internal Reflection Fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and the dynamic features of membrane-associated Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane bound Dvl2 is predominantly monomer in the absent of Wnt (mean size 1.10). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.08/μm2 to 0.34/μm2. Wnt3a also leads to increased oligomerization which raises the weighted averaged mean size of Dvl2 complexes to 1.4; with 65% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of Dvl2 at the membrane caused by increased affinity of Dvl2 for Fzd, the Dvl2 and Fzd binding is independent of LRP5/6. The oligomerized Dvl2 complexes have greatly increased dwell time, 2~3 minutes compared to less than 1 second for monomeric Dvl2. These properties make Dvl a unique scaffold dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.Significance StatementCanonical Wnt signaling is one of the most widely distributed pathways in metazoan development. Despite intense genetic and biochemical study for over 35 years, the major features of signaling across the plasma membrane are still poorly understood. Dishevelled serves as an essential bridge between the membrane receptors and downstream signaling components. Attempts to reconstruct the pathway and analyze its biochemical features in vitro have been hampered by Dishevelled’s tendency to aggregate in vitro and to form large aggregates of dubious significance in vivo. To obtain a molecular understanding of the role of Dvl in Wnt signaling, while circumventing these aggregation problems we have expressed a fluorescent tagged Dishevelled in cells at their physiological concentration and quantified the size distribution of Dishevelled before and after Wnt treatment. We found that limited oligomerization in response to the Wnt ligand is very dynamic and provides a key step of signal transduction.
A Network of Phosphatidylinositol 4,5-bisphosphate Binding Sites Regulate Gating of the Ca2+-activated Cl− Channel ANO1 (TMEM16A)
Authors: Yu Kuai , Jiang Tao , Cui YuanYuan , Tajkhorshid Emad , Hartzell H. Criss 
Published: May 2019

AbstractANO1 (TMEM16A) is a Ca2+-activated Cl− channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Here we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid-protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2. Three of these sites captured 85% of all ANO1 - PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane-cytosol interface found three regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same three sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid sidechains and phosphate/hydroxyl groups on PI(4,5)P2. Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl−ions. We propose a model consisting of a network of three PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.Significance statementMembrane proteins dwell in a sea of phospholipids that not only structurally stabilize the proteins by providing a hydrophobic environment for their transmembrane segments, but also dynamically regulate protein function. While many cation channels are known to be regulated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), relatively little is known about anion channel regulation by phosphoinositides. Using a combination of patch clamp electrophysiology and atomistic molecular dynamics simulations, we have identified several PI(4,5)P2 binding sites in ANO1 (TMEM16A), a Cl− channel that performs myriad physiological functions from epithelial fluid secretion to regulation of electrical excitability. These binding sites form a band at the cytosolic interface of the membrane that we propose constitute a network to dynamically regulate this highly allosteric protein.
Temporal perturbation of Erk dynamics reveals network architecture of FGF2-MAPK signaling
Authors: Blum Yannick , Mikelson Jan , Dobrzyński Maciej , Ryu Hyunryul , Jacques Marc-Antoine , Jeon Noo Li , Khammash Mustafa , Pertz Olivier 
Published: May 2019

AbstractStimulation of PC-12 cells with epidermal (EGF) versus nerve (NGF) growth factors (GFs) biases the distribution between transient and sustained single-cell ERK activity states, and between proliferation and differentiation fates within a cell population. We report that fibroblast GF (FGF2) evokes a distinct behavior that consists of a gradually changing population distribution of transient/sustained ERK signaling states in response to increasing inputs in a dose response. Temporally-controlled GF perturbations of MAPK signaling dynamics applied using microfluidics reveals that this wider mix of ERK states emerges through the combination of an intracellular feedback, and competition of FGF2 binding to FGF receptors (FGFR) and heparan-sulfate proteoglycans (HSPGs) co-receptors. We show that the latter experimental modality is instructive for model selection using a Bayesian parameter inference. Our results provide novel insights into how different receptor tyrosine kinase (RTK) systems differentially wire the MAPK network to fine tune fate decisions at the cell population-level.Microfluidics, Erk Signaling Dynamics, Mechanistic Modelling, Parameter Estimation, Cell Fate Determination
Differential local stability governs the metamorphic fold-switch of bacterial virulence factor RfaH
Authors: Galaz-Davison P. , Molina J.A. , Silletti S. , Komives E.A. , Knauer S.H. , Artsimovitch I. , Ramírez-Sarmiento C.A. 
Published: May 2019

AbstractA regulatory factor RfaH, present in many Gram-negative bacterial pathogens, is required for transcription and translation of long operons encoding virulence determinants. Escherichia coli RfaH action is controlled by a unique large-scale structural rearrangement triggered by recruitment to transcription elongation complexes through a specific DNA sequence within these operons. Upon recruitment, the C-terminal domain of this two-domain protein refolds from an α-hairpin, which is bound to the RNA polymerase binding site within the N-terminal domain of RfaH, into an unbound β-barrel that interacts with the ribosome to enable translation. Although structures of the autoinhibited (α-hairpin) and active (β-barrel) states and plausible refolding pathways have been reported, how this reversible switch is encoded within RfaH sequence and structure is poorly understood. Here, we combined hydrogen-deuterium exchange measurements by mass spectrometry and nuclear magnetic resonance with molecular dynamics to evaluate the differential local stability between both RfaH folds. Deuteron incorporation reveals that the tip of the C-terminal hairpin (residues 125-145) is stably folded in the autoinhibited state (∼20% deuteron incorporation), while the rest of this domain is highly flexible (>40% deuteron incorporation) and its flexibility only decreases in the β-folded state. Computationally-predicted ΔGs agree with these results by displaying similar anisotropic stability within the tip of the α-hairpin and on neighboring N-terminal domain residues. Remarkably, the β-folded state shows comparable stability to non-metamorphic homologs. Our findings provide information critical for understanding the metamorphic behavior of RfaH and other chameleon proteins, and for devising targeted strategies to combat bacterial diseases.SignificanceInfections caused by Gram-negative bacteria are a worldwide health threat due to rapid acquisition of antibiotic resistance. RfaH, a protein essential for virulence in several Gram-negative pathogens, undergoes a large-scale structural rearrangement in which one RfaH domain completely refolds. Refolding transforms RfaH from an inactive state that restricts RfaH recruitment to a few target genes into an active state that binds to, and couples, transcription and translation machineries to elicit dramatic activation of gene expression. However, the molecular basis of this unique conformational change is poorly understood. Here, we combine molecular dynamics and structural biology to unveil the hotspots that differentially stabilize both states of RfaH. Our findings provide novel insights that will guide design of inhibitors blocking RfaH action.
Androgen-regulated transcription ofESRP2drives alternative splicing patterns in prostate cancer
Authors: Munkley Jennifer , Ling Li , Krishnan S R Gokul , Hysenaj Gerald , Scott Emma , Oo Htoo Zarni , Maia Teresa M. , Cheung Kat , Ehrmann Ingrid , Livermore Karen E. , Zielinska Hanna , Thompson Oliver , Knight Bridget , McCullagh Paul , McGrath John , Crundwell Malcolm , Harries Lorna W. , Daugaard Mads , Cockell Simon , Barbosa-Morais Nuno L. , Oltean Sebastian , Elliott David J 
Published: May 2019

AbstractProstate is the most frequent cancer in men. Prostate cancer progression is driven by androgen steroid hormones, and delayed by androgen deprivation therapy (ADT). Androgens control transcription by stimulating androgen receptor (AR) activity, yet also control pre-mRNA splicing through less clear mechanisms. Here we find androgens regulate splicing through AR-mediated transcriptional control of the epithelial-specific splicing regulatorESRP2. BothESRP2and its close paralogESRP1are highly expressed in primary prostate cancer. Androgen stimulation induces splicing switches in many endogenous ESRP2-controlled mRNA isoforms, including a key splicing switch in the metastatic regulatorFLNBwhich is associated with disease relapse.ESRP2expression in clinical prostate cancer is repressed by ADT, which may thus inadvertently dampen epithelial splice programmes. Supporting this,FLNBsplicing was reciprocally switched by the AR antagonist bicalutamide (Casodex®). Our data reveal a new mechanism of splicing control in prostate cancer with important implications for metastatic disease progression.Key pointsTranscriptional regulation of ESRP2 by the androgen receptor controls splice isoform patterns in prostate cancer cells.Splicing switches regulated by the androgen-ESRP2 axis include a splice isoform in theFLNBgene that is a known metastatic driver.Both ESRP1 and ESRP2 are highly expressed in prostate cancer tissue.Ectopic expression of ESRP1 and 2 inhibits prostate cancer cell growth.By repressing ESRP2 expression androgen deprivation therapy (ADT) may dampen epithelial splicing programmes to inadvertently prime disease progression towards metastasis.
Immunosuppressive regimens based on Cyclophospamide or Calcineurin inhibitors: Comparison of their effect in the long term outcome of Primary Membranous Nephropathy
Authors: Stangou Maria , Marinaki Smaragdi , Papachristou Evangelos , Kolovou Chrysanthi , Sambani Erasmia , Zerbala Synodi , Papadea Panagiota , Balafa Olga , Rapsomanikis Karolos-Pavlos , Andrikos Aimilios , Manolakaki Panagiota , Papadopoulou Dorothea , Mitsopoulos Efstathios , Andronikidi Paraskevi-Eyh , Choulitoudi Vasiliki , Moustakas George , Galitsiou Dimitra , Dafnis Eugene , Stylianou Constantinos , Stefanidis Ioannis , Golfinopoulos Spyridon , Panagoutsos Stylianos , Tsilivigkou Maria , Papadogianakis Apostolos , Tzanakis Ioannis , Sioulis Athanasios , Vlachakos Dimitrios , Grapsa Eirini , Spaia Sophia , Kaperonis Nikolaos , Paliouras Christos , Dioudis Christos , Papoulidou Fani , Apostolou Theofanis , Iatrou Christos , Boletis Ioannis , Goumenos Dimitrios , Papagianni Aikaperini 
Published: May 2019

AbstractManagement of the Primary Membranous Nephropathy (PMN) usually involves administration of immunosuppressives. Cyclophosphamide (Cyclo) and Calcineurin Inhibitors (CNIs) are both widely used but only limited data exist to compare their efficacy in long term follow-up.Aimof the present study was to estimate and compare long term effects of Cyclo and CNIs in patients with PMN.Patients-MethodsClinical data, histologic findings and long term outcome were retrospectively studied. The response to treatment and rate of relapse was compared between patients treated with CNIs or Cyclo based immunosuppressive regimens.ResultsTwenty three centers participated in the study, with 752 PMN patients (Mean age 53.4(14-87)yrs, M/F 467/285), followed for 10.1±5.7 years. All patients were initially treated with Renin Angiotensin Aldosterone System inhibitors (RAASi) for at least 6 months. Based on their response and tolerance to initial treatment, patients were divided into 3 groups, group I with spontaneous remission, who had no further treatment, group II, continued on RAASi only, and group III on RAASi+immunosuppression. Immunosuppressive regimes were mainly based on CNIs or Cyclo. Frequent relapses and failure to treatment were more common between patients who had started on CNIs (n=381) compared to those initially treated with Cyclo (n=110), relapse rate: 25.2% vs. 6.4%, p<0.0001, and no response rate: 22.5% vs. 13.6%, p=0.04, respectively.ConclusionsLong term follow up showed that administration of Cyclo in PMN is followed by better preservation of renal function, increased response rate and less frequent relapses, compared to CNIs.
Optimizing the fitting initial condition for the parallel intrinsic diffusivity in NODDI: An extensive empirical evaluation
Authors: Guerrero Jose M. , Adluru Nagesh , Bendlin Barbara B. , Goldsmith H. Hill , Schaefer Stacey M. , Davidson Richard J. , Kecskemeti Steven R. , Zhang Hui , Alexander Andrew L. 
Published: May 2019

AbstractPurposeNODDI is widely used in parameterizing microstructural brain properties. The model includes three signal compartments: intracellular, extracellular, and free water. The neurite compartment intrinsic parallel diffusivity (d‖) is set to 1.7 µm2⋅ms−1, though the effects of this assumption have not been extensively explored. This work seeks to optimize d‖ by minimizing the model residuals.MethodsThe model residuals were evaluated in function of d‖ over the range from 0.5 to 3.0 µm2⋅ms−1. This was done with respect to tissue type (i.e., white matter versus gray matter), sex, age (infancy to late adulthood), and diffusion-weighting protocol (maximum b-value). Variation in the estimated parameters with respect to d‖ was also explored.ResultsResults show the optimum d‖ is significantly lower for gray matter relative to 1.7 µm2⋅ms−1 and to white matter. Infants showed significantly decreased optimum d‖ in gray and white matter. Minor optimum d‖ differences were observed versus diffusion protocol. No significant sex effects were observed. Additionally, changes in d‖ resulted in significant changes to the estimated NODDI parameters.ConclusionFuture implementations of NODDI would benefit from d‖ optimization, particularly when investigating young populations and/or gray matter.
How do brassinosteroids activate their receptors?
Authors: Moffett Alexander S. , Shukla Diwakar 
Published: May 2019

AbstractBrassinosteroids (BRs) are an important class of plant growth hormones which signal through BRI1 and BAK1, leucine-rich repeat receptor-like kinases (LRR-RLKs). When bound to the BRI1 island domain, BRs act as a “molecular glue”, mediating interactions between BRI1 and BAK1 extracellular domains. However, it is unclear how much other factors contribute to BR-induced BRI1-BAK1 association, including stabilization of the BRI1 island domain and large conformational changes in BRI1. We use several molecular dynamics simulation-based methods to explore the contributions of each mechanism to BR-dependent Arabidopsis thaliana BRI1-BAK1 association. We find that specific BR interactions make major contributions to BRI1-BAK1 association free energy. BR binding stabilizes the BRI1 island domain, while BRI1 undergoes a large conformational change to form a secondary interface with BAK1. These results suggest that each mechanism plays a part in BR signal transduction while raising questions about the functional role of conformational dynamics in other LRR-RLKs.
The neural basis of tadpole transport in poison frogs
Authors: Fischer Eva K. , Roland Alexandre B. , Moskowitz Nora A. , Tapia Elicio E. , Summers Kyle , Coloma Luis A. , O’Connell Lauren A. 
Published: May 2019

AbstractParental care has evolved repeatedly and independently across animals. While the ecological and evolutionary significance of parental behaviour is well recognized, underlying mechanisms remain poorly understood. We took advantage of behavioural diversity across closely related species of South American poison frogs (Family Dendrobatidae) to identify neural correlates of parental behaviour shared across sexes and species. We characterized differences in neural induction, gene expression in active neurons, and activity of specific neuronal types in three species with distinct parental care patterns: male uniparental, female uniparental, and biparental. We identified the medial pallium and preoptic area as core brain regions associated with parental care, independent of sex and species. Identification of neurons active during parental care confirms a role for neuropeptides associated with care in other vertebrates as well as identifying novel candidates. Our work is the first to explore neural and molecular mechanisms of parental care in amphibians and highlights the potential for mechanistic studies in closely related but behaviourally variable species to build a more complete understanding of how shared principles and species-specific diversity govern parental care and other social behaviour.
A megaplasmid family responsible for dissemination of multidrug resistance inPseudomonas
Authors: Cazares Adrian , Moore Matthew P. , Grimes Macauley , Emond-Rhéault Jean-Guillaume , Wright Laura L. , Pongchaikul Pisut , Santanirand Pitak , Levesque Roger C. , Fothergill Joanne L. , Winstanley Craig 
Published: May 2019

AbstractMultidrug resistance (MDR) represents a global threat to health. Although plasmids can play an important role in the dissemination of MDR, they have not been commonly linked to the emergence of antimicrobial resistance in the pathogenPseudomonas aeruginosa. We used whole genome sequencing to characterize a collection ofP. aeruginosaclinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicated that 1) these large plasmids comprise a family present in different members of thePseudomonasgenus and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the pangenome of the megaplasmid family is highly flexible and diverse, comprising a substantial core genome (average of 48% of plasmid genes), but with individual members carrying large numbers of unique genes. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.FundingThis work was supported by the International Pseudomonas Genomics Consortium, funded by Cystic Fibrosis Canada [RCL]; and the Secretaría de Educación, Ciencia, Tecnología e Innovación (SECTEI), Mexico [AC].
Single-cell Lineage Tracing by Integrating CRISPR-Cas9 Mutations with Transcriptomic Data
Authors: Zafar Hamim , Lin Chieh , Bar-Joseph Ziv 
Published: May 2019

AbstractRecent studies combine two novel technologies, single-cell RNA-sequencing and CRISPR-Cas9 barcode editing for elucidating developmental lineages at the whole organism level. While these studies provided several insights, they face several computational challenges. First, lineages are reconstructed based on noisy and often saturated random mutation data. Additionally, due to the randomness of the mutations, lineages from multiple experiments cannot be combined to reconstruct a consensus lineage tree. To address these issues we developed a novel method, LinTIMaT, which reconstructs cell lineages using a maximum-likelihood framework by integrating mutation and expression data. Our analysis shows that expression data helps resolve the ambiguities arising in when lineages are inferred based on mutations alone, while also enabling the integration of different individual lineages for the reconstruction of a consensus lineage tree. LinTIMaT lineages have better cell type coherence, improve the functional significance of gene sets and provide new insights on progenitors and differentiation pathways.
Ab-initiodiscovery of tumoricidal oligonucleotides in a DNA sequencing machine
Authors: Mamet Noam , Rusinek Itai , Harari Gil , Shapira Zvi , Amir Yaniv , Lavi Erez , Zamir Adva , Borovsky Noam , Joseph Noah , Motin Maria , Saban Dekel , Bachelet Ido 
Published: May 2019

AbstractWe describe a technique for the rapidab-initiodiscovery of target-tailored tumoricidal DNA oligonucleotides inside an Illumina sequencing chip. By sequencing oligonucleotide pools we generate a physical microfluidic map of hundreds of millions of potential oligo clusters, in which every cluster is mapped to a specific set of spatial coordinates. Tumor cells, pre-loaded with a fluorogenic reporter of apoptosis, are then injected into the chip and monitored over time. Apoptotic tumor cells are identified and analyzed across the entire map, automatically revealing the coordinates of oligos that induced this effect. We demonstrate this method by identifying, within just a few hours, new oligos capable of directly and selectively inducing apoptosis in primary human tumor cells. Such a major capability could lead to a new paradigm of personalized cancer therapy.