Πέμπτη, 4 Φεβρουαρίου 2016

Gastric Helicobacter pylori Infection Affects Local and Distant Microbial Populations and Host Responses

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Sabine Kienesberger, Laura M. Cox, Alexandra Livanos, Xue-Song Zhang, Jennifer Chung, Guillermo I. Perez-Perez, Gregor Gorkiewicz, Ellen L. Zechner, Martin J. Blaser
Helicobacter pylori is a late-in-life human pathogen with potential early-life benefits. Although H. pylori is disappearing from the human population, little is known about the influence of H. pylori on the host’s microbiota and immunity. Studying the interactions of H. pylori with murine hosts over 6 months, we found stable colonization accompanied by gastric histologic and antibody responses. Analysis of gastric and pulmonary tissues revealed increased expression of multiple immune response genes, conserved across mice and over time in the stomach and more transiently in the lungs. Moreover, H. pylori infection led to significantly different population structures in both the gastric and intestinal microbiota. These studies indicate that H. pylori influences the microbiota and host immune responses not only locally in the stomach, but distantly as well, affecting important target organs.

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Teaser

Kienesberger et al. utilize a mouse model to study H. pylori infections over 6 months. They report that H. pylori significantly affects the population structure of the gastric and intestinal microbiota. The infection alters gastric immune and inflammatory responses and causes distant effects via altered hormones and immunity.


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Subcellular Imbalances in Synaptic Activity

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Naoya Takahashi, Chiaki Kobayashi, Tomoe Ishikawa, Yuji Ikegaya
The dynamic interactions between synaptic excitation and inhibition (E/I) shape membrane potential fluctuations and determine patterns of neuronal outputs; however, the spatiotemporal organization of these interactions within a single cell is poorly understood. Here, we investigated the relationship between local synaptic excitation and global inhibition in hippocampal pyramidal neurons using functional dendrite imaging in combination with whole-cell recordings of inhibitory postsynaptic currents. We found that the sums of spine inputs over dendritic trees were counterbalanced by a proportional amount of somatic inhibitory inputs. This online E/I correlation was maintained in dendritic segments that were longer than 50 μm. However, at the single spine level, only 22% of the active spines were activated with inhibitory inputs. This inhibition-coupled activity occurred mainly in the spines with large heads. These results shed light on a microscopic E/I-balancing mechanism that operates at selected synapses and that may increase the accuracy of neural information.

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Takahashi et al. find that, although global GABAergic inhibition counterbalances synaptic excitation on dendritic trees, this balance breaks down at the microscopic level since only large synapses are excited by this inhibition.


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Ligand-Induced Receptor-like Kinase Complex Regulates Floral Organ Abscission in Arabidopsis

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Xiangzong Meng, Jinggeng Zhou, Jiao Tang, Bo Li, Marcos V.V. de Oliveira, Jijie Chai, Ping He, Libo Shan
Abscission is a developmental process that enables plants to shed unwanted organs. In Arabidopsis, the floral organ abscission is regulated by a signaling pathway consisting of the peptide ligand IDA, the receptor-like kinases (RLKs) HAE and HSL2, and a downstream MAP kinase (MAPK) cascade. However, little is known about the molecular link between ligand-receptor pairs and intracellular signaling. Here, we report that the SERK family RLKs function redundantly in regulating floral organ abscission downstream of IDA and upstream of the MAPK cascade. IDA induces heterodimerization of HAE/HSL2 and SERKs, which transphosphorylate each other. The SERK3 residues mediating its interaction with the immune receptor FLS2 and the brassinosteroid receptor BRI1 are also required for IDA-induced HAE/HSL2-SERK3 interaction, suggesting SERKs serve as co-receptors of HAE/HSL2 in perceiving IDA. Thus, our study reveals the signaling activation mechanism in floral organ abscission by IDA-induced HAE/HSL2-SERK complex formation accompanied by transphosphorylation.

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Meng et al. show that the Arabidopsis SERK family receptor-like kinases (RLKs) regulate floral organ abscission via the ligand-induced interaction and transphosphorylation with the HAE and HSL2 receptors and reveal the receptor activation mechanism in an important plant developmental process.


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Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Tony Gutschner, Monika Haemmerle, Giannicola Genovese, Giulio F. Draetta, Lynda Chin
CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ), or homology-directed repair (HDR). While HDR is absent in G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell-cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1, resulting in a cell-cycle-tailored expression with low levels in G1 but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the rate of HDR by up to 87% compared to wild-type Cas9. Future developments may enable high-resolution expression of genome engineering proteins, which might increase HDR rates further, and may contribute to a better understanding of DNA repair pathways due to spatiotemporal control of DNA damage induction.

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Using a protein engineering approach, Gutschner et al. generate a Cas9 fusion protein to control genome editing in time and space. Coupling Cas9 protein levels to cell-cycle dynamics results in higher site-specific integration events.


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Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically Active in Processing Lipid

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Daniel Fraher, Andrew Sanigorski, Natalie A. Mellett, Peter J. Meikle, Andrew J. Sinclair, Yann Gibert
The role of lipids in providing energy and structural cellular components during vertebrate development is poorly understood. To elucidate these roles further, we visualized lipid deposition and examined expression of key lipid-regulating genes during zebrafish embryogenesis. We also conducted a semiquantitative analysis of lipidomic composition using liquid chromatography (LC)-mass spectrometry. Finally, we analyzed processing of boron-dipyrromethene (BODIPY) lipid analogs injected into the yolk using thin layer chromatography. Our data reveal that the most abundant lipids in the embryo are cholesterol, phosphatidylcholine, and triglyceride. Moreover, we demonstrate that lipids are processed within the yolk prior to mobilization to the embryonic body. Our data identify a metabolically active yolk and body resulting in a dynamic lipid composition. This provides a foundation for studying lipid biology during normal or pharmacologically compromised embryogenesis.

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Fraher et al. develop a method to identify lipids separately in the zebrafish yolk and the embryonic body. Lipid usage and content are dynamic in the yolk and embryo during development. The yolk actively processes lipids prior to migration to the body during early embryogenesis.


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ATRIP Deacetylation by SIRT2 Drives ATR Checkpoint Activation by Promoting Binding to RPA-ssDNA

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Hui Zhang, PamelaSara E. Head, Waaqo Daddacha, Seong-Hoon Park, Xingzhe Li, Yunfeng Pan, Matthew Z. Madden, Duc M. Duong, Maohua Xie, Bing Yu, Matthew D. Warren, Elaine A. Liu, Vishal R. Dhere, Chunyang Li, Ivan Pradilla, Mylin A. Torres, Ya Wang, William S. Dynan, Paul W. Doetsch, Xingming Deng, Nicholas T. Seyfried, David Gius, David S. Yu
The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase checkpoint pathway maintains genome integrity; however, the role of the sirtuin 2 (SIRT2) acetylome in regulating this pathway is not clear. We found that deacetylation of ATR-interacting protein (ATRIP), a regulatory partner of ATR, by SIRT2 potentiates the ATR checkpoint. SIRT2 interacts with and deacetylates ATRIP at lysine 32 (K32) in response to replication stress. SIRT2 deacetylation of ATRIP at K32 drives ATR autophosphorylation and signaling and facilitates DNA replication fork progression and recovery of stalled replication forks. K32 deacetylation by SIRT2 further promotes ATRIP accumulation to DNA damage sites and binding to replication protein A-coated single-stranded DNA (RPA-ssDNA). Collectively, these results support a model in which ATRIP deacetylation by SIRT2 promotes ATR-ATRIP binding to RPA-ssDNA to drive ATR activation and thus facilitate recovery from replication stress, outlining a mechanism by which the ATR checkpoint is regulated by SIRT2 through deacetylation.

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Zhang et al. demonstrate that ATRIP deacetylation at conserved lysine 32 by SIRT2 promotes ATR-ATRIP binding to RPA-ssDNA to drive ATR activation and thus facilitate recovery from replication stress.


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PRMT7 Preserves Satellite Cell Regenerative Capacity

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Roméo Sébastien Blanc, Gillian Vogel, Taiping Chen, Colin Crist, Stéphane Richard
Regeneration of skeletal muscle requires the continued presence of quiescent muscle stem cells (satellite cells), which become activated in response to injury. Here, we report that whole-body protein arginine methyltransferase PRMT7−/− adult mice and mice conditionally lacking PRMT7 in satellite cells using Pax7-CreERT2 both display a significant reduction in satellite cell function, leading to defects in regenerative capacity upon muscle injury. We show that PRMT7 is preferentially expressed in activated satellite cells and, interestingly, PRMT7-deficient satellite cells undergo cell-cycle arrest and premature cellular senescence. These defects underlie poor satellite cell stem cell capacity to regenerate muscle and self-renew after injury. PRMT7-deficient satellite cells express elevated levels of the CDK inhibitor p21CIP1 and low levels of its repressor, DNMT3b. Restoration of DNMT3b in PRMT7-deficient cells rescues PRMT7-mediated senescence. Our findings define PRMT7 as a regulator of the DNMT3b/p21 axis required to maintain muscle stem cell regenerative capacity.

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Decline of muscle stem cell function is associated with both intrinsic and extrinsic factors. Blanc et al. show that the protein arginine methyltransferase PRMT7 regulates the p21/DNMT3b axis in muscle stem cells to preserve their intrinsic capacity to self-renew and to fully regenerate muscles in adult mice.


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Loss of Adipose Fatty Acid Oxidation Does Not Potentiate Obesity at Thermoneutrality

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Jieun Lee, Joseph Choi, Susan Aja, Susanna Scafidi, Michael J. Wolfgang
Ambient temperature affects energy intake and expenditure to maintain homeostasis in a continuously fluctuating environment. Here, mice with an adipose-specific defect in fatty acid oxidation (Cpt2A−/−) were subjected to varying temperatures to determine the role of adipose bioenergetics in environmental adaptation and body weight regulation. Microarray analysis of mice acclimatized to thermoneutrality revealed that Cpt2A−/− interscapular brown adipose tissue (BAT) failed to induce the expression of thermogenic genes such as Ucp1 and Pgc1α in response to adrenergic stimulation, and increasing ambient temperature exacerbated these defects. Furthermore, thermoneutral housing induced mtDNA stress in Cpt2A−/− BAT and ultimately resulted in a loss of interscapular BAT. Although the loss of adipose fatty acid oxidation resulted in clear molecular, cellular, and physiologic deficits in BAT, body weight gain and glucose tolerance were similar in control and Cpt2A−/− mice in response to a high-fat diet, even when mice were housed at thermoneutrality.

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Lee et al. show that a loss of adipose fatty acid oxidation (FAO) at thermoneutrality results in defective induction of thermogenic genes and mtDNA stress in BAT. Long-term housing of FAO-deficient mice results in a loss of interscapular BAT; however, body weight gain and glucose tolerance were unaffected.


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The Gut Microbiota Modulates Energy Metabolism in the Hibernating Brown Bear Ursus arctos

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Felix Sommer, Marcus Ståhlman, Olga Ilkayeva, Jon M. Arnemo, Jonas Kindberg, Johan Josefsson, Christopher B. Newgard, Ole Fröbert, Fredrik Bäckhed
Hibernation is an adaptation that helps many animals to conserve energy during food shortage in winter. Brown bears double their fat depots during summer and use these stored lipids during hibernation. Although bears seasonally become obese, they remain metabolically healthy. We analyzed the microbiota of free-ranging brown bears during their active phase and hibernation. Compared to the active phase, hibernation microbiota had reduced diversity, reduced levels of Firmicutes and Actinobacteria, and increased levels of Bacteroidetes. Several metabolites involved in lipid metabolism, including triglycerides, cholesterol, and bile acids, were also affected by hibernation. Transplantation of the bear microbiota from summer and winter to germ-free mice transferred some of the seasonal metabolic features and demonstrated that the summer microbiota promoted adiposity without impairing glucose tolerance, suggesting that seasonal variation in the microbiota may contribute to host energy metabolism in the hibernating brown bear.

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Sommer et al. show that the microbiota and serum metabolites in brown bears differ seasonally between hibernation and active phase. Colonization of mice with a bear microbiota promoted increased adiposity. These findings suggest that seasonal microbiota variation may contribute to metabolism of the hibernating brown bear.


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Synaptophysin 1 Clears Synaptobrevin 2 from the Presynaptic Active Zone to Prevent Short-Term Depression

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Rajit Rajappa, Anne Gauthier-Kemper, Daniel Böning, Jana Hüve, Jürgen Klingauf
Release site clearance is an important process during synaptic vesicle (SV) recycling. However, little is known about its molecular mechanism. Here we identify self-assembly of exocytosed Synaptobrevin 2 (Syb2) and Synaptophysin 1 (Syp1) by homo- and hetero-oligomerization into clusters as key mechanisms mediating release site clearance for preventing cis-SNARE complex formation at the active zone (AZ). In hippocampal neurons from Syp1 knockout mice, neurons expressing a monomeric Syb2 mutant, or after acute block of the ATPase N-ethylmaleimide-sensitive factor (NSF), responsible for cis-SNARE complex disassembly, we found strong frequency-dependent short-term depression (STD), whereas retrieval of Syb2 by compensatory endocytosis was only affected weakly. Defects in Syb2 endocytosis were stimulus- and frequency-dependent, indicating that Syp1 is not essential for Syb2 retrieval, but for its efficient clearance upstream of endocytosis. Our findings identify an SV protein as a release site clearance factor.

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Rajappa et al. identify self-assembly of exocytosed Synaptobrevin 2 and Synaptophysin 1 by homo- and hetero-oligomerization at endocytic zones as a key mechanism mediating release site clearance. Both prevent cis-SNARE complex formation at the active zone and ensuing short-term depression.


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RNF20 Links Histone H2B Ubiquitylation with Inflammation and Inflammation-Associated Cancer

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Ohad Tarcic, Ioannis S. Pateras, Tomer Cooks, Efrat Shema, Julia Kanterman, Hadas Ashkenazi, Hana Boocholez, Ayala Hubert, Ron Rotkopf, Michal Baniyash, Eli Pikarsky, Vassilis G. Gorgoulis, Moshe Oren
Factors linking inflammation and cancer are of great interest. We now report that the chromatin-targeting E3 ubiquitin ligase RNF20/RNF40, driving histone H2B monoubiquitylation (H2Bub1), modulates inflammation and inflammation-associated cancer in mice and humans. Downregulation of RNF20 and H2Bub1 favors recruitment of p65-containing nuclear factor κB (NF-κB) dimers over repressive p50 homodimers and decreases the heterochromatin mark H3K9me3 on a subset of NF-κB target genes to augment their transcription. Concordantly, RNF20+/− mice are predisposed to acute and chronic colonic inflammation and inflammation-associated colorectal cancer, with excessive myeloid-derived suppressor cells (MDSCs) that may quench antitumoral T cell activity. Notably, colons of human ulcerative colitis patients, as well as colorectal tumors, reveal downregulation of RNF20/RNF40 and H2Bub1 in both epithelium and stroma, supporting the clinical relevance of our tissue culture and mouse model findings.

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Ubiquitination of histone H2B (H2Bub1), primarily by the E3 ligase RNF20, is reduced in many advanced cancers. Tarcic et al. report that downregulation of RNF20 and H2Bub1 promotes chronic colonic inflammation and inflammation-associated colorectal cancer in mice and humans, partly by augmenting NF-kB activity and attenuating the antitumoral T cell response.


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Regulation of Memory Formation by the Transcription Factor XBP1

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Gabriela Martínez, René L. Vidal, Pablo Mardones, Felipe G. Serrano, Alvaro O. Ardiles, Craig Wirth, Pamela Valdés, Peter Thielen, Bernard L. Schneider, Bredford Kerr, Jose L. Valdés, Adrian G. Palacios, Nibaldo C. Inestrosa, Laurie H. Glimcher, Claudio Hetz
Contextual memory formation relies on the induction of new genes in the hippocampus. A polymorphism in the promoter of the transcription factor XBP1 was identified as a risk factor for Alzheimer’s disease and bipolar disorders. XBP1 is a major regulator of the unfolded protein response (UPR), mediating adaptation to endoplasmic reticulum (ER) stress. Using a phenotypic screen, we uncovered an unexpected function of XBP1 in cognition and behavior. Mice lacking XBP1 in the nervous system showed specific impairment of contextual memory formation and long-term potentiation (LTP), whereas neuronal XBP1s overexpression improved performance in memory tasks. Gene expression analysis revealed that XBP1 regulates a group of memory-related genes, highlighting brain-derived neurotrophic factor (BDNF), a key component in memory consolidation. Overexpression of BDNF in the hippocampus reversed the XBP1-deficient phenotype. Our study revealed an unanticipated function of XBP1 in cognitive processes that is apparently unrelated to its role in ER stress.

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Using gain- and loss-of-function approaches, Martinez et al. demonstrate that XBP1, a master regulator of the unfolded protein response (UPR), regulates learning and memory-related processes. This function of XBP1 in the nervous system involves the control of BDNF expression in the hippocampus.


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The Prozone Effect Accounts for the Paradoxical Function of the Cdk-Binding Protein Suc1/Cks

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Sang Hoon Ha, Sun Young Kim, James E. Ferrell
Previous work has shown that Suc1/Cks proteins can promote the hyperphosphorylation of primed Cdk1 substrates through the formation of ternary Cdk1-Cks-phosphosubstrate complexes. This raises the possibility that Cks proteins might be able to both facilitate and interfere with hyperphosphorylation through a mechanism analogous to the prozone effect in antigen-antibody interactions, with substoichiometric Cks promoting the formation of Cdk1-Cks-phosphosubstrate complexes and suprastoichiometric Cks instead promoting the formation of Cdk1-Cks and Cks-phosphosubstrate complexes. We tested this hypothesis through a combination of theory, proof-of-principle experiments with oligonucleotide annealing, and experiments on the interaction of Xenopus cyclin B1-Cdk1-Cks2 with Wee1A in vitro and in Xenopus extracts. Our findings help explain why both Cks under-expression and overexpression interfere with cell-cycle progression and provide insight into the regulation of the Cdk1 system.

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Ha et al. find that Cks2 can both promote and repress Cdk1 phosphorylation of Wee1A in a biphasic manner through its bivalent adaptor-like function. This phenomenon is related to the prozone effect in antigen-antibody interaction and to squelching in transcription.


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Gut-Colonizing Bacteria Promote C. elegans Innate Immunity by Producing Nitric Oxide

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Yi Xiao, Fang Liu, Zhigang Zhang, Jie Tang, Cheng-Gang Zou, Ke-Qin Zhang
Many commensal bacteria in the gut are beneficial to the host immune system, but the underlying mechanisms are largely unclear. Using culture-independent Illumina MiSeq sequencing of the bacterial 16S rRNA gene amplicons, we show that bacterial diversity in the intestine of Caenorhabditis elegans, the free-living nematode, is distinct from that in soil. Of these bacteria, Bacillus subtilis is the most prominent species in the worm gut. We demonstrate that B. subtilis confers worm resistance to infection by pathogenic bacteria, such as Pseudomonas aeruginosa, Salmonella enterica, and Enterococcus faecalis, by producing nitric oxide (NO). Deletion of the nos gene, which encodes an NO synthase, reduces the protective effect. NO promotes innate immune responses to P. aeruginosa PA14 by activating a conserved p38 mitogen protein kinase (MAPK) in C. elegans. Our work provides an example of antagonism of commensal bacteria against pathogens and illustrates the importance of commensal bacteria in host immunity.

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B. subtilis is a symbiont that resides in the gut of C. elegans and generates nitric oxide that is essential for the host. Xiao et al. demonstrate that nitric oxide promotes defense against pathogenic bacteria by activating p38 MAPK, demonstrating the importance of commensal bacteria in host immunity.


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5-Hydroxymethylcytosine Marks Sites of DNA Damage and Promotes Genome Stability

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Georgia Rose Kafer, Xuan Li, Takuro Horii, Isao Suetake, Shoji Tajima, Izuho Hatada, Peter Mark Carlton
5-hydroxymethylcytosine (5hmC) is a DNA base created during active DNA demethylation by the recently discovered TET enzymes. 5hmC has essential roles in gene expression and differentiation. Here, we demonstrate that 5hmC also localizes to sites of DNA damage and repair. 5hmC accumulates at damage foci induced by aphidicolin and microirradiation and colocalizes with major DNA damage response proteins 53BP1 and γH2AX, revealing 5hmC as an epigenetic marker of DNA damage. Deficiency for the TET enzymes eliminates damage-induced 5hmC accumulation and elicits chromosome segregation defects in response to replication stress. Our results indicate that the TET enzymes and 5hmC play essential roles in ensuring genome integrity.

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Kafer et al. demonstrate that DNA damage causes the modified DNA base 5-hydroxymethylcytosine (5hmC) to become locally enriched over broad chromosomal domains and that the TET enzymes promote correct chromosome segregation during replication stress.


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Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): J.J. David Ho, Miling Wang, Timothy E. Audas, Deukwoo Kwon, Steven K. Carlsson, Sara Timpano, Sonia L. Evagelou, Shaun Brothers, Mark L. Gonzalgo, Jonathan R. Krieger, Steven Chen, James Uniacke, Stephen Lee
Protein concentrations evolve under greater evolutionary constraint than mRNA levels. Translation efficiency of mRNA represents the chief determinant of basal protein concentrations. This raises a fundamental question of how mRNA and protein levels are coordinated in dynamic systems responding to physiological stimuli. This report examines the contributions of mRNA abundance and translation efficiency to protein output in cells responding to oxygen stimulus. We show that changes in translation efficiencies, and not mRNA levels, represent the major mechanism governing cellular responses to [O2] perturbations. Two distinct cap-dependent protein synthesis machineries select mRNAs for translation: the normoxic eIF4F and the hypoxic eIF4FH. O2-dependent remodeling of translation efficiencies enables cells to produce adaptive translatomes from preexisting mRNA pools. Differences in mRNA expression observed under different [O2] are likely neutral, given that they occur during evolution. We propose that mRNAs contain translation efficiency determinants for their triage by the translation apparatus on [O2] stimulus.

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Ho et al. show that cells rely on a switch in mRNA translation efficiency, and not mRNA levels, to alter protein output on O2 stimulus. Two distinct cap-dependent protein synthesis machineries mediate this process: the normoxic eIF4F and the hypoxic eIF4FH. The O2-regulated eIF4F and eIF4FH generate complex and adaptive translatomes.


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RAB-5- and DYNAMIN-1-Mediated Endocytosis of EFF-1 Fusogen Controls Cell-Cell Fusion

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Ksenia Smurova, Benjamin Podbilewicz
Cell-cell fusion plays essential roles during fertilization and organogenesis. Previous studies in C. elegans led to the identification of the eukaryotic fusion protein (EFF-1 fusogen), which has structural homology to class II viral fusogens. Transcriptional repression of EFF-1 ensures correct fusion fates, and overexpression of EFF-1 results in embryonic lethality. EFF-1 must be expressed on the surface of both fusing cells; however, little is known regarding how cells regulate EFF-1 surface exposure. Here, we report that EFF-1 is actively removed from the plasma membrane of epidermal cells by dynamin- and RAB-5-dependent endocytosis and accumulates in early endosomes. EFF-1 was transiently localized to apical domains of fusion-competent cells. Effective cell-cell fusion occurred only between pairs of cell membranes in which EFF-1 localized. Downregulation of dynamin or RAB-5 caused EFF-1 mislocalization to all apical membrane domains and excessive fusion. Thus, internalization of EFF-1 is a safety mechanism preventing excessive cell fusion.

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Smurova and Podbilewicz find that RAB-5 and dynamin-mediated endocytosis removes the fusogen EFF-1 from the plasma membrane and serves as a negative regulator of cell-cell fusion in C. elegans embryos. Thus, dynamic and transient localization of EFF-1 on the apical plasma membranes is sufficient to merge neighboring cells.


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ARHGAP12 Functions as a Developmental Brake on Excitatory Synapse Function

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): W. Ba, M.M. Selten, J. van der Raadt, H. van Veen, L.-L. Li, M. Benevento, A.R. Oudakker, R.S.E. Lasabuda, S.J. Letteboer, R. Roepman, R.J.A. van Wezel, M.J. Courtney, H. van Bokhoven, N. Nadif Kasri
The molecular mechanisms that promote excitatory synapse development have been extensively studied. However, the molecular events preventing precocious excitatory synapse development so that synapses form at the correct time and place are less well understood. Here, we report the functional characterization of ARHGAP12, a previously uncharacterized Rho GTPase-activating protein (RhoGAP) in the brain. ARHGAP12 is specifically expressed in the CA1 region of the hippocampus, where it localizes to the postsynaptic compartment of excitatory synapses. ARHGAP12 negatively controls spine size via its RhoGAP activity and promotes, by interacting with CIP4, postsynaptic AMPA receptor endocytosis. Arhgap12 knockdown results in precocious maturation of excitatory synapses, as indicated by a reduction in the proportion of silent synapses. Collectively, our data show that ARHGAP12 is a synaptic RhoGAP that regulates excitatory synaptic structure and function during development.

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Ba et al. find that the Rac1 GAP, ARHGAP12, coordinates dendritic spine morphology and synaptic strength via its GAP activity and interaction with CIP4, respectively. ARHGAP12 limits synapse maturation by restricting silent synapses converting to functional synapses in the developing hippocampus.


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Work Done by Titin Protein Folding Assists Muscle Contraction

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Jaime Andrés Rivas-Pardo, Edward C. Eckels, Ionel Popa, Pallav Kosuri, Wolfgang A. Linke, Julio M. Fernández
Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin immunoglobulin (Ig) domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6–8 pN. We use single-molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but as yet unrecognized, contributor to the force generated by a contracting muscle.

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Titin, the largest protein in the human body, is responsible for muscle elasticity, while myosin motors are thought to provide the sole source of contractile energy. Here, we find that titin unfolding occurs at forces below 10 pN and that subsequent refolding can produce substantial amounts of work that assist in muscle contraction.


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KRAS Engages AGO2 to Enhance Cellular Transformation

Publication date: Available online 4 February 2016
Source:Cell Reports
Author(s): Sunita Shankar, Sethuramasundaram Pitchiaya, Rohit Malik, Vishal Kothari, Yasuyuki Hosono, Anastasia K. Yocum, Harika Gundlapalli, Yasmine White, Ari Firestone, Xuhong Cao, Saravana M. Dhanasekaran, Jeanne A. Stuckey, Gideon Bollag, Kevin Shannon, Nils G. Walter, Chandan Kumar-Sinha, Arul M. Chinnaiyan
Oncogenic mutations in RAS provide a compelling yet intractable therapeutic target. Using co-immunoprecipitation mass spectrometry, we uncovered an interaction between RAS and Argonaute 2 (AGO2). Endogenously, RAS and AGO2 co-sediment and co-localize in the endoplasmic reticulum. The AGO2 N-terminal domain directly binds the Switch II region of KRAS, agnostic of nucleotide (GDP/GTP) binding. Functionally, AGO2 knockdown attenuates cell proliferation in mutant KRAS-dependent cells and AGO2 overexpression enhances KRASG12V-mediated transformation. Using AGO2−/− cells, we demonstrate that the RAS-AGO2 interaction is required for maximal mutant KRAS expression and cellular transformation. Mechanistically, oncogenic KRAS attenuates AGO2-mediated gene silencing. Overall, the functional interaction with AGO2 extends KRAS function beyond its canonical role in signaling.

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Teaser

Shankar et al. show that RAS interacts with AGO2, a key component of the RNA-silencing machinery. Interaction of oncogenic KRAS with AGO2 in the endoplasmic reticulum inhibits AGO2 function, elevates mutant KRAS protein levels, and enhances cellular transformation. AGO2 is required for maximal KRAS-mediated oncogenesis.


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